In termites, as in other social insects, pheromones are involved in probably all social activities from simple recognition between social partners to nest building. In termites which collect food at a distance from their nest, the discovery of food is not the achievement of scouts which singly explore the foraging field far from the nest. Exploration is a collective process: a collective trail or gallery grows from the nest or as an extension of an already preexisting network of trails or galleries. The first detailed descriptions of postflight behavior of termites were given by C. Fuller who reported that females produce pheromones during their calling attitude and tandem running. When soliciting mates, termites raise their abdomen exposing the sternal gland. In termites, defensive reactions are coordinated by various signals, either mechanical, such as vibratory movements and head banging or chemical, that is, trail and alarm pheromones.

Abstract Due to its fundamental role in shaping host selection behavior, we have analyzed the chemosensory repertoire of Chrysomela lapponica. This specialized leaf beetle evolved distinct populations which shifted from the ancestral host plant, willow (Salix sp., Salicaceae), to birch (Betula rotundifolia, Betulaceae). We identified 114 chemosensory candidate genes in adult C. lapponica: 41 olfactory receptors (ORs), eight gustatory receptors, 17 ionotropic receptors, four sensory neuron membrane proteins, 32 odorant binding proteins (OBPs), and 12 chemosensory proteins (CSP) by RNA‐seq. Differential expression analyses in the antennae revealed significant upregulation of one minus‐C OBP (ClapOBP27) and one CSP (ClapCSP12) in the willow feeders. In contrast, one OR (ClapOR17), four minus‐C OBPs (ClapOBP02, 07, 13, 20), and one plus‐C OBP (ClapOBP32) were significantly upregulated in birch feeders. The differential expression pattern in the legs was more complex. To narrow down putative ligands acting as cues for host discrimination, the relative abundance and diversity of volatiles of the two host plant species were analyzed. In addition to salicylaldehyde (willow‐specific), both plant species differed mainly in their emission rate of terpenoids such as (E,E)‐α‐farnesene (high in willow) or 4,8‐dimethylnona‐1,3,7‐triene (high in birch). Qualitatively, the volatiles were similar between willow and birch leaves constituting an “olfactory bridge” for the beetles. Subsequent structural modeling of the three most differentially expressed OBPs and docking studies using 22 host volatiles indicated that ligands bind with varying affinity. We suggest that the evolution of particularly minus‐C OBPs and ORs in C. lapponica facilitated its host plant shift via chemosensation of the phytochemicals from birch as novel host plant.

Covering up to September 2016This review reports on natural compounds that derive from the isoxazolinone ring as well as the 3-nitropropanoic acid (3-NPA) moiety. These structural elements occur in compounds that have been identified in plants, insects, bacteria and fungi. In particular, plants belonging to the family of legumes produce such compounds. In the case of insects, isoxazolin-5-one and 3-NPA derivatives were found in leaf beetles of the subtribe Chrysomelina. A number of these natural products have been synthesized so far. In the case of the single compound 3-NPA, several synthetic strategies have been reported and some of the most efficient routes are reviewed. The toxicity of 3-NPA results from its ability to bind covalently to the catalytic center of succinate dehydrogenase causing irreversible inhibition of mitochondrial respiration. As a motif that is produced by many species of plants, leaf beetles and fungi, different detoxification mechanisms for 3-NPA have evolved in different species. These mechanisms are based on amide formation of 3-NPA with amino acids, reduction to β-alanine, ester formation or oxidation to malonic acid semialdehyde. The biosynthetic pathways of 3-NPA and isoxazolin-5-one moieties have been studied in fungi, plants and leaf beetles. In the case of fungi, 3-NPA derives from aspartate, while leaf beetles use essential amino acids such as valine as ultimate precursors. In the case of plants, it is supposed that malonate serves as a precursor of 3-NPA, as indicated by feeding of 14C-labeled precursors to Indigofera spicata. In other leguminous plants it is suggested that asparagine is incorporated into compounds that derive from isoxazolin-5-one, which was indicated by 14C-labeled compounds as well. In the case of leaf beetles it was demonstrated that detection of radioactivity after 14C-labeling from a few precursors is not sufficient to unravel biosynthetic pathways.

Juveniles of the leaf beetles in subtribe Chrysomelina have efficient defense strategies against predators. When disturbed, they transiently expose volatile deterrents in large droplets from nine pairs of defensive glands on their back. Here, we report on an additional line of defense consisting of the non-volatile isoxazolin-5-one glucoside and its 3-nitropropanoyl ester in the larval hemolymph. Because isoxazolin-5-one derivatives were not detectable in related leaf beetle taxa, they serve as a diagnostic marker for the Chrysomelina subtribe. Conjugation of isotopically labelled 3-nitropropionic acid to isoxazolin-5-one glucoside in vivo demonstrates its function as a carrier for the 3-nitropropanoyl esters. The previous identification of characteristic glucosides as precursors of the volatile deterrents underlines the general importance of glucosides for sequestration from food plants, and the subsequent transport in the hemolymph to the defense system. The combination of repellent volatiles with non-volatile toxic compounds in the hemolymph has the potential to create synergistic effects since the odorant stimulus may help predators learn to avoid some foods. The combination of the two defense lines has the advantage, that the hemolymph toxins provide reliable and durable protection, while the repellents may vary after a host plant change.

Charidotella ambita offers a unique opportunity for unambiguously locating its gold reflector by comparing the structure of reflecting and non-reflecting cuticle of the elytron and pronotum. Using light microscopy and TEM, the reflector was located underneath the macrofiber endocuticle just above the epidermis. The reflector is a multilayer comprising up to 50 bilayers alternating high and low density layers parallel to the surface of the cuticle. It is chirped, i.e., showing a progressive decrease in layer thickness from approximately 150 nm-100 nm across its depth. The high density layers in contact with the endocuticle fuse to the last macrofiber when the reflector is interrupted by a trabecula, demonstrating their cuticular nature. Simulated reflectance spectra from models of the multilayer matched the reflection spectra measured on the major gold patch of the elytron of living specimens.Previous reports in adult insects exhibiting metallic colors located their reflector in the upper strata and structures of the cuticle, i.e., epicuticle, exocuticle, scales and hairs. Thus, the endocuticular location of the reflector in C. ambita (and other tortoise beetles) appears unique for adult insects. Gold reflection appears in C ambita only when the synthesis of the macrolayer endocuticle is complete, which may take up to 2 weeks. The development of the gold reflector coincides with the start of mating behavior, possibly suggesting a signaling function in conspecific recognition once sexual maturity has been reached. (C) 2016 Elsevier Ltd. All rights reserved.

A summary of literature, documented observations and field studies finds evidence that mothers actively defend offspring in at least eight species and three genera of Neotropical Chrysomelinae associated with two host plant families. Reports on three Doryphora species reveal that all are oviparous and feed on vines in the Apocyanaceae. Mothers in the two subsocial species defend eggs and larvae by straddling, blocking access at the petiole and greeting potential predators with leaf-shaking and jerky advances. A less aggressive form of maternal care is found in two Platyphora and four Proseicela species associated with Solanaceae, shrubs and small trees. For these and other morphologically similar taxa associated with Solanaceae, genetic distances support morphology-based taxonomy at the species level, reveal one new species, but raise questions regarding boundaries separating genera. We urge continued study of these magnificent insects, their enemies and their defenses, both behavioral and chemical, especially in forests along the eastern versant of the Central and South American cordillera.

Larvae of the Chrysomelina species Phaedon cochleariae, Hydrothassa marginella, Phratora vulgatissima, Gastrophysa viridula, Gastrophysa atrocyanea, Gastrophysa cyanea and Gastrophysa polygoni produce the iridoid chrysomelidial (1) to defend themselves against predators. Feeding experiments with a deuterated precursor ([(2)H(5)]8-hydroxygeraniol 9) and in vitro isotope exchange experiments with defensive secretion in (2)H(2)O revealed differences in the cyclisation of the ultimate precursor 8-oxogeranial (8) to 1, between members of the genus Gastrophysa and all other species. In P. cochleariae, H. marginella and P. vulgatissima 1 is most likely produced by a Rauhut-Currier-type cyclisation via a "transoid dienamine", with loss of a single deuterium atom from C(4) of the precursor. In contrast, members of the genus Gastrophysa cyclise 8 via a "cisoid dienamine" intermediate, with exchange of all three deuterium atoms from the methyl group at C(3). To study whether the different cyclisation modes influence the stereochemistry of 1, the absolute configuration of 1 of the larvae was determined by GC-MS on a chiral column. In accordance with literature (J. Meinwald, T. H. Jones, J. Am. Chem. Soc. 1978, 100, 1883 and N. Shimizu, R. Yakumaru, T. Sakata, S. Shimano, Y. Kuwahara, J. Chem. Ecol. 2012, 38, 29), we found (5S,8S)-chrysomelidial (1) in H. marginella and P. vulgatissima, but P. cochleariae and all investigated members of the genus Gastrophysa synthesise (5R,8R)-chrysomelidial (1).

The inside cover picture shows a proposed mechanism for the biosynthesis of chrysomelidial in leaf-beetle larvae. The sequence implies the formation of a dienamine intermediate that is “transoid” in P. cochleariae and “cisoid” in G. viridula. For further details see the paper by W. Boland et al. on p. 353 ff. We thank Jana Becher for the arrangement of the inside cover.

Glandular chemical defence relying on the action of salicylaldehyde is characteristic for Chrysomela leaf beetle larvae. The salicylaldehyde precursor salicin, sequestered from salicaceous host plants, is deglucosylated and the aglycon further oxidized by a salicyl alcohol oxidase (SAO) to the respective aldehyde. SAOs, key enzymes in salicin-based glandular chemical defence, were previously identified and shown to be of a single evolutionary origin in Chrysomela species. We here identified and characterized SAO of Phratora vitellinae , the only species outside the genus Chrysomela that produce salicylaldehyde as a defensive compound. Although Chrysomela and Phratora are not closest relatives, their SAOs share glucose–methanol–choline oxidoreductase (GMC) affiliation, a specific GMC i subfamily ancestor, glandular tissue-specific expression and almost identical gene architectures. Together, this strongly supports a single origin of SAOs of both Chrysomela and Phratora . Closely related species of Chrysomela and P. vitellinae use iridoids as defensive compounds, which are like salicylaldehyde synthesized by the consecutive action of glucosidase and oxidase. However, we elucidated SAO-like sequences but no SAO proteins in the glandular secretion of iridoid producers. These findings support a different evolutionary history of SAO, related genes and other oxidases involved in chemical defence in the glandular system of salicylaldehyde and iridoid-producing leaf beetle larvae.

We conducted a phylogeographic study on the cold-adapted leaf beetle Chrysomela lapponica, that feeds on willow or birch, by sampling several populations throughout most of the geographic distribution of the species, and by sequencing for each individual one mitochondrial and two nuclear DNA fragments. Patterns of DNA sequence variation from the mitochondrial and nuclear loci, as displayed in the median-joining networks, appear to display contradicting historical signal: a deep genealogical divergence is observed with the mitochondrial genome between the Alpine population and all other populations found in the Euro-Siberian distribution of the species, that is completely absent with both nuclear loci. We use coalescence simulations of DNA sequence evolution to test the hypothesis that this apparent conflict is compatible with a neutral model of sequence evolution (i.e., to check whether the stochastic nature of the coalescence process can explain these patterns). Because the simulations show that this is highly unlikely, we consider two alternative hypotheses: (1) introgression of the mitochondrial genome of another species and (2) the effect of natural selection. Although introgression is the most plausible explanation, we fail to identify the source species of the introgressed mitochondrial genome among all known species closely related to C. lapponica. We therefore suggest that the putative introgression event is ancient and the source species is either extinct or currently outside the geographic range of C. lapponica explored in this study. The observed DNA sequence variation also suggests that a host-plant shift from willow to birch has occurred recently and independently in each of the three birch-feeding populations. This emphasizes further the relative ease with which these beetles can escape their ancestral host-plant specialization on willow, but shows at the same time that host-plant shifts are highly constrained, as they only occur between willow and birch.

Chrysomelid leaf beetles use chemical defenses to overcome predatory attack and microbial infestation. Larvae of Chrysomela lapponica that feed on willow sequester plant-derived salicin and other leaf alcohol glucosides, which are modified in their defensive glands to bioactive compounds. Salicin is converted into salicylaldehyde by a consecutive action of a β-glucosidase and salicyl alcohol oxidase (SAO). The other leaf alcohol glucosides are not oxidized, but are deglucosylated and esterified with isobutyric- and 2-methylbutyric acid. Like some other closely related Chrysomela species, certain populations of C. lapponica shift host plants from willow to salicin-free birch. The only striking difference between willow feeders and birch feeders in terms of chemical defense is the lack of salicylaldehyde formation. To clarify the impact of host plant shifts on SAO activity, we identified and compared this enzyme by cloning, expression, and functional testing in a willow-feeding and birch-feeding population of C. lapponica. Although the birch feeders still demonstrated defensive gland-specific expression, their SAO mRNA levels were 1,000-fold lower, and the SAO enzyme was nonfunctional. Obviously, the loss of catalytic function of the SAO of birch-adapted larvae is fixed at the transcriptional, translational, and enzyme levels, thus avoiding costly expression of a highly abundant protein that is not required in the birch feeders.

Larvae of Chrysomela lapponica (Coleoptera: Chrysomelidae) sequester characteristic O -glucosides from the leaves of their food plants, namely Betula and/or Salix The present study focuses on birch-feeding larvae of C. lapponica from the Altai region in East Kazakhstan. As in other sequestering leaf beetle larvae, the compounds are transported intact via different membrane barriers into the defensive system, followed by glucoside cleavage and subsequent transformations of the plant-derived aglycones. Unlike previous studies with model compounds, we studied the sequestration of phytogenic precursors by analyzing the complex pattern of glucosides present in food plant Betula rotundifolia (39 compounds) and compared this composition with the aglycones present as butyrate esters in the defensive secretion. In addition to the analytic approach, the insect’s ability, to transport individual glucosides was tested by using hydrolysis-resistant thioglucoside analogs, applied onto the leaf surface. The test compounds reach the defensive system intact and without intermediate transformation. No significant difference of the transport capacity and selectivity was observed between larvae of birch-feeding population from Kazakhstan, and previous results for larvae of birch-feeding population from the Czech Republic or willow-feeding populations. Overall, the transport of the phytogenic glucosides is highly selective and highly efficient, since only minor compounds of the spectrum of phytogenic glucoside precursors contribute to the limited number of aglycones utilized in the defensive secretion. Interestingly, salicortin 44 and tremulacin 60 were found in the leaves, but no aldehyde or esters of salicylalcohol. Surprisingly, we observed large amounts of free glucose, together with small amounts of 6- O -butyrate esters of glucose ( 27a/b and 28a/b ).

Pheromones play a crucial role in the ecology of dispersal and foraging in termites. Sex-pairing pheromones possess a double role of long-range attraction to unite sexual partners and a short-range or contact attraction to maintain the pair during the tandem behaviour. Sex-pairing pheromones most often comprise a single compound capable of eliciting both behavioural effects. They appear very conservative in their evolution, and their role in the reproductive isolation of sympatric species greatly varies according to species. Species-specific sex-pairing pheromones consist of different major compounds or of a common major compound with species-specific minor components. Foraging is a collective behaviour mainly regulated by trail-following pheromones secreted from only one glandular source, the sternal gland. Trail-following pheromones may he also used by "one-piece" termites to colonise additional food sources. Although trail-following pheromones of termites have a double role of orientation and recruitment, they appear most often composed of only one compound. An alternative hypothesis is given to the postulated existence of a volatile ephemeral compound of recruitment and a long-lasting compound of orientation. Trail-following pheromones appear highly conserved in their chemical evolution (only 8 different pheromones for 60 species), even if a clear separation is observed between basal termites and more derived termites. The major ecological event of the external foraging was not related to a chemical evolutionary step of the trail-following pheromones. Pheromonal parsimony (the utilisation of a same molecule for multiple functions) is common in termites in the behavioural context of dispersal and foraging. The same molecule is used in many species as a sex-pairing pheromone and a trail-following pheromone, depending upon the pheromone concentration and the caste involved.