Source: UNIVERSITY OF CALIFORNIA, RIVERSIDE submitted to
INSECT ECDYSIS AS A VITAL TARGET FOR PEST CONTROL
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0198284
Grant No.
(N/A)
Project No.
CA-R-ENT-7193-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2013
Project End Date
Jun 30, 2018
Grant Year
(N/A)
Project Director
Adams, M.
Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521
Performing Department
Entomology, Riverside
Non Technical Summary
The proposed objectives will be accomplished using a combination of molecular biology, genetics, calcium imaging, and bioassay techniques. Work on Drosophila offers enormous advantages due to availability of genetic tools and transgenic fly lines. RNA silencing using the binary Gal4-UAS expression system allows for facile in vivo evaluation of behavioral and physiological outcomes. Work on moths offers advantages for physiological and endocrinological experiments. We will test for ETH regulation of JH levels in dipterans (Drosophila melanogaster, Aedes aegypti) and lepidopterans (Bombyx mori, Heliothis virescens) using a fluorescent tagging/HPLC methodology developed by our collaborator, Dr. Noriega. To demonstrate RNA silencing in mosquitoes and moths double stranded RNA sequences against the ETH gene will be injected into the hemocoel of larval H. virescens and into adult A. aegypti. To screen for ETH antagonists, peptidomimetic analogs synthesized by collaborator Dr. Nachman will be tested in vitro using heterologous expression of the H. virescens ETH receptor in CHO cells co-expressing an aequorin reporter and in vivo by injection.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2113110100030%
2113110104030%
2113110102040%
Goals / Objectives
The overall goals of the project are to evaluate ecdysis signaling pathways as a physiological target for future pest control strategies. We have discovered the epitracheal endocrine system in insects, which releases ecdysis triggering hormones (ETHs) that program ecdysis, a vital developmental process during insect ontogeny. Interruption of ETH signaling has lethal consequences in moths and flies. Recent preliminary data indicates that ETH also regulates juvenile hormone production. Validation of this hypothesis may have consequences not only for programming of morphogenesis, but also for reproduction. We propose to use a combination of model insects, pest insects, and disease vectors to develop practical methods for disruption of vital developmental and reproductive processes. Our proposed objectives for this funding period are: 1. Test the hypothesis that ETH regulates juvenile hormone levels in insects. 2. Attempt RNA silencing of genes encoding ETH and ETH receptor in flies and moths. 3. Screen peptidomimetic analogs of ETH for receptor antagonist activity in vitro and in vivo in pest insects. Expected outputs include new knowledge about how ETH signaling regulates ecdysis, eclosion, and reproduction and the discovery of novel ETH receptor antagonists.
Project Methods
The proposed objectives will be accomplished using a combination of molecular biology, genetics, calcium imaging, and bioassay techniques. Work on Drosophila offers enormous advantages due to availability of genetic tools and transgenic fly lines. RNA silencing using the binary Gal4-UAS expression system allows for facile in vivo evaluation of behavioral and physiological outcomes. Work on moths offers advantages for physiological and endocrinological experiments. We will test for ETH regulation of JH levels in dipterans (Drosophila melanogaster, Aedes aegypti) and lepidopterans (Bombyx mori, Heliothis virescens) using a fluorescent tagging/HPLC methodology developed by our collaborator, Dr. Noriega. To demonstrate RNA silencing in mosquitoes and moths double stranded RNA sequences against the ETH gene will be injected into the hemocoel of larval H. virescens and into adult A. aegypti. To screen for ETH antagonists, peptidomimetic analogs will be tested in vitro using heterologous expression of the H. virescens ETH receptor in CHO cells co-expressing an aequorin reporter and in vivo by injection.

Progress 07/01/13 to 06/30/18

Outputs
Target Audience:Research scientists, postdoctoral fellows, graduate students, and undergraduate students at conferences. We presented our work at the Annual Society for Neuroscience meetings in 2013, 2015, and 2016, at the Annual Drosophila (Genetics Society of America) Conferences in 2013, 2014, 2015, 2017, and 2018, and at Insect Hormone Conferences in 2017 and 2019. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate and undergraduate students have obtained training in a range of experimental techniques, including RT-PCR, quantitative PCR experiments, RNA-silencing, calcium imaging technologies, and behavior analyses. They also have obtained training in communication of scientific evidence at conferences and in preparation of publications. How have the results been disseminated to communities of interest?Research scientists, postdoctoral fellows, graduate students, and undergraduate students at conferences. We presented our work at the Annual Society for Neuroscience meetings in 2013, 2015, and 2016, at the Annual Drosophila (Genetics Society of America) Conferences in 2013, 2014, 2015, 2017, and 2018, and at Insect Hormone Conferences in 2017 and 2019. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have shown that levels of ETH and ETH receptor (ETHR) transcript fluctuate post eclosion in females and remain significant up to 3 weeks post-eclosion. In males, ETH and ETH receptor transcript levels are quite robust through 3 weeks post-eclosion. We have shown that ablation of Inka cells, the source of ecdysis triggering hormone (ETH) in Drosophila, leads to precipitous drops in juvenile hormone levels in day 4 male and female adults. This agrees with previous data showing that RNA silencing of ETHR in the CA leads to drastically reduced JH levels. Calcium imaging data indicate that the corpora allata, the sole source of juvenile hormone (JH), are significantly more sensitive to ETH in the male than the female. These results provide confirmation that ETH is an important allatotropin in the adult life of this organism (Meiselman et al., 2017). Formation and expression of memories are critical for context-dependent decision making. In Drosophila, a courting male rejected by a mated female subse- quently courts less avidly when paired with a virgin female, a behavioral modification attributed to ''courtship memory.'' Here we show the critical role of hormonal state for maintenance of courtship memory. Ecdysis-triggering hormone (ETH) is essential for courtship memory through regulation of juvenile hormone (JH) levels in adult males. Reduction of JH levels via silencing of ETH signaling genes impairs short-term courtship memory, a phenotype rescuable by the JH analog methoprene. JH-deficit- induced memory impairment involves rapid decay rather than failure of memory acquisition. A critical period governs memory performance during the first 3 days of adulthood. Using sex-peptide-expressing ''pseudo-mated'' trainers, we find that robust courtship memory elicited in the absence of aversive chemical mating cues also is dependent on ETH-JH signaling. Finally, we find that JH acts through dopaminergic neurons and conclude that an ETH-JH- dopamine signaling cascade is required during a critical period for promotion of social-context- dependent memory (Lee et al., 2017). ETH and ETHR transcripts in male and female yellow fever mosquitoes (Aedes aegypti) also fluctuate. Interestingly their levels correlate with fluctuating levels of ecdysteroids. This as yet unpublished work remains ongoing (Ding and Adams, in preparation). We have discovered that peptidergic signaling downstream of ETH promotes fluid clearance from the respiratory system at onset of the ecdysis sequence. During developmental transitions, airway clearance is critically important for achieving optimal gas exchange. As insects develop from embryo to adult, airway clearance occurs episodically each time the molt is completed by performance of the ecdysis sequence, coordinated by a peptide-signaling cascade initiated by ecdysis-triggering hormone (ETH). We find that the neuropeptide Kinin (also known as Drosokinin or Leukokinin) is required for normal respiratory fluid clearance or "tracheal air-filling" in Drosophila larvae. Our findings strongly implicate the neuropeptide Kinin as an important regulator of airway clearance via intracellular calcium mobilization in tracheal epithelial cells of Drosophila (Kim et al., 2018). We also reported that stress-induced elevation of ecdysone in Drosophila females is accompanied by decreased levels of ecdysis triggering hormone (ETH). Depressed levels of circulating ETH lead to attenuated activity of its targets, including juvenile hormone-producing corpus allatum and, as we describe here for the first time, octopaminergic neurons of the oviduct. Elevation of steroid thereby results in arrested oogenesis, reduced octopaminergic input to the reproductive tract, and consequent suppression of ovulation. ETH mitigates heat or nutritional stress-induced attenuation of fecundity, which suggests that its deficiency is critical to reproductive adaptability (Meiselman et al, 2018). Ecdysis triggering hormone receptors (ETHR) regulate the behavioral sequence necessary for cuticle shedding. Recent reports have documented functions for ETHR signaling in adult Drosophila melanogaster. In this study, we report that ETHR silencing in local interneurons of the antennal lobes and fruitless neurons leads to sharply increased rates of male-male courtship. RNAseq analysis of ETHR knockdown flies reveals differential expression of genes involved in axon guidance, courtship behavior and chemosensory functions. Our findings indicate an important role for ETHR in regulation of Drosophila courtship behavior through chemosensory processing in the antennal lobe (Deshpande et al., 2018).

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Lee, S.S., Ding, Y., Karapetans, N., Rivera-Perez, C., Noriega, F.G., Adams, M.E. (2017) Hormonal signaling cascade during and early-Adult Critical Period required for courtship memory retention in Drosophila. Current Biol. 27: 2798-2809. Meiselman, M.R., Kingan, K.G., Adams, M.E. Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation. BMC Biology (2018) 16: 18 DOI 10.1186/s12915-018-0484-9 Deshpande, S.A., Meiselman, M., Hice R.H., Arensburger, P., Rivera-Perez, C., Kim, D.-H., Croft, R.L., Noriega, F.G., and Adams, M.E. Ecdysis triggering hormone receptors regulate male courtship behavior via antennal lobe interneurons in Drosophila. Gen. Comp. Endocrinol. 278: 79-88.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Research scientists, postdoctoral fellows, graduate students at conferences. We presented our work at the Annual Society for Neuroscience Meeting in November, 2016 and at the Drosophila Conference during March 2017. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate and undergraduate students have been trained in RT-PCR experiments, RNA-silencing, and calcium imaging technologies. How have the results been disseminated to communities of interest?We presented our work at the Annual Society for Neuroscience Meeting in November, 2016 and at the Drosophila Conference during March 2017. What do you plan to do during the next reporting period to accomplish the goals?We continue our efforts to perform quantitative PCR to establish accurately transcript levels of ETH and ETHR in both Drosophila and Aedes and to correlate these with ecdysone levels during molting episodes and in reproductive males and females. We will continue our collaborations with Fernando Noriega to monitor juvenile hormone levels in flies and mosquitoes and with Ronald Nachman to further establish structure-activity relationships for ETH agonists and antagonists.

Impacts
What was accomplished under these goals? We have discovered that peptidergic signaling downstream of ETH promotes fluid clearance from the respiratory system at onset of the ecdysis sequence. During developmental transitions, airway clearance is critically important for achieving optimal gas exchange. As insects develop from embryo to adult, airway clearance occurs episodically each time the molt is completed by performance of the ecdysis sequence, coordinated by a peptide-signaling cascade initiated by ecdysis-triggering hormone (ETH). We find that the neuropeptide Kinin (also known as Drosokinin or Leukokinin) is required for normal respiratory fluid clearance or "tracheal air-filling" in Drosophila larvae. Our findings strongly implicate the neuropeptide Kinin as an important regulator of airway clearance via intracellular calcium mobilization in tracheal epithelial cells of Drosophila. We also report that stress-induced elevation of ecdysone in Drosophila females is accompanied by decreased levels of ecdysis triggering hormone (ETH). Depressed levels of circulating ETH lead to attenuated activity of its targets, including juvenile hormone-producing corpus allatum and, as we describe here for the first time, octopaminergic neurons of the oviduct. Elevation of steroid thereby results in arrested oogenesis, reduced octopaminergic input to the reproductive tract, and consequent suppression of ovulation. ETH mitigates heat or nutritional stress-induced attenuation of fecundity, which suggests that its deficiency is critical to reproductive adaptability.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Meiselman, M., Lee, S. S., Tran, R.-T., Dai, H., Ding, Y., Rivera-Perez, C., Wijesekera, T.P., Dauwalder, B., Noriega, F. G., Adams, M. E. (2017) Endocrine network essential for reproductive success in Drosophila melanogaster. Proc. Natl. Acad. Sci. 114(19): E3849-E3858. Lee, S. S., Ding, Y., Karapetians, N., Rivera-Perez, C., Noriega, F. G., Adams, M. E. (2017) Hormonal signaling cascade during an early-adult critical period required for courtship memory retention in Drosophila. Current Biology 27: 2798-2809. Kim, D.-H, Kim, Y.-J., and Adams, M. E. (2018) Endocrine regulation of airway clearance in Drosophila. Proc. Natl. Acad. Sci. doi:10.1073/pnas.1717257115/-/DCSupplemental.


Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Undergraduate and graduate students at the University of California, Riverside. Scientists working on agricultural issues nationally and internationally. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of graduate students and undergraduates in molecular biology, calcium imaging, behavioral analysis. How have the results been disseminated to communities of interest?We presented our work at the Drosophila Research conference in March, 2016, the Society for Neuroscience conference in November, 2016 and the Symposium for Glial-Neuronal Interactions in January, 2017. What do you plan to do during the next reporting period to accomplish the goals?We will test the hypothesis that ETH receptors are expressed in primary sensory neurons and whether loss of ETH signaling affects courtship behaviors. We will examine expression of ETH and ETH receptor genes in the yellow fever mosquito, Aedes aegypti.

Impacts
What was accomplished under these goals? We continue to discover functions for ETH during the adult stage, namely multiple functions in Drosophila associated with reproductive state in both males and females. We discovered that ETH regulates juvenile hormone levels that are required for normal egg production. In males, ETH-JH signaling regulates accessory gland function. Reduction of JH levels through interference of ETH signaling leads to lower egg production when ETH- or ETHR-silenced males are mated with wild-type females. Furthermore, stress elevates ecdysone levels and blocks ETH release from Inka cells. We have found that ETH receptor silencing in the mosquito Aedes aegypti leads to reduced ovarian follicle diameter.

Publications


    Progress 10/01/14 to 09/30/15

    Outputs
    Target Audience:Research scientists, postdoctoral fellows, graduate students at conferences. We presented our work at the Drosophila conference during March 2015 and at the annual symposium for Glial-Neuronal Interactions in January, 2016. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training of graduate students and undergraduates in molecular biology, calcium imaging, behavioral analysis. How have the results been disseminated to communities of interest?We presented our work at the Drosophila conference in March, 2015, the Society for Neuroscience conference in October, 2015, and the Symposium for Glial-Neuronal Interactions in January, 2016. What do you plan to do during the next reporting period to accomplish the goals?We plan to examine further the control of vitellogenesis and ovulation in Drosophila melanogaster and Aedes aegypti.

    Impacts
    What was accomplished under these goals? We have found that, with respect to ecdysis, ETH regulates pre-ecdysis via actions on kinin neurons and ecdysis via actions on bursicon neurons in pupal Drosophila. We continue to discover functions for ETH during the adult stage, namely multiple functions in Drosophila associated with reproductive state in both males and females. We discovered that ETH triggers ovulation and oviduct motility in Drosophila. ETH deficiency leads to egg retention and inviable eggs. ETH appears to signal through octopamine, since silencing of ETH receptors (ETHR) in octopaminergic neurons leads to loss of function phenotypes. ETH deficient males mated with wild-type females leads to lower rates of egg production.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2015 Citation: Kim, D.-H., Han, M.-R., Lee, G., Lee, S.-S., Kim, Y.-J., & Adams, M. E. (2015). Rescheduling Behavioral Subunits of a Fixed Action Pattern by Genetic Manipulation of Peptidergic Signaling. PLoS Genetics, 11(9), e1005513. http://doi.org/10.1371/journal.pgen.1005513


    Progress 10/01/13 to 09/30/14

    Outputs
    Target Audience: Research scientists, postdoctoral fellows, graduate students at conferences. We presented our work at the Drosophila conference during March 2014 and at the annual symposium for Glial-Neuronal Interactions in January, 2015. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate and undergraduate students have been trained in RT-PCR experiments, RNA-silencing, and calcium imaging technologies. How have the results been disseminated to communities of interest? We presented our work at the Drosophila conference during March 2014 and at the annual symposium for Glial-Neuronal Interactions in January, 2015. What do you plan to do during the next reporting period to accomplish the goals? We will attempt to perform quantitative PCR to establish accurately transcript levels of ETH and ETHR in both Drosophila and Aedes and correlate these with reproductive state in both males and females. We will continue our collaborations with Fernando Noriega to monitor juvenile hormone levels in flies and mosquitoes and with Ronald Nachman to further establish structure-activity relationships for ETH agonists and antagonists.

    Impacts
    What was accomplished under these goals? We have shown that levels of ETH and ETH receptor (ETHR) transcript fluctuate post eclosion in females and remain significant up to 3 weeks post-eclosion. In males, ETH and ETH receptor transcript levels are quite robust through 3 weeks post-eclosion. We have shown that ablation of Inka cells, the source of ecdysis triggering hormone (ETH) in Drosophila, leads to precipitous drops in juvenile hormone levels in day 4 male and female adults. This agrees with previous data showing that RNA silencing of ETHR in the CA leads to drastically reduced JH levels. Calcium imaging data indicate that the corpora allata, the sole source of juvenile hormone (JH), are significantly more sensitive to ETH in the male than the female. These results provide confirmation that ETH is an important allatotropin in the adult life of this organism. ETH and ETHR transcripts in male and female yellow fever mosquitoes (Aedes aegypti) also fluctuate. Interestingly their levels correlate with fluctuating levels of ecdysteroids.

    Publications


      Progress 07/01/13 to 09/30/13

      Outputs
      Target Audience: Research scientists, postdoctoral fellows, graduate students at conferences. We presented our work at the annual symposium for Glial-Neuronal Interactions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate and undergraduate students have been trained in RNA silencing methodologies, in behavioral analysis, and in quantification of reproductive functions, including fecundity measurements. How have the results been disseminated to communities of interest? We have presented our results at the annual Symposium for Glial-Neuronal Interactions. What do you plan to do during the next reporting period to accomplish the goals? We will continue to investigate roles for ETH signaling in the adult stage, including regulation of reproductive state, courtship memory, and olfactory functions suspected of being important in adult behaviors.

      Impacts
      What was accomplished under these goals? In collaboration with Dr. Fernando Noriega, we have demonstrated that RNAi knockdown of ETH receptors in the corpora allata of Drosophila results in a drastic decrease in juvenile hormone levels in both males and females. These findings correlate well with calcium imaging data showing that exposure of corpora allata from males and females to ETH results in calcium mobilization. Physiological consequences are reduced egg production in mated females and increased egg production in virgin females. We also have found that control females mated with ETHR-knockdown male flies also produce fewer eggs, suggesting that ETH and JH regulate the male reproductive state. ETHR knockdown also has consequences for courtship behavior, including reduction in courtship memory. We also have discovered that ETHR knockdown in specific circuits of the central nervous system results in aberrant patterns of male courtship behavior. In particular, we have evidence that ETHR is expressed in primary olfactory and gustatory neurons and olfactory interneurons in the antennal lobe. Preliminary results from ETHR knockdown in the mosquito Aedes aegypti suggest that egg production is reduced in this species as well.

      Publications


        Progress 01/01/12 to 12/31/12

        Outputs
        OUTPUTS: Ecdysis triggering hormones (ETHs) are vital endocrine signals that initiate and schedule developmental events at the end of each insect molt, which culminates in ecdysis or shedding of the old cuticle. Events leading to ecdysis include tracheal inflation and performance of a behavioral sequence that allows the animal to escape its old cuticle. In the absence of ETH, lethal ecdysis deficiency occurs. We have shown that ETH triggers a downstream peptide-signaling cascade within the central nervous system that coordinates tracheal inflation and successive behaviors. ETH acts directly on a variety of central peptidergic ensembles that activate respiratory dynamics and central pattern generators. We have associated the peptide kinin with tracheal air filling and pre-ecdysis behavior. During the past year, we have shown that activation of kinin neurons using the temperature-sensitive TRP-A1 channel leads to premature tracheal air filling, confirming a role for kinin peptide in this process. Furthermore, activation of CCAP/MIP/AST-CC-bursicon neurons using the temperature-sensitive TRP-M8 channel causes premature ecdysis behavior, providing confirmation that this central peptidergic ensemble activates ecdysis behavior. In some species (e.g., Drosophila), endocrine Inka cells, the sole source of ETH, persist into adulthood. This raises the interesting question regarding possible functions for ETH in the adult stage, during which no further ecdysis occurs. During the past year, we have generated preliminary data indicating that ablation of Inka cells, the sole source of ETH, leads to reduced egg viability in Drosophila. Furthermore, silencing the ETH receptor during the adult stage causes a number of defects, including male-specific reduction in juvenile hormone levels, elevation of male-male courtship behavior, and aberrant male-female courtship behavior. Dissemination of this knowledge has been accomplished through presentations at conferences during the past year. PARTICIPANTS: Dr. Ron Nachman, USDA-ARS supplied peptidomimetic compounds that were evaluated using aequorin-based luminescence assays. Graduate student Ryan Arvidson performed the assays. Furthermore, Mr. Arvidson provided training to other graduate students and undergraduates in the use of luminescence assays for pharmacological profiling of receptors. Dr. Fernando Noriega of Florida International University provided data on juvenile hormone levels in ETH receptor silenced adult fruit flies. TARGET AUDIENCES: Seminars and lectures at conferences were given - Entomology Congress in South Korea, August, 2012. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

        Impacts
        We showed that ETH triggers a downstream peptide signaling cascade in the central nervous system that controls sequential ecdysis-related events, including tracheal air filling and ecdysis behavior. Data generated during the past year indicates that ETH signaling persists to the adult stage, where it regulates courtship behavior and egg viability. Knowledge of these signaling molecules provides insights into the physiology of ecdysis and reproduction, both of which are vital physiological processes in insect pests and disease vectors. These findings provide a basis for future design of environmentally sound and safe insect control agents.

        Publications

        • No publications reported this period


        Progress 01/01/11 to 12/31/11

        Outputs
        OUTPUTS: Ecdysis triggering hormones (ETHs) are vital endocrine signals that initiate and schedule developmental events at the end of each insect molt, which culminates in ecdysis or shedding of the old cuticle. Events leading to ecdysis include tracheal inflation and performance of a behavioral sequence that allows the animal to escape its old cuticle. In the absence of ETH, lethal ecdysis deficiency occurs. We have shown that ETH triggers a downstream peptide-signaling cascade within the central nervous system that coordinates tracheal inflation and successive behaviors. ETH acts directly on a variety of central peptidergic ensembles that activate respiratory dynamics and central pattern generators. We have associated the peptide kinin with tracheal inflation and pre-ecdysis behavior. Ablation of kinin neurons leads to incomplete tracheal inflation and disruption in the timing of pre-ecdysis behavior. Similarly, a kinin receptor hypomorph exhibits defects in tracheal inflation. A distinct peptidergic ensemble that expresses a cocktail of the peptides CCAP, MIP, and bursicon appears to be involved in ecdysis and post-ecdysis behaviors. Ablation of these neurons results in lethal ecdysis deficiency characterized by absence of ecdysis and post-ecdysis behaviors. Calcium imaging studies show that, following ETH exposure, kinin neurons become active within 1-2 min and continue to be active for ~ 10 min. Immediately following cessation kinin neuron activity, activity is initiated in CCAP/MIP/bursicon neurons. We are currently investigating the causes of the delay in activity of this peptidergic ensemble. In some species (e.g., Drosophila), endocrine Inka cells, the sole source of ETH, persist into adulthood. This raises the interesting question regarding possible functions for ETH in the adult stage, during which no further ecdysis occurs. We have preliminary evidence that ETH signaling is involved in male courtship behavior. Dissemination of this knowledge has been accomplished through publication in the scientific literature and presentations at conferences. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

        Impacts
        We showed that ETH triggers a downstream peptide signaling cascade in the central nervous system that controls sequential ecdysis-related events, including tracheal inflation and a behavioral sequence. In the absence of these downstream signaling events, tracheal inflation and ecdysis of the old cuticle are compromised, leading to lethal consequences. Knowledge of these signaling molecules provides insights into the physiology of ecdysis, a vital physiological system in insect pests and disease vectors. These findings provide a basis for future design of environmentally sound and safe insect control agents.

        Publications

        • Zitnan, D., Adams, M. E. (2012) Neuroendocrine regulation of ecdysis. In: Insect Endocrinology, (Gilbert, L. I., Ed.) Academic Press, San Diego.


        Progress 01/01/10 to 12/31/10

        Outputs
        OUTPUTS: Ecdysis triggering hormones (ETHs) from endocrine Inka cells initiate a series of physiological and behavioral events at the end of the molt culminating in ecdysis, which is shedding of the old cuticle. The timely initiation and scheduling of the ecdysis sequence is vital to the survival of insects. We characterized ETH signaling molecules and downstream release of hormones as potential molecular targets for future insect control agents in pest insects (Heliothis virescens), disease vectors (Aedes aegypti), model organisms (Drosophila melanogaster, Manduca sexta, Bombyx mori) and in representatives of diverse arthropods. All insects tested produce ETHs. Immunohistochemical staining revealed Inka cells on the tracheal surface of diverse insects. DNA sequences encoding putative ETH receptors were deduced from available genomes of several hemimetabolous and holometabolous insects. In all insects examined, the ETHR gene encodes two subtypes of the receptor (ETHR-A and ETHR-B). We report for the first time the presence of putative ETHs and ETHRs in genomes of other arthropods, including the tick (Arachnida) and water flea (Crustacea). Our results provide further evidence of structural and functional conservation of ETH-ETHR signaling. We discovered the eclosion hormone receptor, and described it as a receptor guanylyl cyclase BdmGC-1 and its isoform BdmGC-1B in the Oriental fruit fly Bactrocera dorsalis. The B form exhibits the conserved domains and putative N-glycosylation sites found in BdmGC-1, but possesses an additional 46-amino acid insertion in the extracellular domain and lacks the C-terminal tail of BdmGC-1. Combined immunolabeling and in situ hybridization reveal that BdmGC-1 is expressed in Inka cells and neighboring cells of the epitracheal gland. Heterologous expression of BdmGC-1 in HEK cells leads to robust increases in cGMP following exposure to low picomolar concentrations of EH. The B-isoform responds only to higher EH concentrations, suggesting different physiological roles of these cyclases. We propose that BdmGC-1 and BdmGC-1B are high- and low-affinity EH receptors, respectively. Dissemination of this knowledge has been accomplished through publication in the scientific literature and presentations at conferences. PARTICIPANTS: Kuang-Hui Lu, Dept. Entomology, National Chung Hsing University, Taiwan, Republic of China Dusan Zitnan, Institute of Life Sciences, Slovak Academy of Sciences, Bratislava, Slovakia TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

        Impacts
        We documented the expression of ETH signaling molecules in diverse arthropods and discovered the first eclosion hormone receptor in the Oriental fruit fly. Knowledge of these signaling molecules provides insights into the physiology of ecdysis, a vital physiological system in insect pests and disease vectors. These findings provide a basis for future design of environmentally sound and safe insect control agents.

        Publications

        • Mykles, D. L., Adams, M. E., Gade, G., Lange, A. B., Marco, H. G., et al., (2010). Neuropeptide action in insects and crustaceans. Physiol Biochem Zool. 83, 836-46.
        • Roller, L., Zitnanova, I., Dai, L., Simo, L., Park, Y., et al., (2010). Ecdysis triggering hormone signaling in arthropods. Peptides. 31, 429-41. Epub 2009 Nov 29.
        • Chang, J. C., Yang, R. B., Adams, M. E., Lu, K. H., (2009). Receptor guanylyl cyclases in Inka cells targeted by eclosion hormone. Proc Natl Acad Sci U S A. 106, 13371-6. Epub 2009 Jul 28.


        Progress 01/01/09 to 12/31/09

        Outputs
        OUTPUTS: Heliothis virescens is an economic pest insect that infests a multitude of crops, including tobacco, cotton, and corn. In this investigation, H. virescens serves as a pest insect model for ecdysis, the process of shedding the cuticle at the end of each larval developmental stage, or instar. Ecdysis is initiated upon release of a peptide called ecdysis-triggering hormone (ETH) from specialized endocrine cells called Inka cells. ETH binds to ETH receptors (ETHRs), which are G-protein coupled receptors (GPCRs) expressed in a subset of neurons in the insect nervous system, activating a cascade of peptidergic ensembles that lead to the general behavioral sequence culminating in ecdysis (Kim et al., 2006). The mRNA transcript encoding the ETH precursor in H. virescens was cloned and found to contain an open reading frame (ORF) for a putative polypeptide that is assumed to be cleaved into pre-ecdysis triggering hormone (PETH), ETH, and ETH-associated peptide (ETH-AP). Based on sequence homology between these hormones in related insects, H. virescens PETH (Hev PETH) is an 11 amino acid peptide identical to PETH in related Lepidoptera. HevETH is a 25 amino acid peptide with a high degree of similarity to ETH in other Lepidoptera, and ETH-AP has only N-terminal similarity to its homologues in other species. ETH receptors in H. virescens (HevETHR-A and HevETHR-B) contain 565 and 553 amino acids respectively, and exhibit 100% sequence identity in the first 215 N-terminal amino acids. They show a high degree of homology with ETHR isoforms of other Lepidopteran insects. All Lepidopteran ETHRs are predicted to couple with Gαq proteins downstream upon ligand activation, leading to Ca2+ release from intracellular stores. We have co-expressed ETHRs with the calcium reporter aequorin as a basis for screening peptidomimetic agonists and antagonists. Pharmacological characterizations of HevETHR-A and HevETHR-B revealed that both receptor isoforms show high affinity for PETH and ETH from other insect species, with approximately 100-fold less activity shown for hormones or compounds that lack 1) the presence of a "PRX" C-terminal motif, 2) C-terminal amidation, or 3) four or more highly polar residues in the last nine C-terminal amino acids. PARTICIPANTS: Melvin Wei completed a Masters Thesis in the graduate program in Cell, Molecular and Developmental Biology (CMDB Program) at UC Riverside. His training in the CMDB Program provided led to his placement in a biotech company in the San Diego area. TARGET AUDIENCES: Pending completion of this project, publications will provide dissemination of the information into the scientific literature. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

        Impacts
        Molecular cloning, PCR, HPLC, and mass spectrometry developed in this project helped to identify ETH ligands and receptors in the pest insect, H. virescens. Knowledge of the cDNA sequences of ETH receptors led to development of a cell line-based assay suitable for screening of compounds acting as agonists or antagonists. Wei, M. (2007). Characterization of the Ecdysis-triggering Hormone and Receptors in Heliothis virescens.

        Publications

        • Wei, M. (2007). Characterization of the Ecdysis-triggering Hormone and Receptors in Heliothis virescens. M. S. Thesis


        Progress 01/01/08 to 12/31/08

        Outputs
        OUTPUTS: The ecdysis sequence is a series of developmental events at the end of each molt in insects that allows for respiratory inflation, escape from the old cuticle, and advancement to the next stage. It is initiated and scheduled by release of ecdysis triggering hormones (ETHs) from Inka cells of the epitracheal endocrine system. In Drosophila, expression of the ETH gene is vital ecdysis; deletion of the gene produces lethal consequences early in the life history of the animal. We describe for the first time the ETH signaling system in the yellow fever mosquito, Aedes aegypti. ETH-immunoreactive Inka cells occur at branch points of major tracheal trunks and loss of staining is coincident with ecdysis. Peptides (AeaETH1, AeaETH2) purified from extracts of pharate 4th instar larvae have -PRXamide C-terminal amino acid sequence motifs similar to ETHs previously identified in moths and flies. Injection of synthetic AeaETHs induces premature ecdysis behavior in pharate larvae, pupae and adults. Two functionally distinct subtypes of ETH receptors (AeaETHR-A, AeaETHR-B) of A. aegypti are identified and show high sensitivity and selectivity to ETHs. Increased ETHR transcript levels and behavioral sensitivity to AeaETHs arising in the hours preceding the 4th instar larva-to-pupa ecdysis are correlated with rising ecdysteroid levels, suggesting steroid regulation of receptor gene expression. Our description of natural and ETH-induced ecdysis in A. aegypti should facilitate future approaches directed toward hormone-based interference strategies for control of mosquitoes as human disease vectors. These results were disseminated by poster presentation at Genetics Society annual meeting and by publication in General and Comparative Endocrinology. PARTICIPANTS: The postdoctoral trainee on this project was Li Dai. The trainee acquired expertise in immunohistochemistry, quantitative PCR, high performance liquid chromatography, mass spectrometry, and peptide synthesis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

        Impacts
        Molecular cloning, PCR, HPLC, and mass spectrometry developed in this project helped to identify ETH ligands and receptors. Immunohistochemical and in situ hybridization techniques were crucial to the identification of Inka cells. Quantitative PCR was use to quantify expression of ETH and ETH receptor genes; enzyme immunoassay was used to quantify steroid levels. Knowledge of the cDNA sequences of ETH receptors will now be used to screen for antagonist peptidomimetics. These studies show that genes encoding ETH and its receptors are likely under steroid regulatory control.

        Publications

        • Dai, L. and Adams, M. E. (2009). Ecdysis triggering hormone signaling in the yellow fever mosquito Aedes aegypti. Gen. Comp. Endocrinol. In press.


        Progress 01/01/07 to 12/31/07

        Outputs
        Ecdysis is a complex developmental event in insects that occurs at the end of each molt. It is initiated and scheduled by release of ecdysis triggering hormones (ETHs) from Inka cells of the epitracheal endocrine system. ETH triggers activity in many downstream peptidergic neurons, including those that release the hormone bursicon late in ecdysis. We published the predicted precursor sequences of bursicon subunits (Masburs and Maspburs) in the tobacco hawkmoth Manduca sexta. Distinct developmental patterns of mRNA transcript and subunit-specific protein labeling of burs and pburs as well as CCAP in neurons of the ventral nervous system were observed in pharate larval, pupal and adult stages. A subset of bursicon neurons located in thoracic ganglia of larvae express ecdysis triggering hormone receptors, suggesting they are direct targets of ETH. Projections of bursicon neurons within the CNS and to neurohemal secretory sites are consistent with both central signaling and circulatory hormone functions. Intrinsic cells of the corpora cardiaca contain pburs transcripts and pburs-like immunoreactivity, while burs transcripts and burs-like immunoreactivity were absent in these cells. Recombinant bursicon is a multi-functional hormone that induces both wing expansion and tanning. These results were disseminated by poster presentation at international conferences (e.g., Society for Neuroscience) and by publication in the Journal of Comparative Neurology.

        Impacts
        Molecular cloning and PCR expertise developed in this project helped to identify bursicon subunits. Immunohistochemical and in situ hybridization techniques were crucial to the identification of bursicon neurons. Identification and biological activity of bursicon in the tobacco hawkmoth, Manduca sexta. Knowledge of the cDNA sequences of bursicon subunits and the neurons that express them will now be used to identify homologous sequences in H. virescens and A. aegypti.

        Publications

        • Dai, L., Dewey, E. M., Zitnan, D., Luo, C. W., Honegger, H. W., Adams, M. E. (2008). Identification, developmental expression, and functions of bursicon in the tobacco hawkmoth, Manduca sexta. J. Comp. Neurol. 506(5):759-774.


        Progress 01/01/06 to 12/31/06

        Outputs
        Ecdysis in insects occurs at the end of each molt, and is initiated by release of pre-ecdysis triggering hormone (PETH) and ecdysis triggering hormone (ETH) from Inka cells. During the past year, we made progress in the characterization of ETH signaling molecules in the tobacco budworm, HELIOTHIS VIRESCENS. We identified the cDNA transcript encoding PETH, ETH, and ETH-associated peptide. PETH in H. VIRESCENS is identical to its homolog in MANDUCA SEXTA and BOMBYX MORI. H. VIRESCENS ETH is a 25 amino acid peptide very similar to previously described homologs in M. SEXTA and B. MORI. Two subtypes of ETH receptors (ETHR-A, ETHR-B) in H. VIRESCENS have been cloned, sequenced, and expressed in mammalian CHO cells to establish their pharmacological profiles. These receptors arise through alterative splicing of 3-prime exons. ETHR-A shows similar sensitivity to PETH (EC50= 318 nM) and HevETH (EC50=158 nM). ETHR-B is more sensitive to PETH (EC50= 20 nM) and HevETH (EC50= 43 nM). Interestingly, both receptor subtypes are most sensitive to DROSOPHILA peptides ETH1 and ETH2. Since it is known from work in DROSOPHILA that deletion of the ETH gene is lethal, the development of a functional assay for H. VIRESCENS ETH receptors provides the means to begin screening for new pest control agents designed as ETH antagonists.

        Impacts
        Identification and pharmacological profiling of ecdysis-triggering hormones and receptors in the tobacco budworm, HELIOTHIS VIRESCENS.

        Publications

        • Kim, Y. J., Zitnan, D., Cho, K. H., Schooley, D. A., Mizoguchi, A. and Adams, M. E. (2006). Central peptidergic ensembles associated with organization of an innate behavior. Proc Natl Acad Sci U S A. 103, 14211-14216.
        • Kim, Y. J., Zitnan, D., Galizia, C. G., Cho, K. H. and Adams, M. E. (2006). A command chemical triggers an innate behavior by sequential activation of multiple peptidergic ensembles. Curr. Biol. 16, 1395-1407.


        Progress 01/01/05 to 12/31/05

        Outputs
        Insect molting, regulated by steroid hormones, culminates in ecdysis, which is under the control of peptide ecdysis triggering hormones (ETH). Deletion of the ETH gene in Drosophila causes lethal ecdysis deficiencies at the end of the first molt. During the past year, we have made progress in defining ecdysis triggering hormone signaling molecules in the tobacco budworm, Heliothis virescens and the yellow fever mosquito, Aedes aegypti. We have obtained a partial sequence of the Heliothis virescens ETH receptor subtype B (ETHR-B); attempts to define the full length sequence of ETHR-B are in progress. Long range PCR of the ETH receptor subtype A (ETHR-A) also is underway. In addition, we have obtained the complete amino acid sequence of the H. virescens ecdysis triggering hormone (ETH) using single cell mass spectrometry. The sequence of pre-ecdysis triggering hormone (PETH) also has been determined, and is identical to PETH of M. sexta and B. mori. The amino acid sequences of ETH1 and ETH2 in Aedes aegypti have been determined from the genome database, which has become available recently. These peptides are very similar to those of the malaria mosquito, Anopheles gambiae. ETH antagonists have been developed against Manduca sexta ETH and PBAN receptors. These will be tested in A. aegypti and H. virescens during the next year.

        Impacts
        Identification of ecdysis-triggering hormones and receptors in the tobacco budworm, Heliothis virescens and the yellow fever mosquito, Aedes aegypti.

        Publications

        • Zitnan, D. and Adams, M. E. (2005) Neuroendocrine regulation of Insect Ecdysis. In: Comprehensive Molecular Insect Science, (Gilbert, L. I., Iatrou, K., and Gill, S. S., Eds.) Elsevier Pergamon, New York, pp. 1-60.


        Progress 01/01/04 to 12/31/04

        Outputs
        At the end of each instar, Aedes aegypti sheds its cuticle through a precisely timed sequence of behaviors. Hormonal regulation of these behaviors has been described in the moths Manduca sexta and Bombyx mori and the fly, Drosophila melanogaster. Ecdysis-triggering hormones (ETHs) released by endocrine Inka cells initiate the behavioral sequence by activating motor programs in the central nervous system (CNS). We have described Inka cells at branch points of major tracheal trunks of A. aegypti. ETHs purified from Inka cell extracts show conserved C-terminal amino acid sequence motifs with ETHs of moths and flies. Injection of synthetic mosquito ETH induced ecdysis behavior in pharate larvae, pupae and adults. We have cloned and functionally expressed two subtypes of ETH receptors (ETHR) in A. aegypti. ETH receptors show show high affinity and selectivity for ETH when expressed in Chinese hamster ovary cells using luminescence as a reporter for calcium elevation. Developmental expression profiles of ETHRs were demonstrated by qPCR, and shown to coincide with elevated steroid levels. Increasing levels of ETHR in the CNS correspond to appearance of behavioral sensitivity in animals injected with ETHs. Our description of natural and ETHs-induced ecdysis sequence in A.aegypti should facilitate future approaches to the control of mosquitos as human disease vectors.

        Impacts
        Identification of ecdysis-triggering hormones in mosquitos.

        Publications

        • Kim, Y. J., Spalovska-Valachova, I., Cho, K.-H., Zitnanova, I., Park, Y., Adams, M. E. and Zitnan, D. (2004) Corazonin receptor signaling in ecdysis initiation. Proc. Natl. Acad. Sci. 101: 6704-6709.


        Progress 01/01/03 to 12/31/03

        Outputs
        Ecdysis triggering hormones (ETHs) initiate the ecdysis behavioral sequence in insects at the end of each instar. The absence of ETHs leads to lethal developmental deficiencies at the first larval ecdysis. We are developing approaches for disruption of ETH signaling in pest insects (Heliothis virescens) and disease vectors (Aedes aegypti). Efforts to obtain the sequence of Aedes ETH peptides by PCR are ongoing. The availability of Anopheles ETH1 and ETH2 sequences from the public genome database led us to prepare them by chemical synthesis. Injection of Anopheles ETH1 and ETH2 into Aedes larvae induces pre-ecdysis and ecdysis behavior. Manduca and Drosophila ETHs also induce ecdysis behaviors in Aedes, although the temporal parameters of the behaviors differ from those induced by the mosquito peptides. RT-PCR using primer sequences designed from Drosophila and Manduca ETH receptors resulted in identification of the first ETH receptor (subtype B) in Aedes. Efforts are underway to express the receptor in a heterologous system for pharmacological characterization. A new peptide signal causing release of ETH from Manduca Inka cells was identified as corazonin. We are testing whether this peptide also causes ETH release in mosquitos.

        Impacts
        Ecdysis triggering hormones initiate ecdysis in mosquitos.

        Publications

        • Kim, Y. J., Spalovska-Valachova, I., Cho, K.-H., Zitnanova, I., Park, Y., Adams, M. E. and Zitnan, D. 2004.Corazonin receptor signaling in ecdysis initiation. Proc. Natl. Acad. Sci. (In Press).
        • Adams, M E. 2003. Insect Ecdysis as a Vital Target for Pest Control