Study On The Olfactory Mechanism Of CO₂ Sensing In Labial Palp Of Hyphantria Cunea

Hyphantria cunea is a worldwide quarantine pest with a wide range of transmission and strong adaptability,can survive in both low and high temperature environments,and has a strong reproductive capacity.It can reproduce three generations a year,and each female moth can lay more than 1000 eggs,with a high hatching rate.During the larval stage,it undergoes a period of overeating,with a wide range of hosts,and increased destructive power to trees.Due to the above biological characteristics and few natural enemies,once invaded,it is easy to cause great losses to the local ecological environment and economy.Research has found that carbon dioxide(CO2)can have an impact on herbivorous insects,and exploring the molecular mechanisms by which insects perceive CO2 is of great significance.Currently,most research has focused on model insects,so it is particularly important to search for CO2 sensing receptors for Hyphantria cunea.Research has found that adult Lepidoptera insects use their labial palp to sense CO2,which they use to detect CO2 on the surface of plants.Currently,there is also little research on the molecular mechanism of insect perception of CO2.The model organism Drosophila melanogaster has two CO2 receptors that participate in the perception,while Anopheles gambiae has three CO2 receptors that are expressed in the maxillary palps.The Helicoverpa armigera in Lepidoptera also has three CO2 receptors that are expressed in the labial palps,indicating that different species have different CO2 sensing mechanisms.At present,the CO2 sensing mechanism of the important forestry pest,Hyphantria cunea,among the non-model insects is not clear,so we have made the following research on its CO2 sensing mechanism,and the results are as follows:1.Through screening and bioinformatics analysis of the annotated information in the genome and transcriptome database of Hyphantria cunea,44 candidate GRs genes were identified,with a similarity of 35%to 92%among Lepidoptera species.Using the GRs of other Lepidoptera insects,such as Bombyx mori,Manduca sexta,Plutella xylostella,Heliconius melpomene,Pieris rapae,Helicoverpa armigera,Helicoverpa assulta,Dendrolimus punctatus,and Danaus plexippus,as well as two model organisms,Drosophila melanogaster and Anopheles gambiae,which contain genomic data,and Hyphantria cunea,the phylogenetic tree was constructed.It was found that Hyphantria cunea has four gustatory receptor families,including bitter receptor,sugar receptor,fructose receptor,and CO2 receptor.HcunGR1-3 were distributed in the CO2 receptor branch,while the HcunGR16 were distributed in the fructose receptor branch;HcunGR4-10,HcunGR12-14 were distributed in the sugar receptor branch;HcunGR11 and HcunGR44 were distributed in the bitter receptor branch.2.Determine the expression patterns of HcunGRs in different tissues of adults based on transcriptome data.Among them,HcunGR1,HcunGR2,and HcunGR3 are specifically expressed in the labial palps,which may be related to CO2 perception;HcunGR16 and HcunGR19 are specifically expressed in the antennae,which may be related to olfactory perception;In addition,HcunGR5,HcunGR17 and HcunGR27 are also expressed in non-olfactory organs such as the head,chest,abdomen,and legs,which may be related to other biological functions.Q-PCR verified the expression levels of three genes,HcunGR1,HcunGR2,and HcunGR3,in different tissues.Among the labial palp,HcunGR1>HcunGR3>HcunGR2 was expressed,and HcunGR2 was also highly expressed in the thorax of adults and female moth antennaes.3.The genes HcunGR1,HcunGR2,and HcunGR3 were cloned and obtained.At the same time,the heterologous expression of these three genes in Xenopus oocytes was studied using voltage clamp.NaHCO3 solution was used to replace CO2 gas dissolved in a liquid environment,and the concentration of dissolved CO2 was calibrated by measuring the pH of the NaHCO3 solution.The results showed that oocytes expressing HcunGR1,HcunGR2,or HcunGR3 alone could not be activated by high concentrations of CO2 after excluding the effect of Na+.The expression systems of HcunGR1+HcunGR3 and HcunGR1+HcunGR2+HcunGR3 in coexpression combinations could be specifically activated by high concentrations of CO2,with channel currents of 330 nA and 242 nA,respectively,and showed concentration dependence.At the same time,the results showed that the response of the ternary system(HcunGR1+HcunGR2+HcunGR3)was smaller than that of the binary system(HcunGR1+HcunGR3).It was speculated that HcunGRl and HcunGR3 were the decisive genes for the perception of CO2 in Hyphantria cunea,and HcunGR2 might participate in it and play a regulatory role.4.immunohistochemical staining was used to study the whole brain and labial palp of Hyphantria cunea,the results showed that the neuronal projection mechanism of the labial palp was bilateral projection,and the main projection area of the neurons was a single glomerulus located at the dorsal rear of the antennal lobe.We named it DP1,and a part of it projected onto the gnathal ganglion.Through 3D modeling of DP1,we found that there is a difference in its volume between male and female,and the volume of female moth DP1 is significantly larger than that of male moth.We speculate that this may be related to female moth specific behaviors such as oviposition selection and larval information recognition.5.Based on the electrophysiological and Y-tube behavioral experiments of the antennae and labial palp of Hyphantria cunea,it was found that both the labial palp and antennae of Hyphantria cunea can be activated by CO2 in the concentration range of 1%to 10%.With the increase of CO2 concentration,the response of the antennae(labial palp)also increases,and the electrophysiological response of the labial palp to CO2 is significantly greater than that of the antennae,proving that the labial palp are the main organ for sensing CO2 in Hyphantria cunea.The results of a Y-type olfactometer showed that CO2 within this range had a significant attraction to the Hyphantria cunea adult,and its attraction rate increased with the increase of concentration,ranging from 53.33%to 68.33%.In summary,this study has conducted a systematic study on the peripheral molecular mechanism and secondary neural projection mechanism of Hyphantria cunea’s perception of CO2,and explored the function of CO2 in the life activities of Hyphantria cunea in combination with electrophysiological and behavioral experiments.The relevant results have enabled us to better understand the peripheral and secondary neural projection mechanism of the non model insect Hyphantria cunea’s perception of CO2,as well as preliminarily explore the specific impact of CO2 on the behavioral aspects of Hyphantria cunea,On the basis of revealing the olfactory mechanism of non model insects’ perception of CO2,clarify the impact of CO2 concentration changes on insect behavior,thereby providing more rigorous theoretical support for advanced pest management in the context of the increasing greenhouse effect.