Multiple Levels of Synergistic Collaboration in Termite Lignocellulose Digestion

In addition to evolving eusocial lifestyles, two equally fascinating aspects of termite biology are their mutualistic relationships with gut symbionts and their use of lignocellulose as a primary nutrition source. Termites are also considered excellent model systems for studying the production of bioethanol and renewable bioenergy from 2nd generation (non-food) feedstocks. While the idea that gut symbionts are the sole contributors to termite lignocellulose digestion has remained popular and compelling, in recent years host contributions to the digestion process have become increasingly apparent. However, the degree to which host and symbiont, and host enzymes, collaborate in lignocellulose digestion remain poorly understood. Also, how digestive enzymes specifically collaborate (i.e., in additive or synergistic ways) is largely unknown. In the present study we undertook translational-genomic studies to gain unprecedented insights into digestion by the lower termite Reticulitermes flavipes and its symbiotic gut flora. We used a combination of native gut tissue preparations and recombinant enzymes derived from the host gut transcriptome to identify synergistic collaborations between host and symbiont, and also among enzymes produced exclusively by the host termite. Our findings provide important new evidence of synergistic collaboration among enzymes in the release of fermentable monosaccharides from wood lignocellulose. These monosaccharides (glucose and pentoses) are highly relevant to 2nd-generation bioethanol production. We also show that, although significant digestion capabilities occur in host termite tissues, catalytic tradeoffs exist that apparently favor mutualism with symbiotic lignocellulose-digesting microbes. These findings contribute important new insights towards the development of termite-derived biofuel processing biotechnologies and shed new light on selective forces that likely favored symbiosis and, subsequently, group living in primitive termites and their cockroach ancestors

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Comparative genomics of small RNA regulatory pathway components in vector mosquitoes

<p>Abstract</p> <p>Background</p> <p>Small RNA regulatory pathways (SRRPs) control key aspects of development and anti-viral defense in metazoans. Members of the Argonaute family of catalytic enzymes degrade target RNAs in each of these pathways. SRRPs include the microRNA, small interfering RNA (siRNA) and PIWI-type gene silencing pathways. Mosquitoes generate viral siRNAs when infected with RNA arboviruses. However, in some mosquitoes, arboviruses survive antiviral RNA interference (RNAi) and are transmitted via mosquito bite to a subsequent host. Increased knowledge of these pathways and functional components should increase understanding of the limitations of anti-viral defense in vector mosquitoes. To do this, we compared the genomic structure of SRRP components across three mosquito species and three major small RNA pathways.</p> <p>Results</p> <p>The <it>Ae. aegypti, An. gambiae </it>and <it>Cx. pipiens </it>genomes encode putative orthologs for all major components of the miRNA, siRNA, and piRNA pathways. <it>Ae. aegypti </it>and <it>Cx. pipiens </it>have undergone expansion of Argonaute and PIWI subfamily genes. Phylogenetic analyses were performed for these protein families. In addition, sequence pattern recognition algorithms MEME, MDScan and Weeder were used to identify upstream regulatory motifs for all SRRP components. Statistical analyses confirmed enrichment of species-specific and pathway-specific cis-elements over the rest of the genome.</p> <p>Conclusion</p> <p>Analysis of Argonaute and PIWI subfamily genes suggests that the small regulatory RNA pathways of the major arbovirus vectors, <it>Ae. aegypti and Cx. pipiens</it>, are evolving faster than those of the malaria vector <it>An. gambiae </it>and <it>D. melanogaster</it>. Further, protein and genomic features suggest functional differences between subclasses of PIWI proteins and provide a basis for future analyses. Common UCR elements among SRRP components indicate that 1) key components from the miRNA, siRNA, and piRNA pathways contain NF-kappaB-related and Broad complex transcription factor binding sites, 2) purifying selection has occurred to maintain common pathway-specific elements across mosquito species and 3) species-specific differences in upstream elements suggest that there may be differences in regulatory control among mosquito species. Implications for arbovirus vector competence in mosquitoes are discussed.</p

Rapid transcriptional plasticity of duplicated gene clusters enables a clonally reproducing aphid to colonise diverse plant species

Background: The prevailing paradigm of host-parasite evolution is that arms races lead to increasing specialisation via genetic adaptation. Insect herbivores are no exception and the majority have evolved to colonise a small number of closely related host species. Remarkably, the green peach aphid, Myzus persicae, colonises plant species across 40 families and single M. persicae clonal lineages can colonise distantly related plants. This remarkable ability makes M. persicae a highly destructive pest of many important crop species. Results: To investigate the exceptional phenotypic plasticity of M. persicae, we sequenced the M. persicae genome and assessed how one clonal lineage responds to host plant species of different families. We show that genetically identical individuals are able to colonise distantly related host species through the differential regulation of genes belonging to aphid-expanded gene families. Multigene clusters collectively upregulate in single aphids within two days upon host switch. Furthermore, we demonstrate the functional significance of this rapid transcriptional change using RNA interference (RNAi)-mediated knock-down of genes belonging to the cathepsin B gene family. Knock-down of cathepsin B genes reduced aphid fitness, but only on the host that induced upregulation of these genes. Conclusions: Previous research has focused on the role of genetic adaptation of parasites to their hosts. Here we show that the generalist aphid pest M. persicae is able to colonise diverse host plant species in the absence of genetic specialisation. This is achieved through rapid transcriptional plasticity of genes that have duplicated during aphid evolution

Analysis of 67 RNA-seq datasets from various tissues at different stages of a model insect, Manduca sexta

Background: Manduca sexta is a large lepidopteran insect widely used as a model to study biochemistry of insect physiological processes. As a part of its genome project, over 50 cDNA libraries have been analyzed to profile gene expression in different tissues and life stages. While the RNA-seq data were used to study genes related to cuticle structure, chitin metabolism and immunity, a vast amount of the information has not yet been mined for understanding the basic molecular biology of this model insect. In fact, the basic features of these data, such as composition of the RNA-seq reads and lists of library-correlated genes, are unclear. From an extended view of all insects, clear-cut tempospatial expression data are rarely seen in the largest group of animals including Drosophila and mosquitoes, mainly due to their small sizes.Results: We obtained the transcriptome data, analyzed the raw reads in relation to the assembled genome, and generated heatmaps for clustered genes. Library characteristics (tissues, stages), number of mapped bases, and sequencing methods affected the observed percentages of genome transcription. While up to 40% of the reads were not mapped to the genome in the initial Cufflinks gene modeling, we identified the causes for the mapping failure and reduced the number of non-mappable reads to <8%. Similarities between libraries, measured based on library-correlated genes, clearly identified differences among tissues or life stages. We calculated gene expression levels, analyzed the most abundantly expressed genes in the libraries. Furthermore, we analyzed tissue-specific gene expression and identified 18 groups of genes with distinct expression patterns.Conclusion: We performed a thorough analysis of the 67 RNA-seq datasets to characterize new genomic features of M. sexta. Integrated knowledge of gene functions and expression features will facilitate future functional studies in this biochemical model insect.Peer reviewedBiochemistry and Molecular BiologyEntomology and Plant Patholog

Gene families of cuticular proteins analogous to peritrophins (CPAPs) in Tribolium castaneum have diverse functions

Citation: Jasrapuria S, Specht CA, Kramer KJ, Beeman RW, Muthukrishnan S (2012) Gene Families of Cuticular Proteins Analogous to Peritrophins (CPAPs) in Tribolium castaneum Have Diverse Functions. PLoS ONE 7(11): e49844. doi:10.1371/journal.pone.0049844The functional characterization of an entire class of 17 genes from the red flour beetle, Tribolium castaneum, which encode two families of Cuticular Proteins Analogous to Peritrophins (CPAPs) has been carried out. CPAP genes in T. castaneum are expressed exclusively in cuticle-forming tissues and have been classified into two families, CPAP1 and CPAP3, based on whether the proteins contain either one (CPAP1), or three copies (CPAP3) of the chitin-binding domain, ChtBD2, with its six characteristically spaced cysteine residues. Individual members of the TcCPAP1 and TcCPAP3 gene families have distinct developmental patterns of expression. Many of these proteins serve essential and non-redundant functions in maintaining the structural integrity of the cuticle in different parts of the insect anatomy. Three genes of the TcCPAP1 family and five genes of the TcCPAP3 family are essential for insect development, molting, cuticle integrity, proper locomotion or fecundity. RNA interference (RNAi) targeting TcCPAP1-C, TcCPAP1-H, TcCPAP1-J or TcCPAP3-C transcripts resulted in death at the pharate adult stage of development. RNAi for TcCPAP3-A1, TcCPAP3-B, TcCPAP3-D1 or TcCPAP3-D2 genes resulted in different developmental defects, including adult/embryonic mortality, abnormal elytra or hindwings, or an abnormal ‘stiff-jointed’ gait. These results provide experimental support for specialization in the functions of CPAP proteins in T. castaneum and a biological rationale for the conservation of CPAP orthologs in other orders of insects. This is the first comprehensive functional analysis of an entire class of cuticular proteins with one or more ChtBD2 domains in any insect species

Formation of rigid, non-flight forewings (elytra) of a beetle requires two major cuticular proteins

Citation: Arakane Y, Lomakin J, Gehrke SH, Hiromasa Y, Tomich JM, Muthukrishnan S, et al. (2012) Formation of Rigid, Non-Flight Forewings (Elytra) of a Beetle Requires Two Major Cuticular Proteins. PLoS Genet 8(4): e1002682. https://doi.org/10.1371/journal.pgen.1002682Insect cuticle is composed primarily of chitin and structural proteins. To study the function of structural cuticular proteins, we focused on the proteins present in elytra (modified forewings that become highly sclerotized and pigmented covers for the hindwings) of the red flour beetle, Tribolium castaneum. We identified two highly abundant proteins, TcCPR27 (10 kDa) and TcCPR18 (20 kDa), which are also present in pronotum and ventral abdominal cuticles. Both are members of the Rebers and Riddiford family of cuticular proteins and contain RR2 motifs. Transcripts for both genes dramatically increase in abundance at the pharate adult stage and then decline quickly thereafter. Injection of specific double-stranded RNAs for each gene into penultimate or last instar larvae had no effect on larval–larval, larval–pupal, or pupal–adult molting. The elytra of the resulting adults, however, were shorter, wrinkled, warped, fenestrated, and less rigid than those from control insects. TcCPR27-deficient insects could not fold their hindwings properly and died prematurely approximately one week after eclosion, probably because of dehydration. TcCPR18-deficient insects exhibited a similar but less dramatic phenotype. Immunolocalization studies confirmed the presence of TcCPR27 in the elytral cuticle. These results demonstrate that TcCPR27 and TcCPR18 are major structural proteins in the rigid elytral, dorsal thoracic, and ventral abdominal cuticles of the red flour beetle, and that both proteins are required for morphogenesis of the beetle's elytra