Janus Kinase Mediates Faster Recovery From Sevoflurane Anesthesia Than Isoflurane Anesthesia in the Migratory Locusts.

Inhalation anesthetics isoflurane and sevoflurane have been widely used in clinical practice for anesthesia. However, the molecular mechanisms underlying the faster recovery from sevoflurane anesthesia than isoflurane anesthesia remain largely undetermined. Herein, we use RNA-seq, RNA interference, quantitative real-time PCR and western blotting to explore the mechanisms of recovery from isoflurane and sevoflurane anesthesia in the migratory locusts. Although the migratory locusts show similar anesthetic responses to these two chemicals in corresponding half-maximal effective concentrations (EC50s), the recovery from sevoflurane anesthesia is significantly faster than that for isoflurane anesthesia after 30 min of anesthetic exposure. Transcriptome analysis shows that those transcripts involved in cytoskeletal components, Janus kinase (JAK) pathway and cuticle protein are differentially expressed in locust brains in response to isoflurane and sevoflurane. RNAi knockdown confirms that Actin, Myosin-like protein 84B (Mlp84B), JAK and cuticle protein NCP56 do not affect anesthetic response of the locusts to these two chemical anesthetics. Moreover, actin, Mlp84B and NCP56 do not affect differential recovery from isoflurane and sevoflurane anesthesia, whereas RNAi knockdown of JAK and its partner STAT5B does not affect anesthetic recovery from isoflurane but elongates recovery duration from sevoflurane anesthesia. Thus, JAK may mediate faster recovery from sevoflurane anesthesia than from isoflurane anesthesia in the migratory locust. This finding provides novel insights into the molecular mechanism underlying faster recovery from sevoflurane anesthesia than isoflurane anesthesia.

Translocator protein mediates olfactory repulsion.

Translocator protein (TSPO, 18kDa), which was previously known as a peripheral-type benzodiazepine receptor, is associated with psychiatric disorders and acts as a neuroimaging biomarker. However, its function and mechanism in modulating behaviors are less well-known. Herein, we found that TSPO in migratory locusts shows conserved protein traits and is expressed at high levels in the brains. The expression levels of tspo mRNA and protein were higher in brains of solitary locusts than those in gregarious locusts, whereas the mRNA and protein expression levels remained stable during crowding and isolation, suggesting that the expression level of TSPO is potentially associated with behavioral phenotype of solitary locusts. Moreover, tspo RNAi knockdown in the brains of solitary locusts decreased their olfactory repulsion. After RNAi knockdown of tyramine receptor (TyR) in the brains of solitary locusts, RNA-seq analysis identified that a functional class of receptors, which included tspo, was downregulated significantly. Moreover, tspo mRNA and protein expression levels were downregulated and upregulated after TyR RNAi knockdown and activation, respectively. tspo RNAi knockdown in the brains of solitary locusts induced the attractive response and inhibited the function of tyramine (TA)-TyR in inducing olfactory repulsion. In gregarious locusts, tspo RNAi knockdown inhibited the function of TA-TyR inducing olfactory repulsion. This study confirms that TSPO acts as a crucial effector protein in TA-TyR signaling to modulate olfactory repulsion. Furthermore, this study provides a novel mechanism by which TSPO functionally connects a G-protein-coupled receptor and a mitochondria membrane protein in modulating olfactory repulsion.

Retinoid X receptor modulates olfactory attraction through Gα signaling in the migratory locusts.

Animals communicate with each other in aggregating for survival and adaptation. Solitary locusts show an olfactory transition from repulsion to attraction in aggregation. However, the molecular mechanism underlying this transition is less well known. In this study, we explored differentially expressed transcripts (DETs) during locust aggregation and identified that a functional class of general metabolism encompassed the largest number of DETs among all analyzed gene classes. Within this functional class of general metabolism, oxidoreductase mediates synthesis of retinoic acid (RA) from vitamin A and other metabolites derived from carbohydrates. The expression levels of retinaldehyde hydroxylase 1 (raldh1) and retinoid X receptor (rxr), which are two crucial genes for RA synthesis and signaling, were upregulated during 4 h of crowding. Knockdown of raldh1 and rxr by RNA interference (RNAi) in the brains resulted in the loss of olfactory attraction. Moreover, inhibition of RXR by RNAi resulted in downregulated expression of Gna14, a member of the Gα subfamily that transduces signals in G protein-coupled receptor (GPCR) pathways. Abrogating RXR signaling and Gna14 by RNAi knockdown inhibited the function of dopamine receptor 1 (DopR1) and octopamine receptor α1 (OctαR1) in modulating olfactory attraction. RXR signaling is essential for DopR1 and OctαR1 to mediate olfactory attraction. This study showed that RXR signaling mediates attraction by Gα signaling and confirmed a novel link between nuclear receptor RXR and the membrane receptor GPCRs in modulating olfactory attraction.

A retinal-binding protein mediates olfactory attraction in the migratory locusts.

Cellular retinaldehyde-binding protein (CRALBP) is abundantly expressed in retina and its mutations are associated with visual impairments. The functions of CRALBP are less known in extra retinal tissues. Herein, we study the function of CRALBP in modulating olfactory behaviors in gregarious and solitary locusts. The expressions of cralbp mRNA and protein were enriched in locust brains and antennae. RNAi knockdown of cralbp in gregarious locusts decreased their attractive response to gregarious volatiles. RNA-seq and quantitative PCR confirmed that cralbp mRNA and protein expression levels were upregulated and downregulated after octopamine receptor α1 (OctαR1) activation and inhibition, respectively. Gene network analysis revealed that cralbp is the core hub gene in the interactive network among differentially expressed transcripts (DETs) resulting from activating and inhibiting OctαR1. Moreover, cralbp RNAi knockdown inhibited the induction of olfactory attraction by octopamine (OA)-OctαR1 signaling. CRALBP helped to transmit OA signals to mediate olfactory attraction response to guaiacol and veratrole, which are two odorant components in gregarious volatiles. This study suggested that CRALBP may act as a novel effector protein in OctαR1 signaling to mediate olfactory attraction. This study indicated that CRALBP modulates olfactory attraction in extra retina tissues and retinaldehyde metabolism may be crucial for olfactory attraction modulation.

Dop1 enhances conspecific olfactory attraction by inhibiting miR-9a maturation in locusts.

Dopamine receptor 1 (Dop1) mediates locust attraction behaviors, however, the mechanism by which Dop1 modulates this process remains unknown to date. Here, we identify differentially expressed small RNAs associated with locust olfactory attraction after activating and inhibiting Dop1. Small RNA transcriptome analysis and qPCR validation reveal that Dop1 activation and inhibition downregulates and upregulates microRNA-9a (miR-9a) expression, respectively. miR-9a knockdown in solitarious locusts increases their attraction to gregarious volatiles, whereas miR-9a overexpression in gregarious locusts reduces olfactory attraction. Moreover, miR-9a directly targets adenylyl cyclase 2 (ac2), causing its downregulation at the mRNA and protein levels. ac2 responds to Dop1 and mediates locust olfactory attraction. Mechanistically, Dop1 inhibits miR-9a expression through inducing the dissociation of La protein from pre-miR-9a and resulting in miR-9a maturation inhibition. Our results reveal a Dop1-miR-9a-AC2 circuit that modulates locust olfactory attraction underlying aggregation. This study suggests that miRNAs act as key messengers in the GPCR signaling.

Two dopamine receptors play different roles in phase change of the migratory locust.

The migratory locust, Locusta migratoria, shows remarkable phenotypic plasticity at behavioral, physiological, and morphological levels in response to fluctuation in population density. Our previous studies demonstrated that dopamine (DA) and the genes in the dopamine metabolic pathway mediate phase change in Locusta. However, the functions of different dopamine receptors in modulating locust phase change have not been fully explored. In the present study, DA concentration in the brain increased during crowding and decreased during isolation. The expression level of dopamine receptor 1 (Dop1) increased from 1 to 4 h of crowding, but remained unchanged during isolation. Injection of Dop1 agonist SKF38393 into the brains of solitary locusts promoted gregarization, induced conspecific attraction-response and increased locomotion. RNAi knockdown of Dop1 and injection of antagonist SCH23390 in gregarious locusts induced solitary behavior, promoted the shift to repulsion-response and reduced locomotion. By contrast, the expression level of dopamine receptor 2 (Dop2) gradually increased during isolation, but remained stable during crowding. During the isolation of gregarious locusts, injection of Dop2 antagonist S(-)-sulpiride or RNAi knockdown of Dop2 inhibited solitarization, maintained conspecific attraction-response and increased locomotion; by comparison, the isolated controls displayed conspecific repulsion-response and weaker motility. Activation of Dop2 in solitary locusts through injection of agonist, R(-)-TNPA, did not affect their behavioral state. Thus, DA-Dop1 signaling in the brain of Locusta induced the gregariousness, whereas DA-Dop2 signaling mediated the solitariness. Our study demonstrated that Dop1 and Dop2 modulated locust phase change in two different directions. Further investigation of Locusta Dop1 and Dop2 functions in modulating phase change will improve our understanding of the molecular mechanism underlying phenotypic plasticity in locusts.

Octopamine and tyramine respectively regulate attractive and repulsive behavior in locust phase changes.

Aggregative and solitary behaviors are universal phenomena in animals. Interestingly, locusts (Locusta migratoria) can reversibly transit their behavior between gregarious and solitary phase through conspecific attraction and repulsion. However, the regulatory mechanism of neurotransmitters underlying attraction and repulsion among locusts remains unknown. In this study, we found gregarious and solitary locusts were attracted or repulsed respectively by gregarious volatiles. Solitary locusts can transform their preference for gregarious volatiles during crowding, whereas gregarious locusts avoided their volatiles during isolation. During crowding and isolation, the activities of octopamine and tyramine signalings were respectively correlated with attraction- and repulsion-response to gregarious volatiles. RNA interference verified that octopamine receptor α (OARα) signaling in gregarious locusts controlled attraction-response, whereas in solitary ones, tyramine receptor (TAR) signaling mediated repulsion-response. Moreover, the activation of OARα signaling in solitary locusts caused the behavioral shift from repulsion to attraction. Enhancement of TAR signaling in gregarious locusts resulted in the behavioral shift from attraction to repulsion. The olfactory preference of gregarious and solitary locusts co-injected by these two monoamines displayed the same tendency as the olfactory perception in crowding and isolation, respectively. Thus, the invertebrate-specific octopamine-OARα and tyramine-TAR signalings respectively mediate attractive and repulsive behavior in behavioral plasticity in locusts.

The locust genome provides insight into swarm formation and long-distance flight.

Locusts are one of the world's most destructive agricultural pests and represent a useful model system in entomology. Here we present a draft 6.5 Gb genome sequence of Locusta migratoria, which is the largest animal genome sequenced so far. Our findings indicate that the large genome size of L. migratoria is likely to be because of transposable element proliferation combined with slow rates of loss for these elements. Methylome and transcriptome analyses reveal complex regulatory mechanisms involved in microtubule dynamic-mediated synapse plasticity during phase change. We find significant expansion of gene families associated with energy consumption and detoxification, consistent with long-distance flight capacity and phytophagy. We report hundreds of potential insecticide target genes, including cys-loop ligand-gated ion channels, G-protein-coupled receptors and lethal genes. The L. migratoria genome sequence offers new insights into the biology and sustainable management of this pest species, and will promote its wide use as a model system.

Serotonin enhances solitariness in phase transition of the migratory locust.

The behavioral plasticity of locusts is a striking trait presented during the reversible phase transition between solitary and gregarious individuals. However, the results of serotonin as a neurotransmitter from the migratory locust Locusta migratoria in phase transition showed an alternative profile compared to the results from the desert locust Schistocerca gregaria. In this study, we investigated the roles of serotonin in the brain during the phase change of the migratory locust. During the isolation of gregarious nymphs, the concentration of serotonin in the brain increased significantly, whereas serotonin receptors (i.e., 5-HT 1 , 5-HT 2 , and 5-HT 7 ) we identified here showed invariable expression patterns. Pharmacological intervention showed that serotonin injection in the brain of gregarious nymphs did not induced the behavioral change toward solitariness, but injection of this chemical in isolated gregarious nymphs accelerated the behavioral change from gregarious to solitary phase. During the crowding of solitary nymphs, the concentration of serotonin in the brain remained unchanged, whereas 5-HT 2 increased after 1 h of crowding and maintained stable expression level thereafter. Activation of serotonin-5-HT2 signaling with a pharmaceutical agonist inhibited the gregariousness of solitary nymphs in crowding treatment. These results indicate that the fluctuations of serotonin content and 5-HT 2 expression are results of locust phase change. Overall, this study demonstrates that serotonin enhances the solitariness of the gregarious locusts. Serotonin may regulate the withdrawal-like behavioral pattern displayed during locust phase change and this mechanism is conserved in different locust species.

CSP and takeout genes modulate the switch between attraction and repulsion during behavioral phase change in the migratory locust.

Behavioral plasticity is the most striking trait in locust phase transition. However, the genetic basis for behavioral plasticity in locusts is largely unknown. To unravel the molecular mechanisms underlying the behavioral phase change in the migratory locust Locusta migratoria, the gene expression patterns over the time courses of solitarization and gregarization were compared by oligonucleotide microarray analysis. Data analysis revealed that several gene categories relevant to peripheral olfactory perception are strongly regulated in a total of 1,444 differentially expressed genes during both time courses. Among these candidate genes, several CSP (chemosensory protein) genes and one takeout gene, LmigTO1, showed higher expression in gregarious and solitarious locusts, respectively, and displayed opposite expression trends during solitarization and gregarization. qRT-PCR experiments revealed that most CSP members and LmigTO1 exhibited antenna-rich expressions. RNA interference combined with olfactory behavioral experiments confirmed that the CSP gene family and one takeout gene, LmigTO1, are involved in the shift from repulsion to attraction between individuals during gregarization and in the reverse transition during solitarization. These findings suggest that the response to locust-emitted olfactory cues regulated by CSP and takeout genes is involved in the behavioral phase change in the migratory locust and provide a previously undescribed molecular mechanism linked to the formation of locust aggregations.

Modulation of behavioral phase changes of the migratory locust by the catecholamine metabolic pathway.

The migratory locust, Locusta migratoria, shows a striking phenotypic plasticity. It transitions between solitary and gregarious phases in response to population density changes. However, the molecular mechanism underlying the phase-dependent behavior changes remains elusive. Here we report a genome-wide gene expression profiling of gregarious and solitary nymphs at each stadium of the migratory locust, and we identified the most differentially expressed genes in the fourth stadium of the two phases. Bioinformatics analysis indicated that the catecholamine metabolic pathway was the most significant pathway up-regulated in the gregarious phase. We found pale, henna, and vat1, involved in dopamine biosynthesis and synaptic release, were critical target genes related to behavioral phase changes in the locusts. The roles of these genes in mediating behavioral changes in the gregarious individuals were confirmed by RNAi and pharmacological intervention. A single injection of dopamine or its agonist initiated gregarious behavior. Moreover, continuous and multiple injections of a dopamine agonist coupled with crowding resulted in more pronounced gregarious behavior. Our study thus provides insights into the relationships between genes and behavior in phase transition of this important pest species.

De novo analysis of transcriptome dynamics in the migratory locust during the development of phase traits.

Locusts exhibit remarkable density-dependent phenotype (phase) changes from the solitary to the gregarious, making them one of the most destructive agricultural pests. This phenotype polyphenism arises from a single genome and diverse transcriptomes in different conditions. Here we report a de novo transcriptome for the migratory locust and a comprehensive, representative core gene set. We carried out assembly of 21.5 Gb Illumina reads, generated 72,977 transcripts with N50 2,275 bp and identified 11,490 locust protein-coding genes. Comparative genomics analysis with eight other sequenced insects was carried out to identify the genomic divergence between hemimetabolous and holometabolous insects for the first time and 18 genes relevant to development was found. We further utilized the quantitative feature of RNA-seq to measure and compare gene expression among libraries. We first discovered how divergence in gene expression between two phases progresses as locusts develop and identified 242 transcripts as candidates for phase marker genes. Together with the detailed analysis of deep sequencing data of the 4(th) instar, we discovered a phase-dependent divergence of biological investment in the molecular level. Solitary locusts have higher activity in biosynthetic pathways while gregarious locusts show higher activity in environmental interaction, in which genes and pathways associated with regulation of neurotransmitter activities, such as neurotransmitter receptors, synthetase, transporters, and GPCR signaling pathways, are strongly involved. Our study, as the largest de novo transcriptome to date, with optimization of sequencing and assembly strategy, can further facilitate the application of de novo transcriptome. The locust transcriptome enriches genetic resources for hemimetabolous insects and our understanding of the origin of insect metamorphosis. Most importantly, we identified genes and pathways that might be involved in locust development and phase change, and may thus benefit pest management.

Characterization and comparative profiling of the small RNA transcriptomes in two phases of locust.

BACKGROUND: All the reports on insect small RNAs come from holometabolous insects whose genome sequence data are available. Therefore, study of hemimetabolous insect small RNAs could provide more insights into evolution and function of small RNAs in insects. The locust is an important, economically harmful hemimetabolous insect. Its phase changes, as a phenotypic plasticity, result from differential gene expression potentially regulated at both the post-transcriptional level, mediated by small RNAs, and the transcriptional level. RESULTS: Here, using high-throughput sequencing, we characterize the small RNA transcriptome in the locust. We identified 50 conserved microRNA families by similarity searching against miRBase, and a maximum of 185 potential locust-specific microRNA family candidates were identified using our newly developed method independent of locust genome sequence. We also demonstrate conservation of microRNA*, and evolutionary analysis of locust microRNAs indicates that the generation of miRNAs in locusts is concentrated along three phylogenetic tree branches: bilaterians, coelomates, and insects. Our study identified thousands of endogenous small interfering RNAs, some of which were of transposon origin, and also detected many Piwi-interacting RNA-like small RNAs. Comparison of small RNA expression patterns of the two phases showed that longer small RNAs were expressed more abundantly in the solitary phase and that each category of small RNAs exhibited different expression profiles between the two phases. CONCLUSIONS: The abundance of small RNAs in the locust might indicate a long evolutionary history of post-transcriptional gene expression regulation, and differential expression of small RNAs between the two phases might further disclose the molecular mechanism of phase changes.

LocustDB: a relational database for the transcriptome and biology of the migratory locust (Locusta migratoria).

BACKGROUND: The migratory locust (Locusta migratoria) is an orthopteran pest and a representative member of hemimetabolous insects for biological studies. Its transcriptomic data provide invaluable information for molecular entomology and pave a way for the comparative research of other medically, agronomically, and ecologically relevant insects. We developed the first transcriptomic database of the locust (LocustDB), building necessary infrastructures to integrate, organize, and retrieve data that are either currently available or to be acquired in the future. DESCRIPTION: LocustDB currently hosts 45,474 high-quality EST sequences from the locust, which were assembled into 12,161 unigenes. It, through user-friendly web interfaces, allows investigators to freely access sequence data, including homologous/orthologous sequences, functional annotations, and pathway analysis, based on conserved orthologous groups (COG), gene ontology (GO), protein domain (InterPro), and functional pathways (KEGG). It also provides information from comparative analysis based on data from the migratory locust and five other invertebrate species, including the silkworm, the honeybee, the fruitfly, the mosquito and the nematode. The website address of LocustDB is http://locustdb.genomics.org.cn/. CONCLUSION: LocustDB starts with the first transcriptome information for an orthopteran and hemimetabolous insect and will be extended to provide a framework for incorporating in-coming genomic data of relevant insect groups and a workbench for cross-species comparative studies.