[in Japanese]

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Since 1962, a long run project of the studies on the seasonal prevalence and JEV infection of the vector mosquito, Culex tritaeniorhynchus-vishnui complex, has been performed at a farm village near Fukuoka City, Uchihashi, Kasuyagun, Fukuoka Prefecture. In the present paper the results obtained in 1964 and 1965 are fully presented. The results and logical conclusions are summarized as follows : 1) Seasonal prevalence of the vector mosquito population : The abundant season of the vector mosquito was in the period from mid-July to mid-August in 1964 and in the period from late July to late August in 1965. 2) Natural infection of vector mosquitoes : The mosquito infections of the two years were very similar in the time of infection, the length of infection period and the isolation rate. Main period of the mosquito infection did not exceed four to five weeks, despite the fact that the vector mosquito population remained satisfactory for about eight weeks. This strongly suggests that the natural decrease of the infected mosquitoes may be due to the scarcity of amplifying hosts but not to the decline of the total population of vector mosquitoes. 3) Correlation between the mosquito infection and the epidemic of human encephalitis : Aside from the scattered occurrence of reported cases, the regular outbreak of confirmed cases yearly followed on the onset of the mosquito infection with the time lag of about two weeks, and the length of the epidemic corresponded approximately with that of the mosquito infection. A special attention should be paid to the difference between the mosquito infection in 1963 and those in 1964 and 1965 as of the epidemiological importance, because the epidemic of human encephalitis was far larger in the former year than in the latter years. The time of the mosquito infection was distinctly later in 1963 than in the subsequent two years. The significance of this problem will be elucidated in further studies.

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Faunistic and ecological surveys of medically important mosquito species of Japan were made by the author during the period from 1961 to 1966. A total of 5, 200 larval habitatsin 542 localities covering all the prefectures of Japan were surveyed, and altogether about 60 species were collected. The specimens were identified by mature larvae or by adults. The mosquito species were classified tentatively into 24 groups according to their taxonomic and ecological status, and the geographic distribution, type of larval habitats and the relation with associated species were discussed for each species or group. The results are summarized in Table 1 to 3.

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There are many papers dealing with the natural enemies of mosquito larvae. However, quantitative observations of their predative activity have not yet been presented in those works. The authors studied the predators on larvae of Culex tritaeniorhynchus summorosus Dyar, a vector of Japanese encephalitis virus, in the rice paddy field in Okayama, Japan, and determined their predative capacity in the laboratory. The names of predators and the maximum predative activity presented as "observed take-number of mosquito larvae" per day were given as follows : Cloeon dipterum (Mayfly) 5 nymphs 2/10, Aciagrion hisopa (Damselfly) 5 nymphs 2/10, Ceriagrion melanurum (Damselfly) 5 nymphs 2/5, Ceriagrion sp. (Damselfly) 5 nymphs 1/5, Copera annulata (Damselfly) a nymph 3/5, Crocothemis servilia (Dragonfly) a nymph 10/10 and 47/50, Sympetrum sp. (Dragonfly) a nymph 10/10 and 64/100, Anax parthenope julius (Dragonfly) a nymph 50/50 and 154/200, Sigara substriata (Water boatmen) 5 larvae 2/5, Laccotrephes japonensis (Water scorpion) a nymph 10/10, Ranatra chinensis (Water stick-insect) an adult 10/10, Notonecta triguttata (Back swimmer) 3 adults 50/50 and 154/200, Paraplea japonica 5 nymphs 3/5, Sternolophus rufipes (Water scavenger beetle) 5/5 and 15/20, Hydaticus grammicus (Water beetle) a larva 10/10 and Misgurnus fossilis an adult 5/5.

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In order to know the efficiency of insecticide residual spray to Culex tritaeniorhynchus summorosus adults, the observations of the resting behavior of this species were carried out in the suburbs of Kawasaki, Japan, in the summer of 1967. The adult indoor resting collections were made in daytime by the hand-capture in human dwellings, a pig pen, poultry houses, glocery stores, store barns and so. The collections were identified by species and sorted out according to the blood digestion stages. Indoor resting densities of this species were rather low. Otherwise, the adult outdoor resting collections were undertaken from natural shelters, i. e. grass, crop field, paddy field and so. A number of adults were found in grass in daytime. The resting habit on walls of a pig pen which will accompany the biting activity towards pigs was observed throughout a night. The nocturnal rhythm with a light trap collection, which was hung just overhead the pigs, showed one peak at 21 : 00-22 : 00. It is interesting that the nocturnal rhythm of biting activities was almost similar to the catching rhythm of the light trap. The number of resting mosquitoes on a wall increased rapidly from about 19 : 30, and showed a peak at 24 : 00〜1 : 00. More unfed mosquitoes than fed ones were observed on the wall in the first half of the night, and the tendency retrogressed at about 3 : 00. From about that time, the resting mosquitoes decreased rapidly, perhaps owing to getting out of the pig pen. It seemed the resting in the pig pen at night was rather short and temporary. It may be concluded that this species is rather exophily throughout daytime and night.

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Esterase inhibition by organophosphates or carbamates in the resistant houseflies has been studied by the thin layer electrophoresis developed by Dr. Ogita (1962). When the flies belonging to various colonies, such as RP, FC, 203d, Hokota, Dream-Island, 15th dumping Island and Matsudo dumping site, had been treated by topical application with fenthion, fenitrothion, dichlorvos and Nestyn, 0.1μg per female fly, these insecticides inhibited markedly faster migrating esterase bands E_2 and E_4, while diazinon or malathion failed to inhibit E_4 band. Oxon-type organophosphates such as isopropyl diazoxon were effective against the diazinon resistant colonies and strongly inhibited E_2 and E_4 bands. The mixtures of fenthion, fenitrothion, trichlorfon or Nestyn added with dichlorvos at a ratio of 1 : 1 clearly inhibited E_2 and E_4 esterases of the resistant flies. On the other hand, the mixtures of organophosphate (fenthion, fenitrothion or dichlorvos) and carbamate (Sevin, Dimetilan or Meobal) failed to inhibit E_4 esterase band.

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Both adults and larvae of the flesh fly, Boettcherisca peregrina originally collected from Inokashira of Tokyo in 1966 and from Gyoda of Saitama Prefecture in 1965, were tested for γ-BHC resistance after being reared in laboratory. The final instar larvae were tested undre "the dilution contact method" in which the larvae were exposed for 48 hours to a thin layer of desired concentration of insecticide emulsion. The adults were assessed by the topical application test. The LC_<50> value in the larval tests was 3, 800 ppm for Inokashira colony and was 82 ppm for the Gyoda colony. The γ-BHC resistance of the Inokashira colony was enough high as to be 200 times than that of a susceptible colony. The Gyoda colony, on the contrany, showed only 4.3 times of the value as the resistance ratio. However, these resistant two colonies were found to be still susceptible to organophosphorus insecticides such as diazinon and fenthion. The LD_<50> value of the adult flies was 1.6μg per female fly in the Inokashira colony and was 0.11μg in the Gyoda colony.

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Studies on cross resistance among several insecticides was carried out using diazinon-resistant and -susceptible colonies, malathion-resistant and -susceptible colonies, and fenitrothion-resistant and -susceptible colonies of Culex pipiens s. l. larvae. A formula was introduced to calculate the extent of cross resistance. The ratio of cross resistance of chemical B to chemical A (RCR_<B-A>) is shown by the following formula : [numerical formula] in which B max. and B min. are the maximum and minimum LD-50's of the chemical B, respectively, and B_A-max. is the LD-50 of B in the maximum Aresistant colony. In other words, B max. and B min. are the LD-50's in the homozygous resistant and homozygous susceptible colonies, respectively. The RCR can be given only when the values of B max. and B min. have been obtained. In the present studies, the RCR could be calculated only among three chemicals, i. e. diazinon, malathion and fenitrothion, because the maximum LD-50's of the other chemicals were not yet be given. The results are listed in Table. According to the results, diazinon-resistance and fenitrothion-resistance were completely crossed with each other, and malathion-resistance was crossed with diazinon-resistance of fenitrothion-resistance only in a lower level (c. f. RCR=) and vice versa. It was also concluded that diazinon-, malathion- or fenitrothion-resistance was not crossed with DDT-resistance, and diazinon- or fenitrothion-resistance was not crossed with dieldrin- or lindane-resistance. However, it seemed to be curious that the malathion-resistant and -susceptible colonies both selected from a mixed population showed similar and higher resistance to dieldrin than the original susceptible population.

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1) Laboratory tests were conducted on sterilizing effects of the chemosterilants, Metepa and Hempa, with a laboratory colony of the mosquito Culex pipiens molestus Forskal by a contact method. The adults of either of both sexes were brought to contact for 3 hours with the residue of the chemicals which had been sprayed on the filter paper in acetone solution and dried. The number of females which laid eggs, the average number of eggs per raft, and the sterility rate of the eggs in total were recorded. 2) After exposing the adults of both sexes to the residual deposit of 50ml of 0.1 percent solution of Metepa per square meter on filter paper for 3 hours, the sterility rate of eggs laid was 99.9 percent in average, while little effect was shown at 0.1 percent solution. 3) Hempa, when tested by the same method at a concentration of 1.0 percent, caused 99.7 percent sterility. However, this compound at concentration of 0.1 percent or less did not give satisfactory effects. 4) Another series of experiments were conducted to observe the differnce in the effectiveness of the compounds among sexes. Both chemosterilants were found effective when treated on the females, but ineffective on the males. 5) When both males and females were exposed to the residue made on the filter paper by spraying 2.5 percent acetone solution of Metepa at a dose of 50ml per square meter, 5 minutes contact was found enough to give nearly 100 percent sterility. With Hempa 3 hours contact was required to cause 93.3 percent sterility. It was shown that Metepa had a more immediate effect than Hempa. 6) The 50 percent sterilizing time (ST-50) of Metepa when both sexes were treated was estimated to be between 2 and 5 minutes under the described conditions, while that of Hempa turned out to be around 43 minutes. 7) The deposit of either Metepa or Hempa on the filter paper treated at a dose of 50ml of 2.5 percent acetone solution per square meter was tested on the sterilizing effect with both sexes of the mosquito at intervals up to 11 weeks after the spraying. The continuous and rapid loss of the effectiveness was observed with Metepa from over one week onward. After 6 weeks Metepa caused only 8.0 percent of sterility. On the other hand Hempa showed still 95.1 percent sterility effect even at 11 weeks, although a slight reduction in the effect was observed during 2 to 4 weeks from the spraying. 8) It was concluded that by the contact method of test on Culex pipiens molestus Forskal, Metepa showed effect but the activity of the deposit did not last long enough, while Hempa had a slower effect but were longer in the residual activity of the deposit.

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Control experiments of Culex tritaeniorhynchus were carried out in a pig pen and in laboratory. In rapidity of knockdown assessed by Busvine-Nash method (WHO standard method), KT-50s of ronnel 1% emulsion and fenitrothion 0.5% emulsion were approximately 120 minutes each. In the film contact tests to the residues for limited time, the contact time for killing 50% of the mosquitoes was 2.75 minutes in ronnel and 9.0 minutes in fenitrothion. The resting time to the adult mosquitoes for blood sucking on pigs and human arms were estimated to be 5 min., 5 sec. and 10 min., 54 sec. respectively. Mortality of the fed and the unfed Culex tritaeniorhynchus which were resting on walls of the pig pen were 93.5% and 76.2% respectively, if all of the pigs had been sprayed with ronnel several hours before. When ronnel was applied not only to the pigs but also to all over the innor walls of the pen, the mortality of the fed and the unfed raised to 94.6% and 95.2%, respectively.

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From the two plateaux demonstrated on a log time-probit percentage knockdown line (lt-p line) with the German cockroach, Blattella germanica, the composition ratio of three phenotypes of dieldrin resistance, i. e. homozygous susceptibles (SS), heterozygous hybrids (RS) and homozygous resistants (RR), is detectable. On the lt-p lines of chlordane and lindane, similar two plateaux were also shown. The composition ratio of three phenotypes with above three chlorinated hydrocarbon insecticides, i. e. dieldrin, chlordane and lindane, was evaluated to give the same value with each other. Therefore, it was concluded that the resistance to these three chemicals was completely crossed, and that perhaps the same gene was responsible to the resistance of the three chemicals. On the other hand, the resistance in the German cockroach to dieldrin, chlordane and lindane was concluded not to be crossed with three organo-phosphorus compounds, i. e. diazinon, fenthion and fenitrothion, as the result of the lt-p line tests.

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Studies were carried out on the transition of the configuration ratio of three phenotypes, i. e. SS, RS and RR, or the gene frequency of dieldrin resistance in 7 closed laboratory colonies of Blattella germanica (L.), by analzing the log time-probit percentage knockdown lines. All colonies were shown to decrease markedly the resistant gene ratio during the rearing in closed populations and, for example, R ratio of the Kanda colony decreased from 38.8% to 1.1% in 2 years' rearing. Comparative studies were also carried out on the difference in the fecundity between SS and RR colonies separated both from the same colony of B. germanica (L.). No difference was shown in the number of larvae hatched from one female. The interval of the oviposition from the emergence to the 1st oviposition as well as that from the 1st to the 2nd oviposition had no difference between two colonies, but that of the RR colony from the 2nd to the 3rd was significantly longer than that of the SS colony. The longevity of adult females in the RR clony was significantly longer that of the SS colony. These differences in fecundity, however, were presumed not to be the main factor of the decrease of gene frequency of the dieldrin resistance in closed populations.

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In January and March 1967, overwintering mosquitoes were collected in many different types of caves in Izu Peninsula and Chiba Prefecture. The numbers and species collected were 65♀♀ Culex tritaeniorhynchus summorosus, 16♀♀ and 1♂ C. kyotoensis, 242♀♀ C. orientalis, 45♀♀ C. pipiens pallens, 4♀♀ C. vorax and 1♀C. bitaeniorhynchus. It is evident that caves are important sites for overwintering of adult mosquitoes, especially for the vector of Japanese encephalitis virus, Culex tritaeniorhynchus summorosus. Trials for the isolation of Japanese encephalitis virus from these mosquitoes with the intracerebral inoculation into suckling mice and the plaque assay in the chick embryo cell culture were all negative. Results of the examination of ovariole relics indicated that all of 46 Culex tritaeniorhynchus summorosus dissected were nulliparous.

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