Paola Riolo

The sensory structures of the antennal flagellum in Hyalesthes obsoletus (Hemiptera: Fulgoromorpha: Cixiidae): A functional reduction?

Despite their relevance as harmful pests on plants of economic importance, Hemiptera Fulgoromorpha have been poorly studied as regards their antennal sensory structures. In particular, the flagellum has been neglected and, therefore, to date there are no data on its structural organization and sensory equipment. In order to fill this gap, we carried out a study on the sensillum types and distribution on the flagellum of the planthopper Hyalesthes obsoletus Signoret, an efficient vector of the stolbur phytoplasma, the cause of various crop diseases. In this cixiid species the antenna is composed of three segments, the scape, an enlarged pedicel and a long flagellum. This latter is made of a single segment and presents a basal, bulb-like enlargement from which two processes arise, a short spur and a long arista. Combining scanning electron microscopy, transmission electron microscopy and focused ion beam investigations, we discovered the presence of a total number of 6 sensilla, belonging to 4 different types: a single scolopidium extending from the bulb to the arista, three sensilla styloconica within the cuticular spur and two different sensilla coeloconica inside the bulb. As far as structural data can suggest, these sensilla might be involved in the perception of mechanical stimuli (possibly air-borne vibrations), temperature and humidity variations and CO(2) concentration. The strong reduction in sensillum number in this species is discussed as possible functional specialization of the flagellum itself. The ultrastructure of the sensilla in the flagellum of a species of Fulgoromorpha is here presented for the first time.

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.

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.

Perception of Host Plant Volatiles in Hyalesthes obsoletus: Behavior, Morphology, and Electrophysiology

The Palearctic planthopper Hyalesthes obsoletus is the natural vector of the grapevine yellow disease Bois noir. Grapevine is an occasional host plant of this polyphagous planthopper. To deepen our knowledge of the role of plant volatile organic compounds for H. obsoletus host plant searching, we carried out behavioral, morphological, and electrophysiological studies. We tested the attraction of H. obsoletus to nettle, field bindweed, hedge bindweed, chaste tree, and grapevine by using a Y-shaped olfactometer. The results showed a significant attraction of male H. obsoletus to chaste tree, and of the females to nettle. Male H. obsoletus were repelled by odor from hedge bindweed. Ultrastructural studies of the antennae showed at least two types of olfactory sensilla at the antennal pedicel: plaque organs and trichoid sensilla. Volatile organic compounds from nettle and chaste tree were collected, and the extracts were analyzed by coupling gas-chromatography to both mass-spectrometry and electroantennography. The volatile organic compounds that elicited electrophysiological responses in male and female antennae were identified. These findings are discussed with respect to behavior of H. obsoletus males and females in the field.

Getting insight into the prevalence of antibiotic resistance genes in specimens of marketed edible insects.

This study was aimed at investigating the occurrence of 11 transferable antibiotic resistance (AR) genes [erm(A), erm(B), erm(C), vanA, vanB, tet(M), tet(O), tet(S), tet(K), mecA, blaZ] in 11 species of marketed edible insects (small crickets powder, small crickets, locusts, mealworm larvae, giant waterbugs, black ants, winged termite alates, rhino beetles, mole crickets, silkworm pupae, and black scorpions) in order to provide a first baseline for risk assessment. Among the AR genes under study, tet(K) occurred with the highest frequency, followed by erm(B), tet(S) and blaZ. A high variability was seen among the samples, in terms of occurrence of different AR determinants. Cluster Analysis and Principal Coordinates Analysis allowed the 11 samples to be grouped in two main clusters, one including all but one samples produced in Thailand and the other including those produced in the Netherlands.

Natural phytoplasma infection of four phloem-feeding Auchenorrhyncha across vineyard agroecosystems in central-eastern Italy.

ABSTRACT The seasonal variations of grapevine yellow phytoplasma were investigated in four phloem-feeding planthopper and leafhopper species that are vectors of plant disease agents. In total, 1,148 wild specimens were collected from three vineyard agroecosystems in the Marche region (central-eastern Italy), from May to September 2008, and analyzed using polymerase chain reactionrestriction fragment-length polymorphism methods. Of 525 Euscelis lineolatus Brullé, 25.1% were positive for aster yellow phytoplasma (16SrI-C, 16SrI-B subgroups) and stolbur phytoplasma (16SrXII-A subgroup; Vergilbungskrankheit type I [VK-I]). Of 368 Hyalesthes obsoletus Signoret, 19.3% were positive for the 16SrXII-A subgroup (VK-I, VK-II; mainly according to their host plant). Of 146 Neoaliturus fenestratus (Herrich-Schäffer), 15.1% were positive for the 16SrI-C and 16SrI-B subgroups, and 7.3% of 109 Psammotettix alienus (Dahlbom) were positive for the 16SrI-B subgroup. The total inoculation efficiency in the feeding medium assays was 57.1% for P. alienus, 44.7% for E. lineolatus, 44.4% for N. fenestratus and 33.9% for H. obsoletus. All of the phytoplasma subgroups identified in the insect bodies were also detected in their feeding media. Detection of stolbur phytoplasma in E. lineolatus feeding media strengthens the hypothesis that it is a candidate vector of Bois noir disease causal agent. The phytoplasma subgroups detected in the Auchenorrhyncha species showed variations according to season and/or vineyard agroecosystem. This study highlights the different specificities of these phytoplasma-Auchenorrhyncha species relationships, and suggests a primary role of the entire vineyard agroecosystem in the epidemiology of grapevine yellow phytoplasma diseases.

Genetic Variability of Stolbur Phytoplasma in Hyalesthes obsoletus (Hemiptera: Cixiidae) and its Main Host Plants in Vineyard Agroecosystems

ABSTRACT Bois noir is an economically important grapevine yellows that is induced by ‘Candidatus Phytoplasma solani’ and principally vectored by the planthopper Hyalesthes obsoletus Signoret (Hemiptera: Cixiidae). This study explores the ‘Ca. P. solani’ genetic variability associated to the nettle—H. obsoletus and bindweed—H. obsoletus systems in vineyard agroecosystems of the central-eastern Italy. Molecular characterization of ‘Ca. P. solani’ isolates was carried out using polymerase chain reaction/ restriction fragment length polymorphism to investigate the nonribosomal vmp1 gene. Seven phytoplasma vmp-types were detected among the host plants- and insect-associated field-collected samples. The vmp1 gene showed the highest polymorphism in the bindweed—H. obsoletus system, according to restriction fragment length polymorphism analysis, which is in agreement with nucleotide sequence analysis. Five vmp-types were associated with H. obsoletus from bindweed, of which one was solely restricted to planthoppers, with one genotype also in planthoppers from nettle. Type V12 was the most prevalent in both planthoppers and bindweed. H. obsoletus from nettle harbored three vmp-types, of which V3 was predominant. V3 was the only type detected for nettle. Our data demonstrate that planthoppers might have acquired some ‘Ca. P. solani’ profiles from other plant hosts before landing on nettle or bindweed. Overall, the different vmp1 gene rearrangements observed in these two plant hosts-H. obsoletus systems might represent different adaptations of the pathogen to the two host plants. Molecular information about the complex of vmp-types provides useful data for better understanding of Bois noir epidemiology in vineyard agroecosystem.

Lures for red palm weevil trapping systems: aggregation pheromone and synthetic kairomone.

BACKGROUND The optimisation of the lure is essential for the implementation of trapping systems to control insect pests. In this work, the response of the red palm weevil (RPW), Rhynchophorus ferrugineus Olivier, to increasing emission rates of its aggregation pheromone (ferrugineol) and the efficacy of a convenient synthetic kairomone based on fermentation odours (ethyl acetate and ethanol) have been evaluated in different years and locations along the Mediterranean basin. RESULTS In general, although capture data and emission had noticeable variability among locations, significantly fewer RPW were captured in pyramidal Picusan® traps with the lowest ferrugineol emission rates tested (0.6-3.8 mg day-1 ). Captures increased rapidly with ferrugineol emission up to 4-5 mg day-1 ; then, higher emission rates did not improve or reduce captures, up to the highest emission rate tested of 50.9 mg day-1 . Thus, there is no evidence of an optimum release rate corresponding to a maximum of RPW catches. Traps baited with the synthetic kairomone (1:3 ethyl acetate/ethanol) captured 1.4-2.2 times more total weevils than traps baited only with ferrugineol. Moreover, in most of the locations, the synthetic blend was at least as effective as the local coattractants used (plant material + molasses). CONCLUSIONS Ferrugineol emission rate can vary in a wide range without significantly affecting RPW response. Coattractants based on fermenting compounds, ethyl acetate and ethanol, are able to improve the attractant level of ferrugineol and could be employed to replace non-standardised natural kairomones in RPW trapping systems after further optimisation of their proportions and doses.

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.