Bacterial infection in the blue crab, Callinectes sapidus: course of infection and histopathology

JOUR\AL

OF INVERTEBRATE

Bacterial

PATHOLOGY

28. 25-36

(1976)

Infection in the Blue Crab, Callinectes Course of Infection and Histopathology PHYLLIS

sapidus:

T. JOHNSON

U.S. Department of Commerce, National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Middle Atlantic Coastal Fisheries Center, Pathobiology Investigations, Oxford. Maryland 21654 Received

May

15. 1975

During the summer, groups of blue crabs, Callinecfes sapidus. collected in commercial crab traps in Chincoteague Bay, Virginia, often undergo heavy mortalities during the first week to 10 days in the laboratory. Gram-negative bacteria are seen in hemolymph and tissues of many of the sick and dying crabs. The bacterial infections appear to be acquired during capture and transport, suggesting that potentially pathogenic bacteria in water or on the exoskeleton may be introduced into tissues by wounding or other means during the stressful conditions suffered at that time. The pathology caused by bacterial infection includes diminution in numbers of hemocytes. reduced clotting ability of the hemolymph, and progressive formation of hemocyte aggregations with necrotic centers in the heart, arteries, and hemal sinuses and spaces. By the third day, aggregations, often with many bacteria visible in the centers. occur especially in the gills, antenna1 gland, and Y organ. There are large premortem plasma clots in some animals. The focal and massive necroses that occur may be due to hypoxia resulting from obstruction of hemolymph How by cellular aggregations and plasma clots and to toxic products of necrotic cells and/or bacteria.

INTRODUCTION Over the past 3 years, investigations of diseases and other pathological conditions of blue crabs, Callinectes sapidus, have been in progress at the National Marine Fisheries Service Laboratory, Oxford, Maryland. One aspect has been an inquiry into the marked mortalities commercially trapped crabs may undergo while in the traps and during their first week or 10 days of captivity, especially during the summer months. The known biological stressors contributing to mortalities in captive animals include paramoebiasis (“gray crab disease”) and bacterial infection. Paramoebiasis is a cause of mortalities at Chincoteague Bay in shedding tanks used for production of soft crabs (Sprague et al., 1969), and bacterial infection was implicated as a cause of similar mortalities in tanks located on Chesapeake Bay and its tributaries (Krantz et al., 1969). Physical factors also contribute to stress and include the process of capture and handling, crowding in traps and/or holding tanks, transport out of water, exposure to elevated temperatures, 25 Copyright All rights

c 1976 by Academic Press. of reproduction in any form

Inc. reserved.

and wounding by fellow captives. Additionally, the Chincoteague crabs studied at the Oxford Laboratory are forced to adapt suddenly to a salinity below that of their normal environment. During the warmer months, bacterial infection is a major factor in mortalities of freshly collected Chincoteague blue crabs kept in the Oxford Laboratory. The present report describes the histopathology and course of bacterial infection in captive crabs based on observations of both naturally acquired and experimental infections. Comparisons are given between the “high risk” groups of commercially trapped crabs and others collected by gentler means. MATERIALS

AND

METHODS

Techniques of maintenance, surveillance of the animals, and examination of tissues were similar for all groups studied unless otherwise indicated. “Naturally” occurring infections. Observations were based on gross and histological examination of tissues and organs from more

‘6

PHYLLIS

than 300 crabs collected from Chincoteague Bay during May-September, 1974-75. The crabs were taken in baited commercial crab traps, packed into wooden bushel baskets on shipboard, and later transported by car to this laboratory. The time out of water varied from 4 to 8 hr according to the time an individual crab was removed from a trap. At this laboratory, the crabs were maintained in large tanks provided with a flow-through water system. Salinity of water in the natural environment, Chincoteague Bay, is approximately 25% while that of the water at this laboratory during the summer months is between 8 and 11%. Normal summer water temperatures both at Chincoteague and Oxford are 20-30” C. The crabs were juvenile females and small males ranging in width from 7.0 to 13.5 cm (mean 10.5 cm). Many animals had suffered wounds and/or autotomized limbs during their stay in the traps, while being placed in the baskets on the boat, and during their time in the baskets. They underwent further trauma while being placed into the laboratory tanks. In addition, they were subjected to high air temperatures during holding and transport to the laboratory, particularly in July and August. In the laboratory, crabs were checked several times daily, and as many moribund or sick animals as possible were dissected. Occasionally, dead animals were also dissected. “Death” was assumed if the heart was not beating and muscle did not react when the central nervous system was stimulated mechanically. Before dissection, hemolymph was withdrawn from the pericardial sinus or the heart, using a 21-gauge hypodermic needle and a plastic syringe. A drop of hemolymph was placed on a slide, covered with a coverslip, and examined with phase contrast optics. Great variation in hemocyte numbers occurs in normal blue crabs (Sawyer et al., 1970). By rule of thumb, hemolymph with O-20 hemocytes present per high-dry field was considered to have an abnormally low number of circulating hemocytes. Blood with more than 50 hemocytes per fiefd was considered normal. To de-

T. JOHNSON

termine clotting time, hemolymph remaining in the syringe was examined after 10 min. Hemolymph that was still liquid at the end of 10 min was considered to have impaired clotting ability. The following tissues from each of the crabs were fixed in Helly’s solution and processed routinely for paraffin sectioning: heart, gill, hepatopancreas, gonad, muscle, midgut ceca, midgut, antenna1 gland, brain, thoracic ganglion, Y organ, epidermis, and hemopoietic tissue. Tissues were stained by several methods: PAS counterstained with Weigert’s iron-chloride hematoxylin; alcian blue used at pH 2.2 and counterstained with Kernechtrot (Pearse, 1960); the Feulgen reaction counterstained with picric acid and methyl blue (Farley, 1969); and Brown and Brenn’s stain for bacteria in tissues (Sanders, 1972). Sections of tissues of crabs infected with the gram-positive coccus, Aerococcus viridans var. homari. served as controls for the latter stain. The photographs were made of tissue sections stained by the Feulgen technique. Since the course of infection was similar in all groups of crabs containing bacterially infected animals, detailed observations in this paper are limited to two collections made on August 20 and September 17, 1974. Concerning the August collection, water temperatures during the first 6 days in the laboratory were 25.426.5”C. On days 7-40, temperatures decreased from a high of 28.0” C to 19.O”C. With the September collection, temperatures were 21.0-24.5”C the first 6 days and decreased from 21 .O”C to 11 .O” C during days 7-37. Comparative material. Approximately 150 trapped and trawled crabs were taken during April-September, 1973-74, from three tributaries of Chesapeake Bay: The Tred Avon River adjacent to this laboratory, the Nanticoke River, and the Miles River. These crabs were carefully handled, and time out of water was never longer than 2 hr. Two hundred and seventy dredged and trapped Chincoteague crabs, collected during October-April, 1973-75, afforded information from the cooler months.

BACTERIA

IN

As a comparison of the types and extent of mortalities that occur in groups of trawled and trapped crabs, 122 carefully handled trawled crabs and 190 commercially trapped crabs were taken from Chincoteague Bay on September 9, 1975, and maintained in the laboratory under similar conditions. The trawled crabs were out of water approximately 6 hr, while trapped animals were out of water 448 hr. During the first 12 laboratory days, all dead and dying animals of this series were bled and dissected, and gross pathology was observed. In addition, antennal gland, heart, gill, epidermis, hepatopancreas, and hemopoietic tissue were processed for histological examination from 56 trapped and 11 trawled crabs. Shedding tank mortalities. In July, 1974, severe mortalities were experienced in crabs being maintained in a commercial shedding tank located near the Oxford Laboratory. Three recently dead or moribund animals in late premolt condition were received for examination. They were dissected and tissues prepared for histological examination. Experimental infection. The inoculum was freshly drawn hemolymph that contained numerous motile vibrio-like bacteria. The hemolymph was removed from an animal trapped at Chincoteague on September 9, 1975, and dead on the seventh day in the laboratory, with gross pathology consistent with bacterial infection. Seven healthy crabs that had been collected from Chincoteague and kept in the laboratory water system for 54 days were injected with 0.1 ml of hemolymph per animal. After swabbing the area with 70% alcohol, the inoculum was introduced through the dorsal aspect of the intersegmental membrane that lies between the carapace and the most posterior leg. Four controls, collected by trawl on September 9 and kept 7 days in the laboratory, were injected with the same amount of sterile sea water. OBSERVATIONS

AND

RESULTS

Infections in August andseptember

Crabs

Heavy mortalities occurred in both groups during their first 6 days in the laboratory,

BLUE

CRAB

21

and occasional bacteremia was detected during microscopical examination of fresh hemolymph from crabs bled on days O-3 (Tables 1, 2). Clotting of the hemolymph of most of the animals was absent or slow and incomplete. Correlation between the stage of the molting cycle and the extent of hemolymph clotting was lacking. In the entire series, circulating hyaline and granular hemocytes were less numerous than in disease-free animals collected during summer and fall. In heavily infected crabs, circulating hemocytes were virtually absent (O-5 per field). Paramoeba perniciosa. the cause of paramoebiasis, was found in 8 of the 38 crabs collected in August (Table 1). The single infected animal from day 0 and one of the two from day 1 had no evidence of concomitant bacterial infection. Both Paramoebainfected crabs dissected on days 2-3 did have bacterial infections. The single Paramoebainfected crab dissected from the September series on day 3 also had a bacterial infection (Table 2). Bacteria seen in fresh blood were motile, gram-negative rods. In tissues stained by the Brown and Brenn technique, the bacteria stained poorly; those that stained were a pale pink. In control slides of crab tissues infected with the gram-positive coccus, the bacteria stained a deep blue. Bacteria stained bright pink or red with Kernechtrot, and this stain was superior to others for differentiation. More than one species of bacterium was involved, judging from difference in sizes of bacteria from individual infected animals. For example, some infections consisted of organisms with the typical morphology and motility of vibrios, while in other crabs short, straight, broad rods or short, thin rods predominated. Gross pathology. Two of the moribund infected animals from the August collection had extensive premortem plasma (acellular) clots in the anterodorsal and frontal blood sinuses (Table 3), while freshly drawn blood from the hearts of these animals clotted incompletely. Seven of eight dead animals also showed extensive anterior clotting upon dissection, a sign which I have not seen in ani-

necrosis

in two.

bIncluding

62 15 19 ..I 6b 0 0 0 0 0

1 2 3 4 5 6 7-12 13 14-15 16 17-37

nMassivc

Number dead, not examined

Laboratory day

dead

O/2

on days 4-5.

015 O/2

215

o/5

o/2

those

o/3

213

o/3

515

S/5

415

O/6

Hemocyte aggregations in gills

of Bacterial

616

Nodules present

Pathology

316

-. Bacteria in tissue sections

O/l O/5

Oil O/5

m

212

l/2

1 1 0 4 1 Oil o/5

o/4 5/10 616 lo/lo

Hemocyte aggregations in gills

o/4 7110 416 9110

Nodules present

o/4 6110 6/b 9110

Bacteria in tissue sections

of Bacterial

-a 5 18 32 4 13c

Number dead, not examined

aNot recorded. bMassive necrosis in one. %cluding ones dead on day 5.

0 1 2 3 4 5 6 7 8 9-15 16-28 30-4 1

Laboratory day

Pathology

TABLE Infection September

crabs

Oil O/5

O/Z

314 l/10 516 g/10

o/2

o/5

O/5 O/2

o/3

415

3/P

o/3

616

316

Clotting-incomplete or lacking

in dissected

Aggregations and degeneration. antenna1 gland

Pathology

2 in Blue Crabs,

Oil O/S

2/2b

o/4 4llOb 516 lO/lO

crabs

Ctotting incomplete or lacking

August -_ in dissected

in Blue Crabs, Pathology

1

Aggregations and degeneration, antenna1 gland

TABLE Infection

012

O/S

o/3

315

616

Few mature granulocytes

l/l l/5

O/5 O/Z

o/3

115

216

Bacteria in fresh blood

O/l O/S

012

116 o/to

516 8llO

l/2

l/4

2110

s/10

Bacteria in fresh blood

214

Few mature granulocytes

o/2

o/5

o/3

l/S

O/6

Paramoeba present

l/1 2/5

O/2

l/10

116

l/4 2110

Paramoeba present

:

2

z

2 F 3 i

-J

kd

BACTERIA

Course Nodules

1 3 2

6

Heart

118 416 4/10 l/2

TABLE Infection

of Bacterial

and hemocyte

Gills

s/s

Y organ

3/S

516

616 lO/lO 212

lO/lO 212

Nodules

Laboratory day

1 3 13

Heart

of Bacterial

and hemocyte

Gills

39

CRAB

3 in Blue Crabs,

August

Hepatopancreas tubules abnormal

Hepatopancreas rosettes abnormal

Acellular internal clots

5/8

218

5/S

316

316

116

S/l0

216 4/10

3/10

3/10

112

o/2

o/2

112

mals without a bacterial infection. Premortem clotting did not occur in the September group. In Callinectes. the posterior pair of legs is modified for swimming, with the distal two segments flattened and paddlelike. These segments will be called paddles henceforth. In sick animals of the August series, cloudy areas, which proved to be aggregations of hemocytes, were sometimes visible through the semitransparent cuticle of the distal segment. Therefore, paddles were systematically checked in the September collection. Paddles were clear in all animals dissected on day 1, but cloudy areas were present in all animals dissected on day 3 and in one of two on day 13 (Table 4). Other gross pathology seen in affected animals included enlarged chalky-white areas caused by aggregations of hemocytes in lamellae and stems of the gills. Similar areas were found in other tissues, particularly the antenna1 gland and Y organ. Course ofinfection. Microscopical examination of stained tissue sections showed a progression of events which apparently began on laboratory day 0 in most or all of Course

BLUE

aggregations

Antenna1 gland (including necrosis)

Laboratory day

IN

TABLE Infection

3/S

the crabs and had run its acute course by day 6 (Tables l-4). Groups of hemocytes, composed of a small center of cells with pycnotic or karyorrhectic nuclei and surrounded by normal hemocytes, were present on day 0. The groups were sometimes ensheathed by flattened granular or hyaline hemocytes (Fig. 1) and were most common in the heart (Fig. 2). By day 1, many of these small aggregations had hyaline centers, containing several pycnotic nuclei or chromatin fragments, and often were surrounded by a single layer of encapsulating hyaline hemocytes (Fig. 3). These will be referred to henceforth as nodules. Granulocytes were rare, both in blood vessels and in hemal spaces and often had pycnotic nuclei. Hyaline hemocytes were sometimes similarly affected. By day 2, gill lamellae and stems contained nodules and increasingly large aggregations of hemocytes (Fig. 4). The lamellar epithelium was apparently sloughed on lamellae distended with hemocyte aggregations, and these aggregations, or thromboemboli, occasionally caused stasis of hemolymph flow, so that portions of some lamellae were distended with essentially acellular plasma (Fig. 5). There 4 in Blue Crabs.

September

aggregations

Antenna1 gland (including necrosis)

Y organ

Hepatopancreas tubules abnormal

Hepatopancreas rosettes abnormal

Hemocyte aggregations in paddle

616

616

516

316

016

216

O/f3

31.5 212

515

515 l/2

315 O/2

415

415

515

o/2

o/2

l/2

112

FIG. 1. A small group of degenerate hemocytes in the heart of CaNinectes supidur. Note granulocytes (G) among the surrounding hemocytes. The line = 10 pm (Figs. 3.4.7-I I, 13 to same scale). FIG. 2. Lower magnification of Fig. I. showing groups of aggregating hemocytes in the heart. The line = 100 pm (Figs. 5,6. 12 to same scale). FIG. 3. Nodule in the heart, Necrotic cells with pycnotic nuclei form the center, surrounded by a thin layer of flattened hyaline hemocytes. FIG. 4. Gill lamellae and stem, distended with aggregations of hemocytes. FIG. 5. Gill lamella distended with hemolymph. FIG. 6. Large aggregations of hemocytes in clotted blood. Note the degenerate centers. surrounding areolar zone. and peripheral normal hemocytes.

FIG. 7. Abnormally swollen rosettes of fixed phagocytes (R) between the hepatopancreatic acini of Callinectes sapidus. FIG. 8. Normal rosettes (R)of fixed phagocytes. Compare with Fig. 7. FIG. 9. Large necrotic areas (N), with pycnotic nuclei, in the Y organ. FIG. IO. Normal Y organ. Compare with Fig. 9. FIG. 11. Degeneration in the antenna1 gland. The end sac (E) is disrupted and the labyrinthal epithelium (L) is thin, with pycnotic nuclei. FIG. 12. Massive necrosis of the antenna1 gland. Normal structure is entirely lost. Breaks in the tissue are artifacts. FIG. 13. Normal architecture of the antenna1 gland. Compare with Fig. I I. (E), End-sac epithelium; (L), labyrinthal epithelium: (H), intraepithelial hemal space.

32

PHYLLIS

was no visible involvement of the nephrocytes (athrocytes) which occur in large numbers in the gill stem and scattered through the lamellae. The thickened gill epithelium (“salt gland”) that is active in ion transport was often normal in appearance, but sometimes evidence of degeneration was present. Hemocyte aggregations in hemal spaces and blood vessels appeared as separate nodules forming a central mass surrounded by an areolar layer of degenerating hemocytes. Peripherally, normal rounded hemocytes were usually present (Fig. 6). A marked encapsulative response was lacking. Bacteria were visible in many of the aggregations, both intra- and extracellularly, and in the cytoplasm of nearby hyaline hemocytes, which often had pycnotic nuclei. These hyaline hemocytes occasionally contained so many bacteria that the nucleus was flattened against the cell membrane. Hyaline hemocytes also occasionally contained pycnotic nuclei of one or two phagocytized hemocytes. Nodules and cell aggregations were variously present in the blood sinuses and arteries throughout the crab, decreasing in the heart by day 3 but with a general increase in the gills, antenna1 gland, and Y organ (Tables 3 and 4). It has already been remarked that, while hemocyte aggregations were lacking in paddles of the September animals dissected on day 1, sick crabs all had such aggregations by day 3 (Table 4). The organs afected. The hepatopancreas was variously affected in the sick crabs (Tables 3 and 4). Massive sloughing and decrease in thickness of the acinar epithelium, approaching a cuboidal condition in some acini, paucity of mitoses in the apical acinar epithelia, and evidence of karyolysis occurred with varying severity. Fixed phagocytes present in rosettelike groupings in connective tissue strands among the acini were often swollen, vacuolate, and with nuclei exhibiting karyorrhexis, pycnosis, or karyolysis (Fig. 7). Figure 8 shows normal rosettes of phagocytes. In some heavily infected animals, phagocytized bacteria were visible in the fixed phagocytes and large aggregations

T. JOHNSON

of hemocytes caused distention of many of the interacinar spaces. The Y organ, by day 3, was affected in most of the crabs (Tables 3 and 4). Aggregations of hemocytes in hemal spaces and arterioles impinged on the glandular tissue, probably causing focal “infarction,” which sometimes led to extensive focal necroses of the Y-organ tissue (Fig. 9). Normal Y organ is shown in Figure 10. The antenna1 gland exhibited degeneration and presence of hemocyte aggregations. By the third laboratory day, all dissected animals were affected to some degree (Tables 3, 4). When hemocyte aggregations occurred in the hemal spaces between the involutions of the labyrinthal epithelium and in spaces between the interdigitated labyrinthal and end-sac epithelia, the end sac often became degenerate and disorganized, with invading hemocytes present in the disorganized tissues (Fig. 11). The labyrinthal epithelium of some crabs was acidophilic and thinner than normal and had pycnotic or karyorrhectic nuclei, suggesting coagulative necrosis. The degenerative process was usually multifocal, but some glands were almost completely necrotic (Fig. 12). Figure 13 shows the normal relationships of parts of the antenna1 gland. Mitotic activity in hemopoietic tissue varied and could not be correlated with severity or duration of infection. In 3-day infections, some pycnotic nuclei were observed in that tissue. Other tissues and organs were visibly affected mainly when hemocyte aggregations were present in the surrounding hemal spaces. Hemocytes with pycnotic nuclei were often present in small blood vessels and sinuses of the brain and thoracic ganglion and in the spongy glial tissue lying just within the neural lamella. Small nodules also occurred sporadically in the central nervous system, and granulocytic invasion of nerves was common in animals with granulocytes. Comparison of Trapped and Trawled Crabs By day 12, 80% (152/190) of the trapped crabs and 23% (28/122) of the trawled crabs were dead. Deaths had virtually ceased by

BACTERIA

IN

day 9 in both groups. Examination of hemolymph and gross examination of organs and tissues of 135 of the trapped crabs allowed a tentative diagnosis of bacterial disease in 115 (85%) of them. This figure agrees well with the percentage of bacterial infection found in trapped animals that were also prepared for detailed histological examination: 86% (.56/65). Of 11 trawled crabs that were examined both grossly and microscopically, five were diagnosed as being bacterially infected (45 %). Paramoebiasis and cannibalism of molting animals contributed to deaths in this group.

BLUE

CRAB

33

day. Aggregations of hemocytes with nodular centers and containing numerous bacteria were present in the heart and gills and in the fixed phagocytes present in the connective tissues of the hepatopancreas. Three of the control animals were well at the end of 21 days, when the experiment was terminated. The fourth control animal died 24 hr postinoculation with advanced bacterial infection probably contracted 10 days earlier during capture.

DISCUSSION The data presented indicate that most or all of the “naturally” infected crabs acquired Miscellaneous Comparative Material their bacterial infections during the stress of Three of the 270 Chincoteague crabs capture and transport, not before. Not only collected during the cooler months had bac- did the pathological process follow a terial infections. The two taken in April and predictable pattern, beginning with the first March had been in the laboratory 1 and 4 day of capture, but crabs dying from experidays, respectively. The third animal, col- mentally induced infections fit into this patlected in November, had been in the laboratern. Tubiash and Krantz (1970) found blue tory 9 days. crabs were rapidly killed by injected Vibrio Of 150 carefully handled crabs taken from parahaemolyticus as well as some other tributaries of Chesapeake Bay in Apri-Sepspecies of marine vibrios, and in a survey of tember, 1973-74, only one had a bacterial in- bacterial flora of Chesapeake Bay, Kaneko fection. This animal was an adult female and Colwell (1973) found that vibrios, includcollected in August and kept 10 days in the ing V. parahaemolyticus, are associated with laboratory before death. external surfaces of zooplankton. The external surfaces of a species of California Shedding Tank Mortalities crab also have a rich and varied bacterial Two of the three crabs concerned had ex- flora, including both vibrios and pseudotensive postmortem plasma clots throughout monads (Johnson, unpubl.). It perhaps can be the hemal sinuses, and all had nodules within assumed that a similar bacterial flora occurs the heart, gill lamellae, and antenna1 gland. on the exoskeleton of Callinectes. Potentially Bacteria were visible in nodules from two of pathogenic bacteria may have been present the animals. in large numbers on the external surface of the crabs investigated and may have entered Experimental Infection through wounds or by other routes not ordiApproximately 48 hr postinoculation, all narily breached in nonstressed animals. crabs receiving the bacterially infected heRecently, it was stated that 82% of 290 molymph were dead or moribund. Six of the blue crabs collected from Chincoteague Bay seven had extensive plasma clots. The by trapping and dredging and in “acceptable seventh crab was heavily infected with condition” for marketing had bacteria in the paramoebiasis, which causes loss of clotting hemolymph, including V. parahaemolyticus ability of the blood during the terminal in 21% of the samples (Tubiash et al., 1975; stages. Histopathology in all crabs was in Sizemore et al., 1975). Colwell et al. (1975) keeping with that seen in the “naturally” say that, “Clearly the hemolymph of most infected animals on their second laboratory healthy blue crabs is not sterile.” Tubiash .et

34

PHYLLIS

al. (1975) remarked that the crabs they sampled were stressed and traumatized during collection. The results given here suggest that, before assuming that healthy blue crabs normally have bacteria in the hemolymph, it would be necessary to repeat the experiments of Tubiash et al. (1975), using crabs that suffered only the stress of being singly and carefully removed from the water and that were then bled immediately. One must question whether any animal could tolerate successfully the presence of a known pathogen, such as V. parahaemolvticus, within its body fluids and tissues. Bang (1970) observes that the blood of normal invertebrates is sterile and that animals with transient infections might be misinterpreted as being “normal” carriers of the organism if the infection is light or be discarded as sick if the infection is heavy. Bang says, “They [the infected animals] have rarely been held long enough to determine the final outcome.” Sindermann (1971) has reviewed work showing that bacteremias in lobsters and other invertebrates often occur in animals immediately after capture. The marked differences I found in mortality between heavily stressed crabs (commercially trapped in the summer) and less stressed groups (trawled or collected in cooler months) enforce the statements of Bang (1970) and Sindermann (1971). Deaths and sickness in crabs with some histopathology typical of bacterial infections but with few or no demonstrable bacteria in tissues or blood and minimal hemocytic reaction are not understood at present. Endotoxin alone can cause similar effects in Limulw (Levin, 1967; Shirodkar et al., 1958). Also, it is not assumed that bacterial infection was the invariable cause of death in animals that were not examined microscopically. Paramoebiasis, physiological stress, or a combination of several factors may have been responsible. Some crabs were undergoing the normal stress produced by the premolt stage of the molting cycle, and Chincoteague crabs were forced to adapt to a much lower salinity than in their natural environment. Although osmotic stress might

T.

JOHNSON

have subtly enhanced bacterial pathogenicity, C. sapidus is an efficient osmoregulator, and gross inability to osmoregulate was not demonstrable in crabs collected during the warm months of the year. Blue crabs are less able to osmoregulate at low temperatures and failure of osmoregulation, indicated grossly by swelling of the paddles and intersegmental membranes, appears to be a major cause of death in winter crabs held at low salinities (Johnson, unpubl.). Except in heavy infections, phagocytized bacteria were not seen in nonaggregated hemocytes. The granulocytes and hyaline hemocytes with pycnotic nuclei may, however, have phagocytized small numbers of bacteria and been injured by products released during digestion of the bacteria. Endotoxin is known to be involved in the formation of cell aggregates (“cellular clots”) in the horseshoe crab, Limulus (Bang, 1956; Levin, 1967) and endotoxin released after death of phagocytized bacteria may have caused degeneration in the blue crab cells. Cantacuzene (1925) found that infections of gram-negative bacteria in the crab, Carcinus maenas. caused disappearance of granulocytes, followed by diminution in numbers of remaining hemocytes and by the lack of clotting ability. In many respects, the course of bacterial infection in Callinectes was similar to that in Carcinus. Cantacuzene did not discuss the fate of the disappeared hemocytes in infected Carcinus. but probably they were involved in cell aggregations such as those produced following injection of endotoxin in Limulus and like those described in this paper. In the present case, cellular aggregation (“clotting”) apparently was dependent on the presence of necrotic hemocytes, which could not be proven to have phagocytized bacteria. As aggregations became larger and associated groups or masses of bacteria appeared, they became like those described in Limulus infected with gram-negative bacteria (Bang, 1956; Levin, 1967). The appearance and genesis of the hemocyte aggregations in Callinectes were also similar to “nodule” formation in insects, where hemocytes aggregate around foreign

BACTERIA

IN

material, microorganisms, necrotic hemocytes, etc. (Salt, 1970). The acellular premortem clots found in some animals of the present series may have been endotoxin mediated as in Linlulus (Levin, 1967; Bang, 1970). Dinimution of numbers of nodules and cell aggregations in the heart and increased numbers in the antenna1 gland, gills, and Y organ, with increasing duration of infection, might be explained partially by the following suppositions. A greater volume of blood probably passes through the heart than any other single organ or tissue, and bacteria might first be apprehended in numbers there. Small aggregations of hemocytes, forming in the heart and elsewhere and moving through the blood vessels and sinuses, would tend, as cells degenerated centrally, to attract further hemocytes, increasing the mass of the aggregations and depleting the number of circulating hemocytes. As the aggregations increased in size, they would be likely to lodge in constricted areas. It is important physiologically that large amounts of hemolymph pass through the gills (for gas exchange and osmoregulation), through the antenna1 gland (for ionic regulation and excretion of wastes), and through the Y organ (a neurohemal organ involved in the molting cycle and in other imperfectly understood functions). One could, therefore, postulate that emboli would be selectively directed by patterns of hemolymph flow into sinuses and arteries associated with those organs. As remarked, the aggregations caused marked distention of the gill lamellae and occasional obstruction of the flow of hemolymph. Focal necroses in tissues and organs and the occasional massive degeneration and necrosis that occurred might have been due in part to cell aggregations acting as thromboemboli and reducing blood flow through the arteries and hemal spaces, thus causing ischemic tissue damage. Another possible cause of these necroses could have been acellular clotting of the hemolymph, which would have accomplished the same end as the cellular emboli. Toxic products of cell and/or bacterial dissolution also could have affected the

BLUE

CRAB

33

antenna1 gland and fixed phagocytes in the interstitial tissue of the hepatopancreas particularly. It should also be noted that apparently more than one species of bacterium was involved in the blue crab infections. Some bacteria may have been more virulent than others, so that host reaction depended on pathogenic differences in the infecting organisms. If any of the animals in the August and September series that survived beyond day 16 had been infected with bacteria, evidence of such infections no longer remained. ACKNOWLEDGMENTS I am grateful to M. W. Newman for helpful comments on the manuscript, to L. C. Smith and D. H. Wright for histologic preparation of tissues. and to G. E. Ward, Jr., who obtained the crabs.

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36

PHYLLIS

Education and Welfare, Public Health Service, National Institutes of Health, Bethesda, Md. SAWYER. T. K., Cox, R., AND HIGGINBOTTOM, M. 1970. Hemocyte values in healthy blue crabs, Callinectes sapidus. and crabs infected with the amoeba, Patamoeba perniciosa. J. Invertebr. Pathol., 15, 440-446. SHIRODKAR,

M. V., BANG, F. B., AND WARWICK, A. 1958. Antibacterial action of Limulus blood in an in vitro system. Biol. Bull., 115,341. SINDERMA~N, C. J. 1971. Internal defenses of Crustacea: A review. Fish. Bull. 69.455-489. SIZEMORE, R. K., COLWELL. R. R., TUBIASH, H. S., A\D LOVELACE, T. E. 1975. Bacterial flora of the he-

T. JOHNSON molymph of the blue crab, L’allinectes sapidus; merical taxonomy. Appl. Microbial., 29, 393-399.

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V., BECKETT, R. L., ASD SAWYER,T. K. 1969. A new species of Paramoeba (Amoebida. Paramoebidae) parasitic in the crab Callinecres sapidus. J. Invenebt. Pathol., 14. 167- 174.

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TUBIASH, H. S., AUD KRANTZ, G. E. 1970. Experimental bacterial infection of the blue crab, Callinectes sapidus. Bacreriol. Proc.. 1970, G80 (Abstract). TUBIASH. H. S., SIZEMORE, R. K.. ASD COLWELL. R. R. 1975. Bacterial flora of the hemolymph of the blue crab, Callinecfes sapidus: Most probable numbers. Appl. Microbial.. 29,388-392.