Two transcriptional cascades orchestrate cockroach leg regeneration.

The mystery of appendage regeneration has fascinated humans for centuries, while the underlying regulatory mechanisms remain unclear. In this study, we establish a transcriptional landscape of regenerating leg in the American cockroach, Periplaneta americana, an ideal model in appendage regeneration studies showing remarkable regeneration capacity. Through a large-scale in vivo screening, we identify multiple signaling pathways and transcription factors controlling leg regeneration. Specifically, zfh-2 and bowl contribute to blastema cell proliferation and morphogenesis in two transcriptional cascades: bone morphogenetic protein (BMP)/JAK-STAT-zfh-2-B-H2 and Notch-drm/bowl-bab1. Notably, we find zfh-2 is working as a direct target of BMP signaling to promote cell proliferation in the blastema. These mechanisms might be conserved in the appendage regeneration of vertebrates from an evolutionary perspective. Overall, our findings reveal that two crucial transcriptional cascades orchestrate distinct cockroach leg regeneration processes, significantly advancing the comprehension of molecular mechanism in appendage regeneration.

Two sex pheromone receptors for sexual communication in the American cockroach.

Volatile sex pheromones are vital for sexual communication between males and females. Females of the American cockroach, Periplaneta americana, produce and emit two sex pheromone components, periplanone-A (PA) and periplanone-B (PB). Although PB is the major sex attractant and can attract males, how it interacts with PA in regulating sexual behaviors is still unknown. In this study, we found that in male cockroaches, PA counteracted PB attraction. We identified two odorant receptors (ORs), OR53 and OR100, as PB/PA and PA receptors, respectively. OR53 and OR100 were predominantly expressed in the antennae of sexually mature males, and their expression levels were regulated by the sex differentiation pathway and nutrition-responsive signals. Cellular localization of OR53 and OR100 in male antennae further revealed that two types of sensilla coordinate a complex two-pheromone-two-receptor pathway in regulating cockroach sexual behaviors. These findings indicate distinct functions of the two sex pheromone components, identify their receptors and possible regulatory mechanisms underlying the male-specific and age-dependent sexual behaviors, and can guide novel strategies for pest management.

A single gene integrates sex and hormone regulators into sexual attractiveness.

Sex differentiation and hormones are essential for the development of sexual signals in animals, and the regulation of sexual signals involves complex gene networks. However, it is unknown whether a core gene is able to connect the upstream regulators for controlling sexual signal outputs and behavioural consequences. Here, we identify a single gene that integrates both sex differentiation and hormone signalling with sexual attractiveness in an insect model. CYP4PC1 in the German cockroach, Blattella germanica, controls the rate-limiting step in producing female-specific contact sex pheromone (CSP) that stimulates male courtship. As revealed by behavioural, biochemical, molecular, genetic and bioinformatic approaches, in sexually mature females, CYP4PC1 expression and CSP production are coordinately induced by sex differentiation genes and juvenile hormone (JH) signalling. In adult males, direct inhibition of CYP4PC1 expression by doublesexM binding in gene promoter and lack of the gonadotropic hormone JH prevent CSP production, thus avoiding male-male attraction. By manipulating the upstream regulators, we show that wild-type males prefer to court cockroaches with higher CYP4PC1 expression and CSP production in a dose-dependent manner, regardless of their sex. These findings shed light on how sex-specific and high sexual attractiveness is conferred in insects.

Evaluation of Reference Genes for Transcriptional Profiling in Two Cockroach Models.

The German cockroach, Blattella germanica, and the American cockroach, Periplaneta americana are the most common and synanthropic household pests of interest to public health. While they have increasingly served as model systems in hemimetabolous insects for studying many biological issues, there is still a lack of stable reference gene evaluation for reliable quantitative real-time PCR (qPCR) outputs and functional genomics. Here, we evaluated the expression variation of common insect reference genes, including the historically used actin, across various tissues and developmental stages, and also under experimental treatment conditions in these two species by using three individual algorithms (geNorm, BestKeeper, and NormFinder) and a comprehensive program (RefFinder). RPL32 in B. germanica and EF1α in P. americana showed the overall lowest variation among all examined samples. Based on the stability rankings by RefFinder, the optimal but varied reference genes under specific conditions were selected for qPCR normalization. In addition, the combination of RPL32 and EF1α was recommended for all the tested tissues and stages in B. germanica, whereas the combination of multiple reference genes was unfavorable in P. americana. This study provides a condition-specific resource of reference gene selection for accurate gene expression profiling and facilitating functional genomics in these two important cockroaches.