Effects of temperature and [His7]-corazonin on the body darkening in Locusta migratoria

Abstract.  [His⁷]-corazonin is a neuropeptide that induces dark coloration in locusts. This study examined the effect of temperature on body colour in the migratory locust, Locusta migratoria . L. Injection of a low dose (1 pmol) of [His⁷]-corazonin caused albino nymphs to develop dark coloration at 25–34 °C, but little darkening occurred at 38 and 42 °C. However, injection of a high dose (10 pmol) induced darkening even at these high temperatures. Transfer of nymphs injected with 1 pmol of [His⁷]-corazonin from 30 to 42 °C, and vice versa, indicated that temperature influenced darkening at any time after injection. Measurements of the luminance of the pronotum were made using commercially available computer software to follow continuous changes in darkening of the body. The body colour of nymphs injected with [His⁷]-corazonin was reddish-brown at 25 °C, blackish at 30 and 34 °C, and greyish or whitish at 38 and 42 °C. High temperature also suppressed darkening in a normal (pigmented) strain. Most nymphs transferred from 30 to 42 °C during the first three stadia developed a light colour in the fifth stadium without the striking black patterns that are typically manifested in gregarious nymphs at lower temperatures. Such individuals developed black patterns in the fifth stadium when injected with [His⁷]-corazonin at a mid stage of the previous stadium. These results indicate that high temperature may induce light body coloration by suppressing the release of [His⁷]-corazonin in normal locusts.     

The dark-color inducing neuropeptide, [His(7)]-corazonin,causes a shift in morphometic characteristics towards the gregarious phase in isolated-reared (solitarious) Locusta migratoria

The neurohormone, [His⁷]-corazonin is known to induce dark color in the cuticle and epidermis of Locusta migratoria. In the present study, we examined the effects of this hormone on development and morphometrics in two strains, albino and normal, of this locust under isolated conditions. Injection of [His⁷]-corazonin induced dark color in both strains. In either strain, [His⁷]-corazonin injected at the second and third instars did not affect duration of nymphal development or the number of nymphal instars. The shape of the pronotum was more convex in isolated-reared animals than in crowd-reared ones, and injection of [His⁷]-corazonin caused isolated-reared animals to develop a less convex pronotum in the normal strain injected at a high dose (1 nmol×2) but not in the albino strain injected at a low dose (50 pmol×2). [His⁷]-corazonin injected into isolated-reared nymphs caused a shift in 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) towards values typical for crowd-reared (gregarious) individuals of the two strains. This study demonstrated for the first time that [His⁷]-corazonin affected morphometric characteristics in L. migratoria.     

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 .     

Mass spectrometric evidence for the deficiency in the dark-color-inducing hormone,.

A factor present in the brain and corpus cardiacum responsible for the induction of dark colour in Locusta migratoria was recently isolated and identified from the corpora cardiaca of normally pigmented locusts. The purification of this factor, designated as [His7]-corazonin was monitored using an albino mutant from a laboratory colony of an Okinawa (Japan) strain. In this study, we provide unequivocal mass spectrometric evidence that the brain and the corpora cardiaca of this albino Locusta mutant are deficient in [His7]-corazonin. Previously, [His7]-corazonin was shown to be responsible for the induction of dark body colour patterns as observed in crowded locusts. Using nanoflow-liquid chromatography-mass spectrometry, we demonstrated that this dark colour-inducing hormone is, however, present in the corpora cardiaca of solitary locusts (Schistocerca gregaria). Arch.     

Phase-related morphological changes induced by [His7]-corazonin in two species of locusts, Schistocerca gregaria and Locusta migratoria (Orthoptera: Acrididae)

The effects of a neurohormone, [His(7)]-corazonin, on phase-related morphological traits (F/C and E/F ratios; F = length of the hind femur, C = maximum width of the head; E = length of fore wing) were re-examined in the desert locust, Schistocerca gregaria Forsk?l. The F/C ratio was significantly different between adults with five and six nymphal instars, respectively, indicating that they need to be analysed separately. Injections of the synthesized peptide (1 nmol) into individually-reared (solitary) nymphs at the second and third instars caused a shift in classical morphometric ratio towards the value typical for crowded (gregarious) individuals in both sexes. The E/F ratio, which is smaller in solitary locusts than in gregarious ones, was also influenced significantly by injections of [His(7)]-corazonin into individually-reared locusts. The effect of [His(7)]-corazonin on E/F ratios was shown more clearly when the nymphs were injected at a higher dose (2 nmol) at the beginning of the third instar. Single injections of the peptide into individually-reared nymphs at different instars revealed that the earlier the injection the larger the 'gregarizing' effects of the peptide on F/C and E/F ratios. The same tendency was also detected in Locusta migratoria Linnaeus. These results supported the hypothesis that [His(7)]-corazonin plays an important role in the control of phase polymorphism in locusts.     

Behavioural differences in Locusta migratoria associated with albinism and their relation to [His7]-corazonin

Abstract. The neuropeptide [His⁷]-corazonin induces black colouration in locusts, a feature typically occurring in gregarious animals. To date, the function of this neuropeptide in relation to phase transition has not been fully clarified. Unanswered questions on its involvement in behavioural and morphometrical phaseshifting and possible temperature-induced regulation still remain. In the African locust, Locusta migratoria migratorioides (Reiche and Fairmaire) (Orthoptera: Acrididae), the gregarious form possesses mainly a brown colouration with typical intense black pigmentation. To find a possible behavioural function, an albino L. migratoria mutant, deficient in [His⁷]-corazonin and thus lacking the typical gregarious colour, was used. Between these two phenotypes, nonphase specific behavioural differences in a phase-dependent behavioural set-up were found. Additionally, a wild-type of the Okinawa strain was screened behaviourally and located in comparison to the other groups. Treatment of albinos with corazonin induced normal phenotype behaviour. The degree of interference of corazonin in relation to these findings and a possible effect at the level of visual perception is discussed.     

飞蝗变型及体色多型的内分泌控制机理

飞蝗具变型现象,散居型和群居型在形态特征、生理机能、行为及体色等方面存在明显差异。保幼激素已被证实能诱导飞蝗散居型绿色的出现。近年,从飞蝗心侧体成功地分离了一种神经肽——黑化诱导激素([His^7]-corazonin),并用白化型进行检测,证实了其对体色黑化作用的活性。不同时间对飞蝗若虫注射不同剂量的[His^7]-corazonin,能诱导出现除绿色以外的散居型体色,如浅褐色、褐色、赤褐色、黑色等;也能诱导出现群居型的体色,即黑色配以橘黄色的底色。而且,对散居型若虫注射[His^7]-corazonin能诱导其形态向群居型转换。这些研究证实[His^7]-corazonin对飞蝗的变型有着重要的控制作用,但又不是唯一的。     

Effects of [His(7)]-corazonin on the phase state of isolated-reared (solitarious) desert locusts,Schistocerca gregaria

[His⁷]-corazonin is a neuropeptide produced in the pars lateralis of the brain. It is stored in the corpora cardiaca and probably released from there. The only well-documented effect in locusts is increased melanization of the cuticle. We investigated whether this hormone might also be causally related to changes in behavior and morphometrics that, like melanization, occur during crowding-induced gregarization. Solitary fourth-instar nymphs of Schistocerca gregaria (Forsk.) were injected thrice with 1 nmol [His⁷]-corazonin. After molting to the 5th stadium their behavioral phase state was measured in an arena assay and analyzed using multiple logistic regression analysis. The hormone was found not to induce behavioral phase changes. Upon reaching adulthood, morphometrics shifts occurred towards values typical for crowd-reared and regregarized animals. Our results thus indicate that [His⁷]-corazonin is not involved in behavioral gregarization but may participate in morphometrical phase change.     

Endocrine mechanisms controlling body-color polymorphism in locusts.

The present article reviews recent published and unpublished findings on the hormonal mechanisms for the control of body-color polymorphism in locusts. Emphasis is placed on the dark color-inducing factors and their role in the induction of various types of body coloration observed under different environmental conditions. Implantation of corpora cardiaca (CC) taken from normal nymphs of Locusta migratoria induced dark color in nymphs of an albino strain. Using the albino strain for the bioassay, a neuropeptide, [His7]-corazonin, was identified as a dark color-inducing factor for L. migratoria and Schistocerca gregaria. In the former, depending upon the dose and timing of the injection, this peptide and juvenile hormone developed various body colors looking like those found in nature. The body coloration characteristic of gregarious forms was also induced in isolated albino nymphs and field-collected solitary nymphs. In S. gregaria, on the other hand, the peptide induced black patterns, but the orange or yellow background color observed in gregarious forms was not induced when the peptide was injected into solitary individuals. [His7]-corazonin also induced darkening in other grasshoppers and locusts, but not in katydids. Albino L. migratoria developed dark color when implanted with brains or CC taken from other insects belonging to 10 major insect orders, but not with those from Coleoptera. [His7]-corazonin or a similar compound is widespread among insects and plays a pivotal role in controlling body color in some species and presumably other physiological roles in other species. Arch.     

The endocrine control of phase transition: some new aspects

Abstract. The present article summarizes some recent findings relating to the underlying mechanism of phase transition in locusts, from the nonswarming solitarious phase to the swarming gregarious phase. These phases differ in many traits, such as colouration, morphometrics and behaviour. The most comprehensive theory at present to explain the switch from the nonswarming to the swarming form is that the locusts are brought together by the heterogeneity of the environment. They gather at preferential structures and food plants and physical contact then stimulates individuals to gregarize. Phase change can also be transferred across generations by maternal pheromones. The endocrine regulation of phase polymorphism is still not fully understood. The role of ecdysteroids has been studied, so far with no final conclusion. It is remarkable that the prothoracic glands persist longer in isolated-reared adults, which implies that these glands continue to play a role, although they no longer release important amounts of ecdysteroids. Juvenile Hormone, without any doubt, induces certain solitarious characteristics, such as green colouration, but is not the primary causal factor. A real breakthrough was the discovery of [His⁷]-corazonin, made possible by using a novel assay system, the Okinawa albino mutant of Locusta migratoria , which was known to be deficient in this hormone. This peptide, which is produced in the brain and is most likely released via the corpora cardiaca, promotes the gregarious black pigmentation. It also plays a role in morphometrical phase change as well as in behavioural alterations. Corazonin is apparently quite an important peptide not only in locusts, but also in insects in general.     

Age-dependent response of adults of a homochrome grasshopper, Oedipoda miniata, to the dark-colour-inducing neurohormone (DCIN) of locusts

Abstract The effect of the dark-colour-inducing neurohormone (DCIN = [His⁷]-corazonin) of locusts was investigated in field-collected young and old adults of the grasshopper, Oedipoda miniata (Pallas) (Orthoptera: Acrididae). This species shows homochromy, but neither green-brown, nor phase-dependent colour polymorphism. By injecting graded doses of synthetic DCIN in 2 µL of olive oil, young adults were tested within a week of their last moult, and old adults 3.5 months later, a few weeks before natural termination of their reproductive summer diapause. Colour changes were followed for 28 days after injection. Darkening of the young adults was considerable, but their response to DCIN is more moderate than that of conspecific nymphs, by exhibiting a higher threshold, slower response and weaker maximal response. Old adults also show a clear effect, but their response is even slower and less marked than the response of the young adults. It is concluded that the response to DCIN decreases from nymphs to young adults and it is further decreasing with ageing of the adults.     

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.     

Functional Analysis of Conserved Histidines in Choline Acetyltransferase by Site-Directed Mutagenesis

Abstract: The choline acetyltransferase (ChAT) reaction involves the transfer of the acetyl group of acetyl-CoA to choline, in which an active site histidine is believed to act as a general acid/base catalyst. A comparison of the deduced amino acid sequences of the enzyme from Drosophila , pig, rat, and Caernohabditis elegans revealed three conserved histidines: Drosophila His²⁶⁸, His³⁹³, and His⁴²⁶. Each of these histidines was replaced by a leucine and a glutamine, and the kinetic properties of each of the recombinant mutant enzymes were determined. The mutations yielded active His²⁶⁸Leu-ChAT, His^Z68Gln-ChAT, and His³⁹³Gln-ChAT and inactive His³⁹³Leu-ChAT, His⁴²⁶Leu- ChAT, and His⁴²⁶Gln-ChAT. The kinetic constants K_m(CoA), K_{m(acetyloholine)}. and V_max were essentially the same for all of the active mutants. When the integrity of the CoASAc binding site was investigated in the inactive mutants, the data suggested that the binding site in His³⁹³Leu-ChAT is disrupted but conserved in His⁴²⁶Leu-ChAT and His⁴²⁶Gln- ChAT. These results suggest that His⁴²⁶ is an essential catalytic residue and could serve as an acid/base catalyst.     

Identification of

The neuropeptides inducing dark color in albino nymphs of the migratory locust Locusta migratoria were isolated from the larval brain of the silkworm, Bombyx mori and from the adult corpora cardiaca (CC) of the cricket Gryllus bimaculatus, respectively, and their amino acid sequences identified. The two peptides isolated from the two different species are identical to [Arg(7)] corazonin, a neuropeptide known to be present in a cockroach and others. This peptide induces a dark color in albino nymphs of L. migratoria at fmol levels, and a high dose of >/=100 pmol caused albino locusts to turn completely black, but it influenced neither body color nor metamorphosis in B. mori and G. bimaculatus. Therefore, the physiological functions of [Arg(7)] corazonin in the silkworm and the cricket remain unknown. The present study demonstrated the usefulness of the albino strain of L. mirgatoria as a specific bioassay system for this peptide.     

Key Residues Defining the μ-Opioid Receptor Binding Pocket: A Site-Directed Mutagenesis Study

Abstract: Structural elements of the rat μ-opioid receptor important in ligand receptor binding and selectivity were examined using a site-directed mutagenesis approach. Five single amino acid mutations were made, three that altered conserved residues in the μ, δ, and κ receptors (Asn¹⁵⁰ to Ala, His²⁹⁷ to Ala, and Tyr³²⁶ to Phe) and two designed to test for μ/δ selectivity (Ile¹⁹⁸ to Val and Val²⁰² to Ile). Mutation of His²⁹⁷ in transmembrane domain 6 (TM6) resulted in no detectable binding with [³H]DAMGO (³H-labeled d -Ala², N -Me-Phe⁴,Gly-ol⁵-enkephalin), [³H]bremazocine, or [³H]ethylketocyclazocine. Mutation of Asn¹⁵⁰ in TM3 produces a three- to 20-fold increase in affinity for the opioid agonists morphine, DAMGO, fentanyl, β-endorphin_1–31, JOM-13, deltorphin II, dynorphin_1–13, and U50,488, with no change in the binding of antagonists such as naloxone, naltrexone, naltrindole, and nor-binaltorphamine. In contrast, the Tyr³²⁶ mutation in TM7 resulted in a decreased affinity for a wide spectrum of μ, δ, and κ agonists and antagonists. Altering Val²⁰² to Ile in TM4 produced no change on ligand affinity, but Ile¹⁹⁸ to Val resulted in a four- to fivefold decreased affinity for the μ agonists morphine and DAMGO, with no change in the binding affinities of κ and δ ligands.     

Genetic and hormonal control of melanization in reddish–brown and albino mutants in the desert locust Schistocerca gregaria

The genetic and hormonal control of body colouration is investigated using two recessive genetic mutant strains, the reddish–brown (RB) mutant and an albino mutant, as well as a normal (pigmented) strain of the desert locust Schistocerca gregaria. The colour patterns of the RB nymphs are similar to those of a normal strain, although the intensity of the melanization is weaker in the former. Reciprocal crosses between the RB and albino mutants produce only normal phenotypes in the F₁ generation. In the F₂ generation, the normal, RB and albino phenotypes appear in a ratio of 9 : 3 : 4, indicating that two Mendelian units might determine the appearance of dark body colour and the intensity of melanization, respectively. In other words, at least two steps of regulation might be involved in the expression of body colour. Injections of [His⁷]‐corazonin, a neuropeptide inducing dark colour in this locust, fail to induce dark colour in albino nymphs but show a dose‐dependent darkening in RB nymphs in the range, 10 pmol to 1 nmol. Some RB nymphs become indistinguishable from normal individuals after injection of the peptide. Implantation of corpora cardiaca (CC) taken from RB mutants into other RB individuals induces darkening in the latter and CC from RB, albino and normal strains have similar dark colour‐inducing activity when implanted into albino Locusta migratoria. These results suggest the possibility that the RB mutant gene regulates the intensity of melanization, possibly through controlling the pathway of pigment biosynthesis associated with [His⁷]‐corazonin.     

在转Bt基因棉压力下棉铃虫和异色瓢虫的波动性不对称

以取食转Bt基因棉的棉铃虫和取食转Bt基因棉上的蚜虫的异色瓢虫为研究对象,结合各自的适合度参数考察了波动性不对称（fluctuating asymmetry, FA）在不同种群中各对称性状间的表现情况。结果表明： 处理组棉铃虫种群的前、中、后足腿节,前翅肘横脉,前翅副室短径,后翅翅长均表现出FA;与对照种群比较,前足腿节与后翅翅长的FA值较高。 适合度参数测定结果显示,棉铃虫卵孵化率与成虫寿命显著低于对照种群,而成虫产卵量和幼虫发育历期与对照种群差异不显著。异色瓢虫取食了转Bt基因棉上的蚜虫后对其适合度各参数及各虫龄的捕食功能反应均无不良影响,其后翅翅长表现出FA,但与对照种群的FA值无显著差异。FA的大小与种群适合度间存在一定的联系,尚需做进一步的分析。     

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.     

Nerve Growth Factor, Epidermal Growth Factor, and Insulin Differentially Potentiate ATP-Induced [Ca2+]i Rise and Dopamine Secretion in PC12 Cells

Abstract: To study how growth factors affect stimulus-secretion coupling pathways, we examined the effects of nerve growth factor (NGF), epidermal growth factor (EGF), and insulin on ATP-induced [Ca²⁺]_i rise and dopamine secretion in PC12 cells. After a 4-day incubation of cells, all three factors increased ATP-induced dopamine secretion significantly. We then examined which step of ATP-induced secretion was affected by the growth factors. Cellular levels of dopamine-β-hydroxylase and catecholamines were increased by NGF treatment but were not affected by EGF or insulin. The ATP-induced [Ca²⁺]_i rise was also enhanced after growth factor treatment. The EC₅₀ of ATP for inducing [Ca²⁺]_i rise and dopamine secretion was increased by NGF treatment but not by treatment with EGF or insulin. Accordingly, the dependence on [Ca²⁺]_i of dopamine secretion was increased significantly only in NGF-treated cells. Our results suggest that for EGF- and insulin-treated PC12 cells, the increase in secretion is mainly due to increased potency of ATP in inducing [Ca²⁺]_i rise. NGF treatment not only increased the potency of ATP but also decreased the Ca²⁺ sensitivity of the secretory pathway, which as a result becomes more tightly regulated by changes in [Ca²⁺]_i.     

The inhibition of lipid synthesis in vitro in the locust, Schistocerca gregaria, by factors from the corpora cardiaca

ABSTRACT. The rate of lipid synthesis from [¹⁴C]acetate in fat body from Schistocerca americana gregaria has been studied in vitro. Maximum incorporation is found on days 6–10 in adults and day 4 of the fifth stadium. The label appeared in the fatty acid components of triacyl-glycerol, diacylglycerol and phospholipid.
Lipid synthesis in vitro was inhibited by extracts of corpora cardiaca, and such inhibition was most marked (up to 85%) in fat bodies from insects at stages where fatty acid synthesis was greatest. HPLC separation of corpora cardiaca extracts gave several active fractions of which the most active was adipokinetic hormone 1 (AKH-1).