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Daniele Simoneschi

on 19 March 2014

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Transcript of Zebrafish

Control counting vs. 50 /mL

Control counting vs. 33.3 /mL

Embryos were treated acutely at 33 /mL and 50 /mL malathion 52, 76, and 96 hpf
Assessment of Cardiotoxicity and Effects of Malathion on the Early Development of Zebrafish ( )
Using Computer Vision for Heart Rate Quantification

There are several methods to assess cardiac functions in zebrafish embryos:

1. Stopwatch counting

2. Micro pressure system

3. Laser Doppler microscope technique

4. Electrocardiogram

Department of Biology, Manhattanville College, Purchase, NY 10577

In partial fulfillment of a Senior Thesis Project under the supervision of Professor Nancy E. Todd

April, 2013
Cheng (2007) reported that insecticides, such as carbaryl (Sevin), work as acetylcholinesterase inhibitors, ultimately affecting zebrafish embryonic development (Todd and Van Leeuwen, 2002) and heart regularity.
Previous studies at Manhattanville (Lombardo, Elliott, Campagna) showed delays in hatching from malathion similar to carbaryl
Daniele Simoneschi
Zebrafish as a Model Organism
freshwater fish belonging to the family Cyprinidae (Dahm, 2006)

model organism in scientific research:
Carbaryl and Malathion
Malathion, a common organophosphate insecticide, has also been proven to act as an acetylcholinesterase inhibitor.
Malathion is an organophosphate insecticide widely used in agriculture, public health pest control, and public recreation areas.

Acute exposure to extremely high levels of malathion will cause:

- skin and eye irritation - cramps
- excessive sweating - nausea
- seizures - diarrhea
- death

Malathion present in untreated water is converted to malaoxon.

Malathion, therefore, should not be used in waters that may be used as
a source for drinking water, or any upstream waters.
Diethyl 2-[(dimethoxyphosphorothioyl)sulfanyl] butanedioate
Quaternary structure of acetylcholinesterase
Cholinesterase is a family of enzymes that catalyze the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a cholinergic neuron to return to its resting state after activation.
Informational Background
Malathion Background
Gland Cell
ZHE1 and ZHE2
Pituitary Gland
Cholinergic System
Possible Mechanism
But how do we detect zebrafish’s heart rate and assess malathion cardiotoxicity?
Malathion, acting as an acetylcholinesterase inhibitor, induces bradycardia in zebrafish embryos
-> requires skillful operator
-> expensive
-> labor-intensive
-> time-consuming
Current Methods
We have developed a non-invasive technique by video recording under stereomicroscope and examining video samples with our custom made software (FishBeat), to determine the heart rate of zebrafish embryos.
Our Method
Video is divided into frames
How FishBeat Works: Specifics
Heart region is cropped and converted to greyscale images

Consecutive frames are subtracted from each other
How FishBeat Works: Specifics
A bitmap of the most active heart areas is generated
How FishBeat Works: Specifics
How FishBeat Works: Specifics
For each pixel we compute the average intensity frequency and we count the number of downward crossings
Beats per second
The frequency value at the maximum probability indicates the average number of beats per second
How FishBeat Works: Specifics
A probability distribution is computed by counting the number of pixels at the same frequency
Beats per second
Stopwatch counting VS. FishBeat

59 beats
61 beats
Stopwatch counting VS. FishBeat

Heartbeat was determined before any chemical exposure
Eggs were, then, exposed to the corresponding concentration of malathion for 10 min.
Eggs were allowed to stabilize in aged tap water for 30 s in a single well of a 6-well culture plate
Heartbeat was counted for 15 s.
Malathion was subsequently removed and embryos were exposed for 10 min to aged tap water.
Control counting vs. 50 /mL

1. Experimental observations have successfully demonstrated that FishBeat is able to detect the heart rate of zebrafish embryos.

2. Malathion acts as an acetylcholinesterase inhibitor, inducing bradycardia in zebrafish embryos.

3. Malathion decreased the heart rate at all hpf; increased concentrations of malathion induced a more significant bradycardia.
Future Investigation
Hypothesis #2: Natural occurring concentrations of Malathion in water will affect zebrafish embryonic development by acting as acetylcholinesterase inhibitor, ultimately causing a chronic elevation of acetylcholine within the hatching gland cells.
By recreating a natural aquatic environment in which determined dilutions of Malathion have been applied, we originally speculated the following:

Hypothesis #1: Natural occurring concentrations of Malathion in water will directly inhibit the egg hatching enzymes (ZHE1 and ZHE2) of zebrafish.
Improving FishBeat by releasing a new version containing enhanced tools including:

- Autodetection of the heart
- Improved stability, accuracy, and analysis time
- Detection of the embryonic developmental stage

Future FishBeat applications both at Manhattanville and elsewhere:

- Fruit flies (Drosophila melanogaster)
- Marine sediments’ accumulation and changes in floor detection

Literature Cited
Cheng, S. H., Hui, M. N. Y., and C. C. Lin. 2007. Toxicity and Cardiac Effects of Carbaryl in Early Developing Zebrafish (Danio rerio) Embryos. Toxic. and Appl. Pharmac. 222: 159-168.
Dahm, R. 2006. The Zebrafish Exposed. Am. Sci. 94: 446-453.
Denucé, J. M. and Willemse, M. T. M. 1969. Hatching Glands in the Teleosts, Brachydanio rerio, Danio malabaricus, Moenkhausia oligolepis, and Barbus schuberti. Develop., Growth, and Differ. 15: 169-177.
Denucé, J. M., Meijer, R. C., and Schoots, A. F. M. 1983. Dopaminergic Regulation of Hatching in fish Embryos. Develop. Bio. 100: 59-63.
Iuchi, I., Yamagarni, K., and M. Yamamoto. 1982. Presence of Active Hatching Enzyme in the Secretory Granule of Prehatching Medaka Embryos. Develop., Growth, and Differ. 24: 135-143.
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., and Schilling, F. 1995. Stages of embryonic development of the zebrafish. Develop. Dynam. 203: 253-310.
Lin, Y., Ton. C., and Willett, C. 2006. Zebrafish as a Model for Developmental Neurotoxicity Testing. Birth Def. Res. 76: 553-
National Pesticide Information Center (NPIC). 2001. Malathion Fact Sheet. http://npic.orst.edu/ingred/malathion.html
(accessed December 2011).
Todd, N. E. and Van Leeuwen, M. 2002. Effects of Sevin (Carbaryl Insecticide) on Early Life Stages of Zebrafish (Danio rerio).
Ecotox. and Envir. Safety. 53: 267-272.
1. Fast embryonic development
2. Transparent embryo develop outside the mother
3. Administration of drugs directly to the tank
4. Full sequenced genome
5. Availability of mutants
6. Well-understood developmental behaviors
Pixel Intensity
Time (in frames)
Table 1. Mean heart rate of control (manual stopwatch counting) and FishBeat-calculated embryos at 52, 76, and 96 hpf. Values are presented as mean ± SD (n = 26 in each treatment). FishBeat-calculated heart rates showed no significant difference at 52 and 76 hpf (p > 0.05, Student’s t-test). The asterisk indicates significant difference between control and FishBeat-calculated heart rates.
Table 2. Mean heart rate of malathion-exposed (33.3 μg/mL) and control embryos at 52, 76, and 96 hpf. Values are presented as mean ± SD (n = 26 in each treatment). Malathion-exposed embryos showed significant bradychardia at 52 and 76 hpf (p < 0.01, Student’s t-test). Asterisks indicate a significant decrease of heart rate in malathion-exposed embryos.
Table 3. Mean heart rate of malathion-exposed (50 μg/mL) and control embryos at 52, 76, and 96 hpf. Values are presented as mean ± SD (n = 26 in each treatment). Malathion-exposed embryos showed significant bradychardia at all hpf (p < 0.01, Student’s t-test). Asterisks indicate a significant decrease of heart rate in malathion-exposed embryos.
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