Identification of a queen primer pheromone in higher termites.

It is long established that queens of social insects, including termites, maintain their reproductive dominance with queen primer pheromones (QPPs). Yet, the QPP chemistry has only been elucidated in a single species of lower termites. By contrast, the most diversified termite family Termitidae (higher termites), comprising over 70% of termite species, has so far resisted all attempts at QPP identification. Here, we show that the queen- and egg-specific sesquiterpene (3R,6E)-nerolidol acts as the QPP in the higher termite Embiratermes neotenicus. This species has a polygynous breeding system, in which the primary queen is replaced by multiple neotenic queens of parthenogenetic origin. We demonstrate that (3R,6E)-nerolidol suppresses the development of these parthenogenetic queens and thus mimics the presence of mature queen(s). It acts as an airborne signal and may be used to optimize the number of queens, thus being the key regulatory element in the special breeding system of E. neotenicus.

Widespread occurrence of asexual reproduction in higher termites of the Termes group (Termitidae: Termitinae).

BACKGROUND: A decade ago, the mixed reproductive strategy Asexual Queen Succession (AQS) was first described in termites. In AQS species, the workers, soldiers and dispersing reproductives are produced through sexual reproduction, while non-dispersing (neotenic) queens arise through automictic thelytokous parthenogenesis, replace the founding queen and mate with the founding king. As yet, AQS has been documented in six species from three lineages of lower (Rhinotermitidae) and higher (Termitinae: Termes group and Syntermitinae) termites. Independent evolution of the capacity of thelytoky as a preadaptation to AQS is supported by different mechanisms of automixis in each of the three clades. These pioneering discoveries prompt the question on the extent of thelytoky and AQS in the diversified family of higher termites. RESULTS: Here, we investigated the capacity of thelytoky and occurrence of AQS in three species from the phylogenetic proximity of the neotropical AQS species Cavitermes tuberosus (Termitinae: Termes group): Palmitermes impostor, Spinitermes trispinosus, and Inquilinitermes inquilinus. We show that queens of all three species are able to lay unfertilized eggs, which undergo thelytokous parthenogenesis (via gamete duplication as in C. tuberosus) and develop through the transitional stage of aspirants into replacement neotenic queens. CONCLUSIONS: The breeding system in P. impostor is very reminiscent of that described in C. tuberosus and can be characterized as AQS. In the remaining two species, our limited data do not allow classifying the breeding system as AQS; yet, also in these species the thelytokous production of neotenic females appears to be a systematic element of reproductive strategies. It appears likely that the capacity of thelytokous parthenogenesis evolved once in the Termes group, and may ultimately be found more widely, well beyond these Neotropical species.

(3R,6E)-nerolidol, a fertility-related volatile secreted by the queens of higher termites (Termitidae: Syntermitinae).

The queens of advanced social insects maintain their reproductive monopoly by using exocrine chemicals. The chemistry of these "queen pheromones" in termites is poorly known. We show that primary queens of four higher termites from the subfamily Syntermitinae (Embiratermes neotenicus, Silvestritermes heyeri, Labiotermes labralis, and Cyrilliotermes angulariceps) emit significant amounts of the sesquiterpene alcohol (E)-nerolidol. It is the dominant analyte in queen body washes; it is present on the surface of eggs, but absent in kings, workers, and soldiers. In E. neotenicus, it is also produced by replacement neotenic queens, in quantities correlated with their fertility. Using newly synthesised (3R,6E)-nerolidol, we demonstrate that the queens of this species produce only the (R) enantiomer. It is distributed over the surface of their abdomen, in internal tissues, and in the haemolymph, as well as in the headspace of the queens. Both (R) and (S) enantiomers are perceived by the antennae of E. neotenicus workers. The naturally occurring (R) enantiomer elicited a significantly larger antennal response, but it did not show any behavioural effect. In spite of technical difficulties encountered in long-term experiments with the studied species, (3R,6E)-nerolidol remains among eventual candidates for the role in queen fertility signalling.

Identification and Enantiodivergent Synthesis of (5 Z,9 S)-Tetradec-5-en-9-olide, a Queen-Specific Volatile of the Termite Silvestritermes minutus.

The queens of social insects differ from sterile colony members in many aspects of their physiology. Besides adaptations linked with their specialization for reproduction and extended lifespan, the queens also invest in the maintenance of their reproductive dominance by producing exocrine chemicals signaling their presence to the nestmates. The knowledge of the chemistry of queen-specific cues in termites is scarce. In addition to the contact recognition based on cuticular hydrocarbons, long-range signals mediated by volatiles are expected to participate in queen signaling, especially in populous colonies of higher termites (Termitidae). In queens of the higher termite Silvestritermes minutus (Syntermitinae), we have detected a previously undescribed volatile. It is present in important quantities on the body surface and in the headspace, ovaries, and body cavity. MS and GC-FTIR data analyses led us to propose the structure of the compound to be a macrolide 10-pentyl-3,4,5,8,9,10-hexahydro-2 H-oxecin-2-one. We performed enantiodivergent syntheses of two possible enantiomers starting from enantiopure ( S)-glycidyl tosylate. The synthetic sequence involved macrolide-closing metathesis quenched with a ruthenium scavenging agent. The absolute and relative configuration of the compound was assigned to be (5 Z,9 S)-tetradec-5-en-9-olide. Identification and preparation of the compound allow for investigation of its biological significance.

Metabolomic and transcriptomic data on major metabolic/biosynthetic pathways in workers and soldiers of the termite Prorhinotermes simplex (Isoptera: Rhinotermitidae) and chemical synthesis of intermediates of defensive (E)-nitropentadec-1-ene biosynthesis.

Production of nitro compounds has only seldom been recorded in arthropods. The aliphatic nitroalkene (E)-nitropentadec-1-ene (NPD), identified in soldiers of the termite genus Prorhinotermes, was the first case documented in insects in early seventies. Yet, the biosynthetic origin of NPD has long remained unknown. We previously proposed that NPD arises through the condensation of amino acids glycine and/or l-serine with tetradecanoic acid along a biosynthetic pathway analogous to the formation of sphingolipids. Here, we provide a metabolomics and transcriptomic data of the Prorhinotermes simplex termite workers and soldiers. Data are related to NPD biosynthesis in P. simplex soldiers. Original metabolomics data were deposited in MetaboLights metabolomics database and are become publicly available after publishing the original article. Additionally, chemical synthesis of biosynthetic intermediates of NPD in nonlabeled and stable labeled forms are reported. Data extend our poor knowledge of arthropod metabolome and transcriptome and would be useful for comparative study in termites or other arthropods. The data were used for de-replication of NPD biosynthesis and published separately (Jirosová et al., 2017) [1].

Sex-Pairing Pheromones in Three Sympatric Neotropical Termite Species (Termitidae: Syntermitinae).

Termite colonies are almost always founded by a pair of winged dispersers, in spite of the high costs and low success rates inherent in independent colony foundation. The dispersal flights of imagoes from natal colonies are followed by mate search, mediated by sex-pairing pheromones. Here, we studied the chemistry of sex-pairing pheromones and the related aspects of mate search in winged imagoes of two facultatively parthenogenetic species, Embiratermes neotenicus and Silvestritermes minutus, and an additional species from the same subfamily, Silvestritermes heyeri. All three species are widespread in the Neotropics, including the rainforests of French Guiana. After the dispersal flight and spontaneous loss of wings, females expose their hypertrophied tergal glands situated under abdominal tergites VIII - X. The females are attractive to males and, upon direct contact, the two sexes form characteristic tandems. Chemical analyses indicated that the females secrete species-specific combinations of unbranched, unsaturated C12 primary alcohols from the tergal glands, (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (approx. 200 pg per female) and (3Z)-dodec-3-enol (185 pg) in E. neotenicus, (3Z,6Z)-dodeca-3,6-dien-1-ol (3500 pg) in S. heyeri, and (3Z,6Z)-dodeca-3,6-dien-1-ol (300 pg) and (3Z)-dodec-3-enol (50 pg) in S. minutus. (3Z,6Z,8E)-Dodeca-3,6,8-trien-1-ol and (3Z,6Z)-dodeca-3,6-dien-1-ol act as major pheromone components in the respective species and mimic the function of female tergal gland extracts in electrophysiological and behavioral experiments. Biologically relevant amounts of the third compound, (3Z)-dodec-3-enol, elicited non-significant reactions in males of E. neotenicus and S. minutus, and slight synergistic effects in males of S. minutus when tested in combination with the major component.

Co-option of the sphingolipid metabolism for the production of nitroalkene defensive chemicals in termite soldiers.

The aliphatic nitroalkene (E)-1-nitropentadec-1-ene (NPD), reported in early seventies in soldiers of the termite genus Prorhinotermes, was the first documented nitro compound produced by insects. Yet, its biosynthetic origin has long remained unknown. Here, we investigated in detail the biosynthesis of NPD in P. simplex soldiers. First, we track the dynamics in major metabolic pathways during soldier ontogeny, with emphasis on likely NPD precursors and intermediates. Second, we propose a hypothesis of NPD formation and verify its individual steps using in vivo incubations of putative precursors and intermediates. Third, we use a de novo assembled RNA-Seq profiles of workers and soldiers to identify putative enzymes underlying NPD formation. And fourth, we describe the caste- and age-specific expression dynamics of candidate initial genes of the proposed biosynthetic pathway. Our observations provide a strong support to the following biosynthetic scenario of NPD formation, representing an analogy of the sphingolipid pathway starting with the condensation of tetradecanoic acid with l-serine and leading to the formation of a C16 sphinganine. The C16 sphinganine is then oxidized at the terminal carbon to give rise to 2-amino-3-hydroxyhexadecanoic acid, further oxidized to 2-amino-3-oxohexadecanoic acid. Subsequent decarboxylation yields 1-aminopentadecan-2-one, which then proceeds through six-electron oxidation of the amino moiety to give rise to 1-nitropentadecan-2-one. Keto group reduction and hydroxyl moiety elimination lead to NPD. The proposed biosynthetic sequence has been constructed from age-related quantitative dynamics of individual intermediates and confirmed by the detection of labeled products downstream of the administered labeled intermediates. Comparative RNA-Seq analyses followed by qRT-PCR validation identified orthologs of serine palmitoyltransferase and 3-ketodihydrosphingosine reductase genes as highly expressed in the NPD production site, i.e. the frontal gland of soldiers. A dramatic onset of expression of the two genes in the first days of soldier's life coincides with the start of NPD biosynthesis, giving further support to the proposed biosynthetic hypothesis.

Smells Like Home: Chemically Mediated Co-Habitation of Two Termite Species in a Single Nest.

Termite nests often are referred to as the most elaborate constructions of animals. However, some termite species do not build a nest at all and instead found colonies inside the nests of other termites. Since these so-called inquilines do not need to be in direct contact with the host population, the two colonies usually live in separate parts of the nest. Adaptations of both the inquiline and its host are likely to occur to maintain the spatial exclusion and reduce the costs of potential conflicts. Among them, mutual avoidance, based on chemical cues, is expected. We investigated chemical aspects of cohabitation between Constrictotermes cavifrons (Nasutitermitinae) and its obligatory inquiline Inquilinitermes inquilinus (Termitinae). Inquiline soldiers produce in their frontal glands a blend of wax esters, consisting of the C12 alcohols (3Z)-dodec enol, (3Z,6Z)-dodecadienol, and dodecanol, esterified with different fatty acids. The C12 alcohols appear to be cleaved gradually from the wax esters, and they occur in the frontal gland, in soldier headspace, and in the walls of the inquiline part of the nest. Electrophysiological experiments revealed that (3Z)-dodecenol and (3Z,6Z)-dodecadienol are perceived by workers of both species. Bioassays indicated that inquiline soldier heads, as well as the two synthetic compounds, are attractive to conspecific workers and elicit an arresting behavior, while host soldiers and workers avoid these chemicals at biologically relevant amounts. These observations support the hypothesis that chemically mediated spatial separation of the host and the inquiline is an element of a conflict-avoidance strategy in these species.

Asexual queen succession in the higher termite Embiratermes neotenicus.

Asexual queen succession (AQS), in which workers, soldiers and dispersing reproductives are produced sexually while numerous non-dispersing queens arise through thelytokous parthenogenesis, has recently been described in three species of lower termites of the genus Reticulitermes. Here, we show that AQS is not an oddity restricted to a single genus of lower termites, but a more widespread strategy occurring also in the most advanced termite group, the higher termites (Termitidae). We analysed the genetic structure in 10 colonies of the Neotropical higher termite Embiratermes neotenicus (Syntermitinae) using five newly developed polymorphic microsatellite loci. The colonies contained one primary king accompanied either by a single primary queen or by up to almost 200 neotenic queens. While the workers, the soldiers and most future dispersing reproductives were produced sexually, the non-dispersing neotenic queens originated through thelytokous parthenogenesis of the founding primary queen. Surprisingly, the mode of thelytoky observed in E. neotenicus is most probably automixis with central fusion, contrasting with the automixis with terminal fusion documented in Reticulitermes. The occurrence of AQS based on different mechanisms of ploidy restoration raises the hypothesis of an independent evolutionary origin of this unique reproductive strategy in individual lineages of lower and higher termites.

Chemical alarm in the termite Termitogeton planus (Rhinotermitidae).

Effective defense is a common characteristic of insect societies. Indeed, the occurrence of specialized defenders, soldiers, has been the first step toward eusociality in several independent lineages, including termites. Among the multitude of defensive strategies used by termite soldiers, defense by chemicals plays a crucial role. It has evolved with complexity in advanced isopteran lineages, whose soldiers are equipped with a unique defensive organ, the frontal gland. Besides direct defense against predators, competitors, and pathogens, the chemicals emitted by soldiers from the frontal gland are used as signals of alarm. In this study, we investigated the chemical composition of the defensive secretion produced by soldiers of the termite Termitogeton planus (Isoptera: Rhinotermitidae), from West Papua, and the effects of this secretion on the behavior of termite groups. Detailed two-dimensional gas chromatography/mass spectrometry analyses of the soldier defensive secretion revealed the presence of four linear and nine monoterpene hydrocarbons. Soldier head extracts, as well as synthetic mixtures of the monoterpenes found in these extracts, elicited alarm behavior in both soldiers and pseudergates. Our results suggest that the alarm is not triggered by a single monoterpene from the defensive blend, but by a multi-component signal combining quantitatively major and minor compounds.

Sphinganine-like biogenesis of (E)-1-nitropentadec-1-ene in termite soldiers of the genus Prorhinotermes.

In 1974, (E)-1-nitropentadec-1-ene, a strong lipophilic contact poison of soldiers of the termite genus Prorhinotermes, was the first-described insect-produced nitro compound. However, its biosynthesis remained unknown. In the present study, we tested the hypothesis that (E)-1-nitropentadec-1-ene biosynthesis originates with condensation of amino acids with tetradecanoic acid. By using in vivo experiments with radiolabeled and deuterium-labeled putative precursors, we show that (E)-1-nitropentadec-1-ene is synthesized by the soldiers from glycine or L-serine and tetradecanoic acid. We propose and discuss three possible biosynthetic pathways.

Nonadecadienone, a new termite trail-following pheromone identified in Glossotermes oculatus (Serritermitidae).

Within the multitude of chemical signals used by termites, the trail marking by means of pheromones is ubiquitous. Chemistry and biology of the trail-following communication have been described in more than 60 species from all families except for the Neotropical Serritermitidae. The chemical ecology of Serritermitidae is of special interest not only as a missing piece of knowledge on the diversity and evolution of isopteran pheromones but also because it may contribute to the debate on the phylogenetic position of this family, which is still unresolved. Therefore, we aimed in this study to identify the trail-following pheromone of the serritermitid Glossotermes oculatus. Based on a combined approach of analytical chemistry, electrophysiology, and behavioral bioassays, we propose (10Z,13Z)-nonadeca-10,13-dien-2-one to be the trail-following pheromone of G. oculatus, secreted by the sternal gland of pseudergates. Thus, we report on a new termite trail-following pheromone of an unexpected chemical structure, a ketone with 19 carbons, contrasting with unsaturated alcohols containing 12 carbons as trail-following pheromones in other advanced termite families. In addition to this unique trail-following pheromone, we also describe the sternal gland in pseudergates as an organ of unusual shape, size, and structure when compared with other isopteran species. These results underline the peculiarity of the family Serritermitidae and prompt our interest in the chemistry of pheromones in the other genus of the family, Serritermes.