Parasitoid attachment ability and the host surface wettability.

Climbing animals such as geckos and arthropods developed astonishing adhesive mechanisms which are fundamental for their survival and represent valuable models for biomimetic purposes. A firm adhesion to the host surface, in order to successfully lay eggs is necessary for the reproduction of most parasitoid insects. In the present study, we performed a comparative investigation on the attachment ability of four parasitoid species (the egg parasitoid Anastatus bifasciatus (Eupelmidae), the aphid parasitoid Aphidius ervi (Braconidae), the fly pupal ectoparasitoid Muscidifurax raptorellus (Pteromalidae) and the pupal parasitoid of Drosophila Trichopria drosophilae (Diapriidae)) with hosts characterized by a surface having different wettability properties. The friction force measurements were performed on smooth artificial (glass) surfaces showing different contact angles of water. We found that attachment systems of parasitoid insects are tuned to match the wettability of the host surface. Sexual dimorphism in the attachment ability of some tested species has been also observed. The obtained results are probably related to different microstructure and chemical composition of the host surfaces and to different chemical composition of the parasitoid adhesive fluid. The data here presented can be interpreted as an adaptation, especially in the female, to the physicochemical properties of the host surface and contribute to shed light on the coevolutionary processes of parasitoid insects and their hosts.

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.

Mechanoecology: biomechanical aspects of insect-plant interactions.

Plants and herbivorous insects as well as their natural enemies, such as predatory and parasitoid insects, are united by intricate relationships. During the long period of co-evolution with insects, plants developed a wide diversity of features to defence against herbivores and to attract pollinators and herbivores' natural enemies. The chemical basis of insect-plant interactions is established and many examples are studied, where feeding and oviposition site selection of phytophagous insects are dependent on the plant's secondary chemistry. However, often overlooked mechanical interactions between insects and plants can be rather crucial. In the context of mechanoecology, the evolution of plant surfaces and insect adhesive pads is an interesting example of competition between insect attachment systems and plant anti-attachment surfaces. The present review is focused on mechanical insect-plant interactions of some important pest species, such as the polyphagous Southern Green Stinkbug Nezara viridula and two frugivorous pest species, the polyphagous Mediterranean fruit fly Ceratitis capitata and the monophagous olive fruit fly Bactrocera oleae. Their ability to attach to plant surfaces characterised by different features such as waxes and trichomes is discussed. Some attention is paid also to Coccinellidae, whose interaction with plant leaf surfaces is substantial across all developmental stages in both phytophagous and predatory species that feed on herbivorous insects. Finally, the role of different kinds of anti-adhesive nanomaterials is discussed. They can reduce the attachment ability of insect pests to natural and artificial surfaces, potentially representing environmental friendly alternative methods to reduce insect pest impact in agriculture.

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.

Mechanoecology and Chemoecology: Physical and Chemical Interactions between Insects and Plants.

Plants and herbivorous insects, as well as their natural enemies such as predatory and parasitoid insects, are united by intricate relationships [...].

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.

Air-entrapping capacity in the hair coverage of Malacosoma castrensis (Lasiocampidae: Lepidoptera) caterpillar: a case study.

The moth Malacosoma castrensis (Lasiocampidae) is commonly found along the Northern Germany coasts, the habitats of which are mainly represented by salt marshes subjected to sea level variations. Surprisingly, terrestrial caterpillars can withstand many hours of being flooded by seawater. The ability to withstand periods of submersion in a terrestrial insect raises the problem of respiration related to avoiding water percolation into the tracheal system. In the present study, we investigated under laboratory conditions the role of water-repellent cuticle structures in oxygen supply in caterpillars of M. castrensis submerged in water. For this purpose, air-layer stability tests using force measurements, and micromorphology of cuticle structures using SEM and fluorescence microscopy, were performed. A plastron appeared when a caterpillar is underwater. The stability, gas composition and internal pressure of the plastron were estimated. The plastron is stabilized by long and scarce hairs, which are much thicker than the corresponding hairs of aquatic insects. Thick and stiff hairs with sclerotized basal and middle regions protrude into the water through the plastron-water interface, while substantial regions of thin and flexible hairs are aligned along the plastron-water interface and their side walls can support pressure in the plastron even below atmospheric pressure. Additional anchoring points between hair's stalk and microtrichia near the hair base provide enhanced stiffness to the hair layer and prevent the hair layer from collapse and water entering between hairs. The advancing contact angle on hairs is more than 90 deg, which is close to the effective contact angle for the whole caterpillar.

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.

Attachment ability of the polyphagous bug Nezara viridula (Heteroptera: Pentatomidae) to different host plant surfaces.

The present investigation tests through friction experiments the attachment ability of adults of the southern green stink bug Nezara viridula L. (Heteroptera: Pentatomidae), a polyphagous insect representing a cosmopolitan pest, on different host plant species characterized by smooth, hairy and waxy surfaces. Surfaces of different tested plants have been studied in Cryo-Scanning Electron Microscope (Cryo-SEM). The load cell force transducer was used to evaluate the potential damage to the insect attachment devices induced by walking on the different leaf surfaces. In case of the plant Phaseolus vulgaris, where insects showed a strong reduction in their adhesion ability during and after walking on the leaf, the damage to the insect by two cultivars with different morphological features and the insect ability to recover after 24 h has been evaluated. The ability to recover notwithstanding the damage to attachment devices, shown by Cryo-SEM investigations, together with the strong attachment forces produced on various plant leaves, characterized by different morphological features, is in agreement with the great adaptability and ecological plasticity of this widely-spread bug species. The present study, increasing our knowledge on the mechanical interaction of this species with different host plant species, can help to develop new strategies to control this insect pest.