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First Record of Aedes (Stegomyia) malayensis Colless (Diptera: Culicidae) in the Lao People's Democratic Republic, Based on Morphological Diagnosis and Molecular Analysis.

The Nakai Nam Theun National Protected Area (NNT NPA), known as the Watershed Management and Protection Authority area bordering Vietnam, is located in Nakai District, Khammuane Province, Lao People's Democratic Republic (PDR). It is an important Southeast Asian biodiversity area, containing mammals, birds, reptiles, amphibians, (1) and insects, including mosquitoes. It is also the home of a number of rare or newly discovered species of animals. (2-5)

Rueda et al (6) reported a total of 101 species from Laos, including newly recorded Aedes (Stegomyia) species collected from Khammuane province, ie Aedes (Stegomyia) albopictus (Skuse) and Ae. (Stg) pseudoscutellaris (Theobald), and those reported in the literature, ie, Ae. (Stg) aegypti (Linnaeus), Ae. gardnerii imitator (Leicester), Ae. (Stg) seatoi Huang, Ae. (Stg) annandalei (Theobald), Ae. (Stg) craggi (Barraud), Ae. (Stg) malikuli Huang, Ae. (Stg) perplexus (Leicester), Ae. (Stg) desmotes (Giles), Ae. (Stg) pseudalbopictus (Borel). (7-9) All voucher specimens of these newly recorded species, as reported by Rueda et al, (6) were deposited in the Smithsonian Institution, National Museum of Natural History, Washington, DC, and in the Entomology Laboratory, Institut Pasteur Laos. Tangena et al (10) added an additional 51 species to the list of Lao mosquito fauna. However, all voucher specimens from that report were, unfortunately, damaged or lost, and we were not able to conduct any further morphological examinations or DNA analysis of those specimens from this study.

The subgenus Stegomyia of genus Aedes has 128 valid species worldwide, (11) with 24 species found in the Greater Mekong subregion of Asia (Cambodia, China, Laos, Myammar, Thailand, and Vietnam), including 12 species from Lao PDR. Several species of subgenus Stegomyia are major vectors of various organisms that cause human infectious diseases such as dengue, yellow fever, chikungunya, Zika viruses, and filariasis. (12-14)

Aedes aegypti is the primary vector of dengue throughout the tropical and subtropical regions of the world. (15) Aedes albopictus is also an important vector in dengue epidemics. (16-18) Aedes malayensis Colless is widely distributed in many parts of Asia, (19) particularly Cambodia, India, Malaysia, Singapore, Taiwan, Thailand, and Vietnam. (11) A recent study showed a high susceptibility of Ae. malayensis and its vectorial capacity for both dengue serotype 2 and chikungunya. (20) This species is recorded for the first time in the Lao PDR. In this study, we confirmed the identification of Ae. malayensis from Laos based on the cytochrome c oxidase subunit I (COI) mitochondrial gene and the morphological diagnostic characteristics of adults, and compared them with Ae. albopictus.

Materials and Methods

Specimen Collection

Larvae of mosquitoes were collected from aquatic habitats along the Nam Noy River (17.768548[degrees]N, 105.381989[degrees]E), Lao PDR (Figure 1) in March 2017, using standard larval dippers (350 ml, 13 cm diam (BioQuip, Rancho Dominguez, CA, USA). They were carefully transferred into a WhirlPak plastic bag (BioQuip) using pipettes, and transported to the laboratory of Institut Pasteur du Laos.

Morphological Identification

Emerged adults were pinned on paper points, each given a unique collection number, properly labeled, and identified using a stereomicroscope (Olympus SZX7, Tokyo, Japan) following the morphological keys of Rattanarithikul et al. (21) Voucher specimens were deposited in the Entomology Collection, Institut Pasteur du Laos, Vientiane, Lao PDR, and the National Mosquito Collections of the Smithsonian Institution, National Museum of Natural History, Washington, DC. Diagnostic characters of Ae. malayensis adults were photographed.

DNA Extraction and Sequencing

Total genomic DNA was extracted from a single whole mosquito using Macherey-Nagel NucleoSpin Tissue (GmbH & Co KG, Duren, Germany) according to manufacturer's instructions. The fragment of mitochondrial cytochrome c oxidase subunit I (mtDNA COI) gene was amplified using the polymerase chain reaction (PCR) Master Mix 2X (Promega Corporation, Madison, WI, USA) utilizing LCO1490 and HCO2198 primers. (22) The PCR protocol consisted of a one minute denaturation at 94[degrees]C and 5 cycles at 94[degrees]C for 40 seconds, 45[degrees]C for 40 seconds and 72[degrees]C for one minute, followed by 30 cycles at 94[degrees]C for 40 seconds, 49[degrees]C for 40 seconds and 72[degrees]C for one minute, and a 5-minute extension at 72[degrees]C. The PCR amplicons were electrophoresed in 1.5% TAE agarose gels stained with GelRed Nucleic Acid Gel Stain (Biotium Inc, Hayward, CA, USA), and the PCR products were then cleaned by adding ExosapIT (USB Co, Cleveland, OH, USA). Samples were placed in the thermocycler and ran at 37[degrees]C for 30 minutes, followed by 80[degrees]C for 15 minutes.

All sequencing reactions were conducted in both directions using the original primers and the Big Dye Terminator Kit v.3.1 (PE Applied Biosystems, Warrington, UK), analyzed on an ABI Prism 3500xL--Avant Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were edited in Sequencher v.5.4.6 (Genes Codes Co, Ann Arbor, MI, USA), and aligned using Geneious 9.16. (23) A bootstrapped (24) Neighbor Joining tree (25) was used based on 1,000 replicates. The evolutionary distances were calculated using the Kimura-2 parameters method (26) conducted in MEGA v.7. (27) All 658 base pair (bp) of the barcode fragment were included in pairwise comparisons. Two sequences of Ae. albopictus were used as an outgroup.


Morphological Diagnosis

The adult Ae. malayensis is very similar to Ae. albopictus, except for the diagnostic characters given below (21):

1. A supraalar area of thorax with a patch of pale scales extended toward the scutellum in Ae. malayensis, and a supraalar area of thorax with spot of pale scales not extended toward the scutellum in Ae. albopictus (Figure 2);

2. Abdominal terga IV-VI with dorsal white bands connected to lateral pale patches in Ae. malayensis, and abdominal terga IV-VI with dorsal white bands separated from lateral spots in Ae. albopictus (Figure 3).

Furthermore, the adults of Ae. malayensis are very similar to adults of Ae. alcasidi Huang, Ae. riversi Bohart and Ingram, and Ae. scutellaris (Walker) and share the following diagnostic characters (19):

1. Midfemur without median white line on anterior surface;

2. Wing with minute basal spot of white scales on costa; and

3. Hindtarsomere 5 entirely white. (19) Unfortunately, we did not have samples of Ae. alcasidi, Ae. riversi, or Ae. scutellaris to compare, but we present the photographs of those diagnostic characters of Ae. malayensis from the Lao PDR (Figure 4).


Larvae (n=31) of Ae. malayensis were collected in the NNT NPA from rock holes and rock pools along the edges of the Nam Noy River, in an open area between the river and the forest. The habitats were not complete ly shaded, but some of them were naturally shaded by the rocks with fresh, clean, and cool water, and without vegetation (Figure 5). Larvae of Ae. albopictus were also collected from the same habitats in association with Ae. malayensis. Adults (n=17) of Ae. malayensis were collected using sweep nets near larval habitats. Adult females were attracted to humans and fed on their blood.

Molecular Characterization

The fragment of COI was sequenced for 4 specimens of Ae. malayensis, and 2 specimens of Ae. albopictus were used as an outgroup (GenBank numbers: MG921172-MG921178). We compared them with the DNA barcode sequences of our samples with those published previously (KY420809, KY420810, KY420811; KR349280, KR349282). (20) The amplicon length of the barcode sequence of Ae. malayensis was consistent at 658 bp (without primers). The base compositions were similar for all specimens, 14.31% G, 15.98% C, 29.22% A, and 40.49% T. The bootstrap consensus tree confirmed the differences observed in the morphology of the Ae. malayensis female adult compared with Ae. albopictus (Figure 6).


Larvae of Ae. malayensis have been found in tree holes in Singapore and Taiwan, and in coconut shells in Vietnam. In Thailand, they were found in rock holes, rock pools, water jars, and bamboo cups, while in Malaysia they were collected from rock pools, bamboo stumps, a coconut shell, and artificial containers. (19) In the Lao PDR, Ae. malayensis was found in rock pools (Figure 5) and rock holes together with Ae. albopictus. This sympatry was also observed in Singapore, Malaysia, Thailand, and Taiwan. (19,28) The larval habitat along the Nam Noy River is approximately 40 km from the nearest urban center of Oudomsouk (17.710971[degrees]N, 105.15086[degrees]E), and could only be accessed by a 3-hour boat trip.

Larvae of Ae. malayensis are morphologically similar to Ae. albopictus and are very difficult to separate from each other. (28) The body ornamentations of the adults and larvae are highly variable. (28) Despite the fact that Ae. albopictus is very similar to Ae. malayensis, the combination of some distinct morphological characters differentiate these 2 species (21) (Figures 2 and 3). Likewise, the adults of Ae. malayensis are very similar to adults of Ae. alcasidi, Ae. riversi, and Ae. scutellaris, (19) particularly the wings, midfemur, and hindtarsomere 5 (Figure 4). Overall, Ae. malayensis can be especially recognized by its abdominal ornamentation (Figure 3).

DNA barcoding of the mitochondrial COI has been an efficient and useful marker for mosquito identification and confirmation of new species. (9) Herein, the observed morphological differences are corroborated with the COI sequences. The COI sequences of Ae. malayensis from Lao PDR were clustered with the COI sequences of the Singapore samples, and distinguished from the Ae. albopictus, thereby confirming the existence of Ae. malayensis in the Lao PDR (Figure 6).

Dengue, chikungunya, Zika, and yellow fever viruses are the most important pathogens associated with the species of subgenus Stegomyia. (14,32) Because of the important role of Aedes (Stegomyia) species in arbovirus transmission, Huang (28) described for the first time the larvae and pupae, and redescribed both adult sexes of Ae. malayensis from samples collected from the type locality in Pulau Hantu, Singapore.

Even though the medical importance of Ae. malayensis was not well known at the time, it was found to have strong anthropophilic behavior in Bo-Pia, Prachuap Khiri Khan, Thailand, (19) and India, (33) as well as our recent observations in Nakai, along the Nam Noy River. In addition, Rosen et al (34) reported that Ae. malayensis was susceptible to all 4 serotypes of dengue virus after oral infection, but the presence of virus in the mosquito saliva was not determined. Moreover, Mendenhall et al (20) compared the vector competence of Ae. albopictus and Ae. malayensis with Ae. aegypti from Singapore after oral infection with dengue serotype 2 and chikungunya viruses and observed the high susceptibility of Ae. malayensis and Ae. albopictus to both arboviruses.

Importantly, the saliva of infected Ae. malayensis contained infectious particles for both viruses. This provided the evidence that Ae. malayensis and Ae. albopictus from Singapore possess all the necessary traits to transmit these arboviruses.

Aedes malayensis remains an understudied species of Stegomyia, although it is found in Singapore, Malaysia, Thailand, Cambodia, Vietnam, Taiwan, China (Hainan), (19,29,35) India, (33) and now in the Lao PDR. Because of the wide distribution of Ae. malayensis in many parts of Asia, (19) its high vector competence, (20,34) and its anthropophilic behavior, (19,33) more studies are warranted on the significance of this Stegomyia species as an arbovirus vector in the Lao PDR.


We are grateful to the staff of the Watershed Management and Protection Authority (WMPA) area who authorized our research in the WMPA area and provided technical support during the fieldwork; the staff of the Public Health Office, Nakai District, Khammuane Province for cooperation and field assistance; and the Institut Pasteur du Laos staff for their support throughout this study. Special thanks go to Dr Sandra Nagaki for reviewing this manuscript and for her valuable comments. We also appreciate the input of Louis Lambrechts of the Insect-Virus Interaction group of the Institut Pasteur de Paris.

This study was partially supported by the US Naval Medical Research Unit Two, work unit number D1428, in support of the Military Infectious Diseases Research Program and Institut Pasteur du Laos.


(1.) Wildlife Conservation Society. Nakai Nam Theun National Protected Area [internet]. 2017. Available at: Saving-Wild-Places/Nakai-Nam-Theun-NPA.aspx. Accessed December 12, 2017.

(2.) Musser GG, Smith AL, Robinson MF, Lunde DP. Description of a new genus and species of rodent (Murinae, Muridae, Rodentia) from the Khammouan Limestone National Biodiversity Conservation Area in Lao PDR. Am Mus Novitates. 2005;3497:1-31.

(3.) Luu VQ, Truong QN, Thomas C, et al. New country records of reptiles from Laos. Biodivers Data J. 2013;1:e1015.

(4.) Luu VQ, Nguyen TQ, Le MD, Bonkowski M, Ziegler T. A new species of karst-dwelling bent-toed gecko (Squamata: Gekkonidae) from Khammouane Province, central Laos. Zootaxa. 2016;4079(1):87-102.

(5.) Luu VQ, Nguyen TQ, Le MD, Bonkowski M, Ziegler T. A new karst dwelling species of the Gekko japonicus group (Squamata: Gekkonidae) from central Laos. Zootaxa. 2017;4263(1): 179-193.

(6.) Rueda LM, Vongphayloth K, Pecor JE, et al. Mosquito fauna of Lao People's Democratic Republic, with special emphasis on the adult and larval surveillance at Nakai District, Khammuane Province. US Army Med Dep J. July-September 2014;25-32.

(7.) Apiwathnasorn C, ed. A List of Mosquito Species in Southeast Asia. Bangkok, Thailand: SEAMEOTROPMED, National Centre of Thailand, Mahidol University; 1985.

(8.) Tsuda Y, Kobayashi J, Nambanya S, et al. An ecological survey of dengue vectors in Central Lao PDR. Southeast Asian J Trop Med Public Health. 2002;33:63-67.

(9.) Vythilingam I, Sidavong B, Thim CS, Phonemixay T, Prompida S, Jeffrey J. Species composition of mosquitoes of Attapeu Province, Lao's People Democratic Republic. J Am Mosq Control Assoc. 2006;22:140-143.

(10.) Tangena JAA, Thammavong P, Malaithong N, et al. Diversity of mosquitoes (Diptera: Culicidae) attracted to human subjects in rubber plantations, secondary forests, and villages in Luang Prabang Province, Northern Lao PDR. J Med Entomol. 2017;1-16.

(11.) Walter Reed Biosystematics Unit. Systematic catalog of Culicidae (database on line). Suitland, MD: Walter Reed Biosystematics Unit, Smithsonian Institution; 2015. Available at: http://www.mosqui Accessed January 3, 2018.

(12.) Peters W. A Colour Atlas of Arthropods in Clinical Medicine. London, UK: Wolfe Publishing Ltd; 1992.

(13.) Huang Y-M. The subgenus Stegomyia of Aedes in the Afrotropical Region with keys to the species (Diptera: Culicidae). Zootaxa. 2004;700:1-120.

(14.) Huang Y-M. Medical entomology studies-XI. The subgenus Stegomyia of Aedes in the Oriental Region with keys to the species (Diptera: Culicidae). Contr Am Entomol Inst. 1979;165(6):1-79.

(15.) Chow VTK, Chan YC, Yong R, et al. Monitoring of dengue viruses in field-caught Aedes aegypti and Aedes albopictus mosquitoes by a type-specific polymerase chain reaction and cycle sequencing. Am J Trop Med Hyg. 1998;58:578-586.

(16.) Paupy C, Ollomo B, Kamgang B, et al. Comparative role of Aedes albopictus and Aedes aegypti in the emergence of dengue and chikungunya in central Africa. Vector Borne Zoonotic Dis. 2010;10(3):259-266.

(17.) Tomasello D, Schlagenhauf P. Chikungunya and dengue autochthonous cases in Europe, 2007-2012. Travel Med Infect Dis. 2013;11(5):274-284.

(18.) Lai S, Huang Z, Zhou H, et al. The changing epidemiology of dengue in China, 1990-2014: a descriptive analysis of 25 years of nationwide surveillance data. BMC Med. 2015;13(1):1-12.

(19.) Huang Y-M. Contributions to the mosquito fauna of Southeast Asia. XIV. The subgenus Stegomyia of Aedes in Southeast Asia. I-The scutellaris group of species. Contr Am Entomol Inst. 1972;9(1):1-109.

(20.) Mendenhall IH, Manuel M, Moorthy M, et al. Peridomestic Aedes malayensis and Aedes albopictus are capable vectors of arboviruses in cities. PLoS Negl Trop Dis. 2017;11(6):e0005667.

(21.) Rattanarithikul R, Harbach RE, Harrison BA, Panthusiri P, Coleman RE, Richardson JH. Illustrated keys to the mosquitoes of Thailand VI. Tribe Aedini. Southeast Asian J Trop Med Pub Health. 2010;41(suppl 1):1-225.

(22.) Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol. 1994;3:294-299.

(23.) Kearse M, Moir R, Wilson A, et al. Geneious Basis: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012;28:1647-1649.

(24.) Felsenstein J. Confidence limits on phylogenies: An approach using the bootstrap. Evolution. 1985;39:783-791.

(25.) Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406-425.

(26.) Kimura M. A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences. J Mol Evol. 1980;16:111-120.

(27.) Kumar S, Stecher G, Tamura K. MEGA7: molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870-1874.

(28.) Huang Y-M. A redescription of Aedes (Stegomyia) scutellaris malayensis Colless and the differentiation of the larva from that of Aedes (S.) albopictus (Skuse). Proc Entomol Soc Washington. 1971;73(1):1-8.

(29.) Wang G, Li C, Guo X, et al. Identifying the main mosquito species in China based on DNA barcoding. PLoS One. 2012;7(10):e47051.

(30.) Linton YM, Pecor JE, Porter CH, et al. Mosquitoes of eastern Amazonian Ecuador: biodiversity, bionomics and barcodes. Mem Inst Oswaldo Cruz. 2013;108(I):100-109.

(31.) Chan A, Chiang LP, Hapuarachchi HC, et al. DNA barcoding: complementing morphological identification of mosquito species in Singapore. Paras Vect. 2014;7:569.

(32.) Huang Y-M. The subgenus Stegomyia of Aedes in the Afrotropical Region. I. The africanus group of species (Diptera: Culicidae). Contr Am Entomol Inst. 1990;26(1):1-90. Available at: get-tr-doc/pdf?AD =ADA511785. Accessed June 14, 2018.

(33.) Tewari S, Hiriyan J, Reuben R. Epidemiology of subperiodic Wuchereria bancrofti infection in the Nicobar Islands, India. Trans R Soc Trop Med Hyg. 1995;89(2):163-166.

(34.) Rosen L, Rozemboom LE, Gubler DJ, Lien JC, Chaniotis BN. Comparative susceptibility of mosquito species and strains to oral and parental infection with dengue and Japanese encephalitis viruses. Am J Trop Med Hyg. 1985;34(3):603-615.

(35.) Colless DH. Notes on the taxonomy of the Aedes scutellaris Group, and new records of A. paullusi and A. albopictus (Diptera: Culicidae). Proc Linn Soc N S W. 1962;312-315. Available at: https://ar Accessed June 14, 2018.

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Maysa T. Motoki, PhD

Elliott F. Miot, MS

Leopoldo M. Rueda, PhD

Khamsing Vongphayloth, MD

Nothasine Phommavanh, BS

Khaithong Lakeomany, BS

Mustapha Debboun, PhD

LCDR Jeffrey C. Hertz, USN

Paul T. Brey, PhD

Dr Motoki and Mr Miot share lead author responsibility for this report.

Dr Motoki is a Research Entomologist of Institut Pasteur du Laos, Vientiane, Lao PDR, and formerly was Postdoctoral Entomologist of the Department of Entomology, Smithsonian Institution, Museum Support Center, Suitland, Maryland.

Mr Miot is a PhD candidate at the Universite Pierre et Marie Curie (UPMC), Cellule Pasteur UPMC, Paris, France, working at the Institut Pasteur du Laos, Vientiane, Lao PDR. He is affiliated with the Insect-Virus Interactions Group, Department of Genomes and Genetics, Institut Pasteur, Paris, France, and the Centre National de la Recherche Scientifique, Unite Mixte de Recherche 2000, Paris, France.

Dr Rueda is an Adjunct Scientist of the Smithsonian Institution and formerly was Research Entomologist and 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.

Dr Vongphayloth is a Medical Doctor and Entomologist of Institut Pasteur du Laos, Vientiane, Lao PDR.

Mr Phommavanh is an Assistant Technical Entomologist of Institut Pasteur du Laos, Vientiane, Lao PDR.

Mr Lakeomany is an Assistant Technical Entomologist of Institut Pasteur du Laos, Vientiane, Lao PDR.

Dr Debboun is Director of the Mosquito and Vector Control Division, Harris County Public Health, Houston, Texas.

LCDR Hertz is Chief of Entomological Sciences, US Naval Medical Research Center--Asia, located at the US Navy Region Center, Sembawang, Singapore.

Dr Brey is a Research Entomologist and Director of Institut Pasteur du Laos, Vientiane, Lao PDR.

Caption: Figure 1. Sampling location of Ae. malayensis in the Lao PDR.

Caption: Figure 2. Morphological comparison of the thorax.

Caption: Figure 3. Morphological comparison of the abdominal terga IV-VI.

Caption: Figure 4. Morphological characters of Ae. malayensis that are similar to Ae. alcasidi, Ae. riversi, and Ae. scutellaris:

Caption: Figure 5. Larval habitats (rock holes) of Ae. malayensis in the Nakai District.

Caption: Figure 6. Bootstrapped NJ tree using COI sequences of Ae. malayensis (MG921172MG921176 = Lao PDR; KY420809-KY420811 = Singapore20) based on 1,000 replicates of Tamura-Nei algorithm. Bootstrap values less than 50% are not shown. Scale bar represents sequence (%) divergence between samples. Ae. albopictus (MG921177-MG921178) was used as an outgroup.
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Author:Motoki, Maysa T.; Miot, Elliott F.; Rueda, Leopoldo M.; Vongphayloth, Khamsing; Phommavanh, Nothasin
Publication:U.S. Army Medical Department Journal
Article Type:Report
Geographic Code:9LAOS
Date:Jan 1, 2018
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