Prevalence, Symbiosis with Rickettsia, and Transmission of Tomato yellow leaf curl virus of Invasive Bemisia tabaci MED Q2 in Japan

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 the present study, we investigated the prevalence of MED Q2 in Japanese agricultural sites and found that its distribution has expanded since it was initially detected in 2013. A polymerase chain reaction ana­lysis revealed that all MED Q2 individuals were infected with Rickettsia. Rickettsia titers increased during nymphal development, presumably in response to the nutritional needs of the host. A fluorescence in situ hybridization ana­lysis revealed that Rickettsia was densely located near Portiera-containing bacteriocytes at all growth stages. Therefore, Rickettsia may 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, thus, is a high-risk agricultural pest. These results provide important insights into the biology and ecology of invasive MED Q2 to effectively control its spread and minimize its impact on crops.

Globalization and international trade facilitate the long-distance movement of non-native organisms beyond their natural geographic range (Chapman et al., 2017; Epanchin-Niell et al., 2021). The invasion of alien species has increased in recent decades (Seebens et al., 2017), with detrimental effects on human health, ecosystems, and economic activities, such as agriculture (Simberloff et al., 2013; Pyšek and Richardson, 2010).

The sweet potato whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a phloem sap-feeding pest of agricultural crops (Stansly and Naranjo, 2010). It comprises genetically distinct groups that exhibit different biological characteristics, such as habitat and susceptibility to insecticides, but are morphologically indistinguishable (Costa and Brown, 1991; Bedford et al., 1994). Based on mitochondrial cytochrome oxidase subunit I (mtCOI) gene sequences, it may be classified into 44 groups (Kanakala and Ghanim, 2019). Although many genotypes are limited to specific regions, Mediterranean (MED) Q (Moya et al., 2001; Boykin et al., 2007) and Middle East-Asia Minor 1 (MEAM1) (Frohlich et al., 1999; De Barro et al., 2000) have spread globally via international transport from their original habitats in the Mediterranean region and the Middle East and Asia Minor, respectively (De Barro and Ahmed, 2011). A common characteristic of these invasive genetic groups is their high insecticide resistance. This may 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 (Brown et al., 1995; Horowitz et al., 2005; Nauen and Denholm, 2005). These genetic groups may cause substantial damage to a wide range of economically important crops by transmitting more than 100 pathogenic plant viruses (Jones, 2003; Hogenhout et al., 2008). Due to these characteristics, the whitefly, represented by these genotypes, is regarded as one of the most seriously invasive species worldwide according to the global invasive species database (Lowe et al., 2000).

MED Q has been classified into three subgroups, Q1, Q2, and Q3, based on mole­cular phylogenetic ana­lyses of the mtCOI gene (Gueguen et al., 2010). MED Q2 originated in the Eastern Mediterranean and has expanded its distribution in Europe and the United States (Tsagkarakou et al., 2007; Chu et al., 2008; Ahmed et al., 2009; Mouton et al., 2012; Parrella et al., 2014; Karut et al., 2017). In recent years, the distribution of MED Q2 has expanded to Asia. MED Q2 was initially discovered in Asia in 2013 at a site in the Kanto region of Japan (Fujiwara et al., 2015). In 2015, MED Q2 was identified at two additional sites in the same district of Japan (Kurata et al., 2016). This included one site in which MED Q2 was not detected in the 2013 survey. In 2018, MED Q2 was initially identified at two sites in northwestern and southeastern South Korea (Guo et al., 2021). These findings suggest that the range of MED Q2 continues to expand.

Similar to other phloem-sap feeding insects, B. tabaci harbors endosymbiotic bacteria that are stably transmitted to offspring via the ovaries (Buchner, 1965; Luan et al., 2018). All genetic groups of B. tabaci contain the primary endosymbiont, Portiera. Portiera is regarded as an ancient infection with an insect ancestor (Baumann et al., 2004; Thao and Baumann, 2004). In addition to Portiera, B. tabaci often harbors different endosymbiotic bacteria. Eight different bacteria, Hamiltonella, Cardinium, Rickettsia, Wolbachia, Arsenophonus, Frischea, Hemipteriphilus, and Candidatus Rickettsia_Torix_Bemisia_tabaci, have been reported to date (Zchori-Fein and Brown, 2002; Nirgianaki et al., 2003; Weeks et al., 2003; Baumann, 2005; Everett et al., 2005; Gottlieb et al., 2006; Bing et al., 2013b; Wang et al., 2020). These bacteria are considered to have been acquired relatively recently and are collectively referred to as secondary symbionts (S-symbionts).

The MED Q1 and MEAM1 genotypes harbor Portiera and Hamiltonella exclusively within bacteriocytes, which are specialized cells for endosymbiosis (Gottlieb et al., 2008; Skaljac et al., 2010; Li et al., 2022). In these genotypes, Portiera and Hamiltonella are regarded as co-obligate symbionts based on virtually universal infection in their original populations (Gueguen et al., 2010; Tsagkarakou et al., 2012; Gnankiné et al., 2013; Bing et al., 2013a; Fujiwara et al., 2015; Zchori-Fein et al., 2014; Marubayashi et al., 2014; Karut et al., 2017) and the decrease in host fitness caused by symbiont elimination (Su et al., 2013, 2014). However, studies conducted on Italian, Israeli, Turkish, and Japanese populations revealed the absence of Hamiltonella in MED Q2 (Chiel et al., 2007; Gueguen et al., 2010; Karut and Tok, 2014; Parrella et al., 2014; Karut et al., 2017; Fujiwara et al., 2015; Kurata et al., 2016). Rickettsia is nearly fixed in all MED Q2 populations, suggesting its important role in replacing Hamiltonella in MED Q2. Previous studies reported various effects of Rickettsia on naturally infected MEAM1, such as increased susceptibility to insecticides (Kontsedalov et al., 2008), a greater capacity to transmit Tomato yellow leaf curl virus (TYLCV) (Kliot et al., 2014), more fitness benefits, and a higher female-biased sex ratio (Himler et al., 2011). In contrast, limited information is available on the effects of Rickettsia on MED Q2.

In the present study, we characterized MED Q2 and its secondary 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 then analyzed the spatiotemporal dynamics of Rickettsia in MED Q2 using quantitative PCR (qPCR) and in situ hybridization. We also exami­ned the retention of TYLCV in the body and the efficiency of its transmission to tomato plants in MED Q2.

Materials and Methods

Insects and plants

Whiteflies were collected from fields in the Kanto region of Japan between 2016 and 2021 (Table S1 and Fig. S1). All samples were immediately stored in 100% acetone for later ana­lyses (Fukatsu, 1999). Laboratory strains of B. tabaci MED Q1 with Portiera, Hamiltonella, and Cardinium (Fujiwara et al., 2023) and MED Q2 with Portiera and Rickettsia (strain Maebashi, Locality no. 14 in Table S1) were used in the present study. The genotypes of these strains and their symbionts were confirmed by sequencing, as previously described (Fujiwara et al., 2015). These strains were maintained on cabbage (Brassica oleracea) leaves at 25±1°C and 40–60% relative humidity in a long-day regimen (16‍ ‍h light, 8‍ ‍h dark), and exhibited a sex ratio of approximately 1:1. Two tomato plants (Solanum lycopersicum), ‘Momotaro’ (Takii) 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). These cabbage and tomato plant species are not listed as endangered species or species at risk of extinction, according to the IUCN Policy Statement and the Convention on International Trade in Endangered Species of Wild Fauna and Flora.

Identification of genetic groups and symbionts of B. tabaci

Experiments and classifications using multiplex PCR were performed as previously described (Kurata et al., 2016). Following the extraction of the total DNA of B. tabaci and its symbionts from the whole body of each insect, it was amplified using three sets of primer mixes (mix for genotyping, mix 1 for symbionts, and mix 2 for symbionts), as listed in Table S2. mtCOI gene sequences were elucidated for 11 representative samples as previously described (Fujiwara et al., 2015) with minor modifications. Partial sequences were amplified using PCR with KOD FX Neo (TOYOBO) using specific primer sets (Table S2). PCR products were gel-purified and sequenced directly. Sequences were assembled using Geneious Prime ver. 2020.0.5 software (Dotmatics). Sequence similarities in the detected genotypes were analyzed using BLAST (Altschul et al., 1990).

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 (Fukatsu, 1999) until DNA extraction. DNA was extracted from individual samples using a NucleoSpin Tissue XS Kit (Takara Bio). 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) with KOD SYBR qPCR Mix (Toyobo) and specific primer sets (Table S2). qPCR conditions were as follows: at 98°C for 2‍ ‍min, followed by 40 cycles at 98°C for 10‍ ‍s, each annealing temperature for 10‍ ‍s, and a final extension at 68°C for 30 s. qPCR with a dissociation curve ana­lysis was conducted using a standard curve method, as previously described (Tsuchida et al., 2014). TYLCV was quantified using the same system targeting v1, with specific primer sets (Table S2). qPCR conditions were as follows: at 98°C for 2‍ ‍min, followed by 40 cycles at 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 previously described (Koga et al., 2009). Fluorochrome-labeled oligonucleotide probes are listed in Table S2. Host cell nuclei were counterstained with 4,6-diamino-2-phenylindole (DAPI). Observations were performed using a laser scanning confocal microscope (LSM880; Carl Zeiss) and analysed using LSM ZEN2 software (Carl Zeiss). The specificity of in situ hybridization was confirmed by the following control experiments: a no-probe control and an RNase digestion control, as previously described (Tsuchida et al., 2014).

Comparison of TYLCV retention in MED Q1 and Q2

Experiments were performed as previously described (Li et al., 2010) 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. 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 (Kurata et al., 2016). 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 in order 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. Insects were allowed to inoculate tomato plants with TYLCV for 48 or 72 h. As described in a previous study (Mehta et al., 1994), we referred to this period as the inoculation access period (IAP) (Table 1). Experiments were repeated ten times. Inoculated plants were kept at 25±1°C and 40–60% relative humidity in a long-day regimen (16‍ ‍h light, 8‍ ‍h dark). After 30‍ ‍d, we checked the plants for symptoms of the disease (i.e., yellowing, leaf curling, and dwarfing). We then collected the youngest leaves from each plant for DNA extraction, following a previously described method (Thomson and Henry, 1995). The presence and type of TYLCV were confirmed using multiplex PCR with the specific primers listed in Table S2. PCR conditions were as follows: at 95°C for 5‍ ‍min, followed by 35 cycles at 98°C for 10‍ ‍s and 55°C for 30‍ ‍s, and a final extension at 68°C for 60 s.

Table 1.

Transmission rates of TYLCV in MED Q1 and Q2.

TYLCV straina	IAPb	Variety of tomato (recipient)	Transmission rate (%)
			PCR detection		Pc	Disease symptom		Pc
			MED Q1	MED Q2		MED Q1	MED Q2
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.

Statistical ana­lysis

The Wilcoxon rank-sum test after the Bonferroni correction was used to evaluate differences in the bacterial titers of Portiera and Rickettsia between the host sexes. A two-way ana­lysis of variance (ANOVA) using generalized linear modeling (GLM) with a Poisson error structure was adopted to examine the effects of the host sex (female or male), the genetic group of the host (MED Q1 or MED Q2), and their interactions on TYLCV titers. Fisher’s exact test was used to investigate differences in the transmission rates of TYLCV between MED Q1 and MED Q2. This ana­lysis was conducted for each TYLCV-IL and TYLCV-Mld strain. All statistical ana­lyses were performed using R software v. 4.2.1. (Ihaka and Gentleman, 1996).

Accession numbers

All sequences elucidated in the present study were deposited in the DDBJ/NCBI/GenBank database under the following accession numbers: LC735029–LC735039 for mtCOI and LC795726 and LC795727 for the 16S rRNA genes of symbionts of the laboratory MED Q2 strain.

Results

Prevalence of MED Q2

We collected B. tabaci samples from 15 sites in the Kanto region of Japan (Fig. S1) between 2016 and 2021. Multiplex PCR detected three genetic groups: MED Q1, MED Q2, and JpL (Table S1). MED Q1 was detected in 14 sites. JpL was only detected in 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 five of the 10 sites, with regional differences ranging between 100% and 1.1% (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 in Maebashi or Yoshioka in the first year of the survey, but was identified in the same sites in the following year (Table S1 and Fig. 1). MED Q2 was found at moderate rates in Isesaki and Maebashi over three years; however, the number of MED Q2 individuals varied among years and sites.

Fig. 1.

Changes in the prevalence of MED Q2 and its infection status with secondary symbionts (S-symbionts) in the Kanto region of Japan. Locality numbers correspond to those in Fig. S1 and Table S1. Data indicate the total number of MED Q2 individuals collected from various crops listed in 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 and MED Q2 was not detected. The infection status of S-symbionts is shown in different colors: light red, infection with Rickettsia and Wolbachia; sky blue, infection with Rickettsia only.

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 harbored Wolbachia. The infection rate‍ ‍of Wolbachia varied between regions or collection dates; Wolbacia was never detected at some sites (no. 14 and 15, Fig. 1). The S-symbionts, Hamiltonella, Cardinium, Arsenophonus, and Hemipteriphilus, were not detected in any of the individuals exami­ned.

Population dynamics of Portiera and Rickettsia in host developmental stages

Portiera titers continued to increase throughout the nymphal stages, peaked in fourth instar nymphs in males and on day 15 in adult females (Fig. 2), and then began to decrease. Rickettsia showed similar population dynamics to Portiera, with titers increasing throughout the nymphal stage in both females and males. The bacterial population was markedly lower in males than in females at all adult stages (Fig. 2). In males, Rickettsia populations decreased rapidly from 1‍ ‍d after adult eclosion, but remained high in females 30‍ ‍d after eclosion.

Fig. 2.

Population dynamics of symbionts in Bemisia tabaci MED Q2. Bacterial titers of Portiera and Rickettsia were measured using a quantitative polymerase chain reaction in terms of 16S rRNA or gltA gene copies insect^–1. 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 titer in an egg was calculated by averaging the acquired values of 10 individuals. Asterisks indicate a significant difference (P<0.001, the Wilcoxon rank-sum test after the Bonferroni correction).

In vivo localization of symbionts in MED Q2

In adult females, Portiera was only detected in bacteriocytes (Fig. 3A and B). In contrast to Portiera, Rickettsia was not detected in bacteriocytes. This distribution pattern was confirmed by observing bacteriocytes dissected from adult females 1‍ ‍d after eclosion (Fig. S2). Rickettsia was densely localized in close proximity to bacteriocytes, and was rarely observed in other tissues (Fig. 3A and B). Similar localization patterns were observed in first-instar, fourth-instar, and adult males 1‍ ‍d after eclosion (Fig. S3).

Fig. 3.

In vivo localization of Portiera (red) and Rickettsia (green) in Bemisia tabaci MED Q2. (A) The abdomen of a female 15‍ ‍d after eclosion. A whole view of B. tabaci is 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 visualized in blue. Yellow arrowhead, Rickettsia, densely localized 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.

Ovaries collected from adult females 5‍ ‍d after eclosion contained eggs at the pre-vitellogenesis stage (Fig. 4A) before the transfer of bacteriocytes. A FISH ana­lysis of ovaries showed that Rickettsia was present in the eggs at this stage (Fig. 4A). On day 15 after adult eclosion, bacteriocytes containing Portiera were detected in the ovaries (Fig. 4B). However, Rickettsia was not detected within or on bacteriocytes. These results indicate that Rickettsia of MED Q2 was not present inside bacteriocytes and was not transmitted to the next generation via bacteriocytes, in contrast to Portiera.

Fig. 4.

Localization of Rickettsia in the ovary of MED Q2. (A) Ovarioles dissected from an adult female 5‍ ‍d after eclosion. (B) A bacteriocyte just after entering the egg in an adult female 15‍ ‍d after eclosion. Only the Rickettsia (white) signal is shown in the left images. The right images show the nuclear DNA (blue) and Portiera (red) signals overlaid on the Rickettsia (green) signal. Yellow dashed lines indicate outlines of the egg. An orthogonal view of Z-stack images is shown. Red and green dashed lines indicate corresponding points in the orthogonal planes.

Retention and transmission of TYLCV in MED Q2

Fig. 5 shows TYLCV titers in B. tabaci after 48‍ ‍h of feeding on TYLCV-infected plants. A two-way ANOVA using GLM showed that TYLCV-IL titers were slightly higher in MED Q2 than in MED Q1. An interaction was observed between sex and genetic groups. In contrast, TYLCV-Mld titers were significantly lower in MED Q2 than in MED Q1 (Fig. 5). Regarding TYLCV-Mld, no interaction was observed between sex and genetic groups.

Fig. 5.

Effects of host sex and genotype on the retention of TYLCV-IL (A) and TYLCV-Mld (B). Box plots represent the distribution of TYLCV titers insect^–1. 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 a two-way ANOVA using a generalized linear model with a Poisson error structure are shown in the box (*P<0.05).

The PCR ana­lysis conducted in transfer experiments showed that TYLCV-IL was transferred to Momotaro or Micro-Tom by MED Q1 and MED Q2 at similar levels under 48-h 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 was slightly less efficient than MED Q1. When transferred to Momotaro for 72 h, MED Q2 efficiency was 100% and was slightly more efficient than MED Q1. Disease symptom results were similar to those of TYLCV PCR detection. No significant differences were found in transmission rates between the MED Q1 and MED Q2 groups (Table 1).

Discussion

MED Q2 was initially detected in 2013 at a site in the Kanto region of Japan (Fujiwara et al., 2015). In 2015, MED Q2 was detected at two additional sites in the same area (Kurata et al., 2016). In the present study, we detected MED Q2 at ten sites in the same district (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) (Fujiwara et al., 2015). These findings suggest that MED Q2 has expanded its distribution area in the Kanto region since 2013; however, MED Q1 is still the dominant strain in Japan. In the present study, MED Q2 was detected at Sites 11 and 14 over multiple years (Table S1 and Fig. 1), suggesting that the MED Q2 population was established here and in the wider area of the Kanto region. However, it is important to note that in this survey, the number of individuals sampled was small at some sites, which may have led to an underestimation of the actual population size of MED Q2. Therefore, additional surveys with larger sample sizes are needed to further clarify the distribution and establishment of MED Q2.

MED Q2 appeared to have expanded its distribution to many countries. In South Korea, MED Q2 was initially identified in 2018 (Guo et al., 2021). In southern Italy, the distribution of MED Q2 has expanded since it was first discovered, and the dominant type changed from MED Q1 to MED Q2 in only a few years (2010–2013) (Parrella et al., 2014). In the 2017–2019 surveys conducted in central and southern Italy and Sicily, MED Q2 accounted for 87.6% of all samples collected from greenhouse crops (Bertin et al., 2021). The high temperature tolerance, insecticide resistance, and female-biased sex ratio of MED Q2 are considered to have contributed to its spread in Italy (Parrella et al., 2014). Since a female-biased sex ratio was not observed in the Japanese MED Q2 strain (see the Materials and Methods section), global warming and insecticide use may be responsible for the expanding distribution of MED Q2 in Japan. To address this possibility, future studies need to investigate the distribution of MED Q2 in regions with varying annual temperatures and insecticide usage across wider areas of Japan. Additionally, to prevent the spread of MED Q2, effective insecticides and other methods must be identified.

The infection status of MED Q2 was previously reported in Israel (Chiel et al., 2007; Gueguen et al., 2010), Turkey (Karut and Tok, 2014; Karut et al., 2017), and Italy (Parrella et al., 2014). MED Q2 in these countries commonly had a high infection rate with Arsenophonus and Rickettsia, but a low infection rate with Wolbachia. The infection status of the Japanese MED Q2 group markedly differed from those of the other groups. In addition to the primary symbiont, Portiera, two types of S-symbionts, Rickettsia and Wolbachia, were detected in Japanese MED Q2 (Fig. 1). The prevalence of Rickettsia infection was 100% throughout all survey periods; Wolbachia was only detected in some samples. Arsenophonus, which is highly prevalent in MED Q2 in other countries, has never been detected in the Japanese MED Q2 population. Possible reasons for this difference are as follows: (1) only MED Q2, which is uninfected with Arsenophonus, has entered Japan, and (2) the population of Arsenophonus-uninfected MED Q2 has increased due to its better adaptability to Japanese field conditions. However, these hypotheses are not mutually exclusive. A more detailed ana­lysis is needed in the future to clarify this issue.

Rickettsia was nearly fixed in MED Q2 populations in all regions investigated in this study. Rickettsia infection may increase the percentage of infected individuals by manipulation of the sex ratio, as demonstrated in MEAM1 (Himler et al., 2011). Another possibility is that Rickettsia promoted survival and reproduction in MED Q2 through nutritional supply, thereby increasing the number of infected individuals in the population. The second possibility is based on its population dynamics in host insects; Rickettsia titers increased during nymphal development and then rapidly decreased in males from 1‍ ‍d after eclosion (Fig. 2). These bacteria 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 (Koga et al., 2003; Sakurai et al., 2005; Simonet et al., 2018, Fujiwara et al., 2023). This phenomenon has been interpreted as the regulation of nutrient-compensating symbionts to meet the dietary needs of host insects. The Rickettsia titer 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 (Fujiwara et al., 2023).

Previous genomic studies on B. tabaci MEAM1 and MED Q1 showed that Portiera alone was unable to synthesize essential amino acids (e.g., lysine) or B vitamins that are deficient in the host diet (Santos-Garcia et al., 2015; Chen et al., 2016; Xie et al., 2018); the enzymes responsible for producing essential amino acids are found across Portiera, a coexisting symbiont (Hamiltonella or Arsenophonus), and B. tabaci. Therefore, metabolic intermediates must be transported between symbionts and the host multiple times for production. These symbionts coexist within the same bacteriocyte (Gottlieb et al., 2008), which appears to be adaptive for the efficient production of essential nutrients via intertwined metabolic pathways (Fujiwara et al., 2023). Incomplete metabolic pathways were found in Portiera for both MEAM1 and MED Q1 (Santos-Garcia et al., 2012; Sloan and Moran, 2012), suggesting that the loss of these enzymes in Portiera occurred before the divergence of these lineages. Therefore, Portiera may have incomplete metabolic pathways in the derived lineage MED Q2. In the present study, Rickettsia was not detected in the bacteriocytes of MED Q2 (Fig. 3, 4, S2, and S3), which is in contrast to the previously reported “confined type” in which Rickettsia localized exclusively within bacteriocytes, as observed in MEAM1 and MED (Gottlieb et al., 2008). Instead, Rickettsia aggregated in regions proximal to Portiera-containing bacteriocytes at all growth stages (Fig. 3A, B, and 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 (Luan et al., 2018). Rickettsia in MED Q2 were detected in pre-vitellogenic oocytes (stage 1 egg as defined in Shan et al., 2021), which is the stage before the bacteriocyte enters the egg. A previous study reported that Rickettsia in MEAM1 attached to a bacteriocyte and was transferred with the bacteriocyte to the developing egg (Shan et al., 2021). However, in MED Q2, we did not observe Rickettsia on 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 needs to be clarified in more detail using confocal and electron microscopy.

The most severe 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 assessing the risk of whiteflies becoming an agricultural pest. According to previous studies, the ability of Israeli MED Q2 to transmit TYLCV is low because it lacks infection with Hamiltonella, the GroEL of which facilitates TYLCV transmission (Morin et al., 1999, 2000; Gottlieb et al., 2010; Ghanim, 2014). 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 showed the higher retention of TYLCV-IL (Fig. 5) as well as higher 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, transmission rates in MED Q2 were as high as those in MED Q1 (Table 1). These results indicate that Japanese MED Q2 is a high-risk agricultural pest, similar to‍ ‍MED Q1, and possesses efficient TYLCV transmis­sion‍ ‍machinery independent of Hamiltonella. Rickettsia may play a critical role in transmission. In a previous study on MEAM1, Rickettsia was suggested to accelerate the uptake of TYLCV into the hemolymph, which may enhance TYLCV transmission to tomato plants (Kliot et al., 2014). Some 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 (Gottlieb et al., 2010). To investigate the potential role of Rickettsia in the high efficiency of TYLCV transmission in Japanese MED Q2, future experiments will require the elimination of Rickettsia through antibiotic treatment and an ana­lysis of its GroEL interaction with TYLCV. A more detailed understanding of the mechanisms underlying TYLCV transmission may lead to the development of more effective control technologies.

Citation

Fujiwara, A., Hagiwara, H., Tsuchimoto, M., and Tsuchida, T. (2025) Prevalence, Symbiosis with Rickettsia, and Transmission of Tomato yellow leaf curl virus of Invasive Bemisia tabaci MED Q2 in Japan. Microbes Environ 40: ME24095.

https://doi.org/10.1264/jsme2.ME24095

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. This work was also supported by the Division of Instrumental Analysis at the University of Toyama and Laboratory for Analytical Instruments, Education and Research Support Center, Gunma University Graduate School of Medicine. The tomato seed clone (TOMJPF00001) was provided by the University of Tsukuba, the 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 the public utilization of an advanced research infrastructure (Program for supporting the introduction of the new sharing system), Grant Numbers JPMXS0420600120 and JPMXS0420600121.

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