EXPERIMENTAL PARASITOLOGY 41, 198-205 (1977)

Probopyrus

pandalicola: of Shrimp

Discontinuous Hemolymph

Ingestion

STEPHEN P. WALKER Department of Zoology, North Carolina State Uniuersity, Raleigh, North Carolina 27607, U.S.A. (Accepted for publication 1 April 1976) WALKER, S. P. 1977. Probopyrus panckdicola: Discontinuous ingestion of shrimp hemolymph. Experimental Parasitology 41, 196-205. Grass shrimp, Palaemonetes pugio, were injected with “C-labelled amino acids to determine hemolymph losses caused by an ectoparasite, the isopod Probopyrus pandalicola (Packard) (Epicaridea; Bopyridae). The female parasites were removed from labelled shrimp at intervals over 36 hr to monitor hemolymph ingestion. The parasites were found to feed discontinuously throughout the host’s molt cycle, ingesting an average of 7-9 J.LIof hemolymph over a 24-hr period. Feeding was curtailed in darkness; probopyrids consumed only 2-3 pl of hemolymph after 12 hr in the dark. In all experiments, a number of parasites did not feed. The results of this study indicate that significant losses of the host’s hemolymph result from the feeding activity of P. pandalicola. The determination of these losses is essential to begin testing the hypotheses that losses of hemolymph inhibit gonadogenesis in the host by either (i) creating a nutrient imbalance or (ii) depleting titers of reproductive hormones. INDEX DESCRIPTORS: Probopyrus pandalicolu (Packard) (Epicaridea; Bopyridae); Isopod; Hematophagous ectoparasite; Pdaemonetes pugio; Grass shrimp; “C-amino acids; Hemolymph 10s~; Discontinuous feeding; Parasitic castration.

Probopyrus pandalicola (Packard) is a bopyrid isopod, ectoparasitic in the branchial chamber of the grass shrimp Palaemonetes pugio. The female parasite feeds with stylet-like mandibles that pierce the inner wall of the branchiostegite margin to suck hemolymph from the host. Hemolymph is ingested by a pharyngeal pumping mechanism similar to that described by Cals (1966) for other epicaridean isopods. The actual amount of hemolymph ingested by any epicaridean parasite is unknown. Consequently, this study was undertaken to measure the removal of hemolymph from the host Paluemonetes pugio by the female bopyrid Probopyrus pandali-

Copyright 1977 by Academic Press. Inc. .4UrightsaP reproductionin any form nserved.

cola through ments.

use of 14C radiotracer

experi-

MATERIALS AND METHODS

A. Collection

and Maintenance

of Animals

Paluemonetes pugio infected with Probopyrus pandalicola were netted from emergent grasses in shallow, ‘brackish water. Shrimp were collected in South Creek, off Sage Point, near the Pamlico Marine Laboratory, Aurora, North Carolina. Prior to experiments, parasitized shrimp were isolated in finger bowls to regulate feeding and monitor molt cycles. Without frequent feedings, many infected shrimp weakened; death followed if food (fish, liver paste, or Artemia) was withheld for more than 5 to 7 days. Nonparasitized

ISSN 0014-4894

ISOPOD’S

DISCONTINUOUS

INGESTION

shrimp withstood fasting for at least 12 days in most cases (unpublished data). Shrimp were maintained at room temperature in brackish water and exposed to a 14 * 1-hr light and 10 2 I-hr dark photoperiod. In most cases, gravid or ovigerous parasites were used for experiments. Immature or nongravid females usually parasitized small shrimp ( < 50 mg) and were not used. Stages in the molt cycle were determined by examining the margins of the uropods (Passano 1960). B. Preparation of Radioisotope Solutions The stock radioisotope solution contained 15 different L-amino acids uniformly labelled with 14C dissolved in 0.1 N HCl (total activity 0.1 mCi/l.O ml; New England Nuclear Co.). When injections were prepared, the stock was mixed 3: 1 with Van Harreveld’s medium (Welsh, Smith, and Kammer, 1968) and buffered at pH 7.2 with 0.10 M Tris buffer. A small amount (5 to 10 ~1) of blue food coloring (FD&C Blue No. 1) was added to the injection solution to aid in observing the release and spread of the injected fluids in the shrimp’s tissues. The standard dosage per shrimp contained 0.3 &i in 2.5 pl of solution. All animal and fluid samples were examined for radioactivity in a Packard TriCarb liquid scintillation spectrometer (Model 314 EX), with a 14C channel efficiency of 62%. All samples were corrected for efficiency and for self-absorption by comparison with known standards. C. Injection Routine Shrimp received single doses of the radioisotope by intramuscular injection in the mid-dorsal abdomen. Following injections, the net dosage (standard dose minus leakage) was determined by collecting samples of the ambient water to measure the leakage of 14C from the injection wound. Injected shrimp were isolated in 50 ml of water from which 0.5-ml water

OF

SHRIMP

199

HEMOLYMPH

samples were taken after the f&t, second, and final hour of each experiment. Measurements revealed that leakage occurred only during the first hour after injections. Consequently, all subsequent determinations were restricted to the first hour, after which shrimp were rinsed and transferred to uncontaminated water. Shrimp with leakage greater than 7570 of the standard dose were discarded. D. Preparation

of Samples for 14C Analysis

Following each exposure to the radioisotope, the male and female pair of probopyrids were removed from each shrimp, weighed, and placed separately in scintillation vials. Shrimp were weighed, and hemolymph samples (5 ~1) were drawn from the pericardial sinus of each shrimp by micropipet. All samples were placed in separate vials and hydrolyzed in 12 N HCl at 40 C. Excess HCl was drawn off with KOH under vacuum; the desiccated samples were resolubilized in 2.5 ~1 of hydroxide of Hyamine 10X (Packard Instruments Co.). Ten milliliters of toluene phosphor (Permafluor; Packard) were added to each sample, and its radioactivity was measured. The 0.5~ml water samples were mixed with a water-absorbent phosphor (Kinard 1957) and counted. E. Estimation

of Hemolymph

Ingestion

Female Probop yrus from intermolt shrimp were killed 1, 2, 4, 8, 12, 18, 24, and 36 hr after the injection of radioisotope into the host. All injections were performed in the morning, with the 1%hr and 36-hr trials terminating in darkness. Estimation of hemolymph ingested per parasite per time period was based on the general formula, ~‘(/.ll) =

dpm per parasite dpm in 1 /*l of hemolymph

*

Male Probopyrrts were examined for radioactivity 24 hr after injections, but were found to contaiu no detectable amounts of radioisotope.

200

STEPHEX

P. WALKER

The loss of label from parasites through the oxidation of the 14C amino acids to 14C0, and the contamination of attached parasites by the leakage of 14C from the injection wound were examined as potential sources of error in estimating hemolymph ingestion, a. Loss of WO, as a source of error. Radioactive COZ was collected from both infected shrimp and detached female probopyrids in groups of two (Smith 1967). Infected shrimp were injected and then maintained in reaction flasks (Kontes Glass Co.) for 24 hr. The CO, was retrieved for I-hr periods 1, 3, 6, 12, 18, and 24 hr after the injections. After 24 hr, hemolymph samples were taken from the shrimp and the parasites were removed and incubated as pairs for a single 6-hr period. Estimates of total 14C0, loss were made by plotting radioactivity against time and determining the area under the curve. b. Leakage of 14C as a source of error. Two groups of parasitized shrimp were exposed for 1 hr to 0.3 ,&i of radioisotope in 50 ml of water. The first group was prepared and counted for radioactivity as described in section D. The parasites of the second group were extracted with trichloroacetic acid (TCA) to determine if any 14C accumulations found in the first group resulted from contamination of the parasite’s

body surface, or from internal accumulations and perhaps incorporation into tissues. Parasites were washed once in warm 100% ethanol, twice in buffered Robinson’s medium, and then individually homogenized in 2.0 ml of Robinson’s medium. The homogenate was twice centrifuged for 1S min at lOOOg, with both supematants removed by pipet and pooled. The pellet was prepared for counting as described for whole parasites. Supematants from five parasites were combined and mixed with an equal volume of 10% TCA. After 1 min, the TCA-insoluble fraction was collected on a membrane filter (0.45 pm, Millipore Instruments), oven-dried at 66 C, placed in a scintillation vial with toluene phosphor, and counted. A 0.5-ml aliquot of the TCAsoluble filtrate was added to a scintillation vial with the water-absorbent phosphor and counted. RESULTS

A. Sources of Error

While considerable I’CO, was released by the infected shrimp after 24 hr, none was detected for the pairs of parasites during the 6-hr collection period (Table I). Individual parasites accumulated significant levels of the radioisotope, despite the absence of 14C0,. It may be concluded that

TABLE A Summary of i4C02 Losses for the Shrimp, Animal

1 2 3 4 6

Weight (me) P

S

2.9 2.4 2.4 3.6 3.1

107 84 08 114 120

Palaemonetes

Xet dose (dpm)

Shrimp wo2 Cd&

27,537 38,454 18,212 31,456 43,008

5471 2278 1657 0920 9800

I pugio, and the Para.sit,e, Probopyrus P’ /‘@

20 0 9 22 23

Parasite (dpm)

200 42 119 50 23

pandalicob

‘CO2 (dpm)

C’ :c

1’36

0

lib

0

Oc

0

a Results from shrimp are expressed as a percentage of the net dosage. Resu1t.sfrom parasilcs are cxpressod as R percentage of the accumulated radioact,ivity for pairs of animals. b Designates measurements which arc lower than background radiuaclivity (82 dpm). c One member of the pair died during the test.

ISOPOD’S DISCONTINUOUS

INGESTION

the measurements of radioactivity in the parasites were not influenced significantly by a loss of radioisotope through respiration. The parasites exposed for 1 hr to 0.3 &i of radioisotope in water contained significant levels of “C ( 16-87 dpm), which evidently were acquired from the ambient water since no radioisotope was detected in the host’s hemolymph (Fig. 1). Internal accumulation of the radioisotope was suggested by the presence of some radioactivity (46 and 49 dpm) in the TCA-insoluble fraction (Table II). However, the major source of 14C appeared in the TCA-soluble fraction, probably as free amino acids. The pellet, consisting primarily of chitin residues, contained no measurable quantities of radioactivity, suggesting that the TCAsoluble compounds were probably ingested and not surface bound to the exoskeleton of the parasite. These results indicate that water contaminated by the leakage of radioisotope is an important source of error in estimating

OF SHRIMP

201

HEMOLYMPH

TABLE

II

The Incorporation of 14Cin Probopgrus pundalicola Females Exposed to 0.3 pCi of L-amino acids in 50 ml of Watera

Total

1)osage

weight

TCA inS,luble fraction

TCA soluble flacticn1

-I

II

IO.2

9,.-l

rng

1ng

(i7,l X6 dpm

46 dpm

10.5dpm

(i7,886 dpm

(SO“;) 4!) dpm

203 dpm

(ly;,)

(70“; ) (81 ‘; 1

u Each group represents five parasites, for which all fractions were pooled before counting. Values in parentheses are percentages of t.he total radioactivity recovered in each parasite group.

hemolymph ingestion. Since leakage from injection wounds was unavoidable, a correction factor of -44 dpm (the test group mean) was incorporated into the formula to adjust for the radioisotope accumulated from the ambient water. The revised formula, 1’(J)

=

44 dpm dpm in 1 ~1 hemolpmph ’

dpnl per parasite

-

is a more precise expression of hemolymph ingestion. B. Shrimp Hemolymph

P

water

.

,

injected

FIG. 1. The means of “C concentration for samples of hemolymph from Pahmonetes pugio and the parasite, Probopyrus pandalicohz, after a 1-hr exposure to the L-amino acids either in the ambient water, or in the blood of the host. Two mean standard errors are shown. B = blood, P = parasite, (N) = number of animals.

as a Source of 14C

One hr after injection, the radioactivity in 1.0 ~1 of hemolymph varied from 5.9 to 32.8 dpm or approximately O.Ol-O.l% of the net dosage. Variation among the net dosages was greater than the variation in hemolymph radioactivity, due in part to the loss of hemolymph during injections, and to the differences in leakage of the radioisotope. Hemolymph radioactivity, when monitored for 24 hr, was maintained at levels similar to those at 1 hr (Fig. 2). The means of hemolymph radioactivity in all groups, except at 36-hr, were not significantly diffcrent from each other (p = .OS). Each female probopyrid then was exposed to a relatively constant oourcc of l’C in shrimp hemolymph for 24 hr. Therefore, hemo-

STEPHEN

P. WALKER

Q 0

FIG. 2. The accumulation of radioactivity by Probopyrus panduZicoZu females as a function of the “C levels in 1.0 pl of hemolymph from the host Pdaemonetus pugio over a 36-hr period. The wide range of values for P. panduZicoZa appears to result from their irregular feeding behavior. - - l - -, shrimp hemolymph; - - 0 - -, Probopyrus.

lymph losses could be determined by measuring the radioactivity of the parasites at the end of each time period. C. Hemolymph

Ingestion

In the first experiment, the loss of hemolymph was monitored for 36 hr. After 1 hr, the females contained levels of radioactivity that did not differ significantly ( p = .05) from the levels in the probopyrids exposed to the water-borne label (Fig. 1). However, the accumulations of 14C observed in the remaining trials were substantially greater, presumably from the ingestion of labelled hemolymph. The curve of 14C accumulated for all parasite groups combined was biphasic (Fig. 2). The initial phase occurred in the first 12 hr of the experimcnt, in which an average of 7-9 ~1 of hemolymph was consumed by the para-

sites (Fig. 3). During hours S-24, no further significant uptake of the radioisotope was detected (p = 0.5). The radioactivity in the 36-hr group of parasites, however, was significantly greater than the pooled mean for hours S-24 (p = .05). This suggests that the second phase of ‘C accumulation occurred between hours 24 and 36. The increase in radioactivity by the 36th hour indicates that hemolymph ingestion had resumed, despite the drop in specific activity of the host’s hemolymph. D. Nemolymph of PhotoperZod

Ingestion

as a Function

The biphasic pattern of 14C uptake in this experiment coincided directly with changes in light conditions in the laboratory. After S-12 hr of feeding in light, a noticeable decline in feeding took place

ISOPOD’S

DISCONTINUOUS

4

INGESTION

d

12

OF

SHRIMP

16

HEMOLYMPH

20

203

24

Hours

FIG. 3. Ca!culated means of hemolymph consumption by Probopyrus pandaZicoZufemales over a 24-hr period. The volumes of hemolymph ingested by the female parasites show great variation, an indication that feeding is discontinuous. The data after 24 hr were not included, owing to the unmonitored drop in radioactivity of the host’s blood.

near the onset of darkness and continued through hour 24. The point at which feeding resumed between hours 24 and 36, after the onset of light, was not determined. It can be concluded, however, that by hour 36, the parasites had re-engaged in feeding. A second experiment was performed to determine if feeding is more extensive in light than in darkness. Parasitized shrimp received standard doses at the beginning of their normal dark period; the parasites were removed after 12 hr in darkness and assayed for radioactivity. The accumulations of W among probopy rids of this group was significantly less (p = .05) than levels of radioactivity in parasites exposed for 12 hr in the light (Fig. 4). This sug-

gests that the plateau between hours 12 and 24 in the first experiment probably represents a period of reduced feeding. Although feeding varied diurnally, the relationship with photoperiod may be only coincidental. The stimulus or stimuli inducing the changes in feeding may have originated exogenously (Le., host behavior) or endogenously, by some physiological process. E. Variability

of Feeding

In a separate experiment, a number of female isopods from shrimp injected with the dye FD&C Blue No. 1 visibly accumulated some dye within their semitransparent intestines. In a few probopyrids no dye

204

STEPHEN

250

E 0” e; :E 1 t x d 100

P. WALKER

host, damaging the newly forming exoskeleton. This indicates that every few days one feeding site is abandoned and another is established. DISCUSSION

Hematophagy has been demonstrated in the bopyrid Probopyrus pandulicola (Packard) by injecting both a blue dye and W-labelled amino acids into the host a :: Pahmonetes pugio, and tracing them to B P Y dark light the parasite. The quantity of hemolymph ingested by the parasites in each time peFIG. 4. Means of radioactivity and calculated means of hemolymph consumption for hobopyms riod varied considerably (i.e., 5-15 ~1 of pandalicolu females after a 1%hr exposure in the hemolymph in the l&hour group). This light and dark. Two mean standard errors are variability could not be explained by corshown. B = blood, P = parasite, V = volume of relating the body weight of parasites with blood, ( N) = sample size. the amount of hemolymph ingested. Furthermore, 18 parasites (of the 67 tested in appeared, while in others, it was present throughout the intestine after 24 hr. In the groups from hours 1 through 24) failed to accumulate any radioactivity from the some cases, the dye had progressed only labelled hemolymph of their hosts. Most through the pharyngeal region of the forelikely, the discontinuous feeding habits of gut, while in others dye was concentrated in a swollen portion of the hindgut near these parasites account for most of the observed variations. the site of male attachment. It was imposP. panddicola females appear to feed sible to determine whether or not the male accumulated any dye, owing to the pres- more actively during the day than at night; ence of heavy pigmentation. Although the however, feeding may cease temporarily at extent of the dye varied throughout the in- any time. The results of two ‘“C assays revealed that hemolymph ingestion is either testine of different probopyrids, which indicates irregular feeding behavior, it is discontinued or reduced in the dark. Although this behavior may reflect some cirnot clear whether the time spent feeding cadian rhythm in P. panahlicolu, bopyrids or the rate of feeding (or both) varied lack both simple and compound eyes among the female probopyrids. The toothed mandibles of the female re- (Kaestner 1970) and stimuli originating from the host, such as diurnal changes in peatedly pierce the host’s branchiostegite leaving a number of scars, furnishing more the content of the host’s hemolymph, may evidence of sporadic feeding. One to sev- direct this behavior rather than photoeral amber-colored punctiform spots (50 to period. Feeding may cease as a result of hemo100 pm in diameter) are clustered on the branchiostegite opposite the mouth of the lymph coagulation at the wounds in the parasite. New scars appear adjacent to the branchiostegite of the host. On occasion, a old ones during the molt cycle of the host, ring of blue viscous material accumulated resulting from the formation of new feed- around the feeding wound in shrimp ining wounds. Following ecdysis of the host, jected with blue dye, suggesting the forone or two scars remain on the new exo- mation of a clot. Until the wound is either skeleton, which suggests that feeding con- reopened or another is formed by the tinues during the premolt stages of the piercing mandibles of the parasite, feeding

ISOPOD’S

DISCONTINUOUS

INGESTION

could be disrupted altogether. This may explain the formation of new wounds every few days. The general magnitude of an average daily loss of hemolymph (7-9 ~1) can be estimated by comparison with the total volume of hemolymph in the host. Based on determinations of hemolymph volume for Crungon vulgaris (Djangmah 1970), another small caridean shrimp, a 200-mg Palaemonetes pugio would have approximately 30 ~1 of hemolymph. Thus, a loss of 8 ~1 would approximate a 2S70 reduction in total volume of hemolymph. This value is, of course, only an approximation since the total volume of hemolymph in Pahmonetes is unknown and considerable variation occurs in the quantities of hemolymph removed. A related calculation reveals that a mature female probopyrid may ingest daily the equivalent of her own body weight in hemolymph. Any substantial reduction in hemolymph volume for Puluemonetes, or rapid gains in weight by Probopyrus, should be offset by their respective osmoregulatory processes. The growth and fecundity of the female Probopyrus pandulicola (25 times the weight of its dwarf male partner and a series of broods totalling several thousand larvae; unpublished data) can only result from an extensive uptake of nutrients in substantial amounts of shrimp hemolymph. If a chronic loss of hemolymph leads to the suppression of gonadogenesis in the host, the mechanism of action remains to be established. Both a reduction in titers of reproductive hormones ( Chamiaux-Cotton 1960) and a nutrient shortage or imbalance (Potts 1906; Tucker 1930; Field 1969) are certainly possible consequences of chronic blood losses of the magnitude demonstrated here. Elucidation of these effects and their role in the mechanism( s) of castration, however, requires additional research.

OF

SHRIMP

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ACKNOWLWGJIWTS

I wish to thank Dr. Phyllis Bradbury for her help and encouragement during this study and Dr. B. J. Copeland for use of facilities at the Pamlico Marine Laboratory, Aurora, North Carolina, U.S.A. HEFERENCES

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and its control. In “The Physiology of Crustacea” (T. H. Waterman, ed. ), Vol. I, pp. 473-535. Academic Press, New York. Porrs, F. A. 1906. The modifications of the sexual characters of the hermit crab caused by the Journal of Miparasite Peltogaster. Quarterly

croscopical Science 50, 599-621. SMITH, D. E. 1967. Location of the estrogen effect on uterine glucose metabolism. Proceedings of the Society for Experimental Biology and Medicine 124, 746-749. TUCKER, B. W. 1930. On the effects of an epicaridean parasite, Gyge branchialis on Upogebia littoralis. Quarterly Journal of Microscopical Science 74, 1-118. WELSH, J., SMITH, R. I., AND KAMhlER, A. 1968.

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Physi-