Maggie Blondeau, Nicholas Danopoulos, Alexandre Pham, Suthan Sinnathurai
Abstract
1) Because of the 78.35% mortality rate of monarch butterflies in the 2016 Monarch Project at Dawson College, a solution to reduce the cross contamination of the sporing parasite (Ophryocystis elektroscirrha; OE) causing the deaths was found. We believe washing hands with common soap will reduce the number of OE spores found on human skin.
2) OE is a protozoan parasite that as a spore on the wings and body of a grown butterfly, is introduced to milkweed plants when the monarch lands on it[2]. OE either kills monarch pupas during metamorphosis, or the monarch emerges with other OE induced consequences (e.g. disfigurement).
3) 3 trials of washing hands after handling OE infected monarchs to reduce the amount of spores attached to the skin were performed.
4) The average amount of OE spores present on fingers before hand wash and after hand wash were 488.3 and 0.66 spores per millimetre squared respectively.
5) There is a significant difference between the amount of spores per millimetre squared before washing hands versus after. This shows how using common hand soap can reduce spores found on fingers by 99.9986%, helping to reduce the cross contamination of OE spores due to human contact.
Introduction
The monarch butterfly (Danaus plexippus) is a species in danger of extinction, with one study showing that there was an 84% decrease in the Eastern North American monarch population between 1996 & 2015. The decline in the population is thought to be due to the use of insecticides, loss of breeding habitat, plants, climate change, and decrease in the amount of milkweed (genus Asclepias)[1]. Humans are the cause for several of these factors, and therefore have the responsibility to help revive the number of butterflies in its population. The 2016 Monarch Project at Dawson College had a 78.35% mortality rate of monarch butterflies, believed to be caused by a parasite called Ophryocystis elektroscirrha (OE). Previous years also experience increased mortality rates, which may have also been caused by this parasite. With such a large mortality rate, the community needs a safer method of handling the monarchs and their environment, due to the easily spreadable nature of OE spores (Figure 1). If a method of diminishing spores is found, it will decrease the number of deaths due to this parasite for future years of this project.
Ophryocystis elektroscirrha (OE)
OE is a protozoan parasite, that as a spore on the wings and body of the grown butterfly, is introduced to milkweed plants when the monarch lands on it. These spores may also cover monarch eggs if they come in contact with an infected butterfly. Spores are then consumed by the monarch caterpillars as they feed on the milkweed. Once ingested, they release sporozoite (the cells which infect the pupa during metamorphosis) as the larva produces digestive chemicals to break down the milkweed. While the caterpillar is in metamorphosis, these cells go through the stages of vegetative schizogony (asexual reproduction) and line the intestinal tract of the pupa. The parasite continues to reproduce until several days before the butterfly emerges from its cocoon, where it then begins to produce spores[2]. Once infected with this parasite, there are three possible outcomes: The pupa dies in its chrysalis, it emerges as a disabled/disfigured butterfly, or the butterfly emerges without any physical irregularities, though it carries the spores to the next milkweed plant it lands on, allowing the cycle to restart[3].
How to Prevent Humans from Spreading Spores
Monarchs may not be the only species which contribute to the spread of OE spores. It is possible for the spores to be carried from one plant to another, or one butterfly to another, due to human cross contamination. Spores attach to human skin, as studies and personal observation suggests[4], therefore if a person works with a milkweed plant, or a butterfly/caterpillar contaminated with OE, there is a potential for the spores to be spread to any other plant or butterfly/caterpillar that the person may work with. This project aims to find a way to prevent, or diminish this cross contamination, as it contributes to the mortality rate of monarch pupae and to the population of disfigured monarch butterflies.
There have been experiments performed to test hand washing to reduce the amount of spores found on a person’s skin, however OE was not the spore in question. One experiment was performed on Clostridium difficile, a sporing organism that can be spread through human cross contamination[5]. One study found that a non-antimicrobial hand wash reduced the amount of spores found on a carriers hands by 78%[6]. This experiment is pertinent, as it proves how a basic store bought hand soap can eliminate the majority of spores found on human hands. Therefore, it may reflect how the experiment of this project will perform, and thus will reduce the cross contamination of OE spores by humans.
The project aims to determine whether the use of store bought hand wash (a common type found in homes and laboratories) will reduce the number of spores on the person’s skin, which in turn would limit the spreading of these spores. This research has never been done before to our knowledge, and therefore the results will help inform everyone working with monarchs and/or milkweed plants. Taking steps towards reducing human cross contamination of OE will help the struggling monarch population. Therefore, this project will help and improve the survival rate of butterflies of the Monarch Project at Dawson College.
Methods
Before the experiment, the participants’ hands were washed (SoftsoapTM) and dried completely. The participants then rubbed the OE infected monarch’s abdomen with their index finger and thumb. Afterwards, one piece of scotch tape was accurately applied to the part of the index finger that came in contact with the monarch. It was then pulled off and stuck to a hemocytometer slide identified that it is the slide used before the final hand washing. Their hands were then washed and dried again. A different piece of scotch tape was applied to the thumb that came in contact with the monarch. The piece of scotch tape was stuck to the hemocytometer slide and identified as the slide used after the final hand washing. There were three participants, and therefore three trials. Error bar analysis[7] was then used to determine whether the two groups are significantly different.
Results
The average amount of spores present on fingers before final hand wash and after final hand wash were 488.3 and 0.66 spores per millimetre squared respectively after 3 trials (Figure 2). The error bar analysis[7] used concluded that the two groups are significantly different, with a gap of 258.01 spores present.
Discussion
The average amount of OE spores present on fingers before final hand wash and after final hand wash were 488.3 and 0.66 spores per millimetre squared respectively (Figure 2). The two groups had varied standard deviations, with the before group’s being 397.2, meaning that the average from all 3 trials were largely varied. This could have been caused by the limited material (i.e. only one OE contaminated monarch was used in all trials). Therefore the majority of the spores may have been removed during the first trial, with the following trials having fewer spores to attach to the skin. In a more preferable case, there would be a different contaminated monarch for each trial. However, the standard deviation of the after group was low, being only 0.57, suggesting that the hand wash (SoftsoapTM) did remove almost every spore present on the finger. The standard errors were also varied, with that of the before group being high, at 229.3. The after group had a standard error of only 0.33. The standard error bar analysis[7] used concluded that the two groups are significantly different, with a gap of 258.01.
The result effectiveness of the hand wash is similar to that of the experiment performed on Clostridium difficile, a sporing organism that can be spread through human cross contamination. The effectiveness of a hand wash on removing C. diff spores was 78%[5], and the percent effectiveness of the hand wash used for this experiment at removing OE spores was 99.9986%. This suggests that certain sporing organisms are removable via hand wash, which is pertinent to all those who work in environments potentially containing sporing parasites.
This study has never previously been performed, and thus this research is an important and impactful addition to the community of individuals who work with monarchs and/or milkweed by preventing the cross contamination of OE spores. The question remains of which components in the hand soap allow for the decrease of spores attached to the oils human skin secretes, as well as if any hand soap will do this, or if specific brands are required.
References
1. Schmitz, B. A. (2016). Pollinator Populations may become Extinct. Wild Ones Journal, 29(3), 16.
2. Sander, S. E., Altizer, S., De Roode, J. C., & Davis, A. K. (2013, August 28). Genetic Factors and Host Traits Predict Spore Morphology for a Butterfly Pathogen. doi:10.3390/insects4030447
3. Altizer, S. M., & Oberhause, K. S. (1998, August 10). Effects of the Protozoan Parasite Ophryocystis elektroscirrha on the Fitness of Monarch Butterflies (Danaus plexippus). Journal of Invertebrate Pathology, 76-77.
4. Smith, E. (2013, December 22). OE – Ophryocystis elektroscirrha – Monarch Butterfly. Butterfly Fun Facts.
5. Gouliouris, T., Brown, N. M., & Aliyu, S. H. (2011). Prevention and treatment of Clostridium difficile infection. Clinical Medicine, 11(1), 75-79.
6. Shrestha, N. (2009, April). C. difficile spore removal via hand washing a challenge. Infectious Disease News. p. 23.
7. Cumming, G., Fiddler, F., & Vaux, D. L. (2007, April 9). Error bars in experimental biology. The Journal of Cell Biology. doi:10.1083
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