The intracellular bacterium Wolbachia uses parasitoid wasps as phoretic vectors for efficient horizontal transmission

Abstract

Facultative bacterial endosymbionts are associated with many arthropods and are primarily transmitted vertically from mother to offspring. However, phylogenetic affiliations suggest that horizontal transmission must also occur. Such horizontal transfer can have important biological and agricultural consequences when endosymbionts increase host fitness. So far horizontal transmission is considered rare and has been difficult to document. Here, we use fluorescence in situ hybridization (FISH) and multi locus sequence typing (MLST) to reveal a potentially common pathway of horizontal transmission of endosymbionts via parasitoids of insects. We illustrate that the mouthparts and ovipositors of an aphelinid parasitoid become contaminated with Wolbachia when this wasp feeds on or probes Wolbachia-infected Bemisia tabaci AsiaII7, and non-lethal probing of uninfected B. tabaci AsiaII7 nymphs by parasitoids carrying Wolbachia resulted in newly and stably infected B. tabaci matrilines. After they were exposed to infected whitefly, the parasitoids were able to transmit Wolbachia efficiently for the following 48 h. Whitefly infected with Wolbachia by parasitoids had increased survival and reduced development times. Overall, our study provides evidence for the horizontal transmission of Wolbachia between insect hosts by parasitic wasps, and the enhanced survival and reproductive abilities of insect hosts may adversely affect biological control programs.

Fig 1. FISH detection of Wolbachia in adult Eretmocerus parasitoids.

The E. sp. nr. furuhashii wasps were selected randomly 24–48 h after visiting Wolbachia-positive AsiaII7 whitefly nymphs. Panels (A-D): Wolbachia in parasitoid with different body poses, it was found both in the mouthparts (M) and ovipositors (OP); panels (E, F): Wolbachia in the parasitoid mouthparts (front view). Left panels (A, C, E): fluorescence in dark field; right panels (B, D, F): fluorescence in bright field.

Fig 2. FISH detection of Wolbachia in AsiaII7 whitefly.

Wolbachia has two distributions in 3^rd instar AsiaII7 nymphs randomly selected from the Eretmocerus rearing cages. Top panels (A, B): in the scattered pattern, Wolbachia was found both in the ovary (OV) and haemolymph (H) of whitefly; bottom panels (C, D): in the confined pattern, Wolbachia was only found in the ovary (OV) of whitefly. Left panels (A, C): fluorescence in dark field; right panels (B, D): fluorescence in bright field.

Fig 3. The prevalence of newly-acquired Wolbachia in AsiaII7 whitefly over five generations.

The proportion on infected whitefly was measured in a population over 5 generations. The population was founded by four pairs of new emerged AsiaII7 whitefly that had been infected with Wolbachia acquired from a contaminated wasp. The proportion of Wolbachia-infected offspring was monitored with PCR. The bars are standard errors. The means and standard errors were estimated using a generalized linear model and are back-transformed from the logit scale. The is no significant heterogeneity in the prevalence among generations (GLMM likelihood ratio test: χ² = 0.92, df = 4, P = 0.92).

Fig 4. The persistence of newly-acquired Wolbachia in parasitoids.

A: Wolbachia detection by PCR using wsp gene in the seven days after the parasitoid visited an infected host. In each PCR reaction Wolbachia DNA was extracted from 10 individuals. B: The decline in Wolbachia transmission rates with time after the wasp is contaminated. Female Eretmocerus wasps were released to parasitize Wolbachia negative AsiaII7 2^nd-3^rd instar nymphs 24, 48, 72 and 96 h after contamination with Wolbachia. The Wolbachia infection rate of whitefly that were visited by wasps but survived to adulthood was detected with PCR. Each point is the transmission rate to ten whiteflies in a single replicate of the experiment. The horizontal bars are the median. There is a significant decline in transmission rates (logistic generalized linear model: z = 3.50, P = 0.0005).

Fig 5. Effects of Wolbachia on whitefly fitness.

Traits affecting the fitness of newly Wolbachia-infected offspring were compared with those Wolbachia uninfected populations under laboratory conditions. A: percent female, B: development time, C: immature survival, D: female adult fecundity and E: female adult longevity. The data are means ± SE.

Fig 6. The effect of horizontal transmission on the spread of Wolbachia through populations.

Simulations were performed to explore the effect of horizontal transmission on the prevalence of Wolbachia. The blue lines are where there was no horizontal transmission (w = 0), the dashed orange line was a low rate (w = 0.01), and the solid orange line a moderate rate of horizontal transmission (w = 0.06). A: The effect of horizontal transmission when there is not effect of Wolbachia on host fitness and no reproductive manipulation (H = 1, F = 1). B: Wolbachia induces cytoplasmic incompatibility (H = 0.1, F = 1). C: Wolbachia carries a fitness benefit (H = 1, F = 1.05). In all cases the starting prevalence was p = 0.01 and the rate of imperfect maternal transmission was μ = 0.03.