S. H. P. Parakrama Karunaratne

Pathogenomics of Culex quinquefasciatus and Meta-Analysis of Infection Responses to Diverse Pathogens

Closing the Vector Circle The genome sequence of Culex quinquefasciatus offers a representative of the third major genus of mosquito disease vectors for comparative analysis. In a major international effort, Arensburger et al. (p. 86) uncovered divergences in the C. quinquefasciatus genome compared with the representatives of the other two genera Aedes aegypti and Anopheles gambiae. The main difference noted is the expansion of numbers of genes, particularly for immunity, oxidoreductive functions, and digestive enzymes, which may reflect specific aspects of the Culex life cycle. Bartholomay et al. (p. 88) explored infection-response genes in Culex in more depth and uncovered 500 immune response-related genes, similar to the numbers seen in Aedes, but fewer than seen in Anopheles or the fruit fly Drosophila melanogaster. The higher numbers of genes were attributed partly to expansions in those encoding serpins, C-type lectins, and fibrinogen-related proteins, consistent with greater immune surveillance and associated signaling needed to monitor the dangers of breeding in polluted, urbanized environments. Transcriptome analysis confirmed that inoculation with unfamiliar bacteria prompted strong immune responses in Culex. The worm and virus pathogens that the mosquitoes transmit naturally provoked little immune activation, however, suggesting that tolerance has evolved to any damage caused by replication of the pathogens in the insects. The genome of a third mosquito species reveals distinctions related to vector capacities and habitat preferences. The mosquito Culex quinquefasciatus poses a substantial threat to human and veterinary health as a primary vector of West Nile virus (WNV), the filarial worm Wuchereria bancrofti, and an avian malaria parasite. Comparative phylogenomics revealed an expanded canonical C. quinquefasciatus immune gene repertoire compared with those of Aedes aegypti and Anopheles gambiae. Transcriptomic analysis of C. quinquefasciatus genes responsive to WNV, W. bancrofti, and non-native bacteria facilitated an unprecedented meta-analysis of 25 vector-pathogen interactions involving arboviruses, filarial worms, bacteria, and malaria parasites, revealing common and distinct responses to these pathogen types in three mosquito genera. Our findings provide support for the hypothesis that mosquito-borne pathogens have evolved to evade innate immune responses in three vector mosquito species of major medical importance.

Characterization of knockdown resistance in DDT- and pyrethroid-resistant Culex quinquefasciatus populations from Sri Lanka

DDT and pyrethroid resistance in Culex quinquefasciatus have been previously reported in Sri Lanka, but the mechanisms involved have yet to be characterized. We report the presence of two mutant alleles of the sodium channel gene, the target site for both DDT and pyrethroid insecticides. Both mutations resulted in classic knockdown resistance (kdr) L1014F mutation because of either an A‐to‐T substitution or an A‐to‐C substitution. We developed two alternative assays to distinguish between the two mutations and used these to screen 214 individuals from nine geographic locations throughout Sri Lanka. Very high levels of kdr mutations were found throughout the country. A predominance of the A‐to‐C mutation was observed over the A‐to‐T with an average allele frequency of 50% and 2%, respectively. In addition to these non‐synonymous kdr substitutions, we also found an indel (TCACA) in the intron downstream of the kdr mutation. After genotyping this indel in 136 individuals, we found no evident correlation between kdr genotypes and intronic indel. The presence of two alternative kdr mutations has implications for the reliance on single molecular diagnostics for detection of resistance in field populations. Furthermore, the high levels of these kdr mutations in C. quinquefasciatus populations throughout Sri Lanka are of concern for the future of pyrethroid‐based control programmes on this island.

Prevalence and breeding habitats of the dengue vectors Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in the semi-urban areas of two different climatic zones in Sri Lanka

Dengue has become the fastest-growing mosquito-borne disease in Sri Lanka and the control of the vectors, Aedes aegypti Linnaeus and Ae. albopictus Skuse, is the most effective way of controlling the disease. A detailed study on vector prevalence has not been recorded from Sri Lanka. Therefore, the present study was undertaken to study the prevalence of both vectors in four semi-urban study sites in two of the most affected districts, namely Kandy (wet zone) and Kurunegala (intermediate zone), by conducting egg surveys (using ovitraps) and larval surveys from June 2007 to May 2008. A total of 82,524 eggs and 2658 larvae of Ae. aegypti and Ae. albopictus were collected. A total of 3699 potential breeding habitats were examined. Ovitrap and larval indices (house, container and Breteau) showed that all four areas are at epidemic risk, especially due to a high abundance of Ae. albopictus. The highest numbers for both the species were from the Kandy sites where dense vegetation, high rainfall and low temperature prevailed. The results showed a high mortality rate during the egg-to-larva transition, suggesting that conducting an egg survey alone would overestimate the vector abundance and the disease risk. For Ae. albopictus, the monthly mean number of eggs showed positive relationships with relative humidity in both districts and with rainfall in the Kandy District. The number of dengue cases in the area had no positive relationships with the abundance of eggs or larval density indices. Discarded receptacles were the most preferred breeding habitat for these dengue vectors. Since the attractiveness (inferred by the calculated risk factors) of most of the breeding habitats was very high, elimination of these breeding sites is essential for the success of dengue control programmes.

Prevalence and insecticide susceptibility of dengue vectors in the district of Batticaloa in eastern Sri Lanka

Unprecedented incidences of dengue have been reported in Sri Lanka in recent years. The district of Batticaloa, which was devastated by the 2004 Asian tsunami, is one of the districts affected by dengue. One option to curtail this disease is to implement appropriate vector control measures. A nine-month study was carried out within the Batticaloa Municipal Council limit from April to December 2008. Larval collections were conducted fortnightly using conventional ovitraps for nine months covering the dry and wet seasons. Ovitraps (indoor and outdoor) were placed in 15 randomly selected houses. The collected larvae were brought to the laboratory and reared under laboratory conditions. The larval forms and emerged adults were identified on the basis of reported morphological descriptions. The identified adults of 2–3 d old were exposed to common insecticides following the WHO protocol. During the study period, a total of 10,685 Aedes aegypti and Ae. albopictus mosquitoes were collected, with the former constituting 57% of the total sample. Both species were collected from indoor and outdoor ovitraps, and their prevalence was recorded throughout the study period. A seasonal shift was observed in the density, with Ae. aegypti predominating during the dry season and Ae. albopictus during the wet season. Both species were highly resistant to 4% DDT and susceptible to 0.25% permethrin. The continuous presence of potential dengue vectors may have contributed to the dengue prevalence in the district. Since both species can oviposit in indoor and outdoor ovitraps, public awareness and participation should be promoted in the vector control programme of the Ministry of Health along with continuous vector surveillance.

Mechanisms of acaricide resistance in the cattle tick Rhipicephalus (Boophilus) microplus in Sri Lanka

High tolerance of ticks to acaricides is increasingly becoming a problem to cattle farmers. Resistance status of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)from two cattle farms of Sri Lanka were determined against different concentrations of pyrethroid permethrin, organophosphate malathion, organochlorine DDT and carbamate propoxur using Larval Packet Test (LPT) as recommended by Food and Agriculture Organization (FAO). Mechanisms of acaricide resistance were studied by conducting biochemical and PCR assays. Tick larvae were tested for the activity levels of acaricide metabolizing enzymes i.e.esterases, glutathione S-transferases (GSTs) and monooxygenases, and for altered target sites i.e.acetylcholinesterase (target site of organophosphates and carbamates) and sodium channel regulatory proteins (target site of pyrethroids and DDT). According to discriminating dosages specified by FAO for ticks both populations were 24-56% resistant to DDT. LC values showed that the both populations were susceptible to permethrin and resistant to malathion. Moderate insensitivity of AChEs and knock-down resistance (kdr) mutations were found as resistance mechanisms. GSTs and monooxygenases were not elevated. The kdr type mutation G72V (G215T in the gene) found in the sodium channel regulatory protein of R. (B.) microplus samples may be responsible for DDT resistance. Systematic and sophisticated insecticide resistance monitoring programmes and a better understanding on the mechanisms which govern resistance development are vital for future tick control programmes.

Distribution and phylogeny of Wolbachia strains in wild mosquito populations in Sri Lanka

BackgroundWolbachia are a group of maternally inherited intracellular bacteria known to be widespread among arthropods. Infections with Wolbachia cause declines of host populations, and also induce host resistance to a wide range of pathogens. Over the past few decades, researchers were curious to use Wolbachia as a biological tool to control mosquito vectors. During the present study, assessment of the prevalence of Wolbachia infections among wild mosquito populations in Sri Lanka where mosquito-borne diseases are a major health concern, was carried out for the first time. DNA was extracted from the abdomens of mosquitoes, collected from seven provinces, and screened for the presence of Wolbachia by PCR using wsp and groE primers. Group-specific and strain-specific primers were used to classify Wolbachia into the supergroups A and B, and into the strains Mel, AlbA and Pip.ResultsA total of 330 individual mosquitoes belonging to 22 species and 7 genera were screened. Eighty-seven mosquitoes (26.36%) belonging to four species (i.e. Aedes albopictus, Culex quinquefasciatus, Armigeres subalbatus and Mansonia uniformis) were positive for Wolbachia infections. Primary vector of the dengue fever, Ae. aegypti was negative for Wolbachia infections while the secondary vector, Ae. albopictus, showed a very high infection rate. The filarial vector C. quinquefasciatus had a relatively high rate of infection. Japanese encephalitis vectors C. gelidus and C. triteaneorynchus, and the Anopheles vectors of malaria were negative for Wolbachia infections. Nine sequences of Wolbachia-positive PCR products were deposited in the GenBank and compared with other available data. Aedes albopictus was infected with both Wolbachia strains A (AlbA) and B (Pip) supergroups. Phylogenetic analysis of the wsp sequences showed two major branches confirming identities obtained from the PCR screening with strain-specific primers.ConclusionWolbachia infections were found only among four mosquito species in Sri Lanka: Aedes albopictus, Culex quinquefasciatus, Armigeres subalbatus and Mansonia uniformis. Sequence data showed high haplotype diversity among the Wolbachia strains.

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

BackgroundAnopheles 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.MethodsMosquito 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.ResultsAll 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.ConclusionsThe 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.

DNA barcoding of morphologically characterized mosquitoes belonging to the subfamily Culicinae from Sri Lanka

BackgroundVectors of mosquito-borne diseases in Sri Lanka, except for malaria, belong to the subfamily Culicinae, which includes nearly 84% of the mosquito fauna of the country. Hence, accurate and precise species identification of culicine mosquitoes is a crucial factor in implementing effective vector control strategies. During the present study, a combined effort using morphology and DNA barcoding was made to characterize mosquitoes of the subfamily Culicinae for the first time from nine districts of Sri Lanka. Cytochrome c oxidase subunit 1 (cox1) gene from the mitochondrial genome and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization.ResultsAccording to morphological identification, the field collected adult mosquitoes belonged to 5 genera and 14 species, i.e. Aedes aegypti, Ae. albopictus, Ae. pallidostriatus, Aedes sp. 1, Armigeres sp. 1, Culex bitaeniorhynchus, Cx. fuscocephala, Cx. gelidus, Cx. pseudovishnui, Cx. quinquefasciatus, Cx. tritaeniorhynchus, Cx. whitmorei, Mansonia uniformis and Mimomyia chamberlaini. Molecular analyses of 62 cox1 and 36 ITS2 sequences were exclusively comparable with the morphological identifications of all the species except for Ae. pallidostriatus and Aedes sp. 1. Although the species identification of Armigeres sp. 1 specimens using morphological features was not possible during this study, DNA barcodes of the specimens matched 100% with the publicly available Ar. subalbatus sequences, giving their species status. Analysis of all the cox1 sequences (14 clades supported by strong bootstrap value in the Neighbor-Joining tree and interspecific distances of > 3%) showed the presence of 14 different species. This is the first available DNA sequence in the GenBank records for morphologically identified Ae. pallidostriatus. Aedes sp. 1 could not be identified morphologically or by publicly available sequences. Aedes aegypti, Ae. albopictus and all Culex species reported during the current study are vectors of human diseases. All these vector species showed comparatively high diversity.ConclusionsThe current study reflects the significance of integrated systematic approach and use of cox1 and ITS genetic markers in mosquito taxonomy. Results of DNA barcoding were comparable with morphological identifications and, more importantly, DNA barcoding could accurately identify the species in the instances where the traditional morphological identification failed due to indistinguishable characters of damaged specimens and the presence of subspecies.

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

BackgroundAlthough 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).MethodsAdult 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).ResultsAnopheles 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.ConclusionsGene 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.

Effect of dengue mosquito control insecticide thermal fogging on non-target insects

Dengue vector control programmes are mainly focused on insecticide fogging/ space spraying to control adult Aedes mosquito vector populations. Due to the diurnal habit of the vectors, spraying is routinely conducted during the day when many other insect species are also active. This study reports the simultaneous effect of fogging on non-target insects by direct counting of knockdown in the insect population. Eight fogging treatments were conducted in two sites in Kurunegala District of Sri Lanka. Pesguard insecticide was sprayed in each treatment for 8 minutes according to the standard methodology and the ‘knockdown insects’ were collected on randomly spread polyethythene sheets (10 m2). A total of 3884 insects (24.3 insects per treatment per m2 belonging to 12 orders were collected and 12.44% of them recovered during a 24-hr recovery period. Diptera was the most affected insect order (36%) followed by Collembola (30%) and Thysanoptera (17%). Out of the 31 mosquitoes (<1%) collected, only two (<0.1%) belonged to the genus Aedes. Body length of 93% of the affected insects ranged from 0.35 mm to 1.8 mm. Positive controls using the WHO standard cage bioassays with the mosquito Ae. albopictus (n = 417) and the stingless bee Trigona iridipennis (n = 122) showed 100% initial knockdown, and 83.5% mosquito and 93.5% bee mortalities after the recovery period. The study shows that insecticide fogging does have a severe effect on non-target insects such as pollinators; therefore, fogging operations should be done in a controlled manner and indiscriminate fogging should be avoided.