扩头蔡白蚁肠道蛋白的鉴定

【目的】本研究旨在分析比较扩头蔡白蚁Tsaitermes ampliceps工蚁前中肠和后肠及其内容物的蛋白构成和表达差异,挖掘降解木质纤维素的相关酶和蛋白。【方法】通过扩头蔡白蚁工蚁的前中肠和后肠及其内容物蛋白的双向电泳,对高表达或高差异表达的47个蛋白点进行MALDI-TOF/MS测序,并进行生物信息学分析。【结果】测序分析发现,扩头蔡白蚁肠道及其内容物蛋白中有结构蛋白13个、调节蛋白9个、白蚁代谢相关蛋白10个、微生物代谢相关蛋白7个。经PD Quest分析发现,在前中肠和后肠有11个蛋白均高表达;仅在前中肠表达的蛋白有12个,主要是白蚁代谢相关蛋白和调节蛋白;仅在后肠表达的蛋白有8个,主要是微生物代谢相关蛋白。整个肠道内参与木质纤维素降解的相关酶有5个,分别是白蚁自身分泌的内源性纤维素酶,细菌产生的内切-β-1,4-葡聚糖酶和过氧化物歧化酶以及原生动物产生的GH11。【结论】白蚁对木质纤维素食物的降解主要在前中肠,后肠对降解产物进一步降解并进行微生物生长代谢。这些降解产物和微生物菌体蛋白为白蚁的肛哺提供营养成分。     

不同饲料饲养的近暗散白蚁工蚁肠道中差异蛋白分析

【目的】本研究旨在通过比较不同木质纤维素含量的饲料对近暗散白蚁Reticulitermesperilucifugus工蚁前中肠和后肠内容物蛋白的组成和表达差异,为揭示白蚁的营养消化吸收机理提供蛋白水平上的依据。【方法】用木质纤维素含量不同的3种饲料(松木、秸秆和滤纸)饲养近暗散白蚁工蚁,然后用双向电泳分析肠道不同部位的内容物蛋白,并对差异蛋白进行MALDI-TOF/MS测序及生物信息学分析。【结果】双向电泳结果发现,同一饲料饲养的近暗散白蚁工蚁前中肠可辨蛋白点数明显多于后肠;不同饲料饲喂的工蚁前中肠和后肠可辨蛋白点数依次是松木饲养的＞滤纸饲养的＞秸秆饲养的。通过对115个蛋白点的测序分析表明,不同饲料饲喂的工蚁前中肠和后肠中的主要差异蛋白为具有催化活性的蛋白质,包括与氨基酸代谢、丙酮酸代谢、碳代谢、氮代谢、TCA循环、糖酵解、糖异生和纤维素降解等相关的酶,以及参与细胞组成和信号传导的蛋白。【结论】木质纤维素含量不同的饲料饲养的近暗散白蚁工蚁肠道中的差异蛋白主要为具有催化活性的蛋白、细胞组成蛋白和信号传导蛋白,表明不同的饲料影响近暗散白蚁的肠道蛋白组成。这些结果为揭示白蚁对木质纤维素的降解机理提供了参考。     

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。     

黄翅大白蚁后肠优势菌大白蚁营发酵菌的全基因组序列分析

【目的】营发酵单胞菌属Dysgonomonas是黄翅大白蚁后肠的第二优势微生物。前期研究中,我们从黄翅大白蚁后肠分离出一种命名为大白蚁营发酵菌的新菌。为深入了解大白蚁营发酵菌在宿主白蚁体内发挥的作用和功能,有必要解析大白蚁营发酵菌的基因组序列信息。【方法】使用Illumina Miseq测序平台获取该菌的全基因组序列,将其全基因组序列经过注释的基因蛋白质序列提交COG和KEGG数据库进行BLASTp比对分析,确定该菌潜在的重要酶类和代谢途径,并对个别纤维素酶活进行检测。【结果】大白蚁营发酵菌整个基因组大小为4655756 bp,GC含量为38.54%,DDBJ数据库登录号为BBXL01000001–BBXL01000078。生物信息学分析结果表明菌株大白蚁营发酵菌具有多个木质纤维素降解酶基因,且具备完整的木质纤维素降解和乙酸、乳酸生成通路。此外发现该菌株中存在与氮源代谢和抵御病原体相关的基因。【结论】本研究首次解析大白蚁营发酵菌的全基因组序列,了解其基因组基本特征,初步探讨了该菌降解木质纤维素的过程,为细菌协助宿主白蚁降解木质纤维素提供了理论基础,同时为该菌可能参与宿主白蚁氮源代谢和抵御病原体入侵提供了依据。     

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。     

三种白蚁的头胸部及消化道结构特征比较

【目的】白蚁具有降解木质纤维素的能力,主要场所在消化道内,因此消化道形态结构的研究具有重要意义。【方法】本文对采自河南省的3种白蚁(平额散白蚁Reticulitermes planifrons Li et Ping、湖南散白蚁Reticulitermes hunanensis Tsai et Peng和双工土白蚁Odontotermes dimorphus Li et Xiao的兵蚁和工蚁头胸部特征及其消化道结构进行了比较。【结果】两种散白蚁兵蚁的头壳、后颏和上颚没有明显差异,但与双工土白蚁差异显著。3种白蚁工蚁的前肠、后肠及消化道平均长度均大于兵蚁,且差异显著(P<0.05),而中肠的差异不显著;双工土白蚁的肠道总长度工蚁/兵蚁比值(1.4倍)大于两种散白蚁(1.1倍);除了嗉囊和囊形胃外,两种散白蚁工蚁的其余消化道各段差异显著(P<0.05),与双工土白蚁相比差异也显著。【结论】工蚁的消化道比兵蚁发达,散白蚁的消化道结构与土白蚁有较大不同,这些结果与工蚁和兵蚁的社会分工有关,同时也表明高等白蚁与低等白蚁相比不仅是采食习惯的差别,自身消化系统结构与分区也有较大不同,并为白蚁的生物防治和生物质资源利用提供了资料。     

鸡枞菌转录组分析揭示其对木质纤维素的降解功能

【目的】探究鸡枞菌是否能降解木质纤维素成分,并理解其与共生白蚁之间的共生关系。【方法】本研究是应用新一代高通量测序技术454 GS FLX Titanium对鸡枞菌的转录组进行测序,挖掘鸡枞菌中能参与降解纤维素和木质素等成分的多样性酶系。【结果】八分之一的RUN测序总共得到了82386条表达序列标签,去除引物和载体等序列后,剩余的54410条序列被拼接成3301条contigs以及3193条singletons。根据序列相似性,将这些unigenes与三大蛋白数据库(Nr数据库、SwissProt数据库、CDD数据库)中的蛋白序列进行BLAST比较,发现有2681条基因与其他生物的已知基因有不同程度的相似性。在鸡枞菌的这些转录产物中,有33条编码可能参与降解纤维素或半纤维素的酶基因,其中包括5种纤维素酶以及28种水解半纤维素、淀粉或几丁质等物质的酶类。更重要的是,还发现了4种漆酶以及一种芳基乙醇氧化酶基因,这些都是能有效降解木质素的酶类。这些结果揭示了鸡枞菌中存在漆酶并可能有效降解植物残渣中的酚化合物。【结论】这些基因的发现说明了鸡枞菌能降解木质素,并能与共生白蚁分泌的纤维素酶协同作用有效降解纤维素。     

Cflac1基因的RNAi对白蚁肠道氧化还原电位响应病原菌感染的影响

台湾乳白蚁Coptotermes formosanus又称为地下白蚁,它以木质纤维素为主食,是广泛分布于全球的经济害虫。地下白蚁对病原微生物强大的免疫防御能力一直是其生物防治应用的瓶颈之一。植食性昆虫的解毒酶在其消化和免疫调节中起着至关重要的作用。针对具有木质素代谢活性的解毒酶在白蚁肠道免疫过程中氧化还原稳态的调控研究对揭示其肠道免疫机制有重要意义。本研究以金龟子绿僵菌Metarhizium anisopliae为免疫诱导物,基于肠道氧化还原态的微电极检测系统,首次对台湾乳白蚁感染真菌后前肠/唾液腺、中肠及后肠中氧化还原电位的变化进行了描述;并利用RNA干扰技术,抑制了具有木质素代谢活性的乳白蚁漆酶基因Cflac1在感染前后的表达,并确定了Cflac1在乳白蚁肠道免疫防御真菌过程中对氧化还原稳态维系的重要作用。本研究丰富了白蚁肠道免疫机制的研究内容,为拓宽白蚁防治新靶点及生物防治技术的研发提供了理论支撑。     

白蚁消化系统转化和降解木质纤维素酶研究进展

木质纤维素是地球上最丰富的有机聚合物,白蚁是古老但进化最成功的高效木质纤维素降解者之一。了解白蚁降解高度抗性植物聚合物的机制对工业上生物质能源转化和生物仿生设计有重要的借鉴和指导价值。白蚁和其共生微生物产生的木质纤维素酶在其转化利用木质纤维素上发挥着重要作用。本文从来源作用方面对白蚁自身及其肠道原虫、细菌和真菌产生的纤维素酶、木聚糖酶和漆酶等酶研究概况进行了总结,对其存在的问题和前景进行了展望。本综述有助于全面了解白蚁消化系统木质纤维素酶的基因种类、来源、分布、表达以及酶活性和功能。     

白蚁-共生微生物系统降解木质纤维素研究进展

开发利用木质纤维素材料能显著增加地球上可再生资源的储备量。白蚁分布广泛,常见于热带和亚热带地区,它们借助细菌、古细菌、真菌等肠道微生物和原生动物协同降解食物中的木质纤维素,在生态系统的碳、氮循环中发挥着十分重要的作用。本文概括了近年来白蚁肠道微生物研究的进展,特别是近年来已被证明的肠道微生物在木质纤维素降解方面的作用,以期为后续研究木质纤维素的降解提供参考信息。     

长足大竹象消化道不同分段菌群异质性及竹木质纤维素降解能力

【目的】长足大竹象Cyrtotrachelus buqueti消化道共生菌群参与了竹纤维素的降解。本研究旨在揭示长足大竹象幼虫消化道不同分段共生菌群异质性及木质纤维素的降解能力。【方法】通过对16S rRNA测序对长足大竹象幼虫消化道分段口器(YB)、前肠(YFG)、中肠(YMG)和后肠(YHG)进行菌群组成分析及功能预测;分析各消化道分段核心属细菌基因组中碳水化合物活性酶(carbohydrate active enzyme, CAZy)基因,预测木质纤维素降解能力;应用口器混合菌(MPJ)、前肠混合菌(FJ)、中肠混合菌(MJ)和后肠混合菌(HJ)悬液体外降解竹笋粉,检测共生菌群的木质纤维素的降解能力。【结果】长足大竹象幼虫消化道菌群多样性分析显示,YFG, YMG和YHG的菌群多样性大于YB,YFG的菌群物种多样性最高,而YB最低。YFG, YMG和YHG样本中厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)和变形菌门(Proteobacteria)相对丰度最高,而YB中变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和放线菌门(Actinobacteria)相对丰度最高。核心属细菌基因组CAZy基因分析表明长足大竹象幼虫消化道中大多数细菌基因组中存在丰富的CAZy基因,且在拟杆菌属Bacteroides细菌基因组中尤为明显,预示其与木质纤维素的降解密切相关。体外降解竹笋粉实验结果表明,长足大竹象幼虫MPJ, FJ, MJ和HJ对竹笋粉纤维素的降解率分别为21.7%, 39.9%, 44.2%和21.0%,对半纤维素降解率分别为72.7%, 52.3%, 65.7%和61.5%,对木质素降解率分别为20.5%, 41.3%, 39.9%和37.9%。【结论】长足大竹象幼虫的消化道菌群的异质性可以影响木质纤维素降解能力,因而这些菌群可以作为分离高效木质纤维素降解菌的重要来源之一。本研究为竹生物质的工业转化利用提供部分参考信息。     

Dual origin of gut proteases in Formosan subterranean termites (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae)

Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts.     

Aerobic state of gut of Nasutitermes exitiosus and Coptotermes lacteus, high and low caste termites

The redox potential and pH of the gut of the termites Nasutitermes exitiosus and Coptotermes lacteus was investigated by feeding the insects with redox dyes and pH indicators. For N. exitiosus the E′₀ (pH 7.0) of the foregut was above +200 mV; the midgut was about +100 to +150 mV and the hindgut was in the region of ?20 to +30 mV. For C. lacteus the fore- and midgut were about +30 to +50 mV and the hindgut about ?20 to +20 mV. The colours of the ingested dyes indicated that the gut was aerobic in both termites. The pH of the whole gut ranged from 6.5 to 7.5 for N. exitiosus and 6.0 to 7.0 for C. lacteus.     

16S rRNA metagenomic analysis of the symbiotic community structures of bacteria in foregut,midgut, and hindgut of the wood-feeding termite <Emphasis Type="Italic">Bulbitermes</Emphasis> sp.

The termite gut is a highly structured microhabitat with physicochemically distinct regions. It is generally separated into the foregut, midgut and hindgut. The distribution of gut microbiota is greatly influenced by varying physicochemical conditions within the gut. Thus, each gut compartment has a unique microbial population structure. In this study, the bacterial communities of foregut, midgut and hindgut of wood-feeding higher termite, Bulbitermes sp. were analyzed in detail via metagenomic sequencing of the 16S rRNA V3-V4 region. While the microbiomes of the foregut and midgut shared a similar taxonomic pattern, the hindgut possessed more diverse bacterial phylotypes. The communities in the foregut and midgut were dominated by members of the group Bacilli and Clostridia (Firmicutes) as well as taxon Actinomycetales (Actinobacteria). The main bacterial lineage found in hindgut was Spirochaetaceae (Spirochaetes). The significant difference among the three guts was the relative abundance of the potential lignin-degrading bacteria, Actinomycetales, in both the foregut and midgut. This suggests that lignin modification was probably held in the anterior part of termite gut. Predictive functional profiles of the metagenomes using 16S rRNA marker gene showed that cell motility, energy metabolism and metabolism of cofactors and vitamins were found predominantly in hindgut microbiota, whereas xenobiotics degradation and metabolism mostly occurred in the foregut segment. This was compatible with our 16S rRNA metagenomic results showing that the lignocellulose degradation process was initiated by lignin disruption, increasing the accessibility of celluloses and hemicelluloses.     

Marked variations in patterns of cellulase activity against crystalline- vs. carboxymethyl-cellulose in the digestive systems of diverse, wood-feeding termites

Abstract.  Throughout the history of studies on cellulose digestion in termites, carboxymethyl-cellulose has been preferably used as a substrate for measuring cellulase activity in termites due to its high solubility. However, carboxymethyl-cellulose degradation is not directly related to digestibility of naturally occurring cellulose because many noncellulolytic organisms can also hydrolyse carboxymethyl-cellulose. To address this issue, a comparative study of microcrystalline cellulose digestion is performed in diverse xylophagous termites, using gut homogenates. For those termites harbouring gut flagellates , the majority of crystalline cellulose appears to be digested in the hindgut, both in the supernatant and the pellet. For Nasutitermes takasagoensis , a termite free of gut flagellates, crystalline cellulose is degraded primarily in the midgut supernatant, and partially in the pellet of the hindgut. The fungus-growing termite Odontotermes formosanus , which also does not possess intestinal flagellates, shows only a trace of crystalline cellulose hydrolysis throughout the gut. Comparison of levels of activity against crystalline cellulose with previously reported levels of activity against carboxymethyl-cellulose in the gut of each termite reveals significant differences between these activities. The results suggest that the hindgut flagellates produce commonly cellobiohydrolases in addition to endo-β-1,4-glucanases, which presumably act synergistically to digest cellulose. Preliminary evidence for the involvement of bacteria in the cellulose digestion of N. takasagoensis is also found.     

Termite-Microbe Symbiotic System and Its Efficient Degradation of Lignocellulose

Termites thrive in the tropics and play an important role in lignocellulose degradation. This ability depends mainly on intestine microbes in the gut, but most of them are so-called unculturable microbes, which can not be cultivated by traditional culture methods. The recent development of molecular approaches such as the PCR method has made it possible to access the enormous numbers of unculturable microbes in the gut of termites.

This review explains our research on the ecological role of the termite, the termite-microbe symbiotic system, and the functions of lignocellulose degradation using various molecular methods. In the future, new technologies such as genomics should make it possible to analyze and utilize unculturable microbial resources in natural environments.     

Physicochemical conditions and microbial activities in the highly alkaline gut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

The soil macrofauna plays an important role in the carbon and nitrogen cycle of terrestrial ecosystems. In order to gain more insight into the role of the intestinal microbiota in transformation and mineralization of organic matter during gut passage, we characterized the physicochemical conditions, microbial activities, and community structure in the gut of our model organism, the humus-feeding larva of the cetoniid beetle Pachnoda ephippiata. Microsensor measurements revealed an extreme alkalinity in the midgut, with highest values (pH > 10) between the second and third crown of midgut ceca. Both midgut and hindgut were largely anoxic, but despite the high pH, the redox potential of the midgut content was surprisingly high even in the largest instar. However, reducing conditions prevailed in the hindgut paunch of all instars (E(h) approximately -100 mV). Both gut compartments possessed a pronounced gut microbiota, with highest numbers in the hindgut, and microbial fermentation products were present in high concentrations. The stimulation of hindgut methanogenesis by exogenous electron donors, such as H(2), formate, and methanol, together with considerable concentrations of formate in midgut and hemolymph, suggests that midgut fermentations are coupled to methanogenesis in the hindgut by an intercompartmental transfer of reducing equivalents via the hemolymph. The results of a cultivation-based enumeration of the major metabolic groups in midgut and hindgut, which yielded high titers of lactogenic, propionigenic, and acetogenic bacteria, are in good agreement not only with the accumulation of microbial fermentation products in the respective compartments but also with the results of a cultivation-independent characterization of the bacterial communities reported in the companion paper (M. Egert, B. Wagner, T. Lemke, A. Brune, and M. W. Friedrich, Appl. Environ. Microbiol. 69:6659-6668, 2003).