Life cycle of Amblyospora dyxenoides sp. nov. in the mosquito Culex annulirostris and the copepod Mesocyclops albicans

JOURNAL

OF INVERTEBRATE

PATHOLOGY

(1988)

s&46-57

Life Cycle of Amblyospora dyxenoides sp. nov. in the Mosquito annulirostris and the Copepod Mesocyclops aibicans A. W. SWEENEY~ANDM. Army

Malaria

Research

Unit,

Ingleburn

Culex

F. GRAHAM

New

South

Wales

2174, Australia

AND

E.I. Gulf

Coast

Mosquito

Research

Laboratory,

U.S.

HAZARD* Department

of Agriculture,

Lake

Charles,

Louisiana

70601

Received February 4, 1987; accepted July 6, 1987 A new species of Microspora, Amblyospora dyxenoides, is described. This parasite has three sporulation sequences: two in Culex annulirostris, the mosquito host, and one in Mesocyclops albicans, the intermediate copepod host. Diplokaryotic meronts in larval oenocytes persist to the adult stage and form binucleate spores in females which are responsible for transovarial transmission to larval progeny. Unlike other described species OfAmblyospora, binucleate spores may also form in adult male mosquitoes. Diplokaryotic cells infect the oenocytes of some male and female larvae which hatch from transovarially infected egg batches. These larvae survive to the adult stage after which binucleate spores develop in the females to initiate another transovarially transmitted cycle. In other male and female larvae which hatch from infected egg batches the parasite invades fat body tissue where it undergoes meiosis during a complex sporulation sequence resulting in the formation of eight haploid meiospores within a sporophorous vesicle. Fat body infected larvae usually die in the 4th instar. Larval meiospores are responsible for horizontal transmission to M. albicans copepods in which the parasite develops in ovarian tissue to form another kind of uninucleate spore. These infections ultimately lead to death of the copepods and the spores are infectious per OS to C. annulirostris larvae. The first mosquito stages resulting from these infections are small cells with a single large nucleus relative to the cytoplasm. It appears that the parasite then returns to the diploid state by cytoplasmic fusion (plasmogamy) of uninucleate gametes to form binucleate cells which later adopt the diplokaryotic arrangement and invade larval oenocytes to complete the life cycle. 0 1988 Academic Press. Inc. KEY WORDS: life cycle; microsporidium; Amblyospora dyxenoides; mosquitoes; Culex annulirostris; intermediate host; copepods; Mesocyclops albicans.

INTRODUCTION Microsporidia of the genus Amblyospora are common parasites of mosquitoes (Hazard and Oldacre, 1975). For many years it has been known that these parasites are transmitted vertically among successive mosquito generations via the ovaries of infected females which pass the infection to their larval progeny (Kellen and Wills, 1962; Kellen et al., 1966; Andreadis, 1983). However, until recently, it was not understood how the parasites were r To whom all correspondence z Deceased.

horizontally transmitted within mosquito populations. Field and laboratory studies carried out in Australia on an Amblyospora sp. infecting the mosquito Culex annulirostris demonstrated that the copepod Mesocycrops albicans is susceptible to the meiospores produced in mosquito larvae via transovarially induced infections with this parasite (Sweeney et al., 1985). Infected copepods develop spores not previously associated with Amblyospora which are responsible for horizontal transmission of the parasite back to C. annulirostris larvae, thereby completing the life cycle. This dis-

should be addressed.

46 0022-201 l/88 $1.50 Copyright All rights

8 1988 by Academic Press, Inc. of reproduction in any form reserved.

LIFE

CYCLE

OF Amblyospora

covery site in ture in scribe

has enabled us to maintain the paracontinuous laboratory in vivo culthe two hosts. In this paper we dethe life cycle of this species of Amblyospora in mosquitoes and copepods. Data on transmission rates of the parasite in the two invertebrate hosts will be presented in a separate report. MATERIALS

AND METHODS

Infected C. annulirostris adults were maintained in 0.03 m3 cages and allowed to mate, After blood feeding, the engorged females were placed into individual gauzecovered plastic vials (40 mm diam x 55 mm high) containing ca. 20 ml of water for oviposition. After the eggs were laid the females were smeared, stained with Giemsa, and examined for the presence of Amblyospora infection. The larval progeny of those found to be infected were reared in separate sibling batches. Copepods were infected by placing them in batches of 200-500 into plastic trays containing 500 ml of water from a swiftflowing freshwater stream near Sydney, Australia. Water from this source is normally used for rearing larvae in our laboratory. Five dead or moribund larvae of C. annulirostris, in which the thorax and abdomen were tilled with meiospores, were added as inoculum. Uninucleate spores were usually observed within ovarian tissue of infected copepods after 6-10 days. For mosquito infection experiments batches of 100-200 lst-instar C. annulirostris larvae were added to trays of copepods infected by the above method and were reared to adulthood after which they were placed in cages for blood feeding and oviposition. For light microscope examination, smears of infected specimens were stained with either Giemsa, Heidenhain’s hemotoxylin, or lactoacetic-orcein according to the methods described by Hazard et al. (1981). For electron microscopy, specimens were fixed in 3% glutaraldehyde (v/v) in 0.2 M cacodylate buffer. Specimens were post-

dyxenoides

sp. nav.

47

fixed in 1% Dalton’s chrome osmium (w/v) and uranyl acetate, dehydrated in acetone. embedded in Spurr’s resin, sectioned, and examined under a Philips 300 electron microscope. RESULTS Infections

in Adult

Mosquitoes

Diplokaryotic meronts associated with oenocytes were observed in newly emerged adults of both sexes (Fig. 2). These developed in both males and females to form binucleate spores which were cylindrical and slightly bent (Figs. 1B, 3 ). However, the formation of spores from the diplokaryotic stages did not proceed synchronously within a batch of infected adults as dissections showed that there was considerable variability in the duration of this developmental process between different individual infected mosquitoes. For example, in one batch of adults examined within 48 hr of emergence, six out of 12 infected females and seven out of 12 infected males had mature spores. On the other hand, some individual specimens of both sexes dissected 2-3 weeks after emergence had only diplokaryotic meronts or immature spores. Mature spores developed in some females prior to a blood meal but in some other infected females mature spores had not formed following oviposition after the first blood meal. Transovuriully Transmitted Mosquito Larvue

Stages in

Some of the larval progeny from egg batches laid by females infected with mature binucleate spores developed patent fat body infections and ultimately produced meiospores. The earliest stages in 1st and 2nd instar larvae were diplokaryotic meronts (Figs. lD, 4, 11) which were sometimes observed within blood cells (Figs. lE, 5). In late 2nd and early 3rd instar larvae the parasite enters the fat body cells where it commences to sporulate. During the early stages of sporogony meiotic configurations of the nuclei similar to those re-

SWEENEY,

-I!

GRAHAM,

AND HAZARD

phorous vesicle forms around the developing sporont (Figs. lG, 12) which then undergoes two mitotic divisions to form T quadrinucleate and octonucleate sporonts \ (Figs. lH-1K). Cytokinesis may take place at either the binucleate, quadrinucleate, or octonucleate stages (Figs. 7-9). The octonucleated sporonts develop into eight uninucleate sporoblasts (Figs. 9, 13) which form eight meiospores (Figs. 10, 14, 15) while still enclosed within the sporophorous vesicle. In the normal course of infection the fat body of the thorax and ab/ domen becomes completely filled with masses of meiospores and the larvae die in LARVAE i the 4th instar. However, some infected $3 larvae die in the pupal stage and in rare instances mature meiospores have been obI served in surviving adults from infected COPEPOOS larval batches. Patent fat body infections develop in larvae of both sexes, the rate of infection in larval progeny of females with binucleate spores varying from 0 to 100%. Some larvae from transovarially infected egg batches do not develop fat body infections but develop benign infections consisting of diplokaryotic stages within the oenocytes. These infected oenocytes persist to the adult stage and lead to the forFIG. 1. Life cycle of Amblyospora dyxenoides. mation of mature binucleate spores in both A-C: Development of binucleate spores in male and males and females which, in the latter infemale C. annulirostris mosquitoes. A, binucleate stance, are able to initiate another transsporonts; B, C, spores. D-M: Transovarially transmitted infections in C. annulirostris larvae. D, diploovarially transmitted cycle. A variable prokaryotic meront in early instar larvae: E, diploportion of male and female larvae from inkaryotic stages in host blood cell; F, diplokaryotic fected egg batches survive uninfected to cells invade fat body; G, meiosis during early stages of the adult stage. In some individuals both fat sporogony in fat body tissue; H, I, binucleate body and oenocytic infections may proceed sporonts; J, quadrinucleate sporont; K, eight sporoblasts within sporophorous vesicle; L, M, meiospores. simultaneously and, in rare cases, this has N-S: Developmental stages in copepods. N, uninuresulted in the emergence of males and fecleate meronts; 0, binucleate stage; P, quadrinucleate males possessing meiospores derived from stage; Q, binucleate and multinucleate sporonts; R, octonucleate sporonts as well as binucleate uninucleate sporoblast; S, uninucleate copepod spore. T-W: Infections in larvae derived from copepods. T, spores derived from oenocytic infections. uninucleate meront; U, gamete; V, fusion of gametes; In some instances none of the larvae from W, dipiokaryotic meront. an egg batch laid by a female with binucleate spores developed meiospore infecfrom these ported by Hazard and Brookbank (1984) tions but some individuals batches had diplokaryotic stages in larval have been observed (Fig. 6) and synaptooenocytes which persisted to the adult nemal complexes have been demonstrated by electron microscopy (Fig. 12). A sporo- stage.

LIFE CYCLE OF Amblyospora

dyxenoides

sp. IWY.

49

FIGS. 2-10. Light micrographs of Amblyospora dyxenoides infecting adults and larvae of Cu/r.r cmnulirostris (Figs. 2, 3, 6 stained with lactoacetic orcein; Figs. 4, 5, 7-9 Giemsa stained: Fig. IO Heidenhain’s hemotoxylin stained). (All x 1800.) Fig. 2. Diplokaryotic cell in adult female; Fig. 3. binucleate spores in adult female; Fig. 4, diplokaryotic meront in first instar larva; Fig. 5. diplokaryotic meronts (m) in blood cell of first-instar larva (hen = host cell nucleus; c = host cell cytoplasm): Fig. 6, early sporont undergoing meiosis; Fig. 7. quadrinucleate sporont without cytoplasmic cleavage; Fig. 8, quadrinucleate sporont in which cytoplasmic cleavage has already occurred; Fig. 9, eight sporoblasts within sporophorous vesicle; Fig. 10, eight meiospores within sporophorous vesicle.

hfections

in Copepods

early as 4 days to as late as 10 days after exposure to larval meiospores and usually Meiospores from patently infected mos- divide by binary fission to form binucleate quite larvae are infectious to Mesocyclups (Figs. 10, 17) and sometimes quadrinual&cans copepods. The first stages ob- cleate cells (Figs. IP, 18). Plasmodia with served in Giemsa smears of infected co- more than four nuclei have been observed pepods were spherical meronts with a only on rare occasions. The early stages single large nucleus (Figs. lN, 16, 25). multiply within the ovarian tissue of the They have been seen in copepods from as copepods and then undergo sporulation.

SWEENEY,

GRAHAM,

AND HAZARD

FIGS. 1 I- 15. Electron micrographs of transovarially transmitted Amb/yospora dyxenoides in Culex annu[irostris larvae. Fig. 11, Diplokaryotic meront (X 12,800); Fig. 12, binucleate sporont (x5200); Fig. 13, octonucleate sporont (x5200); Fig. 14. uninucleate sporoblast (X 13,000); Fig. 15, mature meiospores (x4750). N,, N,, diplokaryotic nuclei; N, nucleus; MG, metabolic granules; PF, polar filament; P, polaraplast; SC, synaptonemal complex: SV, sporophorous vesicle.

LIFE CYCLE OF Amblyospora

dyxenoides

sp.

nov.

51

dyxenoides in Mesocyclops ahcans (all Giemsa FIGS. 16-24. Light micrographs of Amblyospora stained, all x 1800). Fig. 16, uninucleate meront; Fig. 17, dividing meront; Fig. 18, quadranucleate plasmodium; Fig. 19, 20, dividing binucleate sporonts; Fig. 21, trinucleate sporont: Fig. 22, rosette of 8 sporonts; Fig. 23, uninucleate sporoblast; Fig. 24, mature spores.

Binucleate cells sometimes divide to form two uninucleate sporonts in which the dividing cells elongate and the nuclei migrate to opposite poles (Figs. IQ, 19, 20, 26, 27). In some instances multinucleate stages have been observed in which the cytoplasm cleaves in a rosette pattern (Figs. lQ, 22) to form clavate uninucleate sporonts (Figs. lR, 23). Mature copepod spores are lanceolate and sometimes slightly curved (Figs. 24, 30). They are formed within a sporophorous vesicle from 7 to 14 days

after exposure to larval meiospores. Infected copepods have a white localized appearance which is not as prominent as patent fat body infections in mosquito larvae. They may remain alive with mature spores for several days before succumbing to the infection. Copepod-Transmitted Mosquito Stages The earliest stages observed in Giemsa smears of 2nd and 3rd instar mosquito larvae exposed to infected copepods were

52

SWEENEY,

GRAHAM,

AND HAZARD

FIGS. 25-30. Electron micrographs of Amblyospora dyxenoides in Mesocyclops albicans. Fig. 25, uninucleate meronts ( x 7000); Fig. 26, 27, dividing binucleate sporonts (both X 5150); Fig. 28, uninucleate stages (X 6300); Fig. 29, sporoblast (x 9000); Fig. 30, mature spores ( x 4450). N, nucleus, PF, polar filament; P, polaroplast; SV, sporophorous vesicle.

LIFE

CYCLE

OF Amblyospora

small spherical to oval shaped cells with a single large nucleus (Figs. IT, 31, 33). These cells divide by binary fission (Fig. 32). The next developmental stages are pyriform uninucleate cells (Fig. 33) which, in some Giemsa specimens (Figs. 34-36), appeared to undergo plasmogamy. Cytoplasmic fusion seemed to occur in the region adjacent to the nuclei and resulted in the formation of diplokaryotic meronts. These appeared identical to the larval diplokaryotic stages described above (Figs. 4, 5). They multiplied in the larval oenocytes (Fig. 37) and later formed binucleate sporonts and spores in the adults of both sexes. However, in some individual mosquitoes the uninucleate cells persist to the adult stage. Occasionally diplokaryotic cells as well as uninucleate cells were observed together in Giemsa smears of some infected male and female mosquitoes. Copepod derived infections in mosquitoes are expressed in the larval progeny of infected females in the same way as the transovarially transmitted stages described previously: patent fat body infections develop in some male and female larvae; other larvae develop oenocytic infections; and some larvae remain free of infection. SYSTEMATICS Amblyospora dyxenoides sp. nov. Hosts. Culex annulirostris Skuse and Mesocyclops albicans (Smith). Diagnosis. Binucleate spores in adult female C. annulirostris are responsible for

transovarial transmission to larval progeny. Unlike other described species of the genus, mature binucleate spores sometimes develop in females prior to a blood meal and they are also formed in infected males. Transovarially transmitted infections may develop in oenocytes or in fat body tissue in both male and female larvae. Oenocytic infections persist as diplokaryotic meronts to the adult stage where they develop to binucleate spores and commence another transovarially transmitted cycle. In fat

dyxenoides

sp. nav.

53

body infections, the parasite undergoes meiosis during a complex sequence of sporulation stages similar to other species

of Amblyospora which have been studied (Hazard et al., 1979; Hazard and Brookbank, 1984; Andreadis, 1983, 1985a) and ultimately forms haploid uninucleate meiospores in groups of eight enclosed by a

sporophorous vesicle. Meiospores are infectious to M. albicans copepods. Following merogonial multiplication within ovarian tissue of the copepod host, the parasite develops into lanceolate uninucleate spores. These “copepod” spores are infectious to C. annulirostris larvae. The specific epithet dyxenoides means “two hosted” in reference to the discovery of the intermediate host in this species. Type slides. Slides will be deposited with the International Protozoan Type Slide Collection, Smithsonian Institution, Washington, D.C. Spore dimensions. Binucleate spores, 10.6 2 2.8Cl.m x 3.1 2 0.6Cl.m; meiospores, 6.3 -+ 0.4pm x 4.1 + 0.4pm; copepod spores 12.5 t l.Oum x 4.6 I 0.4Cl.m. DISCUSSION The complexity of the life cycles of Amblyospora parasites was first appreciated by workers who observed differences in host-parasite relationships influenced by sex of the host and the expression of transovarially transmitted oenocytic and fat body infections in different mosquito species (Kellen and Wills, 1962; Kellen et al., 1965; Chapman et al., 1966). Understanding of these relationships permitted the laboratory maintenance of Amblyospora in two mosquito species (Culex tarsalis and Culex salinarius) via transmission of the infections through successive female generations. Nevertheless, the complete life cycle (particularly the fate of the uninucleate spores produced in larval fat body infections) remained enigmatic. Chromosome studies by Hazard et al. (1979) showed that meiosis occurred during

54

SWEENEY,

GRAHAM,

AND HAZARD

dyxenoides in C. annulirostris larvae infected via FIGS. 31-37. Light micrographs of Amblyospora copepods. (All Giemsa stained. Figs. 31-36 x2700; Fig. 37 x 1050). 31, uninucleate meront; Fig. 32, dividing meront; Fig. 33, gamete (g) and uninucleate meront (m); Fig. 34, 35, 36, cytoplasmic fusion of gametes; Fig. 37, diplokaryotic meronts in oenocyte (on, oenocyte nucleus).

LIFE CYCLE OF Amblyospora

dyxenoides

sp. nov.

55

sporulation of fat body infections thereby (Hazardia milleri, Culicospora magna, and an undescribed microsporidium in Aedes indicating that the resulting uninucleate spores were haploid meiospores. This led aegypti) in which horizontal transmission was known to occur (Hazard et al., 1985). these authors to speculate on the existence The earliest stages seen during the infecof an alternate host in which the diploid tion in all three parasites were uninucleate state was resumed. In later work, Hazard and Brookbank (1984) made detailed cyto- stages in blood cells which later invaded logical observations which included DNA the hemolymph. Light microscope and measurements of nuclei of the stages pre- electron microscope observations demonstrated that they were gametes, as fusion of ceding and during sporulation in infected the cytoplasm of these cells was seen and larvae. They clearly demonstrated that karyogamy took place in diplokaryotic the nuclei later came together in the diplomeronts of young larvae prior to meiosis karyotic arrangement. In C. magna and the infecting A. aegypti these and sporulation. Furthermore, this study microsporidium fully described the unusual meiosis of stages developed into binucleate spores in Amblyospora which involves the mingling adult females which are similar to those of of chromosomes of two nuclei at pachytene Amblyospora and which also are responand provided conclusive proof that the sible for transovarial transmission to the larval spores are haploid. next generation. These studies suggested Andreadis and Hall (1979a) undertook the route by which horizontal transmission mathematical analysis of transmission rates of Amblyospora could proceed from hapand survival rates of C. salinarius infected loid to the diploid condition. with an Amblyospora sp. Based on a reAt the same time as these studies in the duction in egg hatch of 52% and a transmisUnited States, the intermediate copepod sion rate averaging 90% they concluded host of Amblyospora dyxenoides was disthat transovarial transmission is not suffi- covered in Australia (Sweeney et al., 1985). cient to maintain the parasite in the mos- The parasite was transmitted successfully quito population and they postulated that via copepods to an uninfected laboratory the existence of an alternate host provided colony of C. annulirostris. These infections a mechanism for parasite maintenance. developed into binucleate spores in adults More recently, in field epizootiological in- and the larval progeny developed fat body vestigations of Amblyospora infecting the infections of typical Amblyospora meiounivoltine mosquito Aedes stimulans, An- spores which were infectious to copepods. dreadis (1985a) reported the presence of The finding of the intermediate host has uninucleate cells in early instar larvae completed the last major link in the life which developed in gastric cecal cells and cycle of Amblyospora. The Australian in oenocytes to form binucleate spores in work on A. dyxenoides in C. annulirostris adult females. The incidence of these has been confirmed by transmitting A. calistages increased in the larval population as fornica infecting C. tarsalis to Mesocythe larvae grew from 1st to 4th instar. This claps leukarti and Macrocyclops alhidus was the first documented proof of hori(Becnel, 1986) and by Andreadis (1985b) zontal transmission in Amblyospora and al- who transmitted Amhlyospora sp. infecting though the source of these infections was Aedes cantator to the copepod Acanthocynot known, the author speculated that they cfops vernafis. So it is clear that the inwere due to an undiscovered alternate volvement of copepods in Amblyospora life host. cycles is of wide significance. During the same period laboratory Laboratory maintenance of Amblyostudies were conducted in Louisiana of spora infecting C. annulirostris in mosquito three mosquito parasitic microsporidia and copepod hosts has revealed the major

56

SWEENEY,

GRAHAM,

developmental sequences of the parasite in these two invertebrates. The copepod cycle involves relatively simple merogonial multiplication by binary and multiple fission and ultimately results in the formation of uninucleate spores. There was no evidence of formation of gametes and karyogamy which indicates that the copepod spores are haploid. The horizontally transmitted Amblyosporu stages in the mosquito host are similar to Giemsa-stained gamonts and gametes described by Hazard et al. (1985) for C. magna, H. milleri, and Microsporidium sp. in A. aegypti. The light micrographs of Giemsa smears shown in Figs. 34, 35, and 36 would appear to indicate cytoplasmic fusion of adjacent gametes. This interpretation, that gametogenesis and plasmogamy take place in larval infections of Amblyospora derived from spores formed in copepods, is consistent with the observations of Hazard et al. (1985) and Becnel (1986) for horizontal transmission of other microsporidia and would seem to be the mechanism by which the parasite returns from the haploid to the diploid state. However, this needs to be confirmed by ultrastructural observations to compare the early larval stages of Amblyospora with those of the other microsporidia studied by Hazard and co-workers. Subsequent development of copepod derived infections in mosquitoes is similar to transovarially acquired infections. Diplokaryotic cells multiply in larval oenocytes and persist to the adult stage and then form binucleate spores which infect the ovaries. The persistence of uninucleate cells to the adult stage in some individual mosquitoes implies that the parasite is not always synchronized with development of the host. It may be that infections acquired in late-instar larvae do not have sufficient time for their development to be completed before adult emergence. Our observations are based on newly emerged adults and on females which have completed the first gonotrophic cycle. It is not yet known whether uninucleate stages in adults are

AND

HAZARD

able to continue their development to diplokaryotic stages and finally to form binucleate spores during subsequent gonotrophic cycles. The problems of microsporidian taxonomy have been reviewed by Hazard et al. (1981) who pointed out the lack of knowledge of life cycles, which are extremely variable and complex in some groups of microsporidia, as well as the difficulties of determining adequate diagnostic criteria for identification and taxonomic placement. These problems are particularly evident in Amblyospora as there is considerable variability between different mosquito species in the expression of fat body and oenocytic infections according to sex of the host. Nevertheless, the morphologies of the spores and vegetative stages in the different mosquito hosts are very similar. Workers have been increasingly reluctant to assign species names to Amblyospora because of these basic similarities in morphology and the inability to perform transmission experiments between hosts. The revelation of the complete life cycle has obvious implications for these problems as it should now be possible to conduct transmission experiments to define the limits of the host range of different Amblyospora species. Of the mosquito sequences of Amblyosporu which have been investigated, that which most closely resembles the development of A. dyxenoides is Amblyospora sp. infecting A. cantator (see Andreadis, 1983). Both parasites may develop either fat body or oenocytic infections in both male and female larvae and adults. However, unlike A. dyxenoides, the species infecting A. cant&or does not form mature binucleate spores in males or in females prior to a blood meal. It would be of considerable interest to compare the limits of specificity of A. dyxenoides in copepods and mosquitoes with that of A. californica, the type species of the genus, which infects C. tarsalis. Both mosquito hosts share common biological

LIFE CYCLE OF Amblyosporu

characteristics and C. tarsalis occupies an ecological niche in North America similar to that of C. annulirostris in Australia. The two mosquitoes are widespread and abundant during summer months in the temperate areas of their respective distribution ranges. Both are involved in transmission of human arboviruses: C. tarsalis being a vector of Western encephalitis and St. Louis encephalitis, whereas C. annulirostris is a vector of Australian encephalitis. Both mosquitoes breed in a wide range of clean and polluted habitats and are similar in appearance, being robust, dark Cufex each with a median white band on the proboscis. Amblyospora development in these two mosquitoes differs in that transovarially transmitted infections of A. californica result in oenocytic stages in female C. tarsalis larvae and fat body infections in male larvae, whereas both sexes may develop either type of infection in larvae of C. annulirostris infected with A. dyxenoides. Although cross infection experiments between these two parasites and their natural hosts have not yet been conducted, it is considered that these differences are sufficient to warrant a formal description of the Australian species at this time. ACKNOWLEDGMENTS This paper is published with the approval of the Director General of Army Health Services. The research received financial support from the UNDPIWHOI World Bank Special Programme for Research and Training in Tropical Diseases, from the Australian National Health and Medical Research Council, and from the Australian National Diseases Control Programme. We thank Mr. J. J. Becnel and Dr. T. G. Andreadis for critical review of the manuscript.

REFERENCES ANDREADIS, T. G. 1983. Life cycle and epizootiology of Amblyospora sp. (Microspora; Amblyosporidae) in the mosquito. Aedes cantutor. J. Protozool., 30, 509-518. ANDREADIS, T. G. 1985a. Life cycle, epizootiology, and horizontal transmission of Amblyospora (Microspora: Amblyosporidae) in a univoltine mosquito, Aedes stimulans. J. Invertebr. Pathol., 46, 31-46. ANDREADIS, T. G. 1985b. Experimental transmission of a microsporidian pathogen from mosquitoes to an

alternate USA,

51

sp. nov.

dyxenoides

copepod

host. Proc.

Natl.

Acad.

Sci.

82, 5514-5511.

ANDREADIS, T. G., AND HALL, D. W. 1979a. Significance of transovarial infections of AmbIyosporu sp. (Microspora: Thelohaniidae) in relation to parasite maintenance in the mosquito Cutler sulinuriru. J Invertebr.

Pathol.,

34, 152- 157.

ANDREADIS, T. G., AND HALL, D. W. 1979b. Development, ultrastructure and mode of transmission of Amb/yospora sp. (Microspora) in the mosquito. .1. Protozool..

26, 444-452.

BECNEL, J. J. 1986. Microsporidian sexuality in culitine mosquitoes. In “Fundamental and Applied Aspects of Invertebrate Pathology” (R. A. Samson. J. M. Vlak, and Dick Peters, Eds.,) pp. 331-334. Fourth Int. Colloquium Invertebr. Pathol.. Veldhoven, The Netherlands. CHAPMAN, H. C., WOODWARD, D. B., KELLEN, W. R., AND CLARK, T. B. 1966. Host parasite relationships of Thefohuniu associated with mosquitoes in Louisiana (Nosematidae: Microsporidia). J. /)Ivertebr.

Puthol..

8, 452-456.

HAZARD, E. 1.. ANDREADIS. T. G., JOSLYN, D. J.. AND ELLIS, E. A. 1979. Meiosis and its implications in the life cycles of AmbIyospora and ParatheIohank (Microspora). J. Pnrasitol., 65, 117- 122. HAZARD, E. I., AND BROOKBANK, J. W. 1984. Karyogamy and meiosis in an Amhlyospora sp. (Microspora) in the mosquito, Culex salinarius Coquillet. J. Invertebr. Pathol., 44, 3- 1 I HAZARD, E. I., ELLIS. E. A., AND JOSLYN. D. J. 1981. Identification of microsporidia. In “Microbial Control of Pests and Plant Diseases, 1970-1980” (H. D. Burges, Ed.), pp. 163-182. Academic Press, New York. HAZARD, E. I., FUKUDA. T.. AND BECNEL, J. J. 1985. Gametogenesis and plasmogamy in certain species of Microspora. /. Invertehr. Pathol.. 46, 63-69. HAZARD, E. I.. AND OLDACRE. S. W. 1975. “Revision of Microsporidia (Protozoa) Close to Thelohania, with Descriptions of One New Family, Eight New Genera and Thirteen New Species.” U.S. Dep. Agric. Tech. Bull. 1530, 104 pp. KELLEN, W. R.. CHAPMAN, H. C.. CLARK, T. B., AND LINDEGREN, J. E. 1965. Host-parasite relationships of some Thelohania from mosquitoes (Nosematidae: Microsporidia). J. lnverfebr. Parho(. . 7, 161-166. KELLEN. W. R., CHAPMAN, H. C.. CLARK, T. B.. AND LINDEGREN, J. E. 1966. Transovarian transmission of some Thelohania (Nosematidae: Microsporidia) in mosquitoes of California and Louisiana. J. Invertebr.

Pathol.,

8, 355-359.

KELLEN, W. R., AND WILLS, W. 1962. The transovarian transmission of Thelohania californica Kellen and Lipa in Culex tars&s Coquillett. J. Insect Pathol., 4, 321-326. SWEENEY, A. W., HAZARD, E. I.. AND GRAHAM, M. F. 1985. Intermediate host for an Amblyosporu sp. (Microspora) infecting the mosquito, C~lex annulirostris. J. Invertebr. Parhol., 46, 98- 102.