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.

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.

不同提取方法对东亚飞蝗酚氧化酶活性的测定与比较(英文)

[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.

培养基和温度对2株治蝗绿僵菌生长和毒力的影响

研究了不同培养基及培养温度对治蝗绿僵菌(Metarhizium anisopliae)菌株LA和LD菌落面积、产孢量及毒力的影响,结果表明:LA在SMAY和PPDA培养基上的菌落面积较大,在SDAY上次之,在PCA上最小,且在SMAY培养基上的产孢量最高;LD在SMAY、PPDA和SDAY培养基上的菌落面积较大,在PCA上较小,且也在SMAY培养基上的产孢量最高;LA在30℃下生长较快,而LD则在25℃生长较快,但2种菌株均在25℃下产孢量较高;在SMAY上培养的LA、LD对东亚飞蝗(Locusta migratoria manilen-sis(Meyen))毒力较高,LT50分别是4.3212d和4.8902d。

Characterization of Carboxylesterase Associated with Malathion Insensitivity in the Field Population of the Oriental Migratory Locust

Carboxylesterases （CarEs） from two field populations of the oriental migratory locust, Locusta migratoria manilensis （Meyen）, were examined to try to understand their contribution to malathion insensitivity. The CarEs activities in Wudi population （WD） were 1.75- and 1.50-fold significantly higher than those in Huangliu population （HL） when a-naphthyl acetate （a-NA） and [3-naphthyl acetate were used as substrates, respectively. Such elevated CarEs activities presented in the WD could be because of an increased staining intensity of the a-NA-hydrolyzing CarEs as shown on the nondenaturing polyacrylamide gel electrophoresis. Inhibition studies of CarEs using paraoxon and malaoxon indicated that CarE activities in the HL were more strongly inhibited than those in the WD. Furthermore, a 449-bp DNA fragment of CarE was obtained from L. migratoria manilensis. Hemiquantity reverse transcription-polymerase chain reaction analysis showed that CarE gene expression level in the WD was higher than that in the HL. The higher CarE activities and the increased CarE mRNA level in the WD appeared to be associated with decreased susceptibility to malathion in the WD due to the application of organophosphorus insecticides.

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.

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.

西藏飞蝗蝗卵的孵化

蝗灾,历来是农业生产上重要的自然灾害之一。其来势猛,损失惨重。治理蝗灾,必须要在了解飞蝗的孵化、取食、产卵及迁移等习性的基础上,方能行之有理、施之有效。西藏飞蝗作为飞蝗属中分布于我国的一特有亚成份,其形态特征较早就有记载。如 Uvarov 曾于1921年和1936年根据采自西藏的蝗虫标本先后报道西藏地区发现飞蝗,但其亚种未确定。一直到1963年,我国的陈永林先生才给予正式确定为一新亚种即:Locusta migratoria tibetensis Chen。

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.

蝗虫的爪与爪垫在与基体表面附着时所发挥的作用

在我国的新疆、内蒙等地,以及世界其他地区,每年都会爆发蝗虫灾害。据香港《文汇报》报道,澳大利亚自2010年4月上旬爆发了严重的蝗虫灾害(图1),席卷了澳洲东南部的昆士兰州、新南威尔士州、维多利亚州和南澳大利亚州等四个州,覆盖约50万平方公里的区域,造成的农业经济损失达20亿澳元。

东亚飞蝗(Locusta migratoria manilensis Meyen)在中国的发生动态

东亚飞蝗系相当典型的草原型昆虫,它具有草原型昆虫的生活特征,如对温湿度有较大的适应范围,喜光,运动器官发达,能作远距离迁移,有群集习性等。主要分布于中国东部农业区边缘的草原地带,故易于造成农业大灾害,为中国历代著名大害虫之一。

亚州飞蝗(Locusta migratoria Меуеn)消化道中苯基硫酸酯酶的活性

飞蝗是我国农业上主要害虫之一,测定其消化道中的某些酶的活性对於抗蝗药剂的设计、合成和应用可以提供一些有用数据。一.实验方法取飞蝗的前肠及中肠,将它们分别作成丙酮粉,再用此粉的甘油提取液与硫酸苯基钾作用,然后以此色法观察其苯基硫酸酯酶的活性。

Alternative relay regulates the adenosine triphosphatase activity of Locusta migratoria striated muscle myosin

Locust(Locusta migratoria)has a single striated muscle myosin heavy chain(Mhc)gene,which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon.The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain,that is,the N-terminal SH3-like domain,one lip of the nucleotide-binding pocket,the relay,and the converter.Here,we investigated the role of the alternative regions on the motor function of locust muscle myosin.Using Sf9-baculovirus protein expression system,we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin(HMM),including the major isoform in the thorax dorsal longitudinal flight muscle(FL1)and 4 isoforms expressed in the abdominal intersegmental muscle(AB1 to AB4).Among these 5 HMMs,FL1-HMM displayed the highest level of actin-activated adenosine triphosphatase(ATPase)activity(hereafter referred as ATPase activity).To identify the alternative region(s)responsible for the elevated ATPase activity of FL1-HMM,we produced a number of chimeras of FL1-HMM and AB4-HMM.Substitution with the relay of AB4-HMM(encoded by exon-14c)substantially decreased the ATPase activity of FL1-HMM,and conversely,the relay of FL1-HMM(encoded by exon-14a)enhanced the ATPase activity of AB4-HMM.Mutagenesis showed that the exon-14a-encoded residues Gly474 and Asn509 are responsible for the elevated ATPase activity of FL1-HMM.Those results indicate that the alternative relay encoded by exon-14a/c play a key role in regulating the ATPase activity of FL1-HMM and AB4-HMM.

A neural m^(6)A pathway regulates behavioral aggregation in migratory locusts

RNA N6-methyladenosine(m^(6)A),as the most abundant modification of messenger RNA,can modulate insect behaviors,but its specific roles in aggregation behaviors remain unexplored.Here,we conducted a comprehensive molecular and physiological characterization of the individual components of the methyltransferase and demethylase in the migratory locust Locusta migratoria.Our results demonstrated that METTL3,METTL14 and ALKBH5 were dominantly expressed in the brain and exhibited remarkable responses to crowding or isolation.The individual knockdown of methyltransferases(i.e.,METTL3 and METTL14)promoted locust movement and conspecific attraction,whereas ALKBH5 knockdown induced a behavioral shift toward the solitary phase.Furthermore,global transcriptome profiles revealed that m^(6)A modification could regulate the orchestration of gene expression to fine tune the behavioral aggregation of locusts.In summary,our in vivo characterization of the m^(6)A functions in migratory locusts clearly demonstrated the crucial roles of the m^(6)A pathway in effectively modulating aggregation behaviors.

Peculiar influence of linearly polarized spectrum illumination patterns on the sensitivity characteristics of locust response to polarized light

This study investigated the influence of different linearly polarized spectrum lights on locusts polartactic response characteristics linearly polarized vector sensitivity mode and polartactic response by using linearly polarized spectrum vector light module and experimental device.The objective was to clarify the vector sensitivity characteristics and functional effect of linearly polarized light spectrum intensity on locusts polartactic response,determine the influence specificity of linearly polarized spectrum illumination properties on locusts polarization-related behavior.When spectrum and illumination were constant,locusts polartactic response,presenting the response feature of sine and cosine function change specificity,was related to spectrum attribute.The visual acuity effect stimulated by violet spectrum was the best,whereas the optical distance modulation effect induced by orange spectrum was the strongest.When illumination was enhanced,locusts vector sensitivity mode shifted to present the specific sensitivity prompted by light intensity at long distance and inhibited by light intensity at short distance.Moreover,the regulating function of violet spectrum was the strongest,and the regulatory mutation effect of orange spectrum was the least significant.Simultaneously,locusts polartactic sensitivity to 300°vector at 100 lx,whereas to 240°vector at 1000 lx of linearly polarized violet light was the strongest.Locusts polartactic aggregation and visual tendency sensitivity to 90°vector at 100 lx,whereas to 270°vector at 1000 lx of linearly polarized violet light was the strongest.The heterogeneous regulation function of different linearly polarized spectrum couplings with light intensity led to significant variations in locusts vector sensitivity mode.This was derived from the antagonistic and specific tuning characteristics of locusts polartactic vision,reflecting the integrated output effect of locusts vector dependence regulated by linearly polarized spectrum intensity attribute.The findings were significant for the construction of pest polarization induction light sources and the investigation of the sensitive physiology pathway of locusts polarization vision.

Adipokinetic hormone signaling regulates adult dehydration resistance in the migratory locust

Drought events have become more severe under climate change,and this can pose a major threat to the survival of various organisms.The molecular mechanisms involved in dehydration resistance are not well known.Here,adults of the migratory locust,Locusta migratoria,were subjected to food-mediated dehydration,and adipokinetic hormone(AKH)signaling was found to play a key role in regulating dehydration resistance.Specifically,dehydration shortened the lifespan,increased the body weight loss,and reduced the water loss rate in adult locusts.Global transcriptome profiles revealed variations in tissue-specific gene expression between dehydration-resistant locusts and normal locusts.Importantly,dehydration selection and exposure induced prominent expression of AKH genes in the retrocerebral complex of adult locusts.Furthermore,individual knockdown of AKH1,AKH2,or AKH receptor(AKHR)accelerated water loss and shortened the lifespan of adult locusts under dehydration conditions,and trehalose supplementation ameliorated the negative effects caused by interference with AKH or AKHR.These findings demonstrated that AKH/AKHR signaling-dependent trehalose metabolism plays a crucial role in regulating locust dehydration resistance and thus provide novel insights into the regulatory mechanism underlying drought resistance.