The Pupae Bionomics Of Apocheima Cinerarius Erschoff

Apocheima cinerarius Erschoff(Lepidoptera : Geometridae)is one of the main pests of Populus euphratica forest in Xinjiang desert,and it poses a threat to various forest and fruit plants such as poplar,willow,elm,jujube,pear,apple,and mulberry trees.A.cinerarius is a univoltine obligate diapause insect.The pupae oversummer and overwinter in the soil under the soil of the canopy is up to 10 months.There have been few reports on the biological characteristics of A.cinerarius during the pupal stage,but there are obvious appearance changes in different pupal stages.Therefore,exploring the biological characteristics of the pupal stage of A.cinerarius and clarifying the changes of gene expression in the pupal stage are not only helpful to understand the adaptability of A.cinerarius to the ecological environment,improve the level of population prediction,but also help to clarify the developmental regulation mechanism of pupal stage of A.cinerarius.This provides a scientific basis for formulating effective comprehensive management strategies.The results are as follows :1.External morphological characteristics of A.cinerarius pupaThe middle of the front edge of the 8th abdominal segment web of the female pupa has a ’ Y ’-shaped groove,which forms an inverted triangle with the rear edge of the 7th abdominal segment web,while the male pupa does not have this feature.Additionally,the female pupa reproductive pore is connected with the oviposition pore to form a crack,and the two sides are relatively flat without protrusions,while the male pupa has a longitudinal crack in the center of the 9th abdominal segment web,and there are semi-circular tumor-like protrusions on both sides.The relative length between the thoracic dorsal plate of the female pupa is smaller than that of the male pupa,while the thoracic dorsal plate of the male pupa was the widest,and its trailing edge was obviously protruded outward.These features can be used to quickly,non-destructively,and accurately distinguish between male and female of A.cinerarius pupae.However,there are no significant differences in length and width between female and male of A.cinerarius pupae.2.Developmental characteristics of A.cinerarius pupaAccording to the combination of field sampling and greenhouse rearing,the color changes of the pupal cuticle were systematically observed.The pupal cuticles were observed using a stereo microscope,and images were captured using ZEN 2 software.The RGB values of the pupal cuticles were measured using the color picker tool in Photoshop software.The results showed that under natural conditions,the color of the pupal cuticle gradually deepened from yellow-brown to reddish-brown and then gradually turned to black-brown with the development of A.cinerarius pupa.The RGB values of the color change process showed a decreasing trend from large to small.In the early pupation stage,the pupal cuticle is generally yellow-brown,with a slightly light green color on the head,corresponding to the maximum RGB value.From May to June,the color of the pupal cuticle deepens,and from June to July,the color change from reddish-brown to black-brown becomes more evident,and black-brown color occupied most of the pupal stage,during which the RGB values reach the minimum and show a horizontal fluctuation trend.By unshaving the male and female pupae at different developmental stages,the color change rule of the pupae after removing the pupal shell was similar to that of the pupal shell.At the beginning of May,the pupal shell was light yellow liquid,and the appendages had not yet begun to differentiate.The pupae in June have developed a complete body,but did not form a formed internal genitalia.From September to December,the body color changed from reddish brown to dark brown.In order to further clarify the structure of the internal reproductive organs and the developmental stages of ovarian development in female and male pupae.The anatomical results showed that :(1)The dissected pupae clearly revealed two sets of four ovarioles,with the number of eggs and their volume gradually increasing inside the ovarioles and the color of the eggs deepened to a dark yellow,and the mating pouch,fertilization pouch,accessory gland pouch,and accessory glands became more distinct and increased in size.The testes and seminal vesicles of male pupae gradually enlarged and matured,and the accessory glands grew thicker.The color of the accessory glands deepened.(2)The internal reproductive organs of female pupae included one pair of ovaries,one pair of lateral oviducts,one fertilization pouch,accessory glands,an ovipositor,a mating pouch,and one median oviduct,as well as eight symmetrical ovarian tubules.The internal reproductive organs of male pupae included one testis,one vas deferens,one seminal vesicle,and one pair of accessory glands,as well as one ejaculatory duct.(3)The ovarian development in female pupae was preliminarily classified into five stages: undifferentiated stage(Stage I),milky transparent stage(Stage II),yolk deposition stage(Stage III),mature and ready to lay eggs stage(Stage IV),and final oviposition stage(Stage V).In September,the ovaries of female pupae were primarily at Stage I or II.In October and November,most of the ovaries were at Stage II or III.After December,the majority of the ovaries reached Stage IV.(4)The pupal stage was divided into four stages based on the color change of the pupal cuticle,the color of the internal larval body,and the development of the internal reproductive system.The four stages were as follows: Stage 1 pupa in May,Stage 2 pupa from early June to late August,Stage 3 pupa from early September to late November,and Stage 4 pupa from early December to late February.3.Transcriptome analysis of the pupal diapause of A.cinerariusTranscriptome sequencing of A.cinerarius pupal stage was conducted using the high-throughput Illumina Hi Seq platform.The results showed that a total of 287,231 unigenes were obtained,with a total sequence length of 175.63 Mb and an average length of 611.46 bp per sequence.Among them,60,595,27,968,30,829,23,939,54,871,and 40,225 unigenes were annotated in the Nr,GO,KEGG,Pfam,egg NOG,and Swiss-prot databases,respectively.For female pupae,a comparison between May and October revealed 4,905 upregulated genes,10,323 downregulated genes,and 15,228 differentially expressed genes,showing the most significant differential expression.A comparison between May and June showed 56 upregulated genes,392 downregulated genes,and 448 differentially expressed genes,indicating the least significant difference.For male pupae,a comparison between May and October showed 5,111 upregulated genes,3,963 downregulated genes,and 9,074 differentially expressed genes.A comparison between May and August revealed 23 upregulated genes,229 downregulated genes,and 252 differentially expressed genes,indicating the least significant difference.Among them,the ALP,CAT,POD,SOD,TRE,LDH,PK,FAR,CYC,and PDHAα genes showed significant differences between different stages.The FPKM values of PK,CAT,SOD,and POD genes increased during the summer diapause period,while the FPKM values of PDHAα,FAR,TRE,LDH,and CYC genes increased during the winter diapause period.The ALP gene in female pupae showed increased FPKM values during the summer diapause and decreased FPKM values during the winter diapause,while the opposite pattern was observed in male pupae.Among the 10 genes,the CYC gene had the highest FPKM value,and the LDH gene had the lowest FPKM value.