The Mitochondrial Phosphatase PTPMT1 is Required for the Proper Growth Rate in the Red Flour Beetle, Tribolium castaneum.

Protein tyrosine phosphatase, mitochondrial 1 (PTPMT1) is a mitochondrial phosphatase that is highly conserved in animals. Functional analyses using knockout animals have revealed a variety of physiological roles of PTPMT1 in vertebrates and insects. However, because of the high lethality of knockout in these animals, the roles of PTPMT1 in the later postembryonic development remain relatively obscure. In the present study, using the RNA interference technique, we analyzed PTPMT1 functions in later larval stages of the red flour beetle, Tribolium castaneum. PTPMT1 was expressed in both anterior and posterior parts of the body constitutively without obvious fluctuations from the middle larval instar through pupation. The PTPMT1-knockdown larvae injected with PTPMT1 double-stranded RNA at the middle instar showed a prolonged larval period, which was mainly caused by an extra larval molt. On the other hand, the increase in adult body length was subtle in the PTPMT1-knockdown T. castaneum, and the head capsule width was smaller than that of the control animals at the same larval instar. The expression levels of genes encoded by the mitochondrial genome were reduced in PTPMT1-knockdown larvae, indicating that PTPMT1 plays an important role in mitochondrial function in T. castaneum, like in other species. By contrast, the expression levels of a juvenile hormone (JH)-biosynthetic gene and a JH-signaling gene were rather increased in the PTPMT1-knockdown larvae, which may have been caused indirectly by the reduction of larval growth rate. Altogether, these findings indicate that PTPMT1 is required for the proper growth rate via some mitochondrial physiological role in T. castaneum larvae.

A mitochondrial phosphatase PTPMT1 is essential for the early development of silkworm, Bombyx mori.

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.

Molecular mechanism underlying juvenile hormone-mediated repression of precocious larval-adult metamorphosis.

Juvenile hormone (JH) represses precocious metamorphosis of larval to pupal and adult transitions in holometabolous insects. The early JH-inducible gene Krüppel homolog 1 (Kr-h1) plays a key role in the repression of metamorphosis as a mediator of JH action. Previous studies demonstrated that Kr-h1 inhibits precocious larval-pupal transition in immature larva via direct transcriptional repression of the pupal specifier Broad-Complex (BR-C). JH was recently reported to repress the adult specifier gene Ecdysone-induced protein 93F (E93); however, its mechanism of action remains unclear. Here, we found that JH suppressed ecdysone-inducible E93 expression in the epidermis of the silkworm Bombyx mori and in a B. mori cell line. Reporter assays in the cell line revealed that the JH-dependent suppression was mediated by Kr-h1. Genome-wide ChIP-seq analysis identified a consensus Kr-h1 binding site (KBS, 14 bp) located in the E93 promoter region, and EMSA confirmed that Kr-h1 directly binds to the KBS. Moreover, we identified a C-terminal conserved domain in Kr-h1 essential for the transcriptional repression of E93 Based on these results, we propose a mechanism in which JH-inducible Kr-h1 directly binds to the KBS site upstream of the E93 locus to repress its transcription in a cell-autonomous manner, thereby preventing larva from bypassing the pupal stage and progressing to precocious adult development. These findings help to elucidate the molecular mechanisms regulating the metamorphic genetic network, including the functional significance of Kr-h1, BR-C, and E93 in holometabolous insect metamorphosis.

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor.

The Drosophila Zinc Finger Transcription Factor Ouija Board Controls Ecdysteroid Biosynthesis through Specific Regulation of spookier.

Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C2H2-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly Drosophila melanogaster. Expression of ouib is predominantly limited to the PG, and genetic null mutants of ouib result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, ouib mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, spookier. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of spok by binding to a specific ~15 bp response element in the spok PG enhancer element. Most remarkable, the developmental arrest phenotype of ouib mutants is rescued by over-expression of a functionally-equivalent paralog of spookier. These observations imply that the main biological function of Ouib is to specifically regulate spookier transcription during Drosophila development.

Krüppel Homolog 1 Inhibits Insect Metamorphosis via Direct Transcriptional Repression of Broad-Complex, a Pupal Specifier Gene.

The Broad-Complex gene (BR-C) encodes transcription factors that dictate larval-pupal metamorphosis in insects. The expression of BR-C is induced by molting hormone (20-hydroxyecdysone (20E)), and this induction is repressed by juvenile hormone (JH), which exists during the premature larval stage. Krüppel homolog 1 gene (Kr-h1) has been known as a JH-early inducible gene responsible for repression of metamorphosis; however, the functional relationship between Kr-h1 and repression of BR-C has remained unclear. To elucidate this relationship, we analyzed cis- and trans elements involved in the repression of BR-C using a Bombyx mori cell line. In the cells, as observed in larvae, JH induced the expression of Kr-h1 and concurrently suppressed 20E-induced expression of BR-C. Forced expression of Kr-h1 repressed the 20E-dependent activation of the BR-C promoter in the absence of JH, and Kr-h1 RNAi inhibited the JH-mediated repression, suggesting that Kr-h1 controlled the repression of BR-C. A survey of the upstream sequence of BR-C gene revealed a Kr-h1 binding site (KBS) in the BR-C promoter. When KBS was deleted from the promoter, the repression of BR-C was abolished. Electrophoresis mobility shift demonstrated that two Kr-h1 molecules bound to KBS in the BR-C promoter. Based on these results, we conclude that Kr-h1 protein molecules directly bind to the KBS sequence in the BR-C promoter and thereby repress 20E-dependent activation of the pupal specifier, BR-C. This study has revealed a considerable portion of the picture of JH signaling pathways from the reception of JH to the repression of metamorphosis.

A role for Taiman in insect metamorphosis.

Recent studies in vitro have reported that the Methoprene-tolerant (Met) and Taiman (Tai) complex is the functional receptor of juvenile hormone (JH). Experiments in vivo of Met depletion have confirmed this factor's role in JH signal transduction, however, there is no equivalent data regarding Tai because its depletion in larval or nymphal stages of the beetle Tribolium castaneum and the bug Pyrrhocoris apterus results in 100% mortality. We have discovered that the cockroach Blattella germanica possesses four Tai isoforms resulting from the combination of two indels in the C-terminal region of the sequence. The presence of one equivalent indel-1 in Tai sequences in T. castaneum and other species suggests that Tai isoforms may be common in insects. Concomitant depletion of all four Tai isoforms in B. germanica resulted in 100% mortality, but when only the insertion 1 (IN-1) isoforms were depleted, mortality was significantly reduced and about half of the specimens experienced precocious adult development. This shows that Tai isoforms containing IN-1 are involved in transducing the JH signal that represses metamorphosis. Reporter assays indicated that both T. castaneum Tai isoforms, one that contains the IN-1 and another that does not (DEL-1) activated a JH response element (kJHRE) in Krüppel homolog 1 in conjunction with Met and JH. The results indicate that Tai is involved in the molecular mechanisms that repress metamorphosis, at least in B. germanica, and highlight the importance of distinguishing Tai isoforms when studying the functions of this transcription factor in development and other processes.

Importance of juvenile hormone signaling arises with competence of insect larvae to metamorphose.

Juvenile hormone (JH) postpones metamorphosis of insect larvae until they have attained an appropriate stage and size. Then, during the final larval instar, a drop in JH secretion permits a metamorphic molt that transforms larvae to adults either directly (hemimetaboly) or via a pupal stage (holometaboly). In both scenarios, JH precludes metamorphosis by activating the Kr-h1 gene through a JH receptor, Methoprene-tolerant (Met). Removal of Met, Kr-h1, or JH itself triggers deleterious precocious metamorphosis. Although JH is thought to maintain the juvenile status throughout larval life, various methods of depleting JH failed to induce metamorphosis in early-instar larvae. To determine when does JH signaling become important for the prevention of precocious metamorphosis, we chose the hemimetabolous bug, Pyrrhocoris apterus, and the holometabolous silkworm, Bombyx mori. Both species undergo a fixed number of five larval instars. Pyrrhocoris larvae subjected to RNAi-mediated knockdown of Met or Kr-h1 underwent precocious adult development when treated during the fourth (penultimate) instar, but younger larvae proved increasingly resistant to loss of either gene. The earliest instar developing minor signs of precocious metamorphosis was the third. Therefore, the JH-response genes may not be required to maintain the larval program during the first two larval instars. Next, we examined Bombyx mod mutants that cannot synthesize authentic, epoxidized forms of JH. Although mod larvae expressed Kr-h1 mRNA at severely reduced levels since hatching, they only entered metamorphosis by pupating after four, rarely three instars. Based on findings in Pyrrhocoris and Bombyx, we propose that insect postembryonic development is initially independent of JH. Only later, when larvae gain competence to enter metamorphosis, JH signaling becomes necessary to prevent precocious metamorphosis and to optimize growth.

Hormonal regulation and developmental role of Krüppel homolog 1, a repressor of metamorphosis, in the silkworm Bombyx mori.

Juvenile hormone (JH) has an ability to repress the precocious metamorphosis of insects during their larval development. Krüppel homolog 1 (Kr-h1) is an early JH-inducible gene that mediates this action of JH; however, the fine hormonal regulation of Kr-h1 and the molecular mechanism underlying its antimetamorphic effect are little understood. In this study, we attempted to elucidate the hormonal regulation and developmental role of Kr-h1. We found that the expression of Kr-h1 in the epidermis of penultimate-instar larvae of the silkworm Bombyx mori was induced by JH secreted by the corpora allata (CA), whereas the CA were not involved in the transient induction of Kr-h1 at the prepupal stage. Tissue culture experiments suggested that the transient peak of Kr-h1 at the prepupal stage is likely to be induced cooperatively by JH derived from gland(s) other than the CA and the prepupal surge of ecdysteroid, although involvement of unknown factor(s) could not be ruled out. To elucidate the developmental role of Kr-h1, we generated transgenic silkworms overexpressing Kr-h1. The transgenic silkworms grew normally until the spinning stage, but their development was arrested at the prepupal stage. The transgenic silkworms from which the CA were removed in the penultimate instar did not undergo precocious pupation or larval-larval molt but fell into prepupal arrest. This result demonstrated that Kr-h1 is indeed involved in the repression of metamorphosis but that Kr-h1 alone is incapable of implementing normal larval molt. Moreover, the expression profiles and hormonal responses of early ecdysone-inducible genes (E74, E75, and Broad) in transgenic silkworms suggested that Kr-h1 is not involved in the JH-dependent modulation of these genes, which is associated with the control of metamorphosis.

Transcriptional regulation of juvenile hormone-mediated induction of Krüppel homolog 1, a repressor of insect metamorphosis.

The Krüppel homolog 1 gene (Kr-h1) has been proposed to play a key role in the repression of insect metamorphosis. Kr-h1 is assumed to be induced by juvenile hormone (JH) via a JH receptor, methoprene-tolerant (Met), but the mechanism of induction is unclear. To elucidate the molecular mechanism of Kr-h1 induction, we first cloned cDNAs encoding Kr-h1 (BmKr-h1) and Met (BmMet1 and BmMet2) homologs from Bombyx mori. In a B. mori cell line, BmKr-h1 was rapidly induced by subnanomolar levels of natural JHs. Reporter assays identified a JH response element (kJHRE), comprising 141 nucleotides, located ∼2 kb upstream from the BmKr-h1 transcription start site. The core region of kJHRE (GGCCTCCACGTG) contains a canonical E-box sequence to which Met, a basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) transcription factor, is likely to bind. In mammalian HEK293 cells, which lack an intrinsic JH receptor, ectopic expression of BmMet2 fused with Gal4DBD induced JH-dependent activity of an upstream activation sequence reporter. Meanwhile, the kJHRE reporter was activated JH-dependently in HEK293 cells only when cotransfected with BmMet2 and BmSRC, another bHLH-PAS family member, suggesting that BmMet2 and BmSRC jointly interact with kJHRE. We also found that the interaction between BmMet2 and BmSRC is dependent on JH. Therefore, we propose the following hypothesis for the mechanism of JH-mediated induction of BmKr-h1: BmMet2 accepts JH as a ligand, JH-liganded BmMet2 interacts with BmSRC, and the JH/BmMet2/BmSRC complex activates BmKr-h1 by interacting with kJHRE.

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.

Identification of the Bombyx red egg gene reveals involvement of a novel transporter family gene in late steps of the insect ommochrome biosynthesis pathway.

Ommochromes are one of the major pigments involved in coloration of eggs, eyes, and body surface of insects. However, the molecular mechanisms of the final steps of ommochrome pigment synthesis have been largely unknown. The eggs of the silkworm Bombyx mori contain a mixture of ommochrome pigments, and exhibit a brownish lilac color. The recessive homozygous of egg and eye color mutant, red egg (re), whose eggs display a pale orange color instead of normal dark coloration, has been long suggested to have a defect in the biosynthesis of the final ommochrome pigments. Here, we identify the gene responsible for the re locus by positional cloning, mutant analysis, and RNAi experiments. In the re mutants, we found that a 541-bp transposable element is inserted into the ORF of BGIBMGA003497-1 (Bm-re) encoding a novel member of a major facilitator superfamily transporter, causing disruption of the splicing of exon 9, resulting in two aberrant transcripts with frameshifts yielding nonfunctional proteins lacking the C-terminal transmembrane domains. Bm-re function in pigmentation was confirmed by embryonic RNAi experiments. Homologs of the Bm-re gene were found in all insect genomes sequenced at present, except for 12 sequenced Drosophila genomes, which seemed to correlate with the previous studies that have demonstrated that eye ommochrome composition is different from other insects in several Dipterans. Knockdown of the Bm-re homolog by RNAi in the red flour beetle Tribolium castaneum caused adult compound eye coloration defects, indicating a conserved role in ommochrome pigment biosynthesis at least among holometabolous insects.

Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the 'Black Box' of the ecdysteroid biosynthesis pathway.

In insects, the precise timing of molting and metamorphosis is strictly guided by a principal steroid hormone, ecdysone. Among the multiple conversion steps for synthesizing ecdysone from dietary cholesterol, the conversion of 7-dehydrocholesterol to 5beta-ketodiol, the so-called 'Black Box', is thought to be the important rate-limiting step. Although a number of genes essential for ecdysone synthesis have recently been revealed, much less is known about the genes that are crucial for functioning in the Black Box. Here we report on a novel ecdysteroidgenic gene, non-molting glossy (nm-g)/shroud (sro), which encodes a short-chain dehydrogenase/reductase. This gene was first isolated by positional cloning of the nm-g mutant of the silkworm Bombyx mori, which exhibits a low ecdysteroid titer and consequently causes a larval arrest phenotype. In the fruit fly, Drosophila melanogaster, the closest gene to nm-g is encoded by the sro locus, one of the Halloween mutant members that are characterized by embryonic ecdysone deficiency. The lethality of the sro mutant is rescued by the overexpression of either sro or nm-g genes, indicating that these two genes are orthologous. Both the nm-g and the sro genes are predominantly expressed in tissues producing ecdysone, such as the prothoracic glands and the ovaries. Furthermore, the phenotypes caused by the loss of function of these genes are restored by the application of ecdysteroids and their precursor 5beta-ketodiol, but not by cholesterol or 7-dehydrocholesterol. Altogether, we conclude that the Nm-g/Sro family protein is an essential enzyme for ecdysteroidogenesis working in the Black Box.

UDP-glycosyltransferases from the UGT73C subfamily in Barbarea vulgaris catalyze sapogenin 3-O-glucosylation in saponin-mediated insect resistance.

Triterpenoid saponins are bioactive metabolites that have evolved recurrently in plants, presumably for defense. Their biosynthesis is poorly understood, as is the relationship between bioactivity and structure. Barbarea vulgaris is the only crucifer known to produce saponins. Hederagenin and oleanolic acid cellobioside make some B. vulgaris plants resistant to important insect pests, while other, susceptible plants produce different saponins. Resistance could be caused by glucosylation of the sapogenins. We identified four family 1 glycosyltransferases (UGTs) that catalyze 3-O-glucosylation of the sapogenins oleanolic acid and hederagenin. Among these, UGT73C10 and UGT73C11 show highest activity, substrate specificity and regiospecificity, and are under positive selection, while UGT73C12 and UGT73C13 show lower substrate specificity and regiospecificity and are under purifying selection. The expression of UGT73C10 and UGT73C11 in different B. vulgaris organs correlates with saponin abundance. Monoglucosylated hederagenin and oleanolic acid were produced in vitro and tested for effects on P. nemorum. 3-O-ß-d-Glc hederagenin strongly deterred feeding, while 3-O-ß-d-Glc oleanolic acid only had a minor effect, showing that hydroxylation of C23 is important for resistance to this herbivore. The closest homolog in Arabidopsis thaliana, UGT73C5, only showed weak activity toward sapogenins. This indicates that UGT73C10 and UGT73C11 have neofunctionalized to specifically glucosylate sapogenins at the C3 position and demonstrates that C3 monoglucosylation activates resistance. As the UGTs from both the resistant and susceptible types of B. vulgaris glucosylate sapogenins and are not located in the known quantitative trait loci for resistance, the difference between the susceptible and resistant plant types is determined at an earlier stage in saponin biosynthesis.

Function, diversity, and application of insect juvenile hormone epoxidases (CYP15).

Juvenile hormones (JHs) represent a family of sesquiterpenoid hormones in insects, and they play a key role in regulating development, metamorphosis, and reproduction. The last two steps of the JH biosynthetic pathway, epoxidation and methyl esterification of farnesoic acid to JH, are insect specific, and thus have long been considered a promising target for biorational insecticides. Recently, the enzymes involved in the last two steps have been molecularly identified: JH acid methyltransferase catalyzes the esterification step and the cytochrome P450 CYP15 enzyme catalyzes the epoxidation step. In this review, we describe the recent progress on the characterization of JH biosynthetic enzymes, with special focus on the function and diversity of the CYP15 family. CYP15 genes have evolved lineage-specific substrate specificity and regulatory mechanisms in insects, which appear to be associated with the lineage-specific acquisition of unique JH structure and function. In addition, the lack of CYP15 genes in crustacean (Daphnia pulex) and arachnid (Tetranychus urticae) species, whose genomes have been fully sequenced, may imply that CYP15 enzymes are an evolutionary innovation in insects to use the epoxide forms of methylated farnesoid molecules as their principal JHs. Molecular identification and characterization of CYP15 genes from broad taxa of insects have paved the way to the design of target-specific, biorational anti-JH agents.

Determination by LC-MS of juvenile hormone titers in hemolymph of the silkworm, Bombyx mori.

Juvenile hormone (JH) I, II and III in the hemolymph of the silkworm, Bombyx mori were quantified by liquid chromatography-mass spectrometry (LC-MS). JHs were treated with methanol and trifluoroacetic acid to convert into JH methoxyhydrines (JH-MHs). The key to the analytical condition for JH-MHs was the addition of 5 µM sodium acetate to the eluting solution. Each JH-MH was observed as the sodium adduct ion with good sensitivity. This improved method enabled the titration of JH I, II and III in hemolymph of the silkworm to be monitored from the 3rd instar through to the early pupal stage. A peak of JH I was observed immediately after ecdysis in the 3rd and 4th instar stages. The JH I titer sharply decreased on day 1 and reached the lowest level before ecdysis, but there was no peak at the beginning of the 5th stadium, and no apparent increase was observed until pupation.

Reinvestigation of the sex pheromone of the wild silkmoth Bombyx mandarina: the effects of bombykal and bombykyl acetate.

Sex pheromone investigations of the domesticated silkmoth, Bombyx mori (Lepidoptera: Bombycidae), helped elucidate the molecular and physiological fundamentals of chemical communication in moths, yet little is known about pheromone evolution in bombycid species. Therefore, we reexamined the sex pheromone communication in the wild silkmoth, Bombyx mandarina, which is considered ancestral to B. mori. Our investigations revealed that (a) B. mandarina females produce (E,Z)-10,12-hexadecadienol (bombykol), but not (E,Z)-10,12-hexadecadienal (bombykal) or (E,Z)-10,12-hexadecadienyl acetate (bombykyl acetate), which are pheromone components in other bombycid moths; (b) antennae of male B. mandarina respond strongly to bombykol as well as to bombykal and bombykyl acetate; and (c) bombykal and bombykyl acetate strongly inhibit attraction of B. mandarina males to bombykol in the field. The present study clarifies the evolution of pheromone communication in bombycid moths.

The Number of Larval Molts Is Controlled by Hox in Caterpillars.

Animals with exoskeletons molt for further growth. In insects, the number of larval (or nymphal) molts varies inter- and intra-specifically, and it is widely accepted that the variation in the number of larval molts is an adaptive response to diverse environmental conditions.1-5 However, the molecular mechanism that underlies the variety and plasticity in the number of larval molts is largely unknown. In the silkworm, Bombyx mori, there are strains that molt three, four, or five times, and these numbers are determined by allelic variation at a single autosomal locus, Moltinism (M).6-9 Here, we demonstrate that the Hox gene Sex combs reduced (Scr) is responsible for the phenotypes of the M locus. Scr is selectively expressed in the larval prothoracic gland (PG), an endocrine organ that produces molting hormones.2Scr represses the biosynthesis of molting hormones in the PG, thereby regulating the incremental increase in body size during each larval instar. Our experiments consistently suggest that the differential expression levels of Scr among the three M alleles result in different growth ratios that ultimately lead to the different number of larval molts. Although the role of Hox genes in conferring segmental identity along the body axis and in molding segment-specific structure later in development has been well established,10-13 the present study identifies an unexpected role of Hox gene in hormone biosynthesis. This new role means that, in addition to shaping segment-specific morphology, Hox genes also drive the evolution of life history traits by regulating animal physiology.

Genome-wide assessment and development of molecular diagnostic methods for imidacloprid-resistance in the brown planthopper, Nilaparvata lugens (Hemiptera; Delphacidae).

BACKGROUND: The brown planthopper, Nilaparvata lugens (Stål), is one of the most notorious pests of rice throughout Asia. The brown planthopper has developed high resistance to imidacloprid, a member of neonicotinoid insecticides. Several genes and mutations conferring imidacloprid resistance in N. lugens, especially in eastern and southeastern Asia populations, have been reported. Thus, the key mechanisms of imidacloprid resistance need to be examined. RESULTS: RNA-seq analyses revealed that only one cytochrome P450 monooxygenase gene, CYP6ER1, was commonly upregulated in the five resistant strains tested. Sequences of CYP6ER1, which were highly expressed in the imidacloprid-resistant strains, contained a three-nucleotide deletion in the coding region, and amino acid substitutions and deletion, compared to that in an imidacloprid-susceptible strain. RNAi-mediated gene knockdown of CYP6ER1 increased imidacloprid susceptibility in a resistant strain. Further, we established two simple and convenient PCR-based molecular diagnostic methods to detect the CYP6ER1 locus with the three-nucleotide deletion. Using these methods, the resistance of F2 progenies derived from the crosses of F1 siblings from susceptible and resistant parents was analyzed, showing that the imidacloprid resistance had a relationship to the CYP6ER1 locus with the three-nucleotide deletion. CONCLUSION: The overexpression of a variant CYP6ER1 with amino acid substitutions and deletion was involved in imidacloprid resistance in N. lugens. Based on these findings, molecular diagnostic methods have been developed and are promising tools for monitoring imidacloprid resistance in paddy fields. © 2020 Society of Chemical Industry.

Krüppel homolog 1, an early juvenile hormone-response gene downstream of Methoprene-tolerant, mediates its anti-metamorphic action in the red flour beetle Tribolium castaneum.

Juvenile hormone (JH) prevents ecdysone-induced metamorphosis in insects. However, our knowledge of the molecular mechanisms of JH action is still fragmented. Krüppel homolog 1 (Kr-h1) is a JH-inducible transcription factor in Drosophila melanogaster (Minakuchi, C., Zhou, X., Riddiford, L.M., 2008b. Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster. Mech. Dev. 125, 91-105). Analysis of expression of the homologous gene (TcKr-h1) in the beetle Tribolium castaneum showed that its transcript was continuously present in the larval stage but absent in the pupal stage. Artificial suppression of JH biosynthesis in the larval stage caused a precocious larval-pupal transition and a down-regulation of TcKr-h1 mRNA. RNAi-mediated knockdown of TcKr-h1 in the larval stage induced a precocious larval-pupal transition. In the early pupal stage, treatment with an exogenous JH mimic (JHM) caused formation of a second pupa, and a rapid and large induction of TcKr-h1 transcription. JHM-induced formation of a second pupa was counteracted by the knockdown of TcKr-h1. RNAi experiments in combination with JHM treatment demonstrated that in the larval stage TcKr-h1 works downstream of the putative JH receptor Methoprene-tolerant (TcMet), and in the pupal stage it works downstream of TcMet and upstream of the pupal specifier broad (Tcbr). Therefore, TcKr-h1 is an early JH-response gene that mediates JH action linking TcMet and Tcbr.