Inhibition of fungal growth in thermoregulating locusts,Locusta migratoria,infected by the fungus Metarhizium anisopliae var acridum

The locust, Locusta migratoria, has the capacity to develop a behavioural fever which reduces fungal infection by Metarhizium anisopliae var acridum. We investigated hemocyte and blastospore kinetics in infected insects under conditions that did or did not allow thermoregulation. Hemocyte concentrations were severely reduced in inoculated insects that did not thermoregulate but remained similar to those of controls in inoculated insects that were allowed to thermoregulate. Reductions in hemocyte counts were accompanied by an increase in the concentration of blastospores. In non-thermoregulating insects, circulating blastospores were first observed two days post-inoculation and had heavily colonized the hemolymph by day 5; in contrast, no blastospores were recovered from hemolymph of inoculated-thermoregulating insects. We used fluorescein isothiocyanate (FITC)-labelled silica beads to examine in vivo phagocytosis in thermoregulating and non-thermoregulating locusts. In the absence of fungus, a greater proportion of beads were engulfed by hemocytes in thermoregulating than in non-thermoregulating locusts early (4 and 24h) after bead injection, but the proportions were similar thereafter. In infected locusts, phagocytosis in non-thermoregulating insects was progressively impaired; such impairment, however, was not observed in challenged, thermoregulating insects. Our results suggest that thermoregulation helped keep fungal growth in check, apparently through the maintenance of hemocyte population levels and the direct inhibition of blastospore propagation by elevated temperatures.     

Haemolymph levels of Schistocerca gregaria ion transport peptide and ion transport-like peptide

Abstract.  Titres of ion transport peptide (ITP) and ion transport-like peptide (ITP-L) in the haemolymph of the desert locust Schistocerca gregaria Forskål are investigated using a combination of enzyme-linked immunosorbent assay and reversed-phase high-performance liquid chromatography (HPLC). The estimated circulating levels of these two peptides are used to investigate their putative physiological roles. Haemolymph levels of ITP-L are significantly higher in fed and exercise-stressed locusts than in starved insects, suggesting a role for ITP-L in postfeeding osmoregulation and metabolism associated with stress. The higher titres of ITP-L in the final nymphal stadium could be related to increased levels of ecdysteroids over this same period. In addition detection of ITP-L in the brain and corpus cardiacum of the locust demonstrates for the first time that the ITP-L is expressed in these tissues. Immunoreactivity to antibodies raised against ITP is significantly higher in fed locusts compared with starved insects. However, >95% of this immunoreactivity elutes later than synthetic ITP when separated by HPLC. The results suggest that this more hydrophobic immunoreactivity is neither native ITP nor a metabolic breakdown product of ITP. Haemolymph levels of immunoreactivity to ITP during the last nymphal stadium are similar to ITP-L but, unlike ITP-L, there is no measured increase in immunoreactivity to ITP due to exercise stress.     

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

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

Role of Olfactory and Visual Cues in the Attraction/Repulsion Responses to Conspecifics by Gregarious and Solitarious Desert Locusts

Desert locusts [Schistocerca gregaria Forskål (Orthoptera, Acrididae)] change phase in response to population density: solitarious insects avoid one another, but when crowded they change to the gregarious phase and aggregate. The attraction/repulsion responses of gregarious and solitarious locusts maintain phase differences in locust populations. Despite considerable research, the cues for aggregation are poorly understood; moreover, the repulsion response of solitarious locusts has not previously been investigated. This study analyzes the role of visual and olfactory stimuli in triggering these different responses to conspecifics. Isolation-reared insects were repelled by both olfactory and visual stimuli from other locusts. Crowd-reared insects were attracted by the combination of olfactory and visual cues. In addition, olfactory stimuli affected other behaviors in both phases, and behavioral differences between isolation- and crowd-reared locusts were clear even in the absence of conspecifics. The sensory and neurological mechanisms underlying these responses are not well understood and will form the basis for neurobiological investigations of locust phase.     

Feeding causes the appearance of a factor in the haemolymph that stimulates protein synthesis

Soon after a locust (Locusta migratoria) begins to feed, an increase in protein synthesis can be detected in the animal. Isolation of fat body shows that this tissue synthesizes protein at a faster rate in recently fed animals than it does in fasting insects. Fasting locusts injected with haemolymph from fed insects increased protein synthesis when compared with locusts injected with haemolymph from fasting locusts. The factor causing this increase was present in the haemolymph within 5 min of feeding. Feeding or direct contact with the food was not essential to increase protein synthesis. Exposure of fasting locusts to feeding insects was sufficient to elevate the rates of protein synthesis in the fasting animals.The increase inprotein synthesis was not a result of general excitation or an increase in the concentration of tryptophan or isoleucine in the haemolymph. Ecdysteroid titres were uniformly low during the first ten days of adult life. Gel filtration of the fed haemolymph revealed a low molecular weight fraction (about 600 daltons) which stimulated protein synthesis upon injection into fasting locusts.     

Regulation of water ingestion by the locust, Chortoicetes terminifera: the effect of injections into the haemolymph

Adults of the locust Chortoicetes terminifera which had been deprived of water for 24 h took larger 'meals' of water than insects which had been deprived for 2 h. Water or dilute solutions of NaCl injected into the kaemcoel reduced the amount of water ingested by 24-h, deprived locusts. Injections of more concentrated solutions of NaCl (0.5 m) or glycerol (1.0m) increased the water intake of 2-h deprived locusts. Water injections into 24-h deprived locusts did not reduce their responsiveness to sucrose and it is concluded that haemolymph osmotic pressure plays an important role in determining the specific responsiveness of C. terminifera to water. The time course of the effects of injection indicates that the effects of haemolymph osmotic pressure on water responsiveness are mediated neurally. Injections of water into 24-h deprived insects caused a marked reduction in the sizes of their meals of seedling wheat. It is suggested that the mechanisms responsible for the regulation of water intake might limit total intake when locusts have access only to fresh vegetation of high water content.     

The role of the glandular lobes of the corpora cardiaca during flight in Locusta

ABSTRACT. Flight performance in Locusta is reduced following severance of the major afferent nerves to the corpora cardiaca or removal of the glandular lobes of the corpora cardiaca. These operations prevent the release of adipokinetic hormone and the consequent mobilization of stored lipid. However, locusts deprived of about 90% of their glandular lobe tissue, while flying poorly, did mobilize lipid. It is suggested that the remaining glandular parenchyma cells are capable of secreting enough hormone to stimulate lipid mobilization, but that the concentration may be inadequate to encourage lipid utilization. After removal of all the glandular lobe parenchyma, the blood carbohydrate concentration was temporarily depressed. Nevertheless flight performance was equally poor, both when haemolymph carbohydrate levels were low and when they had returned to normal. After the injection of trehalose into operated control locusts and locusts deprived of their glandular lobes, flight was still markedly poorer in the operated insects, even though the injection of trehalose prevented adipokinetic hormone release in the intact locust. It seems that the poor flight performance of locusts deprived of their glandular lobes cannot be fully explained by the simple absence of adipokinetic hormone.     

Invasion of the pathogenic fungus Metarhizium anisopliae through the guts of germfree desert locusts,Schistocerca gregaria

Less than 1% of an ingested inoculum of the pathogenic fungus Metarhizium anisopliae was retained for long enough (ca. 24 h) in the gut of the desert locust, Schistocerca gregaria, for germination and penetration to have occurred. The residual inoculum did not initiate an infection in guts of fed conventional or axenic locusts. However, symptoms of mycosis (hyphal bodies in the haemolymph, fungal penetration of the hindgut intima and epithelium, tetanic paralysis) were consistently observed in axenic but not conventional locusts which were starved post-inoculation.It is concluded that the antifungal toxin produced by the gut bacteria defends the desert locust against gut invasion by Metarhizium anisopliae during periods of starvation when the physical defences, prominent in fed insects, are less apparent.     

Cowpea aphid (Aphis craccivora) associated with different host plants has different facultative endosymbionts

Maternally inherited facultative endosymbiotic bacteria are common among insects, including many polyphagous insect herbivores. To investigate whether symbiont infection is structured by host plant in the polyphagous aphid Aphis craccivora Koch, pyrosequencing and diagnostic PCR were performed on 26 populations from two different host plants, alfalfa (Medicago sativa) or black locust (Robinia pseudoacacia). Results indicated that Aphis craccivora harbours distinctly different microbial communities in alfalfa versus locust. The facultative symbiont Hamiltonella was found only in aphids collected from alfalfa, and the facultative symbiont Arsenophonus was found only in aphids from locust. Hamiltonella is known to protect aphids against hymenopteran parasitoids, whereas the phenotypic effects of Arsenophonus in aphids are unknown. Correspondingly, a screen of the aphid samples for hymenopteran DNA indicated that Hamiltonella‐bearing alfalfa populations of A. craccivora experienced lower parasitism than Arsenophonus‐bearing locust populations. This study contributes to the growing body of evidence that correlative associations between bacterial endosymbionts and host plants may be a common phenomenon in polyphagous herbivores, and suggests that microbial symbionts have the potential to act as drivers for observed ecological differences among host‐associated populations of polyphagous insects.     

Functional Characterization of the Short Neuropeptide F Receptor in the Desert Locust,Schistocerca gregaria

Whereas short neuropeptide F (sNPF) has already been reported to stimulate feeding behaviour in a variety of insect species, the opposite effect was observed in the desert locust. In the present study, we cloned a G protein-coupled receptor (GPCR) cDNA from the desert locust, Schistocerca gregaria. Cell-based functional analysis of this receptor indicated that it is activated by both known isoforms of Schgr-sNPF in a concentration dependent manner, with EC₅₀ values in the nanomolar range. This Schgr-sNPF receptor constitutes the first functionally characterized peptide GPCR in locusts. The in vivo effects of the sNPF signalling pathway on the regulation of feeding in locusts were further studied by knocking down the newly identified Schgr-sNPF receptor by means of RNA interference, as well as by means of peptide injection studies. While injection of sNPF caused an inhibitory effect on food uptake in the desert locust, knocking down the corresponding peptide receptor resulted in an increase of total food uptake when compared to control animals. This is the first comprehensive study in which a clearly negative correlation is described between the sNPF signalling pathway and feeding, prompting a reconsideration of the diverse roles of sNPFs in the physiology of insects.     

Fate and effects of the trehalase inhibitor trehazolin in the migratory locust (Locusta migratoria)

Trehalose is the main haemolymph sugar in many insect species. To be utilized trehalose must be hydrolysed into its glucose units by trehalase (EC 3.2.1.28). Inhibitors of trehalase have attracted interest as possible pesticides and tools for studying the regulation of trehalose metabolism in insects. To make full use of these inhibitors requires knowledge of their fate and effects in vivo. To this end we have measured trehazolin in locusts using a method based on the specific inhibition of a trehalase preparation. After injection of 20 μg, trehazolin decreased in haemolymph with a half-life of 2.6 days and after 10 days almost 95% had disappeared. Trehazolin did not reach the intracellular water space of locust tissues, but appeared with full inhibitory potency in locust faeces, suggesting that it was not metabolized, but quantitatively eliminated via the gut. Haemolymph trehalose increased transiently upon trehazolin injection, it was maximal after 3 days, then decreased and reached control level after 10 days. Inhibition of flight muscle trehalase by trehazolin was prolonged and still conspicuous 21 days post injection, suggesting that trehazolin inhibits trehalase activity irreversibly in vivo and that recovery requires de novo enzyme synthesis.     

博落回生物碱对蝗虫成虫毒力试验简报

目的:测定博落回生物碱对蝗虫成虫的毒力作用,为利用药用植物提取物杀灭蝗虫的无公害防治提供参考。方法:用文献法提取博落回果实中的生物碱,制备博落回生物碱溶液;采用喷雾法将博落回生物碱溶液喷施于蝗虫成虫;进行了博落回生物碱溶液对蝗虫成虫的田间小面积试验;用计算机软件SPSS11.5进行统计学分析。结果:在96小时内,博落回生物碱对蝗虫成虫的LD50为29.568mg/L,95%可信限为2.5563～69.473mg/L。田间小面积施药3天后,每平方米内虫口减退达90%左右,持效时间7天。结论:博落回溶液对蝗虫成虫具有一定的杀灭作用。     

Selection by phage display of a variant mustard trypsin inhibitor toxic against aphids

The mustard trypsin inhibitor, MTI-2, is a potent inhibitor of trypsin with no activity towards chymotrypsin. MTI-2 is toxic for lepidopteran insects, but has low activity against aphids. In an attempt to improve the activity of the inhibitor towards aphids, a library of inhibitor variants was constructed and cloned into the pRlac3 phagemid vector. The library of 9.3 x 107 independent colonies was created by randomisation of a stretch of five consecutive codons in the reactive site. Repeated selection rounds against bovine trypsin and chymotrypsin allowed the identification of novel, MTI-2 derived, antitrypsin and antichymotrypsin inhibitors. Chy8, the selected variant with highest affinity for bovine chymotrypsin (Ki = 32 nm versus >1000 nm for the wild-type) represents the strongest known recombinant chymotrypsin inhibitor of the MTI-2 family. It is highly toxic to nymphs of the aphid Acyrthosiphon pisum, and moderately toxic to nymphs of Aphis gossypii and Myzus persicae. The LC50 of 73 microg ml-1 towards A. pisum is the lowest value known among chymotrypsin inhibitors. The aphicidal activity of Chy8 was improved eightfold compared to the wild-type inhibitor. This demonstrates, for the first time, that bovine chymotrypsin provides a useful template to select engineered proteins highly toxic against these aphids. The selected gene will allow the development of transgenic crops that are protected against sucking insect pests.     

The germ-free culture of desert locusts, Schistocerca gregaria

A technique is described for rearing germ-free desert locusts. Axenic insects appeared to develop and reproduce normally, therefore it is concluded that the gut bacterial flora does not contribute significantly to locust nutrition. However, it is suggested that the gut flora may benefit its host by contributing to the locust's defences against attack by pathogenic microorganisms. The results of morphometric measurements are discussed in terms of the bacterial origin of the so called phase pheromone, locustol. No evidence was found to support the hypothesis that microbial activity restricts gut carbohydrase.     

Density-dependent aposematism in the desert locust

The ecological processes underlying locust swarm formation are poorly understood. Locust species exhibit phenotypic plasticity in numerous morphological, physiological and behavioural traits as their population density increases. These density-dependent changes are commonly assumed to be adaptations for migration under heterogeneous environmental conditions. Here we demonstrate that density-dependent nymphal colour change in the desert locust Schistocerca gregaria (Orthoptera: Acrididae) results in warning coloration (aposematism) when the population density increases and locusts consume native, toxic host plants. Fringe-toed lizards (Acanthodactylus dumerili (Lacertidae)) developed aversions to high-density-reared (gregarious-phase) locusts fed Hyoscyamus muticus (Solanaceae). Lizards associated both olfactory and visual cues with locust unpalatability, but only gregarious-phase coloration was an effective visual warning signal. The lizards did not associate low rearing density coloration (solitarious phase) with locust toxicity. Predator learning of density-dependent warning coloration results in a marked decrease in predation on locusts and may directly contribute to outbreaks of this notorious pest.     

Identification of indole glucosinolate breakdown products with antifeedant effects on Myzus persicae (green peach aphid)

The cleavage of glucosinolates by myrosinase to produce toxic breakdown products is a characteristic insect defense of cruciferous plants. Although green peach aphids ( Myzus persicae ) are able to avoid most contact with myrosinase when feeding from the phloem of Arabidopsis thaliana , indole glucosinolates are nevertheless degraded during passage through the insects. A defensive role for indole glucosinolates is suggested by the observation that atr1D mutant plants, which overproduce indole glucosinolates, are more resistant to M. persicae , whereas cyp79B2 cyp79B3 double mutants, which lack indole glucosinolates, succumb to M. persicae more rapidly. Indole glucosinolate breakdown products, including conjugates formed with ascorbate, glutathione and amino acids, are elevated in the honeydew of M. persicae feeding from atr1D mutant plants, but are absent when the aphids are feeding on cyp79B2 cyp79B3 double mutants. M. persicae feeding from wild-type plants and myrosinase-deficient tgg1 tgg2 double mutants excrete a similar profile of indole glucosinolate-derived metabolites, indicating that the breakdown is independent of these foliar myrosinases. Artificial diet experiments show that the reaction of indole-3-carbinol, a breakdown product of indol-3-ylmethylglucosinolate, with ascorbate, glutathione and cysteine produces diindolylmethylcysteines and other conjugates that have antifeedant effects on M. persicae . Therefore, the post-ingestive breakdown of indole glucosinolates provides a defense against herbivores such as aphids that can avoid glucosinolate activation by plant myrosinases.     

Cannibalism can drive the evolution of behavioural phase polyphenism in locusts

During outbreaks, locust swarms can contain millions of insects travelling thousands of kilometers while devastating vegetation and crops. Such large-scale spatial organization is preceded locally by a dramatic density-dependent phenotypic transition in multiple traits. Behaviourally, low-density 'solitarious' individuals avoid contact with one another; above a critical local density, they undergo a rapid behavioural transition to the 'gregarious phase' whereby they exhibit mutual attraction. Although proximate causes of this phase polyphenism have been widely studied, the ultimate driving factors remain unclear. Using an individual-based evolutionary model, we reveal that cannibalism, a striking feature of locust ecology, could lead to the evolution of density-dependent behavioural phase-change in juvenile locusts. We show that this behavioural strategy minimizes risk associated with cannibalistic interactions and may account for the empirically observed persistence of locust groups during outbreaks. Our results provide a parsimonious explanation for the evolution of behavioural plasticity in locusts.     

Successful parasitation of locusts by entomopathogenic nematodes is correlated with inhibition of insect phagocytes

The entomopathogenic nematodes Heterorhabditis megidis and Steinernema feltiae turned out to be successful antagonists of the orthopteran insects Locusta migratoria and Schistocerca gregaria. The death rate of locusts maintained on nematode-inoculated sand was remarkably high. Even dosages as low as one nematode per cubic centimeter of sand killed approximately 50% of the locusts within 10 days. The impact of parasitation on locusts' immune defense was closely investigated for L. migratoria parasitized by H. megidis. Adult locusts died within 30-35 h after being fed with 50 infective H. megidis juveniles. Within the first 30 h after ingestion of the nematodes, locust hemolymph was assayed for alterations in the humoral and cellular defense components and for the presence of the nematode-associated Photorhabdus luminescens bacteria. Humoral defense was generally low without any correlation to the state of parasitation. There was no detectable activity against Escherichia coli and only little lysozyme-like activity against Micrococcus luteus. In contrast, cellular defense components were strongly influenced by parasitation. Most interestingly, the phagocytic capacity of the hemocytes was already hampered 12 h after oral application of the nematodes, whereas considerable hemocyte death occurred not earlier than 24 h after feeding. The nematode-associated bacteria could be detected in hemolymph of some of the nematode-fed locusts as early as 3 h after feeding and in all hemolymph samples after 24 h. Supernatants from isolated P. luminescens cultures were able to inhibit the L. migratoria phagocytes in vitro; thus the successful parasitation appears to be dependent on an inhibition by bacteria-released compounds rather than on overloading or simply killing of the phagocytic active hemocytes.     

A broadly tuned odorant receptor in neurons of trichoid sensilla in locust,Locusta migratoria

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. Odorant receptors (ORs) on the membrane of chemosensory neurons are believed to be key molecules in sensing exogenous chemical cues. ORs in different species of insects are diverse and should tune a species to its own specific semiochemicals relevant to their survival. The orthopteran insect, locust (Locusta migratoria), is a model hemimetabolous insect. There is very limited knowledge on the functions of locust ORs although many locust OR genes have been identified in genomic sequencing experiments. In this paper, a locust OR, LmigOR3 was localized to neurons housed in trichoid sensilla by in situ hybridization. LmigOR3 was expressed as a transgene in Drosophila trichoid olfactory neurons (aT1) lacking the endogenous receptor Or67d and the olfactory tuning curve and dose-response curves were established for this locust receptor. The results show that LmigOR3 sensitizes neurons to ketones, esters and heterocyclic compounds, indicating that LmigOR3 is a broadly tuned receptor. LmigOR3 is the first odorant receptor from Orthoptera that has been functionally analyzed in the Drosophila aT1 system. This work demonstrates the utility of the Drosophila aT1 system for functional analysis of locust odorant receptors and suggests that LmigOR3 may be involved in detecting food odorants, or perhaps locust body volatiles that may help us to develop new control methods for locusts.     

Diversity of locust gut bacteria protects against pathogen invasion

Diversity–invasibility relationships were explored in the novel context of the colonization resistance provided by gut bacteria of the desert locust Schistocerca gregaria against pathogenic bacteria. Germ‐free insects were associated with various combinations of one to three species of locust gut bacteria and then fed an inoculum of the pathogenic bacterium Serratia marcescens. There was a significant negative relationship between the resulting density of Serratia marcescens and the number of symbiotic gut bacterial species present. Likewise there was a significant inverse relationship between community diversity and the proportion of locusts that harboured Serratia. Host mortality was not negatively correlated with resistance to gut‐invasion by Serratia marcescens, although there were significantly more deaths among pathogen fed germ‐free insects than tri‐associated gnotobiotes. The outcome is consistent with the predictions of community ecology theory that species‐rich communities are more resistant to invasion than species‐poor communities.