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| − | <h1><b>Introduction</b></h1>
| + | <i>Varroa</i> |
| − | <p> Honeybees are incredibly important pollinators responsible for the abundance and diversity of our food. Unfortunately, honeybees are in trouble: the fittingly named <i>Varroa destructor</i> (from here on ‘<i>Varroa</i>’ or ‘<i>Varroa</i> mite’)weakens the bees and spreads diseases. iGEM Wageningen UR 2016 aims to save the bees from <i>Varroa.</i></p>
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| − | <p> Honeybees can pollinate an enormous range of plants through active collection of pollen<sup><a href="#fn1" id="ref1">1</a></sup>, making them important for pollination of crops. Apples, almonds and numerous other crops rely heavily on bee pollination, tying the diversity of our diet to the well-being of these insects<sup><a href="#fn1" id="ref1">1</a></sup>. Therefore, it is of key importance to keep bees alive and healthy.</p>
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| − | <p> Unfortunately bees are not doing well at all. There has been a sustained loss of bee colonies in the western world for at least 10 years. In 2014-2015, US beekeepers lost over 40% of their colonies<sup><a href="#fn2" id="ref2">2</a></sup>! Such numbers are unacceptable!</p>
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| − | </section>
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| − | <section id="varroa">
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| − | <h1><i>Varroa destructor</i></h1>
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| − | <p> What causes the loss of bee colonies? According to beekeepers we have spoken to and as has been suggested in scientific studies<sup><a href="#fn3" id="ref3">3,</a><a href="#fn4" id="ref4">4,</a><a href="#fn5" id="ref5">5</a></sup>, the most important factor is the mite <i>Varroa destructor</i>. <i>Varroa</i> mites feed on haemolymph (‘bee-blood’) weakening the bees, while spreading serious diseases like the deformed wing virus. Beekeepers and bee researchers stress that the first and most important step to save the bees should be to control <i>Varroa</i> more effectively. </p>
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| − | <figure>
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| − | <img src="https://static.igem.org/mediawiki/2016/6/64/T--Wageningen_UR--varroa10x.jpg">
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| − | <figcaption>Figure 1. 10x magnification of a <i>Varroa destructor</i>, taken with a stereo microscope.</figcaption>
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| − | </figure><br/>
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| − | <p> Currently in the Netherlands and other European countries, beehives are treated against <i>Varroa</i> with a combination of organic products consisting of thymol, formic acid and oxalic acid. These compounds can contaminate beeswax and honey<sup><a href="#fn6" id="ref6">6</a></sup>. More importantly, beekeepers are wary that such compounds can be harmful to bees and humans when the concentration used is too high<sup><a href="#fn7" id="ref7">7,</a><a href="#fn8" id="ref8">8</a></sup>. On the other hand, too low concentrations fail to kill <i>Varroa</i> and facilitate resistance<sup><a href="#fn9" id="ref9">9</a></sup>. The hobbyist character of beekeepers further adds to the concerns that accompany <i>Varroa</i> treatment. Beekeepers often care for their bees in their spare time and might not have time, resources or the experience to use existing treatments in the intended manner. </p> </section>
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| − | <section id="BeeT">
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| − | <h1><b>Bee T</b></h1>
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| − | <p>We propose a <i>Varroa</i> specific treatment through the use of engineered bacteria that releases toxin in a specified and regulated manner. The released toxin is not harmful to bees or humans. To do so, the bacteria will employ a network of regulation mechanisms to produce the toxin only when mites are present and when enough bacteria are present to effectively kill the mite. Other mechanisms are intended to strictly confine the bacteria to the treated hive, preventing them from spreading and mixing with natural ecosystems. </p></section>
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| − | <section id="Specificity">
| + | |
| − | <h1><b>Specificity</b></h1>
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| − | <p>No <i>Varroa</i> specific toxin was available when we started. There were however results <sup><a href="#fn10" id="ref10">10</a></sup> suggesting that a crystal protein produced by a <i>Bacillus</i>-like strain is able to specifically kill <i>Varroa</i> mites. It proved impossible to acquire the strains that were tested. Therefore we have tried to identify other bacteria that can produce such a protein. Our efforts yielded one interesting candidate link to Varroa isolates text (read more here). </p>
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| − | <p>To improve our chances of finding a <i>Varroa</i> specific toxin, we have also attempted to modify an existing crystal toxin to specifically target <i>Varroa</i>. Additionally we tried to actually create a protein domain able to bind <i>Varroa</i> gut cells. This domain could then be used to make existing protein toxins target <i>Varroa</i> mites. Read more here to see how we made and tested these domains. </p>
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| − | <p>Testing natural and ‘home-made’ crystal toxins on <i>Varroa</i> mites proved very problematic. Their parasitic nature makes them incredibly vulnerable for laboratory experiments. To facilitate testing of the toxins, we developed a new <i>in vitro</i> method for testing <i>Varroa</i> toxicity. This method involves the use of vesicles created form <i>Varroa</i> mites and release of fluorophores from these vesicles. Read the whole story here.</p>
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| − | <section id="Regulation">
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| − | <h1><b>Regulation</b></h1>
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| − | <p>As constant low levels of toxin facilitate resistances<sup><a href="#fn11" id="ref11">11</a></sup>, we wanted to only produce toxic proteins when <i>Varroa</i> mites are near the bacteria. Therefore we created and tested two systems were protein expression is dependent on the presence of mites. These systems are based on Guanine and B12. Curious how those relate to mite presence? Read it here.</p>
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| − | <p>Another measure to prevent low concentrations of toxin was to only produce toxin when enough bacteria are present to produce substantial amounts of the protein. This also minimized effects of the toxin on bacterial growth. To further increase the growth and survival of our bacteria, we designed a system to sustain a subpopulation of non-producers. Both systems were modelled to assess their functioning and how they would combine.</p>
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| − | <p>Bacteria might not be able to grow very well in beehives. Therefore, regulation based on bacterial density may not be the best approach for toxin expression. As an alternative we have created a toggle switch that combines the mite sensing with B12 or Guanine with one of our safety measures: vulnerability of BeeT to light.</p>
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| − | <section id="Biocontainment">
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| − | <h1><b>Biocontainment</b></h1>
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| − | <p>We wanted to limit BeeT to the treated hive. This prevents unknown effect the bacteria could have on larger ecosystems. The first, most intuitive mechanism here is making BeeT dependent on the shade that Beehives provide. Incorporated as part of the toggle-switch we have attempted to create an optogenetic kill-switch, killing BeeT when it is exposed to light. To better understand this system a model was constructed.</p>
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| − | <p>As a final biocontainment, we envisioned BeeT to depend on the presence of an artificial amino acid. Such a system had already been constructed. We have tried to improve this system, causing DNA breakdown when the bacteria run out of artificial amino acid. </p>
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| − | <section id="references">
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| − | <h1>References</h1>
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| − | <ol class="references"> <!-- Use ol for numbered list, ul for bullet points-->
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| − | <a id="fn1" href=http://pubs.acs.org/doi/abs/10.1021/es101468w>1.</a> Spivak, M., Mader, E., Vaughan, M., & Euliss Jr, N. H. (2010). The Plight of the Bees†. Environmental science & technology, 45(1), 34-38. <a href="#ref1" title="Jump back to footnote 1 in the text.">↩</a>
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| − | <br><br>
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| − | <a id="fn2" href=http://www.tandfonline.com/doi/full/10.1080/00218839.2016.1153294>2.</a> Seitz, N., Traynor, K. S., Steinhauer, N., Rennich, K., Wilson, M. E., Ellis, J. D., ... & Delaplane, K. S. (2016). A national survey of managed honey bee 2014–2015 annual colony losses in the USA. Journal of Apicultural Research, 1-12. <a href="#ref2" title="Jump back to footnote 2 in the text.">↩</a>
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| − | <a id="fn3" href=http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131611>3.</a> Van Der Zee, R., Gray, A., Pisa, L., & De Rijk, T. (2015). An observational study of honey bee colony winter losses and their association with <i>Varroa destructor</i>, neonicotinoids and other risk factors. PloS one, 10(7), e0131611. <a href="#ref3" title="Jump back to footnote 3 in the text.">↩</a>
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| − | <a id="fn4" href=http://www.apidologie.org/articles/apido/full_html/2010/03/m09161/m09161.html>4.</a> Genersch, E., Von Der Ohe, W., Kaatz, H., Schroeder, A., Otten, C., Büchler, R., ... & Meixner, M. (2010). The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies. Apidologie, 41(3), 332-352. <a href="#ref4" title="Jump back to footnote 4 in the text.">↩</a>
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| − | <a id="fn5" href=http://www.apidologie.org/articles/apido/abs/2010/04/m09037/m09037.html>5.</a> Guzmán-Novoa, E., Eccles, L., Calvete, Y., Mcgowan, J., Kelly, P. G., & Correa-Benítez, A. (2010). <i>Varroa destructor</i> is the main culprit for the death and reduced populations of overwintered honey bee (<i>Apis mellifera</i>) colonies in Ontario, Canada. Apidologie, 41(4), 443-450. <a href="#ref5" title="Jump back to footnote 5 in the text.">↩</a>
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| − | <a id="fn6" href=http://www.tandfonline.com/doi/full/10.1080/10412905.2015.1076741>6.</a> Serra Bonvehí, J., Ventura Coll, F., & Ruiz Martínez, J. A. (2016). Residues of essential oils in honey after treatments to control <i>Varroa destructor</i>. Journal of Essential Oil Research, 28(1), 22-28.
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| − | <a href="#ref6" title="Jump back to footnote 5 in the text.">↩</a>
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| − | <a id="fn7" href=http://www.tandfonline.com/doi/full/10.1080/10412905.2015.1076741>7.</a> Bonnafé, E., Alayrangues, J., Hotier, L., Massou, I., Renom, A., Souesme, G., ... & Armengaud, C. (2016). Monoterpenoid‐based preparations in beehives affect learning, memory, and gene expression in the bee brain. Environmental Toxicology and Chemistry. <a href="#ref7" title="Jump back to footnote 7 in the text.">↩</a>
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| − | <a id="fn8" href=http://onlinelibrary.wiley.com/doi/10.1002/ps.3539/full>8.</a> Charpentier, G., Vidau, C., Ferdy, J. B., Tabart, J., & Vetillard, A. (2014). Lethal and sub‐lethal effects of thymol on honeybee (<i>Apis mellifera</i>) larvae reared <i>in vitro</i>. Pest management science, 70(1), 140-147.
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| − | <a href="#ref8" title="Jump back to footnote 8 in the text.">↩</a>
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| − | <a id="fn9" href=http://www.biosecurity.govt.nz/files/pests/varroa/control-of-varroa-guide.pdf>9.</a> Goodwin, M., & Van Eaton, C. (2001). Control of <i>Varroa</i>. A guide for New Zealand Beekeepers. New Zealand Ministry of Agriculture and Forestry (MAF). Wellington, New Zealand.
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| − | <a href="#ref9" title="Jump back to footnote 9 in the text.">↩</a>
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| − | <a id="fn10" href=https://doi.org/10.1007/s13592-014-0288-z >10.</a> Alquisira-Ramírez, E. V., Paredes-Gonzalez, J. R., Hernández-Velázquez, V. M., Ramírez-Trujillo, J. A., & Peña-Chora, G. (2014). In vitro susceptibility of Varroa destructor and Apis mellifera to native strains of Bacillus thuringiensis. Apidologie, 45(6), 707–718.
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| − | <a href="#ref10" title="Jump back to footnote 10 in the text.">↩</a>
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| − | <a id="fn11" href=https://doi.org/10.1038/nbt.2597 >11.</a> Tabashnik, B. E., Brévault, T., & Carrière, Y. (2013). Insect resistance to Bt crops: lessons from the first billion acres. Nature Biotechnology, 31(6), 510–521.
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| − | <a href="#ref11" title="Jump back to footnote 11 in the text.">↩</a>
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