Jitka Štáfková

Queens of eusocial insects maintain their reproductive monopoly through queen pheromones. We recently identified the sesquiterpene alcohol (3R,6E)-nerolidol (RNERO) as the queen pheromone in the termite Embiratermes neotenicus and characterized a terpene synthase, EneoC, that produces RNERO in vitro. Here, we aimed to (i) determine whether EneoC is responsible for RNERO biosynthesis in vivo, (ii) analyze its expression and protein abundance across castes in the context of terpenoid biosynthesis and endocrine regulation, (iii) estimate the physiological costs of RNERO production based on investment in EneoC, (iv) assess whether eggs actively produce RNERO, and (v) localize RNERO biosynthesis in queens.We report a pronounced queen-specific upregulation of EneoC at transcript and protein levels. Using RNA interference, we demonstrate that EneoC produces RNERO in vivo and show that it is among the most abundant proteins in queens. EneoC is exclusively expressed in queen epidermis all over the body surface, specifically within structures derived from mitochondria in class 1 secretory cells. We identify an enrichment of additional terpenoid biosynthesis enzymes in queens, including the farnesyl diphosphate synthase EneoB, expected to supply substrate for EneoC. Both enzymes exhibit similar protein abundance patterns and contain mitochondrial targeting sequences, suggesting their co-localization within modified mitochondria in class 1 cells. Absence of EneoC in eggs indicates that eggs acquire RNERO from queens rather than via endogenous biosynthesis. Taken together, our findings establish EneoC as a key enzyme underlying the reproductive dominance of E. neotenicus queens, which entails substantial physiological costs, consistent with predictions for honest fertility signaling.

Termites produce the most diverse array of terpenoids among terrestrial metazoans, comprising over 200 structures. However, their biosynthesis has not yet been elucidated. Here, we identify a gene family which arose through a series of duplications of geranylgeranyl pyrophosphate synthase in the common ancestor of Neoisoptera, the terpene-producing termite lineage. We functionally characterized several proteins from this rich GGPPS-like family as terpene synthases generating biologically relevant sesqui- and diterpenes. These include the queen pheromone (3 R ,6 E )-nerolidol in Embiratermes neotenicus and the presumed precursor of polycyclic defensive diterpenes ( E , E , E )-neocembrene in Nasutitermes takasagoensis . We report significant enrichment of transposable elements in the GGPPS-like genomic loci, study the selection pressures acting in the evolution of the GGPPS-like family, and highlight an amino acid site crucial for cyclization capacity and enantiospecificity of the characterized enzymes. We conclude that we have identified an enzyme family that facilitated the emergence of the remarkable richness of termite terpenoids.

Termites produce the most diverse array of terpenoids among metazoans, comprising over 200 structures. However, their biosynthesis has not yet been elucidated. Here, we identify a gene family which arose through the duplication of geranylgeranyl pyrophosphate synthase in the common ancestor of Neoisoptera, the terpene-producing termite lineage. We functionally characterized several proteins from this family as terpene synthases generating biologically relevant sesqui-and diterpenes. These include the queen pheromone (3 R ,6 E )-nerolidol in Embiratermes neotenicus and the precursor of polycyclic defensive diterpenes ( E , E , E )-neocembrene in Nasutitermes takasagoensis . We explore transposable element-mediated genomic mechanisms and selection pressures acting in the evolution of this gene family and report an amino acid site crucial for cyclization capacity as well as the enantiospecificity of the characterized enzymes. We conclude that we have identified an enzyme family underlying the remarkable richness of termite terpenoids, which likely contributed to the ecological success of Neoisoptera.

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.

Malaria-causing Plasmodium parasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of two Anopheles species and a Plasmodium berghei fluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and Indian Anopheles species and propose that the mosquito complement-like system contributes to the species-specific dynamics of Plasmodium invasion. The traversal of the mosquito midgut cells is one of the critical stages in the life cycle of malaria parasites. Motile parasite forms, called ookinetes, traverse the midgut epithelium in a dynamic process which is not fully understood. Here, we harnessed transgenic reporters to track invasion of Plasmodium parasites in African and Indian mosquito species. We found important differences in parasite dynamics between the two Anopheles species and demonstrated a role of the mosquito complement-like system in regulation of parasite invasion of the midgut cells.

Malaria-causingPlasmodiumparasites traverse the mosquito midgut cells to establish infection at the basal side of the midgut. This dynamic process is a determinant of mosquito vector competence, yet the kinetics of the parasite migration is not well understood. Here we used transgenic mosquitoes of twoAnophelesspecies and aPlasmodium bergheifluorescence reporter line to track parasite passage through the mosquito tissues at high spatial resolution. We provide new quantitative insight into malaria parasite invasion in African and IndianAnophelesspecies and demonstrate that species-specific kinetics ofPlasmodiuminvasion is shaped by the mosquito complement-like system.Author Summary: The traversal of the mosquito midgut cells is one of the critical stages in the life cycle of malaria parasites. Motile parasite forms, called ookinetes, traverse the midgut epithelium in a dynamic process which is not fully understood.Here, we harnessed transgenic reporters to track invasion ofPlasmodiumparasites in African and Indian mosquito species. We found important differences in parasite dynamics between the two anopheline species and demonstrated an unexpected role of mosquito complement-like system in regulation of parasite invasion.

Pyruvate is a key product of glycolysis that regulates the energy metabolism of cells. In Trypanosoma brucei, the causative agent of sleeping sickness, the fate of pyruvate varies dramatically during the parasite life cycle. In bloodstream forms, pyruvate is mainly excreted, whereas in tsetse fly forms, pyruvate is metabolized in mitochondria yielding additional ATP molecules. The character of the molecular machinery that mediates pyruvate transport across mitochondrial membrane was elusive until the recent discovery of mitochondrial pyruvate carrier (MPC) in yeast and mammals. Here, we characterized pyruvate import into mitochondrion of Trypanosoma brucei. We identified mpc1 and mpc2 homologs in the T. brucei genome with attributes of MPC protein family and we demonstrated that both proteins are present in the mitochondrial membrane of the parasite. Investigations of mpc1 or mpc2 gene knock-out cells proved that T. brucei MPC1/2 proteins facilitate mitochondrial pyruvate transport. Interestingly, MPC is expressed not only in procyclic trypanosomes with fully activated mitochondria but also in bloodstream trypanosomes in which most of pyruvate is excreted. Moreover, MPC appears to be essential for bloodstream forms, supporting the recently emerging picture that the functions of mitochondria in bloodstream forms are more diverse than it was originally thought.