Symbiotic fungi produce laccases potentially involved in phenol degradation in fungus combs of fungus-growing termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

Symbiotic Fungi Produce Laccases Potentially Involved in Phenol Degradation in Fungus Combs of Fungus-Growing Termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

Comparison of gut-associated and nest-associated microbial communities of a fungus-growing termite (Odontotermes yunnanensis)

Abstract The digestion of cellulose by fungus-growing termites involves a complex of different organisms, such as the termites themselves, fungi and bacteria. To further investigate the symbiotic relationships of fungus-growing termites, the microbial communities of the termite gut and fungus combs of Odontotermes yunnanensis were examined. The major fungus species was identified as Termitomyces sp. To compare the micro-organism diversity between the digestive tract of termites and fungus combs, four polymerase chain reaction clone libraries were created (two fungus-targeted internal transcribed spacer [ITS]– ribosomal DNA [rDNA] libraries and two bacteria-targeted 16S rDNA libraries), and one library of each type was produced for the host termite gut and the symbiotic fungus comb. Results of the fungal clone libraries revealed that only Termitomyces sp. was detected on the fungus comb; no non-Termitomyces fungi were detected. Meanwhile, the same fungus was also found in the termite gut. The bacterial clone libraries showed higher numbers and greater diversity of bacteria in the termite gut than in the fungus comb. Both bacterial clone libraries from the insect gut included Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, Nitrospira, Deferribacteres, and Fibrobacteres, whereas the bacterial clone libraries from the fungal comb only contained Firmicutes, Bacteroidetes, Proteobacteria, and Acidobacteris.     

Genetic diversity of termite-egg mimicking fungi “termite balls” within the nests of termites

Antagonistic or mutualistic interactions between insects and fungi are well-known, and the mutualistic interactions of fungus-growing ants, fungus-growing termites, and fungus-gardening beetles with their respective fungal mutualists are model examples of coevolution. However, our understanding of coevolutionary interactions between insects and fungi has been based on a few model systems. Fungal mimicry of termite eggs is one of the most striking evolutionary consequences of insect–fungus associations. This novel termite–fungus interaction is a good model system to compare with the relatively well-studied systems of fungus-growing ants and termites because termite egg-mimicking fungi are protected in the nests of social insects, as are fungi cultivated by fungus-growing ants and termites. Recently, among systems of fungus-growing ants and termites, much attention has been focused on common factors including monoculture system for the ultimate evolutionary stability of mutualism. We examined the genetic diversity of termite egg-mimicking fungi within host termite nests. RFLP analysis demonstrated that termite nests were often infected by multiple strains of termite egg-mimicking fungi, in contrast to single-strain monocultures in fungus combs of fungus-growing ants and termites. Additionally, phylogenetic analyses indicated the existence of a free-living stage of the termite egg-mimicking fungus as well as frequent long-distance gene flow by spores and subsequent horizontal transmission. Comparisons of these results with previous studies of fungus-growing ants and termites suggest that the level of genetic diversity of fungal symbionts within social insect nests may be important in shaping the outcome of the coevolutionary interaction, despite the fact that the mechanism for achieving genetic diversity varies with the evolutionary histories of the component species.     

培菌白蚁菌圃微生物降解木质纤维素的研究进展

白蚁及其共生微生物协同降解植物细胞壁的机理一直被世界各国科学家所关注。培菌白蚁作为高等白蚁,相比低等食木白蚁具有更多样化的食性,其利用外共生系统“菌圃”,对多种植物材料进行处理。本文综述了菌圃微生物降解木质纤维素的研究进展,以期为深入研究菌圃中木质纤维素降解过程及其机制,并挖掘利用菌圃降解木质纤维素的能力及仿生模拟菌圃开发新的生物质利用系统提供参考。培 菌白蚁在其巢内利用由植物材料修建的多孔海绵状结构——“菌圃”来培养共生真菌鸡枞菌Termitomyces spp.,形成了独特的木质纤维素食物降解和消化策略,使木质纤维素在培菌白蚁及其共生微生物协同作用下被逐步降解。幼年工蚁取食菌圃上的共生真菌菌丝组成的小白球和老年工蚁觅得食物并排出粪便堆积到菌圃上成为上层菌圃。这一过程中,被幼年工蚁取食的共生真菌释放木质素降解酶对包裹在植物多糖外部的木质素屏障进行解聚。菌圃微生物(包括共生真菌)对解聚的木质素基团进一步降解,将多糖长链或主链剪切成短链,使菌圃基质自下而上被逐步降解。最后下层的老熟菌圃被老年工蚁取食,其中肠的内源酶系及后肠微生物将这些短链进一步剪切和利用。因此,蚁巢菌圃及其微生物是培菌白蚁高效转化利用木质纤维素的基础。化学层面的研究表明,菌圃能够实现对植物次生物质解毒和植 物纤维化学结构解构。对共生真菌相关酶系的研究显示可能其在菌圃的植物纤维化学结构和植物次生物质的降解中发挥了作用,但不同属共生真菌间其效率和具体功能不尽相同。而菌圃中的细菌是否发挥了作用和哪些细菌类群发挥了作用等仍有待进一步的研究。相比于低等食木白蚁利用其后肠共生微生物降解木质纤维素,培菌白蚁利用菌圃降解木质纤维素具有非厌氧和能处理多种类型食物两大优势,仿生模拟菌圃降解木质纤维素的机制对林地表面枯枝落叶的资源化利用具有重要意义。     

Occurrence of fungi in combs of fungus-growing termites (Isoptera: Termitidae, Macrotermitinae)

Fungus-growing termites cultivate their mutualistic basidiomycete Termitomyces species on a substrate called a fungal comb. Here, the Suicide Polymerase Endonuclease Restriction (SuPER) method was adapted for the first time to a fungal study to determine the entire fungal community of fungal combs and to test whether fungi other than the symbiotic cultivar interact with termite hosts. Our molecular analyses show that although active combs are dominated by Termitomyces fungi isolated with direct Polymerase Endonuclease Restriction - Denaturing Gradient Gel Electrophoresis (PCR-DGGE), they can also harbor some filamentous fungi and yeasts only revealed by SuPER PCR-DGGE. This is the first molecular evidence of the presence of non-Termitomyces species in active combs. However, because there is no evidence for a species-specific relationship between these fungi and termites, they are mere transient guests with no specialization in the symbiosis. It is however surprising to notice that termite-associated Xylaria strains were not isolated from active combs even though they are frequently retrieved when nests are abandoned by termites. This finding highlights the implication of fungus-growing termites in the regulation of fungi occurring within the combs and also suggests that they might not have any particular evolutionary-based association with Xylaria species.     

Intracolony variation of bacterial gut microbiota among castes and ages in the fungus-growing termite Macrotermes gilvus

The fungus-growing termites Macrotermes cultivate the obligate ectosymbiontic fungi, Termitomyces. While their relationship has been extesively studied, little is known about the gut bacterial symbionts, which also presumably play a crucial role for the nutrition of the termite host. In this study, we investigated the bacterial gut microbiota in two colonies of Macrotermes gilvus, and compared the diversity and community structure of bacteria among nine termite morphotypes, differing in caste and/or age, using terminal restriction fragment length polymorphism (T-RFLP) and clonal analysis of 16S rRNA. The obtained molecular community profiles clustered by termite morphotype rather than by colony, and the clustering pattern was clearly more related to a difference in age than to caste. Thus, we suggest that the bacterial gut microbiota change in relation to the food of the termite, which comprises fallen leaves and the fungus nodules of Termitomyces in young workers, and leaves degraded by the fungi, in old workers. Despite these intracolony variations in bacterial gut microbiota, their T-RFLP profiles formed a distinct cluster against those of the fungus garden, adjacent soil and guts of sympatric wood-feeding termites, implying a consistency and uniqueness of gut microbiota in M. gilvus. Since many bacterial phylotypes from M. gilvus formed monophyletic clusters with those from distantly related termite species, we suggest that gut bacteria have co-evolved with the termite host and form a microbiota specific to a termite taxonomic and/or feeding group, and furthermore, to caste and age within a termite species.     

You eat what you find – Local patterns in vegetation structure control diets of African fungus‐growing termites

Fungus‐growing termites and their symbiotic Termitomyces fungi are critically important carbon and nutrient recyclers in arid and semiarid environments of sub‐Saharan Africa. A major proportion of plant litter produced in these ecosystems is decomposed within nest chambers of termite mounds, where temperature and humidity are kept optimal for the fungal symbionts. While fungus‐growing termites are generally believed to exploit a wide range of different plant substrates, the actual diets of most species remain elusive. We studied dietary niches of two Macrotermes species across the semiarid savanna landscape in the Tsavo Ecosystem, southern Kenya, based on carbon (C) and nitrogen (N) stable isotopes in Termitomyces fungus combs. We applied Bayesian mixing models to determine the proportion of grass and woody plant matter in the combs, these being the two major food sources available for Macrotermes species in the region. Our results showed that both termite species, and colonies cultivating different Termitomyces fungi, occupied broad and largely overlapping isotopic niches, indicating no dietary specialization. Including laser scanning derived vegetation cover estimates to the dietary mixing model revealed that the proportion of woody plant matter in fungus combs increased with increasing woody plant cover in the nest surroundings. Nitrogen content of fungus combs was positively correlated with woody plant cover around the mounds and negatively correlated with the proportion of grass matter in the comb. Considering the high N demand of large Macrotermes colonies, woody plant matter seems to thus represent a more profitable food source than grass. As grass is also utilized by grazing mammals, and the availability of grass matter typically fluctuates over the year, mixed woodland‐grasslands and bushlands seem to represent more favorable habitats for large Macrotermes colonies than open grasslands.     

培菌白蚁与鸡枞菌研究进展

培菌白蚁起源于非洲,蚁巢内具有复杂的社会分工.培菌白蚁依靠独特的蚁巢结构维持内部稳态和气体循环.菌圃是白蚁培育鸡枞菌的场所.鸡枞菌隶属于担子菌亚门,但其传播方式和生活史具有区别于其它担子菌的特点.鸡枞菌协助白蚁进行植物纤维的消化,白蚁则为鸡枞菌提供合适的生长环境,并控制鸡枞菌的遗传结构.培菌白蚁和鸡枞菌形成紧密的共生关...     

First record of the termite ectoparasite Laboulbeniopsis termitarius thaxter in Japan

Laboulbeniopsis termitarius, an ectoparasitic fungus of termites, was harvested from the body surface of Reticulitermes speratus (Isoptera: Rhinotermitidae), collected in Uji, Kyoto Prefecture, Japan. This is the first record of this fungus from Japan. Three to eighteen termite workers from 500 individuals were infected among the eight colonies investigated. From our results, several hundred termite are required for the survey of this ectoparasitic fungi in Japan. The temperature effect on the relationship between the infection rate and the host activity was discussed.     

白蚁及共生微生物木质纤维素水解酶的种类

白蚁是热带生态系统重要的木质纤维素降解者。白蚁种类丰富,可分成高等白蚁和低等白蚁,食性也具有各自特点。白蚁自身可以产生纤维素酶,主要是GHF9的内切葡聚糖酶(EG),也有β-葡萄糖苷酶(GB)。低等白蚁共生的原虫中已发现丰富的纤维素酶基因,属于GHF5,7和45。同时还有其他相关功能基因,如木聚糖酶和果胶类物质水解酶。高等白蚁肠道中没有共生原虫。高等培菌白蚁可以利用共生蚁巢伞属真菌促进木质纤维素降解,真菌可以产生纤维素酶,果胶质水解酶类、木聚糖酶,同时还产生可能与木质素分解相关的一种漆酶,但是从分子水平,关于共生真菌纤维素水解酶的研究还较少。白蚁肠道已分离出许多具有木质纤维素降解能力的菌株,最近的研究也发现了大量细菌纤维素酶基因。白蚁-共生系统丰富的木质纤维素水解酶类为发展生物方法开发纤维素乙醇这一思路提供有价值的资源。     

Termite-egg mimicry by a sclerotium-forming fungus

Mimicry has evolved in a wide range of organisms and encompasses diverse tactics for defence, foraging, pollination and social parasitism. Here, I report an extraordinary case of egg mimicry by a fungus, whereby the fungus gains competitor-free habitat in termite nests. Brown fungal balls, called 'termite balls', are frequently found in egg piles of Reticulitermes termites. Phylogenetic analysis illustrated that termite-ball fungi isolated from different hosts (Reticulitermes speratus, Reticulitermes flavipes and Reticulitermes virginicus) were all very similar, with no significant molecular differences among host species or geographical locations. I found no significant effect of termite balls on egg survivorship. The termite-ball fungus rarely kills termite eggs in natural colonies. Even a termite species (Reticulitermes okinawanus) with no natural association with the fungus tended termite balls along with its eggs when it was experimentally provided with termite balls. Dummy-egg bioassays using glass beads showed that both morphological and chemical camouflage were necessary to induce tending by termites. Termites almost exclusively tended termite balls with diameters that exactly matched their egg size. Moreover, scanning electron microscopic observations revealed sophisticated mimicry of the smooth surface texture of eggs. These results provide clear evidence that this interaction is beneficial only for the fungus, i.e. termite balls parasitically mimic termite eggs.     

Carbon mineralization by termites in tropical forests,with emphasis on fungus combs

A role of termites in decomposition processes was quantitatively evaluated in a dry evergreen forest (DEF) in Thailand, using respiration rates and biomasses of fungus combs as well as of termites themselves. The termite population and fungus combs mineralized 11.2% of carbon (C) in the annual aboveground litterfall (AAL) by their respiration. Fungus combs were responsible for a major part (7.2% of the AAL) of the C mineralization mediated by termites. For comparison, fractions of AAL mineralized by respiration from termite populations and fungus combs were estimated for tropical forests and savannas where termites have been well studied, assuming that there is the same ratio as for the DEF between biomass of fungus combs and abundance of fungus growers. Termites in dry tropical forests (annual rainfall<2,000 mm) are shown to mineralize about 10% of C in the AAL by respiration from their populations and fungus combs, and their ecological impact in savannahs is comparable in this aspect. A significant negative correlation between fraction of AAL and annual rainfall demonstrates that the importance of termites in decomposition processes is greater in dry tropical forests than in moist tropical forests. Considering that fungus combs contributed significantly to AAL mineralization in most of the tropical forests and savannas, fungus growers are a much more influential group than previously expected in tropical ecosystems.T. Abe deceased on 27 March 2000     

Diversity of fungus‐growing termites (Macrotermes) and their fungal symbionts (Termitomyces) in the semiarid Tsavo Ecosystem,Kenya

Fungus‐growing termites of the subfamily Macrotermitinae together with their highly specialized fungal symbionts (Termitomyces) are primary decomposers of dead plant matter in many African savanna ecosystems. The termites provide crucial ecosystem services also by modifying soil properties, translocating nutrients, and as important drivers of plant succession. Despite their obvious ecological importance, many basic features in the biology of fungus‐growing termites and especially their fungal symbionts remain poorly known, and no studies have so far focused on possible habitat‐level differences in symbiont diversity across heterogeneous landscapes. We studied the species identities of Macrotermes termites and their Termitomyces symbionts by excavating 143 termite mounds at eight study sites in the semiarid Tsavo Ecosystem of southern Kenya. Reference specimens were identified by sequencing the COI region from termites and the ITS region from symbiotic fungi. The results demonstrate that the regional Macrotermes community in Tsavo includes two sympatric species (M. subhyalinus and M. michaelseni) which cultivate and largely share three species of Termitomyces symbionts. A single species of fungus is always found in each termite mound, but even closely adjacent colonies of the same termite species often house evolutionarily divergent fungi. The species identities of both partners vary markedly between sites, suggesting hitherto unknown differences in their ecological requirements. It is apparent that both habitat heterogeneity and disturbance history can influence the regional distribution patterns of both partners in symbiosis.     

Multipartite symbioses in fungus-growing termites (Blattodea: Termitidae,Macrotermitinae) for the degradation of lignocellulose

Fungus-growing termites are among the most successful herbivorous animals and improve crop productivity and soil fertility. A range of symbiotic organisms can be found inside their nests. However, interactions of termites with these symbionts are poorly understood. This review provides detailed information on the role of multipartite symbioses (between termitophiles, termites, fungi, and bacteria) in fungus-growing termites for lignocellulose degradation. The specific functions of each component in the symbiotic system are also discussed. Based on previous studies, we argue that the enzymatic contribution from the host, fungus, and bacteria greatly facilitates the decomposition of complex polysaccharide plant materials. The host–termitophile interaction protects the termite nest from natural enemies and maintains the stability of the microenvironment inside the colony.     

培菌白蚁菌圃和粪便微生物多样性分析

【背景】培菌白蚁是属于白蚁科的一类与鸡枞菌属真菌共生的高等白蚁,其与体内肠道微生物和体外菌圃微生物形成三维共生体系。【目的】分析培菌白蚁菌圃和粪便的微生物多样性,并与肠道微生物进行比较。【方法】通过Illumina MiSeq高通量测序方法对培菌白蚁菌圃和粪便样品进行细菌16S rRNA基因和真菌ITS测序分析。【结果】高通量测序获得培菌白蚁菌圃和粪便样品细菌和真菌的有效序列和OTU数目。5个样品细菌OTU数目在90-199之间,而真菌OTU在10-58之间,细菌的种类多样性明显大于真菌。不论是细菌还是真菌,粪便样品的OTU数目多于菌圃样品。经物种分类分析,菌圃样品主要优势细菌是变形菌门(Proteobacteria),其相对含量超过82.4%;其次是拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes);粪便样品中优势细菌为拟杆菌门,其次是变形菌门,粪便优势菌属为别样杆菌属和营发酵单胞菌属,这与培菌白蚁肠道菌多样性组成一致。培菌白蚁菌圃和粪便样品共生真菌主要为担子菌门(Basidiomycota)和子囊菌门(Ascomycota)。菌圃优势真菌为鸡枞菌属(Termi...     

Exploring the Potential for Actinobacteria as Defensive Symbionts in Fungus-Growing Termites

In fungus-growing termites, fungi of the subgenus Pseudoxylaria threaten colony health through substrate competition with the termite fungus (Termitomyces). The potential mechanisms with which termites suppress Pseudoxylaria have remained unknown. Here we explore if Actinobacteria potentially play a role as defensive symbionts against Pseudoxylaria in fungus-growing termites. We sampled for Actinobacteria from 30 fungus-growing termite colonies, spanning the three main termite genera and two geographically distant sites. Our isolations yielded 360 Actinobacteria, from which we selected subsets for morphological (288 isolates, grouped in 44 morphotypes) and for 16S rRNA (35 isolates, spanning the majority of morphotypes) characterisation. Actinobacteria were found throughout all sampled nests and colony parts and, phylogenetically, they are interspersed with Actinobacteria from origins other than fungus-growing termites, indicating lack of specificity. Antibiotic-activity screening of 288 isolates against the fungal cultivar and competitor revealed that most of the Actinobacteria-produced molecules with antifungal activity. A more detailed bioassay on 53 isolates, to test the specificity of antibiotics, showed that many Actinobacteria inhibit both Pseudoxylaria and Termitomyces, and that the cultivar fungus generally is more susceptible to inhibition than the competitor. This suggests that either defensive symbionts are not present in the system or that they, if present, represent a subset of the community isolated. If so, the antibiotics must be used in a targeted fashion, being applied to specific areas by the termites. We describe the first discovery of an assembly of antibiotic-producing Actinobacteria occurring in fungus-growing termite nests. However, due to the diversity found, and the lack of both phylogenetic and bioactivity specificity, further work is necessary for a better understanding of the putative role of antibiotic-producing bacteria in the fungus-growing termite mutualistic system.     

高效降解木质纤维素的白蚁肠道微生物组

木食性白蚁是自然界木质纤维素的高效降解者,在长期进化过程中白蚁与其肠道微生物组协同作用发展出不同的纤维素降解机制。木食性白蚁具有分别来源于白蚁和共生微生物的两套纤维素酶系统。在低等白蚁中,木质颗粒经过白蚁前、中肠分泌的内源性酶初步消化后,在后肠共生鞭毛虫中被降解为乙酸、二氧化碳和氢。高等木食性白蚁在进化中丢失了鞭毛虫,木质颗粒经白蚁自身分泌的酶初步消化后,在后肠大量共生细菌的帮助下被有效降解。培菌类白蚁利用其菌圃中的蚁巢伞菌和肠道微生物协同作用降解木质纤维素。共生微生物在白蚁的氮素固定与循环、中间产物代谢及纤维素降解等过程中发挥了重要作用。学习和模拟白蚁高效降解木质纤维素的体系,对生物质能源的产业化发展具有积极的意义。     

Fungus-growing termites originated in African rain forest

Fungus-growing termites (subfamily Macrotermitinae, Isoptera) cultivate fungal crops (genus Termitomyces, Basidiomycotina) in gardens inside their colonies. Those fungus gardens are continuously provided with plant substrates, whereas older parts that have been well decomposed by the fungus are consumed (cf.). Fungus-growing termites are found throughout the Old World tropics, in rain forests and savannas, but are ecologically dominant in savannas. Here, we reconstruct the ancestral habitat and geographical origin of fungus-growing termites. We used a statistical model of habitat switching repeated over all phylogenetic trees sampled in a Bayesian analysis of molecular data. Our reconstructions provide strong evidence that termite agriculture originated in African rain forest and that the main radiation leading to the extant genera occurred there. Because extant savanna species are found in most genera, this moreover suggests that the savanna has repeatedly been colonized by fungus-growing termites. Furthermore, at least four independent "out-of-Africa" migrations into Asia, and at least one independent migration to Madagascar, have occurred. Although fungus growing by termites is ecologically most successful under the variable, unfavorable conditions of the savanna, it seems to have evolved under the more constant and favorable conditions of the rain forest.