Journal of Invertebrate Pathology 112 (2013) 83–87
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Transfer of a parthenogenesis-inducing Wolbachia endosymbiont derived from Trichogramma dendrolimi into Trichogramma evanescens Masaya Watanabe a,1, Daisuke Kageyama b, Kazuki Miura a,c,⇑ a
Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashi Hiroshima 739-8511, Japan National Institute of Agrobiological Sciences (NIAS), 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan c National Agricultural Research Center for Western Region, 6-12-1 Nishifukatsu, Fukuyama, Hiroshima 721-8514, Japan b
a r t i c l e
i n f o
Article history: Received 17 August 2012 Accepted 25 September 2012 Available online 11 October 2012 Keywords: Cell culture Infection density Microinjection Parasitoid Trichogramma Wolbachia
a b s t r a c t Wolbachia, which are maternally transmitted endosymbionts, are considered to have moved horizontally between invertebrate hosts multiple times. However, it is not well understood how easily Wolbachia are transmitted horizontally between different hosts and how frequently horizontally-transmitted Wolbachia become established in their new hosts. We transferred a parthenogenesis-inducing Wolbachia endosymbiont derived from the parasitic wasp Trichogramma dendrolimi to Trichogramma evanescens. Specifically, Wolbachia was cultivated in a mosquito cell line and the Wolbachia-infected cells were microinjected into uninfected T. evanescens. Among 276 pupae inoculated with Wolbachia-infected cells, 65 adults emerged (G0). Diagnostic PCR demonstrated that 25 of 37 G0 females (68%) were Wolbachia-positive. Among isofemale lines established from G0 females, the proportions of infected lines were 80% (20 of 25) in G1 and 100% (18 of 18) in G2. In an isofemale line, infection was stably maintained for more than 10 generations. These results indicate invasion of Wolbachia into the germline of the recipient insect. Quantitative PCR demonstrated that the Wolbachia titer in the recipient host was significantly lower than that in the native host. The absence or very low number, if any, of parthenogenetically-reproducing individuals in the recipient host may be caused by the low Wolbachia titer. The Wolbachia titer in the recipients was lower in G11 than in G5, suggesting a decline in the density. Together with a previous report, our study may imply that Wolbachia in Trichogramma species are highly adapted to their hosts, which hinders robust expression of the Wolbachia phenotype in non-native host species. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Wolbachia pipientis (also referred to as Wolbachia) is a bacterium belonging to the alpha-subdivision of Proteobacteria and is the most ubiquitous endosymbiont among arthropods and filarial nematodes (Zug and Hammerstein, 2012). Various effects on insect reproduction, such as cytoplasmic incompatibility, parthenogenesis induction, male killing, and feminization, have attracted particular attention, but at present, very little is known about their underlying mechanisms (Werren et al., 2008). Wolbachia is maternally inherited within the host lineage and does not spread in an infectious manner. However, the topological incongruence observed between the phylogeny of Wolbachia and that of its hosts
⇑ Corresponding author at: National Agricultural Research Center for Western Region, 6-12-1 Nishifukatsu, Fukuyama, Hiroshima 721-8514, Japan. Fax: +81 84 924 7893. E-mail address:
[email protected] (K. Miura). 1 Present address: National Institute of Agrobiological Sciences (NIAS), 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan. 0022-2011/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2012.09.006
suggests that horizontal transmission of Wolbachia has occurred repeatedly in the evolutionary timescale (possibly via parasitism, predation, or cannibalism) (Werren et al., 2008). At present, it is not well understood how often Wolbachia are transmitted horizontally between different hosts and how frequently horizontally-transmitted Wolbachia become established in their new hosts. To date, experimental transfer of Wolbachia endosymbionts between different host species has been repeatedly achieved (Braig et al., 1994; Grenier et al., 1998; Walker et al., 2011). However, the extent to which Wolbachia transfer is feasible is not precisely understood, because successful cases are more favorably published. In the present study, we transinfected a parthenogenesis-inducing Wolbachia derived from the parasitic wasp T. dendrolimi into the OK94 strain of Trichogramma evanescens. Since T. evanescens OK94 is highly tolerant of high temperatures (Inoue and Kubo, 2001) and thus a promising candidate for use in greenhouses as a natural enemy against various lepidopteran pests, induction of parthenogenesis would reduce the cost of rearing and contribute to the mass production of this favorable insect.
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2. Materials and methods 2.1. Insects The Wolbachia-infected strain of T. dendrolimi and uninfected T. evanescens OK94 were reared on UV-irradiated Ephestia kuehniella eggs at 25 ± 1 °C and 65 ± 20% relative humidity under a 16-h light/8-h dark photoperiod. T. evanescens OK94 is a strain derived from a single female collected in the field, and has been maintained in the laboratory for more than 12 years. 2.2. Cell line A cell line, NIAS-AeAl-2, derived from Aedes albopictus was provided by Dr. Hiroaki Noda in the National Institute of Agrobiological Sciences, Tsukuba, Japan (Noda et al., 2002). The insect cell line was maintained in IPL-41 culture medium (Life Technologies Japan Ltd., Tokyo, Japan) supplemented with 4–5% fetal bovine serum (Cosmo Bio Co. Ltd., Tokyo, Japan) at 25–28 °C using 50-ml plastic culture vessels without CO2 gas incubation (Noda et al., 2002). 2.3. Inoculation of Wolbachia into the cell line Wolbachia derived from T. dendrolimi (wDen) was inoculated into the NIAS-AeAl-2 cell line as previously described (Kubota et al., 2005). After T. dendrolimi female adults were surface-sterilized with 70% ethanol for a few minutes, the ovary was dissected in a droplet of IPL-41 medium and then crushed with insect pins in a new droplet of medium containing NIAS-AeAl-2 cells. The droplet containing the ovary fragment homogenate was used as the inoculum and introduced into a 50-ml plastic culture vessel containing the cells. The vessel was kept at 25 ± 1 °C until the cells became confluent, at which point a portion of the cells (approximately 20%) was introduced into a new culture vessel and provided with fresh medium. This handling of the cells was usually performed once every 7–10 days. During the maintenance of the inoculated cell culture, uninfected healthy cells were sometimes added. The inoculated cell culture was observed using an inverted phase contrast microscope (TMD300; Nikon, Tokyo, Japan). 2.4. Detection of Wolbachia in the inoculated cell culture To confirm active proliferation of Wolbachia in the cell culture, the cells were sampled every 2–3 days. After centrifugation of the cells at 1100 g for 5 min, the supernatant was removed, and the cell pellet was resuspended in 32 ll of STE buffer (5 N NaCl, 500 nM EDTA pH 8.0, 1 M Tris–HCl pH 8.0) and 2 ll of proteinase K (0.5 mg/ml). This mixture was sequentially incubated at 56 °C for 2 h and 99.9 °C for 3 min. Diagnostic PCR for Wolbachia infection was performed using the Wolbachia-specific primers wsp81F and wsp691R that amplify a partial sequence of the wsp gene (Braig et al., 1998). Quantification of Wolbachia titers was performed by fluorescence real-time quantitative PCR (see below). 2.5. Microinjection of Wolbachia-infected cells into T. evanescens The wDen-infected A. albopictus cells after cultivation for more than 3 months was used as a donor for microinjection. The cells constituting a confluent monolayer were gently removed from the 25-ml flask and the volume of 1.5 ml (containing cells and IPL-41 medium) was centrifuged at 1100 g for 5 min. The supernatant was removed and the cell pellet resuspended in 200-ml SPG buffer (218 mM sucrose, 3.8 mM KH2PO4, 7.2 mM K2HPO4, 4.9 mM L-glutamate, pH 7.2) (Xi and Dobson, 2005) was used as a donor for microinjection within 30 min.
An uninfected strain of T. evanescens OK94 was used as a recipient for microinjection. At 25 °C, this insect usually pupates at 9– 10 days after oviposition. The pupation can be recognized by dark pigmentation of the E. kuehniella eggs. E. kuehniella eggs in which fully developed wasp pupae (with red eyes) were recognizable were aligned on double-sided tape using a paintbrush. Microinjection needles were made from GD-1 borosilicate glass capillaries (Narishige, Tokyo, Japan) using a PN-3 needle puller (Narishige). Using a disposable razor blade, the tip of a needle was cut to make it approximately 7 lm in diameter. The Wolbachia-containing SPG buffer described above was microinjected into the ventral side of T. evanescens pupae by penetrating the chorion of E. kuehniella eggs using an M152 3D-micromanipulator (Narishige) and an IM-6 microinjector (Narishige) under a TMD300 dissecting microscope (Nikon, Tokyo, Japan).
2.6. Detection of Wolbachia in the recipient wasps Isofemale lines were established from each of the microinjected T. evanescens females. After mass-mating and oviposition, all individuals in each isofemale line were squashed in a 1.5-ml microtube and subjected to the above-mentioned proteinase K treatment. Diagnostic PCR for Wolbachia infection performed as described above allowed us to detect the presence or absence of Wolbachia in each line.
2.7. Nucleotide sequencing To confirm that the detected Wolbachia in the recipient wasps were wDen, the PCR products of the partial sequence of the Wolbachia wsp gene were directly sequenced using a BigDye DNA Sequencing Kit ver. 3.0 (PE Applied Biosystems, Tokyo, Japan). The sequencing products were evaluated using an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems).
2.8. Quantitative PCR Quantitative PCR was carried out with real-time detection of the 16S rRNA gene of Wolbachia in the cell line ( A. albopictus), the native host ( T. dendrolimi) and the new host ( T. evanescens) using an ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems). Whole A. albopictus cells in the vessels were harvested at intervals of several days, and PCR templates were prepared with the above-mentioned proteinase K treatment. When the bottom of the vessel was covered with a confluent cell monolayer (9 days after inoculation), the cells were diluted fivefold. Six adults were harvested from the native host T. dendrolimi (female). Six adults were harvested from the recipient host (T. evanescens; female) in each of the G5 and G11 generations. The Wolbachia 16S rRNA gene was amplified by PCR using the primers Triwol_16S-8F (50 -GCGCGTAGGCTGGTTAATAAGT-30 ) and Triwol_16S-153R (50 -TGGGTGTTCCTCCTAATATTTACGA-30 ), and a single-stranded DNA probe RT16S (50 -TCCCGAGGCTTAACCTTGGAATTGCT-30 ) as previously described (Kubota et al., 2005). The Wolbachia density data obtained by quantitative PCR were subjected to statistical analyses using R ver. 2.4.0 software (R Development Core Team, 2005). As some of the data sets did not exhibit a normal distribution and/or homogeneous variance, we adopted a generalized linear model (McCullagh and Nelder, 1989) for Gaussian, inverse Gaussian, gamma, or negative binomial distributions, which was selected according to the Akaike information criterion.
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3. Results
4. Discussion
3.1. Establishment of a wDen-infected NIAS-AeAl-2 cell line
4.1. Transinfection and maintenance of Wolbachia in insect cell culture
Diagnostic PCR consistently showed stable maintenance of Wolbachia in the cell culture. Quantitative PCR showed that the Wolbachia titer increased exponentially from days 1–9 and from days 10–18 in the cell culture (Fig. 1). The speed of the Wolbachia proliferation is likely to be comparable to that of the insect cells.
Cultivation of Wolbachia in insect cell lines has previously been documented (Dobson et al., 2002; Kageyama et al., 2008; Kubota et al., 2005; Noda et al., 2002). In the present study, a Wolbachia strain derived from T. dendrolimi (wDen) was successfully proliferated and maintained in the mosquito cell line NIAS-AeAl-2. This is the second report of cultivation of Wolbachia derived from Trichogramma wasps in insect cell cultures following Kubota et al. (2005), who reported cultivation of Wolbachia from T. kaykai (wKay).
3.2. Transinfection of wDen into T. evanescens Among 276 pupae inoculated with wDen-infected cells, 65 individuals (37 females and 28 males) emerged as adults (Table 1). Among the 37 females, 25 were positive for Wolbachia (Table 1, G0). Among 25 isofemale lines produced by the Wolbachia-positive females, 20 were Wolbachia-positive in G1 (Table 2). In G2, all 18 lines were positive for Wolbachia (Table 2). Direct sequencing demonstrated that the partial sequence of the wsp gene (555 bp) in the recipient T. evanescens was identical to that in T. dendrolimi, confirming that the transferred Wolbachia was wDen. 3.3. High vertical transmission and absence of detectable phenotype in the recipient host In G4 and G10, 24 and 11 unmated females were individually allowed to lay eggs on a sufficient number of E. kuehniella eggs for 48 h. All of their offspring (average of about 15 individuals per brood) were males. The presence of Wolbachia in their mothers (24 and 11 individuals, respectively) was individually confirmed by PCR. These findings indicated extremely low or no penetrance of Wolbachia (induction of thelytokous parthenogenesis) in the recipient insects. In addition, the presence of Wolbachia in all the examined individuals indicates high vertical transmission of Wolbachia in the recipient host (>95.8% in G4; >90.9% in G10). 3.4. Reduced titer of Wolbachia in the recipient host The Wolbachia titers were estimated by quantitative PCR in the G5 and G11 broods (Fig. 2). Compared with the Wolbachia titer in the native host T. dendrolimi, the Wolbachia titers in the recipient host (T. evanescens) were significantly lower. Furthermore, the Wolbachia titer in G11 was significantly lower than that in G5, suggesting a gradual decrease in the Wolbachia density in the recipient host.
4.2. Wolbachia invasion in the host germline via somatic microinjection The high vertical transmission of Wolbachia and maintenance of Wolbachia for more than 10 generations showed that wDen invaded the germline of T. evanescens. This is consistent with the presence of Wolbachia for 26 generations in the recipient species in a previous study, in which Wolbachia derived from Trichogramma pretiosum was transferred to a naturally-uninfected strain of T. dendrolimi (Grenier et al., 1998). Microinjection of Wolbachia into somatic tissues does not always establish infection in the germline (e.g., Kageyama et al., 2008). Therefore, the germline of Trichogramma spp. may relatively easily accept Wolbachia infection. Invasion of Wolbachia into the germline via somatic microinjection is also known in Drosophila (Frydman et al., 2006) and planthoppers (Kang et al., 2003; Kawai et al., 2009). 4.3. Highly adapted relationship between Wolbachia and its host? The Wolbachia density was consistently lower in the recipient host (T. evanescens) than in the native host (T. dendrolimi). It has been repeatedly observed in insects and insect cell lines that transferred Wolbachia exhibit a weaker phenotype or lower density in the new host compared with the native host (Hughes et al., 2012; Ikeda et al., 2003; Riegler et al., 2004; Rigaud et al., 2001). Conversely, pathogenic Wolbachia wMelPop in Drosophila melanogaster exhibited a high initial density and virulence in a new host, Drosophila simulans (McGraw et al., 2002). In the recipient host, the Wolbachia titer in G11 was significantly lower than that in G5, which may indicate a gradual decrease in the Wolbachia density. Despite successful invasion of the germline, the genetic background of T. evanescens may not be optimal for the proliferation of wDen. However, the high vertical transmission of wDen in the recipient host (i.e., all 24 and 11 females were infected in G4 and G11, respectively) tempts us to envisage an alternative view, in which the density of Wolbachia in T. dendrolimi is harmful to T. evanescens and the decreased density of Wolbachia is the result of selection for less virulent Wolbachia that ensure high transmission efficiency in T. evanescens (McGraw et al., 2002). More detailed experiments may reveal which of the two hypotheses (i.e., genetic incompatibility versus selection for lower virulence) is correct. In any case, it seems likely that Wolbachia in Trichogramma species are highly adapted to their hosts. 4.4. Low penetrance or absence of Wolbachia phenotype in the recipient host?
Fig. 1. Proliferation of Wolbachia derived from T. dendrolimi (wDen) in the cell line NIAS-AeAl-2. The relative numbers of Wolbachia-specific 16S rRNA fragments within a culture vessel on days 1, 3, 6, 9, 10, 12, 15, and 18 compared with the number on day 10 are shown. One-fifth of the contents was transferred to a new culture vessel and diluted with new medium on day 10.
Despite the maintenance of wDen for more than 10 generations in the recipient host, approximately 500 individuals produced by 35 unmated females were all males (approximately 15 males were examined in each brood). This observation may suggest that the
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Table 1 Survivorship and infection status of T. evanescens microinjected with Wolbachia.
a
Trial
No. of microinjected pupae
No. of emerged adults (female:male)
Survival rate
Proportion of infected adults (n)a
1 2 3 4 5 Total
38 58 60 60 60 276
10 11 13 10 21 65
0.26 0.19 0.22 0.17 0.35 0.24
0.33 1.00 0.46 0.71 0.85 0.68
(3:7) (3:8) (11:2) (7:3) (13:8) (37:28)
(3) (3) (11) (7) (13) (37)
Only females were examined for infection. Wolbachia-positive females were used as founders of isofemale lines.
Table 2 Wolbachia infection in isofemale lines established using microinjected T. evanescens females. Trial
1 2 3 4 5 Total
G1
G2
No. of infected lines (n)
Proportion
No. of infected lines (n)
Proportion
0 (1) 3 (3) 3 (5) 5 (5) 9 (11) 20 (25)
0.00 1.00 0.60 1.00 0.82 0.80
– 1 (1) 3 (3) 5 (5) 9 (9) 18 (18)
– 1.00 1.00 1.00 1.00 1.00
study) might be sufficiently long for such selection to act and let Wolbachia to lose the ability to cause thelytokous parthenogenesis. It has been reported in D. melanogaster that the densities of Wolbachia (i.e., the strain wMelPop) and the expression of the phenotype (i.e., cytoplasmic incompatibility) were reduced when reintroduced after cultivation in the A. albopictus cell line (McMeniman et al., 2008). Another possibility is that the genetic differences between the native host ( T. dendrolimi) and the new host (T. evanescens), although closely related, are attributed to the loss of the Wolbachia phenotype. It has been reported that the transfer of Wolbachia between closely related host species may change their phenotype (Jaenike, 2007; Sasaki et al. 2002, 2005). 4.5. Significance of publication of the results of Wolbachia transfers Fig. 2. Relative titers of Wolbachia wDen in the native host T. dendrolimi (solid bar) and the recipient host T. evanescens (open bars). The relative numbers of Wolbachiaspecific 16S rRNA fragments per single insect compared with that in the native host T. dendrolimi are shown. The sample sizes are given in parentheses. The different letters above the bars indicate significant differences (P < 0.05, generalized linear model with Bonferroni corrections).
transferred Wolbachia did not induce thelytokous parthenogenesis in the recipient insect. Alternatively, it may represent very low penetrance of the transferred Wolbachia. If this was the case, less than 0.2% (one of 500 individuals) of the offspring would be diploid, i.e., parthenogenetically-produced females. This value is not significantly different from the result of Grenier et al. (1998), who found that only 6 of 2490 offspring (0.24%) were females in the transinfected species (Fisher’s exact probability test, P > 0.05). As Grenier et al. (1998) speculated, the very low expression of thelytokous parthenogenesis in the recipient insects may be caused by the reduced Wolbachia density, which was demonstrated in our quantitative PCR assays. It is possible that the threshold Wolbachia density for inducing thelytokous parthenogenesis lies between the density in the donor insect and that in the recipient insect. However, other hypotheses can explain the loss of parthenogenesis induction in the new host. One possibility is that the cultivation of Wolbachia in a cell line of the mosquito A. albopictus, a species distantly related to the native host, has relaxed the selection for the ability to cause thelytokous parthenogenesis. Three months (i.e., cultivation period of Wolbachia in the cell line in this
At present, it seems likely that Wolbachia transferred between closely related hosts are more likely to induce a stronger phenotype and higher vertical transmission than those transferred between distantly related hosts (Kawai et al., 2009; Sakamoto et al., 2005; Van Meer and Stouthamer, 1999; Walker et al., 2011; Zabalou et al., 2004). However, even Wolbachia transferred between closely related hosts can express a phenotype that was weak or undetectable (Grenier et al., 1998; this study), while Wolbachia transferred between distantly related hosts expressed a strong phenotype (Kang et al., 2003). Therefore, the question arises as to the condition that allows a sufficiently high density and high penetrance of Wolbachia. At present, the number of reports on Wolbachia transfers is too small to systematically understand the relationship between the combination of Wolbachia and their hosts and the expression of their phenotype. This is largely caused by publication bias, in that researchers tend to publish their results with a clear phenotype. In this sense, publication of more work on Wolbachia transfers between various host combinations is encouraged to systematically understand the condition of Wolbachia required to express its phenotype, which would contribute to better understanding of Wolbachia-induced host manipulations. Acknowledgment We thank Dr. Hiroaki Noda, National Institute of Agrobiological Sciences, for providing the mosquito cell line NIAS-AeAl-2.
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