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