A neural m6A pathway regulates behavioral aggregation in migratory locusts.

RNA N6-methyladenosine (m6A), as the most abundant modification of messenger RNA, can modulate insect behaviors, but its specific roles in aggregation behaviors remain unexplored. Here, we conducted a comprehensive molecular and physiological characterization of the individual components of the methyltransferase and demethylase in the migratory locust Locusta migratoria. Our results demonstrated that METTL3, METTL14 and ALKBH5 were dominantly expressed in the brain and exhibited remarkable responses to crowding or isolation. The individual knockdown of methyltransferases (i.e., METTL3 and METTL14) promoted locust movement and conspecific attraction, whereas ALKBH5 knockdown induced a behavioral shift toward the solitary phase. Furthermore, global transcriptome profiles revealed that m6A modification could regulate the orchestration of gene expression to fine tune the behavioral aggregation of locusts. In summary, our in vivo characterization of the m6A functions in migratory locusts clearly demonstrated the crucial roles of the m6A pathway in effectively modulating aggregation behaviors.

Egg size-dependent embryonic development in the desert locust, Schistocerca gregaria.

Phenotypic plasticity in body size is a product of modification of the developmental pathway. Although hatchlings of the desert locust, Schistocerca gregaria, show egg size-dependent plasticity in body size, it remains unclear how embryogenesis during egg development regulates final embryonic body size. To determine the developmental pathway causing body size variation at hatching, we examined egg and embryonic development at the early, middle, and late egg developmental stages in S. gregaria by comparing small and large eggs. Crowd-reared females produced larger eggs than isolated-reared females. The daily egg developmental rate was similar between small and large eggs: eggs dramatically absorbed external water after days 3 to 7 and nearly doubled the initial egg weight at the late stage of day 12. Morphological measurements of eggs and embryos at different days after oviposition revealed that large eggs were longer than small eggs throughout developmental stages. However, embryo length was similar between small and large eggs at the early stage (anatrepsis). Embryos begin to absorb yolk into their bodies after blastokinesis. The size of large-egg embryos increased significantly from the middle stage (katatrepsis) due to absorption of more yolk than small eggs. Egg length and embryo length were conspicuously larger in large eggs than in small eggs on day 12 of late katatrepsis. These results suggest that egg size did not influence the egg developmental rate and initial embryo size. Large eggs had more yolk and space, resulting in larger final embryos than small eggs. The amount of yolk and size of eggshells during katatrepsis could play a key role in determining hatchling body size in S. gregaria.

Myoinhibitory peptides in the central complex of the locust Schistocerca gregaria and colocalization with locustatachykinin-related peptides.

The central complex in the brain of insects provides a neural network for sensorimotor processing that is essential for spatial navigation and locomotion and plays a role in sleep control. Studies on the neurochemical architecture of the central complex have been performed especially in the fruit fly Drosophila melangoaster and the desert locust, Schistocerca gregaria. In several insect species, myoinhibitory peptides (MIPs) are involved in circadian control and sleep-wake regulation. To identify neurons that might underlie these functions, we investigated the distribution of MIPs in the central complex of the locust. In silico transcript analysis suggests the presence of eight different MIPs in the desert locust. Through immunolabeling, we identified five systems of central-complex neurons that express MIP-like peptides. Two systems constitute columnar neurons of the protocerebral bridge and the lower division of the central body, while the other three systems are columnar neurons (two systems) and tangential neurons (one system) of the upper division of the central body. The innervation pattern and cell count of two systems of columnar neurons revealed the existence of 18 instead of 16 columns of the protocerebral bridge. Immunostaining of preparations containing intracellularly stained single cells allowed us to further specify subtypes of labeled columnar neurons. Double-label experiments showed that three systems of MIP-immunostained columnar neurons are also locustatachykinin-immunoreactive. No colocalization was found with serotonin immunostaining. The data provide novel insights into the architecture of the locust central complex and suggest that MIPs play a prominent role within the central-complex network.

A journey towards an integrated understanding of behavioural phase change in locusts.

Behavioural phase change initiates and functionally couples the suite of traits that comprise density-dependent polyphenism in locusts. Here I provide a semi-expurgated account of my 25-year research journey studying behavioural phase transition in the desert locust. The journey spans continents, involves a cast of extraordinary colleagues, and travels across levels of biological organisation from deep within the nervous system of individual locusts to mass migration and the evolution and population dynamics of swarming.

Prothoracicostatic Activity of the Ecdysis-Regulating Neuropeptide Crustacean Cardioactive Peptide (CCAP) in the Desert Locust.

Accurate control of innate behaviors associated with developmental transitions requires functional integration of hormonal and neural signals. Insect molting is regulated by a set of neuropeptides, which trigger periodic pulses in ecdysteroid hormone titers and coordinate shedding of the old cuticle during ecdysis. In the current study, we demonstrate that crustacean cardioactive peptide (CCAP), a structurally conserved neuropeptide described to induce the ecdysis motor program, also exhibits a previously unknown prothoracicostatic activity to regulate ecdysteroid production in the desert locust, Schistocerca gregaria. We identified the locust genes encoding the CCAP precursor and three G protein-coupled receptors that are activated by CCAP with EC50 values in the (sub)nanomolar range. Spatiotemporal expression profiles of the receptors revealed expression in the prothoracic glands, the endocrine organs where ecdysteroidogenesis occurs. RNAi-mediated knockdown of CCAP precursor or receptors resulted in significantly elevated transcript levels of several Halloween genes, which encode ecdysteroid biosynthesis enzymes, and in elevated ecdysteroid levels one day prior to ecdysis. Moreover, prothoracic gland explants exhibited decreased secretion of ecdysteroids in the presence of CCAP. Our results unequivocally identify CCAP as the first prothoracicostatic peptide discovered in a hemimetabolan species and reveal the existence of an intricate interplay between CCAP signaling and ecdysteroidogenesis.

One hundred years of phase polymorphism research in locusts.

One hundred years ago in 1921, Sir Boris Uvarov recognized that two locust species are one species but appearing in two different phases, a solitarious and a gregarious phase. As locust swarms are still a big problem affecting millions of people, basic research has tried to understand the causes for the transition between phases. This phenomenon of phase polymorphism, now called polyphenism, is a very complex multifactorial process and this short review will draw attention to this important aspect of insect research.

Chronic electrical stimulation reduces reliance on anaerobic metabolism in locust jumping muscle.

Chronic electrical stimulation (CES) is a well-documented method for changing mammalian muscle from more fast-twitch to slow-twitch metabolic and contractile profiles. Although both mammalian and insect muscles have many similar anatomical and physiological properties, it is unknown if CES produces similar muscle plasticity changes in insects. To test this idea, we separated Schistocerca americana grasshoppers into two groups (n = 37 to 47): one that was subjected to CES for 180 min each day for five consecutive days and one group that was not. Each group was then electrically stimulated for a single time period (0, 5, 30, 60, or 180 min) before measuring jumping muscle lactate, a characteristic of fast-twitch type fibers. At each time point, CES led to a significantly reduced jumping muscle lactate concentration. Based on similar short-term CES mammalian studies, the reduction in lactate production was most likely due to a reduced reliance on anaerobic metabolism. Thus, longer stimulation periods should result in greater aerobic enzymatic activities, altered myosin ATPase, and shift fiber types. This is the first study to use electrical stimulation to explore insect muscle plasticity and our results show that grasshopper jumping muscle responds similarly to mammalian muscle.

Malpighamoeba infection compromises fluid secretion and P-glycoprotein detoxification in Malpighian tubules.

Malpighian tubules, analogous to vertebrate nephrons, play a key role in insect osmoregulation and detoxification. Tubules can become infected with a protozoan, Malpighamoeba, which damages their epithelial cells, potentially compromising their function. Here we used a modified Ramsay assay to quantify the impact of Malpighamoeba infection on fluid secretion and P-glycoprotein-dependent detoxification by desert locust Malpighian tubules. Infected tubules have a greater surface area and a higher fluid secretion rate than uninfected tubules. Infection also impairs P-glycoprotein-dependent detoxification by reducing the net rhodamine extrusion per surface area. However, due to the increased surface area and fluid secretion rate, infected tubules have similar total net extrusion per tubule to uninfected tubules. Increased fluid secretion rate of infected tubules likely exposes locusts to greater water stress and increased energy costs. Coupled with reduced efficiency of P-glycoprotein detoxification per surface area, Malpighamoeba infection is likely to reduce insect survival in natural environments.

Direct and indirect effects of plant diversity and phenoxy herbicide application on the development and reproduction of a polyphagous herbivore.

Widespread application of synthetic pesticides and loss of plant diversity are regarded as significant drivers of current global change. The effects of such phenomena on insect performance have been extensively studied separately, yet the interactions of these two drivers have been poorly explored. Here, we subjected the polyphagous grasshopper Pseudochorthippus parallelus (Zetterstedt, 1821) to a full-lifecycle field experiment with 50 cages containing experimental plant communities differing in grass species richness (2 vs. 8 grass species), half of them treated with a phenoxy herbicide commonly employed to control broadleaf plants in grasslands. We measured plant elemental content as a proxy for plant physiology, and a wide range of insect traits in both female and male grasshoppers. In females, grass diversity increased herbivory, insect nitrogen content and egg load, while herbicide reduced herbivory but increased the number of offspring, likely mediated by altered plant community composition. In males, grass diversity also increased herbivory, had positive effects on fat body, muscle volume and lifespan, and negative effects on body mass. Herbicide negatively affected herbivory in both females and males. Overall, plant diversity and herbicides may shift resource allocation in generalist terrestrial insect herbivores, indicating complex and unexpected effects of human-induced environmental change.

The desert locust, Schistocerca gregaria, plastically manipulates egg size by regulating both egg numbers and production rate according to population density.

Egg-size adjustment is one of the important plastic life-history traits for animals living in heterogeneous environments. The adaptive investment hypothesis predicts that mothers should increase progeny size according to certain cues predicting adverse future conditions of their offspring. However, reproductive resources are limited, and females have to simultaneously reduce egg number to allocate more resources to increase size. It remains unclear how single individuals alter egg size and number according to temporally heterogeneous environments. In the present study, we examined how desert locusts, Schistocerca gregaria, plastically alter egg size and number according to population density. We also investigated the trans-generational maternal effects on progeny characteristics as well as their own maternal physiological response (oviposition interval). Females kept in crowded conditions laid significantly larger and heavier eggs by reducing clutch size (number of eggs per egg pod) compared to isolated females, suggesting the existence of a reproductive trade-off between the two traits. The crowding-forced isolated females induced concerted changes not only in egg size but also in egg number tending towards those characteristics of gregarious control, implying that single individuals showed trade-off when egg size was increased. Double-blind testing confirmed the rapid crowding effects on egg size. Females also responded to crowding by extending the oviposition interval. As the oviposition interval extended, egg size increased, but clutch size decreased. Eggs from crowding-forced isolated females began to produce gregarious-phase type hatchlings (large and black) instead of solitarious-phase type ones (small and green). These results suggested that S. gregaria plastically manipulate egg size by regulating egg numbers and egg production rate, and indicated the presence of trans-generational maternal effects on progeny phase.

Revealing hidden density-dependent phenotypic plasticity in sedentary grasshoppers in the genus Schistocerca Stål (Orthoptera: Acrididae: Cyrtacanthacridinae).

Comparative quantification of reaction norms across closely related species in a clade is rare, but such a study can reveal valuable insights into understanding how reaction norms evolve along phylogeny. The grasshopper genus Schistocerca Stål (Orthoptera: Acrididae: Cyrtacanthacridinae) is an ideal group to study the evolution of density-dependent phenotypic plasticity because it includes both swarming locusts and non-swarming sedentary grasshoppers, which show varying degrees of plastic reaction norms in many traits. The swarming locusts exhibit locust phase polyphenism in which cryptically colored and solitary individuals can transform into conspicuously colored and highly gregarious individuals in response to increases in population density. The sedentary grasshoppers do not swarm in nature, and thus it has been assumed that they have little or no expression of plastic reaction norms in many traits, except for color, which has been shown to be a phylogenetically conserved trait. In this study, we have quantified density-dependent reaction norms in behavior, color, body size, and morphometric ratio in the nymphs of four sedentary species within Schistocerca by conducting explicit rearing experiments to induce potential phenotypic changes in response to isolation and crowding. In contrast to our previous assumption, we find that all four species show a certain level of density-dependent plastic reaction norms, which implies that these sedentary species have hidden reaction norms that can only be induced experimentally, some components of which must be phylogenetically conserved. Furthermore, we demonstrate that rearing density differentially affects the expression of reaction norms in different species, suggesting that different reaction norms must have followed independent evolutionary trajectories.

New ways to acquire resistance: imperfect convergence in insect adaptations to a potent plant toxin.

Evolution of insensitivity to the toxic effects of cardiac glycosides has become a model in the study of convergent evolution, as five taxonomic orders of insects use the same few similar amino acid substitutions in the otherwise highly conserved Na,K-ATPase α. We show here that insensitivity in pyrgomorphid grasshoppers evolved along a slightly divergent path. As in other lineages, duplication of the Na,K-ATPase α gene paved the way for subfunctionalization: one copy maintains the ancestral, sensitive state, while the other copy is resistant. Nonetheless, in contrast with all other investigated insects, the grasshoppers' resistant copy shows length variation by two amino acids in the first extracellular loop, the main part of the cardiac glycoside-binding pocket. RT-qPCR analyses confirmed that this copy is predominantly expressed in tissues exposed to the toxins, while the ancestral copy predominates in the nervous tissue. Functional tests with genetically engineered Drosophila Na,K-ATPases bearing the first extracellular loop of the pyrgomorphid genes showed the derived form to be highly resistant, while the ancestral state is sensitive. Thus, we report convergence in gene duplication and in the gene targets for toxin insensitivity; however, the means to the phenotypic end have been novel in pyrgomorphid grasshoppers.

The puzzle of locust density-dependent phase polyphenism.

Locust density-dependent phase polyphenism presents a quintessential example of environmentally induced plasticity. Almost a century of research has yielded ample knowledge regarding the multitude of ecological, physiological, and molecular phase-dependent characteristics. This short review highlights the considerable advances that have been made in our understanding of the locust's extreme plasticity and the highly complex nature of the phase phenomenon. Several challenges in locust research resulting from this unique complexity are also presented. It is concluded that the joint, interdisciplinary collaborative efforts, already underway, hold the promise of translating our ample knowledge into a complete solution to untangling the locust phase puzzle.

Respiratory gas levels interact to control ventilatory motor patterns in isolated locust ganglia.

Large insects actively ventilate their tracheal system even at rest, using abdominal pumping movements, which are controlled by a central pattern generator (CPG) in the thoracic ganglia. We studied the effects of respiratory gases on the ventilatory rhythm by isolating the thoracic ganglia and perfusing its main tracheae with various respiratory gas mixtures. Fictive ventilation activity was recorded from motor nerves controlling spiracular and abdominal ventilatory muscles. Both hypoxia and hypercapnia increased the ventilation rate, with the latter being much more potent. Sub-threshold hypoxic and hypercapnic levels were still able to modulate the rhythm as a result of interactions between the effects of the two respiratory gases. Additionally, changing the oxygen levels in the bathing saline affected ventilation rate, suggesting a modulatory role for haemolymph oxygen. Central sensing of both respiratory gases as well as interactions of their effects on the motor output of the ventilatory CPG reported here indicate convergent evolution of respiratory control among terrestrial animals of distant taxa.

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.

Re-examination of the maternal control of progeny size and body color in the desert locust Schistocerca gregaria: Differences from previous conclusions.

The desert locust shows conspicuous phase polyphenism of various traits in response to crowding conditions. Gregarious females lay larger eggs that produce black hatchlings, whereas solitarious females lay smaller eggs that produce green hatchlings. Previous studies have shown that changes in egg size and hatchling body color occurred easily in the laboratory upon exposure of the female parent to crowding or isolation for as few as 2 days. Based on these observations, these studies concluded that female adults perceive crowding stimuli with their antennae and require light for perception of the stimuli, with contact chemicals present on the integument of sexually mature males that are responsible for the crowding effect. We undertook this study to identify the chemical structures of the reported contact substances, which remain unknown to date. However, we could not reproduce the main results reported in the aforementioned studies and found that egg size and hatchling body color did not alter easily, even after crowding or isolation of the female parent for 2 days or longer. We were not able to observe a change in the progeny crowding characteristics by stimulating the antennae of female adults through either physical contact with male adults or using hexane extracts of their body surfaces. Similarly, the importance of light for gregarization or solitarization was also ruled out. These results indicate that some of the conclusions of previous studies should be reconsidered.

A ß-carotene-binding protein carrying a red pigment regulates body-color transition between green and black in locusts.

Changes of body color have important effects for animals in adapting to variable environments. The migratory locust exhibits body color polyphenism between solitary and gregarious individuals, with the former displaying a uniform green coloration and the latter having a prominent pattern of black dorsal and brown ventral surface. However, the molecular mechanism underlying the density-dependent body color changes of conspecific locusts remain largely unknown. Here, we found that upregulation of ß-carotene-binding protein promotes the accumulation of red pigment, which added to the green color palette present in solitary locusts changes it from green to black, and that downregulation of this protein led to the reverse, changing the color of gregarious locusts from black to green. Our results provide insight that color changes of locusts are dependent on variation in the red ß-carotene pigment binding to ßCBP. This finding of animal coloration corresponds with trichromatic theory of color vision.

Enantioselective Synthesis and Activity of All Diastereoisomers of ( E)-Phytal, a Pheromone Component of the Moroccan Locust, Dociostaurus maroccanus.

The Moroccan locust, Dociostaurus maroccanus (Thunberg, 1815) (Orthoptera: Acrididae), is a polyphagous pest capable of inflicting large losses in agriculture under favorable environmental and climatic conditions. Currently, control of the pest relies solely on the application of conventional insecticides that have negative effects on the environment and human safety. In the search for a more rational, environmentally acceptable approach for locust control, we have previously reported that ( Z/ E)-phytal (1) is a male-produced candidate sex pheromone of this acridid. This molecule, with two stereogenic centers at C-7 and C-11, has four different diastereomers along with the Z/ E stereochemistry of the double bond at C-2. In this paper, we present for the first time the enantioselective synthesis of the four diastereomers of ( E)-phytal and their electrophysiological and behavioral activity on males and females. Our results demonstrate that the ( R, R)-phytal is the most active diastereomer in both assays, significantly attracting females in a double-choice Y olfactometer, and confirming the previous chromatographic assignment as component of the sex pheromone of the Moroccan locust.

Juvenile hormone-dependent Kazal-type serine protease inhibitor Greglin safeguards insect vitellogenesis and egg production.

In addition to preventing insect metamorphosis, juvenile hormone (JH) is known to stimulate aspects of insect reproduction. However, the molecular mechanisms of JH action in insect reproduction remain largely unknown. By reanalyzing the transcriptomic data from adults and other developmental stages of the migratory locust Locusta migratoria, we identified a gene coding for Kazal-type protease inhibitor, previously named Greglin. Greglin is specifically expressed in adult females and most abundant in the fat body and ovaries. Interestingly, Greglin is among the top 3 of highly expressed genes in adult female locusts, after 2 vitellogenin ( Vg) genes. Greglin is induced by JH and expressed at remarkably high levels in the vitellogenic stage. Knockdown of Greglin in adult female locusts results in accelerated degradation of serine protease substrate and significantly reduced levels of Greglin protein in hemolymph and ovaries. The consequent phenotypes include blocked oocyte maturation, arrested ovarian growth and shrunken follicular epithelium, as well as declines in egg number and hatchability. The data provide the first evidence, to our knowledge, that JH-dependent Greglin is involved in locust vitellogenesis and oocyte maturation likely by protecting vitellogenesis and other forms of yolk precursors from proteolysis. The result offers new insights into the regulation of JH and function of protease inhibitors in insect vitellogenesis, oocyte maturation and fecundity.-Guo, W., Wu, Z., Yang, L., Cai, Z., Zhao, L., Zhou, S. Juvenile hormone-dependent Kazal-type serine protease inhibitor Greglin safeguards insect vitellogenesis and egg production.