Prevalence, symbiosis with Rickettsia, and transmission of Tomato yellow leaf curl virus of invasive Bemisia tabaci MED Q2 in Japan

Prevalence, symbiosis with Rickettsia, and transmission of Tomato yellow leaf curl virus of invasive Bemisia tabaci MED Q2 in Japan | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Prevalence, symbiosis with Rickettsia, and transmission of Tomato yellow leaf curl virus of invasive Bemisia tabaci MED Q2 in Japan Akiko Fujiwara, Hiroki Hagiwara, Maiko Tsuchimoto, Tsutomu Tsuchida This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3976000/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Apr, 2025 Read the published version in Microbes and Environments → Version 1 posted You are reading this latest preprint version Abstract The whitefly, Bemisia tabaci , is a notorious insect pest that transmits plant pathogenic viruses to a wide range of economically-important crops. An invasive genetic group of B. tabaci , Mediterranean Q2 (MED Q2), has recently spread to Europe, USA, and Asia. In this study, we investigated the prevalence of MED Q2 in Japanese agricultural sites and found that its distribution has expanded since it was first detected in 2013. Polymerase chain reaction analysis revealed that all MED Q2 individuals were infected with Rickettsia . Rickettsia titres increased during nymphal development, presumably in response to the nutritional needs of the host. Fluorescence in situ hybridisation analysis revealed that Rickettsia was densely located near Portiera -containing bacteriocytes at all growth stages. Rickettsia may therefore play an important role, such as supplying nutrients to the host, in cooperation with Portiera . Transfer experiments indicated that MED Q2 was as effective a vector for Tomato yellow leaf curl virus as MED Q1 and is therefore a high-risk agricultural pest. These results provide important insights into the biology and ecology of the invasive MED Q2 to effectively control its spread and minimise its impact on crops. Biological sciences/Zoology/Entomology Biological sciences/Microbiology/Bacteria/Symbiosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Globalisation and international trade facilitate the long-distance movement of non-native organisms beyond their natural geographic range [ 1 , 2 ]. The invasion of alien species has increased in recent decades [ 3 ], with detrimental effects on human health, ecosystems, and economic activities such as agriculture [ 4 , 5 ]. The sweet potato whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a phloem-sap-feeding pest of agricultural crops [ 6 ]. It comprises genetically distinct groups that exhibit different biological characteristics, such as habitat and susceptibility to insecticides, although they are morphologically indistinguishable [ 7 , 8 ]. Based on the mitochondrial cytochrome oxidase subunit I (mtCOI) gene sequences, they can be classified into 44 groups [ 9 ]. Although many genotypes are limited to certain regions, Mediterranean (MED) Q [ 10 , 11 ] and Middle East-Asia Minor 1 (MEAM1) [ 12 , 13 ], have spread globally via international transport from their original habitats in the Mediterranean region and the Middle East and Asia Minor, respectively [ 14 ]. A common characteristic of these invasive genetic groups is their high insecticide resistance. This could explain why they have expanded their range in agricultural fields and greenhouses, where higher amounts of insecticides are used, and have replaced existing indigenous species [ 15 – 17 ]. These genetic groups can cause substantial damage to a wide range of economically important crops by transmitting over 100 pathogenic plant viruses [ 18 , 19 ]. Because of these characteristics, the whitefly, represented by these genotypes, is considered one of the most seriously-invasive species in the world [ 20 ]. MED Q was classified into three subgroups, Q1, Q2, and Q3, using molecular phylogenetic analyses of the mtCOI gene [ 21 ]. MED Q2 originated in the Eastern Mediterranean and has expanded its distribution in Europe and the United States [ 22 – 27 ]. In recent years, the distribution of MED Q2 has expanded to Asia. The first MED Q2 in Asia was discovered in 2013 at a site in the Kanto District of Japan [ 28 ]. In 2015, MED Q2 was identified at two additional sites in the same district of Japan [ 29 ]. This included one site in which MED Q2 was not detected in the 2013 survey. In 2018, MED Q2 was first identified at two sites in northwestern and southeastern South Korea [ 30 ]. These results suggest that the range of MED Q2 continues to expand. Similar to other phloem-sap feeding insects, B. tabaci harbours endosymbiotic bacteria that are stably transmitted to the offspring via the ovaries [ 31 , 32 ]. All genetic groups of B. tabaci contain a primary endosymbiont, Portiera . It is believed that Portiera has an ancient infection with an insect ancestor [ 33 , 34 ]. In addition to Portiera , B. tabaci often harbours different endosymbiotic bacteria. To date, eight different bacteria, Hamiltonella , Cardinium , Rickettsia , Wolbachia , Arsenophonus , Frischea , Hemipteriphilus , and RiTBt, have been reported [ 35 – 42 ]. These bacteria are thought to have been acquired relatively recently and are collectively referred to as secondary symbionts (S-symbionts). The MED Q1 and MEAM1 genotypes harbour Portiera and Hamiltonella exclusively within bacteriocytes, which are specialised cells for endosymbiosis [ 43 – 45 ]. In these genotypes, Portiera and Hamiltonella are considered co-obligate symbionts, based on the virtually universal infection observed in their original populations [ 21 , 27 , 28 , 46 – 50 ] and the decrease in host fitness caused by symbiont elimination [ 51 , 52 ]. However, studies carried out in Italian, Israeli, Turkish, and Japanese populations have shown that Hamiltonella is not detected in MED Q2, which is closely related to the MED Q1 genotype [ 21 , 26 – 29 , 53 , 54 ]. Instead, Rickettsia is nearly fixed in all MED Q2 populations, suggesting its important role in replacing Hamiltonella in MED Q2. Previous studies have reported various effects of Rickettsia on B. tabaci , such as increased susceptibility to insecticides [ 55 ], increased capacity to transmit Tomato yellow leaf curl virus (TYLCV) [ 56 ], increased fitness benefits, and female bias [ 57 ]. However, these studies were conducted using the MEAM1 genotype. Therefore, little is known about the effects of Rickettsia on the MED Q2. In this study, we characterised MED Q2 and its symbiont, Rickettsia . We conducted a detailed survey on the distribution of MED Q2 and its infection status with symbiotic bacteria in the Japanese population. We also analysed the spatiotemporal dynamics of Rickettsia in MED Q2 using quantitative polymerase chain reaction (PCR) and in situ hybridisation. Finally, we analysed the retention of TYLCV in the body and the efficiency of its transmission to tomato plants in MED Q2. Results Prevalence of MED Q2 We collected B. tabaci samples from 15 sites in the Kanto District of Japan (Supplementary Fig. S1 ) between 2016 and 2021. Multiplex PCR detected three genetic groups: MED Q1, MED Q2, and JpL (Supplementary Table S1 ). MED Q1 was detected in all 15 sites. JpL was detected at only two sites (localities 14 and 15). MED Q2 was detected in 10 of the 15 sites. The identification of MED Q2 was confirmed using partial mtCOI sequencing. MED Q2 was detected at high frequencies at many sites, with regional differences ranging from 100–1.1% (Supplementary Table S1 ). Multi-year surveys were conducted at three sites in Gunma Prefecture: Isesaki (site 11), Maebashi (site 14), and Yoshioka (site 15). MED Q2 was not detected at Maebashi or Yoshioka in the first year of the survey but was detected at the same sites in the following year (Supplementary Table S1 and Fig. 1 ). MED Q2 was found at Isesaki and Maebashi at moderate rates over the three years. Endosymbiotic microbiota of MED Q2 Multiplex PCR detected Rickettsia in all individuals in the MED Q2 population, in addition to Portiera (Fig. 1 ). Some MED Q2 also harboured Wolbachia . The infection rate of Wolbachia varied between regions or collection dates; Wolbacia was never detected at some sites (nos.14 and 15, Fig. 1 ). The S-symbionts, Hamiltonella , Cardinium , Arsenophonus , and Hemipteriphilus , were not detected in any of the individuals. Population dynamics of Portiera and Rickettsia in host developmental stages Portiera titre continued to increase throughout the nymphal stages, reaching its peak at the fourth instar nymphs in males and at 15th-day-adult in females (Fig. 2 ), and then began to decrease. The Rickettsia showed similar population dynamics to Portiera . This increased throughout the nymphal stage in both females and males. The bacterial population was substantially lower in males than in females at all adult stages (Fig. 2 ). In males, Rickettsia populations decreased rapidly from 1 d after adult eclosion, whereas in females the population size remained high 30 d after eclosion. In vivo localization of the symbionts in MED Q2 In adult females, Portiera was detected only in bacteriocytes (Fig. 3 A and 3 B). In contrast to Portiera , Rickettsia was not detected in the bacteriocytes. This distribution pattern was confirmed by observing bacteriocytes dissected from adult females one day after eclosion (Supplementary Fig. S2). Instead, Rickettsia was densely localised in close proximity to the bacteriocytes (Fig. 3 B). Similar localisation patterns were observed in first-instar, fourth-instar, and adult males one day after eclosion (Supplementary Fig. S3). Ovaries collected from adult females 5 d after eclosion contained eggs at the pre-vitellogenesis stage (Fig. 4 A) before the transfer of the bacteriocytes. Fluorescence in situ hybridization (FISH) analysis of the ovaries showed that Rickettsia was present in the eggs at this stage (Fig. 4 A). On the 15th day after adult eclosion, bacteriocytes containing Portiera were detected in the ovaries (Fig. 4 B). However, Rickettsia was not detected within or on the bacteriocytes. These results indicate that Rickettsia of MED Q2 is not present inside bacteriocytes and is not transmitted to the next generation via bacteriocytes, as Portiera is. Retention and transmission of TYLCV in MED Q2 Figure 5 shows the TYLCV titre in B. tabaci after 48 h of feeding on TYLCV-infected plants. Two-way analysis of variance (ANOVA) using generalised linear modelling (GLM) showed that TYLCV-IL titres tended to be higher in MED Q2 than in MED Q1 but were not statistically different. An interaction between sex and genetic groups was observed. In contrast, the TYLCV-Mld titres were significantly lower in MED Q2 than in MED Q1 (Fig. 5 ). For TYLCV-Mld, no interaction between sex and genetic group was observed. PCR analysis conducted in the transfer experiments showed that TYLCV-IL was transferred to Momotaro or Micro-Tom by MED Q1 and MED Q2 at comparable levels under 48-h inoculation access period (IAP) conditions (Table 1). When IAP was extended to 72 h in Momotaro, the transmission rate of TYLCV-IL increased in both MED Q2 and MED Q1. When TYLCV-Mld was transferred to Micro-Tom under 48-h IAP conditions, MED Q2 tended to be less efficient than MED Q1, but the differences were not statistically significant. When transferred to Momotaro for 72 h, MED Q2 efficiency was 100% and tended to be slightly more efficient than MED Q1. The disease symptom results were comparable to those of TYLCV PCR detection. No significant differences in the transmission rates were found between the MED Q1 and MED Q2 groups (Table 1). Discussion MED Q2 was first detected in 2013 at a site in Kanto District, Japan [ 28 ]. In 2015, MED Q2 was detected at two additional sites in the same area [ 29 ]. In the present study, we detected MED Q2 at ten sites in the same district (Supplementary Table S1 ). In a previous study, we also investigated two sites, Isesaki (locality no. 11) and Maebashi (locality no. 14) but did not detect MED Q2 (0/12 and 0/6 individuals, respectively) [ 28 ]. This suggests that MED Q2 has expanded its distribution area in Kanto District since 2013. MED Q2 was detected at Sites 11 and 14 over multiple years (Supplementary Table S1 and Fig. 1 ), suggesting that the MED Q2 population was established here and in the wider area of the Kanto District. MED Q2 appeared to have expanded its distribution to many countries. In South Korea, MED Q2 was first identified in 2018 [ 30 ]. In southern Italy, the distribution of MED Q2 has expanded since it was first discovered, and the dominant type was replaced MED Q1 by MED Q2 in only a few years (2010–2013) [ 26 ]. In the 2017–2019 surveys conducted in central and southern Italy and Sicily, MED Q2 accounted for 87.6% of the total samples collected from greenhouse crops [ 58 ]. The high temperature tolerance and insecticide resistance of MED Q2 are thought to have contributed to its spread in Italy [ 26 ]. Therefore, global warming and insecticide use may be responsible for the expanding distribution of the MED Q2 in Japan. To prevent the spread of MED Q2, effective insecticides and other methods must be identified. The infection status of MED Q2 has been previously reported in Israel [ 21 , 53 ], Turkey [ 27 , 54 ], and Italy [ 26 ]. The MED Q2 in these countries commonly indicated a high infection rate with Arsenophonus and Rickettsia , but a low infection rate with Wolbachia . The infection status of the Japanese MED Q2 group differed substantially from those of the other groups. In addition to the primary symbiont, Portiera , two types of S-symbionts, Rickettsia and Wolbachia , were detected in the Japanese MED Q2 (Fig. 1 ). The prevalence of Rickettsia infection was 100% throughout all survey periods; Wolbachia was only detected in some of the samples. Interestingly, Arsenophonus , which is highly prevalent in MED Q2 in other countries, has never been detected in the Japanese MED Q2 population. The possible reasons for this difference are as follows: (1) only MED Q2, which is uninfected with Arsenophonus , has entered Japan, and (2) Arsenophonus -uninfected MED Q2 has increased their population due to its better adaptability under Japanese field conditions. However, these hypotheses are not mutually exclusive. In future, a detailed analysis is necessary to clarify this issue. Rickettsia was nearly fixed in MED Q2 populations in all regions, suggesting possible important contributions to survival, reproduction, and so on. The importance of Rickettsia in MED Q2 is also suggested by its population dynamics in host insects; Rickettsia titres increased during nymphal development and then rapidly decreased in males from 1 d after eclosion (Fig. 2 ). These bacterial population dynamics have been observed in many obligate symbionts of various insect hosts, including Buchnera in the pea aphid ( Acyrthosiphon pisum ), Sodalis in cereal weevils ( Sitophilus oryzae ), and Portiera and Hamiltonella in B. tabaci MEAM1 [ 59 – 62 ]. This phenomenon has been interpreted as the regulation of nutrient-compensating symbionts to meet the dietary needs of host insects. The Rickettsia titre in females remained at higher levels for a longer duration than that in males (Fig. 2 ). This suggests that symbiotic populations are regulated differently depending on the sex of the host. It is conceivable that higher Rickettsia levels in females evolved to ensure vertical transmission, as proposed for the obligate symbiont Hamiltonella in the MEAM1 of B. tabaci [ 62 ]. Previous genomic studies on B. tabaci MEAM1 and MED Q1 have shown that Portiera alone is unable to synthesise essential amino acids that are deficient in the host diet [ 63 – 65 ]; and the enzymes responsible for producing essential amino acids are found across Portiera , a coexisting symbiont ( Hamiltonella or Arsenophonus ), and B. tabaci . Hence, metabolic intermediates must be transported between the symbionts and the host multiple times for production. These symbionts coexist within the same bacteriocyte [ 43 ], which seems to be adaptive for the efficient production of essential nutrients via intertwined metabolic pathways [ 62 ]. Incomplete metabolic pathways were found in Portiera for both MEAM1 and MED Q1 [ 66 , 67 ], suggesting that the loss of the enzymes in Portiera occurred before the divergence of these lineages. Therefore, it is likely that Portiera has incomplete metabolic pathways in the derived lineage MED Q2. In the present study, Rickettsia was not detected in the bacteriocytes of MED Q2 (Figs. 3 , 4 ; Supplementary S2 and S3), in contrast to the previously reported "confined type" in MEAM1 and MED Q2 [ 43 ]. Instead, Rickettsia aggregated in the regions proximal to Portiera -containing bacteriocytes at all growth stages (Fig. 3 ; Supplementary Fig. S3). The physical proximity of Rickettsia and Portiera in MED Q2 suggests a close interaction via nutrient metabolism. To identify their metabolic pathways and test this possibility, genomic analyses of Rickettsia and Portiera in MED Q2 are required. In MED Q1 and MEAM1, the co-obligate symbionts Portiera and Hamiltonella are passed on to the next generation by transferring a whole bacteriocyte bearing the symbionts to the developing egg [ 32 ]. Rickettsia in MED Q2 were detected in pre-vitellogenic oocytes (stage 1 egg as defined in [ 68 ]), which is the stage before the bacteriocyte enters the egg. A previous study reported that Rickettsia in MEAM1 attached to a bacteriocyte and transferred with the bacteriocyte to the developing egg [ 68 ]. However, in MED Q2, we did not observe Rickettsia on the bacteriocytes in eggs (Fig. 4 ). This result suggests that there is a mechanism in MED Q2 for the transmission of Rickettsia into eggs without the involvement of bacteriocytes. In the future, the invasion process of Rickettsia into early developing eggs should be clarified in detail using confocal and electron microscopy. The most serious damage to crops caused by B. tabaci is due to the transmission of TYLCV. Therefore, the ability to transmit TYLCV is a critical factor in determining the risk of whiteflies becoming an agricultural pest. According to previous studies, Israeli MED Q2 has low ability to transmit TYLCV because it lacks infection with Hamiltonella , whose GroEL facilitates TYLCV transmission [ 69 – 72 ]. This study showed that Japanese MED Q2, which is free of Hamiltonella infection, is an effective vector for both TYLCV-IL and TYLCV-Mld, similar to Hamiltonella -infected MED Q1. MED Q2 tended to have higher TYLCV-IL retention (Fig. 5 ) and transmission rates to plants (Table 1) than MED Q1. TYLCV-Mld retention was significantly lower in MED Q2 than in MED Q1 (Fig. 5 ). However, the transmission rates in MED Q2 were as high as those in MED Q1 (Table 1). These results indicate that the Japanese MED Q2 is a high-risk agricultural pest, comparable to MED Q1, and possesses efficient TYLCV transmission machinery independent of Hamiltonella . Rickettsia may play a critical role in the transmission. In a previous study on MEAM1, Rickettsia was suggested to accelerates the uptake of TYLCV into the haemolymph, which may enhance TYLCV transmission to tomato plants [ 56 ]. Certain proteins, such as GroEL, produced by Rickettsia , may be involved in the transmission efficiency of TYLCV in MED Q2, similar to Hamiltonella GroEL in MED Q1 [ 71 ]. To investigate the potential role of Rickettsia in the high efficiency of TYLCV transmission in the Japanese MED Q2, future experiments will require the elimination of Rickettsia through antibiotic treatment and analysis of its GroEL interaction with TYLCV. Elucidation of the mechanisms underlying TYLCV transmission may lead to the development of more effective control technologies. Materials and Methods Insect and plants Whiteflies were collected from fields in the Kanto region of Japan between 2016 and 2021 (Supplementary Table S1 and Fig. S1 ). All samples were immediately stored in 100% acetone until further use [ 73 ]. Laboratory strains of B. tabaci MED Q1 with Portiera , Hamiltonella and Cardinium [ 62 ] and MED Q2 with Portiera and Rickettsia (strain Maebashi, Locality no.14 in Supplementary Table S1 ) were used in this study. These strains were maintained on cabbage ( Brassica oleracea ) leaves at 25 ± 1°C and 40–60% relative humidity in a long-day regimen (16L: 8D). Two tomato plants ( Solanum lycopersicum ), 'Momotaro' (Takii, Kyoto, Japan) and 'Micro-Tom' (provided by the National Bio-Resource Project (NBRP) tomato program at the University of Tsukuba, Japan), were cultivated under the same temperature and humidity conditions as cabbage and used in infection experiments with TYLCV. TYLCV-Israel and Mild strains (TYLCV-IL and TYLCV-Mld) were infected and maintained in tomato plants (cv. House-Momotaro) (Takii). All methods involving plants were carried out in accordance with relevant guidelines. Identification of genetic groups and the symbionts of B. tabaci Experiments and classification using multiplex polymerase chain reaction (PCR) were performed as previously described [ 29 ]. The total DNA of B. tabaci and its symbionts were individually extracted and amplified using three sets of primer mixes (mix for genotyping, mix 1 for symbionts, and mix 2 for symbionts), as listed in Supplementary Table S2. The mtCOI gene sequences were determined for 11 representative samples as previously described [ 28 ] with minor modifications. Partial sequences were amplified using PCR with KOD FX Neo (TOYOBO, Osaka, Japan) using specific primer sets (Supplementary Table S2). PCR products were gel-purified and sequenced directly. Sequences were assembled using Geneious Prime ver.2020.0.5 software (Dotmatics, Boston, USA). The sequence similarity of the detected genotypes was analysed using BLAST [ 74 ]. All sequences determined in this study were deposited in the DDBJ/NCBI/GenBank database under the following accession numbers: LC735029 – LC735039 for mtCOI and LC795726 and LC795727 for 16S rRNA genes of the symbionts of the laboratory MED Q2 strain. Quantitative PCR (qPCR) Eleven individuals of the MED Q2 laboratory strain were collected at each developmental stage from egg to adult. Eggs and first to fourth instar nymphs were collected without distinguishing between males and females because the sex of whiteflies is indistinguishable at immature stages. At the adult stage, whiteflies of both sexes were collected separately. Samples were preserved in 100% acetone [ 74 ] until DNA extraction. DNA was individually extracted from the samples individually using a NucleoSpin Tissue XS Kit (Takara Bio, Shiga, Japan). DNA was extracted from the eggs of 10 individuals. Portiera and Rickettsia were quantified in terms of 16S rRNA or gltA gene copies using the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, California, USA) with KOD SYBR qPCR Mix (Toyobo, Osaka, Japan) and specific primer sets (Supplementary Table S2). The qPCR conditions were 98°C for 2 min, followed by 40 cycles of 98°C for 10 s, each annealing temperature for 10 s, and a final extension at 68°C for 30 s. The qPCR with dissociation curve analysis was conducted using a standard curve method, as described previously [ 75 ]. TYLCV was quantified using the same system targeting v1 , with specific primer sets (Supplementary Table S2). The qPCR conditions were 98°C for 2 min, followed by 40 cycles of 98°C for 10 s and 65°C for 10 s, and a final extension at 68°C for 30 s. Fluorescence in situ hybridization (FISH) Whole-body or dissected insect tissue specimens were fixed in Carnoy’s solution (EtOH: chloroform: glacial acetic acid, 6:3:1), bleached in 6% hydrogen peroxide in EtOH, and subjected to whole-mount FISH, as described [ 76 ]. Fluorochrome-labelled oligonucleotide probes are listed in Supplementary Table S2. The host cell nuclei were counterstained with 4,6-diamino-2-phenylindole (DAPI). Observations were performed using a laser scanning confocal microscope LSM880 (Carl Zeiss, Germany) and analysed using LSM ZEN2 software (Carl Zeiss, Germany). The specificity of in situ hybridisation was confirmed by the following control experiments: a no-probe control and an RNase digestion control, as previously described [ 75 ]. Comparison of TYLCV retention in MED Q1 and Q2 The experiments were performed as previously described [ 77 ] with modifications. Approximately 30 adult whiteflies (3–4 d after eclosion) of MED Q1 or MED Q2 were released on each of the two TYLCV (IL or Mld)-infected tomato plants in a plastic container with an insect-proof mesh. The insects were allowed to feed on the plants for 48 h to acquire TYLCV. Total DNA was extracted from each individual of B. tabaci using a simple extraction method as previously described [ 29 ]. The amount of TYLCV in the samples was measured using qPCR, as described above. Transmission of TYLCV Approximately 200 adult whiteflies (MED Q1 or MED Q2), 3–4 d after eclosion, were released on each of the two TYLCV (IL or Mld)-infected tomato plants in a plastic container with an insect-proof mesh. Insects were allowed to feed on the plants for 48 h to acquire the TYLCV strain. Ten adults were collected and transferred to a healthy tomato plant (cv. Momotaro, or Micro-Tom) with three true leaves. The insects were allowed to inoculate tomato plants with TYLCV for 48 h or 72 h. As in a previous study [ 78 ], we refer to this period as the inoculation access period (IAP) (Table 1). The experiments were repeated ten times. The inoculated plants were kept at 25 ± 1°C and 40–60% relative humidity in a long-day regimen (16L:8D). After 30 d, we checked the plants for symptoms of the disease (i.e. yellowing, leaf curling, and dwarfing). Subsequently, we collected the youngest leaves from each plant for DNA extraction, following the method described [ 79 ]. The presence and type of TYLCV were determined using multiplex PCR with the specific primers listed in Supplementary Table S2. The PCR conditions were 95°C for 5 min, followed by 35 cycles of 98°C for 10 s and 55°C for 30 s, and a final extension at 68°C for 60 s. Statistical analysis The Wilcoxon rank-sum test after Bonferroni correction was used to evaluate differences in the bacterial titres of Portiera and Rickettsia between the host sexes. A two-way ANOVA using a GLM with a Poisson error structure was adopted to evaluate the effect of host-sex (female or male), genetic group of the host (MED Q1 or MED Q2), and their interactions on TYLCV titres. Fisher's exact test was used to assess differences in the transmission rates of TYLCV between MED Q1 and MED Q2. This analysis was performed for each TYLCV-IL and TYLCV-Mld strain. All statistical analyses were performed using R software v. 4.2.1. [ 80 ]. Declarations Acknowledgements We thank D. Kawabata, H. Sakai, H. Uga, I. Kabasawa, K. Honda, K. Ikeda, K. Nakamura, K. Ohnishi, K. Tanaka, K. Taniguchi, K. Yokoyama, M. Watanabe, N. Murakami, S. Miki, S. Shimizu, T. Shiraishi, Y. Katayanagi, Y. Nakazawa, and Y. Utsuno for collecting whitefly samples; N. Haruyama for providing B.tabaci MED Q1; J. Ohnishi for providing TYLCV-infected tomato plants; and K. Kawabe, M. Abe, and N. Murakami for their technical assistance. Tomato seed clone (TOMJPF00001) was provided by University of Tsukuba, Tsukuba Plant Innovation Research Center, through the NBRP of the MEXT/AMED, Japan. Part of this study was supported by a GUCFW Research Grant for Regional Cooperation on Food Science and Wellness (to A.F.) and by JSPS KAKENHI Grant number 18K05673 (to T.T.). A.F. was supported by the Leading Initiative for Excellent Young Researchers (LEADER) Program. This work was the result of using research equipment shared in the MEXT Project for promoting public utilisation of advanced research infrastructure (Program for supporting the introduction of the new sharing system), Grant Numbers JPMXS0420600120 and JPMXS0420600121. Author contributions A.F. and T.T. designed the research; A.F., H.H., M.T., and T.T. performed the research and analysed the data; A.F. and T.T. wrote the paper. Data availability statement All data are included in the manuscript and supplementary sections. 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Table Table 1 Transmission rate of TYLCV in MED Q1 and Q2 Transmission rate (%) TYLCV Variety of tomato PCR detection Disease symptom strain a IAP b (recipient) MED Q1 MED Q2 p c MED Q1 MED Q2 p c IL 48 h Momotaro 50 50 1 50 50 1 IL 48 h Micro-Tom 60 70 1 60 70 1 IL 72 h Momotaro 70 90 0.582 60 80 0.629 Mld 48 h Micro-Tom 30 10 0.582 30 10 0.582 Mld 72 h Momotaro 70 100 0.211 70 100 0.211 a IL, TYLCV-Israel strain; Mld, TYLCV-Mild strain b Inoculation access period c The results of Fisher's exact test are shown. Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.pdf Cite Share Download PDF Status: Published Journal Publication published 30 Apr, 2025 Read the published version in Microbes and Environments → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3976000","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":278333901,"identity":"ebe383c9-ee72-4a4d-bf69-133087750575","order_by":0,"name":"Akiko Fujiwara","email":"data:image/png;base64,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","orcid":"","institution":"Gunma University","correspondingAuthor":true,"prefix":"","firstName":"Akiko","middleName":"","lastName":"Fujiwara","suffix":""},{"id":278333902,"identity":"f8b2a76e-c099-4b5c-9114-fdb968a716ad","order_by":1,"name":"Hiroki Hagiwara","email":"","orcid":"","institution":"Gunma University","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Hagiwara","suffix":""},{"id":278333903,"identity":"29175a15-d2b4-433b-a51e-d44524ca8b73","order_by":2,"name":"Maiko Tsuchimoto","email":"","orcid":"","institution":"University of Toyama","correspondingAuthor":false,"prefix":"","firstName":"Maiko","middleName":"","lastName":"Tsuchimoto","suffix":""},{"id":278333904,"identity":"42a7968d-b512-4626-a780-6dd8b092e51b","order_by":3,"name":"Tsutomu Tsuchida","email":"","orcid":"","institution":"University of Toyama","correspondingAuthor":false,"prefix":"","firstName":"Tsutomu","middleName":"","lastName":"Tsuchida","suffix":""}],"badges":[],"createdAt":"2024-02-21 14:48:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3976000/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3976000/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1264/jsme2.ME24095","type":"published","date":"2025-05-01T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52622475,"identity":"a2a8be2d-385f-4fec-8039-ab094e208647","added_by":"auto","created_at":"2024-03-13 17:14:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":31674,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the prevalence of MED Q2 and its infection status with secondary symbionts (S-symbionts) in Kanto District, Japan. Locality numbers correspond to those in Supplementary Fig. S1 and Table S1. The data indicates the total number of MED Q2 individuals collected from various crops listed in Supplementary Table S1. The percentage of MED Q2 (number of individuals/total number of whiteflies examined) is indicated by the size of the pie chart. The percentage value is at the top of the pie chart. The black dot indicates that the survey was conducted but MED Q2 was not detected. The infection status of the S-symbionts is shown in different colours: light red, infection with \u003cem\u003eRickettsia\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e; sky blue, infection with \u003cem\u003eRickettsia\u003c/em\u003e only.\u003c/p\u003e","description":"","filename":"Figs1.png","url":"https://assets-eu.researchsquare.com/files/rs-3976000/v1/d7de659b599c24786ae67a2e.png"},{"id":52622479,"identity":"b593099d-8642-49dd-9b96-a549b1fb6d23","added_by":"auto","created_at":"2024-03-13 17:14:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24135,"visible":true,"origin":"","legend":"\u003cp\u003ePopulation dynamics of symbionts in \u003cem\u003eBemisia tabaci\u003c/em\u003e MED Q2. Bacterial titres of \u003cem\u003ePortiera\u003c/em\u003e and \u003cem\u003eRickettsia \u003c/em\u003ewere measured using quantitative polymerase chain reaction in terms of 16S rRNA or \u003cem\u003egltA\u003c/em\u003e gene copies per insect. Each dot represents an individual; grey circles, eggs and nymphs; filled circles, adult females; open circles, adult males; n = 10 for eggs, n = 11 for others. The symbiont titre in an egg was calculated by averaging the acquired values of 10 individuals. The asterisks indicate statistically significant differences (P \u0026nbsp;\u0026lt; 0.001, Wilcoxon rank sum test after Bonferroni correction).\u003c/p\u003e","description":"","filename":"Figs2.png","url":"https://assets-eu.researchsquare.com/files/rs-3976000/v1/8fd3a43ab49c5c474740db22.png"},{"id":52622481,"identity":"ac8df6ad-108d-48a4-88c0-4656393e9f22","added_by":"auto","created_at":"2024-03-13 17:14:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1400270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo \u003c/em\u003elocalization of \u003cem\u003ePortiera\u003c/em\u003e (red) and \u003cem\u003eRickettsia\u003c/em\u003e(green) in \u003cem\u003eBemisia tabaci\u003c/em\u003e MED Q2. (A) The abdomen of a female 15 d after eclosion. A whole view of \u003cem\u003eB. tabaci \u003c/em\u003eis shown in the lower right. The area enclosed by the black square was observed. (B) Enlarged image of the area indicated by the blue square in (A). Host nuclear DNA is visualised in blue. Yellow arrowhead, \u003cem\u003eRickettsia,\u003c/em\u003e densely localised in close proximity to bacteriocytes. In (B), orthogonal views of Z-stack images are shown; red and green dashed lines indicate corresponding points in the orthogonal planes.\u003c/p\u003e","description":"","filename":"Figs3.png","url":"https://assets-eu.researchsquare.com/files/rs-3976000/v1/d12fcfb8b1b096610efb938e.png"},{"id":52622477,"identity":"6c130a79-5f8c-4700-8a3b-4992fe48f1b3","added_by":"auto","created_at":"2024-03-13 17:14:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":719489,"visible":true,"origin":"","legend":"\u003cp\u003e. Localization of \u003cem\u003eRickettsia\u003c/em\u003ein the ovary of MED Q2. (A) Ovarioles dissected from an adult female 5 d after eclosion. (B) Bacteriocyte just after entering the egg in an adult female 15 d after eclosion. Only the \u003cem\u003eRickettsia\u003c/em\u003e (white) signal is shown in the left images. The right images show the nuclear DNA (blue) and \u003cem\u003ePortiera\u003c/em\u003e (red) signals overlaid on the \u003cem\u003eRickettsia\u003c/em\u003e (green) signal. Yellow dashed lines indicate outlines of the egg. Orthogonal view of Z-stack images is shown. Red and green dashed lines indicate corresponding points in the orthogonal planes.\u003c/p\u003e","description":"","filename":"Figs4.png","url":"https://assets-eu.researchsquare.com/files/rs-3976000/v1/4e7c4ea49a8debe0c48f3202.png"},{"id":52622476,"identity":"e5cd898e-2751-4bb9-9a48-7a74f1bc39dd","added_by":"auto","created_at":"2024-03-13 17:14:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22426,"visible":true,"origin":"","legend":"\u003cp\u003eInfluence of host sex and genotype on the retention of TYLCV-IL (A) and TYLCV-Mld (B). The box plots represent the distribution of TYLCV titres per insect. Each dot represents an individual value. Blue circles, MED Q1; red triangles, MED Q2; Six and 10 individuals per group were used for the TYLCV-IL and TYLCV-Mld experiments, respectively. The results of two-way ANOVA using a generised linear model with a Poisson error structure are shown in the box (*P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figs5.png","url":"https://assets-eu.researchsquare.com/files/rs-3976000/v1/2c3349acdfbfaa2d32f23bd4.png"},{"id":83563579,"identity":"6c3c83b4-630b-4a8a-a2a3-08fe044ae2ba","added_by":"auto","created_at":"2025-05-28 14:22:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3196646,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3976000/v1/4f9cc751-9602-4b2f-ba2e-50cbde60a79f.pdf"},{"id":52622480,"identity":"56518a9d-6507-41b3-8098-be8c0cb13e87","added_by":"auto","created_at":"2024-03-13 17:14:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12825563,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3976000/v1/0345fece23f98acb401c42e2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Prevalence, symbiosis with Rickettsia, and transmission of Tomato yellow leaf curl virus of invasive Bemisia tabaci MED Q2 in Japan","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobalisation and international trade facilitate the long-distance movement of non-native organisms beyond their natural geographic range [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The invasion of alien species has increased in recent decades [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], with detrimental effects on human health, ecosystems, and economic activities such as agriculture [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe sweet potato whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e (Hemiptera: Aleyrodidae) is a phloem-sap-feeding pest of agricultural crops [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It comprises genetically distinct groups that exhibit different biological characteristics, such as habitat and susceptibility to insecticides, although they are morphologically indistinguishable [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Based on the mitochondrial cytochrome oxidase subunit I (mtCOI) gene sequences, they can be classified into 44 groups [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Although many genotypes are limited to certain regions, Mediterranean (MED) Q [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and Middle East-Asia Minor 1 (MEAM1) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], have spread globally via international transport from their original habitats in the Mediterranean region and the Middle East and Asia Minor, respectively [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A common characteristic of these invasive genetic groups is their high insecticide resistance. This could explain why they have expanded their range in agricultural fields and greenhouses, where higher amounts of insecticides are used, and have replaced existing indigenous species [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These genetic groups can cause substantial damage to a wide range of economically important crops by transmitting over 100 pathogenic plant viruses [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Because of these characteristics, the whitefly, represented by these genotypes, is considered one of the most seriously-invasive species in the world [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMED Q was classified into three subgroups, Q1, Q2, and Q3, using molecular phylogenetic analyses of the \u003cem\u003emtCOI\u003c/em\u003e gene [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. MED Q2 originated in the Eastern Mediterranean and has expanded its distribution in Europe and the United States [\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In recent years, the distribution of MED Q2 has expanded to Asia. The first MED Q2 in Asia was discovered in 2013 at a site in the Kanto District of Japan [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In 2015, MED Q2 was identified at two additional sites in the same district of Japan [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This included one site in which MED Q2 was not detected in the 2013 survey. In 2018, MED Q2 was first identified at two sites in northwestern and southeastern South Korea [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. These results suggest that the range of MED Q2 continues to expand.\u003c/p\u003e \u003cp\u003eSimilar to other phloem-sap feeding insects, \u003cem\u003eB. tabaci\u003c/em\u003e harbours endosymbiotic bacteria that are stably transmitted to the offspring via the ovaries [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. All genetic groups of \u003cem\u003eB. tabaci\u003c/em\u003e contain a primary endosymbiont, \u003cem\u003ePortiera\u003c/em\u003e. It is believed that \u003cem\u003ePortiera\u003c/em\u003e has an ancient infection with an insect ancestor [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In addition to \u003cem\u003ePortiera\u003c/em\u003e, \u003cem\u003eB. tabaci\u003c/em\u003e often harbours different endosymbiotic bacteria. To date, eight different bacteria, \u003cem\u003eHamiltonella\u003c/em\u003e, \u003cem\u003eCardinium\u003c/em\u003e, \u003cem\u003eRickettsia\u003c/em\u003e, \u003cem\u003eWolbachia\u003c/em\u003e, \u003cem\u003eArsenophonus\u003c/em\u003e, \u003cem\u003eFrischea\u003c/em\u003e, \u003cem\u003eHemipteriphilus\u003c/em\u003e, and RiTBt, have been reported [\u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39 CR40 CR41\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. These bacteria are thought to have been acquired relatively recently and are collectively referred to as secondary symbionts (S-symbionts).\u003c/p\u003e \u003cp\u003eThe MED Q1 and MEAM1 genotypes harbour \u003cem\u003ePortiera\u003c/em\u003e and \u003cem\u003eHamiltonella\u003c/em\u003e exclusively within bacteriocytes, which are specialised cells for endosymbiosis [\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In these genotypes, \u003cem\u003ePortiera\u003c/em\u003e and \u003cem\u003eHamiltonella\u003c/em\u003e are considered co-obligate symbionts, based on the virtually universal infection observed in their original populations [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR47 CR48 CR49\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e] and the decrease in host fitness caused by symbiont elimination [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. However, studies carried out in Italian, Israeli, Turkish, and Japanese populations have shown that \u003cem\u003eHamiltonella\u003c/em\u003e is not detected in MED Q2, which is closely related to the MED Q1 genotype [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Instead, \u003cem\u003eRickettsia\u003c/em\u003e is nearly fixed in all MED Q2 populations, suggesting its important role in replacing \u003cem\u003eHamiltonella\u003c/em\u003e in MED Q2. Previous studies have reported various effects of \u003cem\u003eRickettsia\u003c/em\u003e on \u003cem\u003eB. tabaci\u003c/em\u003e, such as increased susceptibility to insecticides [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], increased capacity to transmit \u003cem\u003eTomato yellow leaf curl virus\u003c/em\u003e (TYLCV) [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], increased fitness benefits, and female bias [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. However, these studies were conducted using the MEAM1 genotype. Therefore, little is known about the effects of \u003cem\u003eRickettsia\u003c/em\u003e on the MED Q2.\u003c/p\u003e \u003cp\u003eIn this study, we characterised MED Q2 and its symbiont, \u003cem\u003eRickettsia\u003c/em\u003e. We conducted a detailed survey on the distribution of MED Q2 and its infection status with symbiotic bacteria in the Japanese population. We also analysed the spatiotemporal dynamics of \u003cem\u003eRickettsia\u003c/em\u003e in MED Q2 using quantitative polymerase chain reaction (PCR) and \u003cem\u003ein situ\u003c/em\u003e hybridisation. Finally, we analysed the retention of TYLCV in the body and the efficiency of its transmission to tomato plants in MED Q2.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePrevalence of MED Q2\u003c/h2\u003e \u003cp\u003eWe collected \u003cem\u003eB. tabaci\u003c/em\u003e samples from 15 sites in the Kanto District of Japan (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) between 2016 and 2021. Multiplex PCR detected three genetic groups: MED Q1, MED Q2, and JpL (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). MED Q1 was detected in all 15 sites. JpL was detected at only two sites (localities 14 and 15). MED Q2 was detected in 10 of the 15 sites. The identification of MED Q2 was confirmed using partial \u003cem\u003emtCOI\u003c/em\u003e sequencing. MED Q2 was detected at high frequencies at many sites, with regional differences ranging from 100\u0026ndash;1.1% (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Multi-year surveys were conducted at three sites in Gunma Prefecture: Isesaki (site 11), Maebashi (site 14), and Yoshioka (site 15). MED Q2 was not detected at Maebashi or Yoshioka in the first year of the survey but was detected at the same sites in the following year (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). MED Q2 was found at Isesaki and Maebashi at moderate rates over the three years.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEndosymbiotic microbiota of MED Q2\u003c/h2\u003e \u003cp\u003eMultiplex PCR detected \u003cem\u003eRickettsia\u003c/em\u003e in all individuals in the MED Q2 population, in addition to \u003cem\u003ePortiera\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Some MED Q2 also harboured \u003cem\u003eWolbachia\u003c/em\u003e. The infection rate of \u003cem\u003eWolbachia\u003c/em\u003e varied between regions or collection dates; \u003cem\u003eWolbacia\u003c/em\u003e was never detected at some sites (nos.14 and 15, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The S-symbionts, \u003cem\u003eHamiltonella\u003c/em\u003e, \u003cem\u003eCardinium\u003c/em\u003e, \u003cem\u003eArsenophonus\u003c/em\u003e, and \u003cem\u003eHemipteriphilus\u003c/em\u003e, were not detected in any of the individuals.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePopulation dynamics of\u003c/b\u003e \u003cb\u003ePortiera\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eRickettsia\u003c/b\u003e \u003cb\u003ein host developmental stages\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003ePortiera\u003c/em\u003e titre continued to increase throughout the nymphal stages, reaching its peak at the fourth instar nymphs in males and at 15th-day-adult in females (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and then began to decrease. The \u003cem\u003eRickettsia\u003c/em\u003e showed similar population dynamics to \u003cem\u003ePortiera\u003c/em\u003e. This increased throughout the nymphal stage in both females and males. The bacterial population was substantially lower in males than in females at all adult stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In males, \u003cem\u003eRickettsia\u003c/em\u003e populations decreased rapidly from 1 d after adult eclosion, whereas in females the population size remained high 30 d after eclosion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003elocalization of the symbionts in MED Q2\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn adult females, \u003cem\u003ePortiera\u003c/em\u003e was detected only in bacteriocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast to \u003cem\u003ePortiera\u003c/em\u003e, \u003cem\u003eRickettsia\u003c/em\u003e was not detected in the bacteriocytes. This distribution pattern was confirmed by observing bacteriocytes dissected from adult females one day after eclosion (Supplementary Fig. S2). Instead, \u003cem\u003eRickettsia\u003c/em\u003e was densely localised in close proximity to the bacteriocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similar localisation patterns were observed in first-instar, fourth-instar, and adult males one day after eclosion (Supplementary Fig. S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOvaries collected from adult females 5 d after eclosion contained eggs at the pre-vitellogenesis stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) before the transfer of the bacteriocytes. Fluorescence \u003cem\u003ein situ\u003c/em\u003e hybridization (FISH) analysis of the ovaries showed that \u003cem\u003eRickettsia\u003c/em\u003e was present in the eggs at this stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). On the 15th day after adult eclosion, bacteriocytes containing \u003cem\u003ePortiera\u003c/em\u003e were detected in the ovaries (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, \u003cem\u003eRickettsia\u003c/em\u003e was not detected within or on the bacteriocytes. These results indicate that \u003cem\u003eRickettsia\u003c/em\u003e of MED Q2 is not present inside bacteriocytes and is not transmitted to the next generation via bacteriocytes, as \u003cem\u003ePortiera\u003c/em\u003e is.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eRetention and transmission of TYLCV in MED Q2\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the TYLCV titre in \u003cem\u003eB. tabaci\u003c/em\u003e after 48 h of feeding on TYLCV-infected plants. Two-way analysis of variance (ANOVA) using generalised linear modelling (GLM) showed that TYLCV-IL titres tended to be higher in MED Q2 than in MED Q1 but were not statistically different. An interaction between sex and genetic groups was observed. In contrast, the TYLCV-Mld titres were significantly lower in MED Q2 than in MED Q1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). For TYLCV-Mld, no interaction between sex and genetic group was observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePCR analysis conducted in the transfer experiments showed that TYLCV-IL was transferred to Momotaro or Micro-Tom by MED Q1 and MED Q2 at comparable levels under 48-h inoculation access period (IAP) conditions (Table\u0026nbsp;1). When IAP was extended to 72 h in Momotaro, the transmission rate of TYLCV-IL increased in both MED Q2 and MED Q1. When TYLCV-Mld was transferred to Micro-Tom under 48-h IAP conditions, MED Q2 tended to be less efficient than MED Q1, but the differences were not statistically significant. When transferred to Momotaro for 72 h, MED Q2 efficiency was 100% and tended to be slightly more efficient than MED Q1. The disease symptom results were comparable to those of TYLCV PCR detection. No significant differences in the transmission rates were found between the MED Q1 and MED Q2 groups (Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMED Q2 was first detected in 2013 at a site in Kanto District, Japan [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In 2015, MED Q2 was detected at two additional sites in the same area [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the present study, we detected MED Q2 at ten sites in the same district (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In a previous study, we also investigated two sites, Isesaki (locality no. 11) and Maebashi (locality no. 14) but did not detect MED Q2 (0/12 and 0/6 individuals, respectively) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This suggests that MED Q2 has expanded its distribution area in Kanto District since 2013. MED Q2 was detected at Sites 11 and 14 over multiple years (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), suggesting that the MED Q2 population was established here and in the wider area of the Kanto District.\u003c/p\u003e \u003cp\u003eMED Q2 appeared to have expanded its distribution to many countries. In South Korea, MED Q2 was first identified in 2018 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In southern Italy, the distribution of MED Q2 has expanded since it was first discovered, and the dominant type was replaced MED Q1 by MED Q2 in only a few years (2010\u0026ndash;2013) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In the 2017\u0026ndash;2019 surveys conducted in central and southern Italy and Sicily, MED Q2 accounted for 87.6% of the total samples collected from greenhouse crops [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The high temperature tolerance and insecticide resistance of MED Q2 are thought to have contributed to its spread in Italy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, global warming and insecticide use may be responsible for the expanding distribution of the MED Q2 in Japan. To prevent the spread of MED Q2, effective insecticides and other methods must be identified.\u003c/p\u003e \u003cp\u003eThe infection status of MED Q2 has been previously reported in Israel [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], Turkey [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], and Italy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The MED Q2 in these countries commonly indicated a high infection rate with \u003cem\u003eArsenophonus\u003c/em\u003e and \u003cem\u003eRickettsia\u003c/em\u003e, but a low infection rate with \u003cem\u003eWolbachia\u003c/em\u003e. The infection status of the Japanese MED Q2 group differed substantially from those of the other groups. In addition to the primary symbiont, \u003cem\u003ePortiera\u003c/em\u003e, two types of S-symbionts, \u003cem\u003eRickettsia\u003c/em\u003e and \u003cem\u003eWolbachia\u003c/em\u003e, were detected in the Japanese MED Q2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The prevalence of \u003cem\u003eRickettsia\u003c/em\u003e infection was 100% throughout all survey periods; \u003cem\u003eWolbachia\u003c/em\u003e was only detected in some of the samples. Interestingly, \u003cem\u003eArsenophonus\u003c/em\u003e, which is highly prevalent in MED Q2 in other countries, has never been detected in the Japanese MED Q2 population. The possible reasons for this difference are as follows: (1) only MED Q2, which is uninfected with \u003cem\u003eArsenophonus\u003c/em\u003e, has entered Japan, and (2) \u003cem\u003eArsenophonus\u003c/em\u003e-uninfected MED Q2 has increased their population due to its better adaptability under Japanese field conditions. However, these hypotheses are not mutually exclusive. In future, a detailed analysis is necessary to clarify this issue.\u003c/p\u003e \u003cp\u003e \u003cem\u003eRickettsia\u003c/em\u003e was nearly fixed in MED Q2 populations in all regions, suggesting possible important contributions to survival, reproduction, and so on. The importance of \u003cem\u003eRickettsia\u003c/em\u003e in MED Q2 is also suggested by its population dynamics in host insects; \u003cem\u003eRickettsia\u003c/em\u003e titres increased during nymphal development and then rapidly decreased in males from 1 d after eclosion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These bacterial population dynamics have been observed in many obligate symbionts of various insect hosts, including \u003cem\u003eBuchnera\u003c/em\u003e in the pea aphid (\u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e), \u003cem\u003eSodalis\u003c/em\u003e in cereal weevils (\u003cem\u003eSitophilus oryzae\u003c/em\u003e), and \u003cem\u003ePortiera\u003c/em\u003e and \u003cem\u003eHamiltonella\u003c/em\u003e in \u003cem\u003eB. tabaci\u003c/em\u003e MEAM1 [\u003cspan additionalcitationids=\"CR60 CR61\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. This phenomenon has been interpreted as the regulation of nutrient-compensating symbionts to meet the dietary needs of host insects. The \u003cem\u003eRickettsia\u003c/em\u003e titre in females remained at higher levels for a longer duration than that in males (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This suggests that symbiotic populations are regulated differently depending on the sex of the host. It is conceivable that higher \u003cem\u003eRickettsia\u003c/em\u003e levels in females evolved to ensure vertical transmission, as proposed for the obligate symbiont \u003cem\u003eHamiltonella\u003c/em\u003e in the MEAM1 of \u003cem\u003eB. tabaci\u003c/em\u003e [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious genomic studies on \u003cem\u003eB. tabaci\u003c/em\u003e MEAM1 and MED Q1 have shown that \u003cem\u003ePortiera\u003c/em\u003e alone is unable to synthesise essential amino acids that are deficient in the host diet [\u003cspan additionalcitationids=\"CR64\" citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]; and the enzymes responsible for producing essential amino acids are found across \u003cem\u003ePortiera\u003c/em\u003e, a coexisting symbiont (\u003cem\u003eHamiltonella\u003c/em\u003e or \u003cem\u003eArsenophonus\u003c/em\u003e), and \u003cem\u003eB. tabaci\u003c/em\u003e. Hence, metabolic intermediates must be transported between the symbionts and the host multiple times for production. These symbionts coexist within the same bacteriocyte [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], which seems to be adaptive for the efficient production of essential nutrients via intertwined metabolic pathways [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Incomplete metabolic pathways were found in \u003cem\u003ePortiera\u003c/em\u003e for both MEAM1 and MED Q1 [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], suggesting that the loss of the enzymes in \u003cem\u003ePortiera\u003c/em\u003e occurred before the divergence of these lineages. Therefore, it is likely that \u003cem\u003ePortiera\u003c/em\u003e has incomplete metabolic pathways in the derived lineage MED Q2. In the present study, \u003cem\u003eRickettsia\u003c/em\u003e was not detected in the bacteriocytes of MED Q2 (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Supplementary S2 and S3), in contrast to the previously reported \"confined type\" in MEAM1 and MED Q2 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Instead, \u003cem\u003eRickettsia\u003c/em\u003e aggregated in the regions proximal to \u003cem\u003ePortiera\u003c/em\u003e-containing bacteriocytes at all growth stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Supplementary Fig. S3). The physical proximity of \u003cem\u003eRickettsia\u003c/em\u003e and \u003cem\u003ePortiera\u003c/em\u003e in MED Q2 suggests a close interaction via nutrient metabolism. To identify their metabolic pathways and test this possibility, genomic analyses of \u003cem\u003eRickettsia\u003c/em\u003e and \u003cem\u003ePortiera\u003c/em\u003e in MED Q2 are required.\u003c/p\u003e \u003cp\u003eIn MED Q1 and MEAM1, the co-obligate symbionts \u003cem\u003ePortiera\u003c/em\u003e and \u003cem\u003eHamiltonella\u003c/em\u003e are passed on to the next generation by transferring a whole bacteriocyte bearing the symbionts to the developing egg [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. \u003cem\u003eRickettsia\u003c/em\u003e in MED Q2 were detected in pre-vitellogenic oocytes (stage 1 egg as defined in [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]), which is the stage before the bacteriocyte enters the egg. A previous study reported that \u003cem\u003eRickettsia\u003c/em\u003e in MEAM1 attached to a bacteriocyte and transferred with the bacteriocyte to the developing egg [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. However, in MED Q2, we did not observe \u003cem\u003eRickettsia\u003c/em\u003e on the bacteriocytes in eggs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This result suggests that there is a mechanism in MED Q2 for the transmission of \u003cem\u003eRickettsia\u003c/em\u003e into eggs without the involvement of bacteriocytes. In the future, the invasion process of \u003cem\u003eRickettsia\u003c/em\u003e into early developing eggs should be clarified in detail using confocal and electron microscopy.\u003c/p\u003e \u003cp\u003eThe most serious damage to crops caused by \u003cem\u003eB. tabaci\u003c/em\u003e is due to the transmission of TYLCV. Therefore, the ability to transmit TYLCV is a critical factor in determining the risk of whiteflies becoming an agricultural pest. According to previous studies, Israeli MED Q2 has low ability to transmit TYLCV because it lacks infection with \u003cem\u003eHamiltonella\u003c/em\u003e, whose GroEL facilitates TYLCV transmission [\u003cspan additionalcitationids=\"CR70 CR71\" citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. This study showed that Japanese MED Q2, which is free of \u003cem\u003eHamiltonella\u003c/em\u003e infection, is an effective vector for both TYLCV-IL and TYLCV-Mld, similar to \u003cem\u003eHamiltonella\u003c/em\u003e-infected MED Q1. MED Q2 tended to have higher TYLCV-IL retention (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) and transmission rates to plants (Table\u0026nbsp;1) than MED Q1. TYLCV-Mld retention was significantly lower in MED Q2 than in MED Q1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). However, the transmission rates in MED Q2 were as high as those in MED Q1 (Table\u0026nbsp;1). These results indicate that the Japanese MED Q2 is a high-risk agricultural pest, comparable to MED Q1, and possesses efficient TYLCV transmission machinery independent of \u003cem\u003eHamiltonella\u003c/em\u003e. \u003cem\u003eRickettsia\u003c/em\u003e may play a critical role in the transmission. In a previous study on MEAM1, \u003cem\u003eRickettsia\u003c/em\u003e was suggested to accelerates the uptake of TYLCV into the haemolymph, which may enhance TYLCV transmission to tomato plants [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Certain proteins, such as GroEL, produced by \u003cem\u003eRickettsia\u003c/em\u003e, may be involved in the transmission efficiency of TYLCV in MED Q2, similar to \u003cem\u003eHamiltonella\u003c/em\u003e GroEL in MED Q1 [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. To investigate the potential role of \u003cem\u003eRickettsia\u003c/em\u003e in the high efficiency of TYLCV transmission in the Japanese MED Q2, future experiments will require the elimination of \u003cem\u003eRickettsia\u003c/em\u003e through antibiotic treatment and analysis of its GroEL interaction with TYLCV. Elucidation of the mechanisms underlying TYLCV transmission may lead to the development of more effective control technologies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eInsect and plants\u003c/h2\u003e \u003cp\u003eWhiteflies were collected from fields in the Kanto region of Japan between 2016 and 2021 (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). All samples were immediately stored in 100% acetone until further use [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Laboratory strains of \u003cem\u003eB. tabaci\u003c/em\u003e MED Q1 with \u003cem\u003ePortiera\u003c/em\u003e, \u003cem\u003eHamiltonella\u003c/em\u003e and \u003cem\u003eCardinium\u003c/em\u003e [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] and MED Q2 with \u003cem\u003ePortiera\u003c/em\u003e and \u003cem\u003eRickettsia\u003c/em\u003e (strain Maebashi, Locality no.14 in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were used in this study. These strains were maintained on cabbage (\u003cem\u003eBrassica oleracea\u003c/em\u003e) leaves at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 40\u0026ndash;60% relative humidity in a long-day regimen (16L: 8D). Two tomato plants (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e), 'Momotaro' (Takii, Kyoto, Japan) and 'Micro-Tom' (provided by the National Bio-Resource Project (NBRP) tomato program at the University of Tsukuba, Japan), were cultivated under the same temperature and humidity conditions as cabbage and used in infection experiments with TYLCV. TYLCV-Israel and Mild strains (TYLCV-IL and TYLCV-Mld) were infected and maintained in tomato plants (cv. House-Momotaro) (Takii). All methods involving plants were carried out in accordance with relevant guidelines.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIdentification of genetic groups and the symbionts of\u003c/b\u003e \u003cb\u003eB. tabaci\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExperiments and classification using multiplex polymerase chain reaction (PCR) were performed as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The total DNA of \u003cem\u003eB. tabaci\u003c/em\u003e and its symbionts were individually extracted and amplified using three sets of primer mixes (mix for genotyping, mix 1 for symbionts, and mix 2 for symbionts), as listed in Supplementary Table S2. The \u003cem\u003emtCOI\u003c/em\u003e gene sequences were determined for 11 representative samples as previously described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] with minor modifications. Partial sequences were amplified using PCR with KOD FX Neo (TOYOBO, Osaka, Japan) using specific primer sets (Supplementary Table S2). PCR products were gel-purified and sequenced directly. Sequences were assembled using Geneious Prime ver.2020.0.5 software (Dotmatics, Boston, USA). The sequence similarity of the detected genotypes was analysed using BLAST [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. All sequences determined in this study were deposited in the DDBJ/NCBI/GenBank database under the following accession numbers: LC735029 \u0026ndash; LC735039 for \u003cem\u003emtCOI\u003c/em\u003e and LC795726 and LC795727 for 16S rRNA genes of the symbionts of the laboratory MED Q2 strain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative PCR (qPCR)\u003c/h2\u003e \u003cp\u003eEleven individuals of the MED Q2 laboratory strain were collected at each developmental stage from egg to adult. Eggs and first to fourth instar nymphs were collected without distinguishing between males and females because the sex of whiteflies is indistinguishable at immature stages. At the adult stage, whiteflies of both sexes were collected separately. Samples were preserved in 100% acetone [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e] until DNA extraction. DNA was individually extracted from the samples individually using a NucleoSpin Tissue XS Kit (Takara Bio, Shiga, Japan). DNA was extracted from the eggs of 10 individuals. \u003cem\u003ePortiera\u003c/em\u003e and \u003cem\u003eRickettsia\u003c/em\u003e were quantified in terms of 16S rRNA or \u003cem\u003egltA\u003c/em\u003e gene copies using the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, California, USA) with KOD SYBR qPCR Mix (Toyobo, Osaka, Japan) and specific primer sets (Supplementary Table S2). The qPCR conditions were 98\u0026deg;C for 2 min, followed by 40 cycles of 98\u0026deg;C for 10 s, each annealing temperature for 10 s, and a final extension at 68\u0026deg;C for 30 s. The qPCR with dissociation curve analysis was conducted using a standard curve method, as described previously [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. TYLCV was quantified using the same system targeting \u003cem\u003ev1\u003c/em\u003e, with specific primer sets (Supplementary Table S2). The qPCR conditions were 98\u0026deg;C for 2 min, followed by 40 cycles of 98\u0026deg;C for 10 s and 65\u0026deg;C for 10 s, and a final extension at 68\u0026deg;C for 30 s.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFluorescence\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003ehybridization (FISH)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWhole-body or dissected insect tissue specimens were fixed in Carnoy\u0026rsquo;s solution (EtOH: chloroform: glacial acetic acid, 6:3:1), bleached in 6% hydrogen peroxide in EtOH, and subjected to whole-mount FISH, as described [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Fluorochrome-labelled oligonucleotide probes are listed in Supplementary Table S2. The host cell nuclei were counterstained with 4,6-diamino-2-phenylindole (DAPI). Observations were performed using a laser scanning confocal microscope LSM880 (Carl Zeiss, Germany) and analysed using LSM ZEN2 software (Carl Zeiss, Germany). The specificity of \u003cem\u003ein situ\u003c/em\u003e hybridisation was confirmed by the following control experiments: a no-probe control and an RNase digestion control, as previously described [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eComparison of TYLCV retention in MED Q1 and Q2\u003c/h2\u003e \u003cp\u003eThe experiments were performed as previously described [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e] with modifications. Approximately 30 adult whiteflies (3\u0026ndash;4 d after eclosion) of MED Q1 or MED Q2 were released on each of the two TYLCV (IL or Mld)-infected tomato plants in a plastic container with an insect-proof mesh. The insects were allowed to feed on the plants for 48 h to acquire TYLCV. Total DNA was extracted from each individual of \u003cem\u003eB. tabaci\u003c/em\u003e using a simple extraction method as previously described [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The amount of TYLCV in the samples was measured using qPCR, as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTransmission of TYLCV\u003c/h2\u003e \u003cp\u003eApproximately 200 adult whiteflies (MED Q1 or MED Q2), 3\u0026ndash;4 d after eclosion, were released on each of the two TYLCV (IL or Mld)-infected tomato plants in a plastic container with an insect-proof mesh. Insects were allowed to feed on the plants for 48 h to acquire the TYLCV strain. Ten adults were collected and transferred to a healthy tomato plant (cv. Momotaro, or Micro-Tom) with three true leaves. The insects were allowed to inoculate tomato plants with TYLCV for 48 h or 72 h. As in a previous study [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e], we refer to this period as the inoculation access period (IAP) (Table\u0026nbsp;1). The experiments were repeated ten times. The inoculated plants were kept at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and 40\u0026ndash;60% relative humidity in a long-day regimen (16L:8D). After 30 d, we checked the plants for symptoms of the disease (i.e. yellowing, leaf curling, and dwarfing). Subsequently, we collected the youngest leaves from each plant for DNA extraction, following the method described [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. The presence and type of TYLCV were determined using multiplex PCR with the specific primers listed in Supplementary Table S2. The PCR conditions were 95\u0026deg;C for 5 min, followed by 35 cycles of 98\u0026deg;C for 10 s and 55\u0026deg;C for 30 s, and a final extension at 68\u0026deg;C for 60 s.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Wilcoxon rank-sum test after Bonferroni correction was used to evaluate differences in the bacterial titres of \u003cem\u003ePortiera\u003c/em\u003e and \u003cem\u003eRickettsia\u003c/em\u003e between the host sexes. A two-way ANOVA using a GLM with a Poisson error structure was adopted to evaluate the effect of host-sex (female or male), genetic group of the host (MED Q1 or MED Q2), and their interactions on TYLCV titres. Fisher's exact test was used to assess differences in the transmission rates of TYLCV between MED Q1 and MED Q2. This analysis was performed for each TYLCV-IL and TYLCV-Mld strain. All statistical analyses were performed using R software v. 4.2.1. [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank D. Kawabata, H. Sakai, H. Uga, I. Kabasawa, K. Honda, K. Ikeda, K. Nakamura, K. Ohnishi, K. Tanaka, K. Taniguchi, K. Yokoyama, M. Watanabe, N. Murakami, S. Miki, S. Shimizu, T. Shiraishi, Y. Katayanagi, Y. Nakazawa, and Y. Utsuno for collecting whitefly samples; N. Haruyama for providing \u003cem\u003eB.tabaci\u003c/em\u003e MED Q1; J. Ohnishi for providing TYLCV-infected tomato plants; and K. Kawabe, M. Abe, and N. Murakami for their technical assistance. Tomato seed clone (TOMJPF00001) was provided by University of Tsukuba, Tsukuba Plant Innovation Research Center, through the NBRP of the MEXT/AMED, Japan. Part of this study was supported by a GUCFW Research Grant for Regional Cooperation on Food Science and Wellness (to A.F.) and by JSPS KAKENHI Grant number 18K05673 (to T.T.). A.F. was supported by the Leading Initiative for Excellent Young Researchers (LEADER) Program. This work was the result of using research equipment shared in the MEXT Project for promoting public utilisation of advanced research infrastructure (Program for supporting the introduction of the new sharing system), Grant Numbers JPMXS0420600120 and JPMXS0420600121.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.F. and T.T. designed the research; A.F., H.H., M.T., and T.T. performed the research and analysed the data; A.F. and T.T. wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are included in the manuscript and supplementary sections.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information (including a Competing Interests Statement)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThese authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eChapman, D., Purse, B. 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Secondary endosymbionts of Turkish \u003cem\u003eBemisia tabaci\u003c/em\u003e (Gennadius) populations. \u003cem\u003ePhytoparasitica\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 413-419 (2014).\u003c/li\u003e\n \u003cli\u003eKontsedalov, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The presence of \u003cem\u003eRickettsia\u003c/em\u003e is associated with increased susceptibility of \u003cem\u003eBemisia tabaci\u003c/em\u003e (Homoptera: Aleyrodidae) to insecticides. \u003cem\u003ePest\u0026nbsp;\u003c/em\u003e\u003cem\u003eManag\u003c/em\u003e\u003cem\u003e. Sci\u003c/em\u003e. \u003cstrong\u003e64\u003c/strong\u003e, 789-792 (2008).\u003c/li\u003e\n \u003cli\u003eKliot, A., Cilia, M., Czosnek, H. \u0026amp; Ghanim, M. Implication of the bacterial endosymbiont \u003cem\u003eRickettsia\u003c/em\u003e spp. in interactions of the whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e with \u003cem\u003eTomato yellow leaf curl virus\u003c/em\u003e. \u003cem\u003eJ. 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Soc. Lond. B.\u003c/em\u003e \u003cstrong\u003e270\u003c/strong\u003e, 2543-2550 (2003).\u003c/li\u003e\n \u003cli\u003eSakurai, M., Koga, R., Tsuchida, T., Meng, X. Y. \u0026amp; Fukatsu, T. \u003cem\u003eRickettsia\u003c/em\u003e symbiont in the pea aphid \u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e: novel cellular tropism, effect on host fitness, and interaction with the essential symbiont \u003cem\u003eBuchnera\u003c/em\u003e. \u003cem\u003eAppl. Environ. Microbiol\u003c/em\u003e. \u003cstrong\u003e71\u003c/strong\u003e, 4069-4075 (2005).\u003c/li\u003e\n \u003cli\u003eSimonet, P.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e Bacteriocyte cell death in the pea aphid/\u003cem\u003eBuchnera\u003c/em\u003e symbiotic system. \u003cem\u003ePNAS\u003c/em\u003e. \u003cstrong\u003e115\u003c/strong\u003e, E1819-E1828; 10.1073/pnas.1720237115 (2018).\u003c/li\u003e\n \u003cli\u003eFujiwara, A., Meng, X. Y., Kamagata, Y. \u0026amp; Tsuchida, T. 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Spectr\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, e0468422; 10.1128/spectrum.04684-22 (2023).\u003c/li\u003e\n \u003cli\u003eChen, W.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The draft genome of whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e MEAM1, a global crop pest, provides novel insights into virus transmission, host adaptation, and insecticide resistance. \u003cem\u003eBMC Biol\u003c/em\u003e. \u003cstrong\u003e14\u003c/strong\u003e, 110; 10.1186/s12915-016-0321-y (2016).\u003c/li\u003e\n \u003cli\u003eXie, W.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The invasive MED/Q \u003cem\u003eBemisia tabaci\u003c/em\u003e genome: a tale of gene loss and gene gain. \u003cem\u003eBMC Genomics\u003c/em\u003e. \u003cstrong\u003e19\u003c/strong\u003e, 68; 10.1186/s12864-018-4448-9 (2018).\u003c/li\u003e\n \u003cli\u003eSantos-Garcia, D., Vargas-Chavez, C., Moya, A., Latorre, A. \u0026amp; Silva, F. J. 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New insights into the transovarial transmission of the symbiont \u003cem\u003eRickettsia\u003c/em\u003e in whiteflies. \u003cem\u003eSci. China Life Sci\u003c/em\u003e. \u003cstrong\u003e64\u003c/strong\u003e, 1174-1186 (2021).\u003c/li\u003e\n \u003cli\u003eMorin, S.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e A GroEL homologue from endosymbiotic bacteria of the whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e is implicated in the circulative transmission of Tomato Yellow Leaf Curl Virus. \u003cem\u003eVirology\u003c/em\u003e \u003cstrong\u003e256\u003c/strong\u003e, 75\u0026ndash;84 (1999).\u003c/li\u003e\n \u003cli\u003eMorin, S., Ghanim, M., Sobol, I. \u0026amp; Czosnek, H. The GroEL protein of the whitefly \u003cem\u003eBemisia tabaci\u0026nbsp;\u003c/em\u003einteracts with the coat protein of transmissible and nontransmissible begomoviruses in the yeast two-hybrid system. \u003cem\u003eVirology\u003c/em\u003e \u003cstrong\u003e276\u003c/strong\u003e, 404-416 (2000).\u003c/li\u003e\n \u003cli\u003eGottlieb, Y.\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e The transmission efficiency of \u003cem\u003eTomato yellow leaf curl virus\u003c/em\u003e by the whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e is correlated with the presence of a specific symbiotic bacterium species. \u003cem\u003eJ. Viro\u003c/em\u003e\u003cem\u003el\u003c/em\u003e. \u003cstrong\u003e84\u003c/strong\u003e, 9310-9317 (2010).\u003c/li\u003e\n \u003cli\u003eGhanim, M. A review of the mechanisms and components that determine the transmission efficiency of \u003cem\u003eTomato yellow leaf curl virus\u003c/em\u003e (Geminiviridae; \u003cem\u003eBegomovirus\u003c/em\u003e) by its whitefly vector. \u003cem\u003eVirus Res\u003c/em\u003e. \u003cstrong\u003e186\u003c/strong\u003e, 47-54 (2014).\u003c/li\u003e\n \u003cli\u003eFukatsu, T. Acetone preservation: a practical technique for molecular analysis. \u003cem\u003eMol. Ecol\u003c/em\u003e. \u003cstrong\u003e8\u003c/strong\u003e, 1935-1945. (1999).\u003c/li\u003e\n \u003cli\u003eAltschul, S. F., Gish, W., Miller, W., Myers, E. W. \u0026amp; Lipman, D. J. Basic local alignment search tool. \u003cem\u003eJ. Mol. Biol\u003c/em\u003e. \u003cstrong\u003e215\u003c/strong\u003e, 403-410 (1990).\u003c/li\u003e\n \u003cli\u003eTsuchida, T., Koga, R., Fujiwara, A. \u0026amp; Fukatsu, T. Phenotypic effect of \u0026quot;\u003cem\u003eCandidatus\u003c/em\u003e Rickettsiella viridis,\u0026quot; a facultative symbiont of the pea aphid (\u003cem\u003eAcyrthosiphon pisum\u003c/em\u003e), and its interaction with a coexisting symbiont. \u003cem\u003eAppl. Environ. Microbiol\u003c/em\u003e. \u003cstrong\u003e80\u003c/strong\u003e, 525-533 (2014).\u003c/li\u003e\n \u003cli\u003eKoga, R., Tsuchida, T. \u0026amp; Fukatsu, T. Quenching autofluorescence of insect tissues for \u003cem\u003ein situ\u003c/em\u003e detection of endosymbionts. \u003cem\u003eAppl. Entomol. Zool\u003c/em\u003e. \u003cstrong\u003e44\u003c/strong\u003e, 281-291 (2009).\u003c/li\u003e\n \u003cli\u003eLi, M., Hu, J., Xu, F. C. \u0026amp; Liu, S. S. Transmission of \u003cem\u003eTomato Yellow Leaf Curl Virus\u003c/em\u003e by two invasive biotype and a Chinese indigenous biotype of the whitefly \u003cem\u003eBemisia tabaci\u003c/em\u003e. \u003cem\u003eInt. J. Pest Manag\u003c/em\u003e. \u003cstrong\u003e56\u003c/strong\u003e, 275-280 (2010).\u003c/li\u003e\n \u003cli\u003eMehta, P., Wyman, L. A., Nakhla, M. K. \u0026amp; Maxwell, D. P. Transmission of tomato yellow leaf curl geminivirns by \u003cem\u003eBemisia tabaci\u003c/em\u003e (Homoptera: Aleyrodidae). \u003cem\u003eJ. Econ. Entomol\u003c/em\u003e. \u003cstrong\u003e87\u003c/strong\u003e, 1291-1297 (1994).\u003c/li\u003e\n \u003cli\u003eThomson, D. \u0026amp; Henry, R. Single-step protocol for preparation of plant tissue for analysis by PCR. \u003cem\u003eBiotechniques\u003c/em\u003e \u003cstrong\u003e19\u003c/strong\u003e, 394-397 (1995).\u003c/li\u003e\n \u003cli\u003eIhaka, R. \u0026amp; Gentleman, R. R: A language for data analysis and graphics. \u003cem\u003eJ. Comput. Graph. Stat\u003c/em\u003e. \u003cstrong\u003e5\u003c/strong\u003e, 299-314 (1996).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 Transmission rate of TYLCV in MED Q1 and Q2\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"673\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.806835066864785%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"8.469539375928678%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"19.61367013372957%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"54.82912332838038%\" colspan=\"6\"\u003e\n \u003cp\u003eTransmission rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.280832095096582%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.27637444279346%\" colspan=\"2\"\u003e\n \u003cp\u003eTYLCV\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.61367013372957%\"\u003e\n \u003cp\u003eVariety of tomato\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.436849925705793%\" colspan=\"2\"\u003e\n \u003cp\u003ePCR detection\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.280832095096582%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"2.0802377414561666%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"23.031203566121842%\" colspan=\"2\"\u003e\n \u003cp\u003eDisease symptom\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.280832095096582%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.792284866468842%\"\u003e\n \u003cp\u003estrain \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.456973293768545%\"\u003e\n \u003cp\u003eIAP \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.584569732937684%\"\u003e\n \u003cp\u003e(recipient)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003eMED Q1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003eMED Q2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.077151335311573%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.72106824925816%\"\u003e\n \u003cp\u003eMED Q1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003eMED Q2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.792284866468842%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.456973293768545%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.584569732937684%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.077151335311573%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.72106824925816%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.792284866468842%\"\u003e\n \u003cp\u003eIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.456973293768545%\"\u003e\n \u003cp\u003e48 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.584569732937684%\"\u003e\n \u003cp\u003eMomotaro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.077151335311573%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.72106824925816%\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.792284866468842%\"\u003e\n \u003cp\u003eIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.456973293768545%\"\u003e\n \u003cp\u003e48 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.584569732937684%\"\u003e\n \u003cp\u003eMicro-Tom\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.077151335311573%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.72106824925816%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.792284866468842%\"\u003e\n \u003cp\u003eIL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.456973293768545%\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.584569732937684%\"\u003e\n \u003cp\u003eMomotaro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e0.582\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.077151335311573%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.72106824925816%\"\u003e\n \u003cp\u003e60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e0.629\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.792284866468842%\"\u003e\n \u003cp\u003eMld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.456973293768545%\"\u003e\n \u003cp\u003e48 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.584569732937684%\"\u003e\n \u003cp\u003eMicro-Tom\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e0.582\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.077151335311573%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.72106824925816%\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e0.582\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"9.792284866468842%\"\u003e\n \u003cp\u003eMld\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.456973293768545%\"\u003e\n \u003cp\u003e72 h\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.584569732937684%\"\u003e\n \u003cp\u003eMomotaro\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e0.211\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"2.077151335311573%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.72106824925816%\"\u003e\n \u003cp\u003e70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\n \u003cp\u003e0.211\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60.326894502228825%\" colspan=\"5\"\u003e\n \u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eIL, TYLCV-Israel strain; Mld, TYLCV-Mild strain\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.280832095096582%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"2.0802377414561666%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.738484398216938%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.292719167904904%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.280832095096582%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.833827893175076%\" colspan=\"3\"\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003e Inoculation access period\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"2.077151335311573%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.72106824925816%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.275964391691394%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.270029673590504%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"60.326894502228825%\" colspan=\"5\"\u003e\n \u003cp\u003e\u003csup\u003ec\u003c/sup\u003e The results of Fisher\u0026apos;s exact test are shown.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.280832095096582%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"2.0802377414561666%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.738484398216938%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"11.292719167904904%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"7.280832095096582%\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3976000/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3976000/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe whitefly, \u003cem\u003eBemisia tabaci\u003c/em\u003e, is a notorious insect pest that transmits plant pathogenic viruses to a wide range of economically-important crops. An invasive genetic group of \u003cem\u003eB. tabaci\u003c/em\u003e, Mediterranean Q2 (MED Q2), has recently spread to Europe, USA, and Asia. In this study, we investigated the prevalence of MED Q2 in Japanese agricultural sites and found that its distribution has expanded since it was first detected in 2013. Polymerase chain reaction analysis revealed that all MED Q2 individuals were infected with \u003cem\u003eRickettsia\u003c/em\u003e. \u003cem\u003eRickettsia \u003c/em\u003etitres\u003cem\u003e \u003c/em\u003eincreased during nymphal development, presumably in response to the nutritional needs of the host. Fluorescence\u003cem\u003e in situ \u003c/em\u003ehybridisation analysis revealed that \u003cem\u003eRickettsia\u003c/em\u003e was densely located near \u003cem\u003ePortiera\u003c/em\u003e-containing bacteriocytes at all growth stages. \u003cem\u003eRickettsia\u003c/em\u003emay therefore play an important role, such as supplying nutrients to the host, in cooperation with \u003cem\u003ePortiera\u003c/em\u003e. Transfer experiments indicated that MED Q2 was as effective a vector for \u003cem\u003eTomato yellow leaf curl virus\u003c/em\u003e as MED Q1 and is therefore a high-risk agricultural pest. These results provide important insights into the biology and ecology of the invasive MED Q2 to effectively control its spread and minimise its impact on crops.\u003c/p\u003e","manuscriptTitle":"Prevalence, symbiosis with Rickettsia, and transmission of Tomato yellow leaf curl virus of invasive Bemisia tabaci MED Q2 in Japan","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-13 17:14:09","doi":"10.21203/rs.3.rs-3976000/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f53a5529-8a4a-445c-bf8e-7e6e36fb5e3c","owner":[],"postedDate":"March 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29316857,"name":"Biological sciences/Zoology/Entomology"},{"id":29316858,"name":"Biological sciences/Microbiology/Bacteria/Symbiosis"}],"tags":[],"updatedAt":"2025-05-28T14:22:23+00:00","versionOfRecord":{"articleIdentity":"rs-3976000","link":"https://doi.org/10.1264/jsme2.ME24095","journal":{"identity":"microbes-and-environments","isVorOnly":true,"title":"Microbes and Environments"},"publishedOn":"2025-05-01 00:00:00","publishedOnDateReadable":"May 1st, 2025"},"versionCreatedAt":"2024-03-13 17:14:09","video":"","vorDoi":"10.1264/jsme2.ME24095","vorDoiUrl":"https://doi.org/10.1264/jsme2.ME24095","workflowStages":[]},"version":"v1","identity":"rs-3976000","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3976000","identity":"rs-3976000","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}