Emergence of terpene chemical communication in insects: Evolutionary recruitment of isoprenoid metabolism.

Insects have evolved a chemical communication system using terpenoids, a structurally diverse class of specialized metabolites, previously thought to be exclusively produced by plants and microbes. Gene discovery, bioinformatics, and biochemical characterization of multiple insect terpene synthases (TPSs) revealed that isopentenyl diphosphate synthases (IDS), enzymes from primary isoprenoid metabolism, are their likely evolutionary progenitors. However, the mutations underlying the emergence of the TPS function remain a mystery. To address this gap, we present the first structural and mechanistic model for the evolutionary emergence of TPS function in insects. Through identifying key mechanistic differences between IDS and TPS enzymes, we hypothesize that the loss of isopentenyl diphosphate (IPP) binding motifs strongly correlates with the gain of the TPS function. Based on this premise, we have elaborated the first explicit structural definition of isopentenyl diphosphate-binding motifs (IBMs) and used the IBM definitions to examine previously characterized insect IDSs and TPSs and to predict the functions of as yet uncharacterized insect IDSs. Consistent with our hypothesis, we observed a clear pattern of disruptive substitutions to IBMs in characterized insect TPSs. In contrast, insect IDSs maintain essential consensus residues for binding IPP. Extending our analysis, we constructed the most comprehensive phylogeny of insect IDS sequences (430 full length sequences from eight insect orders) and used IBMs to predict the function of TPSs. Based on our analysis, we infer multiple, independent TPS emergence events across the class of insects, paving the way for future gene discovery efforts.

Ancient origin and conserved gene function in terpene pheromone and defense evolution of stink bugs and hemipteran insects.

Insects use diverse arrays of small molecules such as metabolites of the large class of terpenes for intra- and inter-specific communication and defense. These molecules are synthesized by specialized metabolic pathways; however, the origin of enzymes involved in terpene biosynthesis and their evolution in insect genomes is still poorly understood. We addressed this question by investigating the evolution of isoprenyl diphosphate synthase (IDS)-like genes with terpene synthase (TPS) function in the family of stink bugs (Pentatomidae) within the large order of piercing-sucking Hemipteran insects. Stink bugs include species of global pest status, many of which emit structurally related 15-carbon sesquiterpenes as sex or aggregation pheromones. We provide evidence for the emergence of IDS-type TPS enzymes at the onset of pentatomid evolution over 100 million years ago, coinciding with the evolution of flowering plants. Stink bugs of different geographical origin maintain small IDS-type families with genes of conserved TPS function, which stands in contrast to the diversification of TPS genes in plants. Expanded gene mining and phylogenetic analysis in other hemipteran insects further provides evidence for an ancient emergence of IDS-like genes under presumed selection for terpene-mediated chemical interactions, and this process occurred independently from a similar evolution of IDS-type TPS genes in beetles. Our findings further suggest differences in TPS diversification in insects and plants in conjunction with different modes of gene functionalization in chemical interactions.

Genotyping-by-sequencing analysis of Orobanche crenata populations in Algeria reveals genetic differentiation.

Crenate broomrape (Orobanche crenata Forsk.) is a serious long-standing parasitic weed problem in Algeria, mainly affecting legumes but also vegetable crops. Unresolved questions for parasitic weeds revolve around the extent to which these plants undergo local adaptation, especially with respect to host specialization, which would be expected to be a strong selective factor for obligate parasitic plants. In the present study, the genotyping-by-sequencing (GBS) approach was used to analyze genetic diversity and population structure of 10 Northern Algerian O. crenata populations with different geographical origins and host species (faba bean, pea, chickpea, carrot, and tomato). In total, 8004 high-quality single-nucleotide polymorphisms (5% missingness) were obtained and used across the study. Genetic diversity and relationships of 95 individuals from 10 populations were studied using model-based ancestry analysis, principal components analysis, discriminant analysis of principal components, and phylogeny approaches. The genetic differentiation (F ST) between pairs of populations was lower between adjacent populations and higher between geographically separated ones, but no support was found for isolation by distance. Further analyses identified four genetic clusters and revealed evidence of structuring among populations and, although confounded with location, among hosts. In the clearest example, O. crenata growing on pea had a SNP profile that was distinct from other host/location combinations. These results illustrate the importance and potential of GBS to reveal the dynamics of parasitic weed dispersal and population structure.

TCF21+ mesenchymal cells contribute to testis somatic cell development, homeostasis, and regeneration in mice.

Testicular development and function rely on interactions between somatic cells and the germline, but similar to other organs, regenerative capacity declines in aging and disease. Whether the adult testis maintains a reserve progenitor population remains uncertain. Here, we characterize a recently identified mouse testis interstitial population expressing the transcription factor Tcf21. We found that TCF21lin cells are bipotential somatic progenitors present in fetal testis and ovary, maintain adult testis homeostasis during aging, and act as potential reserve somatic progenitors following injury. In vitro, TCF21lin cells are multipotent mesenchymal progenitors which form multiple somatic lineages including Leydig and myoid cells. Additionally, TCF21+ cells resemble resident fibroblast populations reported in other organs having roles in tissue homeostasis, fibrosis, and regeneration. Our findings reveal that the testis, like other organs, maintains multipotent mesenchymal progenitors that can be potentially leveraged in development of future therapies for hypoandrogenism and/or infertility.

Regulation of meiotic progression by Sertoli-cell androgen signaling.

Androgen receptor (AR) signaling in Sertoli cells is known to be important for germ-cell progression through meiosis, but the extent to which androgens indirectly regulate specific meiotic stages is not known. Here, we combine synchronization of spermatogenesis, cytological analyses and single-cell RNAseq (scRNAseq) in the Sertoli-cell androgen receptor knockout (SCARKO) mutant and control mice, and demonstrate that SCARKO mutant spermatocytes exhibited normal expression and localization of key protein markers of meiotic prophase events, indicating that initiation of meiotic prophase is not androgen dependent. However, spermatocytes from SCARKO testes failed to acquire competence for the meiotic division phase. ScRNAseq analysis of wild-type and SCARKO mutant testes revealed a molecular transcriptomic block in an early meiotic prophase state (leptotene/zygotene) in mutant germ cells, and identified several misregulated genes in SCARKO Sertoli cells, many of which have been previously implicated in male infertility. Together, our coordinated cytological and scRNAseq analyses identified germ-cell intrinsic and extrinsic genes responsive to Sertoli-cell androgen signaling that promotes cellular states permissive for the meiotic division phase.

Gametogenesis: A journey from inception to conception.

Gametogenesis, the process of forming mature germ cells, is an integral part of both an individual's and a species' health and well-being. This chapter focuses on critical male and female genetic and epigenetic processes underlying normal gamete formation through their differentiation to fertilization. Finally, we explore how knowledge gained from this field has contributed to progress in areas with great clinical promise, such as in vitro gametogenesis.

Identification of Differentially Methylated Sites with Weak Methylation Effects.

Deoxyribonucleic acid (DNA) methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect differentially methylated cytosines (DMCs) among treatments. Most statistical methods for DMC detection do not consider the dependency of methylation patterns across the genome, thus possibly inflating type I error. Furthermore, small sample sizes and weak methylation effects among different phenotype categories make it difficult for these statistical methods to accurately detect DMCs. To address these issues, the wavelet-based functional mixed model (WFMM) was introduced to detect DMCs. To further examine the performance of WFMM in detecting weak differential methylation events, we used both simulated and empirical data and compare WFMM performance to a popular DMC detection tool methylKit. Analyses of simulated data that replicated the effects of the herbicide glyphosate on DNA methylation in Arabidopsis thaliana show that WFMM results in higher sensitivity and specificity in detecting DMCs compared to methylKit, especially when the methylation differences among phenotype groups are small. Moreover, the performance of WFMM is robust with respect to small sample sizes, making it particularly attractive considering the current high costs of bisulfite sequencing. Analysis of empirical Arabidopsis thaliana data under varying glyphosate dosages, and the analysis of monozygotic (MZ) twins who have different pain sensitivities-both datasets have weak methylation effects of <1%-show that WFMM can identify more relevant DMCs related to the phenotype of interest than methylKit. Differentially methylated regions (DMRs) are genomic regions with different DNA methylation status across biological samples. DMRs and DMCs are essentially the same concepts, with the only difference being how methylation information across the genome is summarized. If methylation levels are determined by grouping neighboring cytosine sites, then they are DMRs; if methylation levels are calculated based on single cytosines, they are DMCs.

Herbicide injury induces DNA methylome alterations in Arabidopsis.

The emergence of herbicide-resistant weeds is a major threat facing modern agriculture. Over 470 weedy-plant populations have developed resistance to herbicides. Traditional evolutionary mechanisms are not always sufficient to explain the rapidity with which certain weed populations adapt in response to herbicide exposure. Stress-induced epigenetic changes, such as alterations in DNA methylation, are potential additional adaptive mechanisms for herbicide resistance. We performed methylC sequencing of Arabidopsis thaliana leaves that developed after either mock treatment or two different sub-lethal doses of the herbicide glyphosate, the most-used herbicide in the history of agriculture. The herbicide injury resulted in 9,205 differentially methylated regions (DMRs) across the genome. In total, 5,914 of these DMRs were induced in a dose-dependent manner, wherein the methylation levels were positively correlated to the severity of the herbicide injury, suggesting that plants can modulate the magnitude of methylation changes based on the severity of the stress. Of the 3,680 genes associated with glyphosate-induced DMRs, only 7% were also implicated in methylation changes following biotic or salinity stress. These results demonstrate that plants respond to herbicide stress through changes in methylation patterns that are, in general, dose-sensitive and, at least partially, stress-specific.