N. Pradeep Kumar

DNA Barcodes Can Distinguish Species of Indian Mosquitoes (Diptera: Culicidae)

Abstract Species identification of mosquitoes (Diptera: Culicidae) based on morphological characteristics remains often difficult in field-collected mosquito specimens in vector-borne disease surveillance programs. The use of DNA barcodes has been proposed recently as a tool for identification of the species in many diverse groups of animals. However, the efficacy of this tool for mosquitoes remains unexplored. Hence, a study was undertaken to construct DNA barcodes for several species of mosquitoes prevalent in India, which included major vector species. In total, 111 specimens of mosquitoes belonging to 15 genera, morphologically identified to be 63 species, were used. This number also included multiple specimens for 22 species. DNA barcode approach based on DNA sequences of mitochondrial cytochrome oxidase gene sequences could identify 62 species among these, in confirmation with the conventional taxonomy. However, two closely related species, Ochlerotatus portonovoensis (Tiwari & Hiriyan) and Ochlerotatus wardi (Reinert) could not be identified as separate species based on DNA barcode approach, their lineages indicating negligible genetic divergence (Kimura two-parameter genetic distance = 0.0043).

DNA barcoding for identification of sand flies (Diptera: Psychodidae) in India

About 50 species of sand flies have been reported to be prevalent in India. We explored the utility of the DNA barcode approach towards species identification of these medically important insects. A total of 62 specimens belonging to seven morphologically identified species of two genera, Phlebotomus and Sergentomyia, collected from Puducherry Union Territory, Maharashtra and Rajasthan states of India were subjected to the analysis. Neighbor‐joining (NJ) analysis of DNA barcode sequences identified the individuals of seven morphological species into eight distinct species, as presented in the designed NJ tree. This methodology delineated morphologically identified species, S. bailyi, into two genetically isolated groups. Also, this study characterizes DNA barcodes of P. argentipes and P. papatasi, the vector species of leishmaniasis in India, for the first time.

DNA Barcodes indicate members of the Anopheles fluviatilis (Diptera: Culicidae) species complex to be conspecific in India.

Anopheles fluviatilis, a major vector of malaria in India has been described as a complex of three sibling species members, named as S, T and U, based on variations in chromosomal inversions. Also, ribosomal DNA markers (repetitive Internal Transcribed Spacer 2 (ITS2) and 28S D3 region) were described to differentiate these three sibling species members. However, controversies prevail on the genetic isolation status of these cryptic species. Hence, we evaluated this taxonomic incongruence employing DNA barcoding, the well established methodology for species identification, using 60 An. fluviatilis sensu lato specimens, collected from two malaria endemic eastern states of India. These specimens were also subjected to sibling species characterization by ITS2 and D3 DNA markers. The former marker identified 31 specimens among these as An. fluviatilis S and 21 as An. fluviatilis T. Eight specimens amplified DNA fragments specific for both S and T. The D3 marker characterized 39 specimens belonging to species S and 21 to species T. Neither marker identified species U. Neighbor Joining analysis of mitochondrial cytochrome c oxidase gene 1 sequences (the DNA barcode) categorized all the 60 specimens into a single operational taxonomic unit, their Kimura 2 parameter (K2P) genetic variability being only 0.8%. The genetic differentiation (FST) and gene flow (Nm) estimates were 0.00799 and 62.07, respectively, indicating these two ‘species’ (S & T) as genetically con‐specific intermixing populations with negligible genetic differentiation. Earlier investigations have refuted the existence of species U. Also, this study demonstrated that An. fluviatilis and the closely related An. minimus could be taxonomically differentiated by the DNA Barcode approach (K2P = 5.0%).

Temporal distribution and behaviour of sand flies (Diptera: Psychodidae) in a cutaneous leishmaniasis focus of the Kani Tribe settlements in the Western Ghats, India.

The temporal distribution of sand flies in relation to environmental factors was studied in the Kani tribe settlements located on the southernmost part of the Western Ghats, Kerala, India, between June 2012 and May 2013. This area is known for occurrence of cutaneous leishmaniasis (CL) cases. Employing hand-held aspirator, light trap and sticky-trap collection methods, a total of 7874 sand fly specimens, comprising 19 species was collected. Sergentomyia baghdadis was predominant species, followed by Phlebotomus argentipes. Sand fly abundance was significantly higher indoors (χ(2)=9241.8; p=0.0001) than outdoors. Mean density of P. argentipes in human dwellings, cattle sheds and outdoors was 7.2±2.9, 27.33±21.1 and 0.64±0.2 females/per man-hour (MHR), respectively. No sand fly species other than P. argentipes was obtained from cattle sheds. Although, sand fly populations were prevalent throughout the year, their abundance fluctuated with seasonal changes. Multiple regression analysis with backward elimination indicated that the increase in precipitation and relative humidity contributed to a significant positive association with the increase in sand fly abundance, while the increase in temperature showed no association. Fully engorged female sand flies tested for blood meal source showed multiple host-blood feeding. Analysis of resting populations of sand flies collected from human shelters indicated that the populations were found maximum on interior walls at 6-8 and >8 ft height, including ceiling during summer (F=83.7, df=6, p=0.001) and at the lower half of the wall at 0 and 0-2 ft height, during monsoon season (F=41.4, df=6, p=0.001). In cooler months, no preference to any height level (F=1.67, df=6, p=0.2) was observed. Proportion of females sand flies with Sellas classification of abdominal stages, namely full-fed, half-gravid and gravid females did not vary significantly (t=1.98, p=0.13827) indoors, confirming their endophilic behaviour. Risk of CL transmission in these tribal settlements is discussed.

Sergentomyia (Neophlebotomus) monticola, a new species of sand fly (Diptera: Psychodidae) from the Western Ghats, Thiruvananthapuram District, Kerala, India

Sergentomyia (Neophlebotomus) monticola, a new species of sand fly (Diptera: Psychodidae), from the Kani tribal settlements, Thiruvananthapuram District, Kerala, southern India was described. These settlements were located in the Western Ghats, which is one of the 25 biodiversity hotspots in the world. Morphological characters of male and female specimens of Sergentomyia (Neophlebotomus) monticola were described with illustrations and its taxonomic position is defined within the genus. The DNA barcode analysis showed that both male and female specimens of the species were belonging to a single taxonomic category. The genetic distance with the most similar taxonomic neighbour was 14.61%, which confirms its distinctness from its congeners. Voucher specimens of the new species were deposited at the museum, Vector Control Research Centre (Indian Council of Medical Research), Puducherry, India, Zoological Survey of India, India and Smithsonian National Museum of Natural History (NMNH), Washington, D.C., USA.

Development of a PCR methodology to distinguish species members of Culex vishnui subgroup (Diptera: Culicidae), based on DNA Barcodes

“DNA Barcoding” has emerged as a very efficient tool for species identification of diverse group of animals and plants (Stoeckle & Hebert, 2008). This methodology was proposed as a universal approach for species identification about a decade back (Hebert et al, 2003). Realizing its importance towards description of global biodiversity, a global consortium of DNA Barcoding of life (CBOL) was launched under the auspices of the Smithsonian Institution, U.S.A. and about 0.175 million species of plants and animals have been DNA barcoded so far, globally (Barcode of life data systems [BOLD]) (Ratnasingham & Hebert, 2007). Utility of DNA Barcoding methodology for species identification of economically important insects, the mosquitoes have been recorded (Cywinska et al., 2006; Kumar et al., 2007; Kumar et al., 2009; Rajavel et al., 2015). Culex vishnui subgroup of mosquitoes prevalent in Southeast Asian countries are important vectors of arboviral diseases such as Japanese encephalitis, Chandipura virus, etc. Three major species of this subgroup Culex vishnui, Culex pseudovishnui, and Culex tritaeniorhynchus are reported to be prevalent in India (Reuben et al., 1969; Sirivanakarn, 1975). The taxonomic identification of these species had been extremely difficult or impossible in adult stages and the larval exuviae were normally used for the same (Bram, 1967). Later, Reuben et al. (1994) described adult morphological characteristics, towards their species identification. However, these being very minute (scale distribution and color of the proboscis,

Domestic dogs as reservoir hosts for Leishmania donovani in the southernmost Western Ghats in India

The peripheral blood samples from domestic dogs (n=47) and wild rats (n=25) in the Kani Tribe settlements, located southernmost part of the Western Ghats, Thiruvananthapuram district, Kerala, India were examined for Leishmania infection. This area is known for cases of leishmaniasis with cutaneous manifestations and sandfly abundance. The tribes domesticate dogs to protect them from untoward activities of wild animals. Leishmania donovani parasite DNA was detected only from 6.4% (n=3) of the blood samples collected from the domestic dogs by amplification of the diagnostic kinetoplast mini-circle DNA and PCR-RFLP analysis of the UTR region of heat shock protein 70 (hsp70) gene. None of the blood samples collected from rats was positive. Through sequencing, L. donovani infection among dogs was confirmed. The DNA sequences generated for hsp70 were deposited with the GenBank. The GenBank accession numbers of these samples are KR905363, KR905364 and KR905365 for hsp70 genes. The results indicated that the DNA isolates from dog blood samples matched precisely with that of our earlier isolates from skin lesions of Kani tribes and also from P. argentipes vector. Thus, the role of dogs as reservoirs for L. donovani parasite in the Kani tribe settlements is confirmed.

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.

Bionomics of Mansonioides mosquitoes in relation to community structure of hydrophytes/breeding habitats in Cherthala, Kerala

Three species of Mansonioides vectors viz.,Ma. annulifera, Ma. uniformis andMa. indiana were found in Cherthala taluk, Kerala which is one of the endemic areas due toB. malayi. The immatures of Mansonioides thrive mainly in association with macrophytic hydrophytes such asP. stratiotes, S. molesta andE. crassipes in perennial habitats (ponds, channels/ canals etc.,) andI. miliaceae in seasonal habitats (fallow lands etc.) Breeding potential was higher (130.19) in clean ponds withP. stratiotes, compared to that of polluted ones (40.69). However, the polluted habitats infested with the same host plants were found to be the most productive forMa. annulifera, with an average daily adult emergence rate of 601/100 sq.m.). The clean habitats played a major role in the contribution ofMa. uniformis, whereS. molesta in the perennial habitats and I. miliaceae in the seasonal fallow lands were the favourable plants contributing a daily output of 12.5/100 sq.m and 221.81/100 sq.m. respectively.E. crassipes infested polluted habitats formed the major source forMa. indiana, the emergence rate being 13.89/100 sq.m. The perennial habitats supported mainly the breeding ofMa. annulifera (70.82%), whereas the seasonal habitats contributed the major chunk ofMa. uniformis (92.54%) andMa. indiana (71.43%). The bionomics of Mansonioides mosquitoes are thus shown to be greatly influenced by the community structure of hydrophytes and also the nature of breeding habitats.