Endocrine modulation of primary chemosensory neurons regulates Drosophila courtship behavior.

The decision to engage in courtship depends on external cues from potential mates and internal cues related to maturation, health, and experience. Hormones allow for coordinated conveyance of such information to peripheral tissues. Here, we show Ecdysis-Triggering Hormone (ETH) is critical for courtship inhibition after completion of copulation in Drosophila melanogaster. ETH deficiency relieves post-copulation courtship inhibition (PCCI) and increases male-male courtship. ETH appears to modulate perception and attractiveness of potential mates by direct action on primary chemosensory neurons. Knockdown of ETH receptor (ETHR) expression in GR32A-expressing neurons leads to reduced ligand sensitivity and elevated male-male courtship. We find OR67D also is critical for normal levels of PCCI after mating. ETHR knockdown in OR67D-expressing neurons or GR32A-expressing neurons relieves PCCI. Finally, ETHR silencing in the corpus allatum (CA), the sole source of juvenile hormone, also relieves PCCI; treatment with the juvenile hormone analog methoprene partially restores normal post-mating behavior. We find that ETH, a stress-sensitive reproductive hormone, appears to coordinate multiple sensory modalities to guide Drosophila male courtship behaviors, especially after mating.

Parasitoid wasp venom re-programs host behavior through downmodulation of brain central complex activity and motor output.

The parasitoid wasp Ampulex compressa hunts down its host, the American cockroach (Periplaneta americana), and envenomates its brain to make it a behaviorally compliant food supply for its offspring. The primary target of the wasp sting is a locomotory command center called the central complex (CX). In the present study, we employ, for the first time, chronic recordings of patterned cockroach CX activity in real time as the brain is infused with wasp venom. CX envenomation is followed by sequential changes in the pattern of neuronal firing that can be divided into three distinct temporal phases during the 2 h interval after venom injection: (1) reduction in neuronal activity for roughly 10 min immediately after venom injection; (2) rebound of activity lasting up to 25 min; (3) reduction of ongoing activity for up to 2 h. Long-term reduction of CX activity after venom injection is accompanied by decreased activity of both descending interneurons projecting to thoracic locomotory circuitry (DINs) and motor output. Thus, in this study, we provide a plausible chain of events starting in the CX that leads to decreased host locomotion following brain envenomation. We propose that these events account for the onset and maintenance of the prolonged hypokinetic state observed in stung cockroaches.

Parasitoid wasp venom manipulates host innate behavior via subtype-specific dopamine receptor activation.

The subjugation strategy employed by the jewel wasp is unique in that it manipulates the behavior of its host, the American cockroach, rather than inducing outright paralysis. Upon envenomation directly into the central complex (CX), a command center in the brain for motor behavior, the stung cockroach initially engages in intense grooming behavior, then falls into a lethargic sleep-like state referred to as hypokinesia. Behavioral changes evoked by the sting are due at least in part to the presence of the neurotransmitter dopamine in the venom. In insects, dopamine receptors are classified as two families, the D1-like and the D2-like receptors. However, specific roles played by dopamine receptor subtypes in venom-induced behavioral manipulation by the jewel wasp remain largely unknown. In the present study, we used a pharmacological approach to investigate roles of D1-like and D2-like receptors in behaviors exhibited by stung cockroaches, focusing on grooming. Specifically, we assessed behavioral outcomes of focal CX injections of dopamine receptor agonists and antagonists. Both specific and non-specific compounds were used. Our results strongly implicate D1-like dopamine receptors in venom-induced grooming. Regarding induction of hypokinesia, our findings demonstrate that dopamine signaling is necessary for induction of long-lasting hypokinesia caused by brain envenomation.

Parasitoid Jewel Wasp Mounts Multipronged Neurochemical Attack to Hijack a Host Brain.

The parasitoid emerald jewel wasp Ampulex compressa induces a compliant state of hypokinesia in its host, the American cockroach Periplaneta americana through direct envenomation of the central nervous system (CNS). To elucidate the biochemical strategy underlying venom-induced hypokinesia, we subjected the venom apparatus and milked venom to RNAseq and proteomics analyses to construct a comprehensive "venome," consisting of 264 proteins. Abundant in the venome are enzymes endogenous to the host brain, including M13 family metalloproteases, phospholipases, adenosine deaminase, hyaluronidase, and neuropeptide precursors. The amphipathic, alpha-helical ampulexins are among the most abundant venom components. Also prominent are members of the Toll/NF-κB signaling pathway, including proteases Persephone, Snake, Easter, and the Toll receptor ligand Spätzle. We find evidence that venom components are processed following envenomation. The acidic (pH∼4) venom contains unprocessed neuropeptide tachykinin and corazonin precursors and is conspicuously devoid of the corresponding processed, biologically active peptides. Neutralization of venom leads to appearance of mature tachykinin and corazonin, suggesting that the wasp employs precursors as a prolonged time-release strategy within the host brain post-envenomation. Injection of fully processed tachykinin into host cephalic ganglia elicits short-term hypokinesia. Ion channel modifiers and cytolytic toxins are absent in A. compressa venom, which appears to hijack control of the host brain by introducing a "storm" of its own neurochemicals. Our findings deepen understanding of the chemical warfare underlying host-parasitoid interactions and in particular neuromodulatory mechanisms that enable manipulation of host behavior to suit the nutritional needs of opportunistic parasitoid progeny.

Endocrine regulation of airway clearance in Drosophila.

Fluid clearance from the respiratory system during developmental transitions is critically important for achieving optimal gas exchange in animals. During insect development 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. Disruption of Kinin signaling leads to defective air-filling during all larval stages. Such defects are observed upon ablation or electrical silencing of Kinin neurons, as well as RNA silencing of the Kinin gene or the ETH receptor in Kinin neurons, indicating that ETH targets Kinin neurons to promote tracheal air-filling. A Kinin receptor mutant fly line (Lkrf02594 ) also exhibits tracheal air-filling defects in all larval stages. Targeted Kinin receptor silencing in tracheal epithelial cells using breathless or pickpocket (ppk) drivers compromises tracheal air-filling. On the other hand, promotion of Kinin signaling in vivo through peptide injection or Kinin neuron activation through Drosophila TrpA1 (dTrpA1) expression induces premature tracheal collapse and air-filling. Moreover, direct exposure of tracheal epithelial cells in vitro to Kinin leads to calcium mobilization in tracheal epithelial cells. Our findings strongly implicate the neuropeptide Kinin as an important regulator of airway clearance via intracellular calcium mobilization in tracheal epithelial cells of Drosophila.

Endocrine network essential for reproductive success in Drosophila melanogaster.

Ecdysis-triggering hormone (ETH) was originally discovered and characterized as a molt termination signal in insects through its regulation of the ecdysis sequence. Here we report that ETH persists in adult Drosophila melanogaster, where it functions as an obligatory allatotropin to promote juvenile hormone (JH) production and reproduction. ETH signaling deficits lead to sharply reduced JH levels and consequent reductions of ovary size, egg production, and yolk deposition in mature oocytes. Expression of ETH and ETH receptor genes is in turn dependent on ecdysone (20E). Furthermore, 20E receptor knockdown specifically in Inka cells reduces fecundity. Our findings indicate that the canonical developmental roles of 20E, ETH, and JH during juvenile stages are repurposed to function as an endocrine network essential for reproductive success.

Ecdysis triggering hormone receptors regulate male courtship behavior via antennal lobe interneurons in Drosophila.

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.

Stress-induced reproductive arrest in Drosophila occurs through ETH deficiency-mediated suppression of oogenesis and ovulation.

BACKGROUND: Environmental stressors induce changes in endocrine state, leading to energy re-allocation from reproduction to survival. Female Drosophila melanogaster respond to thermal and nutrient stressors by arresting egg production through elevation of the steroid hormone ecdysone. However, the mechanisms through which this reproductive arrest occurs are not well understood. RESULTS: Here we report that stress-induced elevation of ecdysone 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. CONCLUSIONS: Our findings indicate that, as a dual regulator of octopamine and juvenile hormone release, ETH provides a link between stress-induced elevation of ecdysone levels and consequent reduction in fecundity.

Ampulexins: A New Family of Peptides in Venom of the Emerald Jewel Wasp, Ampulex compressa.

The parasitoid wasp Ampulex compressa injects venom directly into the brain and subesophageal ganglion of the cockroach Periplaneta americana, inducing a 7 to 10 day lethargy termed hypokinesia. Hypokinesia presents as a significant reduction in both escape response and spontaneous walking. We examined aminergic and peptidergic components of milked venom with HPLC and MALDI-TOF mass spectrometry. HPLC coupled with electrochemical detection confirmed the presence of dopamine in milked venom, while mass spectrometry revealed that the venom gland and venom sac have distinct peptide profiles, with milked venom predominantly composed of venom sac peptides. We isolated and characterized novel α-helical, amphipathic venom sac peptides that constitute a new family of venom toxins termed ampulexins. Injection of the most abundant venom peptide, ampulexin 1, into the subesophageal ganglion of cockroaches resulted in a short-term increase in escape threshold. Neither milked venom nor venom peptides interfered with growth of Escherichia coli or Bacillus thuringiensis on agar plates, and exposure to ampulexins or milked venom did not induce cell death in Chinese hamster ovary cells (CHO-K1) or Hi5 cells ( Trichoplusia ni).

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.

Secretory competence in a gateway endocrine cell conferred by the nuclear receptor ßFTZ-F1 enables stage-specific ecdysone responses throughout development in Drosophila.

Hormone-induced changes in gene expression initiate periodic molts and metamorphosis during insect development. Successful execution of these developmental steps depends upon successive phases of rising and falling 20-hydroxyecdysone (20E) levels, leading to a cascade of nuclear receptor-driven transcriptional activity that enables stage- and tissue-specific responses to the steroid. Among the cellular processes associated with declining steroids is acquisition of secretory competence in endocrine Inka cells, the source of ecdysis triggering hormones (ETHs). We show here that Inka cell secretory competence is conferred by the orphan nuclear receptor ßFTZ-F1. Selective RNA silencing of ßftz-f1 in Inka cells prevents ETH release, causing developmental arrest at all stages. Affected larvae display buttoned-up, the ETH-null phenotype characterized by double mouthparts, absence of ecdysis behaviors, and failure to shed the old cuticle. During the mid-prepupal period, individuals fail to translocate the air bubble, execute head eversion and elongate incipient wings and legs. Those that escape to the adult stage are defective in wing expansion and cuticle sclerotization. Failure to release ETH in ßftz-f1 silenced animals is indicated by persistent ETH immunoreactivity in Inka cells. Arrested larvae are rescued by precisely-timed ETH injection or Inka cell-targeted ßFTZ-F1 expression. Moreover, premature ßftz-f1 expression in these cells also results in developmental arrest. The Inka cell therefore functions as a "gateway cell", whose secretion of ETH serves as a key downstream physiological output enabling stage-specific responses to 20E that are required to advance through critical developmental steps. This secretory function depends on transient and precisely timed ßFTZ-F1 expression late in the molt as steroids decline.

Low voltage-activated calcium channels gate transmitter release at the dorsal root ganglion sandwich synapse.

A subpopulation of dorsal root ganglion (DRG) neurons are intimately attached in pairs and separated solely by thin satellite glial cell membrane septa. Stimulation of one neuron leads to transglial activation of its pair by a bi-, purinergic/glutamatergic synaptic pathway, a transmission mechanism that we term sandwich synapse (SS) transmission. Release of ATP from the stimulated neuron can be attributed to a classical mechanism involving Ca(2+) entry via voltage-gated calcium channels (CaV) but via an unknown channel type. Specific blockers and toxins ruled out CaV1, 2.1 and 2.2. Transmission was, however, blocked by a moderate depolarization (-50 mV) or low-concentration Ni(2+) (0.1 mM). Transmission persisted using a voltage pulse to -40 mV from a holding potential of -80 mV, confirming the involvement of a low voltage-activated channel type and limiting the candidate channel type to either CaV3.2 or a subpopulation of inactivation- and Ni(2+)-sensitive CaV2.3 channels. Resistance of the neuron calcium current and SS transmission to SNX482 argue against the latter. Hence, we conclude that inter-somatic transmission at the DRG SS is gated by CaV3.2 type calcium channels. The use of CaV3 family channels to gate transmission has important implications for the biological function of the DRG SS as information transfer would be predicted to occur not only in response to action potentials but also to sub-threshold membrane voltage oscillations. Thus, the SS synapse may serve as a homeostatic signalling mechanism between select neurons in the DRG and could play a role in abnormal sensation such as neuropathic pain.

Receptor guanylyl cyclases in Inka cells targeted by eclosion hormone.

A signature of eclosion hormone (EH) action in insect ecdysis is elevation of cGMP in Inka cells, leading to massive release of ecdysis triggering hormone (ETH) and ecdysis initiation. Although this aspect of EH-induced signal transduction is well known, the receptor mediating this process has not been identified. Here, we describe a receptor guanylyl cyclase BdmGC-1 and its isoform BdmGC-1B in the Oriental fruit fly Bactrocera dorsalis that are activated by EH. 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. 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.

Suppression of host nocifensive behavior by parasitoid wasp venom.

The parasitoid wasp Ampulex compressa envenomates the brain of its host the American cockroach (Periplaneta americana), thereby making it a behaviorally compliant food supply for its offspring. The target of venom injection is a locomotory command center in the brain called the central complex. In this study, we investigate why stung cockroaches do not respond to injuries incurred during the manipulation process by the wasp. In particular, we examine how envenomation compromises nociceptive signaling pathways in the host. Noxious stimuli applied to the cuticle of stung cockroaches fail to evoke escape responses, even though nociceptive interneurons projecting to the brain respond normally. Hence, while nociceptive signals are carried forward to the brain, they fail to trigger robust nocifensive behavior. Electrophysiological recordings from the central complex of stung animals demonstrate decreases in peak firing rate, total firing, and duration of noxious-evoked activity. The single parameter best correlated with altered noxious-evoked behavioral responses of stung cockroaches is reduced duration of the evoked response in the central complex. Our findings demonstrate how the reproductive strategy of a parasitoid wasp is served by venom-mediated elimination of aversive, nocifensive behavior in its host.

Regulation of Drosophila Long-Term Courtship Memory by Ecdysis Triggering Hormone.

Endocrine state is an important determinant of learning and memory in animals. In Drosophila, rejection of male courtship overtures by mated females leads to an aversive response manifested as courtship memory. Here we report that ecdysis triggering hormone (ETH) is an obligatory enabler of long-term courtship memory (LTM). ETH deficiency suppresses LTM, whereas augmented ETH release reduces the minimum training period required for LTM induction. ETH receptor knockdown either in the mushroom body (MB) γ lobe or in octopaminergic dorsal-anterior-lateral (DAL) neurons impairs memory performance, indicating its direct action in these brain areas. Consistent with these findings, brain exposure to ETH mobilizes calcium in MB γ lobe neuropils and DAL neurons. ETH receptor (ETHR) knockdown in the corpus allatum (CA) to create juvenile hormone (JH) deficiency also suppresses LTM, as does knockdown of the JH receptor Met in the MB γ lobe, indicating a convergence of ETH and JH signaling in this region of the brain. Our findings identify endocrine-enabled neural circuit components in the brain that are critical for persistent behavioral changes resulting from aversive social experience.

A command chemical triggers an innate behavior by sequential activation of multiple peptidergic ensembles.

BACKGROUND: At 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. RESULTS: To 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. CONCLUSIONS: Our findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles.

Potent activity of a PK/PBAN analog with an (E)-alkene, trans-Pro mimic identifies the Pro orientation and core conformation during interaction with HevPBANR-C receptor.

The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a multifunctional role in an array of important physiological processes in insects, including regulation of sex pheromone biosynthesis in moths. A cyclic PK/PBAN analog (cyclo[NTSFTPRL]) retains significant activity on the pheromonotropic HevPBANR receptor from the tobacco budworm Heliothis virescens expressed in CHO-K1 cells. Previous studies indicate that this rigid, cyclic analog adopts a type I beta-turn with a transPro over residues TPRL within the core PK/PBAN region. An analog containing an (E)-alkene, trans-Pro mimetic motif was synthesized, and upon evaluation on the HevPBANR receptor found to have an EC(50) value that is not statistically different from a parent C-terminal PK/PBAN hexapeptide sequence. The results, in aggregate, provide strong evidence for the orientation of Pro and the core conformation of PK/PBAN neuropeptides during interaction with the expressed PBAN receptor. The work further identifies a novel scaffold with which to design mimetic PBAN analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting PK/PBAN-regulated pheromone signaling systems.

Ecdysis triggering hormone signaling in the yellow fever mosquito Aedes aegypti.

At the end of each developmental stage, the yellow fever mosquito Aedes aegypti performs the ecdysis behavioral sequence, a precisely timed series of behaviors that culminates in shedding of the old exoskeleton. Here we describe ecdysis triggering hormone-immunoreactive Inka cells located at branch points of major tracheal trunks and loss of staining 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 induced 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.

Molecular cross-talk in a unique parasitoid manipulation strategy.

Envenomation of cockroach cerebral ganglia by the parasitoid Jewel wasp, Ampulex compressa, induces specific, long-lasting behavioural changes. We hypothesized that this prolonged action results from venom-induced changes in brain neurochemistry. Here, we address this issue by first identifying molecular targets of the venom, i.e., proteins to which venom components bind and interact with to mediate altered behaviour. Our results show that venom components bind to synaptic proteins and likely interfere with both pre- and postsynaptic processes. Since behavioural changes induced by the sting are long-lasting and reversible, we hypothesized further that long-term effects of the venom must be mediated by up or down regulation of cerebral ganglia proteins. We therefore characterize changes in cerebral ganglia protein abundance of stung cockroaches at different time points after the sting by quantitative mass spectrometry. Our findings indicate that numerous proteins are differentially expressed in cerebral ganglia of stung cockroaches, many of which are involved in signal transduction, such as the Rho GTPase pathway, which is implicated in synaptic plasticity. Altogether, our data suggest that the Jewel wasp commandeers cockroach behaviour through molecular cross-talk between venom components and molecular targets in the cockroach central nervous system, leading to broad-based alteration of synaptic efficacy and behavioural changes that promote successful development of wasp progeny.

Identification, developmental expression, and functions of bursicon in the tobacco hawkmoth, Manduca sexta.

During posteclosion, insects undergo sequential processes of wing expansion and cuticle tanning. Bursicon, a highly conserved neurohormone implicated in regulation of these processes, was characterized recently as a heterodimeric cystine knot protein in Drosophila melanogaster. Here we report the predicted precursor sequences of bursicon subunits (Masburs and Maspburs) in the moth Manduca sexta. Distinct developmental patterns of mRNA transcript and subunit-specific protein labeling of burs and pburs as well as crustacean cardioactive peptide 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 expresses ecdysis-triggering hormone (ETH) receptors, suggesting that 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, whereas burs transcripts and burs-like immunoreactivity were absent in these cells. Recombinant bursicon induces both wing expansion and tanning, whereas synthetic eclosion hormone induces only wing expansion.