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Phototaxis in ​Zophobas morio ​larvae

Stephanie Finley and Summer Stevenson

Lakehead University

M. Zettek

EDUC - 4200

26 February 2017
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Introduction

In this study we will observe the impact of light stimuli on ​Zophobas morio​ (​Z. morio​)

larvae. ​Z. morio, ​also known as superworms,​ ​are a member of the Tenebrionidae family. This

family of insects was named ​Tenebrio,​ which translated means ‘seeker of dark places,’ due to

their nocturnal behaviour (Jaeger, 1955). It is important to study the behaviour of ​Z. morio ​larvae

as they are a commonly reared insect in various industries.

The main use for ​Z. morio ​larvae is in pet trade as they are high in protein thus making

them optimal for food for various pets (Miao, 2011). ​Due to their low-cost, population

abundance, and macronutrient profile, studies suggest that ​Z. morio ​larvae are a​ viable insect to

be used for biodiesel production as they have the potential for use as feedstock for applications

in energy production (Leung et al. 2012). The larvae can also be used to assist in the

degradation of various plastics as larvae can grow normally on a diet of bran and various

plastics (Miao, 2011). ​Z. morio ​larvae are also being explored as an option for livestock feed

and as a replacement of fish meal in fish farming as they provide effective nutrition while

keeping costs down (​Abd Rahman Jabir et al. 2012)

It is important to examine ​Z. morio ​larvae in various conditions as they can be affected

by multiple factors that may impact rearing attempts. In knowing how these larvae behave in

different conditions, rearing can be optimized by providing optimal environments, feeds, and

handling and harvesting techniques. For example, by knowing how the worms react to light, the

environment can be set up to enhance potential rearing outcome.

An organism's response to light stimuli is also described as phototaxis, -taxis referring to

the “innate behaviour in which an organism responds to a factor by moving towards or away

from it,” (Chen et al. 2012). If an organism exhibits phototaxis, the response can either be

negative, in which it moves away from light, or positive, in which it moves towards the light.
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“Light has properties of intensity, wavelength and degree of polarization and can be a powerful

tool of environmental influence on insects in a physiological, behavioural and ecological way

including phototaxis” (Erens et al. 2012). As burrowers, ​Z. morio ​larvae are not exposed to large

amount of light in their natural habitats and previous studies have shown that both adults and

larvae have negative phototaxis (Miao, 2011). Therefore, we hypothesize that ​Z. morio​ larvae

will demonstrate negative phototaxis when placed in an arena where the option of light and

darkness are both present. If this is true, then more larvae will move towards the dark side of

their enclosure when light is present.

This study was controlled by having consistent temperature, humidity, and field

enclosure to ensure this did not alter worm behaviour. The independent variable was the

presence of light and the dependent variable that was studied was the ​Z. morio ​larvae.

Materials and Methods

The behavioural trials measuring the phototactic response of ​Z. morio​ larvae were

conducted in a dark room in a private home in Thunder Bay, Ontario over the course of two

days (January 27 & 28, 2018). 45 larvae were monitored in both near-dark and light conditions

during the experiment. The ​Z. morio ​larvae were obtained from a local pet store and were stored

in a translucent container filled with wheat bran. We were unable to identify the packaging date

of the larvae and they appeared to be in various stages of development. The larvae were

randomly divided into 3 groups of 15 individuals and placed in separate containers filled with

wheat bran.

The trials were conducted in an enclosure made from a shoebox. Any openings in the

box were sealed using black electrical tape to ensure that no larvae escaped during the

experiment. A piece of cardboard was used to cover roughly ⅓ of the top of the open box in

order to create a dark refuge inside the enclosure. A desk lamp with a 60W ‘Soft White’ LED
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bulb was positioned directly overhead (see figures 1, 2, & 3). An LED bulb was chosen over an

incandescent bulb due to its low running heat in order to maintain the same temperature in both

the uncovered and covered areas of the enclosure. Each group of larvae were transferred into

clear glass containers in order to minimize the amount of wheat bran contamination in the

enclosure and increase the efficiency of transfer during trials.

The experiment was conducted in the same room between the hours of 14:00-20:00 and

the ambient temperature fluctuated between 20℃-21℃ during each trial. Group 1 was placed in

the middle of the enclosure with the assistance of a shaded light. The light was immediately

turned off and the larvae were left in the dark for 10 minutes. After 10 minutes the shaded light

was illuminated and the larvae in the uncovered area were quickly counted and recorded. The

overhead lamp was then turned on for 10 minutes and the larvae were reset to be in the middle

of the enclosure. Once the 10 minutes were finished the larvae in the uncovered area were

counted, recorded, and reset. This process was repeated once more. Once Group 1 completed

2 rounds the larvae were returned to their container with wheat bran and the remaining groups

underwent the same trial.

Figure 1: Enclosure in natural light Figure 2: Enclosure in near-darkness Figure 3: View of inside of enclosure in
with overhead lamp illuminated near-darkness with overhead lamp
illuminated
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Results

Across groups (of 15 individuals) we found that the mean of larvae in the covered area

was 3.8 (25.56%) when near-dark and 10 (66.67%) when exposed to the 60W light (see figure

4). In each trial there were consistently more worms in the covered section during the light

events (table 1) which would suggest that the larvae prefer dark environments and will move

towards them in response to light stimuli. In order to determine if there was a significant

difference between these two conditions we input the data (table 1) into a paired two-tailed

t-test.

Figure 4: The mean of ​Z. morio​ larvae in both the covered and uncovered sections of the enclosure during near-dark
and light events.

Table 1: Number of ​Z. morio ​larvae in covered area after being exposed to near-darkness or 60W light for 10
minutes.
Trials Near-Dark 60 W Bulb Difference

1 2 7 -5

2 4 9 -5

3 5 13 -8

3 8 11 -3

5 2 10 -8

6 2 10 -8
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A paired two-tailed t-test established that the p-value < 0.05 (p-value = 0.00087). Based

on this evidence we can reject the null hypothesis (​H​o​: there is no difference between

distribution of ​Z. morio ​larvae during light and dark events) and tentatively accept the alternative

hypothesis (​H​A:​ ​there is a difference in the distribution of ​Z. morio ​larvae during light and dark

events). This means that the distribution of larvae was not due to chance and supports the

assumption that larvae demonstrate negative phototaxis by moving to dark environments during

events of light stimuli.

Discussion

When placed in an arena where the option of light and darkness are both present, ​Z.

morio ​larvae move towards darkness which supports our hypothesis that ​Z. morio ​larvae

respond with negative taxis to light stimuli. Along with this, we observed that larvae were also

much more active during the count after periods of light than in periods of dark. We found that

when the light was on, larve tried to climb up the sides of the enclosure which was not the case

in the near-dark environment.

These observations reflect the natural history of ​Z. morio ​and the Tenebrionidae family.

In their natural habitat, this species prefers dark, damp, and humid areas and is a burrower that

pupates under the soil, consuming decaying vegetation, leaves, and tree bark during both larva

and adult stages (Ward’s Science, 2013). In laboratory settings ​Tenebrio molitor ​(​T. molitor​;

another member of the Tenebrionidae family) are known to bury themselves in their respective

substrate when exposed to light (Cloudsley-Thompson, 1953).

By conducting the experiment in an enclosure without a substrate the larvae were

forced to respond in a different manner than burrowing. This could explain why the larvae were

expending more energy in response to light stimulus as they were looking for a place to burrow,

not just a place that was dark. While burrowing may also have been important during near-dark
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events, the larvae may have selected to remain still instead of expending more energy to locate

a burrowing spot.

During our first phase of experimental design we tried lining the enclosure with damp

paper towel in order to mimic a more natural environment and found that the larvae would

burrow under the towel regardless of light or dark events. This provides further evidence that

burrowing is one of the primary behaviours of ​Z. morio ​larvae. However, we did remove the

paper towels as the burrowing limited our ability to draw conclusions about movement towards

or away from light sources as we had to disrupt larval position during counting events. It would

have also been extremely challenging to monitor the change in humidity levels over the course

of the experiment without relative air humidity measurement tools.

We were also unable to conduct the trials in true darkness as we lacked a dark room,

photometer, and night vision tools that would have allowed us to do so. Therefore, we cannot

draw any conclusions about the behaviour of ​Z. morio ​larvae in a true control environment (total

darkness). Our results are also contingent on the wavelength and intensity of the bulb used

during the trails. While negative phototaxis in ​Z. morio ​larvae is widely accepted, as well as

supported by our experiment, we do not know the implications of different colours of light or light

intensities. Future research should be done to examine the impacts of these different forms of

light. However, previous research has found that ​T. molitor ​adults and larvae have a visible

range that lies between < 350 and 700 m​μ​ ​(Yinon, 1970) which accommodates most lights

readily available for insect rearing. Therefore it is highly likely that negative phototaxis will still

be exhibited in typical insect rearing conditions.

We were also unable to control the life stages of the larvae used during the trials. While

it is likely that all larval stages exhibit negative phototaxis there hasn’t been extensive research

on adult beetles ​(Cloudsley-Thompson, 1953)​. It is not uncommon for larval stages of insects to
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be negatively phototactic while adults are positively phototactic and other members of the

Tenebrionidae family present positive phototaxis during their adult stage (Duehl et al., 2011).

This is something that should be uncovered as positively phototactic insects are much easier to

spot and capture than their negatively phototactic counterparts.

This study illustrates the importance of minimizing light pollution in ​Z. morio ​rearing

facilities when handing or examining larvae. Light will act as a deterrent which will make finding

larvae difficult and may also cause undue stress to the organisms. Perhaps this can also shed

some light on potential harvesting methods that aim to reduce stress and burrowing behaviours

by reducing direct light exposure.

Works Cited

Abd Rahman Jabir, M.D., Razak, S.A., & Vikineswary, S. (2012). Nutritive potential and

utilization of superworm (Zophobas morio) meal in the diet of Nile tilapia (Oreochromis

niloticus) juvenile.​ African Journal of Biotechnology​, ​11​(24), 6592-6598.

Chen, Y., Luo, C.W., Kuang, R.P., Li, H.W., Chen, Z., & Liu, Y.J. (2012). Phototactic behavior of

the armand pine bark weevil, ​Pissodes punctatus. ​Journal of Insect Science,

13​(3), 1-10.

Cloudsley-Thompson, J. L. (1953). Studies in diurnal rhythms. IV. Photoperiodism and geotaxis

in ​Tenebrio molitor ​L. (Coleoptera: Tenebrionidae). ​Physiological Entomology, 28​(10-12),

117-132.

Duehl, A. J., Cohnstaedt, L. W., Arbogast, R. T., & Teal, P. E. (2011). Evaluating light attraction

to increase trap efficiency for Tribolium castaneum (Coleoptera: Tenebrionidae). ​Journal

of Economic Entomology​, ​104​(4), 1430-1435.

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Erens, J., Es van, S., Haverkort, F., Kapsomenou, E., & Luijben, A. (2012). ​A Bug’s Life:

Large-scale insect rearing in relation to animal welfare. ​Wageningen University​.

Leung, D., Yang, D., Li, Z., Zhao, Z., Chen, J., & Zhu, L. (2012). Biodiesel from ​Zophobas morio

larva oil: Process optimization and FAME characterization. ​Industrial & Engineering

Chemistry Research​, 51(2), 1036-1040.

Jaeger, E.C. (1955). ​A source-book of biological names and terms​. Springfield, Ill., Thomas.

Miao, S.J. (2011). Study on biological characteristics of ​Zophobas morio​ and its function on

plastic degradation​. ​Retrieved from http://www.globethesis.com/?t=2143360305974610.

Ward’s Science (2013). ​Superworms​. Retrieved from

https://www.sargentwelch.com/www.sargentwelch.com/images/Superworms.pdf

Yinon, U. (1970). The visual mechanisms of ​Tenebrio molitor​: some aspects of the spectral

response. ​J​ournal of Experimental Biology​, ​53​(1), 221-229.