Investigation of three enzymes and their roles in the embryonic development of parthenogenetic Haemaphysalis longicornis

Abstract

Background: The tick Haemaphysalis longicornis exhibits two separate reproductive populations: bisexual and parthenogenetic, which have diploid and triploid karyotypes, respectively. The parthenogenetic population can undergo engorgement without copulation and produce viable female-only offspring with a longer incubation period than the bisexual population. Three enzymes, cathepsin B, cathepsin D and acid phosphatase, were found to be involved in vitellin degradation during the embryonic development of bisexual H. longicornis. However, the expression and activity profiles of these enzymes during the embryonic development of parthenogenetic ticks remain unknown. In the present study, the transcriptional expression profile, enzyme activity and roles in embryogenesis of the three enzymes during the embryonic development of parthenogenetic H. longicornis were investigated.

Methods: Quantitative real-time polymerase chain reaction (qPCR) and fluorescence detection were used to analyze the dynamic changes in the three enzymes during embryogenesis. The roles of the three enzymes during embryogenesis were also explored using RNA interference (RNAi).

Results: The three enzymes were all expressed during embryonic development in parthenogenetic H. longicornis. The expression of cathepsin B was highest on day 15, whereas that of cathepsin D was highest on day 3 and the peak of acid phosphatase expression occurred on day 9. The activity of cathepsin B was highest on day 3 and lowest on day 5, then gradually increased and remained stable. Cathepsin D activity was highest on day 1 and showed a gradually decreasing trend, whereas acid phosphatase showed the opposite trend and reached a peak on day 23. RNA interference experiments in engorged female ticks revealed that there was no significant difference in the number of eggs laid, but the hatching rate of the eggs was significantly decreased.

Conclusion: The three enzymes all play important roles in embryonic development of H. longicornis, but the expression patterns and changes in the activity of the enzymes in the bisexual and parthenogenetic populations are different. The results will help a better understanding of the similarities and differences underlying embryonic development in the bisexual and parthenogenetic populations and contribute to the future exploration of the development of the parthenogenetic population of H. longicornis.

Keywords: Acid phosphatase; Cathepsin B; Cathepsin D; Embryogenesis; Haemaphysalis longicornis; Parthenogenesis.

Fig. 1

qPCR amplification products analysis of cathepsin B (a), cathepsin D (b) and acid phosphatase (c) of the parthenogenetic H. longicornis

Fig. 2

Dynamic changes of cathepsin B during embryonic development of the parthenogenetic H. longicornis. a Gene transcripts of cathepsin B. b Activity of cathepsin B. The levels of gene mRNA expression were normalised against the mRNA of β-actin and the error bars represent the mean ± SE values and the letters (a, b, c, d) labels represent significant differences (P ≤ 0.0001)

Fig. 3

Dynamic changes of cathepsin D during embryonic development of parthenogenetic H. longicornis. a Gene transcripts of cathepsin D. b Activity of cathepsin D. The levels of gene mRNA expression were normalised against the mRNA of β-actin and the error bars represent the mean ± SE values and the letters (a, b, c, d) labels represent significant differences (P ≤ 0.0001)

Fig. 4

Dynamic changes of acid phosphatase during embryonic development of parthenogenetic H. longicornis. a Genes transcripts of acid phosphatase. b Activity of acid phosphatase. The levels of gene mRNA expression were normalised against the mRNA of β-actin and the error bars represent the mean ± SE values and the letters (a, b, c, d, e, f) labels represent significant differences (P ≤ 0.0001)

Fig. 5

RNAi PCR amplification products analysis of cathepsin B (a), cathepsin D (b) and acid phosphatase (c) of parthenogenetic H. longicornis

Fig. 6

Assessment of genes silencing efficiency of cathepsin B (a), cathepsin D (b) and acid phosphatase (c) of parthenogenetic H. longicornis. The levels of gene mRNA expression were normalised against the mRNA of β-actin and the error bars represent the mean ± SE values; asterisks indicate represent significant differences (P ≤ 0.05)

Fig. 7

DsRNA-mediated knockdown of transcripts for cathepsin B (CB), cathepsin D (CD) and acid phosphatase (AP) affect oviposition (a) and eggs’ hatchability (b) of parthenogenetic H. longicornis. The error bars represent the mean ± SE values and the letter (a, b, c) labels represent significant differences (P ≤ 0.0001)