CRISPR Cas9 mediated knockout of sex determination pathway genes in Aedes aegypti.

The vector role of Aedes aegypti for viral diseases including dengue and dengue hemorrhagic fever makes it imperative for its proper control. Despite various adopted control strategies, genetic control measures have been recently focused against this vector. CRISPR Cas9 system is a recent and most efficient gene editing tool to target the sex determination pathway genes in Ae. aegypti. In the present study, CRISPR Cas9 system was used to knockout Ae. aegypti doublesex (Aaedsx) and Ae. aegypti sexlethal (AaeSxl) genes in Ae. aegypti embryos. The injection mixes with Cas9 protein (333 ng ul-1) and gRNAs (each at 100 ng ul-1) were injected into eggs. Injected eggs were allowed to hatch at 26 ± 1°C, 60 ± 10% RH. The survival and mortality rate was recorded in knockout Aaedsx and AaeSxl. The results revealed that knockout produced low survival and high mortality. A significant percentage of eggs (38.33%) did not hatch as compared to control groups (P value 0.00). Highest larval mortality (11.66%) was found in the knockout of Aaedsx female isoform, whereas, the emergence of only male adults also showed that the knockout of Aaedsx (female isoform) does not produce male lethality. The survival (3.33%) of knockout for AaeSxl eggs to the normal adults suggested further study to investigate AaeSxl as an efficient upstream of Aaedsx to target for sex transformation in Ae. aegypti mosquitoes.

CRISPR/Cas9 mediated sex-ratio distortion by sex specific gene editing in Aedes aegypti.

Aedes aegypti is a principal vector for several viruses including dengue virus, chikungunya virus and zika virus. Economic burden of mosquito-borne diseases, relative failure of traditional control strategies and the resistance development against insecticides enforces towards genetic manipulation of Ae. aegypti. Hence, a key gene doublesex (Aedsx) which regulate sex differentiation and alternatively splices to form male and female specific transcripts (AedsxM and AedsxF ). CRISPR/Cas9 technique was employed to sex specifically disrupt the female-specific isoforms, AedsxF1 and AedsxF2 , both of which were shown to be expressed only in female mosquitoes. Targeting of dsxF at the developmental stage has resulted in various phenotypic anomalies of adult females. The rate of adult mutation phenotype was recorded between 29 and 37% along with anomalies of wing size, proboscis length and reduction in the sizes of pre-blood-meal and after blood-meal ovaries in dsxF1 and dsxF2 microinjected groups, respectively. These findings can be correlated with reduced fecundity rate of Go female, where AedsxF1 and AedsxF2 groups showed reduction rate in range of 23-31%. Furthermore, hatching inhibition rate of 28 to 36% was also observed in G1 generation when compared to the wildtype. Overall, these results demonstrated that AedsxF disruption has resulted in multiple female traits disruption including decreased fertility of the female that could directly or indirectly associated with reproduction and its disease transmitting abilities. All these findings suggesting that CRISPR works to alter the developmental pathways as predicted, and therefore this method potentially gives us the basis for the sex-ratio distortion system as genetic control approach for the management of this vector.

Insecticidal and Genotoxic effects of some indigenous plant extracts in Culex quinquefasciatus Say Mosquitoes.

Five different weed plants viz. Convulvulus arvensis, Chenopodium murale, Tribulus terrestris, Trianthema portulacastrum, and Achyranthes aspera were investigated for their entomocidal and genotoxic effects against Culex quinquefasciatus mosquitoes. High mortality was observed at 72 hours in a dose dependent manner. Among all the tested plants, A. aspera was found highly significant which showed 100% mortality at 250 ppm after 72 hours with LC50 of 87.46, 39.08 and 9.22 ppm at 24, 48, respectively. In combination with Bacillus thuringiensis israelensis (Bti); A. aspera also caused 100% mortality at 250 ppm concentration after 72 hours (LC50 8.29 ppm). Phytochemical analysis of all the tested weed plants showed the presence of flavonoids, saponins, tannins, steroids, cardiac glycosides, alkaloids, anthrequinones and terpenoids. Random Amplification of Polymorphic DNA-Polymerase chain reaction (RAPD-PCR) and comet assay were performed to assess the genotoxic effect of A. aspera but no change in DNA profile was observed. Furthermore, FTIR showed the presence of phenolic compounds in A. aspera extract. It is suggested that certain phenolic compounds such as flavonoids modulate the enzymatic activity and, hence, cause the death of larvae of Cx. quinquefasciatus. Altogether, current study would serve as an initial step towards replacement of synthetic insecticides to plant-microbe based biopesticide against Culex mosquitoes in future.

Insecticidal, biological and biochemical response of Musca domestica (Diptera: Muscidae) to some indigenous weed plant extracts.

In the current study; insecticidal, growth regulation, oviposition deterrence and repellency of petroleum ether extracts of Azadirachta indica, Penganum harmala, Datura stramonium, Tribulus terrestris and Chenopodium murale against 2nd instar larvae of housefly was investigated. Five different concentrations (5%, 10%, 15%, 20% and 25%) were used through larval feeding and the mortality data was recorded after 24, 48 and 72 hrs. Highest mortality was induced by P. harmala (63.87%) followed by D. stramonium (62.78%), A. indica (53.84%), T. terrestris (41.86%) and C. murale (4.09%) after 72 h at 25% concentration, respectively. Increased mortality was observed with increased time duration and concentration. Longest larval duration (9.33 ±â€¯0.33 days) and pupal duration (7.33 ±â€¯0.33 days) days) was recorded in larvae treated with 25% concentration of P. harmala which also caused a decrease in the activity of AChE, ACP, AKP, α-Carboxyl, and ß-Carboxyl enzymes. However, at 25% concentration, C. murale showed highest oviposition deterrence activity (81.88%) followed by D. stramonium (79.58%). In comet assay test, at highest concentration (25%) the mean comet tail lengths represented by Penganum harmala, Datura stramonium and Azadirachta indica (Reference plant) were 10.20 ±â€¯0.49, 9.20 ±â€¯0.37 and 7.80 ±â€¯0.49 µm while percent DNA damage was 10.56 ±â€¯0.77, 10.67 ±â€¯1.62 and 8.11 ±â€¯0.85% respectively compared to controls cells. Phytochemical analysis indicated the presence of flavonoids, steroids, saponins, cardiac glycosides, tannins, alkaloids, terpenoids and anthraquinones. Fourier Transform Infrared spectroscopy (FTIR) analysis revealed the presence of phenolic flavonoids, saponins, tannins as major functional groups. Further studies are needed to explore and thus, to incorporate weed plant extracts for the management of house flies.