Identification of Metabolically Active Bacteria in the Gut of the Generalist <em>Spodoptera littoralis</em> via DNA Stable Isotope Probing Using ¹³C-Glucose

Abstract

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction 1 , boosting the immune response 2 , pheromone production 3 , as well as nutrition, including the synthesis of essential amino acids 4, among others.Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13 Cglucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA 5 . The incorporation of 13 C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled ( 12 C) one. In the end, the 13 Clabeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12 C-unlabeled similar one 6 . Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, 13 C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet. Video LinkThe video component of this article can be found at

Biodiversity and Activity of the Gut Microbiota across the Life History of the Insect Herbivore Spodoptera littoralis

“…S. littoralis larvae were hatched from eggs and reared on artificial diet as previously described 41 . Plastic cabinets with the diet and the larvae were kept at 23–25 °C under a regime of 16 h illumination and a dark period of 8 h. The emerged adults were supplied with a sucrose solution.…”

“…For sample processing, all insects were first rinsed three times in sterile water, surface-sterilized in 70% ethanol for 30 s and rinsed again in sterile water. The whole gut tissue was dissected from each individual and homogenized for nucleic acid extraction, as previously described 41 . After dissection, the typical vitellogenic ovariole was observed in the mature female.…”

“…Bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) was performed using a Roche 454 FLX instrument with Titanium reagents as described previously 41 . Basically, the hypervariable V1–V3 segment in the 16S rDNA was amplified using the fusion primer set Gray28F (5-GAGTTTGATCNTGGCTCAG-3) and Gray519r (5-GTNTTACNGCGGCKGCTG-3) extended with the respective primer Adaptor A/B and sample-specific multiplex identifiers (MID).…”

Gut microbiota metabolic potential correlates with body size between mulberry‐feeding lepidopteran pest species

“…Mature silkworms were collected on the third day after moulting to the fifth (last) instar. To collect the whole gut, larvae were twice surface‐sterilized in 70% ethanol for 1 min each time, then rinsed in sterile water three times, and finally dissected on ice as described previously . Gut tissues were stored at −80 °C until further processing.…”

Draft Genome Sequence of Enterococcus mundtii SL 16, an Indigenous Gut Bacterium of the Polyphagous Pest Spodoptera littoralis

“…Briefly, the normal larvae were washed and sedated on ice for at least 1 h to anesthetize them. Then the whole gut sections were dissected from larvae using a fine Vannas scissor and forceps under a binocular microscope (Shao et al, 2013). The fresh gut tissues were put into phosphate buffered saline (PBS: 137 mM NaCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , and 2.7 mM KCl) and homogenized by hand with a sterile pestle.…”