Environmental ameliorations and politics in support of pollinators. Experiences from Europe: A review.

At least 87% of angiosperm species require animal vectors for their reproduction, while more than two-thirds of major global food crops depend on zoogamous pollination. Pollinator insects are a wide variety of organisms that require diverse biotic and abiotic resources. Many factors have contributed to a serious decrease in the abundance of populations and diversity of pollinator species over the years. This decline is alarming, and the European Union has taken several actions aimed at counteracting it by issuing new conservation policies and standardizing the actions of member countries. In 2019, the European Green Deal was presented, aiming to restore 100% of Europe's degraded land by 2050 through financial and legislative instruments. Moreover, the Common Agricultural Policies have entailed greening measures for the conservation of habitats and beneficial species for more than 10 years. The new CAP (CAP 23-27) reinforces conservation objectives through strategic plans based on eco-schemes defined at the national level by the member countries, and some states have specifically defined eco-schemes for pollinator conservation. Here, we review the framework of EU policies, directives, and regulations, which include measures aimed at protecting pollinators in agricultural, urban, and peri-urban environments. Moreover, we reviewed the literature reporting experimental works on the environmental amelioration for pollinators, particularly those where CAP measures were implemented and evaluated, as well as studies conducted in urban areas. Among CAP measures, several experimental works have considered the sowing and management of entomophilous plants and reported results important for environmental ameliorations. Some urban, peri-urban and wasteland areas have been reported to host a considerable number of pollinators, especially wild bees, and despite the lack of specific directives, their potential to contribute to pollinator conservation could be enhanced through targeted actions, as highlighted by some studies.

The Odorant-Binding Proteins of the Spider Mite Tetranychus urticae.

Spider mites are one of the major agricultural pests, feeding on a large variety of plants. As a contribution to understanding chemical communication in these arthropods, we have characterized a recently discovered class of odorant-binding proteins (OBPs) in Tetranychus urticae. As in other species of Chelicerata, the four OBPs of T. urticae contain six conserved cysteines paired in a pattern (C1-C6, C2-C3, C4-C5) differing from that of insect counterparts (C1-C3, C2-C5, C4-C6). Proteomic analysis uncovered a second family of OBPs, including twelve members that are likely to be unique to T. urticae. A three-dimensional model of TurtOBP1, built on the recent X-ray structure of Varroa destructor OBP1, shows protein folding different from that of insect OBPs, although with some common features. Ligand-binding experiments indicated some affinity to coniferyl aldehyde, but specific ligands may still need to be found among very large molecules, as suggested by the size of the binding pocket.

Proteinase pattern of honeybee prepupae from healthy and American Foulbrood infected bees investigated by zymography.

American foulbrood disease (AFB) is the main devastating disease that affects honeybees' brood, caused by Paenibacillus larvae. The trend of the research on AFB has addressed the mechanisms by which P. larvae bacteria kill honeybee larvae. Since prepupae could react to the infection of AFB by increasing protease synthesis, the aim of this work was to compare protease activity in worker prepupae belonging to healthy colonies and to colonies affected by AFB. This investigation was performed by zymography. In gel, proteolytic activity was observed in prepupae extracts belonging only to the healthy colonies. In the prepupae extracts, 2D zimography followed by protein identification by MS allowed to detect Trypsin-1 and Chymotrypsin-1, which were not observed in diseased specimens. Further investigations are needed to clarify the involvement of these proteinases in the immune response of honeybee larvae and the mechanisms by which P. larvae inhibits protease production in its host.

Chemical Communication and Reproduction Partitioning in Social Wasps.

Social wasps encompass species displaying diverse social organization regarding colony cycle, nest foundation, caste differences (from none to significant dimorphism) and number of reproductive queens. Current phylogenetic data suggests that sociality occured independently in the subfamily Stenogastrinae and in the Polistinae+Vespinae clade. In most species, including those with the simplest social organization, colony reproduction is monopolised by a single or few females. Since their nest mates can also develop ovaries and lay eggs, dominant females must somehow inhibit them from reproducing. Physical interactions in the form of open aggression or, usually, ritualised dominance by the fertile females contribute to fertility inhibition in several species, but it is unlikely to function in large colonies. In the latter case, reproduction within the colony is likely to be regulated through pheromones. Relatively little is known about these semiochemicals. Studies on all the three social wasp subfamilies, revealed that cuticular hydrocarbon components differ in abundance between egg-laying and not egg-laying females and that their composition depends on fertility status. In several species, females have been reported to manifestly react towards females with activated ovaries, but there is little evidence to support the hypothesis that fertile individuals are either recognized through their CHC composition, or that over-represented CHC constituents can inhibit fertility. Moreover, very little information exists on the possibility that exocrine glands release fertility signals or chemicals inhibiting fertility.

Diversity, abundance, and sex-specific expression of chemosensory proteins in the reproductive organs of the locust Locusta migratoria manilensis.

Chemosensory proteins (CSPs) are small soluble proteins often associated with chemosensory organs in insects but include members involved in other functions, such as pheromone delivery and development. Although the CSPs of the sensory organs have been extensively studied, little is known on their functions in other parts of the body. A first screening of the available databases has identified 70 sequences encoding CSPs in the oriental locust Locusta migratoria manilensis. Applying proteomic analysis, we have identified 17 of them abundantly expressed in the female reproductive organs, but only one (CSP91) in male organs. Bacterially expressed CSP91 binds fatty acids with a specificity for oleic and linoleic acid, as well as medium-length alcohols and esters. The same acids have been detected as the main gas chromatographic peaks in the dichloromethane extracts of reproductive organs of both sexes. The abundance and the number of CSPs in female reproductive organs indicates important roles for these proteins. We cannot exclude that different functions can be associated with each of the 17 CSPs, including delivery of semiochemicals, solubilization of hormones, direct control of development, or other unknown tasks.

Cyclic Acetals as Novel Long-Lasting Mosquito Repellents.

The use of skin repellents against hematophagous mosquitoes is an important personal protection practice wherever these insects are abundant and where they are vectors of diseases. DEET and Icaridin are the major synthetic insect repellents in commercial formulations and are considered the most effective. Here, we tested against the mosquito Aedes albopictus several cyclic hydroxyacetals synthesized by acetalization of commercially available aliphatic carbonyl compounds (ranging from C3 to C15) with either glycerol, 1,1,1-trismethyloletane, or 1,1,1-trismethylolpropane and compared their efficacy with commercial repellents. We found that several hydroxyacetals were comparable with DEET and Icaridin both in terms of the required dose and repellence duration, while a few performed better. For those most active, toxicity was investigated, finding that a few of them were less cytotoxic than DEET and less prone to permeate through cell layers. Therefore, such results indicate that novel safe mosquito repellents could be developed among cyclic hydroxyacetals.

Chemosensory proteins as putative semiochemical carriers in the desert isopod Hemilepistus reaumurii.

The order Isopoda contains both aquatic and terrestrial species, among which Hemilepistus reaumurii, which lives in arid environments and is the most adapted to terrestrial life. Olfaction has been deeply investigated in insects while it has received very limited attention in other arthropods, particularly in terrestrial crustaceans. In insects, soluble proteins belonging to two main families, Odorant Binding Proteins (OBPs) and Chemosensory Proteins (CSPs), are contained in the olfactory sensillar lymph and are suggested to act as carriers of hydrophobic semiochemicals to or from membrane-bound olfactory receptors. Other protein families, namely Nieman-Pick type 2 (NPC2) and Lipocalins (LCNs) have been also reported as putative odorant carriers in insects and other arthropod clades. In this study, we have sequenced and analysed the transcriptomes of antennae and of the first pair of legs of H. reaumurii focusing on soluble olfactory proteins. Interestingly, we have found 13 genes encoding CSPs, whose sequences differ from those of the other arthropod clades, including non-isopod crustaceans, for the presence of two additional cysteine residues, besides the four conserved ones. Binding assays on two of these proteins showed strong affinities for fatty acids and long-chain unsaturated esters and aldehydes, putative semiochemicals for this species.

Cooperative interactions between odorant-binding proteins of Anopheles gambiae.

To understand olfactory discrimination in Anopheles gambiae, we made six purified recombinant OBPs and investigated their ligand-binding properties. All OBPs were expressed in bacteria with additional production of OBP47 in the yeast Kluveromyces lactis. Ligand-binding experiments, performed with a diverse set of organic compounds, revealed marked differences between the OBPs. Using the fluorescent probe N-phenyl-1-naphthylamine, we also measured the binding curves for binary mixtures of OBPs and obtained, in some cases, unexpected behaviour, which could only be explained by the OBPs forming heterodimers with binding characteristics different from those of the component proteins. This shows that OBPs in mosquitoes can form complexes with novel ligand specificities, thus amplifying the repertoire of OBPs and the number of semiochemicals that can be discriminated. Confirmation of the likely role of heterodimers was demonstrated by in situ hybridisation, suggesting that OBP1 and OBP4 are co-expressed in some antennal sensilla of A. gambiae.

Advances in mosquito repellents: effectiveness of citronellal derivatives in laboratory and field trials.

BACKGROUND: Several essential oils, including citronella (lemongrass, Cymbopogon sp., Poaceae), are well-known mosquito repellents. A drawback of such products is their limited protection time resulting from the high volatility of their active components. In particular, citronella oil protects for <2 h, although formulations with fixatives can increase this time. RESULTS: We synthesized hydroxylated cyclic acetals of citronellal, the main component of citronella, to obtain derivatives with lower volatility and weaker odour. The crude mixture of isomers obtained in the reaction was tested under laboratory conditions for its repellency against two mosquito species, the major malaria vector Anopheles gambiae and the arbovirus vector Aedes albopictus, and found to be endowed with longer protection time with respect to DEET (N,N-diethyl-meta-toluamide) at the same concentration. Formulated products were tested in a latin square human field trial, in an area at a high density of A. albopictus for 8 h from the application. We found that the performance of the citronellal derivatives mixture is comparable (95% protection for ≤3.5 h) with those of the most widespread synthetic repellents DEET and Icaridin, tested at a four-fold higher doses. CONCLUSIONS: Modifying the hydrophilicity and volatility of natural repellents is a valuable strategy to design insect repellents with a long-lasting effect. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Differential expression of odorant-binding proteins in the mandibular glands of the honey bee according to caste and age.

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) mediate both perception and release of chemical stimuli in insects. The genome of the honey bee contains 21 genes encoding OBPs and 6 encoding CSPs. Using a proteomic approach, we have investigated the expression of OBPs and CSPs in the mandibular glands of adult honey bees in relation to caste and age. OBP13 is mostly expressed in young individuals and in virgin queens, while OBP21 is abundant in older bees and is prevalent in mated queens. OBP14, which had been found in larvae, is produced in hive workers' glands. Quite unexpectedly, the mandibular glands of drones also contain OBPs, mainly OBP18 and OBP21. We have expressed three of the most represented OBPs and studied their binding properties. OBP13 binds with good specificity oleic acid and some structurally related compounds, OBP14 is better tuned to monoterpenoid structures, while OBP21 binds the main components of queen mandibular pheromone as well as farnesol, a compound used as a trail pheromone in the honey bee and other hymenopterans. The high expression of different OBPs in the mandibular glands suggests that such proteins could be involved in solubilization and release of semiochemicals.

The double nature of 1,5-diaminonaphthalene as matrix-assisted laser desorption/ionization matrix: some experimental evidence of the protonation and reduction mechanisms.

1,5-Diaminonaphthalene (DAN) has been described as an interesting and effective matrix for matrix-assisted laser desorption/ionization (MALDI) experiments in positive ion mode, being able to activate in-source decomposition phenomena and, when employed for the analysis of proteins containing disulphide bridge(s), being able to activate reduction processes, resulting in disulphide bridge cleavage. The mechanisms of the DAN reactivity have been studied in detail, and the results indicate that the reduction properties of the matrix are of a radical nature. In the present study the structure of the reactive species produced by DAN, responsible for its reductive properties, has been investigated by accurate mass measurements and tandem mass spectrometry (MS/MS) experiments. Contrary to what is usually observed by laser irradiation of other MALDI matrices (with the sole formation of the MH(+) ion of the matrix), DAN leads to the formation of odd-electron molecular ions M(+•) . This can be rationalized by the occurrence of two photon pooling processes, due to the low ionization energy of DAN. Thus the M(+•) ion of DAN can be considered responsible for both analyte protonation and disulphide bond reduction and some mechanisms are proposed for this behaviour.

Lipocalins in Arthropod Chemical Communication.

Lipocalins represent one of the most successful superfamilies of proteins. Most of them are extracellular carriers for hydrophobic ligands across aqueous media, but other functions have been reported. They are present in most living organisms including bacteria. In animals they have been identified in mammals, molluscs, and arthropods; sequences have also been reported for plants. A subgroup of lipocalins, referred to as odorant-binding proteins (OBPs), mediate chemical communication in mammals by ferrying specific pheromones to the vomeronasal organ. So far, these proteins have not been reported as carriers of semiochemicals in other living organisms; instead chemical communication in arthropods is mediated by other protein families structurally unrelated to lipocalins. A search in the databases has revealed extensive duplication and differentiation of lipocalin genes in some species of insects, crustaceans, and chelicerates. Their large numbers, ranging from a handful to few dozens in the same species, their wide divergence, both within and between species, and their expression in chemosensory organs suggest that such expansion may have occurred under environmental pressure, thus supporting the hypothesis that lipocalins may be involved in chemical communication in arthropods.

Mapping the expression of soluble olfactory proteins in the honeybee.

Chemical communication in insects is mediated by soluble binding proteins, belonging to two large families, Odorant-binding Proteins (OBPs) and Chemosensory Proteins (CSPs). Recently, evidence has been provided that OBPs are involved in recognition of chemical stimuli. We therefore decided to investigate the expression of OBPs and CSPs in the honeybee at the protein level, using a proteomic approach. Our results are in agreement with previous reports of expression at the RNA level and show that 12 of the 21 OBPs predicted in the genome of the honeybee Apis mellifera and 2 of the 6 CSPs are present in the foragers' antennae, while the larvae express only three OBPs and a single CSP. MALDI mass spectrometry on crude antennal extracts and MALDI profiling on sections of antennae demonstrated that these techniques can be applied to investigate individual differences in the expression of abundant proteins, such as OBPs and CSPs, as well as to detect the presence of proteins in different regions of the antenna. Finally, as part of a project aimed at the characterization of all OBPs and CSPs of the honeybee, we expressed 5 OBPs and 4 CSPs in a bacterial system and measured their affinity to a number of ligands. Clear differences in their binding spectra have been observed between OBPs, as well as CSPs.

Increased immunocompetence and network centrality of allogroomer workers suggest a link between individual and social immunity in honeybees.

The significant risk of disease transmission has selected for effective immune-defense strategies in insect societies. Division of labour, with individuals specialized in immunity-related tasks, strongly contributes to prevent the spread of diseases. A trade-off, however, may exist between phenotypic specialization to increase task efficiency and maintenance of plasticity to cope with variable colony demands. We investigated the extent of phenotypic specialization associated with a specific task by using allogrooming in the honeybee, Apis mellifera, where worker behaviour might lower ectoparasites load. We adopted an integrated approach to characterize the behavioural and physiological phenotype of allogroomers, by analyzing their behavior (both at individual and social network level), their immunocompetence (bacterial clearance tests) and their chemosensory specialization (proteomics of olfactory organs). We found that allogroomers have higher immune capacity compared to control bees, while they do not differ in chemosensory proteomic profiles. Behaviourally, they do not show differences in the tasks performed (other than allogrooming), while they clearly differ in connectivity within the colonial social network, having a higher centrality than control bees. This demonstrates the presence of an immune-specific physiological and social behavioural specialization in individuals involved in a social immunity related task, thus linking individual to social immunity, and it shows how phenotypes may be specialized in the task performed while maintaining an overall plasticity.

Semiochemicals for intraspecific communication of the fig weevil Aclees sp. cf. foveatus (Coleoptera: Curculionidae): a first survey.

The fig tree weevil Aclees sp. cf. foveatus (Coleoptera: Curculionidae), introduced in Italy in 2005, is currently causing significant economic and environmental losses to fig tree nurseries and orchards in Central Italy. Fig damages are due to the adults feeding on leaves and fruits, and to the galleries dug by the xylophagous larvae in the trunk, which lead the plants to death. To date, no chemical or biological control methods resulted to be effective against this invasive pest. In order to gain information about possible semiochemicals involved in mate recognition and choice, both the volatile organic compounds (VOCs) and the epicuticular lipids of male and female specimens were analysed. VOCs emissions of specimens were characterized essentially by monoterpenes, while epicuticular lipids contained long chained 2-ketones, alkanes, alkenes, including some methyl alkenes, and several fatty acid propyl esters. The attractiveness of reconstituted VOCs blends of the two sexes was tested in electrophysiological and behavioural assays in laboratory conditions. Both the male and the female reconstituted VOCs drove a significant response towards individuals of the opposite sex, thus demonstrating features of sexual attractants. Our results suggest a possible application of VOCs blends as pheromonic attractants in field monitoring and mass trapping of Aclees sp. cf. foveatus.

Polistes dominulus (Hymenoptera, Vespidae) larvae show different cuticular patterns according to their sex: workers seem not use this chemical information.

During reproductive phase, larvae of male and female are intermingled in nest of social wasps. Workers care for and feed larvae that gives them an opportunity to bias investment with respect to sex, or even to kill some larvae, if they can distinguish between immature males and females. Cuticular hydrocarbon (CHC) mixtures are the most studied cues for species, nestmate, and caste recognition in social Hymenoptera. In this study, we investigate the paper wasp Polistes dominulus to see if male and female larvae show different patterns of CHCs and if workers are able to discriminate between male and female larvae on this basis. We performed gas chromatography-mass spectrometry analysis on cuticular extracts of larvae, and then we genotyped them to assign sex. We found sex-based variation in CHC-profiles sufficient for discrimination. However, our behavioral assays do not support the view that adults discriminate between male and female larvae within nests.

Natural biocide disrupts nestmate recognition in honeybees.

Honeybee colonies are under the threat of many stressors, biotic and abiotic factors that strongly affect their survival. Recently, great attention has been directed at chemical pesticides, including their effects at sub-lethal doses on bee behaviour and colony success; whereas the potential side effects of natural biocides largely used in agriculture, such as entomopathogenic fungi, have received only marginal attention. Here, we report the impact of the fungus Beauveria bassiana on honeybee nestmate recognition ability, a crucial feature at the basis of colony integrity. We performed both behavioural assays by recording bee guards' response towards foragers (nestmate or non-nestmate) either exposed to B. bassiana or unexposed presented at the hive entrance, and GC-MS analyses of the cuticular hydrocarbons (CHCs) of fungus-exposed versus unexposed bees. Our results demonstrated that exposed bees have altered cuticular hydrocarbons and are more easily accepted into foreign colonies than controls. Since CHCs are the main recognition cues in social insects, changes in their composition appear to affect nestmate recognition ability at the colony level. The acceptance of chemically unrecognizable fungus-exposed foragers could therefore favour forager drift and disease spread across colonies.

Rapid assay of topiramate in dried blood spots by a new liquid chromatography-tandem mass spectrometric method.

Topiramate (TPM) is a new antiepiletic drug with efficacy in several types of seizures. Therapeutic drug monitoring of TPM is essential for effective patient management. The aim of this study was to evaluate the use of dried blood spot (DBS) specimens to determinate the TPM levels during the treatment. Advantages of DBS include short collection time, low invasiveness, ease and low cost of sample collection, transport and storage. Performance comparison between this method and the commercially available fluorescence-polarization immunoassay (FPIA) was made. The analysis was performed in selected reaction monitoring (SRM) mode. The calibration curve in matrix using D(12)-topiramate was linear in the concentration range of 0.0166-1.66mg/L (0.5-50mg/L in DBS) of topiramate with correlation coefficient value of 0.9985. In the concentration range of 0.5-50mg/L, the coefficients of variation in DBS were in the range 2.13-11.85% and the accuracy ranged from 93.93% to 110.67%. There was no significant differences between the concentrations (ranging 0.5-50mg/L) measured both FPIA on venous samples and LC-MS/MS assay on simultaneous DBS samples. The sensitivity and specificity of tandem mass spectrometry allow now high throughput topiramate analysis (the improvement was three times in comparison with FPIA). This new assay has favourable characteristics being highly precise and accurate. FPIA also proved to be precise and accurate, but is not always suitable for the sample collection in neonates in whom obtaining larger blood samples is not convenient or possible.

Proteomic analysis of chemosensory organs in the honey bee parasite Varroa destructor: A comprehensive examination of the potential carriers for semiochemicals.

We have performed a proteomic analysis on chemosensory organs of Varroa destructor, the honey bee mite, in order to identify putative soluble carriers for pheromones and other olfactory cues emitted by the host. In particular, we have analysed forelegs, mouthparts (palps, chelicera and hypostome) and the second pair of legs (as control tissue) in reproductive and phoretic stages of the Varroa life cycle. We identified 958 Varroa proteins, most of them common to the different organs and stages. Sequence analysis shows that four proteins can be assigned to the odorant-binding protein (OBP)-like class, which bear some similarity to insect OBPs, but so far have only been reported in some Chelicerata. In addition, we have detected the presence of two proteins belonging to the Niemann-Pick family, type C2 (NPC2), which have also been suggested as semiochemical carriers. Biological significance: The mite Varroa destructor is the major parasite of the honey bee and is responsible for great economical losses. The biochemical tools used by Varroa to detect semiochemicals produced by the host are still largely unknown. This work contributes to understand the molecular basis of olfaction in Varroa and, more generally, how detection of semiochemicals has evolved in terrestrial non-hexapod Arthropoda. Moreover, the identification of molecular carriers involved in olfaction can contribute to the development of control strategies for this important parasite.