Effect of repeated hemolymph sampling on growth, mortality, hemolymph protein and parasitism of oysters, Crassostrea virginica

Camp. Biorhem. Physiol. Vol. 85A, No. 3, pp. 465470, 1986 Printed in Great Britain

0300-9629186$3.00 + 0.00 Per~amon Journals Ltd

EFFECT OF REPEATED HEMOLYMPH SAMPLING ON GROWTH, MORTALITY, HEMOLYMPH PROTEIN AND PARASITISM OF OYSTERS, CRASSOSTREA VIRGINICA SUSAN

Shellfish

Research

E.

FORD

Laboratory, New Jersey Agricultural Experiment Station, Norris, NJ 08349, USA. Telephone: (609) 785-0074

Rutgers

University,

Port

(Received 12 February 1986)

Abstract-l. A method for repeated sampling of hemolymph from bivalve molluscs was evaluated by bleeding oysters, Crussostrea oirginica, from the adductor muscle through a notch in the shell, at 2-week and l-month intervals over a 4-month period. 2. Hemolymph protein concentrations in oysters bled monthly did not change significantly from initial or final control levels; however, levels in oysters sampled bi-weekly were consistently lower than those of the l-month group during the entire experiment. 3. Increased bleeding frequency had no measurable effect on growth or final mortality, but did increase within-oyster variability in hemolymph protein concentrations and was associated with increased parasitism by Haplosporidium nelsoni (MSX)

INTRODUCTION

affords the possibility of examining changes in the abundance or appearance of parasites or hemocytes as well as alteration of serum or cellular constituents in individual oysters subjected to experimental manipulation. It is critical, however to determine whether a repeated assay of this type would measurably debilitate the animal or in any way cause changes in the parameters being investigated. Reported here are the results of an experiment designed to determine whether repeated sampling of blood from individual oysters, Crassostrea virginica, in sufficient quantities for biochemical or histological analyses, would affect growth, survival, total hemolymph protein (THP) concentration, or infection levels after exposure to Haplosporidium nelsoni (MSX) (Haskin et al., 1966; Levine et al., 1980) a parasite that causes serious mortalities of C. virginica in Delaware and Chesapeake Bays (Ford and Haskin, 1982; Andrews, 1984). The experiment was also intended to document the extent of individual variability, over time, in THP and to determine whether changes in THP concentrations were correlated with fluctuations in ambient food conditions.

Biochemical assays of bivalve molluscs with tightly opposed valves usually require sacrificing animals because the shell must be removed to reach the soft tissues. This prevents repeated sampling of the same individuals during the course of an experiment. The ability to sample individuals more than once, however, would increase experimental and statistical flexibility and could reduce the number of animals needed for an assay. It would also provide a measure of temporal variation within the same individual. Removal of part of one valve provides access to soft tissues of living bivalves (Feng, 1965), but obviously compromises the experimental animal and prevents its being returned to the field between observations. On the other hand, small pieces of shell taken from the growing edge are quickly repaired without seriously diminishing the shell’s protective function (Loosanoff and Nomejko, 1955; personal observation). Hemolymph can then be drawn from the adductor muscle sinus (Feng et al., 1971) through the notch without sacrificing the mollusc. Hemolymph can be withdrawn from some bivalves such as the soft shell clam Mya arenariu without shell notching, since the valves do not shut tightly (Farley et al., submitted). Repeated bleeding to monitor the “internal” status of individual bivalves during the course of an experiment is an attractive bioassay because changes in hemolymph or hemocyte composition may be an indicator of physiological condition (Feng, 1965; Bayne, 1973; Riley, 1976; Allen, 1977; Thompson, 1977; Douglass, 1977), especially of disease (Mackin, 195 1; Cheng, 1969; Feng and Canzonier, 1970; Feng et al., 1970; Douglass, 1977; Cooper et al., 1982a,b; Mulvey and Feng, 1982; Ford, 1986). In the case of parasitic diseases, for instance, blood diagnosis

MATERIALS

AND METHODS

uirginica) were randomly H. nelsoni- mortality resistant strain (Haskin -and Ford, 1979) that had been maintained in the harbor at Cape May. N.J.. U.S.A. from setting in 1980 until the experiment began in June, 1983. Seventy oysters were cleaned of fouling organisms, numbered and weighed. The shell margins of 40 individuals were notched with a cutting disk adjacent to the adductor muscle to provide an opening into the mantle cavity just large enough for a 23-gauge needle. The notch was repaired in all oysters within a day or two and with almost no disturbance Uninfected oysters (Crassosfrea selected from a hatchery-reared,

465

466

SUSAN E. FORD

to the inner shell surface, even after repeated notching at the same site. During the experiment, oysters were maintained in a tray on the intertidal flats of Delaware Bay (Cape Shore) near Cape May, where they were examined ar least weekiy. Infection pressure by H. nelsoni is usually heavy at this location beginning in June of each year (Haskin and Ford, 1979); the use of resistant oysters was intended to minimize parasite-caused deaths in experimental animals. Twenty of the notched oysters were bled monthly (Group lM), on 5 July, I and 30 August. The second 20 were bled at approx. 2-week intervals (Group 2W) at six dates between 16 June and 30 August (Fig. IA). At each sampling date beginning on 16 June, all oysters were removed from the tray at early low tide around mid-day and placed in a running sea-water tank in the laboratory. Within 2-3 hr, each oyster scheduled for sampling was removed from the tank and bled immediately. These procedures were taken to minimize possible effects on hemolymph protein of tidal variation such as that documented for feeding and digestion (Morton, 1970 and 1971), of circadian cycles (Boucard er ul., 1985), or of prolonged shell closure. The volume of blood

collected was adjusted

for oyster size: 0.2-0.3 ml from the smallest oysters (25.--35 g total animal wt) to 0.40.5 ml from the largest (8&l IS g). Thirty control oysters were handled in the same manner but were neither notched nor bled until mid-October when surviving oysters in all three groups were bled and fixed for histological examination. All oysters were weighed monthly and at the final sample date in midOctober. The total volume of hemolymph that can be collected from the adductor muscle of oysters weighing l&12Og (total animal wt) ranges between 0.5 and 5.5ml (Ford, unpublished). In general, only large oysters provide large volumes, while small volumes are collected from all sizes. Thus, it is difficult to estimate the fraction of total blood volume represented by the 0.24.5 ml collected in the present experiment; however, in most oysters it was probably between 15 and 2504. Hemolymph was centrifuged to remove cells and total protein (THP) was determined, on the day of collection, according to the method of Bradford (1976) using gamma globulin as a reference (Bio-Rad). Salinity, temperature, and chlorophyil-a concentrations near the holding trays were determined weekly or bi-weekly during the experiment (W. J. Canzonier, pers. comm.). Histological preparation and examination for H. nelsoni followed Ford and Haskin (1982).Non-predation mortalities were calculated for each sampling interval and cumulated over the course of the experiment (Haskin and Ford, 1979). Relative growth constants for all oysters still alive at the end of the experiment were computed according to Wilbur and Owen (1963). Differences between THP means were tested with oneway analysis of variance (ANOVA), with log transformation for unequal variances when necessary; withinand between-oyster variation was compared using two-way ANOVA without replication and differences among proportions were tested with contigency table analysis (Sokal and Rohlf, 1981). Variation is reported as & one standard deviation (SD) from the mean.

Total he~~l~~ph protein (THP) The initial hemolymph sample, collected 16 June. contained an average of 9.8 ( + 3.7 SD) mg/ml of gamma globulin equivalent-protein. Mean THP concentration in oysters bled monthly (Group IM) ded

,

I

I

i

I D

a

ChloroOhyll

JUW

July

August

A

Septenbw

Cktobel

Fig. 1. (A) Growth, (B) nonpredation mortality, and (C) hemoiymph protein (THP) concentration in experimental oysters and (D) chlorophyll-~ levels in ambient water. Hemolymph protein is expressed as gamma globulin equivalents. Cumulative nonpredation mortality excludes oysters killed by the oyster drill. Total weight includes the shell. Vertical lines are 95% confidence intervals. Only Group 2W (twice monthly bleeding) oysters were bled on 19 June, but are considered representative of Group IM (monthly bleeding) at the same date.

creased only slightly from this level during the remainder of the ex~~ment (Fig. 1A). Oysters bled every 2 weeks (Group 2W), however, showed a gradual loss of THP until early August when concentrations reached a mean of 5.0 ( + 2.6) mg/ml, approx. half the initial level. From then until the end of August, THP levels in Group 2W were significantly lower (P < 0.05) than those in Group IM (Fig. 1A). By mid-October, when neither of the experimental groups had been bled for 6 weeks, THP in group IM (8.6 2 2.1 mg/ml) was virtually identical with that in the control group, bled for the first time, while THP in Group 2W (8.8 + 2.0 mg/ml), (6.9 + 2.2 mg/ml) was significantly less than controls (P < 0.05). Mean THP concentration was consistently higher (P < 0.05, Wilcoxon Signed-Ranks Test for paired observations in Group 2W) in oysters that survived the entire period than in those that died during the experiment, including those that were killed by the oyster drill Urosalpinx cinerea (Table 1). There was no significant correlation (Groups IM and 2W com-

Effect of repeated bleeding on oysters Table

I, Comparison of total

Date

hemolyph

protein

in oysters that survived did not

Bi-Weeklv SurviYors n g sd

Bleeding Non-Survivors’ i sd n

June

19

12

lo.0

2.9

8

9.4

4.9

July

5

12

8.6

3.7

8

7.8

3.1

July

16

12

7.5

2.6

6

7.7

2.8

Aug.

1

12

5.2

2.5

6

4.7

3.0

Aug.

16

12

5.8

2.7

6

4.8

3.0

Aug.

30

12

6.4

1.4

4

3.9

bined) between protein concentration and size of oysters in the initial samples (N = 40, r = 0.02), or between mean protein and growth constants in individuals that survived until the end of the experiment (N = 46; r = 0.23). Variab~Iit~ in THP measurements

Within-oyster (i.e. sample-to-sample) variation in THP was compared with between-oyster variation for all experimental animals alive on October 17. Between-oyster variation at each sampling date was significant (P < 0.01) in both groups (Group 1M: df= 12, F = 5.232; Group 2W: df = 13, F = 4.973); within-oyster variation was significant in Group 2W (df = 6; F = 7.159), but not in Group 1M (df = 3; F = 0.819). Within-oyster variability in Group 2W was also significant (df = 3; F = 3.268; P < 0.05) when the analysis was restricted to dates when Group IM was also sampled, showing that increased variance was associated with the repeated bleeding method itself and was not due to the fact that a 2-week examination schedule detected fluctuations overlooked by a l-month schedule. Growth and mortality Two

of the 20 Group 2W oysters died after the

Table 2. Relationship

the experiment

Monthlv Survivors n I sd

2.8

‘Oysters that were alive at the time of sampling on I7 October

467 with those that

BleedinK Non-Survivors~ n 2 sd

14

10.6

3.9

6

7.2

2.6

14

9.2

3.5

6

7.7

4.5

14

9.6

4.0

5

8.1

3.4

but did not survive

until the end of the experiment

second bleeding, but other than these, no deaths occurred in any group until late August. At the conclusion of the experiment, non-predation losses of 15-20%, were essentially the same for all groups (Fig. IB). Oysters killed by U. cinerea were excluded from this calculation; however, no significant differences were found among treatments when these individuals were included in the analysis (df= 4; G = 1.442). Growth was also similar in all treatments (Fig. 1C). The apparent large weight gain in all groups toward the end of the experiment resulted from the deaths of slow-growing oysters rather than to real growth during that interval. Parasitism

At the end of the experiment in October, H. nelsoni was diagnosed in 5 (25%) of the 20 surviving control oysters, 5 (36%) of 14 Group IM oysters, and 6 (50%) of 12 Group 2W oysters. Although parasitism appeared to increase with the frequency of bleeding, there were no significant differences among groups (df= 2; G = 2.062; P > 0.05). All infections were localized in the gill and not considered lethal (Ford and Haskin, 1982); but because no tissues from moribund or dead oysters were recovered for diagno-

between total hemolymph parasitism, and repeated

protein (THP) levels, H. nelsoni bleeding

THP Conoentratioo’ N Groups

July

5

October

f7

1.8b

l&2

H. nelsoni --

+’

11

9.8

+ 2.ga

6.5

f

&. nelsoni

-

15

9.5

f 4*ga

8.6

f 2.5a

Control j!.

nelsoni

+

5

not

sampled

7.9

f 2.1ajb

a.

nelsoni

-

15

not

sampled

8.4

f 3.0a

1SD. THP levels with different letter superscripts are statistically different at the P = 0.05 confidence level. ‘Parasite diagnosis in October sample. ‘Mean +

SUSAN

468

sis during the experiment, it is uncertain whether the parasite contributed to the observed mortality. Further analysis of the interrelationships between bleeding, H. nelsoni parasitism, and THP concentrations was made by comparing THP levels in early July and in mid-October in oysters diagnosed, in the final sample, as H. nelsoni-positive or -negative (Table 2). Data from Groups 1M and 2W were pooled because there were no significant differences in THP concentrations between them at these dates (Fig. IA). In the 5 July sample (before patent H. nelsoni would be expected) there was no difference between individuals that eventually did or did not become patently infected. By the time patent H. nelsoni were found in mid-October, however, oysters that had been repeatedly bled and were infected had significantly lower THP concentrations (6.5 + 1.8 SD mg/ml) compared to unifected oysters (8.6 k 2.5 mg/ml) and also to all individuals in the 5 July sample (9.8 + 2.9 mg/ml). In contrast, protein levels in control oysters, bled for the first time in October when the parasite diagnosis was made, were statistically the same in infected and uninfected individuals (Table 2). Hydrography

and nutrients

The temperature was 22°C in mid-June, rose to about 28°C at the end of July, and then decreased to 18°C by mid-October. Salinity increased gradually from about 18 ppt (parts per thousand) at the beginning of the experiment to 24ppt at the end. Chlorophyll-a concentrations were generally above 30mg/m3 for the first month of the experiment and reached a peak of 107 mg/m3 on 7 July (Fig ID). All readings after mid-July were below 20 mg/m’ except for a brief period in late August. DISCUSSION

Repeated sampling of hemolymph from individual oysters at 2-week intervals significantly reduced total hemolymph protein concentrations and increased individual variability compared to sampling at l-month intervals. Neither treatment affected growth or final mortality. There was an apparent trend towards higher prevalence of the parasite Haplosporidium nelsoni with increased bleeding frequency, but differences among treatments were not significantly different. One explanation for the reduction of THP concentration in oysters bled twice a month is that the volume of fluid removed at each bleeding was not replaced and was accompanied by a continuous loss of serum protein. A much more likely alternative, since oysters are osmoconformers (Galtsoff, 1964; Lynch and Wood, 1966), is that an influx of sea-water restored the original hemolymph volume and diluted the remaining protein. This explanation is supported by the fact that there was no increase in the difficulty of obtaining blood from the same individuals during the experiment. Also, the average decrease in THP concentration between successive Group 2W samples from 19 June to 1 August was 19% (range 8-34%). This was in the same range as the estimated proportion of blood volume collected (15-25%) and would indicate little net biosynthesis during this period.

E.

FORD

Dilution of hemolymph by reduced ambient salinity was suggested by the work of Swift and Ahmed (1983) who found protein concentrations reduced by up to 50% in the hemolymph of oysters held, unfed, for 3 weeks in sea-water at 12 and 18 ppt compared to those at 24ppt. The reduction in THP concentrations in oysters bled twice monthly in the present study compared to the relative stability in those bled once a month suggests that the rate of synthesis of serum proteins was rapid enough to restore protein concentrations within a month, but not within 2 weeks. In this context, however, it is interesting that no change in THP was found between the initial Group 2W sample taken at the time oysters were moved from water at 32 ppt in Cape May Harbor and the initial Group 1M sample taken when oysters had been in water at 18-19 ppt at the Cape Shore for 2 weeks. If dilution of serum protein occurred in this instance, the initial concentration was restored within 2 weeks. These oysters were feeding on natural phytoplankton, in contrast to those used by Swift and Ahmed (1983) suggesting that lack of food may inhibit synthesis of hemolymph proteins. Feng (1965) reported that hemocyte counts were unaffected in oysters repeatedly bled from the heart. Although he removed much smaller quantities (0.05 ml) than in the present study, he did so at more frequent intervals; in one experiment, a cumulative volume of 0.35 ml was taken from oysters 8815 cm in height during a 4-hr period. Becker (1972) measured glucose in blood sampled from freshwater snails, Biomphalaria glabrata, at 20-min intervals and found levels ranging from 0.1 to 1.7 mg/ml in the same individual, with no trend toward higher or lower values with repeated bleeding. The relatively short periods between bleedings in both of these studies probably did not allow either dilution or possible effects on biosynthesis to have measurable effects. Great variation exists in blood metabolite concentration among individual molluscs subjected to the same experimental conditions (Cheng, 1969; Douglass, 1977; Swift and Ahmed, 1983; Ford, 1986) as well as among those collected in nature (Bayne, 1973; Thompson, 1977). Results of repeated sampling in the present study suggest that some of this variability results from temporal fluctuations within the same individual. Both between- and within-oyster variation support earlier conclusions (Thompson, 1977; Ford, 1986) that blood components are not well regulated and that molluscs can tolerate large fluctuations without apparent harm. Nevertheless, the consistently lower THP levels in oysters that died during the experiment compared to those that survived indicates that a minimum level of THP is associated with “good health” and that some regulatory mechanism may exist that directly or indirectly maintains it. A regulatory mechanism may explain the apparent “stabilization” of THP levels in Group 2W oysters after 1 August. Chlorophyll-a levels did not indicate an increase in food, and, in fact, there was no clear evidence that THP concentrations were related to fluctuations in chlorophyll-a. Rather, net synthesis must have increased at this time to restore protein (if dilution was occurring) or to prevent continued loss of protein (if dilution was not occurring). The raw

Effect of repeated bleeding on oysters

materials could have come from increased consumption or assimilation of available food, diversion or retention of assimilated substrates in the hemolymph, or catabolism of stored protein (Bayne, 1973). Two results of the study suggested an association between repeated bleeding and the development of H. nelsoni parasitism. First, H. nelsoni prevalence appeared to increase with sampling frequency, although numbers were small and differences not statistically significant. Second, the oysters that had been bled repeatedly and were diagnosed as H. ne~so~i-positive had significantly lower THP concentrations than did oysters diagnosed as H. nelsoni-negative. This result is significant because all H. nelsoni infections were localized in the gill and previous studies (Douglass, 1977; Ford, 1986) demonstrated that THP is reduced only in systemically infected oysters. In fact, no effect of localized H. nelsoni infections was found in control oysters, which had not been bled before diagnosis was made. Although these results are by no means conclusive, the possibility cannot be discounted that repeated bleeding led to increased susceptibility to infection. Frequent bleeding, for instance, might have reduced numbers of hemocytes engaged in defense activities, lowered concentrations of a “protective molecule”, or simply presented a mechanical trauma that lowered defense capabilities. Neither humoral nor cellular defense mechanisms have been clearly identified in oysters infected with H. nelsoni (Douglass, 1977; Ford, 1986); however, some evidence exists that “stressors” such as siltation and infestation by the mud worm Polydoru are associated with elevated H. nelsoni infections (unpublished data, this laboratory). The trauma hypothesis is strengthened by the finding that within-oyster variation was magnified by high-frequency bleeding and, by implication, that the process was an adversity that exaggerated the oysters’ inability to regulate hemolymph constitutents. The results of the experiment presented here indicate that repeated bleeding of oysters, and probably of other molluscs, may cause sublethal changes in experimental animals, even though it may not increase mortality (Cooper et al., 1982~). If this technique is to be used, the frequency of bleeding or perhaps the volume of blood collected, should be carefully adjusted to minimize damage to the experimental animals, particularly if they are held in the laboratory without food or with only minimum rations, With the appropriate controls, however, repeated hemolymph sampling should be a valuable approach to experimental studies of bivalve molhtscs. Acknowle~gentents-The

author

thanks

H. Haskin

for

assistance in bleeding oysters; C. Rizzo for histological examinations; and B. Barber, W. Canzonier, A. Farley, A. F&eras.

L. Fritz, H. Haskin, and S. Kanaley for helpful

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