Linking lignocellulosic dietary patterns with gut microbial Enterotypes of Tsaitermes ampliceps and comparison with Mironasutitermes shangchengensis.

Tsaitermes ampliceps (lower termites) and Mironasutitermes shangchengensis (higher termites) are highly eusocial insects that thrive on recalcitrant lignocellulosic diets through nutritional symbioses with gut dwelling prokaryotes and eukaryotes. We used denaturing gradient gel electrophoresis and a 16S rRNA clone library to investigate i) how microbial communities adapt to lignocellulosic diets with different cellulose and lignin content, ii) the differences in the dominant gut microbial communities of the 2 types of termites. The results indicated that gut microbiota composition in T. ampliceps was profoundly affected by 2-week diet shifts. Comparison of these changes indicated that Bacteroidetes and Spirochaetes act in cellulose degradation, while Firmicutes were responsible for lignin degradation. Additionally, Proteobacteria consistently participated in energy production and balanced the gut environment. Bacteroidetes may function without hindgut protozoans in higher termites. The diversity of enteric microorganisms in M. shangchengensis was higher than that in T. ampliceps, possibly because of the more complicated survival mechanisms of higher termites.

Cellulolytic activity and structure of symbiotic bacteria in locust guts.

Locusts are able to digest the cellulose of Gramineae plants, resulting in their being considered as major crop pests. To illustrate the mechanism involved in cellulose digestion, the cellulolytic activity and zymography in the gut contents of 16 locust species were determined using carboxymethyl cellulose (CMC) as substrate. The diversity of gut symbiotic bacteria was studied using denaturing gradient gel electrophoresis (DGGE). The results showed that high CMC activity was present in Acrididae gut fluid (mean 356.4 U/g proteins). Of the 5 locust species, Oxya chinensis had the highest diversity of intestinal symbiotic bacteria, characterized by the DGGE profile containing more than 20 bands of 16S rRNA. Klebsiella pneumoniae, in the gut of Locusta migratoria manilensis, was identified as the most abundant symbiotic bacterium by DNA sequencing, with a relative abundance of 19.74%. In comparison, Methylobacterium sp was the most dominant species in the Atractomorpha sinensis gut, with a relative abundance of 29.04%. The results indicated that the cellulolytic enzymes and gut microbial communities probably reflected their phylogenetic relationship with different locust species and associated feeding strategies.

Evaluation of cellulolytic activity in insect digestive fluids.

Efficient and low-cost cellulolytic enzymes are urgently needed to degrade recalcitrant plant biomass during the industrial production of lignocellulosic biofuels. Here, the cellulolytic activities in the gut fluids of 54 insect species that belong to 7 different taxonomic orders were determined using 2 different substrates, carboxymethyl cellulose (CMC) (approximating endo-ß-1,4-glucanase) and filter paper (FP) (total cellulolytic activities). The use of CMC as the substrate in the zymogram analysis resulted in the detection of distinct cellulolytic protein bands. The cellulolytic activities in the digestive system of all the collected samples were detected using cellulolytic activity analysis. The highest CMC gut fluid activities were found in Coleoptera and Orthoptera, while FP analysis indicated that higher gut fluid activities were found in several species of Coleoptera and Lepidoptera. In most cases, gut fluid activities were higher with CMC than with FP substrate, except for individual Lepidoptera species. Our data indicate that the origin of cellulolytic enzymes probably reflects the phylogenetic relationship and feeding strategies of different insects.

In vitro mutagenesis of Xanthomonas campestris alpha-amylase gene by partially replacing deoxythymidine triphosphate with 5-bromo-2'-deoxyuridine-5'-triphosphate using a PCR technique.

Three mutants of the wild type alpha-amylase gene from Xanthomonas campestris pv. campestris 8004 were obtained using a PCR technique in which deoxythymidine triphosphate (dTTP) was partially replaced by 5-bromo-2'-deoxyuridine-5'-triphosphate (BrdUTP), at an optimal dTTP:BrdUTP ratio of 1000:1. Of thre three mutants that were obtained and which were sequenced, one mutant with 40 times higher activity than the wild type alpha-amylase gene product was obtained by using primary PCR products as a template for a second PCR reaction.