Protein kinase C mediates juvenile hormone-dependent phosphorylation of Na+/K+-ATPase to induce ovarian follicular patency for yolk protein uptake.

In oviparous animals, vitellogenesis is prerequisite to egg production and embryonic growth after oviposition. For successful insect vitellogenesis and oogenesis, vitellogenin (Vg) synthesized in the fat body (homologue to vertebrate liver and adipose tissue) must pass through the intercellular channels, a condition known as patency in the follicular epithelium, to reach the surface of oocytes. This process is controlled by juvenile hormone (JH) in many insect species, but the underlying mechanisms remain elusive. Previous work has suggested the possible involvement of Na+/K+-ATPase in patency initiation, but again, the regulatory cascade of Na+/K+-ATPase for patency initiation has been lacking. Using the migratory locust Locusta migratoria as a model system, we report here that RNAi-mediated knockdown of gene coding for Na+/K+-ATPase, inhibition of its phosphorylation, or suppression of its activity causes loss of patency, resulting in blocked Vg uptake, arrested oocyte maturation, and impaired ovarian growth. JH triggers G protein-coupled receptor (GPCR), receptor tyrosine kinase (RTK), phospholipase C (PLC), inositol trisphosphate receptor (IP3R), and protein kinase C (PKC) to phosphorylate Na+/K+-ATPase α-subunit at amino acid residue Ser8, consequently activating Na+/K+-ATPase for the induction of patency in vitellogenic follicular epithelium. Our results thus point to a previously unidentified mechanism by which JH induces the phosphorylation and activation of Na+/K+-ATPase via a signaling cascade of GPCR, RTK, PLC, IP3R, and PKC. The findings advance our understanding of JH regulation in insect vitellogenesis and oogenesis.

Cold acclimation improves chill tolerance in the migratory locust through preservation of ion balance and membrane potential.

Most insects have the ability to alter their cold tolerance in response to temporal temperature fluctuations, and recent studies have shown that insect cold tolerance is closely tied to the ability to maintain transmembrane ion gradients that are important for the maintenance of cell membrane potential (Vm). Several studies have therefore suggested a link between preservation of Vm and cellular survival after cold stress, but none has measured Vm in this context. We tested this hypothesis by acclimating locusts (Locusta migratoria) to high (31°C) and low temperature (11°C) for 4 days before exposing them to cold stress (0°C) for up to 48 h and subsequently measuring ion balance, cell survival, muscle Vm, and whole animal performance. Cold stress caused gradual muscle cell death, which coincided with a loss of ion balance and depolarization of muscle Vm The loss of ion balance and cell polarization were, however, dampened markedly in cold-acclimated locusts such that the development of chill injury was reduced. To further examine the association between cellular injury and Vm we exposed in vitro muscle preparations to cold buffers with low, intermediate, or high [K+]. These experiments revealed that cellular injury during cold exposure occurs when Vm becomes severely depolarized. Interestingly, we found that cellular sensitivity to hypothermic hyperkalaemia was lower in cold-acclimated locusts that were better able to defend Vm whilst exposed to high extracellular [K+]. Together these results demonstrate a mechanism of cold acclimation in locusts that improves survival after cold stress: increased cold tolerance is accomplished by preservation of Vm through maintenance of ion homeostasis and decreased K+ sensitivity.

Wright-Giemsa staining to observe phagocytes in Locusta migratoria infected with Metarhizium acridum.

Hemocytes are the first line of defense in the invertebrate immune system. Understanding their roles in cellular immunity is important for developing more efficient mycoinsecticides. However, the exact classification of hemocytes has been inconsistent and the various types of phagocytes in Locusta migratoria are poorly defined. Herein, the Wright-Giemsa staining method and microscopy were employed to characterize the hemocytes of L. migratoria following infection by Metarhizium acridum. Hemocytes were classified into four types, including granulocytes, plasmatocytes, prohemocytes, and oenocytoids, based on size, morphology, and dye-staining properties. Each type of hemocyte was classified into several subtypes according to different ultrastructural features. At least four subtypes of granulocytes or plasmatocytes, including small-nucleus plasmatocytes, basophil vacuolated plasmatocytes, homogeneous plasmatocytes, and eosinophilic granulocytes, carried out phagocytosis. The percentage of total phagocytes increased two days after infection by M. acridum, then gradually declined during the next two days, and then increased sharply again at the fifth day. Our data suggested that plasmatocytes and granulocytes may be the major phagocytes that protect against invasion by a fungal pathogen in L. migratoria. Total hemocytes in locusts significantly increased in the initial days after infection and decreased in the late period of infection compared to controls. In the hemocoel, hyphal bodies were recognized, enwrapped, and digested by the phagocytes. Then, the broken hyphal pieces were packaged as vesicles to be secreted from the cell. Moreover, locusts might have a sensitive and efficient cellular immune system that can regulate phagocyte differentiation and proliferation before fungi colonize the host hemolymph.

Development of primary cell cultures using hemocytes and phagocytic tissue cells of Locusta migratoria: an application for locust immunity studies.

Insect cell cultures played central roles in unraveling many insect physiological and immunological processes. Regardless, despite imminent needs, insect cell lines were developed primarily from Dipteran and Lepidopteran orders, leaving many important insects such as Orthopteran locusts under-represented. Besides the lack of cell lines, the slow progress in development of in vitro techniques is attributed to poor communications between different laboratories regarding optimized primary cell cultures. Therefore, we report here about methods developed for primary cell culture of Locusta migratoria hemocyte and phagocytic tissue cells by which we could maintain viable hemocytes in vitro for over 5 d and phagocytic tissue cells for over 12 d. 2-Mercaptoethanol and phenyl-thiourea supplements in Grace's medium together with addition of fetal bovine serum 30 min after cell seeding resulted in a successful setup of the primary cell cultures and a week-long survival of the hemocytes and phagocytic tissue cells in vitro.

Neurons without dendrites?--A novel type of neurosecretory cell in locusts.

Small-diameter nerves were found that are associated with the lateral peripheral nerves of the unfused abdominal ganglia of locusts. Such small nerves were observed in about 30% of all cases in Locusta migratoria, more than 60% in Schistocerca gregaria. Retrograde staining of these small nerves showed two somata in the posterior, lateral, and ventral region of an abdominal ganglion. These cells give rise to the small nerves that accompany the big lateral nerves and, on their surface, form putative neurohaemal release sites. Astonishingly the cells do not form any dendritic ramifications within the neuropile of the ganglia.

De novo transcriptome analysis of wing development-related signaling pathways in Locusta migratoria manilensis and Ostrinia furnacalis (Guenée).

BACKGROUND: Orthopteran migratory locust, Locusta migratoria, and lepidopteran Asian corn borer, Ostrinia furnacalis, are two types of insects undergoing incomplete and complete metamorphosis, respectively. Identification of candidate genes regulating wing development in these two insects would provide insights into the further study about the molecular mechanisms controlling metamorphosis development. We have sequenced the transcriptome of O. furnacalis larvae previously. Here we sequenced and characterized the transcriptome of L. migratoria wing discs with special emphasis on wing development-related signaling pathways. METHODOLOGY/PRINCIPAL FINDINGS: Illumina Hiseq2000 was used to sequence 8.38 Gb of the transcriptome from dissected nymphal wing discs. De novo assembly generated 91,907 unigenes with mean length of 610 nt. All unigenes were searched against five databases including Nt, Nr, Swiss-Prot, COG, and KEGG for annotations using blastn or blastx algorithm with an cut-off E-value of 10-5. A total of 23,359 (25.4%) unigenes have homologs within at least one database. Based on sequence similarity to homologs known to regulate Drosophila melanogaster wing development, we identified 50 and 46 potential wing development-related unigenes from L. migratoria and O. furnacalis transcriptome, respectively. The identified unigenes encode putative orthologs for nearly all components of the Hedgehog (Hh), Decapentaplegic (Dpp), Notch (N), and Wingless (Wg) signaling pathways, which are essential for growth and pattern formation during wing development. We investigated the expression profiles of the component genes involved in these signaling pathways in forewings and hind wings of L. migratoria and O. furnacalis. The results revealed the tested genes had different expression patterns in two insects. CONCLUSIONS/SIGNIFICANCE: This study provides the comprehensive sequence resource of the wing development-related signaling pathways of L. migratoria. The obtained data gives an insight into better understanding the molecular mechanisms involved in the wing development in L. migratoria and O. furnacalis, two insect species with different metamorphosis types.

Retinoic acid as a survival factor in neuronal development of the grasshopper, Locusta migratoria.

Based on experience with cell cultures of adult insect neurons, we develop a serum-free culture system for embryonic locust neurons. Influences of trophic substances on survival and neurite outgrowth of developing neurons are investigated. For the first time, a positive trophic effect of 9-cis retinoic acid (9-cis RA) was shown in vitro on embryonic neurons of an insect. We observed longer cell survival of 50 % developmental stage neurons in cultures supplemented with 0.3 nM 9-cis RA. Furthermore, an influence on neuron morphology was revealed, as the addition of 9-cis RA to cell culture medium led to an increase in the number of neurites per cell. Although an RA receptor gene, LmRXR (Locusta migratoria retinoid X receptor), was expressed in the central nervous system throughout development, the influence of 9-cis RA on neuronal survival and outgrowth was restricted to 50 % stage embryonic cells.

A pair of motion-sensitive neurons in the locust encode approaches of a looming object.

Neurons in the locust visual system encode approaches of looming stimuli and are implicated in production of escape behaviours. The lobula giant movement detector (LGMD) and its postsynaptic partner, the descending contralateral movement detector (DCMD) compute characteristics of expanding edges across the locust eye during a loom and DCMD synapses onto motor elements associated with behaviour. We identified another descending interneuron within the locust ventral nerve cord. We named this neuron the late DCMD (LDCMD) as it responds later during an approach, with the firing rate peaking at about the time of collision. LDCMD produced lower amplitude, broader action potentials that were associated with an afterhyperpolarization, whereas DCMD action potentials showed a brief afterhyperpolarization often followed by an afterdepolarization. Within the mesothoracic ganglion, the primary LDCMD axon located adjacent to the DCMD axon, was thinner and lacked collateral projections to the lateral region of the neuropil. When compared with DCMD, LDCMD fired with fewer spikes during a loom and showed weaker habituation to repeated approaches. Coincidence of LDCMD and DCMD firing increased during object approach. Our findings indicate the presence of an additional motion-sensitive descending neuron in the locust that encodes temporally distinct properties of an approaching object.

Is juvenile hormone involved in the maternal regulation of egg size and progeny characteristics in the desert locust?

The role of juvenile hormone (JH) in the maternal regulation of progeny characteristics was examined in the desert locust, Schistocerca gregaria. Female adults of this species are known to produce smaller but more eggs when reared in isolation than do those reared in a group. Eggs laid by isolated females develop green hatchlings typical of solitarious forms, whereas those laid by the latter produce black hatchlings typical of gregarious forms. Topical application of a juvenile hormone analog (JHA), fenoxycarb, or implantation of corpora allata (CA) taken from the migratory locust, Locusta migratoria, caused crowded S. gregaria females to deposit smaller eggs, but did not have a significant effect on the number of eggs per egg pod except at high doses of JHA. The production of smaller eggs by treated and untreated crowded females was closely associated with earlier deposition of the egg pods and shorter oviposition intervals. However, neither JHA application nor CA implantation influenced the progeny characteristics in actively reproducing aged females under crowded conditions, while untreated control females started producing smaller and more eggs upon transfer to isolated conditions. These results may suggest that JH is not directly involved in the maternal regulation of phase-dependent progeny characteristics.

Development of the subsoephageal body cells and the pericardiac cells during embryogenesis with diapause in Locusta migratoria (Linnaeus 1758) (Orthoptera: Acrididae).

During Locusta migratoria embryogenesis, the yolk is progressively degraded and the resulting metabolites are released in the haemolymph. We researched the organs possibly involved in the uptake of haemolymphatic proteins. Among organs originated from mesoderm, the SOB (suboesophageal bodies) situated in the embryonic head are remarkable by a very early acquisition of differentiated cytological characters, while most other cells of the embryo are undifferentiated. The SOB quite disappear before hatching. Just before katatrepsis stage, the other organs derived from mesoderm begin to differentiate, including the PC (pericardial cells) which take over from the SOB. These cells, situated in thorax and abdomen, are developed during the dorsal close of embryo. The development and the ultrastructural changes of the SOB cells and of the PC were studied during an embryogenesis with diapause. The morphology of embryos which enter diapause is comparable with that of a continuous development at the beginning of katatrepsis. However, the cells of SOB and PC cells suffer from remarkable changes not only physiologically but cytologically. At the beginning of diapause, the proteosynthetic activity practically disappears in the SOB cells and the lysis areas appear. Nevertheless, the exchanges between these cells and the haemolymph still remain important. For the period of cold, which is necessary to the resumption of development, the aspect of the SOB cells changes and in particular the areas of lysis become less wide. When the embryo reopens its development, the SOB cells show a proteosynthetic activity and the areas of lysis disappear. The changes of the SOB cells and of the PC cells are regularized during the resumption of the development: the SOB cells which had again taken a normal activity start to regress from the stage VII on, while the PC cells take over.

Neuronal connections between central and enteric nervous system in the locust, Locusta migratoria.

The number and location of neurons, in the central nervous system, that project into the frontal connective was studied in the locust by using retrograde neurobiotin staining. Staining one frontal connective revealed some 70 neurons in the brain. Most of these were located within both tritocerebral lobes. Additional groups of neurons were located within the deutocerebrum and protocerebrum. Some 60 neurons were labelled in the suboesophageal ganglion. These formed nine discernable populations. In addition, two neurons were located in the prothoracic ganglion and two neurons in the first abdominal neuromere of the metathoracic ganglion. Thus, some 250 neurons located within the head ganglia, and even neurons in thoracic ganglia, project into the ganglia of the enteric nervous system. This indicates that the coordination between the central and enteric ganglia is much more complex than previously thought. With the exception of some previously described dorsal unpaired median neurons and a few motor neurons in the head ganglia, the identity and function of most of these neurons is as yet unknown. Possible functions of the neurons in the thoracic ganglia are discussed.

Origin and development of unusual insect muscle tension receptors.

This work describes the origin and development of about 200 tension receptor cells located around the anterior attachment site of the locust ovipositor muscle and their migration to their final position on the muscle fibres. The locust ovipositor muscle is the only insect system in which more than 100 tension receptor cells are associated with a single muscle. Neuronal precursors of tension receptors are first detectable by horseradish peroxidase immunohistochemistry in fourth instar larvae. Precursors consist of cell clusters (doublets, triplets and quadruplets) located on the anterior attachment site of the muscle. In the early fifth larval stage, cell clusters are absent, although a few sensory neurons that lie embedded between the muscle fibres are apparent. These neurons send their dendrites towards the anterior end of the muscle fibres and their axons posteriorly. By the fourth day of the fifth larval stage, a large number of cell clusters appears on the anterior muscle attachment site. In addition to these assemblies, cells have been identified that extend long processes running exactly along the lateral margin of the attachment site. These cells are thought to provide navigating cues for migrating tension receptors, since they are absent in later stages. By the end of the fifth larval stage, most of the clusters gradually disappear and increasing numbers of differentiated neurons embedded between the muscle fibres become visible. We conclude that the majority of tension receptors develop during the last larval stage from precursors situated on the muscle apodeme. They then migrate from the apodeme to their final place on the muscle fibres where they assume an appropriate orientation.

Natural variation in the thermotolerance of neural function and behavior due to a cGMP-dependent protein kinase.

Although it is acknowledged that genetic variation contributes to individual differences in thermotolerance, the specific genes and pathways involved and how they are modulated by the environment remain poorly understood. We link natural variation in the thermotolerance of neural function and behavior in Drosophila melanogaster to the foraging gene (for, which encodes a cGMP-dependent protein kinase (PKG)) as well as to its downstream target, protein phosphatase 2A (PP2A). Genetic and pharmacological manipulations revealed that reduced PKG (or PP2A) activity caused increased thermotolerance of synaptic transmission at the larval neuromuscular junction. Like synaptic transmission, feeding movements were preserved at higher temperatures in larvae with lower PKG levels. In a comparative assay, pharmacological manipulations altering thermotolerance in a central circuit of Locusta migratoria demonstrated conservation of this neuroprotective pathway. In this circuit, either the inhibition of PKG or PP2A induced robust thermotolerance of neural function. We suggest that PKG and therefore the polymorphism associated with the allelic variation in for may provide populations with natural variation in heat stress tolerance. for's function in behavior is conserved across most organisms, including ants, bees, nematodes, and mammals. PKG's role in thermotolerance may also apply to these and other species. Natural variation in thermotolerance arising from genes involved in the PKG pathway could impact the evolution of thermotolerance in natural populations.

Temperature-sensitive gating in a descending visual interneuron, DCMD.

Activity in neural circuits can be modified through experience-dependent mechanisms. The effects of high temperature on a locust visual interneuron (the descending contralateral movement detector, DCMD) have previously been shown to be mitigated by prior exposure to sub-lethal, elevated temperatures (heat shock, HS). Activity in the DCMD is reduced at high temperature in naïve animals (control), whereas HS animals show a maintained spike count at all temperatures. We examined whether this finding was due to direct effects of temperature on visual processing, or whether other indirect feedback mechanisms were responsible for the observed effect in the DCMD. Activity in the DCMD was elicited using a computer-generated looming image, and the response was recorded extracellularly. The temperature of visual processing circuits contributes directly to HS-induced plasticity in the DCMD, as maintaining the brain at 25 degrees C during a thoracic temperature ramp eliminated the high frequency activity associated with HS. Removing ascending input by severing the thoracic nerve cord reduced DCMD thermosensitivity, indicating that indirect feedback mechanisms are also involved in controlling the DCMD response to increased thoracic temperature. Understanding how thermosensitive feedback within the locust affects DCMD function provides insight into critical regulatory mechanisms underlying visually-guided behaviors.

Fatty-acid-binding protein from the flight muscle of Locusta migratoria: evolutionary variations in fatty acid binding.

Intracellular lipid-binding proteins have evolved from a common ancestral gene with the appearance of mitochondrial oxidation, to guarantee, for example, transport of fatty acids through the aqueous cytosol to their site of utilization. The mammalian forms of these lipid carriers are structurally well-characterized and have been categorized, on the basis of sequence similarities and several typical ligand-binding features, into four subfamilies. Only a single complex structure of an invertebrate fatty-acid-binding protein (FABP) has been reported to date, which reveals a unique ligand-binding arrangement yet unknown in vertebrate FABPs. In the present study, the structure of a second invertebrate FABP (locust muscle) complexed with a fatty acid has been determined on the basis of intermolecular NOE connectivities between the protein and the uniformly (13)C-enriched oleate ligand. The resulting ligand conformation, although resembling the closely related mammalian heart- and adipocyte-type FABPs, is characterized by certain binding features that differ significantly from the typical hairpin-turn ligand shapes of the latter forms. This is primarily due to an alanine-to-leucine substitution in locust FABPs that produces a steric hindrance for ligand binding. A comparison with an FABP from tobacco hornworm larvae furthermore demonstrates that certain amino acid substitutions that appear to be specific for invertebrates decidedly influence the binding arrangement inside the protein cavity. Hence, as a result of these evolutionary variations, invertebrate FABPs may display a much greater diversity in intracellular lipid binding than observed for the mammalian transport proteins, thus possibly providing new insights for the design of modified lipid carriers.

Effect of cooling rates on the cold hardiness and cryoprotectant profiles of locust eggs.

To examine the relationship between cooling rate and cold hardiness in eggs of the migratory locust, Locusta migratoria, the survival rates and cryoprotectant levels of three embryonic developmental stages were measured at different cooling rates (from 0.05 to 0.8 degrees C min(-1)) in acclimated and non-acclimated eggs. Egg survival rate increased with decreasing cooling rate. The concentration of cryoprotectants (myo-inositol, trehalose, mannitol, glycerol, and sorbitol) increased in non-acclimated eggs, but varied significantly in response to different cooling rates in acclimated eggs. The acclimation process (5 degrees C for 3 days) did not increase eggs resistance to quick cooling ("plunge" cooling and 0.8 degrees C min(-1)). Earlier stage embryos were much more sensitive than later stage embryos to the same cooling rates. Time spent at subzero temperatures also had a strong influence on egg survival.