Proc. Nati. Acad. Sci. USA Vol. 76, No. 9, pp. 4425-4428, September 1979 Botany Experimental hybridization of Coelomomyces dodgei and Coelomomyces punctatus (fungal systematics/hybrids/sporangia/biological control/mosquitoes) BRIAN A. FEDIERICI Divisibn of Biological Control, Department of Entomology, University of California, Riverside, California 92521 Communicated by John N. Couch, July 5, 1979 ABSTRACT Two closely related fungi, Coelomomyces dodgei and C. punctatus (Chytridiomycetes Blastocladiales), showing considerable potential for mosquito control, have been hybridized. Both species have life cycles involving an obligate alteration of sexual and asexual generations between an inter- mediate copepod host and a definitive mosquito host, respec- tively. Hybridization was accomplished by making reciprocal crosses of gametes derived from infected copepods. In both cases, C. dodgei 9 X C. punctatus 6 and C. punctatus 9 X C. dodgei 6, zygotes infective for mosquito larvae were formed. Most sporangia produced by the hybrids in infected larvae were not typical of either parental species but resembled C. dodgei more than C. punctatus. Additionally, the majority of the spo- rangia formed in several larvae exhibited surface structures similar to C. lativittatus, indicating this species may be a nat- urally occurring hybrid of C. dodgei and C. punctatus. The ex- perimental methods described provide a system to aid in the study of taxonomic relationships among closely related species of Coelomomyces, and furthermore they may contribute to the development of strains efficacious for mosquito control. Mosquitoes continue to be the most medically important group of insects affecting humans. Their ability to transmit the agents of viral, protozoan, and filarial diseases has made them the target of a variety of control strategies. Although chemical in- secticides have been effective in suppressing mosquito popu- lations and the incidence of disease they transmit for almost four decades, the development of insecticide resistance in many mosquito species, the high cost of newer insecticides, and concern about environmental pollution have stimulated re- newed interest in alternative methods of control, particularly biological control using natural enemies. There is considerable interest in developing fungi of the genus Coelomomyces (class Chytridiomycetes order Blasto- cladiales) as biological control agents because, unlike many other mosquito pathogens, several species of this genus are known to periodically cause epizootics resulting in mortalities greater than 90% in larval populations (1-3). There are ap- proximately 40 described species of Coelomomyces, most of which have been reported as obligate parasites of mosquito larvae (4). Within the past few years it has been demonstrated that several species require an intermediate crustacean host to complete their life cycles (5-9). Moreover, through experiments with C. psorophorae, C. dodgei, and C. punctatus (5, 10), the life cycle has been determined to be of the Euallomyces type with gametophyte and sporophyte generations alternating, respectively, between an intermediate copepod host and a definitive mosquito host. In this life cycle, important to un- derstanding the experiments described below, the sporophyte that develops in the mosquito larva forms sporangia (= meiosporangia), which, after larval death, release haploid meiospores. These meiospores infect a copepod, within which each develops into a gametophyte that upon maturation cleaves and forms thousands of haploid gametes. Shortly thereafter, the copepod dies as a result of infection, the gametes escape from the carcass, and opposite mating types fuse, forming biflagellate zygotes. Each zygote seeks out and infects a mosquito larva, in which it forms a sporophyte, thereby completing the life cycle. In the gametophytic phase of C. dodgei and C. punctatus, gametangia and gametes of opposite mating type are of dif- ferent colors, one being amber and the other bright orange. These have been designated female and male, respectively, and it has been demonstrated experimentally that they are incapable of infecting mosquito larvae (10). Except for the system reported by Couch (1), prior to the elucidation of this life cycle it had proven extremely difficult to achieve mosquito infection in the laboratory, making critical studies on the biology of these fungi and evaluation of their control potential virtually impossible. Determination of the important role the copepod played in the life cycles of C. pso- rophorae, C. punctatus, and C. dodgei led to the establishment of routine methods (5, 8, 11) for maintaining in vivo cultures of these species. The existence of a sexual phase and the striking sexual di- morphism in C. dodgei and C. punctatus provided an oppor- tunity to begin studies on the systematics and genetics of this interesting and potentially important fungal group through hybridization experiments. This paper reports the results of initial studies in which these two species have been hybrid- ized. MATERIALS AND METHODS In vivo cultures of C. dodgei and C. punctatus were the source of the gametes used in these experiments. These species are maintained in the laboratory by using the copepod Cyclops vernalis as the intermediate host and larvae of the mosquito Anopheles quadrimaculatus as the definitive host. Methods for maintaining these fungi and the copepod and mosquito colonies have been published (8, 11). For the experiments reported here these methods were modified in the following manner. Cope- pods were infected by combining biWO-10,00 nauplii, 48-72 hr old, in 300 ml of a weak salt solution [(mg/liter) NaCl (100), KC1 (4), CaCl2 (6)] with ca 3 X 104 sporangia discharging meiospores. Six hours later the mixture was transferred to 12 liters of medium and the copepods were reared by standard methods (8). Infected specimens were removed 7-10 days later and separated into groups according to the mating type of the gametothyte(s) [i.e., amber (9), orange (d), or both amber (9) and orange (d)] developing in the hemocoel. To ensure that each copepod contained a gametophyte(s) of only one mating type the thallus (or thalli) developing within the hemocoel was ex- amined carefully at X400 with phase microscopy. Individual 4425 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 76 (1979) _1 FIG. 1. Scanning electron micrographs illustrating surface stru'dire of sporangia of C. punctatus, C. dodgei, and hybrids produced in reciprocal crosses of these two species. (X1240 bar = 10 am.) (a-c) Sporangia typical of C. punctatus (d) variant sporangium of C. punctatus possessing elongated pits more characteristic of C. dodgei (e-g) sporangia typical of C. dodgei (h) variant sporangium of C. dodgei possessing pits char- acteristic of C. punctatus (i-I) sporangia produced by hybrids formed by crossing amber (9) gametes of C. punctatus with orange (d) gametes of C. dodgei (m-p) sporangia produced by hybrids formed by crossing amber (9) gametes of C. dodgei with orange (d) gametes of C. punctatus. Note the variation in the hybrids as compared with the parental species. 4426 Botany: Federici