A unique primary structure of RDL (resistant to dieldrin) confers resistance to GABA-gated chloride channel blockers in the two-spotted spider mite Tetranychus urticae Koch.

The primary structure of the second transmembrane (M2) segment of resistant to dieldrin (RDL), an ionotropic γ-aminobutyric acid receptor (GABAR) subunit, and the structure-function relationships in RDL are well conserved among insect species. An amino acid substitution at the 2' position in the M2 segment (Ala to Ser or Gly) confers resistance to non-competitive antagonists (NCAs) of GABARs. Here, a cDNA encoding RDL was cloned from the two-spotted spider mite Tetranychus urticae Koch. Unlike insect homologs, native TuRDL has His at the 2' position (H305) and Ile at 6' (I309) in the M2 segment and is insensitive to NCAs. Single and multiple mutations were introduced in the M2 segment of TuRDL, and the mutant proteins were expressed in Xenopus oocytes and examined for the restoration of sensitivity to NCAs. The sensitivity of a double mutant (H305A and I309T in the M2 segment) was greatly increased but was still considerably lower than that of insect RDLs. We therefore constructed chimeric RDLs consisting of TuRDL and Drosophila melanogaster RDL and examined their sensitivities to NCAs. The results show that the N-terminal region containing the Cys-loop as well as the M2 segment confers functional specificity; thus, our current understanding of the mechanism underlying NCA binding to GABARs requires reappraisal.

Everyone on Their Own! Individualization of Synaptic Boutons.

Animals associate different odors with good and bad memories. This memory formation requires high neuronal plasticity. In this issue of Neuron, Bilz et al. (2020) demonstrate that associative learning modulates individual synaptic boutons of the intrinsic neurons of the memory center of Drosophila.

Third-Order Neurons in the Lateral Horn Enhance Bilateral Contrast of Odor Inputs Through Contralateral Inhibition in Drosophila.

The survival and reproduction of Drosophila melanogaster depends heavily on its ability to determine the location of an odor source and either to move toward or away from it. Despite the very small spatial separation between the two antennae and the redundancy in sensory neuron projection to both sides of the brain, Drosophila can resolve the concentration gradient by comparing the signal strength between the two antennae. When an odor stimulates the antennae asymmetrically, ipsilateral projection neurons from the first olfactory center are more strongly excited compared to the contralateral ones. However, it remains elusive how higher-order neurons process such asymmetric or lateralized odor inputs. Here, we monitored and analyzed for the first time the activity patterns of a small cluster of third-order neurons (so-called ventrolateral protocerebrum neurons) to asymmetric olfactory stimulation using two-photon calcium imaging. Our data demonstrate that lateralized odors evoke distinct activation of these neurons in the left and right brain hemisphere as a result of contralateral inhibition. Moreover, using laser transection experiments we show that this contralateral inhibition is mediated by presynaptic neurons most likely located in the lateral horn. Finally, we propose that this inhibitory interaction between higher-order neurons facilitates odor lateralization and plays a crucial role in olfactory navigation behavior of Drosophila, a theory that needs to be experimentally addressed in future studies.

Odor mixtures of opposing valence unveil inter-glomerular crosstalk in the Drosophila antennal lobe.

Evaluating odor blends in sensory processing is a crucial step for signal recognition and execution of behavioral decisions. Using behavioral assays and 2-photon imaging, we have characterized the neural and behavioral correlates of mixture perception in the olfactory system of Drosophila. Mixtures of odors with opposing valences elicit strong inhibition in certain attractant-responsive input channels. This inhibition correlates with reduced behavioral attraction. We demonstrate that defined subsets of GABAergic interneurons provide the neuronal substrate of this computation at pre- and postsynaptic loci via GABAB- and GABAA receptors, respectively. Intriguingly, manipulation of single input channels by silencing and optogenetic activation unveils a glomerulus-specific crosstalk between the attractant- and repellent-responsive circuits. This inhibitory interaction biases the behavioral output. Such a form of selective lateral inhibition represents a crucial neuronal mechanism in the processing of conflicting sensory information.

Pheromones mediating copulation and attraction in Drosophila.

Intraspecific olfactory signals known as pheromones play important roles in insect mating systems. In the model Drosophila melanogaster, a key part of the pheromone-detecting system has remained enigmatic through many years of research in terms of both its behavioral significance and its activating ligands. Here we show that Or47b-and Or88a-expressing olfactory sensory neurons (OSNs) detect the fly-produced odorants methyl laurate (ML), methyl myristate, and methyl palmitate. Fruitless (fru(M))-positive Or47b-expressing OSNs detect ML exclusively, and Or47b- and Or47b-expressing OSNs are required for optimal male copulation behavior. In addition, activation of Or47b-expressing OSNs in the male is sufficient to provide a competitive mating advantage. We further find that the vigorous male courtship displayed toward oenocyte-less flies is attributed to an oenocyte-independent sustained production of the Or47b ligand, ML. In addition, we reveal that Or88a-expressing OSNs respond to all three compounds, and that these neurons are necessary and sufficient for attraction behavior in both males and females. Beyond the OSN level, information regarding the three fly odorants is transferred from the antennal lobe to higher brain centers in two dedicated neural lines. Finally, we find that both Or47b- and Or88a-based systems and their ligands are remarkably conserved over a number of drosophilid species. Taken together, our results close a significant gap in the understanding of the olfactory background to Drosophila mating and attraction behavior; while reproductive isolation barriers between species are created mainly by species-specific signals, the mating enhancing signal in several Drosophila species is conserved.

Structures and functions of insect arylalkylamine N-acetyltransferase (iaaNAT); a key enzyme for physiological and behavioral switch in arthropods.

The evolution of N-acetyltransfeases (NATs) seems complex. Vertebrate arylalkylamine N-acetyltransferase (aaNAT) has been extensively studied since it leads to the synthesis of melatonin, a multifunctional neurohormone prevalent in photoreceptor cells, and is known as a chemical token of the night. Melatonin also serves as a scavenger for reactive oxygen species. This is also true with invertebrates. NAT therefore has distinct functional implications in circadian function, as timezymes (aaNAT), and also xenobiotic reactions (arylamine NAT or simply NAT). NATs belong to a broader enzyme group, the GCN5-related N-acetyltransferase superfamily. Due to low sequence homology and a seemingly fast rate of structural differentiation, the nomenclature for NATs can be confusing. The advent of bioinformatics, however, has helped to classify this group of enzymes; vertebrates have two distinct subgroups, the timezyme type and the xenobiotic type, which has a wider substrate range including imidazolamine, pharmacological drugs, environmental toxicants and even histone. Insect aaNAT (iaaNAT) form their own clade in the phylogeny, distinct from vertebrate aaNATs. Arthropods are unique, since the phylum has exoskeleton in which quinones derived from N-acetylated monoamines function in coupling chitin and arthropodins. Monoamine oxidase (MAO) activity is limited in insects, but NAT-mediated degradation prevails. However, unexpectedly iaaNAT occurs not only among arthropods but also among basal deuterostomia, and is therefore more apomorphic. Our analyses illustrate that iaaNATs has unique physiological roles but at the same time it plays a role in a timezyme function, at least in photoperiodism. Photoperiodism has been considered as a function of circadian system but the detailed molecular mechanism is not well understood. We propose a molecular hypothesis for photoperiodism in Antheraea pernyi based on the transcription regulation of NAT interlocked by the circadian system. Therefore, the enzyme plays both unique and universal roles in insects. The unique role of iaaNATs in physiological regulation urges the targeting of this system for integrated pest management (IPM). We indeed showed a successful example of chemical compound screening with reconstituted enzyme and further attempts seem promising.

N-acetyltransferase (nat) is a critical conjunct of photoperiodism between the circadian system and endocrine axis in Antheraea pernyi.

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16:8 (LD) and LD12:12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4 °C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNA(NAT) caused dysfunction of photoperiodism. dsRNA(PER) upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNA(NAT) decreased melatonin while dsRNA(PER) increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNA(NAT), to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

Serotonin receptor B may lock the gate of PTTH release/synthesis in the Chinese silk moth, Antheraea pernyi; a diapause initiation/maintenance mechanism?

The release of prothoracicotropic hormone, PTTH, or its blockade is the major endocrine switch regulating the developmental channel either to metamorphosis or to pupal diapause in the Chinese silk moth, Antheraea pernyi. We have cloned cDNAs encoding two types of serotonin receptors (5HTRA and B). 5HTRA-, and 5HTRB-like immunohistochemical reactivities (-ir) were colocalized with PTTH-ir in two pairs of neurosecretory cells at the dorsolateral region of the protocerebrum (DL). Therefore, the causal involvement of these receptors was suspected in PTTH release/synthesis. The level of mRNA(5HTRB) responded to 10 cycles of long-day activation, falling to 40% of the original level before activation, while that of 5HTRA was not affected by long-day activation. Under LD 16:8 and 12:12, the injection of dsRNA(5HTRB) resulted in early diapause termination, whereas that of dsRNA(5HTRA) did not affect the rate of diapause termination. The injection of dsRNA(5HTRB) induced PTTH accumulation, indicating that 5HTRB binding suppresses PTTH synthesis also. This conclusion was supported pharmacologically; the injection of luzindole, a melatonin receptor antagonist, plus 5th inhibited photoperiodic activation under LD 16:8, while that of 5,7-DHT, induced emergence in a dose dependent fashion under LD 12:12. The results suggest that 5HTRB may lock the PTTH release/synthesis, maintaining diapause. This could also work as diapause induction mechanism.

A target-specific feeding toxicity of ß(1) integrin dsRNA against diamondback moth, Plutella xylostella.

Integrin is a cell-surface protein consisting of α and ß heterodimers. A predicted amino acid sequence of an integrin subunit of the diamondback moth, Plutella xylostella, was highly homologous to other lepidopteran ß1 subunits and possessed essential functional domains. The ß1 integrin of P. xylostella (ßPx1) was expressed in all developmental stages of P. xylostella. It was also expressed in all tested tissues including hemocyte, fat body, gut, and epidermis of last instar. When ßPx1 expression was suppressed by injection of dsRNA specific to ßPx1 (dsRNA(ßPx1)), the treated larvae exhibited significant suppression in immune response and also suffered significant larval mortality. When dsRNA(ßPx1) was orally fed to young larvae, it suppressed the expression of âPx1 and resulted in a significant mortality. By contrast, a dsRNA specific to ß1 subunit of Spodoptera exigua gave little adverse effects on ßPx1 expression and larval development when it was treated by injection or oral administration, though these two genes showed 71% sequence homology. These results suggest a target-specific RNA interference of dsRNA(ßPx1), which causes significant mortality to P. xylostella by feeding treatment.

RNA interference of ß1 integrin subunit impairs development and immune responses of the beet armyworm, Spodoptera exigua.

Integrin is a cell surface protein that is composed of α and ß heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2862 bp) of a ß1 subunit integrin (ßSe1) was cloned from the beet armyworm, Spodoptera exigua. Phylogenetic analysis showed that ßSe1 was clustered with other insect ß integrin subunits with the highest amino acid sequence identity (98.3%) to ß1 of Spodoptera litura. Structural analysis of the deduced amino acid sequence indicated that ßSe1 possessed all functional domains known in other insect ß1 integrins. RT-PCR analysis showed that ßSe1 was expressed in all developmental stages and all tested tissues of S. exigua. Its expression was further upregulated in hemocytes by injections of various microbes from quantitative RT-PCR analysis. Injection of double-stranded ßSe1 RNA (dsRNA(ßSe1)) into late instar S. exigua suppressed ßSe1 expression and resulted in significant reduction in pupal weight. The dsRNA(ßSe1) injection significantly impaired hemocyte-spreading and nodule formation of S. exigua in response to bacterial challenge. Furthermore, oral ingestion of dsRNA(ßSe1) induced reduction of ßSe1 expression in midgut and resulted in significant mortality of S. exigua during immature development. These results suggest that ßSe1 plays crucial roles in performing cellular immune responses as well as larval development in S. exigua.