Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, ornamentals, and legumes worldwide. Besides damage caused by feeding, it transmits several tospoviruses. Identification of T. palmi at an early stage is crucial in implementing appropriate pest management strategies. Morpho-taxonomic identification of T. palmi based on the adult stage is time-consuming and needs taxonomic expertise. Here, we report a rapid, on-site, field-based assay for identification of T. palmi based on recombinase polymerase amplification (RPA), its first application in insects. RPA primers designed based on 3′ polymorphisms of the Internal Transcribed Spacer 2 region efficiently discriminated T. palmi without any cross-reactivity to other predominant thrips species. RPA was performed with crude DNA, extracted from single T. palmi simply by crushing in sterile distilled water and could be completed within 20 min by holding the reaction tubes in the hand. The assay was further simplified by using fluorescent as well as colorimetric dyes thus eliminating the gel-electrophoresis steps. The presence of T. palmi was visualized by a change in color from dark blue to sky blue. The assay was validated with known thrips specimens and found to be effective in diagnosing the presence of T. palmi in natural vegetation. This on-site, rapid assay for diagnosis of T. palmi can be used by non-expert personnel in the field of quarantine and pest management.

Thrips palmi (Thysanoptera: Thripidae) is the predominant tospovirus vector in Asia-Pacific region. It transmits economically damaging groundnut bud necrosis virus (GBNV, family Tospoviridae) in a persistent propagative manner. Thrips serve as the alternate host, and virus reservoirs making tospovirus management very challenging. Insecticides and host plant resistance remain ineffective in managing thrips–tospoviruses. Recent genomic approaches have led to understanding the molecular interactions of thrips–tospoviruses and identifying novel genetic targets. However, most of the studies are limited to Frankliniella species and tomato spotted wilt virus (TSWV). Amidst the limited information available on T. palmi–tospovirus relationships, the present study is the first report of the transcriptome-wide response of T. palmi associated with GBNV infection. The differential expression analyses of the triplicate transcriptome of viruliferous vs. nonviruliferous adult T. palmi identified a total of 2,363 (1,383 upregulated and 980 downregulated) significant transcripts. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed the abundance of differentially expressed genes (DEGs) involved in innate immune response, endocytosis, cuticle development, and receptor binding and signaling that mediate the virus invasion and multiplication in the vector system. Also, the gene regulatory network (GRN) of most significant DEGs showed the genes like ABC transporter, cytochrome P450, endocuticle structural glycoprotein, gamma-aminobutyric acid (GABA) receptor, heat shock protein 70, larval and pupal cuticle proteins, nephrin, proline-rich protein, sperm-associated antigen, UHRF1-binding protein, serpin, tyrosine–protein kinase receptor, etc., were enriched with higher degrees of interactions. Further, the expression of the candidate genes in response to GBNV infection was validated in reverse transcriptase-quantitative real-time PCR (RT-qPCR). This study leads to an understanding of molecular interactions between T. palmi and GBNV and suggests potential genetic targets for generic pest control.

Begomoviruses are transmitted by whitefly (Bemisia tabaci Gennadius, Hemiptera: Aleyrodidae) in a persistent-circulative way. Once B. tabaci becomes viruliferous, it remains so throughout its life span. Not much is known about the copies of begomoviruses ingested and/or released by B. tabaci during the process of feeding. The present study reports the absolute quantification of two different begomoviruses viz. tomato leaf curl New Delhi virus (ToLCNDV, bipartite) and chilli leaf curl virus (ChiLCV, monopartite) at different exposure of active acquisition and inoculation feeding using a detached leaf assay. A million copies of both the begomoviruses were acquired by a single B. tabaci with only 5 min of active feeding and virus copy number increased in a logarithmic model with feeding exposure. Whereas, a single B. tabaci could inoculate 8.21E+09 and 4.19E+11 copies of ToLCNDV and ChiLCV, respectively in detached leaves by 5 min of active feeding. Virus copies in inoculated leaves increased with an increase in feeding duration. Comparative dynamics of these two begomoviruses indicated that B. tabaci adult acquired around 14-fold higher copies of ChiLCV than ToLCNDV 24 hrs post feeding. Whereas, the rate of inoculation of ToLCNDV by individual B. tabaci was significantly higher than ChiLCV. The study provides a better understanding of begomovirus acquisition and inoculation dynamics by individual B. tabaci and would facilitate research on virus-vector epidemiology and screening host resistance.

Chilli leaf curl virus (ChiLCV; genus: Begomovirus), transmitted by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in a persistent-circulative manner, is a major constraint in chilli production. The present study demonstrates for the first time that a topical spray of naked double-stranded RNA (dsRNA) on chilli plants causes mortality and inability to acquire and transmit ChiLCV in B. tabaci. dsRNA targeting heat shock protein 70 (hsp70) and fasciclin 2 (fas2) of B. tabaci Asia II 1 was first assessed under controlled conditions through oral delivery. Hsp70 and fas2 dsRNA resulted in up to 82.22% and 72% mortality of B. tabaci and around 12.4- and 8.5-fold decreases in mRNA levels, respectively, 24 h post-ingestion. ChiLCV copies in hsp70 dsRNA-fed B. tabaci steadily decreased with an increase in dsRNA concentration and were undetectable at a higher concentration of dsRNA. However, ChiLCV copies significantly increased in fas2 dsRNA-fed B. tabaci. Transmission of ChiLCV by B. tabaci was completely inhibited post-24 h feeding on hsp70 dsRNA at 3 μg/mL. Naked hsp70 dsRNA was topically sprayed on ChiLCV-infected chilli plants like an insecticide. 67.77% mortality of B. tabaci, 4.6-fold downregulation of hsp70 mRNA, and 1.34 × 10¹⁵-fold decreased ChiLCV copies in B. tabaci were recorded when adults were exposed to the dsRNA-treated plants under semi-field conditions. Foliar application of naked dsRNA reduced the ChiLCV transmission by 75% without any visible symptoms in the inoculated plants. A total of 2 consecutive sprays of dsRNA provided significant protection to B. tabaci for up to 20 days under semi-field conditions.

Cardamom bushy dwarf virus (CBDV) causes foorkey disease of large cardamom (Ammomum subulatum Roxburgh) in the eastern sub-Himalayan mountains. Although the aphid Micromyzus kalimpongensis Basu (Hemiptera: Aphididae) is known as a vector of CBDV, its behavior in dissemination of CBDV has not been investigated. In the present study, M. kalimpongensis was observed to colonize in higher number on CBDV-infected large cardamom plants compared with the healthy plants in the several plantations in Sikkim and Darjeeling hills. The affinity of M. kalimpongensis to the diseased large cardamom plants was further confirmed in a contained field experiment with intact plant as well as in a laboratory bioassay with the plant extract, where significantly higher number of aphids settled on the diseased plants or extracts compared with the healthy counterparts. Aphids grown on CBDV-infected large cardamom plants had shortened nymphal period and increased longevity and fecundity compared with those grown on the healthy plants. In the contained field experiment, M. kalimpongensis migrated to the CBDV-infected plants, colonized there, acquired CBDV, and once the diseased plants withered, migrated to healthy plants, which eventually became diseased. Our results suggest a general pattern of spread of CBDV by M. kalimpongensis where CBDV-infected plants attract or arrest and stimulate emergence and migration of viruliferous aphids that otherwise are sedentary in the underground plant parts of large cardamom. To our knowledge, this is the first study that shows the influence of a plant virus from the family Nanoviridae in altering behavior of its insect vector that favors its dissemination.