Deep data mining reveals variable abundance and distribution of microbial reproductive manipulators within and among diverse host species Authors

Bacterial symbionts that manipulate the reproduction of their hosts to increase their successful transmission are important factors in invertebrate ecology and evolution. In light of their use as a biological control agent, studying the genomic and phenotypic diversity of reproductive manipulators can improve efforts to control infectious diseases and contribute to our understanding of host-symbiont evolution. Despite the vast genomic and phenotypic diversity of reproductive manipulators, only a handful of Wolbachia strains are used as biological control agents because little is known about the broad scale infection frequencies of these bacteria in nature. Here we develop a data mining approach to quantify the number of arthropod and nematode host species available on the Sequence Read Archive (SRA) that are infected with Wolbachia and other reproductive manipulators such as Rickettsia and Spiroplasma. Across the entire database, we found reproductive manipulators infected 1733 arthropod and 103 nematode samples, representing 121 and 10 species, respectively. We estimated that Wolbachia infects approximately 24% of all arthropod species and 20% of all nematode species. In contrast, we estimated other reproductive manipulators infect 0-8% of arthropod and nematode species. We show that relative Wolbachia density within hosts, titer, is significantly lower than the titer of the other reproductive manipulators. Considering the fitness costs of high titers, low titer may contribute to enabling Wolbachia’s high prevalence across hosts species and mitigate impacts on host biology compared with other reproductive manipulator taxa. Our study demonstrates that data mining is a powerful tool for understanding host-symbiont coevolution and opens an array of previously inaccessible questions for further analysis. Introduction Bacterial symbionts of eukaryotic hosts exhibit an impressive array of phenotypes that interact with host biology. Included among these symbionts are bacteria that alter host reproduction in order to increase their likelihood of transmission to the next host generation [1–4], a strategy termed reproductive manipulation. Depending on the nature of host reproduction, reproductive manipulators are generally transmitted vertically by associating with either the oocyte or developing embryos [5]. Manipulative phenotypes range from strategies that prevent the survival of uninfected offspring, such as cytoplasmic incompatibility, to strategies such as feminization, male killing, and parthenogenesis, that actually change the sex ratio to favor females for overall increased infection rates [1–4]. These drastic manipulations to normal host biology mean that reproductive manipulators can induce reproductive isolation, drive changes in sexuality, and alter reproductive ecology in their hosts [6–8]. While reproductive manipulators undergo vertical transmission to reach the next host generation by definition, many species and strains also exhibit low rates of horizontal transmission between contemporary host species. This combination of transmission strategies coupled with reproductive manipulation serves to quickly increase the prevalence of reproductive manipulators in a host population and to spread rapidly among host species. Additionally, some reproductive manipulators are mutualistic, helping to increase host fecundity, and further increasing infection rates [9-12]. For example, the strain of Wolbachia that infects bed bugs supplements B vitamins lacking in the host’s diet [9] and some strains of Wolbachia protect hosts from viral or parasite infection [10, 11]. Given the impressive abilities of these bacteria to colonize new hosts and impact host fertility and development for their own reproduction, much effort has been spent attempting to estimate the infection frequencies of reproductive manipulators. Due to its wide distribution and presence in model organisms such as Drosophila, Wolbachia is one of the most well studied endosymbionts in general, and is the best studied reproductive manipulator in particular. Estimates of the frequency of Wolbachia infections amongst arthropods range from 11% [13] to 76% [14]. The large variation in estimates is likely an effect of sampling bias and variation in assay sensitivity. For example, PCR amplification of the Wolbachia surface protein gene (wsp) is commonly used to estimate the infection frequency and species distribution of this reproductive manipulator [15– 17]. However, wsp is not present in all Wolbachia strains, and thus, the use of these primer sets biases the data and underestimates infection rates [18]. Other reproductive manipulators in the Cardinium, Arsenophonus, Rickettsia and Spiroplasma clades are reported to occur in 4% to 7% of all species [19]. However, these estimates are less certain than Wolbachia because less data is available for these clades, and similar detection biases might impact frequency estimates. Given that the probability of sampling an infected individual is positively correlated to the prevalence of the endosymbiont and the number of individuals sampled from the host population, undersampling also imposes a barrier to confident detection of low frequency infections. Reproductive manipulators also experience a range of fitness tradeoffs during host growth and development. A symbiont must be present at sufficiently high frequencies within a host to promote successful transmission to subsequent host generations. However, exceedingly high abundance of symbiont cells relative to host cells, termed titer, may impose a significant fitness cost for the host and their endosymbionts [20, 21]. Reproductive manipulators may be more virulent to their hosts when they achieve high titers, and natural selection on symbiont proliferation and host regulation should favor the evolution of intermediate endosymbiont frequencies [22, 23]. This tradeoff between reliable transmission and fitness costs to hosts is therefore an essential component of understanding reproductive manipulator co-evolution. Nonetheless, little is known about the relative abundances of reproductive manipulators within host individuals in large part due to the challenges of collecting these data in high throughput ways. Characterizing the prevalence and distribution of reproductive manipulators could be especially valuable to biomedical researchers using reproductive manipulators to control the spread of human pathogenic viruses such as Zika, Dengue and Chikungunya [24–26]. Currently, only the wMel strain of Wolbachia is being used as a biological control agent [26, 27]. However, the large genetic and phenotypic diversity of reproductive manipulators could suggest different species or strains of bacteria to combat the spread of arboviruses. Additionally, a strategy that opportunizes on cytoplasmic incompatibility, termed the “sterile male technique”, can be used to control mosquito population sizes [28, 29]. As this strategy requires low infection frequencies in females to work [30] and different strains may have compatible rescue abilities [31], it is necessary to understand the diversity and distribution of these bacteria in nature. Cataloguing the distribution and titer of reproductive manipulators builds a foundation to explore reproductive manipulators as powerful and versatile biological control agents. Whole genome sequencing and bioinformatic approaches offer appealing alternatives to conventional PCR-based survey methods to estimate reproductive manipulator infection rates. These methods are not biased by primer selection, are less sensitive to false positives due to contamination, and enable testing of large numbers of samples. Similarly, genome-sequencing is a potentially powerful tool for interrogating endosymbiont titer within host individuals. With Illumina shotgun sequencing, when the genome of a potential host individual is sequenced, the host’s endosymbionts are also sequenced. This makes the publicly-available databases a treasure trove for sampling reproductive manipulators with bioinformatic approaches. For example, the NCBI Sequence Read Archive (SRA) [32] contains ~62,000 sequencing runs for samples tagged as Arthropoda alone. Searching, or mining, genomic sequencing data has been shown to be a cost effective and powerful strategy to detect Wolbachia infections [13, 33]. However prior studies did not include other reproductive manipulators [13, 33] or were focused on a single host species [33]. Indeed, one of the most compelling arguments for using non-targeted, publicly available data is to avoid ascertainment bias towards selecting species already known to harbor reproductive manipulator infections. Here, we develop and apply a computational pipeline to determine the infection status of host samples downloaded from the SRA database and estimate the titer of endosymbionts within positively infected host individuals. Using this approach, we comprehensively survey for reproductive manipulators across diverse host species and estimate their global infection frequencies. We classified 1733 arthropod and 103 nematode samples as infected with a reproductive manipulator, including at least 54 previously unknown infections. Additionally, we show that endosymbiont titer varies systematically across bacterial clades and may partly explain the disparate global distributions of reproductive manipulators. Results and Discussion Arthropod and Nematode Infections in the SRA