Proc. Nat. Acad. Sci. USA Vol. 72, No. 2, pp. 693-696, February 1975

Life History of Coelomomyces psorophorae ( pathogen/Cyclops/sexuality/alternate hosts/alternation of generations) HOWARD C. WHISLER*, STEPHEN L. ZEBOLD*, AND JOSEPH A. SHEMANCHUKt * Department of Botany, University of Washington, Seattle, Wash. 98195; and t Lethbridge Research Station, Canada Department of Agriculture, Lethbridge, Alberta, Canada Communicated by Ralph Emerson, December 9, 1974

ABSTRACT The mosquito parasite, Coelomomyces mosquitoes. These results suggested that Cyclops might be psorophorae (Blastocladiales, Chytridiomycetes) alternates This obligately between the larvae of inornata and the serving as an alternate host for Coelomomyces (13). copepod Cyclops vernalis. Isogametes, derived from paper presents evidence confirming this hypothesis and out- heterothallic, wall-less gametangia which develop in the lines a previously unknown life history. copepod, fuse to produce a diploid zygote that subse- quently infects the mosquito host. Zoospores from the MATERIALS AND METHODS resistant sporangia which are produced in the haemocoel A colony of Culiseta inornata was developed from egg rafts of the mosquito infect the copepod. A tentative life- history is proposed and implications of these discoveries collected from infection sites near Lethbridge and Fincastle, for the biology, , and possible role of Coelo- Alberta, Canada. Rearing techniques were based on those of momyces in biological control are discussed. McLintock (14). The water used for larval rearing and infec- tion trials contained 0.5 g of NaHCO3, 0.25 g of MgSO4. 7H20, The water mold Coelomomyces (Chytridiomycetes) is a host- 0.1 g of KCl, and 0.5 g of Ca(NO3)2*4H20 per liter of distilled specific, obligate parasite of the larvae of mosquitoes, black water, with a final pH of 8.2. Larvae were fed a mixture of , chironomids, and tabanids (1-5). The fungus develops in 60% enriched flour, 25% nonfat dried milk, 10% yeast ex- the haemocoel of the host as a weakly-branched coenocytic tract, and 5% liver powder as required for optimal growth. thallus. At maturity, this vegetative structure differentiates Resistant sporangia (RS) from 10 dead larvae, either from into a number of thick-walled resistant sporangia (RS) (6). field or laboratory infections, were washed in distilled water, Under appropriate conditions, each of these sporangia will collected on NC Millipore filters, placed in individual moist give rise to several hundred posteriorly uniflagellate zoo- chambers, and stored at 5°. When needed, sporangia were spores. The genus includes pathogens of the major genera of transferred to a dilute salt solution (15) and held at 20° under mosquitoes and may cause significant epizootics in mosquito continuous illumination [with cool-white fluorescent light at populations. Preliminary field trials have suggested that 65 foot candles (699.7 lx) ]. After 5 days, 80% of the sporangia Coelomomyces may have potential as a biological control were ready to release zoospores. agent of mosquitoes (7-11). Efforts to explore this possibility Infection trials were typically carried out in plastic pans have encountered significant difficulties when workers at- (27 cm in diameter) containing 1.8 liters of water and held at tempted to establish infected colonies in the laboratory. 20 4 20 in diffuse light. Bottom material (75 cm8) from the Larvae bathed in high densities of zoospores from the RS did colony rearing trays was autoclaved and added to each pan. not become infected. Couch (12) has developed a technique Most mosquito infection trials were initiated with 125 late for mass production of infected larvae but the conditions second-instar larvae and RS from one filter. Larvae were fed leading to infection are complex and include a number of 0.2 g of food every other day. Addition-of the food also initiates living components other than the host and its parasite. zoospore release by lowering the oxygen tension. Our studies have utilized C. psorophorae in Culiseta inornata. A population of Cyclops vernalis from the main infection site This species. of Coelomomyces is a common parasite of mos- near Fincastle, Alberta, has been maintained along with the quitoes in the irrigated regions of southern Alberta (10). mosquito larvae. Infected copepods were reared in 37- by 48- Establishment of an infected colony in the laboratory was cm pans containing the standard water and bottom material achieved by use of the Couch procedure (12) along with addi- and received about two RS-filters per week. tions of various components of the biota from the original Material was prepared for electron microscopy by fixation habitat, e.g., microalgae, , etc. Attempts to stan- with glutaraldehyde and post-fixation in osmic acid (16, 17). dardize infection rates by controlling the environment, density of the RS-zoospores, or age and condition of the host were RESULTS unsuccessful. Attempts to reduce the biotic complexity of our The first indication that copepods might be involved in the gross cultures typically resulted in loss of infection. The Coelomomyces infection problem came from pans which con- source of this problem was revealed when single components tained Cyclops as well as RS and mosquitoes (Table 1,A). If of the complex or "gross" system were added individually to these copepods were serving as an alternate host for Coelo- "clean" pans containing only the fungus and host . momyces, then one might expect that from pans with a When the copepod Cyclops vernalis was added to pans con- history of high infectivity should be able to transmit the dis- taining RS and larvae, infection was obtained. Control pans ease without the addition of other fungal inocula. This pre- lacking this crustacean consistently failed to yield infected sumption was supported when carefully washed copepods from infective pans were added to pans containing mosquito Abbreviation: RS, resistant sporangia. larvae but lacking RS and RS-zoospores (Table 1,B). A direct 693 Downloaded by guest on September 25, 2021 694 Botany: Whisler et al. Proc. Nat. Acad. Sci. USA 72 (1975)

A Three isoga etes j.e FIG.-tFIG. 1. Threeon4*isogametes and one ofpsoro-t88*HC.. mtapsoro- FIG. 2. Section of gametangium of C. psorophorae in Cyclops

phoras. The recently fused zygote with two flagella still display- vernalis. Wall-less gametangium is appressed to host tissue two adjacent nuclear caps and two side bodies (groups of re- (bottom). Cone shaped nucleus (N), nuclear (NC), side body fractile lipid granules). X2070. (SB), and basal mitochondrion (M) characteristic of the of Coelomomyces are evident, but obvious cleavage furrows are search for the fungus in these copepods revealed the presence not evident. X 14,400. of an unknown phase in the life history of Coelomomyces. Some of the animals, which had recently ceased swimming, them to pans holding the mosquito host (Table 1,C). After 5 were packed with flagellated swarm cells that will be referred days many of the larvae contained hyphal bodies which sub- to as planonts (Fig. 1). Dissection of the Cyclops indicated sequently developed into the resistant sporangia typical of C. that these planonts had been cleaved out of wall-less thalli psorophorae. These results confirmed that the fungus had two which had developed in the haemocoel of the host (Fig. alternate hosts, with the spores from the resistant sporangia 2). The active planonts filled all available spaces in the cepha- infecting the copepods, and the planonts from the copepods lothorax, antennules, legs, and abdomen, and after a variable infecting the mosquito. Further evidence for this cycle was period of swarming, they escaped to the external environment obtained by exposing the two hosts to the two types of swarm- through a tear in the host's integument. The animal, typically ers (Table 1,D). The results indicate that the alternation of an adult or near adult (4-5th copepodite) died following this different hosts is obligatory; transmission from copepod to traumatic event. copepod or mosquito to mosquito was not seen. A test of the infectivity of these planonts was made by The planonts from the copepod had either one or two poste- pooling the planonts from several copepods and distributing riorly inserted flagella and their general morphology resembled TABLE 1. Infection trials with Coelomomyces psorophorae

Potential Other Percent Total no. No. tests with infection Trial host components Infection infection host assayed No. tests attempted A Mosquito* + RS/Zoospore, + Cyclops Yes 22 212 3/3 Mosquito + RS/Zoospore No 0 180 0/3 B Mosquito + Cyclops (I)t Yes 54 92 2/2 Mosquito + Cyclops (NI) No 0 123 0/2 C Mosquito + Spores from Cyclops Yes 34 137 3/3 Mosquito No 0 50 0/1 D Cyclops + RS/Zoospore Yes 32 141 § 3/3 Mosquito + RS/Zoospore No 0 327 0/6 Mosquito + Spores from Cyclops Yes 45 292 6/6 Cyclops + Spores from Cyclops No 0 360§ 0/3 Cyclops (Control-no spores) No 0 208§ 0/3 Mosquito (Control-no spores) No 0 300 0/6 E Mosquito Gametes No 0 70 0/611 Mosquito Zygotes and gametes Yes 30 65 6/61 * Mosquito, larvae of Culiseta inornata. t I, Cyclops from pans showing infection. t NI, Cyclops from pans without record of infection. § Adult Cyclops vernalis. ¶ Each test used gametes or gamete/zygote populations from different, individual Cyclops hosts. Tests run in 100 mm X 50 mm crys- talizing dishes. Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 72 (1975) Life History of COelOMOMYCe8 695

that of the zoospores from the RS of C. psorophorae (Fig. 1). were uninucleate and The planonts with a single flagellum A measured 5 X 9,jm, whereas, the biflagellate planonts mea- sured 6 X 12 Mm and contained either one or two nuclei. Although improper cleavage could explain this nuclear difference, sexual reproduction, involving uniflagellate iso- gametes that fuse to form a biflagellate zygote, appeared more likely. This was confirmed by the following observations: (1) Dissection of infected copepods at the earliest sign of planont activity provided only uniflagellate planonts. (2) Biflagellate planonts appeared after a period of swarming either in or out- side the host. (3) Uniflagellate planonts (gametes) were seen to fuse and form a biflagellate zygote. Although most infected copepods released both gametes and zygotes (40-90% zygotes), occasional individuals liberated only gametes which did not undergo fusion. This lack of sexual E competence might be explained on the basis of inhospitable mating conditions or the presence of a mating compatibility system. The latter situation appears to be the case. When the persistently uniflagellate populations were combined, zygotes promptly formed in approximately one-half of the crosses. Reciprocal crosses between five different uniflagellate popula- tions suggest that C. psorophorae is heterothallic, since all gamete populations that were mutually incompatible fused FIG. 3. Life cycle of C. psorophorae. Zygote (A) infects larva with each population of the opposite mating type. of Culiseta inornata (B) leading to development of hyphal bodies, The presence of these pure gamete populations allowed us mycelium and, ultimately, thick-walled resistant sporangia. to test the infectivity of the gamete and zygote against mos- Under appropriate conditions these sporangia (C) release zoo- quito larvae. Larvae in small containers were exposed to spores of opposite mating type (D) which infect the alternate gametes or to populations of planonts containing a preponder- host, Cyclops vernalis (E). Each zoospore develops into a thallus ance of zygotes. The results (Table 1,E) indicate that the and, eventually, gametangia. Gametes of opposite mating type gametes will not infect the larvae, but the zygotes, by infer- (F) fuse either in or outside of the copepod to form the mosquito- ence, can. iifecting zygote. Microscopical observations of populations of fusing gametes number of unique features that distinguish them from other indicate that fusion commences soon after swarming is groups of fungi. These characters include a highly organized initiated. Plasmogamy, which occurs at a relatively oblique zoospore with a membrane-bounded nuclear cap, a resistant angle, is quickly followed by reorientation of the basal bodies sporangium with a multilayered wall, and if sex is involved, and flagella until they are closely parallel. The two flagella of fusion between two motile gametes (18). Coelomomyces con- active zygotes are so tightly appressed that they appear to be a forms to these characteristics. Emerson (19) has noted three single unit. Populations examined during the early stages of different life cycles in the blastocladialean genus Allomyces. fusion contain numerous zygotes with two sets of organelles The Euallomyces type, with independent sporophyte and (e.g., nuclei, nuclear caps and side bodies) arranged in a paral- gametophyte generations; the Cystogenes type with the game- lel or adjacent manner (Fig. 1). The side bodies coalesce first, tophyte reduced to a cyst; and the Brachyallomyces type followed by fusion of the nuclear caps and nuclei. It seems which lacks the gametophyte entirely. The absence of sexu- probable, therefore, that the mosquito larvae are infected by a ality in the latter type is apparently related to the failure of zygote with a diploid nucleus. meiosis in the resistant sporangia. Meiosis occurs in the RS of the first two life history types (20). Subsequent studies DISCUSSION on other members of the Blastocladiales [e.g., Blastocladiella, The existence of alternate hosts in the life cycle of a parasite is Catenaria, Blastocladia (19, 21-24) ] suggest that this pattern a theme that recurs in many of the major groups of parasitic of three life histories is widespread in the order and may be organisms. In the mycological world, the rust fungi appeared recognized as an ordinal characteristic (18). to have a monopoly on this approach to parasitological The development of C. psorophorae seems to fit the Eual- success. It now appears that the Blastocladiales have evolved lomyces type of life cycle and displays a number of similarities their own variation on this theme of heteroecism. The results to the life history of Blastocladiella variabilis (21). Although presented in Table 1 indicate that alternation between mos- proof that meiosis occurs in the RS is needed, the presence of quito and copepod is obligatory. This conclusion is also sup- two vegetative phases separated by isogamy and RS-zoospores ported by the numerous unsuccessful attempts in various provides strong support for the following interpretation of the laboratories to infect mosquitoes with the zoospores from life history of C. psorophorae (Fig. 3). The zygote infects the resistant sporangia. Less study has been devoted to the infec- host mosquito and gives rise to a diploid, coenocytic thallus. tion of the copepods, but the infection rate of our copepod- After 4 days' growth in the body of the larva, the wall-less containing "gross" pans tends to diminish quickly if sporangia mycelium differentiates into thick-walled RS. Thin-walled are not added periodically. This would not be the case if either sporangia, which are found on the sporophyte of Allomyces the gametes or the zygotes were re-infecting the Cyclops host. and other members of the Blastocladiales have not been seen The water molds included in the Blastocladiales possess a in our C. psorophorae. They have been reported, however, in Downloaded by guest on September 25, 2021 696 Botany: Whisler et al. Proc. Nat. Acad. Sci. USA 72 (1975)

mosquitoes infected with other species of Coelomomyces (8). 2. Garnham, P. C. C. & Lewis, D. J. (1959) "Parasites of reference to If the mature resistant sporangia atre placed in appropriate British Honduras with special leishmaniasis," Trans. Roy. Soc. Trop. Med. Hyg. 53, 12-35. conditions, they will germinate by the two-step germination 3. Weiser, J. & VAvra, J. (1964) "Zur Verbreitung der Coelo- process characteristic of the genus. Based on comparisons to momyces-Pilze in europiischen Insekten," Z. Tropenmed. other genera of the order, we assume that meiosis occurs Parasitol. 15, 38-42. during germination of the RS. Preliminary aceto-orcein 4. Koval, E. Z. & Andreeva, R. V. (1971) "On studying pathogenic micoflora of horse flies," Dopov. Akad. Nauk. squashes indicate that nuclear divisions are occurring at this Ukr. RSR Ser. B 11, 1042-1044. time but the problems presented by the small size of the chro- 5. Manier, J.-F., Rioux, J.-A., Coste, F. & Maurand, J. (1970) mosomes call for further work with a variety of cytological "Coelomomyces tuzetae n. sp. (Blastocladiales-Coelomomy- techniques. After infecting the copepod, the RS-zoospore, or cetaceae) parasite des larves de Chironomes (Diptera- Ann. Parasitol. 119-128. meiospore, develops into a haploid thallus which eventually Chironomidae)," 45, 6. Whisler, H. C., Shemanchuk, J. A. & Travland, L. B. differentiates gametes of the appropriate mating type. The (1972) "Germination of the resistant sporangia of Coelo- cell wall is absent during gametogenesis and the isogametes momyces psorophorae," J. Invert. Pathol. 19, 139-147. are in an advanced state of differentiation before cleavage is 7. Walker, A. J. (1938) "Fungal infections of mosquitoes, initiated (Fig. 2). If the host contains thalli of opposite mating especially of Anopheles costalis," Ann. Trop. Med. Parasitol. 32, 231-244. type, their gametes may fuse during the swarming period in- 8. Muspratt, J. (1946) "On Coelomomyces fungi causing high side the host animal; if not, they must seek their mate outside mortality of Anopheles gambiae larvae in Rhodesia," Ann. the copepod. Further development of the cycle depends on the Trop. Med. Parasitol. 40, 10-17. zygote finding and infecting a new mosquito host. Infection 9. Muspratt, J. (1946) "Experimental infection of the larvae of with a Coelo- studies in the laboratory have followed the same fungal mate- Anopheles gambiae (Dipt., Culicidae) momyces fungus," Nature 158, 202. rial from an infected mosquito, through the intermediate 10. Shemanchuk, J. A. (1959) "Note on Coelomomyces psoro- copepod host and back to a new mosquito. This observation phorae Couch, a fungus parasitic on mosquito larvae," precludes the existence of other unknown hosts in this life Can. Entomol. 91, 743-744. history. 11. Laird, M. (1967) "A coral island experiment: a new ap- proach to mosquito control," World Health Organ. Chron. The minimum time to complete the cycle in our rearing 21, 18-26. conditions is approximately 20 days, with 9 days from spore to 12. Couch, J. N. (1972) "Mass production of Coelomomyces, a RS in the mosquito, 4 days for maturation of the RS, and 7 fungus that kills mosquitoes," Proc. Nat. Acad. Sci. USA days from meiospore to gametes in the copepods. 69, 2043-2047. 13. Whisler, H. C., Zebold, S. L. & Shemanchuk, J. A. (1974) The copepod phase of C. psorophorae is remarkably similar "Alternate host for mosquito parasite Coelomomyces," to the Cyclops parasite, Callimastix cyclopis. Originally de- Nature 251, 715-716. scribed as a protozoan parasite of Cyclops (25), this organism 14. McLintock, J. (1952) "Continuous laboratory rearing of has more recently been associated with the Blastocladiales Culiseta inornata (Will.) (Diptera: Culicidae)," Mosquito (26, 27). The few differences that exist between the two genera News 12, 195-201. 15. Whisler, H. C. (1966) "Host-integrated development in the may well relate to different host species and rearing conditions. Amoebidiales," J. Protozool. 13, 183-188. Problems of synonomy and taxonomic priority between 16. Umphlett, C. J. & Olson, L. W. (1967) "Cytological and Coelomomyces (1921) and Callimastix (1912) must await morphological studies of a new species of Phlyctochytrium," resolution of these differences and study of any available type Mycologia 59, 1085-1096. material. 17. Whisler, H. C. & Fuller, M. S. (1968) "Preliminary observa- tions on the holdfast of Amoebidium parsiticum," Mycologia Whether the other forty varieties (1, 28) and species of 60, 1068-1079. Coelomomyces follow the full cycle described here for C. 18. Sparrow, F. K. (1960) Aquatic Phycomycetes (University of psorophorae awaits study. Discovery of "brachy" and "cysto- Michigan Press, Ann Arbor), 2nd ed. "An of the life genes" cycles would not be surprising and these would pre- 19. Emerson, R. (1941) experimental study cycles and taxonomy of Allomyces," Lloydia 4, 77-144. sumably lack the crustacean host. However, the problems of 20. Wilson, C. M. (1952) "Meiosis in Allomyces," Bull. Torrey laboratory domestication encountered by workers with other Bot. Club 79, 139-160. host-parasite combinations could argue for the existence of 21. Harder, R. & S6rgel, G. (1938) "Iber einen neuen plano- long-cycle forms in other species of the fungus. isogamen Phycomyceten mit Generationswechsel und seine phylogenetische Bedeutung," Nachr. Ges. Wiss. The discovery of heteroecism in Coelomomyces has obvious Gottingen 3, 119-127. implications for the potential use of this fungus in biological 22. Couch, J. N. & Whiffen, A. J. (1942) "Observations on the control of mosquitoes and other dipteran hosts. Knowledge of genus Blastocladiella," Amer. J. Bot. 29, 582-591. the copepod involvement has permitted us, after a long period 23. Couch, J. N. (1945) "Observations on the genus Catenaria," of erratic results, to obtain consistently high levels of mosquito Mycologia 37, 163-193. 24. Blackwell, E. M. (1940) "A life cycle of Blastocladia prings- mortality with relatively few infected copepods. The discovery heimii Reinsch.," Trans. Brit. Mycol. Soc. 24, 68-86. of sex and mating type presents a potential tool for examining 25. Weissenberg, R. (1912) "Callimastix cyclopis, n. g., n. sp., the genetics of infectivity and speciation in the genus as well as ein geisseltragendes Protozoon aus dem Serum von Cy- selection for "super killers." clops." Sitzungsber. Ges. Naturfr. Freunde 5, 299-305. 26. Weissenberg, R. (1950) "The development and affinities of We thank Linda B. Travland for assistance in electron micros- Callimastix cyclopis Weissenberg, a parasitic microorganism copy, Dr. Paul L. Mlg for aid in the identification of Cyclops from the serum of Cyclops," Proc. Amer. Soc. Protozool. 1, vernalis, and Dr. John N. Couch for advice on the production of 4-5. infected mosquitoes. The work was partially supported by 27. V~vra, J. & Joyon, L. (1966) "Etude sur la morphologie, le Grant AI 05882 from the National Institutes of Health. cycle evolutif et la position syst6matique de Callimastix 1. Couch, J. N. & Umphlett, C. J. (1963) in Pathology: cyclopis Weissenberg 1912," Protistologia 2, 5-16. An Advanced Treatise, ed. Steinhaus, E. A. (Academic 28. Roberts, D. W. (1974) in Mosquito Control, ed. Bourassa, Press, New York), Vol. 2, pp. 149-188. J.-P. (Univ. of Quebec Press, Montreal), in press. Downloaded by guest on September 25, 2021