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Background In Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015. Methods A survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a Centers for Disease Control and Prevention (CDC) miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to polymerase chain reaction (PCR)-based sequence analysis of the internal transcribed spacer 2 ( ITS2) region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method with the Kimura 2-parameter model on MEGA X version 10.2.2. Results A total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae, was discovered in eastern Hokkaido in 2004. Anopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. hyrcanus group, namely An. belenrae, An. engarensis, An. lesteri, and An. sineroides, have been found in Hokkaido. The results also suggest that An. sinensis, formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido. Conclusion The updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae, described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan. Graphical abstract

Background: In Hokkaido, northern island of Japan, at least seven cases of falciparum malaria were reported by 1951. A survey conducted at that time was unsuccessful in implicating any mosquito species as the possible vector. Although active anopheline mosquito surveillance continued until the middle of the 1980s, there is very limited information on their current status and distribution in Japan. Therefore, this study is an update on the current status and distribution of anopheline mosquitoes in Hokkaido based on a 15-year entomological surveillance between 2001 and 2015.Methods: A survey of mosquitoes was conducted at 22 sites in Hokkaido, Japan, from 2001 to 2015. Adult mosquitoes were collected from cowsheds, lakesides, shrubs, and habitats ranging from open grassland to coniferous forest using a CDC miniature light trap enhanced with dry ice, aspirators, and sweeping nets. Larvae were collected from lakes, ponds, swamps, stagnant and flowing rivers, and paddy fields. All specimens were morphologically identified and subjected to PCR-based sequence analysis of the ITS2 region of rDNA. Phylogenetic trees were reconstructed using the neighbor-joining method. Results: A total of 46 anopheline specimens were used for the phylogenetic analysis. During the survey, a new member of the Anopheles hyrcanus group, An. belenrae Rueda (2005), was discovered in eastern Hokkaido in 2004. Anopheles belenrae has since then been consistently found and confirmed to inhabit only this area of Japan. Four members of the An. hyrcanus group, An. belenrae, An. engarensis, An. lesteri, and An. sineroides, have been found in Hokkaido. The results also suggest that An. sinensis, formerly a dominant species throughout Japan, has become a rarely found species, at least currently in Hokkaido.Conclusion: The updated distribution of anopheline mosquitoes in Hokkaido, Japan, showed considerable differences from that observed in previous surveys conducted from 1969 to 1984. In particular, areas where An. sinensis was previously distributed may have been greatly reduced in Hokkaido. The phylogenetic analysis revealed a novel An. hyrcanus group member identified as An. belenrae, described in South Korea in 2005. It is interesting that An. belenrae was confirmed to inhabit only eastern Hokkaido, Japan.

Japanese encephalitis (JE) remains a public health concern in several countries, and the Culex mosquito plays a central role in its transmission cycle. Culex mosquitoes harbor a wide range of viruses, including insect-specific viruses (ISVs), and can transmit a variety of arthropod-borne viruses (arboviruses) that cause human and animal diseases. The current trend of studies displays enhanced efforts to characterize the mosquito virome through bulk RNA sequencing due to possible arbovirus&ndash, ISV interactions, however, the extent of viral diversity in the mosquito taxon is still poorly understood, particularly in some disease vectors. In this study, arboviral screening and RNA virome analysis of Culex tritaeniorhynchus and C. pseudovishnui, which are part of the Culex vishnui subgroup mosquitoes, were performed. Results from these two mosquito species, known as the major vectors of JE virus (JEV) in Asia, collected in three prefectures in Japan were also compared with the sympatric species C. inatomii. A total of 27 viruses, including JEV, were detected from these Culex mosquitoes. Molecular and phylogenetic analyses of the detected viruses classified 15 of the 27 viruses as novel species, notably belonging to the Flaviviridae, Rhabdoviridae, Totiviridae, and Iflaviridae families. The successful isolation of JEV genotype I confirmed its continuous presence in Japan, suggesting the need for periodic surveillance. Aside from JEV, this study has also reported the diversity of the RNA virome of disease vectors and broadened the knowledge on mosquito virome profiles containing both arbovirus and ISV. Mosquito taxon seemed to contribute largely to the virome structure (e.g., virome composition, diversity, and abundance) as opposed to the geographical location of the mosquito species. This study therefore offers notable insights into the ecology and evolution of each identified virus and viral family. To the authors&rsquo, knowledge, this is the first study to characterize the viromes of the major JE vectors in Japan.

Global warming threatens to increase the spread and prevalence of mosquito-transmitted diseases. Certain pathogens may be carried by migratory birds and transmitted to local mosquito populations. Mosquitoes were collected in the northern Philippines during bird migration seasons to detect avian malaria parasites as well as for the identification of potential vector species and the estimation of infections among local mosquito populations. We used the nested PCR to detect the avian malaria species. Culex vishnui (47.6%) was the most abundant species collected and Cx. tritaeniorhynchus (13.8%) was the second most abundant. Avian Plasmodium parasites were found in eight mosquito species, for which the infection rates were between 0.5% and 6.2%. The six Plasmodium genetic lineages found in this study included P. juxtanucleare -GALLUS02, Tacy7 (Donana04), CXBIT01, Plasmodium species LIN2 New Zealand, and two unclassified lineages. The potential mosquito vectors for avian Plasmodium parasites in the Philippines were Cq. crassipes, Cx. fuscocephala, Cx. quinquefasciatus, Cx. sitiens, Cx. vishnui, and Ma. Uniformis; two major genetic lineages, P. juxtanucleare and Tacy7, were identified.

Knowing the genetic variation that occurs in pathogen populations and how it is distributed across geographical areas is essential to understand parasite epidemiology, local patterns of virulence, and evolution of host-resistance. In addition, it is important to identify populations of pathogens that are evolutionarily independent and thus free' to adapt to hosts and environments. Here, we investigated genetic variation in the globally distributed, highly invasive avian malaria parasite Plasmodium relictum, which has several distinctive mitochondrial haplotyps (cyt b lineages, SGS1, GRW11 and GRW4). The phylogeography of P. relictum was accessed using the highly variable nuclear gene merozoite surface protein 1 (MSP1), a gene linked to the invasion biology of the parasite. We show that the lineage GRW4 is evolutionarily independent of GRW11 and SGS1 whereas GRW11 and SGS1 share MSP1 alleles and thus suggesting the presence of two distinct species (GRW4 versus SGS1 and GRW11). Further, there were significant differences in the global distribution of MSP1 alleles with differences between GRW4 alleles in the New and the Old World. For SGS1, a lineage formerly believed to have both tropical and temperate transmission, there were clear differences in MSP1 alleles transmitted in tropical Africa compared to the temperate regions of Europe and Asia. Further, we highlight the occurrence of multiple MSP1 alleles in GRW4 isolates from the Hawaiian Islands, where the parasite has contributed to declines and extinctions of endemic forest birds since it was introduced. This study stresses the importance of multiple independent loci for understanding patterns of transmission and evolutionary independence across avian malaria parasites. (Less)

Outbreaks of Culex inatomii occurred widely in disaster areas of the tsunami caused by the Great East Japan earthquake in March 2011. Mosquitoes were collected in southern Miyagi Prefecture and northern Fukushima Prefecture in August and September 2011, respectively. In southern Miyagi Prefecture, the average number of adult Cx. inatomii collected by a suction trap baited with 1 kg of dry ice ranged between 69.3 and 132.8 per day in locations within 2.6 km from the coast in tsunami areas, while no Cx. inatomii individuals were collected 8.3 km from the coast, where seawater did not reach, and which therefore escaped the tsunami disaster. There were many ground pools of brackish water of various sizes, and larvae of Cx. inatomii were collected in 48% and 36% of the pools examined in Miyagi Prefecture and Fukushima Prefecture, respectively. Ecological notes on the larval habitats, biting behavior, seasonal prevalence, and reproduction of Cx. inatomii are presented for reference based on ecological studies conducted in Sakata Wetland, Niigata Prefecture, central Japan, in June and August 2011. Ecological factors relating to the current outbreak and the potential medical importance of Cx. inatomii are discussed.

e Tsunami caused by the Great East Japan Earthquake on 11 March 2011 in north-eastern Japan destroyed urban and rural environments completely, including build- ings, dwellings, roads, bridges, agriculturalelds and natural vegetation. �e aer-eects of the Tsunami on the distribution and abundance of mosquitoes were studied in riceeld ar- eas in Miyagi Prefecture, Japan during June to August 2011. Adult mosquitoes were collected by traps enhanced with 1 kg dry ice and the density was compared between theTsunani� area andNo Tsunamiarea. A total of 6,542 adults of 9 species in 4 genera were collected and the following 4 species were dominant: Culex pipiens pallens, Cx. inatomii , Cx. tritaenio- rhynchus and Aedes albopictus. �e high density and expanded distribution of Cx. inatomii was marked. �e adult density of the former 3 dominant species was signicantly higher in the Tsunami area than No Tsunami area, while Ae. albopictus was signicantly abundant in the No Tsunami area. Ground pools in disaster areas contained brackish water with 0.47 and 0.21% average salinity in June and August 2011, respectively. �e percentage of water sam- ples with mosquito larvae increased from 2.7% in June to 79.5% in August, suggesting the expansion of the larval distribution of Cx. inatomii and Cx. tritaeniorhynchus as well as Anopheles sinensis-group.

In Japan, the prevalence of avian Plasmodium in birds and mosquitoes has been partially examined in the temperate and subtropical zones; however, mosquitoes in the Japanese subarctic zone have not been adequately investigated. In this study, mosquito collections and avian Plasmodium detections from the mosquito samples were carried out to demonstrate the avian Plasmodium transmission between vector mosquitoes and birds inhabiting in Kushiro Wetland, subarctic zone of Japan. A total of 5,657 unfed mosquitoes from 18 species and 320 blood-fed mosquitoes from eight species was collected in summer 2008, 2009, and 2010. Three Aedes esoensis that fed on Hokkaido Sika Deer and one unfed Culex pipiens group were found to be positive for avian Plasmodium by polymerase chain reaction. This is the first report of the detection of avian Plasmodium DNA from mosquitoes distributing in the subarctic zone of Japan. The blood meals were successfully identified to captive or wild animals, including seven mammalian species, four bird species, and one amphibian species. These results indicated that infected birds with avian Plasmodium inhabited and direct contacts occurred between the infected birds and mosquitoes in Kushiro Wetland, Hokkaido, Japan.

The 2003-2004 H5N1 highly pathogenic avian influenza (HPAI) outbreaks in Japan were the first such outbreaks in 79 years in Japan. Epidemic outbreaks have been occurring in Southeast Asia, with the most recent in 2010. Knowledge of the transmission route responsible for the HPAI outbreaks in these countries remains elusive. Our studies strongly suggested that field and laboratory studies focusing on mechanical transmission by blow flies should be considered to control H5N1 avian influenza outbreaks, in particular in epidemic areas, where there are high densities of different fly species throughout the year. In this paper, we review these field and laboratory entomological studies and discuss the possibility of blow flies transmitting H5N1 viruses.

We conducted laboratory experiments to verify molecular techniques of avian malaria parasite detection distinguishing between an infected mosquito (oocysts on midgut wall) and infective mosquito (sporozoites in salivary glands) in parallel with blood-meal identification from individual blood-fed mosquitoes prior to application to field survey for avian malaria. Domestic fowl infected with Plasmodium gallinaceum was exposed to a vector and non-vector mosquito species, Aedes aegypti and Culex pipiens pallens, respectively, to compare the time course of polymerase chain reaction (PCR) detection for parasite between competent and refractory mosquitoes. DNA of the domestic fowl was detectable for at least 3 days after blood feeding. The PCR-based detection of P. gallinaceum from the abdomen and thorax of A. aegypti corresponded to the microscopic observation of oocysts and sporozoites. Therefore, this PCR-based method was considered useful as one of the criteria to assess developmental stages of Plasmodium spp. in mosquito species collected in the field. We applied the same PCR-based method to 21 blood-fed C. sasai mosquitoes collected in Rinshi-no-mori Park in urban Tokyo, Japan. Of 15 blood meals of C. sasai successfully identified, 86.7% were avian-derived, 13.3% were bovine-derived. Plasmodium DNA was amplified from the abdomen of three C. sasai specimens having an avian blood meal from the Great Tit (Parus major), Pale Thrush (Turdus pallidus), and Jungle Crow (Corvus macrorhynchos). This is the first field study on host-feeding habits of C. sasai in relation to the potential role as a vector for avian malaria parasites transmitted in the Japanese wild bird community.

During autumn, a large number of adult Culex tritaeniorhynchus Giles suddenly appeared at a park in urban Tokyo. A total of 14,069 females and 2,802 males were collected at rest by sweeping from April to November 2007, of which 99.8% of the females were collected from late September to November. The appearance of a large number of Cx. tritaeniorhynchus started on 22 September, peaked at 1,062 per collection/hr on 13 October, and gradually decreased by the end of November. A total of 565 females were marked and released on 3 October, and the total number of females in the park was estimated to be 545,225 +/- 157,392 by the Lincoln index. No females were collected by human landing collections, and the parous rate of females collected by sweeping was 4.4% (41/936). Dissections showed that 85.7% (120/140) of these females were in reproductive diapause, with an ovariole stage of N or I and a primary over secondary follicular ratioor = 1.5. From January to early April 2008, only 10 overwintering Cx. tritaeniorhynchus females were collected resting. These results indicated that these females were in reproductive diapause and that some overwintered in this urban setting. This is the first field evidence of prediapause seasonal migration of this species from breeding sites to overwintering sites.

Background Malaria infection in mosquitoes is traditionally detected by microscopic examination for Plasmodium oocysts and sporozoites. Although PCR is now widely used, the presence of parasite DNA in a mosquito does not prove that sporogony is achieved. Thus, detection of sporozoites by microscopy is still required to definitively identify vector mosquitoes. The aim of this study was to confirm sporogony of avian Plasmodium spp. in Culex pipiens pallens and C. inatomii caught from the wild. Findings Mosquitoes collected at two sites in Japan were dissected and examined by microscopy for Plasmodium oocysts and sporozoites. DNA was extracted from the midgut and salivary gland of infected mosquitoes, and the infecting Plasmodium species was identified by sequencing 478 bp of cytochrome b. Oocysts, or both oocysts and sporozoites, were found in 3.94 and 0.46 % of C. p. pallens and C. inatomii, respectively. Four (CXPIP09, GRW4, GRW11 and SGS1) and three cytochrome b lineages (CXINA01, CXINA02 and CXQUI01) were confirmed to achieve sporogony in C. p. pallens and C. inatomii, respectively. One mosquito each of C. p. pallens and C. inatomii was co-infected with two different Plasmodium lineages. Conclusions These findings demonstrate that C. p. pallens and C. inatomii are natural vectors of four and three lineages of avian Plasmodium spp., respectively. The data indicate that a systematic procedure combining microscopy and PCR is a feasible and reliable approach to identify natural vectors of wildlife malaria.

We collected 641 small mammals belonging to 17 species of Rodentia and four species of Soricomorpha in Japan, Korea, Russia, Taiwan, and Thailand and investigated the prevalence and genetic diversity of Bartonella species. Apodemus (field mice) and Rattus (rats) were the most-common genera captured, making up 56.0% and 23.1% of the total specimens, respectively. Bartonellae were isolated from 54.6% of the collected animals, and the prevalence varied depending on the host species and the country of origin. The isolates were identified to the species level based on gltA and rpoB sequences. Although most Bartonella species were shared by more than two host species, the distribution patterns of Bartonella species clearly differed among the four most-common host genera: Apodemus, Rattus, Myodes (voles), and Suncus (shrews). The predominant Bartonella species were Bartonella grahamii in Apodemus, Bartonella tribocorum in Rattus, B. grahamii and Bartonella taylorii in Myodes, and an unclassified Bartonella sp. in Suncus.

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.

Several species of captive and wild birds have been found to be infected with various avian blood protozoa in Japan. We investigated the prevalence and transmission of avian malaria parasite and determined the bloodmeal hosts of mosquitoes collected in a zoological garden in Tokyo, Japan, by using the polymerase chain reaction. In total, 310 unfed and 140 blood-fed mosquitoes of seven species were collected by using sweep nets and CDC traps. Bloodmeal identification indicated that mosquitoes had fed on 17 avian and five mammalian species, including captive animals. The results of avian malaria parasite detection from mosquitoes with avian bloodmeals indicated that Culex pipiens pallens Coquillet is a main vector of avian Plasmodium in the current study site and that some captive and wild birds could be infected with avian malaria parasites. Furthermore, the distances between the collection site of blood-fed mosquitoes and the locations of their blood-source captive animals were estimated. Most females with fresh bloodmeals were found within 40 m of caged animals, whereas half-gravid and gravid females were found between 10 and 350 m from caged host animals. We demonstrated that blood-fed mosquitoes can provide useful information regarding the mosquito vector species of avian malaria parasites and allows for noninvasive detection of the presence of avian malaria parasites in bird populations.

Bloodmeal identification and the detection of avian malaria parasite from mosquitoes (Diptera: Culicidae) were carried out by polymerase chain reaction-based methods for field samples collected in coastal areas of Tokyo Bay, Japan, from April to October 2007. The following seven mosquito species were collected: Aedes albopictus (Skuse), Culex pipiens pallens Coquillett, Culex pipiens form molestus Forskal, Culex tritaeniorhynchus Giles, Culex inatomii Kamimura & Wada, Culex bitaeniorhynchus Giles, and Lutzia vorax Edwards. Forty blood-fed mosquitoes were collected and 95% of bloodmeals of Cx. pipiens pallens were avian-derived, whereas only mammalian bloodmeals were identified for Ae. albopictus. Plasmodium DNA was amplified from 65% (15/23) of blood-fed Cx. pipiens pallens and unfed females of Cx. pipiens pallens and Cx. pipiens form molestus with a minimum infection rate of 29.9 and 13.5, respectively. One unfed female of Lt. vorax was also positive for Plasmodium parasites. Five genetically distinct lineages of Plasmodium were identified, with 0.21 to 5.86% sequence divergence. Rinshi-8, the most prevalent lineage at our study site, was identical to the published sequence of Plasmodium relictum-P5.