Recently, there has been an increase in the number of studies on the gut symbionts of xylophagous insects like termites, with the objective of determining their role in the metabolic and digestive processes of these insects. Termites thrive in great abundance in the terrestrial ecosystems. Their ability to degrade lignocellulose gives termites an important role in global carbon recycling^[1]; and thus, lignocellulose-degrading enzymes from the "termite digestome", both termites and their gut symbionts have many potential bioenergy applications that warrant careful consideration^[2]. With the removal of cellulose (74%-99%), hemicellulose (65%-87%) as well as lignin (5%-83%), the digestion of lignocellulose by termites is far more efficient which typically achieving over 95% bioconversion within a day^[3]. Termites have developed a unique microflora of archaea, archaezoa, bacteria, yeasts and probably fungi^[4]. This specialized microflora enables the termites to efficiently digest lignocellulose and led to a tremendous evolutionary success (Figure 1). Of these symbionts, the anaerobic flagellates produce acetate, CO₂ and H₂ from cellulose. Acetate is also formed from CO₂ and H₂ by acetogenic bacteria^[5]. Acetate is absorbed by the gut epithelium and functions as a major carbon source of termites. The heterotrophic bacteria fulfill several functions in the hindgut. They fix dinitrogen and recycle the nitrogen of uric acid, maintain a low redox potential, synthesize amino acids, produce acetate and other lower fatty acids, and protect the hindgut from foreign bacteria^[6]. Yeasts are single-celled fungi and currently they are becoming the organisms of choice in studies aimed at their potential applications^[7]. They are frequent in guts of insects that feed on detritus or wood, and they likely play a part in digestion^[8].

In contrast to previous studies on lignin breakdown, which gave no convincing evidence of microbial degradation of lignin in the termite intestinal tract^[9], the recent investigations have shown that the paunch of termites harbors a significant number of yeasts and bacteria with hemicellulose-degrading activities^[10-11]. Clearly, between 10⁷ and 5×10⁸ yeast cells were found per mL gut content in Zootermopsis angusticollis and Neotermes castaneus^[10]. These yeasts were related to the genera Candida, Pichia, Sporothrix, and Debaromyces. Even though none of these yeasts produced all the required hydrolytic enzymes to completely digest hemicelluloses, they play a significant role in the breakdown of hemicellulose in the termite gut, probably through synergistic activities of their glycolytic enzymes with other gut symbionts (Figure 1). In another study^[12], Debaryomyces hansenii and Sporothrix albicans as well as species of Trichosporon and Rhodosporidium could also be found in the hindgut of the termites from families: Mastotermitidae, Hodotermitidae, Kalotermitidae and Rhinotermitidae. Therefore, one may regard the termite gut as a complex ecosystem with free-living and attached microorganisms.

Cellulose and hemicellulose are digested to a large extent during passage of food through the digestive tract by both lower and higher termites, but with different digestive strategies. In the phylogenetically "lower" termites, the gut symbionts include bacteria and protozoa. Many of protozoa are capable of ingesting wood particles and are cellulolytic^[13]. However, in the phylogenetically "higher" termites which constitute about 75% of all termite species, the gut microbiota essentially consist of bacteria alone. The confirmed role of bacteria in cellulose digestion is still unclear in lower as well as in higher termites, because animals themselves also produce cellulases^[14], and the interesting question: what role do symbionts play? On the other hand, fungi are found in the gut content of termites but do not appear to be involved in cellulose digestion^[14] so another interesting questions are: (ⅰ) does the termites produce a cellulase themselves? (ⅱ) does it ingest fungal enzymes? and (ⅲ) what role do yeasts play?

Termite host tissues, via recent transcriptomic efforts, have been shown to produce a litany of cellulases and hemicellulases, as well as lignases, and contribute to lignocellulose degradation^[3]. The association with the lignin-degrading fungi enables the fungus-cultivating termites to degrade lignocellulose nearly completely, as reflected in the small volume of their final feces^[10]. The key activities attributed to the fungal partner in the mutualistic symbiosis of fungus-cultivating termites are extensive delignification of the substrate and the conversion of plant fiber to fungal biomass^[15]. The "acquired enzyme hypothesis" of Martin and Martin^[16] was confirmed by evidence for an activity within the gut of fungal cellulases ingested by the termites together with the fungus comb material^[17-18]. However, claims that the fungal cellulases are essential for cellulose digestion in the termite guts remain controversial, especially in view of the recently discovered ability of termites to produce their own cellulases^[3].

1 Yeast biodiversity

The "yeast" word is broadly used to represent a fungal growth form described by single-cell microorganisms^[19]. Still despite their widespread presence, it is thought that only 1% of the diversity of yeast species has been described so far, demonstrating that there is a huge number of undescribed species^[20]. At least 1500 yeast species, belonging to the Ascomycota and Basidiomycota phyla, are known^[21]. Of Ascomycota, almost 700 yeast species described in 93 genera are called the "true yeasts" and belong to the class Saccharomycetes^[19]. These genera include Candida, Saccharomyces, Metschnikowia, Pichia, and Kluveromyces^[22]. Cystofilobasidium, Tremella, Cryptococcus, Fellomyces, and Ustilago are yeast genera belonging to Basidiomycota^{[21, 23]}.

Most of the known yeast symbionts harbored in the gut of insects belong to Candida, Pichia, Metschnikowia, Pseudozyma, and Cryptococcus^[21]. In most cases, these symbionts are grown intercellularly in their host guts^[19]. Clearly, true yeasts represent most of the gut symbionts although there are a group of fungal endosymbionts, "yeast-like symbionts" (YLS), that differs from true yeasts. YLS are characterized by evolutionary reduced yeast forms from the Pezizomycotina sub-phylum^[19]. However, the diversity and the association between YLS and insects seem to be more limited comparing with true yeasts and insects. To date, the association between YLS and insects have been observed mainly in some species of planthopper, aphid, and anobiid beetles^[24]. Overall, it appears mutualistic association between true yeast, YLS and insects which can vary with different taxonomic classification^[25]. Generally, the specificity in this association could be correlated to the services provided by the insect to the yeast or vice versa^[24].

2 Evolutionary considerations of the intestinal yeasts

Termites are well known for their specific prokaryotic and eukaryotic gut symbionts^[26]. Despite their wide distribution of the investigated termites in different continents, the majority of yeasts isolated from lower termites comprise a homogeneous assemblage of representatives from the Endomycetales^[27]. Based on a genotypic characterization, Trichosporon mycotoxinivorans sp. nov., was the only Basidiomycetous yeast isolated from lower termites. This yeast species is useful in biological detoxification of various mycotoxins. Additionally, a highly symbiotic yeast consortium in the hindgut of the Pachyiulus flavipes was detected^[28]. The yeast consortia consist almost exclusively of DTZ-group of Ascomycetes identified to be Debaryomyces hansenii, Torulaspora delbrueckii, and Zygowilliopsis californica^[29]. Beside the DTZ-group, other yeast species were minor components in the excrement, and include Pichia membranaefaciens, Trichosporon cutaneum, and Geotrichum candidum. The cell walls of such yeasts probably resist the enzymatic attack in the digestive tract of the insects^[30]. In contrast to lower termites, the symbiotic Basidiomycetous yeast species were commonly detected in the hindgut of the higher termite Odontodermes obesus. These species contained xylose in their cell walls and identified to the genera Tremellaand Filobasidium^[27].

Based on the 18S rDNA sequences and fossil record, Berbee and Taylor^[32] reported that the Endomycetales are a sister group from the filamentous Ascomycetes. Considering a new concept of the carbohydrate composition of yeast cells, sexuality and mating type evolution, Prillinger et al.^[31] interpreted the Endomycetales as primitive fungi which have lost their filamentous stage based on the extinction of the hosts^[33]. However, arguments to the later interpretation were added by Kurtzman^[34]. The presence of Endomycetes yeast symbionts representing the mannose-glucose carbohydrate pattern in lower termite documents that the "Saccharomyces type" is most probably ancestral to the Ustilago, Microbotryum, Protomyces, and Tremella type^[31].

Although intimate associations are known between filamentous Ascomycetes or Agaricales and higher termites, there is no information with respect to the yeast mycoflora in termites and its usefulness to trace the phylogeny of Ascomycetous and Basidiomycetous yeasts. If the speculation of Martynov^[35] about the evolution of termites is correct, coevolution between termites and yeasts may be extremely useful to trace the phylogeny of filamentous Ascomycetous and Basidiomycetous yeasts^[30]. Based on the study of Prillinger et al.^[27] about yeast symbionts in the hindgut of termites, it may be concluded that an amalgamation of information obtained from symbiotic associations, ribosomal DNA sequencing, yeast cell wall sugars, fermentation and the fossil record might be a more reliable indicator for fungal phylogeny than evolutionary trees based on ribosomal DNA sequencing alone to trace fungal phylogeny back to early Paleozoic periods^[30].

3 The possible benefits from the yeast-termite association 3.1 Benefits for the yeasts

Termites can provide yeasts with many benefits (Figure 2). The survival of yeast symbionts is widely demonstrated and is expected to be similar for bacterial symbionts^[8]. Generally, it is considered that symbionts are, first, acquired by ingestion. Then, they colonized the insect guts, and finally being released from feces for dispersion of microorganisms. Depending on the insect behavior, yeast cells can be inoculated in eggs by females or they can be re-ingested by colonization of food sources or through trophallaxis and coprophagy^[8].

Contrary to filamentous fungi, yeasts elaborate spore cell types are able to survive numerous extreme environmental conditions in a laboratory experiment, such as high and low extremes of pH, high salt concentrations, heat shocks, growth at 42 ℃, and ether vapor^[36]. These characters demonstrated that spores are more adapted than vegetative cells to endure environmental harsh conditions^[37]. The cell wall structure of the spore confers their resistance to stresses associated with digestion and allowing the yeasts to be dispersed by oviposition or insect feeding^[38]. Another benefit to yeasts after their consumption by termites is the putative role of digestion in the promotion of outbreeding^[37]. This role is fundamental to increase the probability of adaptation and to maintain genetic variation among the microbial progeny. Frequently, inbreeding between the four ascospores housed in the same ascus is observed during sexual reproduction of yeasts. To release these ascospores and avoid inbreeding, enzymes are required to break up the ascus. It has been proposed that the host enzymes of termite guts accomplish this function by breaking down the haploid ascospore tetrads and promoting outbreeding^[39].

3.2 Benefits for the termites

Yeast symbionts can also provide insects with many benefits (Figure 2). Vage et al.^[19] reported that the role of yeasts in insect nutrition is very important, where insect performance would decrease due to the absence of yeast symbionts. Yeasts are sources of B3 and B5 vitamins, amino acids, proteins, and trace metals that could be easily assimilated through simple digestion. In addition, yeast cells contain about 8.5% nitrogen by dry weight, thus insect feeding on yeasts represents a better nitrogen source than the plant tissue itself^{[19, 40]}. Becher et al.^[41] reported the nutritional importance of yeasts for Drosophila flies during egg maturation and larval development due to sufficient amounts of nitrogen, vitamins, lipids that yeasts provide. Additionally, previous studies reported the nutritional role of nitrogen, vitamins, essential amino acids, and sterols provided by the YLS on Symbiotaphrina and anobiid beetles^[42-43]. Furthermore, the role of yeast symbionts as facilitators of insect host nutrition has been demonstrated^{[19, 40]}. These yeasts play a major role in the production of digestive enzymes that degrade plant lignocellulose into molecules with nutritional value for herbivores. Different types of enzymes are produced by termite-associated yeasts including peptidases, lipases and hydrolases. On the other hand, yeast symbionts in the Lasioderma serricorne, Ernobius mollis and Xestobium piumbeum beetles produce glucosidases lipases, phosphatases and trypsin in order to degrade cellobiose^[44]. Recently, research on the role of yeast enzymes has focused on the degradation of complex polymers into simple sugars that the termite can absorb directly. True yeast species of the genus Candida have the ability to degrade wood components such as cellulose, glucosides, and pectin. Therefore, wood-feeding termites might rely on their associated yeast species to degrade cellulose, and their symbionts produce glucosidases and pectinases^[45].

3.3 Some roles of the symbiotic yeasts isolated from the termite guts and their potential applications

Termite gut symbionts might be interesting due to their pronounced solvent tolerance or may be a future source of industrially useful solvent-tolerant enzymes. In addition these microbiota may detoxify allelochemicals or may be responsible for the biosynthesis of essential compounds such as vitamins, sterols, or nitrogen-containing constituents. Generally these microorganisms may be the source of novel metabolic capabilities such as defense substances and antibiotics or may fixate atmospheric nitrogen. It has been known for many years that enzymes in the gut which originate from ingested fungal tissue or gut microbes may mediate cellulolysis. The data on yeasts in the termite guts are scarce. Nevertheless, the first investigations showed that the yeast isolates occur at high numbers in the guts of lower and higher termites of different systematic positions. Since they have been constantly isolated, they belong most probably to the autochthonous symbiotic microbiota of the paunch. They produce cellulolytic and hemicellulolytic enzymes and may play a significant role in the degradation of lignocellulose besides the protists, which thrive only in lower termites and bacteria^[46].

Termites play a major role in the recycling of photosynthetically fixed carbon. With the aid of their gut symbionts they degrade to a high extent the wood constituents cellulose and hemicellulose. Nevertheless, the microbial species involved in the degradation of cellulose and hemicelluloses have been yet poorly defined before the investigations of Schäfer et al.^[10] and Wenzel et al.^[47]. They identified different yeast strains with cellulose- or xylan-degrading capability. A series of different hemicellulose-degrading yeasts were isolated for the first time from the termite gut^[10]. Some of yeast species; namely Candida blankii, Candida edax, Pichia pini, Sporothrix schenkii and Debaryomyces hansenii var fabryii isolated from the lower termites Mastotermis darwiniensis, Neotermes jouteli, Reticulitermes santonensis, Zootermopsis angusticollis and Zootermopsis nevadensis produced clear zones on agar plates containing insoluble xylan (oat spelt) or dye-linked xylan or solubilized xylan (oat spelt) in liquid media^[27]. Reports on cellulase production by yeast wild-type strains are rare^[48]. Some of the yeast isolates from termites produced hemicellulases^[10] and some were also able to degrade cellulose^[47]. Some cellulolytic yeast strains, Sporothrix schenkii and Trichosporon sp. were isolated from Zootermopsis nevadensis and Mastotermes darwiniensis, respectively^[27]. From a biotechnological perspective, termite gut yeasts constitute promising and mostly untapped sources for potential applications in industrial processes, agriculture, medicine, and bioremediation (Figure 3). A number of approaches based on yeasts from termite guts have been successfully implemented (Table 1).

4 Conclusion

Termite gut symbionts, including other insect gut symbionts, constitute a rich and mostly untapped source of bioactive small molecules as well as a variety of digestive enzymes for potential biotechnological values. In addition to exploiting their metabolic capabilities, it has been suggested that the main roles offered by insect gut symbionts are recognized for a nutritional contribution to their diets, promote insect health as well as to suppress pathogens that might hamper the insect development. Consequently, the study of how insects detect microbes has been focused on insects that depend on microorganisms for their dietary requirements. For this reason in the case of yeast-insect symbiosis, most of the available knowledge was derived from the beetles and Drosophilids, while other insects, such as termites, have been greatly ignored. Hence, the study of termites and the association with their yeast symbionts not only promise to uncover interesting new yeast symbionts but may also prove valuable in the continued efforts to find new sources of biotechnologically important molecules and enzymes. Specifically, targeted searches for compounds with particular application values, such as antimicrobial agents, detoxifying enzymes, industrial enzymes (e.g., xylanases, ligninases, cellulases, etc.) may benefit from being guided by the knowledge on the ecology of insect-symbiont interactions, which would have a potential to predict a promising system for future exploration.