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













                            SHELL DISEASE OF BLUE CRABS, Callinectes sapidus,

                                  IN THE PAMLICO RIVER, NORTH CAROLINA
















                                                                         C



















                                North Carolina Department of Environment,
                                      Health, and Natural Resources

                                      Division of Marine Fisheries
                                        Morehead City, NC 28557


       QL
       444
        D3                                    January 1990
       M343
       1990


0













                              SHELL DISEASE OF BLUE CRABS, Callinectes sapidus,
                                    IN THE PAMLICO RIVER, NORTH CAROLINA
                                                      by
                                                 Sean McKenna

                                                 Maura Jansen 1
                                               Michael G. Pulley






                                     Special Scientific Report Number 51









                                  North Carolina Department of Environment,
                                        Health, and Natural Resources

                                        Division of Marine Fisheries
                                           Morehead City, NC 28557







                                                 January 1990



                                                Property of CSC Library
      




                                     1 North Carolina State University
                          Department of Microbiology, Pathology and Parasitology
                                        School of Veterinary Medicine
                                       Raleigh, North Carolina 27606

                                       US Department of Commerce
                                       NOAA Coastal Service Center Library
                                       2234 South Lobson Avenue
                                       Charleston, SC 29405-2413
 









                                                ABSTRACT



                  Blue crabs with large lesions on their carapace were collected in the
             Pamlico River estuary during the summer and fall of 1987. Diseased crabs were
             most abundant on the south side of the River between Durham and South creeks.
             Infection studies showe  'd a significant difference in the amount of time
             required for lesion development between this area and control sites.
             Chitin-degrading and lipolytic bacteria were cultured from natural and
             experimentally induced lesions.    The etiology of shell disease within the
             Pamlico River estuary is probably stress due to a change in environmental
             quality.   Possible stress-related events include abnormal shell synthesis,
             abnormal shell repair or degradation of the shell in the water column.











                                                TABLE OF CONTENTS



             ABSTRACT   . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .


             TABLE OF CONTENTS   . . . . . . . . . . . . . . . . . . . . . . . . . .


             INTRODUCTION    . . . . . . . . . . . . . . . . . . . . . . . . . . . .


             MATERIALS AND METHODS    . . . . . . . . . . . . . . . . . . . . . . . .            3
                   Trawl Survey  . . . . . . . . . . . ... . . . . . . . . . . . . .             3

                   Commercial Catch   Sampling   . . . . . . . . . . . . . . . . . . .           3

                   Sentinel Study   . . . . . . . . . . . . . . . . . . . . . . . . .            3
                        Experiment   I  . . . . . . . . . . . . . . . . . . . .. . . .           5

                        Experiments 2 and 3      . . . . . . . . . . . . . . . . . . .           5

                   Bacterial and Histological    Studies  . . . . . . . . . . . . . . .          5

             RESULTS  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .              8

                   Trawl Survey  . . . . . . . . . . . . . . . . . . . . . . . . . .             8

                   Commercial Catch   Sampling   . . . . . . . . . . . . . . . . . . .           8

                   Sentinel Study   . . . . . . . . . . . . . . . . . . . . . . . . .           10
             DIS*CUSSION   . . . . . . . . . . . . . . . . . . . . . . . . . . . . .            18

             ACKNOWLEDGMENTS   . . . . . . . . . . . . . . . . . . . .. . . . . . . .           26


             LITERATURE CITED    . . . . . . . . . . . . . . . . . . . . . . . . . .            27









                             INTRODUCTION                      Sandifer      and     Eldridge      1974).
                                                               Although all    sizes    of  animals are
                                                               affected, the more severe cases are
                    Shell   disease is    a ubiquitous         usually observed in adults, which may
               syndrome expressing itself as necrotic          be due to the shorter intermolt period
               lesions on the exoskeleton of aquatic           of juveniles (Sandifer and Eldridge
               crustaceans (Rosen 1970; and Fisher et          1974; Baross et al . 1978), as the
               al.  1976).    This disease has been            disease is not transferred to the new
               observed   in   the   American     lobster      exoskeleton    during    ecdysis     (Rosen
               (Homarus americanus) (Hess 1937; Young          1970).
               and Pearce 1975; Fisher et al. 1976;
               Malloy    1978),     European      lobster
               (Homarus   gammarus)    (Roald   et    al.            Although the etiology of shell
               1981), blue crab (Callinectes sapidus)          disease is uncertain, chitinoclastic
               (Rosen 1967; Cook and Lofton 1973;              bacteria (Vibrio spp., Beneckea spp.
               Sandifer     and      Eldridge       1974;      and Pseudomonas spp.) are most often
               Overstreet,     1978;   Johnson     1983),      associated with      the  lesions     (Hess
               tanner   crab   (Chionoecetes     tanneri)      1937;    Rosen 1967; Cook and Lofton
               (Baross   et    al.  1978),   rock    crab      1973;    Malloy    1978).     Myxobacteria
               (Cancer irroratus) (Young and Pearce            (Anderson and Conroy 1968), psychro-
               1975), penaeid shrimp (Cook and Lofton          philic luminescent bacteria, Photobac-
               1973; Johnson 1978; Overstreet 1979;            terium spp. (Baross et al. 1978) and
               Cipriani   et  al.   1980),    freshwater       fungi   (Rosen   1970)   have also been
               shrimp (Macrobrachium spp. and Palae-           isolated. The polyphenolic epicuticle
               monetes spp.) (Johnson 1977), crawfish          must be breached in order for chinti-
               (Procambarus    spp.)  (Johnson     1977),      noclastic bacteria to attack chitin
               caridean shrimp (Crangon septemspinosa          (Johnson 1983). Rosen (1970) hypothe-
               (Gopalan and Young 1975), and brown             sized that the only way for bacteria
               shrimp    (Crangon   crangon)     (Nottage      to enter was through breaks in the
               1982).                                          epicuticle.    However, Baross et al.
                                                               (1978) believed that microbial degra-
                    The  early stage of shell disease          dation of the epicuticle could be
               manifests itself as numerous puncti-            another portal of entry.        Lipolytic
               form brown marks with reddish-brown             bacteria may also play an       important
               depressed centers (Rosen 1970).         As      role in the penetration and removal of
               the disease progresses, these lesions           the epicuticle (Cipriani et al. 1980).
               deepen    and    eventually       coalesce
               (Johnson 1983).      The edges of the
               lesions may be. black or rimmed in
               blue,   presumably    due   to     melanin            Recently,   blue crabs collected
               deposition     (Johnson    1983).       In      from    the    Pamlico    River,      North
               natural., unstressed environments, the          Carolina    have   been   observed     with
               prevalence of shell     disease is low          large lesions on their    carapace.     The
               (Hess 1937;   Cook and Lofton 1973).            first such animal was     caught between
               However,   in   degraded     environments       Long and Hickory Points on 11 June
               (Young and Pearce 1975; Gopalan and             1987      (Figure     1).       Subsequent
               Young 1975) and under crowded condi-            fishery    dependent sampling by the
               tions such as holding pens and shedd-           Division of Marine Fisheries (DMF)
               ing boxes, the prevalence increases             indicated a trend to the geographic
               (Rosen 1970; Cook and Lofton 1973;              distribution of these animals.          All
               Mallory 1978).   The contagious nature          were caught on the south side of the
               of this disease poses a threat to blue          River    between     Durham   and    South
               crab shedding operations (Rosen 1967;           Creeks.

                                                           1





                                                                  N
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                    Washington










                                              Wbn          upper 4.
                                               P           coose



                                           Hills
                                           Pt.
                                                           Mauls   .P8m,ico                                C*1.
                                                           Pt.











                                                    03
                                                                                                                  Hickory


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                                                                           I Inch
             Figure 1.    Study location and cage sites:                   3.75 miles
                          1 = Core Point
                          2 = Texasgulf
                          3 = Long Point
                          4 = Indian Island









                    The Pamlico River is one of the                 On 17 July 1987,      60 stations
               largest estuaries in North Carolina            were   sampled from Mauls      Point   to
               and contributes significantly to the           Indian Island using a 6.1 m flat
               state's   commercial    shellfish     and      trawl with 19.0 mm bar mesh in the
               finfish landings.    Additionally, this        wings and body and 6.4 mm bar mesh in
               area is used extensively by recrea-            the tail bag.       Tow durations were
               tional  boaters    and  fishermen.      In     five minutes at each station.      Trawls
               recent years the Pamlico River has             were calibrated to travel       69 m   in
               been the site of numerous fish kills,          one minute.     A systematic sampling
               algae   blooms,    anoxia   events    and      design was employed with the River
               outbreaks of fish disease, (ulcerative         being   divided     into grids    of   1.3
               mycosis) (Dykstra et al. 1986; Rader           square kilometers.     All crabs caught
               et al.  1987).    Although the etiolo-         were measured spike      to spike (CW),
               gies of these events are uncertain,            sexed, maturity noted in females and
               they might be indicative of declining          examined for disease.
               water quality in the Pamlico. Some of
               the factors possibly contributing to                   Commercial Catch Sampling
               the decline include freshwater runoff,
               erosion and sedimentation, nutrients,                To   quantify    the  location   of
               heavy metals, salinity and dissolved           disease on the exoskeleton, 58 crabs
               oxygen.  An excellent review of these          captured by commercial fishermen were
               factors is provided by Rader et al.            examined.   Forty were randomly collec-
               (1987). The addition of shell disease          ted on 7 July 1987 and 18 on the 10th
               to the problems of the Pamlico River           of July.      All crabs were measured
               is of great concern to State agencies          (CW), sexed, maturity noted in females
               and the public in general.      Not only       and the location of.lesions were noted
               does shell     disease exemplify the           (Figure 2).
               deterioration of the water quality in
               the Pamlico River but,        it serves                     Sentinel Study
               notice   that    the problems    of    the
               Pamlico are far greater than pre-                    The   purpose    of  the    sentinel
               viously perceived.                             study was to determine if "healthy"
                                                              crabs developed shell disease when
                                                              placed in the River and if we could
                         MATERIALS AND METHODS                differentiate high-risk from low-risk
                                                              sites for disease development.         In
                    The data reported in this paper           addition,    we   wished     to    obtain
               were obtained from four sources: (1) a         information     on     infection   rates,
               trawl survey, (2) a commercial catch           progression    of     the   disease    and
               sampling program, (3) a sentinel study         environmental   factors associated with
               and (4) bacterial     and histological         the disease.     Cages were constructed
               studies.    Methods and results are            out of 2.5 cm x 1.3 cm vinyl coated
               described separately.                          hardware cloth and measured 127 cm I
                                                              x 50.8 cm w x 60.9 cm h.      Each cage
                                                              contained ten individual compartments
                            Trawl Survey                      measuring 25.4 cm 1 x 25.4 cm w x
                                                              60.9 cm h.        Cages  were placed on
                    The objectives of      this survey        the bottom in    depths ranging from 0.9
               were to:   1) determine the geographic         to 1.8 meters.     Core Point and Indian
               distribution    of diseased crabs, 2)          Island were in the apparently low
               investigate the magnitude of infection         risk    region,    while  the    Texasgulf
               and 3)   determine the distribution of         effluent    and  Long   Point    locations
               disease in the population.                     were in the apparently         high risk


                                                         3




























                                                   10




















                             Figure 2. Regions of blue crab exoskeleton.









                                                4









               region (Figure 1). Two cages contain-             exoskeleton was accomplished by use of
               ing 10 crabs apiece were placed at                a blunt     instrument or by allowing
               each site.                                        another crab to bite the treatment
                                                                 animal.     The Mann-Whitney Test wa      s
                                                                 used to examine differences between
               Experiment  1                                     time required     to    develop     lesions
                                                                 and life expectancy of study animals
                                                                 (Zar 1974).
                    Blue crabs were collected in Rose
               Bay from commercial crab pot catches
               (Figure 3).     All were mature males               Bacterial and Histological Studies
               measuring 130-160 mm CW.       Crabs were
               placed in cages on 3 August 1987.        On
               4 August all crabs were measured and                   On 14 September 1987, 37 crabs
               any    extraneous     marks    on     their       were sacrificed for bacterial and
               exoskeletons      were     noted.       The       histological      examinations.       Blue
               following day, the epicuticle      of five        crabs   used   for    these   examinations
               randomly selected crabs from each cage            were taken from the sentinel study
               was   removed    with   a     knife.    All       and crab pots set at each location.
               individuals were scraped on the left              Crabs were     placed   in    individually
               side over the epibranchial and hepatic            labeled bags and kept on ice until
               region      (Figure      4).       Weather        their    return    to   the    lab.     All
               permitting, crabs were checked daily,             individuals were measured (CW), sexed
               fed ad lib with Atlantic menhaden                 and a detailed description of lesions
               (Brevoortia tyrannus) and any type of             noted.     Bacterial     cultures     were
               mark or discoloration noted.       Surface        obtained by dipping     a 1 ul loop mini
               and bottom temperature, salinity and              tip culturette in a modified          salt
               dissolved oxygen were recorded at each            water yeast extract broth (MSWYE)
               station.    Initially, any crab which             (0.1% protease peptone,       0.1% yeast
               ,died   was    replaced    with    another        extract in a three salt solution of
               individual caught in the DMF juvenile             0.4M NACL, 0.028M MgSO       7H 0, 0.01M
               stock assessment program.     The replace         KCL and distilled water) (Cipriani et
               ment animal was treated similarly to              al.   1980),     quickly    touching    the
               the original animal, i.e., control or             perifery of the lesion and streaking
               scraped.                                          agar   plates   containing    salt water
                                                                 yeast extract (SWYE) (1.0% protease
                                                                 peptonei 0.3% yeast extract, 2.0%
               Experiments 2 and 3                               agar 0.4M NACL, 0.028M MgSO 7H 0 and
                                                                 0.01M   KCL),     (ibid).    Plates   were
                                                                 incubated at room temperature and the
                     In   late    August,    the     above       number of colonies noted after 24 and
               procedure was stopped and      replacement        48 hours.    Before streaking the agar
               animals were     treated i n   one of two         plates, the culturettes, were tapped
               ways:   1) removal of the epicuticle              onto   sterile microscope      slides     in
               and exocuticle or 2) fracturing of the            order to obtain gram stains.        Slides
               exoskeleton.    Blue crabs used in    these       were then heat fixed.     The lesion was
               experiments were collected by crab                then excised and placed in Bouins
               pots set at each study site; only                 fixative.    Shells were prepared for
               apparently healthy crabs were         used.       histological examination by fixation
               Animals were marked in the same area              in     neutral      buffered      formalin,
               as described in experiment 1. A       knife       decalcified     in   formic    acid     and
               was used to remove the epicuticle and             stained with hematoxylin and eosin
               exocuticle, while fracturing of the               (H+E).


                                                            5







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                          TAR RIV"R



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                                                                                                      Aor





                                                                                                        PAPIL






                                           p
                                     New Bern


                                         RIVER


                                                          Ak
          0
          I -@
            miles




          Figure 3. Location of Rose Bay.









































                    Figure 4. Location of area either scraped or damaged for Experiments 1    3.








                                                         7











                           RESULTS                         the river had diseased crabs.         There
                                                           was a strong relationship between the
                       Trawl Survey                        number of crabs caught and water depth.
                                                           Sixty-nine percent were caught in
              A total of 1,459 blue crabs was              water less than 1.8 meters, 30.3% in
         examined during      the   course of this         water 1.8 to 3.6 meters and .4% in
         investigation     (Tables     1   and    2).      greater than 3.6 meters.          The same
         Seventy-three (5.0%)      diseased crabs          trend held true for diseased crabs,
         were f ound.    The infection rate for            with   72.5% caught      in the shallowest
         males was 5.1%, 2.5% for immature                 strata, 27.4% caught in the 1.8 to 3.6
         females and 16.2% for mature females.             meter range,while none were caught in
         The  highest    prevalence of       disease       the deep water (Table 3).
         occurred on the south side of the
         River, where 16 of the 26 stations
         (62.0%) had diseased crabs (Figure 5).
         Ninety-four percent of these stations                     Commercial Catch Sampling
         were located between Durham Creek and
         Indian Island. Five (31.2%) of the 16                   Of the 57 diseased crabs exam-
         stations located on the north side of             ined, 28 were males and 29 were mature


         Table 1. Standard statistics for healthy blue crabs captured by trawl
                    17 July 1987 in Pamlico     River, North Carolina.                          Percent
                                               x CW                               Range          of
         Sex                       No.         (mm)         SD          Cv          (mm)        total

         Males                     938       112.01        35.66       31.83        26-203      67.67


         Immature females          386         88.80       29.28       32.97        31-162      27.84

         Mature females             62       157.93        11.68        7.39       122-185       4.47








         Table 2. Standard statistics for diseased blue crabs captured by trawl
                    17 July 1987 in Pamlico River, North Carolina.

                                                                                                Percent
                                               x CW                                 Range        of
         Sex                        No.        (mm)         SD          Cv           (mm)       total

         Males                      51       134.37        28.48       21-10        72-195      69.86

         Immature females           10         94.60       26.40       27.91        62-132      13.69

         Mature females             12       155.40        13.32        8.57       138-183      16.43



                                                      8











                                                                                                                         N
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                                     Washington






                                                                                          5

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                                                                                     wan    OD             upper       t.
                                                                                      N.                   Coose
                                                                              Hills                          0                                              CD
                                                                             Pl.
                                                                                                            0          OParrI*           0           0       0
                                                                                                           Maul$       0          [Co     River              0        0       0        Mixon C
                                                                                                           Pt.                           0           0                                                  0          0
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                                                                                                                                                                                                              Hickory









                                                                                                                                         1 1 ch
                                                                                                                                         3.75 miles


                    Figure 5. Approximate location of stations sampled during
                                            trawl survey. Solid circles indicate stations
                                           where diseased crabs were captured.
                L










         Table 3. Relationship between water depth and the prevalence of healthy and
                    diseased crabs in the Pamlico River, North Carolina.



                                Total                      Total        Total            1
            Depth              number       Total         number      number       CPUE
            strata               of         @number       without     with         without       CPUE1
             (M)             stations     of crabs        disease     disease      disease      diseased

            0 - 1.82             25           1,022         969          53          7.75        0.42

         1.83 - 3.65             22             447         427          20          4.06        0.18

                  .6ro
                                 13              6             6           0         0.09        0.00


          CPUE    catch per unit    effort where   one minute  sample time = one      unit of effort.


         females.    The mean CW for males was              while one individual had lesions in
         141.3 mm and 159.5         mm for mature           areas 6 and 9 and the last in areas 7
         females.   All animals     had lesions on          and 9.    Five of the six females had
         the dorsal surface of the carapace.                lesions in area 6.      Four of those had
         Four males and no mature females had               an additional lesion in areas 7, 8, 9
         lesions    on   the    ventral      surface.       or 10. The other animal with a lesion
         Dorsally, the     highest percentage of            in area 6 was also affected in areas
         lesions was in area 6 for both males               7, 8 and 10.       The   last animal was
         and    females,      42.4%    and      47.2%       affected in areas 7, 9 and 10.
         respectively.     This was followed by
         area 7 (33.3%     males, 22.2% females),                         Sentinel    Study
         area 9 (18.2%     males, 13.9% females),
         area 8 (30.3% males, 8.3% females) and                   The results of     Experiment I are
         area 10 (3.0% males, 8.3% females).                shown in Tables 4-7.           Of the ten
         Three males had lesions on the chela               crabs scraped at Indian Island, seven
         (1 right, 2 left) and five females                 developed    the     disease.    The     mean
        .were affected (3 right, 2 left).        With       number     of     days     required       for
         the exception of a mature female, all              initiation of the disease was 8.7 (SD
         animals had unaffected walking legs.               6.3).     The mean survival time          for
         The affected female had a lesion on                crabs held at Indian Island was 26.2
         the propodite of the fourth walking                days.   All of the scraped crabs held
         leg.                                               at Long Point and Texasgulf developed
                                                            the disease. The mean number of days
              No multiple lesions were noted on             required     for    initiation      of    the
         the ventral surface.     One animal had a          disease was 3.8 (SD 2.89) at Long
         small   lesion on the 5th abdominal                Point and 4.7 (SD 3.27) at Texasgulf.
         segment, another below the right eye,              The mean survival time for Long Point
         the third     in   the subhepatic region           was 27.5 days and 14.7 for Texasgulf.
         and the last animal below the mouth.               Nine of the ten crabs at Core Point
         Five   males    and     six  females    had        developed the disease.         The. average
         multiple    lesions     on    the     dorsal       number of days required for develop-
         surface.     Three of      the males    had        ment     of  the   disease     was 7.7 (SD
         lesions    in both areas        6 and 7,           1.8).     Crabs at this location lived



                                                        10









              Table 4. Results of experiment 1 for blue crabs near Indian Island, P      amlico River,
                         North Carolina.



                                                   Number        No. of days for         No. of days
              Crab &                               of days    first sign of disease*     for pitting
              cage no.      Control    Scraped     alive*       Control      Marked     Control Marked

                   1                      X          10A                          3                NP
               2 - 1           X                     17B            NS                     NP
               3 - 1           X                     55B            NS                     NP
               4 - 1                      X          10B                         NS                NP
               5 - 1           X                     24B            NS                     NP
               6 - 1                      X          17B                         10                NP
               7 - 1           X                     32B            NS                     NP
               8 - 1           X                     49B            NS                     NP
               9 - I                      X          31B                         NS                NP
              10 - 1                      X          41B                         21                NP
               1 - 2                      X            1B                        NS                NP
               2 - 2           X                     13B            NS                     NP
               3 - 2                      X          19B                          6                NP
               4 - 2           X                     40C            NS                     NP
               5 - 2           X                       3B           NS                     NP
               6 - 2                      X          40C                         10                NP
               7 - 2           X                     59C            NS                     NP
               8 - 2                      X          55B                          9                36
               9 - 2           X                     27A            NS                     NP
              10 - 2                      X          10A                          2                NP


                  A: escaped
                  B: died
                  C: terminated
                 NS: no sign of  disease
                 NP: no pitting










       Table 5. Results of experiment 1 for blue crabs near Long Point, Pamlico River,
                  North Carolina.


                                            Number        No. of days for        No. of days
       Crab  &                              of days    first sign of disease*    for pitting*
       cage  no.     Control    Scraped     alive*       Control      Marked    Control Marked

        I  - 3          X                    37B            NS                      NP
        2  - 3                     X         15B                          1                NP
        3  - 3                     X         55B                          1                33
        4  - 3                     X         15A                          6                NP
        5  - 3          X                    38B            NS                      NP
        6  - 3          X                    59C            NS                      NP
        7  - 3                     X         28B                          6                NP
        8  - 3          X                    40C            NS                      NP
        9  - 3                     X         24B                          8                NP
       10  - 3          X                    13B            NS                      NP
        1  - 4          X                      1B           NS                      NP
        2  - 4                     X           9B                         2                NP
        3  - 4                     X         51B                          2                NP
        4  - 4          X                    40B            NS                      NP
        5  - 4                     X         26B                          8                NP
        6  - 4                     X         32B                          1                NP
        7  - 4          X                    26B            NS                      NP
        8  - 4          X                    31B            NS                      NP
        9  - 4          X                    14B            NS                      NP
       10  - 4                     X         28B                          3                NP



           A:  escaped
           B:  died
           C': terminated
           NS: no sign of disease
           NP: no pitting






















                                                  12









              Table 6.   Results of experiment I for blue crabs near Texasgulf effluent station,
                         Pamlico River, North Carolina.


                                                   Number        No. of days for        No. of days
              Crab  &                              of days    first sign of disease*    for pitting
              cage  no.     Control    Scraped     alive*       Control      Marked    Control Marked

               1  - 5          X                      1A           NS                      NP
               2  - 5                     X          59C                         5                35
               3  - 5          X                      3B           NS                      NP
               4  - 5          X                     24B           NS                      NP
               5  - 5                     X          17B                         1                NP
               6  - 5                     X          40C                         5         NP
               7  - 5          X                     21B           NS                      NP
               8  - 5                     X          12B                         5                NP
               9  - 5                     X          10A                         3                NP
              10  - 5          X                      6B           NS                      NP
               1  - 6          X                     23B           NS                      NP
               2  - 6                     X          12A                         10               NP
               3  - 6                     X          33B                         7                22
               4  - 6          X                     40C           NS                      NP
               5  - 6                     X          40C                         1                38
               6  - 6                     X           3B                         1                NP
               7  - 6          X                     15B           NS                      NP
               8  - 6          X                      8B           NS                      NP
               9  - 6                     X          12B                         9                NP
              10  - 6          X                      7A           NS



                   A: escaped
                   B :died
                   C: terminated
                  NS: no sign of disease
                  NP: no pitting





















                                                         13









        Table 7. Results of experiment 1 for blue crabs near Core Point, Pamlico River,
                  North Carolina.



                                             Number       No. of days for         No. of days
        Crab &                               of days   first sign of disease*     for_RiLtin
                                                                                            1@
        cage no.     Control    Scraped      alive*      Control      Marked     Control Marked



         1 - 7           X                    10A            NS                     NP
         2 - 7                     X          19B                          10               NP
         3 - 7                     X            7B                        NS                NP
         4 - 7           X                    28B            NS                     NP
         5 - 7           X                    15B            NS                     NP
         6 - 7                     X          20B                          7                NP
         7 - 7                     X          53B                          7        26
         8 - 7           X                    20B            NS                     NP
         9 - 7           X                    15B            NS                     NP
        10 - 7                     X          35B                          9                NP
         1 - 8                     X          53B                          9        26*
         2 - 8           X                      8B           NS                     NP
         3 - 8           X                    12B            NS                     NP
         4 - 8                     X          26B                          5                NP
         5 - 8                     X          26B                          5                NP
         6 - 8                     X          40C                          9                35
         7 - 8           X                    27B            NS                     NP
         8 - 8                     X          46B                          8                27**
         9 - 8           X                    40B            NS                     NP
        10 - 8           X                    11B            NS                     NP



            A: escaped
            B: died
            G: terminated
           NS: no sign of disease
           NP: no pitting
          *20  days from start of  pitting to healing
          **8  days from start of  pitting to healing

















                                                   14









              an average of 25.6 days.       The above         disease in an average of 31.6 days.
              data reflects the early stages of the            At Core Point, pitting occurred on
              disease, i.e. punctiform brown marks             average in 28.6 days (N = 4).
              with reddish-brown depressed centers.
              There was     a significant (P<0.05)
              difference between the time required                  The results of experiment 2 are
              for lesion -development in the high              summarized in Tables 9-12.     Only crab
              risk area (x_= 4.2 days) and the low             3, cage 5 developed lesions similar to
              risk area (x = 8.1 days).         Due to         ones seen in the high risk area. This
              small   sample   size, analysis between          involved the complete breakdown of the
              individual sites was not attempted.              crystalline matrix of the endocuticle.
              There were no significant differences            Eleven days later new "wound cuticle"
              between the mean survival time of                (Neville 1975) was deposited.         Two
              control or scraped crabs (Table 8).              other   animals    at    the    Texasgulf
              Due to the short cage life of crabs,             location      exhibited       endocuticle
              the progression of the disease could             breakdown (crabs 4 and 10, cage 5),
              not be followed for all individuals.             but  they were either terminated for
              The    last   column    of   Tables     4-7      bacteriological examination (crab 10)
              represents    the    time  required     for      or  died (crab     4)   before    healing
              deepening    and    joining    of    early       occurred.
              lesions. Only one crab at both Indian
              Island and Long'Point developed the                   Experiment     3     involved    the
              latter stages of the disease.          The       fracturing of the exoskeleton.        The
              time required for pitting was 36 days            objective of this experiment was to
              at Indian Island and 33 days for Long            examine the healing process.      A total
              Point.     Three    crabs  at    Texasgulf       of 20 animals were treated: Indian
              developed the latter stages of the               Island (3), Long Point (6), Texasgulf




              Table 8. Results of Mann-Whitney test      for comparisons of mean survival time or
                         blue crabs sentinel experiment in Pamlico River, North Carolina.


                                                                                          Significance
              Comparison                                U                    U1               level
              Low risk area control                                                                2
                vs scraped                              153.5                102.5               NS
              High risk area  3 control
                vs scraped                              87.0                 123.0               NS

              Control low vs high
                risk area                               132.5                107.5               NS

              Scraped low  vs high
                risk area                               127.0                 97.0               NS


              1 Core Point and Indian Island
              2 Not significant at the 0.05 level
              3 Texasgulf and Long Point



                                                         15









       Table 9. Results of experiment 2 for blue crabs near Indian Island, Pamlico
                 River, North Carolina.

                                          Number of days required to*
                                          note changes in scraped area
       Crab and          Date                                                        Days
       cage no.        scraped   Yellow    Yellow-Brown    Brown   Black Pitting alive

       9 - 2             9/1       NS             2            6     NS        8     13C
       4 - 1             9/3       NS           NS           NS        7     NS      11C
       9 - 1             9/5         2          NS           NS      NS      NS        4B
       1 - 1             9/7         4          NS           NS        4     NS    8 shed
       7 - 1             9/7       NS           NS           NS      NS      NS    2 shed
       9 - 1             9/10      NS           NS           NS      NS      NS        4C


           A: escaped
           B: died
           C: terminated
          NS: no sign of  disease



       Table 10. Results of experiment 2 for blue crabs near Long Point, Pamlico River,
                  North Carolina.

                                             Number of days required to*
                                             note changes in scraped area
       Crab and      Date                                                               Days
       cage no.      scraped            Yellow          Brown          Pitting          alive

        5 - 4          8/@l                1               NS             NS              5B
        9 - 4          8/31                1               NS             NS              5B
        5 - 4          9/5                 2               NS               4            12B
        7 - 4          9/5                 2               NS               4            12B
        1 - 4          9/6                NS                 3              5            27C
        7 - 3          9/6                 1               NS               6            19B
        9 - 3          9/7                 2               NS               5            10B
        2 - 4        9/10                  1               NS               2             4C
        9 - 4        9/12                 NS               NS             NS              5C


           A: escaped
           B: died
           C: terminated
          NS: no sign of  disease



                                                 16







              Table 11. Results of experiment 2 for blue crabs near Texasgulf, Pamlico River,
                         North Carolina.

                                                   Number of days required to*
                                                   note changes in scraped area
                                                                 Breakdown
                                                                     of
              Crab and     Date                                crystalline                     Days
              cage  no.    scraped    Yellow     Brown  Pitting matrix     Healing     Blue    alive

               7 -  5         9/1         .2      NS        5        NS        NS       NS       13C
               3 -  5         9/3        6        NS        NS        3        14       20       29C
               4 -  5         9/3        NS.       3        3         7        NS       NS       13B
              10 -  5         9/7        2        NS        NS        2        NS       NS         7C
               3 -  6       9/10         1         2        7        NS        NS       NS       23C
               9 -  6       9/10         2        NS        NS       NS        NS       NS         4C
               8 -  5       8/29         2         3        a        NS        22       NS       35C


                  A: escaped
                  B: died
                  C: terminated
                 NS: no sign of  disease





              Table 12. Results of experiment 2 for blue cra    bs near Core Point, Pamlico River,
                         North .Carolina.        Number of days required to*
                                                 note changes in scraped area
              Crab and     Date                    Yellow-                                     Days
              cage no.   scraped      Yellow       brown         Brown    Pitting   Healing    alive

               6 -  7      8/28          NS          NS           4          NS        NS        20C
               3 -  7      9/1            1          NS           NS         NS        NS          2B
               7 -  8      9/1            1            5          8          8         NS        13B
               3 -  7      9/3            3          NS           6.         6         NS        14C
               1 -  7      9/10           1          NS           NS         2          7        23C
              10 -  7      9/10           1          NS           7          NS         7        10B


                  A: escaped
                  B: died
                  C: terminated
                 NS: no sign of  disease


                                                       17










       (6), Core Point (5).      The mean time                       DISCUSSION
       required   for   deposition   of   wound
       cuticle was 24 days Indian Island (N =
       2), 25 days Long Point (N = 1), 23                  There appeared to   be a trend in
       days Texasgulf (N = 1) and 20 days             the  geographic distribution of disea-
       Core Point (N = 5).    Crabs with large        sed crabs in the Pamlico River.       The
       fractures in which the epidermis was           trend first became evident through
       breached   never   healed    completely.       interviews with local    fishermen    and
       Regardless of location, all fractures          was substantiated with results of the
       increased in size.     The edges of the        trawl survey.   Throughout the course
       fracture became brittle and eventually         of this investigation, fishermen were
       sloughed off.                                  interviewed daily as to the location
                                                      of diseased crabs in their catch.
            Five predominant bacterial colony         For the most part, the capture of
       types    were    isolated:      Type    A      diseased crabs was limited to the
       Pseudomonas spp., Type B        unidenti-      area between Durham and South Creeks.
       fied, Type C Vibrio spp., Type D               Occasionally   diseased    crabs    were
       Vibrio spp. and Type E Proteus spp.            caught outside of this area, but with-
       (Table 13).     Bacterial compositions         in the Pamlico River estuary. The low
       for the various study animals are              frequency of these reports were attri-
       given in Tables 14-17 and for natural          buted to random movements   ' (Judy and
       lesions in Table 18.                           Dudley 1970; Sholar 1983). Blue crabs
                                                      with lesions as severe as those     seen
                                                      in the Pamlico River were examined
            The three environmental       para-       from   Masonboro,    Bogue    and   Core
       meters measured (temperature, salinity         Sounds. The total number of crabs ex-
       and dissolved oxygen) are summarized           amined was eight and in all cases the
       in Table 19.                                   damage was attributed to mechanical






       Table 13. Description of the predominant bacterial colonies isolated from
                  experimental crabs collected in Pamlico River, North Carolina during
                  September, 1987.

                                                   Bacteria type
                         .A               B                C               D              E

       Gram
       reaction

       Shape             rod      stippled rod        curved rod          rod            rod

       Color on        orange          white        opaque white       translucent     white
       SWYE agar                                                           white





                                                 18










           Table 14. Number of bacterial colonies isolated from crabs at Indian Island, Pamlico River, North Carolina,
                      September, 1987.


                                              Bacteria type                            Total number           Description of
           Crab            A            B            C           D             E       of colonies            lesion

             1             0            0            0           0             0           0                  Scraped 9/3
                                                                                                              9/14 1 black spot
                                                                                                              1 mm diameter

             2             11           20           0           0             4           60                 Scraped 9/10
                                                                                                              9/14 no color

             3             0            0            0           0             1*          1                  Scraped 9/1
                                                                                                              9/14 brown and
                                                                                                              little pitting

             4             0            0            0           0             0           0                  Scraped 8/5
                                                                                                              9/14 3 brown spots
                                                                                                              each I mm
      ko
             5             0            0            0           0             0           0                  Scraped 9/12
                                                                                                              9/14 no color

             6             0            -            -           0                                            NAShed 9/9
                                                                                                              9/14 multiple
                                                                                                              rust marks


             7             0            0            0           0             0           0                  Control



             8             0            0            0           0             0           0                  Control




              Plate overgrown with type   E
              75% of plate overgrown with type E, 10% with type B and 10% type C
              N/A not available









          Table 15. Number of bacterial colonies isolated from crabs at Long Point, Pamlico River, North Carolina,
                      September, 1987.


                                            Bacteria type                                     Total number     Description
          Crab            A           B            C           D            E                 of colonies      of lesion

           1              0           0            0           0            0                       0          Scraped 9/12
                                                                                                               9/14 10% area
                                                                                                               yellow

           2              48          48           12          0            0                     120          Scraped 9/12
                                                                                                               9/14 100%
                                                                                                               area yellow

           3              14          14           0           16           1                      45          Scraped 9/12
                                                                                                               9/14 5% area
                                                                                                               yellow

           4              2           19           5           4            0                      29          Scraped 9/10
     NO                                                                                                        9/14 85% yellow
     0                                                                                                         15% brown
                                                                                                               little pitting

           5              0           0            0           0            0                       0          Control

           6              0           0            0           0            0                       0          Control









           Table 16. Number of bacterial colonies isolated from crabs at Texasgulf outfall, Pamlico River, North
                      Carolina, September, 1987.

                                               Bacteria type                           Total number     Description
           Crab            A            B           C           D          E           of colonies      of lesion

            1              0            0           0           0          1*               1           Scraped 8/5 9/14 mottled brown

            2              0            0           0           0          0                0           Scraped 8/5 9/14 little pitting

            3              10           0           10          0          60              100          Crab bit 8/29 9/14 edges
                                                                                                        brown

            4              0            0           0           0          1*               0           Scraped 9/12 9/14 50% yellow

            5              -            0           -           0          0              TNTC          Scraped 9/12 9/14 85% yellow

            6              9           200          12          4          9               300          Scraped 9/1 9/14 10% yellow &
                                                                                                        pitted 90% brown

            7              0            0           0           0          1*               1           Scraped 9/10 9/14 40% yellow

            8              0            6           10          0          1                28          Scraped 9/7 9/14 50% brown &
                                                                                                        soft


            9              0            0           0           0          0                0           Control

           10              0            0           0           0          0                0           Control

           11              0            0           0           0          0                0           Control


             Plate overgrown with type E.
             TNTC to numerous to count, estimated that 90% of     plate covered by type A   +  10% by type C.






           Tabl e 17.  Number of bacterial colonies isolated from crabs at Core Point, Pamlico River, North Carolina,
                       September, 1987.

                                             Bacteria type                          Total number           Description
           Crab            A           B            C           D         E         of colonies            of lesion

             1             0           0            0           0         1*               1           Scraped 8/5 9/14 brown
                                                                                                       & pitted

             2             8           8            5           9         0               30           Fractured 8/28

             3             0           1            1           0         0                2           Scraped 8/28 9/14 area
                                                                                                       brown

             4            70           19           8           0         0               100          Scraped 9/3 9/14 entire
                                                                                                       area brown 5% pitted

             5            15           11           17          34        0               77           Scraped 9/12 9/14 75%
                                                                                                       yellow

       r1a   6            16           4            0           0         0               20           Scraped 9/12 9/14 90%
       DO
                                                                                                       area yellow

             7             2           20           4           1         0               30           Scraped 9/12 9/14 20%
                                                                                                       area yellow

             8             6           4            9           30        0               60           Scraped 9/12 9/14 20%
                                                                                                       area yellow

             9             0           0            0           0         0                0           Control


             10            1           5            6           6         0               20           Control


             11            0           0            0           0         0                0           Control


             12            0           0            0           0         0                0           Control


           *Sparse growth











               Table 18. Number of bacterial colonies isolated from natural lesions of blue
                           crabs from Pamlico River, North Carolina, September, 1987.

                                                                                    Total      Descrip-
                                                                                    number      tion
                                            Bacteria type                             of         of
               Crab         A          B            C          D           E       colonies    lesions


                 1          5          11           3          .23         1          43        Brown
                                                                                                pitted

                 2          5          11           0            0         0          40        Brown &
                                       no                                                       pitted
                            3                       0           12         0          40        Brown &
                                                                                                pitted















               Table 19. Monthly temperature, salinity and dissolved oxygen (x SD) for the four
                           study sites in Pamlico River, North Carolina, August-September, 1987.


                                      Temperature              Salinity             Dissolved oxygen
               Indian Island    -August      September     August     September    August      September
                 Top            28.05 1.0    26.43 0.8    11.45 1.8   12.66 1.3    6.60 0.9    7.61 1.0
                 Bottom         28.02 0.9    26.20 1.0    11.47 1.6   12.71 1.2    6.49 0.9    7.50 1.0

               Long Point
                 Top            28.15 1.2    26.44  1.0    9.90 1.4   11.06 1.5    6.77 1.1    7.60 1.6
                 Bottom         28.18 1.1    26.04  1.3   10.08 1.4   11.06 1.5    6.33 0.9    7.09 1.5

               Texasgulf
                 Top            28.29 1.6    26.63  1.5    8.22 1.8    9.66 2.0    6.64 1.2    6.89 0.7
                 Bottom         28.30 1.5    26.38  1.4    8.93 1.5   10.11 1.4    6.44 1.5    6.91 1.1


               Core Point
                 Top            28.65 1.7    26.54  1.9    8.15 1.0    8.63 1.5    7.01 1.2    7.23 1.1
                 Bottom         28.37 1.5    26.10  1.7    8.26 1.0    8.85 1.5    6.21 1.1    6.17 1.0





                                                           23










        damage, i.e., crab bite.      Observation        influence individual susceptibility to
        of crabs collected in monthly juvenile           shell disease.     Fisher et al. (1976)
        sampling -by     the    Pamlico   District       found that long-term dietary deficien-
        indicated a widespread distribution of           cies were capable of - weakening the
        blue crabs with shell     disease.    How-       defenses provided by the epicuticle.
        ever, in no case did the severity of             Shell   disease    may    be a phenomenom
        lesions compare to those in the study            resulting from poor environment con-
        area.  Lesions were located on the               ditions found in mariculture raceways
        appendages and the ventral surface and           (Cipriani    et   al.  1980),    or  other
        Were more typical of lesions described           heavily fouled waters (Gopalan and
        by Rosen (1970).                                 Young 1975, Young and Pearce 1975).


             The prevalence of shell disease                  The   epicuticle     of   blue  crabs
        was greatest     in   larger males      and      contains lipids, proteins and calcium
        mature females, which probably related           and serves as a first line of defense
        to a longer intermolt period for the             against      microorganisms       (Johnson
        larger males.    Mature females do not           1980).       In      vitro     inoculation
        shed   after     their   terminal     molt       experiments      showed       that      the
        (Milikin       and    Williams       1984).      epicuticle had to be removed in order
        Therefore,   they are unable to rid              to create a typical lesion (Bright et
        themselves of      the  disease    through       al.   1960,   McLeese   1965,   Cook and
        ecdysis (Sandifer and Eldridge 1974;             Lofton 1973, Cipriani et al.         1980,
        Baross et al. 1978).       Other f actors        Nottage 1982).       Rosen   (1970),   Cook
        possibly   contributing    to   the   high       and Lofton (1973), Malloy (1978) and
        prevalence     of  disease    in    mature       Overstreet     (1978) hypothesized that
        females are longevity and the con-               mechanical damage to      the   epicuticle
        tagiou's nature of the disease.         Van      allows     chitinoclastic    bacteria    to
        Engel  (1958) noted that females can             enter.
        live up to two years following their
        terminal molt, thus increasing their
        chance    of     suffering      mechanical            The exoskeleton of crustaceans
        damage to the exoskeleton. Due to the            can   be viewed as a microenvironment.
        contagious nature of the disease and             Through succession, a primary food
        the increased    susceptibility of soft          source may be utilized by more than
        crabs to mechanical damage, a female             one type of bacteria.      The waste pro-
        undergoing her terminal molt has a               duced would be utilized by another
        high probability of infection through            bacterium    with    the   process   being
        prolonged    contact    with   its    mate       repeated   until     all  possible   foods
        especially if the latter is infected.            have been used (Gunsalus and Stanier
                                                         1962). Cipriani et al. (1980) sugges-
                                                         ted that lipolytic bacteria might play
             There     are    three   etiological.       an important role in the penetration
        scenarios for shell disease:       stress,       of the epicuticle.    Once this layer is
        mechanical      damage   and     bacteria.       breached, then chitinoclastic bacteria
        Chemical pollutants cause stress in              might attack the chitin in the exocu-
        marine    animals  (Sindermann    et   al :      ticle. The organic acids which are
        1982),    often     by   decreasing      an      produced as the end-product of chitin
        organism's resistance to disease by              digestion    would   then   dissolve    the
        impairing mechanisms of cellular and             calcium salts     (Rosen   1970).    These
        humoral   immunity (Murchelano 1982).            acids might affect     calcium    deposits
        Nottage     (1982)    hypothesized    that       in both the epicuticle and exocuticle,
        subtle changes in body chemistry may        24   which would allow further microbial









              degration and lead to the breakdown of             digestion (Hood and Meyers 1977).
              the endocuticle.                                   Organic loading and/or water quality
                                                                 might explain the results of experi-
                    Regardless of the mechanism for              ment 1, in which the Long Point and
              penetration of the epicuticle,           the       Texasgulf crabs developed the early
              end result is the breakdown of chitin              stages of the disease more rapidly
              resulting      in    necrotic      lesions.        than the control sites.     Not only did
              Chitinoclastic     bacteria     are    found       the disease develop more rapidly at
              world-wide and play.an important role              these sites,    but the severity of in-
              in the marine carbon and nitrogen                  fection was greater, especially at the
              cycles (Cambell and Williams 1951).                the Texasgulf location.
              These bacteria are most abundant in
              sediments     (Hock   1950,     Seki   1965,
              Donderski 1984) where the majority of                   The various colors of experimen-
              chitin    decomposition      occurs    (Seki       tal lesions (yellow, brown, black and
              1965,     Hood     and    Meyers      1977).       blue)   can   be    attributed    to    two
              Therefore, the benthic         nature     of       factors, melanin deposition and bac-
              blue crabs     predisposes them to these           teria.    Johnson (1983) suggested that
              opportunistic pathogens.                           darkening of the affected area is due
                                                                 to the deposition of melanin. Malloy
                     The bacterial fauna identified              (1978) noted that cratering of          the
              from natural lesions and study animals             lesions on experimental lobsters was
              were similar, with Pseudomonas spp.,               accompanied by the development of a
              Type B bacterium and Vibrio spp. being             yellow slime layer.     Whether this is
              the predominant organisms          present.        the result of bacterial action is not
              Pseudomonas spp. and Vibrio spp. have              known,  but of the 21 crabs that de-
              previously been isolated from animals              veloped this yellow slime, 14 showed
              with shell disease (Cook and Lofton                pitting.   Roald et al. (1981) noted
              1973, Baross et al. 1978, Malloy 1978,             that lesions were limited to the area
              Cipriani et al. 1980, Roald et al.                 within the blue line.       This line de-
              1981).    These findings suggest the               veloped in some animals and was common
              role of chitinoclastic bacteria in                 on crabs with natural lesions.
              this shell      disease outbreak since
              these    genera   often   have    chitinase             The lesions of blue crabs caught
              activity.     The occurrence of Proteus            ouside of the Pamlico River are pro-
              spp. in the bacterial samples has a                bably the result of mechanical damage.
              number of implications.      The tendency          This damage could be a function          of
              of this genus to swarm and overgrow                abrasive action, predator-prey rela-
              the surface of agar plates makes it                tionship or    intraspecific      interac-
              difficult to isolate other bacteria                tions, though abrasive action is the
              (Starr    et   al.   1981).      Therefore,        most likely explanation. This disease
               -quantitative  analysis    of    bacterial        was most prevalent on the appendages
              compositions between sites was not                 and ventral surface.     These areas are
              possible.     In aquatic environments,             most often in contact with the sedi-
              this genus is found in polluted waters             ments and more likely to be damaged
              (Buchanan and Gibbons 1974, Laskin and             through normal activity.
              Lechevalier 1977).        Areas of high
              organic load and poor water quality
              generally contribute to an increase in                  The etiology of shell disease
              numbers of bacteria (Cook and Lofton               within the Pamlico River estuary is
              1973,    Sindermann 1974), while higher            probably stress due to a change in
              overall  bacteria populations generally            environmental     quality.        Possible
              result   in   .a higher rate of chitin       25    stress-related events include abnormal










        shell synthesis, abnormal shell repair           sis due to a chemical        or    mineral
        or degradation of the shell in the               imbalance    resulting    in a structur-
        water colum.     Brine (1978) examined           ally inferior shell.     Thus the shells
        -the chitin content at various loca-             of these animals may be prone to
        tions on the carapace and chelipeds of           mechanical damage or bacterial attack.
        the blue crab.    The dorsal surface of          Cadmium and fluroide levels in the
        the carapace was divided into seven              Pamlico River were found to be higher
        regions, with percentage of chitin               along the southside and in particular
        (dry wgt) averaging 15.7%, while the             in the sediments near the discharge of
        cheliped    sections    averaged      11.6%      Texasgulf (Barry Adams, pers. comm).
        chitin.   The claws are the defensive            Cadmium was found to cause lesions in
        instruments of the crab. The reduc-              the   carapace    of  the   crab    Scylla
        tion in chitin      in the claws is a            serrata (D.M. Dhavale,     pers. comm.).
        function of higher deposition of cal-            Arey (1979) noted that high levels of
        cium salts in the cuticle affording              fluoride could tie up calcium ions,
        greater structural strength to this              which are needed for tanning of the
        appendage     (Brine   1978,     Greenaway       exoskeleton.     Future work should exa-
        1985).   The epibranchial region has a           mine the effect of these substances
        chitin content of 13.7%, the lowest              and other chemicals on shell synthesis
        percentage noted on the dorsal surface           and shell repair.
        of the carapace.     This region corres-
        ponds in part to area 6 of this study.
        If a lower chitin content is indica-                          ACKNOWLEDGMENTS
        tive of an increase in the amount of
        calcium salt deposition,       then this              We wixh to thank Ettles Henry,
        area should be more structurally sound           Jr. of Carolina Seafood and his fish-
        than other areas     on the carapace and         ermen for all their help and coopera-
        less    likely   to   suffer    mechanical       tion throughout the study.        We al so
        damage.    It is interesting to note             want to thank John Charles Singleton
        that crabs caught in the stress zone             and Steve Chandler of J.C. Singleton
        appeared to have weak carapaces that             Construction for their assistance        in
        could     easily     be    broken      with      the construction of the holding pens.
        slight    pressure.     The    possibility       Additionally, the authors are indebted
        exists    that   crabs    in   this    area      to Juanita Tripp and Dee Willis for
        are undergoing abnormal shell synthe-            typing the manuscript.




















                                                  26











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