A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

Resistance to the larvicide temephos and altered egg and larval surfaces characterize salinity-tolerant Aedes aegypti.

Aedes aegypti, the principal global vector of arboviral diseases and previously considered to oviposit and undergo preimaginal development only in fresh water, has recently been shown to be capable of developing in coastal brackish water containing up to 15 g/L salt. We investigated surface changes in eggs and larval cuticles by atomic force and scanning electron microscopy, and larval susceptibility to two widely-used larvicides, temephos and Bacillus thuringiensis, in brackish water-adapted Ae. aegypti. Compared to freshwater forms, salinity-tolerant Ae. aegypti had rougher and less elastic egg surfaces, eggs that hatched better in brackish water, rougher larval cuticle surfaces, and larvae more resistant to the organophosphate insecticide temephos. Larval cuticle and egg surface changes in salinity-tolerant Ae. aegypti are proposed to respectively contribute to the increased temephos resistance and egg hatchability in brackish water. The findings highlight the importance of extending Aedes vector larval source reduction efforts to brackish water habitats and monitoring the efficacy of larvicides in coastal areas worldwide.

Morphological and odorant-binding protein 1 gene intron 1 sequence variations in Anopheles stephensi from Jaffna city in northern Sri Lanka.

Three Anopheles stephensi biotypes have historically been differentiated through variations in the mode numbers of egg ridges and adult spiracular indices. Anopheles stephensi odorant-binding protein 1 gene (AsteObp1) sequences in Iran and Afghanistan have been recently interpreted to suggest that the three biotypes are sibling species. AsteObp1 intron 1 sequences, mode numbers of egg ridges and spiracular indices of An. stephensi in Jaffna city in Sri Lanka were therefore investigated in field-collected mosquitoes and short-term laboratory colonies established from them. AsteObp1 intron 1 sequences revealed the region to be polymorphic with four unique sequences, ASJF1-4, present in both short-term laboratory colonies and field-collected An. stephensi. The spiracular index did not relate to the mode number of egg ridges in Jaffna An. stephensi. The results suggested that numbers of egg ridges, spiracular indices and AsteObp1 intron 1 sequences were not useful for differentiating An. stephensi biotypes in Jaffna. It is proposed that the observed differences between An. stephensi mosquitoes in Jaffna now result from normal population variance in the context of rapidly changing bionomics in India and northern Sri Lanka.

Regional Variation in Dengue Virus Serotypes in Sri Lanka and Its Clinical and Epidemiological Relevance.

Dengue is a significant health concern in Sri Lanka, but diagnosis of the infecting dengue virus (DENV) serotype has hitherto been largely restricted to the Colombo district in the western province. Salinity tolerant Aedes vectors are present in the island's northern Jaffna peninsula, which is undergoing rapid groundwater salinization. Virus serotypes were determined by RT-qPCR in 107 and 112 patients diagnosed by NS1 antigen positivity from the Jaffna district in 2018 and 2019, respectively, and related to clinical characteristics. DENV1 and DENV2 were the most common serotypes in both years. Infections with multiple serotypes were not detected. DENV1 was significantly more prevalent in 2019 than 2018, while DENV3 was significantly more prevalent in 2018 than 2019 among the Jaffna patients. Limited genomic sequencing identified DENV1 genotype-I and DENV3 genotype-I in Jaffna patients in 2018. Dengue was more prevalent in working age persons and males among the serotyped Jaffna patients. DENV1 and DENV2 were the predominant serotypes in 2019 in the Colombo district. However, DENV1 and DENV3 were significantly more prevalent in Colombo compared with Jaffna in 2019. The differences in the prevalence of DENV1 and DENV3 between the Jaffna and Colombo districts in 2019 have implications for dengue epidemiology and vaccination. Salinity-tolerant Aedes vector strains, widespread in the Jaffna peninsula, may have contributed to differences in serotype prevalence compared with the Colombo district in 2019. Significant associations were not identified between virus serotypes and clinical characteristics among Jaffna patients.

Transcriptomic, proteomic and ultrastructural studies on salinity-tolerant Aedes aegypti in the context of rising sea levels and arboviral disease epidemiology.

BACKGROUND: Aedes aegypti mosquito, the principal global vector of arboviral diseases, lays eggs and undergoes larval and pupal development to become adult mosquitoes in fresh water (FW). It has recently been observed to develop in coastal brackish water (BW) habitats of up to 50% sea water, and such salinity tolerance shown to be an inheritable trait. Genomics of salinity tolerance in Ae. aegypti has not been previously studied, but it is of fundamental biological interest and important for controlling arboviral diseases in the context of rising sea levels increasing coastal ground water salinity. RESULTS: BW- and FW-Ae. aegypti were compared by RNA-seq analysis on the gut, anal papillae and rest of the carcass in fourth instar larvae (L4), proteomics of cuticles shed when L4 metamorphose into pupae, and transmission electron microscopy of cuticles in L4 and adults. Genes for specific cuticle proteins, signalling proteins, moulting hormone-related proteins, membrane transporters, enzymes involved in cuticle metabolism, and cytochrome P450 showed different mRNA levels in BW and FW L4 tissues. The salinity-tolerant Ae. aegypti were also characterized by altered L4 cuticle proteomics and changes in cuticle ultrastructure of L4 and adults. CONCLUSIONS: The findings provide new information on molecular and ultrastructural changes associated with salinity adaptation in FW mosquitoes. Changes in cuticles of larvae and adults of salinity-tolerant Ae. aegypti are expected to reduce the efficacy of insecticides used for controlling arboviral diseases. Expansion of coastal BW habitats and their neglect for control measures facilitates the spread of salinity-tolerant Ae. aegypti and genes for salinity tolerance. The transmission of arboviral diseases can therefore be amplified in multiple ways by salinity-tolerant Ae. aegypti and requires appropriate mitigating measures. The findings in Ae. aegypti have attendant implications for the development of salinity tolerance in other fresh water mosquito vectors and the diseases they transmit.

Aedes larval bionomics and implications for dengue control in the paradigmatic Jaffna peninsula, northern Sri Lanka.

BACKGROUND: The larval bionomics of Aedes across the Jaffna peninsula in northern Sri Lanka was investigated to obtain information needed for developing more effective larval source reduction measures to control endemic arboviral diseases. METHODS: The habitats of preimaginal stages of Aedes mosquitoes were surveyed, and ovitrap collections were carried out in densely populated areas of the Jaffna peninsula. Aedes larval productivities were analysed against habitat characteristics, rainfall and dengue incidence. Adults emerging from collected larvae were tested for dengue virus (DENV). RESULTS: Only Aedes aegypti, Ae. albopictus and Ae. vittatus were identified in the field habitat collections and ovitraps. Aedes aegypti was the predominant species in both the field habitat and ovitrap collections, followed by Ae. albopictus and small numbers of Ae. vittatus. Tires and open drains were the preferred field habitats for Ae. aegypti, although larval productivity was higher in discarded plastic containers. The three Aedes species differed in field habitat preferences. Concomitant presence of the three Aedes species was observed in the field habitats and ovitraps. Larval productivities were inversely correlated with the salinity of the field habitat. Rainfall in the preceding month significantly correlated with larval productivity in the field habitats. DENV serotype 2 was detected in Ae. aegypti collected from ovitraps in the city of Jaffna. High Breteau, House and Container indices of 5.1, 5.1 and 7.9%, respectively, were observed in the field habitat surveys and ovitrap indices of up to 92% were found in Jaffna city. CONCLUSIONS: Aedes larval indices in populated areas of the peninsula showed a high potential for dengue epidemics. Unacceptable littering practices, failure to implement existing dengue control guidelines, vertical transmission of DENV in vector mosquitoes and preimaginal development in brackish water and open surface drains, as well as in domestic wells that provide potable water, are serious constraints to the current Aedes larval source reduction methods used to control dengue in the Jaffna peninsula. Similar shortcomings in arboviral disease control are likely present in other resource-constrained tropical coastal zones worldwide.

Molecular forms of Anopheles subpictus and Anopheles sundaicus in the Indian subcontinent.

BACKGROUND: Anopheles subpictus and Anopheles sundaicus are closely related species, each comprising several sibling species. Ambiguities exist in the classification of these two nominal species and the specific status of members of these species complexes. Identifying fixed molecular forms and mapping their spatial distribution will help in resolving the taxonomic ambiguities and understanding their relative epidemiological significance. METHODS: DNA sequencing of Internal Transcribed Spacer-2 (ITS2), 28S-rDNA (D1-to-D3 domains) and cytochrome oxidase-II (COII) of morphologically identified specimens of two nominal species, An. subpictus sensu lato (s.l.) and An. sundaicus s.l., collected from the Indian subcontinent, was performed and subjected to genetic distance and molecular phylogenetic analyses. RESULTS: Molecular characterization of mosquitoes for rDNA revealed the presence of two molecular forms of An. sundaicus s.l. and three molecular forms of An. subpictus s.l. (provisionally designated as Form A, B and C) in the Indian subcontinent. Phylogenetic analyses revealed two distinct clades: (i) subpictus clade, with a single molecular form of An. subpictus (Form A) prevalent in mainland India and Sri Lanka, and (ii) sundaicus clade, comprising of members of Sundaicus Complex, two molecular forms of An. subpictus s.l. (Form B and C), prevalent in coastal areas or islands in Indian subcontinent, and molecular forms of An. subpictus s.l. reported from Thailand and Indonesia. Based on the number of float-ridges on eggs, all An. subpictus molecular Form B were classified as Species B whereas majority (80%) of the molecular Form A were classified as sibling species C. Fixed intragenomic sequence variation in ITS2 with the presence of two haplotypes was found in molecular Form A throughout its distribution. CONCLUSION: A total of three molecular forms of An. subpictus s.l. and two molecular forms of An. sundaicus s.l. were recorded in the Indian subcontinent. Phylogenetically, two forms of An. subpictus s.l. (Form B and C) prevalent in coastal areas or islands in the Indian subcontinent and molecular forms reported from Southeast Asia are members of Sundaicus Complex. Molecular Form A of An. subpictus is distantly related to all other forms and deserve a distinct specific status.

Anopheline bionomics, insecticide resistance and transnational dispersion in the context of controlling a possible recurrence of malaria transmission in Jaffna city in northern Sri Lanka.

BACKGROUND: Malaria was eliminated from Sri Lanka in 2013. However, the influx of infected travelers and the presence of potent anopheline vectors can re-initiate transmission in Jaffna city, which is separated by a narrow strait from the malaria-endemic Indian state of Tamil Nadu. METHODS: Anopheline larvae were collected from different habitats in Jaffna city and the susceptibility of emergent adults to DDT, malathion and deltamethrin investigated. RESULTS: Anopheline larvae were found in wells, surface-exposed drains, ponds, water puddles and water storage tanks, with many containing polluted, alkaline and brackish water. Anopheles culicifacies, An. subpictus, An. stephensi and An. varuna were identified in the collections. Adults of the four anopheline species were resistant to DDT. Anopheles subpictus and An. stephensi were resistant while An. culicifacies and An. varuna were possibly resistant to deltamethrin. Anopheles stephensi was resistant, An. subpictus possibly resistant while An. varuna and An. culicifacies were susceptible to malathion. DNA sequencing showed a L1014F (TTA to TTC) mutation in the IIS6 transmembrane segment of the voltage-gated sodium channel protein in deltamethrin-resistant An. subpictus-a mutation previously observed in India but not Sri Lanka. CONCLUSION: Anopheles subpictus in Jaffna, like An. stephensi, may have recently originated in coastal Tamil Nadu. Besides infected overseas travelers, wind- and boat-borne carriage of Plasmodium-infected anophelines across the Palk Strait can potentially reintroduce malaria transmission to Jaffna city. Adaptation to diverse larval habitats and resistance to common insecticides in anophelines are identified as potential problems for vector control should this happen.

Development of the major arboviral vector Aedes aegypti in urban drain-water and associated pyrethroid insecticide resistance is a potential global health challenge.

BACKGROUND: Aedes aegypti were found developing in the water in open public drains (drain-water, DW) in Jaffna city in northern Sri Lanka, a location where the arboviral diseases dengue and chikungunya are endemic. METHODS: Susceptibilities to the common insecticides dichlorodiphenyltrichloroethane (DDT), malathion, propoxur, permethrin and deltamethrin and activities of the insecticide-detoxifying enzymes carboxylesterase (EST), glutathione S-transferase (GST) and monooxygenase (MO) were compared in adult Ae. aegypti developing in DW and fresh water (FW). RESULTS: DW Ae. aegypti were resistant to the pyrethroids deltamethrin and permethrin, while FW Ae. aegypti were susceptible to deltamethrin but possibly resistant to permethrin. Both DW and FW Ae. aegypti were resistant to DDT, malathion and propoxur. Greater pyrethroid resistance in DW Ae. aegypti was consistent with higher GST and MO activities. CONCLUSIONS: The results demonstrate the potential for insecticide resistance developing in Ae. aegypti adapted to DW. Urbanization in arboviral disease-endemic countries is characterized by a proliferation of open water drains and therefore the findings identify a potential new challenge to global health.

Anthropogenic Factors Driving Recent Range Expansion of the Malaria Vector Anopheles stephensi.

The malaria vector Anopheles stephensi is found in wide tracts of Asia and the Middle East. The discovery of its presence for the first time in the island of Sri Lanka in 2017, poses a threat of malaria resurgence in a country which had eliminated the disease in 2013. Morphological and genetic characterization showed that the efficient Indian urban vector form An. stephensi sensu stricto or type form, has recently expanded its range to Jaffna and Mannar in northern Sri Lanka that are in proximity to Tamil Nadu state in South India. Comparison of the DNA sequences of the cytochrome oxidase subunit 1 gene in An. stephensi in Jaffna and Mannar in Sri Lanka and Tamil Nadu and Puducherry states in South India showed that a haplotype that is due to a sequence change from valine to methionine in the cytochrome oxidase subunit 1 present in the Jaffna and Mannar populations has not been documented so far in Tamil Nadu/Puducherry populations. The Jaffna An. stephensi were closer to Tamil Nadu/Puducherry populations and differed significantly from the Mannar populations. The genetic findings cannot differentiate between separate arrivals of the Jaffna and Mannar An. stephensi from Tamil Nadu or a single arrival and dispersion to the two locations accompanied by micro-evolutionary changes. Anopheles stephensi was observed to undergo preimaginal development in fresh and brackish water domestic wells and over ground cement water storage tanks in the coastal urban environment of Jaffna and Mannar. Anopheles stephensi in Jaffna was resistant to the common insecticides deltamethrin, dichlorodiphenyltrichloroethane and Malathion. Its preimaginal development in wells and water tanks was susceptible to predation by the larvivorous guppy fish Poecilia reticulata. The arrival, establishment, and spread of An. stephensi in northern Sri Lanka are analyzed in relation to anthropogenic factors that favor its range expansion. The implications of the findings for global public health challenges posed by malaria and other mosquito-borne diseases are discussed.

Susceptibility to common insecticides and detoxifying enzyme activities in Anopheles sundaicus (sensu lato) after cessation of indoor residual spraying of insecticides in the Jaffna Peninsula and its surroundings in northern Sri Lanka.

BACKGROUND: Sri Lanka has been malaria-free since 2013 but re-introduction of malaria transmission by infected overseas travelers is possible due to a prevalence of potent malaria vectors. Knowledge of the insecticide resistance status among Anopheles vectors is important if vector control has to be reintroduced in the island. The present study investigated the insecticide susceptibility levels and resistance mechanisms of Anopheles sundaicus (sensu lato) (previously classified as Anopheles subpictus species B) an important malaria vector in the Jaffna Peninsula and it surroundings in northern Sri Lanka after indoor residual spraying of insecticides was terminated in 2013. RESULTS: Species-specific PCR assays identified An. sundaicus (s.l.) in four locations in the Jaffna and adjacent Kilinochchi districts. Bioassays confirmed that An. sundaicus (s.l.) collected in Kilinochchi were completely susceptible to 0.05% deltamethrin and 5% malathion and resistant to 4% dichlorodiphenyltrichloroethane (DDT), whereas those from Jaffna were relatively susceptible to all three insecticides. Kilinochchi populations of An. sundaicus (s.l.) showed significantly higher glutathione S-transferase activity than population from Jaffna. However, Jaffna An. sundaicus (s.l.) had significantly higher Propoxur-resistant acetylcholinesterase activity. Activities of non-specific esterases and monooxygenases were not significantly elevated in An. sundaicus (s.l.) collected in both districts. CONCLUSIONS: The susceptibility to malathion and deltamethrin in An. sundaicus (s.l.) suggests that they can be still used for controlling this potential malaria vector in the Jaffna Peninsula and adjacent areas. Continuing country-wide studies on other malaria vectors and their insecticide susceptibilities are important in this regard.

Genotype and biotype of invasive Anopheles stephensi in Mannar Island of Sri Lanka.

BACKGROUND: Anopheles stephensi, the major vector of urban malaria in India, was recently detected for the first time in Sri Lanka in Mannar Island on the northwestern coast. Since there are different biotypes of An. stephensi with different vector capacities in India, a study was undertaken to further characterise the genotype and biotype of An. stephensi in Mannar Island. METHODS: Mosquito larvae were collected in Pesalai village in Mannar and maintained in the insectary until adulthood. Adult An. stephensi were identified morphologically using published keys. Identified adult An. stephensi were molecularly characterized using two mitochondrial (cox1 and cytb) and one nuclear (ITS2) markers. Their PCR-amplified target fragments were sequenced and checked against available sequences in GenBank for phylogenetic analysis. The average spiracular and thoracic lengths and the spiracular index were determined to identify biotypes based on corresponding indices for Indian An. stephensi. RESULTS: All DNA sequences for the Mannar samples matched reported sequences for An. stephensi from the Middle East and India. However, a single nucleotide variation in the cox1 sequence suggested an amino acid change from valine to methionine in the cox1 protein in Sri Lankan An. stephensi. Morphological data was consistent with the presence of the Indian urban vector An. stephensi type-form in Sri Lanka. CONCLUSIONS: The present study provides a more detailed molecular characterization of An. stephensi and suggests the presence of the type-form of the vector for the first time in Sri Lanka. The single mutation in the cox1 gene may be indicative of a founder effect causing the initial diversification of An. stephensi in Sri Lanka from the Indian form. The distribution of the potent urban vector An. stephensi type-form needs to be established by studies throughout the island as its spread adds to the challenge of maintaining the country's malaria-free status.

Population genomics reveals that an anthropophilic population of Aedes aegypti mosquitoes in West Africa recently gave rise to American and Asian populations of this major disease vector.

BACKGROUND: The mosquito Aedes aegypti is the main vector of dengue, Zika, chikungunya and yellow fever viruses. This major disease vector is thought to have arisen when the African subspecies Ae. aegypti formosus evolved from being zoophilic and living in forest habitats into a form that specialises on humans and resides near human population centres. The resulting domestic subspecies, Ae. aegypti aegypti, is found throughout the tropics and largely blood-feeds on humans. RESULTS: To understand this transition, we have sequenced the exomes of mosquitoes collected from five populations from around the world. We found that Ae. aegypti specimens from an urban population in Senegal in West Africa were more closely related to populations in Mexico and Sri Lanka than they were to a nearby forest population. We estimate that the populations in Senegal and Mexico split just a few hundred years ago, and we found no evidence of Ae. aegypti aegypti mosquitoes migrating back to Africa from elsewhere in the tropics. The out-of-Africa migration was accompanied by a dramatic reduction in effective population size, resulting in a loss of genetic diversity and rare genetic variants. CONCLUSIONS: We conclude that a domestic population of Ae. aegypti in Senegal and domestic populations on other continents are more closely related to each other than to other African populations. This suggests that an ancestral population of Ae. aegypti evolved to become a human specialist in Africa, giving rise to the subspecies Ae. aegypti aegypti. The descendants of this population are still found in West Africa today, and the rest of the world was colonised when mosquitoes from this population migrated out of Africa. This is the first report of an African population of Ae. aegypti aegypti mosquitoes that is closely related to Asian and American populations. As the two subspecies differ in their ability to vector disease, their existence side by side in West Africa may have important implications for disease transmission.

Mosquito vectors developing in atypical anthropogenic habitats: Global overview of recent observations, mechanisms and impact on disease transmission.

The major mosquito vectors of human diseases have co-evolved with humans over a long period of time. However, the rapid growth in human population and the associated expansion in agricultural activity and greater urbanisation have created ecological changes that have had a marked impact on biology of mosquito vectors. Adaptation of the vectors of malaria and important arbovial diseases over a much shorter time scale to the new types of preimaginal habitats recently created by human population growth and activity is highlighted here in the context of its potential for increasing disease transmission rates. Possible measures that can reduce the effects on the transmission of mosquito-borne diseases are also outlined.

DNA barcoding of Sri Lankan phlebotomine sand flies using cytochrome c oxidase subunit I reveals the presence of cryptic species.

Sri Lanka is known for high diversity of phlebotomine sand flies and prevalence of cutaneous and visceral leishmaniasis; a disease vectored by sand flies. The taxonomy of phlebotomine sand flies is complicated and often the diversity is over/underrated. The current study aims to use the cytochrome c oxidase gene subunit 1 (COI) sequence and formulate a barcode for the sand fly species in Sri Lanka. A total of 70 samples comprising seven species morphologically identified and collected from dry zone districts of Hambantota, Anuradhapura, Vavuniya, Trincomalee and Jaffna were processed. Neighbour-joining (NJ) tree created using the sequences revealed the species identity is compatible with the current morphology based identification. Further the analysis delineated morphologically identified Se. bailyi, Se babu babu and Se babu insularis into genetically distinct groups.

Karyotypic assignment of Sri Lankan Anopheles culicifacies species B and E does not correlate with cytochrome oxidase subunit I and microsatellite genotypes.

BACKGROUND: The identification of species B and E in the Anopheles culicifacies complex in the Indian subcontinent has been based on Y-chromosome karyotype. Since no detectable variations were previously found in DNA markers commonly used for sibling species identification, further molecular characterization using cytochrome oxidase subunit I (COI) and microsatellite markers was carried out on Y-chromosome karyotyped Anopheles culicifacies specie B and E from Unnichchai, Kallady and Ranawarunawa in Sri Lanka. FINDINGS: COI sequence analysis (n = 22) revealed the presence of nine unique haplotypes with six in each species. Three haplotypes were shared by both species. The two sibling species had a pairwise FST value of 1.338 (p < 0.05) with the number of migrants (Nm) value <1. The genetic structure analysis resulted in two genetic clusters not 100% associated with karyotypes. While none of the species B were incorrectly assigned two were inconclusive. Five out of 26 specimens karyotyped as species E were incorrectly assigned, while further 9 were inconclusive. CONCLUSIONS: The new molecular data support the existence of two genetically different populations of the Culicifacies Complex in Sri Lanka that are not associated with the Y-chromosome karyotype. Detailed analysis with more microsatellite markers and assortative mating experiments are needed to establish the presence of the two genetically distinct populations and relate them to Y-chromosome morphology.

Bionomic aspects of the Anopheles subpictus species complex in Sri Lanka.

Anopheles subpictus Grassi s.l. (Diptera: Culicidae) functions as a secondary malaria vector to Anopheles culicifacies Giles s.l. (Diptera: Culicidae) in Sri Lanka. The taxon A. subpictus is reported to exist as a species complex comprising four sibling species (A-D) that can be differentiated through polytene chromosome banding patterns and stage-specific morphometric traits in India. Based on the morphological characteristics described for the Indian Subpictus Complex, the presence of all four sibling species has been described in Sri Lanka. As sibling species show distinct bio-ecological characteristics that are important for devising appropriate vector control measures, a study was carried out in six districts in the dry zone of Sri Lanka. The results confirm the presence of all four sibling species, with species C predominating in inland areas and species B in coastal areas. Species C and D were indoor-resting and indoor-feeding, while species B was outdoor-resting with no significant preference for indoor- or outdoor-resting. Species B showed distinct morphological variation in the ornamentation of wings and palpi. Blood meal analysis revealed that species B, C, and D can feed on humans as well as cattle. The differential bio-ecological traits shown by the members of the Subpictus Complex are important for developing appropriate vector control measures in Sri Lanka.

Biological differences between brackish and fresh water-derived Aedes aegypti from two locations in the Jaffna peninsula of Sri Lanka and the implications for arboviral disease transmission.

The mainly fresh water arboviral vector Aedes aegypti L. (Diptera: Culicidae) can also undergo pre-imaginal development in brackish water of up to 15 ppt (parts per thousand) salt in coastal areas. We investigated differences in salinity tolerance, egg laying preference, egg hatching and larval development times and resistance to common insecticides in Ae. aegypti collected from brackish and fresh water habitats in Jaffna, Sri Lanka. Brackish water-derived Ae. aegypti were more tolerant of salinity than fresh water-derived Ae. aegypti and this difference was only partly reduced after their transfer to fresh water for up to five generations. Brackish water-derived Ae. aegypti did not significantly discriminate between 10 ppt salt brackish water and fresh water for oviposition, while fresh water-derived Ae. aegypti preferred fresh water. The hatching of eggs from both brackish and fresh water-derived Ae. aegypti was less efficient and the time taken for larvae to develop into pupae was prolonged in 10 ppt salt brackish water. Ae. aegypti isolated from coastal brackish water were less resistant to the organophosphate insecticide malathion than inland fresh water Ae. aegypti. Brackish and fresh water-derived Ae. aegypti however were able to mate and produce viable offspring in the laboratory. The results suggest that development in brackish water is characterised by pertinent biological changes, and that there is restricted genetic exchange between coastal brackish and inland fresh water Ae. aegypti isolates from sites 5 km apart. The findings highlight the need for monitoring Ae. aegypti developing in coastal brackish waters and extending vector control measures to their habitats.

Molecular characterization of the malaria vector Anopheles barbirostris van der Wulp in Sri Lanka.

BACKGROUND: Anopheles barbirostris is a vector of malaria in Sri Lanka. The taxon exists as a species complex in the Southeast Asian region. Previous studies using molecular markers suggest that there are more than 4 distinct clades within the An. barbirostris complex in Southeast Asia. The present study characterizes Sri Lankan An. barbirostris using mtDNA cytochrome oxidase subunit I (COI) and ribosomal RNA internal transcribed spacer 2 (ITS2) gene sequences. FINDINGS: DNA was extracted from morphologically identified An. barbirostris specimens from Sri Lanka, the COI and ITS2 regions amplified and their sequences analysed by comparison with other GenBank entries. Maximum likelihood trees suggested that Sri Lankan An. barbirostris constitute a different molecular type most closely related to clade I. CONCLUSIONS: Considering the uncorrected p distances between the clade I and Sri Lankan specimens it is fair to assume that the specimens collected from widely separated locations in Sri Lanka with morphology characteristic of An. barbirostris s.l. form a new molecular type with close resemblance to An. barbirostris s.s from Indonesia and Thailand.