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Communities of microbes that inhabit the changing hindgut landscape of a subsocial beetle

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Abstract

Microbes that have adopted endosymbiotic life styles not only have evolved to live in specialized habitats within living organisms, but the living habitats also have evolved to accommodate them. The hindgut of the passalid beetle (Odontotaenius disjunctus) is lined with a cuticle that undergoes dramatic topographic changes during the life cycle of the beetle. This manuscript addresses the changes that have been observed in time and space for the cuticular landscape of the hindgut as well as for the microbial communities within the hindgut. Microbial identity is based on morphology, culture, and extrapolation from previously reported passalid gut inhabitants.

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... Desde el siglo pasado, se ha venido estudiando la simbiosis existente entre microorganismos endosimbióticos y animales (Suh et al., 2003; Suh et al., 2005). Sin embargo, el conocimiento acerca de este tipo de interacciones es aun muy poca (Nardi et al., 2006 ). Estudios realizados con larvas de coleópteros han demostrado que hay un alto número de especies de bacterias y levaduras presentes en su tracto digestivo, representando un potencial muy importante para la biotecnología (Boekhout, 2005). ...
... De manera análoga, los organismos detritívoros , aquellos que se alimentan de materia orgánica en descomposición (García et al., 2004), requieren de microcomunidades en el tracto digestivo, compuestas generalmente de bacterias y hongos (levaduras y otros ascomycetes), que permiten la absorción de ciertos nutrientes. El degradar los compuestos que sus hospedadores son incapaces de digerir, indica que los microorganismos presentes en el tracto digestivo de dichos animales también juegan un papel importante en la digestión de la materia orgánica (Boekhout, 2005; Nardi et al., 2006). En los agroecosistemas, la biodiversidad puede ser clasificada según el papel que juega cada organismo en la actividad agrícola. ...
... Las relaciones entre microbios e invertebrados detritívoros se deben considerar un factor importante en el sostenimiento del suelo. Además debido a las posibles aplicaciones de los microorganismos simbióticos en la agricultura (ya sea en el mantenimiento del suelo , o en el control microbiológico de plagas y enfermedades), esta microbiota requiere ser reconocida (Boekhout, 2005; Nardi et al., 2006 ). El estudio del tipo de microorganismos presentes en el tracto digestivo de insectos detritívoros, se convierte en el primer paso para la realización de estudios de exploración de la edafofauna y de inventarios preliminares de microorganismos. ...
Article
Available information about the symbiotic relationships between endosymbiotic microorganisms and insects is scarce. In this context, detritivorous organisms are of great importance in soil formation, decomposition of organic matter and nutrient cycling. Due to these facts, and its potential use in agrobiotechnology, studying the relationships of symbiotic microorganisms and detritivore insects is a matter of interest. Based on literature we standardized a protocol for dissecting the digestive tract of detritivorus beetles and moth larvae. Eight bacteria morphotypes were isolated from the beetle larva (Family: Scarabaeidae) and four morphotypes of bacteria and three yeast morphotypes, were isolated from a lepidopteran larva (family: Noctuidae). To explore their possible use as antagonist organisms, in vitro tests were performed co-cultivating those with a Fusarium oxysporum isolate. The largest inhibition percent was obtained with a bacterium extracted from the beetle. Unexpectedly, two bacteria promoted F. oxysporum growth, one taken from the beetle and other from the lepidopteran. These results state bases for future research on symbiotic relationships between insect detritivore larvae and microorganisms, and its possible future applications to agriculture. La información disponible acerca de las relaciones simbióticas entre microorganismos endosimbióticos e insectos es escasa, dentro de lo cual los organismos detritívoros son de gran importancia en la formación del suelo, ciclado de nutrientes y descomposición de materia orgánica. Por ello estudiar las relaciones de insectos detritívoros y microorganismos simbióticos, es interesante por su potencial uso en el campo agrícola y su aplicación biotecnológica. Para ello, a partir de estudios de varios autores se estandarizó un protocolo de disección del tracto digestivo de larvas de coleópteros y lepidópteros detritívoros. Así se aislaron ocho morfotipos de bacterias del tracto digestivo de una morfoespecie de coleóptero (familia: Scarabaeidae) y cuatro morfotipos de bacterias y tres de levaduras del tracto digestivo de un lepidóptero (familia: Noctuidae). Por su potencial aplicación, se realizaron pruebas de antagonismo in vitro de estos aislados sobre Fusarium oxysporum. El mayor porcentaje de inhibición se obtuvo con una bacteria extraída del coleóptero. Sorpresivamente se obtuvieron dos bacterias que promovían el crecimiento de F. oxysporum, una extraída del coleóptero, y otra del lepidóptero. Este trabajo deja bases para realizar futuras investigaciones acerca de las relaciones simbióticas entre larvas de insectos detritívoros y sus microorganismos, y las posibles futuras aplicaciones en la agricultura.
... Beetles acquire their microbiota mainly from the materials on which they feed; many of these microorganisms could have adapted to the gut environment and over time, developed mutually beneficial relationships with their host [10,11]. This beneficial microbiota can be vertically transmitted from parents to offspring, a phenomenon that has been documented in insects such as aphids and whiteflies, but not fully characterized in beetles [12,13,14]. ...
... This beneficial microbiota can be vertically transmitted from parents to offspring, a phenomenon that has been documented in insects such as aphids and whiteflies, but not fully characterized in beetles [12,13,14]. Vertical transmission of beneficial microbes may be particularly important to the Passalidae family, given that these beetles exhibit a subsocial behavior in which adults provide care for larvae sharing the same decomposing log, and even two or more generations can overlap in the same niche [11,15]. ...
... Fungi are also well known members of insect gut microbial communities, including beetles [11,53]. In this study, we isolated filamentous fungi from 23 different families; however this observed diversity may be representing the intrinsic flora of the beetle gut, or transitory associants passing through the intestinal tract. ...
Article
Full-text available
Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus Streptomyces (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, β and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.
... However, the nature of gut microbiota-host associations can be highly variable across insects, suggesting differences in the type and extent of benefits provided to the host (Engel and Moran, 2013). For example, termites (Brune and Dietrich, 2015), social bees , cockroaches (Tinker and Ottesen, 2016) or certain ant (Hu et al., 2018;Sanders et al., 2017) and beetle species (Shukla et al., 2018a;Ceja-Navarro et al., 2019;Nardi et al., 2006) harbor specialized and dense gut microbial communities, representing longstanding microbiotahost associations. Conversely, fruit flies or mosquitoes are mostly colonized by transient and relatively sparse communities that are acquired from the environment (Wong et al., 2013;Bost et al., 2018;Coon et al., 2016b;Pais et al., 2018). ...
... In wood-feeding passalid beetles (Odontotaenius disjunctus), distinctive microbial communities across gut compartments enable a spatially separated, step-wise lignocellulose deconstruction: lignocellulose depolymerization starts in the midgut and continues in the posterior hindgut, facilitated by the availability of oxygen and the presence of diverse microbe-encoded enzymes. In contrast, fermentation of sugars occurs in the anterior hindgut under anoxic conditions (Ceja-Navarro et al., 2019;Nardi et al., 2006;Ceja-Navarro et al., 2014). In both termites and wood-feeding beetles, symbionts and digested nutrients are transferred to adult and larval nestmates via anal-oral trophallaxis. ...
... The case of the termites also illustrates that the digestion of plantderived polysaccharides occurs step-wise, and is not only divided between different symbiotic partners but also across gut compartments (Tokuda et al., 2018;Tokuda et al., 2012). This is confirmed by studies on wood-feeding passalid beetles, honey bees and also turtle ants (see Fig. 2) (Ceja-Navarro et al., 2019;Nardi et al., 2006;Ceja-Navarro et al., 2014;Zheng et al., 2019;Lanan et al., 2016;Hu et al., 2018). ...
Article
Insects are the most diverse group of animals and colonize almost all environments on our planet. This diversity is reflected in the structure and function of the microbial communities inhabiting the insect digestive system. As in mammals, the gut microbiota of insects can have important symbiotic functions, complementing host nutrition, facilitating dietary breakdown or providing protection against pathogens. There is an increasing number of insect models that are experimentally tractable, facilitating mechanistic studies of gut microbiota–host interactions. In this Review, we will summarize recent findings that have advanced our understanding of the molecular mechanisms underlying the symbiosis between insects and their gut microbiota. We will open the article with a general introduction to the insect gut microbiota and then turn towards the discussion of particular mechanisms and molecular processes governing the colonization of the insect gut environment as well as the diverse beneficial roles mediated by the gut microbiota. The Review highlights that, although the gut microbiota of insects is an active field of research with implications for fundamental and applied science, we are still in an early stage of understanding molecular mechanisms. However, the expanding capability to culture microbiomes and to manipulate microbe–host interactions in insects promises new molecular insights from diverse symbioses.
... While insect detritivores, such as termites, beetles, crickets, and cockroaches, have guts that are longer than their body lengths and are often folded and convoluted to fit within their body cavities (Brune, 2014(Brune, , 1998Nardi et al., 2006;Ulrich et al., 1981), the alimentary canals of millipedes are straight along their entire lengths and do not exceed the lengths of their bodies. Like all arthropods guts, millipede guts are delineated into three distinct regions e foregut, midgut, hindgut e with the midgut exceeding both the hindgut and foregut in length. ...
... The surface topography can be smooth or sculpted in complex landscape features such as spines, plates, pores, and folds. The hindgut surface of many arthropods is vastly expanded with bulbs formed by invaginations of hindgut epithelium into the lumen and numerous cuticular bristles covering the surfaces of these bulbs (Nardi et al., 2006;Czolij et al., 1985;Ulrich at al., 1981;Bignell, 1980). While microbes adhere to cuticle of millipede hindguts, they pass through the midgut lumina, rarely establishing stable contact with the microvilli that line these lumina (Figs. ...
... Regional differentiation of landscapes along the lengths of hindguts has been described for the passalid beetle, Odontotaenius disjunctus (Ceja-Navarro et al., 2014;Nardi et al., 2006) and higher termites (Czolij et al., 1985;Bignell et al., 1980). Enumeration of the molecular characteristics of microbes inhabiting the complex landscapes of different species of arthropod guts, however, has not often been related to the ultrastructure of the habitats occupied by these microbes. ...
Article
The gut lumen of the arthropod detritivore provides hospitable and multifaceted environments for diverse assemblages of microbes. Many microbes, including trichomycetes fungi, bacteria, and archaea establish stable, adherent communities on the cuticular surface secreted by the hindgut epithelium. Regional differences in the surface topography within the hindgut of a given millipede are reflected in differing and diverse microbial assemblages. The spirostreptid millipede Cambala speobia is a detritivore found on the floors of Texas caves. This millipede species has a very circumscribed distribution in North America and a diet confined to the limited litter that accumulates on floors of these caves while the common julid millipede Cylindroiulus caeruleocinctus, an introduced European species, feeds on the diverse litter found in organic soils throughout North America. In both millipedes, the gut lumens are inhabited along their entire lengths by microbes, with the highest microbial densities in the hindguts. The anterior third of the hindgut with its distinctive six-fold symmetry is lined by cuticle having fine polarized scales, and the posterior-most third is lined by smooth cuticle. Trichomycetes only inhabit the anterior third of the hindgut, and scattered patches of filamentous bacteria along with their smaller adherent microbes occupy the posterior third. The densest populations of microbes inhabit the central region of the hindgut. Over the cuticular surface of this hindgut region, uniformly distributed indentations mark possible channels for nutrient and water exchange between the hindgut lumen and host hemolymph. Films of microbes are adherent to the cuticle that lines the hindgut while those microbes in the remainder of the gut (i.e., foregut + midgut) represent mostly unattached inhabitants.
... Adults also envelop the larvae at the time of metamorphosis to pupae with a covering of frass and pre-digested wood, the pupal chamber, that becomes the first meal for juveniles as they emerge (Reyes-Castillo, 1970;Tallamy and Wood, 1986). Such behavior suggests that horizontal transfer of microbes is required for the complete metamorphosis from larvae to adults, and apparently larvae cannot survive when fed only sterilized pulverized rotten wood (Pearse et al., 1936;Reyes-Castillo, 1970;Nardi et al., 2006;Berkov et al., 2007;Rao et al., 2007). These findings emphasize the important role of the passalid gut microbiota in the digestion of the substrate in woodfeeding insects. ...
... The biology, ecology and physiology of passalids are best studied for Odontotaenius disjunctus, a common species in the southeastern United States (Pearse et al., 1936;Roberts, 1952;Bryan, 1954;Hiznay and Krause, 1955;Ferguson and Land, 1961;Robertson, 1962;Collings, 1966;Burnett et al., 1969;Ward, 1971;Delfinado and Baker, 1975;Dismukes and Mason, 1975;Schuster, 1975;Gibson, 1977;Rains and Dimock, 1978;Buchler et al., 1981;Mason et al., 1983;Tafuri and Tafuri, 1983;Wit et al., 1984;Sawvel et al., 1992;MacGown and MacGown, 1996;King and Fashing, 2007;Punzo, 2007;Jackson et al., 2009;Wicknick and Miskelly, 2009). Nardi et al. (2006) characterized the morphological and cellular transformations of the hindgut region of the digestive system from larva to adult of O. disjunctus, and described the physical distribution and arrangement of the microbiota in the gut compartments. ...
... The digestive system of an adult O. disjunctus is often over 10 cm long and a complex organ, at least twice as long as the length of an individual. The three regions of the gut (foregut, midgut and hindgut) are conspicuously differentiated and, in addition, the hindgut has readily distinguishable anterior and posterior compartments (Nardi et al., 2006). The gut regions also differ in their physiological conditions of O 2 , CO 2 and pH (Ceja-Navarro et al., 2013). ...
Article
Full-text available
The gut of insects is a productive environment for discovering undescribed species of yeasts, and the gut of wood-feeding insects of several families is especially rich in yeasts that carry out the fermentation of cellobiose and xylose. Passalid beetles (Passalidae, Coleoptera) live in dead wood that they ingest as their primary food source. We report the isolation, molecular identification and physiological characterization of 771 yeast cultures isolated from the gut of 16 species of passalids collected in nine localities in Guatemala. Ascomycete yeasts were present in the gut of every passalid studied, and the xylose-fermenting (X-F) yeasts Scheffersomyces shehatae and Scheffersomyces stipitis were the most abundant taxa isolated. The gut of the beetles also contained undescribed cellobiose-fermenting and X-F species in the Lodderomyces, Scheffersomyces and Spathaspora, and undescribed species in Sugiyamaella clades as well as rare yeast species in the Phaffomyces and Spencermartinsiella clades. Basidiomycete yeasts in the genera Cryptococcus and Trichosporon were also common. The yeast species richness was influenced by the host species and the substrate, and gut-inhabiting yeasts have the ability to survive the differing physiological conditions of several gut compartments.
... reviewed inBrune and Ohkuma, (2011)). The gut microbiome of wood-feeding beetles likely overcomes similar N constraints, and certain beetles have received recent attention regarding their digestive tract morphology and culturable fungal communities (Slaytor, 1992;Suh et al., 2003;Nardi et al., 2006;Klass et al., 2008). One of the most ubiquitous members of the beetle wood-feeding guild in the eastern United States is Odontotaenius disjunctus (Passalidae), a large beetle that can reach B30 mm in length and is common from Florida northward to southern Canada and westward to the Rocky Mountains. ...
... Pichia stipitis), that has the ability to ferment xylose, a significant component of hemicellulose (Shi et al., 2010). In a detailed study of O. disjunctus gut morphology and gut inhabitants,Nardi et al. (2006)divided each gut into four morphologically distinct regions where the gut biota appears to be differentiated as noted previously for fungus-like organisms known as trichomycetes (Lichtwardt et al., 1999). Here we describe the bacterial, archaeal, including N-fixing populations across the four gut regions of O. disjunctus, and characterize the physicochemical properties (pH, O 2 ) and nitrogenase gene expression activity of each. ...
... The continuous movement of the electrode tip through the beetle gut until it reached the opposite side showed profiles of O 2 that mirrored those obtained when entering the gut. By measuring the gut wall thickness of the AHG and PHG from the images reported byNardi et al. (2006)and according to the O 2 profiles determined in this paper, all gut regions are characterized by a rapid transition from microaerophilic to anaerobic conditions in the lumen. The extent of oxygen availability varied by gut region, being 4300 mm into the lumen in the FG and MG when considering gut wall thickness o100 mm compared with approximately 50 and 280 mm for the AHG and PHG, respectively, where gut wall thicknesses were 200 and 110 mm. ...
Article
Full-text available
Coarse woody debris is an important biomass pool in forest ecosystems that numerous groups of insects have evolved to take advantage of. These insects are ecologically important and represent useful natural analogs for biomass to biofuel conversion. Using a range of molecular approaches combined with microelectrode measurements of oxygen, we have characterized the gut microbiome and physiology of Odontotaenius disjunctus, a wood-feeding beetle native to the eastern United States. We hypothesized that morphological and physiological differences among gut regions would correspond to distinct microbial populations and activities. In fact, significantly different communities were found in the foregut (FG), midgut (MG)/posterior hindgut (PHG) and anterior hindgut (AHG), with Actinobacteria and Rhizobiales being more abundant toward the FG and PHG. Conversely, fermentative bacteria such as Bacteroidetes and Clostridia were more abundant in the AHG, and also the sole region where methanogenic Archaea were detected. Although each gut region possessed an anaerobic core, micron-scale profiling identified radial gradients in oxygen concentration in all regions. Nitrogen fixation was confirmed by (15)N2 incorporation, and nitrogenase gene (nifH) expression was greatest in the AHG. Phylogenetic analysis of nifH identified the most abundant transcript as related to Ni-Fe nitrogenase of a Bacteroidetes species, Paludibacter propionicigenes. Overall, we demonstrate not only a compartmentalized microbiome in this beetle digestive tract but also sharp oxygen gradients that may permit aerobic and anaerobic metabolism to occur within the same regions in close proximity. We provide evidence for the microbial fixation of N2 that is important for this beetle to subsist on woody biomass.
... Odontotaenius disjunctus, the passalid beetle, is a wood-feeding insect that is considered one of the most highly subsocial arthropods 11 , with pairs defending log tunnels, remaining with their offspring from egg to adulthood, and demonstrating parent-offspring cooperation for protection and caring of other offspring 12,13 . These beetles are usually found in second-stage decayed wood 14 , where adults grind the lignocellulose substrate into small particles that are ingested and processed through a complex, morphologically differentiated digestive system 15 . Passalid feeding accelerates woody biomass decomposition in forested ecosystems, with each colony member consuming approximately 4.5 times its weight in woody material per day 16,17 . ...
... The external and internal morphology of the adult O. disjunctus digestive tract is characterized by four morphologically differentiated regions (Fig. 1), each consisting of a complex landscape with varying cuticle thickness and unique topography 15 . ...
... These landscapes allow diverse microbial populations to establish, suggesting that such anatomical properties and separation of these compartments may be beneficial to the processes of energy extraction from woody biomass 15 . Isolation and analysis of O. disjunctus' fungal microbiota, particularly in the posterior hindgut region (PHG), has identified a large diversity of yeasts with xylose fermentation capabilities, suggestive of their role in host metabolism [19][20][21][22][23][24] . ...
Article
Full-text available
Beneficial microbial associations enhance the fitness of most living organisms, and wood-feeding insects offer some of the most striking examples of this. Odontotaenius disjunctus is a wood-feeding beetle that possesses a digestive tract with four main compartments, each of which contains well-differentiated microbial populations, suggesting that anatomical properties and separation of these compartments may enhance energy extraction from woody biomass. Here, using integrated chemical analyses, we demonstrate that lignocellulose deconstruction and fermentation occur sequentially across compartments, and that selection for microbial groups and their metabolic pathways is facilitated by gut anatomical features. Metaproteogenomics showed that higher oxygen concentration in the midgut drives lignocellulose depolymerization, while a thicker gut wall in the anterior hindgut reduces oxygen diffusion and favours hydrogen accumulation, facilitating fermentation, homoacetogenesis and nitrogen fixation. We demonstrate that depolymerization continues in the posterior hindgut, and that the beetle excretes an energy- and nutrient-rich product on which its offspring subsist and develop. Our results show that the establishment of beneficial microbial partners within a host requires both the acquisition of the microorganisms and the formation of specific habitats within the host to promote key microbial metabolic functions. Together, gut anatomical properties and microbial functional assembly enable lignocellulose deconstruction and colony subsistence on an extremely nutrient-poor diet. © 2019, The Author(s), under exclusive licence to Springer Nature Limited.
... Investigations of the gut of wood-feeding beetles (Passalidae) recently led to the discovery of several distinct clades of ascomycete yeasts that are consistently associated with these beetles, perhaps as adaptations of the host feeding behavior and habitat (Suh and Blackwell 2004, Suh et al. 2005, Nguyen et al. 2006, Berkov et al. 2007, Grunwald et al. 2010, Hughes et al. 2011, Calderon and Berkov 2012. In particular members of the Sugiyamaella, Scheffersomyces and Spathaspora clades have been found in association with passalids (Zhang et al. 2003, Suh et al. 2004, Nguyen et al. 2006, Houseknecht et al. 2011, as well as other wood-feeding insects and woody plants (Berkov et al. 2007, Grunwald et al. 2010, Hughes et al. 2011. ...
... Investigations of the gut of wood-feeding beetles (Passalidae) recently led to the discovery of several distinct clades of ascomycete yeasts that are consistently associated with these beetles, perhaps as adaptations of the host feeding behavior and habitat (Suh and Blackwell 2004, Suh et al. 2005, Nguyen et al. 2006, Berkov et al. 2007, Grunwald et al. 2010, Hughes et al. 2011, Calderon and Berkov 2012. In particular members of the Sugiyamaella, Scheffersomyces and Spathaspora clades have been found in association with passalids (Zhang et al. 2003, Suh et al. 2004, Nguyen et al. 2006, Houseknecht et al. 2011, as well as other wood-feeding insects and woody plants (Berkov et al. 2007, Grunwald et al. 2010, Hughes et al. 2011. Species of Sugiyamaella are xylose-assimilating yeasts, and members of the Scheffersomyces and Spathaspora clades have attracted attention because they possess the rare ability to ferment D-xylose, which gives them economic potential for production of bioethanol from plant waste residues (Agbogbo andWenger 2006, 2007;Agbogbo and Coward-Kelly 2008). ...
... The isolation of the new X-F yeast isolated from the gut of wood roaches extends the association of yeasts with the rare biochemical ability to ferment D-xylose and wood-feeding insects (Zhang et al. 2003, Suh et al. 2004, Nguyen et al. 2006, Berkov et al. 2007, Grunwald et al. 2010). This finding is in agreement with studies in which the X-F yeasts Sc. stipitis and a FIG. 4. ML consensus tree based on XYL1 using a 489-character matrix of X-F members in the Scheffersomyces stipitis subclade. ...
Article
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The gut of wood-feeding insects is a microhabitat for a specialized community of microbes, including bacteria and several groups of eukaryotes such as nematodes, parabasalids, and fungi. The characterization of gut yeast communities from a variety of insects has shown that certain yeasts often are associated with the insects. The gut of wood-feeding insects is rich in ascomycete yeasts, and in particular xylose-fermenting (X-F) and assimilating yeasts have been consistently present in the gut of lignicolous insects. The objective of this study was the characterization of the yeast flora from the gut of the wood roach Cryptocercus sp. (Blattodea: Cryptocercidae). Five wood roach individuals were collected along the Appalachian Trail near the border between Tennessee and North Carolina, USA. We isolated 18 yeast strains from the wood roaches identified as Sugiyamaella paludigena and Sugiyamaella lignohabitans, xylose-assimilating yeasts, and Scheffersomyces cryptocercus (NRRL Y-48824T = CBS 12658) a new species of X-F yeast. The presence of X-F and certain non X-F yeasts in the gut of the subsocial wood roach Cryptocercus sp. extends the previous findings of associations between certain ascomycete yeasts and lignicolous insects. New combinations were made for 13 asexual members of the Sugiyamaella clade.
... The native P. stipitis strains were originally isolated from insect larvae. Phylogenetically, it is related to the endosymbionts gut microbiota, which inhabits the passalid beetles gut [86] that degrades white rotted hardwood [87]. Genetically and systematically, P. stipitis is predominantly homothallic, haploid and hemiascomycetous yeast [79,[88][89][90] that forms buds along with pseudomycelia during vegetative growth, and two hat-shaped ascospores from each ascus. ...
... The genomes of P. stipitis strains are encoding for xylanase, mannases, cellulases, and other degradative enzymes that enable them to grow and survive in the insect-gut environment of wood-inhabiting [86]. P. stipitis has the ability to ferment xylan, xylose [94,95], and cellobiose and to use all the main sugars that found in wood containing rhamnose and arabinose [96]. ...
Article
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Scarcity of the non-renewable energy sources, global warming, environmental pollution, and raising the cost of petroleum are the motive for the development of renewable, eco-friendly fuels production with low costs. Bioethanol production is one of the promising materials that can subrogate the petroleum oil, and it is considered recently as a clean liquid fuel or a neutral carbon. Diverse microorganisms such as yeasts and bacteria are able to produce bioethanol on a large scale, which can satisfy our daily needs with cheap and applicable methods. Saccharomyces cerevisiae and Pichia stipitis are two of the pioneer yeasts in ethanol production due to their abilities to produce a high amount of ethanol. The recent focus is directed towards lignocellulosic biomass that contains 30–50% cellulose and 20–40% hemicellulose, and can be transformed into glucose and fundamentally xylose after enzymatic hydrolysis. For this purpose, a number of various approaches have been used to engineer different pathways for improving the bioethanol production with simultaneous fermentation of pentose and hexoses sugars in the yeasts. These approaches include metabolic and flux analysis, modeling and expression analysis, followed by targeted deletions or the overexpression of key genes. In this review, we highlight and discuss the current status of yeasts genetic engineering for enhancing bioethanol production, and the conditions that influence bioethanol production.
... Generation of biofuel from such biomass has been thought to be an economical alternative to limited fossil fuel resources (Jeffries, 2006; Jeffries & Shi, 1999; Jeffries et al., 2007). A few earlier studies have reported the presence of gut yeasts in wood-decaying beetles by microscopic observation or by molecular cloning (Nardi et al., 2006; Zhang et al., 2003), and this has been proven by repeated isolation of many phylogenetically distinct yeasts from these beetles (Suh et al., 2003Suh et al., , 2004Suh et al., , 2005 Houseknecht et al., 2011). Interestingly, some of these yeasts, such as Scheffersomyces stipitis, Spathaspora passalidarum and Candida jeffriesii, are able to produce ethanol from xylose, a major component of hemicellulose, which is a backbone of plant cell walls (Nguyen et al., 2006; Suh et al., 2003). ...
... Interestingly, more than half of the yeast isolates from this study were identified as S. illinoinensis or S. stipitis, and most of them were associated with the passalid beetle O. disjunctus (Table 1). The presence of S. stipitis-like yeasts in the gut of O. disjunctus has been reported in a few previous studies based on microscopic observation (Lichtwardt et al., 1999; Suh et al., 2004; Nardi et al., 2006) as well as molecular biological approaches (Suh et al., 2003; Zhang et al., 2003), and the repeated isolation of S. illinoinensis (or S. stipitis) in this study agrees well with previous findings. Although S. illinoinensis and S. stipitis are highly similar to each other in nrRNA gene sequences (Fig. S1), the two species can be clearly differentiated by the sequences of RPB1, RPB2 and XYL1 (Figs 1 and 2 andTable S1; Urbina & Blackwell, 2012). ...
Article
During a survey of yeasts associated with wood-ingesting insects, sixty nine strains in the Scheffersomyces clade and related taxa were isolated from passalid and tenebrionid beetles and the decayed wood inhabited by them. The majority of those yeasts was found capable of fermenting xylose, and was recognized as Scheffersomyces stipitis or its close relative Scheffersomyces illinoinensis which are known to be associated with wood-decaying beetles and rotten wood. Yeasts in 'Scheffersomyces' (= Candida) ergatensis and 'Scheffersomyces' (= Candida) coipomoensis were also frequently isolated. The remaining six strains were identified as four novel species in the genera Scheffersomyces and Candida based on the multilocus sequence analyses of nuclear rRNA genes and four protein-coding genes, as well as other taxonomic characteristics. Two xylose-fermenting species, Scheffersomyces parashehatae f.a., sp. nov. (ATCC MYA-4653T = CBS 12535) and Scheffersomyces xylosifermentans f.a., sp. nov. (ATCC MYA-4859T = CBS 12540), formed a clade with Scheffersomyces shehatae and related Scheffersomyces species. Interestingly, S. xylosifermentans can survive at 40C, which is a rare property among the xylose-fermenting yeasts. Candida broadrunensis sp. nov. (ATCC MYA-4650T = CBS 11838) is a sister taxon of C. ergatensis, while Candida manassasensis sp. nov. (ATCC MYA-4652T = CBS 12534) is closely related to Candida palmioleophila in the Candida glaebosa clade. The multilocus DNA sequence comparisons in this study suggest that the genus Scheffersomyces needs to be circumscribed to the species near S. stipitis (type species) and S. shehatae which can be characterized by the ability to ferment xylose.
... Not only do both sexes provide parental care until adulthood is reached (> 3 months), but adult offspring help parents to maintain the pupal cases of their younger siblings Schuster 1985, Valenzuela-Gonzalez 1993). O. disjunctus creates long galleries lined with the digested wood on which larvae rely for food (Pearse et al. 1936) and from which offspring are likely to acquire wood-digesting gut microbes (Suh et al. 2003, Nardi et al. 2006. O. disjunctus larvae are abundant in galleries during June, July, and ...
... Not only do both sexes provide parental care until adulthood is reached (Ͼ3 mo), but adult offspring help parents to maintain the pupal cases of their younger siblings Schuster 1985, Valenzuela-Gonzalez 1993). O. disjunctus creates long galleries lined with the digested wood on which larvae rely for food (Pearse et al. 1936) and from which offspring are likely to acquire wood-digesting gut microbes (Suh et al. 2003, Nardi et al. 2006. O. disjunctus larvae are abundant in galleries during June, July, and August (Gray 1946). ...
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1. Species incidence is influenced by environmental and intrinsic factors operating at multiple scales. The incidence of a dispersal‐limited beetle, Odontotaenius disjunctus (Coleoptera: Passalidae), was surveyed within hierarchically nested organisational levels of its environment (log sections 2. Three scale‐sensitive aspects of O. disjunctus incidence were evaluated: (i) the extent (52–3600 ha) within which forest cover was most associated with incidence; (ii) the hierarchical level at which environmental variables best predicted incidence; and (iii) the hierarchical level at which incidence exhibited the greatest spatial autocorrelation as a result of intrinsic factors (e.g. dispersal limitation).3. Forest cover best predicted incidence at 225 ha, but accounted for only 1.2% of variation in incidence. Incidence was most sensitive to environmental factors measured at the finest scale (i.e. territories). Incidence was positively associated with moderately decayed wood and increased surface area of logs (9.9% and 3.1% of variance, respectively). When environmental factors were accounted for, spatial autocorrelation in incidence was greatest within subplots and logs, consistent with the hypothesis that intrinsic autocorrelation is associated with O. disjunctus average dispersal distance (4. This study indicates the influences of factors acting at multiple scales, but suggests that environmental conditions at the scale of territories may be most important for species incidence.
... Transtadial transmission was also demonstrated for Phlebotomus duboscqi Neveu-Lemaire (Diptera: Psychodidae) (Volf et al., 2002) and three Anopheles species (Pumpuni et al., 1996). Nardi et al. (2006) observed that many of the cuticular features of the hindgut epithelium morphology of the wood feeding beetle, Odontotaenius disjunctus (Illiger) are conserved during the transition from larva to adult. These conserved anatomical regions could likely retain bacteria during metamorphosis. ...
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The gut bacterial community of a bark beetle, the pine engraver Ips pini (Say), was characterized using culture-dependent and culture-independent methods. Bacteria from individual guts of larvae, pupae and adults were cultured and DNA was extracted from samples of pooled larval guts. Analysis of 16S rRNA gene sequences amplified directly from the gut community suggests that the gut bacterial communities associated with I. pini are relatively simple, compared to many other systems. Six bacterial genera from four classes were detected by culturing gut bacteria from larvae, pupae and adults. Two genera, Pantoea and Stenotrophomonas (γ-Proteobacteria) were found in all life stages of I. pini, consistently in larvae and adults, and less commonly in pupae. Sequences that affiliate with the Enterobactereaceae of the γ-Proteobacteria were found in 95% of the clones sampled. The Enterobactereaceae genera, Pantoea and Erwinia, accounted for 88% of all clone sequences. These results are consistent with previous work indicating that another bark beetle, the southern pine beetle, Dendroctonus frontalis Zimmerman, also has a relatively simple gut flora, compared with wood colonizing insects such as wood borers and termites. The composition and abundance of bacteria associated with different life stages of I. pini are possibly associated with specific functions of the gut bacterial communities of larvae, pupae, and adults.
... This requires specializations for digesting plant cell walls consisting of chemically recalcitrant components such as lignocellulose and various hemicelluloses. A recurrent theme in studies on gut structure and function in scarabaeoids, particularly larvae (Werner, 1926;Wiedemann, 1930;Wildbolz, 1954;Rössler, 1961;Bayon, 1980;Bayon and Mathelin, 1980;Lemke et al., 2003;Egert et al., 2005;Nardi et al., 2006;Andert et al., 2010;Huang et al., 2010;Sheng et al., 2012;Zheng et al., 2012), is that the hindgut, aided by microbial symbionts, is likely to play a key role in the digestion. Several species have a large, sac-like hindgut dilatation (''paunch''), with high microbial abundance, evidently functioning as a fermentation chamber. ...
Article
Most adult dung beetles (Scarabaeidae: Scarabaeinae) feed on fresh, wet dung of larger herbivorous or omnivorous mammals. As refractory plant fragments are selected out before ingestion, the food is presumed easily digestible. However, members of the desert-living scarabaeine genus Pachysoma (probably evolved from an ancestor closely related to the wet-dung feeding genus Scarabaeus) select dry dung pellets and/or plant litter. Thus, they ingest a much higher proportion of structural plant material, which nevertheless appears to be digested rather efficiently. This study investigates morphological modifications of the gut for this digestion in adults of eight Pachysoma species, both pellet and litter feeders. To ascertain hypothesized ancestral conditions, four fresh-dung feeding Scarabaeus species were also examined. The latter have the usual dung beetle gut consisting of a long, simple midgut, followed by an equally simple, much shorter hindgut of the same width. Lengths of midguts (M) and hindguts (H) divided by body length (B) for comparison between species of different size are: 4.9-6.3 (M/B) and 0.7-0.8 (H/B), which is normal for dung feeders. In Pachysoma, lengths are 6.3-6.5 (M/B) and 1.0-1.4 (H/B) in pellet feeders, and 4.4-5.0 (M/B) and 2.0-2.5 (H/B) for litter feeders. Hindguts are still morphologically undifferentiated and of midgut width, but clearly longer, particularly in litter feeders. Presumably, plant fragments in the food are digested, at least partly, in the hindgut. If so, the morphological adaptation is unusual: simple elongation rather than the expansion of part of the hindgut, as found in several other plant- or detritus-feeding scarabaeids. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.
... Scheffersomyces stipitis (S. stipitis), formerly known as Pichia stipitis [1], is a hemiascomycetous yeast, closely related to several yeast endosymbionts of passalid beetles that inhabit and decay white-rotted hardwood [2,3]. It has the highest native capacity for xylose fermentation of any known microbe [4,5]. ...
... The adult epithelial cells show abundant mitochondria in the apical region and infolded membranes with lateral junctions. Such features are associated with the reabsorption of water and ions in several arthropods, including insects, crustaceans and millipedes (Mykles 1979;Phillips et al. 1987;Nardi et al. 2006Nardi et al. , 2009Nardi et al. , 2016Bogataj et al. 2018). Moreover, the fibre structures of the epithelial cells are more developed in adults than in larvae and have also been proposed to be involved in the intracellular transport of water and ions in different crustacean groups (Komuro and Yamamoto 1968;Witkus et al. 1969;Vernon et al. 1974). ...
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Arthropods are the most diversified animals on Earth. The morphology of the digestive system has been widely studied in insects; however, crustaceans have received comparatively little attention. This study describes the hindgut tract of the common spider crab Maja brachydactyla Balss, 1922, in larvae and adults using dissection, light and electron microscopical analyses. The hindgut tract maintains a similar general shape in larvae and adults. Major differences among stages are found in the morphology of epithelial cells and microspines, the thickness of the cuticle and connective-like tissue, and the presence of rosette glands (only in adults). Here, we provide the description of the sub-cellular structure of the folds, epithelium (conformed by tendon cells), musculature, and microspines of the hindgut of larvae and adults of M. brachydactyla. The morphological features of the hindgut of M. brachydactyla are compared with those of other arthropods (Insecta, Myriapoda and Arachnida). Our results suggest that the morphology of the hindgut is associated mainly with transport of faeces. In adults, the hindgut may also exert an osmoregulatory function, as described in other arthropods. At difference from holometabolous insets, the hindgut of M. brachydactyla (Decapoda) does not undergo a true metamorphic change during development, but major changes observed between larval and adult stages might respond to the different body size between life stages.
... Hosts are not the only ones facing nutritional challenges. Para- 298 sites feed on their host by either hijacking food or feeding on theBedhomme et al., 2004; De Roode et al., 2008; Ebert et al., 2000; 310 Nesheim et al., 1978; Ryder et al., 2007; Smith and Holt, 1996), 2005 Kau et al., 2011; Ley et al., 2006; Noverr and 366 Huffnagle, 2004; Shin et al., 2011; Turnbaugh et al., 2008; Vijay 367 Kumar et al., 2010; Wen et al., 2008)Koch and Schmid-Hempel, 2011a), beetles (Egert et al., 2005; Leh 410 man et al., 2009; Nardi et al., 2006; Zhang and Jackson, 2008), flies 411 (Cox and Gilmore, 2007; Ren et al., 2007; Ryu et al., 2008; Shin 412 et al., 2011; Wong et al., 2011), lepidopterans (Pauchet et al., 413 2010; Xiang et al., 2006) and termites (Hongoh et al., 2003). 414 ...
... Hosts are not the only ones facing nutritional challenges. Para- 298 sites feed on their host by either hijacking food or feeding on theBedhomme et al., 2004; De Roode et al., 2008; Ebert et al., 2000; 310 Nesheim et al., 1978; Ryder et al., 2007; Smith and Holt, 1996), 2005 Kau et al., 2011; Ley et al., 2006; Noverr and 366 Huffnagle, 2004; Shin et al., 2011; Turnbaugh et al., 2008; Vijay 367 Kumar et al., 2010; Wen et al., 2008)Koch and Schmid-Hempel, 2011a), beetles (Egert et al., 2005; Leh 410 man et al., 2009; Nardi et al., 2006; Zhang and Jackson, 2008), flies 411 (Cox and Gilmore, 2007; Ren et al., 2007; Ryu et al., 2008; Shin 412 et al., 2011; Wong et al., 2011), lepidopterans (Pauchet et al., 413 2010; Xiang et al., 2006) and termites (Hongoh et al., 2003). 414 ...
... The higher ethanol concentration obtained with S. stipitis, at pH 6.0, also could be attributed in part to its numerous genes for endoglucanases and b-glucosidases [8], along with xylanase, activities [29,30] and because S. carlsbergensis yeast strain used in this study was not able to ferment xylose. ...
... Evidence that supports our hypothesis is: 1) S. shehatae, S. lignicola, and S. insectosa, often found in association with insects, are indistinguishable by morphology and some molecular markers (e.g. SSU and D1/D2 LSU); 2) branch lengths are constrained in the phylogenetic tree (Fig. 2); and 3) gut morphology is modified to enhance the horizontal transmission of gut yeasts across generations, e.g. the posterior hindgut region of O. disjunctus is colonized mainly by filamentous yeasts attached by a holdfast [84]; in addition mycetomes occur in lignicolous cerambycid beetles colonized exclusively by closely related species of S. shehatae [22]. ...
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Many of the known xylose-fermenting (X-F) yeasts are placed in the Scheffersomyces clade, a group of ascomycete yeasts that have been isolated from plant tissues and in association with lignicolous insects. We formally recognize fourteen species in this clade based on a maximum likelihood (ML) phylogenetic analysis using a multilocus dataset. This clade is divided into three subclades, each of which exhibits the biochemical ability to ferment cellobiose or xylose. New combinations are made for seven species of Candida in the clade, and three X-F taxa associated with rotted hardwood are described: Scheffersomyces illinoinensis (type strain NRRL Y-48827(T) = CBS 12624), Scheffersomyces quercinus (type strain NRRL Y-48825(T) = CBS 12625), and Scheffersomyces virginianus (type strain NRRL Y-48822(T) = CBS 12626). The new X-F species are distinctive based on their position in the multilocus phylogenetic analysis and biochemical and morphological characters. The molecular characterization of xylose reductase (XR) indicates that the regions surrounding the conserved domain contain mutations that may enhance the performance of the enzyme in X-F yeasts. The phylogenetic reconstruction using XYL1 or RPB1 was identical to the multilocus analysis, and these loci have potential for rapid identification of cryptic species in this clade.
... The high ethanol yield could be explained because P. stipitis has numerous genes for endoglucanases and β-glucosidases, along with xylanase, mannanase and chitinase activities. These indicate that this yeast could metabolize polysaccharides in the beetle gut [9], where it is usually found as endosymbiont [13]. Ethanol production was significantly influenced (P-value ˂ 0.05) by the severity of the pretreatment as shown in (Fig. 4). ...
... Scheffersomyces stipitis (S. stipitis), formerly known as Pichia stipitis [1], is a hemiascomycetous yeast, closely related to several yeast endosymbionts of passalid beetles that inhabit and decay white-rotted hardwood [2,3]. It has the highest native capacity for xylose fermentation of any known microbe [4,5]. ...
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Fermentation of xylose, the major component in hemicellulose, is essential for economic conversion of lignocellulosic biomass to fuels and chemicals. The yeast Scheffersomyces stipitis (formerly known as Pichia stipitis) has the highest known native capacity for xylose fermentation and possesses several genes for lignocellulose bioconversion in its genome. Understanding the metabolism of this yeast at a global scale, by reconstructing the genome scale metabolic model, is essential for manipulating its metabolic capabilities and for successful transfer of its capabilities to other industrial microbes. We present a genome-scale metabolic model for Scheffersomyces stipitis, a native xylose utilizing yeast. The model was reconstructed based on genome sequence annotation, detailed experimental investigation and known yeast physiology. Macromolecular composition of Scheffersomyces stipitis biomass was estimated experimentally and its ability to grow on different carbon, nitrogen, sulphur and phosphorus sources was determined by phenotype microarrays. The compartmentalized model, developed based on an iterative procedure, accounted for 814 genes, 1371 reactions, and 971 metabolites. In silico computed growth rates were compared with high-throughput phenotyping data and the model could predict the qualitative outcomes in 74% of substrates investigated. Model simulations were used to identify the biosynthetic requirements for anaerobic growth of Scheffersomyces stipitis on glucose and the results were validated with published literature. The bottlenecks in Scheffersomyces stipitis metabolic network for xylose uptake and nucleotide cofactor recycling were identified by in silico flux variability analysis. The scope of the model in enhancing the mechanistic understanding of microbial metabolism is demonstrated by identifying a mechanism for mitochondrial respiration and oxidative phosphorylation. The genome-scale metabolic model developed for Scheffersomyces stipitis successfully predicted substrate utilization and anaerobic growth requirements. Useful insights were drawn on xylose metabolism, cofactor recycling and mechanism of mitochondrial respiration from model simulations. These insights can be applied for efficient xylose utilization and cofactor recycling in other industrial microorganisms. The developed model forms a basis for rational analysis and design of Scheffersomyces stipitis metabolic network for the production of fuels and chemicals from lignocellulosic biomass.
... Very few papers have been concerned with Scarabaeoidea hindgut and histology (Lewis 1926;Becton 1930;Cooper 1938;Umeya 1960Umeya , 1974Miller 1961;Halffter & Matthews 1971;López-Guerrero 2002;Nardi et al. 2006). Umeya (1960) did a comparative study, which may help with the understanding of the cladogenesis processes by comparing the anatomical aspects of adult alimentary tracts in order to establish a correlation between the alimentary tract length and feeding behaviour. ...
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When seen as a whole, the hindgut, more than any other section of the digestive tube, provides insights for the taxonomy of higher-level taxa of Passalidae. This comparative cladistics study utilized parsimony analyses, with 57 terminal taxa (including five outgroups) and 18 characters from the ileum of most genera of passalid beetles (Coleoptera: Passalidae), which resulted in a single tree. Based on the results and the topology of the cladogram, the following classification was adopted: (Aulacocyclinae (Solenocyclinae (Proculinae (Passalinae, Macrolininae)))). Three subfamilies (Solenocyclinae, Proculinae, and Macrolininae) are here elevated from the tribal level to the subfamily level. The subfamily Passalinae is redefined and no longer contains any tribes. The family-group name Leptaulacinae is placed in synonym with Solenocyclinae and the family-group name Ceracupedini is placed in synonymy with Aulacocyclinae.
... Similarly, Warnecke et al. (2007) conducted a metagenomic and functional analysis of the microbial community inhabiting the hindgut of Nasutitermes sp. and documented the presence of various bacterial genes for cellulose and xylan hydrolysis. It is clear from research on beetles that diverse communities of wood-digesting gut microbes are not limited to termites (Zhang, Suh & Blackwell, 2003;Nardi et al., 2006;Urbina, Schuster & Blackwell, 2013). For example, Suh et al. (2003) isolated yeasts from the guts of passalid beetles collected from North and Central America capable of fermenting and assimilating xylose or of hydrolysing xylan, major components of hemicellulose. ...
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The diversity and habitat requirements of invertebrates associated with dead wood have been the subjects of hundreds of studies in recent years but we still know very little about the ecological or economic importance of these organisms. The purpose of this review is to examine whether, how and to what extent invertebrates affect wood decomposition in terrestrial ecosystems. Three broad conclusions can be reached from the available literature. First, wood decomposition is largely driven by microbial activity but invertebrates also play a significant role in both temperate and tropical environments. Primary mechanisms include enzymatic digestion (involving both endogenous enzymes and those produced by endo- and ectosymbionts), substrate alteration (tunnelling and fragmentation), biotic interactions and nitrogen fertilization (i.e. promoting nitrogen fixation by endosymbiotic and free-living bacteria). Second, the effects of individual invertebrate taxa or functional groups can be accelerative or inhibitory but the cumulative effect of the entire community is generally to accelerate wood decomposition, at least during the early stages of the process (most studies are limited to the first 2–3 years). Although methodological differences and design limitations preclude meta-analysis, studies aimed at quantifying the contributions of invertebrates to wood decomposition commonly attribute 10–20% of wood loss to these organisms. Finally, some taxa appear to be particularly influential with respect to promoting wood decomposition. These include large wood-boring beetles (Coleoptera) and termites (Termitoidae), especially fungus-farming macrotermitines. The presence or absence of these species may be more consequential than species richness and the influence of invertebrates is likely to vary biogeographically.
... Scheffersomyces stipitis (formerly known as Pichia stipitis) is a crabtree-negative yeast that habits the gut of wood-decaying beetles (Nardi et al., 2006). Moreover, S. stipitis genome encodes the enzymes and transporters necessary to dissimilate a wide variety of saccharides (Jeffries and Van Vleet, 2009). ...
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Resveratrol is a plant-derived aromatic compound with a wide range of beneficial properties including antioxidant and anti-aging effects. The resveratrol currently available on the market is predominantly extracted from certain plants such as grape and the Japanese knotweed Polygonum cuspidatum. Due to the unstable harvest of these plants and the low resveratrol purity obtained, it is necessary to develop a stable production process of high-purity resveratrol from inexpensive feedstocks. Here, we attempted to produce resveratrol from a wide range of sugars as carbon sources by a using the genetically-engineered yeast Scheffersomyces stipitis (formerly known as Pichia stipitis), which possesses a broad sugar utilization capacity. First, we constructed the resveratrol producing strain by introducing genes coding the essential enzymes for resveratrol biosynthesis [tyrosine ammonia-lyase 1 derived from Herpetosiphon aurantiacus (HaTAL1), 4-coumarate: CoA ligase 2 derived from Arabidopsis thaliana (At4CL2), and stilbene synthase 1 derived from Vitis vinifera (VvVST1)]. Subsequently, a feedback-insensitive allele of chorismate mutase was overexpressed in the constructed strain to improve resveratrol production. The constructed strain successfully produced resveratrol from a broad range of biomass-derived sugars [glucose, fructose, xylose, N-acetyl glucosamine (GlcNAc), galactose, cellobiose, maltose, and sucrose] in shake flask cultivation. Significant resveratrol titers were detected in cellobiose and sucrose fermentation (529.8 and 668.6 mg/L after 120 h fermentation, respectively), twice above the amount obtained with glucose (237.6 mg/L). Metabolomic analysis revealed an altered profile of the metabolites involved in the glycolysis and shikimate pathways, and also of cofactors and metabolites of energy metabolisms, depending on the substrate used. The levels of resveratrol precursors such as L-tyrosine increased in cellobiose and sucrose-grown cells. The results indicate that S. stipitis is an attractive microbial platform for resveratrol production from broad types of biomass-derived sugars and the selection of suitable substrates is crucial for improving resveratrol productivity of this yeast.
... However, in many cases, the transcripts overlapped the region targeted for amplicon analysis, but had low sequence identities to the 16S OTUs. This could be a reflection of the dynamic nature of the midgut community, as has been observed for the gut communities of other xylophagous insects [36], or it could be an artifact of feeding on a non-sterile food source where DNA from microbes ingested during feeding is more abundant than DNA from microbes that are truly residents of the midgut. Consistent with the utility of targeting expressed rRNAs for symbiont community analysis, the taxonomic classification of the rRNAs that were assembled from the transcriptome libraries most strongly resembled the taxonomic assignments obtained from MEGAN analysis of the microbial protein coding genes. ...
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Wood-feeding beetles harbor an ecologically rich and taxonomically diverse assemblage of gut microbes that appear to promote survival in woody tissue, which is devoid of nitrogen and essential nutrients. Nevertheless, the contributions of these apparent symbionts to digestive physiology and nutritional ecology remain uncharacterized in most beetle lineages. Through parallel transcriptome profiling of beetle- and microbial- derived mRNAs, we demonstrate that the midgut microbiome of the Asian longhorned beetle (Anoplophora glabripennis), a member of the beetle family Cerambycidae, is enriched in biosynthetic pathways for the synthesis of essential amino acids, vitamins, and sterols. Consequently, the midgut microbiome of A. glabripennis can provide essential nutrients that the beetle cannot obtain from its woody diet or synthesize itself. The beetle gut microbiota also produce their own suite of transcripts that can enhance lignin degradation, degrade hemicellulose, and ferment xylose and wood sugars. An abundance of cellulases from several glycoside hydrolase families are expressed endogenously by A. glabripennis, as well as transcripts that allow the beetle to convert microbe-synthesized essential amino acids into non-essential amino acids. A. glabripennis and its gut microbes likely collaborate to digest carbohydrates and convert released sugars and amino acid intermediates into essential nutrients otherwise lacking from their woody host plants. The nutritional provisioning capabilities of the A. glabripennis gut microbiome may contribute to the beetles' unusually broad host range. The presence of some of the same microbes in the guts of other Cerambycidae and other wood-feeding beetles suggests that partnerships with microbes may be a facilitator of evolutionary radiations in beetles, as in certain other groups of insects, allowing access to novel food sources through enhanced nutritional provisioning.
... C.J. Mason et al. Journal of Insect Physiology 102 (2017) 27-35 assemblage (Ceja-Navarro et al., 2013;Nardi et al., 2006). Collectively, this suggests that physical and morphological modifications may enable greater differentiation of microbial communities than by different physicochemical gradients occupying those regions. ...
Article
Insect guts harbor diverse microbial assemblages that can be influenced by multiple factors, including gut physiology and interactions by the host with its environment. The Asian longhorned beetle (A. glabripennis; Cerambycidae: Lamiinae) is an invasive tree-killing insect that harbors a diverse consortium of fungal and bacterial gut associates that provision nutrients and facilitate lignocellulose digestion. The physicochemical conditions of the A. glabripennis gut and how these conditions may influence the microbial composition across gut regions are unknown. In this study, we used microsensors to measure in situ oxygen concentrations, pH, and redox potential along the length of the A. glabripennis larval gut from two North American populations. We then analyzed and compared bacterial and fungal gut communities of A. glabripennis within individual hosts along the length of the gut using 16S and ITS1 amplicon sequencing. The A. glabripennis midgut lumen was relatively anoxic (< 0.01 kPa) with a pH gradient from 5.5 to 9, moving anterior to posterior. Redox potential was higher in the anterior midgut relative to posterior regions. No differences in physicochemistry were measured between the two populations of the beetle, but the two populations harbored different communities of bacteria and fungi. However, microbial composition of the A. glabripennis gut microbiota did not differ among gut regions despite physicochemical differences. Unlike other insect systems that have distinct gut compartmentalization and corresponding microbial assemblages, the A. glabripennis gut lacks dramatic morphological modifications, which may explain why discrete microbial community structures were not found along the digestive system.
... Thus, the microbial communities and how they change during the long course of wood debris decomposition are thought to play at least a nutritional role in the ecology of wood feeding insects. There is also evidence of additional relationships between microbes and wood feeding insects, especially within the context of co-evolution of endosymbionts (Suh and Blackwell, 2005;Nardi et al., 2006;Warnecke et al., 2007;Ulyshen, 2016) and fungal ectosymbiosis (Krivosheina, 1991;Mueller et al., 2005;Biedermann and Vega, 2020). ...
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Sociality has independently arisen in several wood-dwelling insect lineages, yet little is understood about how the properties of decaying logs have favored the evolution of cooperative social groups. Here we evaluate the current literature on wood-dwelling social insects to identify the structural, nutritional, and microbial properties of decaying logs that have led to the repeated evolution of social behavior. Wood-tissue is structural resilient, and thus provided an enclosed, defensible nest site for early wood-feeding insect groups. This structural stability enabled the long-term persistence of family groups, and was likely a key feature in the transition toward more complex eusocial societies. The resilient structure and relatively poor nutritional quality of wood also likely provided a stable environment for the evolution of complex mutualisms with prokaryote and fungal symbionts to digest this resource. Parental care likely evolved as a means to protect the valuable nest site and ensure adequate nutrition for offspring in this environment by allowing parents to both provision and transfer microbial symbionts to offspring. Pathogenic microbes are also abundant in nests constructed in wood-tissue, and social adaptations such as allogrooming and nest maintenance may have evolved in response to microbial invaders. In general, the dynamic relationships between insects, microbes, and the wood-tissue that they inhabit was a critical component in the evolution of sociality in this habitat.
... In-depth examination of hindgut-specific microbe interactions remains to be performed. However, it is worth noting that the cuticle of the pyloric region of several insects and related diplopods contains cuticular microspines, which are thought to serve as sites of enriched microbial communities within the intestinal tract (Elzinga 1998;Nardi et al. 2006;). The pylorus is also an immune signaling hub in the insect gut. ...
Article
The insect excretory system contains two organ systems acting in concert: the Malpighian tubules and the hindgut perform essential roles in excretion and ionic and osmotic homeostasis. For over 350 years, these two organs have fascinated biologists as a model of organ structure and function. As part of a recent surge in interest, research on the Malpighian tubules and hindgut of Drosophila have uncovered important paradigms of organ physiology and development. Further, many human disease processes can be modeled in these organs. Here, focusing on discoveries in the past 10 years, we provide an overview of the anatomy and physiology of the Drosophila excretory system. We describe the major developmental events that build these organs during embryogenesis, remodel them during metamorphosis, and repair them following injury. Finally, we highlight the use of the Malpighian tubules and hindgut as accessible models of human disease biology. The Malpighian tubule is a particularly excellent model to study rapid fluid transport, neuroendocrine control of renal function, and modeling of numerous human renal conditions such as kidney stones, while the hindgut provides an outstanding model for processes such as the role of cell chirality in development, nonstem cell-based injury repair, cancer-promoting processes, and communication between the intestine and nervous system.
... Other known representatives of the group of native xylose-fermenting yeastsare, Candida shehatae (teleomorph form is known as Scheffersomyces shehatae), Pachysolen tannophilus, Spathaspora passalidarum, and Ogataea polymorpha [38,128,160,162]. As a rule, most natural xylose-fermenting yeasts inhabit the guts of passalid beetles that degrade white-rotted hardwood [125,130] with the exception of O. polymorpha strains that have been isolated from other natural habitats [46]. S. stipitis is closely related to other yeast endosymbionts of passalid beetles [175]. ...
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This review summarizes progress in the construction of efficient yeast ethanol producers from glucose/sucrose and lignocellulose. Saccharomyces cerevisiae is the major industrial producer of first-generation ethanol. The different approaches to increase ethanol yield and productivity from glucose in S. cerevisiae are described. Construction of the producers of second-generation ethanol is described for S. cerevisiae, one of the best natural xylose fermenters, Scheffersomyces stipitis and the most thermotolerant yeast known Ogataea polymorpha. Each of these organisms has some advantages and drawbacks. S. cerevisiae is the primary industrial ethanol producer and is the most ethanol tolerant natural yeast known and, however, cannot metabolize xylose. S. stipitis can effectively ferment both glucose and xylose and, however, has low ethanol tolerance and requires oxygen for growth. O. polymorpha grows and ferments at high temperatures and, however, produces very low amounts of ethanol from xylose. Review describes how the mentioned drawbacks could be overcome.
... Furthermore, the host can evolve to where tripartite beetlefungi-bacteria mutualism takes place [23,26]. This phenomenon seems to be widespread, although the mechanisms that govern these interactions are still poorly understood [4,27]. ...
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... Although the gut-dwelling microbes of passalids have been studied for well over a century (Pearse et al. 1936;Leidy 1852;Lichtwardt et al. 1999), the biggest advances in understanding have been made in the past 10-20 years, and the findings from these more recent studies are briefly summarized here. It is clear that passalid guts house complex assemblages of microorganisms (including bacteria, Archaea, protists, nematodes, and various fungi) and that the composition of microbial communities varies among gut regions (Nardi et al. 2006). Ceja-Navarro et al. (2014), for example, reported significant compositional differences in bacteria and archaea communities among the four main gut regions (foregut, midgut, anterior hindgut, and posterior hindgut) of O. disjunctus, although the midgut and posterior hindgut communities did not differ from one another. ...
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Consisting of about 1000 species globally, beetles of the family Passalidae feed on decomposing wood in tropical and subtropical forests throughout the world. Passalids live in subsocial family groups within their galleries, characterized by overlapping generations, cooperative brood care, and a complex communication system involving stridulations. In what has been referred to as an “external rumen,” larval passalids feed on the microbe-rich frass and finely chewed wood paste produced by the wood-feeding adults. Endosymbionts found within the guts of passalids include a variety of microbes, including nitrogen-fixing prokaryotes and yeasts that aid in the digestion of wood. In addition to wood consumption, passalids fragment large amounts of wood in the process of creating extensive tunnel systems and are, among saproxylic insects, perhaps rivaled only by termites in their importance to wood decomposition. Although a number of laboratory studies have measured the amount of wood processed by various passalid species, no attempt has been made to quantify their contributions to wood decomposition under natural conditions. Passalids , along with their many microbial and invertebrate associates, are of considerable conservation concern given high levels of endemism and flightlessness. Many species appear sensitive to forest loss and disturbance and they have been used as indicator taxa in the creation of protected natural areas.
... Spathaspora passalidarum NN245 is an unusual yeast that shows a high capacity for fermenting xylose, cellobiose and glucose from pure sugars or from lignocellulosic hydrolysates (Long et al. 2012). The Spathaspora genus (Cadete et al. 2009(Cadete et al. , 2013Lopes et al. 2016) is closely related to the Scheffersomyces clade (Urbina and Blackwell 2012) and shares with it a proclivity for living in the hindgut of larva and adult wood-boring beetles of the family Passalidae (Nardi et al. 2006;Urbina, Schuster and Blackwell 2013). The physiology of S. passalidarum is still poorly understood, even though its genome has been sequenced (Wohlbach et al. 2011). ...
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... It is suggested that these sacs act as a refuge for the hindgut symbionts, where they can be retained safely against the gut flow until released into the hindgut. However, gut yeasts in passalid larvae are sometimes found adhering to the gut wall by means of branched filaments (holdfasts), which helps prevent them washing out (Nardi et al. 2006). ...
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... Arthropod gut symbionts comprise diverse organisms including bacteria, protists, nematodes (Metazoa) and fungi, each developing a particular role within the digestive system of their arthropod hosts (Dexter Dyer 2002, Cafaro 2005, Lichtwardt et al 2001, Nardi et al 2006. Some of these endosymbionts share particular characteristics such as those included within the ecological group ''trichomycetes'', organisms with filamentous structures, branched or not, attached to the chitinous gut lining of their hosts by means of cellular structures or secreted holdfast material (Lichtwardt 1986). ...
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Two strains of a basidiomycetous yeast were derived from an insect trypanosomatid culture isolated from the intestine of a plant bug, Collaria oleosa (Heteroptera: Miridae), collected in Costa Rica. The yeast did not form ballistoconidia but reproduced only by budding. Teliospores were not observed in individual and crossed cultures of each strain. Morphological and other taxonomic characteristics of the yeast were similar to those of the species in the polyphyletic genus Rhodotorula. However, molecular phylogeny inferred from the internal transcribed spacers and D1/D2 region of the large subunit rRNA gene showed that the strains represent a new species placed among the smut fungi in the family Ustilentylomataceae, which includes Aurantiosporium subnitens, Fulvisporium restifaciens, Ustilentyloma fluitans, and Rhodotorula hordea. Given the well distinguished phylogenetic position of this novel species within the Ustilentylomataceae, we propose Microbotryozyma collariae gen. nov., sp. nov. to accommodate the yeast isolated from C. oleosa, with strain American Type Culture Collection MYA-4666(T) (= PRA303-1S = CBS 12537) designated as the type strain.
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Production of fuel ethanol is one of the largest fields of industrial biotechnology. Currently, most of fuel ethanol is produced from conventional feedstock (first-generation ethanol); however, lignocellulose-derived (second-generation) ethanol has great future. Current review describes achievements in construction of the advanced strains of Saccharomyces cerevisiae with increased yield and productivity of ethanol synthesis from glucose. Among producers of second-generation ethanol, review presents data on three species, conventional yeast S. cerevisiae, and two nonconventional yeast species: one of the most efficient natural xylose-fermenting yeast Scheffersomyces stipitis and the thermotolerant yeast Ogataea polymorpha. Improvements of the available strains using methods of rational metabolic engineering and classical selection are discussed.
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The horned passalus (Odontotaenius disjunctus) is one of the most extensively studied saproxylic beetles in the eastern United States. For several decades this species has been the subject of investigations into the behaviors associated with subsociality as well as physiological responses to stress, and, most recently, the composition of its gut microbiome has been closely examined. However, no published study to date has characterized this beetle’s broad-scale population genetic structure. Here, we conducted intensive geographic sampling throughout the southern Appalachian Mountains and surrounding areas and then assessed mitochondrial DNA (mtDNA) sequence variation among individuals. Unexpectedly, we discovered two divergent, yet broadly sympatric, mtDNA clades. Indeed, the magnitude of divergence between- vs. within-clades ranged from 5.9 to 7.5×, depending on the dataset under consideration, and members of the two lineages were often syntopic (i.e., found in the same rotting log). Given the potential implications for past and future studies on behavior, physiology, and the gut microbiome, we developed a simple cost-efficient molecular assay (i.e., polymerase chain reaction restriction fragment length polymorphism; PCR-RFLP) to rapidly determine mtDNA clade membership of O. disjunctus individuals. We suggest that the evolutionary processes that gave rise to the emergence and persistence of divergent sympatric lineages reported here warrant investigation, as this type of spatial-genetic pattern appears to be rare among southern Appalachian forest invertebrates.
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Chapter
Minute organisms, including yeasts with diverse physiological capabilities, make possible the existence of arthropods, especially insects, the most speciose group of organisms on Earth. The yeast growth form occurs throughout most of the fungal kingdom and is often associated with insects. Fungi and insects evolved together in the same habitats where casual associations certainly occurred early in their shared geological history and yeast attractants for insects developed over their lives together. Examples of their interactions range from accidental dispersal and the use of yeasts as food to obligate mutualisms described in this chapter. These include discussions of yeast-like symbionts, the use of the wasp gut for yeast outcrossing, extension of endophyte life cycles to include dispersal by insects, the advantage of yeasts in the diet of many insects such as blood-sucking dipterans, and the yeast-like germination of phoretic fungi in ephemeral habitats. Future studies of yeast–insect associations will continue to include species discovery but also approach theoretical questions of sexual and asexual reproduction, host specificity, host switching, advantages of horizontal and vertical dispersal, and studies that include entire interactive communities.
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In this article, the composition and distribution of bacteria associated with the gut of Tenebrio molitor (L.) larvae were investigated using both culture-dependent and culture-independent denaturing gradient gel electrophoresis (DGGE) methods. This work compares bacterial species associated with four different parts of T. molitor larvae gut: foregut, anterior midgut, posterior midgut, and hindgut. Five genera, Weissella, Lactococcus, Rahnella, Cronobacter, and Enterococcus , were isolated using nutrient agar. All of these strains were present in the posterior midgut and hindgut. The strains with milk-clotting activity in selective casein-plates assay were sequenced and identified as species of genera Weissella and Lactococcus, and those with proteolytic activity as Rahnella and Cronobacter, implying that they may be involved in protein utilization. But none of these strains showed cellulolytic activity. In DGGE experiment, 19 isolated bands belonging to nine taxa ( Spiroplasma , Lactococcus, Lactobacillus, Bacillus, an uncultured Bacillaceae , Clostridium, Enterobacter, Pantoea, and an uncultured Clostridium ) were extracted and identified from DGGE gels. These species could be assigned to three phyla Tenericutes, Firmicutes, and Proteobacteria. According to the DGGE analysis, the bacterial communities of the four gut regions exhibited some differences, with the hindgut showing the highest bands abundance and diversity. The genus S piroplasma, which is generally regarded as pathogen or male-killing bacteria in insects, had a high abundance in the gut environment, their potential role is worthy of a further study.
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Among the many parasitic or commensalistic symbionts of Passalidae (Coleoptera) are fungi that live within their hindgut and on the exoskeleton of the beetles and their parasitic mites. Three Eccrinales (Trichomycetes) include Leidyomyces attenuatus (= Enterobryus attenuatus), Passalomyces compressus (= Enterobryus compressus), and an unnamed species originally described by Heymons and Heymons in 1934. Leidyomyces attenuatus has been found in populations throughout the range of Passalidae in the Americas, whereas P. compressus and the Heymons' eccrinid are reported only from Neotropical passalid beetles. A new genus and species of branched fungus, Enteroramus dimorphus, lives in the hindgut of the common eastern North American passalid, Odontotaenius disjunctus. In axenic culture the fungus converts to a yeastlike growth form. Externally, both the beetles and their mites carry parasitic thalli of many species of Rickia (Ascomycota: Laboulbeniales). The probability that many fungi from Passalidae remain unreported worldwide is discussed.
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Insect associations with fungi are common and may be casual or highly specific and obligate. For example, more than 40 fungal species are as-sociated with the coffee berry borer (Hypothenemus hampei, Coleoptera: Cur-culionidae; Pérez et al. 2003) and about the same number with the subterranean termite Reticulitermes flavipes (Zoberi and Grace 1990; table 9.1). In one system 28 species of yeasts were isolated from the external parts of Drosophila serido and 18 species, including some not found on the external surfaces, from their crop (Morais et al. 1994; table 9.1). In relatively few cases a specific role for the fungus has been identified, as is the case for associations with ants (chapter 7), termites (chapter 8), and bark beetles (Chapter 11; Six 2003). These associations imply that different species are living together, reinforced by specific interactions, a concept popularized as symbiosis by de Bary (1879). Symbiotic associations have been classified as ectosymbiotic when the symbiont occurs outside the body of the host or endosymbiotic when the symbiont occurs internally, either intra-or extracellularly (Steinhaus 1949; Nardon and Nardon 1998; Margulis and Chapman 1998). Several interesting symbiotic associations occur between insects and yeasts. In all cases that are well studied, the benefit that accrues for the insect is better understood than the benefit to the yeasts. The term "yeast" is used to describe a particular fungal growth form (Steinhaus 1947; Alexopoulos et al. 1996). These predominantly unicellular ascomycetes di-vide by budding at some point in their life cycle (e.g., Saccharomyces). A sur-prising number of yeasts, however, also produce filamentous hyphae. At present, almost 700 species in 93 genera (Barnett et al. 2000) have been described in the ascomycete class Saccharomycetes, a group known informally as "true yeasts." True yeasts lack specialized sex organs, and sexual spores (ascospores) are produced in 211 Table 9.1. Yeasts internally isolated from insects. Insect Species Order: Family Yeast Location (Species) a Reference Stegobium paniceum Coleoptera: Anobiidae Mycetomes Escherich 1900 (= Sitodrepa panicea) (Saccharomyces) b Buchner 1930 Cecae (Torulopsis buchnerii) Gräbner 1954 Mycetome between foregut and midgut Pant and Fraenkel 1954 Mycetomes (Symbiotaphrina buchnerii) Kühlwein and Jurzitza 1961 Mycetomes and digestive tube (Torulopsis buchnerii) Bismanis 1976 Gut cecae (Symbiotaphrina buchnerii) Noda and Kodama 1996 Lasioderma serricorne Coleoptera: Anobiidae Mycetome between foregut and midgut van der Walt 1961; Jurzitza 1964 (Symbiotaphrina kochii) Gams and von Arx 1980 Noda and Kodama 1996
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We review literature on extant detritivores, including cockroaches and termites, and conclude that coprophagy was the key behavior leading to the evolution of hindgut fermentation systems in the stem group of Dictyoptera. Coprophagy exploits concentrated microbial consortia on cellulose based substrates. These microbes are potential mutualists and food, but they also initiate degradation of cellulose, detoxify allelochemicals and soften the substrate, a phenomenon known as the “external rumen”. We suggest that the evolution of a sophisticated hindgut fauna is a process of internalizing this self-assembled microbial community, accompanied by changes in host-microbe interdependence, the source of microbial inoculum for neonates, and host social behavior. Proctodeal trophallaxis evolved from pre-existing intraspecific coprophagous behavior when termite ancestors became subsocial, because the physiology of encystment in oxymonad and hypermastigid flagellates precludes their transfer via cysts in adult feces. The behavior was reinforced by the benefits of using the trophic stages of flagellates as food. The association of these flagellates with the dictyopteran lineage is an ancient one, and may have originated as part of the external rumen in the Carboniferous coal swamps.
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Enteroramus dimorphus from the gut of the passalid beetle Odontotaenius disjunctus was described originally as a yeast-like fungus of unknown taxonomic affiliation. The fungus can be observed in situ, attached by a specialized cell to the beetle hindgut wall. In a recent study of yeast endosymbionts from a variety of beetles, we discovered that E. dimorphus is a member of the Pichia stipitis (Saccharomycetes) clade, known for xylose fermentation and assimilation. The closest relative of E. dimorphus is the PASS1 isolate, repeatedly acquired from passalid beetles in eastern North America from Pennsylvania to Louisiana. In addition to xylose fermentation and assimilation, these yeasts are characterized by the production of hat-shaped ascospores in culture, assimilation of a wide range of sugars, and synthesis of several vitamins. Enteroramus dimorphus, however, can be distinguished from close relatives by several physiological characteristics and rDNA sequences, which vary slightly from the more widespread PASS1 genotype. We present an amended description of E. dimorphus and discuss its symbiotic phase in association with O. disjunctus, including a holdfast that parallels those of unrelated symbiotic yeasts associated with nematodes.
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The diversity of the Insecta is reflected in the large and varied microbial communities inhabiting the gut. Studies, particularly with termites and cockroaches, have focused on the nutritional contributions of gut bacteria in insects living on suboptimal diets. The indigenous gut bacteria, however, also play a role in withstanding the colonization of the gut by non-indigenous species including pathogens. Gut bacterial consortia adapt by the transfer of plasmids and transconjugation between bacterial strains, and some insect species provide ideal conditions for bacterial conjugation, which suggests that the gut is a "hot spot" for gene transfer. Genomic analysis provides new avenues for the study of the gut microbial community and will reveal the molecular foundations of the relationships between the insect and its microbiome. In this review the intestinal bacteria is discussed in the context of developing our understanding of symbiotic relationships, of multitrophic interactions between insects and plant or animal host, and in developing new strategies for controlling insect pests.
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Segmented filamentous bacteria (SFB) are autochthonous bacteria inhabiting the intestinal tracts of many species, including humans. We studied the effect of SFB on the mucosal immune system by monoassociating formerly germfree C3H/HeN mice with SFB. At various time points during 190 days of colonization, fragment cultures of small intestine and Peyer’s patches (PP) were analyzed for total immunoglobulin A (IgA) and SFB-specific IgA production. Also, phenotypic changes indicating germinal center reactions (GCRs) and the activation of CD4 ⁺ T cells in PP were determined by using fluorescence-activated cell sorter analyses. A second group of SFB-monoassociated mice was colonized with a gram-negative commensal, Morganella morganii , to determine if the mucosal immune system was again stimulated and to evaluate the effect of prior colonization with SFB on the ability of M. morganii to translocate to the spleen and mesenteric lymph nodes. We found that SFB stimulated GCRs in PP from day 6 after monoassociation, that GCRs only gradually waned over the entire length of colonization, that natural IgA production was increased to levels 24 to 63% of that of conventionally reared mice, and that SFB-specific IgA was produced but accounted for less than 1.4% of total IgA. Also, the proportion of CD4 ⁺ , CD45RB low T cells, indicative of activated cells, gradually increased in the PP to the level found in conventionally reared mice. Secondary colonization with M. morganii was able to stimulate GCRs anew, leading to a specific IgA antibody response. Previous stimulation of mucosal immunity by SFB did not prevent the translocation of M. morganii in the double-colonized mice. Our findings generally indicate that SFB are one of the single most potent microbial stimuli of the gut mucosal immune system.
Chapter
The gut of wood- and litter-feeding termites harbors a dense and diverse community of prokaryotes that contribute to the carbon, nitrogen and energy requirements of the insects. Acetogenesis from H2 plus CO2 by hindgut prokaryotes supports up to 1/3 of the respiratory requirement of some termite species; and N2-fixing and uric acid-degrading microbes can have a significant impact on termite N economy. Microelectrode studies reveal that hindguts consist of an anoxic lumen surrounded by a microoxic periphery — a finding consistent with the occurrence of both anaerobic and O2-dependent microbial metabolism in hindguts. They also suggest that the enigmatic dominance of acetogens over methanogens as an H2 “sink” reflects a spatial separation of these H2-consuming populations, with the former being closer to sources of H2 production. Isolation of a number of the prokaryotes (including spirochetes, which have proven to be H2/CO2-acetogens) reveals that termite guts are a source of novel microbial diversity. However, molecular biological analyses indicate that much of that diversity is still poorly represented in culture.
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We have regularly cultured yeasts from the gut of certain beetles in our ongoing research. In this study cloned PCR products amplified from the gut contents of certain mushroom-feeding and wood-ingesting beetles in four families (Erotylidae, Tenebrionidae, Ciidae, and Passalidae) were sequenced and compared with culture results. Cultural techniques detected some yeasts present in the gut of the beetles, including a Pichia stipitis-like yeast associated with wood-ingesting passalid beetles. Clone sequences similar to several ascomycete yeasts and Malassezia restricta, a fastidious basidiomycetous yeast requiring special growth media, however, were not detected by culturing. Unexpectedly, phylogenetic analysis of additional clone sequences discovered from passalid beetles showed similarity to members of the Parabasalia, protists known from other wood-ingesting insects, termites, and wood roaches. Examination of all gut regions of living passalids, however, failed to reveal parabasalids, and it is possible that they were parasites in the gut tissue present in low numbers.
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The phylogenetic diversity of parabasalian flagellates was examined based on the sequences of small subunit ribosomal RNA genes amplified directly from the mixed population of flagellates in the hindgut of lower termites. In total, 33 representative sequences of parabasalids were recovered from eight termite species. Fluorescent-labeled oligonucleotide probes specific for certain sequences were designed and used for the in situ identification of parabasalian species by whole-cell hybridization. The hypermastigotes, Pseudotrichonympha grassii, Spirotrichonympha leidyi, and Holomastigotoides mirabile in the hindgut of Coptotermes formosanus, and Spirotrichonympha sp. and Trichonympha spp. in Hodotermopsis sjoestedti were identified. In the phylogenetic tree constructed, the sequences from the termites were dispersed within the groups of known members of parabasalids, reflecting the presence of diverse parabasalids in the hindgut of termites. There were three paraphyletic lineages of hypermastigotes represented by Pseudotrichonympha, Trichonympha, and Spirotrichonympha, in agreement with the morphology-based taxonomic groups. The analysis of the tree-root suggested that the Pseudotrichonympha group is the most probable ancient lineage of parabasalids and that the Trichonympha group is the secondly deep-branching lineage. The Spirotrichonympha group and the Trichomonadida may have emerged later.
Article
Epidermal cells in Calpodes and other insects form basal processes or feet that at first extend axially and later shorten at the same time as the larval segment shortens to the pupal shape. The feet grow into spaces at the surfaces of other cells to make a basal interlacing meshwork of cellular extensions that are combined mechanically by their desmosomal attachments to cell bodies above and to the basal lamina below. Microtubules and microfilaments are linked to these junctions by a reticular fibrous matrix. Gap junctions on the feet may couple cells that are several cell bodies removed from one another. The meshwork is also a sieve separating the hemolymph from the spaces between cells to form an intercellular compartment. Entry to the intercellular compartment is through the sieve made by the negatively charged basolateral cell surfaces that can prevent the entry of positively charged molecules such as cationic ferritin. As the cells become columnar, coincident with the metamorphic change in segment shape, the feet shorten and pack more densely together. At this time the basal lamina buckles axially as if responding to contraction of the feet. Segment shape change involves cell rearrangement and relative cell movement, necessitating the transient loss of plasma membrane plaque attachments to the cuticle apically and the loss of junctions laterally. Gap junctions involute in characteristic vacuoles. The metamorphic reduction in cell surface area coincides with the loss of basolateral membrane in smooth tubes and vesicles and the turnover of the apical surface in multivesicular bodies. New apical plasma membrane plaques and new lateral and basal junctions stabilize the cells in their pupal positions.
Article
The eukraotic cell, the unit of structure of protoctists, plants, fungi, and animals, is not at all homologous to prokaryotic cells. Instead the eukaryotic cell is homologous to communities of microorganisms such as those of the sulfuretum. This research is based on the hypothesis that at least four different interacting community members entered the original associations that, when stabilized, led to the emergence of eukaryotic cells. These are: (1) host nucleocytoplasm (thermoplasma like archaebacteria); (2) mitochrondria (paracoccus or bdellovibryo like respiring bacteria; and (3) plastids (cyanobacteria) and undulipodia. Tubulin like protein was found in the free living spirochete Spirochaeta bajacaliforniensis and in several other spirochetes. The amino acid sequence was to see if the spirochete protein is homologous to the tubulin of undulipodial and mitotic spindle microtubules.
Article
Cell proliferation in Drosophila imaginal discs appears to be regulated by a disc-intrinsic mechanism involving local cell interactions that also control the formation of patterns of differentiation. This growth-control mechanism breaks down in animals homozygous for the mutation lethal (2) giant discs (l(2)gd) which remain as larvae for up to 9 days longer than normal. During this time cell proliferation continues in the imaginal discs as well as in the imaginal rings for the salivary glands, foregut, and hindgut, so that these tissues become greatly overgrown. When wild-type wing discs from mid-third instar larvae were removed and cultured for up to 28 days in wild-type female adult hosts, they grew and terminated growth at a cell number close to that which would be attained in situ by the time of pupariation. On the other hand, wing discs from l(2)gd homozygotes grew rapidly and continuously when cultivated in wild-type hosts, reached an enormous size, and acquired abnormal folding patterns. Overgrowth of mutant imaginal rings also continued during culture of these tissues in wild-type hosts. We conclude that overgrowth in this mutant is due to an autonomous defect in the imaginal primordia, which requires an extended larval period for its expression in situ.
Article
The basal surface in transporting epithelia is infolded in a way that encourages the formation of standing gradients. Many insect cells have a similar infolded reticular system (RS) although they are clearly not transporting epithelia. These cells are like one another metabolically in that they sequester lipid from hemolymph lipophorins (lipid transporting proteins). Dietary lipids enter the hemolymph from the midgut RS which may be an adaptation for lipophorin loading. The plasma membrane reticular system of tissues metabolizing lipids (fat body, wax glands, oenocytes, lenticles) may be an adaptation for lipophorin reception and unloading. Cationic ferritin (pI 8.5) shows all RSs are covered by a lamina functioning as a negatively charged sieve. The basal plasma membrane leading to the RS is also negatively charged. The RS is a container with charged entrances that would be expected to affect the composition of the contents. Midgut cells release lipid particles into their RS. The particles are positively charged since in tracer studies they associate with anionic but not cationic ferritin. Lipophorins are anionic. The electrostatic binding of lipid to lipophorin would make it less anionic and more likely to leave the RS when loaded, thus carrying lipid to the hemolymph. Conversely, at the destination RS, loaded lipophorin would penetrate more easily than unloaded. A change in charge with unloading would be expected to alter the equilibrium between entering and leaving lipophorin, causing protein concentration in the RS of lipid receiving tissues as has been observed in the fat body. Reticular systems may thus be reaction vessels for interactions between carrier proteins and their load.
Article
The colonic epithelium has been examined for ultrastructural evidence of physiological activity. The cells show extensive folding of the apical plasma membrane, associated with mitochondria and an internal coating of particles about 120 A diameter. Anteriorly many apical infoldings are dilated at the tip to form substantial extracellular spaces up to 0.8 micron wide. Narrow intercellular channels are present, opening to the haemolymph side of the epithelium. Pinocytosis is seen frequently at the basal surface. The surface densities of apical plasma membranes were not significantly different in the posterior mid-gut and colon. Similarly the volume densities of mitochondria were equal in the colonic epithelium and rectal pads, but the surface density of outer mitochondrial membranes was greater in the colon. It is suggested that the colon may absorb organic solutes from the gut lumen.
Article
The traditional view of cellulose digestion in animals is that they cannot produce their own cellulase, and so rely on gut microorganisms to hydrolyse cellulose. A classic example of this symbiosis is that between phylogenetically lower termites and the unicellular organisms (protists) that colonize their hindguts: cellulose fermented to acetate by the protists can be used as an energy source by the termite. There is evidence for the production of endogenous cellulase components by termites and other wood-feeding insects; however, an unambiguous origin for such enzymes has not been established, to our knowledge, until now. Here we describe the first insect cellulase-endoding gene to be identified, RsEG, which encodes an endo-beta-1,4-glucanase (EC 3.2.1.4) in the termite Reticulitermes speratus.
Article
Segmented filamentous bacteria (SFB) are autochthonous bacteria inhabiting the intestinal tracts of many species, including humans. We studied the effect of SFB on the mucosal immune system by monoassociating formerly germfree C3H/HeN mice with SFB. At various time points during 190 days of colonization, fragment cultures of small intestine and Peyer's patches (PP) were analyzed for total immunoglobulin A (IgA) and SFB-specific IgA production. Also, phenotypic changes indicating germinal center reactions (GCRs) and the activation of CD4(+) T cells in PP were determined by using fluorescence-activated cell sorter analyses. A second group of SFB-monoassociated mice was colonized with a gram-negative commensal, Morganella morganii, to determine if the mucosal immune system was again stimulated and to evaluate the effect of prior colonization with SFB on the ability of M. morganii to translocate to the spleen and mesenteric lymph nodes. We found that SFB stimulated GCRs in PP from day 6 after monoassociation, that GCRs only gradually waned over the entire length of colonization, that natural IgA production was increased to levels 24 to 63% of that of conventionally reared mice, and that SFB-specific IgA was produced but accounted for less than 1.4% of total IgA. Also, the proportion of CD4(+), CD45RBlow T cells, indicative of activated cells, gradually increased in the PP to the level found in conventionally reared mice. Secondary colonization with M. morganii was able to stimulate GCRs anew, leading to a specific IgA antibody response. Previous stimulation of mucosal immunity by SFB did not prevent the translocation of M. morganii in the double-colonized mice. Our findings generally indicate that SFB are one of the single most potent microbial stimuli of the gut mucosal immune system.
Article
Spirochetes from termite hindguts and freshwater sediments possessed homologs of a nitrogenase gene (nifH) and exhibited nitrogenase activity, a previously unrecognized metabolic capability in spirochetes. Fixation of 15-dinitrogen was demonstrated with termite gut Treponema ZAS-9 and free-living Spirochaeta aurantia. Homologs of nifH were also present in human oral and bovine ruminal treponemes. Results implicate spirochetes in the nitrogen nutrition of termites, whose food is typically low in nitrogen, and in global nitrogen cycling. These results also proffer spirochetes as a likely origin of certain nifHs observed in termite guts and other environments that were not previously attributable to known microbes.
Article
Endodermal or midgut cells have only recently been recognized as the site of pheromone synthesis in bark beetles. Midgut cells are not only specialized for digestion, but they have also been recruited to form isoprenoid compounds that function as pheromone components in Ips pini and Dendroctonus jeffreyi. Male bark beetle midgut cells are competent to produce isoprenoid pheromones after feeding or stimulation by juvenile hormone (JH) III. Competent midgut cells share many ultrastructural features with cells that do not secrete isoprenoid pheromone, but they are distinguished from these by abundant and highly ordered arrays of smooth endoplasmic reticula. During secretion, both midgut cells that produce pheromone and cells that do not are characterized by the presence of apical extrusions (apocrine secretion) rather than the presence of vesicles that fuse with the apical membrane and undergo exocytosis (eccrine secretion). Pheromone-producing cells of the midgut do not represent a population of cells that are distinct from cells involved in digestion. All, or most, midgut cells of male I. pini and D. jeffreyi can secrete pheromones as well as digestive enzymes.
Article
Fixed nitrogen is a limiting nutrient in most terrestrial ecosystems and has been assumed to be supplied almost entirely by free-living bacteria as well as by bacteria living in association with plants. The survival and growth of many arthropods on diets with extremely high carbon to nitrogen (C:N) ratios suggest that these arthropods are not obtaining sufficient nitrogen from their diets but must be obtaining additional nitrogen from some other source(s). Estimates of N(2) fixation have suggested that symbiotic microbes of some arthropod hindguts could be obtaining this additional nitrogen as a result of nitrogen fixation. With the recent availability of antibody and nucleic acid probes, the presence of the enzyme that reduces dinitrogen gas to ammonia (nitrogenase) as well as the presence of its transcripts can be detected and localized with great sensitivity. A preliminary survey of a few detritivores indicates that nitrogen-fixing microbes of diverse forms are widespread in arthropod hindguts. In calculating nitrogen budgets, the possible contributions of nitrogen fixation by symbionts in arthropod guts, however, has been largely ignored. N(2) fixation in arthropod guts, with rates as high as 10-40 kg/ha/year being possible, may represent a significant contribution both to the growth of arthropods and to their ecosystem functions of processing carbon and nitrogen.
Article
During a survey of insect gut micro-organisms, we consistently isolated Pichia stipitis-like yeasts (Fungi: Ascomycota, Saccharomycetes) from the wood-ingesting beetles, Odontotaenius disjunctus and Verres sternbergianus (Coleoptera: Passalidae). The yeasts were isolated from passalid beetles over a wide area, including the eastern and midwestern USA and Panama. Phylogenetic analyses of the nuclear encoded small and large subunit rRNA gene (rDNA) sequences distinguished a well-supported clade consisting of the passalid yeasts and Pichia stipitis, P. segobiensis, Candida shehatae and C. ergatensis. Members of this clade have the ability to ferment and assimilate xylose or to hydrolyse xylan, major components of the polysaccharide, hemicellulose. Sexual reproduction was present in the passalid isolates but was rare among the gut yeasts of other beetles to which they were compared. Minor genetic and phenotypic variation among some of the passalid yeasts was detected using markers from the internal transcribed spacer region of the rDNA repeat unit, morphology, and in vitro metabolic tests. The consistent association of xylose-fermenting yeasts of almost identical genotypes with passalid beetles across a broad geographical distribution, suggests a significant symbiotic association.
Article
We have regularly cultured yeasts from the gut of certain beetles in our ongoing research. In this study cloned PCR products amplified from the gut contents of certain mushroom-feeding and wood-ingesting beetles in four families (Erotylidae, Tenebrionidae, Ciidae, and Passalidae) were sequenced and compared with culture results. Cultural techniques detected some yeasts present in the gut of the beetles, including a Pichia stipitis-like yeast associated with wood-ingesting passalid beetles. Clone sequences similar to several ascomycete yeasts and Malassezia restricta, a fastidious basidiomycetous yeast requiring special growth media, however, were not detected by culturing. Unexpectedly, phylogenetic analysis of additional clone sequences discovered from passalid beetles showed similarity to members of the Parabasalia, protists known from other wood-ingesting insects, termites, and wood roaches. Examination of all gut regions of living passalids, however, failed to reveal parabasalids, and it is possible that they were parasites in the gut tissue present in low numbers.
Article
A novel cellulase [beta-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA belonging to glycoside hydrolase family (GHF) 45 was cloned from the mulberry longicorn beetle, Apriona germari. The cDNA encoding EGase of A. germari (Ag-EGase) is 711 bp long with an open reading frame of 237 amino acid residues. The Ag-EGase was closely related to another beetle, Phaedon cochleariae, cellulase and one symbiotic protist cellulase in the hindgut of the termite Reticulitermes speratus, those belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase. Southern blot analysis of genomic DNA suggested the presence of Ag-EGase gene as a single copy and Northern blot analysis confirmed midgut-specific expression at transcriptional level. Similarly, the Ag-EGase enzyme assay exhibited high activity only in midgut tissue, suggesting that the midgut is the prime site where large quantities of EGase are synthesized for degrading the absorbed cellulose from the diet. The cDNA encoding Ag-EGase was expressed as a 29-kDa polypeptide in baculovirus-infected insect Sf9 cells and the culture supernatants of the recombinant baculovirus-infected cells showed EGase enzyme activity of 15.25 U/ml of medium containing 0.5 x 10(6) cells at 5 days post-infection (p.i.). The enzyme activity of the purified recombinant Ag-EGase expressed in baculovirus-infected insect cells was approximately 992 U per mg of recombinant Ag-EGase. The purified recombinant Ag-EGase showed the highest enzymatic activity at 50 degrees C and pH 6.0, and was stable at 55 degrees C at least for 10 min.
Article
Termites are among the most important cellulose-digesting animals on earth, and are well-known for the symbiotic relationship they have with cellulolytic trichomonad and oxymonad flagellates (unicellular eukaryotes). Perhaps less well-known is the fact that approximately 75% of the approximately 2600 described termite species -- those belonging to the family Termitidae -- do not harbour such flagellates. Unlike most termites from other families, the majority of termitids do not consume wood, feeding instead on soil, leaf litter, fungi, grass, or lichen. Recent years have seen the characterization of the endogenous cellulase enzymes that help termites digest cellulose, from one flagellate-harbouring species (Reticulitermes speratus), as well as one termitid (Nasutitermes takasagoensis). The genes encoding the enzymes in these two termites are similar. However, their site of expression differs markedly -- the salivary glands in R. speratus and the midgut in N. takasagoensis. To investigate this difference further, we performed a comparative study of cellulase expression in various termitid and flagellate-harbouring species, using enzyme assays and reverse transcription polymerase chain reactions. Taxa from phylogenetically basal lineages were consistently found to express endogenous genes specifically in the salivary glands, whilst those from a relatively apical lineage containing termitids expressed cellulases solely in the midgut. Relatively low levels of cellulase activity were found in nonwood-feeding species, while the wood-feeding Coptotermes formosanus -- arguably the most destructive pest species world-wide -- was found to have high levels of activity in all parts of the gut when compared to all other termites. In the light of these results, as well as recently accumulated phylogenetic data, we discuss scenarios for the evolution of cellulose digestion in termites.
Symbiont-mediated detoxification in insect herbivores
  • Dowd
Dowd, P.F., 1991. Symbiont-mediated detoxification in insect herbivores.
Passalus und seine intestinale Flora
  • Heymonds
Heymonds, R., Heymonds, H., 1934. Passalus und seine intestinale Flora. Biologisches Zentralblatt 54, 40–51.
On the existence of entophyta in healthy animals, as a natural condition
  • Leidy
Termite microbial communities
  • Grosovsky