Tomato spotted wilt virus (TSWV) is transmitted in a persistent propagative manner by Frankliniella occidentalis, the western flower thrips. While it is well established that vector competence depends on TSWV acquisition by young larvae and virus replication within the insect, the biological factors associated with frequency of transmission have not been well characterized. We hypothesized that the number of transmission events by a single adult thrips is determined, in part, by the amount of virus harbored (titer) by the insect. Transmission time-course experiments were conducted using a leaf disk assay to determine the efficiency and frequency of TSWV transmission following 2-day inoculation access periods (IAPs). Virus titer in individual adult thrips was determined by real-time quantitative reverse transcriptase-PCR (qRT-PCR) at the end of the experiments. On average, 59% of adults transmitted the virus during the first IAP (2 to 3 days post adult-eclosion). Male thrips were more efficient at transmitting TSWV multiple times compared with female thrips of the same cohort. However, females harbored two to three times more copies of TSWV-N RNA per insect, indicating that factors other than absolute virus titer in the insect contribute to a successful transmission event. Examination of virus titer in individual insects at the end of the third IAP (7 days post adult-eclosion) revealed significant and consistent positive associations between frequency of transmission and virus titer. Our data support the hypothesis that a viruliferous thrips is more likely to transmit multiple times if it harbors a high titer of virus. This quantitative relationship provides new insights into the biological parameters that may influence the spread of TSWV by thrips.

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Tomato spotted wilt virus (TSWV) is an economically important virus that is transmitted in a persistent propagative manner by its thrips vector, Frankliniella occidentalis. Previously, we found that a soluble form of the envelope glycoprotein G(N) (G(N)-S) specifically bound thrips midguts and reduced the amount of detectable virus inside midgut tissues. The aim of this research was to (i) determine if G(N)-S alters TSWV transmission by thrips and, if so, (ii) determine the duration of this effect. In one study, insects were given an acquisition access period (AAP) with G(N)-S mixed with purified virus and individual insects were assayed for transmission. We found that G(N)-S reduced the percent of transmitting adults by eightfold. In a second study, thrips were given an AAP on G(N)-S protein and then placed on TSWV-infected plant material. Individual insects were assayed for transmission over three time intervals of 2 to 3, 4 to 5, and 6 to 7 days post-adult eclosion. We observed a significant reduction in virus transmission that persisted to the same degree throughout the time course. Real-time reverse transcription polymerase chain reaction analysis of virus titer in individual insects revealed that the proportion of thrips infected with virus was reduced threefold when insects were preexposed to the G(N)-S protein as compared to no exposure to protein, and nontransmitters were not infected with virus. These results demonstrate that thrips transmission of a tospovirus can be reduced by exogenous viral glycoprotein.

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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.

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.

The transmission efficiency of Papaya ringspot virus (PRSV) by three aphid vectors (i.e., Aphis gossypii, A. craccivora, and Myzus persicae) was studied. Efficiency was measured by single-aphid inoculation, group inoculation (using five aphids), duration of virus retention, and the number of plants following a single acquisition access period (AAP) to which the aphids could successfully transmit the virus. Single-aphid inoculation studies indicated that M. persicae (56%) and A. gossypii (53%) were significantly more efficient in transmitting PRSV than A. craccivora (38%). Further, in the former two species, the time required for initiation of the first probe on the inoculation test plant was significantly shorter compared to A. craccivora. PRSV transmission efficiency was 100% in all three species when a group of five aphids were used per plant. There was a perceptible decline in transmission efficiency as the sequestration period increased, although M. persicae successfully transmitted PRSV after 30 min of sequestration. A simple leaf-disk assay technique was employed for evaluating the transmission efficiency of three species of aphids. The results of leaf-disk assays also indicated that A. gossypii (48%) and M. persicae (56%) were more efficient PRSV vectors than A. craccivora. Using leaf-disk assays, the ability of individual aphids to inoculate PRSV serially to a number of plants was studied. Following a single AAP on an infected leaf, M. persicae was more efficient than the other two species with 52.5% transmission after the first inoculation access period (IAP). However, its inoculation efficiency significantly decreased with the second and subsequent IAPs. A. gossypii was able to transmit PRSV sequentially up to four successive leaf disks, but with significantly declining efficiency. Since A. gossypii is reported to be the numerically dominant vector in south India in addition to being a more efficient vector capable of inoculating PRSV to multiple plants, it should be the target vector for control strategies.