This work studied the influence of diet on gut bacterial communities of P. americana. Here the 16S rDNAs sequencing approach was used to explore the plasticity of the intestinal flora of Periplaneta americana, comparing wild-types insects with others fed on different diets. The analysis of 16S rRNA sequences showed that the diet had a significant influence on gut microbial communities in this insect. It is important to note that the conditioning of these insects to different diets had no effect on animal survival rate during the study period (data not shown). Our results show clearly that there is a significant increase in Firmicutes presence and also an important decrease in Proteobacteria and Synergistetes in the gut of animals fed cellulose-rich diet (Figure 2).
An association between bacteria and insects during biomass degradation is observed in several models. The leaf-cutter ant Atta colombicai, for example, uses fresh leaves to cultivate a fungus and also bacteria (mainly γ-Proteobacteria of the family Enterobacteriaceae) that produce high numbers of different cellulases and hemicellulases in specialized biodegrading gardens (Suen et al. 2010). In higher termites (that do not have symbiotic protists in the gut), such as Cornitermes cumulans and Nasutitermes sp, a high bacterial diversity is observed, basically composed by the phyla Proteobacteria, Spirochaeta, Bacteroidetes, Firmicutes, Actinobacteria, Fibrobacter and Treponema (Gijzen et al. 1994; Grieco et al. 2012). Phylogenetic analysis of gut bacteria from the low termite Reticulitermes flavipes (that harbors protist symbionts in the gut) and Cryptocercus (wood-feeding cockroach) showed a diverse range of members of major bacterial phyla, such as Proteobacteria, Spirochaetes, Bacteroidetes, Firmicutes, Actinobacteria, Synergistetes and the newly proposed Endomicrobia (Fisher et al. 2007; Berlanga et al. 2009). The gut microbiota of the cockroach Shelfordella lateralis was dominated by members of the Bacteroidetes and Firmicutes (mainly Clostridia), however, Deltaproteobacteria, Spirochaetes and Fibrobacteres, which are abundant members of termite gut communities, were absent in this insect (Schauer et al. 2012).
The impact of diet on intestinal microbiota is also observed in other models such as dogs (Middelbos et al. 2010), snails (Cardoso et al. 2012), cattle (Kong et al. 2010; Hess et al. 2011), sheep (Cunha et al. 2011) and humans (Ley et al. 2006) for example. In the higher termite Nasutitermes takasagoensis, it was demonstrated that the intestinal bacterial community structure is not so stable, varying depending on diet composition. The Spirochaetes was predominant sequences in the wood-feeding termites, whereas Bacteroidetes was more abundant in the gut of xylophagous termites. Firmicutes was predominant sequences in xylose fed termites (Miyata et al. 2007). The analysis of the termite Reticulitermes flavipes gut microbiome submitted to different diets showed that diet, environment and host genetics have important effects over microbiome composition (Boucias et al. 2013). Our results showed that the main Proteobacteria clones associated to wild-types cockroaches are distributed along several genera, including typical bacteria from sludge such as Brucella and Alphaproteobacteria.
The presence of microorganisms in the gut of American cockroach P. americana was initially described by light, scanning and transmission electron microscopy. The results suggested the presence of a complex community including protozoa, bacteria and archaea (Gijzen and Barugahare 1992; Bignell et al. 1977). Previous studies showed that a cellulose rich diet induces an increase in the population of protozoa and also in methanogenesis in the hindgut of this insect 19-(Gijzen et al. 1994; Kane and Breznak 1991). In cockroaches and termites, the protists involved in lignocellulose degradation use not only their own enzymes, but also could use the enzymes originated from its endo and/or ecto symbiotic bacteria (Todaka et al. 2010). The greater complexity of nutrients within the wild-types and sugarcane bagasse diets compared to the simple diet (only cellulose) suggests that a greater repertoire of bacteria may be required to efficiently utilize all of the nutritional components of more complex foods. Results from our laboratory show that these bacterial community alterations associated with changes in dietary composition triggered some changes in the intestinal enzyme profile. For example, there is an important increase on Endo-1,3(4)-β-glucanase and Endo-beta-1,3-1,4 glucanase II activities of anterior intestine of insect fed sugarcane bagasse compared to other two groups (data not shown). An interesting hypothesis is that these specialized bacteria could help to digest complex dietary polymers during the passage through the first segments of the insect’s intestinal tract. After that, host enzymes could digest these bacteria as a nutrient source in the last gut segments as observed in flies with a strong participation of cathepsin-D-like proteases and lysozyme (Lemos and Terra 1991).
The exact role of the cockroach microbiota in biomass degradation still remains unknown. Bacteria from the phylum Bacteroidetes (formerly known as Cytophaga-Flavobacteria-Bacteroides-CFB group) are involved in associations with a wide variety of gut protist species as either intracellular endosymbionts or surface-attached ectosymbionts. These bacteria digest a wide variety of substrates, including complex polymers, such as cellulose and chitin, using various glycosyl hydrolases (Noda et al. 2006; Mahowald et al. 2009). It is important to note that all Bacteroidetes related clones retrieved from sugarcane bagasse fed insects are affiliated with Elizabethkingia miricola. These bacteria are Gram-negative, non-motile, that can utilize several glycosidic substrates such as D-fructose, D-glucose, D-maltose, 2-naphthyl-alpha-D-glucopyranoside, 1-naphthyl-N-acetyl-beta-D-glucosaminide and 2-naphthyl-alpha-L-fucopyranoside (Kim et al. 2005).
In the intestine (including in humans), Firmicutes are strongly involved in fermentation processes and may be partners in many catabolic activities such as those observed in the degradation of glucose to generate several catabolites as lactate, ethanol, H2 and CO2 (Ley et al. 2006; Wüst et al. 2011). These bacteria could also reduce sulfate, degrade volatile fatty acids, such as butyrate and its analogs, and provide H2 to archeal methanogens (Ley et al. 2008; Rivière et al. 2009). Most clones from cockroaches fed on a cellulose-rich diet (Sugarcane Bagasse and Cellulose groups) are distributed in two main distantly related species, Lactobacillus dextrinicus and Enterococcus asini that are examples of typical fecal bacteria (Furet et al. 2009). The Synergistetes phyla includes Gram-negative, rod-shaped bacteria isolated from humans, animals and terrestrial and oceanic bacteria that metabolize amino acids and proteins to provide short-chain fatty acids and sulfate for methanogens and sulfate-reducing bacteria (Vartoukian et al. 2007). Our data do not show the presence of bacteria from the genus Clostridium, which is an important group in the rumen of cattle involved in fiber degradation by the enzymatic complex called cellulosome (Ley et al. 2006). This result is also different from Nasutitermes takasagoensis where there is an important presence of Clostridia on the mixed segments of this higher termite (Tokuda et al. 2000).
The enzymatic repertoire involved in digestion of lignocelullosis in insects could include glycoside hydrolases, laccase, peroxidases and detoxification proteins such as superoxide dismutase and catalase (Scharf and Boucias 2010). Considering that insects like cockroaches and termites perform the pretreatment and hydrolysis under mild conditions within a few millimeters of intestinal tissue, our data demonstrate that there is an important specialization of the microbiota in fiber digestion. Thus, the knowledge about gut microorganisms and their enzymes involved in the pretreatment and hydrolysis of biomass could be useful for new insights related to the development of bioethanol or other high-value products.