D. Sukumaran

Electroantennogram and Behavioral Responses of Culex quinquefasciatus (Diptera: Culicidae) Females to Chemicals Found in Human Skin Emanations

Abstract The southern house mosquito, Culex quinquefasciatus Say (Diptera: Culicidae), is closely associated with humans and is a vector of filariasis. Use of semiochemicals for control of mosquitoes is a novel and potentially ecofriendly control approach. Human skin emanations are known to attract mosquitoes. In the current study, olfactory and behavioral responses of host-seeking female Cx. quinquefasciatus to various chemical components of human skin emanations (carboxylic acids, alcohols, and aldehydes) were evaluated separately at three doses (0.01, 0.1, and 1 μg) for electroantennogram (EAG) and three doses (0.1, 1, and 10 μg) for behavioral assay. Results of EAG studies indicated that all carboxylic acids elicited significant olfactory responses except tetradecanoic acid (C14) and octadecanoic acid (C18). In particular, hexanoic acid (C6) elicited a maximum, eight-fold olfactory response compared with the solvent control. Ethylene glycol and benzyl alcohol exhibited significant EAG and behavioral responses, whereas among aldehydes, nonanal elicited high EAG and behavioral responses, but only at all three doses tested compared with control. Some carboxylic acids elicited significant behavioral responses, attracting ≈75% of females tested toward chemical stimuli in a Y-tube olfactometer.

Resistance Status of the Malaria Vector Mosquitoes, Anopheles stephensi and Anopheles subpictus Towards Adulticides and Larvicides in Arid and Semi-Arid Areas of India

Abstract Susceptibility studies of malaria vectors Anopheles stephensi Liston (Diptera: Culicidae) and An. subpictus Grassi collected during 2004–2007 from various locations of Arid and Semi-Arid Zone of India were conducted by adulticide bioassay of DDT, malathion, deltamethrin and larvicide bioassay of fenthion, temephos, chlorpyriphos and malathion using diagnostic doses. Both species from all locations exhibited variable resistance to DDT and malathion from majority of location. Adults of both the species were susceptible to Deltamethrin. Larvae of both the Anopheline species showed some evidence of resistance to chlorpyriphos followed by fenthion whereas susceptible to temephos and malathion.

Midgut Microbial Community of Culex quinquefasciatus Mosquito Populations from India

The mosquito Culex quinquefasciatus is a ubiquitous species that serves as a major vector for west nile virus and lymphatic filariasis. Ingestion of bloodmeal by females triggers a series of physiological processes in the midgut and also exposes them to infection by these pathogens. The bacteria normally harbored in the midgut are known to influence physiology and can also alter the response to various pathogens. The midgut bacteria in female Cx. quinquefasciatus mosquitoes collected over a large geographical area from India was studied. Examination of 16S ribosomal DNA amplicons from culturable microflora revealed the presence of 83 bacterial species belonging to 31 bacterial genera. All of these species belong to three phyla i.e. Proteobacteria, Firmicutes and Actinobacteria. Phylum Proteobacteria was the most dominant phylum (37 species), followed by Firmicutes (33 species) and Actinobacteria (13 species). Phylum Proteobacteria, was dominated by members of γ-proteobacteria class. The genus Staphylococcus was the largest genus represented by 11 species whereas Enterobacter was the most prevalent genus and recovered from all the field stations except Leh. Highest bacterial prevalence was observed from Bhuj (22 species) followed by Nagrota (18 species), Masimpur (18 species) and Hathigarh (16 species). Whereas, least species were observed from Leh (8 species). It has been observed that individual mosquito harbor extremely diverse gut bacteria and have very small overlap bacterial taxa in their gut. This variation in midgut microbiota may be one of the factors responsible for variation in disease transmission rates or vector competence within mosquito population. The present data strongly encourage further investigations to verify the potential role of the detected bacteria in mosquito for the transmission of lymphatic filariasis and west nile virus. To the best of our knowledge this is the first study on midgut microbiota of wild Cx. quinquefasciatus from over a large geographical area.

Genetic variability in geographical populations of Culex quinquefasciatus Say (Diptera: Culicidae) from India based on random amplified polymorphic DNA analysis.

Genetic variability and environmental factors may influence the refractiveness, propagation of pathogen and transmission of disease. Random amplified polymorphic DNA (RAPD) is one of the widely used molecular markers for population genetic diversity studies. In present study, RAPD is used to ascertain the genetic variability in Culex quinquefasciatus populations collected from various Indian geographical locations. Out of 50 RAPD primers screened, 14 primers exhibited clear, concrete and distinct banding pattern showing up to 100% polymorphism. Primer OPBD3 was tested with DNA of 14 geographical populations from India (including one laboratory population) showed 21 loci representing 14 populations with 100% polymorphism. The genetic diversity among the populations indicated the Shannon index (I) and gene diversity index (H(ST)), 0.48 and 0.31, respectively among the population, displaying rich genetic variation among the Cx. quinquefasciatus populations. Consensus tree showed two clusters indicating the genetic variation among the various geographical populations. The findings of this study may be useful to understand the population variation under different ecological conditions and development of effective vector management strategies.

Variations in life tables of geographically isolated strains of the mosquito Culex quinquefasciatus

Variations in the life tables and other biological attributes of four strains of Culex quinquefasciatus Say (Diptera: Culicidae) from geographically isolated regions of India that had been reared to the fifth generation in the laboratory were assessed under a standardized rearing regime under constant laboratory conditions. Two strains from arid habitats [Jodhpur (JD) and Bikaner (BKN)], one from a semi‐arid inland habitat [Bathinda (BTH)], one from a semi‐arid coastal habitat [Jamnagar (JMN)] and a standard laboratory strain (LAB) were compared. Horizontal life‐table parameters were measured for each strain. Egg mortality ranged from 4.4% (JD and BTH) to 19.5% (BKN). The lowest rate of adult emergence and highest female : male ratio were found in BKN, and the highest rate of adult emergence and lowest female : male ratio were recorded in BTH. The egg‐hatching period was longest in BTH and shortest in LAB. The duration from oviposition to adult emergence was longest in JD and shortest in LAB. Females lived longer than males in all strains. The net reproductive rates (R0) of all field‐derived strains (122.9–162.2) differed significantly between strains and were significantly greater than that of LAB (107.6). Similarly, both the intrinsic rate of increase (rm) and finite rate of increase (λ) were found to be lower in LAB than in the field strains, but the mean generation time (T) and doubling time (DT) were longest in LAB. For several life‐table attributes, JD and BTH clustered together and were more similar to JMN than to BKN and LAB. The results indicate that BTH, BKN and JD can be characterized as r‐strategists, more so than JMN. Overall fecundity increased with age. Differences in annual temperature ranges and mean annual rainfall between locations were positively correlated (r = 0.46–0.97) with egg production, female life expectancy, R0, rm, λ and T. The results suggest that strains of Cx. quinquefasciatus from different geographical areas with contrasting habitats vary in their survival and reproductive strategies accordingly.

Two novel epistatic mutations (E1:K211E and E2:V264A) in structural proteins of Chikungunya virus enhance fitness in Aedes aegypti.

Expansion of CHIKV outbreaks with appearance of novel mutations are reported from many parts of the world. Two novel mutations viz. E1:K211E and E2:V264A in background of E1:226A are recently identified from Aedes aegypti dominated areas of India. In this study, the role of these mutations in modulation of infectivity, dissemination and transmission by two different Aedes species was studied. Mutations were sequentially constructed in CHIKV genome and female Ae. aegypti and Aedes albopictus mosquitoes were orally infected with eight different CHIKV mutants. Double mutant virus containing E1:K211E and E2:V264A mutations in background of E1:226A revealed remarkably higher fitness for Ae. aegypti, as indicated by significant increase in virus infectivity (13 fold), dissemination (15 fold) and transmission (62 fold) compared to parental E1:226A virus. These results indicate that adaptive mutations in CHIKV are leading to efficient CHIKV circulation in Ae. aegypti endemic areas, contributing and sustaining the major CHIKV outbreaks.

Mosquito saliva induced cutaneous events augment Chikungunya virus replication and disease progression.

Chikungunya virus (CHIKV) is transmitted when infected mosquito probes the host skin. While probing, mosquito saliva is expectorated into host skin along with virus which contains cocktail of molecules having anti-hemostatic and immunomodulatory properties. As mosquito saliva is a critical factor during natural arboviral infection, therefore we investigated mosquito saliva induced cutaneous events that modulate CHIKV infection. The effect of mosquito saliva on CHIKV infection was examined through inoculation of suckling mice subcutaneously with either CHIKV alone or uninfected mosquito bite followed by CHIKV. Histopathological evaluation of skin revealed infiltration of transmigrated inflammatory cells. Dermal blood vessels were hyperemic and adnexa showed degenerating lesions. Severe hemorrhage was observed in dermis and hypodermis in mosquito bite+CHIKV group compared to CHIKV group. Analysis of cytokines in skin showed significant downregulation of inflammatory genes like TLR-3, IL-2, IFN-γ, TNF-α and IFN-β in mosquito bite+CHIKV group compared to CHIKV group. In contrast, significant upregulation of anti-inflammatory genes like IL-4 and IL-10 was observed. These early events might have been responsible for increased dissemination of CHIKV to serum and peripheral organs as demonstrated through >10-fold higher viremia, antigen localization, cellular infiltration and degenerative changes. Thus mosquito saliva induced early cellular infiltration and associated cytokines augment CHIKV pathogenesis in a mouse model. This mosquito improved CHIKV mouse model simulates the realistic conditions that occur naturally during infected mosquito bite to a host. It will lead to better understanding of CHIKV pathobiology and promote the evaluation of novel medical countermeasures against emerging CHIKV.

Development of a SYBR green I-based quantitative RT-PCR for Ross River virus: Application in vector competence studies and antiviral drug evaluation

Abstract Ross River virus (RRV) is an emerging Alphavirus and is presently endemic in many parts of Oceania. Keeping in mind its emergence, we developed a molecular detection system and utilized it to study vector competence and evaluate activity of antiviral compounds against RRV. A SYBR Green I-based quantitative RT-PCR for detection of RRV was developed targeting the E2 gene, with a detection limit of 100 RNA copies/reaction. The specificity was confirmed with closely related Alphaviruses and Flaviviruses. The assay was applied to study the vector competence of Indian Aedes aegypti for RRV, which revealed 100% infection and dissemination rate with 75% transmission rate. Viral RNA was found in saliva as early as 3day post infection (dpi). Further application of the assay in antiviral drug evaluation revealed the superior in vitro activity of ribavirin compared to chloroquine in Vero cells. Successful demonstration of this assay to detect RRV in low titre mosquito samples makes it a sensitive tool in vector surveillance. This study also showed that Indian Ae. aegypti are well competent to transmit RRV highlighting the risk of its introduction to naïve territories across continents. Further validation of this assay, revealed its utility in screening of potential antivirals against RRV.

Malaria vector Anopheles culicifacies sibling species differentiation using egg morphometry and morphology

BackgroundThe malaria vector Anopheles culicifacies (sensu lato) is an important malaria vector in Southeast Asia which comprises of five sibling species namely A, B, C, D and E. However, only a few forms have been identified as malaria vectors in various endemic countries. Currently, for the first time egg morphometry and morphology has been used to differentiate the three known vector sibling species of Anopheles culicifacies collected from malaria endemic Madhya Pradesh state of central India.MethodsThe adult An. culicifacies (s.l.) was collected from five districts using standard mosquito collection methods. Adult female mosquitoes were allowed to lay eggs individually. The emerged mosquitoes were identified using allele specific polymerase chain reaction (AS-PCR) to sibling species. Eggs of sibling species A, D and E were studied using scanning electron microscopy (SEM) for morphometric and morphological characteristics.ResultsCurrently AS-PCR identified four known sibling species (B, C, D and E) of An. culicifacies in the study area. The surface morphology and morphometric attributes of the sibling species A, D and E eggs considerably differed from each other. An. culicifacies E had a narrow deck as compared to A and D, while An. culicifacies A had a bigger micropyle with 6–7 sectors as compared to D and E that had 6 sectors. An. culicifacies D had the smallest float (the structure present on sides of the egg surface in which air is filled that help in floating) and the number of ribs was also fewer than for An. culicifacies A and E.ConclusionsThe present study provides the first evidence that in addition to PCR assay, sibling species of An. culicifacies can also be differentiated using morphological and morphometric characteristics of the egg stage. The results also advocate that the sibling species of An. culicifacies are morphologically dissimilar and can be resolved using advanced microscopy.

A review on test methods for insecticidal fabrics and the need for standardisation

Insecticidal fabrics are effective personal protective measures against disease vectors and unlike bed nets, these fabrics can provide protection from day-biting mosquitoes and in outdoor environments. The rapid geographical expansion of day-biting mosquitoes and their role in disease transmission necessitate technological interventions, which can be effectively used during the daytime. There is a renewed interest in insecticidal fabrics mainly due to the recent outbreaks and geographical spread of dengue and chikungunya and with the emerging threat of Zika virus infection. Insecticidal fabrics are useful for protection from night-biting mosquitoes and also in situations were sleeping under a bed net is not possible. They are also effective against other biting arthropods like ticks, mites, tsetse flies, sand flies and body lice. Although long-lasting insecticidal fabrics factory-treated with permethrin are now commercially available for military and civilian use, there are no international guidelines for testing their efficacy. The different methods employed so far for testing bioefficacy, washing and quantification of permethrin are compiled in this review. The future prospects and challenges ahead for long-lasting insecticidal fabrics are discussed in the context of the increased threat from day-biting mosquitoes and the diseases transmitted by them. The review focuses on the need for standardisation of the test methods for ensuring adequate bioefficacy and safety to the user. The differences between long-lasting insecticidal nets and long-lasting insecticidal fabrics are elaborated, and the need for a separate registration and licencing procedure for long-lasting insecticidal fabrics is highlighted. A test procedure for insecticidal fabrics is described, which could be used until internationally accepted guidelines are available.