Tachystatin C

Ants, as eusocial insects, live in dense groups with high connectivity, increasing the risk of pathogen spread and possibly driving the evolution of their antimicrobial immune system. Draft genomes of seven ant species provide a new source to undertake comparative study of their antimicrobial peptides (AMPs), key components of insect innate immunity. By using computational approaches, we analyzed five AMP families that include abaecins, hymenoptaecins, insect defensins, tachystatins, and crustins in ants, which comprise 69 new members. Among them, a new type of proline-rich abaecins was recognized and they are exclusively present in ants. Hymenoptaecins, a family of glycine-rich AMPs from Hymenoptera and Diptera, exhibit variable numbers of intragenic tandem repeats in a lineage-specific manner and all hymenoptaecins in ants have evolved an acidic C-terminal propeptide. In some ant species, insect defensins with the cysteine-stabilized α-helical and β-sheet (CSαβ) fold and tachystatin-like AMPs with the inhibitor cysteine knot (ICK) fold have undergone gene expansion and differential gene loss. Moreover, extensive sequence diversity exists in the C-termini of the defensins and the ICK-type peptides and the n-loop of the defensins. Also, we identified for the first time a crustin-type AMP in ants, which are only known in crustaceans previously. These ant crustins evolutionarily gain an aromatic amino acid-rich insertion when compared with those of crustaceans. Our work not only enlarges the insect AMP resource, but also sheds light on the complexity and dynamic evolution of AMPs in ants.

Antimicrobial peptides, named tachystatins A, B, and C, were identified from hemocytes of the horseshoe crab Tachypleus tridentatus. Tachystatins exhibited a broad spectrum of antimicrobial activity against Gram-negative and Gram-positive bacteria and fungi. Of these tachystatins, tachystatin C was most effective. Tachystatin A is homologous to tachystatin B, but tachystatin C has no significant sequence similarity to tachystatins A and B. Tachystatins A and B showed sequence similarity to omega-agatoxin-IVA of funnel web spider venom, a potent blocker of voltage-dependent calcium channels. However, they exhibited no blocking activity of the P-type calcium channel in rat Purkinje cells. Tachystatin C also showed sequence similarity to several insecticidal neurotoxins of spider venoms. Tachystatins A, B, and C bound significantly to chitin. A causal relationship was observed between chitin binding activity and antifungal activity. Tachystatins caused morphological changes against a budding yeast, and tachystatin C had a strong cell lysis activity. The septum between mother cell and bud, a chitin-rich region, was stained by fluorescence-labeled tachystatin C, suggesting that the primary recognizing substance on the cell wall is chitin. As horseshoe crab is a close relative of the spider, tachystatins and spider neurotoxins may have evolved from a common ancestral peptide, with adaptive functions.

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.