Neither per, nor tim1, nor cry2 alone are essential components of the molecular circadian clockwork in the Madeira cockroach.

Circadian clocks control rhythms in physiology and behavior entrained to 24 h light-dark cycles. Despite of conserved general schemes, molecular circadian clockworks differ between insect species. With RNA interference (RNAi) we examined an ancient circadian clockwork in a basic insect, the hemimetabolous Madeira cockroach Rhyparobia maderae. With injections of double-stranded RNA (dsRNA) of cockroach period (Rm´per), timeless 1 (Rm´tim1), or cryptochrome 2 (Rm´cry2) we searched for essential components of the clock´s core negative feedback loop. Single injections of dsRNA of each clock gene into adult cockroaches successfully and permanently knocked down respective mRNA levels within ~two weeks deleting daytime-dependent mRNA rhythms for Rm´per and Rm´cry2. Rm´perRNAi or Rm´cry2RNAi affected total mRNA levels of both genes, while Rm´tim1 transcription was independent of both, also keeping rhythmic expression. Unexpectedly, circadian locomotor activity of most cockroaches remained rhythmic for each clock gene knockdown employed. It expressed weakened rhythms and unchanged periods for Rm´perRNAi and shorter periods for Rm´tim1RNAi and Rm´cry2RNAi.As a hypothesis of the cockroach´s molecular clockwork, a basic network of switched differential equations was developed to model the oscillatory behavior of clock cells expressing respective clock genes. Data were consistent with two synchronized main groups of coupled oscillator cells, a leading (morning) oscillator, or a lagging (evening) oscillator that couple via mutual inhibition. The morning oscillators express shorter, the evening oscillators longer endogenous periods based on core feedback loops with either PER, TIM1, or CRY2/PER complexes as dominant negative feedback of the clockwork. We hypothesize that dominant morning oscillator cells with shorter periods express PER, but not CRY2, or TIM1 as suppressor of clock gene expression, while two groups of evening oscillator cells with longer periods either comprise TIM1 or CRY2/PER suppressing complexes. Modelling suggests that there is an additional negative feedback next to Rm´PER in cockroach morning oscillator cells.

Analysis of Pigment-Dispersing Factor Neuropeptides and Their Receptor in a Velvet Worm.

Pigment-dispersing factor neuropeptides (PDFs) occur in a wide range of protostomes including ecdysozoans (= molting animals) and lophotrochozoans (mollusks, annelids, flatworms, and allies). Studies in insects revealed that PDFs play a role as coupling factors of circadian pacemaker cells, thereby controlling rest-activity rhythms. While the last common ancestor of protostomes most likely possessed only one pdf gene, two pdf homologs, pdf-I and pdf-II, might have been present in the last common ancestors of Ecdysozoa and Panarthropoda (Onychophora + Tardigrada + Arthropoda). One of these homologs, however, was subsequently lost in the tardigrade and arthropod lineages followed by independent duplications of pdf-I in tardigrades and decapod crustaceans. Due to the ancestral set of two pdf genes, the study of PDFs and their receptor (PDFR) in Onychophora might reveal the ancient organization and function of the PDF/PDFR system in panarthropods. Therefore, we deorphanized the PDF receptor and generated specific antibodies to localize the two PDF peptides and their receptor in the onychophoran Euperipatoides rowelli. We further conducted bioluminescence resonance energy transfer (BRET) experiments on cultured human cells (HEK293T) using an Epac-based sensor (Epac-L) to examine cAMP responses in transfected cells and to reveal potential differences in the interaction of PDF-I and PDF-II with PDFR from E. rowelli. These data show that PDF-II has a tenfold higher potency than PDF-I as an activating ligand. Double immunolabeling revealed that both peptides are co-expressed in E. rowelli but their respective levels of expression differ between specific cells: some neurons express the same amount of both peptides, while others exhibit higher levels of either PDF-I or PDF-II. The detection of the onychophoran PDF receptor in cells that additionally express the two PDF peptides suggests autoreception, whereas spatial separation of PDFR- and PDF-expressing cells supports hormonal release of PDF into the hemolymph. This suggests a dual role of PDF peptides-as hormones and as neurotransmitters/neuromodulators-in Onychophora.

Mutagenesis of odorant coreceptor Orco fully disrupts foraging but not oviposition behaviors in the hawkmoth Manduca sexta.

The hawkmoth Manduca sexta and one of its preferred hosts in the North American Southwest, Datura wrightii, share a model insect-plant relationship based on mutualistic and antagonistic life-history traits. D. wrightii is the innately preferred nectar source and oviposition host for M. sexta Hence, the hawkmoth is an important pollinator while the M. sexta larvae are specialized herbivores of the plant. Olfactory detection of plant volatiles plays a crucial role in the behavior of the hawkmoth. In vivo, the odorant receptor coreceptor (Orco) is an obligatory component for the function of odorant receptors (ORs), a major receptor family involved in insect olfaction. We used CRISPR-Cas9 targeted mutagenesis to knock out (KO) the MsexOrco gene to test the consequences of a loss of OR-mediated olfaction in an insect-plant relationship. Neurophysiological characterization revealed severely reduced antennal and antennal lobe responses to representative odorants emitted by D. wrightii In a wind-tunnel setting with a flowering plant, Orco KO hawkmoths showed disrupted flight orientation and an ablated proboscis extension response to the natural stimulus. The Orco KO gravid female displayed reduced attraction toward a nonflowering plant. However, more than half of hawkmoths were able to use characteristic odor-directed flight orientation and oviposit on the host plant. Overall, OR-mediated olfaction is essential for foraging and pollination behaviors, but plant-seeking and oviposition behaviors are sustained through additional OR-independent sensory cues.

No Evidence for Ionotropic Pheromone Transduction in the Hawkmoth Manduca sexta.

Insect odorant receptors (ORs) are 7-transmembrane receptors with inverse membrane topology. They associate with the conserved ion channel Orco. As chaperon, Orco maintains ORs in cilia and, as pacemaker channel, Orco controls spontaneous activity in olfactory receptor neurons. Odorant binding to ORs opens OR-Orco receptor ion channel complexes in heterologous expression systems. It is unknown, whether this also occurs in vivo. As an alternative to this ionotropic transduction, experimental evidence is accumulating for metabotropic odor transduction, implicating that insect ORs couple to G-proteins. Resulting second messengers gate various ion channels. They generate the sensillum potential that elicits phasic-tonic action potentials (APs) followed by late, long-lasting pheromone responses. Because it is still unclear how and when Orco opens after odor-OR-binding, we used tip recordings to examine in vivo the effects of the Orco antagonist OLC15 and the amilorides MIA and HMA on bombykal transduction in the hawkmoth Manduca sexta. In contrast to OLC15 both amilorides decreased the pheromone-dependent sensillum potential amplitude and the frequency of the phasic AP response. Instead, OLC15 decreased spontaneous activity, increased latencies of phasic-, and decreased frequencies of late, long-lasting pheromone responses Zeitgebertime-dependently. Our results suggest no involvement for Orco in the primary transduction events, in contrast to amiloride-sensitive channels. Instead of an odor-gated ionotropic receptor, Orco rather acts as a voltage- and apparently second messenger-gated pacemaker channel controlling the membrane potential and hence threshold and kinetics of the pheromone response.

In situ tip-recordings found no evidence for an Orco-based ionotropic mechanism of pheromone-transduction in Manduca sexta.

The mechanisms of insect odor transduction are still controversial. Insect odorant receptors (ORs) are 7TM receptors with inverted membrane topology. They colocalize with a conserved coreceptor (Orco) with chaperone and ion channel function. Some studies suggest that insects employ exclusively ionotropic odor transduction via OR-Orco heteromers. Other studies provide evidence for different metabotropic odor transduction cascades, which employ second messenger-gated ion channel families for odor transduction. The hawkmoth Manduca sexta is an established model organism for studies of insect olfaction, also due to the availability of the hawkmoth-specific pheromone blend with its main component bombykal. Previous patch-clamp studies on primary cell cultures of M. sexta olfactory receptor neurons provided evidence for a pheromone-dependent activation of a phospholipase Cß. Pheromone application elicited a sequence of one rapid, apparently IP3-dependent, transient and two slower Ca(2+)-dependent inward currents. It remains unknown whether additionally an ionotropic pheromone-transduction mechanism is employed. If indeed an OR-Orco ion channel complex underlies an ionotropic mechanism, then Orco agonist-dependent opening of the OR-Orco channel pore should add up to pheromone-dependent opening of the pore. Here, in tip-recordings from intact pheromone-sensitive sensilla, perfusion with the Orco agonist VUAA1 did not increase pheromone-responses within the first 1000 ms. However, VUAA1 increased spontaneous activity of olfactory receptor neurons Zeitgebertime- and dose-dependently. We conclude that we find no evidence for an Orco-dependent ionotropic pheromone transduction cascade in M. sexta. Instead, in M. sexta Orco appears to be a slower, second messenger-dependent pacemaker channel which affects kinetics and threshold of pheromone-detection via changes of intracellular Ca(2+) baseline concentrations.

Sequence and expression of per, tim1, and cry2 genes in the Madeira cockroach Rhyparobia maderae.

Most of what we know today about the molecular constituents of the insect circadian clock was discovered in the fruit fly Drosophila melanogaster. Various other holometabolous and some hemimetabolous insects have also been examined for the presence of circadian genes. In these insects, per, tim1, and cry2 are part of a core feedback loop system. The proteins inhibit their own expression, leading to circadian oscillations of mRNA and proteins. Although cockroaches are successfully employed circadian model organisms, their clock genes are mostly unknown. Thus, we cloned putative circadian genes in Rhyparobia maderae (synonym Leucophaea maderae), showing the presence of period (per), timeless 1 (tim1), and mammalian-type cryptochrome (cry2). The expression levels of per, tim1, and cry2 in R. maderae were examined in various tissues and photoperiods employing quantitative PCR. In brains and excised accessory medullae, expression levels of rmPer, rmTim1, and rmCry2 oscillated in a circadian manner with peaks in the first half of the night. Oscillations mostly continued in constant conditions. In Malpighian tubules, no significant oscillations were found. In animals raised in different photoperiods (LD 18:6, 12:12, 6:18), the peak levels of rmPer, rmTim1, and rmCry2 expression adjusted with respect to the beginning of the scotophase. The daily mean of expression levels was significantly lower in short-day versus long-day animals. We suggest that rmPer, rmTim1, and rmCry2 are part of the Madeira cockroach nuclear circadian clock, which can adjust to different photoperiods.

Seasonal regulation of cocaine- and amphetamine-regulated transcript in the arcuate nucleus of Djungarian hamster (Phodopus sungorus).

The hypothalamic neuropeptidergic system involved in the photoperiodic control of energy metabolism in seasonal mammals, is poorly understood. In the present study we examined whether distribution and number of the hypothalamic neuronal cell populations containing cocaine- and amphetamine-regulated transcript (CART) are influenced by different photoperiod and ambient temperature, or by food status in the Djungarian hamster (Phodopus sungorus). Hamsters bred and raised in long day photoperiod at room temperature (16 h light/8h dark at 23 degrees C; LD) were transferred to short day photoperiod and moderate cold (8h light/16 h dark at 16 degrees C; SD). After a 4 weeks acclimation period, uterus and body weight were decreased in SD as compared to controls maintained in LD. The number of CART-immunoreactive cells within the arcuate nucleus (ARC) was significantly higher in SD hamsters compared to LD control. This increase was restricted to the rostro to mid portion of the ARC, specifically in the hypothalamic retrochiasmatic area close to the rostral ARC and in the hypothalamic region lateral to the ARC and ventral to the ventromedial hypothalamic nuclei. In similar hypothalamic regions, food deprivation for 48 h significantly decreased the number of CART-immunoreactive cells in SD hamsters. Shortening of photoperiod combined with lowering of ambient temperature and food deprivation had no effect on the number of CART-immunoreactive cells in the lateral hypothalamic area. These findings suggest that photoperiod and ambient temperature influence energy metabolism potentially by alterations of the CART neuronal system in the rostral portion of the ARC in Djungarian hamsters.