Mealworm pupa and the successful survival on a polystyrene diet: the potential for mealworms to reduce the impact of plastics in the environment

Mealworm pupa and the successful survival on a polystyrene diet: the potential for mealworms to reduce the impact of plastics in the environment

Montana Morgan – Lambton High School

Observations have been made surrounding the ability of mealworms to successfully digest and break down the polymer polystyrene (PS). Two colonies were established in order to compare the progression of darkling beetle larvae; or mealworms, on a usual oat-diet to a PS-diet. This investigation determined that mealworms are able to successfully mature on a diet predominantly based on expanded polystyrene. Furthermore, results showed that larvae on a PS-diet were able to progress into the pupa and adult, or beetle stage of life at a faster rate than their oat-diet counterparts. This highlights the potential of plastic waste management systems to be implemented that utilise mealworms to degrade potentially harmful plastics.

Literature review:

Polystyrene is a synthetic hydrocarbon polymer discovered by Eduard Simon in 1839 (“Polystyrene,” 2020), which poses significant threats to natural ecosystems due to its ability to withstand biodegradation. The main research area of this investigation was to explore new methods of using invertebrates in the form of mealworms in order to provide a solution to the overwhelming volume of PS waste that reaches natural environments. Despite this research area being relatively new, there are several previous studies which explore different aspects of the topic, allowing for this investigation to produce new data that fills current gaps. The work of Yang and coworkers (2020) suggests that “mealworm[s] (Tenebrio molitor) [where] capable of degrading and mineralizing Styrofoam (PS foam)” (Yang et al., 2020). The 28-day investigation found that mealworms which were fed styrofoam as a sole diet were able to live just as well as those fed on a usual diet of oat bran. The study also concluded that the average PS foam consumption rate was 0.14 mg/d, suggeting that this method may be feasible to introduce on a larger scale. Another investigation led by Hughes (“Teens Use Science to Worm through Plastic Waste,” 2016), aimed to discover which type of plastic mealworms could most efficiently digest. The investigation involved feeding mealworms small amounts of Polyethylene terephthalate (PET), polyethylene (PE), Polystyrene (PS) and Polypropylene (PP) sourced from conventional plastic waste. The experiment concluded that the mealworms consumed 64% of the PS, 50% of the PET, 40% of the PP and 29% of the PE. Hughes' experiment is beneficial as it identifies PS as the polymer that mealworms most readily consume, and provided a basis for this

investigation to focus on a single plastic type, although the article may have reduced reliability as it is not from a peerreviewed academic source. A third article by Siva (2020), furthered the research area through identifying the types of microbes present within the mealworm's gut that are able to digest PS. The article acknowledges the severity of PS pollution within the environment, stating that an “An estimated 359 million tons of plastic [was] produced globally in 2018, including 33 million tons of polystyrene” (Beetle Larvae Can Survive on Polystyrene Alone, n.d.). It is also speculated that the mealworm's ability to biodegrade plastics is evolutionary-based. This is due to scavengers, such as mealworms, consuming lignin (wood-based substances) for food, which is highly difficult to break down as lignin is a complex biopolymer that is both hydrophilic and insoluble to most solvents (Lignin - an Overview | ScienceDirect Topics, n.d.). The researchers stated that they were beginning to isolate certain gut bacteria within the mealworms to test their ability to degrade plastic (Beetle Larvae Can Survive on Polystyrene Alone, n.d.). However, this study did not investigate the effectiveness of a PS diet in terms of the mealworm's ability to develop past larvae stage, highlighting the gap that was explored in this investigation.

The ability of invertebrates to successfully digest plastics is not limited to mealworms, with other insect larvae being capable of surviving on a PS diet. For example, Billen and coworkers (2020) observed larvae of the greater wax moth (Galleria mellonella), which seemed to chew and digest polyethylene; although whether they successfully degraded the polyethylene was unknown. This investigation aimed at understanding the mechanisms behind the wax moth’s gut microbes and how it can be applied to technology in order to replicate it in a more efficient manner. This is due to 4-10 tonnes of greater wax moth larvae being needed to digest 1 tonne of plastic (Billen et al., 2020). Such research showed the need for an invertebrate that will be more efficient in digesting plastic, in comparison to wax moths, to make a large-scale replication feasible. This notion leads to Rodrigues’ investigation (2016), which compared the plasticdigesting ability of mealworms to superworms (Zophobas morio). The three week comparative experiment concluded that mealworms largely outperform superworms; with 5 mealworms consuming 1 gram of plastic in two days, which is more than what the superworms could consume in a week (“Teens Use Science to Worm through Plastic Waste,” 2016). Another benefit for the use of mealworms as a method for polystyrene waste control is their existing use as a common food source for other species including a range of birds, reptiles and fish in the agricultural and pet industries. The investigation highlighted the ability of mealworms to consume relatively high quantities of PS in comparison to other invertebrates, indicating that along with their availability, they were the ideal choice for an invertebrate in this investigation.

The possible implications of these findings has also been acknowledged, with the prospect to create a zero-waste sustainable bioremediation cycle. Another study from 2020 highlights the possibility of using mealworms as a source to degrade PS, while also utilising the frass to assist in the growth of plants, specifically dragon fruit cacti (Koh et al., 2020). The study also researched which supplement was best for promoting PS consumption. Three different supplements were studied: bran, cinnamon and table sucrose, with the mealworms mass being calculated after 4 days. Results; shown in figure 2, determined that table sucrose and bran almost doubled the PS consumption from the control, with the consumption rate rising from 1.035 and 1.397 mg / g of worm per day to 1.787 and 2.142, respectively (Koh et al., 2020). The study demonstrated the possibility of a practical application to reduce PS waste entering the natural environment. The question of the possible risk of biomagnification arose, as PS commonly contains pollutants and additives including UV stabilisers, antioxidants and flame retardants (Smith & Taylor, 2002). Furthermore, Browne and coworkers (2013) demonstrated the minimal influence of microplastics on the transfer of pollutants into the tissue of lugworms, a close relative of mealworms. The study found that biochemically clean sand transfers more pollutants into the worm’s tissue than microplastics (Browne et al., 2013). Thus, the potential of mealworms to become an efficient method of PS control, alongside the minimal risk of biomagnification showed strong merit in further research being completed within this area as well as prototype methods being constructed to determine the viability of mealworms being used as a means of polystyrene control.

Figure 1: Average rate of PS consumption (mg / g of worm per day) by superworms (Sw) and mealworms (Mw) with and without food additives (cinnamon, sugar and bran(Koh et al., 2020)

Scientific research question:

To what extent are mealworms able to consume and successfully develop on a diet predominantly based on polystyrene, examining the effect of altered diet on: • Mean larvae body mass • Mortality during larvae and pupa stage • Number of successful maturing adults

Hypothesis:

Null Hypothesis: mealworms on a diet predominantly consisting of polystyrene will be unable to maintain their body mass and therefore not successfully develop into the pupa or eventual adult stage of their life cycle.

Alternative hypothesis: mealworms on a diet predominantly consisting of polystyrene will be able to maintain their body mass and successfully develop into the pupa and eventual adult stage of their life cycle.

Methodology:

Prior to the commencement of the methodology, an ethical report (see Appendix 1) was prepared in order to ensure all considerations including sourcing mealworms and their disposal are successfully met.

Required materials:

Consumables: • 2000 Mealworms - sourced from Petstock • Potato - sourced from Coles • Oatbran (ground rolled oats) - sources from Coles • Polystyrene (disposable cups ground styrofoam) - sourced from Coles.

Non-consumables: • 3x four draw storage cabinets - sourced from Office Works • 8x small plastic containers (chinese take away containers) • Scales • Ruler • Blender • Sieve • Knife • Metal tweezers • Beakers

Colony establishment:

The Methodology accepted to investigate the hypothesis involves creating two colonies of mealworms to compare data. One colony acted as a control, consisting of four sets of approximately 250 mealworms being fed a diet of ground oats (oat-diet) alongside a slice of potato for moisture and trace nutrients. The other colony will consist of the same number of mealworms and slices of potato, but were fed shredded polystyrene (PS-diet) in replacement of an oat-diet. These colonies were maintained within storage draws (as shown in figure 2) with holes drilled along the top to ensure airflow. Each draw was filled with 3-4cm of ground oats or shredded PS for the control and experimental colony respectively.

Figure 2: colony set up of (a) 3 sets of draws for 2 colonies and spares for adults & trials (b) oat-diet draw and (c) PS-diet draw

Data collection methodology:

At the beginning of the investigation, the mass of a random sample of 20 larvae was recorded. The colonies were maintained and monitored for 28 days; which is the approximate time mealworms are in larvae stage(Instructions on Breeding Mealworms, n.d.), by regularly measuring the mass of a random sample of 20 larvae and recording any larvae that progressed to the pupae stage. Throughout the experiment, any larvae or pupa deaths were recorded to provide a measure of the survival of the mealworms. Any surviving mealworms that made it to the adult stage of their life cycle were tallied and separated into 8 small plastic containers (4 PS-diet and 4 Oat-diet) with the intention of establishing additional colonies for potential future research into the impact of a PS-diet on subsequent mealworm generations. The decision to have four sets of approximately 250 mealworms within each colony was chosen to have a sufficiently large colony population to reduce the impact of environmental and observational random error when only sampling a relatively small portion of the population.

Data analysis methodology:

Once this data was collected, a range of techniques were utilised in order to complete a statistical analysis. As the data being collected is primary and quantitative, descriptive statistics such as mean, median and standard deviation were calculated. These measurements are represented graphically in order to highlight trends and patterns present within the data. This statistical data was found for both the control and experimental colony, allowing for a comparison between the two data sets via a two tailed t-test to examine whether the observed differences in the mean body mass of the colonies were statistically significant.

Results:

Effect of altered diet on mean larvae body mass:

The observed body mass of the control (oat-diet) and test (PS-diet) colony tracked over the 28-day period of the study is shown in Table 1. Figure 3 shows the observed trend in the mean sampled body mass throughout the 28-day trial. The lines of best fit for each colony clearly show that the mean sampled body mass of the PS-diet colony is comparable across the period of the study with a slight decline in body mass becoming evident after day 10. A two tailed t-test assuming equal variances with an alpha value of 0.05 (Table 2) showed that the test colony on the PS-diet (M= 52, SD= 3.5) compared to the control colony on the oat-diet (M= 54 , SD= 3.1) was observed to have a statistically significantly different mean body mass across the 28-day study period, t(2.7)= 2.0, p= 0.009. As the p value is smaller than the alpha value, the null working hypothesis was able to be rejected, stating that the mean body mass of the control colony is equal to the test colony. This presents strong evidence that the observed differences in the mean body mass between each mealworm colony is considerable.

Table 1: observed body mass over 28 days based on a random sample of 20 mealworms.

Figure 3: approximate change of mass of mealworm larvae over a 28-day period based on a sample size of 20

Table 2: Summary statistics for the two tailed t-test assuming equal variances with an alpha value of 0.05 comparing mean body mass of the test colony on the PS-diet to the control colony on the oat-diet. (Null hypothesis: mean mass of PS MW = mean mass of oatm

Effect of altered diet on mortality during larvae stage:

Throughout the investigation, the number of mealworm larvae was collected, with the cumulative mortality for the combined 4 trials of the oat-diet and PS-diet colonies being displayed in figure 4. In relation to the control colony, there is a strong positive correlation between the cumulative mortality and time throughout the 28-day trial. This determines that the number of larvae mortality was consistent and above zero during the investigation. In contrast, the PS-diet colony is shown to have a smaller cumulative larvae mortality in comparison to the control colony, alongside increasing in cumulative mortality past the 15th day of the investigation. As well as this, the first larvae found dead within the polystyrene colony was earlier than the first found dead within the control colony. The control colony has a mortality rate of 5.6 deaths per 100 larvae, in comparison to a mortality rate of only 0.5 deaths per 100 larvae for the polystyrene colony. This poses strong evidence for mealworms to successfully mature on a PS-based diet.

Figure 4: graph showing cumulative larvae mortality of both control and test colony over 28 day period

Effect of altered diet on number of successful maturing adults:

Figure 5 shows the cumulative number of successful pupa formed from larvae throughout the 28 day trial. A significant trend shown within figure 5 is that the Polystyrene colony began to form into pupa two days prior to when the control colony first progressed into pupa. As a result of this, at day 28 of the trial, the PS-diet colony had 696 larvae that had become pupa, whereas only 603 pupa were formed during this time within the oat-diet colony. Furthermore, Table 3 depicts the cumulative number of beetles formed within each colony, collating the data from the four replicates of each colony. It is shown that throughout the 28 days, the polystyrene colony produced more beetles, or adult mealworms, in comparison to the control. This shows that under a polystyrene diet, mealworms may be able to more efficiently mature into successful adults in comparison to the control, oat-diet.

Figure 5: graph showing cumulative pupa of control and test colony over 28 day period

Table 3: cumulative number of beetles observed over the 28 day study period.

Discussion:

The impacts of plastic waste on the natural environment are becoming increasingly apparent; with a significant proportion of this waste being polystyrene. Manufacturing and households produced 38.4 and 14.5 thousand tonnes of PS waste respectively within Australia over 2016-17 (Waste Account, Australia, Experimental Estimates, 2016-17 Financial Year | Australian Bureau of Statistics, 2019). This has resulted in an increasing amount of PS entering waste streams each year, ultimately leading to a high risk of PS entering the natural environment. PS waste that enters the environment has been linked to numerous detrimental effects, including chemical leaching, bioaccumulation, alongside a potential choking hazard for wildlife if broken down (Kik et al., 2020). The significant impact of PS on the natural environment is a driving force for this research.

The findings of this investigation successfully indicates the potential for mealworms to break down polystyrene while surviving to maturity; thus reducing the negative impacts of plastics on the environment. This confirms the claim that mealworms are capable of consuming polystyrene posed by Hughes (“Teens Use Science to Worm through Plastic Waste,” 2016). The small amount of potato used for moisture and trace nutrients within this investigation was unlikely to provide a significant source of nutrition for the mealworms on the PSdiet, meaning that they are consuming the polystyrene to maintain their body mass and progress through their lifecycle. This could be confirmed within future investigations by using a damp cotton ball in place of potato slices to avoid providing a secondary food source. Although this aspect of the methodology may reduce the confidence of the conclusions, the results still provide strong evidence that the larvae are capable of maturing on this altered diet. These findings are also consistent with the work of Yang and coworkers (Yang et al., 2020), which similarly determined that mealworm larvae are able to survive on a diet of PS. The same 28-day period trial and control material leading to similar results within both experiments highlights the reliability and validity of this investigation. However, this investigation also raises several new research questions surrounding the apparent increased rate of progression through the life cycle of mealworms in the colony on a PS-diet in comparison to the colony on an Oat-diet. This is consistent with the qualitative observations of the PS-colony larvae being more physically active in comparison to larvae in the oatdiet colony. This may also be a potential selection pressure that could be used to explore the effect of the PS-diet on the microbes within the larvaes GI tract in a future investigation, possibly expanding on the work presented by Siva (2020)(Beetle Larvae Can Survive on Polystyrene Alone, n.d.). Another aspect of the methodology that may impact the results of the investigation is the size of the PS used within the PS-diet colony. The styrofoam cups used in the PS colony are significantly less dense than the mealworms, leading to most larvae settling to the bottom of the containers. This made the process of counting individuals that had moved onto the pupa stage or any mortalities much harder to complete compared to the oat-diet, which remained on the surface of the bedding. This aspect may impede on an accurate reflection of the mortality experienced in the PS-diet colony. However, altering materials to use polystyrene blended more thoroughly into a finer particle size may help avoid this.

Future directions of study available for this investigation include a multigenerational approach, by which the offspring of the PS-diet are raised under the same conditions to determine the long-term implications of the altered diet. This leads to investigations surrounding the functionality of the colony, to determine if the offspring can also live off PS or if a new batch of mealworms has to be introduced in place of each new generation. As well as this, additional research could be placed on the impact of certain additives on the consumption rate of PS, first explored by Koh and coworkers (Koh et al., 2020). A wide variety of additives can be investigated, including cinnamon and table sucrose used within the previous study. This information will be of high value if waste management strategies were to be put in place in order to ensure the efficiency of polystyrene consumption. Furthermore, a real-world simulation could be constructed using waste PS as a food source; instead of an artificial trial, to provide insight into the possibility of mealworms being implemented into areas such as landfills in order to control the plastic waste that may end up in surrounding ecosystems.

Conclusion:

This investigation confidently shows the potential for mealworms to reduce the impact of plastics on the environment. The investigation involved the establishment of two colonies; PS-diet and oat-diet, to compare their ability to successfully mature into adults. It was found that there was a significant statistical difference between the mean body mass of the PS-diet and oat-diet colonies, alongside the establishment of the trend that the PS-diet colony progressed to the pupa and beetle life stages before the control colony. This allowed for the null hypothesis to be rejected, and provides strong evidence in support of the alternative hypothesis. The investigation's findings also highlight several opportunities for further research to be conducted surrounding both multigenerational colonies and the conformation of life stages occurring quicker; alongside the completion of a real-world investigation to determine the plausibility of using mealworms as part of a waste management scheme. Overall, this investigation provided clear evidence that mealworms have the ability to successfully live off a PS-based diet, giving value to the notion that invertebrates could be employed as part of a waste management system in order to combat plastic waste entering natural environments.

Acknowledgements:

I would like to thank Mr Nicholson for his valuable assistance and guidance throughout all stages of the investigation and report.

Reference List:

Beetle Larvae Can Survive on Polystyrene Alone. (n.d.). The Scientist Magazine®. Retrieved November 26, 2020, from https://www.thescientist.com/news-opinion/beetle-larvaecan-survive-on-polystyrene-alone-67251

Billen, P., Khalifa, L., Van Gerven, F., Tavernier, S., & Spatari, S. (2020). Technological application potential of polyethylene and polystyrene biodegradation by macro-organisms such as mealworms and wax moth larvae. Science of The Total Environment, 735, 139521. https://doi.org/10.1016/j.scitotenv.2020.13 9521

Browne, M. A., Niven, S. J., Galloway, T. S., Rowland, S. J., & Thompson, R. C. (2013). Microplastic Moves Pollutants and Additives to Worms, Reducing Functions Linked to Health and Biodiversity. Current Biology, 23(23), 2388–2392. https://doi.org/10.1016/j.cub.2013.10.012

Instructions on Breeding Mealworms. (n.d.). Exotic Nutrition. Retrieved August 18, 2021, from https://www.exoticnutrition.com/how-toraise-mealworms.aspx

Kik, K., Bukowska, B., & Sicińska, P. (2020). Polystyrene nanoparticles: Sources, occurrence in the environment, distribution in tissues, accumulation and toxicity to various organisms. Environmental Pollution, 262, 114297. https://doi.org/10.1016/j.envpol.2020.114 297

Koh, D. W.-S., Ang, B. Y.-X., Yeo, J. Y., Xing, Z., & Gan, S. K.-E. (2020). Plastic agriculture using worms: Augmenting polystyrene consumption and using frass for plant growth towards a zero-waste circular economy. BioRxiv, 2020.05.29.123521. https://doi.org/10.1101/2020.05.29.12352 1

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Smith, S. H., & Taylor, L. T. (2002). Extraction of various additives from polystyrene and their subsequent analysis. Chromatographia, 56(3), 165– 169. https://doi.org/10.1007/BF02493206

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Yang, Y., Wang, J., & Xia, M. (2020). Biodegradation and mineralization of polystyrene by plastic-eating superworms Zophobas atratus. Science of The Total Environment, 708, 135233. https://doi.org/10.1016/j.scitotenv.2019.13 5233

Appendix 1- ETHICAL REPORT:

• Ethical report written prior to the commencement of the investigation, approved by the classroom teacher:

As part of my science extension investigation, which focuses on exploring new methods of plastic waste control, mealworms will be used throughout the investigation in hopes to prove or disprove the predetermined hypothesis. As mealworms; which are the larvae of Darkling beetles, are classified as invertebrates, the restrictions placed within the Animal Research Act of 1985 and Prevention of Cruelty to Animals Act 1979 do not apply. This is due to the definition of an animal within the acts being “a vertebrate animal, and includes a mammal, bird, reptile, amphibian and fish, but does not include a human being.”(Animal Research Act 1985 No 123 - NSW Legislation, n.d.)

The first batch of mealworms was purchased from a petstore, which is allowed under animals in school- NSW department of education (Animals in Schools, n.d.) after the first batch has matured into beetles, the offspring will be kept in an appropriate enclosure until they have reached the larvae stage. Once the mealworms have reached the larvae stage, it is intended that they will be used within the investigation. The original colony of Darkling beetles will be kept for approximately two terms, or until the experiments for the investigation have concluded. As part of the experiments, some of the mealworms will be exposed to a diet of exclusively Polystyrene(PS), instead of the regular diet of oat bran. Although the PS colony will be supplied with moisture in the form of potato slices or damp cotton balls, if an excessive amount of larvae, pupa or beetles were to die, immediate alterations to their diet will be made in order to keep the remaining specimens alive. It is to be expected that there may be an increased mortality rate within the PS colony in comparison to the control colony, however if this were to increase significantly, the remaining PS colony will be moved into an enclosure containing a normal diet of oat bran.

Once the experiment has concluded, it is intended that any Darkling beetle larvae, pupa or beetles will be either given to a pet owner who eats mealworms, given to a petshop, frozen or given to native wildlife. This will depend on how many mealworms remain after the experiments, alongside if pet shops or pet owners will be willing to take them. Regarding freezing the mealworms to death, due to them being invertebrates they do not possess the ability to register drastic changes in temperature. This means that freezing them for around 48 hours (Disposing of Mealworms, n.d.) would be an ethical alternative to disposing of live mealworm specimens. In terms of disposing of dead larvae, pupa or beetles throughout the experiment, they will be placed into bin liners to be disposed of in the garbage.

Reference list:

Animal Research Act 1985 No 123— NSW Legislation. (n.d.). Retrieved February 3, 2021, from https://www.legislation.nsw.gov.au/view/h tml/inforce/current/act-1985-123#sec.3

Animals in Schools. (n.d.). Animals in Schools. Retrieved August 14, 2021, from http://nswschoolanimals.com/

Disposing of mealworms. (n.d.). Chameleon Forums. Retrieved February 3, 2021, from https://www.chameleonforums.com/threa ds/disposing-of-mealworms.3119/