Introduction

Bacterial symbiotic association with insect host is ubiquitous in nature, which confers the host with traits to explore new ecological niches (Sudakaran, Kost, & Kaltenpoth, 2017). Symbiotic bacteria inhabit either intra- or extra-cellularly inside the insect, providing hosts with vital benefits, including nutrition supply, pathogen resistance, assistance in immunity development, among others (Engel & Moran, 2013; McCutcheon, Boyd, & Dale, 2019). Mutualistic symbionts may have various origins, including environmental bacteria or infective parasites (Sachs, Skophammer, & Regus, 2011; Sachs, Skophammer, Bansal, & Stajich, 2013). Despite of distinct evolutionary pathways, the convergent transition to a mutualistic symbiotic lifestyle often involves the loss of virulence factors, degeneration of superfluous functions, and preservation or obtaining of traits beneficial to the host (EWALD, 1987; McCutcheon & Moran, 2012). However, direct evidence showing taxonomic turn-over in insect symbionts are scarce. Alternatively, comparative genomic analysis of mutualistic symbionts with the free-living lineages that are phylogenetically closely related provides a feasible route to examine the evolutionary pattern and adaptive mechanism accompanying the evolutionary transition to mutualism (Boscaro et al., 2013; Zheng, Dietrich, & Brune, 2017).
The honeybees have simple but specific gut symbiotic bacteria from genera of Gilliamella , Snodgrasella , Lactobacillusand Bifidobacterium (Martinson, Moy, & Moran, 2012). They compose more than 95% of the whole gut community, and the association with these five core bacteria can be dated back prior to the divergence of corbiculate bees (i.e., honeybee, bumblebee, stingless bee, and orchid bee) (Kwong et al., 2017). Although bee gut bacteria are not intracellular inhabitant and maternally transferred, they are inheritable among worker bees in the colony through social contacts (Powell, Martinson, Urban-Mead, & Moran, 2014). Along with the establishment of symbiotic association, these bacteria are subjected to genome reduction and evolutionary adaptation (Kwong, Engel, Koch, & Moran, 2014). This adaptive transition increases their fitness to the bee gut niche, but also limits the capacity to thrive in other environments (Ellegaard et al., 2019). As with other symbiotic microbes, honeybee gut bacteria share ancestry with bacterial species carrying varied lifestyles (Segers, Kešnerová, Kosoy, & Engel, 2017). Subsequently, genomic changes are expected to be remarkably different between the transitions from the common ancestry to gut symbionts and to other lifestyles (Tamarit et al., 2015). Thus, the honeybee-gut bacteria system provides a promising model to explore the genomic features underlying lifestyle transition of free-living bacteria to mutualistic gut symbionts.
Apibacter is a genus of bacteria that is prevalent in Apis cerana , Apis dorsata and bumblebee species (genusBombus ), however, they are only sporadically reported inApis mellifera (Kwong et al., 2017; Kwong & Moran, 2016). Our knowledge on Apibacter is primarily based on genome sequences of only four strains till now (Kwong, Steele, & Moran, 2018). Phylogenetic analysis showed that Apibacter spp. form a monophyletic lineage, which is embedded in the Chryseobacterium clade (familyFlavobacteriaceae ) (Kwong & Moran, 2016). The members of theChryseobacterium clade show a variety of lifestyles, including environmental free-living, opportunistic or obligate mammal and bird pathogens (McBride, 2014), of which the genus Chryseobacterium is the dominant member and consist of mostly environmental habitants. But strains of Chryseobacterium gleum and Chryseobacterium indologenes are common clinical isolates, which cause infections in immunocompromised patients (Calderán et al., 2011). Type strains of species from Elizabethkingia and Empedobacter are also opportunistic human pathogens that infect urinary and respiratory tract, causing sepsis, pneumonia and meningitis (Bhat, Priya, Krishnan, & Kanungo, 2016; Gupta, Zaman, Mohan, & Taneja, 2017; Lau et al., 2015; Zaman, Gupta, Kaur, Mohan, & Taneja, 2017). Isolates of generaBergeyella and Weeksella infect mammal respiratory tracts and female genitourinary tract, respectively (Hugo, Bruun, & Jooste, 2006; Lin, Chen, & Liu, 2007; Slenker, Hess, Jungkind, & DeSimone, 2012). Members of Riemerella and Ornithobacterium are important bird pathogens, causing infections to respiratory tract and other organs (McBride, 2014). These bacteria can be transmitted horizontally by aerosol or vertically between host generations. Type species of genera Bergeyella , Weeksella, Riemerella andOrnithobacterium are considered non-free-living, showing a preference to micro-aerophilic conditions, which is different from most other strictly aerobic members of the Chryseobacterium clade (Mavromatis et al., 2011; Van Empel & Hafez, 1999). The common features of the Chryseobacterium clade include proteolytic activity, carbohydrate utilization and multiple antibiotic resistance, causing challenges to the treatment of infection. Protease and biofilm formation are suggested to be important for their pathogenesis (McBride, 2014). However, the genetic mechanisms underlying the pathogenicity remain uncovered.
In this study, we isolated and sequenced the genomes of 14Apibacter strains from the Asian honeybee Apis cerana . Molecular and physiology experiments were conducted to characterize its colonization positions and specificity inside the bee gut. Comparative genomic analyses of honeybee symbiotic Apibacter spp. with phylogenetically related free-living and pathogenic strains from theFlavobacteriaceae family revealed key genomic changes ofApibacter underlying adaptation to mutualistic symbiotic relationship with the honeybee.