Mosquito biosurveillance on Kyushu Island, Japan, with emphasis on anopheles Hyrcanus group and related species (Diptera: Culicidae).
The purpose of our study was to strengthen mosquito-borne disease biosurveillance capability in Japan by acquiring biogeographic vector data from sites identified as having taxonomic and ecological importance, thereby enhancing the knowledge base associated with potential malaria vectors, and incorporating this information as a component of already in-place mosquito surveillance programs, including the Walter Reed Biosystematics Unit's VectorMap/MosquitoMap, and the US Army Public Health Command Regions--North and Pacific mosquito surveillance training programs.
MATERIALS AND METHODS
Mosquito Field Collection and Identification
Specimen collections were conducted from 2006-2013 from various areas within Kumamoto, Fukuoka, Saga and Nagasaki Prefectures, on Kyushu Island, Japan (Figure 1). Additional specimens were previously collected by Dr Motoyoshi Mogi from 1984-2005 from localities in Saga and Nagasaki Prefectures. These prefectures were selected because the taxonomic records for the Anopheles Hyrcanus Group were unclear or had conflicting information regarding previously reported and described species from this region. The Hyrcanus Group includes all known malaria vector species in Japan (5,14) and it is essential to clarify the taxonomy of the group, including geographic distribution records of the group species. The mosquito taxonomic classification used in this paper follows that of Knight and Stone. (19)
Depending on the habitats (rice paddies, irrigation ditches, permanent and temporary pools, other standing water areas (Figures 2 and 3)), larvae were collected using a standard larval dipper (350 ml, 13 cm diameter) or a white plastic larval tray (25 x 20 x 4 cm) (BioQuip, Rancho Dominguez, CA). Each habitat within a location was surveyed for up to one hour or until about 100 larvae were collected. The latitude and longitude of each location was recorded using a hand-held global positioning system (GPS) unit (Garmin International, Olathe, KS) set to the WGS84 datum. Sampling locations were photographed using a digital camera to assist in verifying the accuracy of the habitat description. Collected larvae were placed in plastic Whirl-Pak bags (118 ml, 8x18 cm) (BioQuip, Rancho Dominguez, CA) and filled approximately XA full with water from the collection site. The Whirl-Pak was then tightly closed to retain air, placed in a cooler, and brought to the laboratory where the larvae were directly preserved in 100% ethanol for molecular identification. The remaining larvae were individually link-reared to adult stage, as morphological voucher specimens for this work (Figure 4). Emergent adults were pinned on paper points, each given a unique collection number, and identified using diagnostic morphological characters (Figure 5).
DNA Isolation and Sequencing
For molecular species identification, DNA was isolated from individual larvae, pupae, and adults (1 or 2 legs per adult) by phenol-chloroform extraction, and the PCR amplification protocol, cycling conditions, and direct sequencing were carried out using standard protocol. (17) A fragment of rDNA ITS2 was amplified using the primers 5.8S (5'-ATCACTCGGCTCGTGGATCG-3') and 28S (5'-ATGCTTAAATTTAGGGGGTAGTC-3'). (18) The PCR products were directly sequenced using Big Dye 3.0 (Applied Biosystems, Inc (ABI), Foster, CA) with an ABI 3100 sequencer. Sequences were edited using Sequencher (V4.8, Gene Codes Corporation, Ann Arbor, MI) and aligned in Clustal X.Sequences of An. Hyrcanus Group species (An.sinensis, An. lesteri) are those of previous studies using the primers therein. (17,18) Voucher specimens and collection records will be deposited in the US National Museum of Natural History (USNMNH) of the Smithsonian Institution, Suitland, MD.
The summary of collection localities and larval habitats for Anopheles species (primarily An. sinensis and An. lesteri) from 4 prefectures (Fukuoka, Kumamoto, Nagasaki, Saga) of Kyushu Island, Japan, are presented in the Table (page 18). The map of Kyushu, with collection sites of mosquitoes, is shown in Figure 1. Prior to 2013, larvae of An. sinensis were collected from various habitats either alone or in association with the following Aedes or Culex species: Cx. (Culex) tritaeniorhynchus Giles larvae (in rice fields, irrigation ditches, marsh and drainage areas, ground pits or depressions) in Nagasaki and Kumamoto Prefectures. Aside from An. sinensis, no Anopheles species were collected from any larval habitats in association with Aedes or Culex species. In 2013, other Hyrcanus Group larvae (still to be identified using molecular sequences) were also found in association with the following: Cx. (Cux.) tritaeniorhynchus, Cx. (Cux.) spp.; Ae. (Finlaya) spp.; Ae. (Ochlerotatus) spp. in rice paddies and irrigation ditches in Nagasaki Prefecture (Isahaya, Moriyama, Obama-Unzen, Onakao).
During the 2013 survey of various localities in Kyushu Island, the rice paddies where we collected the larvae and pupae of Anopheles Hyrcanus Group had water pH ranging from 6.68-8.61 (mean, 7.77), millivoltage (175.00-237.00 mV; mean, 215.10) and temperature (30.50[degrees]C-32.80[degrees]C; mean, 32.10[degrees]C). Other water habitats (irrigation ditches, ponds, stream margin, pools, and drainage) that were positive for Anopheles larvae and pupae also exhibited variable pH, mV, and temperatures.
Culicine mosquitoes (nonanophelines) collected from Kyushu Island in 2013 included Ae. (Fin.) japonicus (Theobald) from Moriyama and Nagasaki (artificial containers, shrine stone bowls); Ae. (Fin.) togoi (Theobald) from Isahaya (pond); Ae. (Ste.) albopictus (Skuse) from Moriyama and Nagasaki (artificial containers, drainage ditches, shrine stone bowls, tree stumps or holes, temporary seepage); Cx. (Ocu.) bitaeniorhynchus Giles from Moriyama (drainage ditches); and Cx. (Cux.) tritaeniorhynchus from Moriyama, Obama-Unzen, Hitoyoshi (drainage ditches, irrigation ditches, rice paddies). About 60 mosquito larvae collected from Nagasaki Prefecture could not be identified morphologically, including those in Ae. (Finlaya) from Isahaya; Ae. (Ochlerotatus) from Nomozaki and Onakao; and Cx. (Culex) from Moriyama, Nagasaki, Obama-Unzen, Setoishi, Isahaya, Aikawa, and Onakao. Molecular analysis of those unidentified larval specimens of Aedes and Culex, together with both larvae and adults of An. Hyrcanus Group from 4 prefectures, will be completed in the future.
Among the Anopheles Hyrcanus Group species, An. pullus, An. sinensis, An. lesteri, An. kleini, and An. belenrae are known or potential vectors of vivax malaria in the Korean peninsula and other countries. Anopheles sinensis is the most common anopheline species in Japan, including the Ryukyu Islands. (14) It has long been suspected as the most important vector of malaria in Japan, including Okinawa and Hokkaido. Even though indigenous malaria has disappeared, this vector remains abundant throughout Japan. It is a known vector of malaria in South Korea and China, and it has a wide distribution in Asia. (4,5,8-10,14,20) Anopheles lesteri (= anthropophagus) is a very important vector of malaria in China. To clarify and stabilize the taxon, Rueda and others (6) designated and described the neotype and alloneotype of An. lesteri. This species was suspected to be an important vector of indigenous malaria in Japan, particularly in Hokkaido where it commonly occurs in great numbers. It is also common in the Ryukyu Islands and has been found more frequently in coastal regions in Honshu and Kyushu. (14) Anopheles yatsushiroensis is not known as a vector of indigenous malaria in Japan. Anopheles belenrae (Figure 4B), first recorded in Japan in 2013 from Hokkaido, (15) is a potential vector of vivax malaria in Korea. (21) Plasmodium berghei Vincke and Lips, a nonhuman specific parasite, was first detected from An. belenrae adults in South Korea. (22) The morphological details of the head, thorax, abdomen, wings, and legs of An. belenrae are shown in the Walter Reed Biosystematics Unit's website.* The other Hyrcanus Group species (ie, An. sineroides and An. engarensis), as well as several Anopheles (Anopheles) species (An. bengalensis Puri; An. koreicus Yamada and Watanabe; An. lewisi Ludlow; An. lindsayi japonicus Yamada; An. omorii Sakakibara; An. saperoi Bohart and Ingram; An. yaeyamaensis Somboon and Harbach), are not known vectors of indigenous malaria in Japan.
Most mosquito collections, including Anopheles species, noted by Tanaka and others (14) in 1979, are presently deposited at the National Institute of Infectious Diseases (NIID), Tokyo, Japan, where most of the Hyrcanus Group species were examined by author L. M. Rueda during his visit in 2006. In a recent conversation with the authors, Dr Kyoko Sawabe mentioned that there are some possible specimens of An. yatsushiroensis collected by Dr M. Otsuru in 1951 and 1964 on Kyushu Island now deposited at the NIID, Tokyo. These specimens should be examined for further morphological and molecular analysis to clarify the existence of this species. In 2003, Dr Motoyoshi Mogi inquired to check the type specimens of An. yatsushiroensis from the Department of Parasitology (DP), Faculty of Medicine, Kyushu University, Fukuoka, Kyushu (reported as the depository of An. yasushiroensis types by Miyazaki (12) in 1951). However, Professor Isao Tada (former director of the DP) informed Dr Mogi that no type specimens existed at the DP. It may be useful to designate neotypes for An. yatsushiroensis, if it is proven as a valid species.
Although previous researchers considered An. yatsushi roensis as a synonym of An. pullus, they used Korean specimens to obtain their molecular and morphological data. (23,24) However, because the type locality of An. yatsushiroensis is in Japan, it is necessary to do a genetic comparison of An. pullus from South Korea with the topotypic specimens from Japan to resolve definitively if the two are synonyms or not. In 1951, Miyazaki, (12) who first described An. yatsushiroensis, provided elaborate morphological descriptions, ecology, and distributions of this species. We did not collect An. pullus during our previous collections from 2002-2008 in Japan, and no report indicates the existence of An. pullus in that country. Furthermore, An. pullus is considered a major vector of vivax malaria in the Korean peninsula. (16) Through biosurveillance, it is also interesting to investigate if another major Korean malaria vector, An. kleini, is present in Japan.
In our attempt to recollect specimens of the Hyrcanus Group, particularly An. yatsushiroensis, we recently visited numerous localities and conducted extensive larval collections at various habitats in Nagasaki Prefecture and Kumamoto Prefecture (including Yatsushiro City, the type locality of An. yatsushiroensis reported by Miyazaki (12)) and neighboring areas from 2006 to 2013. Unfortunately, we were not able to collect samples of An. yatsushiroensis from 2006 to 2012.
Although Dr Sawabe mentioned that there are some possible specimens of An. yatsushiroensis deposited at the NIID, Tokyo, we have not examined them yet. Furthermore, more than 200 larvae and adults of An. Hyrcanus Group collected in 2013 from Kumamoto and Nagasaki Prefectures are still being examined and analyzed by morphological and molecular techniques. Molecular data (PCR, sequences) will be reported later, particularly from the 2013 specimens for possible presence of An. yatsushiroensis and other species in An. Hyrcanus Group on Kyushu Island.
This research was performed under a Memorandum of Understanding between the Walter Reed Army Institute of Research and the Smithsonian Institution, with institutional support provided by both organizations.
We express our sincere appreciation to the following: Dr Motoyoshi Mogi for arranging the visits of Dr Rueda to Saga and Fukuoka Prefectures, his help in mosquito collections, and for sharing his mosquito specimens; CPT Robert Moore and SGT J. Santano for their help in collecting mosquito samples from Kumamoto Prefecture; Professor Y. Oneda, for his help in collecting samples and guiding us in locating larval habitats in Akagawa and Takegima, Fukuoka Prefecture and Tosu City, Saga Prefecture. Special thanks go to Dr Noburo Minakawa, particularly for making the arrangements for our visit to Nagasaki, and Dr Kyoko Sawabe for correspondence and invitation to visit and examine the mosquito collections at NIID, Tokyo.
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Leopoldo M. Rueda, PhD
Benedict Pagac, BS
Masashiro Iwakami, BS
Alexandra R. Spring, MS
Maysa T. Motoki, PhD
James E. Pecor, BS
Yukiko Higa, PhD
Kyoko Futami, PhD
Nozomi Imanishi, MS
MAJ Lewis S. Long, MS, USA
COL Mustapha Debboun, MS, USA
Dr Rueda is a Research Entomologist, Principal Investigator, and Acting Chief of the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research, located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland.
Mr Benedict Pagac is the Chief, Entomology Section at the US Army Public Health Command Region-North, Fort George G. Meade, Maryland.
Mr Iwakami is an Entomologist at the US Army Public Health Command-Pacific, Entomology Program. Camp Zama, Japan.
Ms Spring is a Molecular Biologist at the Entomology Section, US Army Public Health Command Region-North, Fort George G. Meade, Maryland.
Dr Motoki is a Postdoctoral Entomologist at the Entomology Department, Smithsonian Institution, Museum Support Center, Suitland, Maryland.
Mr Pecor is a Museum Specialist at the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research, located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland.
Dr Higa, Dr Futami, and Ms Imanishi are Assistant Professors and Graduate Student, respectively, at the Department of Vector Ecology and Environment, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki City, Nagasaki, Japan.
MAJ Long is currently on training leave from his position as Chief of the Walter Reed Biosystematics Unit, Entomology Branch, Walter Reed Army Institute of Research, located at the Smithsonian Institution, Museum Support Center, Suitland, Maryland.
COL Debboun is the Chief of the Department of Preventive Health Services, Academy of Health Sciences, US Army Medical Department Center & School, Fort Sam Houston, Texas. He is also the Chairman of the Army Medical Department Journal Editorial Review Board.
Summary of collection localities and larval habitats for Anopheles (Anophleles) in 4 prefectures of Kyushu Island, Japan. Prefecture Location Grid coordinates Fukuoka Akagawa, Ogori City 33.34975N/130.51352E Fukuoka Takejima, Yasutake- 33.34975N/130.54597E mache, Kurume Kumamoto Amitsu, Uto City 32.69973N/130.60432E Kumamoto Gyokuto City, Tamana 32.91638N/130.62565E County Kumamoto Hitoyoshi 32.22652N/130.77038E Kumamoto Kato shrine, Yatsushiro 32.47998N/130.57202E Kumamoto Lake Ezu 32.77755N/130.73855E Kumamoto Matsubase, Uki City 32.65355N/130.67050E Kumamoto Matsubase, Uki City 32.65625N/130.66788E Kumamoto Matsubase, Uki City 32.65355N/130.67050E Kumamoto Sumiyoshi, Uto City 32.70005N/130.60015E Kumamoto Takasima, Yatsushiro 32.52158N/130.57750E Kumamoto Tamana City, Tamana 32.91638N/130.54523E County Kumamoto Ueki City 32.88017N/130.68148E Kumamoto Uto 32.69660N/130.66845E Kumamoto Uto 32.69660N/130.66845E Kumamoto Yatsushiro 32.50033N/130.61932E Kumamoto Yatsushiro 32.52158N/130.57750E Nagasaki Ariake-cho, Nagasaki 32.69414N/130.32505E Nagasaki Goto, Fukue Island 32.67867N/128.76802E Nagasaki Goto, Fukue Island 32.67867N/128.76802E Nagasaki Goto, Fukue Island 32.67867N/128.76802E Nagasaki Goto, Fukue Island 32.67867N/128.76802E Nagasaki Goto, Fukue Island 32.67867N/128.76802E Nagasaki Isahaya 32.81308N/130.12767E Nagasaki Isahaya 32.81308N/130.12767E Nagasaki Isahaya 32.81308N/130.12767E Nagasaki Isahaya-shi, 32.80759N/130.10737E Kamiimuta, Moriyama-cho Nagasaki Mikawa-machi 32.78590N/129.88779E Nagasaki Moriyama 32.83508N/130.11372E Nagasaki Moriyama 32.83508N/130.11372E Nagasaki Nagasaki 32.77217N/129.86950E Nagasaki Nomozaki 32.58570N/129.75660E Nagasaki Obama-Unzen 32.71354N/130.20073E Nagasaki Obama-Unzen 32.71354N/130.20073E Nagasaki Obama-Unzen 32.71354N/130.20073E Nagasaki Obama-Unzen 32.71354N/130.20073E Nagasaki Onako 32.88408N/129.69598E Nagasaki Togitsu 32.82683N/129.84866E Nagasaki Tsushima 34.17745N/129.29039E Nagasaki Utzutzugawa 32.79328N/129.92803E Nagasaki Utzutzugawa 32.79328N/129.92803E Saga Fukutomi 33.17567N/130.17284E Saga Kase 33.23807N 130.25824E Saga Kinyu 33.24204N 130.29149E Saga Morita 33.09454N 130.10894E Saga Nabeshima 33.27991N 130.26614E Saga Shiroishi 33.17837N 130.14394E Saga Shiroishi 33.17837N 130.14394E Saga Yamato-cho 33.14766N 130.14832E Saga Yamato-cho 33.14766N 130.14832E Saga Tosu City 33.34463N 130.51352E Prefecture Collection Stage Collector date Fukuoka 19-20 Sep 2008 Adult (a) L. M. Rueda, Y. Oneda Fukuoka 19-20 Sep 2008 Adult (a) L. M. Rueda, Y. Oneda Kumamoto 23 Sep 2008 Adult (a) L. M. Rueda, M. Iwakami, J. Santano Kumamoto 22 Sep 2008 Adult (a) L. M. Rueda, M. Iwakami, J. Santano Kumamoto 12 Jul 2013 Larva L. M. Rueda, B. Pagac, M. Iwakami Kumamoto 15 Sep 2008 Adult (a) L. M. Rueda, M. Iwakami, J. Santano Kumamoto 24 Sep 2008 Adult L. M. Rueda, M. Iwakami, J. Santano Kumamoto 30 Aug 2006 Adult (a) M. Iwakami, R. Moore Kumamoto 17 Sep 2008 Adult (a) L. M. Rueda, M. Iwakami, J. Santano Kumamoto 30 Aug 2006 Adult (a) M. Iwakami, R. Moore Kumamoto 22 Sep 2008 Adult (a) L. M. Rueda, M. Iwakami, J. Santano Kumamoto 15 Sep 2008 Adult (a) L. M. Rueda, M. Iwakami, J. Santano Kumamoto 22 Sep 2008 Adult (a) L. M. Rueda, M. Iwakami, J. Santano Kumamoto 22 Sep 2008 Adult (a) L. M. Rueda, M. Iwakami, J. Santano Kumamoto 29 Aug 2006 Adult (a) M. Iwakami, R. Moore Kumamoto 29 Aug 2006 Adult (a) M. Iwakami, R. Moore Kumamoto 11 Jul 2013 Larva, pupa, L. M. Rueda, B. adult (a) Pagac, M. Iwakami Kumamoto 16 Sep 2008 Adult (a) L. M. Rueda, M. Nagasaki 20 Jul 1988 Adult (a) Iwakami, J. Santano Nagasaki 17 Jul 2013 Larva, pupa L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 17 Jul 2013 Larva L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 17 Jul 2013 Adult (a) L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 17 Jul 2013 Larva L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 17 Jul 2013 Adult (a) L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 12 Jul 2013 Adult (a) NG1 Nagasaki 12 Jul 2013 Larva NG1 Nagasaki 12 Jul 2013 Larva NG1 Nagasaki 27 May 2006 Adult T. Yoshio Nagasaki 27 May 1962 Adult NU Nagasaki 9 Jul 2013 Adult (a) L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 9 Jul 2013 Larva L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 20 Jun 1989 Adult M. Mogi Nagasaki 15 Jul 2013 Larva NG2 Nagasaki 10 Jul 2013 Larva L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 10 Jul 2013 Larva L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 10 Jul 2013 Larva L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 10 Jul 2013 Adult (a) L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 16 Jul 2013 Larva NG2 Nagasaki 7-9 Aug 1956; Adult NU 22 Jul 1962 Nagasaki 27 May 1962 Adult NU Nagasaki 9 Jul 2013 Larva L. M. Rueda, B. Pagac, M. Iwakami Nagasaki 9 Jul 2013 Larva L. M. Rueda, B. Pagac, M. Iwakami Saga 13, 20, 28 Aug Adult M. Mogi 2005; 11, 13, 24 Sep 2005; 10 Oct 2005; Saga 17 Sep 2005 Adult M. Mogi Saga 2, 5, 10 Jun 1986 Adult M. Mogi Saga 16 May 1995 Adult M. Mogi Saga 30 May 1985; Adult M. Mogi 14 Jun 1985; 3, 5, 6, 7, 20 Jun 1986; 9 Jun 1990; 7 Jun 1995; 3 Jun 1996 Saga 7 May 2000 Adult M. Mogi Saga 3 Nov 1997; Adult M. Mogi 7 May 2000 Saga 10 Apr 2000 Adult T. Sunahara Saga 5 Jun 1986; Adult T. Sunahara, 24, 25, 26 M. Mogi Apr 2000; 10, 11, 12, 25 May 2000; 24, 25 Apr 2004 Saga 20 Sep 2008 Adult L. M. Rueda, (a) Y. Oneda Prefecture Habitat Collection Anopheles type (b) No. (Anopheles) Species Fukuoka RC JP08-9 sinensis Fukuoka RC KP08-10 sinensis Kumamoto ID, RP JP08-17, 18 sinensis Kumamoto PO JP08-14 sinensis Kumamoto ID, RH, RP JP13-19, 21 Hyrcanus Group (c) Kumamoto DD JP08-2 lesteri Kumamoto LM JP08-19 sinensis Kumamoto RP JP06-2-37A, 40A lesteri Kumamoto RP JP08-7, 8 lesteri Kumamoto RP JP06-2-1A, 2A, sinensis 3A, 4A, 6A, 25A; JP08-5, 6 Kumamoto ID JP08-17B sinensis Kumamoto HD, WT JP08-1, 4 lesteri Kumamoto RP JP08-15, 16 sinensis Kumamoto ID, RP JP08-12, 13 lesteri Kumamoto RP JP06-1-50A, 51A lesteri Kumamoto RP JP06-1-1A, 2A, sinensis 3A, 4A, 5A, 7A, 16A Kumamoto SP JP13-18 Hyrcanus Group (c) Kumamoto RP JP08-3 sinensis Nagasaki RC JPM-5 sinensis Nagasaki ID JP13-31 Hyrcanus Group (c) Nagasaki RP JP13-30 Hyrcanus Group (c) Nagasaki RP JP13-30 Hyrcanus Group (d) Nagasaki RP JP13-32 Hyrcanus Group (d) Nagasaki RP JP13-32 Hyrcanus Group (d) Nagasaki PO JP13-23 Hyrcanus Group (d) Nagasaki PO JP13-23 Hyrcanus Group (d) Nagasaki RP JP13-25 Hyrcanus Group (d) Nagasaki -- JPM-5 sinensis Nagasaki -- JPM-5 sinensis Nagasaki RP JP13-7 Hyrcanus Group (d) Nagasaki RP JP13-8 Hyrcanus Group (d) Nagasaki RP JPM-5 sinensis Nagasaki AC JP13-26 Hyrcanus Group (d) Nagasaki RP JP13-14 Hyrcanus Group (d) Nagasaki RP JP13-15 Hyrcanus Group (d) Nagasaki WC JP13-16 Hyrcanus Group (d) Nagasaki WC JP13-16 Hyrcanus Group (d) Nagasaki RP JP13-29 Hyrcanus Group (d) Nagasaki -- JPM-5 sinensis Nagasaki -- JPM-5 sinensis Nagasaki RP JP13-13 lindsayi japonicus Nagasaki RP JP13-13 Hyrcanus Group (d) Saga LF JPM-22-1 sinensis Saga ID JPM-21-1, -2, -3 sinensis Saga RP JPM-8, 9 lesteri Saga CA JPM-14 sinensis Saga LF JPM-4, 5, 6, 7 lesteri Saga RP JPM-19-1 lesteri Saga NE JPM-14, 15, 19-2, sinensis 19-3, 19-5, 19-5 Saga CA JPM-18 lesteri Saga CA JPM-8, 15, 16, sinensis 17, 18 Saga GT JP08-11 lesteri (a) Field collected larvae or pupae, reared to emerged adults. (b) DD, drainage ditch; HD, hill or road side ditch; ID, irrigation ditch; LM, lake margin; PO, pond; RC, resting at cowshed or cattle barn; RH, rock hole, pool; RP, rice paddy; SP, stream or river margin or pool; WT, water tank, trough or PVC tube waterer. (b1) NG1 indicates L. M. Rueda, B. Pagac, M. Iwakami, Y. Higa, K. Futami, N. Imanishi. NU indicates Nagasaki University, Entomology Collection. NG2 indicates L. M. Rueda, B. Pagac, M. Iwakami, Y. Higa, K. Futami. (b2) CA, road ditch or small canal; GT, ground pit or depression; ID, irrigation ditch; LF, lotus field; NE, caught by insect net; RP, rice paddy. (c) DNA isolation and sequencing still to be completed. (c1) AC, artificial containers (tires, plastic jugs, kettle, etc); CA, road ditch or small canal; ID, irrigation ditch; LF, lotus field; PO, pond; RP, rice paddy; WC, water well/cistern. (d) DNA isolation and sequencing still to be completed.
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|Author:||Rueda, Leopoldo M.; Pagac, Benedict; Iwakami, Masashiro; Spring, Alexandra R.; Motoki, Maysa T.; Pec|
|Publication:||U.S. Army Medical Department Journal|
|Date:||Jul 1, 2014|
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