Unraveling the role of male reproductive tract and haemolymph in cantharidin-exuding Lydus trimaculatus and Mylabris variabilis (Coleoptera: Meloidae): a comparative transcriptomics approach

Abstract

Background Meloidae (blister beetles) are known to synthetize cantharidin (CA), a toxic and defensive terpene mainly stored in male accessory glands (MAG) and emitted outward through reflex-bleeding. Recent progresses in understanding CA biosynthesis and production organ(s) in Meloidae have been made, but the way in which self-protection is achieved from the hazardous accumulation and release of CA in blister beetles has been experimentally neglected. To provide hints on this pending question, a comparative de novo assembly transcriptomic approach was performed by targeting two tissues where CA is largely accumulated and regularly circulates in Meloidae: the male reproductive tract (MRT) and the haemolymph. Differential gene expression profiles in these tissues were examined in two blister beetle species, Lydus trimaculatus (Fabricius, 1775) (tribe Lyttini) and Mylabris variabilis (Pallas, 1781) (tribe Mylabrini). Upregulated transcripts were compared between the two species to identify conserved genes possibly involved in CA detoxification and transport. Results Based on our results, we hypothesize that, to avoid auto-intoxication, ABC, MFS or other solute transporters might sequester purported glycosylated CA precursors into MAG, and lipocalins could bind CA and mitigate its reactivity when released into the haemolymph during the autohaemorrhaging response. We also found an over-representation in haemolymph of protein-domains related to coagulation and integument repairing mechanisms that likely reflects the need to limit fluid loss during reflex-bleeding. Conclusions The de novo assembled transcriptomes of L. trimaculatus and M. variabilis here provided represent valuable genetic resources to further explore the mechanisms employed to cope with toxicity of CA in blister beetle tissues. These, if revealed, might help conceiving safe and effective drug-delivery approaches to enhance the use of CA in medicine.

Molecular Characterization of Kunitz-Type Protease Inhibitors from Blister Beetles (Coleoptera, Meloidae)

“…In a previous paper, NGS-based transcriptome analyses of adult samples of L. trimaculatus and M. variabilis performed by Fratini et al [ 33 ] revealed several proteins involved in the regulation of coagulation from the whole body, accessory gland, and haemolymph. The gene and protein sequences codifying for Bovine Pancreatic Trypsin Inhibitor (BPTI, mRNA: NM_001001554.3; Protein: NP_001001554.2) and Kunitz-Type protease inhibitors of A. glabripennis (mRNA: XM_018715645.1; Protein: P_018571161.1) were blasted against the transcriptome of L. trimaculatus and M. variabilis to identify homologous sequences in these species.…”

“…The transcriptomic analysis conducted on two species of blister beetle [ 33 ] allowed broadening the knowledge about the transcripts expressed in L. trimaculatus and M. variabilis , also opening the possibility to isolate new proteins with potential biotechnological and biomedical functions.…”

“…The BPTI Kunitz protein (mRNA: NM_001001554.3; Protein: NP_001001554.2) and Anaplophora glabripennis Kunitz protein (mRNA: XM_018715645.1; Protein: P_018571161.1) have been utilized by BLAST analyses to retrieve Kunitz protein from the previous transcriptome [ 33 ] carried out on the haemolymph, accessory glands, and total bodies of L. trimaculatus and M. variabilis . The analyses revealed 33 homolog transcripts, among them 15 sequences were selected containing only one Kunitz-type domain.…”

“…RNA was extracted from the whole body with Trizol reagent (Invitrogen, Waltham, MA, USA) as performed previously [ 33 ]. Total RNA (2 μg), purified from the whole body of L. trimaculatus and M. variabilis , and primer random hexamers were utilized in 20 μL reaction volume, according to the manufacturer’s procedures (Super-ScriptIII First-Strand Synthesis System for RT-PCR, Invitrogen).…”

“…Putative KTPI anticoagulants can be identified in transcriptomic resources obtained for two blister beetle species, Lydus trimaculatus and Mylabris variabilis [ 33 ]. Both species belong to the subfamily Meloinae.…”