Establishment of a new cell line from lepidopteran epidermis and hormonal regulation on the genes

Abstract

When an insect molts, old cuticle on the outside of the integument is shed by apolysis and a new cuticle is formed under the old one. This process is completed by the epidermal cells which are controlled by 20-hydroxyecdysone (20E) and juvenile hormone. To understand the molecular mechanisms of integument remolding and hormonal regulation on the gene expression, an epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was established and named HaEpi. The cell line has been cultured continuously for 82 passages beginning on June 30, 2005 until now. Cell doubling time was 64 h. The chromosomes were granular and the chromosome mode was from 70 to 76. Collagenase I was used to detach the cells from the flask bottom. Non-self pathogen AcMNPV induced the cells to apoptosis. The cell line was proved to be an epidermal cell line based on its unique gene expression pattern. It responded to 20E and the non-steroidal ecdysone agonist RH-2485. Its gene expression could be knocked down using RNA interference. Various genes in the cell line were investigated based on their response to 20E. This new cell line represents a platform for investigating the 20E signaling transduction pathway, the immune response mechanism in lepidopteran epidermis and interactions of the genes.

Figure 1. Morphology of the HaEpi cell line established from larval epidermis of H. armigera subcultured 12th passage.

Observed under phase contrast microscope (10×40). Panels a and b, two kinds of epithelial cells; c, fibroblast-like cell; d, spindle-like cell.

Figure 2. Analysis of the growth curve and chromosomes of the cell line.

A, growth curve of epidermal cell line HaEpi in Grace's medium containing 10% fetal bovine serum at 27°C. B, chromosomes of the HaEpi cell line. 10×100.

Figure 3. Cytopathology character of HaEpi cells infected with baculovirus.

Panel a, uninfected HaEpi cells; panel b, HaEpi cells infected with HaSNPV; panel c, HaEpi cells infected with AcMNPV; panel d, DNA ladder of apoptotic HaEpi cells induced by AcMNPV, 1, DNA ladder of apoptotic HaEpi cells, 2, DL2000 DNA marker. Arrow indicates normal cell in a and b, and apoptotic cell in c. Observed 4 days after virus infection. 10×40 magnified.

Figure 4. Semi-quantitative RT-PCR to compare gene expression patterns in the HaEpi cell line and tissues.

5F, feeding 5th instar larvae; 5M, molting 5th instar larvae; HaEpi, HaEpi cell line; Epi, epidermis; Mg, midgut; Fb, fat body; Hc, haemocyte.

Figure 5. Induction and knock down of HHR3 expression in HaEpi cell line.

A, Northern blot to show induced expression of HHR3 by RH-2485. Con, control was equal volume of isopropanal. 18 s rRNA was used as a qualitative and quantitative control for RNA. 10 µg total RNA, 1% gel. 3, 6, 12, 18 and 24 are periods (hour) after induction. B, RT-PCR to show knock down of HHR3 by RNAi, 12 h induction by 20E.

Figure 6. Hormonal regulation on the genes in HaEpi cell line.

A, induced expression of the genes in the cells with 1 µM 20E for various hours; B, gene expression patterns after removal of and post 12 h incubation in 20E. Con, control with equal volume of DMSO.