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Streptococcus thermophilus holds promise as a chassis for producing and secreting heterologous proteins. Used for thousands of years to ferment milk, this species has generally recognized as safe (GRAS) status in the USA and qualified presumption of safety (QPS) status in Europe. In addition, it can be easily genetically modified thanks to its natural competence, and it secretes very few endogenous proteins, which means less downstream processing is needed to purify target proteins, reducing costs. Extracellular degradation of heterologous proteins can be eliminated by introducing mutations that inactivate the genes encoding the bacterium's three major surface proteases. Here, we constructed an inducible expression system that utilizes a peptide pheromone (SHP 1358 ) and a transcriptional regulator (Rgg 1358 ) involved in quorum-sensing regulation. We explored the functionality of a complete version of the system, in which the inducer is produced by the bacterium itself, by synthesizing a luciferase reporter protein. This complete version was assessed with bacteria grown in a chemically defined medium but also in vivo, in the faeces of germ-free mice. We also tested an incomplete version, in which the inducer had to be added to the culture medium, by synthesizing luciferase and a secreted form of elafin, a human protein with therapeutic properties. Our results show that, in our system, protein production can be modulated by employing different concentrations of the SHP 1358 inducer or other SHPs with closed amino acid sequences. We also constructed a genetic background in which all system leakiness was eliminated. In conclusion, with this new inducible expression system, we have added to the set of tools currently used to produce secreted proteins in S. thermophilus , whose myriad applications include the delivery of therapeutic peptides or proteins.

Vomeronasal receptors (VRs) play a crucial role in recognizing pheromones, which are essential for social chemical communication. The male muskrat (Ondatra zibethicus) secretes musk, which contains pheromones as a reproductive signal, and the female can recognize it through the VNO to mediate social communication behavior. This study aimed to identify the genomic information of VRs (OzVRs) in the female muskrat and elucidate their physicochemical properties and evolutionary relationship. Six predominantly expressed OzVR genes were identified using the RACE technique, and a comprehensive analysis was conducted on their gene structure, subcellular distribution, functional predictions, and mRNA levels, revealed that all OzVRs were transmembrane proteins. Phylogenetic analysis clustered OzVR genes into two clades (V1Rs: OzV1R21, OzV1R81, OzV1R105; V2Rs: OzV2R33, OzV2R44, OzV2R60). Physiochemically, OzV1Rs were basic proteins, while OzV2Rs exhibited weakly acidic character. Among them, OzV1R81 and OzV2R44 were identified as hydrophobicitystable proteins, with the remainder categorized as hydrophobicity-unstable proteins. Promoters analysis revealed the involvement of transcription factors and complexes, including Ahr::Arnt, Runx1, Arnt, and TFAP2A, in regulating the expression of the OzVR genes. Conserved domain and motif analyses demonstrated a high conservation of the VRs superfamily in rodents, with many conserved domains linked to pheromone binding. Functional predictions confirmed that OzVRs were associated with pheromones detection. Finally, the expression patterns of OzVR genes in different tissues and seasons indicated that OzVRs have the highest level of expression in the vomeronasal organ, and OzV1Rs notably higher in the breeding season than that in the non-breeding season, however the expression levels of OzV2Rs were higher in the non-breeding season. This study provided insights into the phylogenetic relationships, gene structure, physicochemical properties, promoter binding sites, functions and gene expression patterns of OzVRs, offering a theoretical reference for further examination of VR gene functions and a foundation for understanding chemical signaling mechanisms in the muskrat., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Many animals exchange chemicals during courtship and mating. In some amphibians, sexual chemical communication is mediated by pheromones produced in male breeding glands that are transferred to the female's nostrils during mating. This has been mostly studied in salamanders, despite frogs having similar glands and courtship behaviours suggestive of chemical communication. In Neotropical poison frogs (Dendrobatidae and Aromobatidae), males of many species develop breeding glands in their fingers, causing certain fingers to visibly swell. Many also engage in cephalic amplexus, whereby the male's swollen fingers are placed in close contact with the female's nares during courtship. Here, we investigate the possible roles of swollen fingers in pheromone production using whole-transcriptome sequencing (RNAseq). We examined differential gene expression in the swollen versus non-swollen fingers and toes of two dendrobatid species, Leucostethus brachistriatus and Epipedobates anthonyi, both of which have specialised mucous glands in finger IV, the latter of which has cephalic amplexus. The overwhelming pattern of gene expression in both species was strong upregulation of sodefrin precursor-like factors (SPFs) in swollen fingers, a well-known pheromone system in salamanders. The differentially expressed SPF transcripts in each species were very high (>40), suggesting a high abundance of putative protein pheromones in both species. Overall, the high expression of SPFs in the swollen fingers in both species, combined with cephalic amplexus, supports the hypothesis that these traits, widespread across members of the subfamilies Colostethinae and Hyloxalinae (ca. 141 species), are involved in chemical signalling during courtship., (© 2024 The Author(s). Molecular Ecology published by John Wiley & Sons Ltd.)

Egg activation, representing the critical oocyte-to-embryo transition, provokes meiosis completion, modification of the vitelline membrane to prevent polyspermy, and translation of maternally provided mRNAs. This transition is triggered by a calcium signal induced by spermatozoon fertilization in most animal species, but not in insects. In Drosophila melanogaster, mature oocytes remain arrested at metaphase-I of meiosis and the calcium-dependent activation occurs while the oocyte moves through the genital tract. Here, we discovered that the oenocytes of fruitfly females are required for egg activation. Oenocytes, cells specialized in lipid-metabolism, are located beneath the abdominal cuticle. In adult flies, they synthesize the fatty acids (FAs) that are the precursors of cuticular hydrocarbons (CHCs), including pheromones. The oenocyte-targeted knockdown of a set of FA-anabolic enzymes, involved in very-long-chain fatty acid (VLCFA) synthesis, leads to a defect in egg activation. Given that some but not all of the identified enzymes are required for CHC/pheromone biogenesis, this putative VLCFA-dependent remote control may rely on an as-yet unidentified CHC or may function in parallel to CHC biogenesis. Additionally, we discovered that the most posterior ventral oenocyte cluster is in close proximity to the uterus. Since oocytes dissected from females deficient in this FA-anabolic pathway can be activated in vitro, this regulatory loop likely operates upstream of the calcium trigger. To our knowledge, our findings provide the first evidence that a physiological extra-genital signal remotely controls egg activation. Moreover, our study highlights a potential metabolic link between pheromone-mediated partner recognition and egg activation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Poidevin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Meiosis is essential for generating genetic diversity and sexual spores, but the regulation of meiosis and ascosporogenesis is not clear in filamentous fungi, in which dikaryotic and diploid cells formed inside fruiting bodies are not free living and independent of pheromones or pheromone receptors. In this study, Gia1, a non-pheromone GPCR (G protein-coupled receptor) with sexual-specific expression in Fusarium graminearum , is found to be essential for ascosporogenesis. The gia1 mutant was normal in perithecium development, crozier formation, and karyogamy but failed to undergo meiosis, which could be partially rescued by a dominant active mutation in GPA1 and activation of the Gpmk1 pathway. GIA1 orthologs have conserved functions in regulating meiosis and ascosporogenesis in Sordariomycetes. GIA1 has a paralog, GIP1 , in F. graminearum and other Hypocreales species which is essential for perithecium formation. GIP1 differed from GIA1 in expression profiles and downstream signaling during sexual reproduction. Whereas the C-terminal tail and IR3 were important for intracellular signaling, the N-terminal region and EL3 of Gia1 were responsible for recognizing its ligand, which is likely a protein enriched in developing perithecia, particularly in the gia1 mutant. Taken together, these results showed that GIA1 encodes a non-pheromone GPCR that regulates the entry into meiosis and ascosporogenesis via the downstream Gpmk1 MAP kinase pathway in F. graminearum and other filamentous ascomycetes.

Colletotrichum fungi are a group of damaging phytopathogens with atypical mating type loci (harboring only MAT1-2-1 but not MAT1-1-1 ) and complex sexual behaviors. Sex pheromones and their cognate G-protein-coupled receptors are conserved regulators of fungal mating. These genes, however, lose function frequently among Colletotrichum species, indicating a possibility that pheromone signaling is dispensable for Colletotrichum sexual reproduction. We have identified two putative pheromone-receptor pairs (PPG1:PRE2, PPG2:PRE1) in C. fructicola , a species that exhibits plus-to-minus mating type switching and plus-minus-mediated mating line development. Here, we report the generation and characterization of gene-deletion mutants for all four genes in both plus and minus strain backgrounds. Single-gene deletion of pre1 or pre2 had no effect on sexual development, whereas their double deletion caused self-sterility in both the plus and minus strains. Moreover, double deletion of pre1 and pre2 caused female sterility in plus-minus outcrossing. Double deletion of pre1 and pre2 , however, did not inhibit perithecial differentiation or plus-minus-mediated enhancement of perithecial differentiation. Contrary to the results with pre1 and pre2 , double deletion of ppg1 and ppg2 had no effect on sexual compatibility, development, or fecundity. We concluded that pre1 and pre2 coordinately regulate C. fructicola mating by recognizing novel signal molecule(s) distinct from canonical Ascomycota pheromones. The contrasting importance between pheromone receptors and their cognate pheromones highlights the complicated nature of sex regulation in Colletotrichum fungi., Competing Interests: The author(s) declare no conflict of interest.

Persisters are a small fraction of growth-arrested phenotypic variants that can survive lethal concentrations of antibiotics but are able to resume growth once antibiotics are stopped. Their formation can be a stochastic process or one triggered by environmental cues. In the human pathogen Streptococcus mutans , the canonical peptide-based quorum-sensing system is an inducible DNA repair system that is pivotal for bacterial survival. Previous work has shown that the CSP-signaling peptide is a stress-signaling alarmone that promotes the formation of stress-induced persisters. In this study, we exposed S. mutans to the CSP pheromone to mimic DNA damage conditions and isolated the antibiotic persisters by treating the cultures with ofloxacin. A transcriptome analysis was then performed to evaluate the differential gene expression between the normal stationary-phase cells and the persisters. RNA sequencing revealed that triggered persistence was associated with the upregulation of genes related to several stress defense mechanisms, notably, multidrug efflux pumps, the arginine deaminase pathway, and the Opu/Opc system. In addition, we showed that inactivation of the VicK kinase of the YycFG essential two-component regulatory system abolished the formation of triggered persisters via the CSP pheromone. These data contribute to the understanding of the triggered persistence phenotype and may suggest new therapeutic strategies for treating persistent streptococcal infections.

Male moths utilize their pheromone communication systems to distinguish potential mates from other sympatric species, which contributes to maintaining reproductive isolation and even drives speciation. The molecular mechanisms underlying the evolution of pheromone communication systems are usually studied between closely-related moth species for their similar but divergent traits associated with pheromone production, detection, and/or processing. In this study, we first identified the functional differentiation in two orthologous pheromone receptors, OR14b, and OR16, in four Helicoverpa species, Helicoverpa armigera, H. assulta, H. zea, and H. gelotopoeon. To understand the substrate response specificity of these two PRs, we performed all-atom molecular dynamics simulations of OR14b and OR16 based on AlphaFold2 structural prediction, and molecular docking, allowing us to predict a few key amino acids involved in substrate binding. These candidate residues were further tested and validated by site-directed mutagenesis and functional analysis. These results together identified two hydrophobic amino acids at positions 164 and 232 are the determinants of the response specificity of HarmOR14b and HzeaOR14b to Z9-14:Ald and Z9-16:Ald by directly interacting with the substrates. Interestingly, in OR16 orthologs, we found that position 66 alone determines the specific binding of Z11-16:OH, likely via allosteric interactions. Overall, we have developed an effective integrated method to identify the critical residues for substrate selectivity of ORs and elucidated the molecular mechanism of the diversification of pheromone recognition systems., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Horizontal gene transfer (HGT) mediated by conjugative plasmids greatly contributes to bacteria evolution and the transmission of antibiotic resistance genes (ARGs). In addition to the selective pressure imposed by extensive antibiotic use, environmental chemical pollutants facilitate the dissemination of antibiotic resistance, consequently posing a serious threat to the ecological environment. Presently, the majority of studies focus on the effects of environmental compounds on R plasmid-mediated conjugation transfer, and pheromone-inducible conjugation has largely been neglected. In this study, we explored the pheromone effect and potential molecular mechanisms of estradiol in promoting the conjugative transfer of pCF10 plasmid in Enterococcus faecalis. Environmentally relevant concentrations of estradiol significantly increased the conjugative transfer of pCF10 with a maximum frequency of 3.2 × 10 -2 , up to 3.5-fold change compared to that of control. Exposure to estradiol induced the activation of pheromone signaling cascade by increasing the expression of ccfA. Furthermore, estradiol might directly bind to the pheromone receptor PrgZ and promote pCF10 induction and finally enhance the conjugative transfer of pCF10. These findings cast valuable insights on the roles of estradiol and its homolog in increasing antibiotic resistance and the potential ecological risk., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pheromone communication is an essential component of reproductive isolation in animals. As such, evolution of pheromone signaling can be linked to speciation. For example, the evolution of sex pheromones is thought to have played a major role in the diversification of moths. In the crop pests Spodoptera littoralis and S. litura , the major component of the sex pheromone blend is ( Z , E )-9,11-tetradecadienyl acetate, which is lacking in other Spodoptera species. It indicates that a major shift occurred in their common ancestor. It has been shown recently in S. littoralis that this compound is detected with high specificity by an atypical pheromone receptor, named SlitOR5. Here, we studied its evolutionary history through functional characterization of receptors from different Spodoptera species. SlitOR5 orthologs in S. exigua and S. frugiperda exhibited a broad tuning to several pheromone compounds. We evidenced a duplication of OR5 in a common ancestor of S. littoralis and S. litura and found that in these two species, one duplicate is also broadly tuned while the other is specific to ( Z , E )-9,11-tetradecadienyl acetate. By using ancestral gene resurrection, we confirmed that this narrow tuning evolved only in one of the two copies issued from the OR5 duplication. Finally, we identified eight amino acid positions in the binding pocket of these receptors whose evolution has been responsible for narrowing the response spectrum to a single ligand. The evolution of OR5 is a clear case of subfunctionalization that could have had a determinant impact in the speciation process in Spodoptera species.

Background: Huntiella resides in the Ceratocystidaceae, a family of fungi that accommodates important plant pathogens and insect-associated saprotrophs. Species in the genus have either heterothallic or unisexual (a form of homothallism) mating systems, providing an opportunity to investigate the genetic mechanisms that enable transitions between reproductive strategies in related species. Two newly sequenced Huntiella genomes are introduced in this study and comparative genomics and transcriptomics tools are used to investigate the differences between heterothallism and unisexuality across the genus., Results: Heterothallic species harbored up to seven copies of the a-factor pheromone, each of which possessed numerous mature peptide repeats. In comparison, unisexual Huntiella species had only two or three copies of this gene, each with fewer repeats. Similarly, while the heterothallic species expressed up to 12 copies of the mature α-factor pheromone, unisexual species had up to six copies. These significant differences imply that unisexual Huntiella species do not rely on a mating partner recognition system in the same way that heterothallic fungi do., Conclusion: While it is suspected that mating type-independent pheromone expression is the mechanism allowing for unisexual reproduction in Huntiella species, our results suggest that the transition to unisexuality may also have been associated with changes in the genes governing the pheromone pathway. While these results are specifically related to Huntiella, they provide clues leading to a better understanding of sexual reproduction and the fluidity of mating strategies in fungi more broadly., (© 2023. The Author(s).)

Many animals engage in cannibalism to supplement their diets. Among dense populations of migratory locusts, cannibalism is prevalent. We show that under crowded conditions, locusts produce an anticannibalistic pheromone called phenylacetonitrile. Both the degree of cannibalism and the production of phenylacetonitrile are density dependent and covary. We identified the olfactory receptor that detects phenylacetonitrile and used genome editing to make this receptor nonfunctional, thereby abolishing the negative behavioral response. We also inactivated the gene underlying phenylacetonitrile production and show that locusts that lack this compound lose its protection and are more frequently exposed to intraspecific predation. Thus, we reveal an anticannibalistic feature built on a specifically produced odor. The system is very likely to be of major importance in locust population ecology, and our results might therefore provide opportunities in locust management.

The human fungal pathogen Candida albicans can switch stochastically and heritably between a "white" phase and an "opaque" phase. Opaque cells are the mating-competent form of the species, whereas white cells are thought to be essentially "sterile". Here, we report that glucose depletion, a common nutrient stress, enables C. albicans white cells to undergo efficient sexual mating. The relative expression levels of pheromone-sensing and mating-associated genes (including STE2/3, MFA1, MFα1, FIG1, FUS1, and CEK1/2) are increased under glucose depletion conditions, while expression of mating repressors TEC1 and DIG1 is decreased. Cph1 and Tec1, factors that act downstream of the pheromone MAPK pathway, play opposite roles in regulating white cell mating as TEC1 deletion or CPH1 overexpression promotes white cell mating. Moreover, inactivation of the Cph1 repressor Dig1 increases white cell mating ~4000 fold in glucose-depleted medium relative to that in the presence of glucose. Our findings reveal that the white-to-opaque epigenetic switch may not be a prerequisite for sexual mating in C. albicans in nature., (© 2023. The Author(s).)

Understanding of the neural bases for complex behaviors in Hymenoptera insect species has been limited by a lack of tools that allow measuring neuronal activity simultaneously in different brain regions. Here, we developed the first pan-neuronal genetic driver in a Hymenopteran model organism, the honey bee, and expressed the calcium indicator GCaMP6f under the control of the honey bee synapsin promoter. We show that GCaMP6f is widely expressed in the honey bee brain, allowing to record neural activity from multiple brain regions. To assess the power of this tool, we focused on the olfactory system, recording simultaneous responses from the antennal lobe, and from the more poorly investigated lateral horn (LH) and mushroom body (MB) calyces. Neural responses to 16 distinct odorants demonstrate that odorant quality (chemical structure) and quantity are faithfully encoded in the honey bee antennal lobe. In contrast, odor coding in the LH departs from this simple physico-chemical coding, supporting the role of this structure in coding the biological value of odorants. We further demonstrate robust neural responses to several bee pheromone odorants, key drivers of social behavior, in the LH. Combined, these brain recordings represent the first use of a neurogenetic tool for recording large-scale neural activity in a eusocial insect and will be of utility in assessing the neural underpinnings of olfactory and other sensory modalities and of social behaviors and cognitive abilities., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Carcaud et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Successful colonization of a host requires bacterial adaptation through genetic and population changes that are incompletely defined. Using chromosomal barcoding and high-throughput sequencing, we investigate the population dynamics of Streptococcus pneumoniae during infant mouse colonization. Within 1 day post inoculation, diversity was reduced >35-fold with expansion of a single clonal lineage. This loss of diversity was not due to immune factors, microbiota, or exclusive genetic drift. Rather, bacteriocins induced by the BlpC-quorum sensing pheromone resulted in predation of kin cells. In this intra-strain competition, the subpopulation reaching a quorum likely eliminates others that have yet to activate the blp locus. Additionally, this reduced diversity restricts the number of unique clones that establish colonization during transmission between hosts. Genetic variation in the blp locus was also associated with altered transmissibility in a human population, further underscoring the importance of BlpC in clonal selection and its role as a selfish element., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Most ascomycete fungi, including the fission yeast Schizosaccharomyces pombe, secrete two peptidyl mating pheromones: C-terminally modified and unmodified peptides. S. pombe has two mating types, plus and minus, which secrete two different pheromones, P-factor (unmodified) and M-factor (modified), respectively. These pheromones are specifically recognized by receptors on the cell surface of cells of opposite mating types, which trigger a pheromone response. Recognition between pheromones and their corresponding receptors is important for mate discrimination; therefore, genetic changes in pheromone or receptor genes affect mate recognition and cause reproductive isolation that limits gene flow between populations. Such genetic variation in recognition via the pheromone/receptor system may drive speciation. Our recent studies reported that two pheromone receptors in S. pombe might have different stringencies in pheromone recognition. In this review, we focus on the molecular mechanism of pheromone response and mating behavior, emphasizing pheromone diversification and its impact on reproductive isolation in S. pombe and closely related fission yeast species. We speculate that the "asymmetric" system might allow flexible adaptation to pheromone mutational changes while maintaining stringent recognition of mating partners. The loss of pheromone activity results in the extinction of an organism's lineage. Therefore, genetic changes in pheromones and their receptors may occur gradually and/or coincidently before speciation. Our findings suggest that the M-factor plays an important role in partner discrimination, whereas P-factor communication allows flexible adaptation to create variations in S. pombe. Our inferences provide new insights into the evolutionary mechanisms underlying pheromone diversification.

The enrichment and spread of antibiotic resistance genes (ARGs) induced by environmental chemical pollution further exacerbated the threat to human health and ecological safety. Several compounds are known to induce R plasmid-mediated conjugation through inducing reactive oxygen species (ROS), increasing cell membrane permeability, enhancing regulatory genes expression, and so forth. Up to now, there has been no substantial breakthrough in the studies of models and related mechanisms. Here, we established a new conjugation model using pheromone-responsive plasmid pCF10 and confirmed that five kinds of bisphenols (BPs) at environmentally relevant concentrations could significantly promote the conjugation of ARGs mediated by plasmid pCF10 in E. faecalis by up to 4.5-fold compared with untreated cells. Using qPCR, gene knockout and UHPLC, we explored the mechanisms behind this phenomenon using bisphenol A (BPA) as a model of BPs and demonstrated that BPA could upregulate the expression of pheromone, promote bacterial aggregation, and even directly activate conjugation as a pheromone instead of producing ROS and enhancing cell membrane permeability. Interestingly, the result of mathematical analysis showed that the pheromone effect of most BPs is more potent than that of synthetic pheromone cCF10. These findings provide new insight into the environmental behavior and biological effect of BPs and provided new method and theory to study on enrichment and spread of ARGs induced by environmental chemical pollution.

Competence is one of the most efficient bacterial evolutionary and adaptative strategies by synchronizing production of antibacterial compounds and integration of DNA released by dead cells. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing a short time window of activation in which only part of the population is responsive. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. However, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using CRISPR interference and performed three complementary screens to investigate competence genetic regulation in the human commensal Streptococcus salivarius. We show that initiation of competence increases upon cell wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report a key role for StkP, a serine-threonine kinase known to regulate cell wall homeostasis. We show that StkP controls competence by a mechanism that reacts to peptidoglycan fragments. Together, our data suggest a key cell wall sensing mechanism coupling competence to cell envelope integrity. IMPORTANCE Survival of human commensal streptococci in the digestive tract requires efficient strategies which must be tightly and collectively controlled for responding to competitive pressure and drastic environmental changes. In this context, the autocrine signaling system ComRS controlling competence for natural transformation and predation in salivarius streptococci could be seen as a multi-input device integrating a variety of environmental stimuli. In this work, we revealed novel positive and negative competence modulators by using a genome-wide CRISPR interference strategy. Notably, we highlighted an unexpected connection between bacterial envelope integrity and competence activation that involves several cell wall sensors. Together, these results showcase how commensal streptococci can fine-tune the pheromone-based competence system by responding to multiple inputs affecting their physiological status in order to calibrate an appropriate collective behavior.

It is well established that pheromones are used by insects to transmit information between individuals. However, research has revealed that individual insects can be both the sender and the receiver of some pheromonal signals. It is therefore interesting to consider whether the pheromonal state of an individual insect can exert an effect on itself. In this study, we monitored the sleep activity of single flies exhibiting a mutation that leads to pheromonal deficiency and found that cuticular hydrocarbons (CHs) exerted self-regulatory effects on the amount of sleep experienced by these flies. To identify the physiological significance of this mechanism, we compared the amounts of sleep in individual young flies and individual old flies (flies are known to sleep less as they get older) and compared this data with young and old flies exhibiting mutations that lead to CH reception defects. The differences in the amount of sleep experienced by young and old mutant flies were significantly lower than those of the control flies. Our data show that hydrocarbon signals produced by the cuticle in Drosophila can be self-perceived and regulate the amount of sleep acquired in a maturation-dependent manner., (© 2022 Institute of Zoology, Chinese Academy of Sciences.)

Background: The nose of most animals comprises multiple sensory subsystems, which are defined by the expression of different olfactory receptor families. Drosophila melanogaster antennae contain two morphologically and functionally distinct subsystems that express odorant receptors (Ors) or ionotropic receptors (Irs). Although these receptors have been thoroughly characterized in this species, the subsystem-specific expression and roles of other genes are much less well-understood., Results: Here we generate subsystem-specific transcriptomic datasets to identify hundreds of genes, encoding diverse protein classes, that are selectively enriched in either Or or Ir subsystems. Using single-cell antennal transcriptomic data and RNA in situ hybridization, we find that most neuronal genes-other than sensory receptor genes-are broadly expressed within the subsystems. By contrast, we identify many non-neuronal genes that exhibit highly selective expression, revealing substantial molecular heterogeneity in the non-neuronal cellular components of the olfactory subsystems. We characterize one Or subsystem-specific non-neuronal molecule, Osiris 8 (Osi8), a conserved member of a large, insect-specific family of transmembrane proteins. Osi8 is expressed in the membranes of tormogen support cells of pheromone-sensing trichoid sensilla. Loss of Osi8 does not have obvious impact on trichoid sensillar development or basal neuronal activity, but abolishes high sensitivity responses to pheromone ligands., Conclusions: This work identifies a new protein required for insect pheromone detection, emphasizes the importance of support cells in neuronal sensory functions, and provides a resource for future characterization of other olfactory subsystem-specific genes., (© 2022. The Author(s).)

Unraveling the origin of molecular pathways underlying the evolution of adaptive traits is essential for understanding how new lineages emerge, including the relative contribution of conserved ancestral traits and newly evolved derived traits. Here, we investigated the evolutionary divergence of sex pheromone communication from moths (mostly nocturnal) to butterflies (mostly diurnal) that occurred ~119 million years ago. In moths, it is the females that typically emit pheromones to attract male mates, but in butterflies males emit pheromones that are used by females for mate choice. The molecular bases of sex pheromone communication are well understood in moths, but they have remained relatively unexplored in butterflies. We used a combination of transcriptomics, real time qPCR, and phylogenetics to identify genes involved in the different steps (i.e., production, regulation, and reception) of sex pheromone communication of the butterfly Bicyclus anynana . Our results show that the biosynthesis and reception of sex pheromones relies both on moth-specific gene families (reductases) and on more ancestral insect gene families (desaturases, olfactory receptors, odorant binding proteins). Interestingly, B. anynana appears to use what was believed to be the moth-specific neuropeptide Pheromone Biosynthesis Activating Neuropeptide (PBAN) for regulating sex pheromone production. Altogether, our results suggest that a mosaic pattern best explains how sex pheromone communication evolved in butterflies, with some molecular components derived from moths, and others conserved from more ancient insect ancestors. This is the first large-scale investigation of the genetic pathways underlying sex pheromone communication in a butterfly.

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci., Competing Interests: The authors of this manuscript have the following competing interests: PH is a Research Director at FNRS.

Odorant-binding proteins (OBPs) play essential roles in the functioning of insect peripheral olfactory systems. To fully understand the olfactory roles of OBPs in Halyomorpha halys, an important invasive pest found worldwide, we studied the expression and functional characterization of five OBP-associated genes from H. halys that are clustered in the genome. The tissue distribution of the OBP gene cluster suggests that these genes were enriched in nymph and adult antennae, indicating their possible involvement in the chemosensory process. The different expression levels of the five OBPs in nymph and adult antennae suggest that this gene cluster is regulated independently. Ligand-binding experiments have shown similar specificities of these five OBPs towards several organic compounds, including the alarm pheromone of H. halys (trans-2-decenal), the aggregation pheromone of Plautia stali (methyl (2E, 4E, 6Z)-decatrienoate), and plant volatile compounds (e.g., cis-3-hexenyl benzoate and β-ionone). In particular, trans-2-dodecenal, an alarm pheromone analog, exhibited high affinity to the five OBP proteins and alarm pheromone activity towards H. halys. Thus, this OBP cluster may mediate the response of stink bugs to the both the alarm pheromone and host-related volatiles and could be an interesting target to design novel olfactory regulators for the management of H. halys infestations., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The diploid fungal pathogen Candida albicans has three configurations at the mating type locus (MTL): heterozygous (a/α) and homozygous (a/a or α/α). C. albicans MTL locus encodes four transcriptional regulators (MTLa1, a2, α1, and α2). The conserved a1/α2 heterodimer controls not only mating competency but also white-opaque heritable phenotypic switching. However, the regulatory roles of MTLa2 and α1 are more complex and remain to be investigated. MTLa/a cells often express a cell type-specific genes and mate as the a-type partner, whereas MTLα/α cells express α-specific genes and mate as the α-type partner. In this study, we report that the MTLa2 regulator controls the formation of mating projections through both the a- and α-pheromone-sensing pathways and thus results in the bi-mater feature of "α cells" of C. albicans. Ectopic expression of MTLa2 in opaque α cells activates the expression of not only MFA1 and STE3 (a-pheromone receptor) but also MFα1 and STE2 (α-pheromone receptor). Inactivation of either the MFa-Ste3 or MFα-Ste2 pheromone-sensing pathway cannot block the MTLa2-induced development of mating projections. However, the case is different in MTLα1-ectopically expressed opaque a cells. Inactivation of the MFα-Ste2 but not the MFa-Ste3 pheromone-sensing pathway blocks MTLα1-induced development of mating projections. Therefore, MTLa2 and MTLα1 exhibit distinct regulatory features that control the mating response in C. albicans. These findings shed new light on the regulatory mechanism of bi-mating behaviors and sexual reproduction in C. albicans., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The cuticle of all insects is covered with hydrocarbons which have multiple functions. Cuticular hydrocarbons (CHCs) basically serve to protect insects against environmental harm and reduce dehydration. In many species, some CHCs also act as pheromones. CHCs have been intensively studied in Drosophila species and more especially in D. melanogaster. In this species, flies produce about 40 CHCs forming a complex sex- and species-specific bouquet. The quantitative and qualitative pattern of the CHC bouquet was characterized during the first days of adult life but remains unexplored in aging flies. Here, we characterized CHCs during the whole-or a large period of-adult life in males and females of several wild type and transgenic lines. Both types of lines included standard and variant CHC profiles. Some of the genotypes tested here showed very dramatic and unexpected aging-related variation based on their early days' profile. This study provides a concrete dataset to better understand the mechanisms underlying the establishment and maintenance of CHCs on the fly cuticle. It could be useful to determine physiological parameters, including age and response to climate variation, in insects collected in the wild., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Eurasian spruce bark beetle, Ips typographus, is a destructive pest in spruce forests. The ability of I. typographus to colonise host trees depends on its massive aggregation behaviour mediated by aggregation pheromones, consisting of 2-methyl-3-buten-2-ol and cis-verbenol. Other biologically active compounds such as ipsdienol and verbenone have also been detected in the beetle. Biosynthesis of 2-methyl-3-buten-2-ol and ipsdienol de novo from mevalonate and that of cis-verbenol from α-pinene sequestrated from the host have been reported in preliminary studies. However, knowledge on the molecular mechanisms underlying pheromone biosynthesis in this pest is currently limited. In this study, we performed metabolomic and differential gene expression (DGE) analysis for the pheromone-producing life stages of I. typographus. The highest amounts of 2-methyl-3-buten-2-ol (238 ng/gut) and cis-verbenol (23 ng/gut) were found in the fed male gut (colonisation stage) and the immature male gut (early stage), respectively. We also determined the amount of verbenyl oleate (the possible storage form of cis-verbenol), a monoterpenyl fatty acid ester, to be approximately 1604 ng/mg in the immature stage in the beetle body. DGE analysis revealed possible candidate genes involved in the biosynthesis of the quantified pheromones and related compounds. A novel hemiterpene-synthesising candidate isoprenyl-di-phosphate synthase Ityp09271 gene proposed for 2-methyl-3-buten-2-ol synthesis was found to be highly expressed only in the fed male beetle gut. Putative cytochrome P450 genes involved in cis/trans-verbenol synthesis and an esterase gene Ityp11977, which could regulate verbenyl oleate synthesis, were identified in the immature male gut. Our findings from the molecular analysis of pheromone-producing gene families are the first such results reported for I. typographus. With further characterisation of the identified genes, we can develop novel strategies to disrupt the aggregation behaviour of I. typographus and thereby prevent vegetation loss., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The traits of two subspecies of western honey bees, Apis mellifera scutellata and A.m. capensis, endemic to the Republic of South Africa (RSA), are of biological and commercial relevance. Nevertheless, the genetic basis of important phenotypes found in these subspecies remains poorly understood. We performed a genome wide association study on three traits of biological relevance in 234 A.m. capensis, 73 A.m. scutellata and 158 hybrid individuals. Thirteen markers were significantly associated to at least one trait (P ≤ 4.28 × 10-6): one for ovariole number, four for scutellar plate and eight for tergite color. We discovered two possible causative variants associated to the respective phenotypes: a deletion in GB46429 or Ebony (NC_007070.3:g.14101325G>del) (R69Efs*85) and a nonsense on GB54634 (NC_007076.3:g.4492792A>G;p.Tyr128*) causing a premature stop, substantially shortening the predicted protein. The mutant genotypes are significantly associated to phenotypes in A.m. capensis. Loss-of-function of Ebony can cause accumulation of circulating dopamine, and increased dopamine levels correlate to ovary development in queenless workers and pheromone production. Allelic association (P = 1.824 x 10-5) of NC_007076.3:g.4492792A>G;p.Tyr128* to ovariole number warrants further investigation into function and expression of the GB54634 gene. Our results highlight genetic components of relevant production/conservation behavioral phenotypes in honey bees., Competing Interests: The authors have declared that no competing interests exist.

Competence for natural transformation extensively contributes to genome evolution and the rapid adaptability of bacteria dwelling in challenging environments. In most streptococci, this process is tightly controlled by the ComRS signaling system, which is activated through the direct interaction between the (R)RNPP-type ComR sensor and XIP pheromone (mature ComS). The overall mechanism of activation and the basis of pheromone selectivity have been previously reported in Gram-positive salivarius streptococci; however, detailed 3D-remodeling of ComR leading up to its activation remains only partially understood. Here, we identified using a semirational mutagenesis approach two residues in the pheromone XIP that bolster ComR sensor activation by interacting with two aromatic residues of its XIP-binding pocket. Random and targeted mutagenesis of ComR revealed that the interplay between these four residues remodels a network of aromatic-aromatic interactions involved in relaxing the sequestration of the DNA-binding domain. Based on these data, we propose a comprehensive model for ComR activation based on two major conformational changes of the XIP-binding domain. Notably, the stimulation of this newly identified trigger point by a single XIP substitution resulted in higher competence and enhanced transformability, suggesting that pheromone-sensor coevolution counter-selects for hyperactive systems in order to maintain a trade-off between competence and bacterial fitness. Overall, this study sheds new light on the ComRS activation mechanism and how it could be exploited for biotechnological and biomedical purposes., Competing Interests: Conflict of interest P. H. and Y. F. D. are research directors at FNRS. All other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5'-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of RGA1 , a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of "kinetic proofreading". Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.

The Tup1-Cyc8 corepressor complex of Saccharomyces cerevisiae is recruited to promoters by DNA-binding proteins to repress transcription of genes, including the a-specific mating-type genes. We report here a tup1(S649F) mutant that displays mating irregularities and an α-predominant growth defect. RNA-Seq and ChIP-Seq were used to analyze gene expression and Tup1 occupancy changes in mutant vs wild type in both a and α cells. Increased Tup1(S649F) occupancy tended to occur upstream of upregulated genes, whereas locations with decreased occupancy usually did not show changes in gene expression, suggesting this mutant not only loses corepressor function but also behaves as a coactivator. Based upon studies demonstrating a dual role of Tup1 in both repression and activation, we postulate that the coactivator function of Tup1(S649F) results from diminished interaction with repressor proteins, including α2. We also found that large changes in mating-type-specific gene expression between a and α or between mutant and wild type were not easily explained by the range of Tup1 occupancy levels within their promoters, as predicted by the classic model of a-specific gene repression by Tup1. Most surprisingly, we observed Tup1 occupancy upstream of the a-specific gene MFA2 and the α-specific gene MF(ALPHA)1 in cells in which each gene was expressed rather than repressed. These results, combined with the identification of additional mating-related genes upregulated in the tup1(S649F) α strain, illustrate that the role of Tup1 in distinguishing mating types in yeast appears to be both more comprehensive and more nuanced than previously appreciated., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Efficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the expression of its type 4 secretion system (T4SS) genes, controlled by the P Q promoter. Transcription from the P Q promoter is tightly regulated, partially to limit cell toxicity caused by overproduction of PrgB, a T4SS adhesin. PrgU plays an important role in regulating this toxicity by decreasing PrgB levels. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. We used a combination of in vivo methods to quantify PrgU effects on prgQ transcripts at both single-cell and population levels. PrgU function requires a specific RNA sequence within an intergenic region (IGR) about 400 bp downstream of P Q . PrgU interaction with the IGR reduces levels of downstream transcripts. Single-cell expression analysis showed that cells expressing prgU decreased transcript levels more rapidly than isogenic prgU -minus cells. PrgU bound RNA in vitro without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors for selective binding in vivo . PrgU binding to its IGR target might recruit RNase(s) for targeted degradation of downstream transcripts or reduce elongation of nascent transcripts beyond the IGR. IMPORTANCE Bacteria utilize type 4 secretion systems (T4SS) to efficiently transfer DNA between donor and recipient cells, thereby spreading genes encoding antibiotic resistance as well as various virulence factors. Regulation of expression of the T4SS proteins and surface adhesins in Gram-positive bacteria is crucial, as some of these are highly toxic to the cell. The significance of our research lies in identifying the novel mechanism by which PrgU performs its delicate fine-tuning of the expression levels. As prgU orthologs are present in various conjugative plasmids and transposons, our results are likely relevant to understanding of diverse clinically important transfer systems.

Enterococcus faecalis, a member of the commensal flora in the human gastrointestinal tract, has become a threatening nosocomial pathogen because it has developed resistance to many known antibiotics. More concerningly, resistance gene-carrying E. faecalis cells may transfer antibiotic resistance to resistance-free E. faecalis cells through their unique quorum sensing-mediated plasmid transfer system. Therefore, we investigated the role of probiotic bacteria in the transfer frequency of the antibiotic resistance plasmid pCF10 in E. faecalis populations to mitigate the spread of antibiotic resistance. Bacillus subtilis subsp. natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited pCF10 transfer by suppressing peptide pheromone activity from chromosomally encoded CF10 (cCF10) without inhibiting E. faecalis growth. The inhibitory effect was attributed to at least one 30- to 50-kDa extracellular protease present in B. subtilis subsp. natto . Nattokinase of B. subtilis subsp. natto was involved in the inhibition of pCF10 transfer and cleaved cCF10 (LVTLVFV) into LVTL plus VFV fragments. Moreover, the cleavage product LVTL (L peptide) interfered with the conjugative transfer of pCF10. In addition to cCF10, faecalis-cAM373 and gordonii-cAM373, which are mating inducers of vancomycin-resistant E. faecalis, were also cleaved by nattokinase, indicating that B. subtilis subsp. natto can likely interfere with vancomycin resistance transfer in E. faecalis. Our work shows the feasibility of applying fermentation products of B. subtilis subsp. natto and L peptide to mitigate E. faecalis antibiotic resistance transfer. IMPORTANCE Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, antibiotic resistance genes can undergo efficient intra- and interspecies transfer via E. faecalis peptide pheromone-mediated plasmid transfer systems. Therefore, this study provided the first experimental demonstration that probiotics are a feasible approach for interfering with conjugative plasmid transfer between E. faecalis strains to stop the transfer of antibiotic resistance. We found that the extracellular protease(s) of Bacillus subtilis subsp. natto cleaved peptide pheromones without affecting the growth of E. faecalis, thereby reducing the frequency of conjugative plasmid transfer. In addition, a specific cleaved pheromone fragment interfered with conjugative plasmid transfer. These findings provide a potential probiotic-based method for interfering with the transfer of antibiotic resistance between E. faecalis strains.

Background: Defining the origin of genetic novelty is central to our understanding of the evolution of novel traits. Diversification among fatty acid desaturase (FAD) genes has played a fundamental role in the introduction of structural variation in fatty acyl derivatives. Because of its central role in generating diversity in insect semiochemicals, the FAD gene family has become a model to study how gene family expansions can contribute to the evolution of lineage-specific innovations. Here we used the codling moth (Cydia pomonella) as a study system to decipher the proximate mechanism underlying the production of the ∆8∆10 signature structure of olethreutine moths. Biosynthesis of the codling moth sex pheromone, (E8,E10)-dodecadienol (codlemone), involves two consecutive desaturation steps, the first of which is unusual in that it generates an E9 unsaturation. The second step is also atypical: it generates a conjugated diene system from the E9 monoene C 12 intermediate via 1,4-desaturation., Results: Here we describe the characterization of the FAD gene acting in codlemone biosynthesis. We identify 27 FAD genes corresponding to the various functional classes identified in insects and Lepidoptera. These genes are distributed across the C. pomonella genome in tandem arrays or isolated genes, indicating that the FAD repertoire consists of both ancient and recent duplications and expansions. Using transcriptomics, we show large divergence in expression domains: some genes appear ubiquitously expressed across tissue and developmental stages; others appear more restricted in their expression pattern. Functional assays using heterologous expression systems reveal that one gene, Cpo_CPRQ, which is prominently and exclusively expressed in the female pheromone gland, encodes an FAD that possesses both E9 and ∆8∆10 desaturation activities. Phylogenetically, Cpo_CPRQ clusters within the Lepidoptera-specific ∆10/∆11 clade of FADs, a classic reservoir of unusual desaturase activities in moths., Conclusions: Our integrative approach shows that the evolution of the signature pheromone structure of olethreutine moths relied on a gene belonging to an ancient gene expansion. Members of other expanded FAD subfamilies do not appear to play a role in chemical communication. This advises for caution when postulating the consequences of lineage-specific expansions based on genomics alone.

Social behavior is one of the most fascinating and complex behaviors in humans and animals. A fundamental process of social behavior is communication among individuals. It relies on the capability of the nervous system to sense, process, and interpret various signals (e.g., pheromones) and respond with appropriate decisions and actions. Eusocial insects, including ants, some bees, some wasps, and termites, display intriguing cooperative social behavior. Recent advances in genetic and genomic studies have revealed key genes that are involved in pheromone synthesis, chemosensory perception, and physiological and behavioral responses to varied pheromones. In this review, we highlight the genes and pathways that regulate queen pheromone-mediated social communication, discuss the evolutionary changes in genetic systems, and outline prospects of functional studies in sociobiology., (© 2021 Yan and Liebig; Published by Cold Spring Harbor Laboratory Press.)

In ciliates, with every sexual event the transcriptionally active genes of the sub-chromosomic somatic genome that resides in the cell macronucleus are lost. They are de novo assembled starting from 'Macronuclear Destined Sequences' that arise from the fragmentation of transcriptionally silent DNA sequences of the germline chromosomic genome enclosed in the cell micronucleus. The RNA-mediated epigenetic mechanism that drives the assembly of these sequences is subject to errors which result in the formation of chimeric genes. Studying a gene family that in Euplotes raikovi controls the synthesis of protein signal pheromones responsible for a self/not-self recognition mechanism, we identified the chimeric structure of an 851-bp macronuclear gene previously known to specify soluble and membrane-bound pheromone molecules through an intron-splicing mechanism. This chimeric gene, designated mac-er-1*, conserved the native pheromone-gene structure throughout its coding and 3' regions. Instead, its 5' region is completely unrelated to the pheromone gene structure at the level of a 360-bp sequence, which derives from the assembly with a MDS destined to compound a 2417-bp gene encoding a 696-amino acid protein with unknown function. This mac-er-1* gene characterization provides further evidence that ciliates rely on functional chimeric genes that originate in non-programmed phenomena of somatic MDS recombination to increase the species genetic variability independently of gene reshuffling phenomena of the germline genome., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The exocrine-gland secreting peptide (ESP)gene family encodes proteinaceous pheromones that are recognized by the vomeronasal organ in mice. For example, ESP1 is a male pheromone secreted in tear fluid that regulates socio-sexual behavior, and ESP22 is a juvenile pheromone that suppresses adult sexual behavior. The family consists of multiple genes and has been identified only in mouse and rat genomes. The coding region of a mouse ESP gene is separated into two exons, each encoding signal and mature sequences. Here, we report the origin and evolution of the ESP gene family. ESP genes were found only in the Muridea and Cricetidae families of rodents, suggesting a recent origin of ESP genes in the common ancestor of murids and cricetids. ESP genes show a great diversity in number, length, and sequence among different species as well as mouse strains. Some ESPs in rats and golden hamsters are expressed in the lacrimal gland and the salivary gland. We also found that a mature sequence of an ESP gene showed overall sequence similarity to the α-globin gene. The ancestral ESP gene seems to be generated by recombination of a retrotransposed α-globin gene with the signal-encoding exon of the CRISP2 gene located adjacent to the ESP gene cluster. This study provides an intriguing example of molecular tinkering in rapidly evolving species-specific proteinaceous pheromone genes., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The Chinese oak silkworm, Antheraea pernyi, has not only been semi-domesticated as an important economical insect but also used for genetic research. The female moths of A. pernyi employ a pheromone blend containing (E,Z)-6,11-hexadecadienal (E6,Z11-16:Ald), (E,Z)-6,11-hexadecadienyl acetate (E6,Z11-16:OAc), and (E,Z)-4,9-tetradecadienyl acetate (E4,Z9-14:OAc). While its biosynthesis pathway is largely unknown. By deep sequencing and de novo assembly of sex pheromone gland (PG) transcriptome, we identified 141 candidate genes that are putatively related to pheromone biosynthesis, degradation, and chemoreception in A. pernyi. Gene expression patterns and phylogenetic analysis revealed that two desaturases (AperDES1 and 2), two fatty acid reductase (AperFAR1 and 2), and three acetyltransferase genes (AperACT1, 2 and 3) showed PG-biased or specific expression and were phylogenetically related to genes known to be involved in pheromone synthesis in other species. Furthermore, two carboxylesterases (AperCOE6 and 11) and two chemosensory protein (AperCSP1 and 6) were also expressed specifically or predominantly in the PGs, which might be related to sex pheromone degradation and transportation, respectively. Based on these results, the sex pheromone biosynthesis and metabolic pathway was proposed in A. pernyi. This study provides some crucial candidates for further functional elucidation, and may be used for interfering sexual communication in other Saturniidae pests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mitogen-activated protein kinase (MAPK) pathways are highly conserved from yeast to human and are required for the regulation of a multitude of biological processes in eukaryotes. A pentameric MAPK pathway known as the Fus3 pheromone module was initially characterised in Saccharomyces cerevisiae and was shown to regulate cell fusion and sexual development. Individual orthologous pheromone module genes have since been found to be highly conserved in fungal genomes and have been shown to regulate a diverse array of cellular responses, such as cell growth, asexual and sexual development, secondary metabolite production and pathogenicity. However, information regarding the assembly and structure of orthologous pheromone modules, as well as the mechanisms of signalling and their biological significance is limited, specifically in filamentous fungal species. Recent studies have provided insight on the utilization of the pheromone module as a central signalling hub for the co-ordinated regulation of fungal development and secondary metabolite production. Various proteins of this pathway are also known to regulate reproduction and virulence in a range of plant pathogenic fungi. In this review, we discuss recent findings that help elucidate the structure of the pheromone module pathway in a myriad of fungal species and its implications in the control of fungal growth, development, secondary metabolism and pathogenicity., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The dental cariogenic pathogen Streptococcus mutans coordinates competence for genetic transformation via two peptide pheromones, competence stimulating peptide (CSP) and comX -inducing peptide (XIP). CSP is sensed by the comCDE system and induces competence indirectly, whereas XIP is sensed by the comRS system and induces competence directly. In chemically defined media (CDM), after uptake by oligopeptide permease, XIP interacts with the cytosolic receptor ComR to form the XIP::ComR complex that activates the expression of comX , an alternative sigma factor that initiates the transcription of late-competence genes. In this study, we set out to determine the molecular mechanism of XIP::ComR interaction. To this end, we performed systematic replacement of the amino acid residues in the XIP pheromone and assessed the ability of the mutated analogs to modulate the competence regulon in CDM. We were able to identify structural features that are important to ComR binding and activation. Our structure-activity relationship insights led us to construct multiple XIP-based inhibitors of the comRS pathway. Furthermore, when comCDE and comRS were both stimulated with CSP and XIP, respectively, a lead XIP-based inhibitor was able to maintain the inhibitory activity. Last, phenotypic assays were used to highlight the potential of XIP-based inhibitors to attenuate pathogenicity in S. mutans and to validate the specificity of these compounds to the comRS pathway within the competence regulon. The XIP-based inhibitors developed in this study can be used as lead scaffolds for the design and development of potential therapeutics against S. mutans infections.

The moth Eogystia hippophaecolus (Hua et al.) is a major threat to sea buckthorn plantations in China. Specific and highly efficient artificial sex pheromone traps have been developed and used to control this pest species. However, the biosynthesis of sex pheromones Z7-14: Ac and E3-14:Ac remains poorly understood. We investigated the female pheromone gland transcriptome of E. hippophaecolus and identified two pheromone biosynthesis-activating neuropeptides (PBANs), two pheromone biosynthesis-activating neuropeptide receptors (PBANrs), five acetyl-CoA carboxylases (ACCs), six fatty acid synthases (FASs), 16 Acyl-CoA desaturases (DESs), 26 reductases (REDs), 13 acetyltransferases (ACTs), one fatty acid transport protein (FATP), one acyl-CoA-binding protein (ACBP), and five elongation of very long-chain fatty acid proteins (ELOs) in pheromone biosynthesis pathways. Additionally, we identified 11 odorant-degrading enzymes (ODEs) and 16 odorant-binding proteins (OBPs), 14 chemosensory proteins (CSPs), two sensory neuron membrane proteins (SNMPs), three odorant receptors (ORs), seven ionotropic receptors (IRs), and six gustatory receptors (GRs). 77 unigenes involved in female pheromone biosynthesis, 31 chemoreception proteins and 11 odorant degradation enzymes were identified, which provided insight into the regulation of the pheromone components and pheromone recognition in the sex pheromone gland, and knowledge pertinent to new integrated pest management strategy of interference pheromone biosynthesis and recognition., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

Plasmid-mediated horizontal gene transfer of antibiotic resistance and virulence in pathogenic bacteria underlies a major public health issue. Understanding how, in the absence of antibiotic-mediated selection, plasmid-bearing cells avoid being outnumbered by plasmid-free cells is key to developing counterstrategies. Here, we quantified the induction of the plasmidial sex pheromone pathway of Enterococcus faecalis to show that the integration of the stimulatory (mate-sensing) and inhibitory (self-sensing) signaling modules from the pCF10 conjugative plasmid provides a precise measure of the recipient-to-donor ratio, agnostic to variations in population size. Such ratiometric control of conjugation favors vertical plasmid transfer under low mating likelihood and allows activation of conjugation functions only under high mating likelihood. We further show that this strategy constitutes a cost-effective investment into mating effort because overstimulation produces unproductive self-aggregation and growth rate reduction. A mathematical model suggests that ratiometric control of conjugation increases plasmid fitness and predicts a robust long-term, stable coexistence of donors and recipients. Our results demonstrate how population-level parameters can control transfer of antibiotic resistance in bacteria, opening the door for biotic control strategies., Competing Interests: The authors have declared that no competing interests exist.

Communication mechanisms underlying the sexual isolation of species are poorly understood. Using four subspecies of Drosophila mojavensis as a model, we identify two behaviorally active, male-specific pheromones. One functions as a conserved male antiaphrodisiac in all subspecies and acts via gustation. The second induces female receptivity via olfaction exclusively in the two subspecies that produce it. Genetic analysis of the cognate receptor for the olfactory pheromone indicates an important role for this sensory pathway in promoting sexual isolation of subspecies, in combination with auditory signals. Unexpectedly, the peripheral sensory pathway detecting this pheromone is conserved molecularly, physiologically, and anatomically across subspecies. These observations imply that subspecies-specific behaviors arise from differential interpretation of the same peripheral cue, reminiscent of sexually conserved detection but dimorphic interpretation of male pheromones in Drosophila melanogaster . Our results reveal that, during incipient speciation, pheromone production, detection, and interpretation do not necessarily evolve in a coordinated manner., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Antennae are often considered to be the nostrils of insects. Here, we sequenced the transcriptome of the pheromone gland-ovipositor complex of Helicoverpa assulta and discovered that an odorant receptor (OR) gene, HassOR31 , had much higher expression in the ovipositor than in antennae or other tissues. To determine whether the ovipositor was involved in odorant detection, we co-expressed HassOR31 and its co-receptor, HassORco , in a Xenopus oocyte model system, and demonstrated that the OR was responsive to 12 plant odorants, especially Z-3-hexenyl butyrate. These odorants elicited electrophysiological responses of some sensilla in the ovipositor, and HassOR31 and HassORco were co-expressed within ovipositor sensilla. Two oviposition preference experiments showed that female moths lacking antennae still preferentially selected oviposition sites containing plant volatiles. We suggest that the expression of HassOR31 in the ovipositor of H. assulta helps females to determine precise egg-laying sites in host plants., Competing Interests: RL, LH, JD, CW No competing interests declared, (© 2020, Li et al.)

The small molecules that mediate chemical communication between nematodes-so-called 'nematode-derived-modular-metabolites' (NDMMs)-are of major interest because of their ability to regulate development, behavior, and life-history. Pristionchus pacificus nematodes produce an impressive diversity of structurally complex NDMMs, some of which act as primer pheromones that are capable of triggering irreversible developmental switches. Many of these NDMMs have only ever been found in P. pacificus but no attempts have been made to study their evolution by profiling closely related species. This study brings a comparative perspective to the biochemical study of NDMMs through the systematic MS/MS- and NMR-based analysis of exo-metabolomes from over 30 Pristionchus species. We identified 36 novel compounds and found evidence for the convergent evolution of complex NDMMs in separate branches of the Pristionchus phylogeny. Our results demonstrate that biochemical innovation is a recurrent process in Pristionchus nematodes, a pattern that is probably typical across the animal kingdom., Competing Interests: CD, CW, VT, RS No competing interests declared, (© 2020, Dong et al.)

Under stressful conditions, the early larvae of C. elegans enter dauer diapause, a non-aging period, driven by the seemingly opposite influence of ascaroside pheromones (ASCRs) and steroid hormone dafachronic acids (DAs). However, the molecular basis of how these small molecules engage in competitive crosstalk in coordination with insulin/IGF-1 signaling (IIS) remains elusive. Here we report a novel transcriptional regulatory pathway that seems to operate between the ASCR and DA biosynthesis under ad libitum (AL) feeding conditions or bacterial deprivation (BD). Although expression of the ASCR and DA biosynthetic genes reciprocally inhibit each other, ironically and interestingly, such dietary cue-mediated modulation requires the presence of the competitors. Under BD, induction of ASCR biosynthetic gene expression required DA, while ASCR suppresses the expression of the DA biosynthetic gene daf-36 . The negative regulation of DA by ASCR was IIS-dependent, whereas daf-36 regulation appeared to be independent of IIS. These observations suggest that the presence of ASCR determines the IIS-dependency of DA gene expression regardless of dietary conditions. Thus, our work defines a molecular basis for a novel reciprocal gene regulation of pheromones and hormones to cope with stressful conditions during development and aging.

For high-frequency transfer of pCF10 between E. faecalis cells, induced expression of the pCF10 genes encoding conjugative machinery from the prgQ operon is required. This process is initiated by the cCF10 (C) inducer peptide produced by potential recipient cells. The expression timing of prgB, an "early" gene just downstream of the inducible promoter, has been studied extensively in single cells. However, several previous studies suggest that only 1 to 10% of donors induced for early prgQ gene expression actually transfer plasmids to recipients, even at a very high recipient population density. One possible explanation for this is that only a minority of pheromone-induced donors actually transcribe the entire prgQ operon. Such cells would not be able to functionally conjugate but might play another role in the group behavior of donors. Here, we sought to (i) simultaneously assess the presence of RNAs produced from the proximal (early induced transcripts [early Q]) and distal (late Q) portions of the prgQ operon in individual cells, (ii) investigate the prevalence of heterogeneity in induced transcript length, and (iii) evaluate the temporality of induced transcript expression. Using fluorescent in situ hybridization chain reaction (HCR) transcript labeling and single-cell microscopic analysis, we observed that most cells expressing early transcripts (Q L , prgB , and prgA ) also expressed late transcripts ( prgJ , pcfC , and pcfG ). These data support the conclusion that, after induction is initiated, transcription likely extends through the end of the conjugation machinery operon for most, if not all, induced cells. IMPORTANCE In Enterococcus faecalis , conjugative plasmids like pCF10 often carry antibiotic resistance genes. With antibiotic treatment, bacteria benefit from plasmid carriage; however, without antibiotic treatment, plasmid gene expression may have a fitness cost. Transfer of pCF10 is mediated by cell-to-cell signaling, which activates the expression of conjugation genes and leads to efficient plasmid transfer. Yet, not all donor cells in induced populations transfer the plasmid. We examined whether induced cells might not be able to functionally conjugate due to premature induced transcript termination. Single-cell analysis showed that most induced cells do, in fact, express all of the genes required for conjugation, suggesting that premature transcription termination within the prgQ operon does not account for failure of induced donor cell gene transfer., (Copyright © 2020 American Society for Microbiology.)

The detection and processing of chemical stimuli involve coordinated neuronal networks that process sensory information. This allows animals, such as the model species Drosophila melanogaster , to detect food sources and to choose a potential mate. In peripheral olfactory tissues, several classes of proteins are acting to modulate the detection of chemosensory signals. This includes odorant-binding proteins together with odorant-degrading enzymes (ODEs). These enzymes, which primarily act to eliminate toxic compounds from the whole organism also modulate chemodetection. ODEs are thought to neutralize the stimulus molecule concurrently to its detection, avoiding receptor saturation thus allowing chemosensory neurons to respond to the next stimulus. Here, we show that one UDP-glycosyltransferase (UGT36E1) expressed in D. melanogaster antennal olfactory sensory neurons (OSNs) is involved in sex pheromone discrimination. UGT36E1 overexpression caused by an insertion mutation affected male behavioral ability to discriminate sex pheromones while it increased OSN electrophysiological activity to male pheromones. Reciprocally, the decreased expression of UGT36E1, controlled by an RNAi transgene, improved male ability to discriminate sex pheromones whereas it decreased electrophysiological activity in the relevant OSNs. When we combined the two genotypes (mutation and RNAi), we restored wild-type-like levels both for the behavioral discrimination and UGT36E1 expression. Taken together, our results strongly suggest that this UGT plays a pivotal role in Drosophila pheromonal detection.

Pheromones are ectohormones that play an important role in communication and behavior. Pheromones and pheromone receptor genes are important in mice and other mammals that rely heavily on pheromone cues to survive. Although there is controversy about whether pheromones and pheromone receptor genes have the same importance or are even active in humans, there are some hints that they might have roles in sociosexual behavior and mental disorders. The aim of this qualitative review was to provide an overview of the state of the art regarding pheromones and pheromone receptors in humans and their possible implications in human physiology and pathology. An electronic search was conducted in MEDLINE, PubMed and Scopus databases for articles published in English up to December 2018. The search concerned a possible role of pheromones and pheromone receptors in humans with implications for sociosexual behavior, mental disorders, the menstrual cycle and nutrition. Pheromone communication in humans has not been definitively demonstrated. However, the potential ability of putative pheromones to activate the hypothalamus, which controls the release of many hormones, suggests they could have a role in systemic functions in humans. Future confirmation of the effects of pheromones and pheromone receptors in humans could be useful in the prevention and treatment of various human disorders.

G protein-coupled receptors (GPCRs) comprise the largest class of membrane proteins in the human genome, with a common denominator of seven-transmembrane domains largely conserved among eukaryotes. Yeast is naturally armoured with three different GPCRs for pheromone and sugar sensing, with the pheromone pathway being extensively hijacked for characterising heterologous GPCR signalling in a model eukaryote. This review focusses on functional GPCR studies performed in yeast and on the elucidated hotspots for engineering, and discusses both endogenous and heterologous GPCR signalling. Key emphasis will be devoted to studies describing important engineering parameters to consider for successful coupling of GPCRs to the yeast mating pathway. We also review the various means of applying yeast for studying GPCRs, including the use of yeast armed with heterologous GPCRs as a platform for (i) deorphanisation of orphan receptors, (ii) metabolic engineering of yeast for production of bioactive products and (iii) medical applications related to pathogen detection and drug discovery. Finally, this review summarises the current challenges related to expression of functional membrane-bound GPCRs in yeast and discusses the opportunities to continue capitalising on yeast as a model chassis for functional GPCR signalling studies., (© FEMS 2019.)

Bombykol (EZ) is the single component of the female sex pheromone in the silkmoth Bombyx mori. EZ alone evokes full courtship behaviors from conspecific males; however, its geometric isomer (EE) was consistently detected in the pheromone glands (PG) of 16 B. mori strains and a field population of the wild silkmoth Bombyx mandarina, which also uses EZ as the single pheromone component. We investigated the pheromonal activities of EE using a commercial hybrid strain of B. mori, Kinshu × Showa. The behavioral assay demonstrated that a 10 4 -10 5 -fold higher dose of EE than EZ was able to elicit behavioral responses from males. To elucidate whether the trace contaminant of EZ in the EE standard is responsible for these responses, we examined the responses of male antennae to EE using a gas chromatograph-electroantennographic detector system (GC-EAD). The EE, at high doses elicited marginal responses from the male antennae. We next examined antennal and behavioral responses of B. mori whose BmOR1 gene, which is responsible for the reception of bombykol, was knocked out. The knockout of BmOR1 resulted in the complete loss of antennal and behavioral responses to EE and EZ, demonstrating that if EE itself is active, it induces these responses via the incidental stimulation of BmOR1, not via the stimulation of EE-specific receptors. The existence of EE in the PG of B. mori and B. mandarina is discussed from the viewpoints of pheromone biosynthesis and the evolution of pheromone communication systems., (Copyright © 2020 Elsevier Ltd. All rights reserved.)