Double-Stranded RNA-Mediated Suppression of Trypsin-Like Serine Protease (t-SP) Triggers Over-Expression of Another t-SP Isoform in Helicoverpa armigera.

High diversity of digestive proteases is considered to be the key factor in the evolution of polyphagy in Helicoverpa armigera. Serine proteases (SPs) contribute ~85% of the dietary protein digestion in H. armigera. We investigated the dynamics of SP regulation in the polyphagous pest, H. armigera using RNA interference (RNAi). HaTry1, an isoform of SP, expressed irrespective of the composition of the diet, and its expression levels were directly proportional to the larval growth rate. Therefore, HaTry1 was silenced by delivering 10 and 20 µg concentrations of double-stranded RNA through semi-synthetic diet. This led to a drastic reduction in the target gene transcript levels that manifested in a significant reduction in the larval weight initially, but the larvae recovered in later stages despite continuous dsRNA treatment. This was probably due to the compensatory effect by over-expression of HaTry13 (31-folds), another isoform of SP. Phylogenetic analysis of H. armigera SPs revealed that the over-expressed isoform was closely related to the target gene as compared to the other tested isoforms. Further, silencing of both the isoforms (HaTry1 and HaTry13) caused the highest reduction in the larval weight and there was no larval growth recovery. These findings provide a new evidence of the existence of compensatory effect to overcome the effect of silencing individual gene with RNAi. Hence, the study emphasizes the need for simultaneous silencing of multiple isoforms.

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.

Multiple Mutations on the Second Acetylcholinesterase Gene Associated With Dimethoate Resistance in the Melon Aphid, Aphis gossypii (Hemiptera: Aphididae).

The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is an important cosmopolitan and extremely polyphagous species capable of causing direct and indirect damage to various crops. Insecticide resistance in melon aphids is of particular concern. To determine the basis of resistance, organophosphate (OP)-resistant strains of A. gossypii were obtained by continuous selection with dimethoate in the laboratory, and resistance mechanisms were investigated along with susceptible strains. Three resistant strains LKR-1, LKR-2, and LKR-3 exhibiting 270-, 243-, and 210-fold resistance obtained after 30 generations of selection with dimethoate, respectively, were utilized in this study. The role of acetylcholinesterase (AChE), a target enzyme for OPs and carbamates (CMs), was investigated. AChE enzyme assay revealed that there was no significant change in the activities of AChE in resistant and susceptible strains. However, AChE inhibitory assay showed that 50% of the enzyme activity in resistant strains was inhibited at significantly higher concentration of dimethoate (131.87, 158.65, and 99.29 µmolL(−1)) as compared with susceptible strains (1.75 and 2.01 µmolL(−1)), indicating AChE insensitivity owing to altered AChE. Molecular diagnostic tool polymerase chain reaction-restriction fragment length polymorphism revealed the existence of two consistent non-synonymous point mutations, single-nucleotide polymorphism, viz., A302S (equivalent to A201 in Torpedo californica Ayres) and S431F (equivalent to F331 in T. californica), in the AChE gene Ace2 of resistant strains. Further, cloning and sequencing of a partial fragment of Ace2 (897 bp) gene from susceptible and resistant strains revealed an additional novel mutation G221A in resistant strains, LKR-1 and LKR-2. Susceptible Ace2 genes shared 99.6 and 98.9% identity at the nucleic acid and amino acid levels with resistant ones, respectively. Functional analysis of these point mutations was assessed by in silico docking studies using the modeled wild-type and naturally mutated AChE2. Computational analysis showed that the conformational changes in AChE2 active site due to structural gene substitutions (A302S, S431F, and G221A) significantly reduced the level of ligand (OP-dimethoate, omethoate, and CM-pirimicarb) binding, suggesting that they are potentially associated with resistance development. These results unambiguously suggested that multiple mutations located in the enzyme active site are responsible for AChE insensitivity to dimethoate and are likely the molecular basis for dimethoate resistance in these selected field populations of A. gossypii.

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.

One-step DNA fragment assembly for expressing intron-containing hairpin RNA in plants for gene silencing.

Double-stranded RNA-mediated RNA interference in plants involves generating a construct expressing intron-containing hairpin RNA (ihpRNA), which usually is a cumbersome, multistep process. Here, we describe a simplified method involving single steps of PCR, restriction, ligation, and transformation for assembling an ihpRNA construct for plant transformation. Our method has several advantages over the currently available ones, viz., wider choice of restriction sites and facility for rapid screening of positive clones, among others. We demonstrate the utility of this approach in assembling the tomato phytoene desaturase gene. This simplified DNA fragment assembly strategy for ihpRNA construction facilitates high-throughput gene silencing in plants.

Molecular differences in the mitochondrial cytochrome oxidase I (mtCOI) gene and development of a species-specific marker for onion thrips, Thrips tabaci Lindeman, and melon thrips, T. palmi Karny (Thysanoptera: Thripidae), vectors of tospoviruses (Bunyaviridae).

A quick and developmental-stage non-limiting method of the identification of vectors of tospoviruses, such as Thrips tabaci and T. palmi, is important in the study of vector transmission, insecticide resistance, biological control, etc. Morphological identification of these thrips vectors is often a stumbling block in the absence of a specialist and limited by polymorphism, sex, stage of development, etc. Molecular identification, on the other hand, is not hampered by the above factors and can easily be followed by a non-specialist with a little training. The mitochondrial cytochrome oxidase I (mtCOI) exhibits reliable inter-species variations as compared to the other markers. In this communication, we present the differences in the mtCOI partial sequence of morphologically identified specimens of T. tabaci and T. palmi collected from onion and watermelon, respectively. Species-specific markers, identified in this study, could successfully determine T. tabaci and T. palmi, which corroborated the morphological identification. Phylogenetic analyses showed that both T. tabaci and T. palmi formed different clades as compared to the other NCBI accessions. The implication of these variations in vector efficiency has to be investigated further. The result of this investigation is useful in the quick identification of T. tabaci and T. palmi, a critical factor in understanding the epidemiology of the tospoviruses, their management and also in quarantine.