Expression analysis of glutathione S-transferases and ferritins during the embryogenesis of the tick Haemaphysalis longicornis

Abstract

In the tick life cycle, embryogenesis is the only stage of development wherein no blood meal is required. Nevertheless, even in the absence of a blood meal, which is the source of nutrients as well as the ferrous iron and heme that could cause oxidative stress in ticks, malondialdehyde (MDA) has been reported to increase during this period. Additionally, the knockdown of some oxidative stress-related molecules such as ferritin has resulted in abnormal eggs and embryonic death. Here, we investigate the gene and protein expression profiles of the identified glutathione S-transferases (GSTs) and ferritins (Fers) of the tick H. longicornis during embryogenesis through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blotting, respectively. We also confirm the lipid peroxidation and ferrous iron concentration level using a thiobarbituric acid reactive substances (TBARS) assay. Finally, we attempt to correlate these findings with the events occurring by establishing a staging process in H. longicornis embryos. Lipid peroxidation increased during the course of embryogenesis, as does the amount of GST proteins. On the other hand, the GST genes have high expression at the 1^st day post-oviposition, during the early stage of embryogenesis and at day 10 during the period wherein the germ band is observable. Fer gene expression also starts to increase at day 10 and peaks at day 15. In the ferritin proteins, only the secretory ferritin (Fer2) is detected and constitutively expressed during embryogenesis. Events occurring during embryogenesis, such as energy production and iron metabolism for cellular proliferation and differentiation cause oxidative stress in the embryo. To counteract oxidative stress, it is possible that the embryo may utilize oxidative stress-related molecules such as GSTs and Fer2, which could be either maternally or embryo-derived.

Keywords: Developmental biology; Embryology; Genomics; Oxidative stress; Proteomics; Transcriptome; Veterinary medicine.

Figure 1

Stages of the embryonic development of H. longicornis. Embryos were deshelled and scaled. The embryonic nuclei were stained with DAPI and observed under fluorescence microscopy. The embryos were staged based on the classification system by Santos et al. (2013). Cm: cumulus cells; VF: ventral furrow; GB: germ band; 4L: 4^th set of limbs. Scale bars = 200 μm.

Figure 2

Transcription profiles of (a) GST1, (b) GST2, (c) Fer1, and (d) Fer2 genes during H. longicornis embryogenesis. Total RNA was prepared from embryos harvested at different days post-oviposition. P0 genes were used as the control. Error bar represents the mean ± standard deviation. Significant difference was determined using the Kruskal–Wallis rank test.

Figure 3

Expression profiles of GSTs and ferritins during H. longicornis embryogenesis. Proteins were prepared at different days post-oviposition. The results of Western blotting are shown as representative data of three separate experiments showing the same trend. The protein concentration was determined using a Micro BCA kit and maintained at 50 μg per lane before loading for Western blotting. Full blot images are available as a supplementary material (Figs. S1–S4).

Figure 4

(a) Malondialdehyde concentrations during the embryogenesis of H. longicornis. MDA concentration was determined by TBARS assay. Significant difference was determined using the Kruskal–Wallis rank test. (b) Ferrous iron percentages during H. longicornis embryogenesis. The total iron concentration was determined using an iron assay kit, while the ferrous iron concentration was determined using the same kit without a reducing agent. The ferrous iron percentage is the concentration of ferrous iron divided by the total iron concentration.