1st rotation presentation
TRANSCRIPT
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Rotation in Molecular
Entomology labKounadi Stella
June 2015
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Tissue localization of CCEae3a esterase in Aedes albopictus
larvae
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Aedes albopictus• Major invasive vector • Viral diseases: Dengue fever, chikungunya• Temephos is being used in many countries to
control its population size
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Temephos• Larvicide• Organophosphate insecticide
Organophosphates• affect the nervous system• inactivation acetylcholinesterase activity• disrupts nerve impulses and • kill or disable the insect
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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?
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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)
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Methods• Western blot• Cryosections• Paraffin sections
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Western blot• Dissection of mosquito larvae• Homogenization of tissues• Electrophoresis & immunoblotting
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Cryosections• Fixation for 3, 5 and 7 hours• Problems in tissue cohesion
Paraffin sections• Overnight fixation• Paraffin embedded larvae
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DNA
DNA
CCEaea3a Merge
MergePre-immune serum
Malp. Tub.
Malp. Tub. 10X
20X
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DNA CCEaea3a Merge
Paraffin-sections from larvae specimens
10X
20X
Malp. Tub.
Malp. Tub.
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DNA CCEaea3a Merge
10X
20X
Nerve cord
Nerve cord
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Pre-immune serumDNA Merge
Nerve cord
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ResultsExpression of CCEae3a in• Malpigian tubules• Nerve cord• Carcass
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Analysis of population structure and insecticide resistance in
Culex pipiens mosquitoes from two agricultural ecosystems in
N. Greece
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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
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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)
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Areas of interest • Thessaloniki• Evros
• Use of insecticides for agricultural applications
• development of insecticide resistance
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Methods• DNA extraction from individual mosquitoes • Molecular diagnostic assays for
1. Biotype identification2. Recording of target site mutations• Kdr (L1014F)• F290V• G119S
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Biotype identification Culex pipiens pipiens with two forms : • molestus and • pipiens
Diagnostic PCR for detection of biotype
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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
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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
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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
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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
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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
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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
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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
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