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.     

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.     

Albino corpus cardiacum extracts induce morphometric gregarization in isolated albino locusts, Locusta migratoria, that are deficient in corazonin

Abstract.  The neuropeptide [His⁷]-corazonin, present in the central nervous system and corpus cardiacum, is known to mimic a 'gregarizing' effect on phase-related morphometric ratios (hind femur length/maximum head width and fore wing length/hind femur length) when injected into locusts reared in isolation. However, an albino strain is known to exhibit phase-specific changes in these ratios in response to rearing density, although it is deficient in [His⁷]-corazonin. To examine whether there is a second factor responsible for this phenomenon, perhaps a corazonin-like factor that has lost its dark-colour inducing activity, methanol extracts of corpora cardiaca taken from crowd-reared albino nymphs of Locusta migratoria are injected into isolated-reared second-stadium albino nymphs and reared to adults in isolation. The hind femur length/maximum head width and fore wing length/hind femur length ratios are significantly different from those of control oil-injected counterparts, and shift significantly towards the values typical for crowd-reared gregarized individuals. The results indicate that the corpora cardiaca contain a factor similar to [His⁷]-corazonin, although it has no dark-colour inducing activity.     

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.     

Hormonal control of body-color polymorphism in Locusta migratoria: interaction between

The effects of injection of [His(7)]-corazonin and juvenile hormone (JH) III on the body color in L. migratoria were investigated using albino and normal (pigmented) nymphs. Most albino nymphs turned green in the fourth instar if injected with JH III during the last 2 days of the previous instar. When albino third instar nymphs injected with 10 pmol of [His(7)]-corazonin on different days were treated with 100 μg of JH III on day 3.5, they developed various body colors in the following nymphal instar: those injected with [His(7)]-corazonin during the first 2 days developed very dark green or black color, whereas some of those injected after this period turned green and their legs and ventral side of the body were variously pigmented, the coloration being similar to green solitary individuals often found in the field. Field-collected brown solitary nymphs injected with 1 nmol of [His(7)]-corazonin and kept individually, turned reddish without any black spots in the following nymphal instar when the ecdysis occurred within 1 day after injection. Injection of [His(7)]-corazonin 2 days before the following ecdysis induced black patterns on an orange background color, the coloration characteristic of gregarious forms. Similar injections into field-collected green solitary nymphs also induced black patterns but the rest of their body remained green. These results may indicate that the temporal changes in the hemolymph titers of [His(7)]-corazonin and JH play an important role in the control of body-color polymorphism in this locust.     

The trans-generational phase accumulation in the desert locust: Morphometric changes and extra molting

To understand the underlying trans-generational phase accumulation, a classical morphometric characteristic, the F/C ratio (F, hind femur length; C, maximum head width), of adult desert locusts (Schistocerca gregaria) was monitored over eight consecutive generations. Adult F/C ratios, which are larger in solitarious locusts than in gregarious ones, were negatively correlated to the darkness of body color at hatching. Two successive generations were required for a complete shift from the gregarious (crowd-reared) to the solitarious (isolated-reared) phase and vice versa in the laboratory. That is (1) female adults needed to be exposed to crowded (or isolated) conditions so that their hatchlings would become large (or small) and dark (or green) in color, and (2) the hatchlings then needed to be exposed to crowded (or isolated) conditions for their entire nymphal stage. Solitarious locusts exhibited extra molting that influenced the F/C ratio in the adult stage, but did not exert significant influences on the trans-generational changes in this trait because the incidence was low. The incidence of extra molting was negatively correlated with nymphal survival rates. The morphometric trans-generational changes may be explained without assuming any accumulating internal factor.     

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.     

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.     

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 role of

The effect of [His(7)]-corazonin on the body color in Locusta migratoria was examined by varying the injected dose and the time of injection in both an albino and a normal (pigmented) strain. Albino nymphs injected with a high dose (100pmol) of [His(7)]-corazonin at the beginning of the third instar turned completely black in the following instar, whereas those injected with the same dose in the middle of the instar developed black patterns with an orange background color, the body coloration characteristic of normal gregarious (crowded) individuals. Injection at the end of the third instar induced a reddish color with few black spots. Irrespective of the time of injection of the peptide, most of these individuals became completely black after ecdysis to the fifth instar. A similar result was obtained with a lower dose (1pmol), although the color expressed was lighter. In the normal strain, injection of 1nmol or 100pmol into crowded third instar nymphs also caused most of them to become completely black in the fourth and fifth instars, but a lower dose apparently had no influence. These results suggest that the temporal changes in hemolymph titer of [His(7)]-corazonin are important in the expression of body color in L. migratoria.     

Hormonal control of phase-related changes in the number of antennal sensilla in the desert locust, Schistocerca gregaria: possible involvement of [His7]-corazonin

The effect of [His(7)]-corazonin on the abundance of antennal sensilla in the desert locust, Schistocerca gregaria, was investigated to test the hypothesis that injection of this neuropeptide would mimic a crowding effect. Solitarious locusts (reared in isolation) were injected with [His(7)]-corazonin at the 3rd nymphal instar and the numbers of sensilla on the 2nd, 8th and 14th antennal segments in the adult stage were compared with those for oil-injected solitarious controls or un-injected gregarious locusts (reared in group). The numbers of sensilla on these antennal segments were all reduced significantly after [His(7)]-corazonin injection compared with those for oil-injected controls, but similar to the values for gregarious individuals. Among the four major types of olfactory sensilla, coeloconic, trichoid, basiconic type A and basiconic type B, [His(7)]-corazonin injection influenced the abundance of all but the last type. The effect of [His(7)]-corazonin injection varied with the time of injection; the earlier the injection the larger the effects on the abundance of total antennal sensilla on the 8th segment, although the way in which the injection affected the abundance varied with the sensillum type. A hypothesis explaining how crowding affects the abundance of antennal sensilla and other phase-related characteristics through changes in [His(7)]-corazonin concentrations was proposed.     

Pathogenicity of Two Species of Entomopathogenic Nematodes Against the Greenhouse Whitefly,Trialeurodes vaporariorum (Hemiptera: Aleyrodidae), in Laboratory and Greenhouse Experiments

The greenhouse whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) is a polyphagous pest in greenhouse crops. The efficacy of two entomopathogenic nematodes (EPN), Steinernema feltiae and Heterorhabditis bacteriophora, as biological control agents against T. vaporariorum was evaluated using two model crops typical of vegetable greenhouse productions: cucumber and pepper. Laboratory tests evaluated adults and second nymphal instars for pest susceptibility to different EPN species at different concentrations of infective juveniles (IJ; 0, 25, 50, 100, 150, 200, and 250 IJ per cm²); subsequent greenhouse trials against second nymphal instars on cucumber and pepper plants evaluated more natural conditions. Concentrations were applied in combination with Triton X-100 (0.1% v/v), an adjuvant for increasing nematode activity. In laboratory studies, both life stages were susceptible to infection by the two nematode species, but S. feltiae recorded a lower LC₅₀ than H. bacteriophora for both insect stages. Similarly, in greenhouse experiments, S. feltiae required lower concentrations of IJ than H. bacteriophora to reach the same mortality in nymphs. In greenhouse trials, a significant difference was observed in the triple interaction among nematode species × concentration × plant. Furthermore, the highest mortality rate of the second nymphal instars of the T. vaporariorum was obtained from the application of S. feltiae concentrated to 250 IJ/cm² on cucumber (49 ± 1.23%). The general mortality caused by nematodes was significantly higher in cucumber than in pepper. These promising results support further investigation for the optimization of the best EPN species/concentration in combination with insecticides or adjuvants to reach a profitable control of this greenhouse pest.     

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。     

Mass spectrometric analysis of peptides in brain neurosecretory cells and neurohemal organs in the adult blowfly,Protophormia terraenovae

Neuropeptides in neurosecretory cells of the pars intercerebralis (PI) and pars lateralis (PL) in the brain, and those in the corpus cardiacum–hypocerebral ganglion complex (CC-HG) and corpus allatum (CA) were examined by mass spectrometry and immunocytochemistry in adult females of the blowfly, Protophormia terraenovae. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometry (ESI-Q-Tof MS) and MS/MS, 4 peptides (including myosuppressin and SIFamide) were detected in the PI, 12 peptides (including [Arg⁷]-corazonin and [Arg⁷]-corazonin^3–11) in the PL, 13 peptides (including myosuppressin, [Arg⁷]-corazonin and [Arg⁷]-corazonin^3–11) in the CC-HG, and 6 peptides in the CA. MALDI-TOF MS analysis of each tissue or organ was made in about 20 flies under diapause-inducing (LD 12:12 at 20 °C) and diapause-averting conditions (LD 18:6 at 25 °C). These molecular ion peaks did not distinctively differ between diapause-inducing and diapause-averting conditions. A peptide with an m/z value at 1395.1 was purified from 240 brains and the 2nd–10th amino acids were sequenced as –YRKPPFNGS–, corresponding to a partial sequence of SIFamide. Only two pairs of somata in the PI were immunoreactive to antisera against SIFamide, which were local neurons widely extending fibers throughout the brain neuropils.     

Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively

The phenotypic and genotypic characteristics of seventeen Achromobacter strains representing MLST genogroups 2, 5, 7 and 14 were examined. Although genogroup 2 and 14 strains shared a DNA–DNA hybridization level of about 70%, the type strains of both genogroups differed in numerous biochemical characteristics and all genogroup 2 and 14 strains could by distinguished by nitrite reduction, denitrification and growth on acetamide. Given the MLST sequence divergence which identified genogroups 2 and 14 as clearly distinct populations, the availability of nrdA sequence analysis as a single locus identification tool for all Achromobacter species and genogroups, and the differential phenotypic characteristics, we propose to formally classify Achromobacter genogroups 2, 5, 7 and 14 as four novel Achromobacter species for which we propose the names Achromobacter insuavis sp. nov. (with strain LMG 26845^T [= CCUG 62426^T] as the type strain), Achromobacter aegrifaciens sp. nov. (with strain LMG 26852^T [= CCUG 62438^T] as the type strain), Achromobacter anxifer sp. nov. (with strain LMG 26857^T [= CCUG 62444^T] as the type strain), and Achromobacter dolens sp. nov. (with strain LMG 26840^T [= CCUG 62421^T] as the type strain).     

Corazonin reduces the spinning rate in the silkworm,Bombyx mori

The insect neuropeptide, [Arg⁷]-corazonin was injected into larvae of the silkworm, Bombyx mori to investigate its influence on development and behavior. A single injection of 50 pmol of corazonin into the fourth and fifth instar larvae induced prolongation of the spinning period in all experimental groups except for those injected on day 10 of the fifth instar. The injection also caused a prolongation of the pupal period in some experimental groups, while it had no effect on the timing of larval ecdysis and the length of feeding period of the fifth instar. The spinning period was significantly prolonged even at a low dose of 1 pmol. Both the spinning rate and the rate of increase in hemolymph ecdysteroid level during the spinning stage were reduced by injection of corazonin. However, corazonin injection during days 5-7 of the fifth instar reduced the spinning rate without influencing the ecdysteroid level until the end of day 8, thereafter the rate of increase in hemolymph ecdysteroid level was slower in the corazonin-injected larvae than in the control larvae. Therefore, the suppressed ecdysteroid level observed in the corazonin-injected larvae appears to be a result rather than a cause of the reduced spinning rate. This study is the first published report for the corazonin effect on the behavior in insects.     

Quantitative analysis of herbivore-induced cytosolic calcium by using a Cameleon (YC 3.6) calcium sensor in Arabidopsis thaliana

Ca²⁺ is a key player in plant cell responses to biotic and abiotic stress. Owing to the central role of cytosolic Ca²⁺ ([Ca²⁺]_cyt) during early signaling and the need for precise determination of [Ca²⁺]_cyt variations, we used a Cameleon YC 3.6 reporter protein expressed in Arabidopsis thaliana to quantify [Ca²⁺]_cyt variations upon leaf mechanical damage (MD), herbivory by 3rd and 5th instar larvae of Spodoptera littoralis and S. littoralis oral secretions (OS) applied to MD. YC 3.6 allowed a clear distinction between MD and herbivory and discriminated between the two larvae instars. To our knowledge this is the first report of quantitative [Ca²⁺]_cyt determination upon herbivory using a Cameleon calcium sensor.     

Knockdown of the corazonin gene reveals its critical role in the control of gregarious characteristics in the desert locust

The two plague locusts, Schistocerca gregaria and Locusta migratoria, exhibit density-dependent phase polyphenism. Nymphs occurring at low population densities (solitarious forms) are uniformly colored and match their body color to the background color of their habitat, whereas those occurring at high population densities (gregarious) develop black patterns. An injection of the neuropeptide, corazonin (Crz) has been shown to induce black patterns in locusts and affect the classical morphometric ratio, F/C (F, hind femur length; C, maximum head width). We herein identified and cloned the CRZ genes from S. gregaria (SgCRZ) and L. migratoria. A comparative analysis of prepro-Crz sequences among insects showed that the functional peptide was well conserved; its conservation was limited to the peptide region. Silencing of the identified SgCRZ gene in gregarious S. gregaria nymphs markedly lightened their body color and shifted the adult F/C ratio toward the value typical of solitarious forms. In addition, knockdown of the gene in solitarious nymphs strongly inhibited darkening even after a transfer to crowded conditions; however, these individuals developed black patterns after being injected with the Crz as a rescue treatment. SgCRZ was constitutively expressed in the brains of S. gregaria during nymphal development in both phases. This gene was highly expressed not only in the brain in both phases, but also in the corpora allata in the gregarious phase. This conspicuous phase-dependent difference in SgCRZ gene expression may indicate a functional role in the control of phase polyphenism in this locust.     

Occurrence and genetics of black‐eyed migratory locusts,Locusta migratoria (Orthoptera: Acrididae)

Black‐eyed Locusta migratoria appeared in albino locusts as a result of crossing between a short‐winged strain originating from Tsushima Island, Japan, and an albino strain originating from Okinawa Island. The black eye trait was recessive to the white eye trait because the crosses between black‐ and white‐eyed albino locusts produced only individuals with white eyes in the F₁ generation. In the F₂ generation, black‐ and white‐eyed individuals appeared in a ratio of 1:3, indicating that the black eye trait was controlled by a simple Mendelian unit. The black eye trait showed no genetic association with other traits including wing morph, adult body dimensions and classical morphometric ratios such as hind femur length / head width and forewing length / hind femur length.     

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.