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Olive (Olea europaea L.) is a traditional crop of great socioeconomic importance in the Mediterranean Basin. In 2021, a survey was performed in an Italian nursery located in Central Italy where unusual symptoms and widespread dieback had been observed on young potted olive trees. Twigs and branches of all plants affected by dryness showed the presence of the alien bark beetle Xylosandrus compactus. Woody tissues colonized by X. compactus were transferred into the laboratory and several fungi such as Cladosporium perangustum and Neofusicoccum parvum were then identified. In addition to these species, Geosmithia pallida was also reported for the first time in Italy on this plant. Studies on this bark beetle and its associated fungi are currently in progress to further evaluate potential risks for olive cultivation. Key Words: Olea europea, fungi, bark beetles.

Bark and wood-boring beetles feeding on coniferous and deciduous trees in different forest ecosystems are often associated with various species of fungi. In 2019, widespread attacks of Liparthrum colchicum Semenov (Coleoptera Curculionidae Scolytinae) were observed on Laurus nobilis L. in Tuscany (Italy). Samples of colonized terminal twigs were collected to investigate the presence of phytopathogenic fungi associated with the scolytid. Two different colonies of the Geosmithia genus were identified as Geosmithia pallida and Geosmithia langdonii. To our knowledge this is the first report of G.pallida and G. langdonii associated with L. colchicum on Bay tree, in Italy.

Xylosandrus compactus and Liparthrum colchicum are two Scolytinae recorded for the first time in Italy in 2011 and 2019, respectively. X. compactus is an “ambrosia beetle” causing damages to several plants typical of the Mediterranean maquis through its digging activity on twigs and branches of the host plants. L. colchicum is a “bark beetle”, monophagous on laurel shrubs and trees. During a survey performed in summer of 2020, in “Tenuta Salviati” located in the Migliarino Natural Park (Pisa, Tuscany), adults of X. compactus and L. colchicum were collected from the same branches and apical twigs of Laurus nobilis hedges. The study aimed at characterizing fungi isolated from the external surface of the two scolytids bodies, to evaluate similarities and even their role in fungal spreading in the environment.

Liparthrum colchicumSemenov (Coleoptera Curculionidae Scolytinae) is a relatively unknown monophagous insect species primarily associated with thin dried twigs (about 0.5-1cm in diameter) of Laurel (Laurus nobilis). After being caught in traps located at Italian ports, it was initially considered an intercepted species alien to the country. However, recent findings, suggest that this beetle may have a different origin. Several records from different sites of Tuscany and Latium showed that it could be relatively widespread in Italy. These observations strengthened the hypothesis that this scolytid, harmless to L. nobilis, might be already established or even native to the country. Therefore, we argue that it could have been easily overlooked thus far, as it thrives only in twigs already wilted due to different environmental factors. Moreover, field observations, carried out in the two Italian Regions, evidenced high frequencies of its infestations in Laurel twigs previously colonized by Xylosandrus compactus. Notes on the taxonomy and biology of this species are also reported in the present wo

Edible insects are generally reared on agricultural product-based diets. These feeding substrates may contain pesticides or toxins that are derived from field practices for plant growth and defence and that may represent a health risk to insect consumers. The present study investigated Tenebrio molitor capability to uptake from contaminated substrates three chemical compounds that are employed in agricultural practices: the insecticide deltamethrin (DELTA), the fungicide tebuconazole (TEB) and the plant growth regulator chlormequat chloride (CCC), all with different properties of lipophilicity and potentially harmful for human and animal health. Newly optimised and validated LC-MS/MS methods have been used for the quantification of pesticide residues in larvae samples. Analysis showed that DELTA and TEB are retained by larvae, even after 24 h of fasting, due to their lipophilicity. Larvae fasting period allowed the elimination of 47% of DELTA residues and 78% of TEB ones. Diversely, CCC, the most hydrophilic chemical compound, was completely eliminated after the fasting. The toxicity of pesticides to larvae has been also investigated. The highest mortality of T. molitor larvae (close to 50%) was recorded for the insecticide DELTA, at the dose of 20 mg/kg and for CCC (15%) at the concentration of 4 mg/kg. The safety of insects as food and feed represents an important section in the field of the control of food of animal origin and animal health. Nevertheless, to date, this aspect is not yet completely determined due to the lack of scientific data in this area. By assessing the accumulation rate of some chemicals in reared insects and by evaluating the toxic effects that chemicals may have on larvae, this study could contribute to filling this gap.

The progression of drugs into clinical phases requires proper toxicity assessment in animals and the correct identification of possible metabolites. Accordingly, different animal models are used to preliminarily evaluate toxicity and biotransformations. Rodents are the most common models used to preliminarily evaluate the safety of drugs; however, their use is subject to ethical consideration and elevated costs, and strictly regulated by national legislations. Herein, we developed a novel, cheap and convenient toxicity model using Tenebrio molitor coleoptera (TMC). A panel of 15 drugs—including antivirals and antibacterials—with different therapeutic applications was administered to TMC and the LD50 was determined. The values are comparable with those already determined in mice and rats. In addition, a TMC model was used to determine the presence of the main metabolites and in vivo pharmacokinetics (PK), and results were compared with those available from in vitro assays and the literature. Taken together, our results demonstrate that TMC can be used as a novel and convenient preliminary toxicity model to preliminarily evaluate the safety of experimental compounds and the formation of main metabolites, and to reduce the costs and number of rodents, according to 3R principles.

The angiotensin-converting enzyme (ACE) inhibitory potential of the main protein fractions from Tenebrio molitor larvae (TML) was examined to evaluate their use as a novel antihypertensive functional food. Both fractions contained YAN tripeptide, previously reported as responsible for ACE inhibition. Although YAN has been synthesized and was used as a standard for LC-MS/MS quantification and IC

Molecular assays based on qPCR TaqMan Probes were developed to identify three species of the genus Xylosandrus, X. compactus, X. crassiusculus and X. germanus (Coleoptera Curculionidae Scolytinae). These ambrosia beetles are xylophagous species alien to Europe, causing damages to many ornamental and fruiting trees as well as shrubs. DNA extraction was carried out from adults, larvae and biological samples derived from insect damages on infested plants. For X. compactus, segments of galleries in thin infested twigs were cut and processed; in the case of X. crassiusculus, raw frass extruded from exit holes was used, while DNA of X. germanus was extracted from small wood chips removed around insect exit holes. The assays were inclusive for the target species and exclusive for all the non-target species tested. The LoD was 3.2 pg/µL for the frass of X. crassiusculus and 0.016 ng/µL for the woody matrices of the other two species. Both repeatability and reproducibility were estimated on adults and woody samples, showing very low values ranging between 0.00 and 4.11. Thus, the proposed diagnostic assays resulted to be very efficient also on the woody matrices used for DNA extraction, demonstrating the applicability of the protocol in the absence of dead specimens or living stages.

In pre-hypertension, moderate control of blood pressure (BP) can be obtained by a nutritional approach. The effects of a diet enriched with defatted larvae of the mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (TM) endowed with ACE inhibitory activity was studied in both spontaneously hypertensive rats (SHR) and in the age-matched normotensive Wistar Kyoto strain. These were fed for 4 weeks with standard laboratory rodent chow supplemented with or without TM or captopril. In SHR, the TM diet caused a significant reduction in BP, heart rate and coronary perfusion pressure, as well as an increase in red blood cell glutathione/glutathione disulphide ratio. Rat brain slices of SHR were more resistant to oxidative stress and contained lower levels of inflammatory cytokines, while vascular and liver enzyme-activities were not affected. These results suggest that TM can be considered a new functional food that can lower BP in vivo and thus control cardiovascular-associated risk factors such as hypertension.

The industrial farming of edible insects for food and feed generally employs cultivated plants and derivatives as rearing diets. These feed substrates may contain toxins produced by different species of fungi which cause ad-verse health effects to consumers. Frequently found in cereals and cereal products, mycotoxins aflatoxins, ochra-toxins and fumonisins are harmful to human and animal health. In this study the uptake of aflatoxin B1 (AFB1), ochratoxin A (OTA) and fumonisin B1 (FB1) and their elimination rates were investigated in T. molitor larvae fed on cereal-based diets contaminated with two concentration levels for each mycotoxin. New analytical methods for extraction and quantification of AFB1, OTA and FB1 residues in larvae were developed andvalidated.It has been demonstrated that T. molitor larvae did not accumulate in detectable or dangerous concentrations the three tested mycotoxins at the two tested concentration levels and that a fasting period of 24 hours ensured a suffi-cient elimination rate of AFB1, OTA and FB1 residues. These results represent a preliminary investigation to define an appropriate quality control procedure to assess the safe use of T. molitor for food and feed purposes.

We investigated the chemical composition of the weevil Rhynchophorus ferrugineus larvae, traditionally used as human food in Asia and known worldwide as one of the most significant pest for palm trees. Total fat content and fatty acid composition were analyzed using standard methodologies in (1) weevil larvae reared on apple fruit slices and wild specimens collected from attacked (2) Phoenix canariensis and (3) Syagrus romanzoffiana palm trees. Total fat content was extremely high in all the specimens (ranged between 57.62 and 60.03% based on dry weight). Despite sharing the same prevalent fatty acids (myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, α-linoleic acid, and α-linolenic acid), fatty acid composition of the wild weevil larvae significantly differed from that of the specimens raised on apple fruit, due to the presence of other minor compounds. In general, a good balance between unsaturated fatty acids (∼53.68% of total fatty acids) and saturated fatty acids (∼43.41% of total fatty acids) and a low cholesterol content (74.61–152.32 mg/kg based on dry matter) were detected in all the specimens. Conversely, the weevil larvae did not represent a good source of α-tocopherol (14.17–26.22 mg/kg based on dry matter). The ability of the protein extracts obtained from the weevil larvae to inhibit in vitro the angiotensin-converting enzyme, the main enzyme involved in blood pressure regulation, was also investigated. To simulate gastrointestinal digestion process, protein extracts were hydrolyzed by the gastrointestinal enzymes. A significantly lower IC50(0.588–0.623 mg/ml) was measured in all the protein extracts after enzymatic hydrolysis versus the corresponding crude protein extracts (3.270–3.752 mg/ml). Given that the weevil larvae are able to provide interesting benefits for human health, this study supports their use as human food not just in the native countries where they are traditionally consumed and farmed but also throughout the world.

Fat is the second most abundant component of the nutrient composition of the mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) that represents also an interesting source of PUFA, especially n-6 and n-3 fatty acids, involved in prevention of cardiovascular diseases. This study investigated the possibility of modifying the fat content and the FA composition of yellow mealworms through feeding and how this would be influenced by developmental stages, pupal sex, and generation with the future aim of applying this coleopteran as a diet supplement for human health. Growth rate and cumulative mortality percentage on the different feeding substrates were also evaluated to select the optimal conditions for a mass-raising of this insect species. Despite the different fat content in the six different breeding substrates used, T. molitor larvae and pupae contained a constant fat percentage (>34% in larvae and >30% in pupae). A similar total fat content was found comparing larvae and male and female pupae of the second generation to those of the first generation. On the contrary, FA composition differed both in larvae and pupae reared on the different feeding substrates. However, the exemplars reared on the diets based on 100% bread and 100% oat flour showed SFA, PUFA percentages, and an n-6/n-3 ratio more suitable for human consumption; the diet based on beer yeast, wheat flour, and oat flour resulted in a contemporary diet that most satisfied the balance between a fat composition of high quality and favorable growth conditions.

Thirty-five bacterial strains were selected on the basis of colony morphology from larvae and pupae of Rhynchophorous (Olivier) ferrugineus collected from infested Phoenix canariensis Chabaud trees growing in different Italian locations. 16S rDNA sequencing showed that bacteria isolated from larvae were mostly Gram positive and belonged to Bacillus , Brevibacillus and Paenibacillus genera. Bacteria isolated from pupae were affiliated to Bacillus and five different Gram negative genera ( Alcaligenes , Morganella , Myroides , Providencia and Serratia ). Some strains produced lytic enzymes like chitinase, lecithinase, lipase and protease in vitro. The enthomopathogenic strains belonging to Bacillus thuringiensis, Providencia rettgeri and Serratia marcescens species may be considered of interest as potential biocontrol agents of R. ferrugineus .

The Velvet Longhorned Beetle Trichoferus campestris (Faldermann, 1835) was intercepted for the first time in Italy, in the Naples harbour, during monitoring activities at entry points carried out in the context of the national project ASPROPI. T. campestris is widely polyphagous and able to colonize several woody species in both agricultural and forest environments. Moreover timber and solid wood items can be equally attractive to this pest. This additional report, in an area where other alien insect species had been identified in the past, emphasises how the national monitoring network represents a key element of the alert system designed to quickly counter the accidental introduction and spreading of exotic insect pests.

Beauveria bassiana is one of the most known entomopathogenic fungal species and its entomopathogenic mechanism involves several bioactive metabolites, mainly cuticle-degrading enzymes and toxic molecules, which are predicted to play a key role as virulence factors. In this study six Beauveria bassiana strains (B 13/I03, B 13/I11, B 13/I49, B 13/I57, B 13/I63, and B 13/I64) were assayed against Tenebrio molitor larvae. Enzymatic activity of total proteases and specifically Pr 1 and Pr 2, as well as the production of toxic compounds were investigated in each fungal strain. Toxins were detected both in vitro-in medium filtrates and mycelia-and in vivo-in Tenebrio molitor larvae infected by the fungal strains tested. B 13/I11 and B 13/I63 strains showed the most significant entomopathogenic activity against Tenebrio molitor larvae (cumulative mortality rate 100 and 97%, respectively; average survival time 5.85 and 6.74 days, respectively). A widely variable and fungal strain-dependent enzymatic activity of total proteases, Pr 1 and Pr 2 was found. Beauvericin, beauvericin A and bassianolide resulted the most prevalent toxins detected in the substrates analyzed. It has been found that an increase of beauvericin content in vivo resulted significantly correlated to a decrease of Tenebrio molitor larvae average survival time in entomopathogenic bioassay (inverse correlation). The involvement of beauvericin in B. bassiana entomopathogenic process is confirmed; in vitro analysis of cuticle degrading proteases activity and toxins production in relation to the methods adopted resulted insufficient for a rapid screening to determine the virulence of B. bassiana strains against Tenebrio molitor larvae.

An aberrant specimen of Rhynchophorus ferrugineus (Coleoptera: Dryophthoridae) also known as red palm weevil (RPW), the most economically important insect pest of palms in the world, was found among a batch of conspecifics reared for research purposes. A morphological analysis of this weevil revealed the presence of nematodes associated with a structured cuticle defect of the thorax. These nematodes were not able to be cultured, but were characterized by molecular analysis using 28S and 18S ribosomal DNA and shown to belong to the family Panagrolaimidae (Rhabditida), within a clade of Panagrellus. While most nematodes in the insect were juveniles, a single male adult was partially characterized by light microscopy. Morphometrics showed similarities to a species described from Germany. Excluding the entomopathogenic nematodes (EPN), only five other genera of entomophilic or saprophytic rhabditid nematodes are associated with this weevil. This is the first report of panagrolaimid nematodes associated with this invasive pest. Possible mechanisms of nematode-insect association are discussed.

Hypertension is well known as one of the major risk for cardiovascular diseases which annually affect millions of people. The angiotensin converting enzyme (ACE) plays a key role in blood pressure regulation process. Indeed, hypertension treatment by synthetic ACE inhibitors (e.g. captopril, lisinopril and ramipril) is effective; however, their use can cause serious side effects, such as hypotension, cough, reduced renal function and angioedema. Thus, research was focused on natural ACE inhibitory peptides sources such as foodstuffs and also, more recently, edible insects. In the last decades, ACE inhibitory activity has been detected in protein hydrolysates from insect species belonging to the orders of Coleoptera, Diptera, Hymenoptera, Lepidoptera and also Orthoptera. Further investigations led to identify specific ACE inhibitory peptides from the silkworm Bombyx mori (Lepidoptera: Bombycidae), the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae), the cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae) and also from the weaver ant Oecophylla smaragdina (Hymenoptera: Formicidae). Even if ACE inhibitory activity of these bioactive peptides has been in vitro assayed and is comparable to those of some bioactive peptides derived from other animal protein sources, the in vivo effectiveness of most of these bioactive peptides still needs to be confirmed. The aim of this review is to present an outline of the currently available data on the potential use of insects for hypertension treatment with a focus on the ACE inhibitory peptides identified in these invertebrates to date.

Rhynchophorus palm weevils are large insects belonging to the family Dryophthoridae. All Rhynchophorus species are polyphagous and have a similar life history but some are major pests because of the serious economic damage they cause, in particular to several species of the family Arecaceae. Here we review the natural enemies of Rhynchophorus species in both their native and introduced regions of the world, to assess the possibility of biological control of this taxon. Moreover, particular attention is paid to the well-studied and harmful species Rhynchophorus ferrugineus, about which more information is available, and to its natural enemies in the Mediterranean region, because the impact of this pest in this recently colonized area is particularly remarkable and also the recent trend in species management is looking for indigenous natural enemies. More than 50 natural enemies have been reported to attack Rhynchophorus species, even if most of them are associated to R. ferrugineus (Olivier), highlighting the lack of information on the other species of the genus. Pros and cons of all the biological control agents are then discussed: among the considered organisms, fungi are noteworthy to be considered for inclusion in integrated pest management programs. Overall, our overview underlines the need to increase knowledge on natural enemies of all the species of the genus Rhynchophorus, to isolate more virulent strains and to determine the optimum conditions for the actions of the biocontrol agents.

Beauveria bassiana is one of the most known entomopathogenic fungal species and its entomopathogenic mechanism involves several bioactive metabolites, mainly cuticle-degrading enzymes and toxic molecules, which are predicted to play a key role as virulence factors. In this study six Beauveria bassiana strains (B 13/I03, B 13/I11, B 13/I49, B 13/I57, B 13/I63, and B 13/I64) were assayed against Tenebrio molitor larvae. Enzymatic activity of total proteases and specifically Pr 1 and Pr 2, as well as the production of toxic compounds were investigated in each fungal strain. Toxins were detected both in vitro-in medium filtrates and mycelia-and in vivo-in Tenebrio molitor larvae infected by the fungal strains tested. B 13/I11 and B 13/I63 strains showed the most significant entomopathogenic activity against Tenebrio molitor larvae (cumulative mortality rate 100 and 97%, respectively; average survival time 5.85 and 6.74 days, respectively). A widely variable and fungal strain-dependent enzymatic activity of total proteases, Pr 1 and Pr 2 was found. Beauvericin, beauvericin A and bassianolide resulted the most prevalent toxins detected in the substrates analyzed. It has been found that an increase of beauvericin content in vivo resulted significantly correlated to a decrease of Tenebrio molitor larvae average survival time in entomopathogenic bioassay (inverse correlation). The involvement of beauvericin in B. bassiana entomopathogenic process is confirmed; in vitro analysis of cuticle degrading proteases activity and toxins production in relation to the methods adopted resulted insufficient for a rapid screening to determine the virulence of B. bassiana strains against Tenebrio molitor larvae.

The red palm weevil Rhynchophorus ferrugineus, recently spreading from its native range throughout the world, is one of the most dangerous pests for several palm species. The sterile insect technique (SIT) is a promising environmentally friendly approach for the management of this pest. An essential prerequisite of the SIT is that irradiated males released in nature are as sexually competitive as their wild counterparts. Here we evaluated the potential use of the SIT in red palm weevil males by investigating the sexual competitiveness of gamma-irradiated (Cobalt 60 source, dose 80 Gray) males in the laboratory in a natural-like social context with a balanced sex ratio and by testing their efficacy in impairing female reproduction. Our results showed that irradiation did not affect any of the mating behaviour parameters. However, the male reproductive potential was drastically reduced, as fertility was markedly decreased in females mated with irradiated males. Therefore, an 80 Gray irradiation dose is a promising option for the SIT in the control of the red palm weevil, producing sterile but sexually competitive males when tested in a natural-like mating context.

The attractiveness of different combinations of volatile compounds to several scolytid and cerambycid species, potential vectors of phytoparasitic nematodes of the genus Bursaphelenchus Fuchs, was investigated in pine woods of central Italy. The blend composed of α-pinene, ethanol, ipsenol, ipsdienol and 2-methyl-3-buten-2-ol was very efficient in trapping the cerambycids Monochamus galloprovincialis, Acanthocinus griseus and Arhopalus syriacus and the scolytids Ips sexdentatus, Orthotomicus erosus and Hylurgus ligniperda. This blend is proposed for operational surveys of Bursaphelenchus spp. associated with these beetles. Comparison of the capture levels with cross-vane and funnel traps baited with α-pinene, ethanol, ipsenol, ipsdienol, 2-methyl-3-buten-2-ol and cis-verbenol revealed the higher efficacy of the cross-vane traps for the above-mentioned cerambycids and the scolytid I. sexdentatus but greater effectiveness of the funnel traps in capturing H. ligniperda and O. erosus. Cross-vane traps were more efficient in capturing large numbers of these insects. Data on non-target xylophagous beetles and predator species are also reported.

In the present study we describe the morphology of the male reproductive apparatus and sperm ultrastructure of the red palm weevil - an invasive pest of several palm tree species - as well as the most important steps of spermatogenesis. The reproductive apparatus consists of a pair of testes (each formed by two lobes) a long tube-like accessory gland, a prostate gland and a small accessory gland. Characteristic features of the sperm are 90-100. μm total length, 10. μm nucleus, two mitochondrial derivatives, two accessory bodies, one well-developed puff-like structure and a typical insect 9+9+2 flagellar axoneme. One of the methods used for the biological control of pests is the Sterile Insect Technique (SIT), for the evaluation of which we make a preliminary comparison of the sperm ultrastructure of non-irradiated and irradiated weevils (at a dose of 80 Gray). © 2014 Elsevier Ltd.

Rhynchophorus ferrugineus is considered the worst pest of palm species, and few natural enemies are reported for this parasite in its area of origin. Here, we report the first recovery of the entomopathogenic fungus Metarhizium pingshaense associated with R. ferrugineus from Vietnam. The morphological, biochemical, and toxicological features of this strain were studied and compared with those of another Metarhizium strain associated with this weevil in Sicily (Italy), an area of recent introduction. The potential use of these fungi as biocontrol agents was tested against adult insects in laboratory trials and a similar mortality rate was found. Both strains were able to produce toxins and cuticle-degrading proteases, but they showed dissimilar enzymatic and toxicological profiles, suggesting a different virulence activity.

The stereoselective synthesis of both enantiomers of trifluoro frontalin (-)-(1S,5R)- and (+)-(1R,5S)-8, as well as of diastereomeric monofluoro frontalines (-)-(1R,2R,5R)-18 and (-)-(1R,2S,5R)-20, analogues of the bioactive component of the aggregation pheromone of the Scolytidae insect family, has been accomplished starting from (-)-(1R)- and (+)-(1S)-menthyl (S)-toluene-4-sulfinate as a source of chirality and methyl trifluoroacetate or fluoroacetate, respectively, as sources of fluorine. The C-1 stereocenters were installed via stereoselective epoxidation of beta-sulfinyl ketones 2 and 13 with diazomethane. The bicyclic core was obtained by totally stereocontrolled and chemoselective tandem Wacker oxidation/intramolecular ketalization of the intermediate unsatured sulfinyl diols 5, 15, and 19. Axially fluorinated (-)-20 elicited a strong electroantennographic response in laboratory tests on females of Dendroctonus micans, whereas equatorially fluorinated (-)-18 and the trifluoroanalogue (-)-8 showed modest responses. Field trials using (-)-20 were not indicative owing to the locally scarce population of D. micans, but it showed some attractiveness for other Coleoptera families.