Intertidal oyster reefs as a tool for estuarine rehabilitation and rejuvenation of the Virginia Oyster Fishery Instrumentation for environmental monitoring

[From the U.S. Government Printing Office, www.gpo.gov]

FINAL PRODUCT FY'94 Task 21
VIMSNMRC Intertidol Oyster Reeft- Phase 11

Final Report for t4e programentitled:

Intertidal Oyster Reefs as a Tipol foT Estuarine Rehabilitation
and Rejuvenation of th@ Vir&ia Oyster Fishery

submitted to:

The Commonwealth of Virginia
Department of Environmental Quality
Chesapeake Bay and Coastal Programs
Virgirua Coastal Resources Management Program, P.O. Box 10009
629 East Main Street
Richmond, VA 23240-009
attn.: Ms. Laura McKay
Coastal Projects Coordinator

The School of Marine Science and Virginia Institute of Marine Science
The College of William and Mary
Gloucester Point, VA 23062

and

Virginia Marine Resources Conunission
P.O. Box 756
%SW Newport News, VA 23607-0756

Investigators:
Dr. Roger Mann, Professor of Marine Science (SMS VIMS)
Dr. Frank 0. Perkins, Professor of Marine Science (SMS / VIMS)
Dr. James Wesson, Head, Shellfish Replenishment Program (VMRC)
Ian K. Bartol, M.A., Graduate Research Assistant (SMS / VIMS)

date of report submission: February 14, 1996

This report was funded, in part, by the Virginia Department of Environmental Quality's Coastal
i Resources Management Program through Grant # 470ZO287-01 of the National Oceanic and
Atmospheric Administration, Office of Ocean and Coastal Resource Management, under the
Coastal Zone Management Act of 1972, as amended.
Mao

Intertidal Oyster Reefs as a Tool for Estuarine Environmental Rehabilitation
and Rejuvenation of the Virginia Oyster Fishery.

A General Introduction to the Project and this Report.

The oyster Crassostrea virginica is recognized as both a keystone organism in the
ecology of the Chesapeake Bay and the focus of a substantial commercial fishery. Oyster
reefs developed in recent geological time as the current Chesapeake Bay was inundated by
rising sea level. By early Colonial times oyster reefs had become significant geological
and biological features of the Bay - they were also major navigation hazards. Continuing
harvest pressure since Colonial times have resulted in the transformation and degradation
of the oyster reefs to subtidal "footprints" of former reefs that maintain drastically reduced
populations of oysters. Reef degradation has undoubtedly been exacerbated by companion
environmental degradation and an historical lack of consideration for water quality and
natural resource management. Statements concerning over fishing by John Mercer Brooks
over 100 years ago fell on deaf ears, but are now appreciated, if not entirely heeded. The
past three decades have been defined by decline in the fishery production and the oyster
resource under the added insult of two protistan parasites, Perkinsus marinus ("Dermo")
and Haplosporidium nelsoni ("MSX"). Since the disease organisms are active throughout
most of the growing range of the oyster there have been few sanctuaries in which to plant
oysters or in which naturally occurring oysters could be found in appreciable quantities.
Indeed, these parasites have effectively eliminated oysters from many sections of the Bay.
Despite over 30 years of disease activity the native oysters have developed neither
tolerance nor absolute resistance to these diseases, and do not exhibit any recovery in
disease endemic areas in Virginia. The oyster fishery is in severe decline and there is a
recognized and urgent need to restore the oyster resource: not just for the commercial
fishery but also to provide both the benthic filter feeder that is so pivotal to the ecology of
the Bay (see discussion by Newell, 1989; Mann, Burreson and Baker, 1992) and the
physical structure which provides habitat for a multitude of species, including many of
commercial interest.

The Secretary of Natural Resources for Virginia, in conjunction with the
Commissioner of Marine Resources, appointed a Blue Ribbon Panel in the Fall of 1991 to
develop a comprehensive plan for restoration of the Virginia oyster resource and fishery.
Among the recommendations of the panel was a proposal to investigate the construction of
oyster reefs identical to those present in the Bay before Colonial settlement - the
acknowledged "structure" of the oyster community in its undisturbed (by man) form. The
rationale supporting this proposal was quite simple - create an optimal physical
environment and the oyster will settle and grow in a markedly improved manner compared
to that observed on the current subtidal "reefs" or "rocks". A consideration of the temporal
relief from continual predation pressure afforded by an intertidal location supports this
rationale. Indeed, a simple observation of the distribution of oysters surviving on bridge
and pier pilings in the Bay - about the only remaining oyster populations undisturbed by
harvesting - reveals that the vast majority of surviving oysters in terms of size and number
are intertidal. In all fairness it is important to note that the Blue Ribbon Panel was and is
not the only body to suggest the value of intertidal reefs as a option to assist in restoration
of the oyster resource. Such discussions have been widespread in the academic community
and management agencies, often with participation of federal agencies; however, the
concept lacked critical evaluation in the field. This project was designed to effect that
evaluation.

The program began in May of 1993 with the construction of an intertidal oyster
reef in the Piankatank River, Virginia with funds supplied by the Shellfish Replenishment
Program of the Virginia Marine Resources Commission (VMRC). Monitoring and
experimental work on the site were initiated shortly thereafter by faculty, staff and students
of the Virginia Institute of Marine Science (VIMS) and the School of Marine Science,
Virginia Institute of Marine Science (SMSNIMS), College of William and Mary. Work
continued with combined support of the aforementioned state agencies until September 30,
1993. After this date the Coastal Resources Management Program, administered by the
Commonwealth of Virginia Department of Environmental Quality (DEQ), provided
additional funding for the period October 1, 1993 through September 30, 1995. This
document constitutes the final report for activities effected under DEQ funds.

The report is offered as a series of concise sections addressing aspects of
recruitment, growth, mortality, and disease prevalence and intensity in oysters on the
constructed reef system. Each section is purposefully written as a "stand alone" document
to minimize the requirement for cross reference. Prior to those sections it is appropriate to
offer background details relevant to the study site and reef construction.

The Piankatank River has not supported a commercial oyster fishery for over a
decade; however, it has been the site of a successful seed oyster program managed by the
VMRC shellfish replenishment plan, currently under the direction of one of the
investigators (Wesson). A limited number of "rocks" have had applications on a regular
basis with subsequent harvest of the settled seed after one or two summers of exposure (the
summer being the period of oyster settlement). The temporal and spatial nature of
settlement is well documented by a continuing program at VIMS under the direction of a
second investigator (Mann). Oyster spat Ouvenile and newly settled oysters) counts of up
to 1000 individuals per bushel of shell are commonplace in seed oyster dredging from these
maintained and managed areas. The footprints of the former reefs are well documented
from both historical sources (Baylor Surveys), recent surveys (Haven and co-workers in the
early 1980's, all material on file at both VIMS and VMRC), and continuing work by the
VMRC staff. The reefs are not uniform in shape, and are clearly site specific and related to
local circulation. For the current project we eliminated a costly (in dollars and time) study
of optimal size and shape in relation to hydrography by simply using the naturally evolved
shape illustrated in the historical footprint. The lack of a continuing commercial presence,
the proven history of the site as one of good settlement, the comparatively pristine
environment at the site (there is essentially no industrial and very little agricultural
development in the Piankatank watershed - even residential density is low), the strongly
supportive attitude of waterfront residents to environmentally sound management, and the
inclusion of the study site in the National Estuarine Reserve System combined to make this
a unique and attractive site for the continuing study.

There have been continuing monitoring efforts in the Piankatank for a number of
years prior to initiation of the current project. These are important because they provide a
long terin. data set that supports use of the site and provide a solid comparative background
for new information collected in the current project. VIMS maintains a program to
describe temporal and spatial settlement of oysters in the Virginia subestuaries of the
Chesapeake Bay throughout the summer months from June through late September. At
weekly intervals shell strings, twelve clean oyster shells of a standard size suspended on a
galvanized wire, are deployed at selected sites. Upon retrieval the number of newly settled
oyster spat are counted and the mean presented as an index of competent to settle oyster
larvae present. Temperature and salinity data are collected at all sites at weekly intervals.
The four sites in the Piankatank River (see Figure 1), at Three Branches, Burton's Point,
Palace Bar, and Ginney Point, are all within close proximity to the study site. Data is
reported in terms of a biweekly newsletter to industry and state agencies, and an annual
report through the VIMS / Sea Grant Advisory Service Office. The historical data set is

Figure 1: General location of all extant oyster reefs and shell plantings (effected by the VMRC Replenishment Program) within the Piankatank River. The
location of the oldest of the constructed reefs, immediately north of Roane Point, is marked R. The environmental instrument package for weather and tide data
is at the eastern tip of the reef, The remaining reefs, progressing in a downstream direction, are A: Ginney Point, B: Island Bar, C: Palace Bar, D: Blands
Point, E: Herring Rock, F: Cape Toone, G: Stove Point, H: Cape Toone inshore (small lumps). Oyster spatfall monitoring stations are located at Ginney
Point, Palace Bar, Burtons Point (labelled 1, traditionally used as shell plant on hard sand but not a a natural reef), and Three Branches (labelled J in the lee of
Gwynns Island on a former shell plant).

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A
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D
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maintained on the VIMS computers. A further benthic dredge survey is effected in the Fall
of each year immediately prior to opening of the commercial season on October lst. The
purpose is to assess cumulative settlement and survival of young of the year spat from the
prior summer, and size distribution and total oyster count of the larger size classes. Again,
data are reported through the VIMS / Sea Grant Advisory Service Office. These efforts
continued during the period of the current program and will continue after its completion.
The reef for the current project was constructed in the lower Piankatank River by
mass deployment of clean, fossil shell from a barge at high tide. The adjacent areas are
used in "traditional" subtidal shell planting by the VMRC replenishment Program. The
reef covers an area of approximately 100 X 1000 feet (30 X 300 m), is elevated to five feet
(1.6 m) above the current bottom, has between one and two feet (0.3 - 0.6 m) exposed at
low tide, and has a convex cross sectional profile. Appropriate permits for reef
construction and guidelines for marking the reef perimeter (for navigational purposes) were
obtained from local wetlands boards, VMRC, the U.S. Army Corps of Engineers, and the
Coast Guard. The reef is appropriately marked with large pilings at its upstream and
downstream extremities, and by buoys at regular intervals along its perimeter. It is
important to emphasize that the reef was constructed on a bottom that had been cleaned of
resident oysters by dredging, and that no "seeding" of the reef was attempted. The reef
surface was, therefore, only to be populated by natural settlement from the surrounding
environment and no residual oysters containing potential infections of either Perkinsus
marinus or MSX were present at the beginning of the study. The projected costs of
construction, $110,000, was supplied by the VMRC Replenishment Program from state
funds.

Acknowledgments

As mentioned earlier, this project was effected as a joint effort of SMSNIMS and
VMRC. Although the project proposal had three investigators - Mann, Perkins and Wesson
- other individuals also contributed to its success, and their efforts are gratefully
acknowledged.

Ian Bartol, graduate research assistant at SMSNIMS, used these studies as the
basis of his M.A. thesis work. Ian was the functional manager of this program on a day to
day basis. His work was tireless and of the highest quality. He defended his thesis in
January of 1995, and continued to work on the project until completion of this report - to
which he is a major contributor and acknowledged equal investigator on the cover page. A
copy of Ian's bound thesis was provided to DEQ shortly after the successful defense. Ian is
currently pursuing a Ph.D. degree at SMS / VIMS.

Kenneth Walker of VIMS acted research vessel captain throughout the duration of
the program. He maintained a the highest standards of safety in the field, and was always
accommodating to the vagaries of research timetables, tides and weather. Juanita Walker
provided a unique set of eyes and years of skill to the assessment of oyster disease in the
collected material. Paul Hershberg provided support in the field and in the analysis of
Perkinsus. A number of staff and students also braved the Piankatank elements, notably
Ray Morales, Soraya Moein, Juli Harding and Sandra Brooke. Finally Aswani Volety
assisted in final collation of disease data.

Literature cited.

Mann, R., E. M. Burreson and P. K. Baker. 1991. The decline of the Virginia oyster
fishery in Chesapeake Bay: considerations for introduction of a non-endemic
species, Crassostrea gigas (Thunberg). J. Shellfish Res. 10(2):379-388
Newell, R. I. E. 1989. Ecological changes in the Chesapeake Bay: Are they the result
of overharvesting the American Oyster (Crassostrea virginica)? in:
Understanding the Estuary: Advances in Chesapeake Bay Research.
Chesapeake Research Consortium Publication No. 129: 536-546.

Small-scale patterns of recruitment on a constructed intertidal reef. the role of spatial
refugia

Ian K. Bartol & Roger Mann
School of Marine Science, Virginia Institute of Marine Science, College of William and
Mary, Gloucester Point, VA 23062

ABSTRACT
Traditional oyster repletion activities have utilized a two-dimensional approach to
shell (substrate) deployment to attain maximal coverage in subtidal locations with little
consideration for optimal thickness of deployed shell and tidal elevation. We report
observations on settlement and mortality patterns of oysters on a three-dimensional
structure, a constructed oyster reef in the Piankatank River, Virginia, from June of 1993
through September of 1994. The reef was constructed entirely of oyster shell on the
footprint of an historical reef, and extended from 2.5 m below MLW to 0.75 in above
MLW. The footprint covered an area approximately 150 x. 30 m, with numerous sections,
varying from 2 to 20 M2 in area, exposed at low tide. In both intertidal and subtidal
locations settlement was monitored both at the surface of the reef shells and within the
interstices of the reef at depths of 10 cm. Settlement was greater in subtidal locations, and
no difference in settlement intensity between surface and sub-surface environments was
detected. Survivorship, rates along the intertidal-subtidal continuum varied temporally but
were highest at MLW for most of the year. Oysters which attached to subsurface substrate
benefited primarily from refugia from temperature extremes in intertidal locations and from
relief from predation in subtidal environments. We suggest the moderation of these
biological and physical stresses within the reef interstices is instrumental in increased
survival: even minor submergence within the reef provides relief from scorching summer
and freezing winter air temperatures and furnishes protection from predators, most notably
crabs and flatworms. In practical terms these results proffer an important lesson: reef tidal
elevation and substrate thickness both provide microscale refugia for settlement and
survival of early oyster life history stages.

INTRODUCTION
Traditional oyster replenishment prograrps have focused on spreading thin veneers
of substrate suitable for larval settlement over coastal and estuarine bottoms or over
foundations of less ideal substrates to maximize area coverage. In general, such activities
have been driven by the practicality of deploying very large volumes of shell, a commodity
of increasing value, at greatest cost efficiency and with reasonable speed, usually with the
subsequent intent of retrieving either juvenile (seed) oysters or market size oysters. The
end product of this approach, a two-dimensional subtidal carpet of available substrate, has
little resemblance to the intricate, three-dimensional reef corrimunities which often extended
out of the water at low tide and which oysters once formed naturally in the Chesapeake Bay
before man's intervention. In light of rapidly declining oyster stocks in the Chesapeake
Bay, a concerted effort to re-establish natural oyster communities by constructing artificial
reefs has been made by repletion agencies. The ultimate goal of such projects is to
rejuvenate dwindling local oyster populations.
Presently, we know little about constructing reefs which are most advantageous for
oyster settlement and survival. From the cumulative literature on oyster biology, we know
that reefs grew by accretion over time periods of hundreds to thousands of years in a
process aided substantially by the preferred settlement of metamorphically competent oyster
larvae on shells of the adult oyster. We also know that the physical environment, in the
form of currents, tides, and sedimentary forces, practically dictate the perimeter size and the
features of the reef However, we remain ignorant in a number of details, and as a result

there are a number of practical questions, fundamental to an organized approach to reef
construction, which are without answers. For example, for a known location what size
and shape should the reef be, and can we obtain guidance on this question from current
"footprints" of formerly intertidal reefs? Is tidal elevation an important factor to consider
when constructing reefs? Given that shell is a valuable commodity, can other substrates be
used to construct reefs?- How thick should substrate layers be and how should they be
applied?
In this study, we focus on the issues of substrate thickness and tidal elevation. The
interstices of a "natural" reef system provide both physical and biological refugia. Do sub-
surface environments of constructed reefs provide similar benefits and if so to what extent
are these benefits quantitatively important? Do oysters even settle beneath the reef surface
in these constructed environments? "Natural" reefs also have some vertical dimension,
which allows for the settlement and subsequent survival of dense populations of oysters at
distinct bands along the tidal continuum. Does tidal elevation play a vital role in the
survivorship of oysters on artificial reefs? Do oysters settle in higher numbers at tidal
heights where they benefit from refugia? We address these questions by measuring
settlement and post-settlement mortality of Crassostrea virginica at two substrate levels
(reef surface and 10 cm. below reef surface) and at various tidal heights ranging from +30
cm above mean low water (mid/high intertidal zone) to -90 cm. below MLW (mid subtidal
zone) on a constructed intertidal reef.

STUDY SITE AND EXPERMENTAL PROCEDURES
The location selected for this study was a sandbar known as Palace Bar in the
Piankatank River, Virginia. This site once supported a highly productive intertidal reef
system, but at the time of reef construction was completely devoid of live oysters. Prior to
construction, the site was dredged clean of any live material to minimize residual infective
material containing Perkinsus marinus. Water temperature at the site varied from 0.5 - 301
C, salinity ranged from 8 -20 ppt, and tidal range was small (mean range = 36 cm).
The reef was constructed by the deployment of aged oyster shells off barges using a
high pressure hose. The shells were discharged in an area approximately 150 x 30 m,
which were the approximate footprint dimensions of the historical reef. After completion,
the reef consisted of numerous sections, varying from 2 -20 M2 in area, exposed at low
tide, and extended from 2.5 m below mean low water (MLW) to 0.75 m above MLW. The
majority of the reef, however, did not extend much deeper than 1.0 m. below MLW or
much higher than 0.35 m above MELW.
The reef was sampled in both 1993 and 1994. During the 1993 sampling period 2
of the 12 principal intertidal hummocks were focused on: one on the reef periphery
completely exposed to wave action and currents and a second situated near the middle of
the reef partially shielded from wave action and currents. These mounds were sampled
using a transect approach, whereby samples were collected along upstream and
downstream transects on each of the two mounds during each period of sampling.
Transects were carefully marked on the reef to prevent resampling. Along each transect
four tidal heights were considered: 30 cm above NEW, MLW, 45 cm below MLW, and 90
cm below MLW.
During the 1994 sampling period, after data from the previous year were analyzed
and we had a preliminary understanding of the reef system, a randomized approach was
used which was more geographically expansive and statistically powerful. In this method,
eight mounds were-partitioned into 64 x 20 cm plots using rope and reinforced bars, and
experimental sites were selected randomly across the mounds. Four of the 12 primary
hillocks were not considered because ice scouring during the '93-'94 winter eroded the
mound apices, resulting in the loss of substantial intertidal substrate. In this randomized
approach, three tidal heights were considered: 25 cm above MLW, MLW, and 90 cm.

below MLW. The high intertidal height was lowered slightly to accommodate as many
intertidal mounds as possible in the sampling procedure, and one of the subtidal heights, 45
cm below MLW, was eliminated to incorporate more replication. In addition to tidal height
another factor, substrate level, was considered. To document the effects of substrate level,
samples were collected both at the reef surface and 10 cm below the reef surface.
During both years of sampling, non-destructive and destructive sampling were
employed from June through September to assess settlement and early recruitment within
the reef ecosystem. Non-destructive sampling involved the weekly placement of oyster
shells in open-topped, 64 x 20 cm, rubber coated 1 inch wire mesh trays secured to the reef
surface by reinforced bars. In 1993 a surface layer of 20 shells was placed weekly in
single level trays which were fixed spatially to the reef at all four tidal height designations
along upstream and downstream transects at each of the two mounds. The concave and
convex side of all 20 shells within indiv'idual cages were examined for recently settled
oyster larvae (spat) using a dissecting microscope, and a spat total per cage was recorded.
In 1994 three-tiered trays containing 30 shell upper and lower levels, which were spaced
10 cm apart, and a 40 shell intennediate level were buried into the reef substrate until the
upper level was even with the reef surface. Each week trays were placed at four distinct,
randomly selected plots chosen at all three tidal heights. Both surfaces of shell found in the
upper and lower tiers were examined for spat, and a surface layer spat total and a deep layer
spat total were recorded at all 12 weekly selected plots.
Destructive sampling involved the weekly placement of 64 x 20 cm. quadrats on the
reef surface, the removal of a layer of shell, and the subsequent examination of both shell
surfaces for spat. This sampling technique provided an index of cumulative spatfall on the
actual reef substrate and accounted for any early post-settlement mortality losses. In 1993
the quadrats were placed at all four tidal heights along upstream and downstream transects
chosen on each of the two mounds. To prevent resampling, successive samples collected
over time were taken along transects which were immediately adjacent to previously
sampled transects. During this period only a surface layer spat total per plot was
calculated. Plots used in 1994 destructive sampling were selected randomly across all eight
remaining intertidal mounds. As with 1994 non-destructive samples, four plots were
selected randomly each week at all three tidal heights. At each plot, a surface layer of shell
and a layer 10 cm beneath the reef surface, easily distinguishable from the surface layer by
its brown detrital film, was extracted and examined for spat. This allowed for the
calculation of both weekly surface and weekly deep spat totals for all 12 plots.
To deten-nine if oysters which settled along these spatial gradients would survive,
oysters of various age classes were tracked throughout the fall, winter, and summer
months. On August 12, 1993, oyster larvae were set on clean oyster shells in densities of
5-25 spat per shell at the Virginia Institute of Marine Science Oyster Hatchery. Shells
conta@ining spat were placed in Vexar mesh bags (100 shells per bag), and spat were reared
in hatchery systems to sizes comparable to oysters found on the reef. On September 26,
1993 the mesh bags were placed on the reef at the same 4 tidal heights designations used in
the 1993 settlement monitoring program along two distinct transects on each of the two
mounds. On October 14 and November 11, 1993, and May 5, 1994 25 shells were
haphazardly selected from each bag, which was shaken vigorously prior to selection, and
shells were photographed with an Olympus OM camera equipped with a 50 mm. macro
lens. Recent spat scars on each shell were noted and proportional mortalities (# scars per
shell # scars per shell + # live oysters) were calculated.
Over the summer of 1994, a different method which considered all intertidal
mounds at the reef -site and two year classes of oysters was used to document mortality.
One year class consisted of hatchery oysters set on oyster shell on May 16, 1994 in the
VIMS Oyster Hatchery, whereas the other year class consisted of a well mixed sample of
oysters used in the previous experiment. For each year class, 30 oysters present

collectively on 15 randon-fly picked shells were numbered using paint markers and were
placed on either the upper or lower level of 32 x 20 cm, three-tiered, 1 inch mesh cages.
Both upper and lower levels, which were 10 cm apart, were filled with shell containing live
oysters, but the middle level was filled with 20 shells devoid of live organisms. To keep
densities within the 15 shell assemblages as constant as possible, the physical removal of
oysters in high density communities was sometimes necessary.
At each of the three tidal heights considered in the 1994 settlement study, eight plots
were selected randomly for each year class, and cages were buried into the reef substrate
until the upper layer was even with the reef surface. The cages were held in place with a
reinforced rod. Photographs of labeled oysters were taken in the field with a Nikonos V
camera equipped with a close-up lens and focusing frame at 28 day intervals in June, July,
August, and September. To enhance photographic clarity and reduce fouling, a 3 HP
gasoline powered Homelite water pump was used in the field to clean labeled oysters and
cages. A proportional mortality value per layer of each cage was computed for each
sampling interval.

Statistical analysis:
The argument may be made that 1993 settlement and mortality samples collected
over time were not independent, since successive samples were taken from either spatially
fixed areas, spatially connected plots, or from the same population of organisms. To
account for this, analyses of variance (ANOVA) with repeated measures on time were
performed on each data set. Multivariate repeated measures were performed on settlement
data, and because of missing values, univariate repeated measures were performed on
mortality data. To satisfy assumptions of homogeneity, all settlement data were log (x + 1)
transformed and proportional mortality data were arcsine transformed. When no significant
interactions between the within factor, time, and any other factor were detected, 3-way
fixed factor (factors: tidal height, mound, and time) ANOVAs were performed. Significant
main effects were examined using Student-Newman-Keuls (SNK) tests.
Linear correlation's were performed on surface and deep samples collected in the
1994 settlement and mortality studies to detern-dne if a relationship existed between the two
substrate levels. If no significant relationship was detected in the correlation analysis,
substrate level was treated as a factor in further statistical procedures. When significant
relationships were detected, paired sample t-tests were used to determine if differences
existed between surface and deep samples. A mean value for surface and deep data was
calculated when no significant difference between the substrate levels was detected, and
further analyses were performed on these mean values.
ANOVAs were run on 1994 non-destructive and destructive log (x+1) transformed
settlement data, and all differences between means were revealed using SNK multiple
comparison tests. Multivariate repeated measures ANOVAs were performed on arcsine
transformed mortality data collected in 1994. All significant between factor effects were
analyzed using SNK multiple comparison tests, whereas significant within factor effects
were examined using Newman-Keuls procedure (pp. 527-528, Winer 1991).

RESULTS AND DISCUSSION
The majority of data analyzed in this study suggest that small-scale spatial changes,
such as 30 cm shifts in tidal elevation or 10 cm changes in substrate depth, strongly
influence the processes of oyster settlement and post-settlement survival. Rather than go
into an exhaustive examination of the data, we feel that it would be more constructive, and
hopefully more interesting, to present representative examples from the data which illustrate
and reinforce key points related to microscale effects. For a more comprehensive treatment
of the data, please see Bartol and Mann (1996 a & b, in prep).

Settlement of oyster larvae in a constructed reef environment is heavily dependent
on the tidal elevation of the reef substrate. Within the shallow water (< 3 in) reef system
considered in this study, settlement increased with tidal depth. This is most clearly seen in
the non-destructive settlement studies, where settlement intensities both in 1993 and 1994
were greatest at -90 cm. (Figure 1). This finding is consistent with several other studies
conducted in non-reef environments. For example, greater subtidal settlement rates have
been documented by McDougall (1942) using unglazed hearth tiles, by Chestnut and Fahy
(1953) using shellstrings, and by Roegner and Mann (1990) using hatchery-reared larvae
exposed to field conditions in microcosms. Nichy and Menzel (1967), who placed oysters
on clothmats of mesh within a reef ecosystem, also observed greater settlement/early
recruitment subtidally.
The higher rates of subtidal settlement observed in this study were likely a result of
several factors. Submergence time may have been one. Oyster larvae in the water column
were exposed to subtidal substrates substantially longer than to intertidal substrates, and as
a result, had a wider time window in which to set. Submergence time alone, however, did
not account for the observed differential settlement. Kenny et al. (1990) and Roegner
(1989) found that settlement intensities are not direct functions of submergence time,
especially in the high intertidal zone and the low subtidal zone. Vertical segregation of
oyster larvae in the water column also may have contributed to elevated subtidal sets
because oyster late stage pediveliger larvae are more abundant near the benthos than at the
surface or within the rriidwater region (Carriker 1951, Kunkle 1957, Haskin 1964, and
Baker 1994). Furthermore, because late stage competent to set larvae are photonegative at
the time of settlement (Ritchie and Menzel 1969, Nelson 1953) and prefer areas of lower
wave energy (Ortega 1981), they may have actively sought subtidal habitats where light
intensities and wave stress are reduced.
Surprisingly, no significant differences in settlement were detected between surface
and deep substrates at any of the tidal heights considered (Paired t-tests > .05). One
concern, however, is that low settlement rates (mean weekly destructive/non-destructive
settlement over a three-week settlement period = 0.5 - 3.5 spat per 30 shells) may have
dramatically lowered the statistical power of the paired t-test. Although this may be a true,
a thorough examination of the of the data sets revealed that there was no trend in greater
settlement by layer. For example, of the 36 destructive samples collected, settlement was
greater at the surface 7 times, greater below the surface 9 times, and equal 20 times!
Oyster larvae may have settled 10 cm beneath the reef for a number of reasons.
Some of the oyster larvae may have actively attached to subsurface substrate because again
they prefer darkened conditions when setting (Ritchie and Menzel 1969) and areas of
reduced wave action (Ortega 1981), but also because they seek out environments where
flow is low, crevices are abundant, and substrates are not heavily fouled (Bushek 1988,
Michener and Kenny 199 1). Reef shells found below the surface layer were considerably
less infested with algal growth and barnacles. It is also feasible that because water currents
are substantially reduced beneath the reef surface, the interstices serve as sediment traps
and entrain larvae. Although it is not clear from this study what mechanism, active and/or
passive transport of larvae, is responsible for sub-surface settlement, it is clear that larvae
are capable of settling within the reef interstices and are not impeded by shell down to
depths of 10 cm. This is quite remarkable considering that there may be 20 or more shells
layers within the 10 cm space.
Although oyster larvae are capable of settling beneath the reef surface, do they
survive in these environments? Results from this study suggest that oysters not only
survive in these environments, but survive better there during certain times of the year. For
example, oysters reared in 1993 which resided at the reef surface at the 25 cm tidal height
experienced significantly higher mortalities than oysters residing below the surface from
mid June through mid July (Figure 2). During this period air temperatures were the highest

of the year, averaging just over 28' C. It is likely that oysters beneath the reef surface
benefited from a shading effect from overlying oysters and shell, and as a result resided in
a cooler, moister, more hospitable environment than their surface dwelling cousins. In
natural reefs oysters grow vertically in highly populous clusters, and this colonial existence
provides refuge from solar radiation for all oysters in the community. Since dense
assemblages of vertically growing oysters takes many years to become established, sub-
surface residence may be critical for the survival of intertidal oysters residing in constructed
reef systems.
A further example of beneficial subsurface residence is found at the -90 cm tidal
height. At this height, significantly higher surface mortalities were detected for 1993
reared oysters over the entire three month summer sampling session (Figure 3). Although
the two most deleterious predators, oyster drills and starfish, were absent at the reef site
because of low salinities, the flatworin Stylochus elliptus, the mud crabs Panopeus
herbstii, Eurypanopeus depressus, and Rhithropanopeus harrisii, and the blue crab
Callinectes sapidus were present, and all are known to contribute to substantial oyster
mortalities (Landers and Rhodes 1970, Abbe 1986, Littlewood 1988, Eggleston 1990, and
Baker 1994). These predators were found within cages at surface and deep layers at all
tidal heights, but were most abundant at the reef surface and at subtidal depths based on
field observations. Flatworms and mud crabs were probably the most deleterious because
they were highly abundant at the study site and were not restricted by the mesh of the
experimental cages. Although adult blue crabs may not have been able to enter the cages,
they were able to prey upon the numerous oysters which grew through the cage mesh.
Of the three tidal heights examined, surface residing oysters survived best at MLW
throughout the summer (June-September). For example, oysters belonging to the 1994
year class and dwelling at the MLW tidal height had a cumulative percent mortality of 12%
over the summer compared with mortalities of 22% and 23% recorded at the +25 cm and
the -90 cm tidal heights, respectively (Figure 4a). Interestingly, higher MLW survival was
not observed for oysters residing below the surface. In fact, beneath the reef surface, there
was no detectable difference in mortality along the intertidal-subtidal continuum. This may
because physical and biological environments within the interstices are relatively constant
and stable regardless of tidal elevation.
During the fall oysters situated at MLW had a cumulative percent mortality of 13 %,
which again was significantly lower than mortalities recorded at other tidal heights (Figure
4b). Oysters residing at MLW during the summer and fall probably experienced less
predation pressure than subtidal oysters as a consequence of aerial exposure, but did not
suffer from significant heat and respiratory stress like mid to high intertidal oysters because
of shorter emergence times. This is consistent with the findings of McDougall (1942),
Chestnut and Fahy (1953), McNulty (1953), Nichy and Menzel (1967), Arakawa (1980),
and Littlewood (1988), all of who have found high oyster survival in the mid to low
intertidal zone as a result of lowered predation pressure, physical stresses, sedimentation,
and/or competition for space.
Oysters situated at MLW did not fare as well during the winter months. Mortality
rates at MLW and higher in the intertidal zone were 95-100% , whereas mortality rates at
the -45 and -90 cin tidal heights were on the order of 25% (Figure 5). These mortality
rates, especially at MLW, were likely atypical and a result of the coincidence of an
unusually brutal winter and the presence of a young population of oysters (oysters were 4
months old at the onset of the winter). From December of '93 through March of '94 air
temperatures dropped below freezing 28 days, which is very unusual for Virginia. Oysters
less than 1 years old are especially vulnerable to freezing conditions because they put much
of their energy into growth and maintenance rather than into the storage of glycogen, a
preferred substrate for anaerobic respiration, and thus are less capable of environmental
isolation (Mann and Gallager 1985, Widdows et al. 1989). In a separate study conducted

by the authors over the '94-'95 winter, oysters of a similar age (5 months) and oysters 15
months residing at MLW experienced winter mortalities between 15 and 20%. This is
evidence that the mortality rates observed over the '93 - '94 winter were exceedingly high.
It should be made clear that the above mortalities only reflect oysters at the surface
substrate layer, since oysters beneath the reef surface were not measured during the winter
periods. It was interesting to note, however, that one cage buried 15 cm beneath the reef
surface in the intertidal zone during the '93 -'94 winter, which is not depicted in the graph,
had mortalities of 50%. This is substantially lower than intertidal mortalities recorded at the
surface. Furthermore, visual inspections of "natural set" oysters in underlying intertidal
environments revealed higher below surface survival. These observations suggest that
residence below the reef surface may not only provide refugia from high temperatures and
predators during the summer and fall, but may also provide relief from ice and wind during
the winter months.
To recap briefly, settlement and early recruitment of oyster larvae are greatest
subtidally, and settlement intensities at the reef surface and 10 cm below the surface are
similar. During the summer and fall, survivorship after substrate attachment is maximized
at MLW; during the winter, mortality of surface dwelling intertidal oysters may be
substantial. Survivorship patterns may differ on a smaller spatial scale as well.
Submergence 10 cm within the reef provides an important refuge both for intertidal oysters
during periods of peak solar exposure (June/July) and for subtidal oysters during periods
of intense predation pressure (summer and fall). Furthermore, there is some evidence to
suggest that sub-surface residence may be beneficial for oysters living in the intertidal zone
during the winter months.
In practical terms these results proffer an important lesson: microscale variability
should not be ignored when constructing reef systems. Adding merely 1 in of vertical
topography onto a constructed reef system so that it may extend marginally out of water at
low tide may elevate survival ability substantially, especially if the addition of substrate
provides a spatial refuge from intense predation and fouling. This was clearly
demonstrated in this study during the summer and fall when mortality rates were lowest at
MLW. Unfortunately. since mortalities recorded over the winter were a product of unusual
circumstances, this study fails to provide a representative comparison between summer,
fall, and winter mortalities, which, of course, would be useful in deterinining whether
surnmer/fall survivorship benefits outweigh mortality losses over the winter. As a result,
we cannot provide a definitive answer as to whether building intertidal reefs will maximize
survival. Nonetheless, we have shown that tidal elevation does affect settlement and post-
settlement survival and that determining the tidal elevation at which recruitment is
maximized for a given geographic setting before deciding on a reef elevation is a necessary
exercise if survivorshipis to be maximized. Substrate depth also should be considered.
The veneer level of shell over a base substrate in reef construction should be thick enough
to provide rnicroscale refugia for settlement and survival of early life history stages. Based
on the results of this study, the substrate should be at least 10 cm thick and allow for
subsurface colonization. Finally, the most important advice we offer to reef builders is to
be aware that the issues of settlement and mortality in relation to biological and physical
environments are determined by microscale variability rather than larger scale uniformity,
and the macroscale patterns observed in the field are the sum of these microscale events.

Acknowledgments.

This work was completed as part of the M.A. thesis of Ian Bartol at the College of William
and Mary. Financial support from the Commonwealth of Virginia, Department of
Environmental Quality, Coastal Resources Management Program and the National Oceanic

and Atmospheric Administration is gratefully acknowledged. This is Contribution Number
#### from the Virginia Institute of Marine Science, College of William and Mary.

LITERATURE CITED

Abbe, G. R. 1986. A review of some factors that limit oyster recruitment in
Chesapeake Bay. American Malacological Bulletin. Special Edition No. 3: 59-
70.
Arakawa, K. Y. 1980. Prevention and removal of fouling on cultured oysters. A
handbook for growers. Mar. Sea Grant Tech. Rep. No. 56, 37 pp. University
of Maine, Orono, ME.
Baker, P.K. 1994. Quantification of settlement and recruitment processes in bivalve
mollusks. Ph.D. Dissertation, College of William and Mary, Virginia. 381 pp.
Bushek, D. 1988. Settlement as a major determinant of intertidal oysters and barnacle
distributions along a horizontal gradient. J. Exp. Mar. Biol. Ecol. 122: 1-18.
Carriker, M.R. 195 1. Ecological observations on the distribution of oyster larvae in
New Jersey estuaries. Ecol. Monogr. 21: 19-38.
Chesnut, A.F. and W.E. Fahy. 1953. Studies on the vertical setting of oysters in North
Carolina. Proc. Gulf Carib. Fish. Inst. 5: 106-112.
Eggleston, D.B. 1990. Foraging behavior of the blue crab, Callinectes sapidus , on
juvenile oysters Crassostrea virginica : effects of prey density and size. Bull.
Mar. Sci. 46: 62-82.
Haskin, H.H. 1964. The distribution of oyster larvae in N. Marshall ed. Proc.Symp.
Exp. Mar. Ecol. Narragansett Mar. Lab., Univ. R.I., Occas. Pap. No. 2. Pp
76-80.
Kenny, P. D., W. K. Michener, and D. M. Allen. 1990. Spatial and temporal patterns
of oyster settlement in a high salinity estuary. Journal of Shellfish. Research
9(2): 329-339.
Kunkle, DR. 1957. The vertical distribution of oyster larvae in Delaware Bay. Proc.
Nat. Shellfish. Assoc. 48: 90-91.
Landers, W.S. and R.W. Rhodes, Jr. 1070. Some factors influencing predation by
the flatworm, Stylochus ellipiticus (Girard), on oysters. Ches. Sci. 11: 55-60.
Littlewood, D. T. J. 1988. Subtidal versus intertidal cultivation of Crassostrea
rhizophorae. Aquaculture 75: 59-71.
Mann, R. and S. M. Gallager. 1985. Physiological and biochemical energetics of I
arvae of Teredo navalis L. and Bankia gouldi (Bartsch)(Bivalvia: Teredinidae).
J. Exp. Mar. Biol. Ecol. 85: 211-228.
McDougall, K. D. 1942. Sessile marine invertebrates of Beaufort, N. C. Ecol.
Monogr.. 13: 321-37 1.
McNulty, J. K. 1953. Seasonal and vertical patterns of oyster setting off Wadmalaw
Island, S. C. Contr. Bears Bluff Lab No. 15. 17 pp.
Michner, W.K. and Kenny P.D. 1991. Spatial and temporal patterns of Crassostrea
virginica (Gmelin) recruitment: relationship to scale and substratum. J. Exp.
Mar.Biol. Ecol. 154: 97-121.
Nelson, T. C. 1953. Some observations on the migrations and setting of oyster larvae.
Proc. Natl. Shellfish Assoc. 43: 99-104.
Nichy, F. E. and R. W. Menzel. 1967. Mortality of intertidal and subtidal oysters in
Alligator Harbor, FL. Proc. Natl. Shellfisheries Assoc. 52:33-41.

Ortega, S. 1981. Environmental stress, competition, and dominance of Crassostrea
virginica near Beaufort, North Carolina, USA. Mar. Biol. 62:47-56.
Ritchie, T. P. and R. W. Menzel. 1969. Influence of light on larval settlement of
American oysters. Proc. Nad. Shellfisheries Assoc. 59: 116-120.
Roegner, G. C., and R. Mann. 1990. Settlement patterns of Crassostrea virginica
(Gmelin, 1791) in relation to tidal zonation. Joumal of Shellfish Research 9(2):
341-346.
Roegner, G.C. 1989. Recruitment and growth of juvenile Crassostrea virginica
(Gmelin) in relation to tidal zonation. Master's Thesis, College of William and
Mary, Virginia. 145 pp.
Widdows, J., R. I. E. Newell, and R. Mann. 1989. The effects of hypoxia and anoxia
on survival, energy metabolism, and feeding of oyster larvae. (Crassostrea
virginica, Gmelin). Biol. Bull. 177: 154-166.
Winer, B.J., D.R. Brown, and K.M. Michels. 1991. Statistical principles in
experimental design. McGraw-Hill, Inc.: New York. 1057 pp.

Table 1. Paired t-tests performed on surface and deep substrate layer spat counts for
non-destructive and destructive samples. Separate analyses were performed on each
tidal elevation.

NON-DESTRUCTIVE SAMPLES

Tidal height Mean difference Degrees of freedom t-value p-value
(surface-deep)
+ 25 CM -.333 11 -.886 1.3944
MLW -.083 11 -.321 .7545
-90 CM .333 11 49 1.5940

DESTRUCTIVE SAMPLES

Tidal height Mean difference Degrees of freedom t-value p-value
(surface-deep)
+25 cm -.083 1 1 -.561 1.5863
MLW .083 1 1 .432 1.6742
-90 cm -.500 1 1 -1.149-F-2750

Figure 1. Mean C. virginica spat counts recorded in the 1993 and 1994 non-destructive
settlement studies. Error bars denote+ I S.E.

10- 1993

7.5-

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30 0 -45 -@O

TIDAL HEIGHT (CM RELATIVE TO NILW)

3-Factor ANOVA; F= 9.69, df= 3, 32, p < .0001

4-
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1994

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TIDAL HEIGHT (CM RELATIVE TO MLW)

*2-Factor ANOVA; F= 26.20, df= 2, 27, p< .0001

Figure 2. Mean percent mortalities of C virginica reared in 1993 residing at the + 25
cm tidal height during the June/July sampling period. Error bars denote + 1
S.E.

................
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SUBSTRATE LEVEL

*1-Factor ANOVA; F=4.857, df=1112, p=.0478

Figure 3. Mean cumulative percent mortalities from June through September, 1994 for
the '93 year class oysters residing at the -90 cm tidal height. Error bars
denote +1 S.E.

20 -

.. ........ ......
0 ".*.*.*.*.'...i ........ .......
SURFACE DEEP

SUBSTRATE LEVEL

Figure 4. Cumulative percent mortalities for A) the '94 year class of oysters from June
- September, 1994 and B) 3-week old oysters from September - November,
1993. Error bars denote +1 S.E.

30-
25- T
........... ..........
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TIDAL HEIGHT (CM RELATIVE TO MLW)

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30 0 -45 -90

TIDAL HEIGHT (CM RELATIVE TO MLW)

Figure 5. Cumulative percent mortality of juvenile oysters from November, 1993 -
May, 1994. Error bars denote +1 S.E.

125-

100-

........ ........
........ ...... I.
........ ........
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TTDAL BEIGHT (CM RELATIVE TO MLW)

Small-scale Settlement Patterns of the Oyster Crassostrea virginica on a Constructed
Intertidal Reef

Ian K. Bartol and Roger Mann
School of Marine Science, Virginia Institute of Marine Science, College of William and
Mary, Gloucester Point, VA 23062

ABSTRACT

The construction of artificial reefs resembling those widely present during colonial
times in the Chesapeake Bay, but now absent due to years of overharvesting, may provide
a more ecologically advantageous environment for oyster settlement and subsequent
survival than present subtidal, two-dimensional habitats. We examined settlement
processes on a constructed, 150 x 30 m intertidal reef composed of oyster shell. The reef
was destructively and non-destructively sampled weekly at tidal heights ranging from 30
cm above to 90 cm below mean low water (MLW) and at two substrate levels (reef surface
and 10 cm. below the reef surface). Oysters settled in comparable magnitudes at the surface
of the reef community and within the reef interstices down to depths of 10 cm.
Furthermore, settlement was generally greatest subtidally; however, there were localized
areas within the reef community where conditions were beneficial for intertidal settlement
and where intertidal/subtidal settlement rates were not significantly different. These results
suggest that microscale variations in tidal elevation and substrate depth strongly affect
settlement processes and should not be ignored when constructing reefs.

INTRODUCTION
When colonists arrived in the Chesapeake Bay region during the 1600s, they
encountered a Bay ecosystem littered with intertidal reefs. These unmistakable biological
features, which proliferated in the Chesapeake Bay and tributaries during the last half of the
Holocene interglacial, were important self-renewing food sources for early settlers and
Native Americans alike (Hargis and Haven 1995). As the economic value of the oyster
Crassostrea virginica began to be realized in the mid 1800s, however, commercial
exploitation of the resource began. Years of subsequent overharvesting has transformed
these once massive, aerial exposed communities to mere subtidal, "footprint" structures
which have significantly less vertical dimension and habitat heterogeneity. Disease,
environmental degradation, and poor resource management in the last half century have
expedited this degeneration.
Today, Virginia's oyster population is less than I % of what it was just 35 years ago
(Wesson et al. 1995). Many of the past rejuvenation efforts, which often have involved the
spreading of relatively thin veneers of shell over coastal and estuarine bottom for larval
attachment, now fail to produce healthy and viable adult oyster communities. These efforts
may be ineffective because they revolve around re-creating habitats produced and shaped
by man, rather than focusing on emulating the natural, three-dimensional, intertidal
communities present during colonial times. Since oysters in the Chesapeake Bay resided in
intertidal communities for centuries and were able to withstand significant environmental
and biological stresses, there is probably an ecological and evolutionary advantage to
intertidal, colonial reef existence in the Bay and a return to it may help rejuvenate ailing
oyster stocks.
We know little about the colonization and ecology of C. virginica on intertidal reefs
in the Chesapeake Bay because of their absence for over a century. Thus, we constructed a
150 x 30 m intertidal reef to study C virginica settlement -- the first step in the colonization
process. We paid particular attention to the effects of elevation relative to mean low water

(MLW) because tidal elevation is a major factor distinguishing intertidal reef environments
from current subtidal habitats. We also examined settlement patterns at the surface of the
reef shells and within the interstices of the reef at depths of 10 cm. along the tidal gradient to
determine if substrate depth is a factor to consider when constructing artificial reefs.

MATERIALS AND METHODS
This study was conducted in the Piankatank River, a subestuary of the Chesapeake
Bay located in Virginia, at a site which once supported a highly productive intertidal reef
system, but at the time of reef construction was completely devoid of live oysters. At this
site water temperature varied from 0.5 - 30 C, salinity ranged from 8 - 20 ppt, and tidal
range was small (mean range = 36 cm).
The reef was constructed by the Virginia Marine Resource Commission (VMRC)
by spraying aged oyster shells off barges with a high pressure water canyon. The shells
were broadcast over an area approximately 150 x 30 m, which were the approximate
footprint dimensions of the pre-existing reef system. After completion, the reef consisted
of numerous hummocks varying from 2 to 20 m2in area exposed at low tide. Although the
constructed reef ranged from 0.5 rn above to 3 m below MLW, the vast majority of the
hummocks did not extend much higher than 0.35 rn. above MLW or much deeper than 1.0
m below M[LW.
The constructed reef was sampled in both 1993 and 1994. During the 1993
sampling period 2 of the 12 principal intertidal hummocks were the focus of study: one on
the reef periphery perpendicular to prevailing currents and unprotected from wave action
and currents and a second situated near the middle of the reef parallel to prevailing currents
and partially shielded from wave action and currents. These mounds were sampled using a
transect approach, whereby samples were collected along 2 transects at tidal heights of 30
cm above MLW, MLW, 45 cm below MLW, and 90 cm below MLW.
During the 1994 sampling period, after data from the previous year were analyzed
and we had a preliminary understanding of the reef system, a randomized approach was
used which was more geographically expansive and statistically powerful. In this method a
series of reinforced bars were driven into the reef substrate on 8 of the 12 aerial exposed
hummocks or mounds and connected with rope so as to partition the mounds into 64 x 20
cm plots. This grid system encompassed reef area from the base to the crest of each
mound. Experimental sites were selected randomly across the hummocks. Four of the 12
hummocks were eliminated because ice scouring during the '93 -'94 winter eroded the
mound apexes, resulting in the loss of substantial intertidal substrate. In this randomized
approach, tidal heights of 25 cm above MLW, M[LW, and 90 cm. below MLW were
considered. The high intertidal height was lowered slightly in 1994 to accommodate as
many intertidal mounds as possible in the sampling procedure, and one of the subtidal
heights, 45 cm below MLW, was eliminated to incorporate more replication. Furthermore,
another factor, substrate level or depth within the substrate, was considered. To document
the effects of substrate level, samples were collected both at the reef surface and 10 cm
below the reef surface.
During both years of sampling, non-destructive and destructive sampling were
employed from June through September to assess settlement and early recruitment within
the reef ecosystem. Non-destructive sampling involved the weekly placement of oyster
shells in open-topped, 64 x 20 cm, rubber coated 1 inch wire mesh trays secured to the
reef surface by reinforced bars. In 1993 a surface layer of 20 shells was placed in single
level trays. The trays were situated at all 4 tidal height designations along 2 spatially
separated transects at each of two mounds. The concave and convex side of all 20 shells
within the individual trays were examined for recently settled oyster larvae (spat) using a
dissecting microscope, and a spat total per tray was recorded. In 1994, three-tiered trays
containing 30 shell upper and lower levels, which were spaced 10 cm apart, and a 40 shell

intermediate level were used. Each week 4 plots were selected randomly for each of the 3
tidal heights. At each plot a three-tiered tray was buried into the reef substrate to a depth
where the upper layer of shell was even with the reef surface. Both surfaces of shell
found in the upper and lower tiers were examined for spat, and a surface and deep layer
spat total were recorded at all 12 weekly selected plots.
Destructive sampling involved the weekly placement of 64 x 20 cm quadrats on the
reef surface, the removal of layer of shell, and the subsequent examination of both shell
surfaces for spat. This sampling technique provided an index of cumulative spatfall on the
actual reef substrate minus any early post-settlement mortality losses. Thus, this method
provided an estimate of early recruitment--the number of larvae which had survived from
settlement to the time of sampling (1-6 weeks later). In 1993 the quadrats were placed at all
4 tidal height designations along 2 spatially distinct transects on each of the 2 mounds. To
prevent re-sampling, successive samples collected over time were taken immediately
adjacent to previously sampled plots. During this period only surface layer samples were
excavated and recorded. Plots used in the 1994 destructive sampling were selected
randon-dy across the 8 principal intertidal mounds. As with the 1994 non-destructive
samples, 4 plots were selected randomly each week at all 3 tidal heights. At each plot, a
surface shell layer and a layer 10 cm beneath the reef surface, easily distinguishable from
the surface layer by its brown detrital film, was extracted and examined for spat. This
allowed for the calculation of both weekly surface and weekly deep spat totals for all 12
plots.
In addition to non-destructive and destructive samples, shellstring samples were
collected weekly from June through September during both years of sampling.
Shellstrings consist of 12 single valve oyster shells, each with a hole drilled through the
center, threaded onto galvanized wire. Only the 10 intermediate shells are considered
because the top and bottom shells of the shellstring have a tendency to collect large
numbers of spat, leading to unrepresentative high spat estimates. The shellstrings were
suspended from pilings located at the north and south reef extremities to a depth of 90 cm
below MLW. Both shell surfaces were examined and a spat total per shell was calculated
so that comparisons could be made with non-destructive and destructive samples.
A number of physical variables were measured during this sampling session to aid
in assessing the above processes. Water temperature, salinity, and secchi depth readings
were recorded each week. To develop an idea of current flow at the reef site, chlorine
tablets housed in 20 cm x 20 cm mesh cages and held 10 cm above the bottom by
reinforced rods were deployed during both neap and spring tides. Cages were placed at
plots sampled in the non-destructive settlement study. The chlorine tablets were weighed,
deployed in the field for 48 hours, and weighed again. Differences in chlorine tablet mass
were compared within each tidal weight to construct, in the case of subtidal plots, a
framework of relative flow rates and, in the case of intertidal plots, a model of both wave
intensity and flow rates. Chlorine tablets were used as a surrogate measure for flow
because turbulent diffusion, the major force driving the dissolution rate in the field, in the
benthic boundary layer at a given bottom roughness varies in a positive fashion with
current speed. It was assumed that the flow speed derived from the dissolution rate of
chlorine tablets placed 10 cm above the reef would be proportional to flow conditions at the
surface and 10 cm below.

Statistical Analysis
The argument may be made that 1993 data collected over time were not
independent, since -successive samples were taken from either spatially fixed areas or
spatially connected plots. Thus, analysis of variance (ANOVA) with repeated measures on
time were performed on both the 1993 non-destructive and destructive data sets. To satisfy
the assumptions of homogeneity, all settlement data were log (x + 1) transformed. When

no significant interactions were detected, 3-way fixed factor (factors: tidal height, time,
mound) ANOVAs were performed. Significant main effects were examined using Student-
Newman-Keuls (SNK) tests.
Linear correlation's were performed first on surface and deep samples collected in
the 1994 settlement study to deterrnine if a relationship existed between the two substrate
levels. Significant relationships were detected, therefore paired sample t-tests were used to
determine if differences existed surface and deep samples. A mean value for surface and
deep data was calculated when no significant difference between the substrate levels was
detected, and further analyses were performed on these mean values. ANOVAs were run
on 1994 log (x + 1) transformed non-destructive and destructive settlement data, and a
differences between the means were revealed using SNK multiple comparison tests.
Furthermore, to determine if a functional dependence existed between settlement and water
movement, linear regressions of log (x + 1) transformed non-destructive settlement on
water movement were performed for each tidal height. The assumptions of regressions
were met as determined by residual analysis (Zar 1984).

RESULTS
1993
Settlement lasted 6 weeks in 1993 beginning the week of August 5 and ending the
week of September 16, and settlement intensity was low overall (Figure 1). Although the
settlement period lasted 6 weeks, non-destructive spat counts recorded during weeks 5 and
6 of the settlement period were so small that they were eliminated from statistical models.
Conversely, dramatic increases in destructive settlement magnitudes were observed during
these periods, and thus these weeks were included in destructive sample statistical tests.
No significant interactive effects between the within factor, time, and any other
factor were detected when 1993 destructive and non-destructive data were analyzed using
repeated measures analysis; thus, subsequent 3-way ANOVAs were performed. These
analyses revealed that in non-destructive samples only tidal height had a significant impact
on settlement (Table la), with settlement being greatest at the -90 cm tidal height (Figure
2). In destructive samples, where the entire 6-week settlement period was considered, time
had a significant impact on settlement (Table lb). In general, destructive sample spat
counts increased with time, which is quite different from the pattern observed in non-
destructive samples where spat counts decreased dramatically in weeks 5 and 6 (Figure 3).
Destructive and non-destructive spat counts were also very different during week I of the
settlement period when no previous settlement had occurred. A mound x tidal height
interaction was present in destructive samples. This interaction was a product of spat
counts at the +25 cm tidal height on mound A (mound perpendicular to prevailing currents)
preventing the detection of significant differences in settlement by tidal height (Figure 5).
Conversely, on mound B (mound parallel to prevailing currents) where +25 cm spat counts
were significantly lower than on mound A, significantly greater settlement occurred at
MLW and -90 cm.

1994
The 1994 settlement season lasted only 3 weeks beginning the week of July 15 and
ending the week of July 28. Spat counts detected on shellstrings throughout the 1993
sampling session were the lowest recorded in the last 17 years, with mean cumulative spat
counts of less than I spat per shell over the entire settlement season (Figure 1).
Unfortunately, these low setIdernent magnitudes together with the short settlement season
precluded a meaningful comparison of intertidal spat counts recorded on mound A with
intertidal counts on the other mounds. Field observations did reveal, however, that
noticeably more intertidal oysters were present on mound A than at any of the other 8
mounds present at the site.

Significant correlation's (p<.001) between surface and deep substrate levels were
detected in both non-destructive and destructive samples, and thus paired t-tests were used
to examine the effects of substrate level. Based on these tests, no significant differences in
settlement were detected between surface and deep layers at any of the tidal heights in either
the non-destructive or destructive samples (p>.05, Table 2).
In the non-destructive study, ANOVAs performed on surface/deep settlement
means revealed that both tidal height and time influenced settlement (Table 3a). Settlement
intensity was greatest at the -90 cm tidal height and peaked the week of July 21-28 (Figure
5). Settlement was influenced significantly by time and tidal height in the destructive study
as well, but a significant time x tidal height interaction confounded the effects (Table 3b).
This interaction was a result of spat counts being significantly greater at the -90 cm tidal
height only during the weeks July 21-28 and July 28 - August 4. During the first week of
the settlement season, settlement magnitudes were so low across all 3 tidal heights that no
significant differences were detectable.

Comparison of spat detection methods
There was a clear discrepancy in spat counts between the 3 sampling methods even
though they were all deployed at a depth of 90 cm below MLW. Clearly more spat settled
on shellstrings, which were replaced weekly and suspended in the water column, than on
shells placed at weekly intervals in trays fixed to the reef or on shells sampled destructively
from the reef (Figure 6). Furthermore, non-destructive samples appeared to collect more
spat than destructive samples.

Physical Parameters
Salinity, water temperature, and secchi disk readings recorded during both years of
sampling are presented graphically in Figure 7. In 1993 settlement occurred during a minor
decrease in water clarity (August 5 - 12), whereas the onset of settlement in 1994 coincided
with a rise in water clarity (July 15 - 21) and water temperature (July 15). Flow rate was
greatest at the reef crests and lowest at the reef bases, and there was no linear dependence
of settlement on water flow (p > .234, R2< .23 1).

DISCUSSION
The short, unimodal settlement events recorded in this study coupled with low
overall settlement magnitudes are indicative of a rapidly declining broodstock population in
the Chesapeake Bay, which at present is showing little sign of rejuvenation (Morales-
Alamo and Mann 1995). Bimodal peaks in settlement were recorded not long ago in the
Chesapeake Bay, and it was not uncommon to detect settlement from June through October
at the Piankatank reef site (Virginia Institute of Marine Science annual oyster spatfall
surveys, unpublished data for the period 1970-1995). In 1986, cumulative spat counts at
the site were 376.5 spat/shell, whereas now they are at 0.9 spat/shell . Although there are
probably a number of reasons for this devastating decline ranging from disease to poor
water quality, years of overharvesting where ecologically advantageous intertidal reef
communities were degraded to mere subtidal footprints undoubtedly weakened oyster
stocks considerably. Hargis (1995) offers the opinion that overharvesting was the
principal instigator of the demise of the Chesapeake oyster. Although this is debatable,
there is widespread agreement that oyster reefs are the most optimal ecosystems for oysters
and we need to learn more about oyster ecology on them.
Based on two years of settlement monitoring on a constructed intertidal oyster reef,
it was clear that tidal elevation had a large impact on settlement and early recruitment. The
higher rates of settlement recorded in the subtidal zone than in the intertidal zone are
consistent with several other studies conducted in non-reef environments, such as
McDougall (1942) where unglazed hearth tiles were used as substrate, Chestnut and Fahy

(1953) where shellstrings were utilized, and Roegner and Mann (1990) where hatchery-
reared larvae exposed to field conditions in microcosms were considered. Nichy and
Menzel (1967), who placed oysters on clothinats of mesh within a reef ecosystem, also
observed greater settlement/early recruitment subtidally.
The high rates of subtidal settlement/early recruitment observed throughout most of
this study were likely a result of a number of factors. Submergence time, for instance, may
have contributed to settlement discrepancies observed at different tidal heights. Oyster
larvae in the water column were exposed to subtidal substrates substantially longer than to
intertidal substrates, and as a result, had a wider time window in which to set.
Submergence time alone, however, did not account for the observed differences in
settlement. Kenny et al. (1990) and Roegner (1989) found that settlement intensities were
not direct functions of submergence time, especially in the high intertidal zone where
settlement was lower than predicted and the low subtidal zone where settlement was higher
than predicted. Another factor contributing to elevated subtidal sets may have been vertical
segregation of oyster larvae within the water column because oyster late stage pediveliger
larvae are more abundant near the benthos than at the surface or within the midwater region
(Carriker 1951, Kunkle 1957, Haskin 1964, and Baker 1994). Furthermore, because late
stage competent to set oyster larvae are photonegative at the time of settlement (Cole &
Knight-Jones 1939, Ritchie & Menzel 1969, Shaw et al. 1970) and prefer areas of lower
wave energy (Ortega 1981, Abbe 1986 Bushek 1988), they may have actively sought
subtidal habitats where light intensities and wave stress are reduced.
Although settlement was maximized subtidally for most of the study, the lack of
significant settlement differences between tidal heights at mound A during the 1993 season
suggests that this is not always the case in artificial reef environments. Mound A is the
most unique hummock comprising the reef system. It is more than twice as large, in terms
of substrate aerial exposed at low tide, as any of the mounds at the site, is the first mound-
exposed to tidal influx, is the only mound oriented completely perpendicular to prevailing
currents, and experiences the most intense wave action. These factors, especially wave
action which kept intertidal substrate clean, may have contributed to high intertidal sets. In
1994, we had hoped to compare sets on mound. A with sets on the seven other mounds at
the reef site to determine if intertidal settlement intensities were indeed higher at mound A.
Unfortunately, spat counts were so low and the settlement season was so abbreviated that
no meaningful comparisons could be made. The highest abundance of intertidal oysters
clearly occurs on mound A based on field observations, which certainly suggests that
conditions at mound A are conducive for intertidal settlement. Conversely, results from
this study and field observations indicate that subtidal settlement is no higher at mound A
than at any of the mounds.
Several other studies have found that settlernentlearly recruitment is not always
maximized subtidally. Hidu and Haskin (1972) found that although settlement was greatest
subtidally 1/2 mile offshore at a transitional slope region where tidal flats merge with deep
water, settlement was greatest intertidally in shallow water near the shore. They attributed
the high intertidal sets inshore to rapid rises in water temperature as seawater passed over
heated intertidal substrates and to the presence of dense intertidal adult populations which
released chemical cues. McNulty (1953), using bags of shell left in the field for two
weeks, found higher settlement/early recruitment in the intertidal zone than in the subtidal
zone, and Kenny et al. (1990), using asbestos plates sampled every two weeks in the
summer and four weeks in the winter, found settlement to be similar from 70 cm above
mean low water in the intertidal zone to 30 cm. below mean low water in the subtidal zone.
McNulty and Kenny et al. attributed the high rates of settlement/early recruitment in the
intertidal zone to less predation and more desirable substrate in the intertidal zone.
The lack of detectable differences in settlement/early recruitment (1-3 weeks)
between surface and deep substrates at any of the tidal heights considered was admittedly

unexpected. Our first reaction was that the low settlement rates recorded in this study
dramatically lowered the power of the statistical tests. Although this may be true, a
graphical re-examination of the data by layer revealed no trend in greater settlement for
either substrate depth. Thus, we concluded that larval oyster settlement was not impeded
by shell down to depths of 10 cm on artificial reefs composed of oyster shell.
Unfortunately, there are no studies on the settlement of sessile organisms as a function of
substrate depth to which this study can be compared. The fact that adult oysters are found
in greater number at the surface of established reefs (Bahr and Lanier 1981), however,
suggests that settlement patterns and adult ranges may deviate from one another. This was
documented by Roegner (1989) and Kenny et al. (1990), who both found discrepancies in
the ranges of settlers and adults.
There are several reasons why oyster larvae may select substrate within the
interstices of the reef. Some larvae may settle on sub-surface substrate because again they
prefer darkened conditions when setting (Cole & Knight-Jones 1939, Ritchie & Menzel
1969, Shaw et al. 1970) and areas of reduced wave action (Ortega 1981, Abbe 1986), but
also because they seek out environments such as the reef interstices where flow is low,
crevices are abundant, and substrates are not heavily fouled (Bushek 1988, Michener &
Kenny 1991, Morales-Alamo and Mann 1990). Fouling by algae and encrusting barnacles
beneath the surface was considerably less than that found at the reef surface. A final
explanation for the observed sub-surface settlement may be that the interstices, where flow
was substantially less than that found at the surface, may have served as sediment traps and
entrained larvae. Although it is not clear from this study which active or passive transport
mechanism(s) is/are responsible for sub-surface settlement, it is clear that larvae are capable
of settling within the fabric of the reef and are not impeded by shell down to depths of 10
cm. This is quite remarkable considering there may be 20 or more shell layers within the
10 cm. space.
The fact that settlement estimates from shellstrings, a frequently used method of
estimating oyster abundance's, were greater than both destructive and non-destructive
samples suggests that suspended shellstrings overestimate settlement on sloping reef
bottoms. Baker (1994) also found shellstrings to be unreliable predictors of settlement
magnitudes on adjacent substrates. Even though shellstrings fail to provide accurate
assessment of oyster settlement on actual reef topography, they are efficient and reliable
predictors of the presence of late-stage pediveligers at a given site. This was evident by the
fact that the beginning dates of the settlement season coincided exactly with the detection of
spat in destructive and non-destructive samples. Furthermore, when compared with
shellstrings suspended at other sites, they may be useful tools for determining relative
settlement intensities.
A direct comparison between the two methods to predict post-settlement mortality
rates could not be made because settlement rates during week I of the settlement season
were dissimilar between destructive and non-destructive samples when no previous
settlement had occurred. The discrepancy in settlement rates during week I was probably a
product of two factors: substrate differences and difficulties in spat detection. The higher
degree of fouling and colonization on reef shells compared with that found on shells placed
in trays on a weekly basis may have contributed to lower destructive sample settlement
rates. Bryozoans, colonial ascidians, and certain barnacles, organisms a present at the
reef site, considerably reduce settlement and survival of oysters (Ortega and Sutherland
1989). Difficulties in identifying spat on heavily fouled reef shells was probably another
factor contributing to lower destructive spat counts. During the early weeks of settlement,
spat were small and difficult to detect on the heavily fouled reef shells, but when spat
became larger (weeks 5 and 6 of the 1993 study), they were more visible and thus spat
counts were higher. This was evident by the substantial increases in settlement found in

the destructive samples during weeks 5 and 6 while only negligible amounts of spat were
detected on non-destructively sampled shells and shellstrings.
Mass spawning of oysters occurs at temperatures between 22*C and 23*C (Galtsoff
1964), and larvae generally mature in two to three weeks (Abbe 1986); however, the
current results indicate that settlement did not occur until at least 8 weeks after water
temperatures first rose to this range in 1993 and after 5 weeks during the 1994 sampling
period. The onset of the '93 and '94 settlement seasons did not coincide with any change
in salinity either. The '93 settlement season occurred shortly after a minor drop in water
clarity, and the '94 settlement began during a rise in water clarity. The coincidence of
initial settlement and high turbidity in 1993 was surprising given that Calabrese & Davis
(1966) and Davis and Hidu (1969) both found suspended sediment loads in the water
column to be detrimental to larval development and settlement. The results of this study
suggested that neither water temperature, nor salinity, nor clear water conditions alone can
explain fully the onset of settlement. Instead settlement is triggered by the interaction of
numerous factors such as water temperature, salinity, dissolved oxygen, suspended
sediments, food supply, pollutants, availability of substratum, hydrodynamic factors, light,
and other organisms (Abbe 1996).
Settlement intensity examined at each fidal height/substrate level combination was
not found to be dependent on water movement. The lack of dependence of settlement on
water movement may appear somewhat surprising, given that several researchers have
found settlement to be high in areas of low to moderate flow. For instance, Nelson (1921)
and Roughley (1933) found high concentrations of swimming and setting oyster larvae in
eddies and areas of slack water. Hidu and Haskin (1971) found high settlement in
Delaware Bay at sharp transition zones between high and low current velocities, and
concluded that this was a result of concentrations of larval falling out of suspension as
water flow decreased. Furthermore, in Galveston Bay, Bushek (1988) found oysters
preferentially settled nearshore on pier pilings where current velocities were low. One
exception to the above studies is found in Carriker (1959), who found settlement to be
greatest near faster currents in a sheltered salt-water pond.
The observed independence of settlement and water flow may be a result of two
factors. First, the velocities considered in this experiment may not have varied dramatically
enough for a strong functional relationship to be observed. Second, microscale flow
conditions may have had a larger impact on settlement intensities than mean flow rates
calculated 10 cm above the reef bed. Oyster larvae setting on the underside of surface
substrates and within the fabric of the reef probably experienced dramatically different flow
regimes than those measured 10 cm above the benthos. Microspatial flow measurements
need to be measured to accurately determine if larval settlement is dependent on flow in an
environment such as an artificial reef where flow rates are highly variable on a small scale.
Although none of the physical factors investigated were reliable predictors of the
onset of settlement and settlement intensity did not appear to be strongly correlated with
flow rate, we did learn that vertical elevatioWrelative to both the reef surface and MLW
influences settlement. Oysters settle not just at the surface of reef communities but settle in
comparable numbers within the reef interstices down to depths of 10 cm. This finding is
significant because presently there is debate over which substrates should be used for reef
construction. Many of the proposed reefs are to be composed of crushed clam shell, tile,
or mounds of sediment capped with a thin shell layer. When these substrates are used,
sub-surface interstitial space is limited, precluding oyster community development below
the surface. The sub-surface environment provides biological and physical refugia for
oysters and may be- very important for survival in developmentally young, artificial reef
communities (Bartol and Mann, in prep). Furthermore, settlement on artificial reefs is
generally greatest subtidally; however, there may be localized areas within the community
where conditions are beneficial for intertidal settlement and intertidal/subtidal settlement

rates are comparable. These zones may be important for oyster reef development because
they allow for the rapid establishment of intertidal and not just subtidal environments. The
establishment of oysters in heterogeneous intertidal/subtidal environments, habitats they
once thrived in naturally, assuredly is beneficial for dwindling oyster populations.

LITERATURE CITED

Abbe, G. R. 1986. A review of some factors that limit oyster recruitment in
Chesapeake Bay. American Malacological Bulletin. Special Edition No. 3: 59-
70.
Bahr, L.M. and P. Lanier. 198 1. The ecology of intertidal oyster reefs of the South
Atlantic coast: a community profile. U.S. Fish and Wildlife Service, Office of
Biological Services, Washington, D.C. FWS/OBS-81/15. 105 pp.
Baker, P.K. 1994. Quantification of settlement and recruitment processes in bivalve
mollusks. Ph.D. Dissertation, College of William and Mary, Virginia. 381 pp.
Bushek, D. 1988. Settlement as a major determinant of intertidal oysters and barnacle
distributions along a horizontal gradient. J. Exp. Mar. Biol. Ecol. 122: 1-18.
Calabrese, A. and H.C. Davis. 1966. The pH tolerance of embryos and larvae of
Mercenaria mercemnaria and Crassostrea virginica. Biol. Bull. 131:427-436.
Carriker, M.R. 195 1. Ecological observations on the distribution of oyster larave in
New Jersey estuaries. Ecol. Monogr. 21: 19-3 1.
Carriker, M.R. 1955. Critical review of biology and control of oyster drills
Urosalpinx and Eupleua. United States Fish and Wildlife Service. Special
Scientific Report. Fisheries Service 148: 1-150.
Chesnut, A.F. and W.E. Fahy. 1953. Studies on the vertical setting of oysters in North
Carolina. Proc. Gulf Carib. Fish. Inst. 5: 106-112.
Cole, H.A. and E.W. Knight-Jones. 1939. Some observations and experiments on
the setting behavior of Ostrea edulis. J. Cons. perm. int. Explor. Mer. 14: 86-
105.
Davis, H.C. and H. Hidu. 1969. Effects of turbidity-producing substances in sea water
on eggs and larvae of three genera of bivalve mollusks. Veliger. 11: 316-323.
Galtsoff, P. S. 1964. The American oyster Crassostrea virginica Gmelin. Fish. Bull.
Fish Wildl. Serv. US. 64:1-80.
Hargis, W.J.Jr. and D.S. Haven. 1995. Oyster reefs, their importance and destruction
and guidelines for restoring them. Oyster Reef Habitat Restoration Symposium.
Pp. 27-28.
Haskin, H.H. 1964. The distribution of oyster larvae in N. Marshall ed. Proc. Symp.
Exp. Mar. Ecol. Narragansett Mar. Lab., Univ. R.I., Occas. Pap. No. 2. Pp 76-
80.
Hidu, H. and H. H. Haskin. 197 1. Setting of the related to environmental factors and
larval behavior. Proc. Natl. Shellfisheries Assoc. 61: 35-50.
Kenny, P. D., W. K. Michener, and D. M. Allen. 1990. Spatial and temporal patterns
of oyster settlement in a high salinity estuary. Journal of Shellfish. Research
9(2): 329-339.
Kunkle, D.R. 1957. The vertical distribution of oyster larvae in Delaware Bay. Proc.
Nat. Shellfish. Assoc. 48: 90-91.
McDougall, K. D. 1942. Sessile marine invertebrates of Beaufort, N. C. Ecol.
Monogr. 13: 321-37 1.

McNulty, J. K. 1953. Seasonal and vertical patterns of oyster setting off Wadmalaw
Island, S. C. Contr. Bears Bluff Lab No. 15. 17 pp.
Michner, W.K. and Kenny P.D. 1991. Spatial and temporal patterns of Crassostrea
virginica (Grnelin) recruitment: relationship to scale and substratum. J. Exp.
Mar. Biol. Ecol. 154: 97-12 1.
Morales-Alamo, R. and R. Mann. 1990. Recruitment and growth of oysters on shell
planted at four monthly intervals in the lower Potomac River, Maryland. J.
Shellfish Res. 9: 165-172.
Morales-Alamo, R. and R. Mann. 1995. The status of Virginia's public oyster fishery
1994. Virgina Institute of Marine Science/Marine Res. Spec. Rep. 37 pp.
Nelson, T.C. 192 1. Aids to successful oyster culture: I Procuring the seed. Bull.
N.J. Agric. Coll. Exp. Sta. 351: 1-59.
Nichy, F.E. and R.W. Menzel. 1967. Mortality of intertidal and subtidal oysters in
Alligator Harbor, FL. Proc. Natl. Shellfisheries Assoc. 52: 33-41.
Ortega, S. 1981. Environmental stress, competition, and dominance of Crassostrea
virginica near Beaufort, North Carolina, USA. Mar. Biol. 62:47-56.
Ortega, S. and J.P. Sutherland. 1992. Recruitment and growth of the Eastern oyster,
Crassostrea virginica, in North Carolina. Estuaries. 158-170.
Ritchie, T. P. and R. W. Menzel. 1969. Influence of light on larval settlement of
American oysters. Proc. Natl. ShelUisheries Assoc. 59: 116-120.
Roegner, G.C. 1989. Recruitment and growth of juvenile Crassostrea virginica
(Grnelin) in relation to tidal zonation. Master's Thesis, College of William and
Mary, Virginia. 145 pp.
Roegner, G. C., and R. Mann. 1990. Settlement patterns of Crassostrea virginica
(Gmelin, 1791) in relation to tidal zonation. Journal of Shely7sh Research 9(2):
341-346.
Roughly, T.C. 1933. The life history of the Australian oyster (Ostrea commercialis).
Proc. Linnean Soc. New South Wales. 58: 279-332.
Shaw, R., D.C. Arnold, and W.B. Stallworthy. 1970. Effects of light on spat
settlement of the American oyster (Crassostrea virginica). J. Fisheries Research
Board of Canada. 27: 743-748.
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Virginia. Oyster Reef Habitat Restoration Symposium. Pp. 10- 11.
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Tablela. ANOVA of non-destructive settlement data.

Source df Sum of Squares Mean Square F-Value P-Value
mound 1 .078 .078 1.059 .3111
tidal height 3 2.147 .716 9.692 .0001
time 3 .573 .191 2.588 .0701
mound * tidal height 3 .516 .172 2.331 .0928
mound * time 3 .127 .042 .572 .6377
tidal height * time 9 .819 .091 1.232 .3108
mound * tidal height * time 9 .989 .110 1.489 .1942
Residual 32 2.362 .074
Dependent: non-destructive settlement log (x+l) transformed

Table 1b. ANOVA of destructive settlement data

Source d fSum of Squares Mean Square F-Value P-Value
mound 1 .043 .043 .648 .4248
tidal height 3 1.207 .402 6.017 .0015
time 5 7.583 1.517 22.690 .0001
mound * tidal height 3 .582 .194 2.903 .0443
tidal height * time 15 .878 .059 .875 .5942
mound * time 5 .287 .057 .858 .5164
mound * tidal height * time 15 .423 .028 .422 .9648
Residual 48 3.208 .067
Dependent: destructive settlement log (x+l) transformed

Table 2. Paired t-tests performed on surface and deep substrate layer spat counts for
non-destructive and destructive samples. Separate analyses were performed on each
tidal elevation.

NON-DESTRUCTIVE SAMPLES

Tidal height Mean difference Degrees of freedom t-value p-value
(surface-deep)
+ 25 cm -.333 11 -.886 1.3944 11
MLW -.083 11 -.321 1.7545
-90 CM .333 11 .549 1.5940 1

DESTRUCTIVE SAMPLES

Tidal height Mean difference Degrees of freedom t-value p-value
(surface-deep)
+25 cm -.083 1 1 -.561 1.5863 11
MLW .083 1 1 .432 .6742
-90 cm -.500 1 1 -1.149 .2750

Table 3a. ANOVA of log (x+l) transformed spat counts measured in the non-destructive
settlement study.

Source df Sum of Squares Mean Square F-Value P-Value
week 2 .607 .303 15.807 .0001
tidal height 2 1.006 .503 26.204 .0001
week * tidal height 4 .038 .010 .499 .7366
Residual 27 .518 .019
Dependent: log (x+1) transforned mean spat count

Table 3b. ANOVA of log (x+1) transformed spat counts measured in the destructive settlement
study.

Source df Sum of Squares Mean Square F-Value P-Value
week 2 .295 .148 15.156 .0001
tidal height 2 .832 .416 42.709 .0001
week * tidal height 4 .129 .032 3.299 .0252
Residual 27 .263 .010
Dependent: Log (x+l) transformed mean spat count

Figure 1. Cumulative oyster spat counts per shell detected on shellstrings at Palace Bar,
VA from 1979 - 1994 recorded in the VIMS Spatfall Surveys. Spat counts
were recorded for only a portion of the 1988 settlement season.

CUMULATIVE SPAT COUNT PER SHELL

uj 4@1
CD C) C)

0
0
1979
cn 1980
0 1981-:':-:-:-:
1982-
co
co 1983-
OD

CD
CD 1985 -
0
...................... ..........
- 1986 -
1987 -
*1988
0
0 1989
3 ..................X
1990 -
CD 1991 -
cn 1992 -
1993-1
1994
CL
0
FD

Figure 2. Mean oyster spat counts per 30 shells recorded in the non-destructive study.
Error bars denote +1 S.E.

NON-DESTRUCTIVE SETTLEMENT DATA

10-

7.5-

C14
T
........ ........
5-
........ ........ ........
.....*.. ........ ........
........ ........ ........
........ ........ ........
........ ........
........ ........ ........
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Z 2.5-
F-7-771
........ .......
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...... ........ ........
. ...... ........ ..... ........
...... ........ .......
... . . . . . . . . . . .
0
30 0 -45 -@O

TIDAL HEIGHT (CM RELATIVE TO MLW)

Figure 3. Mean weekly spat counts for both destructive and non-destructive samples
collected from August 12 through September 23, 1993. Error bars denote
+1 S.E.

20-
T

15-

10-

5-
A4

0
1 2 3 4 6

WEEK

El DESTRUCTIVE SAMPLES

NON-DESTRUCTIVE SAWLES

Figure 4. Mean spat counts recorded on mound A and B during the 1993 settlement
season. Error bars denote+ I S.E.

DESTRUCTIVE SETTLEMENT DATA COLLECTED ON MOUND A

12.5-

10-

C14

7.5-
........ ........
........ ........
T
T
........ ........ ...
........ ........ ........
........ ........ ........ ........
........ ........ ...

. ........ ........
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5- ........
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........ ........ ........ ........
........ ........
.. ........ ........
........ ........ ......
.. ........
2.5 . ..... .. ........ .........
........ ........ ........ ........

........ ........ ........
...... .........

........ ........
0-
30 0 -45 -90

TIDAL HEIGHT (CM RELATIVE TO MLW)

DESTRUCTIVE SETTLEMENT DATA COLLECTED ON MOUND B

15-

........ ........
........ ........
C14 10-

........ .....
........ ........
.......... ......
..........
........ ........
........ ... *.... .....
........ ........
........ ........ ........
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u 5- ........
........ ........
........ ........ ........
........ ........ ........
........ ........
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T

.. ......... ... ........
........ ........ ...

........ ....... ........

........ ........ ........ ........
..... ........
........ ........ ........ ........
... ........ ........
0
30 0 -45 -90

TIDAL HEIGHT (CM RELATIVE TO MLW)

Figure 5. Mean spat counts per 30 shells calculated from non-destructive samples for
(A) each of the three tidal heights and for (B) each week of sampling (week 1
= July 15-21, week 2 = July 21 - 28, and week 3 = July 28 - August 4, 1994).
Error bars denote +1 S.E.

MEAN SPAT COUNT PER 30 SHELLS MEAN SPAT COUNT PER 30 S

0 Vt

.................. .....
................... .....

................... .....
...................
.................. .....
........... ..... .....

....................
. ............. ........... ........
.................... ..........

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.................................. .........
..................................... .......
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....... .. ....
....... ........ ...
............
...... ......... 0.............
....... ......................
....... ............... ........
.................................
............................

Figure 6. Cumulative spat counts per shell at the -90 cm tidal height detected on
shellstrings, non-destructive samples, and destructive samples in 1993 and
1994. Error bars denote+ I S.E.

...... shellstring
4-
non-destructive

3- destructive

0 ......
1993 1994

YEAR

Figure 7. Salinity, secchi disk readings, and water temperatures recorded at the reef
site during 1993 and 1994.

WATER TEMPERATURE
(DEGREES CELCIUS) SECCHI DISK DEPTH
(METERS BELOW THE SURFACE

0 tA tA
0 - w 4 (A Lq
6/17- 6/17- 6/17:
711- 7/1 - 7/1-
7/15- 7/15 7/15
7129- cn 7/29 7/29
8/12- 8/12 8/12-
8/26- CD
(D 8/26- ILD 8/26
9/9- w 9/9 - 9/9-
9/23 : 0
- 9/23- 9/23-
10/8- 10/8- 10/8:
10/21- 10121: 10121-
11/4- 11/4 11/4 -
11/18- 11118- 11/18:
12/21 12/2- 12/2-

WATER TEMPERATURE SECCHI DISK DEPTH
(DEGREES CELCIUS) (METERS BELOW SURFACE)

w
'A 'A Cl 00
5/26- 5/26- 5/26
6/2- 6/2- 612
6/9- 6/9- 619
6116- 6/16- 6116-
6123- 6123- 6/23-
C/i &30 - 6130- cn 6130-
7/6- 7/6
7'6:. ILO 7/15- 7h5:
7115
ko
7121- w 7/21- 7/21-
a 7/28- 7/28-
7/28-
> 8t4 - 0 8/4- 814-
8112- 8112- 8/12-
8118- 8118- 8/ 18-
8/26- 8/26- 8/26-
911- 9/1- 9/1-
9/8- 9/8- 918-
9115- 9115 9115-1

The Importance of Small-scale Spatial and Temporal Variation in C virginica Growth and
Mortality on Constructed Intertidal Reefs

Ian K. Bartol and Roger Mann
School of Marine Science, Virginia Institute of Marine Science, College of William and
Mary, Gloucester Point, VA 23062

ABSTRACT
The spreading of thin veneers of shell over coastal and estuarine bottoms, which
provides hard substrate for oyster settlement and subsequent colonization, no longer
guarantees the establishment of C virginica communities in the Chesapeake Bay largely
because of the presence of endemic diseases. The construction of intertidal reefs
resembling those present in the Bay during colonial times, but now absent largely because
of overharvesting, may provide a more ecologically suitable environment for oyster growth
and survival than present, two-dimensional habitats. We constructed a 150 x 30 in intertidal
reef, and during three, 28-day sampling periods in the summer, examined growth and
mortality of two year classes of oysters placed in cages at three tidal heights (25 cm above
MLW (mid intertidal zone), MLW, and 90 cm below MLW (low subtidal)) and at two
substrate depths (reef surface and 10 cm below the reef surface). Oysters grew faster
below than at the surface in the mid intertidal zone, and grew faster at the surface than
below in subtidal environments. Growth was greatest subtidally and oysters at all tidal
elevations grew well from mid August through mid September. In the intertidal zone,
residence below the reef surface provided relief from heat and respiratory stress during
periods of peak solar exposure, and in the subtidal zone, sub-surface residence provided
refugia during periods of high predation. These results suggest that small-scale spatial and
temporal factors have a large impact on oyster reef ecology and should be considered when
constructing reefs.

INTRODUCTION
Artificial reefs provide structure and spatial complexity within marine ecosystems
and facilitate the establishment of diverse communities of organisms. The construction of
artificial reefs for habitat restoration purposes has increased dramatically worldwide, with
reef projects in Australia, Japan, Southeast Asia, the Caribbean, the eastern and northern
Mediterranean basins, the Pacific Islands, and North America (Seaman et al. 1989). Many
of these projects have been conducted to enhance local fish resources, and as a result much
of the artificial reef research pertains to fish ecology (Turner et al. 1969, Buckley 1989,
Grant et al. 1982, Grove 1982, Bohnsack and Sutherland 1985, Relini et al. 1986,
Bombace et al. 1990, Beets and Hixon 1994, Bortone et al. 1994, Fabi and Fiorentird
1994). Recently, several studies have been conducted to determine the effect of artificial
structures on lobster communities (Davis 1987, Cruz et al. 1987, Eggleston et al. 1990,
Lozano-Alvarez et al. 1994, Barshaw and Spanier 1994) and sessile corals, invertebrates
and algae (Riemers and Branden 1994, Downing et al. 1985, Fitzhardinge and Bailey-
Brock 1989, Hixon and Brostoff 1985, Bombace et al. 1994, Relini et al. 1994); however,
few studies have examined how artificial reefs can serve as tools for enhancing oyster
populations.
Traditionally, oyster rejuvenation efforts in the Chesapeake Bay have involved the
deployment of cultch, generally oyster or clam shell, off barges in subtidal locations with
the subsequent intent of retrieving either juvenile (seed) or market size oysters which attach
to the cultch. Shells were often broadcast over large areas to maximize coverage because of
the increasing cost of available substrate, producing subtidal carpets of shell which lacked
vertical relief. These 2-dimensional "artificial reefs" substantially enhance settlement, the

irreversible adherence of oyster larvae to the substrate, by providing clean, hard substrate
for attachment, especially when planted at the correct time (Manning 1952, Abbe 1988,
Morales-Alamo and Mann 1990). In the past these procedures facilitated the establishment
of adult oyster communities; however, in the Chesapeake Bay where disease is highly
problematic, these efforts no longer guarantee adult oyster development. This may in part
be because this two-dimensional approach fails to accurately simulate 3-dimensional reef
communities formed naturally in the Chesapeake Bay before man's intervention.
During colonial times intertidal oyster reefs were unmistakable geological and
biological features of the Bay landscape. In the early 1800's there were approximately 10
miles of intertidal reef in the James River, a subestuary of the Chesapeake Bay (Hargis,
pers. comm.). As the economic value of the oyster began to be realized in the mid to late
1800s commercial exploitation of the resource began. Years of subsequent harvesting
resulted in the transformation of all of these protruding, aerially exposed features in the Bay
to mere low-lying subtidal "footprints" of pre-existing intertidal reefs. This degeneration
was exacerbated by the arrival of two protistan parasites, Haplosporidium nelsoni (MSX)
and Perkinsus marinus (Dermo), and environmental degradation.
I The rationale for re-creating colonial intertidal reef systems for the rejuvenation of
oyster populations is simple; since oysters in the Chesapeake Bay resided in extensive
intertidal reef communities before man's intervention, these environments are probably
ecologically and evolutionary advantageous and a return to them may elevate survival rates.
Therefore, we constructed a 150 x 30 in intertidal reef and monitored growth and mortality
of C virginica within the reef ecosystem. In this study we took a micro rather than a
macro approach to reef ecology, in which we examined growth and mortality within a
narrow tidal range (25 cin above to 90 cm below MLW), at different levels within the reef
structure (at the reef surface and 10 cm below the reef surface), and during three, 28-day
sampling intervals in the summer. We investigated whether small-scale spatial and
temporal variation have an effect on C virginica growth and mortality in constructed reef
settings and whether these variations should be considered in future rejuvenation efforts.

STUDY SITE
This study was conducted in the Piankatank River, a sub-estuary of the Chesapeake
Bay located in Virginia, at a site which once supported a highly productive intertidal reef
system but at the time of reef construction was devoid of live oysters. The Piankatank
River is ideal for artificial reef construction because generally there is a high abundance of
oyster larvae (Morales and Mann 1995), and there is no commercial oyster fishery and
virtually no industry or agricultural development within the watershed. Tidal range at this
site is small (mean range = 36 cm); however, local meteorological events, wind in
particular, often dramatically alter this range from 0 to 1.25 m. The site is relatively
shallow (1-3 meters), and consists of a sandy bottom. During the course of this study
water temperature at the study site varied from 0.5 - 30 'C and salinity fluctuated from 8-20
ppt.

Tidal heights of 25 cm above MLW (mid intertidal zone), MLW, and 90 cm below
MLW (low subtidal zone) were considered. At each of the 8 mounds, which varied in size
and orientation, reinforced bars were driven into the reef shells at aU three tidal height
designations. This allowed for the expedient location of the tidal elevations during
sampling. Substrate found at each tidal height on all eight mounds was marked and
partitioned into 64 x 20 cm plots using rope and reinforced bars. This resulted in the
formation of three distinct bands of substrate (one for each height) which potentially could
be sampled on each of the eight mounds.
To document growth and mortality, two year classes of oysters, both of which
were set and reared for several weeks in the Virginia Institute of Marine Science (VMS)
Oyster Hatchery, were considered. One year class consisted of oysters set on oyster shell
on May 16, 1994 and reared in hatchery systems for three weeks, whereas the other year
class consisted of oysters set on oyster shell on August 12, 1993, reared in hatchery
systems for three weeks, and placed in subtidal cages on the Piankatank Reef until the
commencement of this study. On June 1, 1995 oysters comprising each year class were
retrieved from either the field or the hatchery and mixed thoroughly. For each year class,
the cultch shells were grouped into 48, 15-shell assemblages. The concave, smooth
surface of the 15 shells within each grouping were subsequently marked using paint and
permanent markers. We were careful to make sure at least 30 oysters were present
collectively on the labeled sides of shells comprising each assemblage. To keep densities
as constant as possible, the physical removal of oysters in high density communities was
sometimes necessary. OveraU densities were 2.85 + .35 S.E. oysters per shell for the
1994 year class and 2.95 + .48 S.E. oysters per shell for the 1993 year class.
The labeled sides of shells comprising each 15-shell assemblage were photographed
on slide film using a Nikonos V camera equipped with a close-up lens and focusing frame.
The shell areas of 30 oysters in each assemblage (48 total assemblages per year class) were
calculated using an image analysis system manufactured by Biosonics Inc. (1988), and a
map of the oysters on each shell was constructed so that they could be tracked in future
study. Initial shell areas for 1994 and 1993 oysters were 1. 18 + 10 S.E. cm 2 and 4.3 +
2
.52 S.E. cm , respectively. After being photographed, the 15 shell units were placed on
either the upper or lower level of labeled 32 x 20 cm, three-tiered, I inch mesh cages. The
upper and lower levels were 10 cm apart and separated by an intermediate level of 20
oysterless shells.
For both year classes, eight plots were selected randomly at each of the three tidal
heights. The '93 year class and '94 year class oyster cages were deployed at their
designated plots on June 14 and June 20, 1994, respectively. The cages were buried into
the reef substrate until the upper layer of oysters was level with the reef surface and held in
place with reinforced bars. At 28 day intervals in July, August, and September, oysters
were removed from the cages, cleaned using a 3 HP gasoline powered Homelite water
pump, and photographed with the Nikonos V camera. Using the Biosonics image analysis
system, a mean growth value for each 15 shell assemblage was computed over all three,
28-day sampling intervals. This was accomplished by determining the shell areas of all 30
tests oysters within each grouping, calculating a mean shell area for the assemblage, and
subtracting this value from the mean shell area at the beginning of the period. The areas
were expressed in mm/day. For each 28-day sampling interval a mean proportional
mortality value (# dead oysters at the end of the 28-day intervalffilive oysters at the
beginning of the interval) was also computed.

Physical Parameters
Each week throughout the study, water temperature, salinity, and secchi depth
readings were recorded at the reef site. After completion of the study, chlorine tablets
housed in 20 cm x 20 cm mesh cages held 10 cm above the reef substrate were deployed
during both neap and spring tides at all of the sampled plots. The chlorine tablets were

weighed, deployed in the field for 48 hours, and weighed again. Differences in chlorine
tablet mass at each tidal height were compared to construct in the case of subtidal plots, a
framework of relative flow rates, and in the case of intertidal plots, a combined relative
estimate of the magnitude of both flow rates and wave intensity. Chlorine tablets were
used because they were a cost-effective method of obtaining flow information. The theory
behind using chlorine tablets as a surrogate measure of flow is that turbulent diffusion, the
major force driving the dissolution rate of the tablets in the field, in the benthic boundary
layer at a given bottom roughness will vary in a positive fashion with current speed.

Statistical Analysis
Correlation coefficients were computed for all surface/deep sample pairs and
compared with values in a critical coefficient table to determine if a relationship existed
between surface and deep levels (Zar 1984). When no significant relationship was detected
for a data set, surface and deep samples collected at a given plot were treated
independently. Conversely, when significant relationships were detected, paired sample t-
tests were performed to decipher significant differences. These tests were performed
separately by height on mean values computed over the entire sampling period.
Multivariate repeated measures analyses of variance (ANOVA) were performed
separately by year class on growth and mortality data. To satisfy the subject within group
and subject between group homogeneity of variance assumptions, proportional mortality
data were arcsine transformed; however, it was not necessary to manipulate growth data.
When interactions were detected, lower-level ANOVAs and/or repeated measures analyses
were performed. All significant between factor effects were analyzed using SNK multiple
comparison tests and significant within factor main effects were examined using Newman-
Keuls procedure (pp. 527-528, Winer 1991).
Linear regressions of growth on water movement were performed to determine if a
functional dependence was present. Analyses were performed separately by each year
class, substrate level position, and tidal elevation. The assumptions of the regressions
were met as determined by residual analysis (Zar 1984).

RESULTS
Growth
1993 Oysters
Significant differences in growth rates between substrate levels were detected at two
of the three tidal heights examined (Table 1). At +25 cm growth was greater 10 cm below
the surface than at the surface, and at -90 cm growth was greater at the surface than below.
These discrepancies were consistent throughout the three sampling periods.
Growth for the 1993 oysters residing at the surface was dependent on tidal height
and time (Table 2a). Surface oyster growth at tidal heights of -90 cm and NILW was
greater than growth at +25 cm, and growth was greatest at all heights during the
August/September sampling period (Figure I a, 2a). Surface residing oysters situated at the
-90 cin tidal height also experienced a visible decrease in growth during the July/August
sampling period, although this decline was not statistically significant (time x tidal height
.058) (Figure 3a).
No difference in growth across the three tidal heights was detected for oysters
residing below the surface within the reef interstices (Table 2b); however, growth at the
deep layer increased significantly at all tidal heights during the August/September sampling
period, as was observed at the surface (Figure 2b). A trend in reduced growth during the
July/August sampling period was also apparent (Figure 2b).

1994 Oysters
As was the case with the 1993 year class of oysters, growth at +25 cm was greater
10 cm below the surface than at the surface, and growth at -90 cm was greater at the

surface than in sub-surface environments (Table 1). These differences were consistent
throughout the 3 sampling periods. No difference in growth by substrate level was
detected at NILW.
A significant time x tidal height interaction was present when the 1994 surface
residing oysters were analyzed (p=.003) (Table 3a). Further analyses revealed that during
two of the three sampling periods growth was greatest subtidally. During the June/July
period growth increased significantly with tidal depth, and during the August/September
period growth at the -90 cm was greater than growth at +25 cm (Figure 2c). Greater
subtidal growth was not, however, found during the July/August period, because of a
significant decline in growth at NEW and -90 cm (Figure 2c). Furthermore, growth rates
were high during the August/September period across all tidal heights, especially at +25 cm
where growth was significantly greater during this time. Below the reef surface within the
fabric of the reef, oyster growth was dependent on tidal height and time (Table 3b).
Growth was greatest at -90 cm and was lowest during the July/August period (Figure 2d,
3).

Mortality
1993 Oysters
A tidal height x layer and tidal height x time interaction both were detected when
mortality data from the 1993 year class were analyzed (Table 4a). Lower level analyses
performed to decouple these interactions revealed several interesting findings. At MLW,
oysters residing in the interstices of the reef experienced greater mortalities than oysters at
the surface substrate level, but at -90 cm, oysters residing at the surface had the greatest
mortalities (Figure 4). Of the oysters that resided at the surface, those that were situated at
MLW had significantly lower mortalities than those situated at +25 cm or -90 cm (Figure
4). During the August/September sampling period, the highest mortalities across both
substrate depths were detected at the -90 cm tidal height, whereas during the June/July
sampling period, the highest surface mortalities occurred at +25 cm (Figure 5).
Furthermore, during the June/July period significantly higher mortalities were recorded at
the surface than beneath the surface for oysters situated at +25 cm (Figure 6).

1994 Oysters
A significant time x tidal height interaction and a nearly significant time x tidal
height x substrate level interaction (p=.066) were detected when mortality data from the
1994 year class were analyzed (Table 4b). The low p-value in the three-way interaction
was in part a product of higher surface than below surface mortalities during the June/July
period for oysters at +25 cm (Figure 7). Lower level analyses revealed that the time x tidal
height interaction was a product of mortality during the June/July period being greatest at
+25 cm and during the August/September period being greatest at -90 cm (Figure 8).
Although mortality was not found to be significantly lower at any of the three tidal
heights, a graphical depiction of cumulative percent mortality throughout the sampling
period revealed a definite trend of lower mortality at the MLW tidal height (Figure 9).
Overall mean cumulative mortality ranged from 14.2% to 23.4%, which was higher than
that documented for the older, 1993 oysters.

Physical Factors
Growth of 1993 and 1994 oysters was independent of water flow (p > .05 ; r 2<
.30). Salinity, water temperature, and secchi disk readings recorded during the sampling
period are presented. graphically in Figure 10. During the first sampling period, which
began June 14 for 1993 oysters and June 20 for 1994 oysters, there was a rapid increase in
salinity. Salinity in subsequent periods, however, was relatively constant, fluctuating
between only 15%o and 17%o. Water temperatures during the first two sampling periods (
mid June / mid July and mid July / mid August) ranged from 26'-30*C, and during last

sampling period (mid August / mid September) decreased considerably to 22.5*C. Water
clarity also improved during the last sampling period, which began August 9 for 1993
oysters and August 16 for 1994 oysters. Mean air temperatures were greatest during the
June/July period and lowest during the August/September period for both year classes.

DISCUSSION
The elevated rates of growth detected below the reef surface at +25 cm for both year
classes was probably a product of both increased submergence times and a reduction in
stressful environmental conditions. Since oysters residing in the deep layer were lower in
vertical elevation, they were inundated longer by tidal flow, and as a result could filter feed
for longer periods of time and achieve greater sizes. The attainment of larger size in oysters
with increased submergence time has been demonstrated by Ingle and Dawson (1952),
Burrell (1982), and Roegner (1989). Oysters dwelling in these underlying habitats also
were shaded from direct sunlight and wind , and thus were less susceptible to desiccation
and heat stress. Bahr (1976) found temperatures 6 cm beneath the surface at an intertidal
oyster reef community at Sapelo Island, Georgia, to be 7' C lower than at the surface even
in October. The reduction in atmospheric stress allowed oysters to invest more energy in
growth and less in environmental resistance.
Higher subsurface intertidal rates of growth may not occur in all intertidal reef
communities, however. Bahr and Lanier (1981) found surface residing oysters in intertidal
reef communities in the South Atlantic to have sharper growing edges than oysters in lower
layers and felt this was indicative of faster growth at the surface. This finding may have
been because Bahr and Lanier studied a mature, established reef community, where flow
and spatial constraints below the surface inhibited subsurface growth. Dense assemblages
of oysters, present at the surface of established reef communities increase sedimentation
rates by as much as 8 times (Lund 1957, Haven and Morales-Alamo 1967). These high
rates of biodeposition coupled with well established surface fouling communities severely
restrict flow in underlying layers, making growth more difficult. Furthermore, the
stationary nature of established substrate, which is a product of the clustering of
generations of oysters, makes growth more arduous in underlying layers where space is
limited. Flow and space problems are not as problematic in newly constructed or
developing reef communities, however, because biomass is still low, substrate is still
movable, and interstitial spaces are abundant.
Contrary to what was found at +25 cm, oysters from both year classes situated at -
90 cm grew faster at the reef surface than below. This reversal in growth optimization was
largely because subtidal oysters within the interstices did not benefit from longer
submergence times (both subtidal substrate levels were submerged constantly) and more
favorable environmental conditions (there was no difference in refugia from atmospheric
stresses) fike intertidal oysters. Instead, oysters residing below the surface in the subtidal
zone lived in an environment where flow was somewhat limiting. Unlike the intertidal
zone, where fouling was low and water motion energetic, surface algae and colonizing
infauna were highly abundant in the subtidal zone and overall flow rates were low. These
factors reduced the amount of flow reaching oysters in underlying layers and resulted in
slower growth rates.
Oyster growth in relation to tidal height is quantified in several studies, most of
which have found growth to be greatest in the subtidal zone. Loosanoff (193 2), Ingle and
Dawson (1952), Burrell (1982), and Roegner (1989) as mentioned earlier all found greatest
growth subtidally for C virginica, and Sumner (1981) and Roland and Albreit (1986)
found similar results for C gigas. Gillmor (1982) and Crosby et al. (1991) presented
evidence that C. virginica held in the intertidal zone under certain levels of aerial exposure
were capable of growing as fast or faster per unit immersion time as subtidal oysters. 'Me
reason for this is that periodicity in feeding allows for the more efficient processing of
crude fiber. In both studies, however, overall growth rates per day were still greatest

subtidally because of longer submergence times. In contrast to the above studies,
Littlewood (1988), working with C rhizophorae in Jamaica, found growth in the low
intertidal zone was greater than growth at subtidal depths, and Spencer and Gough (1978)
were unable to detect a difference in growth of either C gigas or 0. edulis held subtidally
and in the low intertidal zone.
Most of the results of this study suggest that oyster growth in artificial reef
environments is maximized subtidally. This may be largely due to longer feeding times
provided by constant submergence (Peterson & Black 1987); however, growth is not
simply a direct function of submergence time. Kenny et al. (1991) found that intertidal
oysters exhibited double the expected decrease in growth than that predicted by immersion
time alone, and Peterson and Black (1988) found that growth rates of a variety of
suspension feeding bivalves found in both the high intertidal and low subtidal'were not
directly proportional to submergence time. Within the intertidal zone, the metabolic stress
associated with emergence may have played a role as well (Dame 1972, Newell 1979). As
a result of harsh environmental conditions in the intertidal zone, intertidal oysters allocate
more energy to environmental resistance and less to somatic growth than subtidal oysters
(Dame 1972, Newell 1979). This translates into reduced growth intertidally.
While significantly greater subtidal growth was detected in most of the data
collected, no significant growth difference between tidal heights was observed for 1993
oysters residing below the surface. This was not unexpected for differences in
submergence time and environmental stress, the major reasons for growth discrepancies
along the tidal gradient, were less pronounced in subtidal and intertidal sub-surface
environments. In the intertidal zone, oysters in sub-surface environments were lower in
vertical elevation, allowing for longer submergence and feeding times than surface
dwelling intertidal oysters. Sub-surface intertidal oysters also could afford to invest energy
in growth since the shaded, moist underlying environment was more hospitable than the
surface. These factors lead to faster intertidal growth. Conversely, flow restrictions in
sub-surface subtidal habitats caused. by overlying fouling communities and sluggish flow
rates lowered growth rates below MLW. This convergence in intertidal and subtidal
growth rates made growth differences less detectable. Interestingly, growth was greatest
subtidally for the 1994 year class residing beneath the surface. This suggests that the
benefits of longer submergence times may outweigh the disadvantages of restricted flow
and that submergence time is an important factor to consider when assessing growth rates.
The depressions in growth during the July/August period observed across both year
classes was perplexing. A dramatic decrease in growth like this is often correlated with
some environmental condition or physiological event. But the data collected in this study
supplied no insight as to which mechanism caused the depression. No drastic fluctuations
in water temperatures or salinity occurred during this period, and although a brief peak in
water clarity took place, water clarity was often higher during the August/September period
when growth was often greatest. The decrease in growth did coincide with peak spawning
time (June through August (Galtsoff 1964)), in which a shift is made from totally somatic
growth to gametic and somatic growth. A prerequisite for this shift is sexual maturity.
Oysters may develop functional gonads at a young age (2 to 3 months) and small size (less
than I cm in height) (Galtsoff 1964). The 1994 oysters were only 1.5 months old by the
onset of the growth depression period, and thus it is highly unlikely that growth reductions
were attributable to gametic energy reallocation. Conversely, this metabolic shift may be
applicable to 1993 oysters, which were about 11 months in July. However, some other
cause which applies to both year classes is more probable. The depression in growth may
have been attributable to the fluctuation of some environmental factor not measured in the
experiment, such as dissolved oxygen or phytoplankton assemblages, or may have been a
result of a combination of a number of factors.
Many oysters considered in this study grew most rapidly during the
August/September period, and all oysters grew well during this time. Salinity probably did

not account for this since it remained relatively constant throughout the sampling period.
But lower water temperatures and reduced sediment loads in the water column, which were
evident from secchi disk readings, may have. Galtsoff (1964) suggested that optimal filter
feeding of oysters occurs between 25 - 26 * C, water temperatures most often encountered
during the August/September period. These temperatures may have elevated growth rates.
Furthermore, turbidity, which was substantially lower during the August/September
period, is highly detrimental to oyster pumping rates, and is closely coupled with growth
since oysters are filter feeders (Galtsoff 1964). Elevated growth rates during this period
for 1993 oysters may have also been partly due to a shift from somatic and reproductive
growth to simply somatic growth.
The observed independence of growth on water motion in the subtidal zone, where
water motion measurements were a reflection of flow rates, may have been because
growth, especially below the reef surface, was more closely coupled with food depletion
on a microscale than with mean flow speed above the reef bed. Loosanoff (1958) and
WaIne (1972) demonstrated that growth increases with elevated levels of flow. Grizzle et
al. (1992) found that growth decreases with increased current flow in one study which
focused on current velocities from 0 - 10 cm/sec, and in another study which considered
velocities from 0 - 5 cm/sec found maximal growth at I cm/sec. In all of these studies,
however, individual oysters in a flume rather than a patch of oysters in the field were
considered. At the level of a group of non-siphonate bivalves localized food depletion
rather than flow rate may have a greater influence on growth. This is because at the
relatively quiescent region near the bed, especially within the fabric of the reef, clearance
rates probably exceed the influx of new particles, creating zones of low food concentration.
These zones which are influenced not only by flow rate but also resident species density
and reef topography assuredly had a large impact on growth. Malouf and Breese (1977)
and Wilson et al. (1992) came to a similar conclusior?, and emphasized that bivalve growth
in the field is more closely correlated with food availability in the water column than with
current velocity.
At intertidal plots, where water movement measurements were a reflection of not
only flow rate but wave action as well, growth was not dependent on water motion either.
Within the intertidal zone one might expect oysters, especially in sub-surface environments,
to benefit from more intense water movement because hydrologic forces serve to flush out
fecal wastes and replenish the stagnant environment with fresh, nutrient rich seawater. We
did not see a positive relationship between water motion and growth, though. Nor did we
see a detrimental effect of wave action on growth as observed on highly energetic coasts
(Ortega 1991) largely because wave energy at our relatively protected experimental site was
not sufficient to be deleterious. We feel that the lack of dependence was again a product of
growth being more closely coupled with the microscale environment than with flow rate or
wave action 10 cm above the reef bed. Food patchiness, while not as significant a problem
as in the subtidal zone where water movement is reduced substantially, still likely had a
larger impact on growth than flow rate or wave action. When completely submerged,
intertidal oysters which have been aerially exposed for a period often filter feed at higher
rates than subtidal oysters which are continually submerge. These elevated feeding rates
may cause clearance rates to exceed particle influx rapidly, especially within subsurface
environments.
Environmental buffering beneath the reef surface was the primary reason why
higher mortality was observed at the surface than below at +25 cm during the June/July
period, the interval with the highest mean air temperature (28.2 ' Q. In established,
natural intertidal oyster reefs such as those found on the coasts of Georgia and South
Carolina, oysters at the reef surface are clustered and tend to grow vertically so that
neighboring oysters may provide shade and protection from the sun and drying winds. At
the constructed reef surface, however, where a colonial adult oyster community had not yet
been established, there was little relief from the harsh effects of solar exposure and high air

temperatures, factors which contribute to oyster mortalities in the intertidal zone. Intertidal
oyster mortalities in environments which experience intense solar exposure but offer little
shade are not uncommon, especially when oysters are young. In Virginia from June
through July, Roegner (1989) found a mortality rates of > 75 % for unshaded juvenile
oysters held in the mid to high intertidal zone, and in South Carolina, Crosby et al. (1991)
and Michener and Kenny (1991) reported summer intertidal mortalities of 80 - 90% for
oysters set on exposed asbestos plates. Oysters residing below the surface in the intertidal
zone were shielded from atmospheric stresses. Overlying reef shell coupled with longer
periods of submergence (as a result of being slightly lower in vertical elevation) made the
underlying environment cooler and moister than surface habitats, and lowered both heat
stress and evaporative water loss.
The fact that mortality of 1993 oysters found at -90 cm was higher at the reef
surface than below throughout the 3 month sampling period suggests that residence within
the reef interstices may be advantageous in the subtidal zone as well. Oysters in sub-
surface subtidal environments benefited from a reduction in predation pressure. The two
most prominent oysters predators in the Chesapeake Bay, the oyster drills, Urosalpinx
cinera and Eupleura caudata, and the seastar, Asterias forbesi, were absent at the reef
site because of low salinities. However, a number of predators were present such as the
flatworm Stylochus ellipticus, the mud crabs Panopeus herbstii, Eurypanopeus
depressus, and Rhithropanopeus harrisii, and the blue crab Callinectes sapidus.
Flatworms near oyster populations cause significant mortalities even though the extent of
the damage is unknown (Landers and Rhodes 1970, Morales et al. 1988, Abbe 1986,
Littlewood 1988, Baker 1994), mud crabs prey upon small oysters (Abbe 1986, Baker
1994), causing mortalities as high as high as 50% (Mackenzie 1981), and predation by
blue crabs (Carriker 1955, Abbe 1986, Roegner 1989, Eggleston 1990a, b, c) is well
documented.
Flatworms, mud crabs, and blue crabs were found within cages at surface and
deep substrate layers and at all tidal heights, but were most abundant at surface substrate
layers and subtidal depths based on field observations. Flatworms and mud crabs were
probably most deleterious for they were not restricted by the mesh of the experimental
cages. Flatworim and mud crabs both were observed within the valves of recently dead
oysters, and in photographs, we noticed the presence of numerous boring holes in test
oyster shells. Blue crabs, which were only able to enter cages during juvenile
developmental stages, were less problematic. Even though entry into the cages was
restricted to smaller crabs, predation on caged oysters may not have been limited to juvenile
forms. Numerous oysters were found growing through the cage mesh and were exposed
to the surrounding environment. These oysters were freely accessible to larger predators
in the area. Furthermore, field observations revealed that blue crabs often laid flat on the
tops of cages and extended their claws inward into the interior of the cages. Although no
direct predation of blue crabs on oysters was observed during these maneuvers, this
behavior may have allowed larger crabs to feed on caged oysters. The higher rates of
subtidal surface mortality in 1993 oysters together with greater observed predator
abundance's suggests that the above predators preferred to prey upon oysters that were
highly accessible and readily available, rather than burrow through the reef topography to
expose and prey upon oysters in the underlying layers.
The lack of a detectable differences in subtidal surface/sub-surface mortality for
1994 oysters was likely a product of the young oyster's reduced tolerance to sedimentation
and hypoxic conditions. Sedimentation was greater in the deep substrate layer than at the
surface. Although this sedimentation was not substantial and had little effect on the 1993
oysters, it may have contributed to mortality of the smaller 1993 oysters found in lower
layers. Even low/moderate sediment rates bury and subsequently kill small oysters
(Mackenzie 198 1, Abbe 1986). Furthermore, periodic conditions of hypoxia may occur in
deep layers because flow is low and often restricted. When hypoxic conditions are present,

oysters need to isolate themselves from their surroundings and if necessary, switch from
aerobic to anaerobic respiration. This switch may be more difficult for oysters less than 1
year of age because young oysters put much of their energy into growth and maintenance
rather than in the storage of glycogen, the preferred substrate for anaerobic respiration
(Galtsoff 1964). As a result young oysters cannot employ anaerobic respiration as
efficiently as older oysters which have larger glycogen reserves. Mann and Gallager
(1985) and Holland and Spencer (1973) found that this is highly probable, given that
nutrient reserves are small and polysaccharides account for only a small proportion of the
total energy reserves during development. Thus, the benefits of reduced predation for
subtidal subsurface residence probably were eradicated by the young oyster's inability to
tolerate sedimentation and periods of hypoxia.
Of the three tidal heights considered, 1993 oysters living at the surface survived
best at MLW; when examined over the entire sampling period, 1994 surface residing
oysters clearly demonstrated the lowest mortality at MLW. Oysters at NILW experience
less predation, sedimentation, and fouling than oysters in subtidal habitats and encounter
less severe atmospheric conditions and diminished respiratory stress than oysters in higher
intertidal regions. Thus, oysters at MLW experience the best of both worlds; they benefit
from lowered predation pressure and fouling as a consequence of aerial exposure, but do
not suffer from severe heat and respiratory stress because they are not exposed for
extended periods of time.
The advantages of surface residence at MLW are similar to the benefits of sub-
surface residence; oysters receive a refuge from predation and experience environmental
buffering. Unlike oysters within the reef interstices, however, surface residing oysters do
not have to contend with restricted water flow or hypoxic conditions, which may both
contribute to mortality. Consequently, at MLW mortality was lower at the surface than
below.
The fact that the highest mortalities were detected at +25 cm during the June/July
period and at -90 cm during the August/September interval suggests that physical factors
dominated during the initial sampling session but biological factors became more
pronounced in the final session. During the June/July period when air temperature were at
their highest, mortality from heat stress in the intertidal zone was more prominent than
mortality from predation and fouling in the subtidal zone. Thus, greater mortalities were
detected in the intertidal zone. However, from mid-August through mid-September, when
air temperatures were reduced (mean air temperature = 24.5 - 25.1 'C) and predators and
subtidal algal growth became more prevalent, biological factors dominated and mortalities
became highest in the subtidal zone. During the final sampling period there were greater
sitings, both in photographs and in the field, of flatworms. Baker (1994), in a study
conducted in August in the York River, Chesapeake Bay, Virginia found numerous
flatworms to be red in color, the color of a stain used to mark test oysters, and concluded
that predation by flatworms was substantial during this time. Elevated flatworm sitings
also coincided with an influx of blue crabs at the reef site, which may have contributed to
elevated mortality rates. Water temperatures during the early part of this period hovered
around 26-27 'C. At these temperatures crabs are voracious feeders and exhibit type II
inversely density dependent predation, whereby partial prey refuges found at lower
temperatures are eliminated (Eggleston 1990 a). In addition to predation, algal growth may
have been a source of mortality. Enteromorpha grew prolifically in the subtidal zone
during the August/September period, and may have disrupted both pumping rates and
respiratory functions.
Often when we seek to evaluate large systems like an intertidal reef ecosystem or a
series of reef structures, we think in terms of the big picture. Will these reefs enhance
survival of the depleted oyster population? What influence do these systems have on the
Chesapeake Bay? What is the most cost-effective method of reef construction. These
questions are important and are often the reason for conducting a specific project.

However, in evaluating and answering these questions we often loose track of the small-
scale variability inherent within the system and simply focus on the larger scale uniformity.
This study demonstrates that microscale spatial (cms) and temporal (months within a
season) factors have a profound impact on the underpinnings of an artificial reef
ecosystem. Residence merely 10 cm beneath the reef substrate may enhance growth at a
particular tidal elevations and substantially elevate survival during periods of intense solar
exposure or predation. Likewise, a shift of 30 cm in tidal elevation may move an oyster
from an environment which is highly conducive for growth and survivorship to an
environment which is highly stressful during certain times of the year.
With these factors in mind, our advice to oyster reef builders is to consider using
substrate porous enough to allow for sub-surface colonization. This will significantly
increase the amount of available habitat per m2 of substrate and will provide- valuable
refugia from physical and biological stresses. This is especiay important in newly
constructed artificial reefs where significant biomass at the reef surface, helpful in the
buffering of outside stresses, has not yet been established. Reef builders should also be
aware of the effects of tidal elevation. Before a reef is constructed, builders should
determine which height along the tidal continuum provides the most advantageous
compromise of desirable factors (i.e. settlement, growth, survivorship, etc.). At the
Piankatank reef site, where the goal was to maximize growth but at the same time minimize
mortality, the low intertidal/high subtidal zone was the most advantageous region for the
surnmer months. Proposed reefs should them be constructed so that there is suitable
substrate within the desirable tidal range. Finally, temporal variation should not be
overlooked. This study has demonstrated that in a matter of weeks, substantial changes in
growth and mortality may occur within a reef ecosystem. Reefs are constantly in a state of
flux not just over years/decades but also over weeks/days, and it is these microtemporal
and microspatial variations that produce the large-scale patterns that we observe in the field

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Table 1. Paired t-tests peformed on mean growth of oysters residing
at the reef surface and 10 cm below the reef surface from June
through September. Separate analyses were performed on each tidal
height for both the 1993 and 1994 year classes.

Paired t-tests run on surface and deep layers for the 1993 oysters.

Tidal height Mean difference Degrees of freedom t-value p-value
(surface-deep)
+25 cm -1.44 6 -3.518 1.0126 11
MLW .137 6 .206 * 8417 11
-90 cm 2.193 7 2.531 _0392 _jj

Paired Nests run on surface and deep layers for the 1994 oysters.

Tidal height Mean difference Degrees of freedom t-value p-value
(surface-de p)
+25 cm :1 .218 17 -3.066 1.0182 11
MLW .381 16 .898 .4037
-90 cm 1.165 17 4.286 1.0036

Table 2a. Multivariate repeated measures ANOVA performed on growth data
from the 1993 year class of oysters residing at the reef surface.

Source df Sum of Squares Mean Square F-Value P-Value
TIDAL HEIGHT 2 88.926 44.463 7.820 .0039
Subject(Group) 17 96.658 5.686
TIME 2 287.401 143.701 61.380 .0001

TIME TIDAL HEIGHT 4 23.727 5.932 2.534 .0581
TIME Subject(Group) 34 79.599 2.341
Dependent: GROWTH

Table 2 b. Multivariate repeated measures ANOVA performed on growth data
from the 1993 year class of oysters residing 10 cm below the reef surface.

Source df Sum of Squares Mean Square F-Value P-Value
TIDAL HEIGHT 2 13.523 6.762 1.559 .2349
Subject(Group) 20 86.753 4.338
TIME 2 121.867 60.934 23.460 .0001

TIME TIDAL HEIGHT 4 6.566 1.641 .632 .6426
TIME Subject(Group) 40 103.893 2.597
Dependent GROWTH

Table 3a. Multivariate repeated measures ANOVA performed on growth data from the
1994 year class of oysters residing at the reef surface.

Source df Sum of Squares Mean Square F-Value P-Value
tidal height 2 107.804 53.902 15.058 .0001
Subject(Group) 20 71.595 3.580
time 2 104.308 52.154 21.707 .0001
time * tidal height 4 47.054 11.763 4.896 .0026
time * Subject(Group) 40 96.103 2.403
Dependent: growth (mml day)

Table 3b. Multivadate repeated measures ANOVA performed on growth data
from the 1994 year class of oysters residing 10 cm below the reef surface.

Source df Sum of Squares Mean Square F-Value P-Value
tidal height 2 22.190 11.095 5.336 .0139
Subject(Group) 20 41.585 2.079
time 2 28.898 14.449 7.197 .0021
time * tidal height 4 15.052 3.763 1.874 .1338
time * Subject(Group) 40 80.308 2.008
Dependent: growth

Table 4a. Multivariate repeated measures ANOVA performed on arcsine transformed
mortality data collected from the 1993 year class of oysters.

Source df Sum of Squares Mean Square F-Value P-Value
TIDAL HEIGHT 2 400.739 200.370 4.019 .0257
LAYER 1 .028 .028 .001 .9811

TIDAL HEIGHT * LAYER 2 717.397 358.698 7.194 .0021
Subject(Group) 40 1994.427 49.861
TIME 2 931.755 465.878 12.660 .0001

TIME * TIDAL HEIGHT 4 453.722 113.430 3.082 .0205

TIME * LAYER 2 36.603 18.302 .497 .6100

TIME * TIDAL HEIGHT * LAYER 4 264.045 66.011 1.794 .1382
TIME * Subject(Group) 80 2943.926 36.799
Dependent: MORTALITY

Table 4b. Multivariate repeated measures ANOVA performed on arcsine transformed
mortality data from the 1994 year class of oysters.

Source df Sum of Squares Mean Square F-Value P-Value
tidal height 2 343.135 171.567 1.656 .2037
layer 1 11.369 11.369 .110 .7422
tidal height * layer 2 18.367 9.183 .089 .9153
Subject(Group) 40 4143.799 103.595
time 2 588.868 294.434 4.403 .0153
time tidal height 4 1066.695 266.674 3.988 .0053
time layer 2 205.856 102.928 1.539 .2208
time tidal height * layer 4 614.294 153.574 2.297 .0662
time Subject(Group) 80 5349.560 66.870
Dependent: mortality

Figure 1. Mean growth rates examined by tidal elevation for the 1993 year class
oysters found at (A) the reef surface and 03) 10 cm below the reef surface. Error
bars denote +1 S.E.

MEAN GROWTH (MM X MM DAY) cu MEAN GROWTH (MM X MM

tQ U3

........................

LA
. . . . LA
......................
... ..................
...............
. . . . . . . . . . . . . . . . . . . . . .
..........
........................ ....
...............
........................ ......

.. . . . . . . . . . . . . . . . . . t7i
. . . . . . . . . . . . . . . . . . . . . . . .
. ........ .......

. . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
... . . . . . . . . . . . . . ....

. . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . .

............... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . .

Figure 2. Mean growth rates examined by tidal height and sampling period for (A) the
1993 year of oysters residing at the reef surface, (B) the 1993 year class of
oysters residing 10 cm below the surface, (C) the 1994 year class of oysters
residing at the surface, and (D) the 1994 year class of oysters residing 10 cm
below the surface. Error bars denote +1 S.E.

1993 SURFACE OYSTERS C) 1994 SURFACE OYSTERS

12.5- 10

T
10- T
7.5- ...
T

T
7.5-

T 5-

5-
T T
0
T ...
z T 2.5-
z
--t 2.5-

... ... ... .... ...

0-
01
25 0 -90 25 0 -90
TIDAL HEIGHT (CM RELATIVE TO MLW) TIDAL HEIGHT (CM RELATIVE TO MLW)

June/July

July/August

AugusttSeptember

1993 DEEP OYSTERS D) 1994 DEEP OYSTERS
10- 8 T
<
7.5- T 6-

T
x T x
T

.... .... .... ...
Z 4-
T
X. ...
T
0
0

Z 2.5- 2-
z ....
< ...

.... .... .... ...

... .... .... .... ....
... ... ... ... ...
... ... .... ...
0- 0
25 0 -90 25 0 -90

TIDAL HEIGHT (CM RELATIVE TO MLW) TIDAL HEIGHT (CM RELATIVE TO MLW)
-.T.- T
T T T T
T

Figure 3. Mean growth rates examined by tidal height of the 1994 year class of
oysters residing below the reef surface. Error bars denote +1 S.E.

1994 DEEP OYSTERS

6-
...........
..........
...........
..........
T
...........
..........
..........
...........
...........
... ..........
........... ...........
........... ........... ..........
...........
...........
........... ........... ...
4 . ............ ........... ... ...
........... ........... ...........
........... ........... ..........
...........
..............
........... ........... ...........
........... ........... ...........
........... ........
............
..............
0
..........
...........
...........
...........
Z 2-
..........
............
..........
........... ...........
........... ..........
...........
.............
...........

..... ... ..........
........... ...........
...........
0-
25 0 -90

TIDAL BEIGHT (CM RELATIVE TO MLW)

Figure 4. Mean cumulative percent mortalities of the 1993 year class of oysters
from June through September, 1994. Error bars denote +1 S.E.

20-

15-
00

> F- 10- E3 suRFAcELEvEL
z . . . .....
..!t T
P

...... ......
...... ......
...... ......
:2 0 DEEP LEVEL
...... ......

> 5 . ......
0

T
...... ......
...... ......

0
25 0 -90

TIDAL HEIGHT (CM RELATIVE TO MLW)
'T

Figure 5. Mean monthly percent mortality for the the 1993 year class of oysters
examined by sampling period and tidal height. Error bars denote + 1 S.E.

JUNE/JULY

tR 2-

......... .........
........ ........

TIDAL HEIGHT (CM RELATIVE TO NfLW)

JULY AUGUST

........ ........
....... .........

......... .........
........ ........
......... ....

......... .........
........ ........
0.5- .........
........ ........
........ ........

....... ........

X
0 .........
25 0 -90

TIDAL HEIGHT (CM RELATIVE TO NILW)

AUGUST SEPTEMBER
15-

10-

- ------ ........
........ .........
......... ........
........ .........
......... ........
........ .........
......... ........
........ .........
......... ........
........ .........
......... ........
...........
0-
25 0 -90

TIDAL HEIGHT (CM RELATIVE TO MLW)

Figure 6. Mean percent mortality at the +25 cm tidal height during the June/July
sampling period for the 1993 year class oysters. Error bars denote +1 S.E.

.................
................
................

.................
.................
4-

................
z

................

................ ...
................. ...

0
Surface Deep

SUBSTRATE LEVEL

Figure 7. Mean percent mortality at the +25 cm tidal height during the June/July
sampling period for the 1994 year class oysters. Error bars denote +I S.E.

20-

15-

.................
.................
.................
.................
.................
.................
.................
.................
.................
10-
.................
.................
.................
..........
................
.................
................
................
* ...............
................
................
................
......... .................
................ ................
.................
................
.................
...............
5-
.................
................. ................
................. .................
................. ................
................. .................
................ ................
................. .................
................ ................
................. .................
................. ................
................. .................
................. ................
................. .................
................ ................
................. .................
................ ................
................. .................
................ ................
.................
o l@*"***"**"*****,*.,*.*,*.,******* I*-,.*..*.-*.-,..*.,.,.
SuLe Deep

SUBSTRATE LEVEL

Figure 8. Mean percent mortality for the 1994 year class oysters examined by tidal
height and sampling period. Error bars denote +1 S.E.

JUNE/JULY
15-

10-

..........
... ........
......... .........

..... ... ........
......... .........
........ ...... * . ........
........ ......... .........
........ ........ ........
......... .........
..... ........ ........
0
25 0 -90

TIDAL HEIGHT (CM RELATIVE NILW)

JULY/AUGUST
10-

7.5-

........ .........
......... ........
5-
........ .........
......... ........

....... ........

2.5-
...... .... ... ...
.. ...... ....
... ........ .........
......... ......... - *......
........ ........ .........
......... .........
........ ........ .........
........ ........ .........
......... . I ....... ........
........ ........ ........
......... ......... ........
........ ........ ......
......... .......
0 X
25 0 -90

TIDAL HEIGHT (CM RELATIVE TO NUM)

AUGUST/SEPTEMBER
15-

10-

.... .........
........ ........
T
......... .........
5-
....... .... ... ........
......... ........ ........
........ ......... .........
......... ........ ........
...........
.... ..... .
........ .........

......... .... .
........ .....

0
0 -90
TIDAL HEIGHr (CM RELATIVE TO MLW)

Figure 9. Mean cumulative percent mortality for the 1994 year class oysters from June
through September, 1994. Error bars denote +1 S.E.

40-
El SURFACE

30- DEEP

20-
...... ......
...... ......
...... ......
...... ......
..... ......

...... ......
.... ......
...... ......
....... ......
.... ......
10-
.... ......
....... ......
...... ...... ......
..... ......
...... ......
...... ......
...... ...... ......
.... ....... ......
...... ...... ......
...... ....... ......
...... ....
.... .......
0
25 0 -90

TTDAL HEIGHT (CM RELATIVE TO MLW)

Figure 10. Salinity, water temperatures, and secchi disk readings recorded at the
Piankatank reef site during the 1994 summer.

SECCHI DISK DEPTH WATER TEMPERATURE (DEGREES CELCIUS) Im Sp
(METERS BELOW SURFACE)
p r- t@ N 00
LA LA (A tA
5/26- 5/26-
5/26- 6f2 - 6/2-
6/2- 6/9- 6/9-
6/9- 6116- 6/16-
6116- 6123- 6/23-
6/23- 6/30- 6/30-
6/30- 7/6- 7/6-
7/6- 7/15-
7/15- 7/15-
7/21 7/21
7/21- 7/28- 7/28-
7/28- 8/4- 8/4-
0 8/4- 8/12- 8/12-
8/12- - 8/18-
8/1 8- 8/18 8/26-
8/26- 8/26- 911-
911- 911- 9/8
9/8- 9/8 9/15
9115-1 9/151-

Temporal and Spatial Patterns of Growth and Mortality on a Constructed Intertidal Reef:
Results from a Year-Long Study

Ian K. Bartol and Roger Mann
School of Marine Science, Virginia Institute of Marine Science, College of William and
Mary, Gloucester Point, VA 23062

ABSTRACT
During pre-colonial times intertidal oyster reefs were unmistakable geological and
biological features of the Chesapeake Bay, but today aerially exposed reefs are absent in the
Bay largely because of commercial exploitation, disease, and environmental degradation.
Reconstructing three-dimensional oyster reef habitats is one frequently proposed but
critically unevaluated approach for rejuvenating ailing oyster populations. In this study, a
sizable intertidal reef was constructed in the Piankatank River, Virginia, and growth and
mortality of two year classes of hatchery-reared C. virginica placed at three tidal elevations
(25 cm above MLW, MLW, and 90 cm below MLW) were monitored for a period of a
year. To develop an overview of mortality of naturally-set oysters at the reef site, three
annual surveys of oyster density at the three tidal elevations were performed. Furthermore,
we present results from a short mortality study conducted over an unusually harsh winter,
in which oysters were placed in bags on the reef at various tidal elevations. The results
indicated that there is considerable spatial and temporal variation in growth and mortality at
the reef, but that when these variations are summed over a year's time, growth is greatest
subtidally and mortality is lowest at NILW, provided weather conditions are not unusually
harsh. More importantly, however, density measurements collected annually and mortality
measurements collected over a year's time suggest that survivorship is still low in reef
settings regardless of tidal elevation. Reefs, especially those constructed of porous
substrate and having some vertical dimensionality, still may be advantageous for oyster
survival because mortality is still likely lower in reefs than in non-reef settings, but based
on data collected here, it will likely take time before we see a dramatic rebound in oyster
stocks as a result of reef construction.

U41RODUCTION
Intertidal oyster reefs, structures aerially exposed during low tide and composed of'
dense assemblages of live oysters, oyster shell, various invertebrate fauna, and mud, were
unmistakable geological and biological features of the pre-colonial Chesapeake Bay. These
complex habitats, which proliferated in the Chesapeake Bay and tributaries during the last
half of the Holocene interglacial, were important self-renewing food sources for early
settlers and Native Americans alike (Hargis and Haven 1995). As the economic value of
the oyster Crassostrea virginica began to be realized in the mid 1800s, however,
commercial exploitation of the resource began. Years of subsequent overharvesting has
transformed these once massive, aerially exposed communities to mere subtidal, "footprint"
structures which have significantly less vertical dimensionality and habitat heterogeneity.
Disease, environmental degradation, and poor resource management in the last half century
have expedited this degeneration.
Today, Virginia's oyster population is less than I % of what it was just 35 years ago
(Wesson et al. 1995) and is in severe jeopardy of collapse. One frequently proposed, but
critically unevaluated approach for increasing oyster stocks, is the construction of intertidal
oyster reefs, environments oysters resided in naturally before man's intervention. The
rationale here is simple; since oysters in the Chesapeake Bay lived in intertidal conununities
for centuries and we're able to withstand significant environmental and biological stresses,
there is likely an ecological and evolutionary advantage to intertidal, colonial reef existence
in the Bay, and a return to it may help rejuvenate ailing oyster stocks.

As a result of the absence of intertidal oyster reefs in the Chespaeake Bay for over
100 years, we know little about intertidal oyster reef ecology in temperate estuaries. In this
study, we examined Crassostrea virginica growth and mortality on a constructed intertidal
reef in the Chesapeake Bay to assess both the oyster's capacity to withstand environmental
and biological stresses and its ability to develop in a temperate estuary, where presently
intertidal reefs are largely absent. Specifically, we measured growth and mortality of two
year classes of oysters placed at three tidal elevations (25 cm above MLW, MLW, and 90
cm below MLW) on the constructed reef for a period of a year.

STUDY SITE
This study was conducted in the Piankatank River, a sub-estuary of the Chesapeake
Bay located in Virginia, at a site which once supported a highly productive intertidal reef
system but at the time of reef construction was devoid of live oysters. The Piankatank
River is ideal for artificial reef construction because generally there is a high abundance of
oyster larvae (Morales and Mann 1995), and there is no commercial oyster fishery and
virtually no industry or agricultural development. Tidal range at this site is small (mean
range = 36 cm). However, local meteorological events, wind in particular, often
dramatically alter this range from 0 to 1.25 in. The site is relatively shallow (1-3 meters),
and consists of a sandy bottom. During the course of this study water temperature at the
study site varied from 0.5 - 30 'C and salinity fluctuated from 8-20 ppt.

MATERIALS AND METHODS
The reef was constructed in June 1993 by the Virginia Marine Resource
Commission (VMRC). The construction procedure involved the deployment of aged
oyster shells off barges using a high pressure water cannon. The shells were broadcast
over an area approximately 150 m x 30 m, which were the approximate footprint
dimensions of the pre-existing reef system. When this study was initiated, the constructed
reef consisted of 12 intertidal hummocks. Only eight of these hummocks were considered
for this study, however, because the remaining four did not protrude sufficiently above the
water surface to allow for the consideration of heights above mean low water (MLW).
Two year classes of oysters were considered, both of which were reared originally
in the Virginia Institute of Marine Science (VIMS) Oyster Hatchery. One year class of
oysters was set on oyster shell on May 16, 1994, whereas the other was set on oyster shell
on August 12, 1993. The oysters belonging to each year class were placed in cages on the
Piankatank reef after spending approximately 3 weeks in the Hatchery.
The '93 year class was used in an experiment to develop a preliminary
understanding of winter mortality at the reef site. For this experiment a total of 100 cultch
shells containing live oysters was placed in each of 12 Vexar mesh bags. On each of two
mounds, which were vastly different in orientation, size, fouling, and flow conditions, a
bag of oysters was placed at tidal heights of 30 cm above MLW, MLW, 45 cm below
MLW, and 90 cm below MLW along two, spatially distinct transects. The bags were
placed on the reef in November, 1993, and in May, 1994 the bags were retrieved, shaken
vigorously, and opened. Twenty-five cultch shells were removed and photographed in sets
of 5 using an Olympus OM camera and a focusing boom. Spat scars on each cultch shell
were noted in the photographs and mean proportional mortalities (# of scars per shell
scars per shell + live oysters) were calculated for each bag of oysters.
After this experiment the oysters were placed in cages and returned to the reef site,
where they were joined by the newly reared '94 year class. On October 10, 1994, all the
oysters were retrieved from the reef and brought back to the lab. At this time, oysters were
removed from the cultch shells until only 1 oyster remained on each shell. Using a drill
press, a hole, through which I of 5 color-coded labels was attached, was excised into 600
individual cultch shells (300 per year class). Shell heights were recorded, and five oysters,

each containing a different colored label, were placed into each of 120, numbered, 15 cm x
15 cm cages.
Instead of considering four tidal heights as was the case in the preliminary study,
three tidal heights (25 cm above MLW, MLW, and 90 cm below MLW) were investigated
for the remainder of the experiment. The high intertidal height was lowered slightly to
accommodate all eight intertidal mounds in the sampling procedure, and one of the subtidal
heights, 45 cm below MLW, was eliminated to incorporate more replication. At each of the
eight mounds, all reef substrate falling within the 3 tidal ranges was partitioned into 64 x 20
cm sections using rope and reinforced bars.
For both the '93 and '94 year classes of oysters, four plots were selected randomly
at each of the three tidal heights. Reinforced bars were driven into the reef substrate at each
plot on October 18, 1994 and served to anchor 5, 15 x 15 cm cages, all containing color
coded oysters from the same year class, to the reef surface. At 28 day intervals from
October, 1994 through October, 1995, with the exception of the December through April
when no measurements were taken, oyster shell heights were collected. Since each oyster
could be defined by its color tag and cage number, individual oysters were tracked and we
were able to calculate monthly growth rates and proportional mortalities (# oysters dead/ #
oysters alive at the beginning of the 28-day interval). Mean growth rates and proportional
mortalities were computed for each quintuple of cages for all sampling times.
In addition to mortality measurements collected during the '93 - '94 winter and
growth and mortality measurements collected from October '94 - October '95, a yearly
survey of oyster density on the reef was conducted. This survey began in September '93,
shortly after the first settlement event was detected on the reef, and ended in September
'95. This information served to compliment data collected during the first two studies and
provided a generalized overview of naturally-set oyster survivorship on the reef In early
September of '93, '94, and '95, four 64 x 20 cm plots were selected randomly at each of
the three tidal heights. A 64 x 20 cm quadrat was placed at each plot and a surface layer of
30 shells was extracted. Densities, expressed as the number of oysters present per 30 shell
quadrat sample, were recorded, and mean densities per tidal height were calculated for each
of the three years.
Multivariate repeated measures analyses of variance (ANOVA) were performed
separately by year class on growth and mortality data. To satisfy the subject within group
and subject between group homogeneity of variance assumptions, proportional mortality
data were arcsine transformed. However, it was not necessary to manipulate growth data.
When interactions were detected, lower-level ANOVAs and/or repeated measures analyses
were performed. All significant between factor effects were analyzed using SNK multiple
comparison tests and significant within factor main effects were examined using Newman-
Keuls procedure (pp. 527-528, Winer 1991).

RESULTS
Significant time x tidal height interactions were detected in both the 1994 and 1993
year class oysters when growth was analyzed (2-factor ANOVA; 1993 oysters: F = 5.92,
df = 12, 48, p < .000 1; 1994 oysters: F = 5.5 8, df = 12, 54, p < .000 1 ). Lower level
analyses revealed that these interactions occurred because growth was not maximized at any
one tidal height consistently throughout the experiment. For the 1993 year class oysters,
growth was greatest at the -90 cm tidal height during the October-November and
November-May periods, and during the August-September period growth was significantly
greater at -90 cm than at +25 cm (Figure 1 A). However, during the May-June period
growth was maximized at MLW, and in the remaining periods (June - August and
September - October) there was no difference in growth according to tidal height, primarily
because of low growth at the -90 cm tidal height (Figure I A). For the 1994 year class
oysters, growth was greatest at -90 cm during the October-November, November-May,
and September - October periods, and during the August-September period growth at both

MLW and -90 cm was greater than at +25 cm (Figure IB). No significant difference in
growth according to tidal height was detected in the May-June, June-July, or July-August
periods again because of a significant drop in growth at the -90 cm tidal height.
Shell height measurements recorded at the end of 1 year in the field indicated that
the largest shell heights were achieved subtidally. For the 1993 year class, shell height
measurements were 55.3 � 2.8 S.E. mm. at the +25 cm tidal height, 58.1 + 1.9 S.E. mm at
the MLW tidal height, and 64.0 + 1.6 S.E. mm at the -90 cm tidal height. For the 1994
year class, final shell height measurements were 44.7 + 1.3 S.E. mm at the +25 cm tidal
height, 50.5 + 3.1 S.E. mm at the MLW tidal height, and 55.9 + 3.0 S.E. mm. at the -90
cm. tidal height (Figure 2).
When mortality data were analyzed a significant time x tidal height interaction was
detected as well for both year classes (2-factor ANOVA; 1993 oysters: F = 9.58, df = 12,
54, p < .0001; 1994 oysters: F = 5.33, df = 12, 54, p < .0001). This was also attributed
to variations over time as to where along the tidal gradient survivorship was maximized.
During the November - May period mortality of 1993 oysters was highest at +25 cm and
lowest at -90 cm, during the May - June period mortality was greater at -90 cm than at
MLW, during the July - August and August - September period mortality was lowest at
+25 cm, and during the September - October period mortality was lowest at MLW (Figure
3A). For the 1994 year class oysters, mortality was greatest at +25 cm during the
November - May period, greatest at -90 cm during the July - August period, and lowest at
MLW during the August - September period (Figure 3B).
Cumulative percent mortality over the I year sampling period was lowest at MLW.
Figure 4, which depicts conservative estimates of cumulative mortality, shows that for
1994 oysters, mortality rates were 76.1 + 2.4 S.E. % at +25 cm, 61.3 + 4.1 S.E. % at
MLW, and 78.2 + 9.3 S.E. % at -90 cm. For 1993 oysters, mortality rates were 55.0 +
7.0 S.E. % at +25 cm, 51.4 + 7.5 S.E. % at MLW, and 63.4 + 4.8 S.E. % at -90 cm after
1 year's time. These values are conservative because we assumed that oysters lost
throughout the study survived. If the assumption that missing oysters did not survive is
made, these estimates would be 7 - 15% higher, but the trend of lowest mortality at MLW
would remain.
During the initial mortality experiment conducted over the '93 - '94 winter,
mortality rates at MLW and higher in the intertidal zone were 95 - 100%. Mortality rates
during this same period were only on the order of 20 - 25% for oysters residing in the
subtidal zone at depths of -45 cm and -90 cm. Substantially lower mortalities at all tidal
heights were recorded during the '94 - '95 winter. The 1993 year class oysters had a mean
mortality of 35.8 + 7.1 S.E. % at +25 cm, 15.1 + 4.4 S.E. % at MLW, and 1.0 + 1.0 % at
-90 cm. during this period, and 1994 oysters had a mean mortality of 57.5 + 5.7 S.E. % at
+25 cm, 20.9 � 6.4 S.E. % at MLW, and 4 + 1.2 S.E. % at -90 cm.
Density measurements collected annually at the Piankatank reef are illustrated in
Figure 5. We could not extrapolate mortality rates directly from the data because of yearly
settlement events, but it is clear from the graph that considerable mortalities occurred within
the intertidal zone from 1993 to 1994 and across all tidal heights from 1994 to 1995.

DISCUSSION
The results of this study suggest that there is substantial temporal and spatial
variation in growth and mortality of oysters on constructed reefs, and when these variations
are summed over a year or years the macroscale patterns observed in the field are
generated. Over the '94 - '95 growth study, growth was not greatest subtidally at each
sampling period, but significantly greater subtidal growth from August through May was
substantial enough for the largest shell heights to be detected at -90 cm after a year's time.
This occurred despite periods when significant growth differences were undetectable across
tidal heights and periods of significantly greater intertidal growth from May - August.
Faster subtidal than intertidal oyster growth has also been found in an earlier study
conducted on the reef (see preceding manuscript) and by Loosanoff (1932), Ingle and

Dawson (1952), Burrell (1982), Roegner (1989), Sumner (1981), and Roland and Albrect
(1986).
We suspect the low subtidal growth rates from May to August, when growth at
other tidal heights matched or surpassed growth at -90 cm, was a product of the allocation
of more energy towards gonadal tissue production and reproduction. Sexual maturity in
Crassostrea virginica happens after at least 3 months of age, and peak spawning generally
occurs from June through August, when water temperatures fall within optimal ranges for
reproduction (Galtsoff 1964, Abbe 1986). In May, oysters belonging to the '93 and '94
year classes were 13 months and 22 months, respectively. Thus, they were old enough to
undergo somatic/reproductive growth reallocation. Oysters in the intertidal zone may have
delayed or shortened the period of high reproductive energy allocation so that they could
put more energy into their own growth and survival. This may have been necessary,
especially at +25 cm, to withstand the high physical stresses associated with periods of
aerial exposure. Oysters situated at MLW, which encounter much less physical stress and
experience longer submergence times than those at +25 cm, may have been able to take
greater advantage of periods devoted predominantly to somatic growth than oysters
residing higher in the intertidal zone. As a result, growth at M]LW was high at either the
beginning or end of the spawning period, even surpassing that observed at -90 cm.
Significantly lower rates of mortality at MLW during the August-September and/or
September-August periods coupled with relatively low mortalities in the remaining months
allowed oysters at MLW to experience the lowest mortality during the '94 - '95 study.
High mortality rates at +25 cm from November through May, when air temperatures were
low, and during the June-July, August-September, and September-October periods, when
air temperatures were high, contributed to the high overall mortality rates at +25 cm.
Similarly, high subtidal mortality rates from June through October, when mortality from
predation and subtidal fouling were elevated, generated high overall mortality rates at -90
cm. An elevated incidence of disease for oysters residing at -90 cm relative to oysters
situated higher in the intertidal zone may have contributed to high subtidal mortalities as
well (see disease discussion).
The low mortality rates at MLW relative to those detected at +25 cm and -90 cm
probably were a result of these oysters experiencing less predation, fouling, and possibly
disease incidence than oysters in subtidal habitats and encountering less severe atmospheric
conditions than oysters in higher intertidal regions. Oysters at MLW, therefore, experience
the best of both worlds; they benefit from lowered predation pressure, fouling, and disease
as a consequence of aerial exposure, but do not suffer from severe heat, cold, or
respiratory stress because they are not exposed for extended periods of time. High
survivorship at MLW was also detected in the study conducted over the summer of 1994
(see previous manuscript).
The high rates of mortality detected at +25 cm and at MELW over the '93 - '94
winter together with the substantial drop in oyster density in the intertidal zone from 1993 -
1994 suggest that survivorship may not always be maximized at MLW. These exorbitant
mortality rates, especially at MLW, were likely atypical and a result of the coincidence of an
unusually brutal winter and the presence of a young population of oysters (oysters were 4
months old at the onset of the winter). From December of '93 through March of '94, air
temperatures dropped below freezing 28 days, which is very unusual for Virginia. Oysters
less than 1 years old are especially vulnerable to freezing conditions because they put much
of their energy into growth and maintenance rather than into the storage of glycogen, a
preferred substrate for anaerobic respiration, and thus are less capable of environmental
isolation (Mann and Gallager 1985, Widdows et al. 1989). The substantially lower
mortalities detected during the '94 - '95 winter, especially at MLW, give weight to the
argument that the '93 - '94 mortalities were indeed unusual.
Although the intertidal mortality rates over the '93 - '94 winter are atypical and
survivorship at the reef during a normal year when older oysters are present in the reef

community is high at MLW, the high cumulative mortalities and low density figures
recorded at all tidal heights are disturbing. Oyster which survive to 5 months of age and 14
months of age experience mortalites of at least 50% (based on conservative estimates) by
the time they reach 17 and 26 months, respectively, during an average year in terms of
weather even if they are situated at the most optimal tidal depth for survival (NlLW). If you
factor in unusually harsh weather and begin with younger oysters, which are more
susceptible to predation and environmental stress, these mortalties will be even higher.
Mortality rates in reef settings are still likely to be lower than in non-reef settings,
especially if the reefs have vertical dimensionality, but unfortunately, based on the results
of this study these differences are not so substantial that they lead to dramatically higher
oyster survivorship. Two years after the only significant settlement event on the reef,
which occurred in August '93, oyster densities in the subtidal zone where settlement
intensities were highest went from 45.1 oysters per 30 shells to 3.1 oysters per 30 shells.
These densities are higher than densities on adjacent, 2-dimensional beds of shell, where <
0.5 oysters were present per 30 shells, but nontheless are distressingly low. Based on the
results presented in the previous manuscript, oyster densities may be increased somewhat
by constructing the reef out of substrate porous enough to allow for sub-surface
colonization. But even these efforts may not guarantee the establishment of immediate
adult oyster communities because mortalities in the interstitial environment may be
considerable as well. Presently, with the lack of more cost-effective alternatives, the
construction of 3-dimensional oyster reefs which provide spatial complexity and enhance
survivorship may be the best alternative for rejuvenating oyster stocks; the results of this
study suggest, however, that a dramatic rebound in oyster stocks as a result of reef
construction may take many years.

LITERATURE CITED

Abbe, G. R. 1986. A review of some factors that limit oyster recruitment in
Chesapeake Bay. American Malacological Bulletin. Special Edition No. 3:
Burrell, V. G., Jr. 1982. Overview of the South Atlantic oyster industry. World
Mariculture Soc. Spec. Publ. No. 1: 125-127.
Galtsoff, P.S. 1964. The American oyster Crassostrea virginica Gmelin. Fish. Bull.
Fish. Wild. Serv. US. 64: @ 1-80.
Hargis, W.J. Jr. and D.S. Haven. 1995. Oyster reefs, their importance and destruction
and guidelines for restoring them. Oyster Reef Habitat Restoration Symposium.
Pp. 27-28.
Ingle, R.M. and C.E. Dawson, Jr. 1952. Growth of the American oyster Crassostrea
virginica (Gmelin) in Florida waters. Bull. Mar. Sci. of the Gulf and Carib. 2:
393-404.
Loosanoff, V. L. 1932. Observations on propagation of oysters in James and
Corrotoman Rivers and the seaside of Virginia. Virginia Commission of
Fisheries. 46p.
Mann, R. and S. M. Gallager. 1985. Physiological and biochemical energetics of I
arvae of Teredo navalis L. and Bankia gouldi (Bartsch)(Bivalvia: Teredinidae).
J. Exp. Mar. Biol. Ecol. 85: 211-228.
Morales-Alamo, R. and R. Mann. 1995. The status of Virginia's public public oyster
fishery 1994. Virginia Institute of Marine Science/Mar. Res. Spec. Rep. 37 pp.
Roegner, G.C. 1989. Recruitment and growth of juvenile Crassostrea virginica
(Gmelin) in relation to tidal zonation. Master's Thesis, College of William and
Mary, Virginia. 145 pp.
Roland, W.G. and K. Albrecht. 1989. Growth and survival of Pacific oyster seed in
Baynes Sound, B,C. British Columbia Mariculture Newsletter. 6: 13-18.
Sumner, C. E. 198 1. Growth of Pacific oysters, Crassostrea gigas cultivated in
Tasmania. H. Subtidal Culture. Aust. J. Mar. Freshwater Res. 32: 411-416.
Wesson, J.A., R. Mann, and M.W. Luckenbach. 1995. Oyster restoration efforts in
Virginia. Oyster Reef Habitat Symposium. Pp. 10- 11.
Widdows, J., R.I.E. Newell, and R. Mann. 1989. The effects of hypoxia and anoxia on
survival, energy metabolism, and feeding of oyster larvae (Crassostrea virginica,
Gmelin). Biol. BuIL 177: 154-166.
Winer, B.J., D.R. Brown, and K.M. Michels. 1991. Statistical principles in
experimental design. McGraw-Hill, Inc.: New York. 1057 pp.

Figure 1. Mean shell growth from October '94 through October '95 for A) oysters reared
in 1993 and B) oysters reared in 1994. Error bars denote +1 S.E.

A) 1993 OYSTERS
8-

El +25 CM HEIGHT
6- MLW HEIGHT

-90 CM HEIGHT
0
T
4-

0 z
z

0 <
z

TIME PERIOD

B
1994 OYSTERS
10-

7.5- +25 CM HEIGHT

MLW HEIGHT

-90 CM HEIGHT
5-
T

2.5-

0
z
Cn
ka A
0 0
< z
z < cn

TBE PERIOD

Figure 2. Final shell heights of the 1993 and 1994 year class oysters after being placed on
the reef at 3 different tidal elevations for a year. Error bars denote + I S.E.

70-

65- T

60- T
T 1994
55-
... . ... 1993

50-

45-

40
-100 -50 0 50

MAL HEIGHT (CM RELATIVE TO NILW)

Figure 3. Mean percent mortalities from October '94 through October '95 for A) oysters
reared in 1993 and B) oysters reared in 1994. Error bars denote +1 S.E.

A) 60- 1993 OYSTERS

50-

40- T +25 CM HEIGHT
T

0
MLW HEIGHT
30-

-90 CM HEIGHT

20-

10
T

L.A
0
>
0 -.!@ z
z

0
0
z

TDAE PERIOD

80- 1994 OYSTERS

60- T

+25 CM HEIGHT

40-
MLW HEIGHT

-90 CM HEIGHT

20-

0 lAr

z
z LU
AT

TME PERIOD

Figure 4. Cumulative percent mortality of the 1993 and 1994 year class oysters after being
placed on the reef at three tidal elevations for a year. Error bars denote + 1 S.E.

100-

T
75-
T T
T
...... 1993
T
0 . ...... ......
5
...... ......
...... ...... ......
....... ......
...... ....... ......
1994
...... ......
...... ......
...... ......
...... ...... ......
...... ......
...... ......

...... ....... ......
...... ....... ......
25-

...... ......
...... ......
0 ....... ......
25 10 -9,0

TIDAL HEIGHT (CM RELATIVE TO MLW)

Figure 5. Oyster densities taken in September, 1993, 1994, and 1995. Error bars denote
+ 1 S.E.

50-

40-

SEMMBER'93
30-
C4
SEMMBER '94

cn SEMM13ER '95
20-

0 10-

0-
25 0 -90

z
TMAL HEIGFIT (CM RELATIVE TO MLW)
- t-T

Progression of diseases caused by the oyster parasites, Perkinsus marinus and
Haplosporidium nelsoni, in Crassostrea virginica on Constructed Intertidal Reefs.

Frank 0. Perkins
Department of Zoology, North Carolina State University, Raleigh, NC 27695-7617

H%TTRODUCTION
From May 5, 1994 to December 14, 1995 the progression of diseases caused by the
oyster parasites, Perkinsus marinus and Haplosporidium nelsoni, were evaluated by
periodic sampling of oysters which set in August, 1993 on the artificial reef located in the
Piankatank River. It had been previously established (Mackin, 1962; Haskin and
Andrews, 1988) that oysters most often do not become infected by either parasite in the
first 9 to 12 months of life; therefore, no sampling was conducted until 10 months after
setting. This proved to be a reasonable assumption for the present study since the first P.
marinus infections were not detected until 14 months after setting and the first H. nelsoni
infections at plus 15 months (with the exception of one oyster at plus 13 months). The
infections observed were recorded as a function of 1) prevalence (incidence) and intensity
(weighted incidence), 2) host mortalities, 3) oyster size and age and 4) depth below mean
low water at which the host oyster was found on the reef. The total number of reef oysters
sampled was 3,908.

The study was conducted to determine 1) whether depth below mean low water at
which the oysters resided was significant in determining to what extent the oysters became
infected, 2) at what size and age the oysters became infected to levels which resulted in
significant infections and mortalities, and 3) whether oysters which set in an endemic area
(the Piankatank River) on an artificial reef have lower mortalities than susceptible oysters
from an area in which the parasites are rarely observed (the upper James River). The latter
comparison is only valid when similar age oysters are compared; therefore, the
comparisons are of interest only at the end of the study. The first observations/conclusions
should be of interest to individuals responsible for constructing artificial reefs and the
second observations are of interest to individuals who must determine when to harvest
oysters to avoid excessive losses.

METHODS
Oysters which had set on the reef were sampled every 2 to 4 weeks during the
study period of May 5, 1994 to December 14, 1995. They were obtained by hand or by
using oyster hand tongs, depending an the depth. Six samples of 25 oysters per sample
were obtained for each sample time at two locations on the reef The depths in the first 15
sample times were intertidal and 45 and 90 cm. below mean low water. Due to the ever
increasing difficulty in finding intertidal oysters and the recognition that samples from the
bottom of the reef should be obtained, the intertidal sampling was discontinued and
sampling was initiated from near the bottom of the reef at 167 cm for the 16th sample
(February 10, 1995) until the end of the study. With respect to depth the data is analyzed
in terms of -<45 cm and @90 cm. The paucity of intertidal oysters is believed to have been
due to deaths which occur as a result of exposure to freezing temperatures during the winter
months. The base of the reef was located in 2-3 meters below mean low water.

The observations are expressed in ternis of number of weeks after setting. Most of
the set in 1993 occurred from August 5 to 12. To facilitate the handling of the data herein,
August 12 was selected as the date of set. Whereas another primary set occurred during the
study period in 1994, the set which was followed through the course of the study was the
1993 set.

As a means of comparing the progression of infections in the reef-set oysters with
the progression through a population of susceptible adult oysters, 350 uninfected,
susceptible adult oysters were obtained from the upper James River seed beds (Horsehead
rock) and placed in plastic mesh bags on the Piankatank River reef near the sample sites at
the time of the third sampling of the reef oysters (June 16, 1994). The depth of placement
was about midway between the top and bottom of the reef (ca. 100 cm below mean low
water). At the time of placement a sample of 25 oysters was analyzed for the presence of
the two parasites, using techniques described below. No P. marinus or H. nelsoni cells
were found. There was a possibility that the sampling period would occur when one or
both parasites were in low numbers in the area of the reef. Therefore, in order to confirm
that the parasite detection methodology was being applied properly and to check for
patchiness in distribution of the parasites,, 350 James River oysters from the same
population used on the Piankatank River reef were placed in plastic mesh bags in the York
River behind VIMS, an area in which both diseases are known to be commonly present in
high levels. The treatment of the 350 oysters held at VIMS was the same as described for
the other 350 oysters. Each batch of 350 oysters was sampled until none remained. In the
following spring (April 14, 1995) another batch of 350 oysters from the same James River
site was placed on the reef and 350 at VIMS as in the previous year and sampled until none
remained.

Accumulative mortality data was obtained by counting the number of "boxes"
(shells without tissue) encountered in the course of selecting 150 oysters at the time of each
sampling of the August, 1993 set. Unfortunately, because of a lack of communication,
mortality data was not collected for the final 19 weeks of the study. In retrospect it may
have been better to have also selected about 500 oysters on May 5, 1994 at the beginning of
the study, placed them in trays or bags and sampled them for mortalities during the course
of the study. However, this approach would have presented the difficulty that the oysters
would have been crowded so that transmission of P. marinus infections would have
occurred more readily and the mortality would have been higher than for oysters dispersed
over the reef, yielding misleading results.

Oysters were assayed for the presence of P. marinus using the Ray fluid
thioglycollate medium technique (Ray, 1954) in which samples of gill and digestive gland
were incubated in the medium. Perkins (unpublished data) has determined that use of those
organs reveals the presence of the parasite in very light or light infections more frequently
than when mantle or rectal samples are used. The intensity of infections was recorded
using a modification of the Mackin scale (Mackin, 1962) in which 0= no infection, 1 =very
light, 2=light, 3=light-moderate, 4=moderate, 5=moderate-heavy, and 6=heavy. This
differs from the Mackin scale in that very light is assigned a value of 1 instead of 0.5, thus
the highest 5 values used herein are one unit more than on the Mackin scale.

H. nelsoni was detected by using histological, paraffin-embedded sections stained
in hernatoxylin and eosin. The scale of Burreson et al. (1988) was employed in recording
intensities of infections where 0-- no infection, 1= cells were rare, 2= less than two cells
were seen per field of view using a 40X objective, 3= 2-5 cells per field of view and 4=
more than 5 cells per field of view.

The effects of depth and sampling time (age) of oyster on disease susceptibility
(prevalence and intensity) to P. marinus and H. nelsoni were assessed using logistic
regression analysis (Agresti, 1990).

RESULTS
The observations for Perkinsus marinus prevalence in oysters which had set on the
Piankatank reef are summarized in Figure I for depths of _<45 cm and @90 cm below mean
low water. Weighted prevalences are summarized for the same depths in Fig. 2.
Infections did not start to appear until 14 weeks into the study when the oysters were one
year old. Conversion of sampling elapsed times to ages of oysters and times of year are
found in Table I - For the next 44 weeks until the oysters became a year and 10 months old
the number of infected oysters ranged mostly between 15 and 35% after which the
prevalences rose rapidly in the ensuing 2 months to 100% or nearly 100% where they
remained until the end of the study when the oysters were almost 2 years and 5 months old.
The intensities of infections during the plateau phase remained mostly below very light
until the end of the plateau (plus one year, 10 months old) then rose rapidly to the levels of
moderate to moderate-heavy at the age of 2 years plus 1.5 months old followed by a decline
to between light and light-moderate at the end of the study.

The prevalence of H. nelsoni markedly different from that of P. marinus (Fig. 3).
With the exception of one lightly infected, 50 week old oyster, the onset of H. nelsoni
infections did not occur until the oysters were one year, 6.5 months old as opposed to the
appearance of P. marinus in one year old oysters. Thereafter, the infection prevalences of
H. nelsoni rose rapidly to a maximum of 45% when the oysters were 11 months old. The
infections then declined precipitously to almost 0% when the oysters were 2 years, 1.5
months old followed by a slight rise which remained below 10% for the final 4 months of
the study. The intensities of infections reached a peak at the age of one year, 11 months,
one month before the prevalence peak was reached and declined to almost as rapidly as did
the prevalences (Fig. 4).

A comparison was made between the oysters obtained at _<45 cm and 290 cm below
mean low water using logistic regression to analyze the differences in P. marinus and H.
nelsoni infection prevalences and intensities. It was observed that P. marinus prevalence
was significantly higher (p < 0,0001) in oysters collected from depths @90 cm compared to
those from _<45 cm. Prevalence significantly increased (p < 0.0001) in oysters from all
depths with age of oysters, indicating that continued exposure to P. marinus or increasing
age of oysters results in increased infection. Similar results were observed when P.
marinus infection was expressed as weighted incidence or intensity. P. marinus infection
intensity was significantly higher at the greater depths (p < 0.01) and significantly
increased in oysters from all depths with age (p < 0.0001).

Likewise, oysters collected from @90 cm had a significantly higher prevalence and
intensity of H. nelsoni infections compared to those from _<45 cm (p < 0.0001). In
addition, H. nelsoni prevalence and intensity increased with increasing oyster age (p <
0.0001).

From the cumulative mortality data one can see that the mortalities began at the age
of one year 2 months old, 2 months after P. marinus infections first appeared and 6.5
months after H. nelsoni appeared (with the exception of one infected oyster noted above).
The peak mortality value of 40% observed when the oysters were 2 years old corresponds
to time at which the oysters became 100% infected with P. marinus and 3 weeks after the
peak prevalence value for H. nelsoni (Fig. 5). Unfortunately mortality data is not available
for the last 19 weeks of the study. After the appearance of H. nelsoni, it is not possible to
distinguish between mortalities caused by the two pathogens, nor is it possible to
distinguish those causes from other sources of mortality. This would have been possible
only if approximately daily samples had been obtained in which gapers were selected for

analysis, an impossible task using the resources which were available. Less frequent
sampling would have yielded shells without oyster tissues due to the predation which
occurs by crabs and fish once the oysters are not able to close their valves.
Examination of the oyster size data shows that 4 weeks before the appearance of
first P. marinus infections (10 weeks into the study vs. 14 weeks) there was a decrease in
the rate of growth (Fig. 6). This resembles the findings of Paynter and Burreson (199 1)
where they observed a decrease in the growth rate of juvenile and adult oysters immediately
after or just before infection. Since H. nelsoni did not appear until 7.5 months after the
change in growth rate, that pathogen was not responsible. Whether the high summer
temperatures (Fig. 7) or some other factor such as the oysters' food source was responsible
can not be determined. The salinity was relatively constant (Fig. 7); therefore it is unlikely
that it was responsible. The decline in oyster sizes after the age of 2 years (65 weeks into
the study) is believed to be due to death of the larger oysters resulting from infections of the
two pathogens.

The data for the adult oysters which were imported from the upper James River
seed beds and which represent a disease-susceptible population is used to confirm that the
two pathogens were present in the study area and in the neighboring area of the lower York
River (Figs. 8-11). They were used primarily to indicate presence or absence of H.
nelsoni since its levels fluctuate greatly, some years being nearly absent from the lower
York River region. During the study period, one can see that the pathogens were prevalent
and the patterns of disease organism expression was what one would expect based on the
studies of Andrews (1988) and Haskin and Andrews (1988). 'Me discontinuities in the
curves seen in Figs. 8 to 11 are a result of depletion of the first stock of 350 oysters from
which samples were obtained followed by replenishment with a new stock of 350 oysters.

It is interesting to note that P. marinus infection prevalence was the same in oysters
held in the York River and at the Piankatank River reef during 1994 but in 1995 was
expressed earlier and reached 100% 15 weeks before those held in the York River. One
would have expected that the oysters held in the York River would have shown a stronger
prevalence where the salinities ranged about 5 ppt above those in the Piankatank River and
thus would have presented more favorable salinities for expression of P. marinus. On the
other hand, in 1994 H. nelsoni infections were nearly non-existent in the Piankatank River
reef oysters during 1994, whereas in the York River stock infections were above a
prevalence of 60% during the summer and fall of 1994 (Fig. 10). These observations
reaffirm the necessity of having a stock of susceptible, adult oysters present in a study of
this type where juvenile and young oysters are being observed. Age of the oysters will
play a role determining the prevalence and intensities of infections. In addition, the
patchiness of distribution of H. nelsoni within the Chesapeake Bay is confirmed by the
data.

In 1995 a different picture of H. nelsoni infection distributions in the imported,
adult oysters was observed (Figs. 10 and 11). The prevalences and intensities were quite
similar at the two stations with the infections appearing earlier at the York River station and
lasting longer in the population. Nine more weeks of data was obtained from the York
River stock because the stock at the reef was depleted by mortalities earlier, probably due to
P. marinus infections (Figs. 8 and 9).

The reef oysters at the age of @!2 years old can reasonably be compared to the
imported oysters in terms of response to the diseases. The former is a population which set
in an endemic area and thus had a chance to acquire some immunity to the two diseases, if
capable of such a response. The latter was a stock of 2-4 year old (ages not known, only

estimated) susceptible oysters from an area with little or no prior exposure to the two
pathogens. With respect to P. marinus the prevalences and intensities of infections were
similar in the two groups of oysters at the reef, Therefore, one can not state that the reef-
set oysters were more resistant to the pathogen. On the other hand, the H. nelsoni data
indicates that the reef-set oysters were more resistant to those infections. Imported oysters
at the reef reached a peak of 68 % infections (Fig. 10), whereas the reef oysters peaked at
only 36 and 45%, depending on the depth of residence (Fig. 3). Likewise, the intensities
of H. nelsoni infections reached a mean high of level 2 in imported oysters as opposed to
1.3 in the reef oysters. Therefore, some advantage appears to have been obtained for the
reef-set oysters, if one can neglect age differences.

DISCUSSION
The interpretation of epizootiological data such as that generated in this study is
difficult because of the plethora of factors which dictate to the prevalence and intensities of
infections and mortalities. Most, but probably not all, of the factors are: temperature,
salinity, water quality in terms of anthropogenically-derived chemicals present, density of
oyster populations, residence depth in the water column, patterns of water movement,
oyster age and/or size, genetic strains of oysters involved, physiological condition of the
oysters as dictated by food availability (density and species composition of planktonic food
organisms present) and numbers and levels of other parasitic species causing stress on the
oysters. A further complication is the fact that the reservoir of H. nelsoni infective cells is
unknown and transmission of infections is not from oyster-to-oyster as opposed to P.
marinus where transmission is direct.

Despite these formidable complicating factors, one is able to detect patterns of
oyster mortalities and oyster growth changes which can be related to the levels of P.
marinus and H. nelsoni found in the oysters. The present data set is unique in that it is the
first time a population of naturally set oysters of known age has been assayed in situ in
terms of the progression of infections by P. marinus and H. nelsoni over an extended
period of time, in this case for one year and 8 months. Other epizootiological studies have
involved placing naturally set or hatchery set oysters of known age in containers in an
endemic area or placing adult oysters of unknown ages from non-endemic or marginally
endemic areas into containers in an endemic area. Placement in containers provides a
greater degree of experimental control but closer proximity of oysters can lead to results
different from those in naturally set populations where distances from oyster-to-oyster
varies greatly.

Since infections did not appear until 3 months into the study Q months after May 5,
1994), one can assume that the disease progressions were essentially followed from setting
(August 12, 1993) until the oysters were 2 years and 4.5 months old (December 14, 1995).
Considering the fact that the salinity values recorded during the first 11.5 months after
setting did not go below 10 ppt. and most of the time were @!16 ppt, it is reasonable to
assume that infections did not occur prior to May 5, 1994 when sampling began. This
assumption is based on results of other studies in which is was observed that when a
population does become infected the infections do not disappear unless the salinity drops
below 10 ppt. for an extended period of time.

The following factors are mostly considered independently and related to the
existing knowledge generated from 44 years of studying the epizootiology of P. marinus
and 37 years for H. nelsoni in the Chesapeake Bay. Conclusions are then presented
considering all factors measured. In the following discussion, reference is made to age of

the oysters or elapsed time in the study, whichever is appropriate. The reader is referred to
Table 1 for conversion of age to elapsed time to time of year.

Temperature and Time of Year-
The prevalence and intensities of infections of P. marinus followed generally the
patterns observed in earlier studies (Andrews, 1957; 1988), i.e., a rise in the spring,
peaking in October and November and declining in the winter months into spring. The
temperatures had risen to the range of 25-30'C by the time at which P. marinus appeared in
the reef oyster population (Table 1; Figs. I and 7). Had the oysters been placed on or pre-
existed on the reef from August 12, 1993 as juveniles or adults, one would expect
infections to appear earlier when the temperatures had exceeded ca. 150C, depending on the
temperatures to which the oysters had been exposed in the previous 12 months. The
decline in prevalences in the colder months was gradual with unexpected and unexplained
increases in January and February (2-70C) following by minimum values being expressed
in March to May when temperatures were rising from 8 to 190C. The lag in loss of P.
marinus infections during December to February is consistent with observations from the
earlier studies cited. Intensities of infections were more nearly reflective of previous
reports in that the peaks for the two depths were reached in October and November and
reached a minimum in May of the following year.

As one would expect from the literature, late in the second year of life and early in
the third year after temperatures had exceeded 20'C (in June), infection prevalences and
intensities rose rapidly. The peak in prevalence (ca. 100%) was reached in July-August
when the temperatures were 25 to 3CPC and held at about 100% until the temperatures were
below 150C and the peak in intensities (>5) was reached in September when the
temperature was 23"C and had declined to 2.6 by the end of the study. This is an earlier
than expected time to reach the maximum prevalence value; however, the slight decline in
December is when would expect the decline in prevalences to commence.

The data for H. nelsoni was somewhat surprising in that only one oyster was found
to be infected in the first year of life (Fig. 3) and the population did not otherwise begin to
show infections until the oysters were over 1.5 years old. It possible that this lag can be
attributed to 1) the oysters being young and thus less susceptible as has been reported from
other studies, and 2) the fact that even the susceptible, imported adult oysters did not
acquire very many infections at the Piankatank River reef (Fig. 10) in the first year of reef
oyster life. It was clear that H. nelsoni was present in strength in the nearby York River
(Fig. 10), but not in the reef area, thus illustrating the patchiness in distribution of the
reservoir of infective cells, at least in that part of the Chesapeake Bay. The decline in
prevalence and intensity of H. nelsoni in reef oysters was more precipitous that has been
previously reported using imported susceptible adult oysters (Haskin and Andrews, 1988),
reaching a very low level by August rather than about December; however, the start of the
decline was the same as previously reported.

Salinity-
Consideration of the salinity data vs. prevalences and incidences illustrates the point
made in the first paragraph of this discussion concerning the complexities involved in
determining which factors are responsible for limiting or encouraging expression of the
disease organisms. In the first three sample periods the salinities were unfavorable for
expression of the disease organisms in that it was below 15 ppt., but the lack of expression
observed was probably mainly due to the young age of the oysters. Thereafter, the
salinities were mostly above 15 ppt. and were highly favorable for the pathogens when the
values approximated 20 ppt. and in fact that was the time period when the prevalences rose
markedly. The question which can not be answered is whether the primary factor in

encouraging or permitting a rapid increase in prevalences was temperature, oyster age or
salinity. Most probably the best answer is that all three played synergistic roles.

Age-
This factor has been mentioned above. Other studies have noted that oysters are
refractory to acquiring infections in the first year of life and become increasingly more
susceptible into the second year with significant prevalences, incidences and mortalities
being observed then (Andrews, 1957). In fact that pattern was observed in the present
study (Figs. 1-5). As mentioned above, the complicating factor was the low level of
infection pressure from H. nelsoni in the first year of life at the reef.

Ray (1954) followed the progression of P. marinus infections through a population
of young Louisiana oysters of known age and found that infections occurred as early as 9-
10 weeks old but did not start a progression of ever increasing levels of infection until they
were 3 months old. At the end of 12 months the infections reached a level of 37%
prevalence, much lower than the susceptible, adult oysters assayed (90%). The per cent
mortality and weighed incidences of the young oysters remained low for the duration of the
study. The fact that these observations differ from those obtained in this study could be
due to there being a different strain of P. marinus in Louisiana, the higher salinities found
in the study site around Grand Isle, LA and the presence of larger numbers of infective
cells in the water column. In addition, Perkins (unpublished data) has found that a
diversity of bivalve molluscs will filter P. marinus cells from the ambient water and the
cells can accumulate in the interstitial spaces and lumens of the tissues without multiplying
thus infections, in the strictest sense, may not occur in resistant individuals. Nevertheless,
it is unlikely that infective cells of P. marinus were absent from the waters around the
Piankatank River reef in the first year after setting and some pathogen cells should have
accumulated in the tissues of the young oysters; therefore, there are differences between the
Louisiana and Virginia oysters.

Residence depth in water column-
Of considerable interest is the residence depth of the oysters relevant to mean low
water, because the premise behind construction of artificial reefs is that the survival of
oysters in the presence of P. marinus and H. nelsoni will be enhanced if they are grown in
the more natural environment of an oyster shell reef off the bottom of the estuary. In fact,
as stated in the Results section above, there is reason to believe that the oysters which are
growing at 45 cm or less can be expected to have lower prevalences and intensities of
infections of both diseases. Whether this translates to significant differences in survival of
the oysters was not elucidated in this part of the reef study (see following section on
mortalities).

These conclusions concerning P. marinus differ from those of Quick and Mackin
(1971) who observed no effect of depth on prevalences from intertidal to 3m below mean
low water. More significantly, their weighted incidences (intensities) data showed an
decrease with increasing depth. Their study area was the Atlantic and Gulf of Mexico
coasts of Florida. Similar observations are those of Burrell et al. (1984) who found higher
prevalences and intensities of P. marinus in intertidal oysters than in subtidal oysters,
differing from the conclusions of Mackin (1962) who observed that intertidal oysters have
lower levels of infection. He speculated that this results because the oysters are not
exposed to as many infective cells by virtue of the increased amount of time they are closed
and not feeding as compared to subtidal oysters.

Mortalities-
Although the levels of infections which are adequate to kill oysters are not precisely
known, it has been suggested that a mean level of light (level 1) for P. marinus in a
population of oysters can be considered to signal the beginning of significant mortalities
(Andrews, 1988). In studies of this t)W the many potential causes of mortalities can not
be given relative values, but it may be significant in this study that before H. nelsoni
became a source of mortalities (Fig. 4) and when levels of P. marinus were still mostly
below level one, oyster mortalities had begun to climb (after the age of 58 weeks). It is
suggested that when incidences of P. marinus reach a mean of less than level 1,
mortalities will begin to summate at a relatively constant rate (Fig. 5). As stated, the
problem with this assumption is that other sources of mortalities exist and can not be
separated from those caused by P. marinus. Nevertheless, the accumulative mortality
curve in Fig. 5 matches well with the prevalence and intensity curves of P. marinus (Figs.
I and 2). After 91 weeks of oyster age the prevalence, intensity and mortality curves began
to rise rapidly.

The prevalence and intensity curves for H. nelsoni do not match as well to the
mortality curve, the complication being the earlier onset of infections at the deeper levels
below mean low water. Since the mean levels of infection were mostly below level I
(rare), it is suggested that H. nelsoni played a relatively minor role in contributing to
mortalities.

CONCLUSIONS
1) Perkinsus marinus was more significant as an agent of mortalities than was H. nelsoni.
2) In the first 11 months of life it can be expected that only a very small (insignificant)
number of oysters will become infected with the two species of pathogens on the oyster
reef.
3) Assuming that temperature and salinity values approximate those of the study period,
oyster mortalities from P. marinus can be expected to begin 13 months after setting, rising
most significantly one year, 10 months after setting.
4) Residence depths -<45 cm below mean low water were more favorable at the study site in
terms of prevalences and intensities of infections than residences of @:90 cm.
5) In the second year of oyster life, the epizootiological patterns of disease development (as
a function of temperature, salinity and time of year) for P. marinus and H. nelsoni
approximate those patterns which have been previously described in the literature where
adult oysters were used.
6) H. nelsoni has a patchy distribution in the area of the York and Piankatank Rivers;
therefore, adult, susceptible oysters must be used during a study such as the present one to
determine whether the pathogen is present in sufficient numbers to be a factor.

Table 1. Time scale for sampling times used in the study. The oyster ages are estimated
assurning a se ing im,_ of Aug. 12, 1993
Sampling Date Oyster Age (wks.) Sampling Elapsed Time
(wks.)
May 5, 1994 38 0
May 26 41 3
June 16 44 6

June 30 46 8

July 15 48 10
July 28 50 12
August 12 52 14
August 26 54 16
SeptembeL 8 56 18
September 23 58 20

October 5 60 22

October 20 62 24

November 11 65 27

December 8 69 31

January 12, 1995 74 36
February 10 78 40

March 13 83 45

April 14 87 49
May 11 91 53

June 15 96 58

June 30 98 60

July 13 100 62
July 31 103 65
August 24 106 68
September 18 110 72

October 24 115 77

December 14 122 84

LITERATURE CTFED

Agresti, A. 1990. Categorical data analysis. John Wiley and Sons, New York. pp. 79-
129.
Andrews, J. D. 1988. Epizootiology of the disease caused by the oyster pathogen
Perkinsus marinus and its effects on the oyster industry. Amer. Fish. Soc. Spec.
Publ. 18:47-63.
Andrews, J. D. and W. G. Hewatt. 1957. Oyster mortality studies in Virginia. H. The
fungus disease caused by Dennocystidium marinum in oysters of Chesapeake
Bay. Ecol. Monographs 27:1-26.
Burrell, V. G., Jr., M. Y. Bodo and J. J. Manzi. 1984. A comparison of seasonal
incidence and intensity of Perkinsus marinus between subtidal and intertidal oyster
populations in South Carolina. J. World Maricul. Soc. 15:301-309.
Burreson, E. M., E. Robinson and A. Villaba. 1988. A comparison of paraffin histology
and hemolymph analysis for the diagnosis of Haplosporidium nelsoni (MSX) in
Crassostrea virginica (Gmelin). J. Shellf. Res. 7:19-23.
Haskin, H. H. and J. D. Andrews. 1988. Uncertainties and speculations about the life
cycle of the eastern oyster pathogen Haplosporidium nelsoni (MSX). Amer.
Fish. Soc. Spec. Publ. 18:5-22.
Mackin,J.G. 1962. Oyster disease caused by Dermocystidium marinum and other
microorganisms in Louisiana. Publ. Inst. Mar. Sci., Univ. Texas. 7:132-229.
Paynter, K. T. and E. M. Burreson. 199 1. Effects of Perkinsus marinus infection in the
eastern oyster, Crassostrea virginica: H. Disease development and impact on
growth rate at different salinities. J. Shellf. Res. 10:425-43 1.
Quick, J. A., Jr. and J. G. Mackin. 197 1. Oyster parasitism by Labyrinthomyxa marina
in Florida. Professional Papers Series, No. 13, April, 197 1. Fla. Depart. Nat.
Res., Mar. Res. Lab., St. Petersburg, Fla. pp. 24-26.
Ray, S. M. 1954. Biological studies of Dennocystidium marinum. The Rice Institute
Pamphlet. Special Issue, Nov., 1954. pp. 65-76.

ACKNOWLEDGEMENT

Dr. Aswani K. Volety is thanked for providing the statistical analyses and
construction of the graphs used in this report as well as useful and stimulating discussions
concerning interpretation of the results. The interpretations and conclusions are the
responsibility of Frank 0. Perkins.

LIST OF FIGURES

Figs. 1 & 2- Prevalences (Fig. 1) and intensities (weighted incidences) (Fig. 2) of
Perkinsus marinus infections in Piankatank River reef oysters which set in August, 1993,
represented as a function of oyster age and depth of residence below mean low water (:545
cm and @DO cm).

Figs. 3 & 4- Prevalences (Fig. 3) and intensities (weighted incidences) (Fig. 4) of
Haplosporidium nelsoni infections in Piankatank River reef oysters which set in August,
1993, represented as in Figs. I and 2.

Fig. 5- Cumulative mortalities observed in Piankatank River reef oysters which set in
August, 1993, represented as a function of oyster age.

Fig. 6- Sizes of Piankatank River reef oysters which set in August, 1993 and which were
sampled for disease studies summarized above. Sizes are presented as a function of oyster
ages and depths of residence below mean low water (-<45 cm and @90 cm).

Fig. 7- Temperatures and salinities of Piankatank River water at the sampling site and at
the time oysters were obtained for disease studies.

Figs. 8 to 11- Prevalences and intensities (weighted incidences) of P. marinus and H.
nelsoni for adult oysters imported from the upper James River and placed on the
Piankatank River reef and in the York River behind the Virginia Institute of Marine
Science. Two batches of 700 oysters each were placed at the sites (350 at each site) and
assayed until the populations were depleted by sampling and natural mortalities. The
disease organism data is expressed as a function of site and sampling time in the study.

Prevalence of P. marinus infectio-ns
in Reef Oysters
AM 100 -

0
80

60
C:
40

C: 20

> 0
38 46 52 58 65 78 91 100 110
Oyster age (weeks)

<45cm -a-- >90cm

Figure I

P. marinus infection intensities
in Reef Oysters

6 -

3 . . ....

. ........... - - ----------

0 Mae
38 46 5@ '58 65 78 91 100 110
Oyster age (weeks)

<45cm & >90cm

Figure 2

Prevalence of H. nelsoni
infections in Reef Oysters

50-

0
38 46 52 5'8 '6_5 -7@ 91 100 110
Oyster age (weeks)

<45cm >90cm

Figure 3

H. nelsoni infection intensities
in Reef Oysters

1.4

1.2 . .. ......

0. 8 - ------

00.6
4-4
0.4 - -------- - - -------- - - -

-0.2 - ------- - -- -

0 0 0 0 0 i i -4-4- i i4-
38 46 52 58 65 78 91 100 110
Oyster age (weeks)

<45cm -1w- >90cm

Figure 4

Mortality in Reef Oysters

50 -

30 ---------

0
220
11@@o
0

0
38 46 52 58 65 78 91 100 110
Oyster age (weeks)
- - ---- -----

Figure 5

Sizes of Reef Oysters assayed
for infections

70 -

60 - - ------
E 50
E
40 -

N
30 -

20 -

10
38 46 52 58 65 78 91 100 110
Oyster age (weeks)

<45cm * >90cm

Figure 6

Temperature and salinity profile
during the sampling period

35 -
>I
-#--j
.E 30 . ......
-@-z 2 5 - . . ........ . .
U)
20
15

................................ .... . .... ......
0-10
E
5 -
0
0 6 10 14 18 22 27 36 45 53 60 65 72 84
Sampling time (weeks)
Temperature (C) --A- salinity (pptL-.i

Figure 7
- - - ------- --

Prevalence of P. marinus infections
in imported oysters
Cn
22 100
Cn
0 80

40
-7-

CU
> 0
0 6 10 14 18 22 27 36 45 53 60 65 72 84
Sampling time (weeks)

P.river-.*-VIMS

Figure 8

P. marinus infection intensities
in imported oysters

6 -

5
Z
c:4
0

3
0 . ..........
2

0
0 6 10 14 18 22 27 36 45 53 60 65 72 84
Sampling time (weeks)

PRiver * VIMS

Figure 9

Prevalence of H. nelsoni infections
in imported oysters

100

4-i
0
0 6 10 14 18 22 27 36 45 53 60 65 72 84
Sampling time (weeks)

PRiver VIMS

Figure 10

H. nelsoni infection intensities
in imported oysters

4 -

2 -------

0 01"i it" MIII
0 6 10 14 18 22 27 36 45 53 60 65 72 84
Sampling time (weeks)

P. river w VIMS

Figure 11

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