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The CP2 transcription factors are highly conserved in metazoans, where they are divided into two groups: grainyhead and late SV40 factor (LSF). We traced their evolutionary history in the Hexapoda using over 500 insect transcriptomes, to test the hypothesis that the evolution of holometaboly involved novel isoforms of these genes. All insects appear to express at least one grainyhead and one LSFlike gene, regardless of life cycle, as in most known metazoa. No major evolutionary events in these gene families occurred during the evolution of insects.

The comparative analysis of innate immunity across different insect taxa has revealed unanticipated evolutionary plasticity, providing intriguing examples of immunity-related effector gene expansion and loss. Phasmatodea, the stick and leaf insects, is an order of hemimetabolous insects that can provide insight into ancestral innate immunity genes lost by later insect clades. We injected the stick insect Peruphasma schultei with a mixture of microbial elicitors to activate a strong immune response, followed by RNA-Seq analysis to screen for induced immunity-related effector genes. This revealed a highly diverse spectrum of antimicrobial peptides (AMPs) belonging to the attacin, coleoptericin, defensin, thaumatin, and tachystatin families. In addition, we identified a large group of short, cysteine-rich putative AMPs, some of which were strongly elicited. The immunity-related effector gene repertoire also included c-type and i-type lysozymes and several pattern-recognition proteins, such as proteins that recognize Gram-negative bacteria and peptidoglycans. Finally, we identified 45 hemolymph lipopolysaccharide-binding protein sequences, an unusually large number for insects. Taken together, our results indicate that at least some phasmids synthesize a broad spectrum of diverse AMPs that deserve further in-depth analysis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The Malpighian tubules are the insect excretory organs, responsible for ion and water homeostasis and elimination of nitrogenous wastes. Post-genomic assays suggest they also metabolize and detoxify xenobiotic compounds and have antimicrobial properties. The Phasmatodea have an additional, unique set of excretory organs referred to predominantly as midgut appendices. Their function and how it compares to phasmid and other insect Malpighian tubules is unknown. Hypotheses include carbonic anhydrase activity, calcium and metal cation sequestration, and xenobiotic transport. This work presents the first comparative transcriptomic analysis of the Phasmatodean excretory organs, using the model insect Carausius morosus. I produced de novo transcriptomes of the midgut appendices, midgut wall, and Malpighian tubules, and looked for differentially expressed genes associated with putative organ functions. The appendices differentially and highly express lipid transport and metabolism proteins, and the biomineralization gene otopetrin. The Malpighian tubules differentially and highly express acid phosphatases and multiple transporter types, while appendices express fat-soluble vitamin and peptide transporters. Many defense proteins such as multidrug resistance proteins, ABC transporters, cytochrome P450's, and glutathione-S-transferases were differentially expressed in specific excretory organs. I hypothesize that the appendices and Malpighian tubules both have defensive / xenobiotic metabolism functions, but each likely target different substrates. Phasmid Malpighian tubules excrete as in other insects, while the appendices may predominantly regulate amino acids, fats, and fat-soluble compounds. Lipid metabolism in insects is poorly understood, and the Phasmatodea may thus serve as a model for studying this further.

The Phasmatodea (stick insects) have multiple, endogenous, highly expressed copies of glycoside hydrolase family 9 (GH9) genes. The purpose for retaining so many was unknown. We cloned and expressed the enzymes in transfected insect cell lines, and tested the individual proteins against different plant cell wall component poly- and oligosaccharides. Nearly all isolated enzymes were active against carboxymethylcellulose, however most could also degrade glucomannan, and some also either xylan or xyloglucan. The latter two enzyme groups were each monophyletic, suggesting the evolution of these novel substrate specificities in an early ancestor of the order. Such enzymes are highly unusual for Metazoa, for which no xyloglucanases had been reported. Phasmatodea gut extracts could degrade multiple plant cell wall components fully into sugar monomers, suggesting that enzymatic breakdown of plant cell walls by the entire Phasmatodea digestome may contribute to the Phasmatodea nutritional budget. The duplication and neofunctionalization of GH9s in the ancestral Phasmatodea may have enabled them to specialize as folivores and diverge from their omnivorous ancestors. The structural changes enabling these unprecedented activities in the cellulases require further study., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Stick and leaf insects (Phasmatodea) are an exclusively leaf-feeding order of insects with no record of omnivory, unlike other "herbivorous" Polyneoptera. They represent an ideal system for investigating the adaptations necessary for obligate folivory, including plant cell wall degrading enzymes (PCWDEs). However, their physiology and internal anatomy is poorly understood, with limited genomic resources available., Results: We de novo assembled transcriptomes for the anterior and posterior midguts of six diverse Phasmatodea species, with RNA-Seq on one exemplar species, Peruphasma schultei. The latter's assembly yielded >100,000 transcripts, with over 4000 transcripts uniquely or more highly expressed in specific midgut sections. Two to three dozen PCWDE encoding gene families, including cellulases and pectinases, were differentially expressed in the anterior midgut. These genes were also found in genomic DNA from phasmid brain tissue, suggesting endogenous production. Sequence alignments revealed catalytic sites on most PCWDE transcripts. While most phasmid PCWDE genes showed homology with those of other insects, the pectinases were homologous to bacterial genes., Conclusions: We identified a large and diverse PCWDE repertoire endogenous to the phasmids. If these expressed genes are translated into active enzymes, then phasmids can theoretically break plant cell walls into their monomer components independently of microbial symbionts. The differential gene expression between the two midgut sections provides the first molecular hints as to their function in living phasmids. Our work expands the resources available for industrial applications of animal-derived PCWDEs, and facilitates evolutionary analysis of lower Polyneopteran digestive enzymes, including the pectinases whose origin in Phasmatodea may have been a horizontal transfer event from bacteria.

The stick insects or phasmids (Phamsatodea) have a series of pyriform ampulles with long, thin filaments on the posterior end of their midgut referred to as the "appendices of the midgut." Found only in phasmids, their function had never been determined until now. Their similarity to the Malpighian tubules, which are ubiquitous insect organs of excretion, suggested a similar function. To differentiate between the appendices and the Malpighian tubules and compare functional differences between the two tissue types, vital staining (the injection of histological stains into living organisms) was done in conjunction with light and scanning electron microscopy in multiple phasmid species. The results showed that the appendices originated in the basal phasmids (Timematidae) and grew more numerous in derived species. The appendices stain selectively, notably failing to pick up the indicators of the two known systems of invertebrate excretory function, indigo carmine and ammonium carmine. Appendices sequester stains in the ampule portion before eliminating the compounds into the midgut. We conclude by confirming that the appendices do have an excretory function, but one unlike any other known in invertebrates. Their function is likely cation excretion, playing a role in calcium regulation and/or organic alkaloid sequestration. The appendices must thus be considered distinct organs from the Malpighian tubules., (Copyright © 2013 Wiley Periodicals, Inc.)

High cellulase (endo-beta-1,4-glucanase) activity was detected in the anterior midgut of the walking stick (Phasmatodea) Eurycantha calcarata. The enzyme was isolated and analyzed via mass spectrometry. RT-PCR revealed two endoglucanase genes, EcEG1 and EcEG2. Mascot analysis of the purified enzyme confirms it to be the product of gene EcEG1. Homologous cDNAs were also isolated from a distantly related species, Entoria okinawaensis, suggesting a general distribution of cellulase genes in phasmids. Phasmid cellulases showed high homology to endogenously-produced glycoside hydrolase family 9 (GH9) endoglucanases from insects, especially to those of termites, cockroaches, and crickets. The purified E. calcarata enzyme showed clear antigency against an anti-serum for termite GH9 cellulase, which, together with the sequence homology, further suggests an endogenous origin of the enzyme. This discovery suggests a possible nutritive value for cellulose in the leaf-feeding phasmids, unlike in herbivorous Lepidoptera., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Little is known about the Phasmatodea gut microbial community, including whether phasmids have symbiotic bacteria aiding in their digestion. While symbionts are near ubiquitous in herbivorous insects, the Phasmatodea's distinctively thin body shape precludes the gut enlargements needed for microbial fermentation. High-throughput sequencing was used to characterize the entire microbiota of the fat bodies, salivary glands, and anterior and posterior midguts of two species of walking stick., Results: Most bacterial sequences belonged to a strain of Spiroplasma (Tenericutes) found primarily in the posterior midgut of the parthenogenetic species Ramulus artemis (Phasmatidae). Beyond this, no significant differences were found between the R. artemis midgut sections or between that species and Peruphasma schultei (Pseudophasmatidae). Histological analysis further indicated a lack of bacteriocytes., Conclusions: Phasmids are unlikely to depend on bacteria for digestion, suggesting they produce enzymes endogenously that most other herbivorous insects obtain from symbionts. This conclusion matches predictions based on phasmid anatomy. The role of Spiroplasma in insects warrants further study.

Bergmann's rule states that individuals of a species/clade at higher altitudes or latitudes will be larger than those at lower ones. A systemic review of the known literature on inter- and intraspecific variation in insect size along latitudinal or altitudinal clines was done to see how often such clines appeared and whether they reflected classwide, species-specific, or experimentally biased tendencies. Nearly even numbers of studies showed Bergmann clines and converse-Bergmann clines, where insects get smaller as latitude/altitude increases. In fact, the majority of studies suggested no clines at all. Small ranges may have obscured certain clines, while giant ranges may have introduced artifacts. Researchers examining interspecific patterns found clines less frequently than those examining intraspecific patterns because of variation among species within the clades, which renders interspecific studies unhelpful. Bergmann's rule does not apply to hexapods with nearly the same consistency as it does to endothermic vertebrates. The validity of Bergmann's rule for any group and range of insects is highly idiosyncratic and partially depends on the study design. I conclude that studies of Bergmann's rule should focus within species and look at widespread but contiguous populations to account for all sources of variation while minimizing error.