Novel strains of Tomato Spotted Wilt Orthotospovirus (TSWV) are transmitted by western flower thrips in a context-specific manner

Senthilraja Chinnaiah; Arinder K Arora; Kiran R Gadhave

doi:10.1371/journal.pone.0323037

. 2025 Jul 10;20(7):e0323037. doi: 10.1371/journal.pone.0323037

Novel strains of Tomato Spotted Wilt Orthotospovirus (TSWV) are transmitted by western flower thrips in a context-specific manner

Senthilraja Chinnaiah ^1,^2,³, Arinder K Arora ^2,³, Kiran R Gadhave ^2,^3,^*

Editor: Sumit Jangra⁴

PMCID: PMC12244803 PMID: 40638634

Abstract

Novel resistance breaking (RB) strains of tomato spotted wilt orthotospovirus (TSWV) capable of disrupting single gene resistance in tomato (Sw-5b) and pepper (Tsw) have been reported worldwide. Thrips, a supervector of TSWV, transmit these strains in a suite of specialty and staple food crops across the globe. However, transmission biology of RB strains remains virtually unexplored. We investigated various transmission parameters viz. inoculation efficiency, putative sex-specific differences in inoculation, virus accumulation, and source sink relationships to dissect these interactions. Six novel strains of TSWV, namely Tom-BL1, Tom-BL2, Tom-CA, Tom-MX, Pep-BL and Non-RB, transmitted by western flower thrips (WFT) were used and thrips were allowed four 24h consecutive inoculation accession periods (IAPs). Our results show that most strains were inoculated at all four IAPs, however, their rates differed across IAPs. Overall, WFT had highest inoculation efficiency at the first and lowest at the second IAP. Female thrips carried higher virus titers; however, males were better at inoculating TSWV. Furthermore, we did not find significant positive correlations in virus titers between the tissues used for TSWV acquisition, thrips and thrips-inoculated leaf discs. Males inoculated RB strains at 87% efficiency whereas Non-RB strain at 80% efficiency. Female thrips were 77% and 75% efficient at inoculating RB and Non-RB strains, respectively. This study furnishes new insights into the transmission biology of TSWV RB strains, especially from inoculation and thrips sex perspectives, and provides a baseline for future molecular studies surrounding ever evolving novel TSWV strains.

Introduction

Tomato spotted wilt orthotospovirus (TSWV), also known as Orthotospovirus tomatomaculae, is a type member of the Orthotospovirus genus in the family Tospoviridae. TSWV is recognized as the most widespread plant-infecting RNA virus [1]. It affects more than 1,000 agriculturally valuable crops, including tomatoes and peppers, and causing significant economic losses by reducing both yield and quality of agricultural product [2]. In recent decades, two resistance genes, Sw-5b and Tsw, have been identified in tomato and pepper, respectively, and utilized against TSWV through resistance breeding strategies [3–6]. Tomato and pepper cultivars carrying these genes exhibited strong and prolonged resistance to TSWV. However, the intensive cultivation of resistant cultivars in recent decades has driven the global emergence of resistance-breaking (RB) strains of TSWV, now reported in the United States, Mexico, Italy, Australia, China, Argentina, Spain, Hungary, South Africa, Turkey, South Korea, and Brazil [7–19].

TSWV is transmitted in a persistent and propagative manner by plant-feeding insect vectors, namely thrips (Thysanoptera: Thripidae) [20]. Among different thrips species, Frankliniella occidentalis (Western flower thrip, WTF) is considered as the most effective vector due to its high reproductive rate as well as concealed and polyphagous behavior [21]. After acquisition by the first and early second instar larvae, TSWV replicates in the midgut epithelia and adjacent cells of the midgut intestinal muscles, followed by invasion of the primary salivary glands. Viruliferous adults transmit the virus when they secrete saliva into plants while feeding [21–24]. Although males carry a lower viral titer, they are more efficient vectors than females [25], likely due to their greater mobility, distinct feeding behavior, and minimal leaf scarring [26]. In contrast, sedentary females cause more localized damage, which may obstruct virus movement and reduce transmission efficiency. WFT fails to transmit TSWV when they acquire the virus as adult, because the ingested virus accumulates in amorphous electron dense material in epithelial cells and fails to disseminate to other tissues [22].

TSWV infection has synergistic effects on the F. occidentalis fitness as it influences thrips behavior, and increases fecundity, survival, and longevity [27–29], which has been attributed partly to modulating metabolic and plant defense pathways in plants [30]. Most of these studies have used a single isolate of TSWV, however, we recently investigated the WFT fitness after acquisition of four resistance breaking (RB) and one non-resistance breaking (‘Non-RB’) strain. Our lab in an earlier study found that RB strains significantly increased WFT fitness measured via fecundity (i.e., number of offsprings), and adult period (i.e., first to last day of adulthood) compared to the Non-RB strain and non-viruliferous controls [31]. Furthermore, RB-viruliferous thrips transmitted TSWV more efficiently than the Non-RB strain [31]. The term ‘strain’ rather than ‘isolate’ is used to describe our TSWV strains because they cause distinct symptoms in tomato and/or pepper plants (i.e., host phenotype) and have some distinct genetic mutations [32]. This first-ever study on thrips transmission of RB strains suggested that vector-imposed selection pressures, besides single gene resistant hosts, may play an important role in the emergence and spread of new RB strains.

This follow-up study builds on our previous findings to gain deeper insights into the transmission biology of TSWV RB and non-RB strains. Using sequential inoculation assays—where viruliferous WFT feed on a series of leaf discs—we aim to test whether: (i) viral copy numbers differ across thrips sexes before and after the inoculation access period (IAP), as well as across the four sequentially inoculated leaf discs; (ii) a source-sink relationship exists among virus titers in acquisition leaf tissues, thrips, and thrips-inoculated leaf discs; and (iii) inoculation efficiency of TSWV strains, measured by the percentage of leaf discs infected by male versus female thrips, differs.

Methods

Frankliniella occidentalis colony maintenance

A colony of western flower thrips, F. occidentalis originally obtained from Diane Ullman at UC, Davis was used for transmission experiments. This non-viruliferous WFT colony was maintained on surface-sterilized green bean pods in semi-transparent plastic containers with a lid fixed with insect-proof mesh in the center at 25°C and a 16-h photoperiod as described in our prior study [31].

Maintenance of TSWV strains

A total of five RB and one Non-RB TSWV strains, isolated and characterized previously, were used in this study [17,19,31,32]. All strains were maintained through a cycle of mechanical and thrips transmission in live host plants in insect-proof cages kept in a greenhouse at 25°C and a 12-h photoperiod. Among the five RB strains, four, namely Tom-BL1, Tom-BL2, Tom-CA, and Tom-MX, were isolated from different sources (infected leaf tissue or fruits) and maintained in a tomato cultivar (cv. Celebrity) carrying the Sw-5b gene. Among the four tomato RB strains, Tom-BL1 and Tom-BL2 showed typical symptoms of TSWV such as chlorotic patches, concentric rings, and necrotic spots on leaves, while Tom-CA and MX showed puckering and mosaic molting of leaves. Furthermore, Tom-CA exhibited shoestring symptoms on leaves. The fifth RB strain, Pep-BL, was isolated from pepper and maintained in a cultivar containing the Tsw gene (cv. Procraft). The non-RB strain, which is unable to overcome resistance conferred by Sw-5b or Tsw, was originally isolated from pepper and maintained on the susceptible tomato cultivar ‘Hot-Ty’.

TSWV inoculation

TSWV inoculation experiments comprised of four consecutive IAPs of 24-h were performed using modified procedures from Rotenberg et al [25]. A cohort of ~100 first instar larvae (0–24 hour old) of WFT were allowed to acquire TSWV. For virus acquisition the larvae were given an acquisition access period (AAP) of 72-h on symptomatic leaves from plants (tomato or pepper) infected with TSWV in a petri dish as described by Chinnaiah et al [31] (Fig 1). Six such cohorts were used to acquire one strain each.

Fig 1 — Six cohorts each of approximately, ~ 100, 0-24 h old larvae were allowed a 72–h acquisition access period on symptomatic leaves infected with one of each of five RB strains and one Non-RB strain of TSWV, separately in a transparent petri-dish covered with insect mesh in the center. After 72h AAP, thrips were reared on surface sterilized green beans until adulthood. On second day after adult-eclosion, individual female and male (n = 10 each sex/strain) thrips were transferred to a sterile 2 ml centrifuge tube containing leaf disc (1 × 1 cm), separately and allowed to inoculate the virus for 24 h. Strain specific tomato or pepper leaf discs from TSWV negative plants confirmed via qRT-PCR were used. After 24 hours leaf disc (1 × 1 cm) was replaced, and insects were allowed a second IAP of 24 hours. Insects were given 4 consecutive IAPs to inoculate virus, successively. After each IAP, inoculated leaf discs were incubated on sterile water for 48- h in 96 well microtiter plate. Following incubation, TSWV copy number from inoculated leave discs were quantified through RT-qPCR. Further, TSWV copies was quantified from a subset of adults prior to IAP (n = 10/sex/strain) and post-IAP (from the individual insects used for consecutive inoculations event).

Following AAP, thrips were kept on sterilized clean bean pods until adulthood. On second day after adult-eclosion, total of 10 individual female and male (n = 10 each/strain) thrips, separated based morphometric characters [33], were transferred to a sterile 2 ml centrifuge tube (1 thrip/tube) containing leaf disc (1 × 1 cm) of either resistant tomato (cv. Celebrity [for Tom strains]) or pepper (Cv. Procraft [for Pep-BL strain]) or susceptible tomato (cv. Hot-Ty [for Non-RB strain]) and allowed to inoculate the virus for 24-h. A small piece (2 × 2 cm) of tissue paper was placed in the centrifuge tube to absorb moisture. The leaf discs were used one per thrips and each IAP, resulting in a total of 10 replicates/sex/IAP/strain for a total of 20 leaf discs/IAPs/strains. After 24-h of inoculation access period (IAP), leaf discs were replaced with fresh leaf discs to start the next 24 hours IAP, and inoculated leaf discs were floated on sterile nucleus free water for 48-h in a 96 well microtiter plate with an appropriate space between the samples. After the incubation, water from the leaf disc was wiped with sterile Kim wipes and leaf discs were stored at −80⁰C until the extraction of RNA. Likewise, four consecutive IAPs (IAP-I, II, III, and IV) were performed for all the six strains simultaneously (Fig 1).

RNA extraction and TSWV quantification

Total RNA from individual leaf discs was extracted using Tri-Reagent (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacture’ s protocol and stored at −80˚C until further use. Total RNA from individual female and male thrips (n = 10 for each sex) was extracted immediately after acquisition and completion of four consecutive IAPs using Quick Extract solution (Lucigen, Middleton, WI) as described by Chinnaiah et al [31].

A one-step RT-qPCR was performed using 20-μl reaction mixture containing 5-μl of TaqMan Fast Virus 1-Step Master Mix [4X] (Thermo Fisher Scientific, Waltham, MA, United States), 1-μl probe [20X] ([6 ~ FAM] CAGTGGCTCCAATCCT[BHQ1a~Q]) and 1-μl primer pair [10 pmol of each] (5′-AGAGCATAATGAAGGTTATTAAGCAAAGTGA-3′) and (5′-GCCTGACCCTGATCAAGCTATC-3′) targeting nucleocapsid (N) gene using QuantStudio 7 Pro system (Applied Biosystems, Waltham, MA, United States) with the following conditions: 50°C for 10 min, holding at 94°C for 5 min, followed by 40 cycles of 94°C for 10 s and 60°C for 30 s [31]. One ng RNA was used in each RT-qPCR to maintain the comparability between treatments. Virus copy numbers/ng of RNA were estimated using standard curve generated by known copy of tenfold serially diluted plasmid containing TSWV N gene [31].

Statistical analysis

All experimental data were analyzed using R version 3.6.0 [34]. Shapiro-wilk test was performed for virus copy number data to assess the assumption of normality and homogeneity [35]. Since the virus copy number data did not conform to normality, and was over-dispersed, a generalized linear mixed model (GLMM) from “glmmTMB” package [36] was used for viral copy analysis across different treatment groups. The best fit model was chosen based on the lowest AIC value to avoid overfitting. IAP wise, virus copy numbers in leaf discs were analyzed both separately by female and male thrips and combining copy number across different strains. Virus copy numbers of RB strains were compared with Non-RB strain using post-hoc Dunnett test using “DescTools” package [37]. Virus copy number in leaf disc inoculated by males and females within the strains was compared using student’s t-test. Furthermore, virus copies in leaf disc between four IAPs within the strain and between sexes were also analyzed using GLMM and treatment means were compared through post-hoc Tukey test using “multcomp” package [38]. A regression analysis was performed to determine a relationship between TSWV copy numbers in thrips, infected tissues, and thrips-inoculated leaf discs. Percent inoculation efficiency was analyzed using generalized linear mode (glm) followed by post-hoc Tukey test.

Results

Inoculation of different TSWV strains by WFT

Tom-BL1.

Both male and female thrips inoculated significantly higher copies of Tom-BL1 in IAP-I compared to IAP-II, III, and IV (P < 0.001; Fig 2a). Female thrips inoculated lowest virus numbers in IAP-II and males inoculated the lowest during IAP-III, followed by a slight increase in the IAP-IV for both sexes (Fig 2a).

Further, strain Tom-BL1 copy numbers inoculated by both females and males in IAP-I were significantly higher when compared to Non-RB copy numbers inoculated by females (P < 0.001; S1a Fig.) or males (P < 0.001; S1b Fig). When inoculation by males and females was combined together, Tom-BL1 titers were inoculated in significantly higher number during IAP-I (P < 0.001; S1c Fig) and IAP-IV (P = 0.004; S4c Fig) compared to Non-RB. A significant difference in Tom-BL1 copy number inoculated by females as compared to males was observed only in IAP-III (P = 0.015; S3d Fig).

Tom-BL2.

Virus copy number of Tom-BL2 inoculated by females and males was significantly higher in IAP-I than other IAP-I & II (P < 0.001; Fig 2b) and IAP-III (P < 0.05; Fig 2b). The lowest virus copies were inoculated at IAP-III and then increased slightly at IAP-IV for both females and males.

Furthermore, strain Tom-BL2 was inoculated in significantly higher copies by both females and males than Non-RB females (P < 0.001, P < 0.05; S1a Fig) or males (P < 0.001; S1b Fig) in IAP-I. Copy numbers of Tom-BL2 inoculated by males were significantly higher as compared to Non-RB copy numbers inoculated by females in IAP-II (P = 0.023; S2a Fig) and males in IAP-IV (P = 0.040; S4a Fig) IV, respectively. In addition, Tom-BL2 was inoculated in significantly higher numbers than Non-RB when F. occidentalis sex was disregarded in IAP-I (P < 0.001; S1c Fig) and IAP-IV (P = 0.004; S4c Fig). When inoculation was compared between sexes, females inoculated Tom-BL2 at a higher copy number than males in IAP-I (P = 0.042; S1d Fig).

Tom-CA.

For Tom-CA strains, significantly different number of virus copies were inoculated between the IAPs by both female and male (Fig 2c). The female in IAP-II inoculated the lowest virus copies and was significantly different from first and fourth IAP (P < 0.05, P < 0.01; Fig 2c), whereas male inoculated the lowest copy numbers at IAP-III and was statistically different from IAP-IV (P < 0.001; Fig 2c).

No significant difference in Tom-CA copy numbers was observed from Non-RB females and males in any of the IAPs (S1, S2, S3, S4 Fig). However, Tom-CA was inoculated in significantly higher numbers than Non-RB strain when F. occidentalis sex was disregarded in IAP-IV (P = 0.004; S4c Fig)

Tom-MX.

For the strain Tom-MX, lowest copy numbers were inoculated during second and first IAPs for females and males, respectively. However, no statistical difference was found between IAPs in females (P = 0.363; Fig 2d) and males (P = 0.384; Fig 2d).

No significant differences were found between viral copy numbers inoculated by males and females with Non-RB strain (S1, S2, S3, S4 Fig). Additionally, comparison between virus copy numbers inoculated by males and females did not show any significant difference in all the IAPs (S1d, S2d, S3d, S4d Fig).

Pep-BL.

For the Pep-BL strain, lowest copy numbers were observed during second and third IAPs for females and males, respectively. However, no statistical difference was found between IAPs of females (P = 0.067) or males (P = 0.384; Fig 2e).

Pep-BL, however, was inoculated at significantly lower copy numbers by males than Non-RB strain inoculated by females in IAP-I (P < 0.05; S1a Fig). In addition, Pep-BL copy numbers inoculated by males were significantly higher than those inoculated by females of same strain in IAP-II (P = 0.017; S2d Fig).

Non-RB.

For the Non-RB strain, female inoculated significantly higher virus copies at the first IAP compared to second and third IAPs (P < 0.01; Fig 2f). No significant difference was found in copy numbers inoculated by males between the IAPs (P = 0.912; Fig 2f).

Further Non-RB copy numbers inoculated by males were significantly higher than females only in IAP-II (P = 0.047; S2d Fig).

TSWV copies in adult thrips

Pre-IAP.

When compared to Non-RB strain copy numbers in infected female thrips, the males carried lower copies of Tom-CA (P = 0.016) and Pep-BL strain (P = 0.028; Fig 3a), and males carried higher copies of Tom-BL2 strain compared to Non-RB strain copies in male (P < 0.001; Fig 3b). No significant difference was observed when TSWV copies of other RB strains carried by males or female were compared to copies of Non-RB strain carried by males or females (Figs 3a & 3b). When data were combined for males and females, the virus copies of Tom-BL2 were the highest in F. occidentalis adults and therefore significantly different from Non-RB strain copies carried by thrips (P = 0.05, Fig 3c). No statistical difference was observed between males and females for any strain (Fig 3d).

Post-IAP.

The highest and lowest virus copies were found in females of Tom-BL1 and Pep-BL strain, respectively and copy numbers in both treatments were significantly different from Non-RB strain copy numbers in females (P < 0.01, P < 0.05, Fig 4a) and males (P < 0.05, Fig 4b). When data were combined for males and females, the RB strain Tom-BL1 found to have higher virus copies; while Pep-BL had the lowest virus copies in thrips and were statistically different when compared to Non-RB strain (P < 0.05, Fig 4c). None of the other strains significantly differed from Non-RB strain (Fig 4c). No statistically significant difference was found between males and females carrying different TSWV strains (Fig 4d).

Fig 4 — Post-IAP average copy number of different RB-TSWV strains carried by male and female F. *occidentalis* compared to Non-RB strain carried by either **(a)** female; or **(b)** male; (c) Post-IAP average copy number of different strains harbored by *F. occidentalis* (male and female combined) compared to Non-RB strain carried by *F. occidentalis* (male and female combined); (d) Post-IAP average copy number of different TSWV strains carried by female and male compared within the strains. Asterisks indicate significant differences at α = 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001).

Relationship of TSWV copies between source plant, insects, and inoculated leaf discs

Regression analysis revealed no correlation between virus copy numbers observed in female (F = 3.47; P = 0.135; R²= 0.46) (Fig 5a) or male (F = 3.61; P = 0.130; R²= 0.47) (Fig 5b) thrips and virus copies present in plant tissues that were used for virus acquisition. Similarly, there was no correlation between virus titers present in female (F = 1.27; P = 0.321; R² = 0.24) (Fig 5c) or male (F = 2.50; P = 0.188; R² = 0.38) (Fig 5d) thrips and virus copy numbers from thrips-inoculated leaf discs.

TSWV inoculation efficiency of F. occidentalis

Overall, F. occidentalis inoculated TSWV at a higher efficiency at the first IAP (i.e., IAP-I) regardless of sex or strain. Percent inoculation decreased at the second IAP and the reduction was relatively higher for females compared to males, although no statistical difference was found. The inoculation efficiency remained low for III-IAP for most strains before it increased again for IAP-IV (Fig 6).

F. occidentalis males inoculated Tom-BL1 (80% male vs 40% female) and Tom-CA (90% male vs 60% female) with higher efficiency than female at IAP-II (Fig 6a, 6c). However, at IAP-III, a steep increase in female efficiency over male was observed, which then eventually went up to 90–100% for both strains at IAP-IV. Tom-BL2 was inoculated with 100% efficiency by males during IAP-I and II. While the inoculation efficiency of females was substantially lower at IAP-II (50%) (Fig 6b), it went up to 90% at IAP-IV after reaching the same levels as males (30%) at IAP-III. Males inoculated Tom-MX at consistently higher rates across all four IAPs (90–100%), whereas females were relatively less efficient, particularly at second and forth IAPs (70% each) (Fig 6d). Unlike other strains, Pep-BL inoculation at the first IAP was lower than 90% (80% male vs 60% female), which then increased by 10% in males and decreased by 10% in females at IAP-II before reaching 70% for both sexes. Despite the lower initial inoculation rate at the first IAP, females turned out to be more efficient than males at the fourth IAP (90% females vs 80% males) (Fig 6e). Both sexes inoculated Non-RB strain at 90–100% efficiency at IAP-I, after which efficiency dropped steeply in females to 50% vs 80% in males at IAP-II, followed by the recovery to 60% at IAP-III and 90% at IAP-III by females (Fig 6f).

Though there were differences in the inoculation of different strains across IAPs by males and females, no statistical differences were found. In most IAPs, male thrips inoculated virus either at a higher or similar rates to female thrips in all the RB strains, except for IAP-III or IV of Tom-CA, Pep-BL or Non-RB strains (Fig 6). Overall, the average % TSWV inoculation efficiency of adults at four IAPs infected with RB strains was 92, 72, 70, and 92%, while for Non-RB strain it was 95, 65, 65, and 85%, regardless of sex (S5 Fig). Furthermore, inoculation efficiency of females and males for RB strains was 77 and 87%, respectively, which was higher than that of females and males infected with Non-RB strain, 75 and 80%, respectively (S6 Fig). The inoculation efficiency in males surpassed that in females across the strains when all IAPs were combined. Furthermore, Tom-CA was inoculated with an efficiency of 92.5% by male thrips, while PepBL/Tom-BL2 had an inoculation efficiency of 67.5% by females, marking the highest and lowest inoculation efficiencies, respectively. (S7 Fig).

Discussion

Over the years, Sw-5b and Tsw resistant tomato and pepper cultivars have provided resistance and served as the first line defense against TSWV [3,4]. However, the worldwide emergence of novel resistance breaking strains is rendering this single gene resistance only partly effective and, in some instances, non-effective [39]. Despite this, there are virtually no studies investigating transmission biology of multiple strains in a greater detail. Building further on our prior work [31], we comprehensively assessed various aspects of transmission, namely, inoculation efficiency between sexes and virus accumulation of six novel strains of TSWV by western flower thrips, a predominant vector of TSWV. We found that TSWV is inoculated by WFT in all four IAPs. However, the efficiency of inoculation varied at each IAP and between sexes in a context-specific manner.

While thrips were able to consistently inoculate TSWV at each IAP, inoculation efficiency at each IAP was not consistent. A typical observed trend across most if not all strains was high inoculation at the first IAP, followed by a decline in the second, and finally the recovery in the third and fourth IAP. This is possibly due to a limited number of TSWV virions being available in the salivary vesicles [40] as a result of a lag to replenish new virus after the first IAP. The first IAP occurred immediately after adult eclosion which may be why the first IAP had higher inoculation efficiency. A window of 24-48h may be needed for TSWV to propagate in thrips, which plausibly led to increased efficiency at the third and fourth IAPs. This is consistent with a prior study by Van de wetering et al. [26] which reported that TSWV multiplies in adult thrips after their emergence and reached maximum titers in 4-day old thrips. The dynamics of virus titers before and after the inoculation access period (IAP) varied in a context-specific manner for Tom-BL1, Tom-BL2, and Pep-BL strains, but remained stable for the Tom-MX strain when compared to the Non-RB strain. Overall, the largely unchanged post-IAP virus titers across all strains indicate that virus levels do not significantly fluctuate after four successive inoculation events. This suggests that thrips, irrespective of sex, remain viruliferous throughout their lifespan—supporting earlier observations by Ullman et al. [41] and Nagata et al. [42].

Across all strains, male thrips were able to transmit TSWV higher than females, which is consistent with prior reports in this pathosystem [25,26]. It has been speculated that male feeding behavior, and higher mobility than females facilitate higher transmission rate. Females, on the contrary, due to their sedentary nature of feeding produce scars at the site of feeding i.e., necrotic spots, which prevent virus replication and impedes inoculation or transmission [43]. Interestingly, the TSWV silencing suppressor protein, NSs, which suppresses plant RNAi silencing machinery to facilitate TSWV infection, has been reported only in saliva of female thrips during feeding [44]. This was expected to facilitate TSWV inoculation by females better than males, but we didn’t observe this in our study. This is possibly because mobility and feeding behavior were likely to be key contributors, more than NSs, in determining inoculation efficiency. Furthermore, mounting of plant defenses via RNAi is likely to have been compromised in leaf discs as opposed to entire plants.

The thrips were able to acquire all strains of TSWV, however, the numbers of virus particles accumulated in the thrips varied (Fig 5 and 6). Glycoproteins, Gn and Gc on the TSWV M segment are known to be involved in virus binding to thrip gut, before virions enter the cells probably via receptor mediated endocytosis [45,46]. The differences in interaction of virus strains, particularly their glycoproteins with TSWV-interacting proteins (TIPs) of the vector likely to have impacted TSWV propagation [47]. A recent study reported that infection of TSWV in thrips resulted in increased expression of a cytochrome P450 monooxygenases gene -CYP24, which suppressed the insect immune system and helped with the virus propagation. RNAi of NSs in thrips depleted the CYP24 transcripts while RNAi of N and NSm transcripts failed to reduce CYP24 expression, highlighting a potential role of NSs in suppressing thrip immune system [48]. Pep-BL, an originally pepper infecting strain, has several unique point mutations in the NSs compared to tomato infecting RB strains and Non-RB strains (unpublished data) which likely to impact Pep-BL accumulation in F. occidentalis since its titer was lower than other strains (Fig 3 and 4). These differences in virus titer in the adult thrips could be partly attributed to difference in the NSs protein.

Overall, when data from both female and male thrips were combined, the RB strains showed significantly higher inoculation efficiency than the Non-RB strain. While these differences were not statistically different, their biologically implications are not clear. Based on prior studies on source-sink relationship, virus titer in plants and in the insects were expected to impact virus acquisition and inoculation, respectively. However, we did not observe any correlation between virus titer in plant tissue used for acquisition (source), thrips, and thrips-inoculated tissues (sink). This suggests that regardless of the number of viral copies acquired by thrips, the virus propagation in the vector influences titer available for inoculation. However, a threshold for successful inoculation (minimum number of copies needed to be acquired for successful inoculation and subsequent transmission) remain unknown. This could be further demonstrated by Pep-BL and Tom-MX strains. Although the number of virus particles inoculated by thrips were low, both strains were consistently inoculated at higher frequency (% inoculation) by thrips. This warrants a follow up study in which whether inoculation of leaf discs would translate into higher TSWV transmission in plants needs to be studied. Our prior study showed that RB strains increase the fitness of thrips compared to Non-RB strain. This follow up study suggests that this is likely due to the marginal increase in inoculation efficiency we observed in the present study. Additional factors include enhanced nutrient profiles and modified primary metabolism and defense response upon TSWV infection increasing vector fitness in terms of increased vector colonization and offspring [30].

For our experiments we used different plant cultivars as strains were maintained based on their resistance breaking ability in our laboratory. The virus inoculation experiment in the leaf disc lasted for only 24 hours as our focus was to determine inoculation efficiency of different strains by WFT. We believe that due to a short duration of experiment our results were not affected by genetic makeup of varieties. However, in future detailed studies can be conducted to compare acquisition and inoculation efficiency of WFT between different cultivars.

Transmission biology of TSWV RB strains, despite their worldwide emergence over the past decade, remains unexplored area of research. This work offers novel insights into thrips sex and inoculation parameters as potential determinants of TSWV RB transmission. However, more comprehensive investigations at transcriptional, protein and metabolic levels are needed to deepen our understanding of the intricate TSWV-thrips interactions and to devise tailored strategies for their management.

Supporting information

S1 Fig. TSWV copies.ng^-1 of RNA inoculated by Frankliniella occidentalis in IAP-I.

Average copy number of different TSWV strains transmitted by female (n = 10) and male (n = 10) thrips, and compared to Non-RB strain transmitted by either (a) female; or (b) male (c) average copy number of different strains inoculated by F. occidentalis (male and female combined) compared to Non-RB strain inoculated by F. occidentalis (male and female combined) (d) comparison of average copy number of different TSWV strains inoculated by female vs male within the strains. Asterisks indicate significant differences at α = 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001).

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S2 Fig. TSWV copies.ng^-1 of RNA inoculated by Frankliniella occidentalis in IAP-II.

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S3 Fig. TSWV copies.ng^-1 of RNA inoculated by Frankliniella occidentalis in IAP-III.

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S4 Fig. TSWV copies.ng^-1 of RNA inoculated by Frankliniella occidentalis in IAP-IV.

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S5 Fig. Inoculation efficiency of RB strains in four IAPs by Frankliniella occidentalis (TomBL1, Tom-BL2, Tom-CA, Tom-MX, and Pep-BL2 combined) compared with Non-.RB.

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S6 Fig. Overall percent inoculation efficiency of RB strains (TomBL1, Tom-BL2, Tom-CA, Tom-MX, and Pep-BL2 combined) compared to Non-RB strains by female and male Frankliniella occidentalis.

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S7 Fig. Percent inoculation efficiency of different strains by male and females of Frankliniella occidentalis after combining all the events.

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Acknowledgments

We thank undergraduate researchers Justice Crowder, Cayla Moore, and Jewels Hernandez for their help with thrips colony maintenance.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

Texas A&M AgriLife Research Insect Vectored Diseases Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

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PLoS One. doi: 10.1371/journal.pone.0323037.r001

Decision Letter 0

Sumit Jangra

Dear Dr. Gadhave,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: The manuscript “Novel strains of tomato spotted wilt orthotospovirus (TSWV) are transmitted by western flower thrips in a context-specific manner” reports on the differences of thrips (Frankliniella occidentalis - WFT) transmission efficiencies among several resistance-breaking (RB) TSWV strains. This study follows a previous investigation reporting positive effects of the same RB strains on the WFT fitness. This new study provides evidence that WFT transmits some RB strains at a higher rate than non-RB strains, and WFT males showed to be more efficient vectors than females.

The study deals with the understudied role of thrips vectors in spreading TSWV RB strains, but the obtained results are not so outstanding to be considered for publication on PLOS ONE. Also, the paper needs an extensive revision of both English language and data organisation. I would suggest the authors to re-write the paper and to consider its publication on a journal which is limited to the field of plant viruses.

INTRODUCTION:

Line 41: replace “produce” with “production”.

Lines 45-48: the sentences are redundant, re-write a unique sentence.

Line 50: “TSWV is transmitted in a persistent…”

Line 52: “…is considered the most effective vector due to its high reproductive rate as well as concealed and polyphagous behaviour.”.

Line 55: replace “infected” with “viruliferous”.

Lines 57-58: rephrase the sentence, the English is poor. Replace “transmitters” with “vectors”.

Line 58: “was attributed to their mobility, feeding behaviours, and less leaf scar productions”: explain how these parameters can influence the transmission of TSWV.

Lines 62-63: explain how TSWV influences the insect behaviour. The same for the aminoacid content: how the virus can affect the aa content? Also, some of the references 27-31 do not concern WFT: please check.

Lines 65-68: please, write a unique sentence and improve the English language. What does “adult period” mean? How did you measure it?

Line 69: “RB-viruliferous thrips transmitted TSWV more efficiently than the Non-RB strains”.

Lines 70-72: this sentence should be moved earlier in the ms: it is not the first time you mention the RB strains.

Lines 75-83: both English language and content should be improved, it is very hard to understand the meaning of the paragraph. For example, the sentence: “differences in inoculation rates of males and females to transmit different strains” does not make any sense. Also: which is the difference between point (i) and (iii)?

MATERIALS AND METHODS

Line 98: Replace “Of” with “Among the”.

Line 99: typical and characteristic: redundant.

Line 103-4: The sentence is not correct, rephrase.

Line 111: remove “in previous study”.

Line 135: inoculate the virus

Line 141-2: it is not clear what did you stored: the leaf discs, the water???? Rephrase

Lines 150-5: what about the volumes/concentrations of reagents? What about the PCR conditions?

RESULTS

Line 177: rephrase the title: you cannot inoculate a virus titre and insects do not inoculate viruses but they transmit viruses.

The Results section and the figures are very confusing. It is difficult to read and follow the description of the experimental results and to catch the final aim of the study (which should be the assessment of differences between the transmission features of RB and not-RB TSWV strains, if I have understood well….).

I strongly suggest to re-organise the section and to perform the statistical analyses differently. For example, I would suggest to report the main results in the following order:

1. Acquisition efficiency = virus titre in WFT before IAP. First, assess if there are any differences between male and female within each strain. Second, cumulate data of female and males for each RB strain, and for each RB strain assess if the virus titre is significantly lower/higher than non-RB strain.

2. Transmission efficiency = virus titre in the inoculated plants at 4 IAPs. First, assess if there are any differences between male and females within each strain and then cumulate the data when you can. Secondly, compare the virus titre of each RB strain with that of non-RB strain. Third, highlight at which IAP you measured the highest titres and the biggest differences.

3. Virus titre in WFT after IAP: why did you perform this analysis? Which is the aim? Did you want to measure the replication rates of the different virus strains within the vector? I do not think is related to the rest of the study. As well, I cannot see any comment on this in the Discussion section.

Figures: when you are comparing several items in the same graph, use letters (a,b,c etc…) instead of asterisks to indicate significant differences.

Figure 2: the figure lacks the most important result of this study: the significant differences in transmission rates between each RB strain and the non-RB strain.

DISCUSSION:

The Discussion needs to be deeply revised considering the changes I suggested in the Results.

I just highlight two main things that appeared wrong to me:

Lines 373-4: the sentence is not true for all the RB strains.

Lines 383-4: the sentence is contradictory.

Reviewer #2: All the experiments have been conducted in a meticulous manner. The manuscript has been well written. There are very few minor corrections. It may be amended for further improvement.

Line 19-23: Kindly break the sentence and rewrite for better understanding.

Line 24: Inoculated ? or transmitted? Kindly rewrite the sentence.

Line 25: Inoculation efficiency ? or transmission efficiency? Kindly revise through out the text.

Line 39-41: Kindly break the sentence and rewrite for better understanding.

Line 48: United states

Line 50: Modify “TSWV is transmitted by plant-feeding thrips in a persistent and propagative manner”

Line 70: The authors have mentioned the use of isolate instead of strain. However, there are some side heads with the word “strain” Kindly clarify. Modify if necessary.

Line 338: Correct “possibly”

Line 353: “…saliva of the female thrips”

Line 361-363: Rewrite the sentence for better understanding.

The discussion part may be strengthened more.

Reviewer #3: This manuscript addresses an important and timely topic—understanding the transmission biology of resistance-breaking (RB) strains of Tomato spotted wilt virus (TSWV) by Frankliniella occidentalis (Western Flower Thrips). The current work provides insights into inoculation efficiency, virus titers, and sex-specific transmission differences between RB and non-RB strains. The study is well-structured, and the results and objectives are clearly presented. It is mostly in good shape for publication and no further experiments are needed; however, a minor revision is required to improve the quality of the current version.

Minor revision:

98 Provide reference for the TSWV strains used in this study

107 What is the significance of using consecutive IAPs of 24h? How would the outcome vary if the IAPs were 24h, 48h, 72h, and 96h post-eclosion? How would the outcome vary between these scenarios? In the field conditions, transmission occurs in both the scenarios. Add a note in the discussion section.

121 What leaf disks were used? Are they different for tomato and pepper strains? Were they pre-tested for TSWV?

132 Provide reference for insect morphometric characters.

195 Please correct this statement. Virus copy number of Tom-BL2 inoculated by females and males was significantly higher in IAP-I than other IAP-I & II (P<0.001; Fig. 2b) and IAP-III (P<0.05; Fig.2b).

363. The word- plausible is used frequently. Consider using alternative terms to improve readability.

375 The regression analysis reveals no correlation between virus titer in plant tissue used for acquisition (source), thrips, and thrips-inoculated tissues (sink). Have you considered other factors, such as the spatial distribution of the virus, tissue heterogeneity, and sensitivity of virus quantification? Please explain this in detail.

The URLs for some references are leading to a different source. For example: reference 31 and 32. Please verify and correct these, check the remaining references for accuracy.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Kishorekumar Reddy

**********

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PLoS One. 2025 Jul 10;20(7):e0323037. doi: 10.1371/journal.pone.0323037.r002

Author response to Decision Letter 1

16 May 2025

Dr Sumit Jangra, Ph.D.

Academic Editor

PLOS One

May 15, 2025

Dear Dr Jangra,

We sincerely thank you and reviewers for their thoughtful, constructive, and insightful feedback, which has substantially improved the quality and clarity of our manuscript: Novel strains of tomato spotted wilt orthotospovirus (TSWV) are transmitted by western flower thrips in a context-specific manner. We have carefully addressed each of the concerns raised, including clarifications of the text, detailed explanations of the experimental design, and justifications for our analytical approach. Our point-by-point responses are provided below, with revisions indicated in blue. We greatly appreciate your time, effort, and valuable input throughout this review process.

In this study, we conducted an in-depth investigation into the transmission biology of tomato spotted wilt virus (TSWV), a globally significant agricultural pathogen, and its primary vector, the Western flower thrips, a widespread and damaging pest. The novelty of our work lies in two key aspects. First, this is only the second study—building upon our previous research—to examine the transmission biology of novel resistance-breaking strains of TSWV, which have emerged globally in recent years. Second, we delve into how these novel strains are transmitted by thrips in a context-specific manner, focusing on three critical determinants of transmission: TSWV inoculation efficiency, the sex of the thrips and source-sink relationships between virus titers.

We believe this manuscript aligns well with the journal's interdisciplinary scope, as it examines the intricate vector-virus interactions within a globally significant pathosystem. Given that TSWV is among the top ten most economically significant plant viruses worldwide, our findings are likely to attract the interest of a broad, interdisciplinary audience and contribute to advancing research in this field. The paper is not currently being considered for publication elsewhere. All authors have contributed to its production, and all have agreed on the final version. We hereby declare that there is no conflict of interest for any of the authors.

This research was funded by Texas A&M AgriLife Research Insect Vectored Diseases Grant. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Response to reviewer 1:

We thank Reviewer #1 for their thoughtful comments and constructive feedback. While we fully agree that the role of thrips in disseminating RB strains remains largely unexplored, we respectfully differ with their opinion regarding the manuscript’s suitability for publication in PLOS ONE. Consistent with the perspectives of Reviewers #2 and #3, we believe that the novelty of our findings, the interdisciplinary nature of the work spanning entomology, virology, and molecular biology, and the broad scientific scope of PLOS ONE make this manuscript well-suited for the journal. We have addressed all of Reviewer #1’s comments in a detailed, point-by-point manner. Where our interpretation differs, we have provided a clear and well-supported rationale, aligning with the constructive perspectives shared by Reviewers #2 and #3. We have taken great care in preparing this manuscript, and we appreciate the recognition of its clarity and rigor by the other reviewers. That said, all reviewer comments have been carefully considered, and we have made additional revisions to further enhance the manuscript’s clarity. A detailed explanation of our data organization strategy follows below.

INTRODUCTION:

Line 41: replace “produce” with “production”.

L41 The “produce” in this context is referred to agricultural produce such as fruits, vegetables etc. grown and sold for human consumption. “Yield and quality of production” would change the meaning as production is referred to the process of production rather than the end produce. To bring the clarity, the word “agricultural” is added.

Lines 45-48: the sentences are redundant, re-write a unique sentence.

L45-48 The sentence is rewritten.

Line 50: “TSWV is transmitted in a persistent…”

L49: Text is rephrased

Line 52: “…is considered the most effective vector due to its high reproductive rate as well as concealed and polyphagous behaviour.”

L51-52. Text is rephrased as suggested.

Line 55: replace “infected” with “viruliferous”.

L55: Text is rephrased

Lines 57-58: rephrase the sentence, the English is poor. Replace “transmitters” with “vectors”.

L56-58: Text is rephrased to improve clarity.

Line 58: “was attributed to their mobility, feeding behaviours, and less leaf scar productions”: explain how these parameters can influence the transmission of TSWV.

L56-59. An explanation of this is now provided in the revised text.

Lines 62-63: explain how TSWV influences the insect behaviour. The same for the amino acid content: how the virus can affect the aa content? Also, some of the references 27-31 do not concern WFT: please check.

Previous studies have shown that TSWV infection, both directly in the vector and indirectly through the plant host, enhances vector fitness by increasing behavior activity, fecundity, and longevity. For instance, Maris et al. (2004) reported that TSWV-infected plants were more attractive to western flower thrips (WFT) and led to higher offspring numbers. In terms of indirect effects, TSWV-infected plants were found to contain up to 15 times more free amino acids compared to healthy plants, potentially boosting egg production (Shrestha et al., 2012). Additionally, TSWV-infected WFT exhibited increased longevity and survival, and infected leaf discs were more attractive than healthy ones (Ogada et al., 2012). Furthermore, Nachappa et al. (2020) reported that TSWV infection enhances vector fitness by modulating host plant metabolic and defence pathways. All of these references are specific to thrips/TSWV pathosystem and have therefore been cited in the text (27-31). A description is slightly revised to improve clarity (L64).

Lines 65-68: please, write a unique sentence and improve the English language. What does “adult period” mean? How did you measure it?

“Adult period” is a commonly used entomological term refers to a period in days from the first day of adulthood through the death of adult insect. We reared adult thrips on clean bean plants from the first day of adulthood until their death, recording the number of days each adult lived (n = 10). This term is clearly defined in the text (L68-69).

Line 69: “RB-viruliferous thrips transmitted TSWV more efficiently than the Non-RB strains”.

L70: The sentence is rephrased.

Lines 70-72: this sentence should be moved earlier in the ms: it is not the first time you mention the RB strains.

L71. This is the first mention of the term 'strain' in regard to our strains in the main body of the manuscript. So, we believe this sentence is appropriately placed.

L76-83. We’ve restructured this paragraph to bring more clarity.

MATERIALS AND METHODS

Line 98: Replace “Of” with “Among the”.

L98. Text is rephrased as suggested.

Line 99: typical and characteristic: redundant.

L99. Only ‘typical’ is retained as suggested.

Line 103-4: The sentence is not correct, rephrase.

L103-105. Text is rephrased as suggested.

Line 111: remove “in previous study”.

L111. Text is removed as suggested.

Line 135: inoculate the virus

L134. Text is rephrased as suggested.

Line 141-2: it is not clear what did you stored: the leaf discs, the water???? Rephrase

L141. Text is rephrased as suggested.

Lines 150-5: what about the volumes/concentrations of reagents? What about the PCR conditions?

L150-157: These details had been provided in our prior publication Chinnaiah et al. (2023). However, they have been included in the text.

RESULTS

Line 177: rephrase the title: you cannot inoculate a virus titre and insects do not inoculate viruses but they transmit viruses.

Line 178: The text has been rephrased in alignment with Reviewer #2’s comments. We have been deliberate in our use of terminology to ensure accurate representation of our findings and to minimize the potential for misinterpretation. Specifically, we use the term “inoculation efficiency” rather than “transmission efficiency” because the thrips inoculated virus into leaf discs, not whole plants—a distinction critical to our experimental design, which focused on quantifying virus titers. This should not be conflated with the broader term “transmission biology”, which we use to refer to the full range of parameters assessed in this study, including inoculation efficiency, sex-specific differences in inoculation, virus accumulation, and source-sink relationships. These terminological distinctions have been applied consistently throughout the revised manuscript.

I strongly suggest to re-organise the section and to perform the statistical analyses differently. For example, I would suggest to report the main results in the following order:

The objective of our study, as clearly outlined in the Abstract and Introduction, was to investigate multiple transmission parameters—namely, inoculation efficiency, putative sex-specific differences in transmission, virus accumulation, and source–sink relationships—to better understand the transmission biology of RB and Non-RB strains. With this framework, we conducted a comprehensive analysis and presented the complex dataset in a clear, stepwise fashion across multiple figures. For example, in Figure 3, we examined virus titers of different strains acquired by each sex and compared them to the Non-RB strain acquired by females (Fig. 3a) and males (Fig. 3b). We further compared cumulative virus titers between RB and Non-RB strains by pooling data from both sexes (Fig. 3c), and analyzed sex-specific differences within each strain (Fig. 3d). We believe this sequential and logical data presentation enhances clarity and accessibility for readers—a sentiment echoed by Reviewers #2 and #3. To further aid navigation, Figure 1 provides a methodological overview; Figure 2 shows virus titers in leaf discs across four inoculation events; Figures 3 and 4 present pre- and post-IAP virus titers in thrips; Figure 5 illustrates source–sink relationships; and Figure 6 details inoculation efficiency dynamics across events and between sexes.

We defined inoculation efficiency based on the percentage of leaf discs successfully inoculated, not on virus titers (see revised Introduction for clarification). This parameter is clearly presented as percent inoculation efficiency in Figure 6. To maintain readability and avoid overwhelming the main text with excessive data, we have provided extensive supporting information in the supplementary files for readers who wish to explore the dataset in greater depth. For example, Supplementary Figure 5 presents percent inoculation data pooled across all strains for males and females, compared across different IAPs. In Supplementary Figure 7, we combined all IAPs to compare percent inoculation efficiency between sexes across strains. Additionally, we analyzed virus titers transmitted during each IAP: Supplementary Figures 1–4 show differences in virus copy numbers inoculated by males versus females during each IAP, along with cumulative analyses by sex for each inoculation event.

Viruliferous WFT transmit TSWV throughout their lifespan, as the virus replicates within the vector. In our study, we quantified virus titers post-IAP to assess whether WFT remain viruliferous after four consecutive transmission events, and whether there is a substantial reduction in virus titers over time. We thank Reviewer #1 for pointing out the absence of a brief discussion on this aspect. We have now addressed this in the revised manuscript (lines 345–351).

Figures: when you are comparing several items in the same graph, use letters (a,b,c etc…) instead of asterisks to indicate significant differences.

We intentionally used asterisks (*, **, ***) rather than letter annotations (a, b, c), as the asterisks allow us to indicate specific levels of statistical significance—p < 0.05, p < 0.01, and p < 0.001, respectively. This level of detail cannot be conveyed as precisely using letter-based groupings.

Figure 2: the figure lacks the most important result of this study: the significant differences in transmission rates between each RB strain and the non-RB strain.

This information is provided in Supplementary figures 1-4 due to the intricate nature of this data. The key findings from these figures have been presented in Results section.

DISCUSSION:

The Discussion needs to be deeply revised considering the changes I suggested in the Results.

The missing text on Post-IAP in discussion as suggested by Reviewer 1 is added.

I just highlight two main things that appeared wrong to me:

Lines 373-4: the sentence is not true for all the RB strains.

L379-380: The text is rephrased as suggested.

Lines 383-4: the sentence is contradictory.

L390-391: The text is modified for clarity.

Reviewer #2: All the experiments have been conducted in a meticulous manner. The manuscript has been well written. There are very few minor corrections. It may be amended for further improvement.

We sincerely thank Reviewer #2 for their compliments and constructive feedback. Their thoughtful assessment and supportive remarks are much appreciated. We have carefully addressed the minor corrections to further improve the manuscript.

Line 19-23: Kindly break the sentence and rewrite for better understanding.

L21: Sentence is split into two for better understandin

PLoS One. doi: 10.1371/journal.pone.0323037.r003

Decision Letter 1

Sumit Jangra

Dear Dr. Gadhave,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

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Academic Editor

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Partly

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #3: Yes

**********

The authors addressed most of the issues raised in the first version of the manuscript. I keep my opinion that the inoculation efficiency analysed for different RB strains, male vs female thrips, and different IAPs should be firstly put in correlation with the acquisition efficiency: if the thrips differently acquired TSWV at the above-mentioned conditions this would influence the results of the inoculation efficiency experiments. I cannot see any consideration in Results, Discussion, and Figure 2 about the comparison of pre- and post-IAP results.

I still have some minor comments:

INTRODUCTION:

Line 41: to the best ok my knowledge, “produce” is a verb not a noun. The authors should replace it with a noun, like product or production.

References 27-31: not all the references refer to F. occidentalis whereas the sentence in the text refers to this species only. This should be solved.

Line 78: “where viruliferous WFT feed on a series of leaf discs”

Line 103: “The non-RB strain, which is unable to overcome resistance conferred by Sw-5b or Tsw,”

Figures: you may use the letter annotation and indicate the level of significance into the legend, this would simplify the readability of the figures.

Line 382: the sentence: “Overall, when the females and males combined, RB strains inoculated with higher efficiency than the Non-RB stain.” still lacks the meaning.

Reviewer #3: (No Response)

**********

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Reviewer #1: No

Reviewer #3: Yes: Kishorekumar Reddy

**********

PLoS One. 2025 Jul 10;20(7):e0323037. doi: 10.1371/journal.pone.0323037.r004

Author response to Decision Letter 2

18 Jun 2025

Response to reviewer 1:

The manuscript “Novel strains of tomato spotted wilt orthotospovirus (TSWV) are transmitted by western flower thrips in a context-specific manner” reports on the differences of thrips (Frankliniella occidentalis - WFT) transmission efficiencies among several resistance-breaking (RB) TSWV strains. This study follows a previous investigation reporting positive effects of the same RB strains on the WFT fitness. This new study provides evidence that WFT transmits some RB strains at a higher rate than non-RB strains, and WFT males showed to be more efficient vectors than females.

We thank Reviewer 1 for recognizing the novel aspects of our study and for acknowledging how prior comments were addressed. We agree that acquisition efficiency is a critical factor influencing inoculation outcomes. To minimize variability in virus acquisition, we used synchronized cohorts of thrips larvae (both male and female), all subjected to identical acquisition access periods (AAPs) on the same infected leaf tissue.

While acquisition efficiency was measured separately for each group and strain, our primary objective was to assess relative inoculation efficiency between sexes and IAP events within each strain under standardized acquisition conditions. Thus, any observed differences in inoculation efficiency across RB strains, sexes, or IAP events likely reflect post-acquisition dynamics rather than differences in acquisition itself.

For these reasons, we present the data from an “inoculation-centric” perspective, as it directly reflects the central step in transmission, while still accounting for acquisition in a controlled and robust manner.

INTRODUCTION:

Line 41: to the best ok my knowledge, “produce” is a verb not a noun. The authors should replace it with a noun, like product or production.

L41: The word “produce” is replaced with “product”

References 27-31: not all the references refer to F. occidentalis whereas the sentence in the text refers to this species only. This should be solved.

L64. Following this suggestion, the previous reference number 29, which was the only one referring to F. fusca, has been removed.

Line 78: “where viruliferous WFT feed on a series of leaf discs”

L78. The text is rephrased.

Line 103: “The non-RB strain, which is unable to overcome resistance conferred by Sw-5b or Tsw,”

L103-104. The text is rephrased as suggested.

Figures: you may use the letter annotation and indicate the level of significance into the legend, this would simplify the readability of the figures.

We sincerely appreciate the reviewer’s suggestion. However, we believe the current figures effectively convey the significant differences between treatments and their levels in a clear and accessible manner. Including both letter annotations within the figures and significance levels in the legend may compromise overall readability and visual clarity. We have aimed to strike a balance between detail and interpretability and feel the current format best serves that purpose.

Line 382: the sentence: “Overall, when the females and males combined, RB strains inoculated with higher efficiency than the Non-RB stain.” still lacks the meaning.

L382-383. The text has been revised for more clarity.

We sincerely thank Reviewer #3, Dr. Kishorekumar Reddy, for recommending our manuscript for publication.

Attachment

Submitted filename: Cover letter.pdf

pone.0323037.s010.pdf^{(313.7KB, pdf)}

PLoS One. doi: 10.1371/journal.pone.0323037.r005

Decision Letter 2

Sumit Jangra

Novel strains of tomato spotted wilt orthotospovirus (TSWV) are transmitted by western flower thrips in a context-specific manner

PONE-D-25-17574R2

Dear Dr. Gadhave,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0323037.r006

Acceptance letter

Sumit Jangra

PONE-D-25-17574R2

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. TSWV copies.ng^-1 of RNA inoculated by Frankliniella occidentalis in IAP-I.

(TIF)

pone.0323037.s001.tif^{(697.6KB, tif)}

S2 Fig. TSWV copies.ng^-1 of RNA inoculated by Frankliniella occidentalis in IAP-II.

(TIF)

pone.0323037.s002.tif^{(5.4MB, tif)}

S3 Fig. TSWV copies.ng^-1 of RNA inoculated by Frankliniella occidentalis in IAP-III.

(TIF)

pone.0323037.s003.tif^{(700.3KB, tif)}

S4 Fig. TSWV copies.ng^-1 of RNA inoculated by Frankliniella occidentalis in IAP-IV.

(TIF)

pone.0323037.s004.tif^{(769.7KB, tif)}

S5 Fig. Inoculation efficiency of RB strains in four IAPs by Frankliniella occidentalis (TomBL1, Tom-BL2, Tom-CA, Tom-MX, and Pep-BL2 combined) compared with Non-.RB.

(TIF)

pone.0323037.s005.tif^{(3.1MB, tif)}

S6 Fig. Overall percent inoculation efficiency of RB strains (TomBL1, Tom-BL2, Tom-CA, Tom-MX, and Pep-BL2 combined) compared to Non-RB strains by female and male Frankliniella occidentalis.

(TIF)

pone.0323037.s006.tif^{(2MB, tif)}

S7 Fig. Percent inoculation efficiency of different strains by male and females of Frankliniella occidentalis after combining all the events.

(TIF)

pone.0323037.s007.tif^{(778.2KB, tif)}

Attachment

Submitted filename: Cover letter.pdf

pone.0323037.s010.pdf^{(313.7KB, pdf)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Novel strains of Tomato Spotted Wilt Orthotospovirus (TSWV) are transmitted by western flower thrips in a context-specific manner

Senthilraja Chinnaiah

Arinder K Arora

Kiran R Gadhave

Roles

Abstract

Introduction

Methods

Frankliniella occidentalis colony maintenance

Maintenance of TSWV strains

TSWV inoculation

Fig 1. Visual depiction of methods used in sequential inoculation of resistance breaking (RB) and Non-RB TSWV strains by Frankliniella occidentalis.

RNA extraction and TSWV quantification

Statistical analysis

Results

Inoculation of different TSWV strains by WFT

Tom-BL1.

Fig 2. Comparison of TSWV copies.ng-1 of RNA inoculated by Frankliniella occidentalis between IAPs for each strain.

Tom-BL2.

Tom-CA.

Tom-MX.

Pep-BL.

Non-RB.

TSWV copies in adult thrips

Pre-IAP.

Fig 3. TSWV copies.ng-1 of RNA in adult Frankliniella occidentalis prior to inoculation access period.

Post-IAP.

Fig 4. TSWV copies.ng-1 of RNA in adult Frankliniella occidentalis post inoculation access period.

Relationship of TSWV copies between source plant, insects, and inoculated leaf discs

Fig 5. Relationship between TSWV copies present in source plants, in viruliferous Frankliniella occidentalis, and inoculated leaf discs.

TSWV inoculation efficiency of F. occidentalis

Fig 6. Percent inoculation of different strains of TSWV by Frankliniella occidentalis over four consecutive IAPs.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Sumit Jangra

Roles

Author response to Decision Letter 1

Decision Letter 1

Sumit Jangra

Roles

Author response to Decision Letter 2

Decision Letter 2

Sumit Jangra

Roles

Acceptance letter

Sumit Jangra

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Fig 2. Comparison of TSWV copies.ng^-1 of RNA inoculated by Frankliniella occidentalis between IAPs for each strain.

Fig 3. TSWV copies.ng^-1 of RNA in adult Frankliniella occidentalis prior to inoculation access period.

Fig 4. TSWV copies.ng^-1 of RNA in adult Frankliniella occidentalis post inoculation access period.