Crossing experiments supporting the specific status of Anopheles maculatus chromosomal form K

There are 3 recognized chromosomal forms (B, E, K) in the taxon of Anopheles maculatus, 1 of the 8 members of the Anopheles maculatus group. Previous studies suggested that forms B and E are cytotypes of the species, but genetic characteristics of form K are unknown. The present study used crossing experiments, and polytene chromosomes of the ovarian nurse cell in F1 hybrids to show that form K is genetically distinct from forms B and E. In addition, postzygotic genetic incompatibility between form K and An. sawadwongporni, An. dravidicus, and An. pseudowillmori are demonstrated. In all crosses, hybrid males were sterile, with atrophied testes and accessory glands, or partially sterile with abnormal spermatozoa. The hybrid females showed varying degrees of atrophied ovaries. The ovarian nurse cell polytene chromosomes of the F1 hybrid females displayed approximately 70% to almost complete asynapsis. The results provide clear evidence that form K should no longer be regarded as a cytotype of An. maculatus. The present study supports previous suggestions that form K represents another species of the Maculatus group.     

Insecticide-induced behavioral responses in two populations of Anopheles maculatus and Anopheles sawadwongporni, malaria vectors in Thailand

Behavioral responses of 2 wild-caught populations of Anopheles maculatus (Theobald) and Anopheles sawadwongporni Rattanarithikul and Green to operational field doses of DDT (2 g/m2) and permethrin (0.5 g/m2) were characterized using an excito-repellency test system. Both test populations, collected from animal quarters at Ban Pu Teuy, Sai Yok District, Kanchanaburi Province, western Thailand, were found completely susceptible to DDT and permethrin. Specimens from 2 test populations quickly escaped from direct contact with treated surfaces from 2 insecticides compared with paired controls. Noncontact repellency response to DDT was significantly pronounced in An. sawadwongporni (P < 0.05) and comparatively weak in An. maculatus, but it was statistically greater than individually paired controls (P < 0.05). We conclude that contact irritancy is a major behavioral response of both field populations when exposed directly to DDT and permethrin, whereas noncontact repellency to DDT also produced a significant escape response in An. sawadwongporni.     

Excito-repellency of deltamethrin on the malaria vectors, Anopheles minimus, Anopheles dirus, Anopheles swadiwongporni, and Anopheles maculatus, in Thailand

This study compared the behavioral avoidance responses of 4 mosquito malaria vectors, Anopheles minimus, Anopheles dirus, Anopheles maculatus form B, and Anopheles swadiwongporni, to deltamethrin, the primary insecticide used for indoor residual spraying for malaria vector control in Thailand. Six test populations. representing 4 laboratory colonies and 2 wild-caught populations, were observed during and after exposure to deltamethrin at the operational dose (0.02 g active ingredient/m2) in excito-repellency escape chambers. The laboratory colonies included a deltamethrin-susceptible colony and a deltamethrin-resistant colony of An. minimus species A, 1 colony of An. dirus species B, and 1 colony of An. maculatus form B. The 2 wild-caught populations included An. swadiwongporni and members of the An. dirus complex. Times to escape by female mosquitoes during 30 min of exposure to deltamethrin-treated papers were observed in all populations and compared to nontreated paired controls in contact and noncontact test configurations. Strong behavioral avoidance was observed in the deltamethrin-resistant colony of An. minimus, followed by An. swadiwongporni and An. maculatus. The slowest escape response was observed in the colony of An. dirus species B. All 6 populations of Anopheles showed marked contact irritancy to deltamethrin compared to paired controls and noncontact repellency trials, in both controlled laboratory colonies and field-caught populations. The degree of repellency was less profound than irritancy but, in most cases, produced a significant escape response compared to paired controls. Avoidance behavior appears to be an innate behavior of mosquitoes, as indicated by the general avoidance response detected in all 4 species, regardless of deltamethrin susceptibility status, age, or nutritional and physiological status. Excito-repellency assays of the type described in this study should become an integral part of the overall assessment of an insecticide's ability to control disease transmission in any given area.     

Cytogenetic evidence for two species within the current concept of the malaria vector Anopheles leucosphyrus in Southeast Asia

Karyotypes and crossing relationships were investigated for three allopatric populations of Anopheles leucosphyrus in Southeast Asia: South Kalimantan, Sumatra and Thailand. The mitotic karyotypes of these populations were similar to those previously observed in other species of the An. leucosphyrus group. Populations from Thailand and South Kalimantan exhibited telocentric and subtelocentric sex chromosomes, respectively, with a distinctive band of intercalary heterochromatin in the X chromosome. Strikingly different submetacentric X and Y chromosomes were observed in the population from Sumatra, and it seems likely that the evolution of these chromosomes occurred through the acquisition of constitutive heterochromatin. Sterile F1 males were observed in crosses between the Sumatra population and the populations from South Kalimantan and Thailand. No genetic incompatibility was observed in crosses between the latter two populations. We believe that the present concept of An. leucosphyrus includes two allopatric species, one inhabiting Borneo, West Malaysia and southern Thailand and one confined to Sumatra.     

PCR-RFLP method for the identification of four members of the Anopheles annularis group of mosquitoes (Diptera: Culicidae)

The Anopheles annularis group of mosquitoes is widely distributed in Southeast Asia and may be locally important as malaria vectors. Members of this group are morphologically very similar and often difficult to distinguish, particularly A. nivipes and A. philippinensis. We report the sequence analysis of the rDNA internal transcribed spacer 2 (ITS2) and Domain-3 (D3) regions of the four members of the A. annularis group -A. nivipes, A. philippinensis, A. annularis and A. pallidus - and a method for their molecular identification. No intraspecies sequence variation was detected among the specimens, while interspecific sequence differences were greater for ITS2 than the D3 regions. Comparison of the D3 sequences of the four species revealed two SmaI restriction sites in A. nivipes, but only one site in A. philippinensis, A. annularis and A. pallidus. The ApaI site was present in both A. philippinensis and A. pallidus, while an NcoI site was present in A. pallidus only. Restriction digestion of the PCR products of D3 fragment individually with SmaI, ApaI and NcoI produced a distinctive pattern for all the four species. We present, for the first time, a PCR-RFLP method to distinguish the four members of the A. annularis group of mosquitoes.     

Speciation and phylogeography of the Southeast Asian Anopheles sundaicus complex.

Anopheles sundaicus s.l. is a malaria vector in coastal areas of Southeast Asia. Previous studies showed at least four distinct species within the complex. The present study investigated the phylogeography and the status of A. sundaicus s.l. populations from Cambodia, Thailand, Malaysia and Indonesia with regard to A. sundaicus s.s. from Sarawak, Malaysian Borneo and A. epiroticus in Vietnam and Thailand. Three lineages recovered by analyses of Cyt-b and COI (mtDNA) confirmed the presence of A. sundaicus s.s. in Malaysian Borneo, the distribution of A. epiroticus from southern Vietnam to peninsular Malaysia, and recognised a distinct form in Indonesia that is named A. sundaicus E. The phylogenetic and demographic analyses suggest that the three species were separated during the Early Pleistocene (1.8-0.78 Myr) and experienced bottlenecks followed by a genetic expansion in more recent times. Based on the results and knowledge of the biogeography of the area, we hypothesise that the combination of cyclical island and refugium creation was the cause of lineage isolation and bottleneck events during the Pleistocene.     

Population genetics of the malaria vector Anopheles aconitus in China and Southeast Asia

Anopheles aconitus is a well-known vector of malaria and is broadly distributed in the Oriental Region, yet there is no information on its population genetic characteristics. In this study, the genetic differentiation among populations was examined using 140 mtDNA COII sequences from 21 sites throughout Southern China, Myanmar, Vietnam, Thailand, Laos and Sri Lanka. The population in Sri Lanka has characteristic rDNA D3 and ITS2, mtDNA COII and ND5 haplotypes, and may be considered a distinct subspecies. Clear genetic structure was observed with highly significant genetic variation present among population groups in Southeast Asia. The greatest genetic diversity exists in Yunnan and Myanmar population groups. All population groups are significantly different from one another in pairwise Fst values, except Northern Thailand with Central Thailand. Mismatch distributions and extremely significant F_s values suggest that the populations passed through a recent demographic expansion. These patterns are discussed in relation to the likely biogeographic history of the region and compared to other Anopheles species.     

Comparative susceptibility of Southeast Asian Anopheles mosquitoes to the simian malaria parasite Plasmodium cynomolgi.

Seven Anopheles species/isolates were compared with Anopheles dirus (control) for susceptibility to Plasmodium cynomolgi B strain. The mean numbers of oocysts in paired replicates of An. dirus and An. takasagoensis were not significantly different. The remaining test species had significantly fewer mean numbers of oocysts than Anopheles dirus (P less than 0.01). Anopheles dirus had the highest percentage of mosquitoes infected with P. cynomolgi sporozoites (82%). Of the test groups, Anopheles dirus B and An. takasagoensis had the highest percentage of mosquitoes with sporozoites, 77 and 78%, respectively. Fewer than 50% of Anopheles maculatus E and An. maculatus B (NN isolate) had sporozoites in the salivary glands. Anopheles maculatus B (HK isolate) and Anopheles philippinensis were the least susceptible, with fewer than 30% having sporozoites in the salivary glands.     

Dengue emergence and adaptation to peridomestic mosquitoes

Phylogenetic evidence suggests that endemic and epidemic dengue viruses (DENV), transmitted among humans by the anthropophilic mosquitoes Aedes aegypti and Ae. albopictus, emerged when ancestral, sylvatic DENV transmitted among nonhuman primates by sylvatic Aedes mosquitoes adapted to these peridomestic vectors. We tested this hypothesis by retrospectively examining evidence for adaptation of epidemic and endemic versus sylvatic strains of DENV-2 to Ae. albopictus and Ae. aegypti. First and second-generation offspring of mosquitoes from different geographic regions in the Americas and Southeast Asia were tested for their susceptibility to epidemic/endemic and sylvatic DENV-2 isolates from West Africa, Southeast Asia, and Oceania. Both Aedes species were highly susceptible (up to 100% infected) to endemic/epidemic DENV-2 strains after ingesting artificial blood meals but significantly less susceptible (as low as 0%) to sylvatic DENV-2 strains. Our findings support the hypothesis that adaptation to peridomestic mosquito vectors mediated dengue emergence from sylvatic progenitor viruses.     

Population genetic structure of Anopheles maculatus in Thailand

Anopheles (Cellia) maculatus Theobald is a major malaria vector in southern Thailand and peninsular Malaysia, and previous population genetic studies suggested that mountain ranges act as barriers to gene flow. In this study, we examine the genetic variance among 12 collections of natural populations in southern Thailand by analyzing 7 microsatellite loci. Based on analysis of molecular variance (AMOVA), three geographic populations of An. maculatus are suggested. The southern population exists in western Thailand north of 12 degrees north latitude. Mosquitoes to the south fall into two genetic populations: 1) the middle southern collections located on the west side of the Phuket mountain range between 8 degrees and 10 degrees north latitude, and 2) the southern collections located on the east of the Phuket mountain range located between approximately 6.5 degrees and 11.5 degrees north latitude. AMOVA revealed significant genetic differentiation between northern and middle southern and southern populations. The middle southern population was moderately differentiated from the southern population. Furthermore, gene flow was restricted between proximal collections located on different sides of the Phuket mountain range. Collections separated by 50 km exhibited restriction of gene flow when separated by geographic barriers, whereas greater gene flow was evident among collections 650 km apart but without geographic barriers.     

西藏察隅县营区蚊虫的组成及分布特征

目的 调查察隅县营区室内外蚊虫的组成及其空间分布情况。方法 采用二氧化碳诱蚊灯法和帐诱法,对院落、畜圈外周和野外林地3种栖息环境内的蚊虫进行调查取样、分类鉴定和计数,所获资料分别应用数量和分布型进行统计分析。结果 本次调查共捕获蚊类2亚科4属6种共822只,其中,伪杂鳞库蚊数量最多,占捕获总数的86.25%;其次是多斑按蚊和骚扰阿蚊,分别占5.47%和5.23%;在不同栖息环境捕获次数中,人房中最高是伪杂鳞库蚊,占分布类型0.476,畜圈周围较高的是带足按蚊、多斑按蚊和骚扰阿蚊,占分布类型0.750、0.818和0.615,刺扰伊蚊仅在林地捕获。结论 伪杂鳞库蚊不仅在数量上占优势,并偏好入室活动;提示在防治时室内采取滞留喷洒,室外重点进行孳生地治理;带足按蚊和多斑按蚊偏向于在畜圈活动,畜圈应是该2种蚊虫的重点防治区域。     

Morphological differentiation in Anopheles maculatus of Thailand accompanied with genetical divergence assessed by hybridization

Anopheles maculatus of Thailand were distinguished morphologically into densely and sparsely scaled types and cross-mating experiments between these types revealed a substantial amount of genetic divergence. Progeny of wild females of An. maculatus collected throughout Thailand from north to south were examined morphologically with respect to adult tergal pale scales. Northern progeny broods could be separated into densely scaled (willmori) and sparsely scaled (maculatus) types, or W and M types, respectively. Using these two types of An. maculatus from Chiang Rai and Nakhon Phanom, cross-mating experiments were conducted, which resulted in sterile F1 male hybrids in one direction of cross and very low survival rate of F1 male hybrids in the reciprocal cross. However, southern broods could not be separated clearly because of intergradation of scaling. These results indicate that Thai An. maculatus involves at least 2 forms differentiated morphologically, as well as by incomplete reproductive isolation.     

Evidence for a new sibling species of Anopheles minimus from the Ryukyu Archipelago, Japan

The Anopheles minimus complex is known to comprise at least 2 sibling species (A and C) in Thailand and Vietnam. This study investigated the specific status of An. minimus on Ishigaki Island, the Ryukyu Archipelago, Japan using morphological and genetic analyses. Morphological studies revealed that almost all (99.5%) of the adult mosquitoes are characterized by the humeral pale spot on the costa of their wings, a character that partially differentiates species A and C elsewhere. A high frequency (81.4%) have a pale fringe spot at the tip of vein 1A, a character rarely observed in other An. minimus populations. Significant seasonal variation in the size of wild An. minimus mosquitoes on the island was observed, with the largest size in the winter. Scanning micrographs of the cibarial armature of females from Ishigaki Island revealed that over 90% had cone filaments clearly differing in shape from those of species A or C. The Giemsa-stained metaphase karyotypes of larval brain cells were somewhat similar to those of species A, with a few exceptions, but were very different from those reported for species C. Crossing experiments between species A (CM strain) from Thailand and the progeny of An. minimus from Ishigaki Island (ISG strain) revealed postzygotic genetic incompatibility, although no prezygotic isolation. Hybrid progeny were only obtained from CM female x ISG male. F2 hybrid progeny were not obtained, since the hybrid males were sterile or almost sterile with atrophied testes or abnormal spermatozoa, although the polytene chromosomes of hybrid larvae showed synapsis. The hybrid females backcrossed with either CM or ISG males laid eggs with significantly lowered fertility and viability. The sequence for the D3 region of the 28S gene of ribosomal DNA of the ISG strain differed from those of species A and C. In addition, sequence data from Vietnamese mosquitoes suggest that the An. minimus complex may contain additional species. The morphological, cytogenetic, molecular, and hybridization evidence together suggest the existence of another sibling species of the An. minimus complex on Ishigaki Island, which is provisionally designated An. minimus species E.     

Suggesting new species? Comments on "Evidence for a new species of Anopheles minimus from the Ryukyu Archipelago, Japan"

Species recognition and identification are crucial for the planning and evaluation of vector control. In recent years, considerable effort has been made to clarify the species composition of vector taxa. The danger exists, however, that new species are suggested on little evidence and that intraspecific variation is insufficiently surveyed. We discuss these topics on the basis of recent studies on the Anopheles minimus complex, one of the most widespread vector groups in Southeast Asia.     

Bionomics, taxonomy, and distribution of the major malaria vector taxa of Anopheles subgenus Cellia in Southeast Asia: an updated review.

There is high diversity of Anopheles mosquitoes in Southeast Asia and the main vectors of malaria belong to complexes or groups of species that are difficult or impossible to distinguish due to overlapping morphological characteristics. Recent advances in molecular systematics have provided simple and reliable methods for unambiguous species identification. This review summarizes the latest information on the seven taxonomic groups that include principal malaria vectors in Southeast Asia, i.e. the Minimus, Fluviatilis, Culicifacies, Dirus, Leucosphyrus, and Sundaicus Complexes, and the Maculatus Group. Main issues still to be resolved are highlighted. The growing knowledge on malaria vectors in Southeast Asia has implications for vector control programs, the success of which is highly dependant on precise information about the biology and behavior of the vector species. Acquisition of this information, and consequently the application of appropriate, sustainable control measures, depends on our ability to accurately identify the specific vectors.     

How reliable is the humeral pale spot for identification of cryptic species of the Minimus Complex?

The Anopheles minimus Complex Theobald (Diptera: Culicidae) is composed of the 3 sibling species A, C, and E. The malaria vectors An. minimus A and C are distributed over the Southeast Asian region, whereas species E is restricted to the Ryukyu Japanese Islands. Because species A and C can be sympatric and present specific behaviors and have a role in malaria transmission, it is important to differentiate them. The literature mentioned the presence of a presector pale spot on the wing costa of An. minimus A, whereas species C may exhibit both presector and humeral pale spots. However, the reliability of their diagnostic power has not been established over large temporal and geographic surveys. From the analyses of 9 populations throughout Southeast Asia, including published data and field populations from 2 sites in Thailand, we showed that the wing patterns present spatial and temporal variations that make these two morphological characters unreliable for the precise identification of An. minimus A and C. Therefore, molecular identification remains the most efficient method to obtain an unambiguous differentiation of these 2 species. Correct species identification is essential and mandatory for any relevant study on the Minimus Complex and for the application of successful control strategies.     

Problems and potential of researching epidemiological transition: examples from Southeast Asia

The concept of epidemiological transition is now quite widely recognized, if not so widely accepted. The transition appears to progress at varying speeds and to different extents spatially; it seems that there can be considerable international, regional and local variations in its progress. The paper examines this contention in the case of a number of countries in Southeast Asia, principally Hong Kong, Malaysia and Thailand. Drawing on evidence from this region, the paper highlights the importance when researching epidemiological transition of the time period under consideration; socio-cultural variations; the nature and quality of data, and spatial scale. It makes some suggestions as to the potential of the concept of epidemiological transition in health care planning and development studies.     

Cytogenetic evidence for a fifth species within the taxon Anopheles dirus in Thailand

Crossbreeding and chromosomal evidence are presented for the existence of a fifth sibling species within the taxon of Anopheles dirus in Thailand. The new species is morphologically identifiable as Anopheles balabacensis "Fraser's Hill form." Structural differences in mitotic chromosomes and extensive asynapsis in hybrid polytene chromosomes indicate that significant genetic divergence exists between this species and its closest relatives, An. dirus species A, B, C and D and An. balabacensis.     

Geographic subdivision of the range of the malaria parasite Plasmodium vivax

We examined geographically distinct isolates of Plasmodium vivax and categorized them according to developmental success in Anopheles albimanus. We found that parasites from Central America and Colombia form a group distinct from those of Asia. New World isolates have a distinct chromosomal translocation and an episomal variation in the open reading frame (ORF) 470 DNA sequence that distinguishes them from the other isolates tested. Old World types of P. vivax were introduced into the Americas, and a remnant of this lineage remains in P. simium. It is indistinguishable from Old World P. vivax to the extent determinable by using our encoded markers and the examination of its developmental pattern in mosquitoes. The cohesive characteristics that separate types of P. vivax are predictors of range and potential for transmission and hence require taxonomic distinction.     

Isolation and molecular characterization of Banna virus from mosquitoes, Vietnam

We isolated and characterized a Banna virus from mosquitoes in Vietnam; 5 strains were isolated from field-caught mosquitoes at various locations; Banna virus was previously isolated from encephalitis patients in Yunnan, China, in 1987. Together, these findings suggest widespread distribution of this virus throughout Southeast Asia.