Phylogenetic analysis of 16S rRNA isolated clones from microflora of larval gut microbes consisted of many diverse microbial species and many of them have yet to be characterized. The clones were grouped under four major bacterial classes: Gamma Proteobacteria, Actinobacteria, Cocci, Bacilli and the new one proposed to be‘‘Endomicrobia’’ (out groups). All of the known species among these isolates were either strict or facultative anaerobes that have been found and frequently isolated from animal intestines. Most of the insect gut consists of high population of facultative anaerobe microbes that are anoxic having low redox potential. Similar anaerobes isolated from gut microbiota of the firebug was classified under bacterial phylum Actinobacteria and Firmicutes that supplement nutrition required for normal growth [20], [21]. The presence of strict and facultative anaerobic organisms in silkworm gut is a reasonable expectation. Five of the clones were grouped under Gammaproteobacteria that include Pesudomonas Auriginosa, Citrobacter koseri, Aeromonas hydrophila, Psuedomonas mendocina and Klabsiella pneumonia. The presence of these class of microbes have been recently reported in gut microbes of insects that play a crucial role. [22] Reported the isolation of gut symbiont microbes belonging to gammaproteobacteria from shield bug Graphosoma lineatum. Symbiotic bacteria isolated from oriental fruit fly Bactrocera dorsalis showed enhanced insecticide resistance gammaproteobacteria [23]. Comparative analysis of gut microbiota form various races of mosquitoes showed the presence of microbes belonging to gammaproteobacteria and Actinobacteria [24]. The gut contents in millipedes were classified to be bacteria of the gamma subclass of Proteobacteria and the Actinobacteria. Microbes isolated from silkworm gut microbes in the present study showed similar trend with groups classified under both gammaproteobacteria and actinobacteria. These microbes might have role in either symbiosis and resistance. Nitrogen fixation mediated by gut bacteria is one of the crucial aspects for termite symbiosis since termites feed on nitrogen poor wood. Comparative analysis of the gut bacteria of termites and wood-feeding lower termites showed a diverse population of 142 genera, off which nitrogen-fixing bacteria were highly dominant in the wood-feeding termites [25]. Metatranscriptome analysis of subterranean termite gut showed microbes that play direct roles in nitrogen fixation, amino acid biosynthesis, and lignocellulose digestion [26]. Citrobacter freundii and Enterobacter agglomerans nitrogen-fixing bacteria have been previously isolated from several kinds of termites [27], [28]. The isolated microbes that are categorized under gammaproteobacterial cluster include Citrobacter, Pseudomonas, Klebsiella and Aeromonas. These clones might play a role in nitrogen fixation. Some of the Nocardia sp are also found to be a nitrogen fixer [29] and isolate form silkworm gut microbe Nocardia cyriacigeorgica might also play a key role as nitrogen fixer. The phylum Bacilli takes part in earlier and intermediate steps of polymer degradation. Microbes residing in the guts of produce a variety of hydrolytic enzymes. Earlier studies have showed that Paenibacillus ICGEB2008 isolated from the gut of the cotton bollworm produced several biomass-degrading enzymes, including cellulases and hemicellulases [30], [31]. Bacillus are predominant with titres of up to 10 7 CFU/ml gut contents [32], and play a major role in the guts of invertebrates in the first and second step of the degradation of polymeric material under oxygen limitation. In this study, Bacillus licheniformis from the silkworm gut could produce cellulolytic enzymes that might aid in the breakdown of mulberry aiding good absorption of nutritional material. On the other hand, Bacillus subtilis might produce enzyme lipase that could involve in breakdown of fats to fatty acids and glycerol that are important to both male and female larva. Earlier studies on isolation of lipases from silkworm showed to have antiviral activity against Nucleopolyhedrovirus [33]. While other genera’s isolate from the silkworm gut microbes including Staphylococcus, Klebsiella, Pseudomonas and Aeromonas are lipase producing bacteria. The lipase-producing bacterial community depends upon type of food materials provided. The results provide an evidence that diet has a significant impact on gut microbial community. Cocci, particularly streptococcus epidermis has been previously thought to be causing skin diseases. Recent evidence has been developed involving the word “commensal” meaning one organism benefiting without causing no harm to the other (commensalism) or both find organism beneficial (mutualism and proto-cooperation). Streptococcus epidermis is such an organism that plays an active role in host defense having symbiotic relationship [34]. The cocci obtained from silkworm gut could play a similar role of symbiotic relationship.
Insects possess an efficient immune system that allows them to deal with pathogenic infections. The defense system lies in gut microbiota that serves immunity by either producing antimicrobial peptide or by innate immune system. The innate immunity system may be carried out by serious of mechanism [35]. [36]showed primed immune response in silkworm triggered by ingested bacteria leading to systemic infection tolerance. This was carried out by injecting heat killed microbes including P. aeruginosa, heat-killed S. marcescens cells and heat-killed C. albicans cells that showed tolerance against P. aeruginosa [36]. The strains isolated from gut microflora could play an important role in governing immune system in silkworm. Particularly with regard to Bacilli phylum that has shown studies pertaining to cause immunity in few systems. Bacillus licheniformis investigation in animal models particularly mouse showed prevention of asthma development [37]. Similarly, B. licheniformis derived bio surfactant showed modulation of immune response Aeromonas hydrophila in fish [38]. The other strain Bacillus subtilis has also shown immune in various systems. [39] proved probiotic Bacillus subtilis strain to stimulate immune system for common infectious disease in elderly period by increasing salivary SIgA and serum IFN-gamma levels. Pseudomonas mendocina on other hand has been found to synthesis medium-chain-length polyhydroxyalkanoate (PHAMCL) and alginate oligosaccharides (AO). Alginate oligosaccharide has many biological activities, such as antioxidation, anticoagulation, and immune regulation [40]. Similarly nano-vaccine developed using outer membrane protein (OmpW) of Aeromonas hydrophila showed a dose dependent Immunity in Rohu fish(Labeo rohita) [41]. Recent research enlightened Pseudomonas aeruginosa as immune elicitor by secreting type II protease IV functions in Arabidopsis [42]. Protease IV activates pathway involving G protein signaling in immune function. Proteins play a major role in bringing about immune system in various insects [43]. In fact, it has been found that silkworm cocoon consists of many proteins that are of immune related. Eventually [44] isolated proteins from B. mori cocoon that inhibited the germination of Beauveria bassiana spores. Overall, it is important to identify proteins that are expressed in gut microflora by the identified micro-organisms and its role in providing immunity. The protein identification could also enlighten its role on various growth factors, nutrition and immunity. In our experiment, we have identified proteins of gut microbes by two-dimensional electrophoresis combined with MALDI-TOF-MS, and MS spectrum. 5th instar larvae were used to isolate the proteins since most of biochemical metabolism dramatically changes during this period. We identified 7 protein spot, off which highest score was found in SGP6 protein (score 71) and SGP5 protein (score 70). Most of the proteins identified were found to be related with metabolism process and innate immunity, which were similar with the proteins identified from other insects.
RNase H (SGP6) was the major protein with score of 70. It was found to be highly expressed in gut of silkworm PM when compared with CSR2 race. RNase H is an endoribonuclease that catalyzes cleavage of ribonucleic acid through hydrolytic mechanism. It specifically degrades the RNA strand in RNA-DNA hybrids. The enzyme plays a huge role in microorganisms to provide immune system against invading pathogens by disrupting their RNA-DNA hybrid. It is found in all organisms ranging from archaea, bacteria and eukaryote. In a recent research by [45] in M. smegmatis enlightened RNAse H to inhibit mycobacterium infection activity. The enzyme also plays a role in protection of organism against UV and oxidation damage. RNase H enzyme also contributes in growth and development of the organism. They carry out metabolism of the RNA primers of Okazaki fragments formed during lagging strand in DNA replication during transcription. An endonuclease R1Bm element was isolated from silkworm Bombyx mori, which is widely distributed retrotransposons that had high sequence-specific similarity to human L1 retrotransposon sequence. These endonucleases belong to general class of E. coli Exo III having ribonuclease H activity and could in principle be important for retrotransposition [46]. Earlier in silkworm, it was observed that the midgut digestive juice possessed an RNase activity that degrades dsRNA genome of the cytoplasmic polyhedrosis virus (CPV) [47]. The work by [48] also produced similar results wherein 41 kDa RNAse was isolated against polyhedrosis virus from B. mori that showed similarity to bovine thymus endoribonuclease H. RNase H isolated from the silkworm in this study might either play a role in immune system or growth and development. Further charecterization of this protein could lead us in better understanding of its exact role in the insect gut.
The next highest score was recorded for Aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase subunit C (SGP5). Higher expression of aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase was found in the gut of bivoltine silkworm larvae pure race CSR2. Aminotransferase in general are enzymes that hydrolyze amino acids particularly glumatine or asparagines to obtain ammonia that are further used by enzyme itself for further catabolism/reactions [49]. Aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase catalyses formation of correctly charged Asn-tRNA(Asn) or Gln-tRNA(Gln) through the transamidation reaction in organisms that lack either or both of asparaginyl-tRNA or glutaminyl-tRNA synthetases. The reaction utilizes glutamine or Asparagine along with ATP for protein synthesis. In Helicobacter pylori, the heterotrimeric tRNA-dependent amidotransferase (GatCAB) is essential for protein biosynthesis because it catalyzes the conversion of misacylated Glu-tRNA(Gln) and Asp-tRNA(Asn) into Gln-tRNA(Gln) and Asn-tRNA(Asn) [50]. Few of heterotrimeric amidotransferase genes encoded by gatA, gatB, and gatC genes were identified in Chlamydia trachomatis genome. This genes were found to play an important role in amino acid synthesis and proper folding of proteins [51]. The enzyme thus isolated could play an important role of protein synthesis in gut microbiota. [52] found aspartic proteinases to be widely distributed among plants. The entire role of aspartic proteinase is yet to be established. It is thought to be involved in protein processing or degradation in various stages of plant development. In present experiment, higher expression of aspartyl protein was observed in bivoltine silkworm when compared with multivoltine silkworm. Generally, silkworm larvae were fed on mulberry leaves alone. The aspartyl protein content could be recovered from mulberry leaves since mulberry leaves are rich source of aspartyl protein compared with glutamyl protein.
The other protein identified was thioredoxin (SGP 1) with a score of 61. Thoredoxin have a huge role in this environmental system ranging from plants, bacteria to human system. In plants, it catalyzes reversible disulfide-bond formation to regulate structure and function of many proteins. It involves in metabolism, gene expression, growth, and development under different environmental conditions [53]. Thiol-dependent redox enzyme plays a central role in rapid acclimation of chloroplast metabolism based on light availability [54]. In humans, Thioredoxin-1 (TRX1) protein provides anti-oxidant and anti-inflammatory effects by up-regulation of pro-inflammatory cytokines [55]. In Bombyx mori, BmTrx has been shown to protect against oxidative stress caused by extreme temperatures and microbial infection. The amino acid sequence indicated dithiol/disulfide active site residues (CGPC) to be conserved when compared among insect species [56]. Similar results were confirmed by [57] wherein BmTrx protects against oxidative stress caused by extreme temperatures and microbial infection as well as by intracellularly generated reactive oxygen species during metabolism. On the other hand thioredoxin peroxidase BmTPx play a protective role against oxidative stress caused by temperature and viral infection [58]. Thioredoxin peroxidases (Tpxs) play important role in protecting organisms against toxicity of reactive oxygen species (ROS) and regulating intracellular signal transduction in Apis cerana cerana [59]. Many of the antioxidant enzymes including catalases and peroxidases are able to quench oxidants that provide line of defense [60]. This result was consistent with previous observations indicating that of insect antioxidant enzymes, Gryllotalpa orientalis SOD1 [61], G. orientalis Prx [61], BmTPx [58], Bombus ignitus Txl [62], B. ignites SOD1 [62], and G. orientalis ATX1 [63] were up-regulated during microbial infection. Our present results suggest that the new protein of Trx could play a key role in protecting from microbial infection and environmental stress.
The next score of protein identified from gut microflora is Phosphoribosyl-ATP pyrophosphotase (PRPP) with 56. PRPP is required for synthesis of purine and pyrimidine nucleotides, for pyridine nucleotide cofactor NAD (P) and for synthesis of amino acids histidine and tryptophan [64]. In Aspergillus nidulans, this enzyme is encoded by Prs gene catalyses the reaction of ribose-5-phosphate and adenine ribonucleotide triphosphate (ATP) and has central importance in cellular metabolism [65]. In Mycobacterium smegmatis, the gene PrsA encoding for the synthesis phosphoribosylpyrophosphate (PRPP) was found as a key metabolite for several biosynthetic pathways including those for histidine, tryptophan, nucleotides and decaprenylphosphoryl-arabinose, an essential precursor for the mycobacterial cell wall biosynthesis [66]. In silkworm especially tassar silk worm Antheraea mylitta, analysis of transcripts expressed in one and fifth instar silk glands showed phosphoribosylpyrophosphate gene as house-keeping gene to carry out cellular metabolism [67]. Similar results were obtained by identified phosphoribosylpyrophosphate synthetase associated protein in insect skeletal muscle of silkworm. In the present study, higher expression of Phosphoribosyl-ATP pyrophosphatase was observed in PM race when compared to CSR2. The enzyme might govern cellular metabolism and could be involved in increasing survival rate of PM larvae. Earlier studies have shown that PM has higher disease resistant ability compared with other breeds. In our findings, survival rate was significantly increased in all groups treated with arginine, histidine and their mixtures. [68] have reported oral supplementation of arginine, histidine and their mixtures resulted in stimulatory effect on survival rate of the silkworm B. mori.
The next identified protein from CSR 2 race was to be Urease subunit gamma (SGP3) that scored for 55. Urease catalyzes hydrolysis of urea to form two molecules of ammonia and one molecule of carbon dioxide [69] and [70]. In general, urease protects bacteria in acidic environments by neutralizing acids [71]. In animals, it is being used as convenient quantitative measure of nitrogen recycling. Particularly in European hare (Lepus europaeus), urease activity has been found to be high in winter periods for proper recycling of nitrogen from diet [72]. In silkworm, urease is important for the nitrogen metabolism of silkworms because the formation of ammonia is assimilated into silk protein. The enzymes are obtained from mulberry leaves and are not synthesized by silkworm itself. Mulberry leaves treated with cowpea seed powder and feeding it to fifth instar larvae of multivoltine cross breed race of silkworm, Bombyx mori showed enhanced production of midgut enzymes including Protease, Amylase, Trehalase, Sucrase and Urease [73]. Transgenic silkworm lines developed through incorporation of artificial gene showed expression of urease which played a major role in nitrogen metabolism [74]. In other bacteria such as Bacillus species, urease has been found in the process bio calcification [75]. In order for healthy production of eggs/offsprings, calcium is utilized by the silkworms. Urease involving in calcium accumulation might govern a potential offspring development in silkworms. The calcium crystals produced provides harder shell for silkworm eggs [76]. Urease genes and genes encoding proteins are also involved in formate synthesis and also protects the cells by counteracting low pH resulting from formate metabolism. pH sensitivity is an important factor for the production of silk protein in silkworm. The silk proteins are stored in glands and transported where it undergoes conformational changes in response to pH and converted to beta sheet fibers from alpha helical soluble conformations [77]. Thus, the protein could possibly indirectly participate in formation of silk fibres. Another possible role urease could play in silkworm gut system could be providing immunity. Plant urease has been shown to provide immunity in plants since the past twenty years. In this context, urease isoforms have been isolated from seed of Canavalia ensiformis (Jack Bean) that provide resistance to insects and fungi. When administered orally, ureases are toxic for insects that provide cathepsin-like peptidases (hemipterans) and trypsin-like peptidases (dipterans) for digestion [78]. To counterpart H. pylori infection in humans, an secretory system have been developed that consists of Ure B gene (Urease) constructed along with signal peptide bombyxin from B. mori. The expression system was found to be higher in terms of production and would aid in the large-scale expression, yield of UreB in silkworm larvae [79]. In fact, the oral immunization of silkworm pupae powder containing recombinant UreB of Helicobacter pylori provided therapeutic effects against Helicobacter pylori infection when tested in mice [80].
The other protein identified in PM silkworm gut microflora was Phenylacetic acid degradation protein (SGP2) which scored for 49. The protein family belong to thioesterase superfamily and are found in phenylacetic acid degradation [81]. They have been thought to play a major role as thioesterases in ring opening. The crystal structure of a Phenylacetic acid (PhAc) degradation protein PaaG was derived from Thermus thermophiles at 1.85 A and was found to carry out ring opening reaction via an isomerase like mechanism [82]. Organisms using aromatic compounds as their growth substrate require energy to breakdown the aromatic ring system. In anaerobic conditions, activation by CoA- thioester formation carries out energy driven reduction of aromatic rings. The biochemical mechanism of exact ring opening has been elucidated from Pseudomonas putida [83]. Intermediates are processed as CoA thioesters and the aromatic ring of phenylacetyl-CoA becomes activated to a ring 1,2- epoxide by oxygenase. The reactive non- aromatic epoxide is isomerized to a seven-member O-heterocyclic enol ether, an oxepin followed by hydrolytic ring cleavage and β-oxidation steps leading to acetyl-CoA and succinyl-CoA. A similar function could be correlated with the identified protein from silkworm gut microflora.
The least score of 27 was identified for L-serine dehydratase (SGP4). It was isolated from the gut of silkworm bivoltine (CSR2) pure race. L-serine ammonia-lyase is a member of β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes that catalyze conversion of L-serine (l-threonine) to pyruvate (α-ketobutyrate) and ammonia [84]. It was confirmed while solving its crystal structure from Rhizomucor miehei at 1.76 A. The enzyme has been found to be a serine dehydratase and plays similar role as identified above [85]. It plays an important role in gluconeogenesis during starvation and high-protein diets. In silkworm, knockout of a single gene causes large scale change in metabolic pathway. The levels of proteins involved in glycolysis/gluconeogenesis, pentose phosphate pathway, and glycine-serine biosynthetic pathway remains down-regulated [86]. The modification leads to redistribution of nutrients leading to increase in pupal weight. Similarly, the process of gluconeogenesis has also been found in thermal parthenogenesis in domesticated silkworm Bombyx mori [87]. Hence the protein identified could play a role of gluconeogenesis during stress conditions for its survival and production of pupa.