Physical characterization and sequence identification of the ovary maturating parsin. A new neurohormone purified from the nervous corpora cardiaca of the African locust (Locusta migratoria migratorioides.

A novel neurohormone, which anticipates ovarian maturation, was recently purified using liquid chromatography from the African locust nervous corpora cardiaca. Both its function and production by the pars intercerebralis of Locusta migratoria lead to its name, the ovary maturating parsin (Lom OMP). In this study, the Lom OMP was physically and chemically characterized. Its multiply charged ion spectrum was interpreted as two peaks of quite equal size having molecular masses of 6923.4 Da (major peak) and 6907.3 Da. The Lom OMP presented no periodic secondary structure according to the far ultraviolet circular dichroism spectrum obtained. It is composed of 65 amino acids and included a high concentration of alanine but is devoid of cysteine, isoleucine, methionine, lysine and threonine. The amino acid sequence indicated only one microheterogeneity, observed at position 26, consisted in the replacement of serine by alanine. The calculated Mr of the two acidic isoforms (calculated pHi = 4.87) were found to be in agreement with mass spectrometry measurements. When compared to the sequence libraries, the Lom OMP, the first insect gonadotropic neurohormone, was revealed as an unique protein.     

Restricted occurrence of Locusta migratoria ovary maturing parsin in the brain-corpora cardiaca complex of various insect species

Ovary maturing parsin (OMP) is a gonadotrophic molecule previously isolated from the neurosecretory lobes of the corpora cardiaca of Locusta migratoria (acridian Orthoptera). A polyclonal antiserum directed against the two biologically active domains of the L. migratoria (Lom) OMP was used to investigate the occurrence of Lom OMP-like substances in brain-corpora cardiaca complexes of other insect species. Using immunohistochemistry, specimens of 40 different insect species belonging to 13 insect orders were tested. The Lom OMP-like substance was strictly limited to specimens of insect species belonging to the Acridae. It occurred in non-basophilic cells of the pars intercerebralis that project to the corpora cardiaca, as in Locusta. Although the antiserum only detected Lom OMP-like material in the Acridae, it is possible that related molecules exist in other insects. The antiserum may be very specific for domains of the Lom OMP molecule that have not been highly conserved during evolution or possibly these domains are not accessible to the antiserum in other insects.     

Immunochemical analysis of the distribution of the new ovary maturating neurohormone during development of the African locust,Locusta migratoria

Summary Following our prior identification of a gonadotropic neurohormone isolated from the neurosecretory lobe of the corpora cardiaca of the African locust, we have raised a polyclonal antiserum against this new molecule. In the present paper, we characterize this antiserum using enzyme-linked immunosorbent assay and Western blotting. The latter procedure reveals that the immune serum specifically recognizes the neurohormone, which we have termed ovary maturating parsin. Immunohistochemistry, enzyme-linked immunosorbent assay and Western blotting were used to analyze the distribution of this gonadotropic neurohormone throughout the central nervous system during development. It is produced only by the type-B neurosecretory cells of the pars intercerebralis-corpora cardiaca system and is present both in males and females throughout life from embryo to adult. This permanent expression suggests that the neurohormone may have functions other than its primary direct gonadotropic role in females.     

Ovary maturing parsin and diuretic hormone are produced by the same neuroendocrine cells in the migratory locust, Locusta migratoria

In the migratory locust, the CRF-related diuretic hormone that stimulates fluid secretion by the Malpighian tubules, and the ovary maturing parsin, a neurohormone able to stimulate oogenesis, are produced by the same neuroendocrine cells of the pars intercerebralis in the brain.     

Isolation and characterisation of two chymotrypsins from the midgut of Locusta migratoria

Two chymotrypsin isozymes (CTR 1 and CTR 2) from the midgut lumen of Locusta migratoria have been identified and purified. MALDI-TOF mass spectrometry revealed an M_r of 22 679 (±30) for CTR 1 and 22 592 (±30) for CTR 2. Both chymotrypsins hydrolysed S-(Ala)₂ProPhe-pNA (CTR 1: K_m=0.29±0.01 mM, V_max=83.0±1.4 U/mg; CTR 2: K_m=0.42±0.01 mM, V_max=48.9±1.1 U/mg) and S-(Ala)₂ProLeu-pNA (CTR 1: K_m=0.50±0.04 mM, V_max=1.7±0.1 U/mg; CTR 2: K_m=1.12±0.08 mM, V_max=11.4±0.6 U/mg), but neither enzyme hydrolysed BTpNA, S-Phe-pNA, Ac-Leu-pNA or S-(Ala)₃-pNA. CTR 1 and CTR 2 activities were effectively inhibited by AEBSF, PMSF, TPCK, chymostatin, SBTI and BPTI. Using S-(Ala)₂ProPhe-pNA as the substrate, CTR 1 gave optimal activity between pH 8.0 and 10.0, while CTR 2 was optimally active over the range pH 8.0–11.0. The N-terminal 15 amino acids of the purified chymotrypsins were determined, revealing their unique sequences which are also different from another, previously characterised Locusta chymotrypsin.     

不同食料植物对东亚飞蝗肠道细菌状况的影响

目的比较在相同的环境条件下不同食料植物对东亚飞蝗肠道细菌状况的影响。方法以墨西哥玉米、野生马唐为食料植物饲喂东亚飞蝗,在其成虫肠道内和粪沙中分离纯化细菌,获得22株菌株,分别对其培养性状、菌体形态、染色反应和生理生化性状进行系统研究。结果上述22个菌株分别属于稀有杆菌属（Rarobact-er）、短状杆菌属（Brachybacterium）、棒杆菌属（Corynebacterium）、棍状菌属（Clavibacter）、葡萄球菌属（Staphylococ-cus）、丙酸杆菌属（Propionibacterium）、埃希菌属（Eschrichia）、气微杆菌属（Aercmicrobium）、沙雷菌属（Serratia）、克雷伯菌属（Klebsiella）、沙门菌属（Salmonella）、志贺菌属（Shigella）、口腔球菌属（Stomatococcus）、短杆菌属（Brevibacteri-um）、地杆菌属（Terrbacter）、微小杆菌属（Exiguobacterium）、皮杆菌属（Dermabacter）、短小杆菌属（Curtobacterium）、纤维单胞菌属（Cellulomonas）、微杆菌属（Microbacterium）、乳杆菌属（Lactobacillus）和凝结芽胞杆菌属（Bacilluscoag-ulans）。结论不同食料植物对东亚飞蝗肠道细菌种类和数量有很大的影响。     

Detection and determination of Locusta migratoria trypsin by radioimmunoassay

Polyclonal antibodies were produced against trypsin-like enzyme TLE-2, one of two trypsins isolated from the midgut cecae of the locust Locusta migratoria. Using those antibodies a heterologous RIA was developed. The specificity of the antibodies was demonstrated by immunoblot technique and cross-reactivity experiments. Positive blot reaction with the antiserum was observed with locust trypsins and binding competition was obtained only with the second trypsin from the locust, TLE-1. Trypsins from bovine and hog as well as trypsins from Tenebrio molitor and Tribolium castaneum and locust chymotrypsin did not inhibit TLE-2 binding to the antiserum even at concentrations 1,000-fold greater than that of locust TLE-2. The concentrations of TLE-2 in cecal fluid, cecal wall, hemolymph, and fat-body were 2,790, 60.4, 6.4, and 0.7 ng per mg tissue, respectively. © 1992 Wiley-Liss, Inc.     

Isolation and sequencing of two FMRFamide-related peptides from the gut of Locusta migratoria L

Two FMRFamide-related peptides (FaRPs) have been isolated and sequenced from the whole gut of Locusta migratoria L. Peptides were extracted from 500 locust whole guts and separated using reversed-phase high performance liquid chromatography (RP-HPLC). Fractions containing FMRFamide-like immunoreactive (FLI) material were identified using radioimmunoassay (RIA). Sequencing of fractions, using tandem mass spectrometry (MALDI-TOF MS/MS), revealed the myosuppressin previously isolated from the locust CNS, SchistoFLRFamide (PDVDHVFLRFamide), and a novel extended RFamide (LWENLRFamide). The isolation of SchistoFLRFamide from midgut tissue supports the hypothesis that this myosuppressin is released locally from FLI processes over the gut and/or from endocrine-like midgut cells to play a role in the regulation of digestion.     

Control of the horizontal flight-course by air-current sense organs in Locusta migratoria

ABSTRACT. The role of the air-current sense organs of Locusta migratoria (the antennae and the hair fields on the frons and vertex of the head) in control of the horizontal flight-course in relation to the air was investigated in locusts flying tethered on a torque gauge. The antennae were apparently not of importance in the control of flight direction in the horizontal plane. If the hair fields were stimulated by an air current from either side, however, a torque on the vertical body axis was generated by the beating wings after a reaction time of at least 0.1 s. In free flight this would result in a yaw, bringing the animal back in line with the air stream. This torque reaction will stabilize the flight direction in the horizontal plane via a negative feedback mechanism, the hair fields above the lateral ocelli being the feedback receptors. In free flying locusts a torque on the vertical body axis can be generated by both the beating wings and the abdomen working as a rudder when bent to either side. The wings, however, are dominant in the reflex stabilization of the flight-course.     

不同来源飞蝗后肠微生物群落高通量测序分析

对来源于田间与室内飞蝗种群肠道细菌群落的454高通量测序(HTS)结果进行了分析,共分析鉴定出OTU(operational taxonomic unit)66个,其中室内飞蝗种群41个,田间飞蝗53个。全部OTU归属于6个门,9个纲,11个目,14个科,20个属。室内种群共含有12个属,其中优势类群为乳球菌属(Lactococcus)和拉乌尔菌属(Raoultella),分别占总种群的13.3%和36.1%。田间飞蝗鉴定出14个属,优势类群为芽胞杆菌属(Bacillus),占总种群的18.8%。田间飞蝗后肠菌落的Ace、Chao和Shannon指数均高于室内种群,而Simpson指标呈相反结果。研究表明来源于田间的飞蝗肠道细菌群落的丰富度和多样性均高于室内饲养的种群。分析结果为今后开展不同来源飞蝗肠道微生物资源研究提供依据。     

Transport of d-galactose by the gastrointestinal tract of the locust, Locusta migratoria

Due to exoskeleton, the absorption of nutrients in adult insects takes place across the gastrointestinal tract epithelium. In most physiological studies, sugar intestinal absorption has been described as a diffusional process and to date no sugar transporter has been cloned from the digestive tract of insects. In the present work, the existence of a saturable transport system for galactose in the gastric caeca of Locusta migratoria is clearly demonstrated. This transport shows a relatively high affinity for galactose (apparent K0.5=2-3 mM) and is inhibited by glucose, 2-deoxyglucose and with less potency by fructose and alpha-methyl-d-glucoside. The absence of sodium or the presence of phloridzin hardly affects galactose absorption, indicating that it is not mediated by a SGLT1-like transporter. The absence of K+, Cl-, Mg2+ and Ca2+ or changes in the pH do not modify galactose absorption either. Nevertheless, phloretin, cytochalasin B and theophylline (inhibitors of facilitative transporters) decrease sugar uptake around 50%. Xenopus laevis oocytes microinjected with poly A+ RNA isolated from gastric caeca show sodium-independent galactose uptake that is three times higher than in non-injected oocytes, further supporting the existence of a mRNA coding for at least one equilibrative sugar transporter in L. migratoria gastric caeca.     

Structure predicts synaptic function of two classes of interneurons in the thoracic ganglia of Locusta migratoria

Summary The relationship between synaptic function and structure was examined for 32 spiking interneurons (13 inhibitory and 19 excitatory) in the meso- and metathoracic ganglia of the locust, Locusta migratoria. In no instance was the structure of an excitatory interneuron similar to that of an inhibitory interneuron. However, 12 of the 13 inhibitory interneurons shared a number of structural features, namely a ventromedially located soma, axon(s) projecting into contralateral connective(s), and a laterally bowed primary neurite. Structurally the excitatory interneurons formed a more heterogeneous group. Even so, 12 of the 19 had a combination of structural features in common, namely laterally located somata and axon(s) projecting into contralateral connective(s). The clear differences in structure of the two main groups of inhibitory and excitatory interneurons suggest that other neurons with structures similar to members of these two groups can be classified as inhibitory and excitatory, respectively. Thus we propose that structure predicts synaptic function for two distinct groups of interneurons in the thoracic ganglia of locusts. Present address: Department of Biology, McGill University, Montreal, Qubeck, Canada     

东亚飞蝗外生殖器的显微结构

利用扫描电镜技术观测了东亚飞蝗Locusta migratoria manilensis(Meyen)雌雄个体外生殖器的显微构造,描述了雌雄两性外生殖器的具体结构以及其表面上感受器的分布情况。雌性的外生殖器由背、腹产卵瓣构成,雄性的外生殖器由外骨骼所包被的阳具基背片和阳具复合体构成。在雌雄两性的外生殖器表面着生有大量的感受器,包括毛形、刺形、锥形,主要是机械感受器,还有少量化学感受器。     

Evidence for crustacean cardioactive peptide-like innervation of the gut in Locusta migratoria

Hindguts from female Vth instar larvae, young adults (1-2 days) and old adults (>10 days) are equally sensitive to the crustacean cardioactive peptide (CCAP), with changes in contraction occurring at a threshold concentration of 10(-9)M and maximal responses observed at concentrations ranging between 10(-7) and 5x10(-6)M. An immunohistochemical examination of the gut of Locusta migratoria with an antiserum raised against CCAP revealed an extensive network of CCAP-like immunoreactive processes on the hindgut and posterior midgut via the 11th sternal nerve arising from the terminal abdominal ganglion. Anterograde filling of the 11th sternal nerve with neurobiotin revealed extensive processes and terminals on the hindgut. Retrograde filling of the branch of the 11th sternal nerve which innervates the hindgut with neurobiotin revealed two bilaterally paired cells in the terminal abdominal ganglion which co-localized with CCAP-like immunoreactivity. Results suggest that a CCAP-like substance acts as a neurotransmitter/neuromodulator at the locust hindgut.     

飞蝗羧酸酯酶基因LmCarE25的体外重组表达

羧酸酯酶(Carboxylesterase,CarE)是一类在生物中广泛分布的多功能家族酶系,在昆虫抗药性形成机制中发挥重要作用。为探讨飞蝗Locusta migratoria(Meyen)羧酸酯酶基因LmCarE25的生物学功能,本文采用荧光实时定量PCR技术进行研究,发现其在飞蝗成虫各组织部位均有表达,其中胃盲囊、翅和肌肉中表达量较低,中肠、马氏管和脂肪体表达量较高。本文尝试在大肠杆菌和昆虫细胞体外重组表达该酶,构建重组表达载体pET32a-LmCarE25,SDS-PAGE结果显示,LmCarE25可在大肠杆菌BL21(DE3)和JM109中表达,蛋白分子量约为60 ku,但为包涵体;将LmCarE25基因插入真核表达载体pFastBacHTA中,借助Bac-to-Bac体系获得重组Bacmid,以sf9细胞系作为宿主细胞表达目的蛋白,Western-Blot检测结果显示,LmCarE25获得可溶性表达。上述结果为进一步深入研究飞蝗羧酸酯酶家族功能提供了基础资料。     

Organ-specific distribution of the calcium sensor CaMKII in Locusta migratoria

The Ca(2+)/calmodulin-dependent kinase CaMKII is a key signaling component in Ca(2+)-dependent physiological processes. The expression and function of CaMKII in insect brain is well documented but less investigated for other tissues of insects. The present study demonstrates that in the locust Locusta migratoria CaMKII is widely expressed in various tissues. Relatively high expression levels of CaMKII were found in the brain, upper part of the digestive tract (pharynx, esophagus), and the flight and leg muscles. The different expression patterns of CaMKII in various tissues, as well as different molecular masses of CaMKII between 48 and 60 kDa indicate a tissue-specific expression of CaMKII variants. The expression was monitored with a polyclonal anti-(rat)CaMKII antibody. About 60% of total CaMKII activity in flight muscle cells is associated to the myofibril-rich, particulate fraction suggesting an important role of CaMKII in sarcomeric function.     

Evidence for the association of FMRFamide-related peptides with the spermatheca of Locusta migratoria

The association of FMRFamide-related peptides (FaRPs) with the spermatheca of Locusta migratoria was demonstrated using radioimmunoassay and immunohistochemical techniques. The physiological effects of various FaRPs on the neurally evoked contractions of the spermatheca were also examined. FMRFamide-like immunoreactivity (FLI) was demonstrated in processes and cell bodies situated in the VIIIth (terminal) abdominal ganglion. These included an anterior, central and posterior pair of ventral cell bodies positioned near the midline of the ganglion, in addition to two bilaterally paired dorsal cell bodies in the posterior region of the VIIIth abdominal ganglion. Two axons displaying FLI proceed down the ventral ovipositor nerve (VON) and into the receptaculum seminis nerve which innervates the anterior regions of the spermatheca. FLI was also noted in processes on the spermathecal muscle with the highest density occurring on the spermathecal sac and coil duct. FaRPs applied to the spermathecal muscle included GQERNFLRFamide, NFIRFamide, ADDRNFIRFamide, YGGFMRFamide, FMRFamide, ADVGHVFLRFamide and SchistoFLRFamide (PDVDHVFLRFamide). Dose-dependent physiological effects were only noted for FMRFamide, ADVGHVFLRFamide and SchistoFLRFamide. FMRFamide led to a dose-dependent increase in the amplitude of neurally evoked contractions with a threshold of approximately 5 x 10(-7) M. SchistoFLRFamide, and ADVGHVFLRFamide, had an inhibitory effect, decreasing the amplitude of neurally evoked spermathecal contractions.