Robert Hanus

Chemical warfare in termites

The rapid development of analytical methods in the last four decades has led to the discovery of a fascinating diversity of defensive chemicals used by termites. The last exhaustive review on termite defensive chemicals was published by G.D. Prestwich in 1984. In this text, we aim to fill the gap of the past 25 years and overview all of the relevant primary sources about the chemistry of termite defense (126 original papers, see Fig. 1 and online supplementary material) along with related biological aspects, such as the anatomy of defensive glands and their functional mechanisms, alarm communication, and the evolutionary significance of these defensive elements.

Analysis of insect cuticular hydrocarbons using matrix-assisted laser desorption/ionization mass spectrometry.

Insect cuticular hydrocarbons (CHCs) were probed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry with a lithium 2,5-dihydroxybenzoate matrix. CHC profiles were obtained for 12 species of diverse insect taxa (termites, ants, a cockroach, and a flesh fly). MALDI spectra revealed the presence of high molecular weight CHCs on the insect cuticle. Hydrocarbons with more than 70 carbon atoms, both saturated and unsaturated, were detected. When compared with gas chromatography/mass spectrometry (GC/MS), MALDI-TOF covered a wider range of CHCs and enabled CHCs of considerably higher molecular weight to be detected. Good congruity between GC/MS and MALDI-TOF was observed in the overlapping region of molecular weights. Moreover, a number of previously undiscovered hydrocarbons were detected in the high mass range beyond the analytical capabilities of current GC/MS instruments. MALDI was shown to hold potential to become an alternative analytical method for insect CHC analyses. The ability of MALDI to discriminate among species varying in the degree of their relatedness was found to be similar to GC/MS. However, neither MALDI-MS nor GC/MS data were able to describe the phylogenetic relationships.

(E,E)-α-Farnesene, an Alarm Pheromone of the Termite Prorhinotermes canalifrons

The behavioral and electroantennographic responses of Prorhinotermes canalifrons to its soldier frontal gland secretion, and two separated major components of the secretion, (E)-1-nitropentadec-1-ene and (E,E)-α-farnesene, were studied in laboratory experiments. Behavioral experiments showed that both the frontal gland secretion and (E,E)-α-farnesene triggered alarm reactions in P. canalifrons, whereas (E)-1-nitropentadec-1-ene did not affect the behavior of termite groups. The alarm reactions were characterized by rapid walking of activated termites and efforts to alert and activate other members of the group. Behavioral responses to alarm pheromone differed between homogeneous and mixed groups, suggesting complex interactions. Antennae of both soldiers and pseudergates were sensitive to the frontal gland secretion and to (E,E)-α-farnesene, but soldiers showed stronger responses. The dose responses to (E,E)-α-farnesene were identical for both soldiers and pseudergates, suggesting that both castes use similar receptors to perceive (E,E)-α-farnesene. Our data confirm (E,E)-α-farnesene as an alarm pheromone of P. canalifrons.

Beyond cuticular hydrocarbons: evidence of proteinaceous secretion specific to termite kings and queens

In 1959, P. Karlson and M. Lüscher introduced the term ‘pheromone’, broadly used nowadays for various chemicals involved in intraspecific communication. To demonstrate the term, they depicted the situation in termite societies, where king and queen inhibit the reproduction of nest-mates by an unknown chemical substance. Paradoxically, half a century later, neither the source nor the chemical identity of this ‘royal’ pheromone is known. In this study, we report for the first time the secretion of polar compounds of proteinaceous origin by functional reproductives in three termite species, Prorhinotermes simplex, Reticulitermes santonensis and Kalotermes flavicollis. Aqueous washes of functional reproductives contained sex-specific proteinaceous compounds, virtually absent in non-reproducing stages. Moreover, the presence of these compounds was clearly correlated with the age of reproductives and their reproductive status. We discuss the putative function of these substances in termite caste recognition and regulation.

Explosive Backpacks in Old Termite Workers

As Neocapritermes taracua termites age, they develop a suicidal toxic apparatus that bursts during aggressive encounters. By nature, defensive behavior is risky. In social insects, such behavior is more likely to occur in individuals whose potential for other tasks is diminished. We show that workers of the termite Neocapritermes taracua develop an exceptional two-component suicidal apparatus consisting of copper-containing protein crystals, stored in external pouches, and internal salivary glands. During aggressive encounters, their bodies rupture, and the crystals react with the salivary gland secretion to produce a toxic droplet. Both the amount of defensive substances and the readiness to explode increase with workers’ age, as their food-collecting ability declines.

Identification by GC-EAD of the two-component trail-following pheromone of Prorhinotermes simplex (Isoptera, Rhinotermitidae, Prorhinotermitinae)

GC/MS analysis confirmed that neocembrene is the major component of the trail pheromone in the three species of the termite genus Prorhinotermes (P. simplex, P. canalifrons, P. inopinatus). In addition, EAG and GC-EAD experiments with P. simplex strongly suggest that dodecatrienol is a quantitatively minor component but a qualitatively important component of this trail pheromone. Trail-following bioassays confirmed the two-component nature of the trail pheromone. This is the first report of the use of the GC-EAD for the identification of trail pheromone in termites. These original results underline once again the special phylogenetic status of the Prorhinotermitinae among Rhinotermitidae.

A comparison of HPLC/APCI-MS and MALDI-MS for characterising triacylglycerols in insects: species-specific composition of lipids in the fat bodies of bumblebee males.

Two mass spectrometric methods for analysing triacylglycerols (HPLC/APCI-MS and MALDI-MS) were used and compared in terms of the relevance of the data for further biostatistical evaluation. While MALDI-MS is simpler and significantly faster, the time-consuming and labour-intensive HPLC/APCI-MS provides more complete information about the lipid components. However, both methods provide well-comparable results concerning the grouping of specimens belonging to different species when evaluated with multivariate exploratory approaches. The compositions of triacylglycerols in the fat bodies of males in 11 bumblebee species (Bombus terrestris, B. lucorum, B. lapidarius, B. pratorum, B. sylvarum, B. ruderatus, B. pomorum, B. subterraneus, B. campestris, B. bohemicus, and B. rupestris) were found to be species-specific.

The frontal gland in workers of Neotropical soldierless termites

The presence of the frontal gland is well established in termite soldiers of Rhinotermitidae, Serritermitidae, and Termitidae. It is one of their main defensive adaptations or even an exclusive weapon. The gland was also occasionally reported in alate imagoes, but never in the worker caste. Here, we report the first observation of a frontal gland in workers of several Neotropical and one African species of Apicotermitinae. The ultrastructure of Aparatermes cingulatus and Anoplotermes nr. subterraneus is described in detail. In these two species, the gland is well-developed, functional and consists of class 1 secretory cells. The presence of envelope cells, wrapping the gland, is an unusual feature, as well as the presence of several zonulae adherens, connecting neighbouring glandular cells. The frontal gland of workers is homologous to this organ in soldiers and imagoes, as evidenced by the same position in the head and its connection to the same muscle. However, the defensive role of the frontal gland in workers remains to be confirmed.

Developmental pathways of Glossotermes oculatus (Isoptera, Serritermitidae): at the cross-roads of worker caste evolution in termites

SUMMARY The onset of a specialized (“true”) worker caste is a crucial step in the evolution of termite societies. Such workers, permanently excluded from wing development, repeatedly evolved from totipotent immatures, called “false” workers or pseudergates. In the family Rhinotermitidae, the presence of true workers and the level of specialization of this caste are highly variable, and key taxa illustrate transitional situations providing clues about worker evolution. Here we focused on the status of working immatures of Glossotermes oculatus, from the family Serritermitidae, now thought to represent either the sister‐group of the Rhinotermitidae or a basal lineage nested within them. Contrary to previous assumptions, we show that the apterous immatures performing worker tasks in G. oculatus are the source of the single wing‐budded nymphal instar preceding the alate. Consequently, they qualify as pseudergates rather than true workers. However, the sex ratio is strongly male biased in pseudergates and soldiers, which is a trait usually restricted to termites with true workers. We therefore argue that pseudergates of G. oculatus are close to a point where the species could easily shift toward the differentiation of a true worker caste, and that G. oculatus pinpoints a new possible route for the evolution of true workers from pseudergates.

Sexual communication in the termite Prorhinotermes simplex (Isoptera, Rhinotermitidae) mediated by a pheromone from female tergal glands

We studied the post-flight behavior and sex attraction in imagoes of the termite Prorhinotermes simplex (Rhinotermitidae, Prorhinotermitinae). Pairing is mediated by the secretion from tergal glands, exposed by females in a calling posture and highly attractive to males. Analysis of extracts of these glands by means of gas chromatography with electroantennographic detection indicated a chromatographic area corresponding to an intense physiological response of males. The retention characteristics of this area proved to be identical with those of (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol. Electroantennographic and behavioral assays revealed that units of picograms of the compound represent a stimulus qualitatively and quantitatively equivalent to one female tergal gland. Thus, we hypothesize that (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol is a major component of the female sex pheromone in P. simplex.