Introduction

Wolbachia are maternally inherited alpha-proteobacteria known to infect a wide range of arthropods (Werren et al. 1995; Jeyaprakash and Hoy 2000). Recent meta-analysis estimated that >65% of insect species harbour Wolbachia, making it among the most abundant intracellular bacteria genera so far discovered, infecting at least 106 insect species alone (Hilgenboecker et al. 2008). The success of Wolbachia can be attributed in large part to its ability to manipulate the reproduction of its host to promote the spread of infection into the host population. The effects of Wolbachia infection on reproduction include: feminization of genetic males (Rousset et al. 1992); parthenogenetic induction, which results in the development of unfertilized eggs (Stouthamer et al. 1993); the killing of male progeny from infected females (Hurst et al. 1999); and sperm-egg incompatibility, which is referred to as cytoplasmic incompatibility (CI) (Bourtzis et al. 1996). Although variable in strategy, the multiple mechanisms by which Wolbachia manipulates host reproduction are similar in that they provide infected female hosts with a reproductive advantage relative to uninfected females. In addition to the effect on reproduction, Wolbachia can also influence the fitness of the host. The Wolbachia-host interaction ranges along a continuum from mutualism (Bandi et al. 1999; Dedeine et al. 2001; Dobson et al. 2002, 2004) to commensalism (Hoffmann et al. 1996, Charlat et al. 2004) and parasitism (Hoffmann et al. 1990; Stevens and Wade 1990; Snook et al. 2000; Champion de Crespigny and Wedell 2006). Fitness costs or benefits conferred by the symbiont also hinder or promote the spread of infection (Hoffmann et al. 1990; Turelli and Hoffmann 1995; Dobson et al. 2002).

In previous studies, Wolbachia have been found to induce CI in the two-spotted spider mite Tetranychus urticae Koch, with the variability of CI expression ranging from no CI to complete CI, including either female embryonic mortality or male conversion types of CI (Breeuwer 1997; Perrot-Minnot et al. 2002; Vala et al. 2002; Gotoh et al. 2003, 2007). A few studies also examined the effect of Wolbachia on the fitness of T. urticae. Wolbachia in mites collected from cucumber plants did not affect longevity, but infected females produced smaller clutch sizes, a more daughter-biased sex ratio and had decreased F1 mortality (Vala et al. 2003). In the L strain collected from Rose-bay, the fecundity of infected females in the first 7 days was 80–100% less than that in cured females (Perrot-Minnot et al. 2002).

Prior study in our laboratory showed that Wolbachia was widely distributed in Chinese populations of T. urticae. All populations were found to be infected with Wolbachia, and the infection rate was between 2.5 and 85% (Xie et al. 2006). How Wolbachia manipulate the reproduction of T. urticae in China, and whether their relationship is beneficial or harmful are still unclear. China has a widely varying topography and different climatic conditions. High genetic differentiation between Chinese populations of T. urticae has been revealed by microsatellite markers (Li et al. 2009). We chose three populations from the north to south of China to represent Chinese populations: Liaoning (LN), Jiangsu (JS) and Hunan (HN) populations. To understand the relationship between Wolbachia and T. urticae in China, we measured the strength of CI, female ratio, fecundity, survival and development time between infected and uninfected strains under laboratory conditions.

Materials and methods

Mite populations

Three populations, in which Wolbachia was previously detected, were used in this study (Xie et al. 2006). Their locations, host plants, collection dates and abbreviations are summarized in Table 1. Mites were reared on leaves of the common bean (Phaseolus vulgaris L.) placed on a water-saturated sponge mat in Petri dishes (diameter 9) at 25 ± 1°C, 60% relative humidity and with a L16–D8 photoperiod. All experiments were carried out under these conditions.

Table 1 Collection history, locality and host plant data of Tetranychus urticae Koch from China
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DNA extraction, PCR amplification and sequencing

DNA was extracted by homogenizing a single female adult in a 25-μl mixture of STE buffer (100 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and proteinase K (10 mg/ml, 2 μl) in a 1.5-ml Eppendorf tube. The mixture was incubated at 37°C for 30 min and later at 95°C for 5 min. The samples were centrifuged briefly and were used immediately for PCR reactions or stored at −20°C for later use.

All PCR reactions were run in 25 μl buffer using the TAKARA Taq kit (no. R001B; Takara Co., Ltd.): 16.3 μl H2O, 2.5 μl 10× buffer, 1.5 μl of 2.5 mM dNTP, 1.5 μl of 25 mM MgCl2, 0.2 μl Taq (1 U), 2 μl sample and 1 μl primers (20 pmol each). The primers used in this study were for the Wolbachia wsp gene (Zhou et al. 1998): 5′-TGG TCC AAT AAG TGATGA AGA AAC-3′ and 5′-AAA AAT TAA ACG CTA CTC CA-3′. Reactions were cycled 35 times at 94°C for 30 s, 52°C for 45 s and 72°C for 1 min. Reagent negative and positive controls were included in the reactions. The PCR products were electrophoresed on a 1.0% agarose gel in TBE/EtBr for 40 min at 60 mA, and then photographed on a UV transilluminator. The techniques used here are a modified version of the methods reported by Johanowicz and Hoy (1996) and Gomi et al. (1997).

The PCR product was cloned into a pGEM-T vector (Promega). The template DNA was amplified by PCR using M13–20 and reverse primers. The sequence was determined by the dye terminator sequencing method with a DNA Sequencer (model 377 and 3700; PE Applied Biosystems).

Preparation of infected and uninfected lines

In order to cross infected and uninfected individuals, 100% infected and 100% uninfected lines were prepared for each population. One female from the teleiochrysalis stage was allowed to lay eggs without being crossed with males. The eggs were reared until adulthood (males). After the males reached sexual maturity they were backcrossed with the mother. After crossing, the female adults were transferred to new leaf discs and were allowed to lay eggs for 3–5 days. A female was checked for Wolbachia infection by PCR amplification. The eggs were separately reared on new leaf discs depending on the infection status of the mother. The above process was continued for three to four generations until a 100% infected population was obtained.

Uninfected lines were established by treating infected lines with tetracycline. Small leaf discs (ca. 3 cm2) from the common bean were placed on a cotton bed soaked in tetracycline solution (0.1%, w/v) in Petri dishes (9 cm in diameter) and kept for 24 h before they were used for rearing newly hatched larvae. Distilled water was added daily to keep the cotton beds wet. The cotton and the leaf discs were replaced every 4 days. Four to eight generations later, mites were checked by PCR to confirm that the lines were free of Wolbachia. These lines were maintained in a mass-rearing environment without antibiotic for about four generations (2 months) before use to avoid potential side effects of the antibiotic treatment.

Cross experiments

In order to determine reproductive compatibility in intra-population crosses, four cross combinations were carried out: uninfected females were crossed with uninfected males, uninfected females were crossed with infected males, infected females were crossed with uninfected males, and infected females were crossed with infected males. Infected colonies were designated as ‘W’ and antibiotic-cured colonies as ‘U’. Female teleiochrysalids, the last developmental stage before adult emergence, were placed with two males on the same leaf disk. We used 1-day-old virgin males produced as a cohort by groups of females isolated as teleiochrysalids. This procedure was designed to avoid the potential decrease of the Wolbachia effect due to male ageing or repeated consecutive matings. Males were discarded 2 days after the females reached adulthood, and mated females were allowed to oviposit for 5 days. Eggs on leaf discs were checked daily to determine the hatchability, survival rate in immature stages and sex ratio (% daughters). Fecundity was estimated as the total number of eggs laid in the first 5 days. Data were analyzed with one-way analysis of variance (ANOVA), and means were compared using the Tukey-HSD test (SPSS 13.0). To normalize the data, log transformation was used for the number of eggs laid per female, and arcsine square root transformation was used for egg hatchability, survival rate and female ratio.

Survival assessment

Differences in host longevity were observed in comparisons of the different Wolbachia infection types. We measured age-specific survival of the U and W lines by placing 8 virgin females and 8 virgin males of the same infection status of the same population on the same leaf. Three leaves were used for each Wolbachia infection status per population. The leaves were monitored every day, and dead females were removed and counted until all females had died. Survivor curves for individual hosts were compared using the Kaplan-Meier method and log-rank test (Dobson et al. 2004).

Development time assessment

The effect of Wolbachia on the development time of mites of the three populations was assessed. Thirty Wolbachia-infected or uninfected females were placed on a leaf disk and allowed to lay eggs for 8 h. The eggs were moved to new small leaf disks individually. The small disks were monitored every 8 h, and the stage of the mite was recorded until adulthood. The development time of every stage was calculated. The distributions of development times were non-normal, even after attempts to transform the data. We used non-parametric Mann-Whitney U tests to estimate the effects of infection.

Results

Sequences of Wolbachia strains

We amplified a 552 base pair (bp) fragment of the wsp gene from the three Wolbachia strains infecting T. urticae. Wsp sequences were submitted to the GenBank database (GenBank numbers GU014539, GU014541, GU014542). Wolbachia strains in the LN and HN populations of T. urticae had an identical wsp gene sequence. The wsp sequence of the Wolbachia strain in the JS population had 99.5% similarity (3 different nucleotides out of 552 nucleotides) to the sequence in the LN and HN populations.

Strength of cytoplasmic incompatibility

The results of intra-population crosses between infected and cured individuals of the three populations are presented in Table 2. The HN population showed strong unidirectional CI. The hatchability of eggs, survival rate in the immature stage and sex ratio in the cross between uninfected females and infected males (U/W) were significantly lower than those of other crosses (U/U, W/U and W/W).

Table 2 Compatibility of crosses between Wolbachia-infected (W) and antibiotic-treated (U) colonies of Liaoning (LN), Jiangsu (JS) and Hunan (HN) populations of Tetranychus urticae
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Wolbachia showed an intermediate level of CI in the LN population. The LNU/LNW cross resulted in significantly reduced hatchability, survival rate at the immature stage and sex ratio among the four combinations, but the hatchability and sex ratio in this incompatible cross were higher than in the HN and JS populations.

The JS population showed no CI. No reduction in egg hatchability and survival rate at immature stages was observed among the four combinations. The sex ratios of the cross JSW/JSU and the cross JSW/JSW were similar, but the values were significantly different from those in the JSU/JSU and JSU/JSW crosses; that is, infected females produced a higher daughter-biased sex ratio. Since Wolbachia are maternally transmitted and males are an evolutionary dead end for maternally inherited infections, more female offspring would promote the spread of infection.

Fecundity

The effects of infection on female fecundity were tested by comparing the number of eggs laid in the first 5 days by infected and uninfected females in crosses involving infected and uninfected males. The results are shown in Table 2. No difference in fecundity was observed between infected and uninfected females in the LN and JS populations. In the HN population, a significant effect of female infection was found, with infected females laying more eggs on average than uninfected females, regardless of the infection status of males. Infected females often had the greatest fecundity when mated with infected males. The difference in fecundity between the cross HNW/HNW and cross HNW/HNU was significant. In summary, Wolbachia in the HN population was the only strain that could promote the fecundity of infected females.

Host longevity

The effects of Wolbachia on host longevity were tested by comparing the life span between infected and uninfected females. The results are presented in Fig. 1. In the LN population (Fig. 1a), uninfected females (14.16 ± 0.97) lived two times longer (χ 2 = 28.487, df = 1, P < 0.001) than infected females (7.44 ± 0.65). Wolbachia shortened the longevity of infected females and provided a fitness advantage to uninfected females. As shown in Fig. 1b, infected females (15.17 ± 0.90) were significantly longer lived than uninfected females (9.63 ± 0.72) in the JS population (χ 2 = 27.954, df = 1, P < 0.001); Wolbachia benefited infected females in the JS population. As shown in Fig. 1c, the HN population showed no difference in longevity between infected (11.88 ± 0.86) and uninfected females (11.72 ± 0.66) (χ 2 = 0.827, df = 1, P > 0.05). Our results clearly show that Wolbachia prolonged the longevity of the JS population, shortened the longevity of the LN population and had no effect on the longevity of the HN population.

Fig. 1
figure 1

Comparison of Wolbachia effect on female longevity in LN (a), JS (b) and HN (c) populations. W, Wolbachia infected strains; U, uninfected strains. Survivor curves for individual hosts were compared using the Kaplan-Meier method and log-rank test

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Development time

The effects of infection on the development time of females and males were tested by comparing the development time of each stage of mites with different infection statuses. The development times of females with different infection statuses are presented in Table 3.

Table 3 Development times of infected (W) and uninfected (U) females in Liaoning (LN), Jiangsu (JS) and Hunan (HN) populations of Tetranychus urticae
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In the LN population, Wolbachia shortened the time to adulthood of infected females. Infected females reached adulthood significantly earlier than uninfected females according to Mann-Whitney U tests. In the JS and HN populations, Wolbachia prolonged the time to adulthood of infected females. Time to adulthood was significantly shorter in uninfected than infected females. We noted that the effect of Wolbachia on the development time of each stage was different.

The development duration of males was shorter than females in the three populations. The development time of males of different infection status are presented in Table 4. In the LN population, infected males had a shorter development time than uninfected males. In the JS population, infected males had a longer development time than uninfected males. No difference in development duration between infected and uninfected males was observed in the HN population.

Table 4 Development times of infected (W) and uninfected (U) males in Liaoning (LN), Jiangsu (JS) and Hunan (HN) populations of Tetranychus urticae
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Discussion

Diversity of CI expression

We found diversity of CI expression in Chinese populations of T. urticae. CI was expressed as a reduction in egg hatchability and a male-biased sex ratio in crosses between uninfected females and infected males. This is concordant with the female mortality type of CI (Breeuwer 1997; Vavre et al. 2000). We found that CI manifested not only in embryonic development but also in nymphal development in the LN and HN populations. This is the first record of a Wolbachia infection that can affect the nymphal development of hosts.

Several factors have been identified that influence the expression of CI phenotype, such as environmental factors, including the age of the host and temperature, bacteria or host genes and bacterial density (Clancy and Hoffmann 1998; Poinsot et al. 1998; Reynolds and Hoffmann 2002; Sakamoto et al. 2005). In our experiments, we used 1-day-old virgin males and performed experiments at a constant temperature. Real-time quantitative PCR was performed to estimate the numbers of Wolbachia, and no difference in bacterial density was observed among the three populations (unpublished data); therefore, it is unlikely that environmental factors and bacterial density are the reason for the variability of CI.

The CI-Wolbachia strains in the LN and HN populations of T. urticae had an identical wsp gene sequence, which was the same as the sequence of non-CI-Wolbachia in the R23 population of T. urticae in Japan (AB266837). The wsp sequence of non-CI-Wolbachia strain in the JS population had the same wsp sequence as the CI-Wolbachia in the G1 population (AB266804) and non-CI-Wolbachia in the G14 population (AB266804) of T. urticae in Japan. All these Wolbachia strains belong to the Ori subgroup; therefore, we estimated that the variability of the CI expression among the four Chinese populations of T. urticae was due to the interaction between Wolbachia and host genotypes.

Diversity of fitness effects

In addition to the heterogeneity of CI intensity, Wolbachia bacteria can have variable affects on T. urticae fitness. Although Wolbachia strains in the LN and HN populations shared identical wsp sequences, they apparently affected fitness in different ways. Wolbachia infection had a positive effect on fecundity in the HN population. By contrast, no fecundity change was observed in the other strains studied. The Wolbachia-associated effect of promoting fecundity may depend on the nuclear background of the host. This is the first report of a Wolbachia infection promoting the fecundity of infected females in T. urticae. This result is opposite to that found in T. urticae in France, in which Wolbachia infection decreased fecundity by 80–100% (Perrot-Minnot et al. 2002). Models can predict the selection of Wolbachia variants that increase female fecundity (Stevens and Wade 1990). There may be an “attenuation” of Wolbachia effects, progressing toward a relationship less detrimental to the host. A recent study of a California population of D. simulans showed that Wolbachia has changed from a parasite to a mutualist over the last 20 years, so that infected females now exhibit an average 10% fecundity advantage over uninfected females in the laboratory. This change was accompanied by the rapid evolution of Wolbachia (Weeks et al. 2007). The underlying process leading to the increased fecundity of infected females is unknown, but may be due to Wolbachia or compensatory evolution in the host.

We found both positive and negative effects of Wolbachia infection on longevity. The effect of Wolbachia on the longevity of two-spotted spider mites was investigated in a strain collected from cucumber plants, and no difference in longevity between infected and uninfected females was observed (Vala et al. 2003). Our result is the first report of a Wolbachia infection affecting the longevity of infected female spider mites. Infected females in the JS population were significantly longer lived than uninfected females. Our data indicate that 21% infected females died 5 days after emergence, while only 4% infected females died 5 days after emergence. Wolbachia had no effect on fecundity in this population. This result was in a relatively lower number of offspring produced by uninfected females, and therefore it gradually reduced the number of uninfected individuals in the following generations. The fitness benefits to survival contributed to the maintenance of Wolbachia infection in the JS population in the absence of CI. Wolbachia-associated fitness, which benefits survival, was also found in mosquito and fruit fly (Dobson et al. 2002, 2004; Fry et al. 2004). The Wolbachia-associated fitness cost to survival was only reported in D. melanogaster (Min and Benzer 1997; Fry et al. 2004). The popcorn Wolbachia strain (wMelPop), which has been found to over-replicate within the cells of its host, caused a reduction in host longevity, and the effects strongly depended on the host nuclear background and temperature (Carrington et al. 2010). Since the Wolbachia strains in the LN and HN populations shared identical wsp sequences, we estimated that the Wolbachia-associated effect of shorting longevity in the LN population may depend on the nuclear background of the host. The Wolbachia-associated effect of prolonging longevity in the JS population may depend on the interaction between Wolbachia and host genotypes. Future experiments (introgression crosses between populations) are required to generate a homogeneous host genetic background for the characterization of Wolbachia effects.

We noticed that the development time of uninfected females in the LN population (10.92 days) was longer than in the JS (9.81 days) and HN (9.73 days) populations. Located in the northeastern part of China, Liaoning Province is much colder than Jiangsu and Hunan provinces. In the long process of evolution, the suitable temperature for development of the LN population may be lower than for the JS and HN populations, leading to the variation in development time among the populations. Both positive and negative effects of Wolbachia infection on the development time of hosts were found in this study. Wolbachia in the LN population shortened the development time of infected females, and Wolbachia in the JS and HN populations prolonged the development time. This is the second instance of Wolbachia infection affecting the development time of hosts. The other known instance is the virulent wDmpopcorn Wolbachia strain in D. melanogaster, which can delay the development time of the host (Reynolds et al. 2003). The popcorn Wolbachia strain in Drosophila simulans showed both positive and negative effects on the host depending on the nuclear background (Carrington et al. 2010). Our results also suggest that host background can influence the effect of Wolbachia on development. To clarify whether line divergence is due to Wolbachia, the host nuclear genome, or the interaction between these genomes, we should generate genetically homogeneous strains with different Wolbachia using microinjection methodology or introgression crosses.