AN

EXPERIMENT TO STUDY THE PREFERENCE OF MEALWORMS FOR COLOUR

By Narakhanti

EXPLORATION
1. Research question
Do larvae of Tenebrio molitor have a preference for colour (red, yellow, blue, or green)
as shown by the number observed in quadrants of a choice chamber containing different
coloured oatmeal as food sources after a period of 2 minutes?

2. Background information
Yellow mealworms are the larvae of the darkling beetle, Tenebrio molitor. Beetles are
a group of insects that form the order coleoptera. They are holometabolous insects,
meaning they go through complete metamorphosis throughout their lifetime, which
involves four stages — egg, larva, pupa, and adult.
Mealworms are nutritious insects that are high in both fat and protein (Ghaly and
Alkoaik, 2009), making them a suitable food source for animals such as birds, reptiles, and
even human beings. They are commercially farmed and therefore it would be interesting
to see whether they have a preference for colour in order to optimise their living
conditions. Environmental enrichment is a process used to stimulate different
environment conditions for animals held in human captivity in order to enhance their
well-being (Passos et al, 2014). As nutrition is a vital part of a living thing’s life, it would
be beneficial to see if mealworms prefer a certain colour of food over another. In this
investigation, the colours of the food are changed by staining oatmeal with different food
colourings — blue, green, yellow, and red.
Mealworms are invertebrates, which are known to have poor vision as their eyes are
not as fully developed as vertebrates, however it is unknown whether they are colour
blind. As mealworms are often found in the wild under rotting logs, leaves or rocks, they
prefer dark environment to bright environment. (Darkling Beetle Or Mealworm
Background Information, 2010). Larvae of holometabolous beetles have simple eyes
which are called the stemmata, which are derived from the typical compound eyes of
adult insects (Buschbeck, 2014). These are simple visual organs which allow them to see,
as it contains a retinula, which is composed of either one or two layers of photoreceptive
cells (Koenemann and Jenner, 2005). These cells are sensitive to light so it allows them to
detect light by converting the light energy to electrical signals, which is then sent to the
central nervous system (Carlson and Chi, 1979), enabling them to differentiate between
dark and light. However, there is insufficient research to prove that they are able to detect
colour.

3. Findings from preliminary experiments
3.1 Choice of food source
The first thing done for the preliminary experiment was to decide which food
source to use. This was done by placing 15 mealworms on a tray and putting pieces of
oatmeal in front of them. After a few minutes, 12 of them would chew on the oatmeal.
With this, the experiment can proceed.

3.2 Number of mealworms per trial

Some animals like sheep have gregarious instinct (Cobb, 1999) in which they
would follow each other (herd behaviour). To determine whether the mealworms
should be placed individually or a few at once in the choice chamber, I tested whether
mealworms possess this behaviour by placing 10 mealworms in a tray and observed
where they moved after 2 minutes. 7 of them stayed a group whilst 3 strayed away,
which shows that it would be best to place them individually in the choice chamber to
ensure a fair test.

3.3 Possible bias whereby a mealworm would go in the direction they faced
The direction in which a mealworm faces might affect which direction they go
in, and since this is crucial in a choice chamber experiment, it must be tested. Thus,
15 mealworms were placed in a choice chamber, and the direction in which they
faced, along with the quadrant the mealworm was found in 2 minutes later, was
noted. Out of the 15 mealworms, 10 of them went in the direction they were facing.
Therefore, there is a discernible bias, one that can be avoided by rotating the
direction in which a mealworm faces during each repeat of the experiment to ensure
fair testing.

4. Hypothesis
Mealworms would have a preference for colour because in terms of environment,
they significantly prefer dark environments to light environments (Darkling Beetle Or
Mealworm Background Information, 2010), meaning they are sensitive to brightness and
are able to detect light – suggesting the possibility that they can differentiate in between
colours.
The mealworms would prefer the green coloured oatmeal and would avoid the red-
coloured one. This is because the colour green is the most abundant in nature as it is the
colour of the trees, grass, plants, and therefore would be familiar to them. Whilst they
would avoid the red oatmeal as through evolution, they might associate the colour with
poisonous fruits such as berries in nature.

5. Variables

Variable Method and reason

Independent Colour of oatmeal Four different food colourings were used to
(red, blue, green, stain the oatmeal — red, blue, green, and
yellow) yellow.
Dependent Mealworm The quadrant of a choice chamber where each
preference of colour mealworm was located was noted after 2
in food minutes.
Controlled Choice chamber set The colour of the whole choice chamber is black
up to optimise the conditions as mealworms prefer
dark environments (Darkling Beetle Or
Mealworm Background Information, 2010), and
the equal dimensions of it ensured the fairness
of the experiment.

Controlled Variable (continued) Method and reason

(continued)
Number of One mealworm was placed in a chamber at a
mealworms per trial time as shown by the preliminary experiment,
they possess herd behaviour.
Texture of oatmeal 20 cm3 of water is mixed with a few drops of
food colouring and 10 g of oatmeal. Kept
constant to avoid bias.
Taste of oatmeal The same oatmeal brand was used throughout
the whole experiment and since food colouring
has no flavour, the taste was kept constant.
Mass of oatmeal 10 g of oatmeal was measured by using a
(± 0.1 g) weighing scale. Kept constant to prevent bias.
Direction which each The mealworm would be physically rotated
mealworm faced clockwise as repeats are done by placing them
when placed in the facing a specific colour each time — yellow,
centre green, red, and blue respectively and this was
repeated for all the mealworms.
Time for mealworm The time was measured for 2 minutes in each
to travel in the trial using a stopwatch — this gives enough time
choice chamber and for the mealworm to crawl around and settle
pick a colour onto a specific coloured oatmeal.
Species of The same species of mealworm (Tenebrio
mealworm molitor) was used. This is to ensure the
reliability of the experiment.
Age of mealworm The mealworms obtained was all about the
(length and colour) same age as can be seen by the colour of its skin
and its similar length. Age might have impact on
appetite.
Uncontrolled Temperature The air-conditioning was set to room
temperature (25oC) and monitored by a
thermometer. No action was taken to ensure it
was at the optimum where mealworms would
be most active. It might have fluctuated due to
the weather and room conditions (doors being
opened)

6. Apparatus

No. Apparatus and materials Justification

1. 100 species of Tenebrio molitor larvae Subject of the experiment
2. Black cardboard (flexible) To make main circular wall of choice
chamber

3. Black plastic board As a base of the choice chamber

4. Tape To stick the main circular wall of choice
chamber and dividing walls on the base
5. Ruler To measure choice chamber dimensions
6. Scissors To cut cardboard and plastic board
7. Stapler To join the black flexible cardboard together
to make circular wall of choice chamber
8. 40 g of oatmeal As a food source for the mealworm
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9. 4 x 100 cm glass beaker Used in making of the coloured oatmeal,
one for each colour to avoid mixture of
colours
10. 4 x 20 cm3 of distilled water Used in the oatmeal mixture to make it
slightly moist and dilute the food colouring
11. 4 different colours of food colouring To stain the oatmeal with four colours
(blue, green, yellow, blue) (independent variable)
12. 4 x 3 cm3 pipettes To transfer food colouring to beaker
13. Digital weighing scale (± 0.001 g) To measure mass of oatmeal, the oatmeal is
placed on the weighing boat and then
14. Weighing boat
weighed using the scale
15. Glass stirrer To stir the oatmeal mixture, ensuring an
even texture and colour distribution
16. Spatula To transfer oatmeal from glass beaker to
choice chamber
17. Stopwatch To measure the time a mealworm is placed
inside the choice chamber

7. Methodology
7.1. Preparing coloured oatmeal
1. Pour 3 cm3 of blue food colouring and add distilled water into the glass beaker
until it reaches the 20 cm3 mark.
2. Mix thoroughly using a glass stirrer to ensure the colour spread is even.
3. Put a weighing boat on the digital weighing scale, set it to 0.00 g. Then measure
and transfer 10 g of oatmeal using a spatula to the glass beaker filled with the
coloured water.
4. Mix the oatmeal and the mixture of food colouring and distilled water using a
glass stirrer until the oatmeal has even colouring and consistent texture.
5. Repeat steps 1-4 3 more times using red, green, and yellow food colouring,
resulting in four different coloured oatmeal. Ensure to rinse spatula and glass
stirrer between making each coloured oatmeal to prevent colour transfer.

7.2. Making of choice chamber (refer to diagram 1 below)

1. Measure and cut 48 x 7 cm of black plastic cardboard to make the main circular
wall of the choice chamber, staple it to form a circle. Stick this to the base, which
is the black plastic board, using tape.
2. Measure four 5 x 7 cm pieces of the black plastic board to make the dividing walls
of the choice chamber.
3. Tape the dividing walls in place to create 4 equal sectors. Since the diameter is 48
cm, stick one wall using tape every 12 cm of the circumference.
4. Cut out 16 x 7 cm of black plastic cardboard and use a stapler to form an open-
ended tube. This will act as a central cup to hold each mealworm before each trial
begins.
5. Put all the oatmeal in each section of the choice chamber. Even it out using a
spatula so that it touches the edge of the central circle. This is done to make
certain that the distance from each mealworm to the food is kept constant, and to
ensure that the mealworm would move towards the food.

Diagram 1: A diagram to represent choice chamber

7.3 Experiment
1. Place one mealworm in the central cup — in each trial, ensure that the mealworm
is rotated clockwise such that it goes from yellow to green to red and to blue. This
removes the bias of the mealworm simply going to the chamber that they were
facing.
2. Lift the central cup and start the stopwatch simultaneously.
3. After for 2 minutes, note where the mealworm is by using a tally system.
4. Repeat steps 1-3 100 times for verification using a different mealworm each time.
Ensure that the mealworms used are the same colour and about the same size.
5. Carry out a chi-squared test to test for an association between different coloured
food and mealworm preference.

8. Ethical implications
8.1. Animal welfare
In this experiment, the mealworms are fed food they do not usually consume
on a regular basis. They were given natural whole grain oats (FairPrice brand) instead
of the usual wheat bran. However, the mealworms can easily tolerate this slight

change as the foods are relatively similar. The mealworms were bought commercially
from a farm in a plastic container of wheat bran. It was ensured that for the duration
of the investigation, they had sufficient food source (bran), water (from pieces of an
apple which was changed every few days), and air (by poking holes on top of the
container) to avoid stress on the mealworms. After the experiment, the mealworms
were released back to the wild (a forest next to the school grounds) to allow them to
be back in their natural habitat. During the experiment, the mealworms were picked
up and placed carefully in the choice chamber using hands instead of forceps to
ensure that no harm was done to them.

8.2. Food waste
The oatmeal used in this experiment could have been consumed by humans
so to minimise the leftover, only the required amount of oatmeal was used in the
experiment and only that amount was brought inside the lab, therefore the rest were
not contaminated and can still be used as a source of food. However, not all the
oatmeal used in the experiment was eaten by the mealworms, therefore an amount
of oatmeal (around 40 g) went to waste.

9. Safety implication
Hygiene: Mealworms, like many other living things, are able to carry viruses that may
cause intestinal canthariasis and hymenolepiasis (Medical News Today, 2014), which are
diseases in birds (predator of mealworms) and can transfer to humans. It is therefore
important to maintain good hygiene practice by washing hands after the experiment with
anti-bacterial soap and cleaning the table area used to carry out the experiment.

10. Environmental implication
After the experiment, the mealworms were released to a secondary forest beside the
school. As mealworms are decomposers, they feed on organic material such as dead
leaves and feces of animals (Allott and Mindorff, 2014). They would have a positive effect
on that particular ecosystem by contributing in the disposal of dead organic matter to
avoid excessive accumulation.

ANALYSIS
1. Raw data
1.1 Table
Table 1: A raw data table to show tally of the number of mealworms that was present at
each coloured oatmeal after being placed in the choice chamber for two minutes

Colour of oatmeal Mealworm tally Total

Blue |||| |||| |||| |||| |||| 24

Green |||| |||| |||| |||| ||| 23

Red |||| |||| |||| |||| |||| |||| |||| |||| |||| | 46
Yellow |||| || 7

1.2 Qualitative observations

- 4 mealworms stayed in the centre and 3 mealworms did not touch the food and tried
climbing up the walls instead. They did not make an obvious choice and were
discounted and then repeated (still using the same species).
- Some mealworms moved from one colour to another whilst some would just stay on
the same colour they picked in the beginning.
- Some mealworm would eat the oatmeal whereas others would only stay on that
specific coloured oatmeal

1.3 Image
Image 1: An image to show set up during one of the trials

Main circular wall

Coloured oatmeal

Dividing wall

Mealworm

(Author’s own, taken 15 November 2016)

2. Processed data
2.1 Graph
Graph 1: A bar graph to show the total number of mealworms that was present at
each coloured oatmeal quadrant after being in the choice chamber for 2 minutes

50
45
40
Number of mealworms

35
30
25
20
15
10
5
0
Blue Green Yellow Red
Colour of oatmeal

Graph 1 shows that there is a preference for red coloured oatmeal in the
mealworms, with nearly half of the population picking that colour. The blue and green
coloured oatmeal had almost an equal number of mealworms choosing it, 24 and 23
respectively. In contrast, only 7 mealworms picked the yellow coloured oatmeal,
which is less than 10% of the total population.
From the graph, red is majorly preferred by the mealworms. However, due to
the moderate numbers of mealworms also picking green and blue, with the total
making up 47% of the mealworms, it is unclear whether there is a significant
preference. In order to test for a preference, a chi-squared test was conducted. This
test is used to test for association between the different coloured oatmeal and the
preference of mealworms. It was done because the type of data collected was count
data.

2.2 Chi-squared test (x2)
H0 (null) hypothesis: There is no preference for colour in mealworms.
H1 (alternate) hypothesis: There is a preference for colour in mealworms. This
hypothesis would only be accepted if the level of probability is less than 0.05 on a chi-
squared table for a particular degree of freedom.
total number of mealworms = 25
E =
number of categories
100
=
Table 2: A processed data table to show chi-squared test calculations
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Category Observed Expected O - E (O - E)2

value (O) value (E)
Blue oatmeal 24 25 -1 1 0.04
Green oatmeal 23 25 -2 4 0.16
Red oatmeal 46 25 21 441 17.64
Yellow oatmeal 7 25 -18 324 12.96
Σ = 100
Σ = 30.8

The x2 value is 30.8.
The degree of freedom (df) is calculated by subtracting the number of categories (n) by
one.
df = n - 1 = 4 - 1 = 3

3 Discussion
Looking at a chi-squared table, the critical value at 5% probability for df = 3 is 7.815.
Since the x2 value exceeds the critical value (30.8 > 7.815), the null hypothesis (H0) is
rejected and the alternative hypothesis (H1) is accepted.
The chi-squared test results as shown in Table 2 indicates that the alternative
hypothesis (H1) stating that there is a preference for colour in mealworms is accepted.
Looking at the data (refer to Table 1 and Graph 1), it is clear that the red coloured oatmeal

had the most number of mealworms going into that quadrant of the chamber so it can be
said that mealworms have a preference for red coloured food.
On the other hand, they disfavour the yellow coloured oatmeal. Whereas the blue and
green oatmeal had almost the same number of mealworms (only a difference of one
mealworm) going into that quadrant of the choice chamber so it can be deduced that it
has no preference for these two colours. It is however important to note that 7 out of the
100 mealworms, did not pick a colour and therefore was repeated. The first trial involving
these 7 mealworms were excluded in the calculations.

EVALUATION
1. Conclusion
To conclude, the data obtained from this investigation supported my hypothesis that
mealworms do have a significance preference for colour.
However, it disproved my hypothesis that mealworms would prefer green and avoid
the red coloured oatmeal as the data collected from the experiment shows that nearly
half (46) of the mealworms chose the red coloured one whilst only 7 picked the yellow
one. Thus, there is in fact a preference for the colour red and least interest in yellow,
which suggests the complete opposite to my hypothesis.
The red coloured oatmeal might have been preferred by the mealworms since it is the
darkest colour, and mealworms do prefer darker environments (Darkling Beetle Or
Mealworm Background Information, 2010). Furthermore, the mealworms might see the
colour yellow to be the brightest colour and therefore would avoid it. One other factor
that might have affected the result is the fact that the first 7 mealworms chose the red
coloured one, so there might have been a bias towards it as the first mealworm may have
left a scent trial to follow.
Finally, the green coloured oatmeal was not preferred as mealworms are detritivores
which feed on dead and decaying matter (Melms and Boyer, 2010) so they would mostly
live in dark environments (e.g. under dead leaves, logs, etc) and stay there throughout
their lifetime. In contrast to my hypothesis, they would not be familiar to the colour green
due to their habitat and method of obtaining nutrients.

2. Strengths of investigation
This experiment was done as accurately as possible in a high school laboratory and
with the materials and time available. It was also a very fair experiment because a large
number variables that can potentially affect a mealworm’s behaviour and contribute to
the biases were kept constant. The only factor that was changed was the food colour and
other factors such as texture, taste, consistency, and mass of the food were controlled.
The choice chamber was customised to the size of the mealworms to get results as quickly
as possible, to allow for as many trials as possible within the time constraint, and to
minimise food waste.
Food colouring was used as it is a simple technique to transfer colour to the oatmeal
without having any effect on the taste and scent. By using this method, it was ensured
that the only factor changed was the colour of the oatmeal.
The number of repeats was another strength as it was sufficient to perform a reliable
statistical test (chi-squared test) to show whether mealworms have a significant
preferences colour in food.

3. Weaknesses and improvements of investigation

Limitation Significance Improvement

Contamination of oatmeal High — when a mealworm Change the oatmeal that has
picks a colour and stays in been contaminated by the
that quadrant, it might have mealworm every trial. To
left a scent trial which may minimise the excess waste,
cause the next mealworm to reduce the amount of
pick the same colour as the oatmeal in each chamber.
scent would therefore be This is done to remove the
familiar to them. This is a scent and prevent biased
large bias as mealworms are choices. The cleanliness of
known to have a good sense the choice chamber should
of smell, and rely heavily on also be maintained by wiping
it. any excess dirt or displaced
oatmeal in between each
trial.
No evidence whether High — there was a lack of Since it is known that
mealworms can see colour information regarding the mealworms can differentiate
eyes and vision of between light and dark,
mealworms, and most replace the 4 colours with
importantly whether they different shades of one
can detect colour as opposed colour and see whether there
to only being able to is a preference.
differentiate between levels
of light and darkness, which
is the main purpose of this
experiment.
Reliability of findings Medium — 100 mealworms Do more repeats. To save
is a sample size big enough to time, invest in making
perform a statistical test on another choice chamber so
but it does not completely trials can be done
represent the whole species. simultaneously.
The red food colouring Medium — the colour was Try another brand of red food
resulted in a dark pink- slightly lighter than red which colouring and ensure that it
coloured oatmeal instead of might have affected the would properly stain the
red results. It can be questioned oatmeal red prior to the
whether the same result actual experiment.
would be obtained if the
oatmeal were stained red
instead of dark
pink. However, it is not very
significant as dark pink is still
a shade of colour close to
red.

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Imperfect choice chamber Low — whilst the Use a more accurate

dimensions measurements were taken as measuring tool — a compass
precisely as possible, the instead of a ruler to measure
dimensions of where the diameter and distance
dividers were placed were between each wall. Use glue
inaccurate due to human gun instead of tape to place
error, making some the walls to ensure that the
quadrants bigger than others, size of the quadrants are
which might lead to biases more equal.
when picking a colour.
Uncertainty of measuring Low — the weighing scale Use a burette or a measuring
equipment and beaker have cylinder instead of a beaker
uncertainties might have to measure the 20 cm3 of
caused slight difference in liquid.
consistency of the oatmeal.

4. Extension
As it is now known based on this experiment that mealworms prefer the colour red, it
would be interesting to test whether they can definitely detect colour, or can they only
differentiate between light and dark. An experiment can be done to see if they have a
preference for light or dark coloured food by repeating the same experiment but changing
the independent variable into different shades of red.
Another possible extension would be to perform another environmental enrichment
experiment to see whether mealworms have other preferences in food, such as in
consistency or scent.
An experiment to test consistency preference in food can be done by having different
consistency or texture of a single food. A fruit such as a banana or an apple can be used
to test for preference in consistency, as it can be juiced for different time lengths or
prepared in different ways (e.g. cut blended, mashed, etc) resulting in different
consistencies while keeping other factors constant.
A preference for scent can be tested by soaking the oatmeal in milk and exposing it to
air for different lengths of time, so the milk would rot (the longer the time exposed to air,
the more rotten). This would determine whether mealworms prefer food prefer with a
stronger scent (assuming the more rotten the milk, the stronger the scent).
The same experiment can be tested on other detritivores or an adult Tenebrio molitor
beetle to see whether it would produce the same results. Further research into colour
preference and eyesight of mealworms may lead to more definitive answers as to whether
they can differentiate between colours.

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