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Senior Research: Assessment of AChE inhibition by Malathion on Embryonic Development of Zebrafish

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Leona Cheng

on 23 April 2014

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Transcript of Senior Research: Assessment of AChE inhibition by Malathion on Embryonic Development of Zebrafish

MANHATTANVILLE COLLEGE
SENIOR RESEARCH
RESEARCH
BACKGROUND
ACKNOWLEDGEMENTS


I would also like to show appreciation to my partner Joan Samantha Corea for assisting embryo breeding and collection. Thank you to Dr. Anna Yeung-Cheung in helping with calculating malathion solution. And lastly, special thanks Professor Nancy E. Todd for all the help and support throughout this entire experience.


HYPOTHESIS
GOAL: Observe the effects of FAV amount of Malathion on zebrafish embryos
EXPERIMENTAL
TIMELINE
DISCUSSION
RESULTS
MAINTENANCE
EXPOSURE
BREEDING + COLLECTION
FUTURE INVESTIGATIONS
quantification of AChE for each group
dopamine, quantifying the amount of dopamine
UNDER THE HOOD
The assessment of acetylcholinesterase inhibition by malathion on the early-stage development of zebrafish
(
Danio rerio
)

Leona Cheng

In Partial Fulfillment of a Senior Research Project Under The Supervision of Dr. Nancy E. Todd

April 2014

Synaptic cleft
acetyl-CoA
acetate
choline
acetylcholine
Choline Acetyl Transferase
CoA
acetylcholinesterase
Choline Carrier
acetylcholinesterase
receptor
Malathion
Acetylcholine
Acetulcholinesterase
Dopamine
Prolactin
ZHE1 and ZHE2
POSSIBLE MECHANICS
post synaptic membrane
Pituitary Gland
Hypothalamus
OP PESTICIDES
Gland Cell
Known to inhibit AChE activity; Malathion, a common insecticide, was proven to act like an AChE inhibitor
Binds irreversibly to cholinesterase; ultimately acting as an AChE inhibitor.
In various studies, organophosphorous pesticides have been known to have an impact on the embryonic development of aquatic life
MALATHION
Common household Insecticide
Organophosphorous (OP)
Malathion present in untreated water is degraded into malaoxon
Final Acute Value (FAV) for freshwater of 0.86 ug of Malathion

ZEBRAFISH
Kingdom: Animalia
Phylum: Chordata
Class: Actinopterygii
Order: Cypriniformes
Family: Cyprinidae

BACKGROUND
Tropical freshwaters
Southeast Asia
6-38 degrees celsius
SCIENTIFIC MODEL
Inexpensive
Small; can be kept in large numbers
High yield in embryos
Transparent embryo
Rapid transition from egg to fry within a 72 hpf

Embryos often used as toxicology models
HATCHING
ENVIRONMENT
PHOTOPERIOD CONTROL

BREEDING TANK
INCUBATOR ENVIRONMENT
MALATHION PREPARATION
0.4 μg/l
0.8 μg/l
PPE
Using a micropipetter, 2μl of malathion purchased from Sigma Aldrich was carefully mixed into a beaker with 300ml of incubated filtered water to create the 0.8μg/l malathion solution
100ml of 0.8μg/l was mixed into a beaker with 100ml of incubated filtered water to create the 0.4μg/l malathion solution

MALATHION EXPOSURE
TRIAL MAINTENANCE
Photoperiod
Oxygenating appropriate water environment

2 MALE FISH
3 FEMALE FISH
STAGING
HATCHING
27°C
FILTERED WATER
ISOLATION OF EMBRYOS VIA 6-WELL PLATED
LAMP
FOOD
WATER TREATMENT
Embryos were staged using
Stages of Embryonic Development of the Zebrafish
(Kimmel, 1995) as a reference

Embryos were observed at
0 hpf
24 hpf
48 hpf
72 hpf
During the observations at the times listed below, hatching of embryos were recorded

Embryos were observed at
0 hpf
12 hpf
24 hpf
36 hpf
48 hpf
60 hpf
72 hpf
84 hpf
96 hpf
Zebrafish purchased from Carolina Biological Company
Light (14:10 light dark)
Heater + Thermometer
Filter
Gravel
Plastic Plants
Card Board Box
Start Right
used to remove chlorine from water

Freeze dried blood worm
Freeze dried brine shrimp
DEVELOPMENT

Danio rerio
from: http://fishbase.org.cn/images/species/Darer_m0.jpg

BACKGROUND
From: http://0.tqn.com/d/chemistry/1/7/l/K/1/malathion.jpg
FIGURE #. Developmental rate of embryos exposed to 0.4 μμug/L and 0.8 μug/L malathion between the hours of 0-24 hpf, 24-48 hpf and 48-72 hpf compared to control
FIGURE #. Developmental rate of embryos exposed to 0.4 μug/L malathion between the hours of 0-24 hpf, 24-48 hpf and 48-72 hpf compared to control
FIGURE 1.Developmental rate of zebrafish embryos exposed 0.8 uμg/L malathion between the hours of 0-24 hpf, 24-48 hpf and 48-72 hpf compared to control
FIGURE #. Developmental rate of embryos exposed to 0.4 μμug/L and 0.8 μug/L malathion between the hours of 0-24 hpf compared to control
FIGURE #. Developmental rate of embryos exposed to 0.4 μμug/L and 0.8 μug/L malathion between the hours of24-48 hpf compared to control
FIGURE #. Developmental rate of embryos exposed to 0.4 μμug/L and 0.8 μug/L malathion between the hours of 48-72 hpf compared to control
DEFORMATIONS
FIGURE 1. Mean percentage of hatching and mortality rate of zebrafish embryos exposed to 0.4 μg/L and 0.8 μg/L malathion between the hours of 0-24 hpf, 24-48 hpf and 48-72 hpf.
FIGURE 2. Mean percentage of hatching and mortality rate of zebrafish embryos exposed to 0.4 μg/L and 0.8 μg/L malathion between the hours of 0-24 hpf
FIGURE 3. Mean percentage of hatching and mortality rate of zebrafish embryos exposed to 0.4 μug/L and 0.8 μug/L malathion between the hours of 24-84 hpf
FIGURE 4. Mean percentage of hatching and mortality rate of zebrafish embryos exposed to 0.4 μg/L and 0.8 μg/L malathion between the hours of 48-72 hpf
FIGURE 5. Mean percentage of hatching and mortality rate of zebrafish embryos exposed to 0.4 uμg/L malathion between the hours of 0-24 hpf, 24-48 hpf and 48-72 hpf
FIGURE 1. Mean percentage of hatching and mortality rate of zebrafish embryos exposed to 0.8 uμg/L malathion between the hours of 0-24 hpf, 24-48 hpf and 48-72 hpf
0.000
0.000
0.000
0.025
0.021
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.025
0.021
Deformations observed with embryos treated with malathion were

Enlarged body
Edema in the cardiovascular area
Various degrees of kinked/arched tail
Small eye diameter
Shortened tail
Lack of pigmentation






Bradycardia
Heart palpitations

0.8μg/l
0.4μg/l
Exposure to malathion 24-48 hpf, will (overload the ACh receptors)
inhibiting the release of egg hatching enzymes
delay in development
Control
0.4μg/l dosed at 0-24hpf
0.4μg/l dosed at 24-48hpf
0.4μg/l dosed at 48-72hpf
0.8μg/l dosed at 0-24hpf
0.8μg/l dosed at 24-48hpf
0.8μg/l dosed at 48-72hpf
** ALL GROUPS DOSED WERE RINSED WITH INCUBATED FILTERED WATER TO REMOVE MALATHION AFTER EXPOSURE
*
*
*
*
*
*
*
*
*
Malathion, at both doses administered at 48-72 hpf, inhibits hatching
secretion of HE from vesicles in the head at those hours
Malathion at both doses administered at 0-24 hpf
displayed delays
development (expected)
hatching
resulted a higher yield for deformations; severe kinked/arched tails

During the 0-24hr period malathion act as AChE inhibitor ultimately causing multiple cascade effects resulting in delay in development but also deformities in the axial skeleton and somite development
-Hanneman and Westerfield (1989) AChE at different times might have different pathways
Studies done at Mville
Cheng
et al
(2007) - carbaryl; similar deformations due to accumulations
Hanneman and Westerfield (1989) - Early expression of AChE at 14 hpf -multiple pathways
Malathion administered at
0-24 hours showed a significance of 0.000 for both doses at all observational hours after exposure
24-48 hours showed a significance of 0.025 (0.4ug) and 0.021 (0.8ug) at the final day of observations

Improvements
perfecting breeding techniques
photographing embryos
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