Contrasting Patterns of Virus Protection and Functional Incompatibility Genes in Two Conspecific Wolbachia Strains from Drosophila pandora

Abstract

Wolbachia infections can present different phenotypes in hosts, including different forms of reproductive manipulation and antiviral protection, which may influence infection dynamics within host populations. In populations of Drosophila pandora two distinct Wolbachia strains coexist, each manipulating host reproduction: strain wPanCI causes cytoplasmic incompatibility (CI), whereas strain wPanMK causes male killing (MK). CI occurs when a Wolbachia-infected male mates with a female not infected with a compatible type of Wolbachia, leading to nonviable offspring. wPanMK can rescue wPanCI-induced CI but is unable to induce CI. The antiviral protection phenotypes provided by the wPanCI and wPanMK infections were characterized; the strains showed differential protection phenotypes, whereby cricket paralysis virus (CrPV)-induced mortality was delayed in flies infected with wPanMK but enhanced in flies infected with wPanCI compared to their respective Wolbachia-cured counterparts. Homologs of the cifA and cifB genes involved in CI identified in wPanMK and wPanCI showed a high degree of conservation; however, the CifB protein in wPanMK is truncated and is likely nonfunctional. The presence of a likely functional CifA in wPanMK and wPanMK's ability to rescue wPanCI-induced CI are consistent with the recent confirmation of CifA's involvement in CI rescue, and the absence of a functional CifB protein further supports its involvement as a CI modification factor. Taken together, these findings indicate that wPanCI and wPanMK have different relationships with their hosts in terms of their protective and CI phenotypes. It is therefore likely that different factors influence the prevalence and dynamics of these coinfections in natural Drosophila pandora hosts.IMPORTANCEWolbachia strains are common endosymbionts in insects, with multiple strains often coexisting in the same species. The coexistence of multiple strains is poorly understood but may rely on Wolbachia organisms having diverse phenotypic effects on their hosts. As Wolbachia is increasingly being developed as a tool to control disease transmission and suppress pest populations, it is important to understand the ways in which multiple Wolbachia strains persist in natural populations and how these might then be manipulated. We have therefore investigated viral protection and the molecular basis of cytoplasmic incompatibility in two coexisting Wolbachia strains with contrasting effects on host reproduction.

Keywords: Dicistroviridae; Wolbachia; antiviral protection; cytoplasmic incompatibility; male killing; virus blocking.

FIG 1

PCR confirmation of the presence of cifA and cifB in wPanCI and wPanMK in Drosophila pandora (A) and PCR confirmation that the cifA (B) and cifB (C) genes are associated with the Wolbachia genomes and not the associated host Drosophila pandora genomes. One percent agarose gel electrophoresis with ethidium bromide staining was used to show DNA fragments amplified by PCR that targeted internal sections of the genes cifA (604 bp) and cifB (431 bp). PCR was conducted on DNA extracted from pools of 5 flies from each fly line. (A) Lanes 3 to 6, PCR products obtained using cifB PCR primers. Lanes 7 to 10, PCR products obtained using cifA PCR primers. Lane 1, 100-bp ladder (NEB); lane 2, empty; lanes 3 and 7, C168-wPanMK; lanes 4 and 8, pl-wPanCI; lanes 5 and 9 Co-wAu (negative control); lanes 6 and 10 w¹¹¹⁸-wMelPop (positive control). There are clear bands in lanes 3 and 4 of approximately 431 bp, showing that C168-wPanMK and pl-wPanMK are positive for the presence of the conserved internal section of cifA. There are also bands of approximately 604 bp in lanes 7 and 8, showing that C168-wPanMK and pl-wPanMK are positive for the presence of the conserved internal region of cifB. (B) PCR products obtained using cifA (604-bp) PCR primers. Lane 1, 100-bp ladder (NEB); lane 2, 10 w¹¹¹⁸-wMelPop (positive control); lane 3, Co-wAu (negative control); lane 4, C168-Tet; lane 5, pl-Tet. The absence of bands in lanes 4 and 5 shows that cifA is not present in the genome of C168 or pl flies. (C) PCR products obtained using cifB (431-bp) PCR primers. Lane 1, 100-bp ladder (NEB); lane 2, 10 w¹¹¹⁸-wMelPop (positive control); lane 3, Co-wAu (negative control); lane 4, C168-Tet; lane 5, pl-Tet. The absence of bands in lanes 4 and 5 shows that cifB is not present in the genome of C168 or pl flies.

FIG 2

Differential expression of the cifA and cifB genes in wPanCI and wPanMK. Total RNA was extracted from 20 paired ovaries of 11- to 14-day-old female flies from three different cohorts of pl-wPanCI and C168-wPanMK Drosophila pandora lines. Relative RNA levels of the cif genes were measured via RT-qPCR and normalized against the host gene, actin88F. Graphs represent the mean and standard error of the mean (SEM) from 4 to 6 independent replicates. The difference between expression of cifA and cifB in the respective fly lines was analyzed using the nonparametric Mann-Whitney test. There is a significant difference between the expression of cifA and cifB in pl-wPanCI fly ovaries (**, P = 0.0022 by the nonparametric Mann-Whitney test). For only one of four samples was cifB expression in C168-wPanMK flies above detectable levels; expression of cifB therefore could not be statistically compared to that of cifA in C168-wPanMK flies.

FIG 3

(A and B) Schematic depiction of the cifB gene (3,501 nucleotides [nt]) and protein (1,167 amino acids [aa]) homologs in wPanCI (A) and of the homologs and deletion in wPanMK (B). The annotated Ulp1-like protease domain has been shown experimentally to be involved in modifications induced by cifB (41). (A) The nucleotide sequence contains nucleotide substitutions (not shown) but no frameshift mutations. The translated protein contains the Ulp1-like protease domain, suggesting that the protein encoded is functional. (B) The nucleotide sequence contains nucleotide substitutions (not shown) and a single nucleotide deletion at nucleotide position 1213. This deletion causes a frameshift and premature stop codon in the sequence, resulting in the truncation of the encoded protein. The truncated protein does not contain the Ulp1-like protease domain, suggesting that this protein is nonfunctional. *, stop codon. The diagram is not to scale. (C) Chromatograph showing raw sequencing data from CLC workbench depicting the single-nucleotide deletion in the wPanMK cifB homolog at nucleotide position 1213.

FIG 4

Cricket paralysis virus (CrPV)-induced mortality and viral accumulation in pl-wPanCI, pl-Tet, C168-wPanMK, and C168-Tet. (A and B) Survival curves derived from the survfit function in R studio (v. 1.0.153). Survival of three cohorts of 4- to 7-day-old females from the pl-Tet (black lines) and pl-wPanCI (gray lines) (A) and the C168-Tet (black lines) and C168-wPanMK (gray lines) (B) lines following challenge with CrPV (10⁹ IU/ml) (dashed lines) and a PBS control (solid lines) was measured. Each cohort contained three vials of 15 flies for each Wolbachia-fly line pairing and treatment group (CrPV and PBS). The coxme function in R studio was used to determine the effect of Wolbachia infection on daily survival and also its effect on CrPV-induced mortality. (A) wPanCI infection had a significant effect on CrPV-induced mortality (P = 0.000032 by the Cox mixed-effects model), decreasing fly survival. (B) wPanMK infection had a significant effect on virus-induced mortality (P = 0.008 by the Cox mixed-effects model), increasing fly survival. (C) RT-qPCR was used to measure the abundance of CrPV RNA normalized to host gene actin88F in pools of at least 3 4- to 7-day-old females from at least 3 separate cohorts at 0 dpi and 2 dpi after challenge with CrPV (10⁹ IU/ml). Graphs show the mean and standard deviation from at least three separate cohorts of flies. A Mann-Whitney unpaired test was used to determine if significant differences exist between the means of the groups. The relative accumulations of CrPV were significantly different between 0 and 2 dpi for each fly line (P < 0.05 by the Mann-Whitney test) but not between Wolbachia and Tet-cured lines at 2 dpi.

FIG 5

Equivalent abundance of Wolbachia in Drosophila pandora infected with wPanCI and wPanMK and high degree of variation in Wolbachia density between individual flies. qPCR was used to measure the relative densities of wPanCI and wPanMK in pools of 5 flies from the pl-wPanCI and C168-wPanMK strains. Pools were collected from three separate cohorts (cohorts A, B, and C), and amplification of the wsp gene was used as the proxy for Wolbachia density and normalized to that of host gene actin88F in qGene. (A) Mean and standard error of the mean (SEM) of Wolbachia abundance. Analysis of the data using a nonparametric Mann-Whitney test indicated no significant difference between the abundances of Wolbachia in pl-wPanCI and C168-wPanMK flies (P = 0.83). The variation in Wolbachia density appears to be greater in pl-wPanCI flies than in C168-wPanMK flies as indicated by a higher range and SEM. (B) The points on the graph represent results from two technical replicates with five different flies from the same cohort. Variation represents variation in the abundance of Wolbachia present in the five different flies sampled from the cohort. This can be seen as a reflection of variation in Wolbachia abundance between individual flies.