Sheena_Melwani Thesis 042312

7/31/2019 Sheena_Melwani Thesis 042312

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Comparison of eye, barbel, and corresponding brain lobe size in the African

cyprinid fishBarbus neumayeri between in-forest and out-forest habitats in

Uganda

Sheena Melwani260309578

Department of Biology, McGill University

BIOL 468

April, 2012Supervisor: Lauren Chapman

Post-doctoral Mentor: Suzanne Gray

Graduate Student Mentor: Vincent Fugre

Abstract: The objective of this study was to describe interpopulational variation in the sensory

morphology of the African cyprinid fish,Barbus neumayeri from two field sites in Western

Uganda: a relatively pristine rainforest stream site and a nearby stream outside the forest in

converted agricultural land. Uganda has suffered severe deforestation, and there is growing

awareness of the effects that forest loss is having on the quality of water in the rivers and

streams. One characteristic of deforested streams in these areas is increased turbidity, which may

affect the visual environment of the fish and lead phenotypic change to improve visual acuity

and/or improve other sensory modalities. In this study, I quantified eye size, barbel length, and

the volume of corresponding optic and olfactory brain lobes ofB. neumayeri from the inforest

and outforest streams. The results show that outforest fish had a larger overall brain mass and a

larger facial lobe suggesting that the brain areas of olfaction are more active in outforest fish and

could correspond with a difference in fish morphology in the two populations. Given that nodifference in barbel length was found, further research is required to determine the

morphological characteristics that an increase in facial lobe volume can be attributed to inB.

neumayeri.


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Introduction

Deforested land that has been converted for agricultural use is a prime contributor to increases in

water turbidity in streams and lakes around the world (Ryan, 1991). Turbidity is a measure of the

amount of suspended particulate matter in the water column, either biotic and/or abiotic.

Research has shown that the amount of sediment being transported globally by rivers has

increased by 2.3 billion tonnes annually as a result of human land-development such as tree

removal and subsequent soil erosion (Syvitski et al., 2005; Donohue & Garcia Molinos, 2009),

and certainly a component of this increase is associated with deforestation. In East Africa, it is

estimated that only 28% of the original rain forests remain today (Martin, 1991). The majority of

land clearing has been for subsistence farming and fuelwood harvest. Uganda has seen an

average forest loss of 86,400 ha each year. This is 2.1% of the forest cover per year from 2000-

2005 (FAO, 2005). In Uganda, out-forest streams (i.e. streams found in deforested, agricultural

land) have been observed to be more turbid than rivers located inside forested areas (Kasangaki

et al., 2008).

Increased turbidity has been linked to multiple ecological changes in aquatic ecosystems

(Donohue & Garcia Molinos, 2009; Kasangaki et al. 2008). For example, an increase in sediment

load has been shown to result in a decrease in total primary production through photosynthesis

due to a decreased transmission of light through the water column (Kirk, 1985; Donohue &

Garcia Molinos, 2009). Increased turbidity has also been linked to reduction in dissolved oxygenavailability in aquatic ecosystems (Bruton, 1985; Donohue & Garcia Molinos, 2009). These

physio-chemical changes can result in changes in ecological interactions such as intra- and

interspecific competition (de Roose et al., 2003) and food web stability (McCann, 2000), due to

the changing availability of resources (Chandler, 1942; Cuker, 1993; Donohue & Garcia

Molinos, 2009).

Effects of increasing turbidity on fishes have been studied extensively, and has been

shown to be detrimental to fish that are visual predators. Increased turbidity decreases the

effectiveness of their visual perception ability due to a decrease in ambient light intensity and

shifts in the colour of underwater light. Therefore, high turbidity levels can diminish feeding

efficiency (Cuker, 1993) and as a result, growth rates of visually predatory fish (Donohue &

Garcia Molinos 2009; Utne-Palm, 2002).


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An organisms physiology and morphology must be considered in the context of its

environment (Bradshaw, 1965). In spite of substantial research on effects of turbidity on fishes,

our understanding of how turbidity may drive phenotypic variation across populations (across

habitats within species) is not well understood. Yet, given how pervasive turbidity is as an

environmental stressor in freshwaters, it is increasingly important that we understand the

ecological and evolutionary consequences. Genetic adaptation and phenotypic plasticity have

been considered as two ways that organisms may adapt to different environments (Schlichting &

Pigliucci, 1998). Natural selection can act on the genetic variation in a population living in the

current environment. Organisms with advantageous traits are more likely to survive and

reproduce offspring with the same advantageous trait. Over a series of generations, this can result

in a population that is locally adapted to environmental conditions (Crispo & Chapman, 2010).

However, it could also be that adaptive phenotypic change can be seen within a generation,

producing a population that is also locally adapted to the environment without genetic change

(i.e. phenotypic plasticity) (Crispo & Chapman, 2010; Bradshaw, 1965).

The objective of this study was to investigate interpopulational variation in the sensory

morphology of the African cyprinid fish,Barbus neumayeri from two field sites in Uganda that

differ in level of turbidity. Specifically, I measured eye size, barbel length, and the volume of

corresponding optic and olfactory brain lobes ofB. neumayeri from in-forest and out-forest

(deforested, converted to agricultural land) stream populations from western Uganda. Previous

observation on cyprinids (minnows) has shown that, in general, the number and length of barbels

are correlated with eye size and habitat (Davis & Miller, 1967; Moore, 1950). In clear water,

minnows are often characterized by large eyes and short barbels, whereas fish in turbid water

tend to have small eyes and long barbels. Furthermore, it was observed that minnows in turbid

water have reduced optic lobes (Davis & Miller, 1967). It is theorized that barbel and eye size is

related to the fishs ability to obtain their food in clear or turbid environments. Methods of

obtaining food will likely vary with the habitat and the effectiveness of the food sensory organs(such as eyes and taste buds) in a way that optimizes food capture and processing (Davis &

Miller, 1967). Since vision is reduced due to the decrease in light penetration in highly turbid

waters, some fish in turbid environments rely on taste buds to locate their food. It is suggested

than an increase in barbel length or size is a compensatory adaptation for reduced vision in turbid


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environments, as an increased barbel length would allow for more effective searching where

vision is limited (Hubbs & Ortenburger, 1929; Davis & Miller, 1967).

With respect to brain development, it has been observed that differences in brain lobe

size can be attributed to functional adaptations. Relative lobe size is correlated with

hyperdevelopment or degeneration of a specific sensory system (Davis & Miller, 1967). A

species lifestyle is reflected in the organization and development of its central nervous system

(Nieuwenhuys et al., 1998). Therefore the structure of brain and allocation to different lobes of

the brain can provide important insight into the biology and the lifestyle for a group of

organisms. The relationship between brain size and relative development of brain areas, and a

spectrum of ecological variables have been studied in by Kotrschal et al., 1998. The relative size

of sensory brain lobes, which reflects sensory specialization, was found to be closely-related to

feeding and the relative development of the integration areas (such as the telencephalon and

cerebellum) was found to be related to differences in microhabitat (Kotrshal et al., 1998). It was

found that the telencephelon brain area is larger in fish that live in spatially structured

environments (Huber et al., 1997).

This study ofB. neumayeri will address the link between human impact and the

morphological variation in fishes from disturbed and undisturbed habitats. We expect to find

evidence to support the idea that as rivers become more turbid due to deforestation, the

morphology of fish living in out-forest streams will change in response to the environment, in

this case, an increase in turbidity. It is possible that due to this anthropological impact affecting

fish morphology and feeding strategy, we could be cascading effects in the food web of the

rivers in West Uganda.

Given previous research on the Cyprinidae family of fish regarding eye size, barbel size

and brain lobe size, and the morphological variation across environmental gradients (Davis &

Miller, 1967; Lindsey et al., 2006), if there is an effect of turbidity on the morphological

characteristics ofB. neumayeri, I hypothesize that I will observe a difference in the

morphological features of the eyes, barbels and corresponding brain lobes in out-forest versus in-

forest streams. Specifically, I predict thatB. neumayeri populations experiencing increased

turbidity (i.e. out-forest streams) will have smaller eyes and optic lobes, and larger barbels and

facial compared to in-forest populations living in relatively clear water. This prediction will be

tested through extraction and measurement ofB. neumayeri barbels, eyeballs and brains from


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two sites in western Uganda with different levels of turbidity to determine if there is a

relationship between turbidity and the size of these traits.

Methods

Study Site and Species

In this study, I used preservedBarbus neumayeri fish collected from the Mikana (MIK) site

within Kibale National Park and from the Outforest Farm (OUTF) site near Kibale National Park

in western Uganda in 2010 and 2011 (Fig. 1). Standard G minnow traps were placed in the water

overnight to collect fish. The fish were euthanized using buffered MS222 in the field and then

preserved in 4% paraformaldehyde and transported to McGill University in Montreal, Canada.

Barbus Neumayeri is a member of the cyprinid family and is widely distributed in Eastern Africa

(Martinez et al., 2011). It lives in a diverse range of environments from turbid swamps to clear

open rivers (Martinez et al, 2011). Previous research has shown population specific variation in

traits related to morphology (Martinez et al., 2011).Barbus neumayeri feeds on small insect

larvae, aquatic plants and detritus (Chapman et al., 1999) and reaches a maximum length of 12.5

cm (Schaack & Chapman, 2003).

Initial Measurements

To obtain standardized measurements, the fish were soaked for 20 minutes in water and then

patted dry prior to photographing and weighing. The preserved fish were weighed on a

microbalance to the nearest 0.1g. To obtain an accurate measurement of standard length, the fish

was pinned onto grid paper (1 side of the square = 2 cm) overlaying a piece of cardboard, next to

a ruler for reference (Fig. 2). The fish was carefully pinned in the nose and in the tail fin so as

not to destroy the barbels or brain. Standard digital pictures of all fish were taken using a Canon

G10 digital camera that was attached to a tripod using a level. Pictures of each fish were captured

against a 2 cm x 2 cm grid and standard length was measured from these photographs usingImageJ.

Barbel Dissection

To determine if the length of the two dominant barbels of fish from Mikana in-forest (MIK) and

out-forest (OUTF) streams differed; the barbels were excised from the right side of the fish using


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a small pair of dissection scissors. A circular piece of fish cheek containing both barbels was cut

off and placed under a dissection microscope. Some barbels did not lie flat under the microscope;

and in cases such as this, the barbels were flattened as much as possible by applying pressure

using modeling clay or pinning the barbel down with a pair of forceps while a picture was taken.

Barbel Pictures and Measurements

A picture of the barbels under the dissection microscope was captured using the program Infinity

Capture (Fig. 3). Using the picture, the length of each of the barbels was recorded using the

program ImageJ. Each barbel was measured from its tip to the point of attachment to the cheek

tissue.

Eye Dissection

To determine the eye diameter, axial length, and the eye diameter to eye axial length ratio of fish

from in-forest and out-forest streams, the right eye was removed from the fish. Under a

dissection microscope, a dull pair of forceps was used to lightly carve around the perimeter of

the eye to detach it from connective tissue. As the tissue was detached a thin film covering the

eye began to come off and was then fully removed with forceps. Once this thin film was

removed, a mark on the dorsal area of the eye was made with a permanent marker in order to

identify the eyes orientation after its removal from the fish. The remaining connective tissue

underneath the eye was pulled off, or cut away with forceps until the optic nerve and surrounding

tissue was fully detached from the eyeball. The eye was then gently removed using forceps. All

other pieces of fat and tissue remaining on the eyeball were carefully removed. The eye was

placed in a beaker of water for five minutes to allow it to rehydrate.

Eye Pictures and Measurements

The eyeball was placed in a divot in modeling clay under a dissection microscope. A picture of

the eyeball was then captured using Infinity Capture as a digital photograph in two different

orientations. The first orientation was a top view of the eye (Fig. 4a). The dorsal edge of the eyewas identified using a pin that pointed at the mark made by the permanent marker before

dissection. The eye was then placed on its side in order to measure its width (Fig. 4b). A picture

was taken of its width with the dorsal side of the eye oriented upwards, towards the camera.

Using these photographs, the diameter of the eyeball was measured using an average of three

diameters. The first diameter measurement was taken from the dorsal marker, extending to a


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point directly across the dorsal marker and the second two measurements were taken across the

diameter of the eyeball from opposite corners of the eyeball. One eye axial length measurement

was taken from the side view of the eyeball, across the width of the eyeball at the dorsal marker.

The eyeball was then placed in paraformaldehyde solution for archiving.

Brain Dissection

To test if the sizes of specific brain components differed between populations,B. neumayeri

brains were extracted from the fish. The left eye was first removed from the fish to sever the

connection between the optic nerve and the brain, which simplified brain extraction. Using a fine

pair of forceps, an incision was made through the backbone of the fish by the dorsal fin, closer to

the anterior end (Fig. 5). The outer layer of scales and skin were removed between the fin and the

skull. The muscle tissue inside the fish was removed with a pair of forceps to expose the spinal

cord leading into the brain area at the top of the head. Once all the tissue was removed, the fish

was removed from under the dissection microscope and placed on a paraffin tray. The fish was

then pinned to the paraffin tray at the head. Two pins were inserted through the ocular spaces,

into the paraffin tray so as not to damage the brain. The posterior end of the fish was also pinned

down to orient the fish in an upright position for the brain extraction. The pinned fish was placed

under the dissection microscope and a shallow incision was made along the length of the head

using either a fine pair of forceps or scalpel. The incision spanned from the top of the skull to the

bottom of the nasal region. This was done very carefully as the brain was directly underneath the

thin layer of skull. The incised area was pulled upwards and to either side of the fish head to

expose the brain tissue. Forceps with an extremely fine tip or a hooked tip were used to remove

the remaining soft tissue from the exposed brain. The connective tissue surrounding the brain

was then carefully carved on both sides of the brain. The exposed spinal cord was then cut at the

posterior end of the skull, with at least 1 cm of spinal cord attached to the brain. The brain was

then lifted out of the skull carefully using forceps, and cleaned of extra tissue. The spinal cord

was cut at a brain landmark apparent on each of theB. neumayeri brains, specifically where the

spinal cord began to widen (Fig. 6).

Brain Pictures and Measurements

Digital photos of the brain were taken in three different orientations so that the volume of each

brain component could be calculated. The brain was first placed on a slide and corrected to be


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level in one plane by balancing it on small, metal pins. It was captured on the dorsal side, ventral

side, and side angle (Fig. 7). Measurements were taken using the digital photos for the length,

width and height for the following brain lobes: telencephelon, optic lobe, cerebellum and facial

lobe using image J. The volume of each brain lobe was calculated using the formula:

. The volume was multiplied by a factor of two for the

telencephelon and optic lobes since their total volume is composed of two symmetrical lobes.

The brains were then weighed on a microbalance to the nearest 0.0001g. The brains were small

and held in preservative. To ensure an accurate weight, every brain was weighed once every day

for three consecutive days, and their masses were averaged.

Statistics

Each of the measurements obtained (standard length (cm), body mass (g), eye diameter (mm),

eye axial length (mm), brain mass (g), and the length width, height of the cerebellum, optic

lobes, telencephalon, and facial lobe (mm)) were log transformed to help the homogeneity of the

variances, as tested with Levines test. ANOVA was used to detect a difference in the standard

length and body mass of the two populations. The statistical test performed on all other measures

was an ANCOVA, with population (in forest-MIK, out forest-OUT) as the fixed factor and a

measure of body size as the covariate. For body weight, eye components and brain mass,

standard length was used as the covariate. For brain components, brain mass was used as a

covariate. When the interaction between the covariate and population was not significant (i.e.

when the slopes of the two populations were homogeneous), the interaction term was removed

from the model. All analyses were performed using SPSS 17.0.

Results

Body Size

I tested for differences in body size (standard length and body mass) between the two

populations using 20 individuals from each population. A t-test showed no significant difference

in the average standard length between the two sites (F(1, 36) = 3.977, p = 0.054), although there

was tendency for Mikana fish to be longer. Outforest (OUTF) fish had a shorter average standard

length of 5.33 cm and Mikana Inforest fish (MIK) fish had an average standard length of 5.85 cm

(Fig. 8). In addition, there was no significant difference detected in body mass of the two


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populations (F(1, 36) = 3.709, p = 0.062), although, again there was a tendency for Mikana

Inforest fish to be heavier. The average weight of the Outforest fish was 3.014 g and the average

weight of the Mikana Inforest fish was 4.030 g (Fig. 10).

An ANCOVA showed a significant relationship between the standard length and mass of fish in

both sites (Outforest farm (OUTF): R2 = 0.964, Inforest Farm (MIK): R2 = 0.819). The intercepts

of the regression lines of the OUTF fish did not differ significantly from MIK (F(1,35) = 0.044,

p = 0.835, Fig. 9).

Brains

In total, six brains from each population were dissected and analyzed. Using an ANCOVA with

log standard length as the covariate, I found that the slope of the regression lines for the brain

mass ofB. neumayeri differed between the two populations; and the regression lines for the body

massbody length relationship were strong and linear (Outfish Farm (OUTF) R2

= 0.969,

Inforest Mikana (MIK) R2= 0.763). There was a significant population effect (F(1,8) = 5.619, p =

0.045), suggesting that OUTF fish have larger brains than MIK fish (Fig. 11). The only brain

component that differed between populations was the facial lobe. Using brain mass as the

covariate, an ANCOVA showed that the slopes of the regression lines for facial lobe volume of

B. neumayeri differed between sites (F(1,8) = 6.493, p = 0.034) and the relationships between

facial lobe size and brain mass were stronger for the Outforest Farm population (Outforest Farm

(OUTF): R2

Linear =0.589, Inforest Mikana (MIK): R2

Linear =0.246, (Fig. 12).

For all other brain components analyzed (telencephelon, optic lobes and cerebellum), with brain

mass as a covariate, there were no significant interaction terms (i.e. population*log standard

length) or population effects. Statistical values for all tests can be found in Table 1.

Eyes and Barbels

Two barbels from 20 fish per population were analyzed, while 10 eyes from individuals from

each population were analyzed. There were no significant differences between populations in

barbel length or eye size (Table 1).

A t-test found that the Eye Diameter to Eye Axial Length Ratio showed a significant difference

between the two populations (ED:AL) F(1,18) = 6.124, p = 0.024, Figure 13).


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Discussion

The objective of this study was to describe interpopulational variation in the sensory morphology

of the African cyprinid,Barbus neumayeri from two field sits, the Outforest stream (OUTF) and

Mikana Inforest farm steam (MIK) that differ in level of turbidity. I did this by quantitatively

comparing barbel length, eye diameter and axial length, and specific brain lobe volume. I

hypothesized that if there was an effect of turbidity on the morphological characteristics ofB.

neumayeri, I would observe a difference in the morphological features of the eyes, barbels, and

corresponding brain lobes in out-forest versus in-forest streams. I predicted thatB. neumayeri

populations experiencing increased turbidity (i.e. out-forest stream) would have smaller eyes and

optic lobes, longer barbels and facial lobes compared to inforest populations living in relatively

clear water. I found a significant difference between the two populations in facial lobe volume,

with the fish from the Outforest population having a larger facial lobes than the Inforest fish.

This was further confirmed by a difference in the overall brain mass between the two populations

with the Outforest fish having heavier brains than the Inforest fish. There was no difference in

the volume of the telencephenlon, optic lobes, cerebellum or in the length of the barbels. There

was also no difference found in the eye size between the two populations.

Body Mass/Size relationship

I tested for a difference in the size of the fish from the Outforest Farm site and the Mikana

Inforest Farm site by looking at the relationship between the mass and length of the fish. There

was no difference in the slopes of bilogarithmic relationship between the mass and length

between the populations. This means that fish of a given standard length in each of the

populations showed no significant difference in mass. This was also confirmed using a t-test

which tested for a difference in average standard length and body mass between the two

populations. Both of these t-tests showed there was no difference between the two populations in

standard length or mass. Therefore, the fish used in this experiment showed a good overlap in

size meaning that they are of a comparable nature. Significant differences cannot be attributed to

a difference in the body size of the fish.

Vision

I predicted that eye size would vary between the clear Inforest and turbid Outforest populations;

however, contrary to my predictions, I found that there was no significant difference in the eye


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size of the two fish populations (See Table 1). There was also no difference in the optic lobe

volume ofB. Neumayeri brains, which is the center of the brain that controls eye function and

processes visual stimuli (See Table 1). Previous research has shown that minnows in turbid

environments have reduced optic lobes compared to minnows in clear water (Davis & Miller,

1967), suggesting that in an environment with reduced visual ability, visual processing centers

are also reduced. However, other research on the scaling of the size of vertebrate eyes using axial

length showed that fish showed a remarkable range in variation in eye size in comparison to all

other vertebrate groups tested (Howland, et al., 2004). This weak relationship between body size

and eye size could be a potential factor in the non-significant difference between Inforest and

Outforest fish because there is no a relationship between body size and eye size to begin with.

Previous research on fish eye size and its relationship to turbid environments has only

been conducted on minnows therefore it is possible that phenotypic plasticity of eye morphology

varies between species. Furthermore, Evans (1952) found that normally, vision plays an

important role in cyprinid activity. However, in turbid aquatic environments, eyes are

compensated for by other sense organs which become more highly developed. In these

environments, the eye may become degenerate (Evans, 1952). This suggests that the effects of

the environment on fish are more obvious in other sense organs than the eyes. I found a

significant difference in the Eye Diameter to Eye Axial Length ratio (ED:AL) in the two

populations however the literature does not specify how this may effect vision. Given that the

size of the fisheyes in the two populations were similar, it is consistent that the associated optic

brain center, the optic lobe, shows no difference in size between the two fish populations, as our

results showed.

Olfaction

I predicted that barbel length would vary between the clear Inforest and turbid Outforest

populations; however I found that there was no difference in the length of Barbel 1 which is the

barbel located closer to the ventral side of the fish. I also found that there was no difference in

the length of Barbel 2, closer to the dorsal side of the fish. However, there was a difference in

overall brain mass between the two fish populations, with the heavier brains belonging to the

Outforest fish. This indicates that there could potentially be areas of hyper-development or

degeneration in one or more sensory lobes inB. Neumayeri brains (See Fig. 11). Further testing


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found that the only brain component that differed between populations was the volume of the

facial lobe, with the Outforest fish having a larger facial lobe (mm3) than Inforest fish (See Fig.

12). This result was in line with my initial hypothesis. Previous research on cyprinid fishes

showed a correlation between the size of barbels and the size of the facial lobe. More

specifically, fish that rely on skin tasting, using their mouth, lips, skin and barbels to forage,

had well developed facial lobes, whereas fish with reduced barbels and a reliance on their eyes

for feeding had reduced facial lobes (Evans, 1952). Therefore, an enlarged facial lobe in the

Outforest fish indicates that there is more activity or use of the taste sensory structures in the

outforest streams. The outforest streams were the turbid environments in this survey. In these

environments, it is more difficult to see underwater as less light is able to penetrate the

suspended sediment in the water (Donohue & Garcia Molinos 2009; Utne-Palm, 2002).

Therefore, fish in these environments rely on their sense of taste in order to obtain food. An

enlarged facial lobe, in comparison to the Inforest stream fish, may reflect a turbid environment.

One possible explanation for the lack of difference in barbel length between populations

could be that although the length of the barbels did not differ between populations, the surface

area or density of taste buds could differ, thereby allowing for more effective tasting. Further

investigation is required to determine if there is a difference in the density of taste buds between

the two populations which could contribute to the increased facial lobe volume. The mouth, lips

and facial skin are other areas on the cyprinid fish that are connected to the facial lobes via

sensory nerves. It may also be helpful to examine the density of taste buds in these areas as well

(Evans, 1952).

It has been found that barbel size largely influences the structure of the hind brain, more

specifically, the facial lobe (Evans, 1952) and so I would continue to further investigate this

difference as it is a primary indicator that human changes in the fresh-water environment in

Western Uganda could have a significant impact on the morphology ofB. neumayeri.

An important note is that these tests were based on a very small sample size with regards

to the eyeballs (NInforest = 10, NOutforest = 10) and brain (NInforest = 6. NOutforest = 6). For future

investigation, I suggest that a larger sample size is obtained for a more robust dataset. In addition

to increasing the sample size of the test populations, I would test fish from additional sites to


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observe if these trends are found over similar habitats (i.e. within turbid environments or clear

water environments) or if certain characteristics are associated with certain locations.

In terms of fish preservation, I suggest that the barbels be removed from the fish in the

field and preserved in a manner that allows them to lie flat. Many of the barbels I worked with

had curled in preservation and made it difficult to obtain accurate measurements. This was also

the case with the measurements of the fish brain. Given that the brains are not evenly distributed

in weight, they were difficult to capture in the same plane, skewing the measurements slightly.

Conclusion

The response of an organism to the environment they live in can occur physiologically,

behaviourally, or morphologically (Bradshaw, 1965). Despite substantial research on the effects

of turbidity on fishes, there is not a strong understanding of how turbidity may drive phenotypic

variation across populations (across habitats within species). It is becoming increasingly

important to understand the ecological and evolutionary consequences of turbidity as it is a

pervasive environment stressor in freshwater environments. My results show that there is a

significant difference brain size between the two fish populations, more specifically in the facial

lobe, with the Outforest fish having a larger facial lobe than Inforest fish. As the facial lobe is the

center for olfaction in the brain, this difference in size suggests that there may be morphological

differences in the olfaction sensory organs for the fish in turbid environments. It is possible that

this is a result of increased turbidity these environments. Further research should be done to

determine if there is a difference in the density of the taste buds in the barbels as there was no

difference found in the length of the barbels between the two populations. There should also be

an investigation of the impact of an increased or decreased Eye Diameter to Axial Length ratio

on the vision of the fish. In the future, the modification of preservation techniques would also

allow for more accurate measurements to be obtained.


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Tables and Figures

Table 1: Statistical Values for ANCOVA and t-test performed onB. neumayeri from two

different sites. N Outforest = 18, N Mikana Inforest = 20. Significant values are bolded.

df F p-value

t-test

Standard Length (cm) 1 3.977 0.054

Mass (g) 1 3.709 0.062

Eye to Diameter

Ratio

1 6.124 0.024*

ANCOVA

Log Mass1

(g) 1 0.019 0.890

Log Brain Mass1

(g) 1 5.619 0.045*

Log Telencephelon2

1 0.599 0.459

Optic Lobe2 1 0.000 0.995

Cerebellum2 1 0.967 0.351

Facial Lobe

2

1 6.493 0.034*

Barbel 1 Length1 1 0.923 0.343

Barbel 2 length1 1 0.050 0.852

Eye Diameter1 1 2.665 0.121

Eye Axial Length1

1 1.348 0.262

Starring indicates a significant interaction effect in addition to a significant population effect.

1

indicates that the covariate used was standard length (cm)2 indicates that the covariate used was brain mass (g)


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Figures

Figure 1

Fig. 1 Mpanga drainage and Dura drainage and surrounding areas showing study sites Outforest (OUTF) and Mikana (MIK)

(Red stars) Western Uganda (Maps provided by Vincent Fugre).


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Figure 2

Pin

2 cm

Measurement reference

Figure 2: Picture ofB. neumayeriwith reference ruler for measuring standard length and

ull length


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Figure 3

Barbel 2

Cheek tissue containing both barbels

Barbel 1


Tip

Figure 3: B. neumayeribarbels 1-bottom and 2-top and measurementlines (yellow line).


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Figure 4 a,b

Figure 4a

Figure 4b


Dorsal Marker

Eyeball

Divot

Figure 4a: B. neumayerieyeball top view orientation and diameter

measurements (yellow and red lines).


Width measurement at dorsal marker

Eyeball

Figure 4b: B. neumayerieyeball side view orientation and axial

measurement (yellow line).


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Figure 5

Incision point

Figure 5: Marker for backbone incision point on B. neumayerifor brain extraction (yellow line).


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Figure 6

Spinal cord cut off point

Figure 6: B. neumayeribrain: Spinal cord cut-off point on B. neumayeribrain (yellow line).


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Figure 7 a,b,c

Cerebellum

Telencephelon

Optic Lobe

Facial Lobe


Figure 7a: B. neumayeribrain lobes: Cerebellum (CC), Telencephelon (TE), Optic

Lobe (TO), Facial Lobe (FL).

Figure 7b: B. neumayeribrain lobes length and width measurements (yellow line):

Cerebellum (CC), Telencephelon (TE), Optic Lobe (TO), Facial Lobe (FL)

measurements: Width (w), Length (l).

FLw

FLl

CCwCCw

TOw

TWw

CCl

TOl

TEl

Figure 7c: B. neumayeribrain lobes height measurement (yellow line): Cerebellum

(CB), Telencephelon (TN), Optic Lobe (OL), Facial Lobe (FL) measurements: Height (h).

TEhTOh

CChFLh


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Figure 8a,b

Figure 8a

Figure 8b

Figure 8b: Estimated marginal means of Log Standard Length (cm)

showing the difference in Log Standard Length ofB. Neumayerisample

population from OUTF and MIK sites.

Figure 8a: The average standard length ofB. Neumayerisample

population from OUTF and MIK sites.


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Figure 9

Figure 9: The relationship between Log mass (g) and Log standard

length (cm) for B. Neumayerisample population from OUTF (Blue) and

MIK (Green) sites.


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Figure 10a,b

Figure 10a

Figure 10b

Figure 10b: Estimated marginal means of Log Mass (g) showing the

difference in Log Mass ofB. Neumayerisample population from OUTF

and MIK sites.

Figure 10a: The average mass (g) ofB. Neumayeri

sample population from OUTF and MIK sites.


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Figure 11a,b

Figure 11a

Figure 11b

Figure 11b: The estimated marginal means of Log Average BrainMass (g) between OUTF and MIK B. Neumayerisample

populations.

Figure 11a: The relationship between Log brain mass (g) and Log

standard length (cm) ofB. Neumayerisample population in

OUTF sample population (Blue) and MIK sample population

(Green).


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Figure 12a,b

Figure 12a

Figure 12b

Figure 12b: The estimated marginal means of Log Facial Lobe Volume (mm3)

between OUTF and MIK B. Neumayerisample populations.

Figure 12a: The relationship between Log brain mass (g) and

Log Facial Lobe Volume (mm3) ofB. Neumayerisample

population in OUTF sample population (Blue) and MIK

sample population (Green).


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Figure 13

Figure 13: The Eye Diameter (ED) to Axial Length (AL) ratio (ED : AL) for OUTF

and MIK B. Neumayerisample populations.

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