Serotonin-immunoreactive neurones in the brain of Locusta migratoria innervating the corpus cardiacum

Summary The serotoninergic innervation of the corpus cardiacum (CC) of Locusta migratoria was investigated using two antisera against serotonin. A dense network of immunoreactive nerve fibres was present in the storage lobe of the CC. Immunopositive fibres only sporadically crossed the border between the storage lobe and the glandular lobe of the CC. Immunopositive fibres entered the storage lobe of the CC via the nervus corporis cardiaci I (NCCI); NCCII was immunonegative. Unilateral retrograde fillings of the NCCI with the fluorescent tracer Lucifer yellow, followed by antiserotonin immunocytochemistry, revealed about 20 double-labelled neurones in the anterior part of the pars intercerebralis. The double-labelled neurones were scattered between fluorescent non-immunoreactive neurones. Additionally, 5–7 neurones labelled only with Lucifer yellow were found at the ventrolateral side of the tritocerebrum. No immunopositive neurones were observed in the hypocerebral ganglion. Immunopositive fibres from neurones in the frontal ganglion ran via the recurrent nerve and the neuropile of the hypocerebral ganglion into the paired oesophageal nerve. At most, a few immunopositive nerve fibres occurred in the cardiostomatogastric nerves II, which connect the storage lobe of the CC with the paired oesophageal nerve at the caudal end of the hypocerebral ganglion.     

Effect of [His7]-corazonin on the number of antennal sensilla in Locusta migratoria

Abstract.  Certain types of antennal sensilla are known to be more abundant in solitarious individuals than in gregarious ones in the migratory locust, Locusta migratoria . We tested the hypothesis that injection of a neurohormone, [His⁷]-corazonin, into isolated-reared nymphs of this species mimics the effect of crowding on the frequencies of various types of antennal sensilla. One nmol of the hormone was injected into nymphs on two occasions, on the third days of the second and third stadia, respectively. Upon adult emergence, the numbers of different types of sensilla on the eighth antennal segment were compared with those of oil-injected controls. [His⁷]-corazonin did not influence the numbers of basiconic sensilla type A, basiconic sensilla type B and trichoid sensilla significantly compared to oil-injected controls. However, the number of coeloconic sensilla was reduced significantly by the hormone injections. Because the length of the antennal segment was not affected by the hormone injection, it appears that the hormone influenced the development of coeloconic sensilla. The results support the hypothesis tested and are consistent with the idea that [His⁷]-corazonin plays an important role in the control of phase polymorphism in L. migratoria .     

A comparison of phase-related shifts in behavior and morphometrics of an albino strain,deficient in [His(7)]-corazonin,and a normally colored Locusta migratoria strain

The albino Okinawa strain of Locusta migratoria is deficient in the neurohormone [His⁷]-corazonin. This peptide induces darkening of the cuticle, one of the typical features of gregarious locusts. As part of a broader study on the possible role of [His⁷]-corazonin in phase transition, we explored whether corazonin-deficiency might be associated with differences in behavior and morphometrics between albino and normal phenotypes of L. migratoria. Using a modification of the logistic-regression assay of behavioral phase state previously derived for Schistocerca gregaria, we found that there were strain dependent behavioral differences between crowd-reared nymphs of the albino Okinawa and the normally colored African strain, with no evidence of the albino strain being obligatorily solitarious. However, upon isolation, a shift towards more solitarious behavior occurs in both strains, even more profoundly in the Okinawa albinos. A shift could also be recorded in morphometrics. The conclusion is that the albino strain, although showing some solitarious features even when crowd-reared, is not, as has been suggested, obligatory solitarious and, as a consequence, the complete absence of corazonin is not sufficient to bring about the solitarious state.     

Phase polymorphism in Locusta migratoria: the relative effects of geographical strains and albinism on morphometrics

An albino strain, which had originated from Okinawa, Japan, and a normally coloured strain, which had originated from West Africa, have been used to study density‐dependent morphometric phase characteristics and their changes in adults of the migratory locust, Locusta migratoria (L.). By repeated crossings we also obtained congenic albinos and normal phenotypes and investigated their morphometrics with increasing West African genome, eventually reaching 99.6% West African and 0.4% Okinawa gene pool. The data were analysed by the classical morphometric ratios (F/C and E/F; F = length of the hind femur, C = maximum width of the head, E = length of the fore wings), as well as by canonical discriminant (multivariate) analysis. The latter was based on measurements of F, C and E (as above), as well as of M (minimum width of the pronotum) and H (maximum height of the pronontum). Okinawa albinos showed more solitarious morphometrics and a smaller amplitude of morphometric phase change than West African normal phenotypes. Both the morphometric ratios and the canonical discriminant analysis demonstrated clearly that these differences were caused primarily by the strain (Okinawa vs. West African). However, the pigmentation (albino vs. normal colouration) also affected morphometric phase differences; albinos showed more solitarious morphometrics and somewhat more restricted morphometric phase change than congenic normal phenotypes. The effect of the pigmentation was considerably smaller than that of the strain. The results refute Nolte's claim that albino locusts constitute an extreme solitarious phase, even under crowding. However, Nolte's less extreme claim, that albino locusts have more solitarious morphometrics than normally coloured locusts, is validated by the present results.     

The dark-colour-inducing neurohormone of locusts: strain-dependent and phase-independent effects on adults of the migratory locust, Locusta migratoria

Abstract.  An albino strain that had originated from Okinawa, Japan, and a normally coloured strain that had originated from West Africa, were used to study the darkening response to injection of graded doses of dark-colour-inducing neurohormone of locusts (DCIN) ([His⁷]-corazonin) of gregarious and solitarious adults of the migratory locust, Locusta migratoria (L.). By repeated crossings, congenic albinos and normal phenotypes were obtained, both with a 99.6% West African genome, and their darkening response was compared with the original Okinawa and West African strains. Within each of these four strains, no difference was found in DCIN-induced darkening between gregarious and solitarious adults despite previous publications in the literature claiming an absence of 'fire-darkening' in gregarious adults of this species. Okinawa albino adults showed a markedly higher darkening response than the other three strains, including albinos with a 99.6% West African genome. This finding demonstrates that the differential darkening response of the Okinawa albinos is caused not by albinism, but by the geographical origin (Okinawa) of the strain. This is the first report of geographical-strain-dependent differences in the response of an insect to a neurohormone. The darkening response of adults reached a maximum on day 10 after injection; subsequently, the dark colour faded slowly. Adults injected 1 day after their moult showed a greater darkening response than those injected after 14 or 28 days.     

Ultrastructural enzyme-cytochemical study of the intrinsic glandular cells in the corpus cardiacum of Locusta migratoria: relation to the secretory and endocytotic pathways,and to the lysosomal system

Summary Ultrastructural aspects of the secretory and the endocytotic pathways and the lysosomal system of corpus cardiacum glandular cells (CCG cells) of migratory locusts were studied using morphological, marker enzyme, immunocytochemical and tracer techniques. It is concluded that (1) the distribution of marker enzymes of trans Golgi cisternae and trans Golgi network (TGN) in locust CCG cells corresponds to that in most non-stimulated vertebrate secretory cell types; (2) the acid phosphatase-positive TGN in CCG cells is involved in sorting and packaging of secretory material and lysosomal enzymes; (3) these latter substances are produced continuously; (4) at the same time, superfluous secretory granules and other old cell organelles are degraded; (5) the remarkable endocytotic activity in the cell bodies and the minor endocytotic activity in cell processes are coupled mainly to constitutive uptake of nutritional and/or regulatory (macro)molecules, rather than to exocytosis; (6) plasma membrane recycling occurs mainly by direct fusion of tubular endosomal structures with the plasma membrane and little traffic passes the Golgi/TGN; and (7) so-called cytosomes arise mainly from autophagocytotic vacuoles and represent a special kind of complex secondary lysosomes involved in the final degradation of endogenous (cell organelles) and exogenous material.     

Muscle activity underlying sexual behaviour in male locusts (Locusta migratoria) after normal and hormonally disturbed development

Abstract. . The activities of three sets of muscles, representing the major components of the abdominal muscle system, were recorded in the pregenital abdominal segments of adult males of Locusta migratoria. The muscular activities were associated with three consecutive actions of the male's mating behaviour: abdominal lowering, S-bending and hooking-up. These three actions depend mainly on the relative coordination of the dorsal versus ventral longitudinal muscles; the activity of the dorsoventral muscles is rather unpredictable, except in the sixth and seventh abdominal segments where they are regularly involved in hooking-up. Studies on male sexual behaviour-linked muscle activity in precocene-induced fifth-instar adultiforms and azadirachtin-induced fifth-instar over-aged nymphs revealed no distinct differences between these creatures and normal mature males. Therefore the muscle activities associated with these adult-specific behavioural acts can be released precociously, i.e. their occurrence is not dependent on a distinct series of instars. Moreover, the results obtained with over-aged nymphs demonstrate that morphogenesis and ethogenesis (i.e. formation of neural circuits controlling adult-specific behaviour) are not closely dependent on each other.     

Effect of Paranosema locustae (Microsporidia) on the behavioural phases of Locusta migratoria (Orthoptera: Acrididae) in the laboratory

The ability of parasites to modify the behaviour of their hosts is a wide spread phenomenon, but the effects of microsporidian parasites on locust behaviour remain unexplored. Here the frequencies of directional changes (ND) and jumping (NJ) per minute of gregarious locusts infected with 2000 spores of the microsporidian parasite Paranosema locustae were significantly different from those of untreated locusts 10 and 16 days after infection, being similar to values for solitary nymphs. In contrast, the behaviour of locusts inoculated with the lower doses of 200 spores/locust was sometimes like that of solitary nymphs. At other times, behaviour was intermediate between solitary and gregarious, i.e. transitional. The rearing density did not affect the turning and jumping behaviour of infected locusts, and their behaviours were similar to those of solitary locusts at 10–16 days after infection. Our study demonstrates that infection with P. locustae may lead gregarious locusts to change some of their behaviour to that typical of solitary locusts.     

Morphology of neurones in the storage part of the corpus cardiacum of Locusta migratoria: no evidence for their involvement in the regulation of adipokinetic cell activity

The storage part of the corpus cardiacum of Locusta migratoria consists of two compartments: a neural part on the haemocoelic side containing neuronal cell bodies that are protected by a blood-brain barrier, and a neurohaemal part adjacent to the aorta. Intracellular filling of the neurones in the neural part with Lucifer yellow followed by confocal laser scanning microscopy has revealed that these neurones can be divided into several classes. None of the neurones has processes extending into the glandular part of the corpus cardiacum. They are, therefore, not directly involved in the regulation of adipokinetic cell activity.     

Co-localization of the adipokinetic hormones I and II in the same glandular cells and in the same secretory granules of corpus cardiacum of Locusta migratoria and Schistocerca gregaria

Summary The immunocytochemical reactivity of the glandular cells of the corpus cardiacum (CCG-cells) of Locusta migratoria and Schistocerca gregaria was investigated at the electron-microscopic level, using the protein A-gold method, with three antisera against fragments of the adipokinetic hormones AKH I and AKH II. This combination of antisera permitted discrimination between anti-AKH I and anti-AKH II immunoreactivity. Fixation in a mixture of 2% glutaraldehyde and 2% formaldehyde, in combination with low-temperature embedding in Lowicryl K₄M, produced the highest and most consistent selective immunogold labelling of the secretory and ergastoplasmic granules. All secretory granules in all CCG-cells investigated possessed a distinct anti-AKH I-immunopositive reaction, whereas most secretory granules showed a weaker anti-AKH II immunoreaction. Ergastoplasmic granules reacted similar to the secretory granules. The average immunolabelling of the secretory granules was higher in the processes than in the cell bodies of the CCG-cells. The results in Schistocerca gregaria were essentially similar to those in Locusta migratoria. It is concluded that (i) the individual CCG-cells synthesize AKH I as well as AKH II; (ii) these hormones coexist in the same ergastoplasmic and secretory granules; and (iii) these granules contain a higher content of AKH I than AKH II.     

Immunocytochemical differentiation between adipokinetic hormone (AKH)-like peptides in neurons and glandular cells in the corpus cardiacum of Locusta migratoria and Periplaneta americana with C-terminal and N-terminal specific antisera to AKH

Summary An immunocytochemical method was used to differentiate between immunoreactive substances in glandular cells in the corpora cardiaca (CC) and in certain cerebral neurons in 2 insect species, Locusta migratoria migratorioides and Periplaneta americana. The staining properties of antisera raised to different parts of the decapeptide adipokinetic hormone (AKH) were compared and their specificity was determined by preabsorption with AKH and related peptides. Antibodies raised to the N-terminal part of AKH (serum 433) and the central and C-terminal part (serum 241) were found to have different staining properties.In the CC of the locust both antisera show a strong immunoreactivity with glandular cells, we therefore suggest that at least one of the compounds revealed is AKH. Some of the glandular cells in the locust and large numbers of glandular cells in the CC of the cockroach are revealed by the N-terminal specific antiserum. On the other hand, neurons in the central nervous system are revealed only by the C-terminal specific antiserum. The possible identity of the various substances revealed by these two antisera is discussed.     

群居型和散居型东亚飞蝗雌成虫飞行肌的超微结构

应用电子显微镜对群居型和散居型东亚飞蝗Locusta migratoria manilensis（Meyen）雌成虫背纵肌进行了比较观察。结果表明：群居型和散居型成虫背纵肌具有类似的亚细胞结构,飞行肌的肌原纤维具有1∶3粗细丝比例,每根粗丝由6根细丝环绕排列成六角形结构。飞行肌的发育和线粒体的形成均是渐进的过程,在不同日龄成虫间存在差异。肌节长度为2.1～3.4 μm;7和10日龄时群居型成虫肌节长度小于散居型;7日龄群居型肌原纤维直径显著大于散居型。背纵肌内线粒体含量约占肌纤维的20%～43%,两型飞蝗之间存在着显著的差异,7日龄时群居型线粒体占肌原纤维的比例高达42.96%,而散居型的只有22.45%;10日龄群居型线粒体含量为41.32%,散居型线粒体29.98%。上述差异可能是东亚飞蝗群居型成虫飞行能力显著强于散居型成虫的重要原因之一。     

飞蝗热休克蛋白70cDNA片段的克隆和序列分析

采用R-PCR方法对海南、河北和辽宁3个飞蝗(Locusta migratoria L．)种群的热休克蛋白70(HSP70)基因cDNA片段进行克隆。在事先优化的条件下,通过简并性上游引物和下游引物扩增出了河北种群飞蝗HSP70基因的604bp cDNA片段(GenBank登录号为AY299637),推导的氨基酸序列包含201个氨基酸残基。分析表明,由飞蝗该片段推导的氨基酸序列与其他昆虫的同源性较高;3个种群该片段的核苷酸序列相似性更高达98．75％。由此推测,飞蝗种群间抗寒性的差异可能不是HSP70的序列变异引起的,而与HSP70的诱导表达有关。     

Identification of a secretomotor centre in the brain of Locusta migratoria,controlling the secretory activity of the adipokinetic hormone producing cells of the corpus cardiacum

Summary After retrograde filling of axons terminating in the glandular lobe of the corpus cardiacum (CC) of Locusta migratoria with cobalt chloride, a paired group of about 15 cobalt containing cells was demonstrated in the lateral area of the protocerebrum. The axons of these cells run via the NCC II into the glandular lobe of the CC. These small neurons have the characteristics of secretory cells; they contain secretory granules of about 1000 Å in diameter. The axon terminals in the glandular lobe, making synaptic contacts with the glandular cells, contain secretory granules of the same size. It is therefore concluded that the cell groups in the protocerebrum control the activity of the glandular cells which produce an adipokinetic hormone. Arborizations of fibers of the lateral secretomotor cells are present in the dorsal neuropile of the protocerebrum, ventral of the mushroom bodies and along the tracts of the NCC I within the brain. It is proposed that these arborizations are sites of synaptic input. It is discussed that the axons of these cells might receive additional synaptic input in the storage lobe of the CC.The localization of cell bodies, the axons of which enter the storage part of the CC is described. The course of the axon tracts of the various cell groups in the protocerebrum and their connections with the NCC I and NCC II are demonstrated.Supported by the Foundation for Fundamental Biological Research (BION) which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). The electron microscopical investigations were performed at the EM-unit of the Faculty of Biology, State University of Utrecht (Director: Prof. Dr. J.C. van de Kamer)The author is greatly indebted to Dr. A.M.Th. Beenakkers and Dr. H.H. Boer for their active interest and helpful advise. Thanks are also due to Mr. H. van Kooten and his staff for making the macro- and microphotographs, to Mr. L.W. van Veenendaal for preparing the electron micrographs and final assistance in the preparation of the photo pages and to Mr. D. Smit, who made the drawings     

Mechanisms of organophosphate resistance in a field population of oriental migratory locust, Locusta migratoria manilensis (Meyen)

The susceptibilities to three organophosphate (OP) insecticides (malathion, chlorpyrifos, and phoxim), responses to three metabolic synergists [triphenyl phosphate (TPP), piperonyl butoxide (PBO), and diethyl maleate (DEM)], activities of major detoxification enzymes [general esterases (ESTs), glutathione S-transferases (GSTs), and cytochrome P450 monooxygenases (P450s)], and sensitivity of the target enzyme acetylcholinesterase (AChE) were compared between a laboratory-susceptible strain (LS) and a field-resistant population (FR) of the oriental migratory locust, Locusta migratoria manilensis (Meyen). The FR was significantly resistant to malathion (57.5-fold), but marginally resistant to chlorpyrifos (5.4) and phoxim (2.9). The malathion resistance of the FR was significantly diminished by TPP (synergism ratio: 16.2) and DEM (3.3), but was unchanged by PBO. In contrast, none of these synergists significantly affected the toxicity of malathion in the LS. Biochemical studies indicated that EST and GST activities in the FR were 2.1- to 3.2-fold and 1.2- to 2.0-fold, respectively, higher than those in the LS, but there was no significant difference in P450 activity between the LS and FR. Furthermore, AChE from the FR showed 4.0-fold higher activity but was 3.2-, 2.2-, and 1.1-fold less sensitive to inhibition by malaoxon, chlorpyrifos-oxon, and phoxim, respectively, than that from the LS. All these results clearly indicated that the observed malathion resistance in the FR was conferred by multiple mechanisms, including increased detoxification by ESTs and GSTs, and increased activity and reduced sensitivity of AChE to OP inhibition.     

Isolation,characterization and cross‐species amplification of eight microsatellite DNA loci in the migratory locust (Locusta migratoria)

Eight polymorphic di‐ and trinucleotide microsatellite loci suitable for population genetic analysis were developed in Locusta migratoria from a partial phagemid genomic library enriched for microsatellite inserts. The expected heterozygosity at these loci ranges from 0.45 to 0.97, with the observed allele numbers varying between nine and 45. The overall microsatellite cloning efficiency in L. migratoria is 14%, suggesting that in migratory locusts, microsatellite sequences are abundant and should provide a valuable and easily accessible source of nuclear markers for genetic studies. These microsatellite loci were highly Locusta‐specific, with only very limited cross‐species applicability.     

飞蝗发育相关基因Omb的克隆、原核表达及时空表达分析

【目的】本研究克隆飞蝗Locusta migratoria发育相关的optomotor-blind(Omb)基因,并对其进行序列和表达分析,旨在更好地了解Omb基因在飞蝗翅发育中的作用及为进一步蝗灾的治理和防治提供新的理论依据。【方法】利用RT-RCR扩增飞蝗Omb基因cDNA序列,采用生物信息学软件分析该基因的核苷酸和氨基酸序列,利用MEGA 6. 0构建昆虫纲分子系统进化树;构建重组表达载体pET-30a/LmOmb,转化到大肠杆菌Escherichia coli BL21(DE3)中,SDS-PAGE及Western blot鉴定重组表达蛋白;基于qPCR技术分析Omb基因在飞蝗不同发育时期及成虫不同组织中的表达谱。【结果】克隆获得飞蝗Omb基因部分cDNA序列,命名为LmOmb(GenBank登录号:MG867658),其长792 bp,编码264个氨基酸,在第37-219位氨基酸之间存在一个T-box superfamily保守结构域。同源序列比对分析表明Lm Omb与褐飞虱Nilaparvata lugens Nl Omb氨基酸序列一致性为93%。在IPTG诱导下目标蛋白以6×His标签融合蛋白的形式在宿主菌中得到稳定表达。荧光定量PCR结果显示LmOmb基因在飞蝗不同发育时期均有表达,其中胚胎期的表达量最高,进入若虫期后表达量下降,且各龄若虫之间表达量相对平稳。Lm Omb基因在雌性和雄性成虫的胸部、足、腹部和翅中都有表达,且雌性和雄性之间表达量明显不同。【结论】LmOmb基因可能参与了飞蝗的胚胎发育。研究结果为进一步研究飞蝗LmOmb的功能提供了依据。     

Projection areas and branching patterns of the tympanal receptor cells in migratory locusts,Locusta migratoria and Schistocerca gregaria

Summary In Locusta migratoria and Schistocerca gregaria, the projection areas and branching patterns of the tympanal receptor cells in the thoracic ganglia were revealed. Four auditory neuropiles can be distinguished on each side of the ventral cord, always located in the anterior part of the ring tract in each neuromere (two in the meta-, one in the meso-, and one in the prothoracic ganglion). Some of the receptor fibres ascend to the suboesophageal ganglion. There are distinct subdivisions within the auditory, frontal metathoracic and mesothoracic neuropiles. The arrangement of the terminal arborisations of the four types of tympanal receptor cells according to their different frequency-intensity responses is somatotopic and similar in the two ganglia. Here the receptor cells of type-1 form a restricted lateroventral arborisation. Cells of type-4 occupy the caudal part with a dorsorostral extension. Cells of type-2 and -3 arborise in a subdivision between both. Most of the stained low-frequency receptors (type-1, -2, and -3) terminate either in the metathoracic or, predominantly, in the mesothoracic ganglion. In contrast, the high-frequency cells (type-4) ascend to the prothoracic ganglion. The receptor fibres of the different types of receptor cells differ in diameter.Abbreviations aRT anterior part of the ring tract - cf characteristic frequency - MVT median ventral tract - SEG suboesophageal ganglion - SMC supramedian commissure - VMT ventral median tract - VIT ventral intermediate tract Supported by the Deutsche Forschungsgemeinschaft; part of program A7 in Sonderforschungsbereich 305 (Ecophysiology)