DOI: 10.5061/dryad.w0vt4b8s9
title: Bacterial supergroup specific 'Cost' of Wolbachia infections in Nasonia vitripennis

description: 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.
description: <b>Sequential mating and sperm depletion of the
 male:</b> 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. <b>Host longevity, progeny family
 size, and fecundity</b> To test whether the
 presence of <i>Wolbachia</i> 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
 <i>Wolbachia</i> 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<b> </b>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
 <i>Wolbachia </i>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.
 <b>Estimation of relative density of
  <i>Wolbachia</i> infections across different developmental
 stages of <i>N. vitripennis</i> </b>
 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<i>.
 </i>Three replicates of 10 larvae or pupae from the three strains
 <i>w</i>A(PU), <i>w</i>B(PU), and
 <i>w</i>A<i>w</i>B(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 <i>Wolbachia</i> across the strains. Amplification
 was done for the <i>Wolbachia</i> hcpA gene (Forward Primer:
 5’-CTTCGCTCTGCTATATTTGCTGC-3’, Reverse Primer:
 5’-CGAATAATCGCAACCGAACTG-3’). The primers were tested
 to amplify both the <i>Wolbachia</i> supergroup A and B
 strains. <i>Nasonia</i> 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 <i>N. vitripennis</i>
 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 <i>Wolbachia</i> was estimated by
 calculating the mean delta threshold cycle (ΔC<sub>q</sub>),
 using the formula:  ΔC<sub>q
 </sub>=<i>1/3</i><sub><i>j=1</i></sub><sup><i>3</i></sup><i>1/3</i><sub><i>i=1</i></sub><sup><i>3</i></sup><i>hcpA-1/3</i><sub><i>i=1</i></sub><sup><i>3</i></sup><i>S6K</i>   where i = number of technical replicates and j = number of biological replicates. 1/ ΔCq was calculated and plotted to show the <i>Wolbachia</i> density across different developmental stages of males and females. The relative <i>Wolbachia</i> density was compared across the three different strains using the Kruskal Wallis <i>H</i> test. Mann Whitney U test was used to compare two different groups with a significance level of 0.05.
description: N/A shows no values.