Synthesis and antiproliferative properties of new hydrophilic esters of triterpenic acids.

To improve the properties of cytotoxic triterpenoid acids 1-5, a large set of hydrophilic esters was synthesized. We choose betulinic acid (1), dihydrobetulinic acid (2), 21-oxoacid 3 along with highly active des-E lupane acids 4 and 5 as a model set of compounds for esterification of which the properties needed to be improved. As ester moieties were used - methoxyethanol and 2-(2-methoxyethoxy)ethanol and glycolic unit (type a-d), pyrrolidinoethanol, piperidinoethanol and morpholinoethanol (type f-h), and monosaccharide groups (type i-l). As a result, 56 triterpenic esters (49 new compounds) were obtained and their cytotoxicity on four cancer cell lines and normal human fibroblasts was tested. All new compounds were fully soluble at all tested concentrations, which used to be a problem of the parent compounds 1 and 2. 16 compounds had IC50 < 10 µM on at least one cancer cell line, 12 compounds had cytotoxicity of <10 µM against at least three of four tested cancer cell lines. The highest activity was found for compound 3c (1.8 µM on MCF7, 2.8 µM on HeLa, and 1.6 µM on G-361 cells) which also had no toxicity on non-cancerous BJ fibroblasts at the highest tested concentration (50 µM). High selective cytotoxicity was also found in compounds 1k, 2k, 3c, and 3i that are ideal candidates for drug development. Therefore, more studies to identify the mechanism of action were performed in case of 1k, 3c, and 3g such as effects on cell cycle and apoptosis. It was found that compounds 3c and 3g can induce apoptosis via caspase-3 activation and modulation of protein Bcl-2 in G-361 cells. In conclusion, compounds 1k, 3c, and 3g show high and selective cytotoxicity, therefore they are significantly better candidates for anti-cancer drug development than the parent acids 1-5.

Molecular Mechanism of the Two-Component Suicidal Weapon of Neocapritermes taracua Old Workers.

In termites, as in many social insects, some individuals specialize in colony defense, developing diverse weaponry. As workers of the termite Neocapritermes taracua (Termitidae: Termitinae) age, their efficiency to perform general tasks decreases, while they accumulate defensive secretions and increase their readiness to fight. This defensive mechanism involves self-sacrifice through body rupture during which an enzyme, stored as blue crystals in dorsal pouches, converts precursors produced by the labial glands into highly toxic compounds. Here, we identify both components of this activated defense system and describe the molecular basis responsible for the toxicity of N. taracua worker autothysis. The blue crystals are formed almost exclusively by a specific protein named BP76. By matching N. taracua transcriptome databases with amino acid sequences, we identified BP76 to be a laccase. Following autothysis, the series of hydroquinone precursors produced by labial glands get mixed with BP76, resulting in the conversion of relatively harmless hydroquinones into toxic benzoquinone analogues. Neocapritermes taracua workers therefore rely on a two-component activated defense system, consisting of two separately stored secretions that can react only after suicidal body rupture, which produces a sticky and toxic cocktail harmful to opponents.

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.

Mutual use of trail-following chemical cues by a termite host and its inquiline.

Termite nests are often secondarily inhabited by other termite species ( = inquilines) that cohabit with the host. To understand this association, we studied the trail-following behaviour in two Neotropical species, Constrictotermes cyphergaster (Termitidae: Nasutitermitinae) and its obligatory inquiline, Inquilinitermes microcerus (Termitidae: Termitinae). Using behavioural experiments and chemical analyses, we determined that the trail-following pheromone of C. cyphergaster is made of neocembrene and (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol. Although no specific compound was identified in I. microcerus, workers were able to follow the above compounds in behavioural bioassays. Interestingly, in choice tests, C. cyphergaster prefers conspecific over heterospecific trails while I. microcerus shows the converse behaviour. In no-choice tests with whole body extracts, C. cyphergaster showed no preference for, while I. microcerus clearly avoided heterospecific trails. This seems to agree with the hypothesis that trail-following pheromones may shape the cohabitation of C. cyphergaster and I. microcerus and reinforce the idea that their cohabitation is based on conflict-avoiding strategies.

Delineating species boundaries using an iterative taxonomic approach: the case of soldierless termites (Isoptera, Termitidae, Apicotermitinae).

Species boundaries are traditionally inferred using morphological characters, although morphology sometimes fails to correctly delineate species. To overcome this limitation, researchers have widely taken advantage of alternative methods such as DNA barcoding or analysis of cuticular hydrocarbons (CHs) profiles, but rarely use them simultaneously in an iterative taxonomic approach. Here, we follow such an approach using morphology, DNA barcoding and CHs profiles to precisely discriminate species of soldierless termites, a diversified clade constituting about one-third of the Neotropical termite species richness, but poorly resolved taxonomically due to the paucity of useful characters. We sampled soldierless termites in various forest types of the Nouragues Nature Reserve, French Guiana. Our results show that morphological species determination generally matches DNA barcoding, which only suggests the existence of three cryptic species in the 31 morphological species. Among them, Longustitermes manni is the only species whose splitting is corroborated by ecological data, other widely distributed species being supported by DNA barcoding. On the contrary, although CHs profiles provide a certain taxonomic signal, they often suggest inconsistent groupings which are not supported by other methods. Overall, our data support DNA barcoding and morphology as two efficient methods to distinguish soldierless termite species.

Comparative study of the labial gland secretion in termites (Isoptera).

Labial glands are present in all castes and developmental stages of all termite species. In workers, their secretion contains a food-marking pheromone and digestive enzymes, while soldier secretion plays a defensive role. However, these functions were studied only in a limited set of species, and do not allow drawing general conclusions. Hence, we have investigated the chemical composition of the labial gland extracts from soldiers and workers in 15 termite species belonging to 6 families using an integrative approach based on proteomic and small-molecule profiling. We confirmed the presence of hydroquinone and cellulase in the labial glands of workers, and we identified new toxic compounds in soldiers and workers of several species. Our results highlight the dual role of labial gland secretion, i.e. the defensive role in soldiers and workers of several termite species, and the digestive function in workers.

Chemistry and anatomy of the frontal gland in soldiers of the sand termite Psammotermes hybostoma.

A great diversity of defensive chemicals has been described in termite soldiers equipped with a unique defensive organ, the frontal gland. Along with the functional diversity of these compounds, reflecting the evolutionary history of particular lineages and their defensive strategies, a considerable degree of chemical variability often occurs among species and populations. Thus, the chemistry of termite defense may provide information on the phylogeny and geographic dispersal of species and populations. In this paper, we report on the anatomy of the frontal gland and on the diversity of soldier defensive chemicals in the sand termite, Psammotermes hybostoma, from nine colonies and five different localities in Egypt. Using gas chromatography-mass spectrometry, a total of 30 sesquiterpene hydrocarbons, or their oxygenated derivatives, were detected, and the chemical identity of most of them identified. In addition, a ketone, an ester, and a diterpene were identified in some colonies. Within colonies, the chemical composition was stable and did not differ among soldier size categories. However, there were pronounced quantitative and qualitative differences in frontal gland chemicals among colonies and geographic locations. The findings are discussed in a broader comparison with other termite taxa.

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.

Chemical communication in termites: syn-4,6-dimethylundecan-1-ol as trail-following pheromone, syn-4,6-dimethylundecanal and (5E)-2,6,10-trimethylundeca-5,9-dienal as the respective male and female sex pheromones in Hodotermopsis sjoestedti (Isoptera, Archotermopsidae).

The trail-following pheromone and sex pheromones were investigated in the Indomalayan termite Hodotermopsis sjoestedti belonging to the new family Archotermopsidae. Gas chromatography coupled to mass spectrometry (GC-MS) after solid phase microextraction (SPME) of the sternal gland secretion of pseudergates and trail-following bioassays demonstrated that the trail-following pheromone of H. sjoestedti was syn-4,6-dimethylundecan-1-ol, a new chemical structure for termite pheromones. GC-MS after SPME of the sternal gland secretion of alates also allowed the identification of sex-specific compounds. In female alates, the major sex-specific compound was identified as (5E)-2,6,10-trimethylundeca-5,9-dienal, a compound previously identified as the female sex pheromone of the termite Zootermopsis nevadensis. In male alates, the major sex-specific compound was identified as syn-4,6-dimethylundecanal, a homolog of syn-4,6-dimethyldodecanal, which has previously been confirmed as the male sex pheromone of Z. nevadensis. The presence of sex-specific compounds in alates of H. sjoestedti strongly suggests for this termite the presence of sex-specific pairing pheromones which were only known until now in Z. nevadensis. Our results showed therefore a close chemical relationship between the pheromones of the taxa Hodotermopsis and Zootermopsis and, in contrast, a clear difference with the taxa Stolotermes and Porotermes, which is in total agreement with the recent creation of the families Archotermopsidae and Stolotermitidae as a substitute for the former family Termopsidae.

Sex pheromone and trail pheromone of the sand termite Psammotermes hybostoma.

Within the complex network of chemical signals used by termites, trail pheromones and sex pheromones are among the best known. Numerous recent papers map the chemical identity and glandular origin of these pheromones in nearly all major isopteran taxa. In this study, we aimed to describe the sex pheromone and the trail pheromone of a poorly known sand termite, Psammotermes hybostoma. We identified (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol (dodecatrienol) as the sex pheromone released by tergal and sternal glands of female imagos and, at the same time, as the trail pheromone secreted from the sternal gland of workers. We conclude that chemical communication in Psammotermes does not differ from that of most other Rhinotermitidae, such as Reticulitermes, despite the presence of a diterpene as a major component of the trail pheromone of Prorhinotermes to which Psammotermes is presumed to be phylogenetically close. Our findings underline once again the conservative nature of chemical communication in termites, with dodecatrienol being a frequent component of pheromonal signals in trail following and sex attraction and, at the same time, a tight evolutionary relationship between the trail following of working castes and the sex attraction of imagos.