Understanding evolution is crucial to modern biology,but most teachers would assume that practical demonstrations of evolution in school laboratories are unfeasible. However, perhaps they have not heard of ‘evolution in a test tube’ and how Wrinkly Spreaders can form the basis for both practical demonstrations of bacterial evolution and further work

Spiers, Andrew J.

Trends in Microbiology

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Andrew J. Spiers

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Getting Wrinkly Spreaders to demonstrate evolution in schools

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 Page 1 of 9 Please reference the published article and not this draft. 1  2  3 A.J. SPIERS (2014). Getting Wrinkly Spreaders to demonstrate evolution in 4 schools. Trends in Microbiology, 22:301-303. 5   6  Page 2 of 9 Trends in Microbiology Science & Society Section, Final Draft 7  8 Getting Wrinkly Spreaders to demonstrate evolution in schools 9 Andrew J. Spiers 10  11 SIMBIOS Centre & School of Science, Engineering and Technology, Abertay 12 University, Bell Street, Dundee, DD1 1HG, UK 13  14 Corresponding author: Spiers, A.J. (a.spiers@abertay.ac.uk) 15 Keywords: Education; Experimental evolution; Microbiology; Microcosms. 16  17 ABSTRACT (49/50 words) 18 Understanding evolution is crucial to modern biology, but most teachers would 19 assume that practical demonstrations of evolution in school laboratories are 20 unfeasible. But perhaps they haven’t heard of ‘evolution in a test-tube’ and how 21 Wrinkly Spreaders can form the basis for both practical demonstrations of 22 bacterial evolution and further work. 23  24 MAIN TEXT (1,306 / 1,200 words) 25 In the absence of a good fossil collection or a nearby natural history museum, 26 the teaching of evolution in schools is largely an exercise in which experimental 27 laboratory-based work is unfeasible (as generally it would be expected to 28 greatly exceed normal school hours). Nonetheless, providing students with a 29 strong understanding of evolution and its broader relevance is essential for the 30 development of a well-rounded biological education. Indeed, the importance of 31  Page 3 of 9 evolutionary theory was famously presented by Theodosius Dobzhansky in 32 1964 as “nothing makes sense in biology except in the light of evolution” [1] 33 (see also [2, 3]). Furthermore, demonstrations of the reality of evolution are also 34 required to combat the insidious penetration of science education by religious 35 fundamentalists and the proponents of intelligent design (e.g. the Dover Panda 36 trial in the USA [4]). 37 Recent attempts to introduce evolution in science classes have focussed on 38 developing ‘thought-provoking’ discussion-based exercises (e.g. [5]) for dulled 39 students who would rather watch David Attenborough interact with extinct 40 animals in CGI-augmented natural history programmes (or more probably the 41 other Attenborough in ‘Jurassic Park’). However, I advocate the use of more 42 exciting bacterial microcosms, and in particular the Wrinkly Spreaders, as 43 means of investigating evolution in secondary schools through practical 44 demonstrations involving basic microbiological techniques. We first presented 45 this as ‘Evolution in a Test-tube : The Rise of the Wrinkly Spreaders’ in [6] and it 46 should probably be accompanied by a very slow progression of minor chords 47 ending with an unresolved diminished 7th (this is a more sophisticated version of 48 leaving out a coffee cup until it develops a surface biofilm, but without the 49 attendant health and safety constraints). 50 Simple artificial environments or microcosms have long been used to 51 investigate aspects of microbial ecology, originating from the early work of 52 Sergei Winogradsky and others in the late 19th and early 20th centuries 53 (reviewed in [7]). More recently they have been used for experimental evolution 54 studies, where the rapid growth of bacteria, large population sizes, and the 55 ease of isolating mutants and storing strains indefinitely at -80°C makes them 56 ideal for investigating various evolutionary processes (reviewed in [8, 9, 10]). In 57 particular, adaptive radiation can be readily demonstrated by quantifying 58 diversification (radiation) by the appearance of mutants in growing populations 59 and determining fitness of mutants compared to the ancestral strain 60 (adaptation). 61 In the peculiar case of Pseudomonas fluorescens SBW25, a non-pathogenic 62 environmental bacterium, radiation in static liquid microcosms results in the rise 63  Page 4 of 9 of a class of adaptive mutants known as the Wrinkly Spreaders (first described 64 by Paul Rainey and Michael Travisano [11] and recently reviewed in [12]). 65 Whilst I happily acknowledge the work of others who have investigated bacterial 66 evolution using different systems (e.g. the long-term Escherichia coli 67 experiments initiated by Richard Lenski, see [13]), none have produced 68 adaptive mutants as spectacular as the Wrinkly Spreader in so little time: 69 typically they appear within three days in static microcosms and may represent 70 ~30% of the population by the fifth day [6].  71 The Wrinkly Spreaders are readily distinguished from the ancestral Pf. SBW25 72 by virtue of their wrinkled colony morphology on agar plates and an altered 73 ecological preference as demonstrated by the formation of a robust biofilm at 74 the air-liquid interface (see Figure 1). In static microcosms, the fitness (W) 75 advantage of the Wrinkly Spreader is ~2.5 greater than the non-biofilm–forming 76 ancestral Pf. SBW25, though in shaken microcosms where biofilms cannot 77 form, the Wrinkly Spreader has no fitness advantage (W ~1) [6]. The value of 78 producing a biofilm is that it allows bacteria to intercept O2 diffusing into the 79 liquid column from the air above, with those in the highly-oxygenated, < 200 µm 80 top layer growing faster than that possible lower down where O2 levels are ≤ 81 0.05% of that found at the surface [14]. Remarkably, it is the early ancestral 82 colonists that establish the O2 gradient that then provides the selective pressure 83 and ecological reward that drives the evolution of the Wrinkly Spreader in this 84 simple environment [14]. 85 Substantial research has been published investigating the underlying molecular 86 biology of the Wrinkly Spreader, providing a satisfying mechanistic explanation 87 linking mutation, ecological preference and fitness (for an introduction to this 88 subject and links to the primary literature, see the review by [12]). In the 89 archetypal Wrinkly Spreader, a single DNA base change (A → T) results in an 90 alteration of an amino acid (Ser → Arg) in the methylesterase (WspF) subunit of 91 the Wsp complex. This and similar chemosensory complexes receive 92 environmental signals and allow the bacteria to respond to changing conditions 93 by modulating the levels of cyclic-di-GMP, an internal second messenger that 94 plays a central role in the regulation of motility, virulence and biofilm formation 95  Page 5 of 9 in many bacteria through a complex signalling network integrating 96 environmental signals and controlling riboswitches, transcription factors and 97 enzyme activities. In Pf. SBW25, the mutation in WspF results in increased 98 levels of cyclic-di-GMP, leading to the over-expression of cellulose and an 99 attachment factor essential for the Wrinkly Spreader phenotype, whilst 100 differences in wrinkleality between individual Wrinkly Spreaders is probably due 101 to differences in the underlying mutations that increase cyclic-di-GMP levels. 102 I recognise a tendency for those who are not biochemists or molecular 103 biologists to skip paragraphs such as the preceding one, but the value of this 104 mechanistic explanation is that it provides extensions into molecular biology, 105 ecology, experimental design and statistics for further discussion and project 106 work (see Figure 2). Although the ‘evolution in a test-tube’ experiments are 107 quantitative and can be rigorously tested with statistics, this type of work has a 108 very obvious visual component, allowing a more qualitative approach based on 109 observations and photography to be taken where appropriate. It is even 110 possible to link the rise of the Wrinkly Spreaders with famous literature, as the 111 Red Queen (a character in Lewis Carroll’s ‘Through the Looking-Glass’) is also 112 an evolutionary hypothesis which proposes that organisms must constantly 113 evolve to survive when pitted against ever-evolving competitors in an ever-114 changing environment. I hesitate to purloin Dobzhansky, but things may make 115 more sense (or fun) in biology when illustrated by Wrinkly Spreaders. 116 Just as the ancestral Pf. SBW25 made the adaptive leap in static microcosms to 117 the Wrinkly Spreader, teachers also need to make some effort to bring this 118 research into secondary schools in order to demonstrate evolution. In a recent 119 survey of UK teachers, equipment access and confidence in techniques were 120 found to be significant limitations in using practical microbiology in schools [15]. 121 However, ‘evolution in a test tube’ does not depend on expensive equipment or 122 complex techniques: it requires an initial inoculum of Pf. SBW25 which is 123 available on request, basic equipment to produce sterile media and for 124 incubation, and skills including sterile technique, serial dilutions and plating-out 125 (as described in [6]), and would be suitable for Scottish Highers or English A or 126 AS-level students (i.e. the last two years of secondary school). We also use 127  Page 6 of 9 Wrinkly Spreaders in BSc undergraduate laboratories as a means to acquire 128 laboratory confidence and basic skills, to generate and analyse replicate data, 129 and to provide a narrative linking mutation, phenotype and fitness (in these 130 students are expected to access and comment on the primary research 131 literature in their final reports). 132 Like many other researchers involved with STEMNET (see Box 1), I am willing 133 to support teachers with practical help and expert advice. Collectively, we need 134 to make sure that the biologists of the future will have a solid understanding of 135 evolution. Like many science disciplines, it is not always easy to provide 136 practical demonstrations or research-based projects for students, but in this 137 instance, it seems that by bringing Wrinkly Spreaders into schools, our students 138 of today can be involved directly in experimental evolution and further enthused 139 by the subject. Who knows, but in a future dominated by synthetic biology and 140 biotechnology, some of these might produce Super Wrinkly Spreaders or 141 indeed, something really quite useful. 142  143 ACKNOWLEDGEMENTS 144 This article was written with support from the SIMBIOS Centre at Abertay 145 University. A. Spiers is also member of the Scottish Alliance for Geoscience 146 Environment and Society (SAGES). 147  148 REFERENCES 149 1  Dobzhansky, D. (1964) Biology, molecular and organismic. Amer. Zoologist 150 4, 443–452 151 2  Griffiths, P.E. (2009) In what sense does ‘nothing make sense except in the 152 light of evolution’? Acta Biotheoretica 57, 11–32 153 3 Losos, J.B. et al. (2013) Evolutionary biology for the 21st century. PLoS Biol. 154 11, e1001466 155  Page 7 of 9 4 DelFattore, J. (2007) Speaking of evolution: the historical context of 156 Kitzmiller v. Dover Area School District. Rutgers J. Law Religion 9.1, 1–81 157 5 Kalinowski, S.T. et al. (2013) Six classroom exercises to teach natural 158 selection to undergraduate biology students. Life Sc. Educ. 12, 483–493 159 6 Green, J.H. et al. (2011) Evolution in a Test-tube : Rise of the Wrinkly 160 Spreaders. J. Biol. Educ. 45, 54–59 161 7 Moshynets, O. et al. (2013) From Winogradsky's column to contemporary 162 research using bacterial microcosms. In Microcosms: Ecology, Biological 163 Implications and Environmental Impact (Harris, C.H. ed), Nova Publishers 164 8 Buckling, A. et al.  (2009) The Beagle in a bottle. Nature 457, 824–829 165 9 Spiers, A.J. (2013) Bacterial evolution in simple microcosms. In 166 Microcosms: Ecology, Biological Implications and Environmental Impact 167 (Harris, C.H. ed), Nova Publishers 168 10 van Ditmarsch, D. and Xavier, J.B. (2014) Seeing is believing: what 169 experiments with microbes reveal about evolution. Trends Microbiol. 22, 2–170 4 171 11 Rainey, P.B. and Travisano, M. (1998) Adaptive radiation in a 172 heterogeneous environment. Nature 394, 69–72 173 12 Spiers, A.J. (2014) A mechanistic explanation linking adaptive mutation, 174 niche change and fitness advantage for the Wrinkly Spreader. Int. J. 175 Evolutionary Biol. 2014: Article ID 675432 176 13 Barrick, J.E. and Lenski, R.E. (2013) Genome dynamics during 177 experimental evolution. Nature Rev. Genet. 14, 827–839 178 14 Koza, A. et al. (2011) Environmental modification and niche construction: 179 Developing O2 gradients drive the evolution of the Wrinkly Spreader. ISME 180 J. 5, 665–673 181  Page 8 of 9 15 Redfern, J. et al. (2013) Practical microbiology in schools: a survey of UK 182 teachers. Trends Microbiol. 21, 557–559 183  184 Figures 185 Figure 1. Adaptive radiation of Pseudomonas fluorescens SBW25 populations 186 in experimental static microcosms gives rise to the Wrinkly Spreader. (A) 187 Wrinkly Spreader colonies (with an irregular circumference) are easily 188 distinguished on agar plates from ancestral Pf. SBW25 which produces smooth 189 rounded colonies. (B) Ancestral Pf. SBW25 colonises the liquid column of static 190 microcosms (left) while the Wrinkly Spreader (right) produces a biofilm at the 191 air-liquid interface, demonstrating an altered niche-preference. (C) Cellulose 192 fibres form the matrix of the Wrinkly Spreader biofilm and can be visualised by 193 fluorescent microscopy (at this low magnification individual bacteria are not 194 detectable). 195  196 Figure 2. Running an ‘evolution in a test-tube’ laboratory session in schools will 197 allow many further extensions into evolution and ecology, molecular biology, 198 experimental design, science communication and awareness. 199  200 Box 1. The STEM Network in the UK 201 • STEMNET (the Science, Technology, Engineering and Mathematics 202 Network) creates opportunities to inspire young people in STEM. 203 • Works with schools, colleges and STEM employers, to enable young 204 people of all backgrounds and abilities to meet inspiring role models, 205 understand real world applications of STEM subjects and experience 206 hands-on STEM activities that motivate, inspire and bring learning and 207 career opportunities to life. 208 • STEMNET delivers three core national programmes, including STEM 209 Ambassadors who volunteer their time and support to promote STEM 210  Page 9 of 9 subjects to young learners in a vast range of original, creative, practical 211 and engaging ways, STEM Clubs to boost enjoyment and learning 212 across STEM outside of the classroom, and the Schools STEM Advisory 213 Network. 214 • Visit www.stemnet.org.uk to find your UK Regional Contact. 215 

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Getting Wrinkly Spreaders to demonstrate evolution in schools

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