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. 2013 May 27:4:6.
doi: 10.5281/zenodo.10894766. eCollection 2013.

Development of an RNAi based microalgal larvicide to control mosquitoes

Anil Kumar  1   2   3 , Songjie Wang  4 , Ruguang Ou  4 , Mustapha Samrakandi  1   5 , Brenda T Beerntsen  4 , Richard T Sayre  1   5
Affiliations

Development of an RNAi based microalgal larvicide to control mosquitoes

Anil Kumar et al. Malariaworld J. .
No abstract available

PubMed Disclaimer

Conflict of interest statement

Competing interests: No competing interests declared.

Figures

Figure 1.
Figure 1.
a: Design of the 3-HKT inverted repeat construct used for Chlamydomonas chloroplast transformation. The two black arrows indicate the binding sites of primers used for PCR confirmation of Chlamydomonas chloroplast transformants. b: PCR confirmation of Chlamydomonas chloroplast transformants. -ve: water control, C: non transgenic parent strain (CC-4147), 1-15: Chlamydomonas chloroplast transformants, +ve: plasmid control as template.
Figure 2.
Figure 2.
Effect of feeding Chlamydomonas chloroplast trans-formants expressing 3-HKT dsRNA on Anopheles stephensi larvae. a) An.stephensi larval mortality observed with Chlamydomonas chloroplast (CC-4147/pCVAC108) transformants expressing 3-HKT dsRNAs. Twenty (1st experiment) or 30 (2nd experiment) newly hatched An. stephensi larvae were reared on algae plus 1/3rd yeast+ sera micron (1/3rd amount of yeast+micron mixture fed to control larvae). Control larvae were reared on non-transgenic algae with a yeast+sera micron mixture diet. Data shown are the average of two experiments. For clone 108-13 results were identical (40% mortality) in both the experiments, hence there is no variation. b) Different phenotypes exhibited by An. stephensi larvae following feeding on transgenic algae producing 3-HKT dsRNA. 1) Dead larvae of An. stephensi reared on transgenic algae. 2) Comparison of a larva exhibiting growth inhibition (red arrow) upon feeding on transgenic algae to a normally developing larva (green arrow) on day 12. 3) Image of a dead pupa (red arrow) and a live pupa (green arrow) in an experimental well with transgenic algae.
Figure 3.
Figure 3.
Confirmation of 3-HKT dsRNA transcription in Chlamydomonas chloroplast transformants. CC4147 chloroplast transformants 13 and 15 were tested for expression of 3-HKT dsRNA by RT-PCR. psbD, a chloroplast gene was used as control in the experiment. Absence of any band in the control PCR experiments for psbD (lanes 2 to 4) and 3-HKT (lanes 9 and 10) indicates that all the RNA samples were free of DNA contamination. Amplification of psbD in lanes 5 to 7 indicates that cDNA synthesis was successful and the absence of the 3-HKT band in CC4147 control (lane 11) was real as it lacks 3-HKT. Presence of the 3-HKT band in CC4147/pCVAC108 clones 13 and 15 confirms that these are transgenics and they transcribe the 3-HKT dsRNA. Loading pattern: 1:100 bp ladder, 2: Parent strain CC-4147 no RT, PCR with psbD primers, 3:108-13 no RT, PCR with psbD primers, 4: CC4147/pCVAC108-15 no RT, PCR with psbD primers , 5: Parent strain CC4147 cDNA, PCR with psbD primers, 6: CC4147/pCVAC108-13 cDNA, PCR with psbD primers, 7: CC4147/pCVAC108-15 cDNA, PCR with psbD primers, 8: Parent strain CC4147 no RT, PCR with 3-HKT primers, 9: CC4147/pCVAC108-13 no RT, PCR with 3-HKT primers, 10: CC4147/pCVAC108-15 no RT, PCR with 3-HKT primers, 11: Parent strain CC4147 cDNA, PCR with 3-HKT primers, 12: CC4147/pCVAC108-13 cDNA, PCR with 3-HKT primers,13: CC4147/pCVAC108-15 cDNA, PCR with 3-HKT primers, 14: 100 bp ladder.
Figure 4.
Figure 4.
Real time analysis to measure steady state levels of 3-HKT transcript among surviving/dead An. stephensi larvae and pupae reared on transgenic Chlamydomonas expressing 3-HKT dsRNA. In each experiment 3-HKT expression was compared and normalised to An. stephensi actin transcript levels. The data shown in each figure is the average of three biological replicates. Each biological replication involved five technical replications for each treatment and control. The mean ΔΔCT values from three biological replicates were subjected to analysis through single factor ANOVA. Asterisks indicate the treatments that had significantly lower 3-HKT transcript levels relative to the parental control, determined by single factor ANOVA (P<0.05). RNA extracted from larvae/pupae reared on nontransgenic Chlamydomonas was used as the control in all experiments. In all experiments A, B, C (as in 108-13A1) etc. refer to the wells from which the larvae/pupae were sourced for analysis and the numbers 1, 2 refer to the first or second larvae/pupae used for analysis from a single well. a) 3-HKT transcript levels observed among surviving larvae reared on clones 108-13 and 108-15. Except for larvae 108-15C1, which did not differ significantly from the control larvae, all other treatments showed significant reduction in 3-HKT transcript levels compared to the parental control. b) 3-HKT transcript levels observed among dead pupae from larvae reared on 108-13. c) 3-HKT transcript levels observed among dead pupae from larvae reared on the 108-15 transgenic Chlamydomonas clone.
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