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.

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.

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.

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。

Comparative analysis of cytochrome P450-like genes from Locusta migratoria manilensis： expression profiling and response to insecticide exposure

The cytochrome P450 monooxygenase （cytochrome P450） gene superfamily comprises many genes that may be involved in the biotransformations of pesticides and other xenobiotics. To date, very little is known about cytochrome P450 genes in the oriental migratory locust, Locusta migratoria manilensis. In this study, we carried out a genomewide analysis ofcytochrome P450 genes of the locust to identify putative cytochrome P450 genes and characterize their expression responses to insecticide exposures. We identified 15 cytochrome P450-1ike genes from a locust expressed sequence tag database （LocustDB）. Reverse transcription polymerase chain reaction （RT-PCR） analysis showed that most cytochrome P450-1ike genes displayed different tissue and developmental stage expression patterns. However, most of them were predominantly expressed in the midgnt, gastric caeca, fatbodies, and/or hindgut. Biochemical analysis showed that cytochrome P450 was differentially affected by three different insecticides. Deltamethrin caused significant inductions in 12 h at LD30 （dose to kill 30% of the tested individuals） in the nymphs, whereas malathion and carbaryl did not have significant effect on cytochrome P450 enzyme activity. Further RT-PCR analysis Showed significant increases of transcriptions of several cytochrome P450 genes in deltamethrin-treated locusts. Thus, the increased cytochrome P450 enzyme activity is likely due to increased transcriptions of multiple cytochrome P450 genes in response to deltamethrin exposure. These results are expected to help us better understand the interactions between insecticides and major detoxification enzymes, and possible changes of the susceptibility to other insecticides in deltamethrin-treated insects at various molecular levels.

Identification and characterization of a gene encoding a UBX domain-containing protein in the migratory locust, Locusta migratoria manilensis

Ubiquitin regulatory X （UBX） domain-containing proteins are believed to func- tion as cofactors for p97/CDC48, an adenosine triphosphatase shown to be involved in mul- tiple cellular processes. In the present study, a full-length complementary DNA （cDNA） of UBX domain-containing gene, termed LmUBX1, was cloned from Locusta migratoria manilensis and characterized, using random amplification of cDNA ends polymerase chain reaction （RACE PCR）, sequence analysis and quantitative real-time PCR. LmUBX1, 1 600 bp in length, is predicted to encode a 446-amino acid protein with a predicted molec- ular weight of 51.18 kDa that contains a central PUB domain and a carboxy-terminal UBX domain. Homology analysis revealed that LmUBX1 has higher similarity to the known UBX domain-containing proteins from insects than from other species. Moreover, based on sequence characteristics and phylogenetic relationships, it is suggested that LmUBX1 can be classified into the UBXD1 subfamily. Expression analysis founded that LmUBX1 exhibited significant expression variations at different developmental stages and in differ- ent tissues, suggesting that the expression of LmUBX1 was highly regulated. Interestingly, its messenger RNA transcript was more abundant in ovary and testis than in other tissues examined, suggesting that it may have more important roles in the reproductive system. In addition, LmUBX1 was differentially expressed in gregarious and solitary locusts and was significantly up-regulated in third and fifth instars of gregarious locusts, implying that LmUBX1 was also likely involved in the phase polyphenisms in L. migratoria manilen- sis. To our knowledge, this is the first report of cloning of a full-length cDNA of UBX domain-containing gene from L. migratoria manilensis.

Optimization of parental RNAi conditions for hunchback gene in Locusta migratoria manilensis （Meyen）

An investigation on the optimization of parental RNA interference （RNAi） conditions for hunchback （hb） gene in Locusta migratoria manilensis （Meyen） was conducted. Double stranded RNA （dsRNA） corresponding to hb gene was injected into haemocoel of female adults ofL. migratoria manilensis. Embryos developed from the eggs laid by the injected adults on the 7th day after eclosion showed observable effects of RNAi for hb. The silencing effect after delivery treatment of dsRNA for hb gene was maintained for more than 21 days. A significant decrease of hb transcripts was further confirmed by Northern blot analysis. The dose of dsRNA/insect at 2μg could trigger 96.9% RNAi effects, while silencing appeared to have no dependence on the size of dsRNA. Results suggest that parental RNAi could be employed to efficiently identify the developmental gene functions in L. migratoria manilensis.

Expression of hunchback during oogenesis and embryogenesis in Locusta migratoria manilensis(Meyen)

hb(hunchback) is a contributing factor in anteroposterior axial patterning of insects.Although the hb function in Locusta migratoria manilensis has been investigated,its expression pattern remains unknown.Here,the mouse polyclonal antibody was produced against Hb fusion protein,and then its expression pattern during oogenesis and embryogenesis of L.migratoria manilensis was examined by immunohistochemical staining.Hb protein was detected in the oocyte nucleus which was positioned centrally within the developing oocyte.The oocyte nucleus gradually moved to the posterior end of the egg along with the oocyte maturing.In freshly laid eggs,Hb formed gradient at the posterior end of the egg,and then hb was expressed as a band in the middle of the blastodisc.As the blastodisc differentiated into the head and trunk,the expression region became wide,which would develop into spatial gnathal and thoracic segments.With abdominal segmentation,the expression domain in the gnathal and thoracic region became faint and eventually faded out,while the Hb expression domain appeared at the posterior growth zone in a discontinuous expression manner.The hb expression pattern of L.migratoria manilensis is greatly similar to that of other locusts,such as Schistocerca americana and another L.migratoria.Compared with other insects,hb expression is conserved in the gnathal and thoracic domains,while divergent in oogenesis and abdomen.

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.

Trehalose and trehalose-hydrolyzing enzyme in the haemolymph of Locusta rnigratoria infected with Metarhizium anisopliae strain CQMa102

Topical application of the Metarhizium anisopliae var. acridum specialist strain CQMa 102 to the locust Locusta migratoria manilensis results in changes of the concentrations of trehalose and glucose in the haemolymph. Micrographs of the locust haemolymph shows Metarhizium anisopliae can effectivly penetrate the external skeleton of locust and after 2 days infection, the hyphae body will appear in the haemolymph of infected insects. The time in decrease of trehalose concentration coincided with that in increase of trehalose-hydrolysing enzyme activity in the haemolymph of the fungus-infected insects. Overlay gel analysis indicated there was considerably more trehalose-hydrolysing activity in the haemolymph of locusts infected by fungus than in controls. A comparable isoform was identified in in vitro culture of the fungus, suggesting a fungal origin for the in vivo enzyme. Haemolymph trehalose decreased significantly during mycosis of locusts by M. anisopliae. All these results suggested that this fungus may take advantage of competing nutrient utilization against the insect by its trehalose-hydrolyzing enzyme secretion. It may provide fundamental knowledge for fungal pathogenesis.

Photoreceptive reaction spectrum effect and phototactic activity intensity of locusts’ visual display characteristics stimulated by spectral light

To reveal the nature of locusts’visual reaction when stimulated by light,and to clarify the regulation characteristics of locusts’phototactic visual physiology and obtain good spectral light features for locusts’phototactic action,this study investigated locusts’visual spectrum response by characterizing their photoreceptive reaction to LED light using an AvaSpec fiber-optic spectrometer system.Locusts’phototactic response to spectral light was compared using this system.The results showed that locusts’visual reaction characterization presents a photo-induced vision spectrum effect and that by offsetting the main wavelength of light and the spectral peak intensity,a time-varying bio-regulation effect emerges.In addition,locusts’visual regulation ability to UV light is higher than that to violet light,whereas their reaction intensity is lower than to violet light,and the visual bio-regulatory force of locusts’visual system absorbing orange light to react sensitively becomes gradually higher than when absorbing green light as time goes on.Moreover,corresponding to nominal illumination with the same radiant energy and a visual spectrum response stimulated by UV,violet,orange,and green light,it appears that the visual spectrum window is symmetrical around 382 nm,400 nm,602 nm,and 530 nm,respectively,with no significant difference between spectral amplitudes and having a time-varying incremental characteristic with amplitude peak width.This indicates that the stimulus intensity of UV,violet,orange,and green light exceeds locusts’visual tolerance,causing them to generate regulation inactivation as a visual physiological reaction,whereas the visual window response effect stimulated by UV light presents an illumination timeliness effect.Simultaneously,time-varying characteristics of locusts’bio-behavior intensity show that light intensity can make up for locusts’visual sensitivity differences at various spectral wavelengths.Presented with differential response time,photosensitive behavior intensity,and induction effect induced by orange light,time is superior for orange light,the stimulation effect caused by violet light is the strongest,and the phototactic synergy effect caused by UV light is the best.