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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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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.
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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.

Mentions: Hatching rate of embryos after miR-286b intervention showed a profound decrease in the number of embryos that hatched after knock-down of miR-286b expression (p < 0.001). This knock-down effect was carried throughout till the development to adults as indicated by the reduced number of larvae developed to pupae and adults in this group. The percentage of mean hatching rate decreased from 74.43% in mimic group to 15.28% in inhibitor group (analyzed by ANOVA, F =191.557 & p < 0.001, Figure 3A).


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)

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

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Show All Figures
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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.
Mentions: Hatching rate of embryos after miR-286b intervention showed a profound decrease in the number of embryos that hatched after knock-down of miR-286b expression (p < 0.001). This knock-down effect was carried throughout till the development to adults as indicated by the reduced number of larvae developed to pupae and adults in this group. The percentage of mean hatching rate decreased from 74.43% in mimic group to 15.28% in inhibitor group (analyzed by ANOVA, F =191.557 & p < 0.001, Figure 3A).

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