Ancestral gene duplication enabled the evolution of multifunctional cellulases in stick insects (Phasmatodea)

Abstract

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.

Acylated Quinic Acids Are the Main Salicortin Metabolites in the Lepidopteran Specialist Herbivore Cerura vinula

“…As the insects' nutritional spectrum is limited to plants of the Salicaceae family (Salix and Populus species), they are regarded as specialist herbivores (Ali and Agrawal 2012;Hintze-Podufal 1970). Numerous metabolic studies (recent examples: Beran et al 2014;Joussen et al 2012;Shelomi et al 2016) have addressed the question whether a specialist herbivore uses a distinct mechanism to cope with the defense of its host plant. In the present publication, we used a double-track strategy: the careful structural identification of metabolic products arising from the diet of the specialist herbivore resulted in an assumption about how the main chemical defense compounds were transformed during digestion.…”

Multifunctional cellulase enzymes are ancestral in Polyneoptera

“…wrighti enzyme in one monophyletic group and the other cockroach enzymes in another monophyletic group. The enzymes were then predominantly clustered by species, not activity as in the Phasmatodea enzymes (Shelomi et al ., ). The exceptions were an enzyme each from Periplaneta , Reticulitermes and Mastotermes , plus the one cellulase from Mantis , that were in a monophyletic clade with one Phasmatodea and one Embioptera GH9 member.…”

“…This species is in the family Timematidae, considered the monophyletic sister group to all other Phasmatodea, although other papers have placed it as a sister group to Embioptera and the rest of the Phasmatodea (Kômoto et al ., ). The GH9 TCR4 was also the only GH9 of the Phasmatodea experiment to not have any detectable cellulolytic or other activity despite successful heterologous expression, and to have no orthologues in the other Phasmatodea species (Shelomi et al ., ). The other Polyneoptera GH9s in the same group also had no detectable enzymatic activity.…”

“…We are confident that most transcripts identified in this paper, including all those that we isolated and heterologously expressed, are endogenous and not produced by symbionts based on their sequence similarity to other demonstrably endogenous insect enzymes, such as those from the asymbiotic Phasmatodea (Shelomi et al ., , ). By the same evidence, we identified at least three enzymes from the 1KITE transcriptomes that show strong sequence similarity to non‐animal genes, and which are probably not endogenous (Shelomi et al ., ).…”

“…S4). However, as the results of this assay differed little to none from the substrate assays, as also observed in prior studies (Shelomi et al ., ), they were not done for the other species. For Antipaluria only we also attempted a quantitative 3,5‐dinitrosalicylic acid assay (Kirsch et al ., ), but with modifications: 15 μl enzyme solution, 12 μl 1% xyloglucan or 2% CMC, and 3 μl 20 mM citrate/phosphate buffer pH 5.0 incubated at 40°C for 2 h. These assays confirmed the activity presence/absence results of the qualitative assays, but the standard error values for the Km value estimations were too high for the quantitative data to be reliable.…”