4 DISCUSSIONS
C. japonica is a kind of forestry pest and has been evaluated for its potential medical value (Chen et al. , 2022). It is meaning to turn this pest, which threatens the development of economic forestry, into a useful resource through in-depth study of its genetic and physiological characteristics, which may help us to promote efficient management solutions and develop a massive rearing technique for obtaining abundant population of C. japonica as economic resources. In this study, we obtained high-quality genome at chromosome level. With the combination of Illumina NovaSeq, PacBio Sequel Ⅱ and Hi-C technology, we obtained that C. japonica has 31 chromosomes, and the genome size is 584.5 MB.
To annotate the genes involved to diapause, we manually annotated the existence of six potential diapause related genes in C. japonica(DH-PBAN ,PTTH, cry1 , cry2 , per and tim ). The absence of per in C. japonica attracted our attention. It is not a unique event, although the DNA sequence of clock genes have been well-conserved. It was also not found of per in Cydia pomonella and Anoplophora glabripennis (Cao et al. , 2021). The absence of cry2 in Drosophila and the absence of cry1 in Tribolium castaneum and Apis mellifera has been reported (Yuan et al. , 2007) (Sandrelli et al. , 2008).per is not a necessary gene for these Lepidoptera insects and may have been lost in the process of evolution. per and timare transcribed promoted by CLK and CYC in the nucleus and translated into proteins in the cytoplasm. At the same time, the concentration of PER and TIM will also reduce the level of CLK and CYC by negative feedback, thus reducing the transcription of per and tim(Tomioka and Matsumoto, no date). However, CRY2 is likely more important negative regulator for transcription of CLK and CYC in some insects and can degrade by light. Therefore, it could be existing some complementary factor of PER in function of circadian regulation pathway. Although,per gene has occurred mutation in evolution, some non-PER insect can develop normally and adapt rhythmic change regularly. S. ricini has the same number of diapause-related genes (in this study) with C. japonica and both absence of per . However,A. pernyi and A. yamamai have different related-diapause gene with two formers. Based on their phylogenetic relationship (Figure 4), it may occur some evolutionary events in rhythmic system. In someper -null flies, they also can into diapause, that suggests per may not involve in initiating by photoperiodic diapause(Saunders, Henrich and Gilbert, 1989).
Genes related to circadian clock are usually expressed in neurosecretory cells in the brain, and these regions also regulate diapause-related hormones gene expression(Meuti and Denlinger, 2013). In non-diapause mutation files of Chymomyza costata , there is a mutation in 5’ leader sequence of tim gene, which suggested TIM plays an important role in it regulation for diapause (Pavelka, Shimada and Kostal, 2003). When per and cry2 were knocked down in bean bug, Riptortus pedestris , it caused the ovarian development of female adults which should have entered diapause in short photoperiod. Similarly, RNA interference (RNAi) was used to interrupt expression of per and cry2 in male adult, which can avoid diapause (Ikeno, Numata and Goto, 2011). Research above indicate that circadian genes can involve in photoperiodic diapause. Hence, for the development of diapause related research of C. japonica , it is important to understand the pathway patterns of circadian genes and diapause-related genes in the genome of the species.
Diapause of resources insects and nature enemies is an important but unaddressed issue. It is a key technical barrier restricting the quantity of products and industrial development in the utilization of resource insects. For example, in the silk industry, the diapause of silkworm will affect the quality and yield of silk. Therefore, the diapause removal of silkworm and its related regulation mechanism have been studied very deeply. If the female adult experiences high temperature during the embryonic period, it will produce diapause eggs (Tsuchiya et al ., 2020). In biological control, the simple acquisition of alternative hosts is the key to the large-scale propagation of natural enemy insects. Diapause also exists in the industrial production of A. pernyi , an important intermediate host of natural enemies such as Trichogrammatidae and Eupelmidae. However, the commercial A. pernyi has been cultivated, there are still some limitations in quality and yield. Not only Lepidoptera, but other natural enemy insects will also be refrigerated to facilitate storage and transportation in biological control, which will cause diapause. The common problem is that the natural enemy insects take a long time to remove diapause, which will affect their control effect on pests. The cocoon of pupa of C. japonica can used to produce natural material. So, it has great potential to explore as a resource insect for its medical value. It is meaningful to know how to regulate the diapause of C. japonica.
The evolution and fluctuation of expression of the gene according to diapause usually caused by environment. However, the critical values of natural conditions for diapause of C. japonica , such as photoperiod and temperature, have not been explored clearly. Therefore, we will quantify the impact of these abiotic factors on diapause and to explore the gene foundations according to biological changes. The expression of each gene in the C. japonica still needs to be explored clearly to better carry out the follow-up verification experiment.
In summary, we firstly reported a high-quality chromosome scale genome of C. japonica and discuss the evolution of diapause related genes. It will provide a valuable database for the future study around resource insect and biomedical materials development.