Reference gene selection for transcriptional profiling in Cryptocercus punctulatus, an evolutionary link between Isoptera and Blattodea

The subsocial life style and wood-feeding capability of Cryptocercus gives us an evolutionary key to unlock some outstanding questions in biology. With the advent of the Genomics Era, there is an unprecedented opportunity to address the evolution of eusociality and the acquisition of lignocellulases at the genetic level. However, to quantify gene expression, an appropriate normalization strategy is warranted to control for the non-specific variations among samples across different experimental conditions. To search for the internal references, 10 housekeeping genes from a gut transcriptome of a wood-feeding cockroach, Cryptocercus punctulatus, were selected as the candidates for the RT-qPCR analysis. The expression profiles of these candidates, including ACT, EF1α, GAPDH, HSP60, HSP70, αTUB, UBC, RPS18, ATPase and GST, were analyzed using a panel of analytical tools, including geNorm, NormFinder, BestKeeper, and comparative ΔCT method. RefFinder, a comprehensive ranking system integrating all four above-mentioned algorithms, rated ACT as the most stable reference gene for different developmental stages and tissues. Expression analysis of the target genes, Hex-1 and Cell-1, using the most or the least appropriate reference genes and a single or multiple normalizers signified this research. Our finding is the first step toward establishing a standardized RT-qPCR analysis in Cryptocercus.

Genetic insights into family group co-occurrence inCryptocercus punctulatus, a sub-social woodroach from the southern Appalachian Mountains

The wood-feeding cockroachCryptocercus punctulatusScudder (Blattodea: Cryptocercidae) is an important member of the dead wood (saproxylic) community in montane forests of the southeastern United States. However, its population biology remains poorly understood. Here, aspects of family group co-occurrence were characterized to provide basic information that can be extended by studies on the evolution and maintenance of sub-sociality. Broad sampling across the species’ range was coupled with molecular data (mitochondrial DNA (mtDNA) sequences). The primary questions were: (1) what proportion of rotting logs contain two or more different mtDNA haplotypes and how often can this be attributed to multiple families inhabiting the same log, (2) are multi-family logs spatially clustered, and (3) what levels of genetic differentiation among haplotypes exist within a log, and how genetically similar are matrilines of co-occurring family groups? Multi-family logs were identified on the premise that three different mtDNA haplotypes, or two different haplotypes among adult females, is inconsistent with a single family group founded by one male–female pair. Results showed that of the 88 rotting logs from which multiple adultC. punctulatuswere sampled, 41 logs (47%) contained two or more mtDNA haplotypes, and at least 19 of these logs (22% overall) were inferred to be inhabited by multiple families. There was no strong evidence for spatial clustering of the latter class of logs. The frequency distribution of nucleotide differences between co-occurring haplotypes was strongly right-skewed, such that most haplotypes were only one or two mutations apart, but more substantial divergences (up to 18 mutations, or 1.6% uncorrected sequence divergence) do occasionally occur within logs. This work represents the first explicit investigation of family group co-occurrence inC. punctulatus, providing a valuable baseline for follow-up studies.

Genome Evolution and Nitrogen Fixation in Bacterial Ectosymbionts of a Protist Inhabiting Wood-Feeding Cockroaches

ABSTRACTBy combining genomics and isotope imaging analysis using high-resolution secondary ion mass spectrometry (NanoSIMS), we examined the function and evolution ofBacteroidalesectosymbionts of the protistBarbulanymphafrom the hindguts of the wood-eating cockroachCryptocercus punctulatus. In particular, we investigated the structure of ectosymbiont genomes, which, in contrast to those of endosymbionts, has been little studied to date, and tested the hypothesis that these ectosymbionts fix nitrogen. Unlike with most obligate endosymbionts, genome reduction has not played a major role in the evolution of theBarbulanymphaectosymbionts. Instead, interaction with the external environment has remained important for this symbiont as genes for synthesis of transporters, outer membrane proteins, lipopolysaccharides, and lipoproteins have been retained. The ectosymbiont genome carried two complete operons for nitrogen fixation, a urea transporter, and a urease, indicating the availability of nitrogen as a driving force behind the symbiosis. NanoSIMS analysis ofC. punctulatushindgut symbionts exposedin vivoto15N2supports the hypothesis thatBarbulanymphaectosymbionts are capable of nitrogen fixation. This genomic andin vivofunctional investigation of protist ectosymbionts highlights the diversity of evolutionary forces and trajectories that shape symbiotic interactions.IMPORTANCEThe ecological and evolutionary importance of symbioses is increasingly clear, but the overall diversity of symbiotic interactions remains poorly explored. In this study, we investigated the evolution and nitrogen fixation capabilities of ectosymbionts attached to the protistBarbulanymphafrom the hindgut of the wood-eating cockroachCryptocercus punctulatus. In addressing genome evolution of protist ectosymbionts, our data suggest that the ecological pressures influencing the evolution of extracellular symbionts clearly differ from intracellular symbionts and organelles. Using NanoSIMS analysis, we also obtained direct imaging evidence of a specific hindgut microbe playing a role in nitrogen fixation. These results demonstrate the power of combining NanoSIMS and genomics tools for investigating the biology of uncultivable microbes. This investigation paves the way for a more precise understanding of microbial interactions in the hindguts of wood-eating insects and further exploration of the diversity and ecological significance of symbiosis between microbes.