Development and formation of wing cuticle based on transcriptomic analysis in Locusta migratoria during metamorphosis

Wings are an important flight organ of insects.Wing development is a complex process controlled by a series of genes.The flightless wing pad transforms into a mature wing with the function of migratory flight during the nymphto-adult metamorphosis.However,the mechanism of wing morphogenesis in locusts is still unclear.This study analyzed the microstructures of the locust wing pads at pre-eclosion and the wings after eclosion and performed the comparative transcriptome analysis.RNA-seq identified 25,334 unigenesand 3,430 differentially expressed genes(DEGs)(1,907 up-regulated and 1,523 down-regulated).The DEGs mainly included cuticle development(LmACPs),chitin metabolism(Lm Idgf4),lipid metabolism-related genes,cell adhesion(Integrin),zinc finger transcription factors(LmSalm,LmZF593 andLmZF521),and others.Functional analysis based on RNA interference and hematoxylin and eosin(H&E)staining showed that the three genes encoded zinc finger transcription factors are essential for forming wing cuticle and maintaining morphology in Locusta migratoria.Finally,the study found that the LmSalm regulates the expression of LmACPs in the wing pads at pre-eclosion,and LmZF593 and LmZF521 regulate the expression of LmIntegrin/LmIdgf4/LmHMT420 in the wings after eclosion.This study revealed that the molecular regulatory axis controls wing morphology in nymphal and adult stages of locusts,offering a theoretical basis for the study of wing development mechanisms in hemimetabolous insects.

Low-density lipoprotein receptor-related protein 2(LRP2)is required for lipid export in the midgut of the migratory locust,Locusta migratoria

Low-density lipoprotein receptor-related protein 2(LRP2)is a multifunctional endocytic receptor expressed in epithelial cells.In mammals,it acts as an endocytic receptor that mediates the cellular uptake of cholesterol-containing apolipoproteins to maintain lipid homeostasis.However,little is known about the role of LRP2 in lipid homeostasis in insects.In the present study,we investigated the function of LRP2 in the migratory locust Locusta migratoria(LmLRP2).The mRNA of LmLRP2 is widely distributed in various tissues,including integument,wing pads,foregut,midgut,hindgut,Malpighian tubules and fat body,and the amounts of LmLRP2 transcripts decreased gradually in the early stages and then increased in the late stages before ecdysis during the nymphal developmental stage.Fluorescence immunohistochemistry revealed that the LmLRP2 protein is mainly located in cellular membranes of the midgut and hindgut.Using RNAi to silence LmLRP2 caused molting defects in nymphs(more than 60%),and the neutral lipid was found to accumulate in the midgut and surface of the integument,but not in the fat body,of dsLmLRP2-treated nymphs.The results of a lipidomics analysis showed that the main components of lipids(diglyceride and triglyceride)were significantly increased in the midgut,but decreased in the fat body and hemolymph.Furthermore,the content of total triglyceride was significantly increased in the midgut,but markedly decreased in the fat body and hemolymph in dsLmLRP2-injected nymphs.Our results indicate that LmLRP2 is located in the cellular membranes of midgut cells,and is required for lipid export from the midgut to the hemolymphand fat body in locusts.

Serine protease inhibitors LmSPN2 and LmSPN3 co-regulate embryonic diapause in Locusta migratoria manilensis(Meyen)via the Toll pathway

Female adults of the migratory locust,Locusta migratoria manilensis(Meyen),can sense seasonal photoperiod changes,which induces embryonic diapause as a key strategy to overwinter.Serine protease inhibitor genes(SPNs)were thought to play key roles during diapause,while few SPNs were functionally characterized.LmSPN2 was one of those genes differentially expressed between diapause and non-diapause eggs;however,its biological function remained to be explored.So,we conducted RNAi knockdown of LmSPN2,resulting in a significant decrease of the egg diapause rate by 29.7%.Using yeast two-hybrid assays,co-immunoprecipitation,and pull-down methods,we found an interaction between LmSPN2 and LmSPN3,which was proved to be mediated by a glutamate(E331)binding site of LmSPN2.RNAi knockdown of LmSPN3 resulted in a significant increase in diapause rate by 14.6%,indicating an inverse function of LmSPN2 and LmSPN3 on diapause regulation.Double knockdown of two SPN genes resulted in a 26.4%reduction in diapause rate,indicating that LmSPN2 was the dominant regulatory signal.Moreover,we found four Toll pathway genes(easter,spätzle,pelle,and dorsal)upregulated significantly after the knockdown of LmSPN2 while downregulated after the knockdown of LmSPN3.Therefore,we speculate that two SPNs regulate diapause through the Toll pathway.Our results indicated that LmSPN2 positively regulates locust egg entry into diapause,while LmSPN3 is a negative regulator of embryonic commitment to diapause.Their interaction is mediated by the binding site of E331 and influences egg diapause through the Toll pathway.This mechanistic understanding of diapause regulation expands our understanding of insect developmental regulation and provides functional targets for developing locust management strategies.

The Phylogenetic Relationships among Four Subspecies of the Genus Locusta Based on Sequences of Three Subunit of Cytochrome Oxidase

[Objective] The aim was to explore the phylogenetic relationships among four subspecies of the genus Locusta.[Method] The sequences of three subunits of cytochrome oxidase of Locusta migratoria tibetensis and Locusta migratoria manilensis were amplified and sequenced（COⅠ 1 539 bp,COⅡ 684 bp,CO Ⅲ 792 bp,with the total of 3 015 bp）.The corresponding sequenses of Locusta migratoria migratoria and Locusta migratoria migratorioides were obtained from GenBank and constructed a multiple alignment.Phylogenic trees of four subspecies of L.migratoria were constructed by Neighbor-Joining,Maximum-parsimony and Bayesian,respectively.[Result] The average content of A ＋ T in three subunits of four subspecies was 69.57%;the third site of codon showed the highest A ＋ T content,and the COⅠ had the highest A ＋ T content（87.6%）;The nucleotide substitution mainly occurred at the third site of codon,and the nucleotide replacement rate of CO Ⅱ was the highest.The second site of codon was conservative,so the replacement rate was in the range of 5.9%-15%.The start codon of COⅠ was CCG or ACG.Genetic distances among four subspecies were ranged from 0.001 to 0.076.The relationship between L.m.tibetensis and Locusta migratoria manilensis was the closest,followed by L.m.migratorioides and L.m.migratorioides,while the genetic distance between L.m.tibetensis and L.m.migratorioides was the largest.[Conclusion] The phylogenetic relationships among four subspecies of Locusta migratoria is L.m.tibetensis,L.m.manilensis,L.m.migratoria,L.m.migratorioides.

Detection and Comparison of Phenoloxidase Activity of Locusta migratoria manilensis (Meyen) by Different Extraction Methods

[Objective] The research aimed to study the activity and dynamic changes of phenoloxidase in Locusta migratoria manilensis（Meyen） .[Method] Two enzyme solutions of insect body and hemolymph of Locusta migratoria manilensis（Meyen） were prepared by different extract methods,the activity and dynamic changes of phenoloxidase in different sites were measured.[Result] The phenoloxidase activity in hemolymph was significantly higher than that in body fluid,and the activity in female individuals was higher than that in male individuals.The phenoloxidase activity in body fluid was gradually enhanced with the prolongation of laying time in air.The phenoloxidase activity in hemolymph of adult was also gradually enhanced with the prolongation of time.[Conclusion] Part of phenoloxidase was existed in tissues and cells of Locusta migratoria manilensis（Meyen） participating in insect development and construction,and the other part was existed in hemolymph playing an immunological and defensive role.The stability of phenoloxidase was very strong,which was the effective guarantee for wide adaptation ability and strong immunity of insect.

Cloning and Functional Analysis of Circadian Gene pdp in Locusta migratoria

[Objective] Through studying mRNA expression levels of circadian gene pdp（Pyruvate dehydrogenase phosphatase） in Locusta migratoria under different conditions to reveal molecular mechanisms of rhythm of Locusta migratoria.[Methods] The prototype of the pdp gene and design primers were found out based on the database of Institute of Zoology,Chinese Academy of Sciences.Using aggregated locust head cDNA as templates,the complete sequence of the gene pdp was obtained.By dividing a day（24 hours） into eight points,fixed-point samples were taken,and the pdp gene expression levels at different time periods were determined by using qRT-PCR;and the pdp gene expression levels with different treatment were also determined.[Results] The circadian gene pdp in Locusta migratoria changed slightly with different treatment.[Conclusion] The study had extremely realistic significance for understanding the rhythm and population characteristics of Locusta migratoria as well as effective insect-resistant.

Effects of Paranosema locustae(Microsporidia) on the development and morphological phase transformation of Locusta migratoria(Orthoptera: Acrididae) through modulation of the neurotransmitter taurine

Neurotransmitters are important in the maintenance of phase transformation of Locusta migratoria(Arthropoda: Orthoptera). Here, the effects of the entomopathogen Paranosema locustae on the neurotransmitter taurine in migratory locusts were studied using biochemical methods. After inoculation with P. locustae, the taurine content of infected locusts significantly declined, but F/C values(ratio between the length of hind femur and the width of the head of locust) increased significantly, compared to healthy locusts. Meanwhile, F/C values of infected locusts that were injected with 2 μg of taurine showed no significant differences from those of healthy locusts, demonstrating that supplemental taurine inhibited the changes in morphological phase caused by P. locustae. Paranosema locustae infection also caused longer developmental durations and lower body weights of locusts, but these changes were unaffected after injection with taurine. These results provided new insights into the mechanisms by which microsporidian parasites affected their locust hosts.

西藏飞蝗(Locusta migratoria tibetensis Chen)耐寒性理化指标

西藏飞蝗是青藏高原上的一种重要害虫。近年来,西藏飞蝗在西藏、青海玉树以及川西高原上危害十分严重。通过对该虫各发育阶段过冷却能力测定,研究了各发育阶段虫体内生理生化物质与过冷却能力之间的关系,揭示了该虫耐寒性规律。结果表明,西藏飞蝗蝗卵的过冷却点和结冰点为最低,分别为-22.02℃和-16.36℃。在1-4龄蝗蝻期,过冷却点随体内水分、糖原和脂肪含量随着虫体的发育而升高,其过冷却点分别为-9.28℃、-8.51℃、-7.18℃、-6.46℃;到5龄蝗蝻时,虫体脂肪和糖原含量达最高值,过冷却点下降为-8.07℃。讨论了虫体脂肪、糖原和甘油含量变化与其耐寒力之间的关系。

沿海蝗区东亚飞蝗(Locusta migratoria manilensis)产卵场所选择的Logistic回归模型

以沿海蝗区南大港水库为研究区域,通过2002和2003两年野外450,50m规则栅格取样获取东亚飞蝗卵块、植物种类及其密度、土壤特性如含盐量、5cm含水量、pH、有机质及地形(阴坡和阳坡)等数据,采用多元Logistic回归模型,运用SAS软件筛选出与飞蝗产卵场所选择密切相关的变量,建立用于预测飞蝗产卵场所选择的Logistic回归模型。结果表明用植株密度(veg_d)和土壤含水量(water)所组建的模型能较好地预测飞蝗产卵选择,log1[P(Y=1)/(1-P(Y=1))]=21.63-76.23/water-5.43log(water)-0.86(veg_d)。利用拟合优度(Goodness of fit)、预测准确性(Predictive accuracy)及模型x2统计(Model chi-square statistic)等指标对模型进行评价的结果表明,所组建的用于预测飞蝗产卵场所选择的Logistic回归模型是可靠的,且能较好地预测事件是否发生。研究结果为区域蝗灾早期预警提供了科学依据和方法,对今后预测飞蝗产卵地点选择及防治决策有较高的实用性和应用价值。

东亚飞蝗Locustamigratoriamanilensis(Meyen)预测预报研究进展

本文对东亚飞蝗的分布及为害进行了简要介绍 ,并对其预测预报研究进展作了全面的总结 ,最后对今后预测预报工作进行了展望。本文认为今后对东亚飞蝗的预测预报分析应紧密结合环境条件的变化 ,应用空间相关分析方法对种群发生规律进行探讨 ,并利用时空分析的结果进行种群空间分布的估计与预测。系统科学理论与技术的应用将会进一步完善现行的东亚飞蝗预测预报方法 ,对我国东亚飞蝗的测报工作产生积极的影响。

Transcriptomic and proteomic analysis of Locusta migratoria eggs at different embryonic stages: Comparison for diapause and non-diapause regimes

Temperate-zone insects typically survive winter by entering diapause. Although many aspects of insect diapause have been studied, the underlying molecular mechanism of insect diapause is not well understood. Here we report the results of the transcriptional and translational differences of migratory locust eggs at different embryonic states using diapause （low temperature） and non-diapause （high temperature） regimes. Compared with non-diapause eggs at 100 degree-days （N2） treatment, 29671 transcripts and 296 proteins were differentially expressed at the diapause maintenance stage （D2）. While compared with 150 degree-days （N3） treatment, 45 922 transcripts and 404 proteins were differentially expressed in the post-diapause stage （D3）. Among them, 51 and 102 transcripts had concurrent transcription and translation profiles in D2 vs. N2 and D3 vs. N3 treatments, respectively. Analysis of Gene Ontology categorized these genes and proteins into three categories： biological processes, cellular components, and molecular functions. Biological pathway analysis indicat- ed that three pathways： （1） insect hormone biosynthesis （KEGG： Map 00981）, （2） the insulin signaling pathway （KEGG： Map 04910）, and （3） the peroxisome proliferator-activated receptor （PPAR） signaling pathway （KEGG： Map 03320） play an important role in locust diapause regulation. Most of these transcripts and proteins were up-regulated in the diapause treatments, and were highly linked to juvenile hormone biosynthesis, insulin and PPAR signaling pathways, suggesting these three pathways may be Jnvolved Jn diapause and development regulation. This study demonstrates the applicability of high-throughput omics tools to identify biochemical pathways linked to diapause in locust egg development. In addition, it reveals that cellular metabolism in diapause eggs is more inactive than in non-diapause eggs, and most of the down-reg- ulated enzymes and pathways are related to reduce energy loss.

Physiological, histopathological and cellular immune effects of Pergularia tomentosa extract on Locusta migratoria nymphs

The migratory locust Locusta migratoria(Orthoptera,Acrididae)is one of the most important pests due to its extensive and severe damage to crops in large parts of Africa and Asia.Biodegradable and ecologically natural products such as bota nical insecticides are emergi ng can didates for replaceme nt of usually applied chemical pesticides.The crude methanolic extract of Pergularia tomentosa(PME)was investigated for their toxicity and physiological aspects on L.migratoria nymphs.Results showed that treatment of newly emerged fourth and fifth instar nymphs resulted in significant mortality and significant repellent activity with an LC50 value of 0.18 and 0.38%,respectively,after seven days of treatment.The PME toxicity was also dem on strated by histopathological cha nges in the alime ntary canal resulting in con siderable disorganization and severe damage of the caeca and proventriculus structure.The extract induced cellular immune reactions which manifested by a significant decrease in the number of the differential haemocyte counts(prohemocytes and plasmatocytes)and important cell lysis.Data of biochemical analyses showed that the PME reduced the activity of acetylcholinesterase and induced the glutathione S-transferases.The neurotoxic effect was confirmed by the histological alterations in the brain structure,particularly in the neurosecretory cells showing typical signs of cell necrosis.

Effects of Glutamate and Na^+ on the Development and Enzyme Activity of the Oriental Migratory Locust,Locusta migratoria manilensis(Meyen) in Successive Generations

Rapid and mass rearing ofLocusta migratoria manilensis is an urgent need to meet the increasing demand for food of people. In this study, the effects of four artificial feeds on the development, reproduction and the activities of detoxification and protective enzymes of L. migratoria manilensis in three successive generations were investigated. The results showed that sucrose and monosodium glutamate （MSG） significantly increased the net reproductive rate （R0） and the intrinsic growth rate （rm） ofL. migratoria manilensis, but sodium chloride （0.17%） suppressed this increase. Furthermore, the artificial feed with sucrose and monosodium glutamate increased the activities of esterase （EST）, acetylcholinesterase （ACHE）, glutathione-S- transferase （GST）, multi-function oxidase （MFO）, phenol oxidase （PO）, catalase （CAT） and peroxidase （POD）, but inhibited the activity of superoxide dismutase （SOD）. However, sodium chloride （0.17%） increased the activities of EST, ACHE, CAT and SOD, and inhibited the activities of MFO, GST, PO and POD. Correlation analysis found that the increasing of PO activity and the decreasing of SOD activities were significantly related with the increasing of the intrinsic growth rate （rm）. The above results indicated that sucrose and monosodium glutamate could promote the development and reproduction of L. migratoria manilensis, but Na＋ inhibit such promotion with the concentration above 0.2%. The activities of PO and SOD can be used as biochemical standard to assess the effect of artificial feed.

东亚飞蝗（Locusta migratoria manilensis）卵巢和胚胎组织的原代培养

用东亚飞蝗４～５龄蝗蝻卵巢组织及孵化１２ｄ的胚胎组织，首次在我们改良的ＴＣ－１９９－ＭＫ和ＴＣ－１００培养基中，２８℃恒温培养，第１０ｄ可见大量透明的梭形细胞从卵巢块周围逸出，第１６０ｄ可见大量圆球状的胚胎细胞分裂成团，来自两种组织的细胞分别培养９０ｄ和３３０ｄ，均贴壁生长，但胚胎细胞贴壁不牢。从培养结果看，它们发展成连续细胞系的潜力很大。

Characteristics and roles of cytochrome b5 in cytochrome P450-mediated oxidative reactions in Locusta migratoria

Cytochrome b5(Cyt-b5)is a small heme protein and known to be involved in a wide range of biochemical transformations,in eluding cytochrome P450 monooxyge nase(CYP)-mediated metabolism of endoge nous and exogenous compo un ds.Studies on Cyt-b5 are more con centrated in mammals,but are relatively rare in in sects.The characteristics and functi on of Cyt-b5 from Locusta migratoria have not been described yet.We sequeneed the full-length cDNA sequenee of Cyt-b5 from L.migratoria(LmCyt-b5)by reverse transcription-PCR(RT-PCR)based on locust transcriptome database.The phylogenetic analysis showed that LmCyt-b5 was closely related to the Cyt-b5 from Blattodea.LmCyt-b5 was highly expressed in ovary,Malpighian tubules,midgut,gastric caeca,and fat bodies.Silencing of LmCyt-b5 had no effect on the susceptibility of L.migratoria to four different insecticides.Suppression of LmCyt-b5 or silencing of both LmCyt-b5 and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin(7-EC).However,coexpression of LmCYP6FD1 with LmCPR and LmCyt-b5 together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of L.mCYP6FD1 with cytochrome P450 reductase(LmCPR)or LmCyt-b5 separately.These results suggest that LmCyt-b5 plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments.Further study is needed to clarify the role of LmCyt-b5 in CYP-mediated catalytic reactions in L.migratoria.

亚洲飞蝗(Locusta migratoria L.)生物学特性及人工饲养技术

阐述了亚洲飞蝗的卵期发育情况,1～5龄幼虫的生长特点,生理特性,各时态持续时间,产卵量及越冬情况等生物学特性。同时根据其特性进行人工饲养,从中掌握亚洲飞蝗的发生规律,为亚洲飞蝗的防治提供参考。

东亚飞蝗Locusta migratoria manilensis(Mey.)膝下器中感觉细胞的超微结构

东亚飞蝗膝下器由具橛感器组成，每一具橛感器主要由三类细胞组成，即：感觉细胞、感橛细胞和冠细胞。感觉细胞位于最近端，细胞核圆且大，细胞质内含有丰富的线粒体、高尔基体、多泡小体等细胞器。感觉细胞向近端发出轴突进入中枢神经系统，向远端发出树突。树突内含有大量的线粒体、纵行微管，树突内最复杂的结构当属纤毛根，从近端到远端依次由远端基体、近端基体、主纤毛根和纤毛小根组成。树突顶端，由远端基体发出一条感觉纤毛，纤毛具有典型的“９＋０”结构。主纤毛根和纤毛小根具有明暗相间横纹结构，两横纹间的间隔距离约为６５ｎｍ。感觉纤毛穿过由感橛细胞形成的感橛空隙，末端进入一高电子密度的顶端细胞外结构——帽。感橛细胞内最明显的特征为具有感橛，感橛细胞围绕着远端树突和感觉纤毛部分，冠细胞紧密地包围着感橛细胞和帽。东亚飞蝗膝下器中同时含有一或两个感觉细胞的具橛感器，这在以往研究报道中是较为少见的。本研究的主要目的在于为以后对此器官的生理功能研究提供形态学的基础材料。

Expression and Characterization of a Sigma-Class Glutathione S-transferase of the Oriental Migratory Locust, Locusta migratoria manilensis (Meyen)

A cDNA encoding a sigma-class glutathione S-transferase of the locust, Locusta migratoria manilensis （LmGSTs1）, was cloned by reverse transcriptase-polymerase chain reaction. The 830 bp-long cDNA encoded a 615 bp open reading frame （204 amino acid polypeptide）, which exhibited the structural motif and domain organization characteristic of GST sigma-class. It revealed 59, 57, 57, and 56% identities to sigma-class GSTs from Blattella germanica, Gryllotalpa orientalis, Nasonia vitripennis, and Pediculus humanus corporis, respectively. A recombinant protein （LmGSTs1） was functionally expressed in Escherichia coli cells in a soluble form and purified to homogeneity. LmGSTs1 was able to catalyze the biotranslation of glutathione with 1-chloro-2,4-dinitrobenzene, a model substrate for GSTs, as well as with p-nitro-benzyl chloride. Its optimal activity was observed at pH 8.0 and at 30℃. Incubation for 30 min at temperatures below 50℃ scarcely affected the activity. The I50 of reactive blue （RB） was 18.5 μmol L-1. In the presence of 0.05 mmol L-1 ethacrynic acid （ECA）, LmGSTs1 showed （81±3）% of the original activities.

Effects of Aqueous Extracts of Seeds of Peganum harmala L. （zygophyllaceae） on 5th Stage Larvae Locusta migratoria cinerascens （Fabricius, 1781） （ Orthoptera： Oedipodinae）

The study has for objective the determination of the efficiency of the aqueous extracts from seeds of Peganum harmala L. on the mortality of the larvas of 5th stage and on the fertility of the female adults of Locusta migratoria cinerascens. For that purpose, a breeding of to custs was realized in th e conditions of labor atories. At hatching, the larvae are fed daily basis lawn Stenotaphrum americanum and a pro tein supplement of wheat b ran. The extraction of the aqueous extract of the seeds of P. harmala is done after maceration in the ethanol, under magnetic stirring using a rotavapor. To determine larval mortality L5, two modes of treatment have been made, one by contact and another by ingestion, using for both treatment 4 doses in a geometric progression, 0.03 mg/mL, 0.06 mg/mL, 0.12 mg/mL and 24 mg/mL. The results showed that the mortality for the doses of 0.12 mg/mL and 0.24 mg/mL, reaches respectively 40% and 60% on the 3rd day, as well for the treatment by contact as by ingestion. But the LD50 for ingestion treatment is lower. It is 0.19 mg/mL contrary to that of the contact treatment （0.19 mg/mL）. The larvae that survived the treatment by ingestion, have suffered morphological changes as well as physiological which consist of a deformation of the wings, delayed of the larval molt, of 6 day s, b locking the fledg ing, the change of the pigmentation as well as an extension of the preoviposition. Fertility was also affected and females lay only twice, a small number of eggs, unlike untreated females which come to lay 3 times with an average of 62.7 eggs/female at first spawning against 50 eggs for the females treated.

苏云金芽孢杆菌抗东亚飞蝗(Locusta migratoria manilensis)的活性

采用毒力生物测定的方法,测定了600株从我国各地土壤和死虫等样品中分离的苏云金芽孢杆菌对东亚飞蝗的毒力.这些菌株几乎分布于已知的70个H血清型.在600株苏云金芽孢杆菌中,有560株(占93.4%)对东亚飞蝗无毒(死亡率小于20%),只有3株(占0.5%)对东亚飞蝗有较高毒性(死亡率大于70%).同时对筛选出的特异性菌株的杀虫活性进行了进一步研究.