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.