Elongation of in vitro cultured ovarian follicles in the mosquito, Culex pipiens pallens.

Ovarian follicles dissected from female Culex pipiens pallens 3 d after a blood meal were cultured in PBS-based medium and morphological changes were recorded as time-lapse movies. The dissected follicles, already reaching stage III, were ovoid in shape, ca 300 µm long, and 180 µm wide. The oocyte had almost finished yolk protein uptake, and the cytoplasm was filled entirely with yolk granules. From 2 to 4 h after the culture started, the follicles started to elongate dramatically to reach a length of 450-550 µm during the next few hours. After culturing for 24-30 h, the follicles had reached stage V, assuming the shape of normal mature eggs with a micropyle apparatus. Since cytochalasin D or colchicine completely or partially inhibited such follicle elongation and maturation, contraction of cytoskeletons made of actin filaments or microtubules may play a significant role in this phenomenon.

Follicular epithelial cell apoptosis of atretic follicles within developing ovaries of the mosquito Culex pipiens pallens.

Follicular atresia, the degeneration of developing follicles, is always incident to normal oogenesis in both oviparous and viviparous animals. Photo- and electron-microscopic observation of degenerating follicles within developing ovaries taken from blood-fed Culex pipiens pallens mosquitoes showed gradual degradation of the internal structures including yolk granules in the oocyte. The epithelial cells, which sometimes incorporated yolk granules from the oocyte along with the shrinkage of the follicle, gradually lost their uniform columnar shape, while their integrity as a covering layer remained. In situ active caspase analysis detected active enzymes in these epithelial regions. In the latest stages of atresia where either the nurse cells or oocyte were lost, the follicle was mainly comprised of irregularly shaped epithelial cells, and some of these cells' nuclei contained condensed chromatin peripherally, one of the characteristics of apoptotic cells. Also terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling treatment indicated that DNA fragmentation occurred in these follicles. It seems likely that in atretic follicles the epithelial cells survive to play key roles in the event, and then finally undergo their own apoptotic cell death so as to give the developmental site to the next follicle in the same ovariole.

Insemination rates of hybrids between Culex pipiens pipiens and Culex pipiens quinquefasciatus or Culex pipiens pallens at high temperature.

Females of Culex pipiens pallens are rarely inseminated at temperatures above 30 degrees C. Insemination and egg-hatching rates (F2) were examined in females of hybrids (F1) between Culex pipiens pipiens and Culex pipiens quinquefasciatus at 25 degrees C and 30 degrees C to examine temperature-dependent reproductive activity. Insemination rates were very high in hybrid females (F1) at 25 degrees C and 30 degrees C, and egg-hatching rate (F2) also was high at both temperatures. Crosses between Cx. p. pipiens and Cx. p. quinquefasciatus did not show insemination rates and egg-hatching rates similar to the rates observed in Cx. p. pallens, suggesting that the hybrids expressed reproductive activity characteristic of Cx. p. quinquefasciatus.

Effects of mating on oogenesis induced by amino acid infusion, amino acid feeding, or blood feeding in the mosquito Anopheles stephensi (Diptera: Culicidae).

Hemocoel infusion of an amino acid mixture at a concentration of 7.5% and 10% (wt:vol) for 24 h rarely activated ovarian development in either mated or unmated females of Anopheles stephensi Liston. Infusion of either concentration mixture for 48 h into unmated females was also scarcely stimulatory, but the same 48 h infusion into mated females resulted in ovarian maturation in 25% (7.5% mixture) or in 78% (10% mixture) of the infused specimens. Similarly, feeding on an amino acid mixture (7.5%) containing sugar induced 44% ovarian maturation among mated females, while only 3% of unmated females showed matured oocytes. The increase of hemolymph amino acid concentration levels may be one of the essential factors for oogenesis of An. stephensi, and mating enhances ovarian development, especially in females ingesting poor meals. However, some additional factor(s) may be included for full development comparable to that induced by blood meals.

Laboratory studies of Ochlerotatus samoanus in association with leaf axils of Freycinetia (Pandanaceae) in Samoa.

Three species of Freycinetia (Pandanaceae) (F. reineckei, F. storkii, and F. hombronii) were tested for oviposition preference of Ochlerotatus samoanus, a vector of filariasis in Samoa. Laboratory tests indicated that F. reineckei was preferred by this mosquito for oviposition. Eggs were preferentially deposited on a peeled or a moist dried leaf. The percentage hatch was highest when eggs were kept moist for longer than 4 days before submersion. Hatching was complete in less than 6 h. These studies allowed us to successfully rear Oc. samoanus in the laboratory, facilitating future studies on the biology and control of this important vector.

Reproductive activity and survival of Culex pipiens pallens and Culex quinquefasciatus (Diptera: Culicidae) in Japan at high temperature.

The egg hatchability, insemination, and longevity of Japanese Culex pipiens pallens Coquillett and Japanese Culex quinquefasciatus Say were compared at 25 and 30 degrees C. Egg hatchability was high in Cx. p. pallens at 25 degrees C, but it was very low at 30 degrees C because almost no females were inseminated at this temperature. In Cx. quinquefasciatus, the egg hatchability and insemination rates were very high, even at 30 degrees C. The longevity of adult females and males was generally shorter in Cx. p. pallens than in Cx. quinquefasciatus at both temperatures. Because high temperatures may restrict the spread of Cx. p. pallens, we suggest that even if this species spreads to Okinawa, the possibility of it becoming established is very low.

Ovarian development induced in decapitated female Culex pipiens pallens mosquitoes by infusion of physiological quantities of 20-hydroxyecdysone together with amino acids.

Infusion of 20-hydroxyecdysone into the hemocoel of unfed decapitated female Culex pipiens pallens mosquitoes, at a very low rate of 500-2000pg per day, often stimulated oögenesis of this species, when the hormone was infused together with amino acids. The hormone alone or amino acids alone showed no such stimulatory effect. Previous reports that using an abdomen ligated immediately after a blood meal for hormone injection reduced the quantity of 20-hydroxyecdysone needed to activate unfed female Aedes aegypti by a few thousand times, are therefore due mainly to a sufficient supply of amino acids from the midgut in the isolated abdomen.