Antennal chemoreceptors in the European ectoparasitoid Sclerodermus cereicollis (Hymenoptera: Bethylidae).

Sclerodermus cereicollis is a European flat wasp ectoparasitoid of some longhorn beetle species. This species is important as a suitable biological control agent against xylophagous pests. To better understand its chemical ecology, the ultrastructure of the antennal sensilla of the adult was studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The sensilla are located mainly in the ventro-medial side of the antennae. We report a clearly sexual dimorphism with respect to antennae length, and to types, number, and distribution of chemosensilla. The antennae in males are significantly longer than those of females. We describe in detail the external and internal structure of different chemoreceptors represented by sensilla placodea, long sensilla basiconica, multiporous sensilla chaetica, grooved sensilla ampullacea, uniporous grooved sensilla chaetica. The potential involvement of the different kinds of chemoreceptors in inter- (mainly sexual recognition and social behavior-kin recognition) or intra-specific communication (mainly host selection) is discussed on the basis of behavioral and electrophysiological investigations performed on other parasitoid species belonging to the same family. Other sensilla with morphology that is not consistent with that of chemoreceptors are represented by grooved pegs, coeloconic pegs, trichoid sensilla. Such detailed ultrastructural investigation of the flagellar chemoreceptors of S. cereicollis, clarifying the number of chemosensory neurons innervating the different sensilla, is crucial for further electrophysiological investigations on this important species. RESEARCH HIGHLIGHTS: Evident sexual dimorphism concerning antennae length, type, number, and distribution of chemosensilla. Long sensilla basiconica (LSB) present only in females could play a role in host location and/or maternal care. Multiporous sensilla chaetica (MSC), significantly longer and mostly represented in males, could play a role in the perception of sexual pheromones. Detailed ultrastructural study is crucial for electrophysiological investigations on this important species.

Sensory pathway in aquatic basal polyneoptera: Antennal sensilla and brain morphology in stoneflies.

Aquatic insects represent a great portion of Arthropod diversity and the major fauna in inland waters. The sensory biology and neuroanatomy of these insects are, however, poorly investigated. This research aims to describe the antennal sensilla of nymphs of the stonefly Dinocras cephalotes using scanning electron microscopy and comparing them with the adult sensilla. Besides, central antennal pathways in nymphs and adults are investigated by neuron mass-tracing with tetramethylrhodamine, and their brain structures are visualized with an anti-synapsin antibody. No dramatic changes occur in the antennal sensilla during nymphal development, while antennal sensilla profoundly change from nymphs to adults when switching from an aquatic to an aerial lifestyle. However, similar brain structures are used in nymphs and adults to process diverging sensory information, perceived through different sensilla in water and air. These data provide valuable insights into the evolution of aquatic heterometabolous insects, maintaining a functional sensory system throughout development, including a distinct adaptation of the peripheral olfactory systems during the transition from detection of water-soluble chemicals to volatile compounds in the air. From a conservation biology perspective, the present data contribute to a better knowledge of the biology of stoneflies, which are very important bioindicators in rivers.

The armoured cuticle of the black soldier fly Hermetia illucens.

We characterise in detail the larval and pupal cuticle of the black soldier fly Hermetia illucens L. (Diptera: Stratiomyidae), a key insect species in circular economy. In particular, we focus on ultrastructure using scanning and transmission electron microscopy, material characterization and composition (elements and minerals) with confocal laser scanning microscope, energy dispersive X-ray microanalysis, powder X-ray diffraction and mechanical properties with nanoindentation measurements. Calcium carbonate crystallizes on the epicuticle as blocks of calcite in the pupal cuticle. Calcium carbonate granules are stored in two specialised Malpighian tubules. CaCO3 is already present in the cuticle of young larval instars, but it is mainly in the form of amorphous calcium carbonate while the amount of calcite increases during larval development. The presence of calcite leads to cuticle hardening. Larval and pupal cuticles contain large amounts of resilin which guarantee cuticle flexibility.

The origin of black and white coloration of the Asian tiger mosquito Aedes albopictus (Diptera: Culicidae).

Micro- and nanostructures of the white and black scales on the tarsi of the mosquito Aedes albopictus are analysed using scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. Reflectance spectra of the white areas are measured. No clear difference is present in the morphology of micro- and nanostructures of black and white scales in SEM and TEM, but black scales contain a dark pigment. The white colour of the scales has a structural origin. The structural white produced by the micro- and nanostructures of the scales on the tarsi of Ae. albopictus appears bright and is angle-dependent, since the reflected light changes according to the angle detection and according to the tarsus orientation. The optical appearance of the scale system of Ae. albopictus has a complex nature and can be explained by the combination of several effects. Among them, multiple refraction and reflection on the micro- and nanostructures of the scales are mainly responsible for the white appearance. The results suggest that mosquito scales, in addition to their superhydrophobic function, produce structural white. The biological role of white and black patches in mate recognition and defensive behaviour in the mosquitoes of the genus Aedes is hypothesized.

Coleoptera claws and trichome interlocking.

The present study tests the hypothesis that the specialized claws with a basal tooth found in some coccinellid beetles represent an adaptation to interlock with flexible unbranched trichomes of different plants. We compared the attachment ability of three Coleoptera species, Chnootriba elaterii, Harmonia axyridis (both Coleoptera: Coccinellidae), and Chrysolina herbacea (Coleoptera: Chrysomelidae) with claws of different shape. The attachment ability of insect individuals with or without claws to a plant with leaves bearing straight non-branched trichomes (Cucurbita moschata) and to a plant with smooth leaves (Prunus laurocerasus) was measured in traction force experiments. Insect attachment ability was also tested on a resin replica of C. moschata leaf, to variate trichome stiffness, and on glass as a reference surface. Centrifugal force tester experiments were performed to compare the attachment ability of the two ladybird species to glass and to the leaf of C. moschata. Natural and artificial substrates were characterized in cryo-SEM. The collected data reveal that plant trichomes can enhance insect attachment to plant surface compared with smooth glass by increasing insect friction force, but this is directly related to the trichome stiffness. To effectively grasp soft trichomes, insects evolved special claws-associated structures, such as the dentate claws observed in Coccinellidae.

Effect of Leaf Trichomes in Different Species of Cucurbitaceae on Attachment Ability of the Melon Ladybird Beetle Chnootriba elaterii.

This study investigates the attachment ability of the oligophagous melon ladybird beetle Chnootriba elaterii to leaves of several Cucurbitaceae species. Using cryo-SEM, we described adult and larva tarsal attachment devices and leaf surface structures (glandular and non-glandular trichomes) in Citrullus lanatus, Cucumis melo, Cucumis sativus, Cucurbita moschata, Cucurbita pepo, Ecballium elaterium, Lagenaria siceraria and Luffa aegyptiaca. Using traction force experiments and centrifugal force tests, we measured the friction force exerted by females and larvae on plant leaves. We observed that Cucurbitaceae glandular trichomes do not affect insect attachment ability at both developmental stages, suggesting some adaptation of C. elaterii to its host plants, while non-glandular trichomes, when they are dense, short and flexible, heavily reduce the attachment ability of both insect stages. When trichomes are dense but stiff, only the larval force is reduced, probably because the larva has a single claw, in contrast to the adult having paired bifid dentate claws. The data on the mechanical interaction of C. elaterii at different developmental stages with different Cucurbitaceae species, combined with data on the chemical cues involved in the host plant selection, can help to unravel the complex factors driving the coevolution between an oligophagous insect and its host plant species.

Cuticular modified air sacs underlie white coloration in the olive fruit fly, Bactrocera oleae.

Here, the ultrastructure and development of the white patches on thorax and head of Bactrocera oleae are analysed using scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. Based on these analyses and measurements of patch reflectance spectra, we infer that white patches are due to modified air sacs under transparent cuticle. These air sacs show internal arborisations with beads in an empty space, constituting a three-dimensional photonic solid responsible for light scattering. The white patches also show UV-induced blue autofluorescence due to the air sac resilin content. To the best of our knowledge, this research describes a specialized function for air sacs and the first observation of structural color produced by tracheal structures located under transparent cuticles in insects. Sexual dimorphism in the spectral emission also lays a structural basis for further investigations on the biological role of white patches in B. oleae.

Tests of search image and learning in the wild: Insights from sexual conflict in damselflies.

Search image formation, a proximal mechanism to maintain genetic polymorphisms by negative frequency-dependent selection, has rarely been tested under natural conditions. Females of many nonterritorial damselflies resemble either conspecific males or background vegetation. Mate-searching males are assumed to form search images of the majority female type, sexually harassing it at rates higher than expected from its frequency, thus selectively favoring the less common morph. We tested this and how morph coloration and behavior influenced male perception and intersexual encounters by following marked Ischnura elegans and noting their reactions to conspecifics. Contrary to search image formation and associative learning hypotheses, although males encountered the minority, male-like morph more often, sexual harassment and clutch size were similar for both morphs. Prior mating attempts or copula with morphs did not affect a male's subsequent reaction to them; males rarely attempted matings with immature females or males. Females mated early in the day, reducing the opportunity for males to learn their identity beforehand. Once encountered, the male-like morph was more readily noticed by males than the alternative morph, which once noticed was more likely to receive mating attempts. Flexible behavior gave morphs considerable control over their apparency to males, influencing intersexual encounters. Results suggested a more subtle proximal mechanism than male learning maintains these color polymorphisms and call for inferences of learning to be validated by behavior of wild receivers and their signalers.

The Antennal Pathway of Dragonfly Nymphs, from Sensilla to the Brain.

Dragonflies are hemimetabolous insects, switching from an aquatic life style as nymphs to aerial life as adults, confronted to different environmental cues. How sensory structures on the antennae and the brain regions processing the incoming information are adapted to the reception of fundamentally different sensory cues has not been investigated in hemimetabolous insects. Here we describe the antennal sensilla, the general brain structure, and the antennal sensory pathways in the last six nymphal instars of Libellula depressa, in comparison with earlier published data from adults, using scanning electron microscopy, and antennal receptor neuron and antennal lobe output neuron mass-tracing with tetramethylrhodamin. Brain structure was visualized with an anti-synapsin antibody. Differently from adults, the nymphal antennal flagellum harbors many mechanoreceptive sensilla, one olfactory, and two thermo-hygroreceptive sensilla at all investigated instars. The nymphal brain is very similar to the adult brain throughout development, despite the considerable differences in antennal sensilla and habitat. Like in adults, nymphal brains contain mushroom bodies lacking calyces and small aglomerular antennal lobes. Antennal fibers innervate the antennal lobe similar to adult brains and the gnathal ganglion more prominently than in adults. Similar brain structures are thus used in L. depressa nymphs and adults to process diverging sensory information.

Variation of attachment ability of Nezara viridula (Hemiptera: Pentatomidae) during nymphal development and adult aging.

The present investigation reports data on the attachment ability of the Southern green stink bug Nezara viridula (Hemiptera: Pentatomidae), a relevant pest in the world, along its whole life cycle. Using a centrifugal force tester, we evaluated a) the differences in the attachment ability among the four active nymphal developmental instars (N2-N5 nymphs) and adult to hydrophilic glass, showing an increased attachment ability during ontogenesis, owing to increased pulvilli size and efficiency; b) the possible role of growth and body shape on insect attachment ability on hydrophilic glass during the intermoult period, revealing that N. viridula nymphs attach stronger (higher safety factor) in the first part of the intermoult period; c) the age-specific differences in the attachment ability of adults of both sexes on hydrophilic glass, showing the best performance at an intermediate age, in agreement with a higher proportion of resilin in comparison with younger or older insects; d) the difference in attachment ability on hydrophilic vs. hydrophobic glass along the insect development, revealing a strong effect of surface hydrophobicity on reducing the attachment of N. viridula nymphs and adults. The results on the attachment ability of a hemimetabolous insect along its life cycle are relevant because they 1) shed light on different adaptations of attachment pads in relation to insect size, shape and age; 2) deepen the knowledge on the functional morphological adaptations, thus potentially contributing to the development of suitable control systems for this important pest insect.

Role of Fruit Epicuticular Waxes in Preventing Bactrocera oleae (Diptera: Tephritidae) Attachment in Different Cultivars of Olea europaea.

The olive fruit fly Bactrocera oleae (Diptera: Tephritidae) is the major pest of cultivated olives (Olea europaea L.), and a serious threat in all of the Mediterranean Region. In the present investigation, we demonstrated with traction force experiments that B. oleae female adhesion is reduced by epicuticular waxes (EWs) fruit surface, and that the olive fruit fly shows a different ability to attach to the ripe olive surface of different cultivars of O. europaea (Arbequina, Carolea, Dolce Agogia, Frantoio, Kalamata, Leccino, Manzanilla, Picholine, Nostrale di Rigali, Pendolino and San Felice) in terms of friction force and adhesion, in relation with different mean values of olive surface wettability. Cryo-scanning morphological investigation revealed that the EW present on the olive surface of the different analyzed cultivars are represented by irregular platelets varying in the orientation, thus contributing to affect the surface microroughness and wettability in the different cultivars, and consequently the olive fruit fly attachment. Further investigations to elucidate the role of EW in olive varietal resistance to the olive fruit fly in relation to the olive developmental stage and environmental conditions could be relevant to develop control methods alternative to the use of harmful pesticides.

Mechanical ecology of fruit-insect interaction in the adult Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae).

Fruit features represent a trade-off between dispersal and protection against frugivore insects. To prevent insect attack, plants evolved chemical and physical barriers, mainly studied in leaves, while limited knowledge is available for fruits, especially concerning mechanical barriers. We used the Mediterranean fruit fly to shed light on the mechanical ecology of insect-fruit attachment in a pest species. We tested the following hypotheses: is there any sexual dimorphism in attachment devices and attachment ability? Can the attachment ability of females of Ceratitis capitata to fruits of various host plants vary according to fruit surfaces with different morphology (smooth, hairy, waxy) or physico-chemical properties? The tarsal attachment devices were studied using Cryo-SEM and TEM. The maximum friction forces of C. capitata females on fruit surfaces of typical host plants were evaluated using a load cell force transducer. The attachment ability of both sexes on artificial surfaces was evaluated using a centrifugal force tester. Our data revealed sexual dimorphism in the size of pulvilli, which are wider in females. A higher friction force is exerted by females in comparison with males, in agreement with the need to firmly adhere to the host plant fruit during oviposition. Among the tested fruits, the stronger friction force was recorded on hairy or rough surfaces while a force reduction was recorded on waxy fruits. To unravel the mechanical ecology of insect-plant interaction between plants and species of Tephritidae can be useful to develop non-chemical methods to control these important crop pests.

Salicylic acid induced by herbivore feeding antagonizes jasmonic acid mediated plant defenses against insect attack.

We recently reported the transcriptomic signature of salicylic acid (SA) and jasmonic acid (JA) biosynthetic and responsive genes in Arabidopsis thaliana plants infested with the herbivore Eurydema oleracea. We demonstrated that insect feeding causes induction of both SA- and JA-mediated signaling pathways. Using transgenic SA-deficient NahG plants, we also showed antagonistic cross-talk between these two phytohormones. To gain more insight into the roles of the SA and JA pathways in plant defenses against E. oleracea, we report here on the dynamics of SA and JA levels in the wild-type genotype Col-0 and the transgenic Arabidopsis NahG mutant that does not accumulate SA. We show that SA strongly accumulates in the wild-type plants after 24 h of herbivore infestation, while JA levels do not change significantly. On the contrary, in the infested NahG plants, SA levels were not affected by E. oleracea feeding, whereas JA levels which were constitutively higher than the wild-type did not significantly change after 6 hours of herbivore feeding. Accordingly, when the wild-type and the jar1-1 mutant (which fails to accumulate JA-Ile) Arabidopsis plants were challenged with E. oleracea in a two-choice arena, the insect fed preferentially on the jar1-1 plants over the wild-type. These data support the conclusion that E. oleracea infestation strongly induces the SA pathway in the wild-type, thus antagonizing JA-mediated plant defenses against herbivory, as a strategy to suppress plant immunity.

Role of chemical cues in cabbage stink bug host plant selection.

The cabbage stink bugs of the genus Eurydema, encompassing several oligophagous species, such as Eurydema oleracea (L.), are known to be important pests of cabbage, broccoli, and other cole crops in Europe. Despite their economic importance, the knowledge regarding the role of chemical cues in host plant selection of these species is very limited. The present investigation on E. oleracea at the adult stage revealed the use of olfaction in host plant selection of this species and demonstrated with behavioural tests that E. oleracea preferred feeding on wild Eruca sativa, rather than on Brassica oleracea. Moreover, ultrastructural data revealed the antennal sensilla of E. oleracea, encompassing single walled and double walled olfactory sensilla, and electroantennographic recordings revealed their sensitivity to several host plant VOCs from E. sativa and B. oleracea. The data shown in the present research may be useful in the development of semiochemical-based strategies or trap crops for the control of this pest in the field.

Structure and biomechanics of the antennal grooming mechanism in the southern green stink bug Nezara viridula.

Insects devote a large amount of time to self-groom to remove foreign material, especially from their sensory appendages. Using various microscopy techniques and behavioural experiments on intact and ablated insects, the present study investigates the antennal grooming of the southern green stinkbug Nezara viridula, which represents a serious pest of different crops in most areas of the world. The antennal grooming behaviour encompasses an action of scraping involving the tibial comb complex (tibial comb + fossula) of both forelegs, generally followed by the tibial comb complex grooming of one leg using the tarsal hairy adhesive pad of the opposite leg (rubbing). From our observations, we can exclude a role in the antennal grooming of other structures such as the foretibial apparatus, while we show an involvement of this last structure in repositioning the stylets inside the labium. The external and internal morphology (cryo-scanning and transmission electron microscopy) and the evidence for the presence of large proportions of the elastic protein resilin (confocal laser scanning microscopy) in some parts of both the tibial comb complex and the foretibial apparatus are shown, and their functional roles are discussed. For the first time we demonstrated here the multipurpose role of the basitarsal hairy adhesive pad that is involved in both antennal grooming and adhesion to the substrate.

The antennae of damselfly larvae.

The larval antennal sensilla of two Zygoptera species, Calopteryx haemorroidalis (Calopterygidae) and Ischnura elegans (Coenagrionidae) are investigated with SEM and TEM. These two species have different antennae (geniculate, setaceous) and live in different environments (lotic, lentic waters). Notwithstanding this, similarities in the kind and distribution of sensilla are outlined: in both species the majority of sensilla types is located on the apical portion of the antenna, namely a composed coeloconic sensillum (possible chemoreceptor), two other coeloconic sensilla (possible thermo-hygroreceptors) and an apical seta (direct contact mechanoreceptor). Other mechanoreceptors, such as filiform hairs sensitive to movements of the surrounding medium or bristles positioned to sense the movements of the flagellar segments, are present on the antenna. Similarities in the antennal sensilla types and distribution are observed also with other dragonfly species, such as Onychogomphus forcipatus and Libellula depressa. A peculiar structure with an internal organization similar to that of a gland is observed in the apical antenna of C. haemorroidalis and I. elegans and it is present also in O. forcipatus and L. depressa. The possible function of this structure is at the moment unknown but deserves further investigations owing to its widespread presence in Odonata larvae.

Antennal responses to volatile organic compounds in a stonefly.

Notwithstanding their long antennae, especially in the adult stage, Plecoptera represent a poorly studied group as regards their sensory structures and their ability to perceive olfactory stimuli is so far totally unknown. A recent investigation on the antenna of Dinocras cephalotes revealed two kinds of putative olfactory sensilla (multiporous single walled sensilla and digitated double walled sensilla). The present electrophysiological study (electroantennography, EAG), in which male and female antennae of D. cephalotes (Plecoptera, Perlidae) have been stimulated with a set of generic odours belonging to different functional groups, shows that Plecoptera can perceive olfactory cues through their antennal sensilla. Indeed, although many chemicals did not elicit any response, high EAG activity has been recorded in response to pentanoic acid, propanal, butyric acid, propionic acid, isoamylamine and ammonia. The response was very similar in both sexes even if propanal elicited a response only in males. EAG dose-responses curves in both males and females showed that EAG responses were similar in males and females and generally increased in amplitude with increasing doses of the chemicals. Behavioural, neuroanatomical and molecular investigations on Plecoptera olfaction, could be particularly interesting not only to increase the knowledge of the adult stonefly behaviour but also to better understand the adaptation of the olfactory sensory system in aquatic insects moving from land to water.

Antennal sensilla of the stonefly Dinocras cephalotes (Plecoptera: Perlidae).

Plecoptera, one of the most primitive groups of Neoptera, are important aquatic insects usually employed as bioindicators of high water quality. Notwithstanding the well-developed antennae of the adult, its sensory abilities are so far not well known. The present paper describes at ultrastructural level under scanning and transmission electron microscopy the antennal sensilla of the adult stonefly Dinocras cephalotes (Plecoptera, Perlidae). Adult males and females show a filiform antenna constituted of a scape, a pedicel and a flagellum composed of very numerous segments with no clear sexual dimorphism in the number and distribution of the antennal sensilla. The most represented sensilla are sensilla trichodea, with different length, whose internal structure reveal their mechanosensory function, sensilla chaetica, with an apical pore, with an internal structure revealing a typical gustatory function, porous pegs representing single-walled olfactory sensilla, digitated pegs with hollow cuticular spoke channels representing double-walled olfactory sensilla, pegs in pits for which we hypothesize a thermo-hygrosensory function. The diversity of described sensilla is discussed in relation to known biological aspects of the studied species. This opens new perspectives in the study of the behavior of these aquatic insects during their adult stage.

Volatile cues can drive the oviposition behavior in Odonata.

Selection for the oviposition site represents the criterion for the behavioral process of habitat selection for the next generation. It is well known that in Odonata the most general cues are detected visually, but laboratory investigations on the coenagrionid Ischnura elegans showed through behavioral and electrophysiological assays that adults were attracted by olfactory cues emitted by prey and that males of the same species are attracted by female odor. The results of the present behavioral and electrophysiological investigations on I. elegans suggest the involvement of antennal olfactory sensilla in oviposition behavior. In particular, I. elegans females laid in the laboratory significantly more eggs in water from larval rearing aquaria than in distilled or tap water. Moreover, the lack of preference between rearing water and tap water with plankton suggests a role of volatiles related to conspecific and plankton presence in the oviposition site choice. I. elegans may rely on food odor for oviposition site selection, thus supporting the predictions of the "mother knows best" theory. These behavioral data are partially supported by electroantennographic responses. These findings confirm a possible role of olfaction in crucial aspects of Odonata biology.

Carbon dioxide detection in adult Odonata.

The present paper shows, by means of single-cell recordings, responses of antennal sensory neurons of the damselfly Ischnura elegans when stimulated by air streams at different CO2 concentrations. Unlike most insects, but similarly to termites, centipedes and ticks, Odonata possess sensory neurons strongly inhibited by CO2, with the magnitude of the off-response depending upon the CO2 concentration. The Odonata antennal sensory neurons responding to CO2 are also sensitive to airborne odors; in particular, the impulse frequency is increased by isoamylamine and decreased by heptanoic and pentanoic acid. Further behavioral investigations are necessary to assign a biological role to carbon dioxide detection in Odonata.