Effects of the parasite MSX (Haplosporidium nelsoni) on oyster (Crassostrea virginica) energy metabolism—II. Tissue biochemical composition

0300-9629/88$3.00+ 0.00 0 1988PergamonPress plc

Camp. &&em. Physiol. Vol. 91A. No. 3, pp. 603-608, 1988 Printed in Great Britain

EFFECTS

OF THE PARASITE MSX (HAPLOSPORIDIUA4 NELSONI) ON OYSTER (CRASSOSTREA VIRGINICA) ENERGY METABOLISM-II. TISSUE BIOCHEMICAL COMPOSITION

BRUCE J. BARBER*, SUSAN E. FORD and HAROLD H. HASKIN Department of Oyster Culture, Cook College, Rutgers University, Shellfish Research Laboratory, PO Box 687, Port Norris, NJ 08349, USA. Telephone: (609) 785-0074 (Received 6 April 1988) Tissue biochemical composition of oysters (Crussostrea uirginicu) from Delaware Bay (USA) was examined from May to November 1985 as a function of infection intensity by the endoparasite, Abstract-l.

Haplosporidium nelsoni (MSX). 2. The lipid, glycogen, protein

and ash content (mg DW) of a standard 100 mm (shell height) oyster was determined for uninfected, epithelial and systemic infection categories. 3. All biochemical components generally decreased in content with increasing MSX infection intensity (and duration). Overall reductions were significant in glycogen [epithelial (P < 0.05) and systemic (P < O.OOl)],protein [systemic (P < 0.05)] and ash [systemic (P < 0.02)] categories. 4. Glycogen was the substrate most readily catabolized to meet the energetic burden posed by MSX. The reduction by MSX of nutrient reserves fitness of surviving oysters is reduced.

affects

INTRODUCIION In all host-parasite relationships, the survival of the parasite depends upon competing successfully with the host for available nutrients. In response, the host makes physiological adjustments or is metabolically altered. In marine bivalves, parasites affect a variety of physiological functions including reduced feeding capability and altered digestion and assimilation efficiencies (Thompson, 1983; Newell and Barber, 1988). In turn, energy metabolism, or the manner in which nutrient reserves are utilized by the host, is altered such that both somatic and germinal production are affected. The end result is that the ecological fitness of the host is compromized (Newell and Barber, 1988). Unknown prior to 1957, the protozoan parasite Haplosporidium nelsoni (MSX) killed 90-95% of the oysters in lower Delaware Bay between 1957 and 1959 and remains enzootic in Delaware and Chesapeake Bays (Haskin et al., 1966; Andrews and Wood, 1967). The history and epizootiology of MSX is reviewed by Andrews (1966) and Ford and Haskin (1982). A typical pattern of infection leading to oyster mortalities has been described by Ford and Haskin (1982) and Ford (1985) as follows. New infections are acquired from June through to October each year. Initially, infections are confined to gill epithelium, suggesting that the infective particles are waterborne. Plasmodia divide and proliferate, eventually breaking through basement membranes. At this point, infections rapidly become systemic as parasites are spread via the circulatory system. Ensuing mortalities occur in late summer and early autumn as infections intensify. Infection levels are usually high over the _ *Present address: Virginia Institute of Marine Gloucester Point, VA 23062, USA.

Science,

other

metabolic

functions

such that the ecological

winter, but mortalities lessen, most likely because of reduced metabolic activity of both host and parasite. Renewed parasite proliferation and oyster mortality accompany rising water temperatures the following spring. The high prevalence levels often recorded in late spring are from infections acquired the previous year. In spite of the obvious ecological impact of MSX on oyster populations and the resultant economic impact on the oyster fishery, relatively little is known about the sublethal effects of MSX on oyster energy metabolism. Newell (1985) found that oysters infected with MSX have significantly reduced clearance rates compared to uninfected oysters. The significantly lower condition index exhibited by infected oysters was attributed to the decline in energy intake associated with reduced clearance (Newell, 1985). Condition index is also related to MSX infection intensity; oysters with systemic infections have a significantly reduced condition index (Barber et al., 1988). Based on a decrease of digestive enzymes (diagnosed histochemically), Eble (1966) suggested that oyster digestion is disrupted in the presence of MSX. These alterations in oyster energy metabolism caused by MSX are reflected in hemolymph constituents. Feng et al. (1970) found levels of total free amino acids to be consistently lower in infected oysters. Ford (1986) found that total serum protein concentration fell sharply and in approximate proportion to MSX infection intensity in oysters with systemic infections. Possibly as an indirect result of the disruption of oyster energy metabolism, infection by MSX significantly reduces fecundity in systemically infected individuals compared to uninfected individuals (Barber et al., 1988). Ford and Figueras (1988) found that infection by MSX delays the gametogenic cycle of oysters. Thus, besides causing 603

BRUCEJ. BARBER ef al.

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mortality, MSX also affects oyster ecological fitness at sublethal levels. This paper is the second in a series intended to systematically define the subethal effects of MSX on oyster energy metabolism. The present study focuses on the effects of MSX on tissue biochemical composition, or the ability of oysters to store nutrients necessary for maintenance, growth, and reproduction. MATERIALSAND METHODS Oysters used for this study were from the 1980 and 1981 year- class produced as part of an ongoing experimental breedina nroaram at Rutgers University (Haskin and Ford, 1979; F&d and Haskin, 1987), and were maintained in trays intertidally at our Cape Shore facility (lower Delaware Bay). Thirty individuals were examined six times between May and November, 1985 for tissue biochemical composition (total lipid, glycogen, protein, and ash) and degree of infection by MSX. Oysters were cleaned of fouling organisms, measured (shell height), and shucked. A standard transverse (dorsal) section of each oyster across gill, stomach, intestine, and digestive diverticula was removed and weighed (wet) prior to fixation for histological processing. The remaining tissue was weighed wet and then dried (lyophilization) so that a dry wt/wet wt ratio could be obtained and used to calculate the dry weight of the entire animal. After weighing, the lyophilized tissue was ground into a powder with a mortar and pestle, and stored in glass vials prior to biochemical analysis. No attempt to separate MSX from oyster tissue was made. Our observations have been that even in heavily infected individuals MSX comprises only a small proportion of total biomass. Total lipid was determined using the gravimetric method of Barnes and Blackstock (1973). A weighed portion of dried, ground tissue (50-75 mg) was mixed with 10 ml of a 2: 1 (v/v) chloroform-methanol mixture in a centrifuge tube using a Virtis homogenizer. Aqueous sodium chloride solution (2 ml, 0.9%) was then added with further mixing and the whole solution allowed to separate overnight in a refrigerator. The lower phase (containing the lipid) was removed with a Pasteur pipet and placed in a tared glass vial. The solvent was allowed to evaporate under a fume hood and the lipid residue was weighed. The upper phase (containing the lipid-extracted tissue) was filtered through a Whatman No. 1 filter and the filter rinsed with 70% ethanol, allowed to dry and stored in the freezer. Glycogen was determined using the phenol-sulphuric acid method of Dubois et al. (1956). A weighed portion (4-5 mg) of the lipid-extracted tissue was placed in a test tube with 3ml distilled water and 5ml concentrated sulphuric acid. The test tubes were covered and left at room temperature overnight to permit complete dissolution of tissue. An aliquot of each was then transferred to another test tube and the volume adjusted to 2 ml with distilled water. To this, 1 ml 10% phenol and 5 ml concentrated

sulphuric acid was added. The solution was mixed and allowed to cool for 30 min while the color developed. Absorbance was read at 490 nm and compared to a standard curve prepared with oyster glycogen. Protein was determined using the Folin phenol method of Lowry et al. (1951). A weighed portion-of dried, ground tissue (5-10 ma) was placed in a test tube with 10 ml of 0.1 N NaOH. Tube contends were covered, mixed, and allowed to stand overnight at room temperature. An aliquot of this (0.25-0.5 ml) was transferred to another test tube along with 5 ml Reagent C. Tube contents were mixed and allowed to stand for 30 min at room temperature. Then 0.5 ml Reagent E was added with immediate mixing. After color had developed for 2 hr, absorbance was read at 750nm and compared to a standard curve prepared with bovine serum albumin, Ash was determined by placing 4&50 mg of dried, ground tissue into a tared crucible and combusting overnight at 450°C. Histological procedures used for the diagnosis of MSX are identical to those of Barber et al. (1988). Tissue sections were fixed in Davidson’s solution, dehydrated, cleared and embedded in paraplast. Six pm sections were stained with iron hematoxylin, acid fuchsin, and aniline blue. Each oyster was then categorized with respect to MSX infection intensity as either uninfected, epithelially infected (gills only) or systemically infected (subepithelial, general). Lipid, glycogen, protein and ash levels (% DW) were multiplied by the dry tissue weight of each oyster to obtain the content of each constituent. Since biochemical content is size dependent, regression analysis was used to calculate the biochemical content of a standard oyster of 1OOmm shell height for each sampling date within each infection category. In the September sample when there was just one uninfected oyster (llomm), its value was used without adjustment. Two-factor analysis of variance (Zar, 1974) was used to examine biochemical content with respect to infection intensity and sampling date. To determine overall effects of infection intensity on biochemical content, the means of differences (all sampling dates) between epithelially infected and uninfected oysters and between systemically infected and uninfected oysters were examined for each constituent using a paired t-test (Zar, 1974).

RESULTS Mean shell height ranged from 93 to 107 mm, with little growth occurring over the study period (Table 1). The distributions of oysters within the respective infection categories and cumulative oyster mortality reflected a typical progression of MSX infection and resultant mortality in 1985 (Table 1) which was described by Barber et al. (1988) Mean tissue dry weight generally increased between May and November in all infection categories (Table 2), contributing to a similar increase in condition index, as reported by Barber et al. (1988). Mean

Table I. Mean shell height (mm), number of oysters in each category of MSX infection intensity (uninfected, epithelially infected and systemically infected), and cumulative mortality (%) on each

sampling date Infection category Date 21 May 25 June 22 July 22 August 19 September 6 November

Shell height 93

SD (mm) 12

Uninf. 21

Epith. 3

Syst. 5

Cumulative mortality (%) 2.9

91 100 102 107 101

16 11 12 14 11

18 8 3 1 13

4 I5 16 9 7

7 I II 20 10

14.5 23.3 32.0 40.4 50.0

Effects of MSX on oyster biochemical composition

605

Table 2. Mean tissue dry weight (g) and lipid, glycogen, protein and ash levels (% DW) of oysters in each category of MSX infection intensity (uninfected, epithelially infected and systemically infected) on each sampling date

21 May Uninf. Epith. syst. 25 June Uninf. Epith. syst. 22 July Uninf. Epith. syst. 22 August Uninf. Epith. Syst. I9 September Uninf. Epith. Syst. 6 November Uninf. Epith. Syst.

Dry wt

SD (g)

% Lipid

I .86 1.27 0.80

0.88 0.42 0.35

12.3 11.7 Il.7

I .82 1.78 I .08

0.51 0.67 0.29

2.70 2.37 I .67

% Glycogen

% Protein

% Ash

18.6 20.2 13.4

41.5 40.5 43.3

10.5 9.9 13.8

10.6 12.2 II.5

27.1 29. I 21.7

46.3 45.8 50.9

8.5 9.1 10.6

0.62 0.97 0.73

13.8 13.7 12.7

38.2 35.6 25.3

34.7 38.2 42.3

6.6 7.1 8.2

3.58 2.30 2.28

0.78 0.93 I .40

14.6 14.3 13.6

32.2 26.8 20.9

37.5 36.4 41.9

6.0 6.7 7.3

2.90 2.54 2.57

I .52 1.52

10.0 12.5 13.6

48.1 43.0 32.1

27.6 36. I 41.7

5.1 6.4 6.7

4.06 3.55 2.50

1.09 2.14 0.87

14.7 13.7 15.2

24.9 20.2 18.9

33.6 37.3 39. I

6.4 6.9 7.6

dry weight was reduced in oysters infected with MSX. In all but the September sample, mean dry weight was inversely correlated with infection intensity (uninfected DW > epithelial DW > systemic DW). Lipid, glycogen, protein and ash levels (% DW) are provided in Table 2 for relative comparisons. Lipid level ranged from 10.0 to 15.2%, glycogen from 13.4 to 48.1%, protein from 27.6 to 50.9% and ash from 5.1 to 13.8%. Comparisons of biochemical component levels between sampling date and infection category were not considered, as a change in one component based on a percentage might change one or more other components. Lipid content (mg lipid contained in a standard 100 mm oyster, Fig. 1) varied significantly as a function of sampling date but not infection category for any one date (P < 0.025, analysis of variance). There was no obvious seasonal pattern in lipid content, although in the two infected categories, there was an increase between May and November. Values were generally between 200-400 mg (in all infection categories) with the exception of the uninfected category

in August (827 mg) and the uninfected (626 mg) and epithelially infected (5 15 mg) categories in November. In May, July, August and November, lipid content decreased with increasing level of infection (uninfected > epithelial infection > systemic infection). However, that relationship did not occur in June and September, as the highest lipid contents in those months were in one of the infected categories. Accordingly the paired t-test indicated that even though lipid content was reduced (overall) by 16% in epithelially infected oysters and 37% in systemically infected oysters, these differences were not statistically significant. Thus MSX infection intensity had no effect on oyster lipid content. Two-factor analysis of variance indicated that glycogen content (Fig. 2) varied significantly as a function of both sampling date and infection category (P c 0.0005). Multiple comparison (Student-Newman-Keuls test) revealed that significant differences in glycogen content occurred between infection categories in all but the May and June samples. A seasonal trend in glycogen content

OlJninfected

OUninfected

Epithelial

Epithelial

lSystemic

lSystemic

1

1

Month-

Month- 1985

1985

Fig. 1, Lipid content (mg DW) of a standard 100mm oyster, Crussosrrea uirginica, uninfected, epithelially infected and sytemically infected by the parasite MSX (Haplosporidium nelsoni), May to November 1985.

Fig. 2. Glycogen oyster, Crussosfrea and systemically losporidium

content (mg DW) of a standard 100 mm oirginica, uninfected, epithelially infected infected by the parasite MSX (Hupnelsoni), May to November 1985.

BRUCE J. BARBERet

al.

DUninfected

OUninfected

Epithelisl n

Epithelial

lsystemic

Month- 1985

-Systemic

1

Month- 1985

Fig. 3. Protein content (mg DW) of a standard 1OOmm oyster, Crassosfrea uirginica,uninfected, epithelially infected and systemically infected by the parasite MSX (Haplosporidiumnelsoni), May to November 1985.

Fig. 4. Ash content (mg DW) of a standard 100 mm oyster, Crussostrea uirginica, uninfected, epithelially infected and systemically infected by the parasite MSX (Haplosporidium nelsoni), May to November 1985.

was evident (all infection categories), with values increasing from about 200mg in May to over

higher values (180-250 mg) occurred in November. There was an inverse relationship between ash content and infection intensity in May, August and November. In June and July ash content was similar in the uninfected and epithelially infected categories but was reduced in the systemically infected category. In September the ash content of all three infection categories was similar. Compared to uninfected oysters, ash content was reduced by 10% (overall) in epithelially infected oysters (not significant) and by 30% in systemically infected oysters (P < 0.02, paired r-test). Thus oysters with systemic MSX infections had a lower ash content.

1000 mg in September and then decreasing in November. There was a distinct relationship between infection category and glycogen content. In all samples uninfected oysters had the highest glycogen content, followed by epithelially infected oysters and then by systemically infected oysters. As indicated by the paired t-test, glycogen content was 22% lower (overall) in epithelially infected oysters than in uninfected oysters (P < 0.05) and 51% lower in systemically infected oysters than in uninfected oysters (P c 0.001). Thus, glycogen content was reduced in oysters in relation to MSX infection intensity. Protein content (Fig. 3) also varied significantly as a function of both sampling date (P < 0.05) and infection category (P < 0.025). Multiple comparison (Student-Newman-Keul’s test) revealed that the protein content of the uninfected category in August was significantly (P < 0.05) greater than both infected categories. With the exception of the August uninfected category (1800 mg) there was little seasonal variation in protein content from May to September (all infection categories), with most values being between 600 and 800mg. Protein content (all infection categories) was slightly higher in November (940-1400). In all but the September sample, protein content was related to infection category, with the uninfected category having the highest protein content, followed by the epithelially infected category and the systemically infected category. Over the entire study period, protein content was 19% lower in oysters with epithelial infections than uninfected oysters (not significant) and 36% lower in oysters with systemic infections than uninfected oysters (P < 0.05, paired t-test). Thus infection by MSX reduced oyster protein content, but only in systemically infected individuals. Two-factor analysis of variance indicated that significant differences in ash content (Fig. 4) were associated with sampling date (P < 0.025) and infection category (P < 0.005). The only within-sample significant difference occurred in May between the uninfected category and both infected categories (Student-Newman-Keul’s test, P < 0.05). Ash content (all infection categories) remained constant (100-200 mg) from May to September. Slightly

DISCUSSION

For marine bivalves, energy taken in that exceeds maintenance metabolism is available for somatic and germinal growth and stored as lipid, carbohydrate and/or protein substrates. These can either be incorporated into cellular structural components or stored as nutrient reserves which are catabolized to meet maintenance energy demands when food is scarce or for the production of gametes. The seasonal acquisition and utilization of nutrient reserves results in an annual “energy storage cycle”, which is integrally related to the gametogenic cycle (Gabbott, 1975, 1983; Bayne, 1976; Sastry, 1979). Parasitism, by competing for available energy, disrupts the normal energy storage cycle of its host either by reducing the amount of energy available for storage (e.g. by inhibiting feeding or digestion) or by utilizing nutrients already stored (Thompson, 1983; Newell and Barber, 1988). Few other studies exist in which the effects of parasitism on bivalve tissue biochemical composition have been examined. Infection of Myths edulis by the copepod Mytilicola intestinalis (Williams, 1969) and the trematode Proctoeces maculatus (Dennis et al., 1974) resulted in little change in tissue biochemical composition under normal environmental conditions. However, both authors concluded that parasitism would have a detrimental affect on host energy metabolism under conditions of environmental stress. Studies conducted on C. uirginica have shown that parasitism has significant effects on tissue biochemical composition. Cheng (1965) histo-

Effects of MSX on oyster

chemically demonstrated a reduction in total fats and Cheng and Burton (1966) found a reduction of glycogen in oysters infected with the trematode Bucephalus sp. Oysters infected with the protozoan Perkinsus marinus exhibit a decrease in total free amino acids (Soniat and Koenig, 1982). The manner in which MSX delibitates its oyster host, eventually leading to death has, for the most part, been speculative. Gross observations, including a loss of condition and pale digestive gland (Farley, 1968) and a cessation of growth (Andrews, 1961), indicated that MSX poses an energetic burden to the oyster, gradually out-competing the oyster for available nutrients. Recently, more specific information on the bioenergetics of the MSX/oyster relationship has become available. Newell (1985) has shown that clearance rate is significantly reduced in heavily infected oysters. Obviously a reduction in food intake will reduce available energy. This would reduce the amount of energy available for growth and energy storage. This is reflected by the fact that both Newell (1985) and Barber et al. (1988) found that oysters infected with MSX had significantly lower condition indexes. The present study takes these results one step further by defining the manner in which tissue biochemical composition (storage of specific energy substrates) is affected by MSX infection. Oyster biochemical composition has been well defined (see Galtsoff, 1964 and Walne, 1970, for reviews). The primary energy storage substrate is glycogen, and its content in oysters is closely linked to the annual reproductive cycle. Glycogen accumulates in the connective tissue of the mantle and labial palps after spawning, and generally remains high until the onset of reproduction, when it is catabolized to fuel gametogenesis. Lipid and protein, although important in other species, have not been found to play a major role in normal oyster energy metabolism. Ash content is primarily indicative of the salinity of the water from which the oyster came. In this study there was very little in the way of seasonal fluctuations in lipid, protein, and ash contents of uninfected oysters. Only the glycogen content varied seasonally, increasing steadily after June, when spawning began (Barber et al., 1988), and continuing to October. Glycogen content was somewhat lower in November, possibly as the result of lower water temperatures and a lower feeding rate. Normal patterns of energy substrate storage are altered in oysters infected with MSX, although it remains unclear whether depletion of a particular reserve is due to active uptake by the parasite, an increase in oyster metabolism due to the fact it is parasitized, a decrease in incoming energy, or some combination of these. Lipid content was reduced in both epithelial and systemic infections but not significantly, verifying the relative unimportance of lipid as an energy substrate in oysters. The fact that glycogen content was (significantly) reduced in oysters with both epithelial and systemic infections indicates the importance of glycogen in oyster energy metabolism. Besides being plentiful, it is readily catabolized, being the only biochemical constituent significantly affected in oysters with lighter epithelial infections. In general, protein in bivalves is catabolized only after other stored substrates (glycogen and

biochemical

composition

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lipid) have been depleted. A significant reduction in oyster protein content was found only in oysters with systemic MSX infections, verifying that its utilization is associated with severe stress. The significant reduction in ash content in systemically infected oysters was most likely to be the result of total dry weight (used in the calculation of content) being lowest in systemically infected oysters even though ash level (%) was greatest in systemically infected oysters on all sampling dates (Table 2). A reduction in the amount of substrate available for catabolism in tissues of C. virginica has been shown to be reflected in other physiological processes. The first of these is a reduction in the level of biomolecules circulating in the hemolymph. Feng et al. (1970) have found that total free amino acids were consistently lower in C. virginica infected with Bucephalus sp. and MSX than in uninfected oysters. Similarly, total serum protein was found to be lower in oysters with systemic MSX infections (Ford, 1986). The second of these is a reduction in fecundity, which is directly dependent on the amount of stored nutrients (Loosanoff, 1965; Bayne, 1976). Barber et al. (1988) have found that relative fecundity is significantly lower in oysters with systemic MSX infections. It is now more evident that H. nelsoni, by presenting an energetic burden, reduces the ecological fitness of C. virginica. This appears to result from a reduction in the amount of energy assimilated and eventually stored and/or an increased demand on nutrients already stored. Resistance to MSX mortality involves the ability to tolerate parasitism (Haskin and Ford, 1979; Ford and Haskin, 1987). This may reflect metabolic adjustments such that disruptions of energy metabolism are minimized. Research is currently being focused in this direction. Acknowledgements-We thank D. O’Connor and L. Ragone for the histological workup. This is NJAES publication D-32504-3-88, supported by state funds and by the New Jersey Department of Environment Protection (Bureau of Shellfisheries) and the New Jersey Commission on Science and Technology (Fisheries and Aquaculture Technology Extension Center). REFERENCES

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