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. 2013 Aug 8;6(1):230.
doi: 10.1186/1756-3305-6-230.

Functional characterization of three MicroRNAs of the Asian tiger mosquito, Aedes albopictus

Santhosh Puthiyakunnon  1 , Yunying Yao, Yiji Li, Jinbao Gu, Hongjuan Peng, Xiaoguang Chen
Affiliations

Functional characterization of three MicroRNAs of the Asian tiger mosquito, Aedes albopictus

Santhosh Puthiyakunnon et al. Parasit Vectors. .

Abstract

Background: Temporal and stage specific expression of microRNAs (miRNAs) in embryos, larvae, pupae and adults of Aedes albopictus showed differential expression levels across the four developmental stages, indicating their potential regulatory roles in mosquito development. The functional characterization of these miRNAs was not known. Accordingly our study evaluated the functional characterization of three miRNAs, which are temporally up-regulated in the various developmental stages of Ae. albopictus mosquitoes.

Methods: miRNA mimics, inhibitors and negative controls were designed and their knock-in and knock-down efficiency were analyzed by qRT-PCR after transfecting the mosquito cell lines C6/36, and also by injecting in their specific developmental stages. The functional role of each individual miRNA was analyzed with various parameters of development such as, hatching rate and hatching time in embryos, eclosion rate in larvae, longevity and fecundity in the adult mosquitoes.

Results: The knock-in with the specifically designed miRNA mimics showed increased levels of expression of miRNA compared with their normal controls. We confirmed these findings using qRT-PCR, both by in vitro expression in C6/36 mosquito cell lines after transfection as well as in in vivo expression in developmental stages of mosquitoes by microinjection. The knock-down of expression with the corresponding inhibitors showed a considerable decrease in the expression levels of these miRNAs and obvious functional effects in Ae. albopictus development, detected by a decrease in the hatching rate of embryos and eclosion rate in larvae and a marked reduction in longevity and fecundity in adults.

Conclusion: This study carried out by knock-in and knock-down of specifically and temporally expressed miRNAs in Ae. albopictus by microinjection is a novel study to delineate the importance of the miRNA expression in regulating mosquito development. The knock-down and loss of function of endogenously expressed miRNAs by the miRNA inhibitors in specific developmental stages had considerable effects on development, but enhancement of their gain of function was not observed on knock-in of these specific miRNAs. Hence, our study indicates that an optimal level of endogenous expression of miRNA is indispensable for the normal development and maintenance of the vectorial population density and pathogen transmissibility of this mosquito vector.

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Figures

Figure 1
Figure 1
Relative expression analysis after miRNA interference with three miRNAs in C6/36 cells by Bulge-loop miRNA qPCR. (A) Relative expression of miR-286b analyzed by ANOVA (F = 72.047, p < 0.001). (B) Relative gene expression of miR-1891 analyzed by ANOVA (F = 146.96, p < 0.001). (C) Relative expression of miR-2942 analyzed by ANOVA (F = 496.035, p < 0.001). * showed a significant decrease (p < 0.001) in expression level in inhibitor group compared with the negative control group (analyzed by statistical method of ANOVA by LSD). ** No significant difference (p > 0.05) in expression level between inhibitor and negative control group (analyzed by statistical method of ANOVA, LSD). In all qPCR quantification, the relative gene expressions from normal controls are calibrated to 1 (not shown in graph). Error bars show the SEM of three biological replicates from three independent cell transfection experiments.
Figure 2
Figure 2
Relative expression analysis after miRNA interference with three miRNAs in developmental stages of Ae. albopictus by Bulge-loop miRNA qPCR. (A) Relative gene expression of miR-1891 in adults analyzed by ANOVA (F = 180.22, p < 0.001) (B) Relative expression of miR-286b in embryo analyzed by ANOVA (F = 32.908, p < 0.001) (C) Relative expression of miR-2942 in larvae analyzed by ANOVA (F = 19.648, p = 0.002). * A significant decrease (p < 0.001) of expression in inhibitor group compared with the negative control group (analyzed by statistical method of ANOVA by LSD). ** No significant difference (p > 0.05) in expression level between inhibitor and negative control group (analyzed by statistical method of ANOVA, LSD). In all miRNA qPCR quantifications, the relative gene expressions from normal controls are calibrated to 1 (not shown in graph). Error bars show the SEM of three biological replicates, each containing one adult male/female.
Figure 3
Figure 3
Functional study of miRNA intervention after miR-mimic and inhibitor injection in Aedes albopictus developmental stages. [A] Hatching rate of embryo after miR-286b injection with mimic, inhibitor, negative control and normal un-injected embryos. * A significant difference (p < 0.001) of inhibitor group with the negative control group in hatching rate of eggs into larvae (statistically analyzed by ANOVA, LSD). The error bar shows the SD of values from three independent experiments, and is statistically evaluated by ANOVA (F = 191.557 & p < 0.001). [B] Daily hatching after embryo injection with miR-286b mimic, inhibitor, negative control and normal un-injected embryo. The 1st hatching day is 2 days after the microinjection. The error bar shows the SD of values from three independent experiments. [C] Eclosion rate in larvae after miR-2942 injection with mimic, inhibitor, negative control and normal un-injected embryo analyzed statistically by ANOVA (F = 38.193 & p < 0.001). * A statistically significant difference (p < 0.001) of inhibitor group with the negative control group and other two groups (statistically analyzed by ANOVA, LSD). A, B, C indicates statistically significant difference (p < 0.001) between the groups (statistically analyzed by ANOVA, LSD). The error bar shows the SD of values from three independent experiments. [D] Fecundity rate after miR-1891 injection. Result analyzed by ANOVA (F = 58.886 and p < 0.001). *A significant decrease in mean eggs per female in inhibitor group (p < 0.001) compared with negative control group (statistically analyzed by ANOVA, LSD) and all other groups observed. The error bar shows the SD of values from three independent experiments.
Figure 4
Figure 4
Longevity (Life table) of male and female adult Ae. albopictus after miR-1891 intervention. (A) Life table of male Ae. albopictus. A significant decrease in survival ratio was shown after miR-1891 inhibitor injection in adults (p < 0.001). Statistical analysis by Kaplan-Meier showed X2 =33.326, p < 0.05 (B) Life table of female Ae. albopictus. Statistical analysis by Kaplan-Meier showed X2 = 4.274, p < 0.05.
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