If you have any problems related to the accessibility of any content (or if you want to request that a specific publication be accessible), please contact us at email@example.com.
In Vivo Gene Knockdown Techniques and the Establishment of Cryopreservation Methods in Culex spp. Mosquitoes
AuthorSandoval Garcia, Roxana
AltmetricsView Usage Statistics
Culex (Cx.) pipiens, known as the common house mosquito (subspecies Cx. pipiens quinquefasciatus and Cx. pipiens pipiens) affects humans and animals through its ability to transmit numerous viruses and parasites such as West Nile Virus (WNV), filarial nematodes, and Plasmodium relictum, the causative agent of avian malaria. RNA interference (RNAi) is an antiviral pathway that is well studied in most arthropods such as Aedes aegypti mosquitoes. In Culex. spp., aspects of the RNAi mechanism are not completely understood, including the role of a potentially antiviral protein, P-element- Induced Wimpy Testis-2 (PIWI2). One of the objectives of this thesis was to knock down PIWI2 gene expression in vivo to investigate its role during virus infection. This was implemented by introducing double-stranded RNA (dsRNA) into mosquitoes to stimulate RNAi-mediated gene silencing of PIWI2. In this study, dsRNA was introduced into adult Cx. quinquefasciatus mosquitoes and pupae via intrathoracic injection or soaking in dsRNA, respectively. However, a significant knockdown of PIWI2 was not achieved using either dsRNA delivery method. Additional experiments testing dsRNA knockdown efficiency in Cx. quinquefasciatus Hsu cells indicated that the dsRNA used for our studies may not have been effective. The next approach would be to conduct further in vivo tests utilizing a different dsRNA production technique and increased quantities of dsRNA. In this study we additionally aimed to contribute to mosquito related research methods. Continuous maintenance of Culex eggs is required for such in vivo studies but is labor-intensive and costly. Thus, the second objective of this thesis was to establish a method for the cryopreservation of Cx. pipiens embryos.Cryopreservation would permit extended storage of Culex mosquito lines without the burden of continual colony rearing. The methodology and effects of dechorionation, permeabilization, and handling on embryo viability were tested. Lab grade sodium hypochlorite and heptane successfully permeabilized 6-9 hr old Culex embryos, but this treatment was lethal. Additional experimentation is needed to determine the optimal conditions for the permeabilization of Culex embryos.