Unexpected High Intragenomic Variation in Two of Three Major Pest Thrips Species Does Not Affect Ribosomal Internal Transcribed Spacer 2 (ITS2) Utility for Thrips Identification.

The mitochondrial cytochrome oxidase I gene (mtCO1) and the ribosomal internal transcribed spacer 2 region (ITS2) are among the most widely used molecular markers for insect taxonomic characterization. Three economically important species of thrips, Scirtothripsdorsalis, Thripspalmi, and Frankliniellaoccidentalis were selected to examine the extent of intragenomic variation within these two marker regions in the family Thripidae, and determine if this variation would affect the utility of markers in thrips molecular diagnostics. For each species, intragenomic (within individual) variation and intergenomic (among individuals) variation was assessed by cloning and sequencing PCR-amplified copies. Intergenomic variation was generally higher than intragenomic variation except in cases where intergenomic variation was very low, as in mtCO1 from S.dorsalis and F.occidentalis. Intragenomic variation was detected in both markers in all three of the thrips species, however, 2-3 times more intragenomic variation was observed for ITS2 than mtCO1 in both S.dorsalis and T.palmi. Furthermore, levels of intragenomic variation were low for both of the genes in F.occidentalis. In all of the three thrips species, no sex-based clustering of haplotypes was observed in either marker. Unexpected high intragenomic variation in ITS2 for two of three thrips species did not interfere with thrips diagnostics. However, caution should be taken in applying ITS2 to certain studies of S.dorsalis and T.palmi when high levels of intragenomic variation could be problematic or confounding. In such studies, mtCO1 may be a preferable marker. Possible reasons for discrepancies in intragenomic variation among genomic regions are discussed.

Within-Plant and Within-Field Distribution Patterns of Asian Bean Thrips and Melon Thrips in Snap Bean.

Asian bean thrips, Megalurothrips usitatus Bagnall, are a serious pest of vegetable crops, especially leguminous crops, across the Asian continent. In Florida, it is a new invasive pest of snap beans. In 2019, it was recorded for the first time in the United States in snap bean (Phaseolus vulgaris) fields. Another thrips species, melon thrips, Thrips palmi Karny, is also a serious pest that affects several vegetable crops. Within-plant and within-field distribution patterns of M. usitatus and T. palmi were determined in snap bean fields in southern Florida. The highest number of both thrips species (Asian bean thrips and melon thrips) in snap beans were in flowers, followed by leaves and pods. Both adults and immatures of these thrips exhibited regular to clumped distribution patterns in bean fields. Several statistical indices showed agreement in the distribution patterns of Asian bean thrips, melon thrips, and larvae, irrespective of sampling units and plot size, in three years of study. In most instances, the distribution of Asian bean thrips and melon thrips was aggregated. This study assessed the optimum sample size to accurately determine the population density of these thrips for management purposes. The results from this study will be useful for implementing targeted management programs against thrips pests, thereby reducing labor costs and time. This information will also help reduce agrochemical use.

Effect of silencing Thrips palmi Btk29A and COL3A1 on fitness and virus acquisition.

Thrips palmi (Thysanoptera: Thripidae) is a major agricultural pest infesting over 200 plant species. Along with direct injury caused by feeding, T. palmi spreads several orthotospoviruses. Groundnut bud necrosis orthotospovirus (GBNV, family Tospoviridae, genus Orthotospovirus) is the predominant orthotospovirus in Asia, vectored by T. palmi. It is responsible for almost 89 million USD losses in Asia annually. Several transcripts of T. palmi related to innate immune response, receptor binding, cell signaling, cellular trafficking, viral replication, and apoptosis are responsive to the infection of orthotospoviruses in thrips. Expression of T. palmi tyrosine kinase Btk29A isoform X1 (Btk29A) and collagen alpha-1(III) chain-like (COL3A1) are significantly regulated post-GBNV and capsicum chlorosis orthotospovirus infection. In the present study, T. palmi Btk29A and COL3A1 were silenced and the effect on virus titer and fitness was assessed. The expression of Btk29A and COL3A1 was significantly reduced by 3.62 and 3.15-fold, respectively, 24 h post-dsRNA exposure. Oral administration of Btk29A and COL3A1 dsRNAs induced 60 and 50.9% mortality in T. palmi. The GBNV concentration in T. palmi significantly dropped post-silencing Btk29A. In contrast, the silencing of COL3A1 led to an increase in GBNV concentration in T. palmi compared to the untreated control. To the best of our knowledge, this is the first report on the effect of silencing Btk29A and COL3A1 on the fitness and GBNV titer in T. palmi.

Groundnut Bud Necrosis Virus Modulates the Expression of Innate Immune, Endocytosis, and Cuticle Development-Associated Genes to Circulate and Propagate in Its Vector, Thrips palmi.

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.

Silencing of Thrips palmi UHRF1BP1 and PFAS Using Antisense Oligos Induces Mortality and Reduces Tospovirus Titer in Its Vector

Thrips palmi (Thysanoptera: Thripidae) is an important pest of vegetables, legumes, and ornamentals. In addition, it transmits several plant viruses. T. palmi genes associated with innate immunity, endocytosis-related pathways, and cuticular development are highly enriched in response to Groundnut bud necrosis orthotospovirus (GBNV, genus Orthotospovirus, family Tospoviridae) infection. As the previous transcriptomic study suggested the involvement of T. palmi UHRF1BP1 and PFAS in GBNV infection, these two genes were targeted for silencing using antisense oligonucleotides (ASOs), and the effects on thrips' fitness and virus acquisition were observed. Phosphorothioate modification of ASOs was carried out by replacing the nonbridging oxygen atom with a sulfur atom at the 3' position to increase nuclease stability. The modified ASOs were delivered orally through an artificial diet. Exposure to ASOs reduced the target mRNA expression up to 2.70-fold optimally. Silencing of T. palmi UHRF1BP1 and PFAS induced 93.33% mortality that further increased up to 100% with an increase in exposure. Silencing of T. palmi UHRF1BP1 and PFAS also produced morphological deformities in the treated T. palmi. GBNV titer in T. palmi significantly declined post-exposure to ASOs. This is the first-ever report of silencing T. palmi UHRF1BP1 and PFAS using modified ASO to induce mortality and impair virus transmission in T. palmi. T. palmi UHRF1BP1 and PFAS would be novel genetic targets to manage thrips and restrict the spread of tospovirus.

Development of a Polymerase Spiral Reaction-Based Isothermal Assay for Rapid Identification of Thrips palmi.

Thrips cause considerable economic losses to a wide range of food, feed, and forest crops. They also transmit several plant viruses. Being cryptic, it is often difficult to distinguish thrips species in crops and large consignments by conventional methods. Melon thrips (Thrips palmi Karny, Thysanoptera: Thripidae) is an invasive insect pest of vegetables, legumes, and ornamentals besides being vector to several viruses. It poses a threat to domestic and international plant biosecurity and can invade and establish in new areas. Here, we report a polymerase spiral reaction (PSR)-based isothermal assay for rapid, sensitive, specific, low-cost, and on-site detection of T. palmi. To the best of our knowledge, this is the first application of PSR in the identification of any insect species. A primer pair designed based on 3'-polymorphism of mtCOIII region can specifically identify T. palmi without any cross-reactivity with predominant thrips species. The assay uses crude lysate of a single thrips saving time and reagents involved in nucleic acid extraction. The presence of T. palmi is visualized by the appearance of bright fluorescence under ultraviolet light or a change in reaction color thus avoiding gel electrophoresis steps. The entire process can be completed in 70 min on-site using only an ordinary water bath. The assay is sensitive to detecting as little as 50 attograms of T. palmi template. The assay was validated with known thrips specimens and found to be efficient in diagnosing T. palmi under natural conditions. The described method will be useful for non-expert personnel to detect an early infestation, accidental introduction to a new area, restrict the spread of diseases and formulate appropriate management strategies.

Progression of Watermelon Bud Necrosis Virus Infection in Its Vector, Thrips palmi.

Thrips are important pests of agricultural, horticultural, and forest crops worldwide. In addition to direct damages caused by feeding, several thrips species can transmit diverse tospoviruses. The present understanding of thrips-tospovirus relationships is largely based on studies of tomato spotted wilt virus (TSWV) and Western flower thrips (Frankliniella occidentalis). Little is known about other predominant tospoviruses and their thrips vectors. In this study, we report the progression of watermelon bud necrosis virus (WBNV) infection in its vector, melon thrips (Thrips palmi). Virus infection was visualized in different life stages of thrips using WBNV-nucleocapsid protein antibodies detected with FITC-conjugated secondary antibodies. The anterior midgut was the first to be infected with WBNV in the first instar larvae. The midgut of T. palmi was connected to the principal salivary glands (PSG) via ligaments and the tubular salivary glands (TSG). The infection progressed to the PSG primarily through the connecting ligaments during early larval instars. The TSG may also have an ancillary role in disseminating WBNV from the midgut to PSG in older instars of T. palmi. Infection of WBNV was also spread to the Malpighian tubules, hindgut, and posterior portion of the foregut during the adult stage. Maximum virus-specific fluorescence in the anterior midgut and PSG indicated the primary sites for WBNV replication. These findings will help to better understand the thrips-tospovirus molecular relationships and identify novel potential targets for their management. To our knowledge, this is the first report of the WBNV dissemination path in its vector, T. palmi.

A rapid field-based assay using recombinase polymerase amplification for identification of Thrips palmi, a vector of tospoviruses.

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.

Commodity risk assessment of Momordica charantia fruits from Suriname.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. M. charantia fruits originating from countries where Thrips palmi is known to occur qualify as high-risk plants. This Scientific Opinion covers the introduction risk for T. palmi posed by fruits of Momordica charantia L. imported from Suriname, taking into account the available scientific information, including the technical information provided by the National Plant Protection Organization of Suriname. The risk mitigation measures proposed in the technical dossier from Suriname were evaluated taking into account the possible limiting factors. An expert judgement is given on the likelihood of pest freedom taking into consideration the potential pest pressure in the field, the risk mitigation measures acting on the pest in the field and in the packinghouse, including uncertainties associated with the assessment. For T. palmi on M. charantia fruits from Suriname, an expert judgement is given on the likelihood of pest freedom following the evaluation of the risk mitigation measures acting on T. palmi, including any uncertainties. The Expert Knowledge Elicitation indicated, with 95% certainty that between 8,652 and 10,000 M. charantia fruits per 10,000 will be free from T. palmi.

Commodity risk assessment of Momordica charantia fruits from Thailand.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. Momordica fruits originating from countries where Thrips palmi is known to occur qualify as high-risk plants. This Scientific Opinion covers the introduction risk for T. palmi posed by fruits of Momordica charantia L. imported from Thailand, taking into account the available scientific information, including the technical information provided by the Department of Agriculture of Thailand. The risk mitigation measures proposed in the technical dossier from Thailand were evaluated taking into account the possible limiting factors. An expert judgement is given on the likelihood of pest freedom taking into consideration the potential pest pressure in the field, the risk mitigation measures acting on the pest in the field and in the packing house, including uncertainties associated with the assessment. For T. palmi on M. charantia fruits from Thailand, an expert judgement is given on the likelihood of pest freedom following the evaluation of the risk mitigation measures acting on T. palmi, including any uncertainties. The Expert Knowledge Elicitation indicated, with 95% certainty that between 9,496 and 10,000 M. charantia fruits/10,000 will be free from T. palmi.

Commodity risk assessment of Momordica charantia fruits from Honduras.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. Momordica fruits originating from countries where Thrips palmi is known to occur qualify as high risk plants. This Scientific Opinion covers the introduction risk for T. palmi posed by fruits of Momordica charantia L. imported from Honduras, taking into account the available scientific information, including the technical information provided by the National Service of Agrifood Health and Safety (SENASA) of Honduras. The risk mitigation measures proposed in the technical dossier from Honduras were evaluated taking into account the possible limiting factors. An expert judgement is given on the likelihood of pest freedom taking into consideration the potential pest pressure in the field, the risk mitigation measures acting on the pest in the field and in the packinghouse, including uncertainties associated with the assessment. For T. palmi on M. charantia fruits from Honduras, an expert judgement is given on the likelihood of pest freedom following the evaluation of the risk mitigation measures acting on T. palmi, including any uncertainties. The Expert Knowledge Elicitation indicated, with 95% certainty that between 9,406 and 10,000 M. charantia fruits per 10,000 will be free from T. palmi.

Commodity risk assessment of Momordica charantia fruits from Sri Lanka.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. Momordica fruits originating from countries where Thrips palmi is known to occur qualify as high-risk plants. This Scientific Opinion covers the introduction risk for T. palmi posed by fruits of Momordica charantia L. imported from Sri Lanka, taking into account the available scientific information, including the technical information provided by the National Plant Quarantine Service of Sri Lanka. The risk mitigation measures proposed in the technical dossier from Sri Lanka were evaluated taking into account the possible limiting factors. An expert judgement is given on the likelihood of pest freedom taking into consideration the potential pest pressure in the field, the risk mitigation measures acting on the pest in the field and in the packing house, including uncertainties associated with the assessment. For T. palmi on M. charantia fruits from Sri Lanka, an expert judgement is given on the likelihood of pest freedom following the evaluation of the risk mitigation measures acting on T. palmi, including any uncertainties. The Expert Knowledge Elicitation indicated, with 95% certainty that between 9831 and 10,000 M. charantia fruits/10,000 will be free from T. palmi.

Commodity risk assessment of Momordica charantia fruits from Mexico.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. Momordica fruits originating from countries where Thrips palmi is known to occur qualify as high risk plants. This Scientific Opinion covers the introduction risk for T. palmi posed by fruits of Momordica charantia L. imported from Mexico, taking into account the available scientific information, including the technical information provided by the National Service of Health, Safety and Agrifood Quality (Senasica) of Mexico. The risk mitigation measures proposed in the technical dossier from Mexico were evaluated taking into account the possible limiting factors. An expert judgement is given on the likelihood of pest freedom taking into consideration the potential pest pressure in the field, the risk mitigation measures acting on the pest in the field and in the packinghouse, including uncertainties associated with the assessment. For T. palmi on M. charantia fruits from Mexico, an expert judgement is given on the likelihood of pest freedom following the evaluation of the risk mitigation measures acting on T. palmi, including any uncertainties. The Expert Knowledge Elicitation indicated, with 95% certainty that between 9,492 and 10,000 M. charantia fruits per 10,000 will be free from T. palmi.

In silico analyses of molecular interactions between groundnut bud necrosis virus and its vector, Thrips palmi.

Groundnut bud necrosis virus (GBNV) is an economically important tospovirus transmitted by Thrips palmi (Thysanoptera: Thripidae). The current understanding of thrips-tospovirus interactions is largely based on the tomato spotted wilt virus-Frankliniella occidentalis relationship. Only limited information is available for the GBNV-T. palmi system. In the present study, available genome data of T. palmi and GBNV were used to predict the protein partners that may play a crucial role in the internalization of GBNV virions into thrips cells. Computational analyses showed that the GBNV precursor glycoprotein bears a signal peptide of 24 amino acids and a secondary cleavage site at position 434-435 separates the amino-terminal mature glycoprotein (GN) from the carboxyl-terminal glycoprotein (GC). Potential interactions of GBNV glycoproteins were predicted with T. palmi enolase, cathepsin, C-type lectin, clathrin and vacuolar ATP synthase subunit E. The in silico analyses suggested that C-type lectin is the primary cellular receptor to interact with GBNV-GN. After receptor binding, virus particles probably enter vector cells by clathrin-mediated endocytosis. This is the first in silico evidence of GBNV-T. palmi protein interaction.

Pest categorisation of Thrips palmi.

The EFSA Panel on Plant Health (PLH) performed a pest categorisation of Thrips palmi (Thysanoptera: Thripidae), for the EU. T. palmi is listed in Annex IAI of 2000/29 EC. Using molecular methods, cryptic speciation has been shown although no new species from the group have been formally described. Here, we consider T. palmi sensu lato as a defined species native to southern Asia, which has spread to tropical and subtropical countries in Asia, the Pacific, North, Central and South America, Africa and Australia. T. palmi has been reported from many different hosts in 20 botanical families; Cucurbitaceae, Solanaceae and Orchidaceae are of primary importance. T. palmi has been intercepted many times on plants from these families. Wild and cultivated hosts are widespread in the EU. However, as a subtropical and tropical species, only a small area of the EU provides climatic conditions where establishment may be possible outdoors. Several host plants are cultivated in glasshouses where conditions may be more favourable for establishment in year-round crops. There have been past outbreaks of T. palmi in EU glasshouses and outdoors in Portugal. T. palmi causes feeding and oviposition damage and populations in Asia are competent vectors of tospoviruses. Impacts could occur on many hosts, especially Cucurbitaceae, Solanaceae and ornamental plants particularly in glasshouses. Phytosanitary measures aim to prevent the entry of T. palmi specifically on cut flowers of Orchidaceae and fruits of Momordica and Solanum melongena. The species meets all the criteria assessed by the PLH Panel to satisfy the definition of a Union quarantine pest, while it does not satisfy all the criteria for it to be regarded as a Union regulated non-quarantine pest (RNQP).