Isoform specific roles of Broad‐Complex in larval development in Leptinotarsa decemlineata

Broad‐Complex (BrC) is a downstream target of both 20‐hydroxyecdysone and juvenile hormone signalling. BrC regulates morphogenetic changes between nymphal instars in hemimetabolans, whereas it controls pupal commitment, pupal morphogenesis and inhibits adult differentiation in holometabolans. Among five BrC cDNAs (Z1–Z4 and Z6) identified in the Colorado potato beetle, we found in this work that Z1, Z2 and Z6 were mainly expressed at the last (fourth) instar and prepupal stages, whereas the levels of Z3 and Z4 increased during the penultimate (third) instar stage, peaked at the last instar larval phase and gradually decreased at the prepupal and pupal periods. When knocking down all BrC isoforms by RNA interference (RNAi) at the penultimate instar stage, around 20% of the resultant larvae remained as moribund beetles. These moribund BrC RNAi larvae were completely or partially wrapped in old cuticle. Likewise, a portion of larvae treated for a single double‐stranded RNA of Z3, Z4 or Z6 displayed a degree of similar aberrancies, increasing in the order of isoforms Z6 < Z3 < Z4. When silencing all BrC isoforms at the last instar period, most of the RNAi larvae did not normally pupate or emerge as adults. Separately silencing each of the five zinc finger domains revealed that approximately 70% of the Z1 RNAi larvae remained as prepupae, around 60% of the Z6 RNAi specimens formed aberrant prepupae or pupae and about 60% of the Z2 RNAi beetles became deformed pupae. After removal of the old exuviae, these deformed larvae in which either Z1, Z2 or Z6 was depleted possessed adult prothorax and mesothorax, developing antenna, mouthparts and wing discs. Moreover, less than 50% of the resultant pupae finally emerged as adults when either of Z1, Z2 or Z6 was knocked down. Therefore, our findings reveal, for the first time, that the two roles of BrC in insect groups (ie directing morphogenetic changes during juvenile development and regulating larval–pupal–adult metamorphosis) are played by different BrC isoforms in Leptinotarsa decemlineata.     

Function of a TGF‐β inducible nuclear protein in the silk gland in Bombyx mori

A TGF‐β inducible nuclear protein 1 (BmTINP1) was cloned from silkworm, Bombyx mori. Polyclonal antibodies against BmTINP1 were produced and subsequently used in immunoblotting and immunohistochemistry analyses. The immunoblotting analyses demonstrated that BmTINP1 was specifically expressed in the anterior silk gland (ASG) and the middle silk gland (MSG) but not in the posterior silk gland (PSG). There were two bands that suggested the existence of an isoform of BmTINP1. The expression profiles of BmTINP1 in ASGs and MSGs were similar, and they manifested a high level of expression throughout the period during which silk gland grew exponentially. Immunohistochemistry results revealed that BmTINP1 was translocated from the nucleus into the cytoplasm when larvae developed from the 4th‐HCS into the 5th instar. 20‐hydroxyecdysone (20E) promotes the translocation, while the methoprene [a juvenile hormone (JH) analog] restrains the process. Our findings indicate that BmTINP1 is involved in silk produce along with the rapid growth of ASGs and MSGs during the last instar larvae, and the process could be regulated by hormones via control of BmTINP1 translocation from the nucleus to the cytoplasm.     

MicroRNA miR‐252 targets mbt to control the developmental growth of Drosophila

Developmental growth is an intricate process involving the coordinated regulation of the expression of various genes, and microRNAs (miRNAs) play crucial roles in diverse processes throughout animal development. The ecdysone‐responsive miRNA, miR‐252, is normally upregulated during the pupal and adult stages of Drosophila development. Here, we found that overexpression of miR‐252 in the larval fat body decreased total tissue mass through a reduction in both cell size and cell number, causing a concomitant decrease in larval size. Furthermore, miR‐252 overexpression led to a delayed larval‐to‐pupal transition with defective anterior spiracle eversion, as well as a decrease in adult size and mass. Conversely, adult flies lacking miR‐252 showed an increase in mass compared with control flies. We found that miR‐252 directly targeted mbt, encoding a p21‐activated kinase, to repress its expression. Notably, co‐overexpression of mbt rescued the developmental and growth defects associated with miR‐252 overexpression, indicating that mbt is a biologically relevant target of miR‐252. Overall, our data support a role for the ecdysone/miR‐252/mbt regulatory axis in growth control during Drosophila development.     

Systemic disruption of the homeostasis of transfer RNA isopentenyltransferase causes growth and development abnormalities in Bombyx mori

Isopentenylation at A37 (i⁶A37) of some transfer RNAs (tRNAs) plays a vital role in regulating the efficiency and fidelity of protein synthesis. However, whether insects, which are well known for their highly efficient protein synthesis machinery, employ this regulatory mechanism remains uninvestigated. In the current study, a candidate tRNA isopentenyltransferase (IPT) gene with three alternative splicing isoforms (BmIPT1–BmIPT3) was identified in Bombyx mori (silkworm). Only BmIPT1 could complement a yeast mutant lacking tRNA IPT. Phylogenetic analysis showed that silkworm tRNA IPT is conserved in the Lepidoptera. BmIPT was expressed in all B. mori tissues and organs that were investigated, but was expressed at a significantly higher level in silk glands of the fourth instar compared to the first day of the fifth instar. Interestingly, BmIPT was expressed at a significantly higher level in the domesticated silkworm, B. mori, than in wild Bombyx mandarina in multiple tissues and organs. Knock‐down of BmIPT by RNA interference caused severe abnormalities in silk spinning and metamorphosis. Constitutive overexpression of BmIPT1 using a cytoplasmic actin 4 promoter in B. mori raised its messenger RNA level more than sixfold compared with nontransgenic insects and led to significant decreases in the body weight and cocoon shell ratio. Together, these results confirm the first functional tRNA IPT in insects and show that a suitable expression level of tRNA IPT is vital for silk spinning, normal growth, and metamorphosis. Thus, i⁶A modification at position A37 in tRNA probably plays an important role in B. mori protein synthesis.     

Functional characterization of ultraspiracle in Leptinotarsa decemlineata using RNA interference assay

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20‐hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non‐drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third–fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L. decemlineata.