FEMS Microbiology Ecology, 91, 2015, fiv022 doi: 10.1093/femsec/fiv022 Advance Access Publication Date: 27 February 2015 Research Article RESEARCH ARTICLE Downloaded from https://academic.oup.com/femsec/article-abstract/91/4/fiv022/598282 by guest on 13 October 2019 Diet shapes the gut microbiota of the omnivorous cockroach Blattella germanica Ana Elena Perez-Cobas´ 1,2,3,# , Elisa Maiques1,# , Alexandra Angelova1, Purificacion´ Carrasco1,Andres´ Moya1,2,3 and Amparo Latorre1,2,3,∗ 1Instituto Cavanilles de Biodiversidad y Biolog´ıa Evolutiva, Universidad de Valencia, 46071 Valencia, Spain, 2Fundacion´ para el Fomento de la Investigacion´ Sanitaria y Biomedica´ de la Comunitat Valenciana (FISABIO), 46020 Valencia, Spain and 3CIBER en Epidemiolog´ıa y Salud Publica´ (CIBEResp), 28029 Madrid, Spain ∗ Corresponding author: Instituto Cavanilles de Biodiversidad y Biolog´ıa Evolutiva, Universitat de Valencia,` 46071 Valencia, Spain. Tel: +34-96-354-3649; Fax: +34-96-354-3670; E-mail [email protected] #A.E.P-C and E.M. contributed equally to this work. One sentence summary: The gut microbiota of the omnivorous cockroach Blattella germanica shows variations in composition of some members of the community when they are fed with diets differing in the protein content. Editor: Cindy Nakatsu ABSTRACT The gut microbiota of insects contributes positively to the physiology of its host mainly by participating in food digestion, protecting against pathogens, or provisioning vitamins or amino acids, but the dynamics of this complex ecosystem is not well understood so far. In this study, we have characterized the gut microbiota of the omnivorous cockroach Blattella germanica by pyrosequencing the hypervariable regions V1–V3 of the 16S rRNA gene of the whole bacterial community. Three diets differing in the protein content (0, 24 and 50%) were tested at two time points in lab-reared individuals. In addition, the gut microbiota of wild adult cockroaches was also analyzed. In contrast to the high microbial richness described on the studied samples, only few species are shared by wild and lab-reared cockroaches, constituting the bacterial core in the gut of B. germanica. Overall, we found that the gut microbiota of B. germanica is highly dynamic as the bacterial composition was reassembled in a diet-specific manner over a short time span, with no-protein diet promoting high diversity, although the highest diversity was found in the wild cockroaches analyzed. We discuss how the flexibility of the gut microbiota is probably due to its omnivorous life style and varied diets. Keywords: insects; omnivorism; intestinal microbiota; synthetic diets; lab-reared versus wild cockroaches INTRODUCTION an endosymbiotic relationship with Blattabacterium strain Bge. This endosymbiotic bacterium inhabits the bacteriocytes, spe- Many insects harbor rich intestinal microbiota, which con- cialized cells in the fat body of the cockroach and plays a vi- tributes to several functions benefiting the host (Engel and tal role in insect nutrition by providing essential amino acids Moran 2013). Additionally, various intracellular mutualistic sym- to its host. In addition, this endosymbiont is involved in nitro- bioses between insects and bacteria have been described, in gen recycling, using uric acid, normally a waste product in ter- which the bacteria participate by contributing positively to host restrial animals, as a source of nitrogen (Lopez-S´ anchez´ et al. physiology (Baumann 2005;Moyaet al. 2008). This is the case 2009;Patino-Navarrete˜ et al. 2013). Cockroaches and termites are of the German cockroach Blattella germanica, an omnivorous in- closely related phylogenetically (Lo et al. 2000; Inward, Beccaloni sect belonging to the family Blattellidae, which has established Received: 24 October 2014; Accepted: 23 February 2015 C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected] 1 2 FEMS Microbiology Ecology, 2015, Vol. 91, No. 4 and Eggleton 2007), and it has been postulated that the acqui- ing that it is highly involved in wood digestion (lignocellulose sition of Blattabacterium took place prior the split of both lin- degradation). Additionally, gut microbiota help fix nitrogen, re- eages (Bandi et al. 1995). However, Blattabacterium has been lost cycle nitrogenous waste and provide essential amino acids to in termites, with the sole exception of the lower termite Mas- the host (Warnecke et al. 2007). These findings contrast to om- totermes darwiniensis (Bandi et al. 1995) whose genome content nivorous cockroaches in which the primary endosymbiont is in- is very similar to that of Blattabacterium from the wood-feeding volved in these functions. However, the role gut microbiota plays cockroach Cryptocercus punctulatus (Neef et al. 2011;Sabreeet al. in the cockroach nitrogen metabolism and other functions is still 2012). Both endosymbionts possess the most reduced genomes unknown. among the Blattabacterium strains, having lost the pathways for This study aims to characterize the gut microbiota of the om- the biosynthesis of several essential amino acids. In terms of nivorous cockroach B. germanica and infers its role in nitrogen phylogeny, C. punctulatus belongs to a group more closely re- metabolism. To do so, we tested the effect of diets containing lated to termites than to other cockroaches (Lo et al. 2000;In- different amounts of protein on the gut microbiota composition Downloaded from https://academic.oup.com/femsec/article-abstract/91/4/fiv022/598282 by guest on 13 October 2019 ward, Beccaloni and Eggleton 2007); thus, most gene losses in at two different moments of the adult life of lab-reared individ- Blattabacterium of C. punctulatus and M. darwiniensis took place uals of B. germanica. We also analyzed the gut microbiota of wild before both lineages split, and could be related to the xy- cockroaches and tested its differences with the lab-reared ones. lophagous nature of these insects (Patino-Navarrete˜ et al. 2013). Nevertheless, Blattabacterium from Panesthia angustipennis, also wood-feeding species that is phylogenetically distant from C. MATERIALS AND METHODS punctulatus and M. darwiniensis, retains the essential amino- Samples, diets and time of sampling acid pathways. This led Tokuda et al. (2013) to postulate that the social behavior of the latter two species, which is gener- The B. germanica individuals used for this study were de- ally found in termites, would favor gene losses related to the rived from a 30-year-old stable lab population held at the In- evolution of high-fidelity transfer of gut microbes, allowing re- stitut de Biologia Evolutiva (CSIC-UPF), Barcelona, Spain. The placement of the lost Blattabacterium functions. Studies show cockroaches were reared at the University of Valencia in cham- o that M. darwiniesis and C. punctulatus share gut microbiota com- bers at 30 C and 70% relative humidity. Only females were used position to a great extent, reflecting both the evolutionary re- for all experiments to exclude potential sex-related bias. The in- lationship between their hosts and the high-fidelity transfer sects were bred in lunchboxes with aeration and fed autoclaved of gut microbes via social behavior (Sabree et al. 2012; Tokuda dog-food croquettes. Water was supplied ad libitum, and lunch- et al. 2013). In these species, the gut microbiota consists mainly boxes were renewed weekly. A minimum of 49 synchronized in- of the phyla Bacteroidetes, Firmicutes (mainly Clostridiales), dividuals (they were born at the same time) were maintained per Spirochaetes and candidate phylum TG1, which are strongly in- box. Three kinds of diets were provided: a complex diet (con- volved in host digestion (Berlanga, Paster and Guerrero 2009; trol diet, CD), for routine maintenance of the colony, and two Sabree et al. 2012). On the other hand, the gut microbiome of chemically defined (synthetic diets) with high-protein content the omnivorous cockroach Periplaneta americana exhibited great- (HP diet) and no-protein content (NP diet) to test the effect of est diversity and abundance than those of M. darwiniesis and C. the excess/absence of protein on the gut bacterial composition punctulatus (Sabree and Moran 2014). (see composition in Supporting Information, Table S1). The CD Higher termites have lost Blattabacterium; thus, its nutrient- consisted of commercial dog-food pellets, whereas synthetic di- provisioning role must have been replaced by gut bacteria. In ets with different protein contents were based on Mullins and fact, comparing termites with cockroaches, the diversity in gut Cochran (1974) with substantial modifications to achieve an ex- bacterial communities seems to be positively correlated with perimental design in which protein content was the main dif- the functionality of Blattabacterium (Sabree and Moran 2014). The ference between diets. Thus, we ensured that the minimal nutri- most abundant members of termites belong to the phyla Bac- tional needs of B. germanica were met (Gordon 1959). Specifically, teroidetes, Firmicutes and Spirochaetes (similar to those found we took into account that its growth is affected by the nitrogen in M. darwiniesis and C. punctulatus), as well as Fibrobacteres, Pro- content of food, but is not very dependent upon the source of it, teobacteria, candidate phylum TG3 and Actinobacteria, the most whereas it is very resistant to avitaminosis (Zabinski 1929). common being Spirochaetes and Fibrobacteres (Warnecke et al. After the last molt (adult day 0), all adult