Coelomomyces dodgei: Establishment of an in vivo laboratory culture

JOURNAL

OF INVERTEBRATE

Coelomomyces

30, 288-297 (1977)

PATHOLOGY

dodgei:

Establishment

of an in Vivo Laboratory

Culture

BRIAN A. FEDERICI Division

of Biological

Control,

University

of Calijornia,

Riverside,

California

92521

AND HAROLDC.CHAPMAN U.S.

Department

of Agriculture,

GulfCoast

Mosquito

Research

Laboratory,

Lake

Charles,

Louisiana

70601

Received July 22, 1976 An in vivo laboratory culture of the fungus Coefomomyces dodgei (Chytridiomycetes: Blastocladiales) was established, using the copepod Cyclops vernalis as an intermediate host and the larvae of the mosquito Anopheles quadrimaculatus as the definitive host. The culture was perpetuated by infecting copepods and mosquitoes using separate procedures. Copepods were infected by being combined with dehiscing sporangia. Patently infected copepods, which contained either light amber, bright orange, or both light amber and bright orange mycelia, were collected daily beginning 12 days later. Mosquitoes were infected by combining 100 first-instar larvae for 48 hr with a mixture of 12 infected copepods, four of each of the above types. The mean rate of infection for the first 100 trials was 41%. When groups of 100 first-, second-, third-, and fourth-instar larvae were exposed to a similar mixture of infected copepods for 48 hr, the mean rates of infection were 37.4,27.0,17X, and 2.4%. respectively. Observations and experimental evidence suggest that the differentially pigmented mycelia found in infected copepods are gametophytes which develop into gametangia that subsequently release gametes of opposite mating types, the light amber gametangia producing female and the bright orange gametangia producing male gametes. Zygotes resulting from the fusion of these gametes lead to the infection of mosquito larvae. Thus, C. dodgei appears to have an Euallomyces type of life cycle with sporophyte and gametophyte generations alternating between mosquito and copepod hosts, respectively, with differentially pigmented sexual structures present in the gametophyte phase.

tablished an in vivo culture of Coelomomyces punctatus in Anopheles quadrimaculatus. Using methods similar to those of Couch (1968, 1972), several other investigators were able to obtain infection of mosquito larvae in the laboratory, but were unable to establish in vivo cultures (Pillai and Woo, 1973; Whisler et al., 1974; Federici and Roberts, 1975). More recently, Whisler et al. (1974, 1975) established a culture of C. psorophorae in Culiseta inornata and discovered that the copepod Cyclops vernalis (Cyclopoida) was an intermediate host’ for this species. Shortly thereafter,

INTRODUCTION

The genus Coelomomyces (Chytridiomycetes: Blastocladiales) consists of approximately 40 known species of aquatic parasitic fungi. Most of these species have been reported from mosquitoes, and several are known to cause epizootics periodically, resulting in high mortalities in larval mosquito populations (Chapman, 1974; Roberts, 1974). There has been substantial interest in this group since the 1930s because these fungi have appeared to have a potential for use in mosquito-control programs. However, until recently, attempts to establish in vivo laboratory cultures for detailed investigations on the biology of Coelomomyces were unsuccessful. Couch (1968, 1972) eventually es-

1 Previously, the copepod has been referred to as an “obligate alternate” host (Whisler et al., 1974, 1975; Federici, 1975; Federici and Roberts, 1976). Although this terminology is appropriate, the more classical 288

Copyright %I 1977 by Academic Press. Inc. All rights of reproduction in any form reserved.

IN VIVO CULTURE

Federici (1975) and Federici and Roberts (1976) reported that C. vernalis was also an intermediate host for a strain of C. punctatus from Louisiana which had been established in their laboratory in A. quadrimaculatus. Even with these recent advances, C. psorophorae var. and C. punctatus are the only species being maintained routinely in the laboratory. A greater understanding of the biology of this fungal group will be obtained by studying a wider variety of species. The present paper describes the establishment of an in vivo laboratory culture of another species, Coelomomyces dodgei, using the copepod Cyclops vernalis and the mosquito Anopheles quadrimaculatus as intermediate and definitive hosts, respectively. Additionally, preliminary data on the relative susceptibility of different larval instars and observations on the development of C. dodgei in C. vernalis are reported. MATERIALS

AND METHODS

Aside from the induction of sporangial dehiscence, all maintenance phases and experimental procedures for the Coelomomyces dodgei culture were conducted in a IO-ft3 walk-in incubator held at 27” t 2”C, with a relative humidity of approximately 80% and a 16-hr photoperiod. Mosquito colonies. Adult mosquitoes of Anopheles quadrimaculatus were held in 2-ft3 cages, and females were offered white mice for blood meals twice weekly. Several split raisins were placed in the cages every week as an additional source of nutrition. Paper cups lined with paper toweling and terms in the parasitological sense, namely, “intermediate” and “definitive” hosts, are used here. This is done for two reasons. First, on the basis of the present study and those cited above, it appears likely that the mosquito is a true definitive host, i.e., a host in which the fungus reaches sexual maturity (undergoes meiosis), while the copepod is a true intermediate host, required for completion of the life cycle, but in which no sexual maturation occurs. Second, these terms have precedence and are widely accepted in the field of animal parasitology.

OF C. DODGEZ

289

containing distilled water were placed in the cages for oviposition. Eggs were collected every 2 days, and, after the larvae hatched, they were reared in 16-liter plastic containers (56 x 44 x 7.5 cm) in 7-10 liter of distilled water on a 3: 1 mixture of powdered Purina Lab Chow and brewer’s yeast diet (100 mg/liter). Additional diet was added as necessary. First-, second-, third-, and fourth-instar larvae are referred to here as L1, Lz, L,, and Lq, respectively. Copepod cultures. Cultures of C. vernalis were reared in 12 liter of medium in the same type of container as described above for rearing mosquito larvae. The cultures were perpetuated by adding 2 liter of an existing culture to 10 liter of distilled water along with 1 g of the laboratory chow-brewer’s yeast diet and 100 ml of an egg yolk solution. The latter was prepared by adding one hard boiled egg yolk to 500 ml of distilled water. Additional food was added weekly. Induction of sporangial dehiscence. Individual patently infected L, larvae, which usually contained from 10,000 to 30,000 sporangiaofc. dodgei (Fig. l), were triturated in a drop of distilled water in a 15-mm-diameter Petri dish. After the majority of the sporangia were liberated from the larva, 4 ml of distilled water was gently added to the dish, dispersing the sporangia until they formed a monolayer (Fig. 2). This procedure was carried out in a room with an average ambient temperature of 23°C under conditions of natural lighting. In most cases, 80% or more of the sporangia dehisced within 48 hr. Establishment

and maintenance

of Coe-

lomomyces

dodgei. The in vivo culture of C. dodgei was initiated at Riverside, California, using C. dodgei-infected larvae of Anopheles crucians. These larvae were collected from the Chloe TNT pond2 which is ’ Dr. J. N. Couch of the University of North Carolina confirmed the identity of the fungus collected from this pond for Chapman and Glenn (1972). The closely related species, C. puncrarus, has never been collected at this site.

290

FEDERICI

AND CHAPMAN

FIG. 1. Sporangia of Coelomomyces dodgei from a fourth-instar larva of Anopheles quadrimaculatus, infected in the laboratory using the procedures described in the text. The wall of the sporangium usually contains pits on one side and bands on the other. Scanning electron micrograph. 1200x. FIG. 2. Sporangia of Coelomomyces dodgei from a fourth-instar larva of Anopheles qaadrimacularus. (a) Resting sporangium in which zoospores have not yet developed. (b) “Go State” sporangium just prior to zoospore release (dehiscence). (c) empty resting sporangium which has discharged its zoospore complement. Fresh mount. 300x. FIG. 3. Cross section through an abdominal segment of an adult of Cyclops vernalis infected with Coelomomyces dodgei. The granular masses (arrows) in the hemocoel are portions of the gametangium and contain completely formed gametes. Epon-Araldite section. 350x.

in the vicinity of Lake Charles, Louisiana, and which has been described previously by Chapman and Glenn (1972). The initial culture was established using the techniques (Method 1) described by Federici and Roberts (1976) for maintaining C. punctatus in the laboratory, whereby the fungus, copepods, and L3 larvae are reared simultaneously in the same container. However, employing C. dodgei in a similar system resulted in routinely low rates of larval infection, usually less than 5%. The following technique, involving separate procedures for copepod and larval infection, was developed. This method has consistently yielded relatively high rates of larval infection. Infection of copepods. A copepod culture was stirred by hand, and 700 ml of medium,

which usually contained several hundred immature and mature copepods, was removed and placed in an enamel pan (29 x 18 x 5 cm) along with 700 ml of distilled water, approximately 5000 dehiscing sporangia, and 5 ml of egg yolk solution. Ten cultures were prepared in this manner at one time on a weekly basis. By examining such cultures daily, it was determined that infected copepods usually began to appear lo-12 days after culture preparation, with the majority appearing between Days 13 and 18. To avoid having to collect infected copepods from separate cultures, the cultures were pooled in a 16-liter plastic container 10 days after preparation. Detection and examination of infected copepods. Several hundred copepods were

IN WV0 60

0”

CULTURE

OF C. DODGEZ

291

r

IO

” II

12

“1

13

14

I5

”

16

17

1’

19

19

’

20

’

21

‘1’

22

23

24

1

25

DAY AFTER DEHISCING SPORANGIA WERE ADDED TO THE COPEPOD CULTURES FIG. 4. Mean number of infected copepods collected daily from the first 10 pooled cultures, from the twelfth through the twenty-fifth day. Each pooled culture consisted of 10 small cultures (1.4L) prepared as described in the text. The small cultures were pooled on Day 10, and collections were initiated on Day 12.

removed from the pooled culture daily and were examined for signs of infection. The copepods were strained from the culture with an 80-mesh net and were rinsed into a white enamel pan. Enough additional distilled water was added, approximately 50 ml, to allow the copepods to move about freely. The copepods were then examined with the aid of a 40-W Tensor lamp for signs of C. dodgei infection (Fig. 3). Infected copepods were usually either light amber, orange, or bright orange. The probable significance of these different color types is discussed below. In cases in which infection was suspected, but was not patent, the copepod was placed on the underside of a 22 x 50-mm coverslip in a drop of water, and, after removal of most of the excess water, it was examined with a phase microscope at magnifications of 100x and 400x. The presence or absence of mycelia was discerned easily at these magnifications. Infection of mosquito larvae. Mosquito larvae were infected by placing them in larval rearing medium from an existing larval culture along with patently infected copepods. More specifically, daily, 100 late L1 larvae were placed in 100 ml of larval rearing medium in a 8 x 20 x 6-cm plastic container along with four light amber, four orange, and four bright orange copepods.

The larvae were fed lightly, and, 48 hr later, they were transferred to 1 liter of fresh larval medium in an enamel pan and were reared normally. During the 48-hr period, C. dodgei killed the copepods and released gametes into the medium, the gametes fused to form mosquito-infective zygotes, and the zygotes infected mosquito larvae. Infections were usually patent in L3 or L, larvae 6- 10 days after the larvae had been combined with the infected copepods. Preliminary data on the relative susceptibility of the different larval instars were obtained by exposing groups of each instar, loo/group, to infected copepods as described above for L, larvae. RESULTS AND OBSERVATIONS

Copepod Infection The rate of patent copepod infection was not determined, but, in general, appeared to be low, probably less than 10%. Nevertheless, the procedures described produced an ample supply of infected copepods for both perpetuation of the culture and experimental purposes. The mean number of infected copepods collected daily from the twelfth to the twenty-fifth day after addition of dehiscing sporangia ranged from 16.3 to 50.5 for the first 10 pooled cultures (Fig. 4). In

292

FEDERICI

0

“1’ IO

II

12

13

” 14

AND CHAPMAN

15

” 16

17

” ‘8

‘9

” 20

21

” 22

23

” 24

25

DAY AFTER DEHISCING SPORANGIA WERE ADDED TO THE COPEPOD CULTURES FIG. 5. Number of infected copepods collected daily from two recent cultures, from the twelfth through the twenty-fifth day. Although these cultures were prepared using the standard method described in the text, the time of occurrence and yield of infected copepods was atypical.

two more recent cultures, much greater numbers were obtained (Fig. 5). Mosquito

Infection

The mean rate of infection for L, larvae in the first 100 trials was 41%. The results of five typical trials are shown in Table 1. In the majority of the trials, most infections became patent (sporangia present) during the L,. Yet, the development of patency during the L3 was not uncommon. Many patently infected L,s molted to the L, and subsequently developed very heavy infection, yielding 40,000-50,000 sporangia/larva. In all such heavily infected larvae, the majority of the fat body was displaced by sporangia, and the larvae being unable to pupate died during this instar. Relative Susceptibility Instars

of Different

Larval

The results for the trials on the relative susceptibility of different larval instars are shown in Table 2. Larval susceptibility decreased as larvae advanced in development. Maintenance

of the in Vivo Culture

Use of these techniques resulted in a constant and more than adequate supply of infected copepods and larvae for maintenance of the culture. Because the copepod cul-

tures were prepared on a weekly basis and were used as a source of infected copepods for at least 2 weeks after they were pooled, there were always two cultures available daily from which infected copepods were collected. This commonly resulted in the production of a total of 100 or more infected copepods per day and at least several hundred per week. As only 12 were normally used per day for larval infection, the majority have been used for experimental purposes or discarded. The same situation existed with infected mosquito larvae. Less than 1% (2/200+) were used weekly to infect copepods. Hence, in addition to its relatively simple maintenance, this system has the capacity for limited mass production of infected mosquito larvae, either by preparing more mosquito infection-producing preparations per day or by exposing more mosquito larvae to greater numbers of infected copepods per preparation. As an example of the latter, 396 infected larvae were obtained when 1000 L, were exposed in a single trial to 30 infected copepods. The relative amounts of the components in this system can be varied, including the numbers and ratios of the different types of infected copepods, and infections will still be obtained although with less consistancy. However, in preliminary trials in which larvae were exposed to only light amber or

IN VIVO

CULTURE TABLE

NUMBER AND TIME OF APPEARANCE OF

PATENTLY

OF C.

293

DODGEI

1 COELOMOMYCES

ANOPHELESQUADRIMACULATUS AFTER EXPOSURETO COPEPODS INFECTED WITH COELOMOMYCESDODGEI

DODGEI-INFECTED

Number of patently infected third- or fourth-instar Trial 1

Trial 2

Day”

L’

L

L

7 8 9

0 0 0

8 17 3

0 30 2

10

0

0

0

28

Subtotal Total

Trial 3

L

LARVAE

OF CYCLOPS

OF

C'ERNALIS

larvae*

Trial 4

Trial 5

L

L

L

L

Lz

L.l

0 7 6

32 0 0

9 1.5 3

8 0

13 14

0 0

20 14

0

1

0

0

1 0

0 0

1 0

32

14

32

27

28

59

46

1 0 9

28 37

0

35 35

a Time in days at which patently infected larvae were detected after larvae were combined with infected copepods. b Results of five typical trials in which 100 L, larvae were exposed to four amber, four orange, and four bright orange copepods: c Larval instar in which patency developed.

bright orange copepods, infections were not obtained. Coelomomyces

dodgei in Cyclops vernalis

Shortly after initiation of the in vivo culture, it was realized that patently C. dodgeiTABLE

2

SUSCEPTIBILITY OF DIFFERENT OF ANOPHELESQLJADRIMACULATUS

INSTAR LARVAE EXPOSED

T~C~EL~M~~~YCE~D~DGEI

Larval instar Replicate” 1

Ll

L

L

L

24 59 32 37 35

71

25

2 3 4 5

10

12

23 17 14

17 16 19

2 3 0 5 2

Mean

37.4

27.0

17.8

2.4

a In each replicate, 100 larvae of each instar were exposed to four amber, four orange, and four bright orange copepods. b Mosquitoes exposed to L,s did not develop patent infections until the pupal stage. These figures represent the number of patently infected pupae.

infected copepods could be recognized by an orange coloration or pigmentation. By microscopical examination, this pigmentation was found to be restricted to the cytoplasm of the mycelia developing within the copepod hemocoel. Healthy copepods normally lacked any distinctive pigmentation. However, the shade and intensity of the pigmentation was observed to vary from a lactescent (milky) orange to a very intense bright orange among different copepods. A series of infected adult copepods representing these different color types was examined under a phase microscope, and it was determined that the bright orange copepods contained only bright orange mycelia, usually in an advanced state of development, while each lactescent orange copepod usually contained either an orange mycelium at an early stage of development, in which there was less pigment than in the more advanced bright orange mycelia, or both bright orange and light amber mycelia. Harder and S&gel (1938; also see Sparrow, 1960) and Emerson and Fox (1940), among others, have demonstrated that orange pigment occurs during the gameto-

294

FEDERICI

AND CHAPMAN

phyte phase in structures designated as male in several other species of fungi of the order Blastocladiales. These findings and the results of Whisler et al. (1975), demonstrating that C. psorophorue produced heterothallic gametangia in infected copepods and that a single copepod could contain gametangia of one or both mating types, suggested that the bright orange and light amber mycelia produced by C. dodgei were gametophytes which produced gametes of opposite mating types. Furthermore, this indicated that infected copepods containing only light amber gametophytes should exist in the cultures of C. dodgei-infected copepods. Several cultures were examined carefully for copepods varying in pigmentation and opacity. In essence, five markedly different types of copepods could be recognized: two healthy and three infected types. The healthy types were: (i) slightly translucent and unpigmented = males, and females without eggs in the uterus; and (ii) opaque with grey pigmentation = females bearing eggs within the uterus. The three opaque infected types were: (i) light amber, previously undetected; (ii) lactescent orange; and (iii) bright orange. Microscopical examination confirmed that, in general, these three color types corresponded with the types of myCelia developing in the hemocoel. The light amber copepods contained mycelia which, under phase microscopy, appeared light amber or unpigmented; the lactescent orange copepods contained either orange mycelia with less pigment than was normally observed in bright orange mycelia or both bright orange and light amber mycelia; and the bright orange copepods contained only bright orange mycelia. The development of the advanced stages of the differentially pigmented gametophytes was followed in several copepods from the time of detection until the escape of gametes from the host. By the time infection was detected, the dichotomously branching gametophyte(s) had usually grown throughout most of the main body hemocoel, and hyphae had begun to penetrate into the

hemocoelic cavities of the leg and antenna1 appendages and the posterior portion of the abdomen. Over the next 24-72 hr, depending on the individual, the hyphae penetrated further into most of these cavities until the gametophyte(s) occupied a major portion of the hemocoel (Fig. 3). At this point, further growth ceased, and the gametophyte, in essence, became a gametangium. The cytoplasm became increasingly granular as gametogenesis occurred. In bright orange gametangia, the pigment was distributed evenly among the gametes and appeared to be localized in the lipid droplets. Gametes which developed in light amber gametangia were unpigmented, even in situations in which bright orange and light amber gametophytes developed in close apposition to one another. After gametogenesis was complete, the gametes began to teem slowly within the gametangium, and a distinct hyaline gametangial wall3 became apparent. The gamete activity increased and eventually the wall ruptured, liberating gametes into the copepod hemocoel. Most copepods were alive and quite active until rupture of the gametangium. However, within a few seconds the copepod died, and within a few minutes the gametes could be observed escaping from the copepod carcass in the vicinity of the dorsal intersegmental abdominal membranes. Gametes could still be differentiated on a color basis at this time. The entire process from the beginning of gamete movement to escape from the copepod usually took approximately 30 min. The fusion of bright orange and unpigmented gametes to form what is most probably the zygote which leads to mosquito infection has been observed, but has not been studied in detail. DISCUSSION

The methods described above have proven successful for the establishment and easy 3 “Wall” here simply means an easily discernible relatively thick covering, but not necessarily a true fungal cell wall. This “wall” is also conspicuous on hyphae as they develop within the copepod hemocoel.

IN WV0

CULTURE

maintenance of C. dodgei in the laboratory and should prove useful for the establishment of other species of Coelomomyces. The development of these techniques was largely empirical, and, although this system provides substantial numbers of infected copepods and larvae on a routine and predictable basis, the data indicate even higher yields will be possible once the parameters which favor high rates of copepod and mosquito infection are determined. As an example, although the methods used for the preparation of pooled copepod cultures were the same, some cultures consistently produced greater numbers of infected copepods daily and usually for a greater number of days (Figs. 4, 5). Furthermore, in some cases, the period of peak production varied markedly from the typical culture (Fig. 5). The copepod colonies used to initiate infected copepod cultures vary in age, and one important factor in successful infection, therefore, may be the average copepod age at the time dehiscing sporangia are introduced to the cultures. Mosquito larvae are most susceptible to infection during the early stages of development, and it is possible that nauplii may be more susceptible than copepodites and adults. In general, copepod infection in most Coelomomyces systems is understood poorly and deserves more intensive study. The wide variation in mosquito infection rates obtained when larvae of the same instar were exposed to infected copepods (Tables 1, 2) further indicates that higher mean infection rates will be realized once the conditions which favor zygote formation and mosquito infection are defined. However, another important parameter possibly responsible for some of this variation is the actual ratio of orange pigmented to unpigmented gametangia that went into each preparation. As stated above, the development of these methods was largely empirical, the objective being to develop a relatively simple method for perpetuating C. dodgei in the laboratory. It was logistically impractical and unnecessary for culture per-

OF C. DODGEI

295

petuation to examine microscopically each copepod used in the preparations to determine the type(s) of gametangia present in the hemocoel. By using 12 copepods, 4 of each color type, the ratio of pigmented to unpigmented gametangia should have been about equal. This was probably true in the case of copepods which were light amber or bright orange; that is, because of the distinctiveness of the pigmentation it is likely that they contained only light amber or bright orange gametangia. However, in the case of the orange copepods, it is probable that some contained only orange gametophytes at an early stage of development, while others contained a mixture of orange and unpigmented gametophytes, resulting in some variation in the number of mosquito-infective zygotes formed in different trials. From this, it is apparent that more accurate bioassay data will only be obtained with more precise methods, e.g., a system in which the number of mosquito-infective zygotes can be determined. The fact that A. quadrimacuhtus was readily susceptible to C. dodgei was particularly interesting because there are no reports of this host-parasite relationship in the literature, and its achievement, even though in the laboratory, casts further doubt on the validity of maintaining C. dodgei and C. punctafus as separate species. Couch (1945) originally described C. dodgei as occurring in A. crucians, A. punctipennis, and A. quadrimacufatus . Shortly thereafter, however, on the basis of more extensive studies, Couch and Dodge (1947) separated C. dodgei into three species, C. dodgei, C. lativittatus, and C. punctatus, based on host associations and sporangial characters. The former two were associated with A. crucians, while the latter occurred in A. quadrimaculatus. But more recent studies on C. dodgei and C. punctatus have demonstrated that these species vary more in host range and sporangial characters than in their descriptions. Chapman and Glenn (1972) reported C. punctatus in A. cruciuns, and the

FEDERICI

296

present

study

AND CHAPMAN

reports C. dodgei in A. . Additionally, Anthony et al. (1971) found that all larvae of A. quadrimaculatus infected with C. punctatus which they examined contained a few sporangia of the C. dodgei type, and all larvae of A. crucians infected with C. dodgei contained some sporangia of the C. punctatus type. Bland and Couch (1973) noted an intergrading of the surface structure of the sporangia of C. dodgei, C. lativittatus, and C. punctatus, and these authors and Anthony et al. (1971) have suggested that cross-infectivity studies should be undertaken to ascertain the taxonomic positions of these species. The availability of in vivo cultures of C. punctatus (Couch, 1972; Federici and Roberts, 1976) and C. dodgei should contribute to the resolution of this problem. Now that copepods have been shown to be intermediate hosts for at least three species of Coelomomyces, copepod host ranges for these and other species should also receive greater attention. The natural intermediate host(s) for C. dodgei and C. punctatus has not been reported. The most interesting and potentially important finding in the current study was the discovery of the apparent differential sexual pigmentation in the gametophytic phases. As noted earlier, several investigators have reported orange pigmentation in structures designated as male in other fungi of the order Blastocladiales with an Euallomycestype of life cycle. Harder and Sbrgel (1938) reported them in Blastocladiella variabilis , and Emerson and Fox (1940) and Emerson (1941) have reported them in Allomyces arbuscula and A javanicus , among others. The presence of orange pigment in some mycelia of C. dodgei in the intermediate copepod host and its lack in others, in conjunction with the studies of Whisler et al. (1975) on C. psorophorae , certainly suggest that C. dodgei has an Euallomyces-type of life cycle with an alternation of sporophyte and gametophyte generations between a definitive mosquito host and an quadrimaculatus

intermediate copepod host, respectively. The sporangia which develop from the sporophyte in the mosquito are most likely meiosporangia, which, upon dehiscence, release meiospores. The haploid meiospores eventually result in infection of copepods within which they produce gametophytes in the hemocoel. Depending on its sexual type, the gametophyte will develop into either a light amber or a bright orange pleomorphic gametangium. Both types may occur within a single copepod. After gametogenesis, by analogy with B. variabilis and A. arbuscula, etc.. in which the orange gametangia produce male gametes, the orange gametangia release male gametes, and the light amber gametangia release female gametes. The zygote which results from the fusion of the male and female gametes leads to mosquito infection and results in the formation of another sporophyte, thereby completing the life cycle. Although there is substantial evidence for this hypothesis in the present investigation, the determination of a complete life cycle for C. dodgei awaits more definitive studies. If the differential pigmentation in the gametophyte phase is confirmed to be sexual, the pigment will provide a valuable sexual marker for many different types of experimental studies, but should be particularly useful in biosystematic investigations. ACKNOWLEDGMENTS We thank Chris Baker and Tom Warr for their able technical r&stance.

REFERENCES ANTHONY, D. W., CHAPMAN, H. C., AND HAZARD, E. I. 1971. Scanning electron microscopy of the sporangia of species of Coelomomyces (Blastocladiales: Coelomomycetaceae). J. Invertebr. Puthol., 17,395403. BLAND, C. E.. AND COUCH, J. N. 1973. Scanning electron microscopy of sporangia of Coelomomyces. Cunad. J. Bat., 51, 1325- 1330. CHAPMAN, H. C. 1974. Biological control of mosquito larvae. Annu. Rev. Entomol., 19, 33-59. CHAPMAN, H. C.. AND GLENN, F. E. 1972. Incidence

IN VIVO CULTURE of the fungus

Coelomomyces punctatus and Coedodgei in larval populations of the mosquito Anopheles crucians in two Louisiana ponds. J. Inraertebr. Pathol., 19, 256-261.

lomomyces

COUCH, J. N. 1945. Revision of the genus Coelomomyces. parasitic in insect larvae. J. Elisha Mitchell Sci. Sot.. 61, 124- 136. COUCH, J. N. 1968. Sporangial germination of Coelomomyces punctatus and the conditions favoring the infection of Anopheles quadrimaculatus under laboratory conditions. In “Proceedings of the Joint U.S.- Japan Seminar on Microbial Control of Insect Pests.” Fukuoka. 1967. pp. 93- 105. COUCH. J. N. 1972. Mass production of Coelomomyces, a fungus that kills mosquitoes. Proc. Nat. Acad.

Sci.

USA,

69, 2043-2047.

COUCH, J. N., AND DODGE, H. R. 1947. Further observations on Coelomomyces, parasitic on mosquito larvae. J. Elisha Mitchell Sri. Sot.. 63, 69-79. EMERSON. R. 1941. An experimental study of the life cycles and taxonomy of Allomyces. Lloydia, 4, 77144. EMERSON, R., AND Fox, D. L. 1940. Gammacarotene in the sexual phase of the aquatic fungus Allomyces. Proc. Royal Sot. London Ser. B, 128(8X), 275-293. FEDERICI, B. A. 1975. Cyclops vernalis (Copepoda: Cyclopoida): An alternate host for the fungus, Coelomomycespunctatus. Ass.. 43, 172- 174.

Proc.

Calif.

Mosq.

Contr.

FEDERICI, B. A., AND ROBERTS, D. W. 1975. Experimental laboratory infection of mosquito larvae with fungi of the genus Coelomomyces. I. Experi-

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ments with Coelomomyces psorophorae var. in taeniorhynchus and Coelomomyces psorophvar. in Culiseta inornata. J. Invertebr. Pathol., 26, 21-27. FEDERICI, B. A., AND ROBERTS, D. W. 1976. Experimental laboratory infection of mosquito larvae with fungi of the genus Coelomomyces. II. Experiments with Coelomomyces punctatus in Anopheles Aedes orae

quadrimaculatus.

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