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

Puthiyakunnon S, Yao Y, Li Y, Gu J, Peng H, Chen X - Parasit Vectors (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Prevention and Control for Emerging Infectious Diseases of Guangdong Higher Institutes, Department of Pathogen Biology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou North Avenue No.1838, Guangzhou, China.

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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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.
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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.

Mentions: The relative expression of three miRNAs after their knock-in and knock-down with their mimics and inhibitors in the insect cell line C6/36, were quantified by Bulge loop miRNA qRT-PCR (Figure 1A-C). The relative expression of three miRNAs after miRNA intervention with mimics and inhibitors were also performed in specific developmental stages of Ae. albopictus and quantified by Bulge loop miRNA qRT-PCR (Figure 2A-C). The U6 primers were used as the controls in both in vitro and in vivo expression. A significant level of knock-in and knock-down efficiency was shown by three miRNA mimics and inhibitors against their miRNA expression in specific developmental stages; embryos, larvae and adults of Ae. albopictus.


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

Puthiyakunnon S, Yao Y, Li Y, Gu J, Peng H, Chen X - Parasit Vectors (2013)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750763&req=5

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.
Mentions: The relative expression of three miRNAs after their knock-in and knock-down with their mimics and inhibitors in the insect cell line C6/36, were quantified by Bulge loop miRNA qRT-PCR (Figure 1A-C). The relative expression of three miRNAs after miRNA intervention with mimics and inhibitors were also performed in specific developmental stages of Ae. albopictus and quantified by Bulge loop miRNA qRT-PCR (Figure 2A-C). The U6 primers were used as the controls in both in vitro and in vivo expression. A significant level of knock-in and knock-down efficiency was shown by three miRNA mimics and inhibitors against their miRNA expression in specific developmental stages; embryos, larvae and adults of Ae. albopictus.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Key Laboratory of Prevention and Control for Emerging Infectious Diseases of Guangdong Higher Institutes, Department of Pathogen Biology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou North Avenue No.1838, Guangzhou, China.

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.

Show MeSH