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Rotation in Molecular
Entomology labKounadi Stella
June 2015
Tissue localization of CCEae3a esterase in Aedes albopictus
larvae
Aedes albopictus• Major invasive vector • Viral diseases: Dengue fever, chikungunya• Temephos is being used in many countries to
control its population size
Temephos• Larvicide• Organophosphate insecticide
Organophosphates• affect the nervous system• inactivation acetylcholinesterase activity• disrupts nerve impulses and • kill or disable the insect
Molecular basis of resistance against temephosIn the Resistant strain:• Increased esterase activity• CCEae3a transcriptionally up-regulated and gene
amplified• Genetic crosses further confirmed the implication
of CCEae3a in the resistance phenotype
Objective of this study :Investigate in which tissues CCEae3a is expressed?
Mosquitoes strains used in this study
Parental: field selected population from Greece (2010)Resistant: mosquitoes after selection of the parental stain in the laboratory for 12 generations with temephos Susceptible: susceptible laboratory stain (origin Malaysia)
Methods• Western blot• Cryosections• Paraffin sections
Western blot• Dissection of mosquito larvae• Homogenization of tissues• Electrophoresis & immunoblotting
Cryosections• Fixation for 3, 5 and 7 hours• Problems in tissue cohesion
Paraffin sections• Overnight fixation• Paraffin embedded larvae
DNA
DNA
CCEaea3a Merge
MergePre-immune serum
Malp. Tub.
Malp. Tub. 10X
20X
DNA CCEaea3a Merge
Paraffin-sections from larvae specimens
10X
20X
Malp. Tub.
Malp. Tub.
DNA CCEaea3a Merge
10X
20X
Nerve cord
Nerve cord
Pre-immune serumDNA Merge
Nerve cord
ResultsExpression of CCEae3a in• Malpigian tubules• Nerve cord• Carcass
Analysis of population structure and insecticide resistance in
Culex pipiens mosquitoes from two agricultural ecosystems in
N. Greece
Objective• Analysis of biotype frequency and distribution of
Culex pipiens mosquitoes in two areas of interest • Analysis of Culex pipiens resistance status to
insecticides • Correlation between biotype and target site
resistance
Culex pipiens • Main vector of West Nile virus • one the most prevalent species in Northern Greece• During 2010, Greece underwent the second largest
WNV epidemic in Europe • WNV outbreak in 2010-2012 in Central Macedonia
(57 deaths)
Areas of interest • Thessaloniki• Evros
• Use of insecticides for agricultural applications
• development of insecticide resistance
Methods• DNA extraction from individual mosquitoes • Molecular diagnostic assays for
1. Biotype identification2. Recording of target site mutations• Kdr (L1014F)• F290V• G119S
Biotype identification Culex pipiens pipiens with two forms : • molestus and • pipiens
Diagnostic PCR for detection of biotype
Kdr (Knockdown resistance) status• Target site resistance mechanism to pyrethroids• mutations in the voltage-gated sodium channel gene
disabling insecticide binding
Kdr diagnostic PCR for detection of L1014F resistant mutationRR RS
F290V resistance status• GTT to TTT• mutation in acetylcholinesterase-1 • Target site resistance to Organophosphates & Carbamates
F290V diagnostic PCR for detection of mutant alleleSS RS
G119S resistance mutation• Target site resistance to Organophosphates, Carbamates
• mutation in acetylcholinesterase-1• PCR/RFLP diagnostic test• Digestion of PCR product with Alul restriction enzyme • The presence of the G119S creates a AluI restriction site in the ace-1
gene of resistant individuals
RSSS
BiotypeBiotype(n=154) Θεσ/κη %pipiens 78,30%molestus 6,00%hybrid 15,70%
Eβρος %pipiens 93%molestus 4%hybrid 3%
Culex pipiens pipiens have a higher representation in both Thessaloniki and Evros
KDR ΚDR (L1014F)Genotype (n=177) Θεσ/κη %RR 44,60%RS 54,30%SS 1%
Eβρος %RR 62,30%RS 36,50%SS 1,20%
Higher frequency of RR mosquitoes in Evros compared to Thessaloniki
L1014C mutation• The available PCR diagnostic cannot
discriminate between L1014F and L1014C
• Sequencing of 15 individuals after PCR amplification
• Development of diagnostic
#mosquitoes KDR mutation1 RR L1014F
14 RR L1014C
F290V & G119SF290V Genotype (n=166) Θεσ/κη %RR 0,00%RS 1,30%SS 99%
Eβρος %RR 0,00%RS 1,10%SS 98,90%
G119SGenotype (n=88) Θεσ/κη %RR 0,00%RS 45,30%SS 55%
Eβρος %RR 0,00%RS 7,80%SS 92,20%
F290V mutationHigher frequency of SS mosquitoes in both Evros and Thessaloniki
G119S mutationNo RR mosquitoesMuch higher RS in Thesssaloniki compared to Evros
These results may be helpful• for surveillance• to adopt a proactive approach for mosquito control
and guide the use of insecticide • to develop strategies for managing the insecticide
resistance