A new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study.

BACKGROUND: The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. METHODS: A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration-response curves for each insecticide-species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. RESULTS: Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species-insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. CONCLUSION: Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide.

Impact of dengue-preventive behaviors on Aedes immature production in Bang Kachao, Samut Prakan Province, Thailand: a cross-sectional study.

BACKGROUND: Controlling sites where mosquitos breed is a key strategy in breaking the cycle of infectious transmission of the dengue virus. Preventive behaviors, such as covering water containers with lids and adding temephos (commercially named Abate sand) in water containers are needed to reduce and control mosquito breeding sites. This study aimed to investigate the impact of dengue-preventive behaviors on Aedes immature production. METHODS: This cross-sectional study used in-person interviews to record occurrence of dengue-preventive behaviors in Bang Kachao, Samut Prakan Province, Thailand. Larval mosquitos in and around houses were observed and recorded, and covered 208 households. RESULTS: It was found that 50% of these households had containers for drinking water and 94% used water containers. Covering water containers with effective lids showed the best success among dengue-preventive behaviors for reducing Aedes immature production. Adding temephos in water containers also was effective. CONCLUSIONS: Such behaviors substantively affected development of Aedes immatures in and around households.

Geometric morphometrics of the scutum for differentiation of trombiculid mites within the genus Walchia (Acariformes: Prostigmata: Trombiculidae), a probable vector of scrub typhus.

The vectors of scrub typhus are the larval stage of trombiculid mites, termed "chiggers". These vectors are very small - the larvae are approximately 0.2 mm in size - and therefore their morphological identification is difficult. Trombiculid mites are widely distributed across Asia and they can be identified at the genus level by the shape, size and setae/sensilla distribution of their dorsal chitin plate (scutum = shield), while morphological identification at the species level requires more mite characteristics. We recently developed a methodology to ascertain paired matched genotype and morphotype of individual chiggers, based on autofluorescence and brightfield microscopy with subsequent molecular identification using the COI gene (approximately 640bp length). However, based on 20 chigger specimens characterised by paired genotypic and morphological data consisting of the four species [Walchia ewingi with 2 subspecies]: Walchia ewingi lupella (n = 9), W. ewingi ewingi (n = 2), W. alpestris (n = 2), W. kritochaeta (n = 5) and W. minuscuta (n = 2) we found evidence of genetic polymorphism and morphological plasticity within the genus Walchia. The phylogenetic inference of the intra-genus relationships within the Walchia spp., based on COI gene (Blankaartia spp. served as outgroup), delineated the five included species by an average interspecific divergence of mean distance 0.218 (0.126 - 0.323). We therefore applied landmark-based and outline-based geometric morphometric (GM) approaches to differentiate Walchia species using scutum measurements. A total of 261 scutum images of Walchia spp. were examined by landmark-based GM (140 chigger specimens) and outline-based GM (121 specimens) techniques. All Walchia spp. showed significant differences in scutum size and shape. W. minuscuta showed the smallest mean scutum size in both techniques. The largest scutum was found in W. ewingi lupella and W. ewingi ewingi by landmark-based and outline-based GM analysis, respectively. The scutum shapes of W. alpestris and W. minuscuta were clearly distinguished from the other species. Cross-validated classification scores were different depending on species and digitizing techniques and landmark-based GM showed better scores than outline-based GM. We conclude that the morphologically closely-related trombiculid mite species can be further differentiated by their scutum features alone, using GM approaches. This technique is a promising tool for the much-needed characterization studies of chiggers and needs evaluation using matched morphometric and genotyping data for other genera of trombiculids.

Autofluorescence microscopy for paired-matched morphological and molecular identification of individual chigger mites (Acari: Trombiculidae), the vectors of scrub typhus.

BACKGROUND: Conventional gold standard characterization of chigger mites involves chemical preparation procedures (i.e. specimen clearing) for visualization of morphological features, which however contributes to destruction of the arthropod host DNA and any endosymbiont or pathogen DNA harbored within the specimen. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a novel work flow based on autofluorescence microscopy was developed to enable identification of trombiculid mites to the species level on the basis of morphological traits without any special preparation, while preserving the mite DNA for subsequent genotyping. A panel of 16 specifically selected fluorescence microscopy images of mite features from available identification keys served for complete chigger morphological identification to the species level, and was paired with corresponding genotype data. We evaluated and validated this method for paired chigger morphological and genotypic ID using the mitochondrial cytochrome c oxidase subunit I gene (coi) in 113 chigger specimens representing 12 species and 7 genera (Leptotrombidium, Ascoschoengastia, Gahrliepia, Walchia, Blankaartia, Schoengastia and Schoutedenichia) from the Lao People's Democratic Republic (Lao PDR) to the species level (complete characterization), and 153 chiggers from 5 genera (Leptotrombidium, Ascoschoengastia, Helenicula, Schoengastiella and Walchia) from Thailand, Cambodia and Lao PDR to the genus level. A phylogenetic tree constructed from 77 coi gene sequences (approximately 640 bp length, n = 52 new coi sequences and n = 25 downloaded from GenBank), demonstrated clear grouping of assigned morphotypes at the genus levels, although evidence of both genetic polymorphism and morphological plasticity was found. CONCLUSIONS/SIGNIFICANCE: With this new methodology, we provided the largest collection of characterized coi gene sequences for trombiculid mites to date, and almost doubled the number of available characterized coi gene sequences with a single study. The ability to provide paired phenotypic-genotypic data is of central importance for future characterization of mites and dissecting the molecular epidemiology of mites transmitting diseases like scrub typhus.

Diversity Of Mosquito Species Ovipositing In Different Zones of Light Intensity Within Limestone Caves In Thailand.

Climate change and human activity affect the geographical and annual distribution and population abundance of mosquitoes. As natural habitats are reduced, it is hypothesized that mosquitoes may seek refuge in more stable environments such as cave habitats. Therefore, we explored the species diversity of mosquitoes exploiting cave habitats in Thailand. Ten species belonging to 4 genera were collected, of which none were considered to be true cave-dwelling species (Troglobiont). The known cavernicolous species, Aedes cavaticus, was observed to oviposit outside of the cave and therefore should be categorized as a subtroglophilic species. Other species were also oviposited inside the cave but should be regarded as trogloxenic species. There was no clear association between environmental factors and mosquito abundance inside the 4 limestone caves, except for Ae. cavaticus, which was positively correlated with rainfall. This study indicates that different biotic or abiotic factors may be involved in mosquito oviposition site selection inside caves.

IDENTIFICATION OF BAT ECTOPARASITE LEPTOCIMEX INORDINATUS FROM BAT-DWELLING CAVE, KANCHANABURI PROVINCE, THAILAND.

Bat bugs are blood-feeding insects of bats or warm blooded animals and humans. Since 2011, Leptocimex spp (Heteroptera: Cimicidae) has been reported in Thailand. However, microscopic examination of Leptocimex spp is complicated, especially when the entire body of the specimen is not available. To confirm the phenotypic identification of L. inordinatus from a limestone bat cave in Thailand, partial fragments of mitochondrial cytochrome c oxidase subunit 1 (COI) gene and 16S mitochondrial ribosomal DNA were PCR amplified and sequenced, which revealed 97% sequence identity with Cimicidae family members, being most similar to Cacodminae gen. sp. and C. vicinus, both bat bugs. Phylogenetic tree construction showed that L. inordinatus has a separate genetic lineage from that of with human bed bugs (Cimex hemipterus or C. lectularius), swallow bugs and other tick species. The presence of L. inordinatus in a bat-dwelling cave frequented by humans presents a potential public health problem requiring attention in particular regarding the possibility of zoonotic transmission of pathogens.

CORRELATION OF HOST SPECIFICITY, ENVIRONMENTAL FACTORS AND ORIENTAL RAT FLEA ABUNDANCE.

Fleas are the vectors of many communicable diseases that are normally found in oriental rats. Climate and environmental changes influence the habitat and migration patterns of vectors. In this study, the oriental rat flea abundance, represented as total flea index, was determined in correlation to host specificity and various environmental factors. The number of hosts and fleas calculated from 3 specific habitats (shipping area, decayed area, and market area) from July 2010 to June 2011. The results showed that the common hosts in the shipping area and decayed area were Rattus rattus and R. exulans, with the total flea indexes of 3.36 and 1.58. R. norvegicus was the most common host identified in the market area. Fleas were virtually absent in rat hosts collected from the market area. Both the density of reservoir hosts and the total flea index were positively correlated with the mean annual rainfall and temperature. These data could be useful for control of rat populations in each specific habitat.

Functional expression and molecular characterization of Culex quinquefasciatus salivary α-glucosidase (MalI).

Salivary α-glucosidases (MalI) have been much less characterized when compared with midgut α-glucosidases, which have been studied in depth. Few studies have been reported on the partial characterization of MalI, but no clear function has been ascribed. The aim of this study is to purify and characterize the recombinant Culex quinquefasciatus (CQ) α-glucosidase expressed in Pichia pastoris. The cDNA encoding mature Cx. quinquefasciatus α-glucosidase gene with polyhistidine tag (rCQMalIHis) was successfully cloned into the expression vector, pPICZαB, designated as pPICZαB/CQMalIHis. The activity of recombinant rCQMalIHis expressed in P. pastoris could be detected at 3.75U/ml, under optimal culture conditions. The purified rCQMalIHis showed a single band of molecular weight of approximately 92kDa on SDS-PAGE. After Endoglycosidase H digestion, a single band at 69kDa was found on SDS-PAGE analysis, suggesting that rCQMalIHis is a glycoprotein. Additionally, tryptic digestion and LC-MALDI MS/MS analysis suggested that the 69kDa band corresponds to the Cx. quinquefasciatus α-glucosidase. Thus, rCQMalIHis is a glycoprotein. The rCQMalIHis exhibited optimum pH and temperature at 5.5 and 35°C, respectively. The catalytic efficiency (kcat/Km) of the purified rCQMalIHis for maltotriose is higher than those for sucrose, maltotetraose, maltose and p-nitrophenyl-α-glucoside, indicating that the enzyme prefers maltotriose. Additionally, the rCQMalIHis is significantly inhibited by d-gluconic acid δ-lactone, but not by Mg(2+), Ca(2+) and EDTA. The rCQMalIHis is strongly inhibited by acarbose with IC50 67.8±5.6nM, but weakly inhibited by glucose with IC50 115.9±7.3mM.

A single-round multiplex PCR assay for the identification of Anopheles minimus related species infected with Plasmodium falciparum and Plasmodium vivax.

This study aimed to develop a single-round multiplex PCR method for the identification of Anopheles minimus complex (An. minimus and Anopheles harrisoni) and Anopheles aconitus subgroup (An. aconitus and Anopheles varuna), and for the simultaneous detection of Plasmodium falciparum and Plasmodium vivax in these vectors. Five primers were created for a single-round multiplex PCR assay to identify four anopheline mosquitoes combined with three Plasmodium primers for the detection of P. falciparum and P. vivax in vectors. The four species of anopheline vectors and two Plasmodium species, P. falciparum and P. vivax, could be identified by the combination of eight primers in the single-round multiplex PCR assay. The amplified species-specific products were 380bp for An. minimus, 180bp for An. harrisoni, 150bp for An. aconitus, 310bp for An. varuna, 276bp for P. falciparum, and 300bp for P. vivax. The sensitivities were 0.5pg/µl (25sporozoites/µl) for P. falciparum DNA and between 0.5 and 5pg/µl (25-250sporozoites/µl) for P. vivax DNA. Furthermore, this developed method could be used to identify field caught An. minimus complex, An. aconitus subgroup from Thailand and Lao PDR. Also, it was successfully used to identify the species An. minimus, An. harrisoni, An. aconitus and An. varuna and to detect and identify P. falciparum and P. vivax in caught anopheline mosquitoes. The sensitivity of this method was high for simultaneous detection of P. falciparum and P. vivax in anopheline mosquitoes.

Comparison of Aedes aegypti (Diptera: Culicidae) resting behavior on two fabric types under consideration for insecticide treatment in a push-pull strategy.

Aedes aegypti (L.), the primary vector of dengue and dengue hemorrhagic fever, breeds and rests predominately inside human dwellings. With no current vaccine available, vector control remains the mainstay for dengue management and novel approaches continue to be needed to reduce virus transmission. This requires a full understanding of Ae. aegypti ecology to design effective strategies. One novel approach is the use of contact irritants at target resting sites inside homes to make the surface unacceptable and cause vectors to escape before biting. The objective of the current study was to observe indoor resting behavior patterns of female Ae. aegypti within experimental huts in response to two fabrics under consideration for insecticide treatment: cotton and polyester. Results indicate that fabric type, coverage ratio of dark to light fabric and placement configuration (vertical vs. horizontal) all influenced the resting pattern of mosquito cohorts. Findings from this study will guide evaluations of a push-pull strategy designed to exploit contact irritant behaviors and drive Ae. aegypti out of homes prefeeding.

Efficacy of three insecticides against Anopheles dirus and Anopheles minimus, the major malaria vectors, in Kanchanaburi Province, Thailand.

We conducted this study to determine the insecticide susceptibility of two malaria vectors, Anopheles dirus and Anopheles minimus from Kanchanaburi Province, Thailand. The mosquitoes were collected and reared under laboratory conditions. The test was carried out on unfed F-1 female mosquitoes using a standard WHO testing protocol. The LD50 and LD90 of deltamethrin in both species were tested for by exposing the mosquitoes to various doses of deltamethrin for 1 hour. The lethal time was also tested among mosquitoes by exposing them to deltamethrin (0.05%), permethrin (0.75%) and malathion (5%), for different exposure times, ranging from 0.5 to 15 minutes. Percent knockdown at 60 minutes and mortality at 24 hours were calculated. The resistance ratio (RR) was determined based on the LD50 and LT50 values. LD50 of deltamethrin against An. dirus and An. minimus were 0.00077% and 0.00066%, respectively. LT50 values for deltamethrin (0.05%), permethrin (0.75%) and malathion (5%) against An. dirus and An. minimus were 1.20, 3.16 and 10.07 minutes and 0.48, 1.92 and 5.94 minutes, respectively. The study revealed slightly increased tolerance by both mosquito species, compared with laboratory susceptible strains, based on LD50 values. The two anopheline species had the same patterns of response to the three insecticides, based on LT50 values, although the LT50 values were slightly higher in the An. dirus population. Both An. dirus and An. minimus were fully susceptible to all the insecticides tested, with 100% mortality at 24 hours post-exposure. Deltamethrin was the most effective insecticide, followed by permethrin and malathion.

Distribution and abundance of Stomoxyini flies (Diptera: Muscidae) in Thailand.

Stomoxyini flies (Diptera: Muscidae) include species of parasitic flies of medical and veterinary importance. The adult flies feed on the blood of mammals and may transmit several parasites and pathogens. We conducted an entomological survey of Stomoxyini flies from different sites in Thailand. Stomoxyini flies were collected at four major types of sites: zoos, livestock farms, wildlife conservation areas and a national park using vavoua traps between November 2010 and April 2011. A total of 3,314 Stomoxyini flies belonging to the genera Stomoxys, Haematobosca, Haematostoma and Haematobia were collected. Eight species were identified: S. calcitrans (46.6%), S. uruma (26.8%), S. pulla (4.3%), S. indicus (0.7%), S. sitiens (0.1%), H. sanguinolenta (11.2 %), H. austeni (0.5%) and H. irritans exigua (9.8%). The diversity of Stomoxyini flies in the livestock farms was higher than the other sites. Altitude correlated with the number of flies. This study provides information that may be useful for Stomoxyini flies control.

Seasonal abundance and bloodfeeding activity of Anopheles dirus sensu lato in western Thailand.

Bloodfeeding activity, host preference, and seasonal abundance of the Anopheles dirus complex in relation to environmental factors were measured during a 2-yr period at Pu Teuy Village, Sai Yok District, Kanchanaburi Province, western Thailand. Anopheles dirus s.l. was found more abundant during the wet season compared with the dry and hot seasons. Bloodfeeding by An. dirus s.l. commenced immediately after sunset with a distinct peak of activity at 2000 h. Anopheles dirus s.l. fed more readily on cattle than on humans, demonstrating a significant but slightly stronger zoophilic behavior (P = 0.02). The biting activity of An. dirus s.l. in Pu Teuy appears to differ from other localities in Thailand, suggesting the existence of significant biological variability within the species complex depending on the local circumstances.

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.

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

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

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.