Olfactory Reception of Host Alarm Pheromone Component by the Odorant-Binding Proteins in the Samurai Wasp, Trissolcus japonicus (Hymenoptera: Scelionidae).

The samurai wasp, Trissolcus japonicus, is the predominant egg parasitoid of the brown marmorated stink bug, Halyomorpha halys, in its native range in China. (E)-2-Decenal is a major component of the alarm pheromone of H. halys, an important invasive insect pest with significant economic importance. T. japonicus can be strongly repelled by (E)-2-decenal, and thus its host location efficiency would be reduced in the field. Better understanding on the molecular basis of olfactory reception of this host alarm pheromone component by T. japonicus may provide opportunities to develop novel approaches to enhance biological control efficacy of the parasitoid against H. halys. We identified six Odorant Binding Proteins (OBPs) from T. japonicus by transcriptome sequencing, within which three classical OBPs were expressed in a heterologous expression system with E. coli, harvested, and then challenged with (E)-2-decenal in binding assay experiments. TjapOBP2 showed the highest binding ability to (E)-2-decenal, compared to TjapOBP1 and TjapOBP3. Our results unambiguously suggest that TjapOBP2 would play an important role in discriminating (E)-2-decenal and could be a possible target for further intervention in the parasitoid-host system.

Insecticide resistance status in the whitefly, Bemisia tabaci genetic groups Asia-I, Asia-II-1 and Asia-II-7 on the Indian subcontinent.

The present study is a summary of the current level of the insecticide resistance to selected organophosphates, pyrethroids, and neonicotinoids in seven Indian field populations of Bemisia tabaci genetic groups Asia-I, Asia-II-1, and Asia-II-7. Susceptibility of these populations was varied with Asia-II-7 being the most susceptible, while Asia-I and Asia-II-1 populations were showing significant resistance to these insecticides. The variability of the LC50 values was 7x for imidacloprid and thiamethoxam, 5x for monocrotophos and 3x for cypermethrin among the Asia-I, while, they were 7x for cypermethrin, 6x for deltamethrin and 5x for imidacloprid within the Asia-II-1 populations. When compared with the most susceptible, PUSA population (Asia-II-7), a substantial increase in resistant ratios was observed in both the populations of Asia-I and Asia-II-1. Comparative analysis during 2010-13 revealed a decline in susceptibility in Asia-I and Asia-II-1 populations of B. tabaci to the tested organophosphate, pyrethroid, and neonicotinoid insecticides. Evidence of potential control failure was detected using probit analysis estimates for cypermethrin, deltamethrin, monocrotophos and imidacloprid. Our results update resistance status of B. tabaci in India. The implications of insecticide resistance management of B. tabaci on Indian subcontinent are discussed.

The First Report of miRNAs from a Thysanopteran Insect, Thrips palmi Karny Using High-Throughput Sequencing.

Thrips palmi Karny (Thysanoptera: Thripidae) is the sole vector of Watermelon bud necrosis tospovirus, where the crop loss has been estimated to be around USD 50 million annually. Chemical insecticides are of limited use in the management of T. palmi due to the thigmokinetic behaviour and development of high levels of resistance to insecticides. There is an urgent need to find out an effective futuristic management strategy, where the small RNAs especially microRNAs hold great promise as a key player in the growth and development. miRNAs are a class of short non-coding RNAs involved in regulation of gene expression either by mRNA cleavage or by translational repression. We identified and characterized a total of 77 miRNAs from T. palmi using high-throughput deep sequencing. Functional classifications of the targets for these miRNAs revealed that majority of them are involved in the regulation of transcription and translation, nucleotide binding and signal transduction. We have also validated few of these miRNAs employing stem-loop RT-PCR, qRT-PCR and Northern blot. The present study not only provides an in-depth understanding of the biological and physiological roles of miRNAs in governing gene expression but may also lead as an invaluable tool for the management of thysanopteran insects in the future.

RNA Interference of Odorant-Binding Protein 2 (OBP2) of the Cotton Aphid, Aphis gossypii (Glover), Resulted in Altered Electrophysiological Responses.

Aphis gossypii (Glover) (Hemiptera: Aphididae) is a highly invasive pest that feeds primarily on phloem resulting in severe economic loss to growers. A. gossypii has cosmopolitan distribution with broad host range, polyphenism, parthenogenetic mode of reproduction, vectoring abilities, and host alteration which has profound influence on its management. Odorant-binding proteins (OBPs) in insects are involved in olfaction, playing a key role in orienting the insect for feeding or oviposition. Recent studies revealed that OBP2 is found in both sensilla trichodea and sensilla basiconica and is preferentially binds to plant volatiles, thus playing crucial roles in host-seeking, detection of oviposition attractants, etc., However, information about the role of OBP2 in A. gossypii (AgOBP2) is still unavailable. In this study, we cloned and characterized OBP2, ortholog from A. gossypii, and the full-length AgOBP2 complementary DNA (cDNA) consisted of 859 bp with an open reading frame of 732 bp. Phylogenetic analysis resulted in grouping of AgOBP2 protein with members of the tribe Aphidini. Further, diet-mediated delivery of double-stranded RNA for AgOBP2 induced silencing, which was evaluated at 48 and 96 h. The reverse transcriptase real-time quantitative polymerase chain reaction (RTq-PCR) results revealed that the level of AgOBP2 messenger RNA (mRNA) was significantly reduced (55-77 %) in dsAgOBP2 treatment after 96 h as compared to the untreated control. The same was reiterated by the electrophysiological responses in the aphids which was reduced (>50 % at 0.25 µg/µl concentration) as compared to the untreated control. Thus, our results showed the potential of gene silencing, possibly to interfere with the odorant perception of A. gossypii for RNAi-mediated pest management. The results from our study provided the first evidence that AgOBP2 play crucial roles in host-seeking, detection of oviposition attractants, etc.; as a result, we suggests that OBP2 could potentially serve as a practicable target for RNAi-mediated gene silencing in hemipteran insect pest control.

Diet-Delivered dsRNAs for Juvenile Hormone-Binding Protein and Vacuolar ATPase-H Implied Their Potential in the Management of the Melon Aphid (Hemiptera: Aphididae).

RNA interference is a sequence-specific gene silencing mechanism mediated by double-stranded RNA (dsRNA), which has been harnessed as a useful tool in devising novel insect pest management strategies for various pests such as melon aphid, Aphis gossypii (Glover). In the current study, we cloned and sequenced juvenile hormone-binding protein (JHBP) and vacuolar ATPase subunit H (V-ATPase-H) from A. gossypii. We also showed the effectiveness of diet-mediated delivery of dsRNA for JHBP and V-ATPase-H, which silenced the above genes and resulted in mortality. The extent of silencing and mortality were similar for both genes up until 96 h. Bioassay results revealed that the target genes were silenced variably, 1.0 µg/µl concentration having a more profound effect than 0.5 and 0.25 µg/µl concentration in reducing the cognate mRNA transcript level. Results indicated a 9.56­73.21% down regulation (across time and concentrations for both the genes) that resulted in the mortality of A. gossypii. Mortality was in the range of 10­63% for both these genes. Thus, the current study demonstrated the potentiality of both JHBP and V-ATPase-H as excellent targets for the management of A. gossypii.

Non-invasive delivery of dsGST is lethal to the sweet potato whitefly, Bemisia tabaci (G.) (Hemiptera: Aleyrodidae).

The sweet potato whitefly, Bemisia tabaci (G.) biotype B (Hemiptera: Aleyrodidae), is one of the most economically important pest, by being a dreaded vector of Geminiviruses, and also causes direct damage to the crops by sucking phloem sap. Glutathione S-transferase (GST) is a large family of multifunctional enzymes that play pivotal roles in the detoxification of secondary allelochemical produced by the host plants and in insecticide resistance, thus regulates insect growth and development. The objective of this study is to show the potential of RNA interference (RNAi) in the management of B. tabaci. RNAi is a sequence-specific gene silencing mechanism induced by double-stranded RNA (dsRNA) which holds tremendous potential in pest management. In this regard, we sequenced the GST from B. tabaci and synthesized approximately 500-bp dsRNA from the above and delivered through diet to B. tabaci. Real-time quantitative PCR (RT-qPCR) showed that continuous application of dsGST at 1.0, 0.5, and 0.25 µg/µl reduced mRNA expression levels for BtGST by 77.43, 64.86, and 52.95 % which resulted in mortality by 77, 59, and 40 %, respectively, after 72 h of application. Disruption of BtGST expression will enable the development of novel strategies in pest management and functional analysis of vital genes in B. tabaci.

DNA barcoding and elucidation of cryptic aphid species (Hemiptera: Aphididae) in India.

Rapid, precise and timely identification of invasive pest insects such as aphids is important and a challenge worldwide due to their complex life cycles, parthenogenetic reproduction, sex and colour morphs. In this respect, DNA barcoding employing a 658 bp fragment of 5' region of the mitochondrial cytochrome oxidase I (CO-I) gene is an effective tool in addressing the above. In the present study, we employed CO-I for discriminating 142 individuals representing 32 species of aphids from India. Sequence analyses revealed that the intraspecific and interspecific distances ranged from zero to 3.8% and 2.31 to 18.9%, respectively. In addition, the study also showed for the first time the prevalence of three cryptic species, namely Brevicoryne brassicae (Linnaeus), Hyperomyzus carduellinus (Theobald) and Brachycaudus helichrysi (Kaltenbach) from India. Our work has clearly demonstrated that DNA barcoding is an efficient and accurate method for identification of aphid species (including cryptic species), an approach that potentially could play an important role in formulating viable pest management strategies, more especially biocontrol.

DNA barcoding and development of species-specific markers for the identification of tea mosquito bugs (Miridae: Heteroptera) in India.

Rapid, accurate, and timely identification of insects as a group is important and challenging worldwide, as they outnumber all other animals in number and diversity. DNA barcoding is a method for the identification of species in a wide range of animal taxa, which uses the 5' region of the mitochondrial cytochrome c oxidase-I (CO-I). Yet another easy, accurate, and economical method of species discrimination is by developing species-specific markers, which produce specific amplicon for the species in question. The method is handy because it is not limited by life stages, sex, polymorphism, and other factors. Herein, we measured the usefulness of CO-I for the species discrimination of mirids in India viz. Helopeltis antonii Signoret, H. thievora Waterhouse, H. bradyi Waterhouse, and Pachypeltis maesarum Kirkaldy in their various life stages. Furthermore, our study showed the utility of species-specific markers in differentiating H. antonii (295) and H. bradyi (514) regardless of their life stages. Analysis of CO-I gene revealed <1% intraspecific divergence for all four species examined, whereas the interspecific distances ranged from 7 to 13%. This study showed that the DNA barcode and species-specific markers will aid the identification of mirids in India and will stand as a decisive tool in formulating integrated pest management (IPM) strategy, quick identification of invasive and cryptic species, haplotypes, biotypes, and other factors, if any.