Targeted disruption of a single sex pheromone receptor gene completely abolishes in vivo pheromone response in the silkmoth

Male moths use species-specific sex pheromones to identify and orientate toward conspecific females. Odorant receptors (ORs) for sex pheromone substances have been identified as sex pheromone receptors in various moth species. However, direct in vivo evidence linking the functional role of these ORs with behavioural responses is lacking. In the silkmoth, Bombyx mori, female moths emit two sex pheromone components, bombykol and bombykal, but only bombykol elicits sexual behaviour in male moths. A sex pheromone receptor BmOR1 is specifically tuned to bombykol and is expressed in specialized olfactory receptor neurons (ORNs) in the pheromone sensitive long sensilla trichodea of male silkmoth antennae. Here, we show that disruption of the BmOR1 gene, mediated by transcription activator-like effector nucleases (TALENs), completely removes ORN sensitivity to bombykol and corresponding pheromone-source searching behaviour in male moths. Furthermore, transgenic rescue of BmOR1 restored normal behavioural responses to bombykol. Our results demonstrate that BmOR1 is required for the physiological and behavioural response to bombykol, demonstrating that it is the receptor that mediates sex pheromone responses in male silkmoths. This study provides the first direct evidence that a member of the sex pheromone receptor family in moth species mediates conspecific sex pheromone information for sexual behaviour

Establishment of tools for neurogenetic analysis of sexual behavior in the silkmoth, Bombyx mori

Background: Silkmoth, Bombyx mori, is an ideal model insect for investigating the neural mechanisms underlying sex pheromone-induced innate behavior. Although transgenic techniques and the GAL4/UAS system are well established in the silkmoth, genetic tools useful for investigating brain function at the neural circuit level have been lacking. Results: In the present study, we established silkmoth strains in which we could visualize neural projections (UASmCD8GFP) and cell nucleus positions (UAS-GFP.nls), and manipulate neural excitability by thermal stimulation (UASdTrpA1). In these strains, neural projections and nucleus position were reliably labeled with green fluorescent protein in a GAL4-dependent manner. Further, the behavior of silkworm larvae and adults could be controlled by GAL4-dependent misexpression of dTrpA1. Ubiquitous dTrpA1 misexpression led both silkmoth larvae and adults to exhibit seizure-like phenotypes in a heat stimulation-dependent manner. Furthermore, dTrpA1 misexpression in the sex pheromone receptor neurons of male silkmoths allowed us to control male sexual behavior by changing the temperature. Thermally stimulated male silkmoths exhibited full sexual behavior, including wing-flapping, orientation, and attempted copulation, and precisely approached a thermal source in a manner similar to male silkmoths stimulated with the sex pheromone. Conclusion: These findings indicate that a thermogenetic approach using dTrpA1 is feasible in Lepidopteran insects and thermogenetic analysis of innate behavior is applicable in the silkmoth. These tools are essential for elucidating the relationships between neural circuits and function using neurogenetic methods

Odorant Concentration Differentiator for Intermittent Olfactory Signals

Animals need to discriminate differences in spatiotemporally distributed sensory signals in terms of quality as well as quantity for generating adaptive behavior. Olfactory signals characterized by odor identity and concentration are intermittently distributed in the environment. From these intervals of stimulation, animals process odorant concentration to localize partners or food sources. Although concentration–response characteristics in olfactory neurons have traditionally been investigated using single stimulus pulses, their behavior under intermittent stimulus regimens remains largely elusive. Using the silkmoth (Bombyx mori) pheromone processing system, a simple and behaviorally well-defined model for olfaction, we investigated the neuronal representation of odorant concentration upon intermittent stimulation in the naturally occurring range. To the first stimulus in a series, the responses of antennal lobe (AL) projection neurons (PNs) showed a concentration dependence as previously shown in many olfactory systems. However, PN response amplitudes dynamically changed upon exposure to intermittent stimuli of the same odorant concentration and settled to a constant, largely concentration-independent level. As a result, PN responses emphasized odorant concentration changes rather than encoding absolute concentration in pulse trains of stimuli. Olfactory receptor neurons did not contribute to this response transformation which was due to long-lasting inhibition affecting PNs in the AL. Simulations confirmed that inhibition also provides advantages when stimuli have naturalistic properties. The primary olfactory center thus functions as an odorant concentration differentiator to efficiently detect concentration changes, thereby improving odorant source orientation over a wide concentration range.UTokyo Research掲載「匂いの濃度を効率的に表現する脳の計算メカニズムの発見」 URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/a-novel-neuronal-mechanism-to-efficiently-code-odorant-concentration/UTokyo Research "A novel neuronal mechanism to efficiently code odorant concentration" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/a-novel-neuronal-mechanism-to-efficiently-code-odorant-concentration

A single amino acid mutation in an ABC transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, \u3cem\u3eBombyx mori\u3c/em\u3e

Bt toxins derived from the arthropod bacterial pathogen Bacillus thuringiensis are widely used for insect control as insecticides or in transgenic crops. Bt resistance has been found in field populations of several lepidopteran pests and in laboratory strains selected with Bt toxin. Widespread planting of crops expressing Bt toxins has raised concerns about the potential increase of resistance mutations in targeted insects. By using Bombyx mori as a model, we identified a candidate gene for a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. BGIBMGA007792-93, which encodes an ATP-binding cassette transporter similar to human multidrug resistance protein 4 and orthologous to genes associated with recessive resistance to Cry1Ac in Heliothis virescens and two other lepidopteran species, was expressed in the midgut. Sequences of 10 susceptible and seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects with the use of transgenesis

Refinement of ectopic protein expression through the GAL4/UAS system in Bombyx mori: Application to behavioral and developmental studies

金沢大学理工研究域自然システム学系Silkmoth, Bombyx mori, is one of the important model insects in which transgenic techniques and the GAL4/UAS system are applicable. However, due to cytotoxicity and low transactivation activity of GAL4, effectiveness of the GAL4/UAS system and its application in B. mori are still limited. In the present study, we refined the previously reported UAS vector by exploiting transcriptional and translational enhancers, and achieved 200-fold enhancement of reporter GFP fluorescence in the GAL4/UAS system. Enhanced protein expression of membrane-targeted GFP and calcium indicator protein (GCaMP5G) drastically improved visualization of fine neurite structures and neural activity, respectively. Also, with the refined system, we generated a transgenic strain that expresses tetanus toxin light chain (TeTxLC), which blocks synaptic transmission, under the control of GAL4. Ectopic TeTxLC expression in the sex pheromone receptor neurons inhibited male courtship behavior, proving effectiveness of TeTxLC on loss-of-function analyses of neural circuits. In addition, suppression of prothoracicotropic hormone (PTTH) or insulin-like peptide (bombyxin) secretion impaired developmental timing and growth rate, respectively. Furthermore, we revealed that larval growth is sex-differentially regulated by these peptide hormones. The present study provides important technical underpinnings of transgenic approaches in silkmoths and insights into mechanisms of postembryonic development in insects. © 2017 The Author(s)

A Single Sex Pheromone Receptor Determines Chemical Response Specificity of Sexual Behavior in the Silkmoth Bombyx mori

In insects and other animals, intraspecific communication between individuals of the opposite sex is mediated in part by chemical signals called sex pheromones. In most moth species, male moths rely heavily on species-specific sex pheromones emitted by female moths to identify and orient towards an appropriate mating partner among a large number of sympatric insect species. The silkmoth, Bombyx mori, utilizes the simplest possible pheromone system, in which a single pheromone component, (E, Z)-10,12-hexadecadienol (bombykol), is sufficient to elicit full sexual behavior. We have previously shown that the sex pheromone receptor BmOR1 mediates specific detection of bombykol in the antennae of male silkmoths. However, it is unclear whether the sex pheromone receptor is the minimally sufficient determination factor that triggers initiation of orientation behavior towards a potential mate. Using transgenic silkmoths expressing the sex pheromone receptor PxOR1 of the diamondback moth Plutella xylostella in BmOR1-expressing neurons, we show that the selectivity of the sex pheromone receptor determines the chemical response specificity of sexual behavior in the silkmoth. Bombykol receptor neurons expressing PxOR1 responded to its specific ligand, (Z)-11-hexadecenal (Z11-16:Ald), in a dose-dependent manner. Male moths expressing PxOR1 exhibited typical pheromone orientation behavior and copulation attempts in response to Z11-16:Ald and to females of P. xylostella. Transformation of the bombykol receptor neurons had no effect on their projections in the antennal lobe. These results indicate that activation of bombykol receptor neurons alone is sufficient to trigger full sexual behavior. Thus, a single gene defines behavioral selectivity in sex pheromone communication in the silkmoth. Our findings show that a single molecular determinant can not only function as a modulator of behavior but also as an all-or-nothing initiator of a complex species-specific behavioral sequence

Precocious Metamorphosis in the Juvenile Hormone–Deficient Mutant of the Silkworm, Bombyx mori

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several “moltinism” mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval–larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval–pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH–deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis

LIM-homeodomain transcription factor Awh is a key component activating all three fibroin genes, fibH, fibL and fhx, in the silk gland of the silkworm, Bombyx mori

In the silkworm Bombyx mori, three fibroin genes, fibroin-heavy-chain (fibH), fibroin-light-chain (fibL) and fibrohexamerin (fhx), are coexpressed only in the posterior silk gland (PSG) cells, while the sericin genes encoding silk glue proteins are expressed in the middle silk gland (MSG) cells. Silk gland factor-2 (SGF-2) is a PSG-specific activator complex of fibH, composed of a LIM-homeodomain protein, Awh, and its cofactors, Ldb and Lcaf. We investigated whether SGF-2 can activate other fibroin genes using transgenic silkworms. The genes for Ldb and Lcaf were expressed ubiquitously in various tissues, while the gene for Awh was expressed strictly specific in PSG of the wild type silkworms. Misexpression of Awh in transgenic silkworms induced ectopic expression of fibL and fhx as well as fibH in MSG. Coincidently with the induction of fibL and fhx by Awh, binding of SGF-2 to the promoter of fibL and fhx was detected in vitro, and SGF-2 binds directly to the fhx core promoter. Ectopic expression of the fibroin genes was observed at high levels in the middle part of MSG. Moreover, fibL and fhx were induced in the anterior silk gland (ASG) of the transgenic silkworms, but fibH was not. These results indicate that Awh is a key activator of all three fibroin genes, and the activity is probably regulated in conjunction with additional factors

Hox transcription factor Antp regulates sericin-1 gene expression in the terminal differentiated silk gland of Bombyx mori

Hox genes are well-known master regulators in developmental morphogenesis along the anteroposterior axis of animals. However, the molecular mechanisms by which Hox proteins regulate their target genes and determine cell fates are not fully understood. The silk gland of Bombyx mori is a tubular tissue divided into several subparts along the anteroposterior axis, and the silk genes are expressed with specific patterns. The sericin-1 gene (ser1) is expressed in the middle silk gland (MSG) with sublocal specificity. Here we show that the Hox protein Antp is a component of the middle silk gland-specific complex, MIC (MSG-intermolt-specific complex), binds to the essential promoter element of ser1, and activates its expression. Ectopic expression of Antp in transgenic silkworms induced the expression of ser1 in the posterior silk gland (PSG), but not in the anterior part of MSG (MSG-A). Correspondingly, a MIC-like complex was formed by the addition of recombinant Antp in extracts from PSG with its cofactors Exd and Hth, but not in extracts from MSG-A. Splicing patterns of ser1 mRNA induced by the ectopic expression of Antp in PSG were almost the same as those in MSG at the fifth instar and altered depending on the induction timing of Antp. Other Hox genes were expressed with sublocal specificity in the silk gland. The Bombyx silk gland might provide a useful system for understanding how Hox proteins select and regulate their target genes. (C) 2013 Elsevier Inc. All rights reserved