Sara Ruschioni

The thermo-sensitive sensilla coeloconica of leaf-cutting ants (Atta vollenweideri).

Social insects show a variety of temperature-guided behaviors. Depending on whether heat reaches the sensillum via air movements (convective heat) or as radiant heat, specific adaptations of thermo-sensitive sensilla are expected. In the present study the morphology and the physiology of thermo-sensitive peg-in-pit sensilla (S. coeloconica) of the leaf-cutting ant Atta vollenweideri were investigated. S. coeloconica are located predominantly in a single cluster on the apical antennomere, and connect to the outside through a small aperture. The sensory peg is double-walled, embedded in a chamber and innervated by three unbranched dendrites. Using tungsten electrodes, activity of the sensory neurons was measured. In most cases, the neuron with the largest spike amplitude responds to changes in air temperature (convective heat) as well as to radiant heat. In response to a drop in air temperature, the neuron shows a phasic-tonic response followed by a complete adaptation within 1 min (cold-sensitive neuron). Based on their morphology and physiology, it is suggested that the S. coeloconica are involved in the recently described thermal orientation behavior of A. vollenweideri leaf-cutting ants.

The microbiota of marketed processed edible insects as revealed by high-throughput sequencing

Entomophagy has been linked to nutritional, economic, social and ecological benefits. However, scientific studies on the potential safety risks in eating edible insects need to be carried out for legislators, markets and consumers. In this context, the microbiota of edible insects deserves to be deeply investigated. The aim of this study was to elucidate the microbial species occurring in some processed marketed edible insects, namely powdered small crickets, whole dried small crickets (Acheta domesticus), whole dried locusts (Locusta migratoria), and whole dried mealworm larvae (Tenebrio molitor), through culture-dependent (classical microbiological analyses) and -independent methods (pyrosequencing). A great bacterial diversity and variation among insects was seen. Relatively low counts of total mesophilic aerobes, Enterobacteriaceae, lactic acid bacteria, Clostridium perfringens spores, yeasts and moulds in all of the studied insect batches were found. Furthermore, the presence of several gut-associated bacteria, some of which may act as opportunistic pathogens in humans, were found through pyrosequencing. Food spoilage bacteria were also identified, as well as Spiroplasma spp. in mealworm larvae, which has been found to be related to neurodegenerative diseases in animals and humans. Although viable pathogens such as Salmonella spp. and Listeria monocytogenes were not detected, the presence of Listeria spp., Staphylococcus spp., Clostridium spp. and Bacillus spp. (with low abundance) was also found through pyrosequencing. The results of this study contribute to the elucidation of the microbiota associated with edible insects and encourage further studies aimed to evaluate the influence of rearing and processing conditions on that microbiota.

Functional bases of host-acceptance behaviour in the aphid parasitoid Aphidius ervi

Abstract The host acceptance behaviour in Aphidius ervi is investigated, assessing the role of both external and internal host‐associated cues, offered to the experimental parasitoids with parafilm‐made aphid dummies. The reaction to internal cues present in the host haemolymph is clearly evident, and its intensity is enhanced by external cues. Parasitoid females lay few eggs in aphid dummies filled with host haemolymph. A significant increase in the number of both oviposition reactions (host stinging) and egg laying is observed only when these dummies are coated with cornicle secretion. However, this enhancement is not observed when the aphid dummies contain distilled water. Thus, the host acceptance behaviour of A. ervi females appears to be controlled by the integration of both external and internal chemical cues. The physiological basis of this behavioural response is investigated with a detailed study on the anatomy and ultrastructure of A. ervi ovipositor. The detection of chemical cues present in the host haemolymph that act as kairomones is made possible by the presence of gustatory sensilla on the tip of the ovipositor. These sensilla consist of porous areas, reached by unbranched dendrites running inside both the lower valves (i.e. first valvulae) and the unpaired upper valve (i.e. second valvulae). The mechanosensory function during oviposition appears to be provided mostly by the basiconic sensilla found on the tip of external valves (i.e. third valvulae). A tentative functional model accounting for the observed oviposition behaviour of A. ervi is proposed.

Insight into the proximate composition and microbial diversity of edible insects marketed in the European Union

In recent years, the idea of exploiting edible insects for their industrial production has attracted the attention of media, research institutions and food industry operators, because of the numerous positive factors associated with this food source. Notwithstanding, insects are still underutilized in Western countries. Moreover, edible insects are carriers of natural microorganisms; hence, safety issues may arise from their industrial production. This study was aimed at providing insight into the proximate composition, with a focus in the fatty acid and amino acid composition, and microbial diversity of some processed edible insects marketed in the European Union. A high content of protein and fat was seen, with values ranging from 59.46 to 46.78 and 35.32 to 15.18%, respectively, with nutritionally valuable characteristics in both the lipid fractions and amino acid profiles. Furthermore, a great variation in microbial counts was seen. Both commensal and potential pathogenic microorganisms ascribed to the genera Pediococcus, Weissella, Streptomyces, Acinetobacter, Agrococcus, Arthrobacter, Naxibacter, Planomicrobium, Rufibacter, Bacillus, Clostridium, Vibrio, Desulfovibrio, Loktanella, Escherichia, Tetrapisispora, Aspergillus, Eurotium, Debaryomyces, and Wallemia, were identified by PCR-DGGE. The high diversity in the chemical composition and microbial profile of the marketed edible insects analyzed suggest a role of both the rearing and processing procedures on these variables. The results overall collected encourage the exploitation of edible insects as a valuable large-scale, animal-based commodity.

Taste detection of the non-volatile isothiocyanate moringin results in deterrence to glucosinolate-adapted insect larvae

Isothiocyanates (ITCs), released from Brassicales plants after hydrolysis of glucosinolates, are known for their negative effects on herbivores but mechanisms have been elusive. The ITCs are initially present in dissolved form at the site of herbivore feeding, but volatile ITCs may subsequently enter the gas phase and all ITCs may react with matrix components. Deterrence to herbivores resulting from topically applied volatile ITCs in artificial feeding assays may hence lead to ambiguous conclusions. In the present study, the non-volatile ITC moringin (4-(α-L-rhamnopyranosyloxy)benzyl ITC) and its glucosinolate precursor glucomoringin were examined for effects on behaviour and taste physiology of specialist insect herbivores of Brassicales. In feeding bioassays, glucomoringin was not deterrent to larvae of Pieris napi (Lepidoptera: Pieridae) and Athalia rosae (Hymenoptera: Tenthredinidae), which are adapted to glucosinolates. Glucomoringin stimulated feeding of larvae of the related Pieris brassicae (Lepidoptera: Pieridae) and also elicited electrophysiological activity from a glucosinolate-sensitive gustatory neuron in the lateral maxillary taste sensilla. In contrast, the ITC moringin was deterrent to P. napi and P. brassicae at high levels and to A. rosae at both high and low levels when topically applied to cabbage leaf discs (either 12, 120 or 1200 nmol moringin per leaf disc of 1cm diameter). Survival of A. rosae was also significantly reduced when larvae were kept on leaves treated with moringin for several days. Furthermore, moringin elicited electrophysiological activity in a deterrent-sensitive neuron in the medial maxillary taste sensillum of P. brassicae, providing a sensory mechanism for the deterrence and the first known ITC taste response of an insect. In simulated feeding assays, recovery of moringin was high, in accordance with its non-volatile nature. Our results demonstrate taste-mediated deterrence of a non-volatile, natural ITC to glucosinolate-adapted insects.

Fine Structure of Antennal Sensilla of Paysandisia archon and Electrophysiological Responses to Volatile Compounds Associated with Host Palms

Paysandisia archon (Lepidoptera: Castniidae) is a serious pest of palm trees. A comprehensive knowledge of the insect olfactory system is essential for the development of efficient semiochemical-based control methods. The olfactory sensilla are located particularly on the antennae, and these can detect plant volatiles that provide important cues for the insects in the search for their host plants. To date, the fine structure of P. archon antennal sensilla studies and their role in host-plant perception have not been investigated in great detail. Using light microscopy and scanning and transmission electron microscopy, the antennae of both sexes of P. archon are described here in detail, according to the different types, quantities and distributions of the sensilla. Six types of sensilla were identified. The most widespread are sensilla trichoidea, sensilla basiconica and sensilla auricilica, which are associated with olfactory function. These have cuticular shafts characterised by numerous pores, and they are innervated by two or three sensory neurons. Sensilla coeloconica, sensilla chaetica and sensilla ampullacea are associated with olfactory or olfactory-thermoreception, mechano-gustatory, and thermo-hygroreception functions, respectively. Moreover, the role of P. archon antennae in locating of the host palms was evaluated using electroantennograms, to monitor responses to ester and terpene compounds previously identified as volatiles of damaged/fermenting palm tissues. P. archon showed responses to all of the synthetic chemicals tested, with greater responses in the females, providing a significant sex*dose effect. Among the compounds tested, ethyl isobutyrate elicited the strongest antenna responses. The fine structure of the cuticular and cellular components of the P. archon antenna sensory equipment is described for the first time. The results of this study form an important starting point and complement physiological and behavioural studies, to provide valuable information of practical importance for the development of efficient semiochemical-based control methods.

Perching mate-locating strategy in Paysandisia archon (Lepidoptera: Castniidae): behavioral and morpho-physiological investigations.

ABSTRACT We studied Paysandisia archon (Burmeister) (Lepidoptera: Castniidae: Castniinae) courtship behavior to provide a detailed qualitative and quantitative description of male and female behaviors. Moreover, to investigate the role of antennal olfaction and visual stimuli in mate-recognition, bio assays with antennectomized adults and dummies were performed. To assess the presence of a pheromone gland in the ovipositor, morphological (using light and scanning electron microscopic techniques), electrophysiological, and chemical investigations were carried out. We observed perching mate-locating behavior of P. archon males, with the female triggering the courtship sequence by approaching the perching male first. The stereotyped courtship sequence is made up of five main steps: female flight, pair flight, alighting close, copulation attempt, and clasping. Our findings suggest that visual cues are important in P. archon courtship behavior, and the role of chemical cues is also discussed. Moreover, we observed a higher antenna cleaning frequency in females than in males. Ovipositor extrusions during courtship appeared not to be related to calling behavior, and histological investigations showed no evidence of glandular tissues involved in sex pheromone production. The P. archon ovipositor consists of the eighth uromere, which forms the ovipositor base, and the 9th and 10th uromeres are fused together and connected to the base by an intersegmental membrane. Overall, 24 compounds were identified from extracts obtained from ovipositors: 9 compounds were detected only in extracts from the ovipositors of 24-h-old virgin females, and not from those of 1-h-old females. None of these compounds elicited any significant electrophysiological responses from male antennae.

The bacterial biota of laboratory-reared edible mealworms (Tenebrio molitor L.): From feed to frass

Tenebrio molitor represents one of the most popular species used for the large-scale conversion of plant biomass into protein and is characterized by high nutritional value. In the present laboratory study, the bacterial biota characterizing a pilot production chain of fresh T. molitor larvae was investigated. To this end, different batches of fresh mealworm larvae, their feeding substrate (wheatmeal) and frass were analyzed by viable microbial counts, PCR-DGGE and Illumina sequencing. Moreover, the occurrence of Coxiella burnetii, Pseudomonas aeruginosa and Shiga toxin-producing E. coli (STEC) was assessed through qualitative real-time PCR assays. Microbial viable counts highlighted low microbial contamination of the wheatmeal, whereas larvae and frass were characterized by high loads of Enterobacteriaceae, lactic acid bacteria, and several species of mesophilic aerobes. Spore-forming bacteria were detected to a lesser extent in all the samples. The combined molecular approach used to profile the microbiota confirmed the low microbial contamination of wheatmeal and allowed the detection of Enterobacter spp., Erwinia spp., Enterococcus spp. and Lactococcus spp. as dominant genera in both larvae and frass. Moreover, Klebsiella spp., Pantoea spp., and Xenorhabdus spp. were found to be in the minority. Entomoplasmatales (including Spiroplasma spp.) constituted a major fraction of the microbiota of one batch of larvae. From the real-time PCR assays, no sample was positive for either C. burnetii or STEC, whereas P. aeruginosa was detected in one sample of frass. Based on the overall results, two sources of microbial contamination were hypothesized, namely feeding with wheatmeal and vertical transmission of microorganisms from mother to offspring. Since mealworms are expected to be eaten as a whole, the overall outcomes collected in this laboratory study discourage the consumption of fresh mealworm larvae. Moreover, microbial loads and the absence of potential pathogens known to be associated with this insect species should be carefully assessed in order to reduce the minimum risk for consumers, by identifying the most opportune processing methods (e.g., boiling, frying, drying, etc.).

Anatomy of the antennal dorsal organ in female of Neodryinus typhlocybae (Hymenoptera: Dryinidae): A peculiar sensory structure possibly involved in perception of host vibration

Neodryinus typhlocybae (Hymenoptera: Dryinidae) is a natural enemy of the planthopper Metcalfa pruinosa, which was introduced from North America into Europe and has become established in various regions as a pest species. Vibrational signals play a crucial role in the communication of M. pruinosa, which appears to be exploited by N. typhlocybae. Scanning and transmission electron microscopy have shown that the antennae of N. typhlocybae females have peculiar and complex sensory structures: deep longitudinal grooves that house long sensilla trichodea, termed here “Antennal Dorsal Organs.” Such structures were not present on male antennae. These sensilla extend for the length of the grooves, without contact with the groove cuticle. Their hair shaft is empty and aporous, and inserted into a specialized socket, underneath which there is a cuticular ampulla‐like chamber. Each sensillum is associated with two sensory neurons: one terminates at the proximal end of the dendritic sheath; the other continues into the sensillum sinus and is enclosed in the dendritic sheath. This second sensory neuron then enters the ampulla‐like chamber through the circular opening, and then terminates with a conspicuous tubular body at the shaft base. The possible involvement of this peculiar structure in the context of host recognition mechanism is discussed. J. Morphol. 277:128–137, 2016.

Contrasting Patterns of Host Adaptation in Two Egg Parasitoids of the Pine Processionary Moth (Lepidoptera: Thaumetopoeidae)

ABSTRACT Adaptation of parasitoids to their phytophagous host is often mediated by environmental conditions and by the food plant of the phytophagous host. Therefore, the host food plant can indirectly affect the survival and fitness of parasitoids that also attack quiescent host stages, such as eggs, in which the resources available to the immature parasitoid stages are limited. Our aim was to investigate how two egg parasitoid species of the pine processionary moth, Thaumetopoea pityocampa (Denis & Schiffermu¨ ller), respond to variations in egg traits at the extremes of a west-to-east geographic gradient in northern Italy. We considered one specialist [Baryscapus servadeii (Domenichini)] and one generalist [Ooencyrtus pityocampae (Mercet)] parasitoid, which reproduce mainly by thelytokous parthenogenesis and are common throughout the whole range of this pest. The size and shell structure of the pine processionary moth eggs were studied under light microscopy and tested experimentally under controlled conditions. We can conclude that 1) the pine processionary moth egg shell thickness is inversely proportional to the parasitism performance; 2) the larger eggs from the pine processionary moth eastern population produce parasitoid females of a larger size, which have greater realized fecundity; 3) the generalist parasitoid performs successfully with either the “home” or “away” (i.e., from both extremes of the geographic gradient) pine processionary moth eggs, which is not the case for the specialist parasitoid. The implications of these responses in the regulation of phytophagous populations are numerous and should be considered in population dynamics studies and pest management programs.