Directional Asymmetry in Wing Length of Drosophila melanogaster

A research proposal presented to

the faculty of the College of Science and Information Technology

Ateneo de Zamboanga University

In partial fulfilment

of the requirements for BFGen Lab

By:

San Pedro, Krizia Corrine C.

Gonzales, Kenneth D.

CHAPTER ONE
 INTRODUCTION

Background

Symmetry exists in nature, explicitly observed throughout many of the varying forms
of metazoic organisms. Bilateral symmetry in particular is shared by the majority of the
living clades under the kingdom animalia, with the exception of echinoderms, poriferans and
cnidarians (Finnerty, 2003). With such abundance of exemplary symmetry, the occurrence of
asymmetry among the subkingdom Bilateria generates interest. Asymmetry is quite often
observed among many organisms as an occurring norm when referring to internal organs,
especially in singularly occurring organs. In contrast to this, asymmetry in the morphological
aspect is scarce and is seen as more of an anomaly, often relating fluctuating asymmetry as a
measure of developmental instability (Ashley J. R. Carter, 2009). Some authors, however,
have claimed that antisymmetry and directional asymmetry may have a significant genetic
basis, thereby rendering these forms of asymmetry useless for studies of developmental
instability, and thus points to the trait as a heritable genetic occurrence (John H. Graham,
1993). Contrarily, additive genetic variation for directional asymmetry presents difficulty in
attempts of demonstrating the phenomenon, primarily resulting in minimal heritability of
asymmetry.

Bilateral symmetry is achieved by the intersection of two orthogonal axes of polarity:

the anterior-posterior (A-P) axis and the dorsal-ventral (D-V) axis. Bilateral organisms are
governed under these axes and develop morphological structures, especially paired structures,
into contrasting pairs of the same structure. Asymmetry also occurs extensively among
organisms even under the subkingdom Bilateria. Asymmetry is an occurring aspect readily
observed among internal organs, primarily in singularly occurring organs such as the
vertebral heart. External, morphological structures however are observed fewer and farther
apart, though still occurring; such that in fiddler crabs, flounders, and gastropods. More
notable forms of asymmetry in organisms are often a product of evolutionary preference,
exhibiting either antisymmetry or an asymmetry shared throughout the species.

Directional symmetry (DA) on the other hand pertains to a significant bias on the
measurements of one side of paired structures that of which is not shared as a feature
throughout the species. With this, DA represents an anomaly in the general phenotype of a
species (Ashley J. R. Carter, 2009). DA is characterized by a symmetry distribution that is
not centered around zero but is biased significantly, towards larger traits either on the left or
the right side (Kotiaho, 2001).

Fluctuating asymmetry (FA) is a particular form of biological asymmetry,

characterized by small random deviations from perfect symmetry of which is based off of a
priori expectation that symmetry is the ideal state of bilaterally paired traits or structures
(Kotiaho, 2001). FA measures deviations from the ideal state of symmetry in bilaterally
paired structures and therefore is thought to reflect the level of developmental and
environmental stress experienced by individuals and populations. In this sense, asymmetry,
often to an extent that is centered around zero, is generally a form of FA and a product of
developmental and environmental stresses rather than a result of genetic anomalies.

Artificial selection shares its importance to studying the genetic and evolutionary
aspects of organisms as it allows us to determine the heritability of certain traits of which are
chosen for the preference of the study. If there is additive genetic variance for the selected
trait, it will respond to the selection, that is, the trait will evolve or continue to be expressed
along the lineage (Conner, 2016). In attempts of exhibiting DA as a genetic and heritable
trait, studies have been made on test organisms in hopes of exploring the phenomenon.
Contrasting ideologies on the concept of DA primarily revolve around it being minimally
expressed in hereditary experiments leading many to argue that DA is simply a canonical
example of a trait without heritable variation (Lewontin, 1974) or a cause of FA. However,
studies on the matter seem to generally present a level of heritability (Santos, 2002). This
existing contradiction in the understanding of asymmetry as a genetic component raises a
need for a pursuit of furthering our understanding on the matter.

Studies done on Drosophila sp. explored morphological structures of the organism

that exhibited a significant potential for additive genetic variation through the observation of
DA traits and their response to artificial selection. In a paper by Pélabon and Hansen (2008),
tests for DA in wing size in insects resulted in over a quarter of the statistical tests and a third
of the species showing significant DA, although the average magnitude is less than 1% of
overall size. In a study conducted by Klingenberg et al. (1998), three representatives of
different Dipteran families revealed minimal but consistent DA in wing morphology. These
results suggest an existing left-right axis in wing morphology as well as presents a potential
for genetic variation for left-right asymmetry to exist in wing traits. The researchers opt to
further explore DA among morphological structures in bilateral organisms through the use of
Drosophila melanogaster cultures harboring specimens exhibiting DA in wing length. By
observing the extent of additive genetic variance on the trait, the researchers opt to determine
the prevalence or absence of DA inheritance in wing lengths among D. melanogaster.

Statement of the Problem

Directional asymmetry continues to be one of the less understood phenomena in

genetic studies. With contradictions on the extent of the involvement of additive genetic
variance on the occurrence of DA, further studies on the matter is of great interest.
Arguments persist on the notion that DA is simply a cause of FA and is primarily a cause of
environmental influences on the development of an organism, this would then suggest no
hereditary potential for such types of asymmetry. Evidences, however, contradict this notion
as studies on directional symmetry patterns a minimal but existing heritability. Drosophila
melanogaster has been a species that represented genetic studies for its efficiency and
practicality. With it being one of the representative species exhibiting a potential for
heritability of DA, it best represents a study regarding the matter. With this, the researchers
seek to further investigate the occurrence of directional asymmetry of morphological
structures of Drosophila melanogaster by exhibiting additive genetic variance through
artificial selection of cultures harboring specimens with directionally asymmetrical wing
length.

Significance of the Study

Studying the heredity and genetics with DA allows us to better understand the role of
asymmetry in bilateral organisms. With FA ought to be primarily an environmental and
developmental anomaly, its use in determining developmental instability proves of
importance. However, tying in DA as a genetic anomaly of which possesses a potential for
additive genetic variance, the role of FA will then require a method in accounting for
heritable DA. Proving or disproving DA’s additive genetic variance will aid in the use of FA
as a measure of developmental instability, either determining its reliance or revealing a need
for improving its methodology.

Scope and Limitations

The study will focus only on wing length asymmetry despite other notable
morphological structures identified to have potential for additive genetic variation due to
restrictions on equipment and strategies allowing only for observations that can be performed
outside of the laboratory settings. As the study will focus on DA, i.e. significant bias on the
measurements of one side of paired structures, it would allow observations of notable
extremes on wing length asymmetry without the use of specialized equipment. Wings are
bilaterally symmetrical structures and therefore are expected to be of similar proportions.
Any observable extremes, pertaining to wings that overlap their accompanying pair, would
easily be sorted out as a notable deviation from the norm, thus representing DA in wing
length.

CHAPTER TWO

LITERATURE REVIEW

In 1960, Maynard Smith and Sondhi published the first recorded directional
asymmetry selection experiment having used Drosophila subobscura. The researchers
focused on the ocelli variation in which an individual fly may possess three at most (Smith &
Sondhi, 1960). With this, they selected two lines. The first was referred to as “Symmetrical”
or the SYM lines in which the selected flies had both the left and right posterior ocelli. On the
other hand, the “Asymmetrical” or the ASY lines including flies that had both anterior and
left posterior ocelli, and lacked the right posterior ocelli. A total of 200 flies were selected
from 12 generations in which 20-30 individuals were selected for generation mating. They
have observed that the SYM line frequencies increased over generations from 14.75% to
64.20% which was due to the successive loss of the anterior ocelli. ASY lines also have
increased significantly as a result of the prevalence of the anterior ocelli. A fluctuation
occurred in the flies only possessing the left posterior ocelli however no trend was evident.
Moreover, single-generation crosses showed no apparent inheritance of the handedness of
ocellus presence. With their observations, Smith and Sondhi (1960) concluded that it is likely
that handedness of individuals is a purely chance phenomenon and is not genetically or
maternally associated.

Directional asymmetry of sternopleural bristle number was selected by Beardmore

(1965) in his experiment on Drosphila melanogaster. Two lines were selected with 30 mating
for each generation. The selection index was the ratio of the number of bristles on the two
sides (left over right) and the lines included an increase in one line and decrease in the other.
His results revealed a significant divergence in directional asymmetry (P=. 03) and
Beardmore (1965) estimated a 3.6% heritability. Although proving the heritability of the trait,
his estimates were based on assumption and were deemed questionable.

In connection, Tunistra et. al. (1990) did a study on an outbred Drosophila

melanogaster population with a scute mutation that decanalize scutellar bristle number. They
performed a selection between left and right bristle numbers accumulating 12 generations
however they found no significant response for directional asymmetry and heritability was at
a negative value.

Purnell and Thompson (1973) used Drosophila melanogaster to examine the

directional asymmetry of wing folding. It was mentioned that the flies at rest hold their wings
against their backs with one over the other, retaining the mannerism for the rest of their lives.
Given this, the researchers selected two lines: one group folding right-over-left, and the other
left-over-right. The lines were selected for 15 generations with over 50 flies of each gender
leaving 5 of them selected to mate for each generation. Overall, data revealed an insignificant
response with - 5.8% heritability (Purnell and Thompson, 1973). There was a significant
response observed at earlier generations however ultimately, the conclusions were based on
ad hoc choice of time periods.

Wing shape of Drosophila melanogaster was also subject to directional asymmetry

studies as initiated by Pelabon et. al. (2006). They observed variation in the shape of left
wings and found direct responses to directional asymmetry under selection. In addition, Rego
et. al. (2006) also discovered increases in the directional asymmetry of wing size in
Drosophila maderiensis and Drosophila subobscura. These studies proved the presence of
genetic variation in line with directional asymmetry however whether these factors generate
additive genetic variance for directional asymmetry within populations was vague.

Carter et. al. (2009) conducted an experiment on wild-type Drosophila melanogaster

highlighting the directional asymmetry of crossvein placement in wings. The basis of their
selection was the relative position of the posterior crossvein on the left and right wings. The
two lines of selection included L-lines which move the crossveins to the left and the R-lines
which moved to the right. There were a total of 15 generations assayed. Their observations
revealed no significant response to selection in directional asymmetry in the mentioned wing
trait as well as no association with heritability.
 Coyne (1987) performed an experiment on the outbred population of Drosophila
melanogaster carrying the mutant eyeless allele (eyr) selecting the directional asymmetry of
eye size. The allele reduces facet number simultaneously dacanalizing symmetry which
creates a variation in facet number asymmetry that can be analyzed qualitatively (Coyne,
1987). For this experiment, he selected three lines: larger left eye, larger right eye, and both
eyes relatively smaller including a control group of the unselected. Overall, a total of 200-500
flies from 30 generations were assayed with 10-20% in each generation selected for mating.
He found out that there was no response for directional asymmetry and his findings had no
clear relevance for the natural populations.

CHAPTER THREE
METHODOLOGY
Research Design
The study mainly employed a qualitative approach to assess the generations of
selected Drosophila melanogaster in each generation.
Mass Breeding
Prior to the selection, the fruit flies were anesthetized by placing the container in the
freezer for about 10 minutes for proper observation of the trait. Number of selected
individuals from each generation were noted. For each generation 10 individuals (five males
and five females) with asymmetrical wings were chosen for mating of the next generation.
The flies were mated in a fresh culture jar. As soon as pupae appear in the culture, parental
generations were removed. The emergence of adult flies prompted the selection stage of the
study.
Selection Procedure
Two lines were selected from cultured Drosophila melanogaster for a proposed total
of three generations representing a small-scale study. The selection lines included those with
equal wing lengths (for both left and right) and those with a wing relatively longer than the
other (either left or right). Wing length were classified as long or short based on qualitative
judgement due to the lack of appropriate equipment. Male and female fruit flies were isolated
to aid the selection for mating. Selection was done only from day one to five upon the
eclosion of adult flies.
Data Analysis
Directional asymmetry was determined with a qualitative approach, comparing one
side of the wing from the other whether one exceeds a given norm. Photographic
documentations of the selected samples were used as reference for analysis of directional
asymmetry. Heritability was measured through the observation of the population size in each
generation.

CHAPTER FOUR
RESULTS AND DISCUSSION

Figure 1. Symmetrical Wings

Figure 2. Asymmetrical Wings

 Flies with Asymmetrical wing lengths

Table 1. Parental and Filial Generation DA Population

Population Filial Isolated Succeeding

Size Generation Population Filial
Size Exhibiting DA Generation
Exhibiting DA

Isolated 10 26 10 0
Parental (F1 generation)

Isolated F1 26 66 0 0
(F2 generation)

F2 66 164 0 2
(F3 generation)

F3 164 NA 2 NA

Parental Generation

From an original population of wild type Drosophila melanogaster, 10 specimens

(5:5- male: female) exhibiting asymmetry in wing length were selected for to observe
heritability of the trait into filial generations.

Filial Generations
 The first succeeding generations (F1= 26 individuals and F2= 66 individuals) yielded
no asymmetry in wing length. The third generation (F3= 164 individuals) only showed 2
specimens exhibiting significant asymmetry in wing length.

Fluctuating Asymmetry

Although artificial selection was done, there is still an observed fluctuation of the
number of individuals per generation in the line with asymmetrical wings. It occurs to have
no association with heritability of the directional asymmetry trait, rather it must be a result of
fluctuating asymmetry. Small deviations from the normal symmetry of body structures is
referred to as fluctuating asymmetry. This is different from directional asymmetry which
imposes a significant deviation from symmetry. This case was more likely to exhibit FA than
DA. The variation may have been caused by environmental stressors which affected the
developmental stage of the fruit flies in captivity. A related study in fluctuating asymmetry
was performed by Vishalakshi (2007) studying the wing traits of Drosophila ananassae.
Morphological traits are significantly affected by poor nutrition and primarily larval density.
Morphological traits were reduced by stress, increasing phenotypic and genetic variability.
The levels of fluctuating asymmetry and positional fluctuating asymmetry were similar in
flies reared on poor and standard media. In contrast, there is a significant difference in both
asymmetry measures in the flies placed at different larval densities for all traits. Moreover,
composite fluctuating asymmetry is higher in their male samples than in females, suggesting
that males are more vulnerable to developmental stress. The results suggest that trait means
are more sensitive to stress than fluctuating-symmetry measures and that the effect of stress is
trait- and sex-specific. In the case of wing length asymmetry, there was no clear association
between sex and the expression of the trait. This is because there are also female individuals
which exhibited the trait found in the prepared culture. This gives a strong correlation to
developmental stress induced by environmental pressures in captivity.

CHAPTER FIVE

CONCLUSION AND RECOMMENDATION

Drosophila melanogaster wings had no response to directional asymmetry. The

results obtained revealed no association with DA rather it was assumed to have been due to
fluctuating asymmetry as induced by developmental stress. Furthermore, as noted from the
number of individuals per generation, the trait seems to not be heritable. To be able to
appropriately observe the heritability of directional asymmetry, more generations must be
observed. Furthermore, the study must employ a quantitative approach to accurately measure
heritability.

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