Aedes Sierrensis Research Articles

Observations on the infection of Aedes sierrensis by a tetrahymenine ciliate

Melanized spots on the cuticles of larvae of Aedes sierrensis were sites of invasion or of attempted invasion by an organism resembling a species of Tetrahymena. Externally the invasion sites were capped by hemispherical membranes or cysts vacated by the invading ciliate. First- and second-instar larvae appeared most susceptible. Several factors limited successful infection by the ciliate such as host activity, host age, and cuticle thickness.

Development and fine structure of Pleistophora sp. (Cnidospora: Microsporida) in the mosquito, Aedes sierrensis

The development and fine structure of a Pleistophora sp. from larvae of Aedes sierrensis was investigated. The usual site of infection was the posterior midgut epithelium, and small uni- and binucleate forms were the first stages observed. There was no evidence of autogamy or the production of uninucleate sporonts. Binucleate sporonts produced prosporoblasts with two and four nuclei. Cellularization of the prosporoblasts resulted in sporoblasts which matured into spores. Heavily infected larvae have less fat body than healthy hosts and usually die when infected in the first or second instar. The pathogen could remain chronic and be carried into the adult host. Although natural infections were low, the pathogen could serve as a biological control agent.

Beauveria tenella as a control agent for mosquito larvae

Beauveria tenella was found to be a naturally occurring pathogen in field populations of Aedes sierrensis larvae. Bioassays showed that B. tenella is also pathogenic for larvae of A. aegypti, A. dorsalis, A. hexodontus, Culex pipiens, C. tarsalis, and Culiseta incidens. Inoculation of 5 × 10 3 or 5 × 10 5 blastospores per milliliter into breeding sites of A. sierrensis produced marked reductions in the number of emerging adults.

The Complete Development of the Sporogonous Stages of Hepatozoon rarefaciens Cultured in a Culex pipiens Cell Line

All vector stages of H. rarefaciens can be grown in a C. pipiens cell line, starting with gametocytes which were obtained from blood of a gopher snake infected with this parasite. After 4 days in culture, oocysts of about 20 u were sufficiently different from the mosquito cells in morphology so that their development could be followed. The increase in size of the oocysts, the growth of sporocysts, and the formation of sporozoites were recorded by photomicrography. The sporogonous cycle was completed in 20 days culture at 23 to 24 C. The mosquito cell line was isolated from embryonated eggs and grown in a medium composed of equal parts of Hsu's, Schneider's, and Grace's insect tissue culture media. Heat-inactivated fetal bovine serum, 10% v/v, was added to the mixture. A subline of C. pipiens cells adapted to grow in Hsu's medium alone containing FBS also supported the growth of the complete cycle of the parasite. Previous papers (Chao and Ball, 1972) reported the in vitro cultivation of certain sporogonous stages of Hepatozoon rarefaciens from Drymarchon corais and of a hemogregarine from Boa constrictor in Grace's cell line (Grace, 1962, 1966) with the substitution of hemolymph of Samia cynthia for that of Antheraea eucalypti. The medium also contained 10% fetal bovine serum (Chao and Ball, 1970). Subsequently, Greene and Charney (1971) and Greene et al. (1972) showed that, in this country, Grace's cell line, which had been called a line of Aedes aegypti, was that of a moth, probably A. eucalypti. Chromosome counts made by us in 1972 indicated that the cells used by us were those of a moth rather than of a mosquito. Development of early sporogonous stages of H. rarefaciens in the hemocoel of Culex tarsalis was described by Ball et al. (1967) and by Ball and Oda (1971). The life history can also be completed in Anopheles albimanus and in Aedes sierrensis. We now report the complete development of sporogonous stages of this parasite in a cell line of the mosquito, Culex pipiens. MATERIALS AND METHODS As previously reported (Chao and Ball, 1972), H. rarefaciens obtained originally from Drymarchon Received for publication 5 December 1972. * Aided by Research Grants NIH AI-00087, NSF GB-14,587, and University of California, Zoology 254. corais was carried in the more easily obtainable gopher snake, Pituophis melanoleucus catenifer. Infected blood, drawn from a gopher snake, was mixed with an equal part of isotonic sodium citrate solution and transferred to a Culex pipiens cell line, which had been initiated in our laboratory. The cell line was grown in 30-ml-capacity Falcon culture flasks. Each flask held 3 ml culture medium and was inoculated with 4 drops (about 0.2 ml) of the citrated blood. The development of the parasite was observed with a Zeiss inverted microscope, and the various stages were recorded by FIGURE 1. Hepatozoon rarefaciens. Vermicules in ce l line culture of Culex pipiens. First halfhour. Arrow points to vermicules. X 235.

Field and laboratory studies on the pathogenicity of the fungus Beauveria bassiana to three genera of mosquitoes

The ability of the fungus Beauveria bassiana to kill mosquito larvae, adults, and eggs was challenged with Culex tarsalis, Culex pipiens, Anopheles albimanus, Aedes aegypti, Aedes sierrensis, and Aedes nigromaculis. B. bassiana was found most effective in killing larvae when applied as a conidial dust to the surface of the water. This was attributed to the primary sites of invasion being the perispiracular lobes of the larval siphon. During May and June of 1966, the mortality of C. pipiens larvae in outdoor test ponds ranged from 70% to 95% following an application of 3 lb. of viable conidia per acre. The Anopheles and Culex larvae tested all proved to be susceptible to the fungus while the Aedes larvae were not. In laboratory tests against the adults of C. tarsalis, C. pipiens, A. aegypti, A. sierrensis, A. nigromaculis, and A. albimanus, conidia of B. bassiana produced 100% mortality within 5 days after exposure, while less than 50% occurred in corresponding controls. Outdoor tests against the adults of A. nigromaculis in screen cages were less successful, yielding only 58% mortality. In this case, the fact that adults rested on the screen walls of their test cages, rather than in the dusted grass that had been provided, may explain the low mortality. Eggs of Culex and Aedes exposed to the conidia of B. bassiana hatched normally.

Embryogeny and Hatching of Aedes sierrensis Eggs (Diptera: Culicidae)

As with several other aedines, hatching of eggs of Aedes sierrensis (Ludlow) is induced by depletion of the dissolved oxygen of the flooding medium; but, in contrast to many of those others, appreciable hatching is obtained only when the dissolved oxygen is reduced to a very low level (0.25 ppm or less). Newly matured eggs undergo a brief increase in the threshold of stimulation necessary to induce hatching. The cause for this was not determined, but moisture conditions (which frequently cause this kind of effect) were ruled out; the altered responsiveness appears to arise spontaneously within the unhatched larva. Embryogeny requires 8–15 days at 25°–27°C; the larval and pupal stages, about 16 days. These developmental rates are much slower than those of A. aegypti , which requires 3–4 days and about 9 days, respectively, for these processes.

Pythium sp. (Phycomycetes:Pythiales) pathogenic to mosquito larvae

A species of the fungus Pythium causing a high rate of mortality in a field collection of Aedes sierrensis was examined for its potential as a biological contol agent. Motile zoospores, the only infective stage, exhibited a strongly positive chemotactic response to wounds of mosquito larvae, but were apparently incapable of penetrating normal cuticle. Wide variation of mortality rate in laboratory tests was a function of harshness of physical treatment which larvae received prior to testing and zoospore concentration.

Further observations on the somatic chromosome cytology of some mosquitoes (Diptera: Culicidae).

The morphology of mitotic chromosomes from brain tissues of young fourth-instar larvae was studied in 6 mosquito species: Eretmapodites chrysogaster Graham, Aedes (Ochlerotatus) sierrensis (Ludlow), A. (Stegomyia) mascarensis MacGregor, A. (S.) polynesiensis Marks, A. (S.) simpsoni (Theobald), and A. (S.) vittatus (Bigot). As with other mosquito species (particularly culicines), differences between individual karyotypes were rather small. In all of these the diploid chromosome number was 6, the complement consisting of 2 relatively large pairs and 1 slightly shorter pair. In several species, prophase chromosomes were frequently polarized; these may possess distinctive, and therefore mappable, chromomere patterns in some species. Among the chromosomal aberrations observed in A. simposoni were a trisomic cell, probably resulting from mitotic nondisjunction, and a rather pronounced heteromorphism of the smallest chromosome in 1 cell. As aedine mosquitoes have more-or-less similar karyotypes, and as their salivary gland chromosomes have not been mapped thus far, examination of the chromosomes of intra- and interspecific hybrids is greatly needed.

Failure to Isolate Microorganisms from within Mosquito Eggs1

By means of standard bacteriological methods, attempts were made to isolate microorganisms from eggs of the following species of mosquitoes: Culex tarsalis Coquillett, Culex quinquefasciatus Say, Anopheles albimanus Wiedemann, Aedes sierrensis (Ludlow), and Aedes aegypti (L.). No microorganisms were recovered from eggs that were sterilized under the dissecting microscrope to insure complete surface contact with the sterilizing agent. Contamination in some experiments with large numbers of eggs was attributed to incomplete surface sterilization and/or to possible invasion of bacteria through damaged egg shell of some of the eggs. Larvae emerged from the uncontaminated eggs continued to live in the sterile medium for many clays.