Plastic biodegradation: do Galleria mellonella larvae - bio-assimilate polyethylene? A spectral histology approach using isotopic labelling and infrared microspectroscopy.

Environmental pollution by non-biodegradable polyethylene (PE) plastics is of major concern, thus, organisms capable of bio-degrading PE are required. The larvae of the Greater Wax Moth, Galleria mellonella (Gm), were identified as a potential candidate to digest PE. In this study, we tested whether PE was metabolized by Gm larvae and could found in their tissues. We examined the implication of the larval gut microbiota by using conventional and axenic reared insects. First, our study showed that neither beeswax nor PE alone favour the growth of young larvae. We then used Fourier-Transform Infrared Microspectroscopy (microFTIR) to detect deuterium in larvae fed with isotopically labelled food. Perdeuterated molecules were found in most tissues of larvae fed with deuterium labelled oil for 72 hours proving that microFTIR can detect metabolization of 1-2 mg of deuterated food. No bio-assimilation was detected in the tissues of larvae fed with 1-5 mg of perdeuterated PED4 for 72 hours and 19-21 days, but micron sized PE particles were found in the larval digestive tract cavities. We evidenced weak bio-degradation of PE films in contact for 24 hours with the dissected gut of conventional larvae; and in the PED4 particles from excreted larval frass. Our study confirms that Gm larvae can bio-degrade PE but can not necessarily metabolize it.

Larval response to frass and guaiacol: detection of an attractant produced by bacteria from Spodoptera littoralis frass

Larval frass in herbivorous lepidopterans is mainly composed of plant-derived material and microbes from the gut. Despite the fact that frass from conspecific larvae repels female moths in Spodoptera littoralis from oviposition, the role of frass volatiles on larval foraging behavior is largely unknown. Here, we show that larvae of S. littoralis walk upwind to larval frass volatiles in a wind tunnel assay. We identified the frass volatile guaiacol (2-methoxyphenol) as key ligand for the S. littoralis odorant receptor (OR) SlitOr59 which we expressed heterologously. We isolated guaiacol-producing bacteria identified as Serratia marcescens from frass of larvae that were fed on cotton, and Enterobacter cloacae, E. ludwigii and Klebsiella sp. from frass derived from cabbage-fed larvae. In addition to guaiacol, we also identified volatiles acetoin, 3-methyl-1-butanol and dimethyl disulfide, in large proportions in headspace collections from the bacteria. A Y-tube olfactometer assay showed that fourth instar S. littoralis larvae are attracted to guaiacol. Moreover, cotton leaves treated with the insecticide Spinosad and guaiacol were highly attractive to the larvae. Our results provide a basis for management of the pest by directly targeting larvae, based on an attract-and-kill strategy. Further studies are needed to test the application of guaiacol for semiochemical-based pest management of Spodoptera pest species.

The olfactory co-receptor IR8a governs larval-frass mediated competition avoidance in a hawkmoth

Finding a suitable oviposition site is a challenging task for a gravid female moth. At the same time, it is of paramount importance considering the limited capability of most caterpillars to relocate to alternative host plants. The hawkmoth, Manduca sexta (Sphingidae), oviposits on solanaceous plants. Larvae hatching on a plant that is already attacked by conspecific caterpillars can face food competition, as well as an increased exposure to predators and induced plant defenses. Here, we show that frass from conspecific caterpillars is sufficient to deter a female M. sexta from ovipositing on a plant and that this deterrence is based on the frass-emitted carboxylic acids 3-methylpentanoic acid and hexanoic acid. Using a combination of genome editing (CRISPR/Cas9), electrophysiological recordings, calcium imaging and behavioral analyses we demonstrate that the ionotropic co-receptor IR8a is essential for acid-mediated frass avoidance in ovipositing hawkmoths.

Confirmation of the Presence of Melitaea Ornata Christoph, 1893 (lepidoptera: Nymphalidae) in Croatia and Bosnia And Herzegovina with Its Host-plants

Various localities in Croatia were visited in 2015, 2016 and 2017 to search for Melitaea ornata Christoph, 1893, which had been reported previously from six locations. Three of these sites were visited in May 2015 and found to be damp meadows and thus most unsuitable for M. ornata. In May 2016 and June 2017, another location (Mt. Sniježnica), given by Koren & Ŝtih (2013) was visited. In 2016, a plant very similar to a known host-plant of M. ornata, Onopordum illyricum L., was found to have been eaten extensively, with copious amounts of larval frass present; however, no larvae were present and no adults seen. In 2017, a male and two females resembling M. ornata were captured at two different locations on this mountain. Also in 2017, a location in the Republic of Bosnia and Herzegovina approximately 8 km southsoutheast of Trebinje (Herzegovina) and 10 km north of Mt. Sniježnica was visited, where a further M. ornata -like female was captured and two egg masses were collected from first year plants of Carduus collinus Waldst. & Kit., the host-plant used by M. ornata in Slovenia. These were returned to the UK and some reared through to adults and confirmed as Melitaea ornata . The butterfly species seen in the Zagreb district, Mt. Sniježnica and Herzegovina are listed.