egg-microinjection-efficient-mating-for-genome-editing-firebrat

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat <em>Thermobia domestica</em>

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

Kyoto University

In the silkworm Bombyx mori, dietary flavonoids are metabolized and accumulate in cocoons, thereby causing green coloration. Classical genetic studies suggest that more than seven independent loci are associated with this trait; however, because of the complex inheritance pattern, none of these loci have been characterized molecularly, and a plausible and comprehensive model for their action has not been proposed. Here, we report the identification of the gene responsible for the Green b (Gb) locus involving the green cocoon trait. In +(Gb) animals, glucosylation at the 5-O position of dietary quercetin did not occur, and the total amount of flavonoids in tissues and cocoons was dramatically reduced. We performed positional cloning of Gb and found a 38-kb deletion in a UDP-glucosyltransferase (UGT) gene cluster associated with the +(Gb) allele. RT-PCR and biochemical studies suggested that deletion of Bm-UGT10286 (UGT) is responsible for Gb and Bm-UGT10286 is virtually the sole source of UGT activity toward the 5-O position of quercetin. Our data show that the regiospecific glucosylation of flavonoids by the quercetin 5-O-glucosyltransferase can greatly affect the overall bioavailability of flavonoids in animals. Furthermore, we provide evidence that flavonoids increase the UV-shielding activity of cocoons and thus could confer an increased survival advantage to insects contained in these cocoons. This study will lead to greater understanding of mechanisms for metabolism, uptake, and transport of dietary flavonoids, which have a variety of biological activities in animals and beneficial effects on human health.

The silkworm Green b locus encodes a quercetin 5-O-glucosyltransferase that produces green cocoons with UV-shielding properties.

It is widely accepted that the anti-metamorphic action of insect juvenile hormones (JHs) is required to inhibit larval-pupal metamorphosis. However, recent studies using RNAi or knockout techniques reveal that larval status may be maintained independently of JHs during the early larval stages. To investigate why larvae of very early instars do not have competence to metamorphose and how they acquire this competence through larval development, we revisited the classic experiments of Piepho (ca. 1930s) and performed implantation assays using the integument of very young larvae of the silkworm, Bombyx mori. Here, we demonstrate that when the integuments of neonate larvae or newly molted second instar larvae are implanted into last instar host larvae, they are able to directly produce pupal cuticle at the time of pupal metamorphosis of the host. To investigate whether the pupal commitment of implants from the neonate first instar larvae is repressed by JHs, the integuments of Met1 knockout larvae lacking a functional JH receptor were implanted into penultimate instar larvae. We found that the implants of Met1 knockout neonate larvae produced patched pupal cuticles after the host larval molt, whereas those of the wild-type strain produced only larval cuticle without any trace of pupal cuticle. Taken together, our results suggest that the epidermis of very early instar larvae can be pupally committed when provided with unidentified blood-borne factor(s) present in final-instar larvae, and that JHs can block the action of that factor(s) to prolong the feeding period until larvae attain a size appropriate for metamorphosis.

Implantation assays using the integument of early stage Bombyx larvae: Insights into the mechanisms underlying the acquisition of competence for metamorphosis.

The insect neuropeptide prothoracicotropic hormone (PTTH) triggers the biosynthesis and release of the molting hormone ecdysone in the prothoracic gland (PG), thereby controlling the timing of molting and metamorphosis. Despite the well-documented physiological role of PTTH and its signaling pathway in the PG, it is not clear whether PTTH is an essential hormone for ecdysone biosynthesis and development. To address this question, we established and characterized a PTTH knockout line in the silkworm, Bombyx mori. We found that PTTH knockouts showed a severe developmental delay in both the larval and pupal stages. Larval phenotypes of PTTH knockouts can be classified into three major classes: (i) developmental arrest during the second larval instar, (ii) precocious metamorphosis after the fourth larval instar (one instar earlier in comparison to the control strain), and (iii) metamorphosis to normal-sized pupae after completing the five larval instar stages. In PTTH knockout larvae, peak levels of ecdysone titers in the hemolymph were dramatically reduced and the timing of peaks was delayed, suggesting that protracted larval development is a result of the reduced and delayed synthesis of ecdysone in the PG. Despite these defects, low basal levels of ecdysone were maintained in PTTH knockout larvae, suggesting that the primary role of PTTH is to upregulate ecdysone biosynthesis in the PG during molting stages, and low basal levels of ecdysone can be maintained in the absence of PTTH. We also found that mRNA levels of genes involved in ecdysone biosynthesis and ecdysteroid signaling pathways were significantly reduced in PTTH knockouts. Our results provide genetic evidence that PTTH is not essential for development, but is required to coordinate growth and developmental timing.

Severe developmental timing defects in the prothoracicotropic hormone (PTTH)-deficient silkworm, Bombyx mori.

Parthenogenesis is an asexual mode of reproduction that plays an important role in the evolution of sex, sociality, and reproduction strategies in insects. Some species of cockroach exhibit thelytoky, a type of parthenogenesis in which female offspring are produced without fertilization. However, the cytological and genetic mechanisms of parthenogenesis in cockroaches are not well understood. Here we provide the first molecular genetic evidence that cockroaches can reproduce through automixis. Using the American cockroach Periplaneta americana, we performed microsatellite analysis to investigate the genetic relationship between parthenogenetically produced nymphs and the parent virgin females, and found that all parthenogenetic offspring were homozygous for autosomal microsatellite markers, whereas the female parents were heterozygous. In addition, flow cytometry analysis revealed that the parthenogenetic offspring were diploid. Taken together, our results demonstrate that P. americana exhibits automixis-type thelytoky, in which diploidy is restored by gamete duplication or terminal fusion. These findings highlight the unique reproduction strategies of cockroaches, which are more varied than was previously recognized.

First molecular genetic evidence for automictic parthenogenesis in cockroaches.

Male moths detect sex pheromones emitted by conspecific females with high sensitivity and specificity by the olfactory sensilla on their antennae. Pheromone binding proteins (PBPs) are highly enriched in the sensillum lymph of pheromone sensitive olfactory sensilla and are supposed to contribute to the sensitivity and selectivity of pheromone detection in moths. However, the functional role of PBPs in moth sex pheromone detection in vivo remains obscure. In the silkmoth, Bombyx mori, female moths emit bombykol as a single attractive sex pheromone component along with a small amount of bombykal that negatively modulates the behavioural responses to bombykol. A pair of olfactory receptor neurons, specifically tuned to bombykol or bombykal, co-localise in the trichodeum sensilla, the sensillum lymph of which contains a single PBP, namely, BmPBP1. We analysed the roles of BmPBP1 using BmPBP1-knockout silkmoth lines generated by transcription activator-like effector nuclease-mediated gene targeting. Electroantennogram analysis revealed that the peak response amplitudes of BmPBP1-knockout male antennae to bombykol and bombykal were significantly reduced by a similar percentage when compared with those of the wild-type males. Our results indicate that BmPBP1 plays a crucial role in enhancing the sensitivity, but not the selectivity, of sex pheromone detection in silkmoths.

In vivo functional characterisation of pheromone binding protein-1 in the silkmoth, Bombyx mori.

The smaller tea tortrix, Adoxophyes honmai, has developed strong resistance to tebufenozide, a diacylhydrazine-type (DAH) insecticide. Here, we investigated its mechanism by identifying genes responsible for the tebufenozide resistance using various next generation sequencing techniques. First, double-digest restriction site-associated DNA sequencing (ddRAD-seq) identified two candidate loci. Then, synteny analyses using A. honmai draft genome sequences revealed that one locus contained the ecdysone receptor gene (EcR) and the other multiple CYP9A subfamily P450 genes. RNA-seq and direct sequencing of EcR cDNAs found a single nucleotide polymorphism (SNP), which was tightly linked to tebufenozide resistance and generated an amino acid substitution in the ligand-binding domain. The binding affinity to tebufenozide was about 4 times lower in in vitro translated EcR of the resistant strain than in the susceptible strain. RNA-seq analyses identified commonly up-regulated genes in resistant strains, including CYP9A and choline/carboxylesterase (CCE) genes. RT-qPCR analysis and bioassays showed that the expression levels of several CYP9A and CCE genes were moderately correlated with tebufenozide resistance. Collectively, these results suggest that the reduced binding affinity of EcR is the main factor and the enhanced detoxification activity by some CYP9As and CCEs plays a supplementary role in tebufenozide resistance in A. honmai.

Genome-wide Identification of Tebufenozide Resistant Genes in the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae).

In Lepidoptera, the roles of period ( per) and the negative feedback involving this gene in circadian rhythm are controversial. In the present study, we established a per knockout strain using TALEN in Bombyx mori, and compared eclosion and hatching rhythms between the per-knockout and wild-type strains to examine whether per is actually involved in these rhythms. The generated per knockout allele was considered null, because it encoded an extensively truncated form of PERIOD (198 aa due to a 64-bp deletion in exon 7, in contrast to 1113 aa in the wild-type protein). In this per knockout strain, circadian rhythms in eclosion and hatching were disrupted. Under LD cycles, however, a steep peak existed at 1 h after lights-on in both eclosion and hatching, and was considered to be produced by a masking effect-a direct response to light. In the per-knockout strain, temporal expression changes of per and timeless ( tim) were also lost. The expression levels of tim were continuously high, probably due to the loss of negative feedback by per and tim. In contrast, the expression levels of per were much lower in the per knockout strain than in the wild type at every time point. From these results, we concluded that per is indispensable for circadian rhythms, and we suggest that the negative feedback loop of the circadian rhythm involving per functions for the production of behavioral rhythms in B. mori.

Involvement of the Clock Gene period in the Circadian Rhythm of the Silkmoth Bombyx mori.

Sex is determined by diverse mechanisms and master sex-determination genes are highly divergent, even among closely related species. Therefore, it is possible that homologs of master sex-determination genes might have alternative functions in different species. Herein, we focused on  (), which is the master sex-determination gene in  and is necessary for female germline development. It has been widely shown that the sex-determination function of  in Drosophilidae species is not conserved in other insects of different orders. We investigated the function of  in the lepidopteran insect  In lepidopteran insects (moths and butterflies), spermatogenesis results in two different types of sperm: nucleated fertile eupyrene sperm and anucleate nonfertile parasperm, also known as apyrene sperm. Genetic analyses using  mutants revealed that the gene is indispensable for proper morphogenesis of apyrene sperm. Similarly, our analyses using  mutants clearly demonstrate that apyrene sperm are necessary for eupyrene sperm migration from the bursa copulatrix to the spermatheca. Therefore, apyrene sperm is necessary for successful fertilization of eupyrene sperm in  Although  is essential for oogenesis in , it also plays important roles in spermatogenesis in  Therefore, the ancestral function of  might be related to germline development.

Dimorphic sperm formation by .

Ommochromes are the major pigments found in the eyes, eggs, wings and epidermis of insects. Here, we report the identification and characterization of the gene responsible for red-1 locus of Tribolium, whose mutants have white eyes due to lack of ommochrome pigments in the eyes. Using a candidate gene approach, we demonstrated that red-1 and peach mutants have molecular defects in the cardinal gene, which encodes a haem peroxidase that is considered to convert 3-hydroxykynurenine into ommochromes in pigment granules. Our experiments showed that the expression pattern of cardinal correlates well with the progression of eye pigmentation during pupal stages. We performed gene editing experiments using the Receptor-Mediated Ovary Transduction of Cargo (ReMOT) Control technique to disrupt the cardinal gene by adult injection, and were able to establish a novel cardinal mutant line. Our complementation test provided definitive genetic evidence that cardinal is located at the red-1 locus. The present study will lead to a greater understanding of the function and diversity of ommochrome pathway genes in insects. Our successful use of ReMOT Control in beetles will facilitate the development of more efficient and versatile systems for insect genome editing by simple adult injection.

Mutations in cardinal are responsible for the red-1 and peach eye color mutants of the red flour beetle Tribolium castaneum.

Juvenile hormone (JH) plays important roles in the control of many biological processes in insects, such as development, reproduction, and polyphenism. JH is primarily produced in the corpora allata (CA) by specific JH biosynthetic enzymes under strict temporal regulation. In a previous study, we identified a novel putative JH biosynthetic gene, protein tyrosine phosphatase, mitochondrial 1 (PTPMT1), from silkworm, Bombyx mori, whose expression is nearly exclusive in the CA and is correlated with JH synthetic activities during late larval development. In this study, to reveal the function of PTPMT1 in vivo, we generated PTPMT1 knockout silkworms using TALEN. In the knockout mutants, no signs indicating defects in JH activity were observed. Instead, PTPMT1 knockout silkworms showed embryonic lethality, developmental arrest, and 3rd-instar lethality not only in mutants lacking total enzymatic activity but also in mutants lacking mitochondrial translocation signals. Moreover, in PTPMT1 knockout embryos, the expression of two genes encoded by the mitochondrial genome, CYTB and ND3, was decreased, indicating a mitochondrial disorder. These results suggested that PTPMT1 plays conserved vital role(s) reported in vertebrates in insect mitochondria.

Article	Year
The silkworm Green b locus encodes a quercetin 5-O-glucosyltransferase that produces green cocoons with UV-shielding properties. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20534444	2010
Implantation assays using the integument of early stage Bombyx larvae: Insights into the mechanisms underlying the acquisition of competence for metamorphosis. Journal of insect physiology\| PubMed ID: 28522416	2017
Severe developmental timing defects in the prothoracicotropic hormone (PTTH)-deficient silkworm, Bombyx mori. Insect biochemistry and molecular biology\| PubMed ID: 28627423	2017
First molecular genetic evidence for automictic parthenogenesis in cockroaches. Insect science\| PubMed ID: 29389065	2019
In vivo functional characterisation of pheromone binding protein-1 in the silkmoth, Bombyx mori. Scientific reports\| PubMed ID: 30202026	2018
Genome-wide Identification of Tebufenozide Resistant Genes in the smaller tea tortrix, Adoxophyes honmai (Lepidoptera: Tortricidae). Scientific reports\| PubMed ID: 30862839	2019
Involvement of the Clock Gene period in the Circadian Rhythm of the Silkmoth Bombyx mori. Journal of biological rhythms\| PubMed ID: 30947602	2019
Dimorphic sperm formation by . Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 31036645	2019
Mutations in cardinal are responsible for the red-1 and peach eye color mutants of the red flour beetle Tribolium castaneum. Biochemical and biophysical research communications\| PubMed ID: 32703438	2020
A mitochondrial phosphatase PTPMT1 is essential for the early development of silkworm, Bombyx mori. Biochemical and biophysical research communications\| PubMed ID: 32773109	2020

Takaaki Daimon

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