Lipases and carboxylesterases affect moth sex pheromone compounds involved in interspecific mate recognition.

Moth sex pheromones are a classical model for studying sexual selection. Females typically produce a species-specific pheromone blend that attracts males. Revealing the enzymes involved in the interspecific variation in blend composition is key for understanding the evolution of these sexual communication systems. The nature of the enzymes involved in the variation of acetate esters, which are prominent compounds in moth pheromone blends, remains unclear. We identify enzymes involved in acetate degradation using two closely related moth species: Heliothis (Chloridea) subflexa and H. (C.) virescens, which have different quantities of acetate esters in their sex pheromone. Through comparative transcriptomic analyses and CRISPR/Cas9 knockouts, we show that two lipases and two esterases from H. virescens reduce the levels of pheromone acetate esters when expressed in H. subflexa females. Together, our results show that lipases and carboxylesterases are involved in tuning Lepidoptera pheromones composition.

Sex pheromone signal and stability covary with fitness.

If sexual signals are costly, covariance between signal expression and fitness is expected. Signal-fitness covariance is important, because it can contribute to the maintenance of genetic variation in signals that are under natural or sexual selection. Chemical signals, such as female sex pheromones in moths, have traditionally been assumed to be species-recognition signals, but their relationship with fitness is unclear. Here, we test whether chemical, conspecific mate finding signals covary with fitness in the moth Heliothis subflexa. Additionally, as moth signals are synthesized de novo every night, the maintenance of the signal can be costly. Therefore, we also hypothesized that fitness covaries with signal stability (i.e. lack of temporal intra-individual variation). We measured among- and within-individual variation in pheromone characteristics as well as fecundity, fertility and lifespan in two independent groups that differed in the time in between two pheromone samples. In both groups, we found fitness to be correlated with pheromone amount, composition and stability, supporting both our hypotheses. This study is, to our knowledge, the first to report a correlation between fitness and sex pheromone composition in moths, supporting evidence of condition-dependence and highlighting how signal-fitness covariance may contribute to heritable variation in chemical signals both among and within individuals.

DNA methylation patterns in the tobacco budworm, Chloridea virescens.

DNA methylation is an important epigenetic modification that is prone to stochastic variation and is responsive to environmental factors. Yet changes in DNA methylation could persist across generations and thus play an important role in evolution. In this study, we used methylation-sensitive amplified fragment length polymorphisms (MS-AFLP) to evaluate whether DNA methylation could contribute to the evolution of the sexual communication signal in the noctuid moth Chloridea virescens. We found that most DNA methylation was consistent across tissues, although some methylation sites were specifically found in pheromone glands. We also found significant DNA methylation differences among families and two pheromone phenotype selection lines, and these differences correlated with genetic variation. Most DNA methylation patterns were inherited, although some sites were subject to spontaneous de novo DNA methylation across generations. Thus, DNA methylation likely plays a role in a wide range of processes in moths. Together, our results present an important initial step towards understanding the potential role of DNA methylation in the evolution of sexual communication signals in moths.

Proximity of signallers can maintain sexual signal variation under stabilizing selection.

How sexual communication systems can evolve under stabilizing selection is still a paradox in evolutionary biology. In moths, females emit a species-specific sex pheromone, consisting of a blend of biochemically related components, to which males are attracted. Although males appear to exert strong stabilizing selection on female pheromone, these blends seem to have evolved rapidly, as evidenced by ~120,000 moth species. Here we propose and test a "proximity model" wherein two females that vary in their relative attractiveness to males, can both benefit from calling in close proximity to each other. In a field study, we show that (1) artificially selected unattractive females can achieve mating rates comparable to attractive females if they signal in close proximity to attractive females, and (2) attractive females benefit from higher mating rates when signalling in close proximity to unattractive females. We propose that frequency-dependent behavioural and spatial interactions can sustain signal variation within populations even when these signals are under stabilizing selection.

Disposable Polydimethylsiloxane (PDMS)-Coated Fused Silica Optical Fibers for Sampling Pheromones of Moths.

In the past decades, the sex pheromone composition in female moths has been analyzed by different methods, ranging from volatile collections to gland extractions, which all have some disadvantage: volatile collections can generally only be conducted on (small) groups of females to detect the minor pheromone compounds, whereas gland extractions are destructive. Direct-contact SPME overcomes some of these disadvantages, but is expensive, the SPME fiber coating can be damaged due to repeated usage, and samples need to be analyzed relatively quickly after sampling. In this study, we assessed the suitability of cheap and disposable fused silica optical fibers coated with 100 µm polydimethylsiloxane (PDMS) by sampling the pheromone of two noctuid moths, Heliothis virescens and Heliothis subflexa. By rubbing the disposable PDMS fibers over the pheromone glands of females that had called for at least 15 minutes and subsequently extracting the PDMS fibers in hexane, we collected all known pheromone compounds, and we found a strong positive correlation for most pheromone compounds between the disposable PDMS fiber rubs and the corresponding gland extracts of the same females. When comparing this method to volatile collections and the corresponding gland extracts, we generally found comparable percentages between the three techniques, with some differences that likely stem from the chemical properties of the individual pheromone compounds. Hexane extraction of cheap, disposable, PDMS coated fused silica optical fibers allows for sampling large quantities of individual females in a short time, eliminates the need for immediate sample analysis, and enables to use the same sample for multiple chemical analyses.

Using DNA-barcoding to make the necrobiont beetle family Cholevidae accessible for forensic entomology.

The beetle family Cholevidae (Coleoptera: Staphylinoidea), sometimes viewed as the subfamily Cholevinae of the Leiodidae, consists of some 1700 species worldwide. With the exception of specialized cave-dwelling species and species living in bird and mammal nests and burrows, the species are generalized soil-dwellers that, at least in temperate regions, are mostly found on vertebrate cadavers. Although they have been regularly reported from human corpses, and offer potential because of many species' peak activity in the cold season, they have not been a focus of forensic entomologists so far. This is probably due to their small size and the difficulty in identifying the adults and their larvae. In this paper, we show that DNA-barcoding can help make this group of necrobiont beetles available as a tool for forensic research. We collected 86 specimens of 20 species of the genera Catops, Fissocatops, Apocatops, Choleva, Nargus, Ptomaphagus, and Sciodrepoides from the Netherlands and France and show that a broad "barcoding gap" allows almost all species to be easily and unambiguously identified by the sequence of the "barcoding gene" cytochrome c oxidase I (COI). This opens up the possibility of adding Cholevidae to the set of insect taxa routinely used in forensic entomology.