Vijay Veer

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.

Next-generation sequencing in clinical virology: Discovery of new viruses

Viruses are a cause of significant health problem worldwide, especially in the developing nations. Due to different anthropological activities, human populations are exposed to different viral pathogens, many of which emerge as outbreaks. In such situations, discovery of novel viruses is utmost important for deciding prevention and treatment strategies. Since last century, a number of different virus discovery methods, based on cell culture inoculation, sequence-independent PCR have been used for identification of a variety of viruses. However, the recent emergence and commercial availability of next-generation sequencers (NGS) has entirely changed the field of virus discovery. These massively parallel sequencing platforms can sequence a mixture of genetic materials from a very heterogeneous mix, with high sensitivity. Moreover, these platforms work in a sequence-independent manner, making them ideal tools for virus discovery. However, for their application in clinics, sample preparation or enrichment is necessary to detect low abundance virus populations. A number of techniques have also been developed for enrichment or viral nucleic acids. In this manuscript, we review the evolution of sequencing; NGS technologies available today as well as widely used virus enrichment technologies. We also discuss the challenges associated with their applications in the clinical virus discovery.

Chryseobacterium culicis sp. nov., isolated from the midgut of the mosquito Culex quinquefasciatus.

A yellow-pigmented bacterial strain, R4-1A(T), isolated from the midgut of the mosquito Culex quinquefasciatus (a vector of lymphatic filariasis), was studied using a polyphasic approach. Cells of the isolate were rod-shaped and stained Gram-negative. A comparison of the 16S rRNA gene sequence of this organism with sequences of type strains of the most closely related species clearly showed an allocation to the genus Chryseobacterium, with the highest sequence similarities (all 97.9 %) to Chryseobacterium jejuense JS17-8(T), C. indologenes ATCC 29897(T), C. arthrosphaerae CC-VM-7(T) and C. aquifrigidense CW9(T). 16S rRNA gene sequence similarities to type strains of other Chryseobacterium species were below 97.5 %. The fatty acid profile of strain R4-1A(T) included the major fatty acids iso-15 : 0, summed feature 4 (comprising iso-15 : 0 2-OH and/or 16 : 1ω7c), iso-17 : 1ω9c and iso-17 : 0 3-OH. DNA-DNA hybridizations with C. jejuense KACC 12501(T), C. indologenes CCUG 14556(T), C. arthrosphaerae CC-VM-7(T) and C. aquifrigidense KCTC 12894(T) resulted in relatedness values of 38.3 % (reciprocal 30.5 %), 29.4 % (32.1 %), 23.2 % (37.2 %) and 29.5 % (47.1 %), respectively. These results and the differentiating biochemical and chemotaxonomic properties show that strain R4-1A(T) represents a novel species, for which the name Chryseobacterium culicis sp. nov. is proposed. The type strain is R4-1A(T) (=LMG 25442(T) =CCM 7716(T)).

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.

Essential oil based polymeric patch development and evaluating its repellent activity against mosquitoes.

Essential oil based insect repellents are environment friendly and provide dependable personal protection against the bites of mosquitoes and other blood-sucking insects. In the present study, optimized mixture of three essential oils was embedded into the ethylcellulose (EC) and polyvinylpyrrolidone (PVP K-30) polymers to develop essential oils based patch type mosquito repellent formulation. The developed formulation was characterized for various physico-chemical properties, oil release efficiency and essential oil-polymer interaction. Repellent activity of the formulation was evaluated against Ae. (S) albopictus mosquitoes and compared with commercially available synthetic insecticide based mosquito repellent cream Odomos(®) in the laboratory. The developed patches were 100% flat and there was no interaction between oil components and the excipients. Patches were smooth, homogenous and provided excellent mosquito repellent activity comparable to Odomos(®) under laboratory condition. Morphological and physico-chemical characterization indicated that the formulation was stable and suitable with the polymeric combination. The patch formulation did not show any inhalation toxicity in experimental Wistar rat. The repellent patches developed and evaluated currently, may provide a suitable, eco-friendly, acceptable and safe alternative to the existing synthetic repellent formulations for achieving protection against mosquitoes.

Insecticide susceptibility and dengue vector status of wild Stegomyia albopicta in a strategically important area of Assam, India

BackgroundDengue vector control programmes are facing operational challenges due to resistance against commonly used insecticides throughout the endemic countries. Recently, there has been appreciable increase in the dengue cases in India, however, no recent data are available on susceptible status of dengue vectors. We have studied the susceptibility level of St. albopicta to commonly used insecticides in India. Adult mosquitoes were tested for the presence of dengue virus.MethodsSt. albopicta larval bioassays were carried out to determine the lethal concentrations (LC10, LC50 and LC99) and the resistance ratios (RR10, RR50 and RR99) for temephos. Susceptibility to 4% DDT, 0.05% deltamethrin and 5% malathion was assessed following standard procedure. Knock-down times (KDT10, KDT50 and KDT99) were estimated and knock-down resistance ratios (KRR10, KRR50 and KRR99) were calculated. VectorTest™ dengue antigen assay was used to detect the dengue virus in the field collected mosquitoes.ResultsIn larval bioassays, the RR ranged from 1.4 (for RR99) to 1.7 (for RR50), which suggested that the tested St. albopicta were susceptible to temephos. There was no deviation among the lethal concentration data from linearity (r2 = 0.61). Adult St. albopicta mosquitoes were resistant to DDT, while fully susceptible to deltamethrin and malathion. The knock-down values (KDT10, KDT50 and KDT99) obtained for DDT displayed straight line in log-dose-probit analysis and follow linear regression model. The KRR99 for DDT was 4.9, which indicated a 4.9 folds increase in knock-down resistance to DDT. However, for malathion and deltamethrin, the KRR99 values were 1.6 and 1.5 respectively suggesting that mosquitoes were knock-down sensitive. None of the mosquito pool was dengue virus positive.ConclusionSt. albopicta showed resistance to DDT and reduced sensitivity to deltamethrin and malathion. This data on insecticide resistance could help public health authorities in India to design more effective vector control measures. More dengue vector specimens need to be scanned to identify the potential dengue vector.

Ixora coccinea Enhances Cutaneous Wound Healing by Upregulating the Expression of Collagen and Basic Fibroblast Growth Factor.

Background. Ixora coccinea L. (Rubiaceae) has been documented for traditional use in hypertension, menstrual irregularities, sprain, chronic ulcer, and skin diseases. In the present study, I. coccinea was subjected to in vitro and in vivo wound healing investigation. Methods. Petroleum ether, chloroform, methanol, and water sequential I. coccinea leaves extracts were evaluated for in vitro antioxidant, antimicrobial, and fibroblast proliferation activities. The promising I. coccinea methanol extract (IxME) was screened for in vivo wound healing activity in Wistar rat using circular excision model. Wound contraction measurement, hydroxyproline quantification, and western blot for collagen type III (COL3A1), basic fibroblast growth factor (bFGF), and Smad-2, -3, -4, and -7 was performed with 7-day postoperative wound granulation tissue. Gentamicin sulfate (0.01% w/w) hydrogel was used as reference standard. Results. IxME showed the potent antimicrobial, antioxidant activities, with significant fibroblast proliferation inducing activity, as compared to all other extracts. In vivo study confirmed the wound healing accelerating potential of IxME, as evidenced by faster wound contraction, higher hydroxyproline content, and improved histopathology of granulation tissue. Western blot analysis revealed that the topical application of I. coccinea methanol extract stimulates the fibroblast growth factor and Smad mediated collagen production in wound tissue.

Molecular phylogenetic study of Culex quinquefasciatus mosquito from different geographical regions of India using 16S rRNA gene sequences.

Culex quinquefasciatus is a major vector of filariasis and various encephalitis in India and worldwide. Vector control remains the most successful strategy for the suppression of mosquito borne diseases. The genetic structure of vector populations in terms of insecticide resistance and susceptibility or refractoriness to infection may possibly vary. To exploit the genetic variability in vector population could pave the path for the alternative strategies in vector management. The sequences of ribosomal RNA molecules have been widely used for such studies. Here, we examined the molecular phylogenetic relationship among the Cx. quinquefasciatus collected from different geographical regions of India, using 16S ribosomal RNA (16S rRNA) gene nucleotide sequences. The distances among the species were measured using Pearson correlation; the Neighbor-Joining (NJ) method was used for the clustering with appropriate bootstrap values using Data Analysis in Molecular Biology and Evolution (DAMBE) software. The results revealed that the populations are genetically diverse. Based on the distance values and the tree topology on the basis of 16S rRNA sequences reflected the clear biogeographical and geoclimatic pattern among the different geographical populations from India.

Culminating anti-malaria efforts at long lasting insecticidal net?

BACKGROUND Long-lasting insecticidal nets (LLINs) are a primary method in malaria control efforts. However, a decline in the biological efficacy and physical integrity over a period of comparatively lesser time than claimed, waning of naturally acquired immunity among regular users and misuse of LLINs are serious concerns. SEARCH AND SELECTION OF LITERATURE The literature for the current review was searched in PubMed, SCOPUS Database and Google using combined search strings of related key-words. Literature with sufficient data and information on the current subject was selected to reach a valid conclusion. FINDINGS The World Health Organization (WHO) has emphasized that LLINs should be considered a public good for people inhabiting malaria endemic settings. LLINs exhibited a cumulative effect on the vector density and may force anthropophilic mosquito vectors to find alternative animal hosts for blood meal. However, the physical integrity and biological activity of LLINs declines faster than the anticipated time due to different operational conditions and the spread of insecticide resistance. LLINs have been successful in reducing malaria incidences by either reducing or not allowing human exposure to the vector mosquitoes, but at the same time, LLINs debilitate the natural protective immunity against malaria parasite. Misuse of LLINs for deviant purposes is common and is a serious environmental concern, as people believe that traditional methods of prevention against malaria that have enabled them to survive through a long time are effective and sufficient. Moreover, people are often ill-informed regarding the toxic effects of LLINs. CONCLUSIONS Specific criteria for determining the serviceable life and guidelines on the safe washing and disposal of LLINs need to be developed, kept well-informed and closely monitored. Malaria case management, environment management and community awareness to reduce the misuse of LLINs are crucial. Focused research on developing effective anti-malarial drugs, vaccines and new insecticides to reduce resistance is imperative to tackle malaria in the future.