Coelomomyces dodgei: Establishment of an in vivo laboratory culture

An in vivo laboratory culture of the fungus Coelomomyces 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, 17.8, 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.     

Coelomomyces opifexi Pillai & Smith (Coelomomycetaceae: Blastocladiales) IV. Host range and relative susceptibility of Aedes australis and Opifex fuscus larvae

Abstract

The host range of Coelomomyces opifexi was investigated. Of the three species of mosquito larvae tested, Aedes australis and Opifex fuscus were susceptible whereas Culex pervigilans was refractory to infection. The results of laboratory infection experiments using inoculum derived from larvae of both species clearly indicated that Ae. australis larvae are more susceptible to infection than O. fuscus; this conclusion is supported by observations of natural infection pools. Of three species of copepods also tested, only Tigriopus sp. cf. angulatus became infected; Cyclops vernalis and C. bicuspidatus proved refractory to infection.     

In vitro studies of Coelomomyces punctatus from Anopheles quadrimaculatus and Cyclops vernalis

Attempts to grow mycelium of Coelomomyces punctatus from Anopheles quadrimaculatus larvae were made using more than 50 combinations of known vertebrate and invertebrate tissue culture media and microbiological media. Growth and/or differentiation of mycelium into sporangia were observed in several media. Significant growth of hyphal fragments and differentiation into young resting sporangia occurred in conditioned Mitsuhashi-Maramorosch insect tissue culture medium. This medium was conditioned by growth for 3 weeks in it of Varma's Anopheles stephensi tissue culture cells and was supplemented with 20% heat-inactivated fetal bovine serum and a synthetic tripeptide, glycyl-histidyl-lysine. Limited growth and elongation of lateral hyphal branches and subsequent development into resting sporangia with typical outer wall markings and pigmentation of mature forms were observed in a modified brain-heart infusion medium. Some media stimulated hyphae to develop into smooth-walled, spherical bodies of size and appearance typical of young sporangium initials but with no further maturity. In most media, no growth or development of mycelium occurred, but the fungus remained alive for 2–4 weeks. Mycelium of C. punctatus dissected from Cyclops vernalis did not grow and develop in any of the media utilized. However, in one case the mycelium differentiated into gametes shortly after dissection into modified brain-heart infusion medium.     

Biotic and abiotic parameters associated with an epizootic of Coelomomyces punctatus in a larval population of the mosquito Anopheles quadrimaculatus.

Biotic and abiotic parameters associated with an epizootic of the fungus Coelomomyces punctatus in larval populations of the mosquito Anopheles quadrimaculatus were investigated for three mosquito breeding seasons (1986-1988) in two adjacent farm ponds in North Carolina. In the first pond, the prevalence of infected larvae averaged 42% (range 0-85%) for collections made weekly from May 1 to November 20, 1986, but larvae did not occur in this pond in 1987. Infection rates in the adjacent pond, sampled during the mosquito breeding seasons of 1987 and 1988, declined from 10.9% (range 0-27.5%) in 1987 to 2.5% (range 0-14.2%) in 1988. Correlation analyses between the number of female copepods and fungal infection rates in sentinel mosquitoes were significant (P < 0.01) for Acanthocyclops robustus but insignificant for eight other species. Infections obtained in sentinel larvae placed in the ponds for 3 hr intervals indicated that C. punctatus infected larvae around sundown. Infection rates for field-collected larvae increased with the stage of larval development. However, experiments with sentinel larvae showed that early instars were more susceptible to infection than later instars, suggesting that the higher infection rates in late instars resulted from individual larvae being infected by two or more zygotes during larval development. Standard multiple regression analyses, used to determine the relationship between seasonal infection rates and water chemistry, weather variables, and the abundance of early and late instar larvae, showed that the abundance of late instars was the only independent variable common to linear models. The models only accounted for 20 and 9% of the variation in larval infection rates for 1987 and 1988, respectively. These results indicate that of the parameters examined, the seasonal abundance of the copepod, A. robustus, was the most important factor (or variable) correlated with the prevalence of mosquito infection.     

Production of the mosquito-parasitic fungus,Coelomomyces dodgei,through synchronized infection and growth of the intermediate copepod host,Cyclops vernalis

High yields of the copepodCyclops vernalis infected with the mosquito-parasitic fungusCoelomomyces dodgei Couch & Dodge were obtained by infecting nauplii in large synchronously developing populations. Exposure of 2000 48 or 72 h old nauplii to 6×10³ sporangia at the time of meiospore release yielded ca. 1500 infected copepods. Based on yields of infected copepods, susceptibility ofC. vernalis toC. dodgei decreased as copepods developed. Infection rates were 75% for copepods exposed as 48 or 72 h old nauplii but declined to 32 and 9.6%, respectively, for those exposed as copepodids or adults. The relevance of these results for domestication of other species ofCoelomomyces and studies on non-target organisms is discussed, and improved procedures for routine production ofC. dodgei are described.     

Evidence for the copepods Acanthocyclops robustus and Mesocyclops edax as competent intermediate hosts for Coelomomyces punctatus during an epizootic in a larval population of the mosquito Anopheles quadrimaculatus.

Field and laboratory experiments were conducted during an epizootic of Coelomomyces punctatus (Chytridiomycetes: Blastocladiales) in a population of the mosquito Anopheles quadrimaculatus in a North Carolina farm pond to examine the interactions of several potential copepod hosts with the mosquito and fungus. The diel vertical migratory behavior of the copepod species Acanthocyclops robustus, Eucyclops serrulatus, Macrocyclops albidus, and Mesocyclops edax were monitored in relation to infection rates in sentinel mosquito larvae. Mosquito infection occurred primarily around dusk, the same period during which A. robustus and E. serrulatus were most abundant near the surface of the pond. However, exposure of A. robustus, E. serrulatus, M. albidus, M. edax, Microcyclops varicans, and Paracyclops poppei to fungal meiospores in the laboratory showed that only A. robustus and M. edax were competent intermediate hosts for C. punctatus. Laboratory studies of the diel periodicity of gametangial dehiscence in A. robustus and M. edax infected with C. punctatus revealed that gamete release and zygote formation also occurred around dusk. The combined results of the laboratory and field studies on copepod abundance, susceptibility to infection, and periodicity of gametangial dehiscence suggest that A. robustus was the principal intermediate host for C. punctatus during the epizootic, though it is probable that M. edax also contributed importantly to the overall rate of larval infection.     

Predation on Mosquito Larvae by Mesocyclops thermocyclopoides (Copepoda: Cyclopoida) in the Presence of Alternate Prey

The cyclopoid copepod Mesocyclops thermocyclopoides, a dominant invertebrate predator in many shallow ponds and temporary water bodies in northern India, feeds on cladocerans, rotifers, ciliates and when present, on mosquito larvae also. We studied in the laboratory the prey consumption rates of the copepod on first and fourth instar larvae of two species of mosquito (Anopheles stephensi and Culex quinquefasciatus) in relation to their density. We also studied its prey selectivity with mosquito larvae in the presence of an alternate prey (the cladocerans‐either Moina macrocopa or Ceriodaphnia cornuta) in different proportions. With either mosquito species, the copepod actively selected Instar‐I larvae, avoiding the Instar‐IV larvae, and with either instar, selected Anopheles stephensi over Culex quinquefasciatus. When prey choice included the cladoceran as an alternate prey, the copepod selected the cladoceran only when the other prey was Instar‐IV mosquito larvae. Our results point to the potential and promise of M. thermocyclopoides as a biological agent for controlling larval populations of vectorially important mosquito species.     

Life cycle of Amblyospora indicola (Microspora: Amblyosporidae), a parasite of the mosquito Culex sitiens and of Apocyclops sp. Copepods

The life cycle of Amblyospora indicola, a parasite of the mosquito Culex sitiens, was revealed by field observations and laboratory infection experiments conducted in Australia. In northern Queensland, infected C. sitiens larvae were often found breeding in association with two cyclopoid copepods: Apocyclops dengizicus and an undescribed species of the same genus. The latter species was found to be an intermediate copepod host of this microsporidium whereas A. dengizicus was not. One complete cycle of the parasite extends over two mosquito generations (by transovarial transmission from females with binucleate spores to their eggs) and by horizontal transmission between mosquitoes and copepods. The latter involves horizontal transmission from mosquitoes to copepods via meiospores produced in larval fat body infections and horizontal transmission from copepods to mosquitoes via uninucleate spores produced within infected copepods. Uninucleate clavate spores were formed in Apocyclops sp. nov. copepods 7-10 days after exposure to larval meiospores and were infectious to larvae of a microsporidian-free colony of C. sitiens. The development of A. indicola within mosquito larvae exposed to infected copepods is similar to that of A. dyxenoides infecting C. annulirostris. It proceeds from stages with a single nucleus to diplokaryotic binucleate cells in oenocytes. These stages persist through pupation to adult emergence after which time a proportion of male mosquitoes and female mosquitoes may develop binucleate spores without the need for a blood meal. A proportion of both male and female larval progeny of infected females with binucleate spores develop patent fat body infections via transovarial transmission and die in the fourth larval instar.(ABSTRACT TRUNCATED AT 250 WORDS)     

How effective are Mesocyclops aspericornis (Copepoda: Cyclopoida) in controlling mosquito immatures in the environment with an application of phytochemicals?

We investigated the susceptibility of Mesocyclops aspericornis and Aedes aegypti larvae to mosquito larvicidal phytochemicals: piperine and eugenol and the predation efficiency of the adult female M. aspericornis on Ae. aegypti larval instar-I and late II at the sublethal concentration of piperine and eugenol. Both prey and predator were susceptible to both the phytochemicals; however, the lethal concentration of either phytochemicals recorded for Ae. aegypti did not exert mortality on the copepods. The predator’s latent time was significantly longer in the phytochemical medium than control. The encounter frequency was significantly higher in control than in the phytochemical medium. For instar-I larvae, the post-encounter attack probabilities were not affected by the tested phytochemicals. However, for instar-II, the post-encounter attack probability was lower in eugenol treatment than in either the control or piperine. The escape and post-escape survival probabilities of larvae were substantially lower in phytochemical treatments than in the control. However, the overall copepod-imposed mortality did not differ significantly between the control and the phytochemical medium. In conclusion, both the phytochemicals modify the copepod feeding behavior without affecting the copepod incurred total larval mortality. Therefore, application of these phytochemicals along with M. aspericornis for mosquito control is advisable.     

Ultrastructural changes in the muscles,midgut, hemopoietic organ,imaginal discs and Malpighian tubules of the mosquito Aedes aegypti larvae infected by the fungus Coelomomyces stegomyiae

Fungi belonging to the genus Coelomomyces can infect mosquito larvae and develop within the larval hemocoel. To examine fungal development, Aedesaegypti larvae infected with Coelomomyces stegomyiae Keilin were fixed, embedded and sectioned for both light and electron microscopy. While fungal hyphae of C. stegomyiae did not invade cells other than the cuticular epithelial cells, they did penetrate a number of tissues including muscles, midgut, hemopoietic organ, imaginal discs, and Malpighian tubules. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gametogenesis inCoelomomyces dodgei Couch (Blastocladiales,Chytridiomycetes)

Summary The ultrastructure of gametogenesis was studied inCoelomomyces dodgei Couch (Blastocladiales, Chytridiomycetes), an obligate parasite of anopheline mosquito larvae and the copepod,Acanthocyclops vernalis. In infected copepods reared under a 16/8 hours light/dark photoperiod at 25 +2 °C., the gametophyte develops over a period of approximately seven days, and gametogenesis is triggered by the onset of the dark period during the last day of development. The initial step of gametogenesis is the elongation of the centriole to form the kinetosome, and measuring time from the onset of the final dark period (0 hours), this occurs prior to the beginning of the light period (8 hours). Subsequently, small vesicles that appear to originate from elements of the rough endoplasmic reticulum (rER) fuse at the distal end of the kinetosome forming the flagellar vesicle into which the axonemal microtubules elongate to form the flagellum (8–12 hours). Similar small vesicles apparently also derived from rER align in planes and fuse to form cleavage furrows which delineate the gamete initials (12–14 hours). As the gamete initials begin forming, the mitochondria within each initial fuse to form a single mitochondrion that associates with the lipid globules and microbodies forming the microbody-lipid globule complex (12–16 hours). The time elapsed between the formation of the flagellar vesicle to the release of mature gametes from the copepod host is about 8.5 hours. No differences were observed in the processes or timing of gametogenesis in male and female gametophytes.     

Epizootiological studies of Amblyospora albifasciati (Microsporidiida: Amblyosporidae) in natural populations of Aedes albifasciatus (Diptera: Culicidae) and Mesocyclops annulatus (Copepoda: Cyclopidae) in a transient floodwater habitat

The epizootiology of the microsporidium Amblyospora albifasciati was studied in natural populations of its definitive host, a multivoltine, neotropical, floodwater mosquito, Aedes albifasciatus, and its intermediate copepod host, Mesocyclops annulatus, in an ephemeral floodwater habitat during a 12-month period. A. albifasciati was enzootic in mosquitoes. Vertically (transovarially) transmitted meiospore infections occurred regularly and were detected in five of eight larval broods but the prevalence of infection was always low, ranging from 0.5 to 6.9% with an overall average of 0.7%. Horizontal transmission of A. albifasciati infection from copepods to mosquitoes was nominal and limited. It was detected at levels of 6.4 to 20% in larval Ae. albifasciatus populations on two occasions, the month of August and late September through early October. The low levels of horizontal transmission of infection to mosquito larvae appeared to be the principal limiting factor that prevented the proliferation of A. albifasciati in Ae. albifasciatus populations. Copepod populations were abundant from May through September and weekly prevalence rates of A. albifasciati averaged over 50% (range = 5.8 to 100%). The moderately high infection rates in M. annulatus copepods were inconsistent with the low prevalence of meiospore infection in Ae. albifasciatus mosquito larvae. Results suggest that either meiospores of A. albifasciati produced in the mosquito host are highly infectious to copepods or they are long-lived and remain viable within the pool as long as some standing water is present. Observations further indicate that A. albifasciati has a significant detrimental impact on M. annulatus copepod populations but minimal impact on larval populations of Ae. albifasciatus at this site.     

Factors affecting horizontal transmission of the microsporidium Amblyospora albifasciati to its intermediate copepod host Mesocyclops annulatus

Factors that directly impact horizontal transmission of the microsporidium Amblyospora albifasciati to its intermediate copepod host, Mesocyclops annulatus were examined in laboratory bioassays. Results were evaluated in relation to life history strategies that facilitate persistence of the parasite in natural populations of its definitive mosquito host, Ochlerotatusalbifasciatus. A moderately high quantity of meiospores from mosquito larvae was required to infect adult female copepods; the IC50 was estimated at 3.6 × 10⁴ meiospores/ml. Meiospore infectivity following storage at 25 °C was detected up to 30 days, while meiospores stored at 4 °C remained infectious to copepods for 17 months with virtually no decline in infectivity. Uninfected female M. annulatus are long-lived; no appreciable mortality was observed in field-collected individuals for 26 days, with a few individuals surviving up to 70 days. The pathological impact of A. albifasciati infection on M. annulatus resulted in a 30% reduction in survivorship after 7 days followed by gradual progressive mortality with no infected individuals surviving more than 40 days. This moderate level of pathogenicity allows for a steady continual release of spores into the environment where they may be ingested by mosquito larvae. Infected female copepods survived in sediment under conditions of desiccation up to 30 days, thus demonstrating their capacity to function as a link for maintaining A. albifasciati between mosquito generations following periods of desiccation. The susceptibility of late stage copepodid M. annulatus to meiospores of A. albifasciati and subsequent transstadial transmission of infection to adult females was established.     

Effects of predation by the copepod Mesocyclops ogunnus on the sex ratios of mosquito Aedes albopictus

Cyclopoid copepods are important predators in many aquatic ecosystems and have been used as biological agents in successful programs to control mosquito larvae. However, the impacts of this predation on adult mosquito populations are still poorly understood. The present study compared the sex ratios and body sizes (measured as wing length) of Aedes albopictus mosquitoes emerging from recipients containing the copepod predator Mesocyclops ogunnus with control situations without this predator. We found that copepod predation significantly biased mosquito sex ratios toward females, and that both the males and females emerging from copepod-containing recipients were significantly larger than control insects. The ecological and epidemiological consequences of the changes induced by copepod predation on mosquito populations are discussed.     

Effect of meiofauna on macrofauna recruitment: settlement inhibition of the polychaete Hydroides elegans by the harpacticoid copepod Tisbe japonica

Various aspects of marine macroinvertebrate ecology cannot be understood without detailed knowledge of larval settlement processes. An important effect underscored during the settlement process is the disturbance of marine invertebrate larvae by predators. We demonstrated that biotic disturbance, comprising physical elimination and mortality due to predation and the behavioral irritation of larvae by the harpacticoid copepod Tisbe japonica, prevent a significant portion of larvae of the polychaete Hydroides elegans from settlement on otherwise suitable substrata. Experiments were performed both in the laboratory and the field showing reproducible significant differences in larval settlement and mortality rates between gender-specific copepod treatments and the control. The trend of decreased larval settlement in the presence of copepods coincided with increased larval mortality in these treatments. In the corresponding field experiments, larval settlement and mortality were similar to the ones obtained under laboratory conditions.     

Intermediate host for an Amblyospora sp. (microspora) infecting the mosquito, Culex annulirostris

Experiments conducted in Australia are described which show that there is an intermediate host involved in the life cycle of an Amblyospora sp. infecting the mosquito, Culex annulirostris. Microsporidian spores produced in the cyclopoid copepod, Mesocyclops albicans, are infectious to laboratory colony C. annulirostris larvae and produce developmental stages identical to those of natural Amblyospora infections in this mosquito. These larval infections result in uninucleate spores in progeny mosquitoes which, in turn, are infectious to M. albicans. Subsequent studies conducted with Amblyospora, copepods, and mosquitoes in the United States to replicate and reconfirm results obtained in Australia were successful. This is the first substantiated evidence of the involvement of intermediate hosts in the life cycles of microsporidia, and the discovery now provides us with information needed to evaluate these organisms as biological control agents for mosquitoes and other disease vectors.     

Coelomomyces opifexi Pillai & smith (Coelomomycetaceae: Blastocladiales) V. Development of the fungus in pupae and adults of Aedes australis

Abstract

Late-instar Aedes australis larvae were experimentally infected with Coelomomyces opifexi, and the subsequent development of the fungal parasite was studied. Five separate experiments involving 1190 infected larvae were conducted. Of these larvae, 114 continued development and pupated; 38 of them contained sporangia. Histological studies indicated that parasite development slowed down, if not ceased, following pupation of infected larvae, and the presence of sporangia in adults reflected the degree of advancement of infection in the larvae at pupation.     

Combination of Mesocyclops thermocyclopoides and Bacillus thuringiensis var. israelensis: a better approach for the control of Aedes aegypti larvae in water containers.

The efficacy of a local Thai-strain of the copepod, Mesocyclops thermocyclopoides and the larvicide, Bacillus thuringiensis var. israelensis (Bti), used jointly and singly, was studied against Aedes aegypti in water containers. In a laboratory test, copepods alone produced mortality of 98-100% in 1st instar larvae of Ae. aegypti at copepod:larvae ratios ranging from 1:1 to 1:4. In an outdoor field simulated experiment that ran for 16 wk, after a single inoculation, the treatment of copepods and Bti combined yielded the better, more sustainable results than the agents used individually. Numbers of mosquito larvae per sample in the combined treatment were zero during the first 8 wk; larval numbers then increased but were maintained at a very low level for the next 4 wk after which the larval numbers increased moderately but still remained below numbers in the control. Bti alone kept the larvae at the zero level for the first 4 wk after which their numbers increased slightly and were at low levels up to 12 wk. Copepods alone maintained larval numbers at a low level as compared with those of the control. During the course of the experiment the larval numbers in the control were greater than 20 per sample. Statistically significant differences were noted among treatment means (F = 23.083, df = 3/60, P<0.01) over the total period of the study. The number of copepods in the joint treatment was significantly higher than in the copepod alone treatment for the first 8 wk (t = -4.97, df = 14, P<0.01). The density of copepods, however, for the whole 16-wk period was not significantly different in these two treatments (t = -1.51, df = 30, P>0.1).     

Biochemistry of mosquito infection: Preliminary studies of biochemical change in Culex pipiens quinquefasciatus following infection with Lagenidium giganteum

The effect of infection of larvae of the mosquito Culex pipiens quinquefasciatus by the fungus Lagenidium giganteum has been studied from a biochemical standpoint. Methods were developed to analyze larval extracts that were essentially free of parasitic material. Biochemical parameters investigated on a per larva basis were protein, and the enzymes o-diphenol oxidase, glutamate transaminase, alkaline phosphatase, trehalase, and chitobiase. The behavior of the total amino acid and sugar pools were also examined. In general it was found that the infected larvae exhibited a significant decrease in rate of synthesis of most of the parameters studied and that the rates eventually fell to zero when compared to healthy control organisms. The progress of mycosis was correlated with visual evidence and it was concluded that the larvae were dying from starvation as a consequence of the utilization of their endogenous reserves by the parasite. Death of the larvae occurred as a general rule, in laboratory conditions, some 48 hr after initiation of infection by the fungus.     

A mosquito parasite from a mosquito predator

The fungus Coelomomyces macleayae has been reported from treehole mosquito larvae of three Aedes subgenera in Australia, Fiji, and the United States. This fungus is now recorded for the first time from a mosquito of the genus Toxorhynchites, the large predatory larvae of which are of some importance in the naturalistic control of associated mosquito pests and vectors. A single parasitized larva of T. rutilus septentrionalis was collected from a magnolia treehole (previously used for A. triseriatus infection experiments) near West Lake, Louisiana, in September, 1971. Larval A. triseriatus and Orthopodomyia signifera were also present but were uninfected.