| The cuticle of different parts of the insect body or the cuticle at different stages of development have different properties,which are caused by differences in the formation of chemical components and molecular interactions in the epidermis,as well as the thickness and arrangement of different structural layers within the epidermis.Termites of different grades of exocuticle pigment deposition and hardening degrees of different levels make them have different appearance characteristics,and there is an inevitable connection between different morphologies and a clear division of labor.Compared with the hard exocuticle,the endocuticle is soft and located under the exocuticle,and the relationship between the endocuticle of termites and the differentiation of grades and the changes in the structure of the epidermis during the development of differentiation are still unclear.In order to explore the relationship between the endocuticle of termites and grade differentiation,this study took the Reticulitermes aculabialis as the research object,firstly,paraffin section and Hematoxylin-eosin staining(HE staining)were used to study the thickness of the exocuticle and endocuticle of larvae,worker nymphs,soldier,adults and neotenic reproductive in the process of grade differentiation;observation of ultrastructure of the endocuticle in the head,thorax and abdomen of workers using transmission electron microscopy(TEM);elucidation of the dynamic changes of expression levels of five endocuticle protein genes in individuals at different developmental stages in the two developmental pathways of the grade differentiation process;and RNA in situ-hybridization(RNA scope)Technology to study the expression localization of endoepithelial protein genes SgAbd-2 and SgAbd-9 in the head of worker.These studies revealed the characteristics of the endocuticle in the development of termite grade differentiation,which provided a basis for indepth elucidating the relationship between the endocuticle and termite grade differentiation and grade function.The main findings and conclusions of this study are as follows:1.A study of the thickness of the exocuticle and endocuticle of termite larvae,worker,nymphs,soldier,adults and neotenic reproductive on the head,thorax and abdomen of termites with R.aculabialis.Through the analysis of HE-stained tissue section data,the results showed that the thickest exocuticle was the head exocuticle of adult(5.68±1.30 μm)and the exocuticle of the thorax(6.31±1.56 μm),and there was no significant difference between the two(P > 0.05);the exocuticle thickness of the adult abdomen was 3.01±0.89 μm,and the exocuticle thickness of the head and thorax was about twice that of the abdomen.The exocuticle thickness of the head of the worker was 1.96±0.34 μm,and the exocuticle thickness of the head of the soldier was2.68±0.21 μm,and there was a significant difference in the exocuticle thickness between the two(P < 0.05).The thickness of the exocuticle of the head of the worker is about 4 times that of the thorax(0.56±0.07 μm)and the thickness of the abdomen(0.48±0.11 μm),and the exocuticle thickness of the head of the soldier is about 3 times that of the thorax(1.18±0.33 μm)and the thickness of the abdomen(0.91±0.16 μm).Larvae,nymphs,and neotenic reproductive have the thinnest exocuticle on the head,thorax,and abdomen,as well as on the thorax and abdomen of worker,and there are no significant differences between them.The thickness of the exocuticle of adult accounts for about 80% of the cuticle,which is the largest of all grades.The thickest endocuticle is the endocuticle of the worker’s head,which is 16.07 ± 5.68 μm thick,which is about 7 times the thickness of the thorax(2.26±0.52 μm)and the thickness of the abdomen(1.82±0.42 μm).After the larvae differentiated into worker and nymphs,the thickness of the endocuticle of the head of the worker was about 4 times that of the endocuticle of the head of the nymph(3.65±0.98 μm),but the thickness of the endocuticle in the thorax and abdomen of the nymph was significantly thicker than that of the thorax and abdomen of the worker.The thickness of the endocuticle of the head of the worker is about 6 times and 4 times that of the soldier ant and the neotenic reproductive,respectively,but there is no significant difference in the thickness of the endocuticle of the thorax and abdomen.The thickness of the endocuticle of the adult head,thorax and abdomen was 0.96±0.18 μm,0.62±0.22 μm,0.48±0.188 μm,respectively,which was the thinnest among individuals with grade development,and the endocuticle thickness of the head,thorax and abdomen of the nymphs was about 4 times,6 times and 5 times that of the adult,respectively.In addition,there was no significant difference in the thickness of the endocuticle between the head,thorax and abdomen of the larvae,and there was no significant difference in the thickness of the endocuticle between the head and the thorax of the larvae.The thickness of the endocuticle in the head of the worker ant accounts for about 84% of the cuticle,accounting for the largest proportion of all grades.These data suggest that if the molting of an nymphs develops into adult,the endocuticle becomes thinner and the exoclticle is extremely thickened.Adults have the thickest exoclticle and the thinnest endocuticle,which increases the hardness of the cuticle and is conducive to protecting the body from external aggression;it is conducive to supporting muscle movement to enhance the ability to fly apart,and the thinning of the endocuticle can also reduce weight and facilitate long-distance flight.After the worker differentiates into soldier or neotenic reproductive,the endocuticle becomes significantly thinner,which may be that the thicker endocuticle of the worker can buffer external pressure when working with the head(foraging for food,building and repairing the nest,taking care of other individuals in the nest group),and the substances rich in the endocuticle may have special physiological functions.Therefore,our results also strongly demonstrate that the epidermal layer structure of termites with R.aculabialis is related to the reproductive pathway and non-reproductive pathway of termite grade differentiation,and the change of endocuticle structure during termite grade differentiation reflects the adaptation of the endocuticle and function of each grade.2.Ultrastructure of the cuticle within worker was observed by transmission electron microscopy(TEM).The ultrastructure observation of the cuticle of the head,throax and abdomen of the worker shows that the surface of the head of the worker has a conical spike structure,and the endocuticle of the head is composed of five horizontal layers,each of which shows a similar degree of electron density.However,no conical spike structure was found on the surface of the thorax and abdomen of the worker ant,and there was no multi-layer structure in the inner epidermis of the thorax and abdomen,only one layer of structure.This study shows that the head of the worker has a special cuticle structure different from that of the thorax and abdomen,and the epidermal protrusion of the conical spikes of the head can make the worker more quickly and efficiently excavate the ant path,and the endocuticle with 5 thick brick-like horizontal layers can withstand external pressure and protect the head from injury.This is the first time that the ultrastructural characteristics of the endocuticle of the head of the worker have been found,which is of great significance for revealing the unique behavioral function of the worker.3.Expression levels of five endocuticle protein genes(3 SgAbd-2,1 SgAbd-9,1 ABD-5)in worker,soldier,nymphs,adults,and neotenic reproductive were compared using qRT-PCR analysis.The results showed that the expression level of the five endocuticle genes in the worker was higher than that of the soldier and adults,and the expression levels of the 3 SgAbd-2 genes,1 SgAbd-9 gene,and 1 ABD-5 gene were 71 times,8 times,23 times,9 times and 7 times that of the soldier,respectively,and 55 times,66 times,11 times,16 times and 2 times that of the adults,respectively.Except for SgAbd-2(unigene0034952)worker expression and neotenic reproductive,the expression level of the remaining 4 genes in the worker was significantly higher than that of the neotenic reproductive,except for Abd-5(unigene0003968)neotenic reproductive and adult expressions,the expression of the remaining 4 genes in the neotenic reproductive was about 7 times,33 times,11 times and 22 times the expression of adults,respectively.The nymphs expression of the five endocuticle protein genes was significantly higher than that of adults,and the expression in the nymphs was about 31 times,181 times,10 times,9 times and 4 times the expression of adults,respectively.Therefore,after the worker differentiate into soldier or neotenic reproductive,the expression level of endocuticle protein genes is significantly reduced;if the nymphs develop into adults through molting,the expression level of endocuticle protein genes decreases.The difference in the expression level of endocutcle protein genes in different grades is consistent with the difference in their endocuticle thickness,and the difference in endocuticle protein gene expression plays a very important role in the formation of termite grade-specific phenotypes,indicating that endocuticle protein is involved in termite grade differentiation and social division of labor.4.The expression localization of endocuticle protein genes SgAbd-2 and SgAbd-9 in the head of worker was studied by RNA in situ hybridization.On the basis of qRT-PCR research,two genes,SgAbd-2(unigene0001350)and SgAbd-9(unigene0016014),were selected to perform the first localization analysis of the expression of endocuticle protein genes in the head of worker by RNA in situ hybridization technology.The results showed that the two endocuticle protein genes of SgAbd-2 and SgAbd-9 were expressed in the epidermis adjacent to the endocuticle of the worker’s head,and the epidermis appeared blue-purple after in situ hybridization by RNA.Endocuticle protein is synthesized and secreted by epidermis,and this study clarified the localization of endocuticle genes SgAbd-2(unigene0001350)and SgAbd-9(unigene0016014)in epidermis,revealing that the formation of the endocutile of termites with the is related to the synthesis of endocuticle protein in epidermis.In summary,the thickness of the endocuticle and exocuticle of individuals will change in the two pathways of termite grade differentiation of the R.aculabialis,and the endocuticle will show dynamic changes in termite grade differentiation.Changes in the thickness of the endocuticle are regulated by the endocuticle protein gene,and a grade-specific epidermis is formed according to the division of labor of termites.The endocuticle is secreted from the epidermis,and the results of RNA in situ hybridization technique show that the endocuticle gene is expressed at the epidermis.In this study,we compared the endocuticle and exocuticle of individuals at different stages of development of nymph pathway(reproductive pathway)and worker pathway,the ultrastructure of the endocuticle of the head of the worker is revealed.The expression levels of five endocuticle protein genes in workers,soldiers,nymphs,adults and neotenic reproductive were analyzed and compared,and the expression levels of endocuticle protein genes in worker were revealed by RNA in situ hybridization technology was revealed for the first time.These studies elucidated the structural characteristics of the endocuticle during termite grade development,for the first time revealed that the endocuticle was related to termite grade differentiation and division.That is,after the worker differentiate into soldier and neotenic reproductive,the endocuticle becomes thinner,which is related to the fact that the worker are the main labor force in the nest and mainly work with their heads;nymphs differentiates into adults,the endocuticle becomes thinner and the exocuticle thickens,which is related to the flight of adults.This study provides a new basis for further study of the relationship between termites and the endocuticle. |
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