Trophic behavior and biting activity of the two sibling species of the Anopheles minimus complex in western Thailand.

The trophic behavior and host preference of two sibling species, Anopheles minimus s.s. (= An. minimus species A) and species C, were observed during a two-year period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, western Thailand. Anopheles minimus s.s. and species C were more prevalent during the hot and wet periods of the year. Both species demonstrated exophagic and zoophilic activities. Feeding activity of An. minimus C was unique compared to An. minimus sensu lato from other localities in Thailand. Outdoor blood feeding by An. minimus C occurred throughout the night with one distinct feeding peak immediately after sunset (1800 h), whereas indoor feeding showed two small peaks at 2000 and 2400 h. The small number of An. minimus s.s collected during this study precluded a determination of peak activity patterns. A better understanding of mosquito behavior related to host and patterns of feeding activity will facilitate and improve the efficiency of vector control operations.     

Biting activity and host preference of the malaria vectors Anopheles maculatus and Anopheles sawadwongporni (Diptera: Culicidae) in Thailand

Adult mosquitoes in the Anopheles maculatus group were surveyed from different regions of Thailand and five different species were morphologically identified, including Anopheles maculatus, Anopheles sawadwongporni, Anopheles notanandai, Anopheles dravidicus, and Anopheles willmori. Blood‐feeding activity and host preference of two species, Anopheles maculatus and Anopheles sawadwongporni, were observed during a one‐year period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, west‐central Thailand. Both species were more prevalent during the wetter period of the year and each had a greater predilection to feed on cattle than humans. Primary feeding activity occurred between 20:00‐23:00 and a smaller peak at 01:00‐03:00. Findings are discussed relative to the importance of these two vectors for malaria transmission in Pu Teuy.     

Population-genetic evidence for two species in Anopheles minimus in Thailand

ABSTRACT. Sympatric occurrence of homozygotes for two electro-morphs controlled by a locus for octanol dehydrogenase, and the absence of heterozygotes, at two localities, indicates two isomorphic species within the taxon Anopheles minimus Theobald in Thailand. This view is supported by significant, relative deficiences of heterozygotes at other electromorphic loci. Gene frequency data are reported for seven electro-morphic loci in An.minimus sensu lato from eleven localities: one of the newly recognized species predominated in all but one locality and the second was confined to two localities. This species pair of An.minimus s.l. was clearly distinguished from An.aconitus Donitz, An.pampanai Biittiker & Beales and An.varuna Iyengar, three species closely related to An.minimus s.l. in the series Myzomyia of Anopheles subgenus Cellia.     

PCR-based methods for identification of species of the Anopheles minimus group: allele-specific amplification and single-strand conformation polymorphism

We report two polymerase chain reaction (PCR)-based methods for distinguishing morphologically similar species based on amplification of a variable region of the 28S gene of ribosomal DNA. The four species we investigated are mosquitoes of the Anopheles minimus group: An. aconitus, An. varuna and An. minimus species A and C. The formally named species are vectors of human malaria parasites in south-east Asia but are difficult to distinguish with certainty on the basis of morphology. Allele-specific amplification was used to differentiate An. minimus A from An. minimus C. This technique has been widely used for the diagnosis of species. Single-strand conformation polymorphisms (SSCPs) were used to separate all four species. This technique, which has seldom been used for species identification, has many advantages: it does not require sequence information beyond that needed for amplification; it is ideally suited for the detection of heterozygotes; it utilizes more of the information in the PCR product than allele-specific amplification; it distinguishes all four species considered here and could easily be extended to other species; previously unknown intraspecific variation and additional species are likely to be detected. Thus, SSCPs provide valuable population genetic information which allele-specific amplification does not.     

Isoenzymatic variability among five Anopheles species belonging to the Nyssorhynchus and Anopheles subgenera of the Amazon region,Brazil

An isoenzymatic comparative analysis of the variability and genetics differentiation among Anopheles species was done in populations of An. (Nys.) intermedius and An. (Ano.) mattogrossensis of the Anopheles subgenus, and of An. darlingi, An. albitarsis and An. triannulatus of the Nyssorhynchus subgenus, with the aim of detecting differences between both subgenera and of estimating the degree of genetic intere specific divergence. Samples from Macap , State of Amap and Janauari Lake, near Manaus, State of Amazonas, were analyzed for eight isoenzymatic loci. Analysis revealed differences in the average number of alleles per locus (1.6-2.3) and heterozygosity (0.060-0.284). However, the proportion of polymorphic loci was the same for An. (Nys.) darlingi, An. (Nys.) triannulatus and An. (Ano.) mattogrossensis (50%), but differed for An. (Nys.) albitarsis (62.5%) and An. (Ano.) intermedius (25%). Only the IDH1 (P > 0.5) locus in all species studied was in Hardy-Weinberg equilibrium. The fixation index demonstrated elevated genetic structuring among species, based on values of Fst = 0.644 and genetic distance (0.344-0.989). Genetic difference was higher between An. (Nys.) triannulatus and An. (Ano.) intermedius (0.989) and smaller between An. (Nys.) albitarsis sensu lato and An. (Nys.) darlingi (0.344). The data show interspecific genetic divergence which differs from the phylogenetic hypothesis based on morphological characters.     

Variability and genetic differentiation among Anopheles (Ano.) intermedius Chagas, 1908 and Anopheles (Ano.) mattogrossensis Lutz & Neiva, 1911 (Diptera: Culicidae) from the Brazilian Amazon

Anopheles (Anopheles) intermedius and Anopheles (Ano.) mattogrossensis are Brazilian anopheline species belonging to the scarcely studied Anopheles subgenus. Few studies have been done on the genetic differentiation of these species. Both species have been found infected by Plasmodium and are sympatric with other anopheline species from the Nyssorhynchus subgenus. Eighteen enzymatic loci were analyzed in larval specimens of An. intermedius and An. mattogrossensis aiming to estimate the variability and genetic differentiation between these species. An. mattogrossensis population showed higher genetic variability (P = 44.4 and Ho = 0.081 +/- 0.031) than that of An. intermedius (P = 33.3 and Ho = 0.048 +/- 0.021). Most analyzed loci showed genotypic frequencies according to Hardy-Weinberg equilibrium, except for LAP1 and LAP2 in An. intermedius, and EST1 and PGM loci in An. mattogrossensis. The genetic distance between these species (D = 0.683) was consistent with the inter-specific values reported for Anopheles subgenus. We verified that the polymorphism and heterozygosity percentile values found in both species and compared to those in the literature, showed no relation between the level of isozyme variability and geographical distribution. The low variability found in these two species is probably more related to the niche they occupy than to their geographic distribution.     

Population genetic structure of the malaria vector Anopheles minimus A in Vietnam

Anopheles minimus A, a major malaria vector in Southeast Asia, is the main target of vector control in this area. The impact of these control measures can be influenced by the population structure of the target species. In rural areas, An. minimus breeds along the banks of small clear-water streams, yet in the suburbs of Hanoi, northern Vietnam, there is an An. minimus population whose immature stages develop in water tanks. This study uses allozyme data (1) to assess the population structure of An. minimus A and (2) to evaluate the taxonomic status of the urban An. minimus population from Hanoi. The population from the suburbs of Hanoi was identified as An. minimus A. Although significant genetic differentiation was observed between rural and urban An. minimus A populations, they have not differentiated substantially by genetic drift. Limited macrogeographical differentiation was observed between two rural populations at distances of more than 1000 km. Consequently, geographical distance is not the primary factor in differentiating An. minimus A populations having the typical breeding ecology. The estimated effective population size is consistent with the moderate macrogeographical differentiation. Furthermore, no genetic structuring was observed between adult mosquitoes having different behaviour. The macrogeographical population structure indicates that genes may spread over large areas, whereas the presence of an 'urban' An. minimus A population shows the ability of this species to adapt to anthropogenic environmental changes.     

Geographical distribution of Anopheles minimus species A and C in western Thailand.

Elucidating vector distribution based on an accurate species identification is important to understanding the nature of the species complex in order to achieve vector control. Morphologically, An. minimus s.l. is difficult to distinguish from both its species complex and its closely related species. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and a single multiplex-allele specific PCR developed for species identification were applied in this study in comparison with morphological identification. Both methods were used, combining with geographical information systems to determine the distribution of An. minimus species A and C. The investigation on the breeding habitats was performed in the malarious area of western Thailand. Anopheles larvae were collected from 36 bodies of water among five districts (Sangkhaburi, Thong Pha Phum, Si Sawat, Muang, and Sai Yok) of Kanchanaburi Province, Thailand. In this study, An. minimus A larvae were present in all study districts but the association differed when focusing on study sites within each district. Although there were many reports of An. minimus A in Ban Phu Rat and Ban Phu Toei villages in Sai Yok District, we did not find the breeding sites of species A in those two areas. An. minimus A and C were found in Ban Phu Ong Ka village in Sai Yok District. The breeding habitats of An. minimus C were present covering 30-40 km of distance in northern part of Sai Yok and this species was also found in the central and southern parts of Si Sawat District.     

Chemically induced behavioral responses in Anopheles minimus and Anopheles harrisoni in Thailand

Behavioral responses of female mosquitoes representing two species in the Minimus Complex exposed to an operational field dose of bifenthrin or DEET (N,N-diethyl-m-toluamide) were described using an excito-repellency test system. Two test populations of An. minimus, one from the field (Tak Province, western Thailand), the other from a long-established laboratory colony, and Anopheles harrisoni collected from Kanchanaburi Province, western Thailand, were used. Results showed that all test populations rapidly escaped after direct contact with surfaces treated with either bifenthrin or DEET compared to match-paired untreated controls. Greater escape response by exposed females to bifenthrin and DEET were observed in the An. minimus colony compared to the two field populations. Field-collected An. minimus demonstrated a more rapid escape response to DEET than to bifenthrin, whereas An. harrisoni showed a converse response. Although fewer females escaped from test chambers without direct contact with treated surfaces compared to contact tests, the spatial repellency response was significantly pronounced in all test populations compared to match-paired controls (P < 0.05). DEET was found to perform as both a contact stimulant and moderate spatial repellent.     

Molecular genetic studies of Anopheles stephensi in Pakistan

Anopheles stephensi Liston s.l. (Diptera: Culicidae) is one of the major vectors of malaria in Pakistan, India, Iran and Afghanistan. In parts of its range this species has shown increases in both relative and absolute abundance in what is hypothesized to be a response to human-mediated environmental change resulting from extensive irrigation. We attempted to detect the molecular genetic signatures of this population instability based on three samples obtained from two villages (149/6R and 111/6R) within an irrigation zone in Punjab Province and from one village (Azakhel) outside the irrigation scheme in Northwest Frontier Province (NWFP), Pakistan, using seven microsatellite loci and 682 basepairs of the mitochondrial CO1 gene. For microsatellite loci, high levels of genetic diversity were observed within populations (mean alleles per locus 10.71-11.57; mean heterozygosity 0.703-0.733). Deviation from Hardy-Weinberg expectations was observed for only two microsatellite loci in 21 tests. No genetic differentiation was observed between populations and average pairwise F(ST) values did not differ significantly from zero for any population pair or either marker system. Tests of population expansion for both mitochondrial and microsatellite loci were inconclusive.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles maculipennis

The Anopheles maculipennis complex includes the most important malaria vectors of the western Palearctic. Anopheles maculipennis s.s., one member of this complex, is a reported vector in the Middle East. Here we describe the isolation of 15 microsatellite polymorphic loci from the An. maculipennis s.s. genome, displaying a high among individual diversity (0.37–0.77) in a sample from France. Three loci displayed a significant departure from Hardy–Weinberg proportions, suggesting a substantial frequency of null alleles. The remaining 12 loci are good candidates for further genetic studies in this species.     

Limited usefulness of microsatellite markers from the malaria vector Anopheles gambiae when applied to the closely related species Anopheles melas

Anopheles melas is a brackish water mosquito found in coastal West Africa where it is a dominant malaria vector locally. In order to facilitate genetic studies of this species, 45 microsatellite loci originally developed for Anopheles gambiae were sequenced in An. melas. Those that were suitable based on repeat number and flanking regions were examined in 2 natural populations from Equatorial Guinea. Only 15 loci were eventually deemed suitable as polymorphic markers in An. melas populations. These loci were screened in 4 populations from a wider geographic range. Heterozygosity estimates ranged from 0.18 to 0.79, and 2.5-15 average alleles were observed per locus, yielding 13 highly polymorphic markers and 2 loci with lower variability. To examine the usefulness of microsatellite markers when applied in a sibling species, the original An. gambiae specific markers were used to amplify 5 loci in An. melas. Null alleles were found for 1 An. gambiae marker. We discuss the pitfalls of using microsatellite loci across closely related species and conclude that in addition to the problem of null alleles associated with this practice, many loci may prove to be of very limited use as polymorphic markers even when used in a sibling species.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles nili

The mosquito Anopheles nili is widespread across tropical Africa and appears to be the major vector of malaria in some rural forested areas of central Africa. Here we describe the isolation of 11 microsatellite polymorphic loci from the A. nili genome, displaying a high among‐individual diversity (0.58–0.96) in samples from west Africa. Two loci displayed a significant departure from Hardy–Weinberg proportions across all samples, suggesting a substantial frequency of null alleles. The remaining nine loci are good candidates for further genetic studies in this species.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles sacharovi

The mosquito Anopheles sacharovi, a member of the A. maculipennis complex, is an important malaria vector in the Middle East. Here we describe the isolation of 15 microsatellite polymorphic loci from the A. sacharovi genome, displaying a high among‐individual diversity (0.30–0.92) in a sample from Turkey. Seven loci displayed a significant departure from Hardy–Weinberg proportions, suggesting a substantial frequency of null alleles. The remaining eight loci are good candidates for further genetic studies in this species.     

Reduced susceptibility to DDT in field populations of Anopheles quadriannulatus and Anopheles arabiensis in Malawi: evidence for larval selection

Abstract.  Bioassays for insecticide resistance in adult mosquitoes were conducted on samples of Anopheles gambiae Giles s.l . (Diptera: Culicidae) species collected as larvae from breeding sites in the lower Shire Valley, Malawi. The results indicate full susceptibility to permethrin, deltamethrin and malathion, but reduced susceptibility to DDT in one sample from Thom (LT₅₀ of 8.39 min for females and 25.09 min for males). Polymerase chain reaction-based species identification of the mosquitoes assayed revealed a mixture of Anopheles arabiensis Patton and Anopheles quadriannulatus (Theobold). The LT₅₀ did not differ significantly between species. Genotyping of the L1014F and L1014S kdr alleles showed all mosquito specimens to be homozygous wild type; thus the reduced susceptibility detected is not attributable to target site insensitivity and instead is likely to be metabolic in nature. Anopheles quadriannulatus is characteristically zoophagic and exophilic. Indeed, of 82 Anopheles collected through knockdown collections within dwellings, only one was An. quadriannulatus and the rest were An. arabiensis . They are unlikely, therefore, to have been exposed to selection pressure arising from insecticide-treated net usage or to DDT indoor residual spraying. Therefore, it is suggested that this example of reduced susceptibility to DDT in An. quadriannulatus reflects selection in the larval stages.     

Biting patterns and seasonal densities of Anopheles mosquitoes in the Cayo District, Belize, Central America with emphasis on Anopheles darlingi.

The present study utilized an experimental hut to conduct human-baited landing collections for characterizing the all-night biting patterns and seasonal densities of adult Anopheles darlingi in the centrally located Cayo District of Belize, Central America. A total of 25 all-night collections (i.e., sunset to sunrise) were conducted from January 2002 to May 2003, capturing a total of 18,878 An. darlingi females. Anopheles darlingi exhibited a bimodal nightly biting pattern with one predominate peak occurring three h after sunset and a smaller peak occurring one h prior to sunrise. Biting females were collected throughout the night in higher densities indoors (9,611) than outside (9,267) the experimental hut (O:I=1.00:1.04). Seasonal adult collections show An. darlingi densities were highest during the transitional months between the end of the wet and beginning of the dry season (January) and the end of the dry season and beginning of the wet season (May). A total of 2,010 An. darlingi females was captured in 31 two-h, human-baited landing collections performed from January to October 2002. Anopheles darlingi monthly population densities were found to have no significant associations with high or low temperatures, precipitation, or river level. However, qualitative data examination indicates an inverse relationship between river level and An. darlingi adult collections suggesting a disturbance of larval habitats. All-night biting and seasonal distribution patterns for other anopheline species are also described. None of the adult specimens collected throughout the entire study tested positive for Plasmodium spp. infection using the VecTest rapid diagnostic kit.     

Cladistic analysis of the subgenus Anopheles (Anopheles) Meigen (Diptera: Culicidae) based on morphological characters

In the present study, we used morphological characters to estimate phylogenetic relationships among members of the subgenus Anopheles Meigen. Phylogenetic analyses were carried out for 36 species of Anopheles (Anopheles). An. (Stethomyia) kompi Edwards, An. (Lophopodomyia) gilesi (Peryassú), Bironella hollandi Taylor, An. (Nyssorhynchus) oswaldoi (Peryassú) and An. (Cellia) maculatus Theobald were employed as outgroups. One hundred one characters of the external morphology of the adult male, adult female, fourth-instar larva, and pupa were scored and analyzed under the parsimony criterion in PAUP. Phylogenetic relationships among the series and several species informal groups of Anopheles (Anopheles) were hypothesized. The results suggest that Anopheles (Anopheles) is monophyletic. Additionally, most species groups included in the analysis were demonstrated to be monophyletic.     

Effect of plant structure on oviposition behavior of Anopheles minimus s.l.

Knowledge of factors that influence oviposition behavior of malaria mosquitoes is critical to vector control measures aimed at larval habitat modifications and source reduction. Anopheles minimus s.l., an important malaria vector in Southeast Asia, generally breeds in clear, unpolluted water along shaded grassy edges of slow-moving streams. The objective of this study was to determine the influence of vegetation and plant structure on An. minimus s.l. ovipositing females. Twenty gravid female mosquitoes per replication were given a choice to lay eggs in bowls surrounded by different combinations of bare soil, grasses, small-leaved plants, and large-leaved plants. An. minimus s.l. females generally preferred to lay eggs in bowls with vegetation. A significantly higher number of eggs were found in bowls with small-leaved plants compared to bowls with grasses (P<0.05). The results suggest that gravid females preferred oviposition habitats in the following order: small-leaved plants > large-leaved plants > grasses > soil. Further studies are needed to determine the possible roles of plant structure and factors such as semiochemicals in the different species of the An. minimus species complex. Knowledge of female oviposition behavior is essential for the development of locally adapted vector control approaches.     

Egg morphology as an indirect method to identify Anopheles benarrochi, Anopheles oswaldoi and Anopheles rangeli (Diptera: Culicidae)

In the Department of Putumayo in southern Colombia, malaria transmission has continued in the absence of the 4 traditional Latin American vector species--Anopheles darlingi, Anopheles nuneztovari, Anopheles albimanus or Anopheles trinkae. Human bait collections yielded Anopheles mosquitoes and a morphological variant of Anopheles benarrochi, the adult females of which can easily be misidentified as Anopheles oswaldoi. Species identification of females of Anopheles in the subgenus Nyssorhynchus is generally difficult due to overlapping morphological characters; therefore, progeny of field collected females were link-reared to assess species identity. Herein a robust method is presented to identify the species Anopheles benarrochi, Anopheles oswaldoi and Anopheles rangeli from southern Colombia, using the morphology of the eggs induced from wild-caught females. Eggs of A. rangeli and A. benarrochi were differentiated on the basis of the anterior crown. In A. rangeli, this feature is positioned apically with high walls. In A. benarrochi, anterior crown is positioned more ventrally with comparatively shorter walls. No crown is present in A. oswaldoi. These differences are clear with the aid of a dissecting microscope and make accurate species determination possible even in field conditions. Egg morphology is shown to be an accurate, albeit indirect, method for the taxonomic determination for the three southern Colombian species and may also be useful in other regions of Latin America where the morphological variant of A. benarrochi is sympatric with A. oswaldoi.     

Isozyme evidence for three sibling species in the Anopheles sundaicus complex from Indonesia

Electrophoretic studies of isoenzymes in three chromosomally distinct forms (A, B and C) of the mosquito Anopheles sundaicus Theobald (Diptera: Culiciae) were undertaken on wild samples collected from six geographically isolated populations in Indonesia. Analyses of 12 enzyme systems comprising 15 loci revealed significant allelic variations, genetic polymorphism, within and among the populations of the An. sundaicus complex. Phylogenetic dendrograms produced by analysis using the Biosys-1 program based on UPGMA methods show that all the populations of form A fall into one cluster, which is closely related to the form C cluster, whereas the populations of form B belong to a more distinct cluster. Allelic frequency and Wright's F statistics of Mpi (mannose phosphate isomerase) are sufficient to identify individuals of each cytological form. This isozyme data correlates with our previous cytological evidence for the existence of three isomorphic species within the taxon An. sundaicus in Indonesia. These three species of the An. sundaicus complex were found together sympatrically at one locality, Asahan in North Sumatra.