Cuticular Hydrocarbon Trails Released by Host Larvae Lose their Kairomonal Activity for Parasitoids by Solidification

Successful host search by parasitic wasps is often mediated by host-associated chemical cues. The ectoparasitoid Holepyris sylvanidis is known to follow chemical trails released by host larvae of the confused flour beetle, Tribolium confusum, for short-range host location. Although the hexane-extractable trails consist of stable, long-chain cuticular hydrocarbons (CHCs) with low volatility, the kairomonal activity of a trail is lost two days after release. Here, we studied whether this loss of kairomonal activity is due to changes in the chemical trail composition induced by microbial activity. We chemically analyzed trails consisting of hexane extracts of T. confusum larvae after different time intervals past deposition under sterile and non-sterile conditions. GC-MS analyses revealed that the qualitative and quantitative pattern of the long-chain CHCs of larval trails did not significantly change over time, neither under non-sterile nor sterile conditions. Hence, our results show that the loss of kairomonal activity of host trails is not due to microbially induced changes of the CHC pattern of a trail. Interestingly, the kairomonal activity of trails consisting of host larval CHC extracts was recoverable after two days by applying hexane to them. After hexane evaporation, the parasitoids followed the reactivated host trails as they followed freshly laid ones. Cryo-scanning electron microscopy showed that the trails gradually formed filament-shaped microstructures within two days. This self-assemblage of CHCs was reversible by hexane application. Our study suggests that the long-chain CHCs of a host trail slowly undergo solidification by a self-assembling process, which reduces the accessibility of CHCs to the parasitoid’s receptors as such that the trail is no longer eliciting trail-following behavior.

Essential Oil-Based Nano-Biopesticides: Formulation and Bioactivity against the Confused Flour Beetle Tribolium confusum

Post-harvest pest control can rely on few approved pesticides and tools; hence, there is a rising interest in new sustainable, eco-friendly approaches. In this study, eight commercial essential oils (EOs) (anise Pimpinella anisum, artemisia Artemisia vulgaris, fennel Foenicum vulgare, garlic Allium sativum, lavender Lavandula angustifolia, mint Mentha piperita, rosemary Rosmarinus officinalis, and sage Salvia officinalis) were selected for their bioactivity and commercial availability, and then formulated in nano-emulsions. Repellency and acute toxicity of the developed nano-formulations were tested against a key stored product pest, Tribolium confusum (Coleoptera: Tenebrionidae). All the developed nano-emulsions presented optimal physical characteristics (droplet dimension = 95.01–144.30 nm; PDI = 0.146–0.248). All the formulations were repellent over time tested against adult beetles, in area preference bioassays. The best repellent was the anise EO-based formulation (RC50 = 0.033 mg). Mortality values from cold aerosol trials showed that the majority of tested EOs caused immediate acute toxicity, and garlic EO nano-emulsion caused the highest mortality of T. confusum adults (LC50 = 0.486 mg/L of air). EO-based nano-insecticides, used as cold aerosol and gel, are promising control methods against stored product pests, which can be integrated and combined with other sustainable biorational approaches.

Rating knockdown of flour beetles after exposure to two insecticides as an indicator of mortality

Knockdown and mortality of adults of the red flour beetle, Tribolium castaneum (Herbst) and the confused flour beetle, Tribolium confusum Jacquelin du Val, were assessed after exposure to two contact insecticides, chlorfenapyr and cyfluthrin, on a concrete surface. Individuals were rated on a scale for knockdown of exposed adults according to their mobility from 1, representing immobilized adults to 5, representing normally moving (similar to the controls). Only cyfluthrin gave immediate knockdown. Adults were rated at 1, 3 and 7 days post-exposure. After the final assessment, adults were discarded and the same procedure was repeated for 5 consecutive weeks with new adults exposed on the same treated surfaces. Despite initial knockdown, many individuals did not eventually die after exposure to cyfluthrin. In contrast, adults exposed to chlorfenapyr were not initially knocked down after exposure but most died after 7 days. These trends were similar during the entire 5-week residual testing period. The storage of the treated dishes in illuminated or non-illuminated conditions did not affect the insecticidal effect of either insecticide. The results of the present study can be further implemented towards the design of a “lethality index” that can serve as a quick indicator of knockdown and mortality rates caused after exposure to insecticides.

The Importance of Methyl-Branched Cuticular Hydrocarbons for Successful Host Recognition by the Larval Ectoparasitoid Holepyris sylvanidis

Cuticular hydrocarbons (CHCs) of host insects are used by many parasitic wasps as contact kairomones for host location and recognition. As the chemical composition of CHCs varies from species to species, the CHC pattern represents a reliable indicator for parasitoids to discriminate host from non-host species. Holepyris sylvanidis is an ectoparasitoid of beetle larvae infesting stored products. Previous studies demonstrated that the larval CHC profile of the confused flour beetle, Tribolium confusum, comprises long chain linear and methyl-branched alkanes (methyl alkanes), which elicit trail following and host recognition in H. sylvanidis. Here we addressed the question, whether different behavioral responses of this parasitoid species to larvae of other beetle species are due to differences in the larval CHC pattern. Our study revealed that H. sylvanidis recognizes and accepts larvae of T. confusum, T. castaneum and T. destructor as hosts, whereas larvae of Oryzaephilus surinamensis were rejected. However, the latter species became attractive after applying a sample of T. confusum larval CHCs to solvent extracted larvae. Chemical analyses of the larval extracts revealed that CHC profiles of the Tribolium species were similar in their composition, while that of O. surinamensis differed qualitatively and quantitatively, i.e. methyl alkanes were present as minor components on the cuticle of all Tribolium larvae, but were absent in the O. surinamensis CHC profile. Furthermore, the parasitoid successfully recognized solvent extracted T. confusum larvae as hosts after they had been treated with a fraction of methyl alkanes. Our results show that methyl alkanes are needed for host recognition by H. sylvanidis.

Evaluation of a Greek Diatomaceous Earth for Stored Product Insect Control and Techniques That Maximize Its Insecticidal Efficacy

Laboratory bioassays were conducted to evaluate the insecticidal efficacy of a diatomaceous earth deposit from Greece, for a wide range of stored product insects. In this context, populations of five different insect species, Tribolium confusum Jacquelin DuVal, the confused flour beetle; Sitophilus oryzae (L.), the rice weevil; Rhyzopertha dominica (F.), the lesser grain borer; Oryzaephilus surinamensis (L.), the sawtoothed grain beetle; Cryptolestes ferrugineus (Stephens), the rusty grain beetle, which cover a major spectrum of insects species of stored products worldwide, were used in the bioassays. The different treatment of diatomaceous earth (DE) rocks (grinding, diatomaceous enrichment, powder granulometry) led to the creation of five types of formulations (namely DE1, DE2, DE3, DE5 and DE6) that exhibited significant fluctuations in their insecticidal efficacy when applied on wheat. In general, some of the modified formulations were found to be very effective against species such as R. dominica and T. confusum that may be difficult to control at the current labeled doses of commercial DE formulations. Overall, our data clearly indicate that this specific Greek deposit has considerable insecticidal properties, which can be further utilized in designing commercial formulations for insect control at the postharvest stages of durable agricultural commodities, provided that the deposit will be modified at specific enrichment and granulometry levels.