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

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)

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

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

Silk Gland Factor-2, Involved in Fibroin Gene Transcription, Consists of LIM Homeodomain, LIM-interacting, and Single-stranded DNA-binding Proteins

SGF-2 binds to promoter elements governing posterior silk gland-specific expression of the fibroin gene in Bombyx mori. Wepurified SGF-2 and showed that SGF-2 contains at least four gene products: the silkworm orthologues of LIM homeodomain protein Awh, LIM domain-binding protein (Ldb), a sequence-specific single-stranded DNA-binding protein (Lcaf), and the silk protein P25/fibrohexamerin (fhx). Using co-expression of these factors in Sf9 cells, Awh, Ldb, and Lcaf proteins were co-purified as a ternary complex that bound to the enhancer sequence in vitro. Lcaf interacts with Ldb as well as Awh through the conserved regions to mediate transcriptional activation in yeast. Misexpression of Awh in transgenic silkworms induces ectopic expression of the fibroin gene in the middle silk glands, where Ldb and Lcaf are expressed. Taken together, this study demonstrates that SGF-2 is a multisubunit activator complex containing Awh. Moreover, our results suggest that the Ldb.Lcaf protein complex serves as a scaffold to facilitate communication between transcriptional control elements

Establishment of Tools for Neurogenetic Analysis of Sexual Behavior in the Silkmoth, <i>Bombyx mori</i>

<div><p>Background</p><p>Silkmoth, <i>Bombyx mori</i>, 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.</p><p>Results</p><p>In the present study, we established silkmoth strains in which we could visualize neural projections (UAS-mCD8GFP) and cell nucleus positions (UAS-GFP.nls), and manipulate neural excitability by thermal stimulation (UAS-dTrpA1). 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.</p><p>Conclusion</p><p>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.</p></div

Control of silkmoth behavior by the thermogenetic approach.

<p>(A–G) The thermosensor protein, dTrpA1, was ubiquitously expressed by crossing <i>Actin A3-GAL4</i> and <i>UAS-dTrpA1</i> strains. (A–C) Thermal stimulation induced systemic contraction in second instar larvae. Phenotypes were analyzed at 23°C and 40°C. The convulsion-like phenotype was observed at 40°C (B) and was reversible by decreasing the temperature to 23°C (C) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113156#pone.0113156.s003" target="_blank">Video S1</a>). (D) Cumulative curve of responsive larvae to incubation temperature. Vertical axis indicates ratio of larvae showing contractile phenotype during the observation time (10 min for each temperature; N = 9). (E–G) A convulsion-like phenotype was induced in male adult moths at 40°C (F), and was reversible at 25°C (G) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113156#pone.0113156.s004" target="_blank">Video S2</a>). (H–J) Sexual behavior was reversibly controlled by thermal stimulation in the male moths with dTrpA1 misexpression in the bombykol receptor neurons (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113156#pone.0113156.s005" target="_blank">Video S3</a>). (K, L) Thermogenetic activation of the bombykol receptor neurons induced pheromone orientation behavior. When the thermal source (heater) was turned on, male transgenic moths began wing-flapping, orienting to the heat source, and attempting copulation, and approached the heat source (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113156#pone.0113156.s006" target="_blank">Video S4</a>).</p