Abstract
The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present study, we focused on the forest cockchafer (Melolontha hippocastani), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP) with 13C cellulose and 15N urea as trophic links, with Illumina MiSeq (Illumina-SIP), we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In 13C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In 15N urea-fed insects, the genera Burkholderia and Parabacteroides were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, 13C cellulose- and 15N urea labeled bacteria. The incorporation of 15N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS). Besides highlighting key bacterial symbionts of the gut of M. hippocastani, this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.
Highlights
Symbiotic associations between insects and gut-dwelling microorganisms, especially bacteria, have long been known (Baumberger, 1919), but it was not until the development of next-generation sequencing techniques that we realized the true extent of the diversity and complexity of such microbial communities (Shi et al, 2010)
Shifting of the DNA toward dense parts (>1.69 g/mL) of the labeled gradient (15N) compared to control (14N) is already observable in the quantification carried out with Helyxyte green. quantitative real-time PCR (qPCR) performed with bacteria-specific primers clarified the increase of 16S rRNA gene copy number in dense pooled fractions of the isotopically labeled gradient (13C or 15N) in comparison to the unlabeled control gradient (12C or 14N), confirming successful labeling for both substrates (Figure 1)
isotope ratio mass spectrometry (IRMS) analyses detected the significant incorporation of 15N in insect tissues (Figure 6). These results strongly indicate the existence of a symbiont-driven nitrogen recycling mechanism in the gut of M. hippocastani, as it happens in planthoppers (Sasaki et al, 1996), termites (Potrikus and Breznak, 1981; Thong-On et al, 2012), and cockroaches (Sabree et al, 2009; Patiño-Navarrete et al, 2014)
Summary
Symbiotic associations between insects and gut-dwelling microorganisms, especially bacteria, have long been known (Baumberger, 1919), but it was not until the development of next-generation sequencing techniques that we realized the true extent of the diversity and complexity of such microbial communities (Shi et al, 2010). A relationship between community composition and host insect taxon and diet has been demonstrated (Colman et al, 2012; Jones et al, 2013; Yun et al, 2014; Guerrero et al, 2016), suggesting crucial roles of the symbionts with regard to insect physiology (Potrikus and Breznak, 1981; Douglas, 2009; Watanabe and Tokuda, 2010). The contribution of insect symbionts in cellulose digestion (Rössler, 1961; Bayon and Mathelin, 1980; Martin, 1983; Anand et al, 2010) and, more recently, in the recycling of nitrogen (Potrikus and Breznak, 1981; Sasaki et al, 1996; Sabree et al, 2009; ThongOn et al, 2012; Ayayee et al, 2014) have been object of extensive research. To which extent the host contributes to the recycling of nitrogen is still unclear; a shared pathway has been reported in the cockroach Blatella germanica (PatiñoNavarrete et al, 2014)
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