Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos

The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. hominivorax from North and Central America. This involved area‐wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making ‘male‐only’ strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro‐apoptotic gene by the tetracycline‐dependent transactivator. Sex‐specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro‐apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro‐apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro‐apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests.     

Bombyx mori cyclin‐dependent kinase inhibitor is involved in regulation of the silkworm cell cycle

Cyclin‐dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin‐dependent kinase (CDK)‐cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654‐bp‐long BmCKI‐L (the longer splice variant) encoding a protein with 217 amino acids and a 579‐bp‐long BmCKI‐S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI‐L and BmCKI‐S contain the Cip/Kip family conserved cyclin‐binding domain and the CDK‐binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181–210. Overexpression of BmCKI‐L or BmCKI‐S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI‐L or BmCKI‐S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI‐L and BmCKI‐S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI‐L overexpression (BmCKI‐L‐OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI‐L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm.     

Dynamics and regulation of glycolysis‐tricarboxylic acid metabolism in the midgut of Spodoptera litura during metamorphosis

Significant changes usually take place in the internal metabolism of insects during metamorphosis. The glycolysis‐tricarboxylic acid (glycolysis‐TCA) pathway is important for energy metabolism. To elucidate its dynamics, the mRNA levels of genes involved in this pathway were examined in the midgut of Spodoptera litura during metamorphosis, and the pyruvate content was quantified. The expression patterns of these genes in response to starvation were examined, and the interaction between protein phosphatase 1 (PP1) and phosphofructokinase (PFK) was studied. The results revealed that the expression or activities of most glycolytic enzymes was down‐regulated in prepupae and then recovered in some degree in pupae, and all TCA‐related genes were remarkably suppressed in both the prepupae and pupae. Pyruvate was enriched in the pupal midgut. Taken together, these results suggest that insects decrease both glycolysis and TCA in prepupae to save energy and then up‐regulate glycolysis but down‐regulate TCA in pupae to increase the supply of intermediates for construction of new organs. The expression of all these genes were down‐regulated by starvation, indicating that non‐feeding during metamorphosis may be a regulator of glycolysis‐TCA pathway in the midgut. Importantly, interaction between PP1 and PFK was identified and is suggested to be involved in the regulation of glycolysis.     

Establishment of transgenic silkworms expressing GAL4 specifically in the haemocyte oenocytoid cells

Insect haemocytes play significant roles in innate immunity. The silkworm, a lepidopteran species, is often selected as the model for studies into the functions of haemocytes in immunity; however, our understanding of the role of haemocytes remains limited because the lack of haemocyte promoters for transgene expression makes genetic manipulations difficult. In the present study, we aimed to establish transgenic silkworm strains expressing GAL4 in their haemocytes. First, we identified three genes with strong expression in haemocytes, namely, lp44, Haemocyte Protease 1 (HP1) and hemocytin. Transgenic silkworms expressing GAL4 under the control of the putative promoters of these genes were then established and expression was examined. Although GAL4 expression was not detected in haemocytes of HP1‐GAL4 or hemocytin‐GAL4 strains, lp44‐GAL4 exhibited a high level of GAL4 expression, particularly in oenocytoids. GAL4 expression was also detected in the midgut but in no other tissues, indicating that GAL4 expression in this strain is mostly oenocytoid‐specific. Thus, we have identified a promoter that enables oenocytoid expression of genes of interest. Additionally, the lp44‐GAL4 strain could also be used for other types of research, such as the functional analysis of genes in oenocytoids, which would facilitate advances in our understanding of insect immunity.     

Shear stress influences the pluripotency of murine embryonic stem cells in stirred suspension bioreactors

Pluripotent embryonic stem cells (ESCs) have been used increasingly in research as primary material for various tissue‐engineering applications. Pluripotency, or the ability to give rise to all cells of the body, is an important characteristic of ESCs. Traditional methods use leukaemia inhibitory factor (LIF) to maintain murine embryonic stem cell (mESC) pluripotency in static and bioreactor cultures. When LIF is removed from mESCs in static cultures, pluripotency genes are downregulated and the cultures will spontaneously differentiate. Recently we have shown the maintenance of pluripotency gene expression of mESCs in stirred suspension bioreactors during differentiation experiments in the absence of LIF. This is undesired in a differentiation experiment, where the goal is downregulation of pluripotency gene expression and upregulation of gene expression characteristic to the differentiation. Thus, the objective of this study was to examine how effectively different levels of shear stress [100 rpm (6 dyne/cm²), 60 rpm (3 dyne/cm²)] maintained and influenced pluripotency in suspension bioreactors. The pluripotency markers Oct‐4, Nanog, Sox‐2 and Rex‐1 were assessed using gene expression profiles and flow‐cytometry analysis and showed that shear stress does maintain and influence the gene expression of certain pluripotency markers. Some significant differences between the two levels of shear stress were seen and the combination of shear stress and LIF was observed to synergistically increase the expression of certain pluripotency markers. Overall, this study provides a better understanding of the environmental conditions within suspension bioreactors and how these conditions affect the pluripotency of mESCs. Copyright © 2012 John Wiley & Sons, Ltd.     

Juvenile hormone regulates the reproductive diapause through Methoprene-tolerant gene in Galeruca daurica

Juvenile hormone (JH) signalling plays an important role in regulation of reproductive diapause in insects. However, its underlying molecular mechanism has been unclear. Methoprene-tolerant (Met), as a universal JH receptor, is involved in JH action. To gain some insight into its function in the reproductive diapause of Galeruca daurica, a serious pest on the Inner Mongolia grasslands undergoing obligatory summer diapause at the adult stage, we cloned the complete open-reading frame (ORF) sequences of Met and other 7 JH signalling-related genes, including JH acid methyltransferase (JHAMT), JH esterase (JHE), JH epoxide hydrolase (JHEH), Krüppel homologue 1 (Kr-h1), vitellogenin (Vg), forkhead box O (FOXO) and fatty acid synthase 2 (FAS2), from this species. GdMet encoded a putative protein, which contained three domains typical of the bHLH-PAS family. Expression patterns of these eight genes were developmentally regulated during adult development. Topical application of JH analogue (JHA) methoprene into the 3-day-old and 5-day-old adults induced the expression of GdMet. Silencing GdMet by RNAi inhibited the expression of JHBP, JHE, Kr-h1 and Vg, whereas promoted the FAS2 expression, which enhanced lipid accumulation and fat body development, and finally induced the adults into diapause ahead. Combining with our previous results, we conclude that JH may regulate reproductive diapause through a conserved Met-dependent pathway in G. daurica.     

The involvement of clathrin‐mediated endocytosis and two Sid‐1‐like transmembrane proteins in double‐stranded RNA uptake in the Colorado potato beetle midgut

RNA interference (RNAi) is a powerful tool in entomology and shows promise as a crop protection strategy, but variability in its efficiency across different insect species limits its applicability. For oral uptake of the double‐stranded RNA (dsRNA), the RNAi trigger, two different mechanisms are known: systemic RNA interference deficient‐1 (Sid‐1) transmembrane channel‐mediated uptake and clathrin‐mediated endocytosis. So far, a wide range of experiments has been conducted, confirming the involvement of one of the pathways in dsRNA uptake, but never both pathways in the same species. We investigated the role of both pathways in dsRNA uptake in the Colorado potato beetle, Leptinotarsa decemlineata, known to have an efficient RNAi response. Through RNAi‐of‐RNAi experiments, we demonstrated the contribution of two different sid‐1‐like (sil) genes, silA and silC, and clathrin heavy chain and the 16kDa subunit of the vacuolar H⁺ ATPase (vha16), elements of the endocytic pathway, to the RNAi response. Furthermore, the sid‐1‐like genes were examined through phylogenetic and hydrophobicity analysis. This article reports for the first time on the involvement of two pathways in dsRNA uptake in an insect species and stresses the importance of evaluating both pathways through a well‐devised reporter system in any future experiments on cellular dsRNA uptake.     

Characterization of layered chondrocyte sheets created in a co‐culture system with synoviocytes in a hypoxic environment

Endeavouring to repair and regenerate articular cartilage using cell sheets, we have previously established a co‐culture system of chondrocytes and synoviocytes, and have reported the successful and rapid production of chondrocyte sheets. In the present study, to examine the effects of oxygen concentration on the chondrocyte sheets, we co‐cultured human articular chondrocytes and human synoviocytes in 2%, 5% and 21% oxygen, and measured chondrocyte metabolic activity and proliferation activities under each condition for 14 days in culture. Layered chondrocyte sheets were also created under each condition and the proteoglycan (PG) level was compared with the gene expression of type I collagen (COL1), COL2, COL27, tissue metallopeptidase inhibitor 1 (TIMP1), fibronectin‐1 (FN1), SRY‐related HMG Box 9 (SOX9), aggrecan‐1 (ACAN), integrin‐α10 (ITGα10), matrix metalloproteinase 3 (MMP3), MMP13 and a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS5). Compared with 5% and 21% oxygen, the 2% condition caused significantly greater cell metabolic activity and proliferation (p < 0.05). The 2% condition produced a 10% greater PG level compared with 21% oxygen (p < 0.05). All conditions increased the expression of chondrocyte‐specific genes, such as COL2, and were associated with low expression levels of catabolic factors, such as MMP3 and MMP13. These observations indicated that the specificity of the chondrocyte sheets was maintained under all conditions. The culture times did not differ between the 5% and 21% conditions. Compared with 21% oxygen, layered chondrocyte sheets rich in extracellular matrix were created 2.85 days earlier in 2% oxygen, which is similar to the level found in deep cartilage. Copyright © 2016 John Wiley & Sons, Ltd.