Bacterial clearance in the California sea hare, Aplysia californica

JOURNAL

OF

INVERTEBRATE

Bacterial

PATHOLOQY

18,

227-239

Clearance

(1971)

in the

Aplysia

California

caltfornica”

Sea Hare, ’

GILBERT B. PAULEY~, STUART M. KRASSNER, AND FAYLLA A. CHAPMAN Center

jar Pathobiology, Department 05Developmental and Cell Biology, 05 Molecular Biology and Biochemistry, University 05 California, California 9.2664 Received

March

and Department Irvine,

18, 1971

Aplysia californica normally contains sterile hemolymph. It is capable of completely clearing in vivo at least 4 marine bacteria from the hemolymph, but wasunable to completely clear the terrestrial bacterium Serratia marcescens during the period tested. Clearance occurred most rapidly at high temperatures (~18-20~C) and was accelerated by previous exposure to the bacterium. The latter indicates some type of primitive anamnestic response. Concomitant with early rapid bacterial clearance was a depression of serum agglutinin titers and a decrease in circulating hemocytes; levels of these two variables later return to normal values. Agglutinin titer levels return to normal values even after 3 bacterial challenges. Hemocyte numbers and agglutinin titers return to preinjection levels more rapidly at higher temperatures. Although A. californica serum exhibited no Iytic activity, opsonic factors which enhanced phagocytosis of chicken red blood cells were found. A mechanism of bacterial clearance by the sea hare involving agglutinin and opsonin activity is hypothesized.

Although marine invertebrat’es are susceptible to a wide variety of fungal, protozoan, and bacterial infect’ions, numerous invertebrates possess cellular and/or humoral defense mechanisms which are effective against microorganisms (Tripp, 1970; Sindermann, 1970). Recently Johnson and Chapman (1970b) have demonstrated in vitro that serum of the California sea hare, Aplysia californica, is not bactericidal. However, this gastropod does contain naturally occurring agglutinins against marine bacteria (Pauley et al., 1972). A study was therefore undertaken to determine the 1 Supported in part. by a NIH lowship 5-FOI-GM38769-02 and 9-440860-23714-3. 2 Part of a dissert,ation by submitted to the Faculty of California in partial fulfillment ments for the degree Doctor of 3 Present Address: National Service Biological Laboratory, 21654

Predoctoral a USPHS

FelGrant

the the

senior author University of of the requirePhilosophy. Marine Fisheries Oxford, Maryland 227

mode of antibact,erial defense in the sea hare by studying in vivo bacterial clearance and correlating this with serum agglutinin activity, tot,al hemocyte numbers, and t’he lack of bactericidal activity. This paper reports the results of these experiments and the probable mechanism of bacterial elimination by A. californica. A small port)ion of this study has appeared in an earlier report (Pauley, 1971). MATERL~L~ AND METHODS Animal collection and main.te?lance.Animals were coIlected south of the Corona de1 Mar game preserve in southern California and maint#ained as previously described (Pauley et al., 1972). Sea hares (~150-250 g) for different experiments were kept in lo-gal aquaria with subsand filters containing fresh seawater maintained at the desired temperature =!=l”C. Bacterial cultures. Five bacteria were used in this study: Gafkya homari (Gh), Serratia marcescens (SM), Micrococcus aquivivus

228

PAULEY,

KRASSNER,

(628), a Pseudowzonas sp. (Fr), and a chromogenic gram negative rod (Ap5Y). The first four bacteria have been characterized in detail by Johnson (1969) and the last organism is described by Pauley et al. (1972). These bacteria were cult,ured, washed, and resuspended to desired concent’rat’ions according to t,he met’hods of Pauley et al. (1972). An inoculum of 1.0 X log bacteria was injected int’o the pedal sinus of each 9. ccdifornz’ca after the pedal surface was cleaned with 70% ethanol, with the exception of AI. aquiviws which was injected at a concentrat.ion of 2.0 X log bacteria. This was done because 1 X log 31. aqzhivus were cleared so rapidly that accurate plat’e counts could not be obt,ained. Inocula of 1.0 X 10’ heat-killed bacteria were used as vaccines. Saline control animals were injected nit’h equal volumes of sterile seawat,er. Xerum. preparation ad analysis. Whole hemolymph was withdrawn from each test animal (Pauley et al., 1972) at- 1 hr, 2 hr, 4 hr, 8 hr, 1 day, 2 days, 4 days, 8 days, 16 days, and 1 month post-inoculation, depending upon the t’est bacteria used. Whole hemolymph was diluted in st,erile seawater to reduce bacterial counts to -600 or less on seawater agar plates (Johnson 196S), although in a few cases higher numbers were found. Three replicate plate counts were made with hemolymph taken from each of two different, sea hares at every sample time. This was done by evenly distributing 0.1 ml of test fluid on agar plates with a glass rod spreader that was dipped in 70% ethanol, flamed, and cooled to room temperature between each use. Plates were then incubated at 26°C for 48 hr, and the individual colonies were counted. Whole undiluted hemolymph was spread on agar pIat,es at each sample time to be cert,ain of the final clearance time, since dilution of very small numbers of bacteria might not be detected, thus giving an erroneous indicat.ion of complete clearance. Hemocyte counts were made according to the method of Pauley and

SND

CHAPMdN

Krassner (in press). Sera Tvere filter sterilized, frozen at -12 or - 18’C, and later used for agglutination assays (Pauley et al., 1972). Except where noted, most tests were performed in duplicate. It. was import.ant Do know whether A. caZijorGca hemolymph is normally sterile; t,herefore, at’ different times, 0.1 ml hemolymph from 5 sea hares chosen at, random was spread on seawater agar plates or on modified agar plates (filtered seawater IS0 ml, cell-free A. cal~ifornica serum 20 ml, Bacto agar 3.0 g, Bacto peptone 1.0 g, and Bacto yeast 0.2 g). Bactericidal action of henzolymph. Hemolymph from 5 different sea hares (held at 17°C) \\-ere tested for bactericidal action against, the 5 bacteria at 13 and at 26°C. The test fluids were kept at. 36 or 13°C for 2 hr prior to inoculation. Three types of test’ solutions were used: (1) st)erile seawater, (2) whole hemolymph cont’aining blood cells, and (3) sera lacking blood cells. Approximat,ely 400 bact,eria were incubat,ed with 0.25 ml test fluid in Kahn tubes for 1, 6, or 24 hr at eibher of the two best temperatures. After the appropriate incubation time, the t.ubes were thoroughly mixed with a vortex mixer, 0.05 ml was removed and spread on seawater agar plates, which were then incubated for 48 hr at 26 or 13°C. Bact’erial growth was recorded as either positive or negative (see Krassner and Flory, 1970). Induced bactericidal response. We attempted to induce a bactericidal effect in A. californica hemolymph by injecting 1.0 X log M. aquivivus into 4 animals held at 20°C. Since this organism is cleared from the sea hare’s hemolymph within 24 hr, hemolymph samples were tested for bact’ericidal activity 1 and 7 days post-injection using t’he assay procedure described in the preceding paragraph. These tests were carried out at 26°C. Opsonixation test. A modificat’ion of the balanced salt solutions (BSS) described by Tripp et al. (1966) and by Cecil (1969) was devised for opsonization studies (Table 1). Sea hare hemolymph was pooled and t’he

BACTERIAL TABLE

CLEARANCE

1

COMPOSITION OF BALANCED 6-4~~ SOLUTION (BSS) USED FOR SEA HARE OPSONIZATION TEST” Quantity

Ingredient NaCl KC1 CaC& (anhydrous) MgCl, (anhydrous) MgSOr (anhydrous) NaHCOa &HP04 (anhydrous) Glucose (dextrose) Trehalose Galactose Phenol red Eagle Essential (50X) Eagle Nonessential (50X) Eagle Essential

19.300 0.670 1.100 2.030 2.940 0.080 0.190 0.500 0.500 0.300 0.005 amino

vitamins

g g g g g g g g g g g

acids 20.00

amino

per liter

ml

acids (100X)

3.50 ml 3.33 ml

a A precipitate of unknown composition forms after adjustment of BSS to the pH of normal sea hare hemolymph (pH 8.6). This is removed by sterilization of the BPS through a Millipore filter (0.45 nm pore size).

cells separated by centrifugation at 2000 rpmj5 min at 4°C. Cells were washed 3 times in BSS (pH S.S), placed on glass slides (4.0 X lo4 washed cells/slide) and allowed to settle in a moist chamber for 1 hr before use. Chicken red blood cells (RBC’s) preserved in Alsevers solution (Colorado Serum Company) were washed three times in 0.15 M NaCl and pretreated in one of t’hree solutions: (1) normal sea hare serum and BSS, 1: 1; (2) boiled sea hare serum and BSS, 1: 1; or (3) BSS alone (cont’rol). Slides with att,ached sea hare blood cells received 0.4 ml of the appropriate fluid containing 1% RBC’s and were incubated in a moist chamber at 19°C for 30 min, 1, or 2 hr, after which excess RBC’s were removed with BSS. The slides were fixed in 100% ethanol and seawater, 1:3, and immediately examined by phase-contrast microscopy because if allowed to dry even after fixation, the cytoplasm of the sea hare cells ruptured, thereby releasing any phagocytosed RBC’s.

IN

SEA

229

HARE

The phagocytic index was determined by 1ocat)ing a cell xvith a phagocytosed RBC and then counting the next 100 sea hare hemocytes found at, random. This test was performed in duplicate and t.he averaged duplicate phagocytic values were reportfed. RESULTS

Hemolymph

Xterility

Except for 3 cases, A. califor?lica hemolymph was sterile. The three exceptions contained a felt isolated colonies and were in hemolymph taken from animals that had not been cleaned with 70% alcohol. It was, therefore, concluded that bacteria found in sea hare hemolymph under normal conditions are contaminants resulting from improper sterile technique. Primary

Bacterial Clearance

All 4 marine bacteria were completely cleared in vivo from sea hare hemolymph. M. aquivivus is the most rapidly cleared bacterium, with complete clearance accomplished in 8 hr at 20°C and in 24 hr at 14°C (Fig. 1). Pseudomonas sp., Ap5Y, and G. homari (lat’ter performed only once) all

TIME IN HOURS

FIG. 1. Levels of ~ficrococcus Aplysia californica hemolymph with 2 X 109 bacteria in animals 20°C.

aquivivus in after injection held at 14 and

230

PAULEY,

KRASSNER,

IND

show similar clearance patterns with clearance occurring most’ rapidly at the higher test temperat,ure (Figs. 2-4). S. mcwcescens exhibited a markedly different. clearance pattern. Although a decrease of one log number of bacteria occurred during the first day, large numbers of bacteria (105) persisted in the hemolymph for at leash 1 month post-injection (Fig. 5). There were always fewer S. marcescens found in animals kept at 18°C than in those maintained at 12°C. Mortalities were observed in only two ex-

CHAPMAN

105 ,o’ z 2 ,03. = z IO*‘@o--

2

4

8

24

48

TIME IN HOURS

”

FIG. 4. Levels of Gu$lcya pathogen, in Aplysia californica injection with 1 X IO9 bacteria 12 and 18°C.

homari, a lobster hemolymph after in animals held at

r

FIG. 2. Levels of Psezdonmms Aplysia californica hemolymph with 1 X log bacteria in animals

sp., strain FR in after injection held at 14 and OL

1I

FIG. 5. Levels of Serratia marcescens, aI1 insect pathogen, in Aplysia caZifo&ca hemolymph after injection of 1 X lo9 bacteria in animals held at 12 and 18°C. Note that bacteria are still present 16 days post inoculation. (Other tests showed high levels of S. marcescens 1 month after injection.)

periments, and in bot,h cases the test bacterium was S. marcescens. In one case, sea hares were exposed to elevated temperatures (WC), and in the other case t,he animals mere subjected to mult,iple bleedings. /

2

4

8

24

TIME IN HOURS

3. Levels of the yellon-, gram negative rod Ap5Y in Aplysia californica hemolymph after injection with 1 X lo8 bacteria in animals held at 12 and 18°C. FIG.

48

Bacterial Agglutinin

Titer

Sea hare serum does not agglutinate S. wzarcescens (Pauley et al., 1972). The gram negat’ive rod Ap5Y causes an immediate

BACTERIAL

CLEARANCE

FIG. 6. Aplysia calijornica agglutinin titers following injection of the chromagenic gram negative rod Ap5Y (1 X lo9 bact,eria), or of sterile seawater (control). Animals were kept at 12 and 18°C. Agglutinin titers of untreated animals are shown on t,hecright.

0

=

m

m

2

4

6

7. Aplysia following injection (1 X lo9 bacteria) in animals kept at of untreated animals FIG.

l

*

9

.

24

m

49

agglutinin titers of Pseudomonas sp., straiu FR or of sterile seawater (cont,rol) 14 and 20°C. Agglutinin titers are shown on t,he right. californica

IN

SEA

231

HARE

whether G. homari caused a similar agglutinin titer depression and return to normal. Therefore, the experiment was performed even though w-e lacked control animals. In the case of M. aquivivus, agglutinin titer depression was noted only at, 14°C and a return to normal levels took place within 4 hr (Fig. 9). This may be relat#ed to the very rapid clearance of 111. aquivivus by A. californica. Agglutinin titers were not increased by prior challenge with any of the bacteria. Titer levels returned to normal values after primary, secondary and tert,iary challenges of Ap5Y made 48 hr apart, indicating that sea hares are capable of quickly replenishing the agglutinin. No difference in normal agglutinin titers were found in sea hares

FIG. 8. Aplysia californica agglutinin titers following injection of Gafllcya homari (1 X log bacteria) in animals kept at 12 and 18°C. Agglutinin titers of untreated animals are shown on the right.

6.3 g

.

20-c LCONiROLl -0-e

7 depression in agglutinin titer, which then 6 NZI1’IIL T .i.s returns to normal levels as the bacteria are g L cleared from the animals’ system (Fig. 6). * 4 m Agglutinin titers return more quickly to 2 normal levels at 18°C (4 hr) than at 12’C . . . (8 hr), which correlates directly with the rate of bacterial clearance at these tempera0 2 4 6 * TIMEIY HOURS tures. A similar pattern of early agglutinin FIG. 9. Aplysia californica agglutinin titers titer depression followed by a return to following injection of Micrococcus aquioivus (2 X normal levels was found with Pseudomonas lo9 bacteria) or of sterile seawater (control). Anisp. (Fig. 7) and G. homari (performed only mals were kept at 14 or 20°C. Agglutinin titers of once, Fig. 8). We were interested in knowing nutreated animals are shown on the right.

232

PAULEY,

KRASSNER,

AND

held at different temperatures. Animals inject’ed wit,h sterile saline did not show agglutinin titer depression at any time during the course of study (see Figs. 6, 7, 9). Total Hemocyfe Xumbew Both gram negative (Figs. 10, 11) and gram positive (Figs. 12, 13) marine bacteria

150

CHAPMAN

depressed the t,ot,al number of circulating hemocytes at all test temperatures. Hemocyte numbers returned to normal levels within 24 hr as the bacteria were eliminated. Sea hares held at, higher temperat.ures usually show a more rapid return to normal hemocyte levels. Surprisingly, the pattern of hemacyte depression n-as also observed

I . .

18’C

(CONTROL)

TIME

FIG.

negative

10. Number rod Ap5Y

of total circulating (1 X IO3 bacteria)

Aplysia californica or of sterile seawat.er

/ ’

0

-\

2O’C

FIG.

11. Number of total circulating sp., strain FR (1 X log bacteria)

hemocytes (control).

4

following injection Animals were kept

of the gram at 12 or 18°C.

,,,,;fmcL~>. . =+ _I

6

i

i

24

8 TIME

nw1zos

HOURS

(CONTROL)

-,,,, /--=-r=

2

IN

Aptysia californica or of sterile seawater

,N

.

48

HOURS

hemocytes (control).

following injection of PseudoAnimals were kept at 14 or 20°C.

BACTERIAL

CLEARANCE

IN

TIME

12. Number of total circulat,ing Aplysia californica aquivivus (2 X 100 bacteria) or of sterile seawater (control). FIG.

TIME

FIG. 13. Number of total circulating Aplysia californica homuri (1 X lo0 bacteria) in animals kept at 12 or 18°C.

in animals injected with S. marcescens(Fig. 14). Control animals injected with sterile seawater did not show any decrease in total hemocyte number (see Figs. 10-12). Even though we lacked animals to run seawater controls with G. homari and S. marcescens, the results are reported here to show that

SEA

!N

233

HARE

HOURS

hemocytes following inject ion of Micrococcus Animals were kept at 14 or 20°C.

IN

HOURS

hemocyt,es following

injection

of Ga$lcya

the hemocyte patterns are the same as injection with the other bacteria. Hemocytes from animals injected with 6’. marcescens contained numerous large red granules in the cytoplasm. Although they were found in all the samples, the granules were especially noticeable 4 hr post-injection and

234

PAULEY,

KRASSNER,

AND

CHAPMAN

with either sterile seawater bacteria. Bactericidal

or heterologous

Action of Hemolymph

A. californica serum had no lytic effect on any of the bacteria tested. S. marcescens,

FIG. 14. Number of total circulating Aplysia caZifornica hemocytes following injection of Serralia marceseens (1 X lo9 bacteria) in animals kept at 12 or 18°C.

‘i 2rr IO’ F

seemed to be most abundant during early 2 ,Oz sample periods (1, 2, and 4 hr). These red granules were never observed in hemocytes 101 from sea hares injected with any of t’he other bacteria, nor were they found in o , 2 4 8 TIME IN HOURS animals injected with seawater. It is assumed FIG. 15. Secondary clearance of chromagenic that t’hey represented phagocytosed S. gram negative rod, Ap5Y, from the hemolymph marcescens. Secondary Bacterial Clearance Bacterial clearance in A. californica may be expedited by a prior inoculation of either live & heat kiiledbacteria 48 hr before the second dose is administered (Fig. 15). Secondary clearance takes place within S hr post-injection (Fig. 15), whereas primary clearance usually takes 24 hr. Accelerated secondary clearance of Ap5Y was detectable as late as 1 month after the primary clearante (t.est performed only once, Fig. 16). Animals held at. 12°C were able t.o clear a secondary dose of Ap5Y as rapidly as sea hares held at 18°C were able to clear a primary injection (Fig. 16). In all the other experiments, animals held at higher temperatures always cleared bacteria more rapidly than did sea hares held at lower temperatures. This accelerated clearance was not associated with any increased agglutinin titers. The specificity of this accelerated cIearance is unknown since we did not have enough animals to make control injections

of Aplysia californica. Secondary inoculat.ion was 48 hr after the primary injection. Control group consisted of previously uninjected animals. The inoculum was 1 X 109 bacteria in all cases. All animals were kept at 18°C.

! 135i , ii lo4 $ ,O,. 5 mlo’” ‘t oi

1

FIG. 16. Secondary clearance of chromogenic gram negative rod, Ap5Y, from the hemolymph of Aplysia californica. Secondary inoculation was 1 month after the primary injection. Experimental animals were kept at 12 or 18°C while control animals (previously uninjected) were kept at 18°C. The inoculum was 1 X log bacteria in all cases.

24

BACTERIAL

CLEARANCE

however, did not grow on plates incubated at 13°C (Table 2), this may be a simple temperature effect as its optimal temperature for growth is 25-30°C (Steinhaus, 1959). No induced bactericidal response was found in serum tested 1 day or 7 days after injection of M. aquivivus (Table 3).

IN

SEA

235

HARE

l

28

I

24

I

20

I

Opsonization Pretreatment of chicken RBC’s with normal or heated A. californica serum enhanced their susceptibility to phagocytosis (Fig. 17). However, the increased phagocytosis was much greater in normal serum. It is apparent from this that there are opsonic factors in the normal serum of A. TABLE EFFECT

OF Aplysia

2

0

californica

Va~ous

HEMOLYMPH

BACTERIAL

Whole hemolymph + bacteria

Serum + bacteria

Test bacteria

Serratia marce8cens Micrococcus aquivivtks Gafflcya homari Pseudomonas sp. Chromagenic gramnegative rod (Ap5Y)

w 5 + + +

._ w w k “2 - _+ + + + + + +

+ + + - -- -

a + = positive bacterial growth no bacterial growth observed.

EFFECT

OF

Micrococcus

Time postimmunization

oh’

Seawater + bacteria

w “g

w 3

+ + + +

+ + + +

+

+ -

present;

-

TABLE 3 Aplysia californica HEMOLYMPH aquivivus, PREVIOUSLY IMMUNIZED WITH M. aquivivuP Serum ii-. aquivivus

Whole hemolymph + M. uquitivus

Seawater iii. aquivivus

1 day

+

+

+

7 days

+

+

+

5+

= positive

bacterial

growth

present.

=

ON

2

I TIME

IN

HOURS

FIG. 17. Phagocytosis of chicken red blood cells (RBC’s) by Aplysia cnlifornica hemocytes following exposure to A. californica normal and heated serum and to balanced salt solution (BSS) control. Percent phagotycosis refers t.o t.he number of hemocytes containing RBC’s/lOO hemocytes counted at random.

californica. In most cases we observed 1 RBC per sea hare hemocyte.

only

DISCUSSION

It is well known that the ambient temperature affects the rate of antibody production in cold-blooded vertebrates (Avtalion, 1969; Tait, 1969). Temperature is also thought to play an important role in invertebrate antibacterial defense mechanisms. Stewart et al. (1969) showed that the mean death time of lobsters (Homarus americanus) infected with G. homari was determined by the ambient temperature even though death always resulted in animals kept. between 3 and 20°C. They reported that no deaths occurred at 1°C although G. homari was still present in the lobsters. McKay and Jenkin (1969) have shown that crayfish (Parachaeraps bicarnatus) immunity to bacteria is related to temperature. In the oyster, Crassostrea virginica, the ambient tempera-

236

PAULEY,

KRASSNER,

ture affects the rate of elimination and the ultimate outcome of bacterial (Feng, 1966) and protozoan (Feng and Stauber, 1968) infections. Our results corroborate these findings; in all cases the ambient temperature influenced the rate of bacterial clearance, but did not influence the final outcome in our animals. This was perhaps due to the small temperature range normally employed in our experiments. In addition, it has been shown that there is a distinct difference in the total number of circulating hemocytes in A. californica held at two different temperatures (Pauley and Krassner, in press). The number of hemocytes available for active phagocytosis may account for the different clearance rates. In one tank, deaths did occur where animals were exposed to 25°C after the cooling system failed. All the A. calijornica in this tank apparently died of a X. marcescens septicemia. This may have been due to t.he temperature moving int.o the optimal growth range (25-30°C) of the bacterium (Steinhaus, 1959). Invertebrate hemolymph bactericidins have been found in insectIs (Briggs, 19.58; Gingrich, 1964; Stephens, 1962), lobsters (Acton et al., 1969; Evans et al., 1968, 1969a), and sipunculids (Evans et al., 196913; Johnson and Chapman, 1970a; Krassner and FIory, 1970). l&Dade and Tripp (1967) reported the presence of lysozyme in the hemolymph of C. virginica which can lyse gram positive bacteria. However, the lack of any bactericidal response has recently been noted in several invertebrates; the oyster, C. virginica (Weinheimer et al., 1969), the snail, Helisoma duryi normale (Cheng, 1969), and t.he eart,hworm, Lumbricus terrestris (Cooper et al., 1969). Since we were unable to demonstrate any nat,ural or induced bactericidal effect in A. califormica, it appears that t’he rapid in vivo elimination of marine bacteria from sea hare hemolymph is not due to a lytic effect. This confirms the recent findings of Johnson and Chapman (1970b).

AND

CHAPMAN

Immunological competence exists in all species of vertebrates studied, including cyclostomes. It is often demonstrated by an accelerated secondary antigen clearance rate which is associated with marked antibody titer elevation. Following injections of phage, increased secondary clearance rates have been observed in the crab, Carcinus maenus (Taylor et al., 1964; Nelstrop et al., 1968) and in the oyster, C. virginica (Acton and Evans, 1968). Humoral antibody was not associated with secondary clearance in either of these invertebrates, and the response is t,hought to be cell mediated. The accelerated secondary clearance of bacteria from A. californica shown in our study points to some type of a primitive anamnestic response, which is probably cell mediated since agglutinin titers were not elevated. One interesting aspect of the memory response in sea hares is that accelerated activity was found as late as 1 month aft.er the primary challenge, whereas many ot,her induced invertebrate responses are more short-lived. Failure of A. califowzica to clear S. mcwcescens in vivo was not due to select#ive phagocytosis by the hemocytes ahhough this process has been observed in the oyster (Bang, 1961) and the sea urchin (Johnson, 1969). Intravenous injections of certain bacteria into vertebrates produce an immediate and profound leucopenia indicative of active phagocytosis (Rogers, 1960). It is, therefore, assumed that S. marcescens is actively taken up by sea hare hemocytes as indicated by the pronounced decrease in circulating hemocytes and by the presence of red granules in the hemocyte cytoplasm following injections of this bacterium. Phagocytosis does not, however, ensure that degradation of t)he bacteria will occur (Rogers, 1960). A situation similar to that of S. marcescens in sea hares has been observed in crabs after injections of G. homari. An immediate depression of circulating hemocytes is accompanied by an active

BACTERIAL

CLEARANCE

phagocytosis of the G. homari; hemocyte counts return to normal within a few hours, but the bacteria are not eliminated (Cornick and Stewart, 1968). Opsonie factors have recently been found in invertebrates: the oyster C. virginica (Tripp and Kent, 1967), the snail Helix aspera (Prowse and Tait, 1969), and the crayfish P. bicarinatus (McKay and Jenkin, 1970). Tripp and Kent (1967) concluded that opsonization in the oyster was due to a natural hemagglutinin present in the hemolymph. Although phagocytosis readily occurs in the absence of opsonins (Wood, 1960), it has been hypothesized that intracellular digestion of ingested bacteria is greatly enhanced by the presence of opsonins (Jenkin, 1963; Miler, 1970). If we assume that the agglutinin and the opsonin found in sea hare serum are the same molecule or group of molecules, this m;ould mean that S. marcescens was not opsonized because it has been shown that it is not agglutinated (Pauley et, al., 1972). S. marcescens would, therefore, be more resistant to intracellular digestion than the four marine bacteria which are readily agglutinated (Pauley et al., 1972). We, therefore, make the assumption Ohat, the natural agglutinin present in the sea hares serves a protective function by acting as an opsonin. The lack of a lytic effect in the serum of A. californica combined with the presence of an opsonin and agglutinin has led us to propose the following mechanism of bacterial clearance in the sea hare. After entering the sea hare, bacteria are quickly agglutinated, as indicated by a dramatic drop in agglutinaphagocytosis ensues, tion titer. Active accompanied by a reduct’ion in the number of circulating hemocytes. Tripp (1956, 1960) observed intracellular digestion of RBC’s and bacteria in C. virginica. Int,racellular digest,ion of the marine bacteria probably occurs in A. californica, and is most likely mediated by lysosomal enzymes similar to those found in other invertebrates (Janoff

IN

SEA

237

HARE

and Hawrylke, 1964; Hearing, 1969). Tripp (1960) has also noted phagocyte migration across epithelial borders leading to elimination of these cells. It is possible that phagocyte migration and elimination take place in the sea hare, which may be how A. californica maintains the status quo when infected with S. marcescens that continue to grow at temperatures of 12 and 18°C. In conclusion, A. californica is capable of rapidly clearing at least 4 marine bacteria from its hemolymph in viva. Clearance is temperature dependent and may be accelerated by previous exposure to the bacterium. During the early rapid clearance period, serum agglutinin titers and the number of circulating hemocytes are depressed, later returning to normal levels as the bacteria are cleared. Although no lytic activity is observable, opsonic factors are present in sea hare hemolymph which are assumed to be the same molecule or group of molecules that cause agglutination of the marine bacteria. ACKNOWLEDGMENTS The authors thank Dr. Gale A. Granger, University of California, Irvine, for his advice and for reviewing the manuscript; we are grateful to Dr. Fernando da Crux, University of California, Irvine, for his help during the early phases of this study. We are indebted to t,he late Director of the Center for Pathobiology, Dr. Edward A. Steinhaus, for his suggestions and encouragement during t.he initial experiments. REFERENCES ACTON, R. ophage uirginica). ACTON, R. E. E. lobster Pathol., AVT,ILION, antibody ory in against

T., AND Eva;us, E. E. 1968. Bactericlearance in the oyster (Crassostrea J. Bacterial., 95, 1260-1266. T., WEINI-IEIMER, P. F., AND EVANS, 1969. A bactericidal system in the Homarus americanus. J. Inuertebr. 13, 463464. R. R. 1969. Temperature effect on production and immunological memcarp (Cyprinta carpio) immunized bovine serum albllmin (BSA). Zrnmunology, 17, 927-931. BANG, F. B. 1961. Reaction to injury in the

238

PAULEY,

oyster (Crassostrea

virginica).

KRASSNER,

Biol.

Bull.,

121,

57-68.

J. D. lepidopterous

1958. Humoral larvae. J. Exp.

BRIGGS,

immunity ZooZ.

138,

in 155-

188. CECIL,

J. T. 1969. Mitosis in cell cultures from cardiac tissue of the surf clam Spisula sotidissima.

J.

Znvertebr.

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