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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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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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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.

Mentions: The effect of miR-1891, on longevity after knock-in and knock-down with miRNA mimic and inhibitor in both male and female Ae. albopictus, were recorded separately for each group and compared with the control groups (Figure 4). Normally, Ae. albopictus under laboratory conditions have a life span of 20–22 days for males and 28–30 days for females. Here we have observed a significant decrease in the life span of both female and male adults in the miR-1891 inhibitor group compared to mimic and control groups (p < 0.001). The mean survival time of males decreased from 16.5 days in the mimic group to 11.33 days and the mean survival time in females reduced from 24.75 days to 13.35 days when miR-1891 expression was knocked-down with the inhibitor.


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)

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

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

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.
Mentions: The effect of miR-1891, on longevity after knock-in and knock-down with miRNA mimic and inhibitor in both male and female Ae. albopictus, were recorded separately for each group and compared with the control groups (Figure 4). Normally, Ae. albopictus under laboratory conditions have a life span of 20–22 days for males and 28–30 days for females. Here we have observed a significant decrease in the life span of both female and male adults in the miR-1891 inhibitor group compared to mimic and control groups (p < 0.001). The mean survival time of males decreased from 16.5 days in the mimic group to 11.33 days and the mean survival time in females reduced from 24.75 days to 13.35 days when miR-1891 expression was knocked-down with the inhibitor.

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