Comparative susceptibility of the copepod Acanthocyclops vernalis to a microsporidian parasite, Amblyospora connecticus, from the mosquito Aedes cantator

JOURNAL

OF INVERTEBRATE

PATHOLOGY

52, 73-77 (1988)

Comparative Susceptibility of the Copepod Acanthocyclops vernalis to a Microsporidian Parasite, Amblyospora connecticus, from the mosquito Aedes cantator THEODORE Department

of Entomology,

G. ANDREADIS

The Connecticut Agricultural Experiment Station, P.O. Box 1106, New Haven, Connecticut 06204

Received September 29, 1987;acceptedFebruary5, 1988

The comparativesusceptibilityof eachdevelopmental stageof the copepod Acanthocyclops connecticus, a microsporidian parasiteof the mosquitoAedes cantator, wasdeterminedin laboratorytransmission tests.Adult femaleandjuvenile copepodidstageA. vernalis werefoundto be susceptible, whileadultmaleandlarvalnaupliistageswerenot. Parasite development is restrictedto the medianovary andpairedlateraloviducts,whichbecomeswollen andnoticeablydistendedin hostfemalecopepods.Ovarianinfectioninterfereswith normalegg productionandappearsto explainthe differentialsusceptibilityof eachhostdevelopmental stage to infection.Observationsof naturallyacquiredinfectionin field populationsof A. vernalis were consistentwith thelaboratorystudies.No infectionsweredetectedin adultmalesor gravidfemales andonly femalecopepodswithout eggsacs(20%,n = 613)werefound to be infected. o 1988 Academic Press, Inc. KEY WORDS: Amblyospora connecticus; Microsporida;Acanthocyclops vernalis; copepod; Aedes cantator; mosquito;susceptibility. vernalis to Amblyospora

INTRODUCTION

plii, copepodids, and adult male and female

Acanthocyclops vernalis to Amblyospora Recent studies (Andreadis, 1985, 1986, connecticus, a microsporidian parasite of 1988; Sweeney et al., 1985, 1988; Becnel, the mosquito Aedes cantutor, and to fur-

1986) have firmly established that cyclopoid copepods are obligate intermediate hosts for mosquito-parasitic microsporidia of the genus Amblyosporu. However, the relative susceptibility of different developmental stages of the copepod host has not been examined. The typical cyclopoid copepod has four distinct developmental stages: an egg, six larval (nauplius) stages, five juvenile (copepodid) stages, and an adult which does not molt. There is considerable sexual dimorphism between adults, and reproduction is always sexual (Wyngaard and Chinnappa, 1982). Information on the susceptibility of each developmental stage is of fundamental importance for conducting basic epizootiological investigations and for future domestication of these polymorphic microsporidia for mosquito control. The objective of this study was to investigate the comparative susceptibility of nau-

ther determine the site(s) of infection within the copepod host. The natural prevalence of A. connecticus in populations of adult A. vernalis was also determined at three field sites, and the results were compared with the laboratory transmission studies. MATERIALS

AND METHODS

Laboratory transmission studies. All A. vernalis copepods and A. connecticus-

infected mosquitoes used in the laboratory transmission studies were collected from three coastal salt marsh breeding pools in Guilford and Milford, Connecticut, during October and November 1986. Copepod stages assayed for susceptibility to A. connecticus included nauplii, copepodids, and adults of both sexes. However, no attempt was made to differentiate the different naupliar (I-VI) or copepodid (I-V) stages prior to exposure. 73 0022-2011B8 $1.50 Copyriaht 0 1588 by Academic AU rights of reproduction

Press, Inc. in my form reserved.

74

THEODORE

G. ANDREADIS

The procedure for exposing males, females, and juvenile copepodids was as follows. Fifty individuals of each developmental stage were placed in a 100 x 80-mm culture dish containing 200 ml of filtered (particle retention 2.5 pm) water from the breeding site and five mascerated 4thinstar larvae of A. cantutor that were grossly infected with mature meiospores of A. connecticus. Copepods were maintained at room temperature (20”-22°C) under a natural photoperiod and were periodically supplied with a small amount of food consisting of bacteria, protozoa, and brewer’s yeast. There were five replicates for each exposure group and an equal number of controls that were treated identically but were not exposed to meiospores. Copepodids were held for 17 days and adults for 14 days. At the end of the exposure period, 10 individuals from each culture dish (5 exposed and 5 control) were removed, smeared on slides, stained with Geimsa stain solution, and examined microscopically (X 1000) for infection. To test the susceptibility of nauplii stages, egg sacs were carefully removed from adult females, individually placed in 1-ozplastic cups containing 10 ml of filtered site water, and allowed to hatch. Sibling nauplii, 24 to 48 hr old, were then collectively transferred to the culture dishes (2 families/dish) and exposed to meiospores as described above with the appropriate controls and same number of replicates. Nauplii were held for 23 days during which time they developed to the copepodid stage. They were then smeared and examined for infection as copepodids. Histology. The site of infection in female host copepods was determined through histological examination of serially sectioned individuals with various degrees of infection. For this study, whole copepods, previously exposed to meiospores, were fured overnight at 4°C in 2.5% (v/v) glutaraldehyde that contained 0.1% (w/v) CaCl, and 1% (w/v) sucrose and was buffered with 0.1 M sodium cacodylate, pH 7.4. Specimens

were postfixed for 2 hr at room temperature in 1% (w/v) 0~0, and stained en bloc overnight in the dark at 4°C with 0.5% (w/v) uranyl acetate in 70% (v/v) ethanol prior to dehydration and embedding in an LX112/araldite mixture. Serial sections were cut at 3-5 pm and examined unstained using phase-contrast microscopy. Host tissues were identified based on published accounts of copepod internal anatomy (Bullough, 1958; Marshall and Orr, 1972; S&ram, 1986). Field studies. The natural prevalence of A. connecticus in field populations of A. vernalis was determined at three coastal salt marsh breeding sites in Guilford and Milford, Connecticut, from March through June 1986. Copepod developmental stages examined included males, gravid females with egg sacs, and females without egg sacs. Each breeding pool was sampled once a week and all collection material was immediately transported to the laboratory. All copepods were identified and categorized. Representative samples (usually 25-50 individuals) of each developmental stage were smeared on microscope slides, stained with Geimsa stain, and examined microscopically for A. connecticus. Because similar observations were made at all three sites, the data were pooled for analysis. RESULTS

was successfully transmitted to adult female and copepodid stage A. vernalis. No infections were detected among adult males or nauplii that were examined as copepodids (Table 1). Infected females lacked egg sacs and their infections were characterized by the presence of sporonts, sporoblasts, and a few mature spores. In contrast, copepodids were infected with meront stages only and were more lightly infected than were adult females. The frequency of transmission of A. connecticus to copepodids, as determined by the prevalence of infection, was slightly more than one-half of that observed in adult females. No infections of any type were A. connecticus

SUSCEPTIBILITY

OF

TABLE Suscemer~~~~

OF DIFFERENT

1

DEVELOPMENTAL

Amblyospora Developmental

stages

75

Acanthocyclops vernalis TO Amblyospora connecticus

STAGES OF connecticus FROM

Exposed

Examined

Number of days held

Nauplius Copepodid Adult male Adult female

Copepodid Copepodid Adult male Adult female

23 17 14 14

found in unexposed control copepods; uninfected females were often observed with paired egg sacs. Histological examination of infected adult female A. vernalis showed numerous developmental stages(meront and sporont) within the median ovary and paired lateral oviducts. These tissues became swollen and noticeably distended owing to the mul-

Acanthocyclops Aedes cuntator

vernalis TO MENX~~RES

Exposed

OF

Unexposed

No.

% Infected

No.

% Infected

50 50 50 50

0 36.0 0 64.0

50 50 50 50

0 0 0 0

tiplication of the microsporidia (Fig. 1). Parasite development was confined to the host cell cytoplasm, and no infections were detected within ovarian nuclei (Fig. 2). There was no sign of infection in any other host tissue; however, the ovarian membranes did eventually rupture and release mature spores into the hemocoel of the host copepod, where they were observed to circulate

FIG. 1. A sag&al section through an adult female Acanthocyclops vernalis showing Amblyospora connecticus infection within the median ovary (Ov) and paired lateral oviducts (Od). G, gut; M, muscle. x 180. FIG. 2. Higher magnification of the same female, showing individual sporonts (S) OfAmblyospora connecticus within a developing copepod egg. M. muscle; N, nucleus; Y, yolk sphere. x 1150.

76

THEODORE

G. ANDREADIS

freely. This usually occurred prior to death of the host. Observations of natural infection of A. connecticus in field populations of adult A. vernalis (Table 2) were consistent with those made in the laboratory. No infections were detected in weekly samples of 179mature males or 472 gravid females (with egg sacs) collected from March through June. Only females without egg sacs were infected; the overall prevalence of infection for these females was 20.2% (n = 613). DISCUSSION

Only copepodid and adult female stage are susceptible to infection when exposed to meiospores of A. connecticus. Nauplii and adult males appear to be refractory and this seems to be directly related, at least in adult hosts, to the site of infection, i.e., ovarian tissue. The differential susceptibility of each host sex is a genuine phenomenon and is further supported by observations of naturally acquired infection in field populations of A. vernalis. It was not possible to determine the sex of infected copepodids in the present study because they could not be induced to molt to the adult stage in the laboratory. However, the rate of transmission of A. connecticus to copepodids was roughly onehalf that obtained with adult females, and if one presumes a one to one sex ratio among these copepodids, then one could conclude that only female copepodids were susceptible as well. This would presuppose the A. vernalis

TABLE PREVALENCE

OF Amblysopora

presence of appropriate gonadal tissue in juvenile copepods, and according to Schram (1986), recognizable gonadal tissue does appear during metamorphosis to the first copepodid stage and progressively develops until the final molt to a sexually mature adult. The absence of gonadal tissue in nauplii stages could also account for their nonsusceptibility, although the possibility that nauplii are unable to ingest the infectious meiospores, which measure 7 x 5 p,m, cannot be entirely ruled out either. It is interesting to note that spores produced in these female copepods are nearly twice as infectious to male as they are to female mosquito larvae, but in mosquitoes, this differential susceptibility appears to result from some unidentified humoral factor rather than host tissue specificity (Andreadis, 1988). The development of A. connecticus appears to be well synchronized with the development of its host, as evidenced by the lack of sporulation in immature copepodid stages. The ecological and evolutionary significance of this phenomenon is apparent from recent epizootiological investigations (Andreadis, unpubl.), which seem to indicate that A. vernalis over-winters as a late stage copepodid and that the copepodid stage serves as the natural overwintering host for A. connecticus. Parasite development in ovarian tissues appears to impede and inhibit normal egg development in A. vernalis. This was evident by the inability of infected females to produce egg sacs in the laboratory trans2

connecticus IN MALE AND FEMALE AT GUILPORD

AND MILFORD,

POPULATIONS CONNECIICIJT, 1986

OF Acantkocyclops

vernalis

Females Males

Collection period

No.

March April May June

119 60 -

Without

% Infected 0 0 -

No. 198 269 125 21

egg sacs % Infected 24.2 21.9 12.0 9.5

With No. 264 172 25 11

egg sacs % Infected 0 0 0 0

SUSCEPTIBILITY

OF Acanthocyclops vernalis TO Amblyospora connecticus

mission studies an by the lack of infection in gravid females (with egg sacs) that were collected from the field. This pathology would seem to eliminate any possible routes of vertical transmission for A. connecticus in A. vernalis, unless female hosts became infected while producing eggs and the microsporidium quickly invaded the egg prior to oviposition, but this has not been observed. On the other hand, by infecting ovarian tissue, the microsporidium does not seriously impede the normal metabolic processes of its host and does gain a selective advantage because it is able to multiply and produce large numbers of spores that will directly facilitate horizontal transmission to the mosquito host and thereby enhance its survival. The differential susceptibility displayed by the various developmental stages of A. vernalis to A. connecticus has important implications with regard to host specificity, future domestication of these microsporidia, and epizootiological investigations that attempt to describe and quantify natural cycles of Amblyospora in field populations. It is not presently known whether differential susceptibility exists in other copepod species that serve as intermediate hosts for other Amblyosporu spp., but this should be investigated in order to clarify whether this phenomenon is universal or unique.

77

REFERENCES AND~EADIS, T. G. 1985.Experimentaltransmission of a microsporidian pathogenfrom mosquitoes to an alternate copepod host. Proc. Natl. Acad. Sci. USA, 82, 5574-5575.

ANDREADIS, T. G. 1986.Lie cycle andepizootiological investigationsof Amblyospora (Microsporida) in the mosquitoAedes cantator andin the intermediatecopepodhost. In “FundamentalandApplied Aspectsof InvertebratePathology”(R. A. Samson, J. M. Vlak, andD. Peters,Eds.),p. 344.Fourth Int. Colloq.Invertebr. Pathol.,Veldhoven,The Netherlands. ANDREADIS, T. G. 1988.Amblyospora connecticus sp.nov. (Microsporida:Amblyosporidae): Horizontal transmission studiesin the mosquito,Aedes cantutor, andformaldescription.J. Invertebr. Pathol., in press. BECNEL, J. J. 1986.Microsporidiansexualityin culitine mosquitoes. In “FundamentalandAppliedAspectsof InvertebratePathology” (R. A. Samson, J. M. Vlak, and D. Peters, Eds.), pp. 331-334. Fourth Int. Colloq.Invertebr. Pathol., Veldhoven, The Netherlands. BULLOUGH, W. S. 1958.“Practical InvertebrateAnatomy.” Macmillan& Co., London. MARSHALL, S. M., ANDORR,A. P. 1972.“The Biologyof the MarineCopepod.”Springer-Verlag, New York. SCHRAM,F. R. 1986.“Crustacea.” Oxford Univ. Press,New York. SWEENEY, A. W., GRAHAM, M. F., AND HAZARD, E. I. 1988.Life cycle of Amblyospora dyxenoides

sp.nov. in the mosquitoCuiexannulirostris andthe copepod Mesocyclops albicans. J. Invertebr. Pathol., 51, 46-57.

A. W., HAZARD,E. I., AND GRAHAM, M. F. 1985.Intermediatehostfor an Amblyospora sp. (Microspora)infectingthe mosquito,Cufex annulirostris. J. Znvertebr. Pathol., 46, 98-102. ACKNOWLEDGMENTS WYNGAARD,G. A., ANDCHINNAPPA,C. C. 1982. Generalbiologyandcytologyof cyclopoids.In “De1 thankP. Moore andD. Stasiakfor their technical velopmentalBiology of FreshwaterInvertebrates” assistance in thefield andlaboratory.I alsothank J. J. Becneland L. A. Magnarellifor their helpful com(F. W. HarrisonandR. R. Cowden,Eds.),pp. 485 533. Liss, New York. mentson the manuscript. SWEENEY,