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

AbstractJuvenile 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

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

AbstractJuvenile 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

Juvenile hormone acid O-methyltransferase in Drosophila melanogaster

Juvenile hormone (JH) acid O-methyltransferase (JHAMT) is the enzyme that transfers a methyl groupfrom S-adenosyl-L-methionine (SAM) to the carboxyl group of JH acids to produce active JHs in thecorpora allata. While the JHAMT gene was originally identified and characterized in the silkwormBombyx mori, no orthologs from other insects have been studied until now. Here we report on thefunctional characterization of the CG17330/DmJHAMT gene in the fruit fly Drosophila melanogaster.Recombinant DmJHAMT protein expressed in Escherichia coli catalyzes the conversion of farnesoic acidand JH III acid to their cognate methyl esters in the presence of SAM. DmJHAMT is predominantlyexpressed in corpora allata, and its developmental expression profile correlates with changes in the JHtiter. While a transgenic RNA interference against DmJHAMT has no visible effect, overexpression ofDmJHAMT results in a pharate adult lethal phenotype, similar to that obtained with application of JHanalogs, suggesting that the temporal regulation of DmJHAMT is critical for Drosophila development

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

Neuropeptide Receptor Transcriptome Reveals Unidentified Neuroendocrine Pathways

Neuropeptides are an important class of molecules involved in diverse aspects of metazoan development and homeostasis. Insects are ideal model systems to investigate neuropeptide functions, and the major focus of insect neuropeptide research in the last decade has been on the identification of their receptors. Despite these vigorous efforts, receptors for some key neuropeptides in insect development such as prothoracicotropic hormone, eclosion hormone and allatotropin (AT), remain undefined. In this paper, we report the comprehensive cloning of neuropeptide G protein-coupled receptors from the silkworm, Bombyx mori, and systematic analyses of their expression. Based on the expression patterns of orphan receptors, we identified the long-sought receptor for AT, which is thought to stimulate juvenile hormone biosynthesis in the corpora allata (CA). Surprisingly, however, the AT receptor was not highly expressed in the CA, but instead was predominantly transcribed in the corpora cardiaca (CC), an organ adjacent to the CA. Indeed, by using a reverse-physiological approach, we purified and characterized novel allatoregulatory peptides produced in AT receptor-expressing CC cells, which may indirectly mediate AT activity on the CA. All of the above findings confirm the effectiveness of a systematic analysis of the receptor transcriptome, not only in characterizing orphan receptors, but also in identifying novel players and hidden mechanisms in important biological processes. This work illustrates how using a combinatorial approach employing bioinformatic, molecular, biochemical and physiological methods can help solve recalcitrant problems in neuropeptide research

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie