Microsatellite-based analysis reveals Aedes aegypti populations in the Kingdom of Saudi Arabia result from colonization by both the ancestral African and the global domestic forms.

The Aedes aegypti (Linnaeus, 1762) mosquito is the main vector of dengue, chikungunya and Zika and is well established today all over the world. The species comprises two forms: the ancestral form found throughout Africa and a global domestic form that spread to the rest of the tropics and subtropics. In Saudi Arabia, A. aegypti has been known in the southwest since 1956, and previous genetic studies clustered A. aegypti from Saudi Arabia with the global domestic form. The purpose of this study was to assess the genetic structure of A. aegypti in Saudi Arabia and determine their geographic origin. Genetic data for 17 microsatellites were collected for A. aegypti ranging from the southwestern highlands of Saudi Arabia on the border of Yemen to the north-west in Madinah region as well as from Thailand and Uganda populations (as representatives of the ancestral African and global domestic forms, respectively). The low but significant level of genetic structuring in Saudi Arabia was consistent with long-distance dispersal capability possibly through road connectivity and human activities, that is, passive dispersal. There are two main genetic groupings in Saudi Arabia, one of which clusters with the Ugandan population and the other with the Thailand population with many Saudi Arabian individuals having mixed ancestry. The hypothesis of genetic admixture of the ancestral African and global domestic forms in Saudi Arabia was supported by approximate Bayesian computational analyses. The extent of admixture varied across Saudi Arabia. African ancestry was highest in the highland area of the Jazan region followed by the lowland Jazan and Sahil regions. Conversely, the western (Makkah, Jeddah and Madinah) and Najran populations corresponded to the global domesticated form. Given potential differences between the forms in transmission capability, ecology and behaviour, the findings here should be taken into account in vector control efforts in Saudi Arabia.

Characterisation of Anopheles species composition and genetic diversity in Meghalaya, northeast India, using molecular identification tools.

Malaria in India is declining, in part due to the use of long-lasting insecticide-treated nets (LLINs) and vector control. Historically, the north-eastern region of India has contributed ~10%-12% of the nation's malaria burden. The important mosquito vectors in northeast India have long been considered to be Anopheles baimaii and An. minimus, both associated with forest habitats. Local deforestation and increased rice cultivation, along with widespread LLIN use, may be changing vector species composition. Understanding if and how vector species composition is changing is critical to successful malaria control. In Meghalaya state, malaria is now at a low level of endemicity with occasional seasonal outbreaks. In a biodiverse setting like Meghalaya, where >24 Anopheles mosquito species have been recorded, accurate morphological identification of all species is logistically challenging. To accurately determine Anopheles species richness in the West Khasi Hills (WKH) and West Jaintia Hills (WJH) districts, adult and larval mosquitoes were collected and identified using molecular methods of allele-specific PCR and cytochrome oxidase I DNA barcoding. In 14 villages across both districts, we identified high species richness, 19 species in total. Molecular findings indicated that An. minimus and An. baimaii were rare, while four other species (An. maculatus, An. pseudowillmori, An. jeyporiensis and An. nitidus) were abundant. Anopheles maculatus was highly prevalent in WKH (39% of light trap collections) and An. pseudowillmori in WJH (45%). Larvae of these four species were found in rice fields, suggesting that land cover change is influencing species composition change. Our results suggest that rice fields might be contributing to the observed abundance of An. maculatus and An. pseudowillmori, which could be playing a role in malaria transmission, either independently due to their high abundance, or in combination with An. baimaii and/or An. minimus.

The Impact, Emerging Needs, and New Research Questions Arising from 12 Years of the Center for the Study of Complex Malaria in India.

The Center for the Study of Complex Malaria in India (CSCMi) was launched in 2010 with the overall goal of addressing major gaps in our understanding of "complex malaria" in India through projects on the epidemiology, transmission, and pathogenesis of the disease. The Center was mandated to adopt an integrated approach to malaria research, including building capacity, developing infrastructure, and nurturing future malaria leaders while conducting relevant and impactful studies to assist India as it moves from control to elimination. Here, we will outline some of the interactions and impacts the Center has had with malaria policy and control counterparts in India, as well as describe emerging needs and new research questions that have become apparent over the past 12 years.

Molecular analysis of knockdown resistance (kdr) mutations in the voltage-gated sodium channel gene of Aedes aegypti populations from Saudi Arabia.

BACKGROUND: The Aedes aegypti mosquito is the primary vector for dengue, chikungunya, yellow fever and Zika viruses worldwide. The first record of Ae. aegypti in southwestern Saudi Arabia was in 1956. However, the first outbreak and cases of dengue fever were reported in 1994, and cases have increased in recent years. Vector control for Ae. aegypti mainly uses pyrethroid insecticides in outdoor and indoor space spraying. The constant use of pyrethroids has exerted intense selection pressure for developing target-site mutations in the voltage-gated sodium channel (vgsc) gene in Ae. Aegypti against pyrethroids-mutations that have led to knockdown resistance (kdr). METHODS: Aedes aegypti field populations from five regions (Jazan, Sahil, Makkah, Jeddah and Madinah) of southwestern Saudi Arabia were genotyped for known kdr mutations in domains IIS6 and IIIS6 of the vgsc gene using polymerase chain reaction (PCR) amplification and sequencing. We estimated the frequency of kdr mutations and genotypes from Saudi Arabia as well as from other countries, Thailand, Myanmar (Southeast Asia) and Uganda (East Africa). We constructed haplotype networks to infer the evolutionary relationships of these gene regions. RESULTS: The three known kdr mutations, S989P, V1016G (IIS6) and F1534C (IIIS6), were detected in all five regions of Saudi Arabia. Interestingly, the triple homozygous wild genotype was reported for the first time in two individuals from the highlands of the Jazan region and one from the Al-Quoz, Sahil region. Overall, nine genotypes comprising four haplotypes were observed in southwestern Saudi Arabia. The median-joining haplotype networks of eight populations from Saudi Arabia, Southeast Asia and East Africa for both the IIS6 and IIIS6 domains revealed that haplotype diversity was highest in Uganda and in the Jazan and Sahil regions of Saudi Arabia, whereas haplotype diversity was low in the Jeddah, Makkah and Madinah regions. Median-joining haplotype networks of both domains indicated selection acting on the kdr-mutation containing haplotypes in Saudi Arabia. CONCLUSIONS: The presence of wild type haplotypes without any of the three kdr mutations, i.e. that are fully susceptible, in Saudi Arabia indicates that further consideration should be given to insecticide resistance management strategies that could restore pyrethroid sensitivity to the populations of Ae. aegypti in Saudi Arabia as part of an integrative vector control strategy.

Novel real-time PCR assay detects widespread distribution of knock down resistance (kdr) mutations associated with pyrethroid resistance in the mosquito, Culex quinquefasciatus, in Thailand.

Susceptibility to pyrethroids in the mosquito Culex quinquefasciatus, the major vector of lymphatic filariasis, is being seriously threatened worldwide. Knockdown resistance (kdr), caused by mutations in the voltage gated sodium channel (VGSC) gene, particularly the L1014F mutation, is an important resistance mechanism. Our aim was to develop a real-time PCR with melt curve analysis to evaluate the distribution of the L1014F mutation in Cx. quinquefasciatus throughout Thailand and to determine the polymorphism pattern of a VGSC gene fragment spanning the L1014F mutation. A total of 3760 females from 18 localities across five regions of Thailand were bio-assayed by exposure to 0.05% deltamethrin WHO papers, showing mortality rates ranging from 2.4% to 83.0%. Genotyping of 753 dead and surviving mosquitoes using our novel real-time PCR assay with melt curve analysis and tetra-primer allele-specific PCR revealed the mutant F1014 allele is closely associated with the deltamethrin resistance phenotype. The L1014F mutation was found at high frequency throughout Thailand, particularly in the North. However, some survivors were homozygous for wild type L1014 allele, which were further sequenced for the IIP-IIS6 region of VGSC gene. The haplotype network of phenotypically characterized individuals indicated the presence of other possible kdr alleles/resistance mechanisms at play including two novel mutations, V978E and D992E. The finding of new putative kdr alleles and widespread distribution of the F1014 allele emphasizes the significant role of kdr mutations in pyrethroid resistance in Thai Cx. quinquefasciatus populations. Monitoring kdr variations and phenotypic resistance is critical for managing resistance in Cx. quinquefasciatus.

Spatial and temporal village-level prevalence of Plasmodium infection and associated risk factors in two districts of Meghalaya, India.

BACKGROUND: Despite declining incidence over the past decade, malaria remains an important health burden in India. This study aimed to assess the village-level temporal patterns of Plasmodium infection in two districts of the north-eastern state of Meghalaya and evaluate risk factors that might explain these patterns. METHODS: Primary Health Centre passive malaria case data from 2014 to 2018 were analysed to characterize village-specific annual incidence and temporal trends. Active malaria case detection was undertaken in 2018 and 2019 to detect Plasmodium infections using PCR. A questionnaire collected socio-demographic, environmental, and behavioural data, and households were spatially mapped via GPS. Adult mosquitoes were sampled at a subset of subjects' houses, and Anopheles were identified by PCR and sequencing. Risk factors for Plasmodium infection were evaluated using bivariate and multivariate logistic regression analysis, and spatial cluster analysis was undertaken. RESULTS: The annual malaria incidence from PHC-based passive surveillance datasets in 2014-2018 was heterogenous but declining across villages in both districts. Active surveillance in 2018 enrolled 1468 individuals from 468 households (West Jaintia Hills) and 1274 individuals from 359 households (West Khasi Hills). Plasmodium falciparum prevalence per 100 people varied from 0 to 4.1% in the nine villages of West Jaintia Hills, and from 0 to 10.6% in the 12 villages of West Khasi Hills. Significant clustering of P. falciparum infections [observed = 11, expected = 2.15, Relative Risk (RR) = 12.65; p < 0.001] was observed in West Khasi Hills. A total of 13 Anopheles species were found at 53 houses in five villages, with Anopheles jeyporiensis being the most abundant. Risk of infection increased with presence of mosquitoes and electricity in the households [Odds Ratio (OR) = 1.19 and 1.11], respectively. Households with reported animals had reduced infection risk (OR = 0.91). CONCLUSION: Malaria incidence during 2014-2018 declined in all study villages covered by the passive surveillance data, a period that includes the first widespread insecticide-treated net campaign. The survey data from 2018 revealed a significant association between Plasmodium infection and certain household characteristics. Since species of Plasmodium-competent mosquito vectors continue to be abundant, malaria resurgence remains a threat, and control efforts should continue.

The current state of training in psychiatry of intellectual disability: perspectives of trainees and trainers.

AIMS AND METHOD: Twelve intellectual disability psychiatry trainee representatives and 13 training programme directors were surveyed to assess the current state of training, to establish what motivated specialty trainees to choose intellectual disability psychiatry, and to explore issues that might affect retention. RESULTS: The combined survey response rate was 83%. All trainees had chosen intellectual disability psychiatry after experience in either their personal or working life. Overall, specialty trainees were satisfied with their training; the majority felt supported to meet training requirements. Trainee isolation was the main concern for current trainees. CLINICAL IMPLICATIONS: Recruitment for specialty training in intellectual disability psychiatry is acknowledged to be a concern for workforce planning and could affect access to and quality of psychiatric care for people with intellectual disability. The results of this survey could be used as a guide to improve efforts to attract trainees. Acknowledging and reducing trainee isolation could improve trainee morale.

A Multiplex PCR Based on Mitochondrial COI Sequences for Identification of Members of the Anopheles barbirostris Complex (Diptera: Culicidae) in Thailand and Other Countries in the Region.

A multiplex-PCR assay based on mitochondrial cytochrome c oxidase subunit I (COI) sequences was developed for identification of five members of the Barbirostris Complex which occur in Thailand: Anopheles barbirostris s.s., An. dissidens, An. saeungae, An. wejchoochotei and An. barbirostris species A3. Anopheles campestris was not included in the assay due to the lack of unequivocal sequences. Allele-specific primers were designed for specific nucleotide segments of COI sequences of each species. Mismatch method and addition of long GC tail were applied for some primers. The assay provided products of 706 bp for An. barbirostris s.s., 238 bp for An. dissidens, 611 bp for An. saeungae, 502 bp for An. wejchoochotei and 365 bp for An. barbirostris A3. The assay was tested using 111 wild-caught female mosquitoes from Bhutan, Cambodia, Indonesia (Sulawesi) and Thailand. The results of the multiplex PCR were in complete agreement with COI sequencing; however, one of three specimens from Bhutan and all 11 specimens from Indonesia were not amplifiable by the assay due to their distinct COI sequences. This, together with the distinct rDNA sequences of these specimens, suggests the presence of at least two additional new species in the Barbirostris Complex.

Integrated systematics of Anopheles subpictus (Diptera: Culicidae) in the Oriental Region, with emphasis on forms in Thailand and Sulawesi, Indonesia.

The Anopheles subpictus complex consists of four species informally designated, based on fixed inversions of polytene chromosomes and morphology, as species A, B, C and D in India. However, recent studies revealed the presence of only species A and B in Sri Lanka. Little is known about the specific identity of the taxon in other countries in Asia. This paper reports the results of a molecular and morphological study of An. subpictus in Thailand and South Sulawesi, Indonesia. The maxillary palpi of most females from Thailand have the apical pale band longer than the subapical dark band, seta 7-I of pupae branched and short, and eggs with 18-25 float ridges. These characters do not agree with those described for species A, B, C and D in India. The females of An. subpictus from South Sulawesi usually have the subapical dark band of the maxillary palpus equal in length to the apical pale band. Phylogenetic analyses of sequences of the internal transcribed spacer 2 (ITS2) region of rDNA and the cytochrome c oxidase subunit I (COI) gene of mtDNA of specimens from Thailand, and South Sulawesi, and from various localities in GenBank, were conducted. ITS2 sequences of specimens from all localities in Thailand were identical, except for a small divergence in specimen from Phang Nga Province. Three distinct COI clades were detected in specimens from Chiang Mai Province in northern Thailand. However, crossing experiments between the three clades revealed no genetic incompatibility, suggesting that they were conspecific. ITS2 and COI sequences of most specimens from Thailand fell in clades other than those of An. subpictus species A and B and An. subpictus from Indonesia (East Nusa Tenggara, Java, South Sulawesi) and the Philippines. ITS2 sequences from South Sulawesi and East Nusa Tenggara were very similar, and fell in a clade consisting of specimen from Phang Nga in southern Thailand and sequences of some specimens from Cambodia and Vietnam, but their COI sequences were distinct. DNA sequences and morphological differences suggest the presence of two species within An. subpictus in Thailand, and more than one species in Indonesia.

Investigation of Relative Development and Reproductivity Fitness Cost in Three Insecticide-Resistant Strains of Aedes aegypti from Thailand.

Knockdown resistance (kdr) and detoxification enzymes are major resistance mechanisms in insecticide-resistant Aedes aegypti throughout the world. Persistence of the resistance phenotype is associated with high fitness of resistance alleles in the absence of insecticide pressure. This study determined the relative fitness cost of three insecticide-resistant strains of Aedes aegypti-PMD, PMD-R, and UPK-R-and a hybrid under similar laboratory conditions in the absence of insecticide. The PMD strain is resistant to DDT with no kdr alleles; the PMD-R is resistant to DDT and permethrin with 1534C homozygous kdr alleles; and UPK-R is resistant to DDT, permethrin, and deltamethrin with 989P + 1016G homozygous alleles. The DDT-resistant PMD strain had the highest fitness compared with the two DDT/pyrethroid-resistant strains (PMD-R and UPK-R) and hybrid. Consistent fitness costs were observed in the DDT/pyrethroid-resistant strains and hybrid, including shorter wing length, reduced egg hatchability, shorter female lifespan, and shorter viability of eggs after storage, whereas no effect was observed on blood feeding rate. In addition, reduced egg production was observed in the PMD-R strain and prolonged developmental time was seen in the UPK-R strain. The corresponding hybrid that is heterozygous for kdr alleles was fitter than either of the homozygous mutant genotypes. This is in accordance with the high frequency of heterozygous genotypes observed in natural populations of Ae. aegypti in Chiang Mai city.

Molecular identification of mosquitoes of the Anopheles maculatus group of subgenus Cellia (Diptera: Culicidae) in the Indonesian Archipelago.

This study reports the molecular differentiation of females of Anopheles maculatus s.l. collected in eight localities on five islands in the Indonesian Archipelago: Hargowilis and Hargotirto villages of Central Java Province, North Kalimantan Province, Sabang off the northern tip of Sumatra Province, Sumba Island of East Nusa Tenggara Province and Sulawesi Province. Analyses based on rDNA (ITS2 and D3) and mtDNA (COII) sequences revealed the presence of An. greeni for the first time in North Kalimantan, and at least one novel (previously unrecognized) species of the Maculatus Group in Central Java (Hargowilis). Despite the similarity of rDNA markers of specimens of An. maculatus s.l. from Central Java and Sulawesi, their COII sequences are highly divergent (3.3%), which might indicate the presence of a further new species. Specimens of An. maculatus s.l. from the other localities had identical rDNA sequences to most An. maculatus s.s. from mainland Southeast Asia, but moderate divergence in their COII sequences (1.2-2.1%). The latter might indicate there are further novel species within the Maculatus Complex. However, as the divergence at COII may be the result of geographical structuring within species related to the historical biogeography of the region, further studies are needed to shed light on this possibility.

Morphological and molecular evidence for a new species of Lutzia (Diptera: Culicidae: Culicini) from Thailand.

We found a species of LutziaTheobald (1903) (Diptera: Culicidae) in Chiang Mai Province and other provinces in northern Thailand which bears morphological and DNA sequence differences from the three species of Lutzia, subgenus Metalutzia Tanaka, previously recorded from Thailand, namely Lt. fuscana (Wiedemann), Lt. halifaxii (Theobald) and Lt. vorax Edwards. The adults of the Chiang Mai form (CM form) have abdominal banding patterns similar to those of Lt. vorax from Japan (which includes the type locality of this species), but differ in having the mediocubital crossvein usually positioned before rather than beyond the radiomedial crossvein. The thoracic and abdominal integument of Lt. vorax larvae is covered with relatively short pointed spicules whereas it is covered by denser, longer and more acutely pointed spicules in the CM form. Some differences are also found in the development of thoracic seta 1-M, and abdominal setae 8-II and 8-III. The pupa of the CM form clearly differs from the pupa of Lt. vorax in having setae 1 and 5 of abdominal segments V and VI branched rather than single. The characters of the wing, larva and pupa of the CM form are similar to those of Lt. fuscana and Lt. halifaxii. However, whereas the phallosome of males of the CM form is similar to males of Lt. vorax and Lt. halifaxii, it is distinct from males of Lt. fuscana. Phylogenetic analysis of mitochondrial cytochrome c oxidase subunits I and II sequences revealed that the CM form falls outside a clade comprised of specimens of Lt. vorax from Japan, Korea, Thailand and Bhutan (Kimura 2-parameter, K2P, genetic distances 3.9-5.6, and 5.1-6.6, respectively). However, the two gene sequences of the CM form are not clearly distinct from clades comprised of sequences from specimens of Lt. fuscana and Lt. halifaxii (K2P 0.2-2.4%). However, based on the combination of morphological and molecular data, the current study provides evidence that the CM form is a previously unrecognized species of the genus Lutzia.

Malaria in Meghalaya: a systematic literature review and analysis of data from the National Vector-Borne Disease Control Programme.

BACKGROUND: Meghalaya, one of eight states in the northeastern region of India, has been reported to carry a high malaria burden. However, malaria surveillance, epidemiology, and vector studies are sparse, and no reviews combining these topics with malaria prevention and control strategies have been published in recent years. Furthermore, no analysis of surveillance data has been published documenting the changes in epidemiology following the first distribution of long-lasting insecticidal nets (LLINs) statewide in 2016. METHODS: A hybrid approach was used to describe the status of malaria in Meghalaya. First, a literature search was performed using the terms 'malaria' and 'Meghalaya'. Second, data were obtained from the Meghalaya State Malaria Control Programme for 2006-2017 for analysis of trends. Data from 3 years 2015-2017 were analysed further by district and year to assess changes in malaria incidence and distribution following the introduction of LLINs. RESULTS/CONCLUSIONS: Like malaria in mainland India, malaria in Meghalaya is complex, with both Plasmodium falciparum and Plasmodium vivax parasites in circulation, multiple Anopheles vector species, and reports of both unusual and severe malaria syndromes across all age groups. Integrated statewide malaria epidemiology, vector, and prevention and control data for Meghalaya are not readily available, and published studies are largely focused on a single topic or a single district or region of the state. Although malaria prevention and control approaches are available, (e.g. spraying, LLINs, personal repellents), their use and effectiveness is also not well characterized in the literature. Analysis of state malaria control programme data indicates that case incidence and related fatalities in Meghalaya have declined over the last decade. This could be attributed to changes in treatment guidelines and/or statewide distribution of effective prevention methods such as LLINs. Since the distribution of more than 900,000 LLINs in 2016, the malaria caseload has declined significantly in most Meghalaya districts, excluding the remote and geographically isolated South Garo Hills. Additionally, the proportion of adult malaria cases (15+ years of age versus children 0-14 years) in most districts was significantly greater following LLIN distribution, which likely reflects common lifestyle practices in these areas (e.g. adults working during night hours; small children in the households receiving priority for bed net protection). While reduction in malaria case incidence and related deaths is clear, the changes in malaria transmission and clinical manifestation have not been characterized. Routine epidemiology and vector surveillance combined with real-time data reporting are essential for the continued reduction and eventual elimination of malaria in Meghalaya.

Genetic diversity and population structure of malaria vector mosquitoes Anopheles subpictus, Anopheles peditaeniatus, and Anopheles vagus in five districts of Sri Lanka.

BACKGROUND: Although Sri Lanka is considered as a malaria-free nation, the threat of re-emergence of outbreaks still remains due to the high prevalence and abundance of malaria vectors. Analysis of population genetic structure of malaria vectors is considered to be one of the vital components in implementing successful vector control programmes. The present study was conducted to determine the population genetic structure of three abundant malaria vectors; Anopheles subpictus sensu lato (s.l.), Anopheles peditaneatus and Anopheles vagus from five administrative districts in two climatic zones; intermediate zone (Badulla and Kurunegala districts) and dry zone (Ampara, Batticoloa and Jaffna districts) of Sri Lanka using the mitochondrial gene, cytochrome c oxidase subunit I (COI). METHODS: Adult mosquitoes of An. subpictus s.l., An. peditaeniatus, and An. vagus were collected from five study sites located in five districts using cattle baited traps and backpack aspirators. Representative samples of each species that were morphologically confirmed were selected from each locality in generating COI sequences (> 6 good quality sequences per species per locality). RESULTS: Anopheles subpictus s.l. specimens collected during the study belonged to two sibling species; An. subpictus 'A' (from all study sites except from Jaffna) and An. subpictus 'B' (only from Jaffna). The results of haplotype and nucleotide diversity indices showed that all the three species are having high genetic diversity. Although a high significant pairwise difference was observed between An. subpictus 'A' and 'B' (Fst> 0.950, p < 0.05), there were no significant genetic population structures within An. peditaeniatus, An. vagus and An. subpictus species A (p > 0.05), indicating possible gene flow between these populations. CONCLUSIONS: Gene flow among the populations of An. peditaeniatus, An. vagus and An. subpictus species A was evident. Application of vector control measures against all mosquito species must be done with close monitoring since gene flow can assist the spread of insecticide resistance genes over a vast geographical area.

Comparative phylogeography of Aedes mosquitoes and the role of past climatic change for evolution within Africa.

The study of demographic processes involved in species diversification and evolution ultimately provides explanations for the complex distribution of biodiversity on earth, indicates regions important for the maintenance and generation of biodiversity, and identifies biological units important for conservation or medical consequence. African and forest biota have both received relatively little attention with regard to understanding their diversification, although one possible mechanism is that this has been driven by historical climate change. To investigate this, we implemented a standard population genetics approach along with Approximate Bayesian Computation, using sequence data from two exon-primed intron-crossing (EPIC) nuclear loci and mitochondrial cytochrome oxidase subunit I, to investigate the evolutionary history of five medically important and inherently forest dependent mosquito species of the genus Aedes. By testing different demographic hypotheses, we show that Aedes bromeliae and Aedes lilii fit the same model of lineage diversification, admixture, expansion, and recent population structure previously inferred for Aedes aegypti. In addition, analyses of population structure show that Aedes africanus has undergone lineage diversification and expansion while Aedes hansfordi has been impacted by population expansion within Uganda. This congruence in evolutionary history is likely to relate to historical climate-driven habitat change within Africa during the late Pleistocene and Holocene epoch. We find differences in the population structure of mosquitoes from Tanzania and Uganda compared to Benin and Uganda which could relate to differences in the historical connectivity of forests across the continent. Our findings emphasize the importance of recent climate change in the evolution of African forest biota.

Effect of Relaxation of Deltamethrin Pressure on Metabolic Resistance in a Pyrethroid-Resistant Aedes aegypti (Diptera: Culicidae) Strain Harboring Fixed P989P and G1016G kdr Alleles.

Mutation of the voltage-gated sodium channel genes or knockdown resistance (kdr) and metabolic resistance in Aedes aegypti (L.) (Diptera: Culicidae) are important resistance mechanisms against pyrethroids. The present study investigated the effect of relaxation of deltamethrin selection pressure on the level of mixed-function oxidases (MFO), when the allele frequency of S989P+V1016G mutations is fixed in a resistant Ae. aegypti strain (UPK-R) from Chiang Mai, Thailand. The mosquitoes were divided into two groups, exposure and nonexposure groups, and maintained for 12 generations in an insectary room. Adults of the exposure group (F3 to F12) were treated with 0.05% deltamethrin-impregnated papers. The median lethal concentrations (LC50) of deltamethrin of larvae were determined by World Health Organization (WHO) bioassay. MFO activity was determined in F0 and F12. The results revealed that there was a decreasing trend of adult mortality rates in the exposure group over time. The larval LC50 values of the exposure group were gradually increased, whereas those of the nonexposure group were gradually decreased. The level of MFO activity in the nonexposure group (F12) was lower than the parent and exposure groups (F12) by 1.5 and 4-fold in the larvae, respectively, and 1.5 and 2.5-fold in the adult females, respectively. However, the frequency of P989+G1016 alleles in both groups was 100% up to F12 when the experiment ended. This study indicates that there was a significant but small reduction in the activity levels of MFOs when pyrethroid selection pressure is relaxed in this kdr strain of Ae. aegypti.

A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

BACKGROUND: Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale. METHODS: A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR). RESULTS: The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory. CONCLUSIONS: Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

Molecular characterization of Anopheline (Diptera: Culicidae) mosquitoes from eight geographical locations of Sri Lanka.

BACKGROUND: Genus Anopheles is a major mosquito group of interest in Sri Lanka as it includes vectors of malaria and its members exist as species complexes. Taxonomy of the group is mainly based on morphological features, which are not conclusive and can be easily erased while handling the specimens. A combined effort, using morphology and DNA barcoding (using the markers cytochrome c oxidase subunit I (COI) gene and internal transcribed spacer 2 (ITS2) region, was made during the present study to recognize anophelines collected from eight districts of Sri Lanka for the first time. METHODS: Cytochrome c oxidase subunit I and ITS2 regions of morphologically identified anopheline mosquitoes from Sri Lanka were sequenced. These sequences together with GenBank sequences were used in phylogenetic tree construction and molecular characterization of mosquitoes. RESULTS: According to morphological identification, the field-collected adult mosquitoes belonged to 15 species, i.e., Anopheles aconitus, Anopheles annularis, Anopheles barbirostris, Anopheles culicifacies, Anopheles jamesii, Anopheles karwari, Anopheles maculatus, Anopheles nigerrimus, Anopheles pallidus, Anopheles peditaeniatus, Anopheles pseudojamesi, Anopheles subpictus, Anopheles tessellatus, Anopheles vagus, and Anopheles varuna. However, analysis of 123 COI sequences (445 bp) (16 clades supported by strong bootstrap value in the neighbour joining tree and inter-specific distances of >3%) showed that there are 16 distinct species. Identity of the morphologically identified species, except An. subpictus, was comparable with the DNA barcoding results. COI sequence analysis showed that morphologically identified An. subpictus is composed of two genetic entities: An. subpictus species A and species B (inter-specific K2P distance 0.128). All the four haplotypes of An. culicifacies discovered during the present study belonged to a single species. ITS2 sequences (542 bp) were obtained for all the species except for An. barbirostris, An. subpictus species B, An. tessellatus, and An. varuna. Each of these sequences was represented by a single species-specific haplotype. CONCLUSIONS: The present study reflects the importance and feasibility of COI and ITS2 genetic markers in identifying anophelines and their sibling species, and the significance of integrated systematic approach in mosquito taxonomy. Wide distribution of malaria vectors in the country perhaps indicates the potential for re-emergence of malaria in the country.

Culex (Culiciomyia) sasai (Diptera: Culicidae), senior synonym of Cx. spiculothorax and a new country record for Bhutan.

Culex (Culiciomyia) spiculothorax was described from Thailand based on the presence of spiculation on the thorax of larvae. Adult females are characterized but are indistinguishable from those of related species, such as Cx. pallidothorax. Phylogenetic analysis of mitochondrial oxidase subunit I (COI) sequences revealed that specimens identified as Cx. spiculothorax from Thailand, Japan and Bhutan form a single clade with Cx. sasai from Japan (Kimura 2-parameter genetic distances 0-0.9%) that is clearly distinct from clades comprised of other species of subgenus Culiciomyia. Attempts to collect Cx. sasai from several locations in Japan were unsuccessful - only larvae with thoracic vesicular-like spicules identified as Cx. spiculothorax were collected. Careful examination of specimens collected near the type locality of Cx. sasai revealed the presence of spicules on the thorax. Based on these findings, Cx. spiculothorax is formally synonymized with Cx. sasai, which replaces the former as the species present in Thailand and is a new country record for Bhutan.