Evaluation of Growth Performance and Environmental Impact of Hermetia illucens Larvae Reared on Coffee Silverskins Enriched with Schizochytrium limacinum or Isochrysis galbana Microalgae.

Hermetia illucens is a promising insect due to its ability to convert low-value substrates as food chain by-products into highly nutritious feed. Its feeding and nutrition are important issues. The aim of this work was to investigate the effect of different substrates consisting of coffee silverskin, a by-product of the roasting process, enriched with different inclusions of microalgae (5%, 10%, 20%, and 25%), Schizochytrium limacinum, and Isochrysis galbana, combined with the assessment of environmental sustainability by LCA. In general, the addition of microalgae led to an increase in larval growth performance due to the higher content of protein and lipids, although S. limacinum showed the best results with respect to larvae fed with coffee silverskin enriched with I. galbana. A higher prepupal weight was observed in larvae fed with 10%, 20%, and 25% S. limacinum; shorter development times in larvae fed with 25% of both S. limacinum and I. galbana; and a higher growth rate in larvae fed with 25% S. limacinum. The 10% S. limacinum inclusion was only slightly different from the higher inclusions. Furthermore, 10% of S. limacinum achieved the best waste reduction index. The greater the inclusion of microalgae, the greater the environmental impact of larval production. Therefore, the addition of 10% S. limacinum appears to be the best compromise for larval rearing, especially considering that a higher inclusion of microalgae did not yield additional benefits in terms of the nutritional value of H. illucens prepupae.

Staphylococcus aureus artificially inoculated in mealworm larvae rearing chain for human consumption: Long-term investigation into survival and toxin production.

The present research reports the results of a long-term study (70 days) of the dynamics of Staphylococcus aureus artificially inoculated in a Tenebrio molitor rearing chain for human consumption. To this end, a rearing substrate consisting of organic wheat middlings was spiked with S. aureus to obtain three initial contamination levels, namely 1 (low level), 5 (medium level) and 7 (high level) Log colony forming unit per gram. Microbial viable counting coupled with metataxonomic analysis were performed to evaluate: i) the persistence and growth of S. aureus in the rearing substrate; ii) the colonization and growth of S. aureus in the insect larvae; iii) the occurrence and load of S. aureus in the frass (excrement from larvae mixed with substrate residues); iv) the presence of S. aureus enterotoxins in the rearing substrate, frass, and larvae. The results of the present study highlighted that wheat middlings contaminated with S. aureus do not represent a suitable environment for the multiplication of the pathogen, irrespective of the initial contamination level. Of note, frass originated from the larvae reared on contaminated wheat middlings might potentially represent a source of S. aureus, with cell loads depending on the initial contamination level. A complex resident microbiota was revealed by metataxonomic analysis. Interestingly, co-occurrence/co-exclusions analysis did not reveal associations between the target microorganism and the microbiota of wheat middlings, larvae, or frass. Considering safety aspects of larvae, the results overall collected suggested that, under the applied conditions, T. molitor represents an inhospitable or even hostile environment for S. aureus, with this latter showing counts below the detection limit in the larvae at the end of the 70-day rearing trial, irrespective of the initial contamination level. The results also suggested that a combination of bactericidal factors, including unfavorable environmental conditions (such as low aw of wheat middlings and frass), might have established in the rearing chain. Finally, the absence of staphylococcal toxins suggests that, even when S. aureus is present at high contamination levels, it is not able to produce toxins in wheat middlings, larvae, or frass.

Fate of Escherichia coli artificially inoculated in Tenebrio molitor L. larvae rearing chain for human consumption.

The edible insect food chain represents a relatively novel food-producing system; hence, associated biological risks still need to be exhaustively evaluated. In the present study, the dynamics of Escherichia coli during the whole living period of Tenebrio molitor larvae (from eggs to pupae) were studied. To this end, a rearing substrate consisting of organic wheat middlings was spiked with E. coli cells at two initial contamination levels: 1 log cfu g-1 (low) and 6 log cfu g-1 (high). Microbial viability counting coupled with metataxonomic analyses was used to assess i) the persistence and growth of E. coli in the rearing substrate (wheat middlings); ii) the colonization and growth of E. coli in the insect larvae; and iii) the occurrence and load of E. coli in the frass (excrement from larvae mixed with substrate residues). The results highlighted a very limited persistence of the pathogen in all analyzed samples. In more detail, the results suggested that when E. coli was present at very low levels in the eggs of the insect, the pathogen was not able to reach concerning levels in the larvae. Moreover, when E. coli was present in the wheat middlings used for rearing, the environmental conditions of the substrate (low aw values) were not favorable for its survival and multiplication, irrespective of the presence of the larvae and their frass. Surprisingly, under the conditions applied in the present study, the larvae fed wheat middlings contaminated with E. coli seemed to be inhospitable or even hostile environments for microbial survival or multiplication. To explain the low levels of E. coli cells in the larvae reared in the present study, many factors can be considered, including the immune response of the host, microbial composition and interactions established in the gut of larvae, and insect species. Of note, part of the major fraction of the microbiota of larvae at the end of rearing was represented by Lactococcus, thus suggesting a possible effect of this lactic acid bacterium on E. coli decay. Further research is needed to better clarify the interactions between E. coli and the insect gut, as well as the interactions established among the target microorganism and those naturally harbored by the insect gut.

Microbial dynamics in rearing trials of Hermetia illucens larvae fed coffee silverskin and microalgae.

In the present study, Hermetia illucens larvae were reared on a main rearing substrate composed of a coffee roasting byproduct (coffee silverskin, Cs) enriched with microalgae (Schizochytrium limacinum or Isochrysis galbana) at various substitution levels. The microbial diversity of the rearing substrates, larvae, and frass (excrement from the larvae mixed with the substrate residue) were studied by the combination of microbial culturing on various growth media and metataxonomic analysis (Illumina sequencing). High counts of total mesophilic aerobes, bacterial spores, presumptive lactic acid bacteria, coagulase-positive cocci, and eumycetes were detected. Enterobacteriaceae counts were low in the rearing diets, whereas higher counts of this microbial family were observed in the larvae and frass. The microbiota of the rearing substrates was characterized by the presence of lactic acid bacteria, including the genera Lactobacillus, Leuconostoc and Weissella. The microbiota of the H. illucens larvae fed Cs was characterized by the dominance of Paenibacillus. H. illucens fed diets containing I. galbana were characterized by the presence of Enterococcus, Lysinibacillus, Morganella, and Paenibacillus, depending on the algae inclusion level, while H. illucens fed diets containing S. limacinum were characterized by high relative abundances of Brevundimonas, Enterococcus, Paracoccus, and Paenibacillus, depending on the algae inclusion level. Brevundimonas and Alcaligenes dominated in the frass from larvae fed I. galbana; the predominance of Brevundimonas was also observed in the frass from larvae fed Schyzochitrium-enriched diets. Based on the results of the present study, an effect of algae nutrient bioactive substances (e.g. polysaccharides, high-unsaturated fatty acids, taurine, carotenoids) on the relative abundance of some of the bacterial taxa detected in larvae may be hypothesized, thus opening new intriguing perspectives for the control of the entomopathogenic species and foodborne human pathogens potentially occurring in edible insects. Further studies are needed to support this hypothesis. Finally, new information on the microbial diversity occurring in insect frass was also obtained.

Occurrence of Antibiotic Resistance Genes in Hermetia illucens Larvae Fed Coffee Silverskin Enriched with Schizochytrium limacinum or Isochrysis galbana Microalgae.

Hermetia illucens larvae are among the most promising insects for use as food or feed ingredients due to their ability to convert organic waste into biomass with high-quality proteins. In this novel food or feed source, the absence of antibiotic-resistant bacteria and their antibiotic resistance (AR) genes, which could be horizontally transferred to animal or human pathogens through the food chain, must be guaranteed. This study was conducted to enhance the extremely scarce knowledge on the occurrence of AR genes conferring resistance to the main classes of antibiotics in a rearing chain of H. illucens larvae and how they were affected by rearing substrates based on coffee silverskin supplemented with increasing percentages of Schizochytrium limacinum or Isochrysis galbana microalgae. Overall, the PCR and nested PCR assays showed a high prevalence of tetracycline resistance genes. No significant effect of rearing substrates on the distribution of the AR genes in the H. illucens larvae was observed. In contrast, the frass samples were characterized by a significant accumulation of AR genes, and this phenomenon was particularly evident for the samples collected after rearing H. illucens larvae on substrates supplemented with high percentages (>20%) of I. galbana. The latter finding indicates potential safety concerns in reusing frass in agriculture.

Physiological responses of Siberian sturgeon (Acipenser baerii) juveniles fed on full-fat insect-based diet in an aquaponic system.

Over the last years, the potential use of Black Soldier Fly meal (BSF) as a new and sustainable aquafeed ingredient has been largely explored in several fish species. However, only fragmentary information is available about the use of BSF meal-based diets in sturgeon nutrition. In consideration of a circular economy concept and a more sustainable aquaculture development, the present research represents the first comprehensive multidisciplinary study on the physiological effects of a BSF diet during sturgeon culture in an aquaponic system. Siberian sturgeon (Acipenser baerii) juveniles were fed over a 60-days feeding trial on a control diet (Hi0) and a diet containing 50% of full-fat BSF meal respect to fish meal (Hi50). Physiological responses of fish were investigated using several analytical approaches, such as gas chromatography-mass spectrometry, histology, Fourier Transformed Infrared Spectroscopy (FTIR), microbiome sequencing and Real-time PCR. While aquaponic systems performed optimally during the trial, Hi50 group fish showed lower diet acceptance that resulted in growth and survival reduction, a decrease in hepatic lipids and glycogen content (FTIR), a higher hepatic hsp70.1 gene expression and a worsening in gut histological morphometric parameters. The low feed acceptance showed by Hi50 group sturgeon highlighted the necessity to improve the palatability of BSF-based diet designed for sturgeon culture.

Addition of Olive Pomace to Feeding Substrate Affects Growth Performance and Nutritional Value of Mealworm (Tenebrio Molitor L.) Larvae.

The well-recognized efficiency of Tenebrio molitor larvae to convert low quality organic matter into a nutritionally valuable biomass was exploited to manage solid wastes coming from the olive oil industry, which represent a severe environmental challenge in the Mediterranean area. Three organic pomace-enriched substrates (mixtures middlings/pomace 3:1, 1:1, and 1:3) were assessed, together with 100% organic wheat flour and 100% organic middlings as control feeds. A feeding substrate made up of 25% olive pomace and 75% wheat middlings appeared to be the best compromise between growth performance (larval and pupal weights, survival rate, development time) and nutritional properties of mealworm larvae. In fact, larvae fed the 3:1 feed showed the highest dry matter (DM) yield (38.05%), protein content (47.58% DM), and essential/non-essential amino acids ratio (1.16). Fat content (32.14% DM) and fatty acid composition were not significantly different than those of larvae fed more pomace-enriched feeds.

Sensory Receptors Associated with the Labial Tip and Precibarium of Philaenus spumarius L. (Hemiptera: Aphrophoride).

The meadow spittlebug, Philaenus spumarius (Linnaeus) (Hemiptera: Aphrophoridae), is an important vector for the xylem-limited bacterium Xylella fastidiosa (Wells, Raju, Hung, Weisburg, Mandelco-Paul, and Brenner), which is associated with olive quick decline syndrome in southern Italy. The mouthparts of Hemiptera have important roles in host plant selection, feeding behavior and for vectoring pathogens that cause plant diseases. In this study, the functional morphology of the sensory structures located on the labium tip and precibarium of P. spumarius was investigated using scanning and transmission electron microscopy. The labium tip is composed of two symmetrical sensory complexes, each with five different types of sensilla: aporous sensilla trichodea type 1 and 2; uniporous sensilla chaetica type 1 and 2; and multiporous sensilla basiconica. The precibarium of P. spumarius has two kinds of sensory structures: bulbous sensilla and papillae sensilla. In particular, two groups of sensilla are located on the epipharynx: a distal group that consists of ten papillae sensilla and a proximal group composed of six papillae sensilla and two bulbous sensilla, while the hypopharynx has only two papillae sensilla. The involvement of these sensory structures in the context of feeding behavior and pathogen transmission is discussed.

Fluid dynamics in the functional foregut of xylem-sap feeding insects: A comparative study of two Xylella fastidiosa vectors.

Xylem sap sucking insects are adapted to ingest fluids under tension. Although much has been learned about such feeding strategy, this adaptation still poses several unresolved questions, including how these insects ingest against strong xylem sap tension. Xylem sap-feeding insects are vectors of the plant pathogenic xylem-limited bacterium Xylella fastidiosa. This bacterium colonizes the cuticular lining of the foregut of vectors in a persistent manner. We used micro-computed tomography and scanning electron microscopy to investigate the foregut morphometry of two X. fastidiosa vector species: Philaenus spumarius and Graphocephala atropunctata (Hemiptera: Aphrophoridae and Cicadellidae, respectively). On the basis of morphometric data, we built a hydrodynamic model of the foregut of these two insect species, focusing on the precibarium, a region previously shown to be colonized by X. fastidiosa and correlated with pathogen acquisition from and inoculation to plants. Our data show that space in the P. spumarius functional foregut could potentially harbor twice as many cells as similar space in G. atropunctata, although the opposite trend has been observed with biological samples. Average flow velocity of ingested fluid depended on the percentage of the cibarium volume exploited for suction: if the entire volume were used, velocities were in the range of meters per second. In contrast, velocities on the order of those found in the literature (about 10 cm/s) were attained if only 5% of the cibarium volume were exploited. Simulated bacterial colonization of the foregut was analyzed in relation to hydrodynamics and pressure needed for insects to ingest. Our model is designed to represent the diameter reduction of the food canal in both insect species when infected with X. fastidiosa. Results indicated that full bacterial colonization significantly increased the mean sap-sucking flow velocity. In particular, the colonization increased the maximum section-averaged velocity in the G. atropunctata more than two times and the net pressure needed to mantain the flow in the precibarium when colonized is relevant (about 0.151 MPa) if compared to a standard xylem sap tension (1 MPa). Bacterial colonization also influenced the sucking process of the G. atropunctata, by hindering the formation of a recirculation zone (or eddy), that characterizd the flow in the distal part of the precibarium when bacteria were absent. On the other hand, considering the pressure the insect must generate to feed, X. fastidiosa colonization probably influences fitness of the G. atropunctata more than that of P. spumarius.

Hermetia illucens in diets for zebrafish (Danio rerio): A study of bacterial diversity by using PCR-DGGE and metagenomic sequencing.

In the present research, bacterial diversity was studied during a 6-month feeding trial utilizing zebrafish (Danio rerio) fed Hermetia illucens reared on different substrates with an emphasis on fish gut bacterial diversity. A polyphasic approach based on viable counting, PCR-DGGE and metagenomic 16S rRNA gene amplicon target sequencing was applied. Two different H. illucens groups were reared on coffee by-products (C) or a mixture of vegetables (S). Viable counts showed a wide variability based on substrate. PCR-DGGE and Illumina sequencing allowed the major and minor bacterial taxa to be detected. Both samples of larvae and their frass reared on the S substrate showed the highest richness and evenness of bacterial communities, whereas zebrafish (ZHC) fed H. illucens reared on substrate C and zebrafish (ZHS) fed H. illucens reared on substrate S had the lowest bacterial richness and evenness. A stimulating effect of bioactive compounds from coffee by-products on the occurrence of Lactobacillaceae and Leuconostoccaceae in H. illucens reared on substrate C has been hypothesized. Zebrafish gut samples originating from the two feeding trials showed complex microbial patterns in which Actinobacteria and Alteromonadales were always detected, irrespective of the diet used. Enterobacteriaceae in fish guts were more abundant in ZHS than in ZHC, thus suggesting an influence of the bioactive compounds (chlorogenic and caffeic acids) in the substrate on Enterobacteriaceae in fish guts. ZHC showed a higher abundance of Clostridia than did ZHS, which was likely explained by stimulating activity on the bacteria in this class by the bioactive compounds contained in H. illucens reared on substrate C. An influence of the microbiota of H. illucens or insect-derived bioactive compounds on the gut microbiota of zebrafish has been suggested. The presence of bacteria consistently associated with zebrafish guts has been found irrespective of the diet, thus attesting to the likely stability of the core fish microbiota.

Functional anatomy of the precibarial valve in Philaenus spumarius (L.).

In phytophagous sap-sucking insects, the precibarial valve plays an important role in sap ingestion. We used light and electron microspcopy to study the morphology and the ultrastructure of the precibarial valve of the meadow spittlebug, Philaenus spumarius (Hemiptera, Aphrophoridae), in order to better understand the operative mechanism of this structure. The precibarial valve revealed to be a complex structure with a bell-like invagination in the middle of the precibarium (on the epipharynx). Unlike the current hypothesis, we propose that the valve opens by dilator muscles and closes through cuticular and fluid tensions, the latter leading to morphological changes to the plane of the valve based on sap flow. Moreover, the presence of a precibarial secretory structure is described for the first time for auchenorrhynchan insects. In light of these observations, functions are hypothesized and discussed for this secretory structure.

Distribution of Transferable Antibiotic Resistance Genes in Laboratory-Reared Edible Mealworms (Tenebrio molitor L.).

In the present study, the distribution of antibiotic resistance genes in laboratory-reared fresh mealworm larvae (Tenebrio molitor L.), their feeding substrates (carrots and wheatmeal), and frass was assessed. Microbial counts on selective media added with antibiotics highlighted the presence of lactic acid bacteria resistant to ampicillin and vancomycin and, more specifically, enterococci resistant to the latter antibiotic. Moreover, staphylococci resistant to gentamicin, erythromycin, tetracycline, and vancomycin were detected. Enterobacteriaceae resistant to ampicillin and gentamicin were also found, together with Pseudomonadaceae resistant to gentamicin. Some of the genes coding for resistance to macrolide-lincosamide-streptogramin B (MLSB) [erm(A), erm(C)], vancomycin [vanA, vanB], tetracycline [tet(O)], and ß-lactams [mecA and blaZ] were absent in all of the samples. For the feeding substrates, organic wheatmeal was positive for tet(S) and tet(K), whereas no AR genes were detected in organic carrots. The genes tet(M), tet(K), and tet(S) were detected in both mealworms and frass, whereas gene aac-aph, coding for resistance to amynoglicosides was exclusively detected in frass. No residues for any of the 64 antibiotics belonging to 10 different drug classes were found in either the organic wheatmeal or carrots. Based on the overall results, the contribution of feed to the occurrence of antibiotic resistance (AR) genes and/or antibiotic-resistant microorganisms in mealworm larvae was hypothesized together with vertical transmission via insect egg smearing.

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.).

Transmission and scanning electron microscopic observations on antennal apical pegs in the wasp species Pimplinae (Insecta: Hymenoptera).

Pimplinae are parasitoids belonging to the family Ichneumonidae that attack and develops inside hidden host; female wasps evolved a peculiar host recognition strategy, that involves the use of self-produced vibrations which are transmitted through the antennae on the substrate and perceived back as an echo using the leg subgenual organ. In this study we investigated, using both scanning and transmission electron microscopy, the antennal tips of a few Pimplinae. In all the investigated species, the antennal tips present peculiar apical pegs with different shape and number, often defining a flattened sole devoid of other antennal structures, such as sensilla. These pegs are present in both sexes with different number and development, are inserted on the antennal wall through an inflexible socket and present a cuticular shaft with cuticle of different thickness. We never found the presence of sensory neurons or glandular epithelium associated with these pegs. Because of their peculiar morphological features, we hypothesize for the antennal apical pegs a role in the context of host searching behavior (in the case of the female through the vibrational sounding strategy), as well as during mating behavior.

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.

Fine structure of antennal sensilla of the spittlebug Philaenus spumarius L. (Insecta: Hemiptera: Aphrophoridae). I. Chemoreceptors and thermo-/hygroreceptors.

The meadow spittlebug, Philaenus spumarius (L.) (Hemiptera: Cercopoidea: Aphrophoridae), is a polyphagous species that transmits Xylella fastidiosa, a bacterium associated with "Olive Quick Decline Syndrome" in Southern Italy. In this study, the morphology and the ultrastructure of the antennal sensilla of P. spumarius were investigated. The antennae consist of three segments: a basal scape, a pedicel and a flagellum composed of a basal enlargement (ampulla) and a long segment (filament). The pedicel bears a single campaniform sensillum while the ampulla houses twelve coeloconic sensilla and three large basiconic sensilla. These latter sensilla show a smooth multiporous external cuticular wall and a total number of 27 sensory neurons per sensillum. The coeloconic sensilla belong to two morphologically distinct types: double-walled and single-walled sensilla. The sensory peg of the double-walled sensilla is smooth at the base and distally has a grooved cuticular surface with pores organized in spoke channels between each ridge. Three sensory neurons enter the lumen while at the basal level, before entering the peg, a fourth sensory neuron is found. The single-walled sensilla show an aporous thick cuticular wall and two sensory neurons entering the sensillar lumen, with a third neuron ending at the sensillum base.

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.

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

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.

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

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 & Schiffermüller), 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.

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.