Wolbachia screening in spiders and assessment of horizontal transmission between predator and prey

ECOLOGY, BEHAVIOR AND BIONOMICS

Wolbachia screening in spiders and assessment of horizontal transmission between predator and prey

Y Yun^{I, II}; Y Peng^I; FX Liu^I; C Lei^II

^ICollege of Life Science, Hubei Univ, Wuhan, China

^IIHubei Key Lab of Insect Resources Utilization and Sustainable Pest Management, College of Plant Science and Technology, Huazhong Agricultural Univ, Wuhan, China

^{Correspondence}

ABSTRACT

Recent studies have revealed that the prevalence of Wolbachia in arthropods is attributable not only to its vertical transmission, but also to its horizontal transfer. In order to assess the horizontal transmission of Wolbachia between predator and prey, arthropods belonging to 11 spider families and six insect families were collected in the same field of rice. The distribution of Wolbachia in these arthropods was detected by diagnostic PCR amplification of the wsp (Wolbachia outer surface protein gene) and 16S rDNA genes. Nurscia albofasciata Strand (Araneae: Titanoecidae), Propylea japonica Thunberg (Coleoptera: Coccinellidae), Paederus fuscipes Curtis (Coleoptera: Staphylinidae), and Nilaparvata lugens Stal (Homoptera: Delphacidae) were infected with Wolbachia. This is the first report of infection of N. albofasciata and P. fuscipes by Wolbachia. No direct evidence indicated the existence of horizontal transmission of Wolbachia between predator and prey.

Keywords: Insect, predation, phylogenetics

Introduction

Wolbachia are alpha-proteobacteria that infect a wide range of arthropods (O'Neill et al 1992, Rousset et al 1992, Werren & O'Neill 1997) and nematodes (Bandi et al 1998) throughout the world. The effects of these bacteria on the reproduction of their hosts (Werren 1997) include cytoplasmic incompatibility, parthenogenesis, male killing, and feminization. Cytoplasmic incompatibility has been reported in insects, mites, and isopods (Yen & Barr 1971, Hoffmann et al 1986, Breeuwer & Werren 1990, O'Neill & Karr 1990). Thelytokous parthenogenesis has been found in haplodiploid wasps (Stouthamer et al 1990), male killing in insects (Hurst et al 1999), and feminization of genetic males in isopods and insects (Rousset et al 1992, Hiroki et al 2002, Negri et al 2008).

Recently, because of the prevalence of Wolbachia in arthropods, an increasing number of studies have examined the modes of transmission of Wolbachia among their arthropod hosts (West et al 1998, Vavre et al 1999, Huigens et al 2000, 2004, Sintupachee et al 2006, Vaishampayan et al 2007). Vertical transfer of Wolbachia is not the only transmission mode, and other modes of transmission, including the horizontal transmission, are known to occur in different hosts of Wolbachia (West et al 1998, Huigens et al 2004, Kittayapong et al 2003, Sintupachee et al 2006, Raychoudhury et al 2009). Although many of these studies proposed that horizontal transmission of Wolbachia occurs between different hosts, most of these inferences are based on molecular phylogenetic methods, and additional ecological proofs for Wolbachia horizontal transmission are needed.

In order to assess the possibility of horizontal transfer of Wolbachia between predator and prey, we evaluated the possible acquisition of Wolbachia by spiders (belonging to 11 spider families) from their possible prey insects (belonging to six insect families) In a field of rice. Wolbachia infection in the individuals collected in this community was detected by PCR amplification of wsp and 16S rDNA fragments. Because Wolbachia transfers from prey to predator have not yet been verified, in this study, the results of molecular phylogenetic and the potential ecological relationship of predation between spiders and insects were expected to provide valuable proof of whether Wolbachia is able to transfer horizontally from prey to predator.

Material and Methods

Arthropod collection and DNA extraction

A total of 317 individuals of arthropods belonging to five orders, 17 families, and 25 species were collected from a 50 × 10 m plots from a field of rice at the Huazhong Agricultural University, Wuhan, Hubei Province, China, from November 2007 to October 2008. All individuals were identified using specific morphological keys (Table 1).

All spiders were placed in labeled vials, taken alive to the laboratory, and kept under controlled conditions (25°C, 70% RH) without any food for three months in order to avoid false positive results of Wolbachia from prey present in the spider's digestive system, before DNA extraction.

The insects were placed in 100% ethanol and stored in -20°C. DNA was extracted from the head tissues of carnivorous species and from the abdomen of phytophagous species. Genomic DNA was obtained by standard phenol-chloroform extraction (Kocher et al 1989). In order to avoid cross-contamination, each individual spider or insect was first dipped in 75% ethanol for 2 min and then washed with distilled water.

PCR amplification and sequencing

Wolbachia infection was tested by carrying out PCR with two primer sets separately: 1) for the outer surface protein (wsp)- wsp81F (5'-TGG TCC AAT AAG TGA TGA AGA AAC-3') and wsp691R (5'-AAA AAT TAA ACG CTA CTC CA-3') (Braig et al 1998); 2) for the 16S rDNA of Wolbachia- 16wol F (5'-TTG TAG CCT GCT ATG GTA TAA CT-3') and 16wol R (5'-GAA TAG GTA TGA TTT TCA TGT-3') (O'Neill et al 1992). PCR reactions using wsp136F (5'-TG AAA TTT TAC CTC TTT TC-3') and wsp691R , or wsp81F and wsp522R (5'-ACC AGC TTT TGC TTG ATA-3') were also used to check if Wolbachia belonged to supergroup A or B, respectively (Zhou et al 1998). PCR was conducted in 30 μl reaction mixtures consisting of 1 μl DNA template, 1.5u Taq, 3.0 μl of 10 × PCR buffer, 1μM of each primer, 0.2 mM dNTPs, and with a final MgCl₂ concentration of 1.5 mM. The thermal cycling profile consisted of 94°C for 4 min, 35 cycles (30 s at 94°C, 30 s at 55°C, 30 s at 72°C), 72°C for 10 min, and then held at 4°C, and PCR product amplification was verified by gel electrophoresis on a 1.0% agarose gel.

The quality of genomic DNA was tested by PCR amplification of 28s rDNA (Werren et al 1995, West et al 1998). Only DNA samples yielding amplification products were used for further analysis. PCR products of 16S rDNA and wsp were purified using a DNA Purification Kit (Promega) before direct sequencing. If direct sequencing failed three times, each PCR product was inserted into the vector pMD18-T according to the manufacturer's protocol (Takara), and transferred into competent cells of Escherichia coli. Positive insert-containing colonies were selected, and at least three clones per individual were sequenced.

Sequence assemblage and phylogenetic analyses

The sequences obtained were aligned with homologous sequences that were deposited at the GenBank by using the CluxtalX 1.83 algorithm (Thompson et al 1997). DNAsp4 (Rozas et al 2003), MEGA3.1 (Kumar et al 2004) and PAUP 4.0b10 (Swofford 1999) were used to analyze all data and to construct the phylogenetic trees. Phylogenies were constructed using both maximum likelihood (ML) and Bayesian inference (BI) approaches. MrModeltest version 2 (Nylander 2002) was used to construct the appropriate models.

The selected models via the standard AIC using MrModeltest 2 were as follows: HKY+I+G for the 876 bp fragment of 16S rDNA, and GTR+G for the 606 bp fragment of wsp genes. ML trees were constructed using PAUP 4.0b10 with 100 replicates of random stepwise addition sequences and tree-bisection-reconnection branch swapping. For the Bayesian analyses, the analysis for each gene consisted of 3,000,000 generations and four chains using MrBayes version 3.0 (Ronquist & Huelsenbeck 2003). Trees were sampled every 100 generations, resulting in 30,000 total trees. The first 3,000 trees (10%) were discarded as "burnin". Bayesian posterior probabilities were calculated using a 50% majority rule consensus. Three independent runs were performed for each dataset.

Results

Infection of Wolbachia

One species of spider, Nurscia albofasciata Strand (Araneae: Titanoecidae), and three species of insects from three families, Propylea japonica Thunberg (Coleoptera: Coccinellidae), Paederus fuscipes Curtis (Coleoptera: Staphylinidae), and Nilaparvata lugens Stal (Homoptera: Delphacidae) were infected with Wolbachia. This is the first report of infection of P. fuscipes and N. albofasciata with Wolbachia. Two individuals of N. albofasciata were infected with supergroup A of Wolbachia, and one individual each of P. japonica, N. lugens, and P. fuscipes were infected with supergroup B (Table 1).

Wolbachia phylogenies

According to the phylogenetic analyses of Wolbachia 16S rDNA and wsp genes (Figs 1, 2), Wolbachia infecting N. lugens, P. fuscipes, and P. japonica all belonged to supergroup B; while Wolbachia infecting N. albofasciata belonged to supergroup A. Wolbachia 16S rDNA sequences from N. lugens showed high nucleotide sequence similarity to those from P. fuscipes (99.7% homology) and P. japonica (98.8% homology). For the wsp sequences from Wolbachia, the homology was 92.2% between N. lugens and P. fuscipes. Furthermore, the Wolbachia wsp sequences in P. fuscipes indicated high nucleotide sequence similarity (99.7% homology) to that from Tetranychus urticae (Acari: Tetranychidae) deposited in GenBank (accession number: AY763428).

Discussion

Wolbachia was not found in any spiders except N. albofasciata, and Wolbachia from this species proved to be distantly related to insect endosymbionts, involving Wolbachia of P. japonica, P. fuscipes, and N. lugens. Thus, our study provided no phylogenetic evidence of horizontal transmission of Wolbachia between spiders and insects.

In this study, 212 individuals of spiders belonging to 11 families were screened for Wolbachia, but only two individuals were positive. The possible causes for the low infection rates of spiders by Wolbachia could be: 1) the sample was not large enough to show the natural infection rate; 2) it is unlikely that Wolbachia shifted from prey to spiders. In this study, samples including both spiders and insects were collected in the same habitat.

Spiders can predate upon many insects. Only three of the seven insect species (belonging to six families) tested in this study were infected with Wolbachia. If Wolbachia can be transferred from infected insects to uninfected spiders by predation, then the incidence of Wolbachia among spiders should be much higher. Concerning Wolbachia horizontal transmission between spiders and insects, Cordaux et al (2001) detected Wolbachia infection in a woodlouse-eating spider Dysdera erythrina (Araneae: Dysderidae), and proposed that the predator-prey route cannot transfer Wolbachia because the symbionts seemed unlikely to survive in the predators' digestive tract. Their suggestion is congruent with the results in this study.

We also argue that the specialized food-intake mechanisms in spiders impede the transmission of Wolbachia. In spiders, digestion is initiated outside the body. After the prey is subdued, spiders regurgitate their digestive fluids from the intestinal tract into the victim, and then suck in a drop of the predigested liquid prey, repeating this process many times (Foelix 1996). Wolbachia is an endosymbiont that cannot live outside its host's cells (Werren 1997). Therefore, the extra-oral digestion system of spiders may be able to destroy the cell structure of the victim.

The phylogenetic analysis of the 16S rDNA and wsp gene fragments indicated a close similarity in nucleotide sequence between Wolbachia in N. lugens and P. fuscipes. Paederus fuscipes and N. lugens have a potential predator-prey relationship. However, wsp or 16S rDNA sequences obtained from them were dissimilar. Previous studies indicated that high rates of recombination have occurred in the wsp gene (Baldo et al 2005, Roy & Harry 2007, Verne et al 2007), and therefore phylogenetic reconstruction according to wsp fragments is not completely reliable. Recently, Multilocus Sequence Typing (MLST) has been an effective means of detecting diversity among strains within a single host, as well as for identifying closely related strains found in different hosts (Baldo et al 2006, Baldo & Werren 2007, Baldo et al 2008). In addition to spiders, other natural enemies of insects are suitable subjects to test the possibility of horizontal transfer of Wolbachia through predation by means of the MLST method, in future studies.

Acknowledgments

We are grateful to Dr Daiqin Li (National University of Singapore) for critical comments on the manuscript. We also thank Dr K D Floate (Lethbridge Research Centre, Agriculture and Agri-Food, Canada) for giving valuable advice on the manuscript. This work was supported by the National Natural Science Foundation of China (No: 30870284).

Received 10 December 2009 and accepted 11 October 2010

Edited by Wesley A C Godoy - ESALQ/USP

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Publication Dates

• Publication in this collection
  09 May 2011
• Date of issue
  Apr 2011

History

• Received
  10 Dec 2009
• Accepted
  11 Oct 2010