Microbial Associates
Bacterial symbionts have been implicated in a range of important roles in insect hosts. In Carabidae, Spiroplasma are known from at least two subfamilies (Lundgren et al. 2007; McManus et al. 2018). However, little research to date has focused on the microbiome of alpine insects (but see: Receveur et al. 2020), let alone documented the role of endosymbionts. Spiroplasma is a common symbiont in insects, with a recent estimation of infection in 8% of arthropod species, although it is rarely detected in beetles (Medinaet al. 2019). Spiroplasma spp. has been observed to have a range of effects in their hosts, including male-killing and protection from parasites, such as wasps and nematodes (Ammar & Hogenhout 2006; Ballinger & Perlman 2019; Harumoto & Lemaitre 2018; Haselkorn & Jaenike 2015; Xie et al. 2010). In this study, a completeSpiroplasma genome was recovered from PacBio sequencing ofN. riversi . This finding coupled with the presence ofSpiroplasma reads in the RNAseq datasets of N. riversiegg, larvae, and adult specimens, imply the prevalence ofSpiroplasma in N. riversi . The detection ofSpiroplasma in egg sample also suggests the endosymbiosis and vertical transmission of this bacterium in N. riversi . However, additional work should characterize the distribution and prevalence ofSpiroplamsa in N. riversi . Furthermore, our phylogenetic analyses places S. sp. NR in Carabid-specific clade ofSpiroplasma isolates. These results suggest thatSpiroplasma may be widespread in Carabidae. Future work that characterizes the prevalence of S. sp. NR in N. riversi , and related Nebria , would help elucidate its role as an endosymbiont.
There are several possible ecological roles that the Spiroplasmamight play in its host. First, analysis of this genome revealed the presence of ankyrin repeats and the OTU domain, which could indicate male-killing and a role as a reproductive parasite (Harumoto & Lemaitre 2018). However, homology to the spaid gene locus was partial, and ankyrin repeats are a common protein-protein interaction motif occurring in proteins with diverse function. No single ORF contained both ankyrin repeats and the OTU domain; however, recent work has demonstrated that spaid is a rapidly evolving locus andspaid- like genes containing only the OTU domain were identified in Spiroplasma and Wolbachia symbionts (Gerth et al. 2020). Our ecological survey data supports a female-skewed sex ratio in N. riversi (Figure S9 ), as might be expected from a long-term infection with a male-killing parasite. Second, because we have observed nematode infections (Gordius sp., Weng and Schoville, personal observation) in N. riversi ,Spiroplasma might have a host defensive role. This role was suggested for another riparian carabid species with Spiroplasma ,Brachinus elongatulus, which is frequently observed to have nematode infections (McManus et al. 2018). However, we did not detect the presence of RIPs in S. sp. NR, which would provide stronger evidence of host defense against nematodes. Third,Spiroplasma might play a role in the production of useful metabolites, which is suggested by the presence of propanoate biosynthesis genes. One possible role of propanoate (also known as propanoic or propionic acid) is serving as a constituent of the reproductive pheromones or defensive chemistry of N. riversi(Attygalle et al. 1992; Francke & Dettner 2005). Propanoate has been found in the defensive gland of at least one carabid beetle (Attygalle et al. 1992), and is considered to have insecticidal properties (Krzyżowski et al. 2020). While it has a pungent smell as a fatty acid volatile, it is also thought to facilitate biosynthesis of strong-smelling benzoquinones (Rocha et al. 2013). Notably,N. riversi seems to complement this pathway (Figure S8 ) by encoding the genes to produce Propanoyl-CoA, which Spiroplasmais predicted to transform into propanoate through propanoyl phosphate.Nebria riversi has an alternative path to producing propanoate, from propanoyl-CoA to propionyl adenylate, which implies there may be different derivatives or functions of propanoate synthesis by S.sp. NR and N. riversi . Fourth, and finally, evidence has shown that the presence of Spiroplasma could help insect hosts to survive over winter (Ebbert & Nault 1994). Future work, including an assessment of the frequency and distribution of Spiroplasma amongN. riversi populations, will be required to distinguish among these alternative endosymbiotic roles.