Introduction

The research of gut microbiota is important for in-depth understanding of relationships between the gut microbiota and its host. Most studies on gut microbiota were performed in mammals, especially in human. However, gut microbiota also exists in insects, functioning on conveying beneficial nutrition and protection to their hosts. In fact, considering the potential disease control, pollination of many food crops, and mediators of biogeochemical cycling (such as nitrogen cycle) for insects in medicine, agriculture and ecology, the intricate relationship of gut microbiota with insect host, such as termites (Isoptera), cockroaches (blatta), bees (Anthophila), black solider fly (Hermetia illucens) etc., has been catching great interests in recent years [1, 2]. From an ecological perspective, gut microbiota play vital roles in the co-evolution of insect symbiotic interactions mediated by secondary metabolites [3, 4]. In agreement, gut microbiota also play crucial roles in the life cycles of insect, including insect growth, development, reproduction, and inevitable damage in the process of metabolism [5,6,7,8,9,10]. The increasing knowledge on the composition and functions of gut microbiota in insects and the link between a balanced gut microbiota and insects could prompt the use of gut microorganisms for insect growth, thus benefiting the environment and further applications on human health.

Considering the unique roles of gut microbiota in insects, this review will mainly encompass features of gut microbiota from insects (composition, diversity and integrity), methods to study gut microbiota, and potential applications of insect gut microbiome. We believe this will prompt the further comprehension of nutrition-transformation relationship of endosymbiont gut microbiota community with their hosts and shed light of a vision of how this knowledge may lead to novel nutrient control strategies.

Features of Gut Microbiota from Insect

Composition of Gut Microbiota in Insects

The insect gut is separated into three major parts, including anterior midgut, posterior midgut, and hindgut. The composition of gut microbiota may be interfered by several factors, including insect development, biochemical conditions in different intestinal regions [11, 12], and the insect ability of acquiring available resources [13]. Among these parts, hindgut of insects, as extension part of the body cavity, contains food waste. As a consequence, it provides gut microbiota with abundant nutrient environment, thus prompting their growth and diversity [14, 15].

Gut microbiota in insect include protists, fungi, archaea and bacteria. In the lower termites, protists occupy more than 90% portion of the hindgut. For example, bacteria and archaea are found in the guts of both lower and higher termites [16]. Researchers reported that the guts of honey bee (Apis mellifera) adult workers are dominated by a distinctive set of nine bacterial species (Five of these, Snodgrassella alvi, Gilliamella apicola, two species of Lactobacillus, and a Bifidobacterium species) [17]. Research also showed that gut microbiota was rarely in direct contact with intestinal epithelial cells due to their special localization. Normally, bacteria residing within the gut appear to be localized to the lumen within the endoperitrophic space, which is delimited by the peritrophic matrix, a chitinous barrier that lines the midgut [18]. It was reported that the insect gut microbiota were dominated by Proteobacteria (62.1% of the total reads, including 14.1% Wolbachia sequences) and Firmicutes (20.7%) [19].

Diversity of Insect Gut Symbionts

The highest ratios of total gut microbial biomass to host mass are found in some litter and wood-feeders, including termites, crickets, and cockroaches. However, insects such as Drosophila, mosquitoes and aphids, contain relatively small communities in relation to host body mass [1, 20]. This may be resulted from highly compartmentalized guts in litter and wood-feeders. The most commonly studied gut bacterial communities in insects are those groups feeding on wood, decaying matter, or detritus, such as termites, cockroaches, crickets, and some beetles [21]. Bacteria and archaea are found in the guts of both lower and higher termites. The most frequently found archaea are methanogens, which are tolerant of hypoxia environment as well as some gut protists such as Dinenympha parva and spirotrichonympha leidyi [22]. The dominant gut microbiota in different insect hosts that feed on different foods, including lower termites, higher termites, cockroaches, crickets, beetles, black soldier fly, drosophila, house fly, bees, butterfly and wasps, were summarized (Table 1). As well known, abundant lignocellulose exits in wood. Although lower and higher termites both feed on wood, the degradative process differed from higher to lower termites. And quite possibly, lignocellulose digestion is conducted by different microbiota in lower and higher termites. In lower termites, this process is mostly accomplished by bacteria, while mostly by protists in higher termites. In fact, research indicated that endoglucanases and β-glucosidases secreted by protists in higher termites might be responsible for the lignocellulose digestion [16]. Different from termites, beetles mostly feed on grass, thus leading to the occupation of Hypocreales, Streptomyces, Firmicutes and some proteobacteria in the gut of beetles [23, 24]. Also, cockroaches possess diverse gut microorganisms because they can feed on feces, utilizing complicated components in the feces.

Table 1 Diversity of gut microbiota derived from different insects
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Bacteria diversity was significantly higher in the guts of insects characterized as omnivores than in those of carnivores or herbivores [19]. Hence, feeding preference of host is a vital factor which impacts the diversity of gut microbiota in the insects. Except some obvious factors such as feeding preference of host, diversity of gut microbiota is also associated with the environmental habitat of the host. The environmental habitat could be divided into sky, ground, underground, and aquatic. Due to the difference of oxygen levels in different habitats, aerobes were more abundant in the gut of the terrestrial insects (sky, ground and underground) than in those of the aquatic insects. Also, the abundance of anaerobes was significantly higher in the guts of aquatic insects than those of terrestrial insects [19]. The abundance variation of anaerobes was most likely related to the levels of oxygen in the different environmental habitats, thus impacting the community diversity of the insect gut microbiota.

16S rRNA sequence is still the widely used standard for classifying gut microbiota. However, although massive amounts of 16S rRNA databases, such as Genomes OnLine Database (GOLD), SILVA, GreenGenes (GG), and the Ribosomal Database Project (RDP), have been developed for taxonomy predictions and annotations [25, 26], classification of the taxonomic in the gut microbiota are limited by the taxonomic depth and the reference databases. More and more researchers refer the SILVA as the best suited database since it contains the largest number of sequences and it is regularly updated. With these powerful datasets, it’s convenient for exploring the gut community structure of insects.

Other than these, beta diversity that incorporates additional information to variation in different species or samples, is now being used by many researchers to describe the relative abundances of species [27]. For instance, age-related changes in the composition of Drosophila gut microbiota were studies, revealing an increase in the relative abundance of Proteobacteria and a decrease in the proportion of Firmicutes [28, 29]. Another example, 16S rRNA sequencing results of Hermetia illucens showed strong difference between the soybean (SD) and insect diet (ID) groups in both type and relative abundance (beta diversity) of microbial species [30]. In particular, Bacteroides plebeius, Elusimicrobium minutum, Alkaliphilus transvaalensis, Christensenella minuta, Vallitalea guaymasensis and Flavonifractor plautii represented the principal contributors of changes in gut microbiota composition of ID group [30]. These application instances demonstrated that beta diversity is a significant criterion revealing the relative abundances of species.

Change of Gut Microbiota During the Different Life Stages of Hosts

Another interesting finding is that the community structure of gut microbiota in hosts could be changed along with the growth of host. For example, Lactobacillus and Bifidobacterium populations decreased significantly along with the aging of short-lived (worker) honey bees, while other bacteria, such as Proteobacteria, Bartonella apis and Acetobacteraceae increased, and consistent with a suite of host senescence markers. In contrast, long-lived (queen) honey bees maintained youthful cellular function with accumulated Lactobacillus firm5, Lactobacillus kunkeei and Bifidobacterium spp., and decreased Proteobacteria, Parasaccharibacterapium and Acetobacteraceae 2.1 [31, 32]. Along with the increased oxidative damage, gram-positive bacteria decrease in workers but increase in queen [31, 33].

In addition, high-throughput sequencing of the gut microbiota also indicated the similar results. Composition of gut bacterial community with variation in V4 region of 16S rRNA gene of Chrysomya megacephala under different ages (eggs, 1-day-old larvae, 5-day-old larvae, pupae, adult females and males) were compared using Illumina MiSeq technology [31, 34]. Their results suggested that gut bacteria in C. megacephala varied across life stages, in which Alphaproteobacteria, Bacilli, Bacteroidia, Betaproteobacteria, Flavobacteriia and Gammaproteobacteria represented the majority in C. megacephala. Eight species were identified to have significantly different abundance between 1-day larvae and 5-day larvae and took 28.95% of shared species between these two groups. Seven of the identified species decreased from 1-day-Larvae to 5-day-Larvae groups, only one species Pseudoclavibacter bifida increased [34]. Gut microbiota community in these honey bees or flies both showed some difference at detailed taxonomic levels across the life stages. Whether larva insect or adult insect, they might share some of core harbored gut bacteria results from overlap of food range.

Gut microbiota in hosts likely originate from insect-vectored plant and animal pathogens or from direct insect pathogens. During the different life stages of insect hosts, these pathogens often also require entry to host cell to complete life cycles (Origin, birth, middle age, old age, death and rebirth), resulting in interplay between hosts, pathogens and host-beneficial gut microbiota [35].

Methods for Exploring Gut Microbiota

Depending on the source of the microbial samples, approaches to determine microorganism taxonomy of insect gut microbiota can be classified into culture-dependent or culture-independent assays (Table 2). Culture-dependent assays focused on enriching previously determined component organisms, which may be used for further characterization. In comparison, culture-independent assays are strongly recommended for the initial taxonomic profiling of microbial communities as many species may not be easily cultured [36].

Table 2 Dominant methods to study gut microbiota
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Culture-Dependent Assays

Studying the composition of gut microbiota helps understand their functions and relationship with the hosts. Normally, microbial characterization is the first step for systemic studies of the gut microbiota [37]. Conventional methods were normally performed by experimental culturing followed up by phenotype characterization using morphological and biochemical characteristics. Notably, the culture-dependent assay encompasses many advantages, such as the availability of pure cultures of bacteria, the ability to study living bacteria for specific functions, and the detection of specific intestinal pathogens [38].

Culture-Independent Assays

The major defects of culture-dependent assay are that many bacteria could not be cultured under laboratory conditions, and the symbiotic relationship among different bacteria is difficult to be studied. Along with the fast development of molecular biotechnologies, culture-independent assays for analyzing the gut microbiota composition have been largely developed, mainly including PCR-based methods, fluorescent in situ hybridization (FISH), flow cytometry, DNA sequencing, etc. Among these PCR-based methods, the quantitative PCR (Q-PCR) is a major representative approach. For example, using PCR primers targeting 16S rRNA genes of the gut microbiota could amplify the 16S rRNA sequences from all gut bacteria. Combining with the subsequent quantification steps using fluorophores or DNA binding dyes, the composition of the gut microbiota could be deciphered. Using this technology, the gut microbiota of honeybee were analyzed and assessed changes in the abundance of major bacterial groups of the honeybee core microbiota [39]. Since Q-PCR assays do not require time-consuming post-PCR procedures, such as gel electrophoresis and staining, effective results could be obtained with reduced contaminations in contrast to traditional PCR analysis [37].

Moreover, non-PCR-based methods, such as FISH and flow cytometry, were also used to aid the exploration of microbial diversity in insects. Fluorescein-labeled oligonucleotide probes targeting 16S rRNA genes are frequently used for FISH analysis. Through hybridization, bacterial cells associated with gut tissues or feces are labeled with a fluorescent probe and can be analyzed by flow cytometry [40]. Recently, due to the fast development of next generation sequencing (NGS) technology in biological research, it became a very powerful method to characterize the diversity of gut microbiota in insects based on its high efficiency and accuracy. The NGS technologies have been applied largely in metatranscriptomics and metagenomics. In recent research, metagenomic and metatranscriptomics technologies were applied for illuminating the diversity of lignocellulolytic enzymes within the termite gut [41]. Metagenomic studies make it possible to explore the potential functions of the specific microorganisms within the gut communities [42]. Besides, it’s also of great significance for investigating key metabolites associated with host-gut microbiota interaction. Moreover, Mass Spectrometry-based metabolomics approaches further facilitated the exploration of the metabolic networks between the hosts and gut microbiota.

Culture-dependent and culture-independent methods have different strength (Table 2). Therefore, new methods that integrated both strategies were also developed to evaluate the effect of gene, protein and metabolite on the composition and function of gut microbiota. For instance, Ogué-Bon, etc., reported that stirred, pH-controlled anaerobic batch cultures in combination with FISH assay could be used to investigate the in vitro effects of galacto-oligosaccharide alone or combined with the probiotic Bifidobacterium bifidum 02 450B on fecal microbiota [43]. Additionally, Lin et al. reported that metabolic capacities of the gut microbiota in a fungus-cultivating Termite through in vitro assays of colonic fermentation with in vitro assays of DGGE and metagenomics [44].

Nutrient Metabolic Process Mediated by Insect Gut Microbiota

Protein Hydrolysis, Sugar Fermentation and Amino Acid Metabolism

Uptake systems and catabolism of proteins/amino acids in gut microbiota bacteria are vital metabolic process, which benefit both gut microbiota and host insects (Fig. 1). Many microbial species such as Clostridium spp., Bacteroides spp., Lactobacillus spp. etc., in gut microbiota contain varieties of proteases involved in catabolism of proteins. In the well characterized proteolytic system of Lactic acid bacteria (LAB), proteinase, peptide transporters and peptidases function cooperatively. In general, protein hydrolysis by LAB is initiated by a cell-envelope proteinase (CEP) that degrades the protein into oligopeptides, which are subsequently taken up by the cells via specific peptide transporters, and further degraded by various intracellular peptidases into shorter peptides and amino acids [45, 46].

Fig. 1
figure 1

Nutrient metabolic process mediated by insect gut microbiota

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As one major source of energy, sugar fermentation is another important metabolic process catalyzed by gut microbiota. It has been reported that LAB acquired the ability to recognize sugars, including xylose, cellobiose, glucose, and fructose [46, 47], thus playing essential roles in Lactic fermentation [45]. Through protein degradation and sugar fermentation, symbiotic gut microbes could provide essential amino acids (EAAs) to their host insects. Birkle et al. reported that the symbiotic bacteria Buchnera could provide tryptophan and other essential amino acids to their aphid hosts due to shortage of EAAs in the food ingredients of aphid [48]. Additionally, gut transcriptomic studies of A. glabripennis revealed the presence of complete bacterial and fungal biosynthetic pathways for several EAAs [49]. The potential mechanism of EAAs synthesis is that nitrogen is recycled and fixed as the form of NH4+, which is then routed into end-products, such as EAAs and non-EAAs [50]. Since wood is a poor resource of nitrogen, this is very important to wood-feeding insects because they are unable to synthesize EAAs de novo.

Lipid Metabolism

Lipid metabolites produced in gut microbes is a source of carbon and energy storage for host insects. Extensive microbial fermentation of polysaccharides exists in the hindgut of insects, in which large amounts of short-chain fatty acids (SCFAs) are produced. Zheng et al. found that microbial metabolism markedly reduces the pH and redox potential in gut through the production of SCFAs. In the hindgut of honey bee, SCFAs including acetate, propionate, and butyrate are main metabolites produced by the resident gut bacteria, which affects its weight [51]. Other than SCFAs, polyhydroxyalkanoates (PHAs) are also a class of important lipid metabolites produced by gut microbes in insects. For instance, PHAs can be transformed into carbon and energy storage by gut microbes in yellow mealworm [52]. Moreover, some new aminoglycolipids were identified from a Deinococcus sp. strain isolated from the gut of queen carpenter ants (Camponotus japonicus), among which four new aminoglycolipids, annotated as deinococcucins A-D, showed functional ability of inducing the quinone reductase production in host cells [53].

Cellulose Digestion and Nitrogen Fixation

Insects which feed on woods usually possess gut bacteria with cellulose digestion ability. For example, Bombyx mori that feeds on mulberry leaves mainly depends on the digestive enzymes produced by its gut bacteria to degrade carbohydrates, such as pectin, xylan, cellulose and starch [54]. Under normal condition, these gut bacteria provide digestive enzymes in a synergic manner and contribute to the larval growth. Similar cases exist in phytophagous and xylophagous insects, which are the most efficient ecosystems to degrade lignocellulose. Studies of gut microbiota of Ergates faber (beetle), Potosia cuprea (chafer), Gromphadorrhina portentosa (cockroach), Locusta migratoria (locust), and Gryllus bimaculatus (cricket) in anaerobic batch reactors indicated that many gut microbiota possess the degradation ability of lignocellulose [55]. Another example is termites, which was recently aimed for converting wood into biofuels due to their strong cellulose degradation ability by their gut microbiota. Degradations of cellulose into hexose and pentose oligomers, and a series of biofuel derivatives are catalyzed by a number of macromolecular complexes, which result from synergism of a number of microbial communities in gut [56].

Other than cellulose digestion, studies to date reported that symbiotic nitrogen fixation was also vital metabolic process for insect nutrition and development. It was identified that the nitrogen nutrition of termites was mainly obtained from their symbiotic gut microbes instead of food [57,58,59]. Interestingly, the nitrogen fixation ability of the gut microbes is determined by termite feeding. Termites that feed on soil generally do not have strong nitrogen fixation ability, while termites that mainly feed on wood have more intestinal bacteria (Citrobacter freundii and Enterobacter agglomerans) with the pronounced biological activities of nitrogen fixation [60]. Similarly, studies also showed that Spirochetes from gut microbiota of termites played important roles in providing carbon, nitrogen, and energy requirements of termite nutrition via acetogenesis and nitrogen fixation [20]. As the symbiotic nitrogen fixation rates were negatively correlated with dietary N, it was suggested that high concentrations of dietary N suppressed symbiotic nitrogen fixation in termites [61]. Also, gut microbiota of dung beetles feed on dung particles with different C/N ratio were characterized and showed that functional gene abundances in larval and adult gut microbiota would reflect differences in their capacities for cellulose digestion and nitrogen metabolism [62].

Vitamin Production

Another benefit from gut microbes is that they can deliver useful metabolites to their host, such as vitamins. A synthetic cycle includes that gut microbiota fix nitrogen into ammonia, which is then assimilated by gut endosymbionts to re-biosynthesize vitamins for insect development [1, 21, 54, 63]. Due to the water-soluble property of B vitamins, they are the major ones produced by gut microbiota in insects. Salem et al. analyzed genes involved in processing of B vitamins through comparative transcriptomic assay. They revealed the differential expression of genes related to the transport and processing of B vitamins, thereby highlighting an important interface for the exchange of symbiont-provided nutritional supplements [64]. Vitamin B deficiency in symbiont-free insects presents that symbiotic gut microbiota undoubtedly play vital role in the supplement of B vitamins for host insects. In addition, research also reported that Wigglesworthia, the mycetocyte symbiont of Glossina brevipalpis requires many vitamins, such as pantothenate (vitamin B5), biotin (vitaminB7), thiamin (vitamin B1), riboflavin and FAD (vitamin B2), pyridoxine (vitamin B6), nicotinamide (vitamin B3) and folate (vitamin B9), as cofactors for its own metabolism [65].

Concluding Remarks

The taxonomic diversity of insects and differences in their habitat and feeding preference cause the diversity of their gut microbiota. At the same time, difference of gut structure also shapes the gut microbiota of insects. The different insect species, such as termites (Isoptera), cockroaches (blatta), bees (Anthophila), black solider fly (Hermetia illucens) etc., encompass different gut microbiota composition due to the habitat and feeding preference of their insect hosts. As a major interface between the host insect and microbes, the gut provides an environment for growth of microbes, which reversely benefit their host insects through providing amino acids, lipid metabolites (SCFAs, PHAs etc.), nitrogen fixation, vitamins, etc.

To dissect the diversity of gut microbiota and their functions in nutritional metabolisms in gut microbiota of insects, culture-dependent methods and culture-independent methods were integrated to analysis. Molecular technologies from the prevailed 16S rRNA profiling of gut communities to newly emerged metagenomic and NGS technologies provide more and more evidence that gut microbiota is critical to the nutritional metabolic process in insects. The increasing knowledge on the composition and functions of gut microbiota in insects and the link between a balanced gut microbiota and insects could prompt the use of gut microorganisms for insect growth, thus benefiting the environment and further application on human health.

Research on functions of gut microbiota in nutritional metabolisms facilitate in-depth understanding of gut microorganisms for insect growth. Under this circumstance, studying the functional genomics of a symbiotic community were focused to profile the symbiotic mechanism of insect gut microbiota. Recently, Frances Blow et al. identified a specialized gut microbiota dominated by the obligate symbiont “Candidatus Erwinia dacicola” in the olive fruit fly Bactrocera oleae. Candidatus, which could supplement dietary nitrogen to the host [66]. Based on this study, they also reconstructed a number of pathways related to nitrogen assimilation within the host. In addition, along with the microbiota identification, functional redundancy between different microbial taxa was observed for genes involved in urea hydrolysis. These studies all indicated that exploring the nutritional metabolism in the insect gut microbiota symbiosis could further understand the symbiotic mechanism of insect gut microbiota. It could be expected that with further understanding the symbiotic relationship between insect and gut microbiota, engineered microbes might be developed to change the insect gut environment to facilitate their growth, metabolism, etc.