Wolbachia is known to alter host reproductive biology for its benefit. It also introduces both positive and negative fitness effects in the vast majority of its host population. Here we describe the effect of maintenance of Wolbachia infection in the parasitoid host Nasonia vitripennis. We have separated the two Wolbachia infections in N. vitripennis to get single lines of Wolbachia supergroup A and supergroup B. We compare the effect of Wolbachia infection on various life-history traits between uninfected, single A, single B, and double infection strains. Contrary to the previous reports, our results suggest that there is a “cost” associated with the maintenance of Wolbachia infection where traits like progeny family size, fecundity, and longevity in females, while the number of copulations performed by males is compromised in Wolbachia infected strains. The double infection of Wolbachia has more detrimental impacts on the host as compared to single infections. The findings of this study raise important questions on the mechanism of survival and maintenance of these reproductive parasites in arthropod hosts.

Sequential mating and sperm depletion of the male:

The males used were obtained from virgin females hosted with one fly pupa for 24 hours and were not given any external sources of nutrition (usually a mixture of sucrose in water) before the experiment. Each male was then mated sequentially with virgin females from the same strain. At the first sign of a male not completing the entire mating behavior (Jachmann and Assem 1996) it was given a rest for half an hour and was subjected to mating again until it stopped mating. The mated females were given a day and each of them was hosted with one fly pupa for 24 hours. The females were removed after 24 hours and the offspring were allowed to emerge and then counted. The average number of copulations and the number of copulations, before sperm depletion, were compared using the Mann-Whitney U test with a significance level of 0.05.

Host longevity, progeny family size, and fecundity

To test whether the presence of Wolbachia has any influence on longevity, emerging adult wasps were kept individually in ria vials at 25⁰C, without any additional nutrition. Survival following emergence was measured by counting the number of dead individuals every 6 hours. Log Rank Statistics was used to identify differences between strains with a significance level of 0.05

To test for the effect of resident Wolbachia infections on the adult progeny family size of virgin and mated females, each female was sorted at the pupal stage and separated into individual ria vials. To enumerate the progeny size of mated females, some of these virgins were offered single males from the same strain and observed till mating was successful. All the females were then hosted individually with one fly pupa for 24 hours. These were kept at 25⁰C for the offspring to emerge which were later counted for progeny size by randomizing the ria vials in a double-blind assay. The differences were compared using the Mann-Whitney U test with a significance level of 0.05.

To investigate if Wolbachia affects the fecundity of the females, emerged females were hosted with one host for 24 hours. The host pupa was placed in a foam plug so that only the head portion of the pupa was exposed and available for the females to lay eggs. The female was removed after 24 hours and the eggs laid were counted under a stereomicroscope (Leica M205 C). The differences in fecundity were compared using the Mann-Whitney U test with a significance level of 0.05.

Estimation of relative density of  Wolbachia infections across different developmental stages of N. vitripennis

To collect the different developmental stages, females were hosted for 4 hours, (instead of 24 hours in the previous experiments), with one host to narrow down the developmental stages of the broods. The larval and pupal stages (from Day 3 to Day 13 for males and from Day 8 to Day 14 for females) were collected every 24 hours. Larval stages for females were not done to avoid any DNA contamination from the males as the two sexes are virtually indistinguishable at the larval stages. Three replicates of 10 larvae or pupae from the three strains wA(PU), wB(PU), and wAwB(PU) were collected for each developmental stage. DNA extraction was done using the phenol-chloroform extraction method, where samples were crushed in 200 µl of 0.5 M Tris-EDTA buffer with 1% sodium dodecyl sulfate (SDS) and 2 µlof 22 mg/ml Proteinase K and incubated overnight at 37°C. DNA was purified with buffer saturated phenol and chloroform-isoamyl alcohol solution (24:1 ratio) and the DNA was precipitated overnight with isopropanol at -20° C. The precipitated DNA pellet was dissolved in 60 µl nuclease-free water. The DNA concentration of the samples was measured using a spectrophotometer (Nanodrop 2000®, Thermo Scientific). The concentrations of all the samples were normalized to 200 ng/µl across the different male and female developmental stage samples to be used for quantitative PCR. CFX96 C1000® Touch Real-time qRT-PCR machine (BioRad) was used to assay the relative density of Wolbachia across the strains. Amplification was done for the Wolbachia hcpA gene (Forward Primer: 5’-CTTCGCTCTGCTATATTTGCTGC-3’, Reverse Primer: 5’-CGAATAATCGCAACCGAACTG-3’). The primers were tested to amplify both the Wolbachia supergroup A and B strains. Nasonia S6K was used as the control gene (Bordenstein and Bordenstein, 2011). The total reaction volume contained 5 μl of iTaq Universal SYBR® Green supermix (BIO-RAD), 0.05 μl each of 10 μM forward and reverse primers, and 200 ng of template DNA for a total volume of 10 μl in each reaction. Uninfected N. vitripennis DNA was used as negative control while DNase-free water was used as non-template control. Reaction conditions included an initial denaturation step of 95°C for 3 minutes followed by 39 cycles of 95°C for 10 seconds, annealing, and amplification at 52°C for 30 seconds. The reaction was run for 40 cycles. All the reactions were performed in triplicates and included a melt curve to check for non-specific amplification. The relative density of Wolbachia was estimated by calculating the mean delta threshold cycle (ΔC_q), using the formula: 

ΔC_q =1/3_j=1³1/3_i=1³hcpA-1/3_i=1³S6K  

where i = number of technical replicates and j = number of biological replicates.

1/ ΔCq was calculated and plotted to show the Wolbachia density across different developmental stages of males and females. The relative Wolbachia density was compared across the three different strains using the Kruskal Wallis H test. Mann Whitney U test was used to compare two different groups with a significance level of 0.05.

N/A shows no values.