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Maintaining water balance is a universal challenge for organisms living in terrestrial environments, especially for insects, which have essential roles in our ecosystem. Although the high surface area to volume ratio in insects makes them vulnerable to water loss, insects have evolved different levels of desiccation resistance to adapt to diverse environments. To withstand desiccation, insects use a lipid layer called cuticular hydrocarbons (CHCs) to reduce water evaporation from the body surface. It has long been hypothesized that the water-proofing capability of this CHC layer, which can confer different levels of desiccation resistance, depends on its chemical composition. However, it is unknown which CHC components are important contributors to desiccation resistance and how these components can determine differences in desiccation resistance. In this study, we used machine-learning algorithms, correlation analyses, and synthetic CHCs to investigate how different CHC components affect desiccation resistance in 50 Drosophila and related species. We showed that desiccation resistance differences across these species can be largely explained by variation in CHC composition. In particular, length variation in a subset of CHCs, the methyl-branched CHCs (mbCHCs), is a key determinant of desiccation resistance. There is also a significant correlation between the evolution of longer mbCHCs and higher desiccation resistance in these species. Given that CHCs are almost ubiquitous in insects, we suggest that evolutionary changes in insect CHC components can be a general mechanism for the evolution of desiccation resistance and adaptation to diverse and changing environments.

Oxytocin/vasopressin-like peptides are important regulators of physiology and social behavior in vertebrates. However, the function of inotocin, the homologous peptide in arthropods, remains largely unknown. Here, we show that the level of expression of inotocin and inotocin receptor are correlated with task allocation in the ant Camponotus fellah. Both genes are up-regulated when workers age and switch tasks from nursing to foraging. in situ hybridization revealed that inotocin receptor is specifically expressed in oenocytes, which are specialized cells synthesizing cuticular hydrocarbons which function as desiccation barriers in insects and for social recognition in ants. dsRNA injection targeting inotocin receptor, together with pharmacological treatments using three identified antagonists blocking inotocin signaling, revealed that inotocin signaling regulates the expression of cytochrome P450 4G1 (CYP4G1) and the synthesis of cuticular hydrocarbons, which play an important role in desiccation resistance once workers initiate foraging.

Sarcophaga peregrina (Robineau-Desvoidy, 1830), a synanthropic flesh fly species found in different parts of the world, is of medical and forensic importance. Traditional methods of inferring developmental age rely on the life stage of insects and morphological changes. However, once the larvae reach the pupal and adult stage, morphological changes would become barely visible, so that the classic method would be invalid. Here, we studied the cuticular hydrocarbon profile of S. peregrina of the whole life cycle from larval stage to adult stage by GC–MS. Sixty-three compounds with carbon chain length ranging from 8 to 36 were detected, which could be categorized into four classes: n-alkanes, branched alkanes, alkenes, and unknowns. As developmental increased, branched alkanes dominant, and the content of high-molecular-weight hydrocarbons is variable, especially for 2-methyl C19, DiMethyl C21, docosane (C22), and tricosane (C23). This study shows that the composition of CHC could be used to determine the developmental age of S. peregrina and aid in postmortem interval estimations in forensic science.

Blowflies (Diptera: Calliphoridae) are of great importance in forensic entomology and in determining the minimum post-mortem interval, as they may be the first group of insects to colonize decomposing remains. Reliable species identification is an essential prerequisite. Classically, morphological characters or DNA sequences are used for this purpose. However, depending on the species and the condition of the specimen, this can be difficult, e.g., in the case of empty fly puparia. Recent studies have shown that cuticular hydrocarbon (CHC) profiles are species-specific in necrophagous taxa and represent another promising tool for identification. However, the population-specific variability of these substances as a function of e.g., local climatic parameters has not yet been sufficiently investigated. The aim of this study was to determine the geographical variation of CHC profiles of the blowfly Calliphora vicina (Robineau-Desvoidy, 1830) depending on different countries of origin. Flies were reared in the United Kingdom, Germany, and Turkey in common garden experiments under ambient conditions. CHC profiles of the resulting adult flies and their empty puparia were analyzed using gas chromatography-mass spectrometry. Data were visualized by principal component analysis and clustered by population. The populations of the United Kingdom and Germany, both having similar climates and being geographically close to each other, showed greater similarities in CHC profiles. However, the CHC profile of the Turkish population, whose climate is significantly different from the other two populations, was very different. Our study confirms the high potential of CHC analysis in forensic entomology but highlights the need to investigate geographical variability in chemical profiles.

Cuticular hydrocarbons (CHCs) are diverse in insects, and include variable classes of cuticular lipids, contributing to waterproofing for insects under desiccation environments. However, this waterproofing function of CHCs is still not well characterized in aphids. In this study, we compared CHC profiles for desiccation-resistant and nonresistant genotypes of the grain aphid, Sitobion avenae (Fabricius), in responses to desiccation. Our result showed that a total of 27 CHCs were detected in S. avenae, and linear alkanes (e.g., n-C29) were found to be the predominant components. Long-chain monomethyl alkanes were found to associate closely with water loss rates in S. avenae in most cases. Resistant genotypes of both wing morphs had higher contents of short-chain n-alkanes under control than nonresistant genotypes, showing the importance of short-chain n-alkanes in constitutive desiccation resistance. Among these, n-C25 might provide a CHC signature to distinguish between desiccation-resistant and nonresistant individuals. Compared with linear alkanes, methyl-branched CHCs appeared to display higher plasticity in rapid responses to desiccation, especially for 2-MeC26, implying that methyl-branched CHCs could be more sensitive to desiccation, and play more important roles in induced desiccation-resistance. Thus, both constitutive and induced CHCs (linear or methyl-branched) can contribute to adaptive responses of S. avenae populations under desiccation environments. Our results provide substantial evidence for adaptive changes of desiccation resistance and associated CHCs in S. avenae, and have significant implications for aphid evolution and management in the context of global climate change.

Chrysomya albiceps (Wiedemann 1819) is one of the most important insects in forensic entomology. Its larval developmental and survival rates are influenced by nutritional resources, temperature, humidity, and geographical regions. The present study investigated the possibility of relying on body size and cuticular hydrocarbon composition as indicators for age estimation of the different larval instars of C. albiceps. Larvae were maintained in standardized laboratory conditions at different experimental temperatures. All larval instars (first, second, and third) were randomly collected for measuring their body sizes and for estimating their cuticular hydrocarbons at different rearing temperatures (30, 35, 40, and 45°C) using gas chromatography–mass spectrometry (GC–MS). Results indicated that the duration of larval stage was temperature dependent as it gradually decreased on increasing the rearing temperature (30, 35, and 40°C) except 45°C at which larval development was ceased. In contrary, larval body size, in terms of length, width, and weight, was temperature dependent as it gradually increased with larval development on increasing rearing temperature except at 45°C at which larval development was ceased. The GC–MS showed a significant difference in the extracted components of cuticular hydrocarbons between different larval instars reared in the same temperature and between the same larval instar that reared at different temperatures. Furthermore, the highest and lowest amounts of cuticular hydrocarbons were detected at 35 and 40°C, respectively. Overall, larval body size and cuticular hydrocarbon components were temperature dependent within the range 30–40°C, which may suggest them as possible reliable age indicators for estimating the postmortem interval in the field of medicolegal entomology.

Group-living individuals experience immense risk of disease transmission and parasite infection. In social and in some non-social insects, disease control with immunomodulation arises not only via individual immune defenses, but also via infochemicals such as contact cues and (defensive) volatiles to mount a group-level immunity. However, little is known about whether activation of the immune system elicits changes in chemical phenotypes, which may mediate these responses. We here asked whether individual immune experience resulting from wounding or injection of heat-killed Bacillus thuringiensis (priming) leads to changes in the chemical profiles of female and male adult red flour beetles, Tribolium castaneum, which are non-social but gregarious. We analyzed insect extracts using GC-FID to study the chemical composition of (1) cuticular hydrocarbons (CHCs) as candidates for the transfer of immunity-related information between individuals via contact, and (2) stink gland secretions, with analysis of benzoquinones as main active compounds regulating ‘external immunity’. Despite a pronounced sexual dimorphism in CHC profiles, wounding stimulation led to similar profile changes in males and females with increases in the proportion of methyl-branched alkanes compared to naïve beetles. While changes in the overall secretion profiles were less pronounced, absolute amounts of benzoquinones were transiently elevated in wounded compared to naïve females. Responses to priming were insignificant in CHCs and secretions. We suggest that changes in different infochemicals after wounding may mediate immune status signaling in the context of both internal and external immune responses in groups of this non-social insect, thus showing parallels to social immunity.

Nasonia is a species complex of four parasitoid wasps. N. vitripennis is cosmopolitan, while the other three species are micro-sympatric with it. This distribution can select distinct species-specific mate recognition capabilities. However, whether Nasonia males can distinguish between hosts with conspecific females and those with heterospecific females is not known. Therefore, we test this hypothesis in a cafeteria-based choice assay and show that N. vitripennis males can distinguish hosts with conspecific wasps against those parasitized by N. giraulti and N. oneida, exhibiting longer search time and distance traversed with faster search speed. We also found that N. longicornis males can distinguish hosts with conspecific wasps, but only against the hosts parasitized by N. oneida. We further investigated the pairwise differences in the cuticular hydrocarbon (CHC) profiles of the parasitized hosts and adult female wasps. The results reveal that males show this ability only when the compounds responsible for differences in adult female CHC profiles are also the key differentiators of the host CHC profiles. The comparative mate searching behaviour of males of all reported species within a genus has rarely been studied. Therefore, this study makes a significant contribution to our understanding of interspecific variation of conspecific-mate searching behaviour.

Long-chain cuticular hydrocarbons (CHC) are key components of chemical communication in many insects. The parasitoid jewel wasps from the genus Nasonia use their CHC profile as sex pheromone and for species recognition. The standard analytical tool to analyze CHC is gas chromatography coupled with mass spectrometric detection (GC/MS). This method reliably identifies short- to long-chain alkanes and alkenes, but CHC with more than 40 carbon atoms are usually not detected. Here, we applied two laser mass spectrometry (MS) techniques, namely direct laser desorption/ionization (d)LDI and silver-assisted (Ag-)LDI MS, respectively, to analyze CHC profiles of N. vitripennis, N. giraulti, and N. longicornis directly from the cuticle or extracts. Furthermore, we applied direct analysis in real-time (DART) MS as another orthogonal technique for extracts. The three methods corroborated previous results based on GC/MS, i.e., the production of CHC with carbon numbers between C25 and C40. However, we discovered a novel series of long-chain CHC ranging from C41 to C51/C52. Additionally, several previously unreported singly and doubly unsaturated alkenes in the C31-C39 range were found. Use of principal component analysis (PCA) revealed that the composition of the newly discovered CHC varies significantly between species, sex, and age of the animals. Our study adds to the growing literature on the presence of very long-chain CHC in insects and hints at putative roles in insect communication.

Social organisation of eusocial insects requires efficient communication among conspecifics, involving various signals. Among them, Cuticular Hydrocarbons Compounds are used like chemical signals for recognition processes. These semiochemical compounds, which can vary qualitatively and quantitatively, form an individual chemical signature carrying identity of each congeners which contribute to the social cohesion of the colony members. In this study, we analysed the chemical signature of workers of the eusocial and invasive Vespidae species, the Yellow-legged hornet, Vespa velutina nigrithorax. The chemical communication system between hornets’ workers is relatively unknown and their social organisation poorly documented. However, a strong chemical heterogeneity between castes and colonies have been previously identified in the Yellow-legged hornet, suggesting a possible chemical diversity between workers. Our results showed a strong chemical heterogeneity mainly explained by their colonial origin, as previously described, but also by their behaviour at a given time. In this study, four behaviours have been reported in the field and could be assigned to a workers’ sub-caste: animal foragers, builders, defenders and material foragers. A chemical separation of individuals into two groups have been observed, where animal foragers exhibit a clear separation of their chemical profiles compared to their counterparts. Also, animal foragers had more alkenes and fewer branched alkanes than the other workers. This exploratory study demonstrates that workers of this invasive hornet species present different cuticular profiles, probably used in both inter and intra-specific recognition phenomena. This is therefore a first step towards understanding the chemical communication involved in the social organisation of hornet workers.