Developmental Biology Special Problem: Benzalkonium Chloride and Drosophila melanogaster (Fruit Fly)...

West Visayas State UniversityCollege of Arts and Sciences

La Paz, Iloilo City

Mutagenic Effects of Benzalkonium Chloride on Drosophila

melanogaster

In Partial Fulfillment of the Requirements In

Bio 206: Development Biology

By

Christopher Jon I. Gaw Put

Mary Grace C. Dumdum

Joanna Rose B. Navarro

BSBS 3B

January 2013

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Table of Contents

Contents Page Number

Introduction to the Study

Research Paradigm

Statement of the Problem

Hypothesis

Significance of the Study

Delimitation of the Study

Review of Related Literature

Methodology

Research Design

Methodology

Materials

Chemicals

Preliminary Activities

Culture Media Preparation

Collecting of Samples

Larva of Drosophila Melanogaster

Experiment Proper

Preparation of Mutagen

Induction of Mutagen

Separation and Transferring of Fruit Flies

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Observation of Visible Mutations

Data Analysis

Results and Discussions

Conclusions

References

Appendix

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Chapter 1

INTRODUCTION TO THE STUDY

Background of the Study

Without genetic variations, some of the basic mechanisms of

evolutionary change cannot operate. Mutations, one of the primary

sources of genetic variation, are the changes in the DNA. A

single mutation can have a large effect.

The fruit fly Drosophila melanogaster is chosen as the

organism of choice because it is a favored organism for the study

of genetic basis of development. It is used extensively in

genetic breeding experiments. It is an ideal testing organism

because it has a short life cycle, exhibits great variability in

inherited characteristics and can be easily raised to produce

large numbers of offspring (Sandhyarani, 2011).

Frequency of mutation in fruit flies has been well

documented. Mutation, as an event that gives rise to heritable

alteration in the genotype, in the fruit flies may be classified

as wild type or a mutant. Visible mutations are easily identified

as the loss of a function or difference in the color of the wild

type fruit fly.

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In this experiment, benzalkonium chloride was used as an

inducer of mutation. It is an antiseptic agent, commonly found in

household products used for cleaning. Previous studies have

showed that benzalkonium chloride is a mutagen as it affects

mammalian somatic cells, yeasts, or bacteria (ScienceLab.com)

This study aims to determine if the 10% concentration of

benzalkonium chloride affects the phenotype of Drosophila

melanogaster. If mutation does occur, this study aims to observe

the different mutant phenotypes and compare phenotypes between

generations.

Research Paradigm

Independent Variable Dependent Variable

Figure 1. Shows the relationship between the independent and

dependent variables of the study

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Phenotype of

Drosophila

melanogaster

Treatment A

10% concentration of

Benzalkonium chloride


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Statement of the Problem

Generally this study will be conducted to determine the

effect of the 10% concentration of benzalkonium chloride to the

phenotype of Drosophila melanogaster.

Specifically this study shall seek to answer the following

questions:

1. What is the effect of the 10% concentration of Benzalkonium

Chloride on the phenotype of Drosophila melanogaster?

Hypothesis

This hypothesis will be tested on this study.

1. Benzalkonium chloride does not significantly affect the

phenotype of Drosophila melanogaster.

Definition of terms

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Significance of the Study

This study would benefit the following:

General public. This study will affirm whether benzalkonium

chloride is indeed a mutagen. This will give information to

people that it is a mutagen and they will be aware of its

possible effects.

This study can also benefit researchers as this can be a

ground for this study.

Delimitation of the Study

This study is limited to assessing the mutagenic effects of

benzalkonium chloride only. Only one concentration will be used

for this study by solving its weight by volume to get its 10%

concentration. There will be 1 trial for this experiment.

Pineapple will be used to collect the fruit flies, and the

offspring of the collected fruit flies will constitute the

parental generation. Sweet potato will then be used as a medium

during the actual conduct of the experiment.

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Chapter 2

REVIEW OF RELATED LITERATURE

Benzalkonium chloride is considered a quaternary ammonium

compound (QACs) which are cationic surfactants that are widely

used as disinfectants (BfR, 2012). In household products, they

are primarily found in fabric softeners, detergents and hair

products (Ferk, 2007). According to ScienceLab.com, benzalkonium

chloride (BAC) is mutagenic for mammalian somatic cells, bacteria

or yeasts. It contains a material which may cause damage to the

kidneys, liver, heart, gastrointestinal tract, cardiovascular

system, and central nervous system. Benzalkonium chloride (BAC)

may affect genetic material and cause adverse reproductive

effects based on laboratory experiments done on animals.

Benzalkonium chloride may be harmful when ingested

accidentally. Animal experiments have indicated that ingestion of

less than 150 gram may be fatal or may produce serious damage to

the health of an individual. It can also produce chemical burns

to the eye and skin following direct contact. Skin contact is

harmful as it may result to systemic effects following

absorption.

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Benzalkonium chloride is an antiseptic agent which must not

be applied to areas which have not been fully rinsed as it is

inactivated by organic compounds. Application of BAC may include

disinfecting instruments and preserving drugs in low

concentration form (chemicalland).

In a study done by Ferk and his team, he studied the

genotoxic effects of benzalkonium chloride (BAC) in mammalian and

plant cells at environmentally relevant concentrations. BAC was

tested in four genotoxicity tests in the Salmonella/microsome

assay with strains TA 98, TA 100 and TA 102, in the single cell

gel electrophoresis (SCGE) assay with primary rat hepatocytes and

in micronucleus (MN) assays with peripheral human lymphocytes and

with root tip cells of Vicia faba. Results show that with BAC, a

moderate but significant effect was found at an exposure of

1.0mg/l. Findings show that this chemical includes moderate but

significant genotoxic effects in eukaryotic cells at

concentrations which are found in wastewaters and indicate that

their release into the environment may cause genetic damage in

exposed organisms. The results show that BAC that was tested

cause DNA migration and MN formation in mammalian and plant-

derived cells but no gene mutation in bacteria.

A disinfectant containing benzalkonium chloride has been

identified as the most probable cause of birth defects and

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fertility problems in caged mice. In the cosmetic safety

database, there is moderate to strong evidence that it is an

immune, skin, and respiratory toxicant. One or more in vitro

tests on mammalian cells exposed to BAC show positive mutation

effects.

According to an article by ET CBC News, scientists at the

University of Ireland in Galway found that when they added the

disinfectant benzalkonium chloride to common bacteria called

Pseudomonas aeruginosa, increasing the amount of germ-fighting

solution over time, the bacteria learned to survive. The

researchers discovered that over time the bacteria became more

efficient at ridding their cells of both the disinfectant and

antibiotic. They also found that a mutation in their DNA made

them resistant to antibiotics.

CHAPTER III

Research Design and Methodology

Research Design

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An experimental method was used in the study to assess the

mutagenic effects of 10% benzalkonium chloride to Drosophila

melanogaster. It consists of a 10% benzalkonium chloride

concentration.

Methodology

Materials

The materials that were utilized are overripe pineapple,

sweet potato, wide-mouth bottles, cheesecloth, rubber bands,

tray, medicine dropper, gas range, pan, fork/spoon, peeler,

water, inoculating loops, cotton, ether/chloroform, dissecting

and compound microscopes, camera, high precision balance and

wild-type fruit flies.

Chemical(s)

The chemical that was used to anaesthetize the Drosophila

melanogaster was chloroform.

Preliminary Activities

Culture Media Preparation

Overripe pineapple (Ananas comosus (l.) merr.) was used as

the culture medium for this experiment. The medium was crushed

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until it is soft and placed in a sterile bottle to ensure a “no-

mold” culture media. This was also be used for the collection of

fruit flies prior to the experiment.

Collecting of Samples

The bottle with the cultured medium was placed beside a

trash bin and was left there for several hours until the bottle

was filled with fruit flies. The cheesecloth was then sterilized

and was used to cover the sanitized bottle which was secured by

the rubber bands. The collected fruit flies were left to breed

until visible larva could be seen.

Larva of Drosophila melanogaster

The larva of the collected fruit flies was used as the

parental generation when it reached its adult stage after six (6)

days.

Preparation of culture medium with mutagen concentration:

In preparing the 10 grams, 10% benzalkonium chloride

concentration, the researchers weighed 1 mL of benzalkonium

chloride via graduated cylinder which was followed by the

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addition of 10 grams mashed sweet potato. The mutagen and the

culture medium were mixed thoroughly and were placed in a wide-

mouth bottle.

Culturing fruit flies and induction of mutagen:

From the earlier collection of wild type fruit flies from

the overripe pineapple, it was cultured using the sweet potato

medium with benzalkonium chloride culture media. The fruit flies

were allowed to mate and their offspring/s were perceived from

larva to adult. Before the offspring develop into adult, the

collected fruit flies were released so that only the parental

generation will remain inside the bottle. The parents were then

allowed to mate until they produced offspring which were our (F1)

generation. When the offspring developed into adult, the females

were separated from the males to prevent them from mating. Their

phenotypes were observed and were also recorded. They were

combined later to mate and their offspring (F2) generation will

again have their phenotypes be observed and recorded.

Separation of Male and Female Virgins Drosophila melanogaster and

Pairing and Transferring of Male and Virgin Female Drosophila

melanogaster to a vial:

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The Drosophila melanogaster that was anesthetized were

transferred in a microscope slide for clear viewing of the

structures of male and female under a microscope. After viewing,

the male and female virgin flies were then paired and transferred

to a vial that contained the cultured medium.

Observation of Visible Mutations

The adult Drosophila melanogaster of the F1 generation and

F2 generation were used in observing the visible mutations under

the aid of a microscope. These mutations were identified based on

the differences in appearance of a wild type fruit fly and a

mutant one.

Data collection and Analysis

All of the recorded data, the observations in the phenotype

were collected for analysis. All the results will be presented in

tabular form and graphs.

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Chapter 4

Results and Discussions

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This study was conducted in order to assess the mutagenic effects

of benzalkonium chloride on Drosophila melanogaster and to

observe and compare subsequent mutations between generations.

Parental Generation: 8 virgin females

5 males

Table 1: Fruit flies in the First Filial Generation (F1

Generation)

Fruit

fly

Number

of

Males:

Male

Frequency:

Percentage: Number

of

Females:

Female

Frequency:

Percentage:

Wild

type

4 0.2 20% 7 0.35 35%

Mutant 0 0 0 9 0.45 45%

Total

number:

4 16

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Graph 1: Fruit flies in the First Filial Generation (F1

Generation)

Wildtype Male20%

Mutant Female45%

Wildtype Female35%

F1 Generation

Our F1 generation showed us that out of the 20 fruit flies,

there were 11 wild type fruit flies and 9 mutant female fruit

flies. Out of the 9 mutant female fruit flies, there were 2 fruit

flies that showed 2 kinds of mutation.

The following graph and table will show you the incidence of

mutation for the F1 generation in terms of its body color and

wing shape.

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Body Number

of

Males:

Male

Frequency:

Percentage: Number

of

Females:

Female

Frequency:

Percentage:

Tan 4 0.2 20% 7 0.35 35%

Yellow 0 0 0 7 0.35 35%

Ebony 0 0 0 2 0.1 10%

Table 2: Incidence of Mutation in the First Filial Generation in terms of body color (F1 Generation)

Graph 2: Incidence of Mutation in the First Filial Generation in terms of body color (F1 Generation)

Tan colored body (Male)20%

Tan colored body (Female)

35%

Yellow colored body (Female)

35%

Ebony colored body (Female)10%

Incidence of Mutations in the body of Fruit Flies (F1 Generation)

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The results of our F1 generation showed us that in terms of

body color, it was found that only females expressed either of

two mutant forms in this generation. Out of the total 20 fruit

flies from the F1 generation, 4 males and 7 females expressed

their tan coloration (wild type fruit fly). While there were, 9

females who showed either yellow or ebony coloration. Out of the

9 mutant fruit flies, 7 expressed the color yellow in their body

and 2 expressed the ebony color.

Parts Number

of

Males:

Male

Frequency:

Percentage: Number

of

Females:

Female

Frequency:

Percentage:

Short

winged

0 0 0 1 0.05 5%

Blistered

wing

0 0 0 1 0.05 5%

Table 3: Incidence of Mutation in the First Filial Generation (F1 Generation)

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The results in our F1 generation showed us that in terms of

wing type and head type, it was found that out of the 9 mutant

female fruit flies, 2 of which showed two kinds of mutation in

terms of its body color and wing type. 1 fruit fly expressed a

short type kind of wing and another 1 expressed a blistered kind

of wing type.

Fruit

fly

Number

of

Males:

Male

Frequency:

Percentage: Number

of

Females:

Female

Frequency:

Percentage:

Wild

type

4 0.09 9% 13 0.32 32%

Mutant 8 0.195 19% 16 0.390 39%

Total

number:

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Table 4: Fruit Flies in the Second Filial Generation (F2 Generation)

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Graph 3: Fruit Flies in the Second Filial Generation (F2 Generation)

Wildtype Male10%

Mutant Male19%Wildtype Female

32%

Mutant Female39%

F2 Generation

Our F2 generation showed us that out of the 41 fruit flies,

there were 17 wild type fruit flies and 24 mutant fruit flies.

Out of the 24 mutant fruit flies, there were 5 fruit flies that

showed 2 kinds of mutation.

The following graph and table will show you the incidence of

mutation for the F2 generation in terms of its body color and

wing shape.

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Table 5: Incidence of Mutation in the Second Filial Generation (F2 Generation)

Body Number

of

Males:

Male

Frequency:

Percentage: Number

of

Females:

Female

Frequency:

Percentage:

Tan 4 0.09 9% 13 0.317 31.7%

Yellow 0 0 0 12 0.29 29%

Ebony 8 0.195 19.5% 4 0.09 9%

Graph 4: Incidence of Mutation in the Second Filial Generation (F2 Generation)

Ebony colored body (Female)10%

Tan colored body (Female)

32%

Ebony colored body (Male)19%

Yellow colored body (Female)

29%Tan colored body (Male)

10%

F2 Generation

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The results of our F2 generation showed us that in terms of

body color, it was found that out of the total 41 fruit flies, 24

mutant fruit flies expressed either yellow or ebony body

coloration. Out of 24 mutant fruit flies, 8 males and 4 females

expressed the ebony coloration. While there were 12 females that

expressed the yellow body coloration.

Discussions:

Abnormal body coloration occurred most likely due to

benzalkonium chloride affecting genes (the yellow genes), located

on the X chromosome, which blocks the synthesis of brown pigment

resulting to fruit flies with yellow bodies. Also, according to

Bruce Alberts (1985), the yellow gene (y) is involved in pattern-

specific melanin pigmentation of the cuticle of the adult fly and

of larval mouth parts of Drosophila melanogaster and has mainly

neural functions. This means that BAC is strong enough to alter

such gene for the phenotypic observation of the mutant fruit

flies that exhibited yellow body coloration. Other abnormal body

coloration is due to a defect in their ebony gene which is

responsible for the building up of the tan colored pigments in

the normal fruit fly. Ebony was found to be exclusively localized

to neuropile and epithelial glial cells (Richardt, 2002). The

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spatial localization of Ebony protein within the adult nervous

system, including the visual system and protocerebrum, was

studied in order to examine the abundance of the protein at

different times of day. Since the ebony gene is defective, this

resulted to the black pigments accumulating all over the body

thus we have obtained an ebony colored body fruit fly. In

addition, according to Wittkopp, True, and Caroll (2002) Ebony is

required to suppress some melanin formation, and is expressed in

cells that will produce both melanized and non-melanized cuticle.

Ectopic expression of Ebony inhibits melanin formation, but

increasing Yellow expression can overcome this effect.

Another mutation that we have observed is with the wing type

of the fruit flies. The short winged fruit flies have a defect in

their vestigial gene on its second chromosome. Flies with short

wing type have a recessive mutation (The Exploratorium, 2013).

Short wing gene is located in chromosome I between 63.5 and 64.

This gene proved to have a very pleiotropic manifestation, on

changes in the wings and the eyes being the most common ones. The

alterations in the wings are manifold; the wings may be stretched

out in an abnormal direction, not backwards as is normal, but

held out from the sides of the body at an angle of 90 degrees.

The wings may exhibit incisions of very different size and

number; if only one incision is present this is usually found in

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the medial margin of the wing, but the medial, lateral and

posterior margin may all have incisions (Eker, 2002).

Blistered wings were also observed on the mutant ebony

Drosophila melanogaster. According to Martin, Zusman, and Li

(1999), a new laminin α chain is assigned to the locus of wing

blister which is essential for embryonic viability and is

involved in processes requiring cell migration and cell adhesion.

Blistered gene in accordance to Fristrom, Gotwals et. al. (1994)

both developmental and genetic analyses suggest that it is

required during metamorphosis for the initiation of intervein

development and the concomitant inhibition of vein development.

A single wingless and legless Drosophila melanogaster was

perceived on the second filial generation. Wingless trait

according to Bishop, Huggler, et. al. (2010) or (mutation H) is

an autosomal recessive gene. Meaning, two copies of an abnormal

gene must be present in order for the disease or trait to

develop. A mutation in a gene on one of the first 22 nonsex

chromosomes leads to wingless trait.

Chapter 5

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Conclusions:

Based on the gathered results, the following conclusions

were made:

The results of the experiment showed that the Benzalkonium

Chloride induces mutation to the F1 generation and F2 Generation

of Drosophila melanogaster.

The observed mutations in this study were: the yellow and

ebony colored body of the fruit fly, and the short winged and

blistered wing type.

References:

Alberts B. (1985). Molecular analysis of the yellow gene (y)

region of Drosophila melanogaster. Retrieved January 14,

2013 from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC391346/pdf/pnas0

0361-0222.pdf

Bundesinstitut fur Risikobewertung. (2012). Health assessment of

benzalkonium chloride residues in food. Retrieved on

December 29, 2012 from:

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http://www.bfr.bund.de/cm/349/health-assessment-of-

benzalkonium-chloride-residues-in-food.pdf

Chemical land (2012).General Description of antiseptic agent.

Retrieved on December 29, 2012 from:

http://chemicalland21.com/lifescience/phar/benzalkonium

%20chloride.htm

Chemwatch (2008). Benzalkonium chloride. Retrieved on December

29, 2012 from: http://datasheets.scbt.com/sc-257126.pdf

Eker R. (2002). The Short wing gene in Drosophila melanogaster

and the effect of temperature on its manifestation.

Retrieved January 14, 2013 from

http://www.ias.ac.in/jarch/jgenet/30/357.pdf

ET CBC News. (2009). Disinfectant use could cause superbugs.

Retrieved on December 30, 2012 from:

http://www.cbc.ca/news/health/story/2009/12/28/disinfectants

-superbugs.html

Ferk F. (2007). Benzalkonium chloride and dimethyldioctadecyl-

ammonium bromide (DDAB), two common quaternanry ammonium

compounds, cause genotoxic effects in mammalian and plant

cells at environmentally relevant concentrations. Retrieved

on December 29, 2012 from:

http://mutage.oxfordjournals.org/content/22/6/363.full.pdf

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http://mutage.oxfordjournals.org/content/22/6/363.full.pdf

http://datasheets.scbt.com/sc-257126.pdf

http://chemicalland21.com/lifescience/phar/benzalkonium%20chloride.htm

http://chemicalland21.com/lifescience/phar/benzalkonium%20chloride.htm

http://www.bfr.bund.de/cm/349/health-assessment-of-benzalkonium-chloride-residues-in-food.pdf

http://www.bfr.bund.de/cm/349/health-assessment-of-benzalkonium-chloride-residues-in-food.pdf


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Fristrom D., Gotwals P., et. al. (1994). Blistered: a gene

required for vein/intervein formation in wings of

Drosophila. Retrieved on January 14, 2013 from

http://dev.biologists.org/content/120/9/2661.full.pdf

Huggler A., Patel G. (2010). Analysis of the Mode of Inheritance

of the Mutant White Eye Trait and Wingless Trait in live

Drosophila melanogaster and the Mapping of Sepia Eye Color

Mutation to Identify the Particular Chromosome on which the

Mutation is Located in Drosophila using Virtual FlyLab.

Retrieved on January 14, 2013 from http://by423-01-fall-

2010.wikispaces.com/BY423-01+Group+1+Report

Martin D., Zusman S., and Li X. (1999). Wing blister, A New

Drosophila Laminin α Chain Required for Cell Adhesion and

Migration during Embryonic and Imaginal Development.

Retrieved January 14, 2013 from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2148222/

Richardt, A., Rybak, J., et al. (2002). Ebony protein in the

Drosophila nervous system: optic neuropile expression in

glial cells. Retrieved January 13, 2012 from

http://www.sdbonline.org/fly/sturtevant/ebony4.htm

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ScienceLab.com (2012).Benzalkonium chloride MSDS. Retrieved on

December 30, 2012 from: http://www/sciencelab.com/msds.php?

msdsId=9923038

The Exploratorium © (2013). Mutant fruit flies. Retrieved

December 30, 2012 from

http://www.exploratorium.edu/exhibits/mutant_flies/mutant_fl

ies.html

Wittkopp PJ., True JR., Carroll SB. (2002). Reciprocal functions

of the Drosophila Yellow and Ebony proteins in the

development and evolution of pigment patterns. Retrieved

January 14, 2013 from

http://www.ncbi.nlm.nih.gov/pubmed/11934851

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http://www/sciencelab.com/msds.php?msdsId=9923038

http://www/sciencelab.com/msds.php?msdsId=9923038


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Appendi

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Normal Wild-type Fruit Flies

Yellow-bodied Fruit flies

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Ebony Fruit flies with blistered wings

Ebony Fruit flies with proportioned body

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Hunchbacked Ebony Fruit flies

Short-winged Fruit fly Wingless and legless Fruit Fly

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Fruit flies with weird antennae

Fruit flies with acute abdomen

Solving for the 10% Benzalkonium Chloride Concentration:

Mass / Volume = 10g x 0.1 = 1 mL of Benzalkonium Chloride mixed with 9 mL of distilled water

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Developmental Biology Special Problem: Benzalkonium Chloride and Drosophila melanogaster (Fruit Fly)...

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