Molecular strategies of the pygmy grasshopper Eucriotettix oculatus adapting to long-term heavy metal pollution.

To study the heavy metal accumulation and its impact on insect exterior and chromosome morphology, and reveal the molecular mechanism of insects adapting to long-term heavy metal compound pollution habitats, this study, in the Diaojiang river basin, which has been polluted by heavy metals(HMs) for nearly a thousand years, two Eucriotettix oculatus populations was collected from mining and non-mining areas. It was found that the contents of 7 heavy metals (As, Cd, Pb, Zn, Cu, Sn, Sb) in E. oculatus of the mining area were higher than that in the non-mining 1-11 times. The analysis of morphology shows that the external morphology, the hind wing type and the chromosomal morphology of E. oculatus are significant differences between the two populations. Based on the heavy metal accumulation，morphological change, and stable population density, it is inferred that the mining area population has been affected by heavy metals and has adapted to the environment of heavy metals pollution. Then, by analyzing the transcriptome of the two populations, it was found that the digestion, immunity, excretion, endocrine, nerve, circulation, reproductive and other systems and lysosomes, endoplasmic reticulum and other cell structure-related gene expression were suppressed. This shows that the functions of the above-mentioned related systems of E. oculatus are inhibited by heavy metal stress. However, it has also been found that through the significant up-regulation of genes related to the above system, such as ATP2B, pepsin A, ubiquitin, AQP1, ACOX, ATPeV0A, SEC61A, CANX, ALDH7A1, DLD, aceE, Hsp40, and catalase, etc., and the down-regulation of MAPK signalling pathway genes, can enhanced nutrient absorption, improve energy metabolism, repair damaged cells and degrade abnormal proteins, maintain the stability of cells and systems, and resist heavy metal damage so that E. oculatus can adapt to the environment of heavy metal pollution for a long time.

The microRNAs let-7 and miR-278 regulate insect metamorphosis and oogenesis by targeting the juvenile hormone early-response gene Krüppel-homolog 1.

Krüppel-homolog 1 (Kr-h1), a zinc-finger transcription factor, inhibits larval metamorphosis and promotes adult reproduction by transducing juvenile hormone (JH). Although the transcriptional regulation of Kr-h1 has been extensively studied, little is known about its regulation at the post-transcriptional level. Using the migratory locust Locusta migratoria as a model system, we report here that the microRNAs let-7 and miR-278 bound to the Kr-h1 coding sequence and downregulated its expression. Application of let-7 and miR-278 mimics (agomiRs) significantly reduced the level of Kr-h1 transcripts, resulting in partially precocious metamorphosis in nymphs as well as markedly decreased yolk protein precursors, arrested ovarian development and blocked oocyte maturation in adults. Moreover, the expression of let-7 and miR-278 was repressed by JH, constituting a regulatory loop of JH signaling. This study thus reveals a previously unknown regulatory mechanism whereby JH suppresses the expression of let-7 and miR-278, which, together with JH induction of Kr-h1 transcription, prevents the precocious metamorphosis of nymphs and stimulates the reproduction of adult females. These results advance our understanding of the coordination of JH and miRNA regulation in insect development.

Rhythmic change of adipokinetic hormones diurnally regulates locust vitellogenesis and egg development.

Adipokinetic hormones (AKHs), the neurohormones synthesized in the insect corpora cardiaca are known to mobilize lipids and carbohydrates for energy-consuming activities including reproduction. However, both inhibitory and stimulatory effects of AKHs on insect reproduction have been reported, and the underlying mechanisms remain elusive. Using the migratory locust, Locusta migratoria, as a model system, we report here that AKHs are expressed in response to rhythmic diel change, and AKH III expression increases markedly at photophase. Diurnal injection of AKH III but not AKH I or AKH II in adult females stimulates vitellogenesis and egg development. In contrast, AKH treatment at scotophase represses female reproduction. RNA interference-mediated knockdown of AKH receptor (AKHR) results in significantly reduced vitellogenin (Vg) expression in the fat body at photophase along with reduced Vg deposition in the ovary. AKHR knockdown also leads to decreased expression of Brummer, triacylglycerol lipase and trehalose transporter, accompanied by suppressed mobilization of triacylglycerol and trehalose. We propose that in addition to stimulating Vg expression at photophase, AKH/AKHR signalling is likely to regulate ovarian uptake of Vg via triacylglycerol mobilization and trehalose homeostasis. This study provides new insights into the understanding of AKH/AKHR signalling in the regulation of insect reproduction.

A locust embryo as predictive developmental neurotoxicity testing system for pioneer axon pathway formation.

Exposure to environmental chemicals during in utero and early postnatal development can cause a wide range of neurological defects. Since current guidelines for identifying developmental neurotoxic chemicals depend on the use of large numbers of rodents in animal experiments, it has been proposed to design rapid and cost-efficient in vitro screening test batteries that are mainly based on mixed neuronal/glial cultures. However, cell culture tests do not assay correct wiring of neuronal circuits. The establishment of precise anatomical connectivity is a key event in the development of a functional brain. Here, we expose intact embryos of the locust (Locusta migratoria) in serum-free culture to test chemicals and visualize correct navigation of identified pioneer axons by fluorescence microscopy. We define separate toxicological endpoints for axonal elongation and navigation along a stereotyped pathway. To distinguish developmental neurotoxicity (DNT) from general toxicity, we quantify defects in axonal elongation and navigation in concentration-response curves and compare it to the biochemically determined viability of the embryo. The investigation of a panel of recognized DNT-positive and -negative test compounds supports a rather high predictability of this invertebrate embryo assay. Similar to the semaphorin-mediated guidance of neurites in mammalian cortex, correct axonal navigation of the locust pioneer axons relies on steering cues from members of this family of cell recognition molecules. Due to the evolutionary conserved mechanisms of neurite guidance, we suggest that our pioneer axon paradigm might provide mechanistically relevant information on the DNT potential of chemical agents on the processes of axon elongation, navigation, and fasciculation.

Effects of FABP knockdown on flight performance of the desert locust, Schistocerca gregaria.

During migratory flight, desert locusts rely on fatty acids as their predominant source of energy. Lipids mobilized in the fat body are transported to the flight muscles and enter the muscle cells as free fatty acids. It has been postulated that muscle fatty acid binding protein (FABP) is needed for the efficient translocation of fatty acids through the aqueous cytosol towards mitochondrial ß-oxidation. To assess whether FABP is required for this process, dsRNA was injected into freshly emerged adult males to knock down the expression of FABP. Three weeks after injection, FABP and its mRNA were undetectable in flight muscle, indicating efficient silencing of FABP expression. At rest, control and treated animals exhibited no morphological or behavioral differences. In tethered flight experiments, both control and treated insects were able to fly continually in the initial, carbohydrate-fueled phase of flight, and in both groups, lipids were mobilized and released into the hemolymph. Flight periods exceeding 30 min, however, when fatty acids become the main energy source, were rarely possible for FABP-depleted animals, while control insects continued to fly for more than 2 h. These results demonstrate that FABP is an essential element of skeletal muscle energy metabolism in vivo.

Jaburetox, a natural insecticide derived from Jack Bean Urease, activates voltage-gated sodium channels to modulate insect behavior.

Jaburetox (Jbtx) is an insecticidal peptide derived from Canavalia ensiformis urease, whose mechanism of action is not completely elucidated. We employed behavioral, electromyographical and electrophysiological protocols to identify the cellular and molecular targets involved in the Jbtx entomotoxicity in cockroaches and locusts. In Nauphoeta cinerea, Jbtx (32 µg/g) altered the locomotory behaviour inducing a significative decrease in the distance travelled followed by a significant increase in stopped time (52 ±â€¯85 cm and 2573 ±â€¯89 s, p < .05, n = 40). Jbtx (8 to 32 µg/g body weight, respectively) also increased the leg and antennae grooming activities (p < .05, n = 40, respectively). Jbtx (8 to 16 µg/g) induced a maximum neuromuscular blockade of 80.72% (n = 6, p < .05) and was cardiotoxic, decreasing the cockroach heart rate. The electrophysiological profiles of both muscle and nerve of L. migratoria showed that Jbtx (2.5 × 10-7 and 2.5 × 10-3 µg/ body weight) induced a significant increase in the amplitude of nerve action potentials (n = 5, p < .05). Voltage clamp analysis of Jbtx (200 nM) applied in Xenopus laevis oocytes heterologously expressed with Nav 1.1 channels showed a significant increase in the sodium currents. In conclusion, this work revealed that the entomotoxic activity of Jbtx involves complex behavioral alterations that begins with an initial activation of voltage-gated sodium channels.

RNAi-mediated knockdown of SPOOK reduces ecdysteroid titers and causes precocious metamorphosis in the desert locust Schistocerca gregaria.

The Halloween gene SPOOK (SPO) is involved in the production of the active metabolite of ecdysteroid, 20-hydroxyecdysone (20E), in insects. A previous study showed that RNAi-mediated knockdown of SPO in Schistocerca gregaria last instar nymphs markedly reduced the hemolymph 20E titer, but did not affect metamorphosis. In the present study, the effects of SPO interference on development were re-examined in this locust. Injections of SPO double-stranded RNA (dsSPO) into nymphs at mid and late instars significantly delayed nymphal development and interfered with molting. The 20E levels of dsSPO-treated nymphs were generally low, with a delayed, small peak, suggesting that disturbance of the 20E levels caused the above developmental abnormalities. A small proportion of the dsSPO-injected nymphs metamorphosed precociously, producing adults and adultoids. Precocious adults were characterized by small body size, short wings with abbreviated venation, and normal reproductive activity. Fourth instar nymphs that precociously metamorphosed at the following instar exhibited temporal expression patterns of ecdysone-induced protein 93F and the juvenile hormone (JH) early-inducible gene Krüppel homolog 1 similar to those observed at the last instar in normal nymphs. Adultoids displayed mating behavior and adultoid females developed eggs, but never laid eggs. JH injection around the expected time of the 20E peak in the dsSPO-injected nymphs completely inhibited the appearance of adultoids, suggesting that appearance of adultoids might be due to a reduced titer of JH rather than of 20E. These results suggest that SPO plays an important role in controlling morphogenesis, metamorphosis, and reproduction in S. gregaria.

Conditional modulation of spike-timing-dependent plasticity for olfactory learning.

Mushroom bodies are a well-known site for associative learning in insects. Yet the precise mechanisms that underlie plasticity there and ensure their specificity remain elusive. In locusts, the synapses between the intrinsic mushroom body neurons and their postsynaptic targets obey a Hebbian spike-timing-dependent plasticity (STDP) rule. Although this property homeostatically regulates the timing of mushroom body output, its potential role in associative learning is unknown. Here we show in vivo that pre-post pairing causing STDP can, when followed by the local delivery of a reinforcement-mediating neuromodulator, specify the synapses that will undergo an associative change. At these synapses, and there only, the change is a transformation of the STDP rule itself. These results illustrate the multiple actions of STDP, including a role in associative learning, despite potential temporal dissociation between the pairings that specify synaptic modification and the delivery of reinforcement-mediating neuromodulator signals.

Susceptibility and potential biochemical mechanism of Oedaleus asiaticus to beta-cypermethrin and deltamethrin in the Inner Mongolia, China.

Oedaleus asiaticus is a highly destructive grass pest in Inner Mongolia, China, and likely developed resistance to pyrethroid insecticides due to their frequent application for control of this locust. In this study, the susceptibility of five field populations of O. asiaticus to two pyrethroid insecticides was investigated. The Wulate Middle Banner (WB) population was the least susceptible, whereas the Ewenki Banner (EB) population appeared to be the most sensitive. The WB population was 3.16 and 5.15-fold less sensitive to beta-cypermethrin and deltamethrin than EB population, respectively. Further, the enzyme activities and mRNA expression levels of carboxylesterase (CarE) and glutathione-S-transferase (GST) were determined and we found that their activities in the WB population were 5.15 and 2.8-fold higher than those in the EB population, respectively. Quantitative real-time PCR (qRT-PCR) analysis demonstrated that the mRNA expression levels of CarE and GST genes were positively correlated with the LD50 in the WB, Siziwang Banner (SB) and EB populations. Our findings suggest that differences in susceptibility to pyrethroids in O. asiaticus might be attributed to the elevated activities and mRNA expression levels of CarE and GST genes.

Neuropathological effect of dimethyl disulfide on neurons of the desert locust Schistocerca gregaria.

Botanical insecticides have introduced a new concept in insecticide research. In response to insect attacks, some plants can release volatile compounds that alter insect metabolism and nervous system activity. In the present study, changes in the electrical activity of chemoreceptors and alteration of the fine structure of metathoracic ganglia of desert locust were examined after acute exposure to dimethyl disulfide (DMDS), a sulfur compound released from Allium porrum. Animals were exposed to 1/4 LC50 of DMDS (0.375 µl/L air) and electrophysiological and electron-microscopical studies were carried out. Application of DMDS showed an increase in the activity of deterrent cells present in tarsal chemosensilla of locust. On the other hand, evident degenerative changes in the neurons, neuroglia, neuropile and synaptic vesicles were observed in the metathoracic ganglia of DMDS-treated animals. These findings revealed that pest control using DMDS might be feasible and future work is highly recommended in this respect.

Lipase activity in insect oral secretions mediates defense responses in Arabidopsis.

How plants perceive herbivory is not yet well understood. We investigated early responses of the model plant Arabidopsis (Arabidopsis thaliana) to attack from the generalist grasshopper herbivore, Schistocerca gregaria (Caelifera). When compared with wounding alone, S. gregaria attack and the application of grasshopper oral secretions (GS) to puncture wounds elicited a rapid accumulation of various oxylipins, including 13-hydroperoxy octadecatrienoic acid, 12-oxo-phytodienoic acid (OPDA), jasmonic acid, and jasmonic acid-isoleucine. Additionally, GS increased cytosolic calcium levels, mitogen-activated protein kinase (MPK3 and MPK6) activity, and ethylene emission but not the accumulation of hydrogen peroxide. Although GS contain caeliferin A16:0, a putative elicitor of caeliferan herbivores, treatment with pure, synthetic caeliferin A16:0 did not induce any of the observed responses. With mutant plants, we demonstrate that the observed changes in oxylipin levels are independent of MPK3 and MPK6 activity but that MPK6 is important for the GS-induced ethylene release. Biochemical and pharmacological analyses revealed that the lipase activity of GS plays a central role in the GS-induced accumulation of oxylipins, especially OPDA, which could be fully mimicked by treating puncture wounds only with a lipase from Rhizopus arrhizus. GS elicitation increased the levels of OPDA-responsive transcripts. Because the oral secretions of most insects used to study herbivory-induced responses in Arabidopsis rapidly elicit similar accumulations of OPDA, we suggest that lipids containing OPDA (arabidopsides) play an important role in the activation of herbivory-induced responses.

Metacridamides A and B, macrocycles from conidia of the entomopathogenic fungus Metarhizium acridum.

Metarhizium acridum, an entomopathogenic fungus, has been commercialized and used successfully for biocontrol of grasshopper pests in Africa and Australia. Its conidia produce two novel 17-membered macrocycles, metacridamides A and B, which consist of a Phe unit condensed with a nonaketide. Planar structures were elucidated by a combination of mass spectrometric and NMR techniques. Following hydrolysis of 1, chiral amino acid analysis assigned the L-configuration to the Phe unit. A crystal structure established the absolute configuration of the eight remaining stereogenic centers in 1. Metacridamide A showed cytotoxicity to three cancer lines with IC50's of 6.2, 11.0, and 10.8 µM against Caco-2 (epithelial colorectal adenocarcinoma), MCF-7 (breast cancer), and HepG2/C3A (hepatoma) cell lines, respectively. In addition, metacridamide B had an IC50 of 18.2 µM against HepG2/C3A, although it was inactive at 100 µM against Caco-2 and MCF-7. Neither analogue showed antimicrobial, phytotoxic, or insecticidal activity.

Chronic accumulation of cadmium and its effects on antioxidant enzymes and malondialdehyde in Oxya chinensis (Orthoptera: Acridoidea).

The accumulation of cadmium (Cd) and its effects on antioxidant enzyme activities and malondialdehyde (MDA) levels of Chinese rice grasshopper (Oxya chinensis) were evaluated under the laboratory conditions. Our results showed that Cd accumulation in O. chinensis exhibited a concentration-dependent increase in both males and females under Cd pollution. Environmental Cd can lead to the absorption of large quantities of Cd, which induces oxidative damage in insects by altering antioxidant defense enzyme systems. Our results demonstrated that Cd stress caused a significant decrease in glutathione peroxidase (GPx) levels and a significant increase in superoxide (SOD) dismutase and catalase (CAT) activities. In the grasshoppers, the MDA content was also enhanced, with an increase in Cd concentrations and a positive correlation between them; for females from second instar nymphs to the adult stage, R(2) was 0.6467, 0.9136, 0.6516, 0.942 and 0.7182, whereas for males, it was 0.6467, 0.8239, 0.9302, 0.7861, 0.8632, respectively. We also observed differences in the effects of Cd between grasshoppers of different developmental stages and genders, which suggested that the insect's developmental stage and sex should be considered when studying enzyme activity.

Memoirs of a locust: density-dependent behavioral change as a model for learning and memory.

A locust outbreak is a stupendous natural phenomenon that remains in the memory of whoever has been lucky (or unlucky) enough to witness it. Recent years have provided novel and important insights into the neurobiology of locust swarming. However, the central nervous system processes that accompany and perhaps even lie at the basis of locust phase transformation are still far from being fully understood. Our current work deals with the memory of a locust outbreak from a new perspective: that of the individual locust. We take locust density-dependent phase transformation - a unique example of extreme behavioral plasticity, and place it within the context of the accepted scheme of learning and memory. We confirm that a short time period of exposure to a small crowd of locusts is sufficient to induce a significant behavioral change in a previously solitary locust. Our results suggest that part of the behavioral change is due to long-term habituation of evasive and escape responses. We further demonstrate that the memory of a crowding event lasts for at least 24h, and that this memory is sensitive to a protein synthesis blocker. These findings add much to our understanding of locust density-dependent phase polyphenism. Furthermore, they offer a novel and tractable model for the study of learning and memory-related processes in a very distinctive behavioral context.

Scanning laser optical tomography resolves developmental neurotoxic effects on pioneer neurons.

Developmental neurotoxic compounds impair the developing human nervous system at lower doses than those affecting adults. Standardized test methods for assessing developmental neurotoxicity (DNT) require the use of high numbers of laboratory animals. Here, we use a novel assay that is based on the development of an intact insect embryo in serum-free culture. Neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons, extending axons along a well-defined pathway to the central nervous system. After exposure to test chemicals, we analyze pioneer neuron shape with conventional fluorescence microscopy and compare it to 3D images, obtained by scanning laser optical tomography (SLOT) and processed by a segmentation algorithm. The segmented SLOT images resolve the 3D structure of the pioneers, recognize pathfinding defects and are thus advantageous for detecting DNT-positive compounds. The defects in axon elongation and pathfinding of pioneer axons caused by two DNT-positive reference compounds (methylmercury chloride; sodium(meta)arsenite) are compared to the biochemically measured general viability of the embryo. Using conventional fluorescence microscopy to establish concentration-response curves of axon elongation, we show that this assay identifies methylmercury chloride and the pro-apoptotic compound staurosporine as developmental neurotoxicants.

Mud-puddling in the yellow-spined bamboo locust, Ceracris kiangsu (Oedipodidae: Orthoptera): does it detect and prefer salts or nitrogenous compounds from human urine?

C. kiangsu adults were observed visiting human urine, especially on hot summer days. The main chemicals in fresh human urine include inorganic salts and CO(NH(2))(2.) When human urine was incubated, NH(4)HCO(3) became the richest nitrogenous compound. The phagostimulants, repellents and attractants in urine were identified here. On the filter papers treated with fresh or incubated urine samples, the 5th instar nymphs and the adults started and continued gnawing around the edges, in contrast to the 3rd and the 4th instar nymphs. The consumed areas were dramatically greater on the filters treated with the urine samples incubated for 3-6 days. The feedings of both male and female adults were also stimulated by several urine-borne components such as NaCl, NaH(2)PO(4), Na(2)SO(4), KCl, NH(4)Cl and NH(4)HCO(3) but not by CO(NH(2))(2). Among them NaCl was the most powerful phagostimulant. The repelling, or attractive/arresting effects of CO(NH(2))(2) and NH(4)HCO(3) were also evaluated by a two-choice test. When exposed to water- and CO(NH(2))(2) solution-immersed filters simultaneously, the adults prefer to stay on water-immersed filter. In contrast, when provided water- and NH(4)HCO(3) solution-treated filters, the adults prefer to stay on NH(4)HCO(3) solution-treated filter. This demonstrated that CO(NH(2))(2) acted as a repellent and NH(4)HCO(3) as an attractant/arrestant. In the bamboo forest, similar feeding behavior was also elicited by NaCl, NH(4)HCO(3) but not by CO(NH(2))(2). Comparing to NaCl solution, a mixed solution of NaCl and CO(NH(2))(2) (1:1) significantly decreased the consumed area of the treated filters whereas a mixed solution of NaCl and NH(4)HCO(3) (1:1) dramatically increased the consumed area. These results demonstrated that the phagostimulatory effect by NaCl was reduced by CO(NH(2))(2) in fresh urine and was enhanced by NH(4)HCO(3) in incubated urine.

Endocrinology of reproduction and phase transition in locusts.

In the last decade, important progress has been made in the experimental analysis of the endocrine mechanisms controlling reproduction and phase transition in locusts. Phase transition is a very fascinating, but complex, phenomenon of phenotypic plasticity that is triggered by changes in population density and can lead to the formation of extremely devastating hopper bands and adult gregarious locust swarms. While some phase characteristics change within hours, others appear more gradually in the next stage(s), or even in the next generation(s). In adults, the phase status also has a major influence on the process of reproduction. A better understanding of how solitarious locusts become gregarious and how this switch affects reproductive physiology may result in novel strategies to fight locust plagues. In this paper, we will review the current knowledge concerning this close interaction between locust phase polyphenism and reproduction.

Different survival of chromium-exposed Oxya chinensis among allozyme genotypes.

The goal of the present study was to compare the different survival of Oxya chinensis exposed to chromium(VI) among allozyme genotypes to gain a better understanding of the relationship between the genetic variations and environmental disturbance. This study analyzed the occurrence of genotypes in O. chinensis population exposed to Cr(VI). O. chinensis samples were collected at Yuanping, Shanxi Province, China and used in acute toxicity tests. Specimens were assigned to Cr(VI) exposure (LD50: 291.0 mg/kg) for 24 h. The genetic composition of both dead and survived specimens was analyzed with horizontal starch gel electrophoresis in four enzymes (GPI, PGM, LDH, and ME). The results indicated that under Cr(VI) exposure, specimens with different genotypes had various mortalities at the four loci in laboratory conditions, and there was a genetic basis for tolerance in O. chinensis during acute exposure to Cr(VI).

Antioxidant enzyme activity in responses to environmentally induced oxidative stress in the 5th instar nymphs of Aiolopus thalassinus (Orthoptera: Acrididae).

The response of antioxidant enzymes to oxidative environmental stress was determined in 5th instar nymphs of Aiolopus thalassinus (Orthoptera: Acrididae) collected from sites with different level of pollution with heavy metals, PO43-, and SO42-. The high polluted site induced higher DNA damage to individuals compared to the control site. The highest values of tail length (TL), tail moment (TM), and percent of DNA in tail (TDNA) were found in the gut of 5th instar nymphs from a high polluted site. Also, protein carbonyls and lipid peroxide levels were significantly higher in insects collected from polluted sites compared to those from the control site. A strong positive correlation between both protein carbonyl and lipid peroxide concentration and the pollution level of the sites was found in all tissues of the insects. The activity of superoxide dismutase (SOD) in the brain of insects collected from the high polluted site was significantly higher than that in the thoracic muscles and gut. We observed strong inhibition of catalase (CAT) activity. This effect was apparently caused by pollutants present at the high polluted site. The level of pollution significantly influenced polyphenol oxidase (PPO) activity in A. thalassinus nymphs in all examined tissues. The highest values were observed in the brain. The relationship between pollution and ascorbate peroxidase (APOX) activity in the examined tissues had no clear tendency. However, the lowest APOX activity was observed in individuals from the low polluted site. Level of pollution of sampling sites, oxidative stress biomarkers, and enzymatic response in A. thalanthsis 5th instar were negatively or positively correlated. Oxidative damage parameters, especially the percent of severed cells, lipid peroxides, and the activity of APOX, can be perceived as good markers of environmental multistress.