“Guan Guan Ju Dove, in the River Continent.” Ladies and gentlemen. “The Book of Verses describes the complex behavior of human love, while love and marriage in the insect world are much simpler.
On July 4, Nature-Ecology and Evolution published the collaborative research results of South China Normal University and Northwest A&F University and other units online, identifying the most critical genes that control the contact pheromone synthesis pathway of German cockroaches, and systematically revealing the molecular mechanism of their sexual attraction – whether there is charisma, sex is fundamental, and hormone levels are indispensable for maintaining sexual attraction.
The research results provide new insights into the production and complex regulation of animal sexual signals. Xavier Bellés, a member of the Royal Spanish Academy of Natural Sciences, said in the review of the paper: “It is clear that this work has gone beyond cockroaches or insects and hints at the general question of how the two sexes of animals arise. ”
Li Sheng’s team Courtesy of the interviewee
One-touch love: The little cockroach’s love skill
“Sexual attraction as the driving force of animal sexual selection includes both the generation and release of sexual signals, and the effective recognition and reception of sexual signals by individuals of the opposite sex. Sex differentiation and hormones are critical to the production of animal sexual signals and involve complex top-down gene regulatory networks. Li Sheng, the corresponding author of the paper and a professor at South China Normal University, told China Science News.
In higher animals, such as humans, behaviors such as love and courtship involve complex signaling systems, including visual, auditory, tactile, and even psychological and social factors.
To simplify the complexity, the scientists chose insects that rely solely on chemical signals to trigger courtship behavior as research objects.
Chen Nan, co-first author of the paper and an associate researcher at South China Normal University, told China Science Daily that Blattella germanica is the most common worldwide household pest of cockroaches, with typical sexual behavior characteristics and clear sexual pheromone components, which is a good model system for studying the chemical communication mechanism of gender.
Fan Yongliang, co-corresponding author of the paper and a researcher at Northwest A&F University, said in an interview with China Science News that German cockroaches carry more than 40 kinds of pathogenic microorganisms, such as E. coli, produce a variety of protein substances that cause human allergies, and are important household health pests. Because of its high resistance and high fecundity, it is very difficult to control.
“Many insects rely on volatile pheromones to search for mates over long distances, using contact pheromones for close courtship recognition. Sex attractants designed with sex pheromones are efficient and green biological control methods, with good application prospects. Fan Yongliang said.
Previously, the German cockroach pheromone has been systematically identified, but the key genes that control its biosynthetic pathway are still unknown. Also, why are only females synthesizing sex pheromones, and sexually mature females have stronger sexual attraction? What upstream signaling pathways are precisely regulated by sexual pheromone synthesis? These questions have not been clearly answered in the past.
As early as 30 years ago, scientists began to “worry” about the “marriage” problem of German cockroaches.
The study found that what insfrequently the males was a mixture of long-chain lipids with up to 29 carbon atoms on the surface of the females, which were very different from the well-known volatile pheromones.
“These lowly volatile sex pheromones must be received through physical contact between male and female, and the male uses the tentacles to perform ‘fencing’ to identify the female sex pheromones, which in turn stimulates the male to courtship and induces sexual mating behavior between the sexes.” Males rotate their bodies 180 degrees and raise their wings as the most typical courtship feature. Chen Nan said that this is like a touch of love.
In 1992, the Blomquist Laboratory of the University of Nevada in the United States used isotope tracing technology to prove at the biochemical level that the most abundant sexually mature female insects had a methylketone, which was generated by a methyl albine through the hydroxylation and oxidation reaction that occurred at C2 position. This hydroxylation reaction is a female-specific step, not in males.
“This step has been thought to have been responsible for a female-specific expression of the P450 gene since 1992, but it has not been identified before.” Li Sheng said.
In 2018, a high-quality genome atlas of the German cockroach was published; In 2019, Fan Yongliang’s team sequenced the transcriptome of German cockroaches at multiple stages of development. “The continuous advancement of sequencing technology and the reduction of costs, as well as the in-depth application of omics methods, gave us the opportunity to verify the function of this P450 gene, CYP4PC1, and the correctness of the hypothesis of that year after 30 years.” Chen Nan said.
Molecular “code” for controlling pheromone synthesis
“In the search for this gene, we encountered some twists and turns.” Chen Nan said.
Originally, the previous view was that the pigment cells under the skin of the abdomen of the German cockroach were the main places for the synthesis of contact pheromones. This led them to start by looking for genes in skin tissue that could be highly expressed to control the synthesis of contact pheromones.
Through large-scale screening of the transcriptome, they found the CYP4PC1 gene in more than 80 P450 gene families.
The next step of verification led to a surprising discovery that the gene is highly expressed in the female’s antennae and wings, and can reach levels hundreds of times that of a day of age at maturity. “Therefore, the female antennae and wings are the main places for the synthesis of contact pheromones. This conclusion overturns previous views. Chen Nan said.
Schematic diagram of the contact pheromone surface distribution of female Cockroaches in Germany, with higher levels of color deeper into the color. Courtesy of respondents
Further studies have found that the CYP4PC1 gene plays a decisive role in the contact pheromone synthesis pathway of German cockroaches. At the same time, the expression of this gene and the content of contact pheromones are synergistically induced by sex differentiation genes and baby-friendly hormone signals.
Li Sheng explained that sex differentiation genes play a role in regulating the development and maintenance of sex characteristics. As one of the two endogenous hormones of insects, juvenile hormone not only inhibits the metamorphosis of insects in juvenile stage, but also acts as a gonadotropin to promote adult reproduction. The concentration and signal of sparing hormones after female feathering increase with the process of sexual maturation.
Chen Nan introduced that their low expression of the CYP4PC1 gene can lead to a significant decrease in the content of contact pheromones in female insects, and male insects no longer show courtship behavior in these female insects. After the introduction of females into the control group, the males still exhibited normal courtship behavior.
“These results suggest that CYP4PC1 is necessary for the synthesis and maintenance of sexual attractiveness of contact pheromones in female worms, and it is highly likely to be involved in the hydroxylation reaction of contact pheromone precursors.” Li Sheng said.
Studies have shown that the hydroxylation step in the contact pheromone synthesis pathway is promoted by sparing young hormones. To further demonstrate that CYP4PC1 controls this decisive hydroxylation step, the team then examined whether the gene was regulated by sparing hormone signaling.
They treated the female with exogenous sparing hormones, which significantly induced the expression of the CYP4PC1 gene; If the sparing hormone signaling gene is suppressed, the sexual attraction of female insects will be greatly weakened.
Thus, the expression of the CYP4PC1 gene is promoted by a signaling pathway triggered by the protected larval hormone, thereby maintaining a high sex pheromone content in females after sexual maturity, stimulating courtship mating in males.
Male insects to male insects “extinguish the lights”
In fact, males also have high levels of contact pheromone precursor compounds. So why can’t males synthesize sex pheromones like females?
To find out, they conducted further research.
Behavioral analysis of competitive courtship. Courtesy of respondents
They found that when adult males who were relatively deficient in hormones supplemented with high doses of exogenous hormones, a small proportion of males attracted males to courtship.
“This suggests that juvenile hormones can induce males to substantially express the CYP4PC1 gene and synthesize contact pheromones, but the effect is still very limited.” Chen Nan said this suggests that there may be more important factors inhibiting the expression of the CYP4PC1 gene in males.
The results showed that the specific expression of CYP4PC1 gene in female insects was regulated by the upstream sex differentiation signaling pathway. In German cockroaches, the intersex gene doublesex produces different proteins dsxF and dsxM, respectively, in male and female adults. Among them, the male-specific product dsxM protein (which belongs to the DMRT family of transcription factors) can bind to the promoter of the CYP4PC1 gene, thereby inhibiting its transcription.
When they inhibit intersex gene expression in male adults, they can lead to the typical homosexual behavior of German cockroaches. For further verification, they made the males’ CYP4PC1 gene low-expression, and homosexual behavior disappeared.
This suggests that in male adults, the dsxM protein inhibits CYP4PC1 gene expression, and the males are relatively deficient in juvenile-sparing hormones, which together inhibit the synthesis of contact pheromones in males, thereby avoiding mutual attraction between wild male cockroaches.
On this basis, the team proposed a theoretical system for the molecular regulation of sexual attraction: the sex differentiation gene dsxM plays a “brake” role in male insects, directly inhibiting CYP4PC1 expression. The intersex gene in the female insect generates dsxF protein, which can remove the brake effect, and the juvenile hormone signal further plays an “accelerator” role in the female insect, promoting the high expression of CYP4PC1 in the sexual maturity process of the female insect, thereby mediating the sexual attraction of sex and age specificity.
German cockroaches have several courtship patterns. a, heterosexual courtship; b, androgenic-androgenic courtship (juvenile hormone treatment); c, males in groups court each other (knock down the dsx gene); D, male-male courtship (knock down the dsx gene). Courtesy of respondents
“So far, we have shown that by manipulating upstream regulators, wild-type males prefer to court cockroaches with higher CYP4PC1 expression and contact pheromone content, and that this preference is independent of the sex of the latter, and that males in the treatment group are even more attractive than sexually mature females.” Li Sheng said that in the past, it was not clear whether core genes could effectively integrate upstream regulatory signals, regulatory signal output and terminal behavior responses.
Their study was the first to find a core gene, CYP4PC1, which integrates the regulatory signaling of sex differentiation genes and the regulatory pathways of endogenous hormones to determine the sexual attraction of insects. “Gender is fundamental, and hormone levels are essential for maintaining sexual attraction.” Fan Yongliang said.
Chen Nan vividly compared: “Why can’t male cockroaches attract male cockroaches?” Because his ‘femininity gene’ is turned off; why male cockroaches prefer sexually mature female cockroaches because of her high ‘endocrine’ levels. ”
During the peer review phase, the three reviewers agreed that the work was an important breakthrough in the field of insect chemical ecology and reproductive physiology: “The authors accurately dissected sex differentiation and the role of gonadotropin signaling in regulatory information synthesis in non-hereditary pattern insects, providing a conceptual framework for understanding the evolutionary study of pheromone synthesis pathways.” “The results of the paper convincingly demonstrate the regulatory role of the key gene CYP4PC1, which is likely to promote the study of key regulatory genes in other animal sex communication systems.”
Unlike existing studies, which have focused mostly on neurological changes, the results also highlight changes in sexual attraction in mediating sexual behaviorThis provides new insights into a better understanding of the mechanisms by which same-sex sexual behavior occurs in animals. (Source: China Science Daily, Li Chen, Zhu Hanbin)
Related paper information:https://doi.org/10.1038/s41559-022-01808-w