Disentangling a Holobiont - Recent Advances and Perspectives in Nasonia Wasps

Abstract

The parasitoid wasp genus Nasonia (Hymenoptera: Chalcidoidea) is a well-established model organism for insect development, evolutionary genetics, speciation, and symbiosis. The host-microbiota assemblage which constitutes the Nasonia holobiont (a host together with all of its associated microbes) consists of viruses, two heritable bacterial symbionts and a bacterial community dominated in abundance by a few taxa in the gut. In the wild, all four Nasonia species are systematically infected with the obligate intracellular bacterium Wolbachia and can additionally be co-infected with Arsenophonus nasoniae. These two reproductive parasites have different transmission modes and host manipulations (cytoplasmic incompatibility vs. male-killing, respectively). Pioneering studies on Wolbachia in Nasonia demonstrated that closely related Nasonia species harbor multiple and mutually incompatible Wolbachia strains, resulting in strong symbiont-mediated reproductive barriers that evolved early in the speciation process. Moreover, research on host-symbiont interactions and speciation has recently broadened from its historical focus on heritable symbionts to the entire microbial community. In this context, each Nasonia species hosts a distinguishable community of gut bacteria that experiences a temporal succession during host development and members of this bacterial community cause strong hybrid lethality during larval development. In this review, we present the Nasonia species complex as a model system to experimentally investigate questions regarding: (i) the impact of different microbes, including (but not limited to) heritable endosymbionts, on the extended phenotype of the holobiont, (ii) the establishment and regulation of a species-specific microbiota, (iii) the role of the microbiota in speciation, and (iv) the resilience and adaptability of the microbiota in wild populations subjected to different environmental pressures. We discuss the potential for easy microbiota manipulations in Nasonia as a promising experimental approach to address these fundamental aspects.

Keywords: Arsenophonus; Nasonia; Wolbachia; host-symbiont interactions; microbiome; symbiosis.

FIGURE 1

(A) Phylogenetic relationships within the Nasonia species complex based on the CO1 gene. The parasitoid wasp Trichomalopsis sarcophagae was used as outgroup. The scale bar indicates substitutions per site. Note the morphological differences in wing size between species and genders (drawing based on Loehlin et al., 2010). (B) Countries in which Nasonia has been observed, based on published records of N. vitripennis [Universal Chalcidoidea Database (Noyes, 2016) and (Raychoudhury et al., 2009, 2010b; Paolucci et al., 2013)]. The gray color indicates countries for which no observations are documented, in most cases due to missing sampling information. For all countries except the US, records state that the observed species was Nasonia vitripennis – however, many of these observations were made before the discovery of the three younger species in the US. Therefore, this global map shows observations of Nasonia without distinguishing between species. (C) Observations of all four Nasonia species in the US and Canada, based on published information (Darling and Werren, 1990; Raychoudhury et al., 2009, 2010a,b). (D) Nasonia life cycle from oviposition to adulthood. Sex-specific differences in ploidy are indicated for adult males and females. Additional sexual dimorphisms include smaller wing size, less pigmented antennae and rounded abdomen in males (open black arrows). Parasitism is representing by a female wasp ovipositing into a fly pupa (closed green arrows: Fly pupa; open green arrows: Ovipositor of the wasp). Embryos are approximately 100 μm by 500 μm in size (closed black arrows). The Nasonia larva and pupa were photographed outside of their fly host. Photo credit: Matthew C. Johnson © 2016

FIGURE 2

Within the Nasonia clade, there are three published sources of hybrid incompatibilities: cytonuclear, cytoplasmic, and microbiota-nuclear incompatibilities. Cytonuclear incompatibilities, or negative interactions between mitochondria and the nuclear genome, are associated with lethality in F₂ males from younger interspecific crosses (N. giraulti and N. longicornis) and near complete lethality in older interspecific crosses (N. vitripennis and N. giraulti or N. longicornis). Hybrid lethality has some plasticity due to environmental factors (Koevoets et al., 2012), but clear cytonuclear incompatibilities that complicate development and gene regulation (Ellison et al., 2008; Niehuis et al., 2008; Koevoets et al., 2012; Gibson et al., 2013) have been genetically mapped across the Nasonia genomes. Cytoplasmic incompatibilities are a consequence of infection with different Wolbachia strains (wA and wB), which causes post-fertilization chromatin defects that result in inviable fertilized eggs (Bordenstein et al., 2001, 2003; Tram and Sullivan, 2002). Finally, microbiota-nuclear incompatibilities result from negative interactions between the microbiota and host genome and lead to hybrid lethality, altered microbial communities and innate immune regulation (Brucker and Bordenstein, 2013). The collective influences of these incompatibilities on Nasonia make it a powerful model for evolutionary and symbiotic studies of speciation and reproductive isolation. How these incompatibilities have evolved relative to each other is an important avenue for future research.

FIGURE 3

(A) Wolbachia-Nasonia associations and phylosymbiosis (modified from Raychoudhury et al., 2009; Brucker and Bordenstein, 2012b,c). Wolbachia acquisitions and subsequent divergence are overlaid on the Nasonia phylogeny. Strains from Wolbachia supergroup A are represented in green, strains from supergroup B in purple. Arsenophonus (+Ar) has been found to infect three species of Nasonia. The microbial community relationships parallel the host phylogeny, indicating species-specific microbiota assemblies that establish phylosymbiosis. This pattern has been observed in males for three species (Brucker and Bordenstein, 2012b, 2013) as well as in females for all four species (R. M. Brucker and S. R. Bordenstein, personal communication). (B) Impact of Wolbachia-induced CI on offspring production. Wolbachia present in males induce a sperm modification that needs to be rescued by the same Wolbachia strain in the fertilized egg for normal offspring production. CI (red background) occurs if the female is uninfected (-) (unidirectional CI) or harbors a different Wolbachia strain (bidirectional CI) and results in male-only (or male-biased) broods due to loss of the paternal chromosomes. Note that although Wolbachia modify male sperm, the symbiont is only maternally transmitted. Offspring will therefore harbor the same Wolbachia strain(s) as their mothers. wA/wB indicate different Wolbachia strains. (C) Impact of Arsenophonus-induced male-killing on offspring production, in combination with Wolbachia-mediated CI. Male-killing results in all-female broods in the absence of CI (white background) and no offspring production in combination with CI (red crosses), since the males that are not affected by CI would be killed by male-killing.

FIGURE 4

The relative abundance of bacterial OTUs observed in male Nasonia throughout development (Brucker and Bordenstein, 2013). The OTUs represented across the three Nasonia species and their S. bullata fly host are dominated by Actinobacteria, Firmicutes, and especially Proteobacteria. Three genera, Providencia, Proteus, and Morganella, are particularly dominant across all samples. However, their relative abundances differ according to host species and developmental stage. The unparasitized S. bullata fly host is similarly dominated by Proteobacteria, specifically the genus Providencia. Emboldened OTUs are observed at higher frequencies in one or more samples. It is important to note that many of the rarer OTUs have also been observed in other studies (Brucker and Bordenstein, 2012b and personal communication).