Odorant binding proteins fromHermetia illucens: potential sensing elements for detecting volatile aldehydes involved in early stages of organic decomposition.

Organic decomposition processes, involving the breakdown of complex molecules such as carbohydrates, proteins and fats, release small chemicals known as volatile organic compounds (VOCs), smelly even at very low concentrations, but not all readily detectable by vertebrates. Many of these compounds are instead detected by insects, mostly by saprophytic species, for which long-range orientation towards organic decomposition matter is crucial. In the present work the detection of aldehydes, as an important measure of lipid oxidation, has been possible exploiting the molecular machinery underlying odour recognition inHermetia illucens(Diptera: Stratiomyidae). This voracious scavenger insect is of interest due to its outstanding capacity in bioconversion of organic waste, colonizing very diverse environments due to the ability of sensing a wide range of chemical compounds that influence the choice of substrates for ovideposition. A variety of soluble odorant binding proteins (OBPs) that may function as carriers of hydrophobic molecules from the air-water interface in the antenna of the insect to the receptors were identified, characterised and expressed. An OBP-based nanobiosensor prototype was realized using selected OBPs as sensing layers for the development of an array of quartz crystal microbalances (QCMs) for vapour phase detection of selected compounds at room temperature. QCMs coated with four recombinantH. illucensOBPs (HillOBPs) were exposed to a wide range of VOCs indicative of organic decomposition, showing a high sensitivity for the detection of three chemical compounds belonging to the class of aldehydes and one short-chain fatty acid. The possibility of using biomolecules capable of binding small ligands as reversible gas sensors has been confirmed, greatly expanding the state-of the-art in gas sensing technology.

Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically Important Chrysomya rufifacies (Diptera: Calliphoridae).

Entomological protocols for aging blowfly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analyzing fly larvae differ, most rely on light microscopy, genetic analysis, or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically important blowfly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, nonbleaching, autofluorescence of larval posterior spiracles. High-content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC.

Evaluation of Salmonella movement through the gut of the lesser mealworm, Alphitobius diaperinus (Coleoptera: Tenebrionidae).

AIMS: The lesser mealworm, Alphitobius diaperinus is an important poultry pest prevalent during production that is capable of vectoring pathogens. This study was undertaken to determine the gut transit time of Salmonella for biosecurity risk analysis of pathogen dispersal into the environment. METHODS: Adult and larval A. diaperinus were exposed to two concentrations of a fluorescently labeled Salmonella enterica for 15, 30, and 60 min time periods then externally disinfected to evaluate internal transfer of Salmonella. The insects were monitored every 30 min over 4 h and evacuated frass (feces) processed for the marker Salmonella. The minimum time monitored was 45 min (15 exposure+30 min time point), and the maximum was 5 h (60 exposure+4 h time point). RESULTS: Adults treated with 10(6) or 10(8) colony-forming units (cfu)/mL, which produced Salmonella positive frass within the 5 h experimental time, displayed a mean gut transit time of 144.4 min (range 90-270 min) and 186.3 min (range 120-300 min), respectively. Larvae treated with 10(6) or 10(8) cfu/mL displayed a mean gut transit time of 172.5 min (range 120-300 min) and 131.7 min (range 60-300 min), respectively. SIGNIFICANCE AND IMPACT OF STUDY: Understanding the sources and contribution of reservoir populations of pathogens in poultry production operations is important for development of biosecurity measures to mitigate their transfer. A. diaperinus are prevalent in production operations and difficult to suppress. Management standards accept the reutilization of litter in which insects survive between flock rotations. Removing litter and spreading it onto nearby fields results in the inadvertent dispersal of beetles. Few studies demonstrating the specific bacterial dispersal capacities of these insects have been performed. This study determined that Salmonella acquired internally, commonly transits the gut, allowed the insect to disperse viable pathogenic bacteria within 2-3 h.