ABCB transporters in a leaf beetle respond to sequestered plant toxins

Phytophagous insects can tolerate and detoxify toxic compounds present in their host plants and have evolved intricate adaptations to this end. Some insects even sequester the toxins for their defence. This necessitates specific mechanisms, especially carrier proteins that regulate uptake and transport to specific storage sites or protect sensitive tissues from noxious compounds. We identified three ATP-binding cassette subfamily B (ABCB) transporters from the transcriptome of the cardenolide-sequestering leaf beetle Chrysochus auratus and analysed their functional role in the sequestration process. These were heterologously expressed and tested for their ability to interact with various potential substrates: verapamil (standard ABCB substrate), the cardenolides digoxin (commonly used), cymarin (present in the species's host plant) and calotropin (present in the ancestral host plants). Verapamil stimulated all three ABCBs and each was activated by at least one cardenolide, however, they differed as to which they were activated by. While the expression of the most versatile transporter fits with a protective role in the blood–brain barrier, the one specific for cymarin shows an extreme abundance in the elytra, coinciding with the location of the defensive glands. Our data thus suggest a key role of ABCBs in the transport network needed for cardenolide sequestration.

Foranotum perforatum gen. et sp. nov.—a new troglobitic darkling beetle (Coleoptera: Tenebrionidae: Kuhitangiinae: Foranotini trib. nov.) from a cave in Southern Zagros, Iran

The troglobitic darkling beetle Foranotum perforatum gen. et sp. nov. from a cave in Southern Zagros Mountains (Iran) is described. This new taxon belongs to the family Tenebrionidae based on the following combination of characters: tarsal formula 5-5-4, antennal insertions concealed from above, antennae 11-segmented, mesocoxal cavities laterally closed by meso- and metaventrite. It is close to Kuhitangia kryzhanovskii Medvedev, 1962 by sharing a similar structure of procoxa (open cavities), labrum (reduced tormal arms) and prothorax (perforating holes on lateral sides of disc). Based on a comprehensive character analysis the subfamily Kuhitangiinae (type genus Kuhitangia Medvedev, 1962) is reestablished. Kuhitangiinae belongs to the pimelioid branch of the family Tenebrionidae based on open mouthparts (mentum small, exposing most of maxillae), mesocoxae externally closed by meso- and metaventrite, metacoxae externally narrowly closed by metaventrite and first abdominal ventrite, tentyrioid structure of abdominal ventrites (absence of membranes between abdominal ventrites 3–5) and absence of defensive glands. The subfamily Kuhitangiinae is divided into following two tribes: Kuhitangiini with a single species Kuhitangia kryzhanovskii Medvedev, 1962 and Foranotini trib. n. with Foranotum perforatum sp. nov. The new tribe differs from Kuhitangiini by the presence of trochanters and epipleura, structure of pronotum, abdominal ventrites and sculpture of meso- and metaventrites.

ABC transporter functions as a pacemaker for sequestration of plant glucosides in leaf beetles

Plant-herbivore interactions dominate the planet’s terrestrial ecology. When it comes to host–plant specialization, insects are among the most versatile evolutionary innovators, able to disarm multiple chemical plant defenses. Sequestration is a widespread strategy to detoxify noxious metabolites, frequently for the insect’s own benefit against predation. In this study, we describe the broad-spectrum ATP-binding cassette transporter CpMRP of the poplar leaf beetle, Chrysomela populi as the first candidate involved in the sequestration of phytochemicals in insects. CpMRP acts in the defensive glands of the larvae as a pacemaker for the irreversible shuttling of pre-selected metabolites from the hemolymph into defensive secretions. Silencing CpMRP in vivo creates a defenseless phenotype, indicating its role in the secretion process is crucial. In the defensive glands of related leaf beetle species, we identified sequences similar to CpMRP and assume therefore that exocrine gland-based defensive strategies, evolved by these insects to repel their enemies, rely on ABC transporters as a key element.

Defensive Glands of the Darkling BeetleMesomorphus villigerBlanchard (Coleoptera: Tenebrionidae)

Massive home invasion by the darkling beetleMesomorphus villigerBlanchard 1853 (Coleoptera: Tenebrionidae) during monsoon season make it a nuisance pest in many regions of south India. Morphology of defensive glands and mode of release and dispersal of the defensive secretion were analysed. Defensive glands were separated from the abdominal sternites by cutting along the posterior margin of the seventh sternite. Glands are evaginations of intersegmental membrane between the seventh and eighth sternites consisting of two long sac-like reservoirs, and glandular secretion is released by exudation and spread through epipleural gutter of elytra. Gradual release of the secretion is a strategy to repel the predators for a longer duration.

Precise RNAi-mediated silencing of metabolically active proteins in the defence secretions of juvenile leaf beetles

Allomones are widely used by insects to impede predation. Frequently these chemical stimuli are released from specialized glands. The larvae of Chrysomelina leaf beetles produce allomones in gland reservoirs into which the required precursors and also the enzymes are secreted from attached gland cells. Hence, the reservoirs can be considered as closed bio-reactors for producing defensive secretions. We used RNA interference (RNAi) to analyse in vivo functions of proteins in biosynthetic pathways occurring in insect secretions. After a salicyl alcohol oxidase was silenced in juveniles of the poplar leaf beetles, Chrysomela populi , the precursor salicyl alcohol increased to 98 per cent, while salicyl aldehyde was reduced to 2 per cent within 5 days. By analogy, we have silenced a novel protein annotated as a member of the juvenile hormone-binding protein superfamily in the juvenile defensive glands of the related mustard leaf beetle, Phaedon cochleariae . The protein is associated with the cyclization of 8-oxogeranial to iridoids (methylcyclopentanoid monoterpenes) in the larval exudates made clear by the accumulation of the acylic precursor 5 days after RNAi triggering. A similar cyclization reaction produces the secologanin part of indole alkaloids in plants.