Holometabolous insects undergo larval moulting and metamorphosis within their life cycle. A cDNA encoding the cathepsin L-like proteinase Ha-cathL has been cloned from Helicoverpa armigera. It has a sequence of 1826 bp and encodes a 550-residue protein with a molecular mass of 63 kDa. Northern blot analysis indicated that Ha-cathL is specifically expressed in haemocytes, with increased expression during larval moulting and metamorphosis. In vivo experimentation revealed that Ha-cathL is up-regulated by 20-hydroxyecdysone. Meanwhile, in situ hybridization and immunocytochemistry revealed that Ha-cathL mRNA is mainly expressed in granulocytes and plasmatocytes. Knock down of cathepsin L by RNA interference results in larvae death before pupation or the formation of a chimeric pupa containing a larval head and thorax, abnormal wings and the pupal abdomen. The reason for this is that the affected haemocytes cannot become granulated, and therefore cannot participate in fat body remodelling and wing development. These facts suggest that Ha-cathL is involved in larval moulting and metamorphosis by participating in the functioning of haemocytes.

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Background: Larval molting and metamorphosis are important physiological processes in the life cycle of the holometabolous insect. We used suppression subtractive hybridization (SSH) to identify genes differentially expressed during larval molting and metamorphosis.

Molting is a very important physiological behavior to arthropods. During molting, integument apolysis occurs, which is the digestion and absorption of the old endocuticle for new cuticle formation. Proteases play critical roles in this process. Molting carboxypeptidase A ( Ha-CPA ) is characterized from Helicoverpa armigera . The Ha-CPA transcript was mainly present in the integument from the 5th instar larvae. In the integument, the transcription level of the gene reached its peak at the 5th instar molting stage and the 6th instar prepupal stage, respectively. The examination of immunohistochemistry revealed that Ha-CPA could distribute into the molting fluid in the molting-and prepupal-stage larvae. The expression of Ha-CPA could be up-regulated by 20-hydroxyecdysone (20E). These facts indicate that Ha-CPA participates in the apolysis of the integument during larval molting and metamorphosis.

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Members of the caspase family play a central and evolutionary role in programmed cell death (PCD), which removes unwanted, damaged and dangerous cells during development to maintain homeostasis. In this paper, we describe the cloning and characterization of a caspase from the cotton bollworm, Helicoverpa armigera, named Hearm caspase-1. The 1,350 bp full-length cDNA contains an 885 bp open reading frame (ORF) that encodes a Hearm caspase-1 proenzyme of 294 amino acids. The deduced protein is highly homologous to Spodoptera frugiperda Sf caspase-1 and Drosophila melanogaster ICE and has the highly conserved pentapeptide QACQG, the recognized catalytic site of caspases, suggesting that it is an effector caspase of the cotton bollworm. Northern blot and RT-PCR analyses demonstrate that Hearm caspase-1 is expressed in embryos and the fat body, midgut and haemocytes of feeding and wandering larvae. Expression of Hearm caspase-1 in the haemocytes appears to be correlated with the pulse of ecdysone, and it is up-regulated by ecdysone agonist RH-2485, implying that Hearm caspase-1 activation is regulated by ecdysone.

Pluripotent stem cells can be created successfully through the inner cell mass (ICM), nuclear transfer, and defined-factor induction. Unfortunately, the epigenetic characteristics of the cells produced are poorly understood. In this article, we compared expression levels of enzymes involved in epigenetic modifications across six pluripotent stem cell lines. Six of the 11 genes evaluated here (Dnmt3a, Dnmt3b, Tet1, Ezh2, Mll1, and Lsd1) showed abnormally low levels of expression in the two germ-line chimeric induced pluripotent stem cell (iPSC) lines. We also conducted locus-specific analysis of DNA methylation at 9 loci. Although iPSCs did express Oct4, the Oct4 promoter region was shown to have a higher level of DNA methylation. The Xist and Line-1 repeating sequences differed relatively little in methylation level across the cell lines, but Peg3, Peg10, and H19 exhibited high degrees of variation in the pattern of DNA methylation. Meg3 in the Dlk1-Dio3 imprinting cluster was incompletely methylated in embryonic stem cells (ESCs) and nuclear transfer (nt) ESCs. However, in germ-line chimeric iPSCs, Meg3 was almost entirely methylated. ESC and ntESC lines showed twice as much Meg3 expression than in the iPSC lines. The genomic 5mC contents detected by reverse-phase high-performance liquid chromatography (HPLC) indicated that, despite their germ-line chimeric abilities, iPSCs remained incompletely reprogrammed, even though no direct evidence is shown here.

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