Probopyrus pandalicola (Packard) (Isopoda; Epicaridea): swimming responses of cryptoniscus larvae in water conditioned by hosts Palaemonetes pugio (Holthuis) (Decapoda; Palaemonidae)

J. Exp. Mar. Biol. Ecol., 1989, Vol. 130, pp. 9-18 Elsevier

9

JEMBE 01285

~~~~0~~~s~~~d~~i~~~~ (Packed)

(Isopoda; ~pi~~idea): swimming responses of cryptoniscus larvae in water conditioned by hosts ~~~~~~~~e~~~pugi@ (Holthuis) (Decapoda; Palaemonidae) Gary Anderson

Department of Bio@ical

and William E. Dale

Sciences, Southern Station, University of Southern ML~I~JI$@, Hattiesburg, Misiwippi, U.S.A.

(Received 23 March 1989; revision received 1 May 1989; accepted 17 May 1989) Abstract: Swimming responses of ~o~p~~p#~~~ic~~Q

(Packard) c~pton~scus larvae to water solutions either conditioned or unconditioned by hosts were determined using a simple Y-tube choice apparatus. Results document that larvae swim at random or downstream with respect to water current in unconditioned control solutions. In the presence of either “crude extract” or “host-metabolite” solution, prepared using definitive hosts P~~ae~~netes pugio (Holthu~s), cryptonisci are strongly rheopositive. However, upstream swimming cryptonisci swam toward the source of the host-conditioned water (i.e., made the correct *choice”) only when it was rather concentrated; when ~‘host-metabo~te” solution was employed, rheopositive parasite larvae swam toward control solution as frequently as toward the experimental solution. We conclude that distance chemoreception may pray a role in host finding by P. pand&&. Key words: Chemoreception; Host finding; ~#~ue~~~e~es~~0~~~~~~~ Swimming behavior

Cryptoniscus larvae of the parasitic isopod Probopyms pand&xla (Packard} are transmitted actively from the intermediate host Acartia tonsa Dana to the definitive host Pa~ae~~~~ge~~~gio(~olth~s). Natural populations of inte~e~ate and de~niti~~ehosts may be separated spatially from one another. A. EQM-Z is holopl~kto~ic and P. pugio larvae are meroplanktonic; both may be dispersed widely within the estuarine circulation Epibenthic postlarvae and adults ofP. pugio are most abundant in shallows near the water’s edge. Laboratory experiments (Anderson, unpubl. data) suggest that either advanced larvae or recently metamorphosed postlarvae of P. ptigio may be successfully infected by parasite larvae but the latter are more apt to survive infection than the former, During the course of laboratory ~vest~gat~ons on the deveiopment of Pr~b~py~ spp. tarvae (Dale, 1979; Anderson & Dale, f98 1; Dale & Anderson, 1982; Anderson, f983), Correspondence address: G. Anderson, Department of Biological Sciences, Box 5018, Southern Station, University of Southern Mississippi, Hattiesburg, MS 39406, U.S.A. ~2~-~81~89~~03.50 0 1989 Elsevier Science Publishers B.V. {Biomedicat Division)

10

G.ANDERSONANDW.E.DALE

we observed that cryptoniscus larvae often (1) swim for considerable distances and lengths of time before infecting definitive hosts and (2) exhibit a dramatic alteration in swimming behavior when in the vicinity of suitable hosts. In culture, after leaving the intermediate host, the cryptoniscus swims rapidly (up to 1 m * min - ‘), almost constantly (for up to 7 days) and in rather straight paths. Apparently, swimming direction in such larvae is random with respect to light, depth, container surface, etc. However, when added to a bowl containing zoea or postlarvae of P. pugio, cryptonisci often swim erratically or in tight circles, especially when in the immediate vicinity of a potential host or a region wherein a potential host has recently been. Similarly, if a drop of crude seawater extract containing homogenized host is pipetted into a culture dish containing a swimming cryptoniscus larva, the modified behavior is exhibited when the larva encounters the extract. Such behavior tends to keep the parasite in the vicinity of the extract. Hence, the behavior of cryptoniscus larvae is reminiscent of the “whirling” activity exhibited by Schistosoma mansoni miracidia in the presence of host extract. This and other chemically mediated aspects of symbiont behavior have been reviewed elsewhere (e.g., Davenport, 1966; McCauley, 1969; Ache, 1974; Derby & Atema, 1980). As a result of our observations, we undertook a series of experiments to evaluate the chemotaxis responses of Probopyrus pandalicola to potential definitive hosts. Specifically, we wished to evaluate the possibilities that (1) P. pandalicola cryptonisci are able to move “upstream” toward either hosts or crude seawater extracts of hosts and (2) cryptonisci are able to discriminate between host factor containing solutions and controls. Some of our results were communicated at an American Society of Zoologists meeting (Anderson & Dale, 1978).

MATERIALS MAINTENANCE

AND

METHODS

OF CULTURES

Parasite larvae were reared from the epicaridium to cryptoniscus stage using A. tonsa as the intermediate host. Cultures were maintained at room temperature (= 22-27 ‘C) and at either 5 or 15x,, S. Copepods were fed daily a mixture of algae, Zsochrysisgalbana Parke, Skeletonema costatum Grev. and Chroomonas salina Wislouch. In as few as 6 days after copepod cultures were inoculated with free-swimming epicaridium larvae, free-swimming cryptoniscus larvae could be removed and used for behavioral assays. Additional details regarding culture methodology and parasite life cycle are provided in Anderson & Dale (1981). BEHAVIORALASSAYPROTOCOL

Chemotaxic responses of cryptoniscus larvae to test solutions were determined using a simple Y-tube choice apparatus modified after Kawashima et al. (1961) (Fig. 1). The apparatus consisted of a horizontal glass Y tube with an inside diameter of 3.5 mm. The

SWIMMING BEHAVIOR OF BOPYRID ISOPOD LARVAE

11

distance from the entry port (where parasite larvae were introduced) to the exit (downstream) is 15 cm. The distance from the entry port to the distal arms beyond the fork of the Y tube is 40 cm. A two-channel peristaltic pump was used to provide for flow of the test solutions within the apparatus. Aerated test solutions were drawn from

entry port exit A==!+

Fig. 1. Diagram showing dimensions of Y-tube choice apparatus used to determine swimming responses ofP. pandulicola cryptonisci to various test solutions. A two-channel peristaltic pump (P) was used to pump aerated test solution from two reservoirs (S) into distal arms of Y tube (R,L). Average flow velocity in proximal arm of Y tube was 57 cm. min I.

reservoirs into the distal arms of the Y tube. Use of the pump insured similar and constant flow rates in both of the distal arms. Using dye, we demonstrated lack of turbulence at the junction of the arms of the Y tube. However, since the inside diameters of the three arms are identical, upstream-s wimming larvae encountered a reduction in current velocity at the junction between proximal and distal arms. Based on measurement of flow volume - time - i, we calculated that the flow rate in the proximal arm of the Y tube averages 57 cm - min - i. For each replicate ( = trial), the pump was turned on, then a single cryptoniscus larva was pipetted into the entry port. The trial lasted ~4 min; it began at the time that the larva entered the proximal arm of the apparatus. No larvae were used for more than one trial. Subsequent to each trial, the apparatus was rinsed with 1 1distilled water. The result of each 4-mm trial was recorded as either “downstream movement” if the larva was swept out of the apparatus or “upstream movement” if the larva entered one of the distal arms of the Y tube. In the latter case, we also recorded which distal arm (right or left) the larva entered. In a few cases (2% of the trials), larvae remained in the test apparatus (somewhere between the entry port and ends of the distal arms) at the te~ination of the trial. These results were omitted from the analyses and that trial was

12

G. ANDERSON

AND W.E. DALE

repeated with a different cryptoniscus. For each treatment (combination of solutions in the reservoirs), 30 trials were made. Hereafter, such a group of 30 trials is denoted as a test. 17 tests were conducted during the present study. Generally, more than one test was performed for each treatment. TEST

SOLUTIONS

Control solutions consisted of Millipore-filtered water (0.45 ~1pore diameter) of 5 or IS%, S. Most tests were conducted using culture water removed from the aquarium in which the parasite larvae had been reared. However, in some cases we used water of a salinity other than that in which the larvae had been reared. Such water was made from the culture water either by dilution with distilled water or by concentration with artificial seawater (Instant Ocean, made according to the manufacturer’s recommendations). No control solutions had been in contact with P. pugio. “Crude host-extract” solutions were made by homogenizing adult grass shrimp in control solution (5 shrimps. I- ’ for the &‘strong” extract and 1 shrimp. l- ’ for the “weak” extract). The solution was subsequently centrifuged (a 10000 rpm for 1 min) and the supematant filtered (0.45 p pore size) prior to use. “Host-metabolite” solution was prepared by maintaining five adult P.pugio in 2 1 control solution for 2 h, then removing the shrimp and filtering the solution as above prior to use. DATA

ANALYSIS

For each of the 17 tests conducted, the null hypothesis below was tested using x2 goodness of fit (Zar, 1984): HoI : swimming direction (upstream or downstream) of cryptonisci is random within the test apparatus. Hence, the expected frequency of cryptonisci s~rnrn~g upstream is 15 for each test. When the critical x2 value was exceeded (P < 0.05), the alternate h~othesis, i.e., movement was nonrandom (and either upstream or downstream), was accepted. It was not possible to distinguish whether downstream movement was the result of a rheonegative response or the result of upstream movement at an average velocity less than the water current velocity. For each test in which either “crude host extract” or “host metabolite” originated from only one of the two test-solution reservoirs, data obtained for the upstreamswimming cryptonisci were reanalysed to test the null hypothesis below: H 02: “Choice” of a distal arm is random with respect to origin of the test solution. Hence, the expected frequency of cryptonisci entering the distal arm containing host-test solution equals one half of the total number of larvae which entered the distai arms during the test. Where appropriate, heterogeneity 31’analyses (Zar, 1984) were conducted to determine if pooling of data for tests utilizing similar treatments was justified.

SWIMMING BEHAVIOR OF BOPYRID ISOPOD LARVAE

13

RESULTS

Of the 17 tests made during the present study, nine were negative controls (the reservoirs of the apparatus contained only brackish water of either 5 or 15x, S) (Table I). For four of these, the null hypothesis was accepted (swimming direction of

P. ~~~~alico~~.Summa~ of results obtained during nine negative control tests to determine s~rnrni~~ direction (upstream or downstream) of cryptaniscus larvae in absence of host factor. s (%o) 15 1s IS 15

15” 5 5

5 5 Heterogeneity

No. upstream

8 14 14 11 3 20 0 0 -I

No. downstream 22 16 16 19 21 10

30 30 23

x2

d.E

6.53* 0.13 0.13 2.13 19.20*** 3.33 30.00*** 30.00*** 8.53** 50.14***

1 1 1 1 I

1 1

1 1 8

Conclusion Downstream Random Random Random Downstream Random Downstream Downstream Downstream Pooling not appropriate

a Larvae reared at 5%,. *P < 0.025, **P -=z0.005, ***P <: 0.001.

larvae was random with respect to current diction); for five tests, the null h~othesis was rejected and the larvae concluded to swim downstream. The highly significant heterogeneity x2 of 50.14 fd.f. = 8, P -=z 0.001) precludes pooling of data obtained during the nine negative control tests. However, in none of these tests was movement concluded to be predom~n~tIy upstream. Hence, in the absence of host solution, cryptoniscus larvae do not swim ups~e~ at a rate fast enough to overcome the average current velocity within the test apparatus. During five tests, the Y tube contained crude host-extract solution (Tabfe II>. In three cases, only one of the distaf arms of the test apparatus contained extract. In these, swimming by cryptonisci was strongly rheopositive (P < 0.001). The heterogeneity x2 of 0.62 (d.f. = 2, P > 0.05) indicated that results of these tests could be pooled, resulting in a highly significant (P< 0.001) x2 of 71.11. The remaining two tests were positive controls (crude extract originated from both distal branches of the Y-tube apparatus). Although in the first test significantly more cryptonisci swam upstream than downstream, in the second the opposite conc~nsion was reached. Data could not be pooied for the two positive controls ~heterogeneity x2 = 35.26; d.f. = 1, P < 0.001). Similarly, pooling of data is unjustified for all five of the tests invoiving crude host extract (heterogeneity x2 - 51.35; d.f. = 1, P < O.~Oi}. However, it may be notewo~hy that of the five tests invoi~ng crude extract the only one for which downstre~ movement of cryptonisci predominated was conducted at a test salinity of 5x,; the others were

14

G. ANDERSON AND W.E. DALE

conducted at 15%,. When results of the test conducted at the low salinity are omitted, pooling of results for the other four tests is just~~able (heterogeneity x2 = 1.73; d.f. = 3, P > OIX). The x2 value for pooled data is 100.83 which is highly significant (d.f, = 1, P < 0.00X). TABLE II P. pff~~~~jco~. Summary ofresultsobtained during live tests to determine swimming direction (upstream or downstream) of cryptoniscus larvae in presence of“crude host extract” (s, strong; w, weak). In first three tests, extract originated from only one of two distal arms of choice apparatus. In latter two tests (positive controls), host extract originated from both distal arms.

s aof

No. upstream

15 (s) 15 (w) 15 (w) Heterogeneity Pooled 15 (s) 5 (wf Heterogeneity

No. downstream .-

27 28 30

3 2 0

85 30 7

5 0 23

x2

d.f. -.

19.20*** 22.53*** 30.00*** 0.62 71.11*** 30.00*** ?3.53** 35.26***

1 1

Conclusion Upstream Upstream Upstream Pooling appropriate Upstream Upstream Downstream Pooling not appropriate

1 2

1 I I

1

+*p< 0.005, ***p < 0.001.

During three tests, ~6host-metabo~ite’~solution was present in the test apparatus (Table III). Cryptonisci were si~i~~~~y rheopositive in two of the tests. ~~0~~ the null hypothesis was accepted for the third test (a positive controf during which parasite larvae were tested at a salinity other than that at which they were reared), results of the TABLE III P. pundulicolu. Summary of results obtained during three tests to determine swimming direction (upstream

or downstream) of cr~ptoniscus larvae in presence of “host-metabolite~ solution. In first two tests, host factor ariginated from only one of two distal arms of the choice apparatus. In third test (a positive control), host factor originated from both distal arms. S (%*) 15 15 15” Heterogeneity Pooled

No. upstream ~.^_ 24 27 20 71

No. downstream _..__ 6 3 10 19

x2

d.f.

10.80** 19.20*** 3.33 3.29 30.04***

1 1 1 2 1

Conclusion Upstream Upstream Random Paoling appropriate Upstream

a Larvae reared at 5X,. **P -C 0.005, ***P < 0.001.

heterogeneity x2 test (x2 = 3.29; d.f. = 2, P > 0.05) pe~tt~ us to pool the results for all three tests, allowing us to conclude that cryptonisci are sign&eantIy rheopositive in the prescence of “host-metabolite” sohrtion (~1’= 30.04; d.f. = 1, P < 0.001).

SWIMMING BEHAVIOR OF BOPYRID ISOPOD LARVAE

15

Table IV sumrn~~~es the results obtained when we tested H,, for the data in Tables IXand III (to determine if u~s~e~-s~rnrn~g cryptonisci entered the distal arm from which host factor, either “crude” or ‘~metabol~te”,originated). ~tematively, they could have entered the “incorrect” arm which contained control solution. Although signifiTABLE IV

P. panda&cola. Summary of results obtained during tests to determine if upstream swimming cryptoniscus

larvae selected “correct” distal branch of choice apparatus (branch from which host factor originated). In first three tests, “crude extract” originated from one of two distal branches; in other two tests, “host metabolite” originated from one of two distal branches (n, total number of cryptoniscus larvae which swam upstream during test).

Heterogeneity Poofed

Correct

Incorrect

n

x2

d.f.

13 19 21

14 3 9

27 28 30

1

53 9 15

32 15 12

85 24 27

27

51

0.04 3.57 4.80* 3.22 5.19** 1.50 0.33 0.37 2.20

Pooled

1 1 2 1 I I 1 1

Conclusion Random Random Correct Pooling appropriate &orrect Random Random Pooling appropriate Random

*P
cantly more cryptonisci made the “correct” choice in only one of the five tests, pooling of data for the three tests involving crude extract is appropriate (heterogeneity x2 = 3.22; d.f. = 2, P > 0.05). Hence, in the presence of crude extract, P. panda&cola cryptonisci swim toward the source of the extract (x2 = 5.19; d.f. = 1, P -C0.025). In contrast, parasite larvae which were rheopositive in the presence of host metabo~te did not swim toward the source of the metabolite (pooled x2 = 0.37; d.f. = 1, P > 0.05).

Results of this investigation document that, whereas in control water infective cryptoniscus larvae of P. pandalicola swim at random or downstream with respect to current direction, in the presence of either “crude extract”, prepared using definitive hosts P. pug& or “host-metabolite water”, within which hosts have been recently retained, c~to~sci are generally strongly rheopositive. When given a choice between water containing host factor and control water, parasite larvae swam toward the source of the host factor (rather than toward contro1 water) only when the former consisted of “crude extract”; there was not a significant tendency for upstre~-s~rnrn~g cryptonisci to swim toward “host-metabo~te” solution. The possibility that ~hemor~eption could be important in host-fmd~g by parasitic

16

G. ANDERSON

AND W. E. DALE

crustaceans has ~t~~~ workers for a number of years. With respect to mo~hoio~c~ correlates, Goudeau (1970) and Nielsen & Stromberg (1973) described ~~as~ct~~ characteristics of aesthetascs in several species of Cryptoniscina, a superfamily of Epicaridea. Nielsen & Stromberg (1973) acknowledged that “well-developed chemosensory equipment is an absolute prerequisite for the successful accomplishment of this life cycle”. Others have speculated about chemoreception in terms of host-finding behavior. In her review of settlement by symbiotic Cirripedia, Lewis (1978) suggested that ecdysone may serve as an attractant for male cyprid larvae, accounting for the phenomenon that virgin externae (but not extemae at other stages of the molt cycle) are quickly colonized by male cyprids as noted in L. Ritchie’s (1977) unpublished work. However, Lewis (1978) acknowledged that generally experimental results demonstrate the importance of contact chemoreception in barnacle settlement but have failed to demonstrate that distance chemoreception is important in orienting toward hosts. Speculation that chemical cues could be used in interspeci~c ~te~elationships between bopyrid isopods and hosts also exists. For example, Beck (1979) suggested the possibility that huwae of bopyrids which attach to mature shrimp or crabs “might be cueing on some sex-specific secretion which might then be present”. Reverberi & Pitotti (1942) [cited by Reinhard (1949) and Danforth (1963)j suggested that cryptoniscus larvae of Zone thorucica may be attracted to female parasites already established on hosts Callianassa laficaudu by a diffusing substance. Similarly, Reinhard (1949, Table 1) and Markham (1974) suggested that chemoreception may operate to attract potential male bopyrid isopods to already established female parasites. Not surprisingly, controlled studies supporting such ideas have not been conducted, probably owing primarily to the difficulties inherent in securing adequate numbers of infective larvae for behavioral assays. Based on our own experience with culture of Probopyrus spp., substantial time and effort are required to obtain large numbers of cryptoniscus larvae. Althou~ the present study provides compelling evidence suppo~~g the notion that chemoreception plays a role in host-ending by the bopyrid isopod P. ~ff~dalico~~,some problems and unanswered questions remain. For example, tests which did not seem to fit within the generalizations drawn for groups of similar tests often involved manipulation of salinity. Hence, the only test in which there was significant downstream movement of cryptonisci in the presence of “crude host extract” was that run at 5x, S (larvae were reared at this salinity). Also, in one of the three tests utilizing “hostmetabolite” solution, swimming direction of cryptonisci was concluded to be random. Although the test was run at 15x,, S, cryptonisci had been reared at 5x,. Certainly, these few examples could simply represent artifacts which could be resolved if addition replicates were conducted. However, the possible interactive effects of salinity may also represent a relevant avenue of future research since parasite larvae are estuarine and salinity fluctuations in their habitat are expected to be commonplace. In Beck’s (1979) study, it was suggested that cryptoniscus larvae of Probo~y~s sp. infesting P. ~~~~dos~~ may swim considerable distances from estuaries harboring the inte~ediate host A. tonsa to the freshwater habitat of the definitive host. The evidence obtained in the

SWIMMING BEHAVIOR OF BUPYRID ISOPOD LARVAE

17

present study which may be relevant to Beck’s study would suggest that parasite larvae of P. pundulicola would not be inclined to swim upstream from brackish to freshwater. However, it may be noteworthy in this regard that the species of parasite on which Beck conducted research is probably not P. pandalicolu but rather a morphologically similar congener, P,floridensis (Dale & Anderson, 1982). Further investigations of host finding behavioral phenomena of P. pandalicola should focus on several areas: (1) An evaluation of various aspects of host specificity would be of interest. For example, do parasite Iarvae respond to extracts of other invertebrates in the same manner that they responded to extracts from P. pugio? Such info~atio~ would indicate if the larvae are responding to an ammo acid or other metaboIite(s) rather than a unique chemical from the normal host. (2) Since few assays were conducted using host factor (either crude extract or metabohte) in which parasite larvae were presented a choice between different solutions in the distal arms of the test apparatus, additional tests are warranted to provide more convincing results. (3) A greater degree of control over host-factor concentratiun (number of shrimp used in the homogenate) et al. (e-g_, age, sex and molt cycle status of hosts used to produce test so~u~ons) should be exercised. (4) Should further work provide additions support for the conch~sion that distance chemor~eption plays a role in orientation of P. p~ndQl~co~~larvae, studies shouId focus on the nature of the chemical factor(s) involved.

REFERENCES Ache, B.W., 1974. The experimental analysis of host location in symbiotic marine invertebrates. in, Syrnbosis in &e sea, edited by W. I?. Vemberg, University of South Carolina Press, Columbia, South Carohna, pp. 45-60. Anderson, G., 1983. U~e~aiio~s on postinfection mortality and growth of ~u~~e~~~~es pugi0 following exposure to the bopyrid isopod Probopyrus ~da~~~ol~~ Am. &of., Vof. 23, p. 942. Anderson, G. & W.E. Dale, 1978. Does chemoreception play a role in hast finding by parasitic bopyrid isopods? Am. Zooi, Vol. 18, p. 620. Anderson, G. & W.E. Dale, 1981.Probopytwpandalicola (Packard)(Isopoda, Epicaridea): morphology and development of larvae in culture. G’rusruceana (Leiden), Vol. 41, pp 143-161. Beck, J.T., 1979. Population interactions between a parasitic castrator, Probopyrus pandalicoIa (Isopoda: Bopyridae), and one ofitsfreshwater shrimp hosts, Palaernonetespaludosus (Decapoda : Caridea). Parasitology, Vol. 79, pp 431-449. Dale, W. E, 1979. Biology and development of Pro!qyrur pundalicola larvae, with comparisons to those of Pro~py~~o~~~~ and Probopm bifhynis (Epicaridea; Isopoda). MSG. thesis, University of Southern Mississ~ppj~ Hattiesburg, Mississippi, 91 pp. Dale, W. E. & G. Anderson, 1982. Comparison of rno~~o~o~e~ of Probo2w-w b~hyn~~ P.~~de~~~ and P. panda&o&z farvae reared in culture (Isopoda, Epicaridea). .F. Cri&zce~ &of. poo& Hole, Mass.), Vol. 2, pp. 392-409. Danforth, C. G., 1963. Bopyridian (Crustacea, Isopoda) parasites Found in the Eastern Pacific of the United States. Ph.D. diss., Oregon State University, Corvallis, Oregon, 110 pp. Davenport, D., 1966. The experimental analysis of behavior in symbioses. In, Symbiosis, Vol. I, edited by S.M. Henry, Academic Press, New York, pp. 381-429. Derby, C. D. % J. Atema, 1980. Induced host odor attraction in the pea crab Pinnotheres maculutus. Viol. BUN. (Wuods Hole, Mass.), Vol. 158, pp. 26-33. Goudeau, M., 1970. Nouvelle description d’fiemioniscus balnni Buchholz, isopode epicaride, au stade de mgle cryptoniscien. Arch. Zool. Exp. G&n., Vol. 111, pp. 41 l-488.

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G. ANDERSON AND W.E. DALE

Kawashima, K., I. Tada & I. Miyazaki, 1961. Host preference of miricidia of Purugonim~ ohirai Miyazaki, 1939 among three species of snails of the genus A&mea. Kyushu J. Med. SC;., Vol. 12, pp. 99-106. Lewis, C. A., 1978. A review of substratum selection in free-living and symbiotic cirripeds. In, Settlement and metamorphosis of marine invertebrate larvae, edited by F.-S. Chia & M. E. Rice, Elsevier, New York, pp. 207-218. Markham, J.L., 1974. A systematic study of parasitic bopyrid isopods in the West Indian faunal region. Ph.D. diss., University of Miami, Coral Gables, Florida, 356 pp. McCauley, J., 1969. Marine invertebrates, chemical signals, and marine products. Lloydia, Vol. 32, pp. 425-437. Nielsen, S.-O. & J.-O. StrSmberg, 1973. Mo~holo~c~ characters of taxonomicai importance in C~ptoniscina (Isopoda, Epicaridea). A scanning electron microscopic study of c~toniscus larvae. Sarsia, Vol. 52, pp. 75-96. Reinhard, E.G., 1949. Experiments on the determination and differentiation of sex in the bopyrid Stegophryxus hyptius Thompson. Biol. Bull. (Woods Hole, Mass.), Vol. 96, pp. 17-31. Reverberi, G. & M. Pitotti, 1942.11ciclo biologic0 e la determinazione fenotipica del sesso di Ione thoracica Montagu, Bopiride parassita di Callianassa Iaticauda Otto. Pubbl. Sm. Zool. Napoli, Vol. 19, pp. 111-184. Zar, J.H., 1984. Biostatistical analysti. Prentice-Hall, Englewood Cliffs, New Jersey, second edition.