Ecological mechanism of climate-mediated selection in a rapidly evolving invasive species.

Recurring seasonal changes can lead to the evolution of phenological cues. For example, many arthropods undergo photoperiodic diapause, a programmed developmental arrest induced by short autumnal day length. The selective mechanisms that determine the timing of autumnal diapause initiation have not been empirically identified. We quantified latitudinal clines in genetically determined diapause timing of an invasive mosquito, Aedes albopictus, on two continents. We show that variation in diapause timing within and between continents is explained by a novel application of a growing degree day (GDD) model that delineates a location-specific deadline after which it is not possible to complete an additional full life cycle. GDD models are widely used to predict spring phenology by modelling growth and development as physiological responses to ambient temperatures. Our results show that the energy accumulation dynamics represented by GDD models have also led to the evolution of an anticipatory life-history cue in autumn.

Morphological and molecular evidence for a new species of Lutzia (Diptera: Culicidae: Culicini) from Thailand.

We found a species of LutziaTheobald (1903) (Diptera: Culicidae) in Chiang Mai Province and other provinces in northern Thailand which bears morphological and DNA sequence differences from the three species of Lutzia, subgenus Metalutzia Tanaka, previously recorded from Thailand, namely Lt. fuscana (Wiedemann), Lt. halifaxii (Theobald) and Lt. vorax Edwards. The adults of the Chiang Mai form (CM form) have abdominal banding patterns similar to those of Lt. vorax from Japan (which includes the type locality of this species), but differ in having the mediocubital crossvein usually positioned before rather than beyond the radiomedial crossvein. The thoracic and abdominal integument of Lt. vorax larvae is covered with relatively short pointed spicules whereas it is covered by denser, longer and more acutely pointed spicules in the CM form. Some differences are also found in the development of thoracic seta 1-M, and abdominal setae 8-II and 8-III. The pupa of the CM form clearly differs from the pupa of Lt. vorax in having setae 1 and 5 of abdominal segments V and VI branched rather than single. The characters of the wing, larva and pupa of the CM form are similar to those of Lt. fuscana and Lt. halifaxii. However, whereas the phallosome of males of the CM form is similar to males of Lt. vorax and Lt. halifaxii, it is distinct from males of Lt. fuscana. Phylogenetic analysis of mitochondrial cytochrome c oxidase subunits I and II sequences revealed that the CM form falls outside a clade comprised of specimens of Lt. vorax from Japan, Korea, Thailand and Bhutan (Kimura 2-parameter, K2P, genetic distances 3.9-5.6, and 5.1-6.6, respectively). However, the two gene sequences of the CM form are not clearly distinct from clades comprised of sequences from specimens of Lt. fuscana and Lt. halifaxii (K2P 0.2-2.4%). However, based on the combination of morphological and molecular data, the current study provides evidence that the CM form is a previously unrecognized species of the genus Lutzia.

Biology of Anopheles saperoi, an Endemic Species in Okinawajima, the Ryukyu Archipelago, Japan.

Biological studies of Anopheles saperoi were conducted using larval and adult mosquito collections in the northern part of Okinawajima of the Ryukyu Archipelago from June 2009 to July 2010. Anopheles saperoi was the most collected species in the northern Okinawajima, except Motobu Peninsula, where it was not collected. The southern distribution of An. saperoi was Sugita Stream, Nago City. Anopheles saperoi was collected throughout the year with reproduction (gonotrophic cycle) observed year-round. Immature densities varied for Hinna and Yona streams, and were negatively affected by precipitation patterns. Human attraction activity of females varied for by study area and collection time and was positively affected by temperature, but negatively by heavy rainfall. The greatest female human attraction activity was observed during 3:00-5:00 p.m., with peak at twilight. Parity rates varied from 23.1% to 83.3% throughout the year.

Blood meal identification and feeding habits of uranotaenia species collected in the ryukyu archipelago.

To know the blood meal in the stomach of Uranotaenia species, blood-fed mosquitoes were collected by 4 methods at different sites in the mountain forest of 3 islands, Amamioshima, Okinawajima, and Iriomotejima in the Ryukyu Archipelago, Japan from 2005 to 2012. One hundred twenty-four blood-fed Uranotaenia mosquitoes of 7 species (Ur. jacksoni, nivipleura, ohamai, yaeyamana, annandalei, lateralis, and macfarlanei) were collected. The collection rates are 0.26, 0.6, 0.31, and 0.66 by black light trap, black light blue with dry ice trap, frog call trap, and sweeping net, respectively. The blood meals of 107 females (86.3%) were successfully identified by a polymerase chain reaction-based method. All Uranotaenia species fed on cold-blooded animals, especially amphibians (99.1%), and notably on frogs. They would feed readily on available frogs in a given region having no close connection with the breeding (calling) season of each frog. They also fed on reptiles (0.9%), but not on warm-blooded animals.

Description of female pupa and larva of Ficalbia ichiromiyagii from Iriomote Island, Ryukyu Archipelago, Japan.

We describe and illustrate for the 1st time the pupa and larva of adult female Ficalbia ichiromiyagii based on specimens collected from a swamp forest in Otomi, Iriomote Island, Ryukyu Archipelago, Japan. The pupa has a trumpet that is 6.25 times its breadth at halfway, with a long meatal cleft line; setae 6-I, II considerably long, usually single or bifid; median caudal seta absent; and paddle edge serrated only on the outer apical half. The larva is characterized by dark pigmentation of the head and siphon, seta I-C elongate and spiniform, usually with a short lateral spine on the inner basal side and siphon with 1 pecten spine and conspicuous seta 1-S at the base. The immature stages were found only in undisturbed jungle swamps with dense vegetation.

Bionomics of the mud lobster-hole mosquito Aedes (Geoskusea) baisasi in the mangrove swamps of the Ryukyu Archipelago, Japan.

The bionomics of the mud lobster-hole mosquito Aedes (Geoskusea) baisasi in the Ryukyu Archipelago, Japan, was studied in the field and in the laboratory. The studies included the natural habitat, seasonal appearance, flight activity, mating behavior, resistance of eggs to desiccation, and breeding periods of the immature stages of this species. The burrow systems made by the mud lobster Thalassina anomala were excellent as breeding and resting habitats for both the immature and adult stages of the mosquito.

New records of mosquito species for different islands of the Ryukyu Archipelago, Japan.

During an 11-year study period (1999-2010) on the islands of the Ryukyu Archipelago, Japan, 20 mosquito species were identified. The results revealed new records of species hitherto not reported on these islands. This contribution updates the mosquito fauna of the various islands of the Ryukyu Archipelago.

First record of avian Plasmodium DNA from mosquitoes collected in the Yaeyama Archipelago, southwestern border of Japan.

We studied the prevalence of avian Plasmodium in 509 mosquitoes of 9 species collected from the Ishigaki and Iriomote islands in the Yaeyama Archipelago, located southwest from the mainland of Japan. Two identical avian Plasmodium lineages were detected from Culex (Culiciomyia) nigropunctatus. Detected lineages were phylogenetically classified into different clade to avian Plasmodium lineages from birds and mosquitoes in the mainland of Japan but identical to a lineage detected from a resident bird, White-breasted Waterken (Amaurornis phoenicurus). This is the first detection of avian Plasmodium DNA from mosquitoes in the Yaeyama Archipelago and suggested that resident birds might have been infected with an avian Plasmodium lineage specific to the studied area and C. nigropunctatus could be the candidate vector mosquito species.

A multiplex PCR-based molecular identification of five morphologically related, medically important subgenus Stegomyia mosquitoes from the genus Aedes (Diptera: Culicidae) found in the Ryukyu Archipelago, Japan.

Internal transcribed spacer regions of ribosomal DNA were sequenced, and new species-specific primers were designed to simplify the molecular identification of five morphologically related subgenus Stegomyia mosquito species--Aedes aegypti, Ae. albopictus, Ae. riversi, Ae. flavopictus, and Ae. daitensis--found in the Ryukyu Archipelago, Japan. Each newly designed primer was able to amplify a species-specific fragment with a different size. Conditions for multiplex PCR were optimized to identify all five species in a single PCR. This method is a convenient tool for entomological field surveys, particularly in arbovirus endemic/epidemic areas where some of these species coexist.

Molecular phylogenetics of Aedes japonicus, a disease vector that recently invaded Western Europe, North America, and the Hawaiian islands.

We used two mitochondrial loci (nicotinamide adenine dinucleotide dehydrogenase subunit 4 and cytochrome oxidase II) and a nuclear locus (28S-D2 spacer) for a total of 1337 bp to evaluate the relationships among the four subspecies of Aedes (Finlaya) japonicus Theobald. Ae. j. japonicus was recently introduced into the United States and has been expanding rapidly. We also included in our analysis a morphologically very closely related species, Aedes (Finlaya) koreicus Edwards, as well as three more distantly related species: Aedes (Finlaya) togoi Theobald, Aedes (Finlaya) hatorii Yamada, and Aedes (Aedimorphus) vexans Meigen. We found that the four subspecies in the Ae. japonicus complex are genetically quite distinct but seem to form a monophyletic group that surprisingly also includes Ae. koreicus, suggesting the need for a taxonomic reconsideration of the group. We also found that the two southern subspecies are more closely related to each other than to any of the remaining subspecies or to Ae. koreicus and may indicate an ancient north-south split of the lineage. Considering the overlap between Ae. j. japonicus and Ae. koreicus, but the stronger association between Ae. koreicus and humans, we are surprised it also has not expanded from its original range. As a proactive reaction to this possibility, we designed and tested a DNA-based rapid assay to differentiate Ae. koreicus from some of the species with which it may be confused in the United States. These Aedes are putative vectors of several important viral encephalitides.

Colonization and biology of the frog-feeding mosquito Uranotaeinia macfarlanei in the Ryukyu Archipelago.

A colony of Uranotaenia macfarlanei, a frog-feeding mosquito, was established in the laboratory. We report the bionomics of the species, as studied in the laboratory colony and in the field on Ryukyu Island, Japan. These include mating activity, feeding and resting habits, manner of oviposition, and egg, larval, and pupal periods.

Mark-release-recapture experiments with Anopheles saperoi (Diptera: Culicidae) in the Yona Forest, northern Okinawa, Japan.

Six mark-release-recapture experiments with Anopheles saperoi Bohart and Ingram were performed in the Yona Forest, northern Okinawa, Japan from June 1998 - November 1999, in order to estimate the gonotrophic cycle, survival, spatial distribution, flight range, and population size of An. saperoi. Adults and immature An. saperoi were collected from the Yona Forest area, taken to the laboratory and maintained under a controlled temperature and humidity in order to get a lager number of mosquitos for the mark-release-recapture experiments. Cohorts of An. saperoi females and males, numbering 3,016, 4,728, 327, and 2,603 for experiments I, II, III, and IV, respectively, were released. Cohorts of An. saperoi females only, numbering 709 and 586 for experiments V and VI, respectively, were also released. At the release site, the An. saperoi were marked with 0.5% fluorescent dye Rhodamine B and released. The recapture rates were 14 (0.93%), 33 (1.40%), 3 (1.83%), 32 (2.46%), 14 (1.97%), and 22 (3.75%) for experiments 1, II, III, IV, V, and VI, respectively. The length of the gonotrophic cycle of the recaptured An. saperoi females was estimated to be about 4 days, through the dissection method. The daily survival rate was estimated to be 0.73 by regression coefficient. The spatial distribution of marked, recaptured An. saperoi was similar to that of unmarked captured An. saperoi. The spatial distribution of the marked, recaptured An. saperoi among the collection site categories was significantly different. The observed differences in the frequency distribution of marked recaptured An. saperoi were considered to be due to the spatial variation of the habitats between the collection sites. The maximum flight range of the recaptured An. saperoi recorded in this study was 0.93 km. The population size of An. saperoi females in the study area was estimated by the Seber method to be 23,841, 1,182, 3,514, 5,679, and 9,238 for experiments I, II, IV, V, and VI, respectively. The estimated population size has a low standard of error using the Seber method, therefore we estimated our population size reasonably well. The population attributes and ecology of An. saperoi in the Yona Forest, in northern Okinawa are discussed.

Habitats and distribution of Anopheles sinensis and associated Anopheles hyrcanus group in Japan.

Mosquito collections were carried out in August 2002 and July 2003 in Japan. Anopheles sinensis of the Hyrcanus Group, Myzomyia Series of Anopheles, was the most common species encountered. The distribution and habitats of 5 Anopheles Hyrcanus Group species are included. Eight species belonging to Aedes, Culex, and Uranotaenia were found associated with An. sinensis in rice paddies and a variety of other larval habitats.

Female Armigeres (Leicesteria) flavus holding an egg raft with her hind legs.

During a mosquito survey in Ulu Gombak, Selangor, Peninsular Malaysia, October 2-16, 2003, we observed a peculiar oviposition habit of Armigeres flavus. This strange behavior is described and illustrated with photographs; although it is well known, no detailed description has been made previously.

A crab-hole mosquito, Ochlerotatus baisasi, feeding on mudskipper (Gobiidae: Oxudercinae) in the Ryukyu Islands, Japan.

Ochlerotatus baisasi was observed and photographed feeding on a mudskipper (Periophthalmus argentilineatus). Origin of blood meals in the stomachs of female mosquitoes collected at burrows of land crabs and mud lobsters in the mangrove forests of Oura, Okinawa, and Komi, Iriomote (the Ryukyu Islands), was determined by enzyme-linked immunosorbent assay to be from a fish source.

The effectiveness of impregnated bed net in malaria control in Laos.

Impregnated bed net (IBN) were used in 366 villages in the central and southern three provinces of Lao PDR from 1999 to 2000. It was confirmed that 81.0% of 40000 bed nets, which were donated by Japanese Grant Aid, were delivered within 2 years. The strengthening of information network systems in anti-malaria and strong relationship between community and local authorities ensured the success of operation in a short period. The number of patients and the slide positive rate of malaria decreased markedly in public health facilities in three provinces after the use of IBN. An entomological survey was conducted in Boualapha district, where malaria is endemic, to investigate the IBN efficacy on malaria vector. The density and parous rate of Anopeles dirus, which is the main malaria vector in the area, were markedly decreased in the village where IBN was used. This mosquito's behavior, which was baiting mainly humans during the time when the inhabitants sleep in the IBN, was considered to be advantageous in preventing malaria infection using by IBN. The area of distribution of A. dirus is similar to the high endemic area of malaria in Lao PDR. Thus, it is expected that the expansion of the IBN program in the southern provinces will lead to successful malaria control in subsequent years.

Knowledge and behavior relating to malaria in malaria endemic villages of Khammouane Province, Lao PDR.

In order to provide basic data for evaluation of malaria control measures, a study on the knowledge and behavior of people regarding prevention of malaria was carried out in 8 malaria endemic villages in Khammouane Province of Lao PDR from 1999 to 2000. The total valid questionnaire respondents were 932, with a mean age of 32.3 +/- 14.9. 43.7% of the respondents were illiterate. About 44% of the respondents suffered from malaria in the past. About 55% of the illiterate group slept in mosquito nets, compared to 75.4% for the educated group. About 29% of the illiterate respondents had knowledge of malaria transmission by mosquito bites, compared to 48.8% for the educated groups. Out of 167 non-impregnated mosquito nets examined in two villages, 13 were in bad condition having holes or leaks and 39 female mosquitos including Anopheles spp were collected in these nets by early morning catches. Knowledge of malaria and behavior in relation to the prevention of malaria were significantly related to educational level. Health education as well as general education must be taken into account for communities in malaria endemic areas to become more involved in malaria control strategies.

An ecological survey of dengue vector mosquitos in central Lao PDR.

An ecological survey of dengue vector mosquitos was carried out in June 2000 in central Lao PDR. Two areas in Khammouane Province, Nongbok and Thakhek, were selected for the survey. Of the 7 mosquito species identified, Aedes aegypti was dominant in both study areas. The container index for Ae. aegypti in Nongbok was 51.8% and was significantly higher than that of Thakhek (40.2%); moreover, significant differences between the study areas were found with records to containers and to the conditions surrounding the houses. The key containers in Nongbok were water jars, whereas drums or small or discarded containers had the highest occurrence rate of Ae. aegypti in Thakhek. Mesocyclops aspericornis was found in large water jars and cement water tanks; no Aedes larvae were found at these sites. Strategy to control dengue vectors in the study areas was discussed.

Investigation of the aedes (Stegomyia) flavopictus complex (Diptera: Culicidae) in Japan by sequence analysis of the internal transcribed spacers of ribosomal DNA.

Aedes (Stegomyia) flavopictus Yamada is widely distributed in Japan and Korea. The species comprises three subspecies based on current morphological taxonomy: Aedes flavopictus in the Palearctic region of Japan, Ae. f downsi Bohart & Ingram from Amami and Okinawa Islands, and Ae. f miyarai Tanaka, Mizusawa & Ingram from Ishigaki and Iriomote Islands of the Ryukyu Archipelago. These subspecies designations are based on observations of a combination of several morphological characters, none of which, by itself is diagnostic for discriminating among the three subspecies. To further study the relationships in this group, we examined the nucleic acid sequence divergence in the internal transcribed spacer regions (ITSI and ITS2) of the ribosomal DNA gene array of Ae. flavopictus individuals collected at five sites from three geographic regions in Japan. Analysis of sequence data by distance and maximum parsimony methods produced phylogenetic trees that showed separation of the specimens into three major clades, corresponding to both subspecies and geographic region. These results were consistent with and support the current classification and geographic distribution of the three subspecies.

Susceptibility of the mosquitoes Anopheles minimus, An. sinensis, and An. saperoi (Diptera: Culicidae) from the Ryukyu Archipelago, Japan, to the rodent malaria Plasmodium yoelii nigeriense.

The susceptibility of three anopheline mosquitoes, Anopheles minimus Theobald, An. sinensis Wiedemann, and An. saperoi Bohart & Ingram, from the Ryukyu Archipelago to the rodent malaria, Plasmodium yoelii nigeriense was examined to find new vectors other than An. stephensi Liston for rodent malaria studies in the laboratory. The survival rate of the mosquitoes after feeding on mice infected with P. y. nigeriense was also examined. The Beech strain of An. stephensi from India was compared with An. minimus from Ishigaki Island, and An. sinensis and An. saperoi from Okinawa Island. Oocysts were first found on day 3 after feeding on mice infected with P. y. nigeriense in An. stephensi, on day 4 in An. minimus and An. saperoi, and day 6 in An. sinensis. From 8 to 14 d after feeding on malaria-positive mice, oocysts were present in 97.2-100% of An. stephensi, 85.7-100% of An. saperoi, 20-74.1% of An. minimus, and 12.5-13.3% of An. sinensis. The duration of oocyst occurrence in An. saperoi was 55 d, the longest among the anopheline mosquitoes used in this study. On day 8 after feeding, sporozoites were found in the salivary glands and heads of all the mosquitoes tested. From the 10th to 16th d, sporozoites were present in the salivary glands of 14.9% (range, 9.1-28.0%) of An. minimus, 47.3% (40.7-58.1) of An. saperoi, and 96.2% (94.1-97.2) of An. stephensi, but were absent in An. sinensis. Anopheles saperoi could be an excellent vector of P. y. nigeriense because it has comparatively high susceptibility and high longevity even after feeding on infected mice.