Cardiaca-corpora Allata Research Articles

Differential gene expression and microRNA profile in corpora allata-corpora cardiaca of Aedes aegypti mosquitoes with weak juvenile hormone signalling

The corpora allata-corpora cardiaca (CA-CC) is an endocrine gland complex that regulates mosquito development and reproduction through the synthesis of juvenile hormone (JH). Epoxidase (Epox) is a key enzyme in the production of JH. We recently utilized CRISPR/Cas9 to establish an epoxidase-deficient (epox−/−) Aedes aegypti line. The CA from epox−/− mutants do not synthesize epoxidated JH III but methyl farneosate (MF), a weak agonist of the JH receptor, and therefore have reduced JH signalling. Illumina sequencing was used to examine the differences in gene expression between the CA-CC from wild type (WT) and epox−/− adult female mosquitoes. From 18,034 identified genes, 317 were significantly differentially expressed. These genes are involved in many biological processes, including the regulation of cell proliferation and apoptosis, energy metabolism, and nutritional uptake. In addition, the same CA-CC samples were also used to examine the microRNA (miRNA) profiles of epox−/− and WT mosquitoes. A total of 197 miRNAs were detected, 24 of which were differentially regulated in epox−/− mutants. miRNA binding sites for these particular miRNAs were identified using an in silico approach; they target a total of 101 differentially expressed genes. Our results suggest that a lack of epoxidase, besides affecting JH synthesis, results in the diminishing of JH signalling that have significant effects on Ae. aegypti CA-CC transcriptome profiles, as well as its miRNA repertoire.

Neuropeptide F regulates feeding via the juvenile hormone pathway in Ostrinia furnacalis larvae.

Feeding by pests is one of the most important reasons for reductions in agricultural crop yield. This study aimed to reveal how juvenile hormone (JH) participates in larval feeding regulation of the Asian corn borer Ostrinia furnacalis. Larvae of O.furnacalis exhibit a daily circadian feeding rhythm, with a peak at ZT18 and a trough at ZT6 under both photoperiod (LD) and constant dark (DD) conditions, which may be eliminated by application of fenoxycarb, a JH active analogue. JH negatively regulates larval feeding as a downstream factor of neuropeptide F (NPF), in which knocking down JH increases larval feeding amount along with body weight and length. The production of JH in the brain-corpora cardiaca-corpora allata (brain-CC-CA) is regulated by brain NPF rather than gut NPF, which was demonstrated in Drosophila larvae through GAL4/UAS genetic analysis. In addition, feeding regulation of JH is closely related to energy homeostasis in the fat body by inhibiting energy storage and promoting degradation. The JH analogue fenoxycarb is an effective pesticide against O.furnacalis, controlling feeding and metabolism. The brain NPF system regulates JH, with functions in food consumption, feeding rhythms, energy homeostasis and body size. This study provides an important basis for understanding the feeding mechanism and potential pest control of O.furnacalis. © 2022 Society of Chemical Industry.

Crustacean cardioactive peptide as a stimulator of feeding and a regulator of ecdysis in Leptinotarsa decemlineata

Crustacean cardioactive peptide (CCAP), a highly conserved amidated neuropeptide, stimulates feeding in Drosophila melanogaster and Periplaneta americana, and regulates pupa-adult transition in Tribolium castaneum and Manduca sexta. In the present paper, we intended to address whether CCAP plays the dual roles in the Colorado potato beetle Leptinotarsa decemlineata. We found that the levels of Ldccap were high in the dissected samples of brain-corpora cardiaca-corpora allata complex and ventral nerve cord, midgut and hindgut in the final (fourth)-instar larvae. A pulse of 20-hydroxyecdysone triggered the expression of Ldccap in the central nervous system but decreased the transcription in the midgut. In contrast, juvenile hormone intensified the expression of Ldccap in the midgut. RNA interference (RNAi)-aided knockdown of Ldccap at the penultimate instar stage inhibited foliage consumption, reduced the contents of trehalose and chitin, and lowered the mRNA levels of two chitin biosynthesis genes (LdUAP1 and LdChSAb). Moreover, around 70% of the Ldccap RNAi larvae remained as prepupae, completely wrapped in the old larval exuviae, and finally died. The remaining RNAi beetles continually developed to severely-deformed adults: most having wrinkled and smaller elytra and hindwings, and shortened legs. Therefore, CCAP plays three distinct roles, stimulating feeding in foraging larval stage, regulating ecdysis, and facilitating wing expansion and appendage elongation in a coleopteran. In addition, Ldccap can be used as a potential target gene for developing novel management strategies against this coleopteran pest.

Mating-Induced Differential Peptidomics of Neuropeptides and Protein Hormones in Agrotis ipsilon Moths.

In many insects, mating induces drastic changes in male and female responses to sex pheromones or host-plant odors. In the male moth Agrotis ipsilon, mating induces a transient inhibition of behavioral and neuronal responses to the female sex pheromone. As neuropeptides and peptide hormones regulate most behavioral processes, we hypothesize that they could be involved in this mating-dependent olfactory plasticity. Here we used next-generation RNA sequencing and a combination of liquid chromatography, matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and direct tissue profiling to analyze the transcriptome and peptidome of different brain compartments in virgin and mated males and females of A. ipsilon. We identified 37 transcripts encoding putative neuropeptide precursors and 54 putative bioactive neuropeptides from 23 neuropeptide precursors (70 sequences in total, 25 neuropeptide precursors) in different areas of the central nervous system including the antennal lobes, the gnathal ganglion, and the corpora cardiaca-corpora allata complex. Comparisons between virgin and mated males and females revealed tissue-specific differences in peptide composition between sexes and according to physiological state. Mated males showed postmating differences in neuropeptide occurrence, which could participate in the mating-induced olfactory plasticity.

Responses of the Housefly, Musca domestica, to the Hytrosavirus Replication: Impacts on Host's Vitellogenesis and Immunity.

Hytrosaviridae family members replicate in the salivary glands (SGs) of their adult dipteran hosts and are transmitted to uninfected hosts via saliva during feeding. Despite inducing similar gross symptoms (SG hypertrophy; SGH), hytrosaviruses (SGHVs) have distinct pathobiologies, including sex-ratio distortions in tsetse flies and refusal of infected housefly females to copulate. Via unknown mechanism(s), SGHV replication in other tissues results in reduced fecundity in tsetse flies and total shutdown of vitellogenesis and sterility in housefly females. We hypothesized that vitellogenesis shutdown was caused by virus-induced modulation of hormonal titers. Here, we used RNA-Seq to investigate virus-induced modulation of host genes/pathways in healthy and virus-infected houseflies, and we validated expression of modulated genes (n = 23) by RT-qPCR. We also evaluated the levels and activities of hemolymph AMPs, levels of endogenous sesquiterpenoids, and impacts of exogenous hormones on ovarian development in viremic females. Of the 973 housefly unigenes that were significantly modulated (padj ≤ 0.01, log2FC ≤ −2.0 or ≥ 2.0), 446 and 527 genes were downregulated and upregulated, respectively. While the most downregulated genes were related to reproduction (embryogenesis/oogenesis), the repertoire of upregulated genes was overrepresented by genes related to non-self recognition, ubiquitin-protease system, cytoskeletal traffic, cellular proliferation, development and movement, and snRNA processing. Overall, the virus, Musca domestica salivary gland hytrosavirus (MdSGHV), induced the upregulation of various components of the siRNA, innate antimicrobial immune, and autophagy pathways. We show that MdSGHV undergo limited morphogenesis in the corpora allata/corpora cardiaca (CA/CC) complex of M. domestica. MdSGHV replication in CA/CC potentially explains the significant reduction of hemolymph sesquiterpenoids levels, the refusal to mate, and the complete shutdown of egg development in viremic females. Notably, hormonal rescue of vitellogenesis did not result in egg production. The mechanism underlying MdSGHV-induced sterility has yet to be resolved.

Physiological roles of trehalose in Leptinotarsa larvae revealed by RNA interference of trehalose-6-phosphate synthase and trehalase genes

Trehalose is proposed to serve multiple physiological roles in insects. However, its importance remains largely unconfirmed. In the present paper, we knocked down either a trehalose biosynthesis gene (trehalose-6-phosphate synthase, LdTPS) or each of three degradation genes (soluble trehalases LdTRE1a, LdTRE1b or membrane-bound LdTRE2) in Leptinotarsa decemlineata by RNA interference (RNAi). Knockdown of LdTPS decreased trehalose content and caused larval and pupal lethality. The LdTPS RNAi survivors consumed a greater amount of foliage, obtained a heavier body mass, accumulated more glycogen, lipid and proline, and had a smaller amount of chitin compared with the controls. Ingestion of trehalose but not glucose rescued the food consumption increase and larval mass rise, increased survivorship, and recovered glycogen, lipid and chitin to the normal levels. In contrast, silencing of LdTRE1a increased trehalose content and resulted in larval and pupal lethality. The surviving LdTRE1a RNAi hypomorphs fed a smaller quantity of food, had a lighter body weight, depleted lipid and several glucogenic amino acids, and contained a smaller amount of chitin. Neither trehalose nor glucose ingestion rescued these LdTRE1a RNAi defects. Silencing of LdTRE1b caused little effects. Knockdown of LdTRE2 caused larval death, increased trehalose contents in several tissues and diminished glycogen in the brain-corpora cardiaca-corpora allata complex (BCC). Feeding glucose but not trehalose partially rescued the high mortality rate and recovered glycogen content in the BCC. It seems that trehalose is involved in feeding regulation, sugar absorption, brain energy supply and chitin biosynthesis in L. decemlineata larvae.

Identification of ten mevalonate enzyme-encoding genes and their expression in response to juvenile hormone levels in Leptinotarsa decemlineata (Say)

The mevalonate pathway is responsible for the biosynthesis of many essential molecules important in insect development, reproduction, chemical communication and defense. Based on Leptinotarsa decemlineata transcriptome and genome data, we identified ten genes that encoded acetoacetyl-CoA thiolase (LdAACT1 and LdAACT2), hydroxymethylglutaryl (HMA)-CoA synthase (LdHMGS), HMG-CoA reductase (LdHMGR1 and LdHMGR2), mevalonate kinase (LdMevK), phospho-mevalonate kinase (LdPMK), mevalonate diphosphate decarboxylase (LdMDD), isopentenyl-diphosphate isomerase (LdIDI) and farnesyl pyrophosphate synthetase (LdFPPS). Nine of these genes (except for LdAACT1) were mainly expressed in the larval brain-corpora cardiaca-corpora allata complex, and adult ovary and testis. The 9 genes were transcribed at high levels right after each ecdysis, and at low levels in the mid instar. Therefore, the 9 genes were indicated to be involved in JH biosynthesis. Moreover, knockdown of a JH biosynthesis gene LdJHAMT to lower JH titer significantly downregulated the transcription of the 9 genes. Ingestion of JH to activate JH signaling also significantly suppressed the expression of the 9 genes. It appears that the accumulation of JH precursors in LdJHAMT RNAi larvae and a high JH titer in JH-fed specimens may cause negative feedbacks to repress the expression of the 9 mevalonate enzyme-encoding genes (excluding LdAACT1) to balance the enzyme quantity in L. decemlineata.

Knockdown of a putative insulin-like peptide gene LdILP2 in Leptinotarsa decemlineata by RNA interference impairs pupation and adult emergence

Five insulin-like peptide LdILP genes were identified in Leptinotarsa decemlineata. All of them contained three exons and two introns, with three genes tandemly arrayed and well separated from the other two. Phylogenetic analysis revealed that the three LdILPs from three tandemly-arrayed genes grouped with TcILP1, whereas the other two resembled with TcILP2 and TcILP4 from Tribolium castaneum. Thus, the five LdILP genes were provisionally named LdILP1a, LdILP1b, LdILP1c, LdILP2 and LdILP4. LdILP2 was widely expressed in several tissues such as the brain–corpora cardiaca–corpora allata (BR–CC–CA) complex, gut and fat body. In contrast, LdILP1a and LdILP1b were only transcribed in BR–CC–CA, LdILP4 was in ovaries, and LdILP1c was in both BR–CC–CA and ovaries. Ingestion of double-stranded RNAs (dsRNAs) targeting LdILP2 (dsLdILP2-1 and dsLdILP2-2) specifically knocked down LdILP2 and upregulated the transcription of both LdInR and Ld4EBP, indicating insulin/insulin-like growth factor signaling pathway (IIS) was inhibited. Approximately 50% of the LdILP2 RNAi larvae did not normally pupate and about 50% of the LdILP2 RNAi pupae did not emerge. Moreover, silencing LdILP2 reduced the expression of a juvenile hormone (JH) biosynthesis gene, lowered JH titer and disturbed JH signaling. Finally, knocking down LdILP2 inhibited an ecdysteroidogenesis gene, decreased 20-hydroxyecdysone (20E) titer, and repressed the expression of two 20E-response genes LdHR3 and LdFTZ-F1. Thus, the IIS pathway is involved in larval–pupal metamorphosis by modification of both JH and 20E signaling in L. decemlineata.

Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae)

The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F1F0ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata–corpora-cardiaca (CC–CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC–CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development.

Homeodomain Protein Scr Regulates the Transcription of Genes Involved in Juvenile Hormone Biosynthesis in the Silkworm.

The silkworm Dominant trimolting (Moltinism, M3) mutant undergoes three larval molts and exhibits precocious metamorphosis. In this study, we found that compared with the wild-type (WT) that undergoes four larval molts, both the juvenile hormone (JH) concentration and the expression of the JH-responsive gene Krüppel homolog 1 (Kr-h1) began to be greater in the second instar of the M3 mutant. A positional cloning analysis revealed that only the homeodomain transcription factor gene Sex combs reduced (Scr) is located in the genomic region that is tightly linked to the M3 locus. The expression level of the Scr gene in the brain-corpora cardiaca-corpora allata (Br-CC-CA) complex, which controls the synthesis of JH, was very low in the final larval instar of both the M3 and WT larvae, and exhibited a positive correlation with JH titer changes. Importantly, luciferase reporter analysis and electrophoretic mobility shift assay (EMSA) demonstrated that the Scr protein could promote the transcription of genes involved in JH biosynthesis by directly binding to the cis-regulatory elements (CREs) of homeodomain protein on their promoters. These results conclude that the homeodomain protein Scr is transcriptionally involved in the regulation of JH biosynthesis in the silkworm.

Peptidomics of Neuropeptidergic Tissues of the Tsetse Fly Glossina morsitans morsitans.

Neuropeptides and peptide hormones are essential signaling molecules that regulate nearly all physiological processes. The recent release of the tsetse fly genome allowed the construction of a detailed in silico neuropeptide database (International Glossina Genome Consortium, Science 344, 380-386 (2014)), as well as an in-depth mass spectrometric analysis of the most important neuropeptidergic tissues of this medically and economically important insect species. Mass spectrometric confirmation of predicted peptides is a vital step in the functional characterization of neuropeptides, as in vivo peptides can be modified, cleaved, or even mispredicted. Using a nanoscale reversed phase liquid chromatography coupled to a Q Exactive Orbitrap mass spectrometer, we detected 51 putative bioactive neuropeptides encoded by 19 precursors: adipokinetic hormone (AKH) I and II, allatostatin A and B, capability/pyrokinin (capa/PK), corazonin, calcitonin-like diuretic hormone (CT/DH), FMRFamide, hugin, leucokinin, myosuppressin, natalisin, neuropeptide-like precursor (NPLP) 1, orcokinin, pigment dispersing factor (PDF), RYamide, SIFamide, short neuropeptide F (sNPF) and tachykinin. In addition, propeptides, truncated and spacer peptides derived from seven additional precursors were found, and include the precursors of allatostatin C, crustacean cardioactive peptide, corticotropin releasing factor-like diuretic hormone (CRF/DH), ecdysis triggering hormone (ETH), ion transport peptide (ITP), neuropeptide F, and proctolin, respectively. The majority of the identified neuropeptides are present in the central nervous system, with only a limited number of peptides in the corpora cardiaca-corpora allata and midgut. Owing to the large number of identified peptides, this study can be used as a reference for comparative studies in other insects. Graphical Abstract ᅟ.

CHARACTERIZATION AND FUNCTIONAL STUDY OF A PUTATIVE JUVENILE HORMONE DIOL KINASE IN THE COLORADO POTATO BEETLE Leptinotarsa decemlineata (Say).

Juvenile hormone diol kinase (JHDK) is an enzyme involved in JH degradation. In the present article, a putative JHDK cDNA (LdJHDK) was cloned from the Colorado potato beetle Leptinotarsa decemlineata. The cDNA consists of 814 bp, containing a 555 bp open reading frame encoding a 184 amino acid protein. LdJHDK reveals a high degree of identity to the previously reported insect JHDKs. It possesses three conserved purine nucleotide-binding elements, and contains three EF-hand motifs (helix-loop-helix structural domains). LdJHDK mRNA was mainly detected in hindgut and Malpighian tubules. Besides, a trace amount of LdJHDK mRNA was also found in thoracic muscles, brain-corpora cardiaca-corpora allata complex, foregut, midgut, ventral ganglia, fat body, epidermis, and hemocytes. Moreover, LdJHDK was expressed throughout all developmental stages. Within the first, second, and third larval instar, the expression levels of LdJHDK were higher just before and right after the molt, and were lower in the intermediate instar. In the fourth larval instar, the highest peak of LdJHDK occurred 56 h after ecdysis. Ingestion of double-stranded RNA (dsRNA) against LdJHDK successfully knocked down the target gene, increased JH titer, and significantly upregulated LdKr-h1 mRNA level. Knockdown of LdJHDK significantly impaired adult emergence. Thus, we provide a line of experimental evidence in L. decemlineata to support that LdJHDK encodes function protein involved in JH degradation.

Characterization of two juvenile hormone epoxide hydrolases by RNA interference in the Colorado potato beetle

In insect, juvenile hormone (JH) titers are tightly regulated in different development stages through synthesis and degradation pathways. During JH degradation, JH epoxide hydrolase (JHEH) converts JH to JH diol, and hydrolyses JH acid to JH acid diol. In this study, two full length LdJHEH cDNAs were cloned from Leptinotarsa decemlineata, and were provisionally designated LdJHEH1 and LdJHEH2. Both mRNAs were detectable in the thoracic muscles, brain–corpora cardiaca–corpora allata complex, foregut, midgut, hindgut, ventral ganglia, Malpighian tubules, fat bodies, epidermis, and hemocytes of the day 2 fourth-instar larvae, and in female ovaries as well as male reproductive organs of the adults. Moreover, both LdJHEH1 and LdJHEH2 were expressed throughout all larval life, with the highest peaks occurring 32h after ecdysis of the final (fourth) instar larvae. Four double-stranded RNAs (dsRNAs) (dsJHEH1-1, dsJHEH1-2, dsJHEH2-1, dsJHEH2-2) respectively targeting LdJHEH1 and LdJHEH2 were constructed and bacterially expressed. Ingestion of dsJHEH1-1, dsJHEH1-2, dsJHEH2-1, dsJHEH2-2, and a mixture of dsJHEH1-1+dsJHEH2-1 successfully knocked down corresponding target gene function, and significantly increased JH titer and upregulated Krüppel homolog 1 (LdKr-h1) mRNA level. Knockdown of either LdJHEH1 or LdJHEH2, or both genes slightly reduced larval weight and delayed larval development, and significantly impaired adult emergence. Therefore, it is suggested that knockdown LdJHEH1 and LdJHEH2 affected JH degradation in the Colorado potato beetle.

Short neuropeptide F (sNPF) is a stage-specific suppressor for juvenile hormone biosynthesis by corpora allata, and a critical factor for the initiation of insect metamorphosis

Molting and metamorphosis are essential events for arthropod development, and juvenile hormone (JH) and its precursors play critical roles for these events. We examined the regulation of JH biosynthesis by the corpora allata (CA) in Bombyx mori, and found that intact brain-corpora cardiaca (CC)–CA complexes produced a smaller amount of JH than that in CC–CA complexes and CA alone throughout the 4th and 5th (last) instar stadium. The smaller amount of synthesis was due to allatostatin-C (AST-C) produced by the brain. The CC synthesized short neuropeptide F (sNPF) that also suppressed the JH synthesis, but only in day 3 4th stadium and after the last larval ecdysis. For the suppression, both peptides prevented the expression of some of the distinct JH biosynthetic enzymes in the mevalonate pathway. Allatotropin (AT) stimulated sNPF expression in the CC of day 1 5th instar stadium, not of day 3 4th; therefore the stage-specific inhibition of JH synthesis by sNPF was partly due to the stimulative action of AT on the sNPF expression besides the stage-specific expression of the sNPF receptors in the CA, the level of which was high in day 2 4th and day 0 5th instar larvae. The cessation of JH biosynthesis in the last instar larvae is a key event to initiate pupal metamorphosis, and both sNPF and AST-C are key factors in shutting down JH synthesis, along with the decline of ecdysone titer and dopamine.

Juvenile Hormone Biosynthesis Gene Expression in the corpora allata of Honey Bee (Apis mellifera L.) Female Castes

Juvenile hormone (JH) controls key events in the honey bee life cycle, viz. caste development and age polyethism. We quantified transcript abundance of 24 genes involved in the JH biosynthetic pathway in the corpora allata-corpora cardiaca (CA-CC) complex. The expression of six of these genes showing relatively high transcript abundance was contrasted with CA size, hemolymph JH titer, as well as JH degradation rates and JH esterase (jhe) transcript levels. Gene expression did not match the contrasting JH titers in queen and worker fourth instar larvae, but jhe transcript abundance and JH degradation rates were significantly lower in queen larvae. Consequently, transcriptional control of JHE is of importance in regulating larval JH titers and caste development. In contrast, the same analyses applied to adult worker bees allowed us inferring that the high JH levels in foragers are due to increased JH synthesis. Upon RNAi-mediated silencing of the methyl farnesoate epoxidase gene (mfe) encoding the enzyme that catalyzes methyl farnesoate-to-JH conversion, the JH titer was decreased, thus corroborating that JH titer regulation in adult honey bees depends on this final JH biosynthesis step. The molecular pathway differences underlying JH titer regulation in larval caste development versus adult age polyethism lead us to propose that mfe and jhe genes be assayed when addressing questions on the role(s) of JH in social evolution.

Peptidomic Analysis of the Brain and Corpora Cardiaca-Corpora Allata Complex in the Bombyx mori

The silkworm, Bombyx mori, is an important economic insect for silk production. However, many of the mature peptides relevant to its various life stages remain unknown. Using RP-HPLC, MALDI-TOF MS, and previously identified peptides from B. mori and other insects in the transcriptome database, we created peptide profiles showing a total of 6 ion masses that could be assigned to peptides in eggs, including one previously unidentified peptide. A further 49 peptides were assigned to larval brains. 17 new mature peptides were identified in isolated masses. 39 peptides were found in pupal brains with 8 unidentified peptides. 48 were found in adult brains with 12 unidentified peptides. These new unidentified peptides showed highly significant matches in all MS analysis. These matches were then searched against the National Center for Biotechnology Information (NCBI) database to provide new annotations for these mature peptides. In total, 59 mature peptides in 19 categories were found in the brains of silkworms at the larval, pupal, and adult stages. These results demonstrate that peptidomic variation across different developmental stages can be dramatic. Moreover, the corpora cardiaca-corpora allata (CC-CA) complex was examined during the fifth larval instar. A total of 41 ion masses were assigned to peptides.

Isolation and functional characterization of an allatotropin receptor from Manduca sexta

Manduca sexta allatotropin (Manse-AT) is a multifunctional neuropeptide whose actions include the stimulation of juvenile hormone biosynthesis, myotropic stimulation, cardioacceleratory functions, and inhibition of active ion transport. Manse-AT is a member of a structurally related peptide family that is widely found in insects and also in other invertebrates. Its precise role depends on the insect species and developmental stage. In some lepidopteran insects including M. sexta, structurally-related AT-like (ATL) peptides can be derived from alternatively spliced mRNAs transcribed from the AT gene. We have isolated a cDNA for an AT receptor (ATR) from M. sexta by a PCR-based approach using the sequence of the ATR from Bombyx mori. The sequence of the M. sexta ATR is similar to several G protein-coupled receptors from other insect species and to the mammalian orexin receptor. We demonstrate that the M. sexta ATR expressed in vertebrate cell lines is activated in a dose-responsive manner by Manse-AT and each Manse-ATL peptide in the rank order ATL-I > ATL-II > ATL-III > AT, and functional analysis in multiple cell lines suggest that the receptor is coupled through elevated levels of Ca 2+ and cAMP. In feeding larvae, Manse-ATR mRNA is present at highest levels in the Malpighian tubules, followed by the midgut, hindgut, testes, and corpora allata, consistent with its action on multiple target tissues. In the adult corpora cardiaca–corpora allata complex, Manse-ATR mRNA is present at relatively low levels in both sexes.

Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle, Tenebrio molitor (Coleoptera, Tenebrionidae)

The genome of Tribolium castaneum encodes two allatostatin [AS type B; W(X)(6)Wamide and AS type C; PISCF-OH] and one allatotropin (AT) precursor, but no AS type A (FGLamide) (Tribolium Genome Sequencing Consortium, 2008: Nature 452:949-955). Here we studied the activity (in vitro) of peptides derived from these precursors on the synthesis/release of juvenile hormone (JH) III. The corpora cardiaca-corpora allata (CC-CA) complexes of adult females of another tenebrionid beetle, the mealworm Tenebrio molitor, were used. Incubating the gland complexes in a medium containing Trica-AS B3 peptide, we showed that the peptide has allatostatic function in T. molitor. The activity of the type C AS depended on the age of the test animals and their intrinsic rate of JH III biosynthesis. The Trica-AS C peptide inhibited the JH release from CA of 3-day-old females with a high intrinsic rate of JH synthesis, but activated JH release from the CA of 7-day-old females with a lower intrinsic rate of JH production. The allatotropin peptide (Trica-AT) also activated the JH release from the CA of 7-day-old females in a dose-dependent and reversible manner. Unexpectedly, a type A AS derived from the precursor of the American cockroach Periplaneta americana (Peram-AS A2b) inhibited the JH release from the CA of younger and older females in the concentration range of 10(-8) to 10(-4) M, and the effects were fully reversible in the absence of peptide. These data suggest a complex role of allatoactive neuropeptides in the regulation of JH III biosynthesis in beetles.

A novel member of the adipokinetic peptide family in a “living fossil”, the ice crawler Galloisiana yuasai, is the first identified neuropeptide from the order Grylloblattodea

This is the first report on the structural identity of a neuropeptide of the insect order Grylloblattodea. A peptide was isolated and sequenced from the retrocerebral corpora cardiaca–corpora allata complex of the ice crawler, Galloisiana yuasai. The sequence of the peptide was deduced from the multiple MS N electrospray mass data as that of an octapeptide: pGlu-Val-Asn-Phe-Ser-Pro-Thr-Trp amide. The retention time on reversed-phase HPLC and the CID MS 2 mass spectra of a synthetic peptide with the same primary structure were exactly the same as of the natural peptide. The sequence represents a novel peptide of the adipokinetic hormone family which contains presently 50 members. The primary structure differs in only one position to a few previously discovered AKHs. A scenario is outlined that makes it likely that the most recently discovered insect order, the Mantophasmatodea, and the Grylloblattodea are closely related.

Spatial expression of the mevalonate enzymes involved in juvenile hormone biosynthesis in the corpora allata in Bombyx mori

The developmental expressions of the mRNA of JH synthetic enzymes have been studied using homogenates of the corpora cardiaca–corpora allata (CC–CA) complexes in Bombyx mori [Kinjoh, T., Kaneko, Y., Itoyama, K., Mita, K., Hiruma, K., Shinoda, T., 2007. Control of juvenile hormone biosynthesis in Bombyx mori: cloning of the enzymes in the mevalonate pathway and assessment of their developmental expression in the corpora allata. Insect Biochemistry and Molecular Biology 37, 808–818]. The in situ hybridization analyses in the CC–CA complex showed that the distribution of the mRNAs of all the mevalonate enzymes and juvenile hormone (JH) acid O-methyltransferase occurred only in the CA cells, indicating that the fluctuations of the enzyme mRNA amounts in the CC–CA complexes were derived solely from the CA. In addition, the size of the CA and their nuclei was not associated with the JH synthetic activity by the CA until the pharate adult. Only female adult CA synthesized JH in B. mori, and the CA and the nuclei were significantly larger than those of male CA which do not synthesize JH.