Sublethal Effects of Four Insecticides Targeting Cholinergic Neurons on Partner and Host Finding in the Parasitic Wasp Nasonia vitripennis.

Lethal and sublethal effects of pesticides on nontarget organisms are one of the causes of the current decline of many insect species. However, research in the past decades has focused primarily on pollinators, although other beneficial nontarget organisms such as parasitic wasps may also be affected. We studied the sublethal effects of the four insecticides acetamiprid, dimethoate, flupyradifurone, and sulfoxaflor on pheromone-mediated sexual communication and olfactory host finding of the parasitic wasp Nasonia vitripennis. All agents target cholinergic neurons, which are involved in the processing of chemical information by insects. We applied insecticide doses topically and tested the response of treated wasps to sex pheromones and host-associated chemical cues. In addition, we investigated the mating rate of insecticide-treated wasps. The pheromone response of females surviving insecticide treatment was disrupted by acetamiprid (≥0.63 ng), dimethoate (≥0.105 ng), and flupyradifurone (≥21 ng), whereas sulfoxaflor had no significant effects at the tested doses. Olfactory host finding was affected by all insecticides (acetamiprid ≥1.05 ng, dimethoate ≥0.105 ng, flupyradifurone ≥5.25 ng, sulfoxaflor ≥0.52 ng). Remarkably, females treated with ≥0.21 ng dimethoate even avoided host odor. The mating rate of treated N. vitripennis couples was decreased by acetamiprid (6.3 ng), flupyradifurone (≥2.63 ng), and sulfoxaflor (2.63 ng), whereas dimethoate showed only minor effects. Finally, we determined the amount of artificial nectar consumed by N. vitripennis females within 48 h. Considering this amount (∼2 µL) and the maximum concentrations of the insecticides reported in nectar, tested doses can be considered field-realistic. Our results suggest that exposure of parasitic wasps to field-realistic doses of insecticides targeting the cholinergic system reduces their effectiveness as natural enemies by impairing the olfactory sense. Environ Toxicol Chem 2023;42:2400-2411. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Age-dependent release of and response to alarm pheromone in a ponerine ant.

Social insect societies are characterized by division of labour and communication within the colony. The most frequent mode of communication is by chemical signals. In general, pheromones elicit specific responses in the receiver, although reactions may vary depending on the receiving individual's physiological or motivational state. For example, it has been shown that pheromones can elicit different responses in morphological worker castes. However, comparably little is known about such effects in worker castes of monomorphic species. Here, we comprehensively studied a monomorphic species showing age polyethism, the thelytokous ant Platythyrea punctata Our analyses revealed that the species' alarm pheromone consists of (S)-(-)-citronellal and (S)-(-)-actinidine, and is produced in the mandibular glands. Ants responded with increased movement activity and increasing ant density towards the pheromone source in whole-colony bioassays, confirming the alarming effect of these compounds. We found age classes to differ in their absolute pheromone content, in the propensity to release alarm pheromone upon disturbance and in their reaction towards the pheromone. Absolute amounts of pheromone content may differ simply because the biosynthesis of the pheromone begins only after adult eclosion. Nonetheless, our results indicate that this clonal species exhibits age-related polyethism in the emission of as well as in the response to its alarm pheromone.

Semiochemicals Mediating Defense, Intraspecific Competition, and Mate Finding in Leptopilina ryukyuensis and L. japonica (Hymenoptera: Figitidae), Parasitoids of Drosophila.

Deciphering the processes driving the evolution of the diverse pheromone-mediated chemical communication system of insects is a fascinating and challenging task. Understanding how pheromones have arisen has been supported by studies with the model organism Leptopilina heterotoma, a parasitoid wasp whose defensive compound (-)-iridomyrmecin also evolved as a component of the female sex pheromone and as a cue to avoid competition with other females during host search. To understand how compounds can evolve from being non-communicative to having a communicative function and to shed light on the evolution of the multi-functional use of iridomyrmecin in the genus Leptopilina, the chemical communication of two additional species, L. ryukyuensis and L. japonica, was studied. We demonstrate that in both species a species-specific mixture of iridoids is produced and emitted by wasps upon being attacked, consistent with their putative role as defensive compounds. In L. ryukyuensis these iridoids are also used by females to avoid host patches already exploited by other conspecific females. However, females of L. japonica do not avoid the odor of conspecific females during host search. We also show that the sex pheromone of female L. ryukyuensis consists of cuticular hydrocarbons (CHCs), as males showed strong courtship behavior (wing fanning) towards these compounds, but not towards the iridoid compounds. In contrast, males of L. japonica prefer their females' iridoids but CHCs also elicit some courtship behavior. The use of iridoid compounds as defensive allomones seems to be common in the genus Leptopilina, while their communicative functions appear to have evolved in a species-specific manner.

Mapping key amino acid residues for the epimerase efficiency and stereospecificity of the sex pheromone biosynthetic short-chain dehydrogenases/reductases of Nasonia.

Males of the parasitic wasp genus Nasonia use blends of chiral hydroxylactones as sex pheromones to attract conspecific females. Whereas all Nasonia species use a mixture of (4R,5S)-5-hydroxy-4-decanolide (RS) and 4-methylquinazoline (MQ) as sex pheromones, Nasonia vitripennis evolved (4R,5R)-5-hydroxy-4-decanolide (RR) as an extra sex pheromone component. We recently identified and functionally characterized three short-chain dehydrogenases/reductases (SDRs) NV10127, NV10128, and NV10129 that are capable of catalyzing the epimerization of RS to RR via (4R)-5-oxo-4-decanolide (ODL) as intermediate. Despite their very high sequence identities of 88-98%, these proteins differ drastically in their ability to epimerize RS to RR and in their stereoselectivity when reducing ODL to RR/RS. Here, in order to unravel the sequence differences underlying these varying functional properties of NV1027, NV10128 and NV10129, we created chimeras of the three enzymes and monitored their catalytic activities in vitro. The results show that a few amino acid changes at the C-termini and active sites of Nasonia vitripennis SDRs lead to substantially altered RS to RR epimerization and ODL-reduction activities. Thus, our study adds to the understanding of pheromone evolution by showing that subtle mutations in key biosynthetic enzymes can result in drastic effects on the composition of chemical signals.

The Post-mating Switch in the Pheromone Response of Nasonia Females Is Mediated by Dopamine and Can Be Reversed by Appetitive Learning.

The olfactory sense is of crucial importance for animals, but their response to chemical stimuli is plastic and depends on their physiological state and prior experience. In many insect species, mating status influences the response to sex pheromones, but the underlying neuromodulatory mechanisms are poorly understood. After mating, females of the parasitic wasp Nasonia vitripennis are no longer attracted to the male sex pheromone. Here we show that this post-mating behavioral switch is mediated by dopamine (DA). Females fed a DA-receptor antagonist prior to mating maintained their attraction to the male pheromone after mating while virgin females injected with DA became unresponsive. However, the switch is reversible as mated females regained their pheromone preference after appetitive learning. Feeding mated N. vitripennis females with antagonists of either octopamine- (OA) or DA-receptors prevented relearning of the pheromone preference suggesting that both receptors are involved in appetitive learning. Moreover, DA injection into mated females was sufficient to mimic the oviposition reward during odor conditioning with the male pheromone. Our data indicate that DA plays a key role in the plastic pheromone response of N. vitripennis females and reveal some striking parallels between insects and mammals in the neuromodulatory mechanisms underlying olfactory plasticity.

Interference of chemical defence and sexual communication can shape the evolution of chemical signals.

According to current evolutionary theory, insect pheromones can originate from extant precursor compounds being selected for information transfer. This is exemplified by females of the parasitoid wasp Leptopilina heterotoma whose defensive secretion consisting mainly of (-)-iridomyrmecin has evolved secondary functions as cue to avoid other females during host search and as female sex pheromone. To promote our understanding of pheromone evolution from defensive secretions we studied the chemical ecology of Leptopilina clavipes. We show here that L. clavipes also produces a defensive secretion that contains (-)-iridomyrmecin as major component and that females use it to detect and avoid host patches occupied by other females. However, the female sex pheromone of L. clavipes consists solely of cuticular hydrocarbons (CHCs) and males did not respond to female CHCs if presented in combination with the defensive secretion containing (-)-iridomyrmecin. This is in contrast to other species of Leptopilina, in which the iridoid compounds have no inhibiting effect or even function as sex pheromone triggering courtship behaviour. This indicates that Leptopilina species differ in the cost-benefit ratio for males searching for females, which might explain the strong divergence in the composition of the sex pheromone in the genus.

Sublethal doses of imidacloprid disrupt sexual communication and host finding in a parasitoid wasp.

Neonicotinoids are widely used insecticides, but their use is subject of debate because of their detrimental effects on pollinators. Little is known about the effect of neonicotinoids on other beneficial insects such as parasitoid wasps, which serve as natural enemies and are crucial for ecosystem functioning. Here we show that sublethal doses of the neonicotinoid imidacloprid impair sexual communication and host finding in the parasitoid wasp Nasonia vitripennis. Depending on the dose, treated females were less responsive to the male sex pheromone or unable to use it as a cue at all. Courtship behaviour of treated couples was also impeded resulting in a reduction of mating rates by up to 80%. Moreover, treated females were no longer able to locate hosts by using olfactory cues. Olfaction is crucial for the reproductive success of parasitoid wasps. Hence, sublethal doses of neonicotinoids might compromise the function of parasitoid wasps as natural enemies with potentially dire consequences for ecosystem services.

Epimerisation of chiral hydroxylactones by short-chain dehydrogenases/reductases accounts for sex pheromone evolution in Nasonia.

Males of all species of the parasitic wasp genus Nasonia use (4R,5S)-5-hydroxy-4-decanolide (RS) as component of their sex pheromone while only N. vitripennis (Nv), employs additionally (4R,5R)-5-hydroxy-4-decanolide (RR). Three genes coding for the NAD+-dependent short-chain dehydrogenases/reductases (SDRs) NV10127, NV10128, and NV10129 are linked to the ability of Nv to produce RR. Here we show by assaying recombinant enzymes that SDRs from both Nv and N. giraulti (Ng), the latter a species with only RS in the pheromone, epimerise RS into RR and vice versa with (4R)-5-oxo-4-decanolide as an intermediate. Nv-derived SDR orthologues generally had higher epimerisation rates, which were also influenced by NAD+ availability. Semiquantitative protein analyses of the pheromone glands by tandem mass spectrometry revealed that NV10127 as well as NV10128 and/or NV10129 were more abundant in Nv compared to Ng. We conclude that the interplay of differential expression patterns and SDR epimerisation rates on the ancestral pheromone component RS accounts for the evolution of a novel pheromone phenotype in Nv.

Drosophila Avoids Parasitoids by Sensing Their Semiochemicals via a Dedicated Olfactory Circuit.

Detecting danger is one of the foremost tasks for a neural system. Larval parasitoids constitute clear danger to Drosophila, as up to 80% of fly larvae become parasitized in nature. We show that Drosophila melanogaster larvae and adults avoid sites smelling of the main parasitoid enemies, Leptopilina wasps. This avoidance is mediated via a highly specific olfactory sensory neuron (OSN) type. While the larval OSN expresses the olfactory receptor Or49a and is tuned to the Leptopilina odor iridomyrmecin, the adult expresses both Or49a and Or85f and in addition detects the wasp odors actinidine and nepetalactol. The information is transferred via projection neurons to a specific part of the lateral horn known to be involved in mediating avoidance. Drosophila has thus developed a dedicated circuit to detect a life-threatening enemy based on the smell of its semiochemicals. Such an enemy-detecting olfactory circuit has earlier only been characterized in mice and nematodes.

A nonspecific defensive compound evolves into a competition avoidance cue and a female sex pheromone.

The evolution of chemical communication and the origin of pheromones are among the most challenging issues in chemical ecology. Current theory predicts that chemical communication can arise from compounds primarily evolved for non-communicative purposes but experimental evidence showing a gradual evolution of non-informative compounds into cues and true signals is scarce. Here we report that females of the parasitic wasp Leptopilina heterotoma use the defensive compound (-)-iridomyrmecin as a semiochemical cue to avoid interference with con- and heterospecific competitors and as the main component of a species-specific sex pheromone. Although competition avoidance is mediated by (-)-iridomyrmecin alone, several structurally related minor compounds are necessary for reliable mate attraction and recognition. Our findings provide insights into the evolution of insect pheromones by demonstrating that the increasing specificity of chemical information is accompanied by an increasing complexity of the chemical messengers involved and the evolution of the chemosensory adaptations for their exploitation.

Divergent diastereoselective synthesis of iridomyrmecin, isoiridomyrmecin, teucrimulactone, and dolicholactone from citronellol.

The iridoid natural products iridomyrmecin, isoiridomyrmecin, teucriumlactone, and dolicholactone were prepared from citronellol using a divergent diastereoselective approach. Key steps include a highly diastereoselective enamine/enal cycloaddition and the selective reduction of masked aldehyde functionalities by ionic hydrogenation.

Stereoselective chemical defense in the Drosophila parasitoid Leptopilina heterotoma is mediated by (-)-iridomyrmecin and (+)-isoiridomyrmecin.

Chemical defense mechanisms are widespread among insects but have rarely been demonstrated in parasitoid wasps. Here, we show that the Drosophila parasitoid Leptopilina heterotoma (Hymenoptera, Figitidae) produces (-)-iridomyrmecin and (+)-isoiridomyrmecin in a cephalic gland, and that these chemicals have a highly repellent effect on ants. Stereoselective synthesis of 4 stereoisomers of iridomyrmecin allowed us to demonstrate that the repellent effect of iridomyrmecins depends on the stereochemistry. Potential food items impregnated with natural doses of (-)-iridomyrmecin were avoided by ants much longer than those impregnated with (+)-iridomyrmecin, (+)-isoiridomyrmecin, or (-)-isoiridomyrmecin, respectively. Quantitative headspace analyses revealed furthermore that females and males of L. heterotoma released iridomyrmecins in higher amounts when confronted with ants. This is the first time, that (-)-iridomyrmecin and (+)-isoiridomyrmecin are reported as natural products. Females synthesize more iridomyrmecins than males, and the most active (-)-iridomyrmecin is produced by females only. We, therefore, hypothesize that this defense mechanism is used mainly by female wasps when foraging for Drosophila larvae on rotten fruits, but also may protect male wasps during dispersal.

A divergent approach to the diastereoselective synthesis of several ant-associated iridoids.

The ant-associated iridoids nepetalactol, actinidine, dolichodial, isoiridomyrmecin, and dihydronepetalactone were prepared from citronellal using a divergent approach. Key features include a three-step synthesis of the individual antipodes of actinidine by a novel tandem cycloaddition/pyridine formation and a facile diastereoselective synthesis of both enantiomers of dolichodial.

Diosphenol-based approach to the a-ring functionalization of advanced Taxol precursors.

Several different approaches to the A-ring functionalization of an advanced, highly functionalized diosphenol precursor to Taxol are described. The first phase of the undertaking consists of an assessment of those reagents conducive to reaction at the enolic oxygen (silylation, methylation, allylation, and acylation). Transformations involving an alternative attack at the enol carbon center (bromination, selenation) have also been defined. Sodium borohydride reduction operates from the beta-face of C-14 as long as the C-1 hydroxyl is not protected so as to offer steric exclusion. Complications associated with various aspects of these methodological undertakings are addressed. The most advanced oxygenation achievements were realized by way of a noteworthy sequence involving epoxidation of the O-methyl ether, methanolysis under mildly acidic conditions, and regioselective oxidation of diol 38 to give 39.

Synthesis of stereoisomeric medium-ring alpha,alpha'-dihydroxy cycloalkanones.

The stereochemical course of the epoxidation of the silyl enol ethers of 2-tert-butyldimethylsilyloxycycloheptanone and -cyclooctanone has been investigated and shown to proceed exclusively anti to the existing alpha-substituent. 2-(Benzyloxy)cyclooctanone behaves similarly, and the presence of a transannular double bond does not alter the outcome. Alpha-ketol rearrangements are seen to operate during ensuing fluoride ion-induced removal of the silyl protecting groups in select examples. The preferred means for generating the cis isomers of the alpha,alpha'-dihydroxy cycloalkanones involves methylenation of the monoprotected trans-dihydroxy ketones with the Nysted reagent, followed by oxidation and subsequent reduction with sodium borohydride. Ozonolysis and fluoride ion-induced desilylation complete the route.

Reactions of alpha,alpha'-dihydroxy ketones with phosgene. Structural requirements for spiro epoxy carbonate formation.

[reaction: see text] Apha,alpha'-dihydroxy ketones having two OH groups that can come into close proximity react with phosgene in the presence of DMAP to produce 1,3-dioxane-2,5-diones. When covalent bonding in this manner is prohibited, alternate reaction pathways can in principle be adopted. Of these, the generation of spiro epoxy carbonates is shown to be feasible in three examples. In other cases, the alpha-ketol rearrangement precedes diacylation.

Effect of 9,10-cyclic acetal stereochemistry on feasible operation of the alpha-ketol rearrangement in highly functionalized paclitaxel (Taxol) precursors.

The convergent, stereocontrolled synthesis of enantiopure stereoisomeric 9,10-cyclic acetals, whose designed role was to serve as potential precursors to Taxol, is reported. These advanced intermediates are multiply functionalized and carry a bridgehead alpha-ketol array which was key to isomerization into the proper framework. In agreement with relative strain energy values obtained by MM3 calculations, a dichotomy was observed between these two families. While the trans-fused acetals failed to undergo bridge migration, their cis counterparts did so efficiently. In fact, isomerization was sufficiently rapid that oxygenation at C2 was now precluded. The operation of several unusual transannular hydride shifts is also detailed.

A delicate balance of energetics. Subtleties associated with alpha-ketol-based bridge migration to afford 9-keto-10beta-p-methoxybenzyloxytaxanes.

The feasibility of the title reaction has been pursued for the purpose of advancing a concise total synthesis of Taxol. Of the two closely related series examined, the first featured an exo-methylene group at C4. The second consisted of an alpha-epoxide at that site. Strikingly, the olefinic construct proved inert to attempted alpha-ketol rearrangement. In contrast, the oxiranyl derivative isomerized smoothly. The reaction sequence associated with arrival at taxane 18 is short (15 steps from a D-camphor derivative) and notably efficient. The thermodynamic issues that are raised by this investigation have been clarified by an assessment of molecular mechanics-derived (MM3) steric energy calculations.

Synthesis and Conformational Properties of the Sterically Crowded D3d -Symmetric all-trans Hexa(spirotetrahydrofuranyl)cyclohexane System.

An unprecedented high barrier to ring inversion [Eq. (1)] prevents 1 from engaging in bifacial complexation with lithium ions. The X-ray crystal structure analysis of this hexaspiro compound corroborates the adoption of a cyclohexane chair with all six C-O bonds projected equatorially (1-eq).