FlyNap as a potential anesthetic agent for the study of mosquito

heart physiology

Weihan Chen and Julián Hillyer Department of Biological Sciences

Vanderbilt University

Hemolymph Circulation In Mosquitoes

•  Function – Transport of

nutrients, wastes, and hormones.

–  Immune surveillance and pathogen killing.

•  Structure – Dorsal vessel.

•  Abdominal heart. •  Thoracic aorta.

Purpose

•  FlyNap (FN) –  Used primarily to anesthetize Drosophila

melanogaster. –  Ingredients

•  50% Triethylamine (active ingredient) •  25% Ethanol •  25% Fragrances

•  Purpose –  Determine whether FlyNap affects

mosquito heart physiology and survival. –  Establish dosage values that are

effective as anesthesia but do not interfere with normal physiological functions.

Heart Parameters Measured Physiological Parameters Definition

Anterograde contraction rate

Beats/second towards the head.

Retrograde contraction rate

Beats/second towards the abdomen.

Total contraction rate Total beats/second. % time anterograde % of time contracting toward

the head. % time retrograde % of time contracting toward

the abdomen. Reversals Number of times the heart

contractions switch directions.

Methods: Experiment 1

Anopheles gambiae

1 min cold anesthesia 10 sec FN 60 sec FN 5 min FN

60 sec recording 60 sec recording 60 sec recording 60 sec recording

Measure Heart Parameters

30 sec FN

60 sec recording

Results

•  Trend of increasing heart rate after short exposure to FlyNap (FN).

•  Prolonged exposure to FlyNap results in a significant decrease in heart rate.

Asterisks show p<0.05 when comparing the marked column with the reference (R) group (Tukey’s).

0

0.5

1

1.5

2

2.5

Cold 10 sec FN 30 sec FN 1 min FN 5 min FN

beat

s/se

cond

Treatments

Anterograde Contraction Rate

0

0.5

1

1.5

2

2.5

Cold 10 sec FN 30 sec FN 1 min FN 5 min FN

beat

s/se

cond

Treatments

Retrograde Contraction Rate

0

0.5

1

1.5

2

2.5

Cold 10 sec FN 30 sec FN 1 min FN 5 min FN

beat

s/se

cond

Treatments

Total Contraction Rate

R R

R

*

* *

Results

•  30 second and 1 min exposure to FlyNap decreased % time anterograde.

Asterisks show p<0.05 when comparing the marked column with the reference (R) group (Tukey’s).

0

10

20

30

40

50

60

70

80

Cold 10 sec FN 30 sec FN 1 min FN 5 min FN

%

Treatments

% Time Anterograde

0

10

20

30

40

50

60

Cold 10 sec FN 30 sec FN 1 min FN 5 min FN

%

Treatments

% Time Retrograde

0 2 4 6 8

10 12 14

Cold 10 sec FN 30 sec FN 1 min FN 5 min FN

# of

reve

rsal

s

Treatments

Reversals

** *

* *

R

R

R

•  Experiment 2 tested the effect of FlyNap on mosquito heart physiology 1 minute post-exposure vs. 1 hour post-exposure.

•  Analysis showed that there was no significant difference between the two time periods.

Experiment 2

Anopheles gambiae

1 min cold anesthesia 30 sec FN 30 sec FN followed by cold anesthesia

60 sec recording 60 sec recording

Measure Heart Parameters

Cold anesthesia followed by 30 sec FN

60 sec recording

Methods: Experiment 3

60 sec recording

Results

•  FN increased contraction rates. •  Exposure to cold + FN and FN +

cold both resulted in the same increase in contraction rates as exposure to FN alone.

Asterisks show p<0.05 when comparing the marked column with the reference (R) group (Tukey’s).

0.000

0.500

1.000

1.500

2.000

2.500

Cold FN Cold FN FN Cold

beat

s/se

cond

Treatments

Anterograde Contraction Rate

0.000

0.500

1.000

1.500

2.000

2.500

Cold FN Cold FN FN Cold

beat

s/se

cond

Treatments

Retrograde Contraction Rate

beat

s/se

cond

Total Contraction Rate

* * * *

* *

R

R

R

* *

*

Results

0 10 20 30 40 50 60 70 80

Cold FN Cold FN FN Cold

%

Treatments

% Time Anterograde

%

Treatments

% Time Retrograde

0 2 4 6 8

10 12 14 16

Cold FN Cold FN FN Cold

# of

reve

rsal

s

Treatments

# of Reversals •  FN exposure decrease % time

anterograde •  FN exposure decreased the

number of reversals.

Asterisks show p<0.05 when comparing the marked column with the reference (R) group (Tukey’s).

R

R

R

* * *

Experiment 4

Cold or 30 sec FN

60 sec recording

1) Injection with PBS or CCAP. 2) 10 min wait

60 sec recording

Measure Heart Parameters

Anopheles gambiae

Results

0.000

0.500

1.000

1.500

2.000

2.500

Cold PBS

Cold CCAP

FN PBS FN CCAP

beat

s/se

c

Treatments

Anterograde Contraction Rate

Pre-Injection 10 Min-Post Injection

0.000

0.500

1.000

1.500

2.000

2.500

Cold PBS

Cold CCAP

FN PBS FN CCAP

beat

s/se

c

Treatments

Retrograde Contraction Rate

Pre-Injection 10 Min-Post Injection

0.000

0.500

1.000

1.500

2.000

2.500

Cold PBS

Cold CCAP

FN PBS FN CCAP

beat

s/se

c

Treatments

Total Contraction Rate

Pre-Injection 10 Min-Post Injection

•  CCAP injection caused a significant increase in contractions for cold anesthetized mosquitoes.

•  This increase is not observed for FN anesthetized mosquitoes.

Asterisks show p<0.05 when comparing the pre and post-injection values (Sidaki’s test).

*

*

*

Results

0 2 4 6 8

10 12 14 16 18

Cold PBS

Cold CCAP

FN PBS FN CCAP

# of

reve

rsal

s

Treatments

# of Reversals

Pre-Injection 10 Min-Post Injection

•  FN exposure decreased % time anterograde.

•  No trend was observed for # of reversals.

Asterisks show p<0.05 when comparing the pre and post-injection values (Sidaki’s test).

*

Methods: Experiment 5

Anopheles gambiae (all females)

Cold Cold + E. coli 30 sec FN + E. coli

Containers Containers Containers

Count Surviving Mosquitoes (14 days)

30 sec FN

Containers

Cold + PBS 30 sec FN+PBS

Containers Containers

Results: Experiment 5

•  30 sec FlyNap exposure has a negative effect on the mosquito’s ability to resist bacteria (green vs. orange lines).

•  FlyNap did not effect mosquito survival after injury (red vs light blue).

Logrank analysis was done on data.

Summary •  Experiment 1:

–  Exposure to Fly-Nap caused increase in heart contraction relative to cold, and decreased the % time mosquito hearts spent contracting anterograde.

•  Experiment 2: –  After exposure to FlyNap, mosquito hearts remained stable.

•  Experiment 3: –  Mosquito heart contraction under cold anesthesia represent basal

contraction rates. –  Fly-Nap exposure increases heart contraction rates.

•  Experiment 4: –  Fly-Nap exposure significantly affected CCAP’s cardioacceleratory

effect on mosquito heart functions. •  Experiment 5

–  FlyNap exposure does not affect a mosquito’s ability to withstand injury.

–  However, FlyNap exposure has a detrimental effect on a mosquito’s ability to survive a bacterial infection.

•  These experiments show that FlyNap is not a viable alternative to cold anesthesia in studies assessing mosquito heart physiology and immune function.

Conclusions

•  Members of the Hillyer Lab – Tania Y Estévez-Lao – Jonas King

– Funding: •  This research was funded by the National

Science Foundation. •  Travel to 2012 ESA Conference was funded

by Vanderbilt University’s Department of Biological Sciences.

Acknowledgements

Background mosquito image obtained from www.vectorbase.org