Semiochemicals and olfactory protein in mosquito control

This thesis reports a biochemical study on the olfactory system in mosquitoes, which pose one of the major threats to human health, in order to devise strategies alternative to insecticides for population control. In particular, the final aim of the research work was the discovery of new mosquito repellents. Our approach has been developed along two parallel lines: a) a biochemical research on the proteins mediating odour perception in mosquitoes, in particular a functional study of Odorant-binding proteins (OBPs) and Chemosensory proteins (CSPs), two classes of polypeptides involved in the detection and recognition of olfactory stimuli in insects. b) a structural comparison of the main components of essential oils behaviourally active on mosquitoes, in order to extract common feature that might provide guidelines for the design of better repellents; The experimental work has been focused on the expression of OBPs and CSPs in mosquitoes. Using a proteomic approach, applied to antennae and pre-adult stages (in particular eggs, larvae and pupae) we have identified OBPs and CSPs that are more likely involved in odour recognition. These proteins were expressed in bacterial system and used in ligand-binding assays with compounds bioactive on mosquitoes. During the last decade, we have witnessed an increasing number of publications dealing with mosquito repellents, most identified in plant essential oils. This large amount of research was stimulated by recent concerns on the safety of DEET, the commercial repellent. We have concluded that the best strategy for designing more efficient repellents is to aim at compounds with lower volatility, that provides a longer permanence on the skin as well as a reduced odour intensity. Based on this idea and taking the structures of some known repellents as templates, we have designed new molecules by introducing additional polar groups in the molecule and/or increasing their molecular weight. The new chemicals have been tested in “warm body” and “human-bait” experiments and several of them proved to be as good to repel mosquitoes as those currently used

Soluble olfactory proteins in insects

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are regarded as carriers of pheromones and odorants in insect chemoreception. These proteins are typically located in antennae, mouth organs and other chemosensory structures; however, members of both classes of proteins have been detected recently in other parts of the body and various functions have been proposed. The best studied of these non-sensory tasks is performed in pheromone glands, where OBPs and CSPs solubilise hydrophobic semiochemicals and assist their controlled release into the environment. In some cases the same proteins are expressed in antennae and pheromone glands, thus performing a dual role in receiving and broadcasting the same chemical message. Several reports have described OBPs and CSPs in reproductive organs. Some of these proteins are male specific and are transferred to females during mating. They likely carry semiochemicals with different proposed roles, from inhibiting other males from approaching mated females, to marking fertilized eggs, but further experimental evidence is still needed. Before being discovered in insects, the presence of binding proteins in pheromone glands and reproductive organs was widely reported in mammals, where vertebrate OBPs, structurally different from OBPs of insects and belonging to the lipocalin superfamily, are abundant in rodent urine, pig saliva and vaginal discharge of the hamster, as well as in the seminal fluid of rabbits. In at least four cases CSPs have been reported to promote development and regeneration: in embryo maturation in the honeybee, limb regeneration in the cockroach, ecdysis in larvae of fire ants and in promoting phase shift in locusts. Both OBPs and CSPs are also important in nutrition as solubilisers of lipids and other essential components of the diet. Particularly interesting is the affinity for carotenoids of CSPs abundantly secreted in the proboscis of moths and butterflies and the occurrence of the same (or very similar CSPs) in the eyes of the same insects. A role as a carrier of visual pigments for these proteins in insects parallels that of retinol-binding protein in vertebrates, a lipocalin structurally related to OBPs of vertebrates. Other functions of OBPs and CSPs include anti-inflammatory action in haematophagous insects, resistance to insecticides and eggshell formation. Such multiplicity of roles and the high success of both classes of proteins in being adapted to different situations is likely related to their stable scaffolding determining excellent stability to temperature, proteolysis and denaturing agents. The wide versatility of both OBPs and CSPs in nature has suggested several different uses for these proteins in biotechnological applications, from biosensors for odours to scavengers for pollutants and controlled releasers of chemicals in the environment

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

Antennal Protein Profile in Honeybees: Caste and Task Matter More Than Age

Reproductive and task partitioning in large colonies of social insects suggest that colony members belonging to different castes or performing different tasks during their life (polyethism) may produce specific semiochemicals and be differently sensitive to the variety of pheromones involved in intraspecific chemical communication. The main peripheral olfactory organs are the antennal chemosensilla, where the early olfactory processes take place. At this stage, members of two different families of soluble chemosensory proteins [odorant-binding proteins (OBPs) and chemosensory proteins (CSPs)] show a remarkable affinity for different odorants and act as carriers while a further family, the Niemann-Pick type C2 proteins (NPC2) may have a similar function, although this has not been fully demonstrated. Sensillar lymph also contains Odorant degrading enzymes (ODEs) which are involved in inactivation through degradation of the chemical signals, once the message is conveyed. Despite their importance in chemical communication, little is known about how proteins involved in peripheral olfaction and, more generally antennal proteins, differ in honeybees of different caste, task and age. Here, we investigate for the first time, using a shotgun proteomic approach, the antennal profile of honeybees of different castes (queens and workers) and workers performing different tasks (nurses, guards, and foragers) by controlling for the potential confounding effect of age. Regarding olfactory proteins, major differences were observed between queens and workers, some of which were found to be more abundant in queens (OBP3, OBP18, and NPC2-1) and others to be more abundant in workers (OBP15, OBP21, CSP1, and CSP3); while between workers performing different tasks, OBP14 was more abundant in nurses with respect to guards and foragers. Apart from proteins involved in olfaction, we have found that the antennal proteomes are mainly characterized by castes and tasks, while age has no effect on antennal protein profile. Among the main differences, the strong decrease in vitellogenins found in guards and foragers is not associated with age

A proteomic investigation of soluble olfactory proteins in Anopheles gambiae

Odorant-binding proteins (OBPs) and chemosensory proteins (CSPs) are small soluble polypeptides that bind semiochemicals in the lymph of insect chemosensilla. In the genome of Anopheles gambiae, 66 genes encode OBPs and 8 encode CSPs. Here we monitored their expression through classical proteomics (2D gel-MS analysis) and a shotgun approach. The latter method proved much more sensitive and therefore more suitable for tiny biological samples as mosquitoes antennae and eggs. Females express a larger number and higher quantities of OBPs in their antennae than males (24 vs 19). OBP9 is the most abundant in the antennae of both sexes, as well as in larvae, pupae and eggs. Of the 8 CSPs, 4 were detected in antennae, while SAP3 was the only one expressed in larvae. Our proteomic results are in fairly good agreement with data of RNA expression reported in the literature, except for OBP4 and OBP5, that we could not identify in our analysis, nor could we detect in Western Blot experiments. The relatively limited number of soluble olfactory proteins expressed at relatively high levels in mosquitoes makes further studies on the coding of chemical messages at the OBP level more accessible, providing for few specific targets. Identification of such proteins in Anopheles gambiae might facilitate future studies on host finding behavior in this important disease vector. © 2013 Mastrobuoni et al

Two Odorant-Binding Proteins Mediate the Behavioural Response of Aphids to the Alarm Pheromone (E)-ß-farnesene and Structural Analogues

Abstract Background: Aphids are agricultural pests of great economical interest. Alternatives to insecticides, using semiochemicals, are of difficult applications. In fact, sex pheromones are of little use as aphids reproduce partenogenetically most of the time. Besides, the alarm pheromone, (E)-ß-farnesene for a great number of species, is difficult to synthesize and unstable in the environment. The search for novel semiochemicals to be used in population control can be efficiently approached through the study of the olfactory system at the biochemical level. Recently odorant-binding proteins (OBPs) have been shown to play a central role in olfactory recognition, thus becoming the target of choice for designing new semiochemicals. Methodology/Principal Findings: To address the question of how the alarm message is recognised at the level of OBPs, we have tested 29 compounds, including (E)-ß-farnesene, in binding assays with 6 recombinant proteins and in behaviour experiments. We have found that good repellents bind OBP3 and/or OBP7, while non repellents present different spectra of binding. These results have been verified with two species of aphids, Acyrthosiphon pisum and Myzus persicae, both using (E)-ß-farnesene as the alarm pheromone. Conclusions: Our results represent further support to the idea (so far convincingly demonstrated only in Drosophila) that OBPs are involved in decoding the chemical information of odorants and pheromones, and for the first time provide such evidence in other insect species and using wild-type insects. Moreover, the data offer guidelines and protocols for the discovery of potential alarm pheromones, using ligand-binding assays as a preliminary screening before subjecting selected compounds to behaviour tests

Espressione e caratterizzazione delle CSP (Chemosensory Protein) nell'ape

RIASSUNTO Questo lavoro di tesi presenta una ricerca effettuata con tecniche di biochimica e di biologia molecolare su proteine solubili coinvolte nella chemiorecezione negli insetti, le Chemosensory protein (CSP). Queste sono proteine solubili di circa 100-120 amminoacidi, presenti nella linfa sensillare degli insetti, dove svolgono una funzione legante verso semiochimici. Alcune di esse sono state trovate anche in altre parti del corpo, come ghiandole produttrici di feromoni, svolgendo in questi casi ancora funzioni legate alla comunicazione chimica. E’ stata anche riportata la presenza di CSP in organi in fase di sviluppo, come nel caso della p10 della blatta, prodotta durante la rigenerazione degli arti, e della CSP5 dell’ape, presente solo nelle uova e negli ovari di regina. In ogni caso, tali proteine costituiscono obiettivi interessanti nel controllo di popolazioni di insetti dannosi alle colture o nocivi per la salute umana, ma anche di insetti utili, come le api, che sono responsabili della impollinazione di molte piante. Una migliore conoscenza di queste proteine e di altre coinvolte nella comunicazione chimica puo’ suggerire azioni mirate per la conservazione di specie utili o la riduzione di quelle dannose. Il lavoro sperimentale, riguarda lo studio delle 6 CSP presenti nel genoma di Apis mellifera. Quattro di queste sono state espresse in sistemi batterici, ottenendo ottime rese e proteine allo stato nativo. Tre di queste proteine, la CSP1, la CSP3 e la CSP4, sono state purificate mediante una serie di passaggi cromatografici su resine a scambio anionico e gel-filtrazione ed utilizzate in saggi di binding con molecole leganti diverse. La CSP1 lega in maniera forte e specifica composti a catena lineare come alcooli ed esteri a 13-14 atomi di carbonio. La CSP3 ha mostrato una selettivita’ simile, legando con alta affinita’ l’1-tridecanolo, accanto ad altri composti, quali benzoati alchilici. E’ noto che alcuni esteri a catena medio-lunga sono stati identificati come componenti di miscele feromonali utilizzate dalle api nei loro rapporti sociali. Non e’ escluso, quindi che sia la CSP1 che la CSP3 siano coinvolte nella percezione di tali messaggi chimici. La CSP4, invece, ha mostrato un comportamento molto diverso, legando composti terpenici di dimensioni realtivamente grandi, quali il retinolo ed il beta-ionone. Utilizzando la spettrometria di massa MALDI alcune CSP sono state identificate nelle antenne di api appartenenti alle diverse caste ed in altri tessuti. Questa rappresenta la prima indagine sulla presenza di proteine olfattive negli insetti effettuata per spettrometria di massa direttamente su tessuti biologici