Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite Reticulitermes flavipes

Termites have developed cellulose digestion capabilities that allow them to obtain energy and nutrition from nutritionally poor food sources, such as lignocellulosic plant material and residues derived from it (e.g., wood and humus). Lower termites, which are equipped with both endogenous (i.e., of termite origin) and symbiotic cellulases, feed primarily on wood and wood-related materials. This study investigated cellulase gene diversity, structure, and activity in the lower termite, Reticulitermes flavipes (Kollar). We initially used a metagenomics approach to identify four genes encoding one endogenous and three symbiotic cellulases, which we refer to as Cell-1, -2, -3 and -4. These four genes encode proteins that share significant sequence similarity with known endoglucanases, exoglucanases and xylanases. Phylogenetic analyses further supported these inferred relationships by showing that each of the four cellulase proteins clusters tightly with respective termite, protozoan or fungal cellulases. Gene structure studies revealed that Cell-1, -3 and -4 are intron-free, while Cell-2 contains the first intron sequence to be identified from a termite symbiont cellulase. Quantitative real-time PCR (qRT-PCR) revealed that the endogenous Cell-1 gene is expressed exclusively in the salivary gland/foregut, whereas symbiotic Cell-2, -3, and -4 are highly expressed in the hindgut (where cellulolytic protists are harbored). Cellulase activity assays mapped the distribution pattern of endoglucanase, exoglucanase and xylanase activity throughout the R. flavipes digestive tract. Cellulase gene expression correlated well with the specific types of cellulolytic activities observed in each gut region (foregut+salivary gland, midgut and hindgut). These results suggest the presence of a single unified cellulose digestion system, whereby endogenous and symbiotic cellulases work sequentially and collaboratively across the entire digestive tract of R. flavipes.     

Functional and translational analyses of a beta-glucosidase gene (glycosyl hydrolase family 1) isolated from the gut of the lower termite Reticulitermes flavipes

This research focused on digestive beta-glucosidases from glycosyl hydrolase family (GHF) 1 from the gut of the lower termite Reticulitermes flavipes. In preceding studies on R. flavipes, we characterized beta-glucosidase activity across the gut and its inhibition by carbohydrate-based inhibitors, and subsequently we identified two partial beta-glucosidase cDNA sequences from a host gut cDNA library. Here, we report on the full-length cDNA sequence for one of the R. flavipes beta-glucosidases (RfBGluc-1), the expression of its mRNA in the salivary gland and foregut, the production of recombinant protein using a baculovirus–insect expression system, optimal recombinant substrate specificity profiles and parameters, and significant inhibition by the established beta-glucosidase inhibitor cellobioimidazole. We also report the partial cDNA sequence for a second gut beta-glucosidase (RfBGluc-2), and show that like RfBGluc-1 its mRNA is localized mainly in the salivary gland. Other results for RfBGluc-1 showing activity against laminaribose, a component of microbial cell walls, suggest that RfBGluc-1 may serve dual functions in cellulose digestion and immunity. These findings provide important information that will enable the testing of hypotheses related to collaborative host–symbiont lignocellulose digestion, and that contributes to the development of next-generation termiticides and novel biocatalyst cocktails for use in biomass-to-bioethanol applications.     

Characterization of four esterase genes and esterase activity from the gut of the termite Reticulitermes flavipes

Four esterase genes and general esterase activity were investigated in the gut of the termite Reticulitermes flavipes. Two genes (RfEst1 and RfEst2) share significant translated identity with a number of insect JH esterases. The two remaining genes (RfEst3 and RfEst4) apparently code for much shorter proteins with similarity to fungal phenolic acid esterases involved in hemicellulose solubilization. All four genes showed consistently high midgut expression. This result was further supported by colorimetric activity assays and Native polyacrylamide gel electrophoresis, which showed significant esterase activity and a number of isoforms in the midgut. The greatest esterase activity and isoform composition were detected when α‐naphthyl propionate was used as a substrate. Moreover, esterase activity and diverse isoforms were present in gut mitochondrial, microsomal, and cytosolic sub‐cellular protein fractions, as well as in the hindgut lumen. These findings reveal an agreement between gut esterase gene expression and activity distributions, and support the idea that R. flavipes gut esterase activity is host (not symbiont)‐derived. In addition, these findings support the hypotheses that termite gut esterases may play important roles in lignocellulose digestion and caste differentiation. This study provides important baseline data that will assist ongoing functional‐genomic efforts to identify novel genes with roles in semiochemical, hormone, and lignocellulose processing in the termite gut. © 2009 Wiley Periodicals, Inc.     

High-quality draft genome sequence of the Opitutaceae bacterium strain TAV1, a symbiont of the wood-feeding termite Reticulitermes flavipes

Microbial communities in the termite hindgut are essential for degrading plant material. We present the high-quality draft genome sequence of the Opitutaceae bacterium strain TAV1, the first member of the phylum Verrucomicrobia to be isolated from wood-feeding termites. The genomic analysis reveals genes coding for lignocellulosic degradation and nitrogen fixation.     

RNA interference in the termite Reticulitermes flavipes through ingestion of double-stranded RNA

RNA interference (RNAi) represents a breakthrough technology for conducting functional genomics research in non-model organisms and for the highly targeted control of insect pests. This study investigated RNAi via voluntary feeding in the economically important pest termite, Reticulitermes flavipes. We used a high-dose double-stranded (ds) RNA feeding approach to silence two termite genes: one encoding an endogenous digestive cellulase enzyme and the other a caste-regulatory hexamerin storage protein. Contrary to results from previous low-dose studies that examined injection-based RNAi, high-dose silencing of either gene through dsRNA feeding led to significantly reduced group fitness and mortality. Hexamerin silencing in combination with ectopic juvenile hormone treatments additionally led to lethal molting impacts and increased differentiation of presoldier caste phenotypes (a phenotype that is not capable of feeding). These results provide the first examples of insecticidal effects from dsRNA feeding in a termite. Additionally, these results validate a high-throughput bioassay approach for use in (i) termite functional genomics research, and (ii) characterizing target sites of conventional and novel RNAi-based termiticides.     

Endocrine control of cuticular hydrocarbon profiles during worker-to-soldier differentiation in the termite Reticulitermes flavipes

The social organization of termites, unlike that of other social insects, is characterized by a highly plastic caste system. With the exception of the alates, all other individuals in a colony remain at an immature stage of development. Workers in particular remain developmentally flexible; they can switch castes to become soldiers or neotenics. Juvenile hormone (JH) is known to play a key role in turning workers into soldiers. In this study, we analyzed differences in cuticular hydrocarbon (CHC) profiles among castes, paying particular attention to the transition of workers to soldiers, in the subterranean termite species Reticulitermes flavipes. CHCs have a fundamental function in social insects as they serve as cues in inter- and intraspecific recognition. We showed that (1) the CHC profiles of the different castes (workers, soldiers, nymphs and neotenics) are different and (2) when workers were experimentally exposed to a JH analog and thus induced to become soldiers, their CHC profiles were modified before and after the worker-presoldier molt and before and after the presoldier-soldier molt.     

Phylogeny of symbiotic methanogens in the gut of the termite Reticulitermes speratus

Abstract The phylogeny of a symbiotic methanogen inhabiting the gut of a lower termite, Reticulitermes speratus , was analysed without cultivation. The small subunit ribosomal RNA gene (ssrDNA) and a 640-bp portion of the gene encoding subunit A of methyl coenzyme M reductase ( mcrA ) were amplified from a mixed-population DNA of the termite gut by polymerase chain reaction and cloned. The nucleotide sequence of the ssrDNA and the predicted amino acid sequence of the mcrA product were compared with those of the known methanogens. Both comparisons indicated that the termite symbiotic methanogen belonged to the order Methanobacteriales but was distinct from the known members of this order.     

A test of seasonal responses to sugars in four populations of the termite Reticulitermes flavipes

Several studies have shown that Reticulitermes termites prefer food with certain types of sugars. However, the specific sugars that were preferred by the termites in each study differed. The difference between the results of these studies might be explained by differences between populations or changes in feeding responses during the active season. To address these variables, we examined the feeding response to a food source food containing glucose, sucrose, or xylose versus a food source without sugar in several populations of termites and observed whether these responses changed during the year. Termites were collected from colonies from four field sites in Missouri during the spring (May and June), summer (July and August), and fall (September and October) and tested for their response to all three sugars under laboratory conditions. Results show there are distinct differences in response to sugars between populations but only a slight seasonal effect.     

High occurrence of colony fusion in a European population of the American termite Reticulitermes flavipes

The coexistence of multiple unrelated reproductives within social insect colonies decreases the relatedness among colony members and therefore challenges kin selection theory. This study investigated the colony genetic structure of a French introduced population of the American subterranean termite Reticulitermes flavipes by analyzing genotypes at eight microsatellite loci and at one mtDNA region. Results revealed that all colonies contained numerous related secondary reproductives, and that 31% of colonies possessed more than two unrelated reproductives. The presence of several unrelated reproductives within colonies of this species is commonly assumed to result from colony fusion. Although such a high occurrence of colony fusion is the highest ever observed in a termite population, it is probable that the available methodology underestimated the detection of colony fusion in French populations. Overall, these results suggest that French colonies might differ strongly from the great majority of American colonies in their capacity to produce secondary reproductives as well as in their ability to merge. The nature and evolutionary origin of these population differences are discussed.     

DNA-based identification of the eastern subterranean termite, Reticulitermes flavipes (Isoptera: Rhinotermitidae)

A DNA-based system for the identification of Reticulitermes flavipes (Koller) was established based on the following criteria: adequate molecular variation, comprehensive specimen sampling, explicit morphological species identification, and cladistic analysis. Termite soldiers were identified by labral shape, and these specimens and associated pseudergates were used in subsequent molecular analyses. Mitochondrial DNA from the AT-rich region was sequenced for 173 R. flavipes, Reticulitermes hageni Banks and Reticulitermes virginicus (Banks) soldiers and pseudergates collected from several widely dispersed Texan localities. Cladistic analysis was performed after exploration of an optimal sequence alignment inclusive of DNA sequences from Texas and published sequences from Georgia and Canada. Among the Texan individuals, 29, 5, and 1 mitochondrial DNA haplotypes were identified respectively with R. flavipes, R. hageni, and R. virginicus. One haplotype from El Paso likely represented a different Reticulitermes species. Species were monophyletic; however, individual relationships were unresolved in a strict consensus of 700 most parsimonious trees. Texas haplotypes were isolated by distance, and a correlation between genetic and geographic distance was observed among the Texas, Georgia, and Canada haplotypes. These results suggest that the methods outlined in this study will allow for the identification of R. flavipes from localities between Texas and Canada.     

The hindgut lumen prokaryotic microbiota of the termite Reticulitermes flavipes and its responses to dietary lignocellulose composition

Reticulitermes flavipes (Isoptera: Rhinotermitidae) is a highly eusocial insect that thrives on recalcitrant lignocellulosic diets through nutritional symbioses with gut‐dwelling prokaryotes and eukaryotes. In the R. flavipes hindgut, there are up to 12 eukaryotic protozoan symbionts; the number of prokaryotic symbionts has been estimated in the hundreds. Despite its biological relevance, this diverse community, to date, has been investigated only by culture‐ and cloning‐dependent methods. Moreover, it is unclear how termite gut microbiomes respond to diet changes and what roles they play in lignocellulose digestion. This study utilized high‐throughput 454 pyrosequencing of 16S V5‐V6 amplicons to sample the hindgut lumen prokaryotic microbiota of R. flavipes and to examine compositional changes in response to lignin‐rich and lignin‐poor cellulose diets after a 7‐day feeding period. Of the ~475 000 high‐quality reads that were obtained, 99.9% were annotated as bacteria and 0.11% as archaea. Major bacterial phyla included Spirochaetes (24.9%), Elusimicrobia (19.8%), Firmicutes (17.8%), Bacteroidetes (14.1%), Proteobacteria (11.4%), Fibrobacteres (5.8%), Verrucomicrobia (2.0%), Actinobacteria (1.4%) and Tenericutes (1.3%). The R. flavipes hindgut lumen prokaryotic microbiota was found to contain over 4761 species‐level phylotypes. However, diet‐dependent shifts were not statistically significant or uniform across colonies, suggesting significant environmental and/or host genetic impacts on colony‐level microbiome composition. These results provide insights into termite gut microbiome diversity and suggest that (i) the prokaryotic gut microbiota is much more complex than previously estimated, and (ii) environment, founding reproductive pair effects and/or host genetics influence microbiome composition.     

Immunomarking reveals food flow and feeding relationships in the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Trophallaxis and feeding relationships in the eastern subterranean termite, Reticulitermes flavipes (Kollar), were examined using a novel marking technique, rabbit IgG protein coupled with an enzyme linked immunosorbent assay (ELISA) to detect the marker. Transfer experiments in small dishes evaluated the trophallactic transfer of the marker from donor workers fed IgG-treated paper to recipient workers or larvae. Worker donors rapidly acquired the marker, and 100% of donors tested positive within 24 h. Trophallactic transfer from donors to recipients was relatively inefficient, and 51 +/- 2% of recipient workers and 31 +/- 2% of recipient larvae tested positive at 72 h. Based on the mean optical density counts, approximately 27% of marker ingested by the donors was passed on to the recipient workers in the first 24 h, 14% to recipient larvae, and 26% to recipient soldiers. The ability of soldiers to feed independently of workers was examined in dish assays. Soldiers showed no significant uptake of the marker when isolated from the workers, and uptake increased significantly when workers were present. The distribution of the marker was further studied in larger colony fragments composed of workers, soldiers, nymphs, and larvae. Marker acquisition by the different castes/developmental stages was highly variable, with workers and nymphs acquiring the marker at a faster rate than soldiers and larvae. The results of this study contribute to our understanding of the foraging ecology and social behavior in R. flavipes. In addition, they may help design improved control programs for subterranean termites based on baits.     

Isolation of cockroach Phe-Gly-Leu-amide allatostatins from the termite Reticulitermes flavipes and their effect on juvenile hormone synthesis

Immunoreactivity to cockroach Diploptera punctata allatostatin-7 (Dippu AST-7) has been demonstrated previously in axons innervating the corpora allata of the termite Reticulitermes flavipes. This peptide and Dippu AST-11 inhibited juvenile hormone (JH) synthesis by corpora allata (CA) of brachypterous neotenic reproductives (secondary reproductives) of termites. The present study shows that R. flavipes CA are also inhibited by Dippu AST-2, AST-5, AST-8, and AST-9 at approximately the same rank order of potency as demonstrated in D. punctata. Another allatostatin from Periplaneta americana (Peram AST-12) also inhibits JH synthesis by R. flavipes CA. Sensitivity to the allatostatins is higher in glands with low rates of JH synthesis than in those with relatively high JH synthetic rates as has been demonstrated in CA from male and female secondary reproductives as well as in those from non-egg-laying and egg-laying females. The identical inhibitory effects of R. flavipes brain extract on CA from both D. punctata and R. flavipes and the isolation and identification of five cockroach allatostatins (Dippu AST-1, AST-2, AST-5, AST-8, and Peram AST-12) from termite brain extract reflect the close relationship between cockroaches and termites.     

Global genetic analysis reveals the putative native source of the invasive termite,Reticulitermes flavipes,in France

Biological invasions are recognized as a major threat to both natural and managed ecosystems. Phylogeographic and population genetic analyses can provide information about the geographical origins and patterns of introduction and explain the causes and mechanisms by which introduced species have become successful invaders. Reticulitermes flavipes is a North American subterranean termite that has been introduced into several areas, including France where introduced populations have become invasive. To identify likely source populations in the USA and to compare the genetic diversity of both native and introduced populations, an extensive molecular genetic study was undertaken using the COII region of mtDNA and 15 microsatellite loci. Our results showed that native northern US populations appeared well differentiated from those of the southern part of the US range. Phylogenetic analysis of both mitochondrial and nuclear markers showed that French populations probably originated from southeastern US populations, and more specifically from Louisiana. All of the mtDNA haplotypes shared between the United States and France were found in Louisiana. Compared to native populations in Louisiana, French populations show lower genetic diversity at both mtDNA and microsatellite markers. These findings are discussed along with the invasion routes of R. flavipes as well as the possible mechanisms by which French populations have evolved after their introduction.     

Phylogenetic diversity and whole-cell hybridization of oxymonad flagellates from the hindgut of the wood-feeding lower termite Reticulitermes flavipes

SSU rRNA genes of oxymonad protists from the hindgut of the wood-feeding termite Reticulitermes flavipes were PCR-amplified using a newly designed oxymonad-specific forward primer and a newly designed reverse primer specific for termite gut flagellates. After cloning, the clone library was sorted into four groups by RFLP analysis and nearly full-length SSU rRNA gene sequences were obtained for representative clones from each group. Phylogenetic analysis revealed that sequences of all four groups formed a monophyletic cluster with the only other existing SSU rRNA gene sequence of oxymonads. Using whole-cell hybridization with clone-specific fluorescently labeled probes, each of the four clone groups could be assigned to a specific morphotype, which were identified as Dinenympha gracilis, Dinenympha fimbriata, and so-far undescribed species of Pyrsonympha and Dinenympha. Our results demonstrate that the morphological variety of oxymonads is not caused by the presence of different developmental stages of the same organism, but that the various morphotypes represent different species.     

Phe-Gly-Leu-amide allatostatin in the termite Reticulitermes flavipes: content in brain and corpus allatum and effect on juvenile hormone synthesis

In the subterranean termite Reticulitermes flavipes, allatostatins (ASTs) with the C-terminus Phe-Gly Leu-amide were localized by immunocytochemistry with antibody against a cockroach AST, Dippu AST-7. AST-immunoreactivity occurred in the corpus cardiacum and corpus allatum and in the lateral and medial neurosecretory cells of the brain that innervate these organs as well as in many other nerve cells of the brain. This was observed in workers, nymphs, soldiers and secondary reproductives. A radioimmunoassay, using anti-Dippu AST-11, demonstrated about 40 fmole equivalents of AST in brains of soldiers and secondary reproductives. The product of the corpora allata in this species was determined to be juvenile hormone III. Its synthesis by corpora allata of secondary reproductives, determined by in vitro radiochemical assay, was inhibited in a dose-dependent fashion by two cockroach allatostatins, Dippu AST-7 and Dippu AST-11. Thus, as in cockroaches and crickets, allatostatin-containing nerves innervate the corpora allata of this termite species and their production of juvenile hormone is inhibited by these neuropeptides.