Young-Joon Kim

A Command Chemical Triggers an Innate Behavior by Sequential Activation of Multiple Peptidergic Ensembles

BACKGROUNDnAt the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system.nnnRESULTSnTo elucidate neural substrates underlying the ecdysis sequence, we identified neurons expressing ETH receptors (ETHRs) in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence.nnnCONCLUSIONSnOur findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles.

Stabilization of the Mass Absorption Cross Section of Black Carbon for Filter-Based Absorption Photometry by the use of a Heated Inlet

In principle, mass concentrations of black carbon (BC) (M BC) can be estimated by the measurement of the light absorption coefficient of BC. Filter-based methods, which quantify the absorption coefficient (b abs) from the change in transmission through a filter loaded with particles, have been widely used to measure M BC. However, reliable determination of M BC has been very difficult because of the large variability in the mass absorption cross section (C abs), which is the conversion factor from b abs to M BC. Coating of BC by volatile compounds and the co-existence of light-scattering particles contribute to the variability of C abs. In order to overcome this difficulty, volatile aerosol components were removed before collection of BC particles on filters by heating a section of the inlet to 400°C. We made simultaneous measurements of b abs by two types of photometers (Particle Soot Absorption Photometer (PSAP) and Continuous Soot Monitoring System (COSMOS)) together with M BC by an EC-OC analyzer to determine C abs at 6 locations in Asia. C abs was stable at 10.5 ± 0.7 m2 g −1 at a wavelength of 565 nm for BC strongly impacted by emissions from vehicles and biomass burning. The stable C abs value provides a firm basis for its use in estimating M BC by COSMOS and PSAP with an accuracy of about 10%. For the quantitative interpretation of the ratio of the C abs to the model-calculated C abs*, we measured C abs for mono-disperse nigrosin particles in the laboratory. The C abs/C abs* ratio was 1.4–1.9 at the 100–200 nm diameters, explaining the ratio of 1.8 for ambient BC.

Two subtypes of ecdysis-triggering hormone receptor in Drosophila melanogaster.

Insect ecdysis is a hormonally programmed physiological sequence that enables insects to escape their old cuticle at the end of each developmental stage. The immediate events leading to ecdysis, which are initiated upon release of ecdysis-triggering hormones (ETH) into the bloodstream, include respiratory inflation and sequential stereotypic behaviors that facilitate shedding of the cuticle. Here we report that the Drosophila geneCG5911 encodes two functionally distinct subtypes of G protein-coupled receptors through alternative splicing (CG5911a and CG5911b) that respond preferentially to ecdysis-triggering hormones of flies and moths. These subtypes show differences in ligand sensitivity and specificity, suggesting that they may play separate roles in ETH signaling. At significantly higher concentrations (>100-fold), certain insect and vertebrate peptides also activate these receptors, providing evidence that CG5911 is evolutionarily related to the thyrotropin-releasing hormone and neuromedin U receptors. The ETH signaling system in insects is a vital system that provides opportunities for the construction of models for the molecular basis of stereotypic animal behavior as well as a target for the design of more sophisticated insect-selective pest control strategies.

The Pheromone Biosynthesis Activating Neuropeptide (PBAN) Receptor of Heliothis virescens: Identification, Functional Expression, and Structure-Activity Relationships of Ligand Analogs

Pheromone biosynthesis activating neuropeptide (PBAN) promotes synthesis and release of sex pheromones in moths. We have identified and functionally expressed a PBAN receptor from Heliothis virescens (HevPBANR) and elucidated structure-activity relationships of PBAN analogs. Screening of a larval CNS cDNA library revealed three putative receptor subtypes and nucleotide sequence comparisons suggest that they are produced through alternative splicing at the 3-end. RT-PCR amplified preferentially HevPBANR-C from female pheromone glands. CHO cells expressing HevPBANR-C are highly sensitive to PBAN and related analogs, especially those sharing the C-terminal pentapeptide core, FXPRLamide (X=T, S or V). Alanine replacements in the C-terminal hexapeptide (YFTPRLamide) revealed the relative importance of each residue in the active core as follows: R5>L6>F2>>P4>T3>>Y1. This study provides a framework for the rational design of PBANR-specific agonists and/or antagonists that could be exploited for disruption of reproductive function in agriculturally important insect pests.

A Neuronal Pathway that Controls Sperm Ejection and Storage in Female Drosophila

In polyandrous females, sperm storage permits competition between sperm of different mates, and in some species females influence the relative fertilization success of competing sperm in favor of a preferred mate [1, 2]. In female Drosophila melanogaster, sperm competition is strongly influenced by the timing of sperm ejection from the uterus [3, 4]. Understanding how female behavior influences sperm competition requires knowledge of the neuronal mechanisms controlling sperm retention and storage, which is currently lacking. Here, we show that D.xa0melanogaster females eject male ejaculates from the uterus 1-6xa0hr after mating with a stereotypic behavior regulated by a brain signaling pathway composed of diuretic hormone 44 (Dh44), a neuropeptide related to vertebrate corticotropin-releasing factor (CRF), and its receptor, Dh44R1. Suppression of Dh44 signals in the brain expedites sperm ejection from the uterus, resulting in marked reduction of sperm in the storage organs and decreased fecundity, whereas enhancement of Dh44 signals delays sperm expulsion. The Dh44 function was mapped to six neurons located in the pars intercerebralis of the brain together with a small subset of Dh44R1 neurons that express the sex-specific transcription factor doublesex. This study identifies a neuronal pathway by which females can control sperm retention and storage and provides new insight into how the female might exercise post-copulatory sexual selection.

translin Is Required for Metabolic Regulation of Sleep

Dysregulation of sleep or feeding has enormous health consequences. In humans, acute sleep loss is associated with increased appetite and insulin insensitivity, while chronically sleep-deprived individuals are more likely to develop obesity, metabolic syndrome, type II diabetes, and cardiovascular disease. Conversely, metabolic state potently modulates sleep and circadian behavior; yet, the molecular basis for sleep-metabolism interactions remains poorly understood. Here, we describe the identification of translin (trsn), a highly conserved RNA/DNA binding protein, as essential for starvation-induced sleep suppression. Strikingly, trsn does not appear to regulate energy stores, free glucose levels, or feeding behavior suggesting the sleep phenotype of trsn mutant flies is not a consequence of general metabolic dysfunction or blunted response to starvation. While broadly expressed in all neurons, trsn is transcriptionally upregulated in the heads of flies in response to starvation. Spatially restricted rescue or targeted knockdown localizes trsn function to neurons that produce the tachykinin family neuropeptide Leucokinin. Manipulation of neural activity in Leucokinin neurons revealed these neurons to be required for starvation-induced sleep suppression. Taken together, these findings establish trsn as an essential integrator of sleep and metabolic state, with implications for understanding the neural mechanism underlying sleep disruption in response to environmental perturbation.

Characterisation and tissue distribution of the PISCF allatostatin receptor in the red flour beetle, Tribolium castaneum

The insect PISCF/allatostatins (ASTs) are pleiotropic peptides that are involved in the regulation of juvenile hormone biosynthesis, are myoinhibitory on the gut and the heart, and suppress feeding in various insects, but their roles in beetles are poorly understood. To provide further insight into the significance of PISCF/ASTs in beetles, the PISCF/AST receptor from Tribolium castaneum has been characterised and its tissue distribution determined. The biological activity of the T. castaneum PISCF/AST (Trica-AS) was also investigated. The Trica-AS receptor shows high sequence homology to other insect PISCF/AST receptors, which are related to the mammalian somatostatin/opioid receptors, a family of G protein-coupled receptors. The Trica-AS receptor was activated in a dose-dependent manner by both Trica-AS and T. castaneum allatostatin double C (Trica-ASTCC) as well as Manduca sexta-allatostatin (Manse-AS). Other allatoregulatory peptides (a FLG/AST, a MIP/AST and an allatotropin) and somatostatin(14) were inactive on this receptor. Receptor transcript levels in tissues, determined by qRT-PCR, were highest in the head and the gut, with variable amounts in the fat body and reproductive organs. There were measurable differences in receptor levels of the head, fat body and reproductive organs between males and females. There was also a widespread distribution of Trica-AS in various tissues of T. castaneum. The Trica-AS peptide precursor was most abundant in the head and there was a significant difference between levels in the heads and reproductive organs of males and females. Whole mount immunocytochemistry localised Trica-AS in the median and lateral neurosecretory cells of the brain, in the corpus cardiacum and throughout the ventral nerve cord. The peptide was also present in midgut neurosecretory cells, but no immunostaining was detected in the reproductive organs or Malpighian tubules. The widespread distribution of both Trica-AS and its receptor suggest this peptide may have multiple roles in beetles. However, Trica-AS had no effect on the spontaneous contractions of the gut or ovaries of T. castaneum but this peptide did stimulate the release of proteases from the anterior midgut of another beetle, Tenebrio molitor. The activation of the Trica-AS receptor by Trica-ASTCC implies a physiological role for this peptide in beetles, which remains to be identified.

Fine-scale mapping of natural variation in fly fecundity identifies neuronal domain of expression and function of an aquaporin.

To gain insight into the molecular genetic basis of standing variation in fitness related traits, we identify a novel factor that regulates the molecular and physiological basis of natural variation in female Drosophila melanogaster fecundity. Genetic variation in female fecundity in flies derived from a wild orchard population is heritable and largely independent of other measured life history traits. We map a portion of this variation to a single QTL and then use deficiency mapping to further refine this QTL to 5 candidate genes. Ubiquitous expression of RNAi against only one of these genes, an aquaporin encoded by Drip, reduces fecundity. Within our mapping population Drip mRNA level in the head, but not other tissues, is positively correlated with fecundity. We localize Drip expression to a small population of corazonin producing neurons located in the dorsolateral posterior compartments of the protocerebrum. Expression of Drip–RNAi using both the pan-neuronal ELAV-Gal4 and the Crz-Gal4 drivers reduces fecundity. Low-fecundity RILs have decreased Crz expression and increased expression of pale, the enzyme encoding the rate-limiting step in the production of dopamine, a modulator of insect life histories. Taken together these data suggest that natural variation in Drip expression in the corazonin producing neurons contributes to standing variation in fitness by altering the concentration of two neurohormones.

Rescheduling Behavioral Subunits of a Fixed Action Pattern by Genetic Manipulation of Peptidergic Signaling.

The ecdysis behavioral sequence in insects is a classic fixed action pattern (FAP) initiated by hormonal signaling. Ecdysis triggering hormones (ETHs) release the FAP through direct actions on the CNS. Here we present evidence implicating two groups of central ETH receptor (ETHR) neurons in scheduling the first two steps of the FAP: kinin (aka drosokinin, leucokinin) neurons regulate pre-ecdysis behavior and CAMB neurons (CCAP, AstCC, MIP, and Bursicon) initiate the switch to ecdysis behavior. Ablation of kinin neurons or altering levels of ETH receptor (ETHR) expression in these neurons modifies timing and intensity of pre-ecdysis behavior. Cell ablation or ETHR knockdown in CAMB neurons delays the switch to ecdysis, whereas overexpression of ETHR or expression of pertussis toxin in these neurons accelerates timing of the switch. Calcium dynamics in kinin neurons are temporally aligned with pre-ecdysis behavior, whereas activity of CAMB neurons coincides with the switch from pre-ecdysis to ecdysis behavior. Activation of CCAP or CAMB neurons through temperature-sensitive TRPM8 gating is sufficient to trigger ecdysis behavior. Our findings demonstrate that kinin and CAMB neurons are direct targets of ETH and play critical roles in scheduling successive behavioral steps in the ecdysis FAP. Moreover, temporal organization of the FAP is likely a function of ETH receptor density in target neurons.

The host-seeking inhibitory peptide, Aea-HP-1, is made in the male accessory gland and transferred to the female during copulation.

Highlights ► A biologically active peptide (Aea-HP-1) has been chemically identified from Aedes aegypti male accessory glands. ► Aea-HP-1 is transferred to the female on copulating. ► Aea-HP-1 is protected from peptide-degrading enzymes of the accessory gland. ► Aea-HP-1 is not a mosquito ‘sex peptide’, but probably alters host-seeking behavior of the post-mated female.