Endosymbiont diversity and evolution across the weevil tree of life

Guanyang Zhang\({}^{\star}\) 11gzhang44@asu.edu School of Life Sciences, Arizona State University, Patrick Browne\({}^{\star}\) \({}^{\#}\) 22padb@envs.au.dk School of Life Sciences, Arizona State University, Geng Zhen\({}^{\star}\) 33gzhang44@asu.edu School of Life Sciences, Arizona State University, Andrew Johnston 44ajohnston@asu.edu School of Life Sciences, Arizona State University, Hinsby Cadillo-Quiroz 55hinsby@asu.edu School of Life Sciences and Swette Center for Environmental Biotechnology at the Biodesign Institute, Arizona State University, and Nico Franz 66nico.franz@asu.edu School of Life Sciences, Arizona State University \(\star\) These authors contributed equally to this work \({}^{\#}\)Current address: Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark



Weevils (Insecta: Coleoptera: Curculionoidea) host diverse bacterial endosymbionts. In his monumental book, ”Endosymbiosis of Animals with Plant Microorganisms”, Buchner (1965) (p. 160) remarked that ”nowhere else aside from the cicadas [Hemiptera: Auchenorrhyncha and Sternorrhyncha] do so many symbiotic sites exist as in this insect family [Curculionidae sensu lato]”. (citation not found: Nardon&Grenier) (p. 181) listed nine morphological types of endocytobiosis (a.k.a, bacterial endosymbionsis) in Curculionidae (excluding Scolytinae, therein as Scolytidae). They lamented on the ubiquity of bacterial endosymbionts in weevils and suggested that ”most if not all Curculionids harbor endocytobiotic bacteria” (p. 189). Despite this long-standing realization, the diversity, evolution and biology of bacterial symbionts of weevils have been poorly documented or understood, especially if compared to Auchenorrhyncha and Sternorrhyncha (”Homoptera”). With 62,000 described species and 220,000 estimated to exist (Oberprieler 2007), weevils are a mega-diverse group of insects and represent a premier system for studying bacterial symbionts.

The astounding diversity of weevils strongly contrasts with the rather small number of species that have been studied for bacterial symbionts. Early research using morphological techniques examined only some 30 genera of weevils (Buchner 1965, (citation not found: Nardon&Grenier), and these typically did not reveal the taxonomic identities of the symbionts. The first molecularly characterized endosymbiont from weevils was SOPE, or ”Sitophilus oryzae Primary Endosymsbiont”. This was later named as ”Candidatus Sodalis pierantonius” (the qualifier ”Candidatus”, indicating the species name is provisional (Murray 1995), is only used at the first occurrence of a name and will be omitted from subsequent occurrences). Sodalis was previously placed in Enterobacteriaceae, which has been split into several families (Adeolu 2016), resulting in its new placement in Pectobacteriaceae of the order Enterobacteriales, in the class Gammaproteobacteria. Sodalis was first described from tsetse flies (Glossinidae, Diptera) (Dale 1999) and has also been found infecting various insects including Aphididae, Cercopidae, Cicadellidae, Pentatomidae, Scutelleridae, (mostly plant sucking insects; Hemiptera), Apidae, Megachilidae (bees; Hymenoptera), Philopteridae (lice; Phthiraptera), and Cerambycidae (longhorn beetles; Coleoptera). Lefevre (2004) described ”Candidatus Nardonella” (Enterobacteriaceae) from a group of dryophthorine weevils (Dryophthorinae/-idae). Subsequent studies found Nardonella from the weevil subfamilies Brachycerinae/-idae (Erirhininae/-idae), Cyclominae, Entiminae and Molytinae (Hosokawa 2010, Hosokawa 2015, Huang 2016, Hirsch 2012, Merville 2013, Conord 2008, White 2015). To date Nardonella has not been found from other insects and its association with weevils is unique among insects. In a series of studies, Toju et al. (2009); Toju et al. (2013); Toju et al. (2010) discovered and documented ”Candidatus Curculioniphilus” (Enterobacteriaceae) from several genera within the Curculioninae, namely, Curculio, Shigizo, Archarius, Koreoculio and Carponinus. Curculioniphilus is also a weevil-specific bacterial lineage. Besides Sodalis, Nardonella and Curculioniphilus other bacterial endosymbionts such as Wolbachia, Rickettsia (Alphproteobacteria), Spiroplasma (Mollicutes), Klebsiella pneumonia (Gammaproteobacteria) have been previously found from weevils. Additionally, several weevils of (Trigonotarsus and Diocarandra) are associated with several distinct and unnamed endosymbionts. Here we provide a succinct summary of the published findings [To do-Guanyang: prepare a table of published symbionts in weevils].

Function of bacterial symbionts of weevils Morales-Jiménez et al. (2013) showed that Raoultella terrigena, a diazotrophic bacteria in the gut of Dendroctonus rhizophagus and D. valens (Scolytinae) has XXX abilities/effects/genes. Bacteria previously reported having nitrogen-fixing capabilities, such as Pantoea agglomerans, Rahnella aquatilis, Stenotrophomonas maltophilia, Raoultella terrigena, have been found in bark beetles (XXX what taxa?) (Bridges 1981)

Weevil taxonomic sampling of previous studies, with a focus on Candidatus Nardonella and Candidatus Curculioniphilus. The taxonomic sampling of weevils in previous studies was limited and scattered.Only XXX families and within the Curculionoidea and XXX subfamilies of the Curculionidae have been studied. An adequate taxonomic sample is crucial for reliable inferences of evolutionary scenarios regarding the acquisition and replacement of endosymbionts.

Evolution of primary symbionts, conservation/replacement.

Research objectives/questions.

[There can/should be a specific number of questions, where some may even have two or more answers that make sense generally (looking at the literature), and so then the paper gets a flavor of hypothesis testing. [see Discussion for a start; it is generally OK to generate some questions with alternative plausible answers where it was always clear to us that one answer is much better than the other] Example: we wondered if all weevils have just ”one species” of Nardonella, or, alternatively, whether there is considerable diversity of Nardonella (a) within and (b) among species of weevils.]

[Nico is wondering whether we can make a case (or not) for having discovered a likely third major ”axis” of evolutionary phenomena needed to properly account for weevil diversification (where host plant and hosting areas are axes 1 & 2, in a simplified SciComm kind of style)?]