How much do small changes actually matter?

Many, many small things in the world add up to big things. I’m sure you’ve heard of some of these:

  • Acts of kindness
  • Pieces of plastic
  • Mutations in bacteria

Wait what?? One of these isn’t like the others…

A brief run-down on bacteria: single-celled organisms that are found everywhere (even on you!). They have a singular chromosome, and occasionally a plasmid – which holds genetic information. On the chromosome is genes – things that code for proteins, and small changes (mutations) in these genes can have drastic effects. Overall, things on this chromosome is termed the genome.

Long term evolution experiments (LTEE) are commonly used to study evolution. Researchers sample parallel populations across thousands of generations, hence bacteria such as Escherichia Coli are often used [2, 3]. Organisms need to adapt to their environment to survive, which allows for divergence of populations, even if they remain in an unchanging environment. These genetic differences then may lead to further adaption [2]. Changes in the genome are believed to cause evolution, but mutations are not always positive for the individual. It is also thought that neutral mutations (ones that are neither beneficial nor harmful) would accumulate at a constant rate [3].

These papers investigated the OVERALL impact of beneficial mutations in E. Coli. Many studies have been done on the how fitness improves over time, but less work has focused on specific genes and how the genetic background of mutations affects gene fitness. Comparatively, Peng et al. looked at a specific gene (pyfK) and the effect of moving the gene into different genetic backgrounds (i.e. other populations of the same bacteria!). Genetic background is simple – just looking at the mutations which came before the one that gave a fitness boost. These mutations may rely on previous sequence changes to work.

Focusing on a specific gene allowed these researchers to directly measure the impact of certain mutations. This was important because it meant we could see how mutation effects change in different backgrounds. It also proves that mutations are not isolated events that randomly improve fitness. Instead, although mutations can have major beneficial effects, this may differ when placed into a new genetic background. They found that the evolved mutations had variable effects in their own background (neutral to +25%) however it varied when placed into the ancestral background [1].

[1] – Peng et al. 2018. Placing two mutations into new strains has differing effects on fitness.

You can see in the picture above (Fig4) that mutation alleles have varying effects when placed into a variety of different genetic backgrounds. The authors chose to take the ‘A301S point mutation’ or ‘deletion mutation’ allele and place in into new strains. This meant that they could compare the evolved alleles, the indel allele and the A301S allele and their effect on fitness.

As a broad generalization, the deletion had the greatest effect on fitness, and the point mutation was less beneficial than the original allele. The A301S point mutation was chosen to be transferred into other strains as it evolved independently in three strains of E. Coli. The deletion mutation was used as a proxy for the insertion mutation which arose [1]. Fitness effects were shown to be affected mainly by the genetic background, rather than the specific mutations being hugely beneficial.

The authors preformed a similar experiment, where they took all the evolved alleles and put them into the ancestor strains, measuring the difference in fitness. This shows a rise in RELATIVE fitness over the first 10-15,000 generations, which begins to drop off after 20,000 generations [1]. From six different populations where mutations had arisen, the pykF allele was taken and reverted to the ancestral gene. Hence the effect of the mutation within pykF could be measured. The evolved allele was placed into the ancestor and fitness effects were measured (Fig5 – below). Whilst pyfK mutations occurred early in the LTEE, the genetic background was less well understood, and so it is hard to understand the relationship between mutations in pyfK and fitness effects of other mutations. However, an understanding of the trend between pyfK and fitness can be inferred [1].

[1] – Peng et al. 2018. How mutations within pyfK can change fitness over time.

Fitness was measured by undertaking competitive fitness assays for both figures. These head to head competitions had relative fitness calculated by growth rate within the environment [1]. Therefore, the fitness effect of the mutation was calculated by “relative fitness minus one”.

Small changes within the genome can influence genes, even if these changes are not in a gene. How well bacteria survive in an unchanging environment is affected by mutations. Evolution is a complex process, with many factors at play, and long-term evolution experiments in bacteria are simply one way to help unravel the mystery of how small changes can have large effects – just like you can. Be kind, be happy and always be open to change.

1.            Peng, F., et al., Effects of Beneficial Mutations in pykF Gene Vary over Time and across Replicate Populations in a Long-Term Experiment with Bacteria. Mol Biol Evol, 2018. 35(1): p. 202-210.

2.            Richard E. Lenski, et al., Long-Term Experimental Evolution in Escherichia coli. I. Adaptation and Divergence During 2,000 Generations. The American Naturalist, 1991. 138(6): p. 1315-1341.

3.            Barrick, J.E., et al., Genome evolution and adaptation in a long-term experiment with Escherichia coli. Nature, 2009. 461(7268): p. 1243-7.

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You’ve heard about measles, you’ve probably even heard about smallpox… but what do you know about polio?

Feeling feverish? It could be the outcome of a fevered excitement to read this article, or it could be polio.
Polio, also known as poliomyelitis (let’s stick with polio), is a highly contagious illness that can, along with fever, induce a paralytic state – one could say it freezes lungs in their tracks. It’s caused by poliovirus, which before vaccines were available was free to have its way with the general populace, particularly during the tail-end of WWII. As is often the case, woes bred woes and with a general decrease in hygiene accompanying an increase in crowding, polio caused many deaths – particularly amongst young children.

Children in an “iron lung” after suffering from polio induced paralysis of the lungs.
https://rarehistoricalphotos.com/children-iron-lung-advent-polio-vaccination-1950/

Fortunately, an effective vaccine was created and in this modern age polio is almost eradicated, with the very illness becoming somewhat of an “ailment of old times”. Less fortunately, there’s been a slight issue with vaccinations…

In essence, the vaccine is causing polio.

Obviously, that’s not ideal.
However, before sharpening any “anti-vaccination” pitchforks, allow me to slip into a little first person prose (the benefits of a blog).

Our fast-paced, high-tech life can make it hard to navigate and sift fact from fiction. The constant plying of information can be overwhelming, with many sources having an agenda of persuasion. For example some sites may simply state “the vaccine is causing polio”, and that could quite understandably leave you with feelings of concern or trepidation when considering whether or not to vaccinate your children.
I would much rather provide you with a summation of information and allow you, the reader, to form your own opinion.

So, if you please, read on for an explanation.

The vaccine is not causing any immunised individuals to develop polio. What’s happening is that in 1988 the W.H.O. (World Health Organisation) made a plan, a very ambitious plan of globally eradicating polio. One challenging part of this was that even remote, impoverished areas needed to be reached – a tough call when staffing and sanitation levels were dubious. The only practical way to implement vaccination in these areas was through use of an oral vaccine – drops taken by mouth.

Within this liquid are a combination of attenuated poliovirus: Type I, II & III. They are called attenuated because their genetic make-up has been altered to the point where they are no longer considered virulent/are not capable of causing polio. However, they are still live viruses, and will for a short time be present in the newly immunised person’s stools. In situations where there is a public sewerage system and good hygiene practices this wouldn’t be an issue, but for some isolated areas this isn’t the case so coupled with many individuals not getting the vaccine, we end up with transmission. As this cycle repeats itself, the attenuated strands are undergoing mutations and eventually we see a reversion to virulence. These variants are referred to as cVDPVs (a mouthful of “circulating vaccine derived polio virus”). Now, any cVDPV infected individuals are at risk of developing polio.

This means that it’s still the unimmunised that are at risk, but that the vaccine is responsible for exposing these unimmunised individuals to the virus. There are a few ways to work around this, one being more thorough immunisation of these remote regions. Another way, and this is the area that my next post focuses on, is identifying how this virulence is regained.

In the mean-time feel free to peruse the following sites for more information on polio, both the illness and the virus.

https://www.medicalnewstoday.com/articles/155580.php
https://www.historyofvaccines.org/timeline#EVT_100314

Update – Following article can be found here: https://thismicrobiallife.wordpress.com/2019/09/13/to-be-or-not-to-be-the-question-of-virulence-as-posed-by-polio/

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Hybrids: a fast track to evolution?

Darwin’s theory of evolution by natural selection is, in my opinion, the most awe-inspiring of scientific theories. Sure, it doesn’t quite have the indispensable quality of gravitational theory in literally holding us to the Earth… But, to look around at the endless forms most beautiful with which we share our planet, and know that every single one of them (and us) shares a common ancestor that lived approximately 4 billion years ago, usually has my brain going something a little like this:

What Darwin got wrong…

Or rather, what he didn’t get quite right.

The branches of Darwin’s classic tree notably extend outwards: simplicity giving rise to greater complexity: one ancestral species giving rise to multiple different species.

Charles Darwin’s hand-drawn “tree of life”
from On the Origin of Species.

But we know that this is not always the case.

Hybrids can be formed through the mating between species (interspecific hybridisation), or between distinct populations/breeds within species (intraspecific hybridisation).

The labradoodle: an intraspecific hybrid of the labrador and poodle. Image credit: thelabradorsite

Life as a hybrid.

Hybridisation has the potential to impact evolution through a variety of ways. After all, the sudden mixing of two dissimilar sets of genetic material has repercussions not otherwise associated with conventional Darwinian evolution. The Nobel Prize winner Barbara McClintock coined the term genome shock, which aptly describes the multitude of potential challenges faced by a genome following hybridisation.

These potential challenges most notably include complications during the phase of cell division where chromosomes are required to pair up; the set of chromosomes the hybrid inherits from each parent may be too different from one another that it interferes with this pairing. In spite of the potential challenges they face, there must be some benefits to being a hybrid, as they can be found throughout the plant, animal and fungal kingdoms.

In fact, the prevalence of hybrid species within the plant kingdom means there’s a good chance that you ate one within the last week (and even the brewer’s yeast used in wine and beer making is a hybrid!)

Or, you may have even admired one in your garden without realising it.
Take the humble sunflower, for instance.

Part of my green-thumb efforts of Summer 2019.

Sunflowers are a crop heavily reliant on hybrid breeding.

Can hybridisation act as an evolutionary stimulus?

A study published just earlier this year used an experimental evolution approach to investigate if naturally-occurring hybridisation can accelerate adaptive evolution in sunflowers.

Experimental evolution studies biological populations under defined environmental conditions and over multiple generations, to understand their evolutionary responses.

But how can someone measure evolution, you ask?

This study tracked 27 traits in a hybrid population, and closely-related non-hybrid control population, of sunflowers. The sunflowers were planted in open fields; the two populations separated by a dense copse of trees to prevent any cross-pollination.

Leaf samples were taken yearly and used in genetic analyses, to track the progression of trait evolution. Seed samples were also taken yearly and stored for usage in a common garden in the final stage of the experiment.

A common garden allows for the comparison of biological populations without the confounding effects of their corresponding environments.

Mitchell et al (2019) Figure 1: the experimental setup.

As neither population was originally from the testing area, the change of the 27 traits over the course of the experiment (which I should add, was eight years long!) in the hybrids relative to the controls, when analysed at each time-point and in the final common garden, would give an indication of how well they were evolving in their new local environment.

If the traits were observed to evolve more rapidly in the hybrid population over the experimental period, this would suggest that hybridisation was accelerating evolution.

What did they find?

Mitchell et al (2019) Figure 2: the evolution of fitness.

Hybrid fitness was shown to increase over time (dark blue and light blue lines), while control fitness (gold line) did not change overall. H. a. texanus (black horizontal line) is a locally-adapted wild sunflower hybrid used as a standard for comparison. The second hybrid population (and the reason for which there are two blue lines) was established to assess the generality of the results.

Fitness is a measure of reproductive success; how well a biological organism or population is adapted to a given environment in order to survive to a reproductive age and to produce offspring.

From the above figure, hybridisation does appear to have accelerated evolution!

So which of the 27 traits evolved most rapidly?

Some key traits associated with
• leaf area
• bud initiation time
• seed maturation time
• flower disk diameter
• herbivore resistance
evolved faster in the hybrids relative to the controls, which could have enabled the hybrids to acquire resources (e.g. sunlight), avoid pest-based damage, and produce viable offspring more effectively: all qualities that would promote the propagation and continuation of the population (which is, of course, the inherent goal of life itself!)

But was it adaptive evolution, or just evolution?

And how can you tell?

Although traits were observed to evolve in the hybrids, this evolution was not necessarily adaptive. To prove adaptive evolution, the researchers needed to show that the traits were under selection, otherwise the evolution could have been driven by additional forces, such as genetic drift or a genetic correlation with other traits under selection.

Selection is the preferential survival and reproduction (or, preferential elimination) of individuals with particular characteristics or features.

Genetic drift is a type of evolution that typically occurs in small populations, whereby certain genetic frequencies change over time due to chance or random processes.

The researchers performed some statistical analyses of the data, focusing on trait selection, and determined that some traits had indeed evolved adaptively.

Mitchell et al (2019) Figure 3: individual trait evolution.

In the above-left figure (3a), colour is used to differentiate whether trait values increased (blue) or decreased (pink), along the shading gradient that corresponds to values derived from the statistical analyses. Traits with an ‘a’ were determined to have evolved adaptively. In the above-right figure (3b), colour is instead used to differentiate the rate of trait evolution and whether it was steeper in the control (orange) or hybrid (blue) population, along the shading gradient. We see in Figure 3b, from the prominent blue shading, that most traits evolved more rapidly in the hybrid populations. In both figures, ‘LBJ’ and ‘BFL’ correspond to the two hybrid populations, and ‘control’ to the control population. A solid black outline of a square denotes a highly statistically significant result, a dashed black outline a statistically significant result, and no outline a non-significant result.

When considering Figure 3a, we see that all adaptively evolved traits are associated with high statistical significance, which gives us confidence in the results. However, sunflower disk diameter (‘DiskDiam’) and overall plant size (‘Volume’) are the only traits for which the hybrid populations both demonstrated adaptive evolution and the control population didn’t. Thus, the title ‘Hybridization speeds adaptive evolution in an eight-year field experiment’ is perhaps a bit of a stretch, and could have done without the inclusion of the word ‘adaptive’, in my opinion.

Some final thoughts.

It is important to remember that the researchers’ conclusions have been drawn from a single study using a single control and two hybrid populations, making it entirely possible that the observed effects are due to species-specific factors at play, or are not completely representative of reality.

Experimental evolution studies in higher organisms (e.g. plants and animals) are impeded by their long generation times; a barrier not encountered in studies of microorganisms such as E. coli, which has a generation time of 15-20 minutes in the lab and has subsequently been used to great effect in the E. coli long-term experimental evolution project. Field-based studies, such as that discussed in this blog post, encounter further barriers in ensuring that conditions are held constant for the duration of the experiment, according to the conditions defined at its outset. Aptly highlighting this was the need for this case study to be terminated after eight years due to a change in land usage at the host site. Conversely, the benefit of field experimental evolution over lab experimental evolution is the ability to study a population in its natural environment, and without the introduction of artifacts that may occur in lab settings.

An artifact, in biological science, is a misleading observation or misrepresentation in the data, introduced by the experimental equipment or techniques utilised.

Scientific knowledge, and its countless applications, would benefit significantly from further field-based experimental evolution studies; particularly those on higher organisms, as well as those on hybrid populations. Such studies are largely under-represented in the literature, as reviewed in Kawecki et al (2012). A follow-up to this case study would also help to clarify and consolidate its results.

As I touched on towards the beginning of this post, hybrid species are found throughout the plant, animal and fungal kingdoms. They are also imperative to the functioning of many industries, and to humanity itself. The more we can learn about hybrids and their evolution, therefore, the greater ability we have to make informed decisions to benefit our ever-increasing global population.


When we consider the data from this study, however, it does suggest that hybridisation accelerates evolution, at least in sunflowers. Thus it appears, in quite a circle-of-life type of way, that the evolutionary processes responsible for shaping sunflower populations able to hybridise with one another, are now being influenced by that very hybridisation.

And for that, I have just three words: science. is. awesome.

Case study paper:

Mitchell, N., Owens, G. L., Hovick, S. M., Rieseberg, L. H., & Whitney, K. D. (2019). Hybridization speeds adaptive evolution in an eight-year field experiment. Scientific Reports, 9(1), 6746. doi:10.1038/s41598-019-43119-4

Further readings on this topic:

Barton, N. H. (2001). The role of hybridization in evolution. Molecular Ecology, 10(3), 551-568. doi:10.1046/j.1365-294x.2001.01216.x

Dimitrijevic, A., & Horn, R. (2018). Sunflower hybrid breeding: from markers to genomic selection. Frontiers in Plant Science, 8, 2238. doi:10.3389/fpls.2017.02238

Kawecki, T. J., Lenski, R. E., Ebert, D., Hollis, B., Olivieri, I., & Whitlock, M. C. (2012). Experimental evolution. Trends in Ecology & Evolution, 27(10), 547-560.
doi:10.1016/j.tree.2012.06.001

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CANCER’S NEXT TOP MODEL?

As a scientist, I want to find a treatment for cancer. To do this I need to perform many experiments testing my new therapy on cancer. I can’t just use human patients, they’re too annoying. How about cancer cells from a tumour and grown in flasks? Sounds good, but through my literature search, I find that cell lines undergo substantial alterations within their genome. That surely won’t do, my therapy targets a specific gene. I need a model which does not undergo such genetic alterations. I probably should look for something else.

Cell Lines

My very own cancer cell line cultures I am currently using

Perhaps it’s because the environment of a flask is too different from a human? How about a mouse?

Mice are mammals, like humans. Sounds good.

If I can place the cancer cells from a human tumour into a mouse, since it is a similar environment, the cancer should not change as excessively. So, I take a sample of cells from a cancerous tumour and surgically graft it into a mouse. Great, now I have an accurate model of cancer. One that I can use to perform experiments on that does not involve those annoying humans. One that will better represent the original tumour than if grown in flasks. One that does not change its genetic make-up over time.

But wait!

Humans and mice seem more similar than humans and flasks. But still wouldn’t the change from a human environment to a mouse also cause changes?

I mean, I like cheese, but not as much as a mouse. Surely this would have an effect.

Apparently not.

It was assumed that Patient-derived Xenografts (PDX’s), where human tumours are surgically grafted into mice, accurately represent the genetics of the human primary tumour.

Well… until recently.

Uri, et al (2017) was not satisfied with this assumption. They set out to test if Patient-derived xenografts accurately represent the genetic landscape of a primary tumour and to compare with two established cancer models; cancer cell lines and cell line derived xenografts (CLDX’s).

Cancer models

Overview of the different cancer cell models.

First, we may need to establish some characteristics of cancer.

We need to view a cancerous tumour not as an individual entity, but as a population of individual cancer cells. The cancer cell population is subjected to evolutionary forces the same as any other population of individuals. Over passages (generations) of cells, certain genes will be lost or gained depending on evolutionary forces.

As you can imagine with a population of animals, if placed in a totally alien environment, the animals would undergo divergent evolution over generations in the new environment. Creating genetically different animals when compared to the original population. A population of cancer cells, in this aspect, is no different. When cancer cells are taken from a human tumour and grown in flasks (Cell Lines) they too will undergo evolution over passages – Gains/losses of DNA via mutations within the genome of cancer cells. This leads to cancer cells with altered genetic landscapes in comparison to the original tumour cells.

This is a current problem for cancer research.

Cancer research relies on models that accurately represent the genetic landscape of the primary tumour. If not, the observed responses may not be applicable to the original cancer in question, otherwise known as – useless.

Currently, cell lines and CLDX’s are known to undergo model specific mutations leading them to be genetically different from the primary tumour over time. It is therefore important for future research to develop a model which accurately represents the primary tumour.

As genetic changes in PDX models over passages had not been directly studied Uri, et al (2017) had to create a catalogue of large genetic alterations – gains or losses of genetic information – between early and late passages of PDX models from other papers. Each study had to include DNA-based genomic measurements from primary tumours and at least one PDX passage. However, this lead to a small data set, too small for a comprehensive analysis.

That won’t do…

To overcome the quantity issue they applied computational inference algorithms on studies with gene expression profiles. In other words, they predicted changes in the genomic landscape by inferring the expression of genes – somewhat concerning. It is unreliable to predict specific losses or gains of DNA based on gene expression. But until a direct study between PDX models and primary tumours is made this will have to do.

Overall, a final data set of 1110 PDX’s representing 24 different cancer types were used (Figure 1). This is a bit misleading as only fractions of this data set were suitable for many of their analyses leading to a much smaller sample size per analysis. This leads to an inability to make specific conclusions, however, general conclusions can be.

Data Set

Figure 1 – Cancer types in the PDX dataset from Uri, et al (2017)

By comparing the passaged models genetic landscapes to the primary tumour’s genetic landscape allowed Uri, et al (2017) to determine if each model still retains – and which best represents – the genetic landscape of the primary tumour. Alterations from the primary tumour’s genetic landscape are considered model-acquired alterations. The greater the amount of model-acquired genetic alterations leads to a poor representation of the primary tumour by that model.

Using the above reasoning the assumption of PDX models retaining the same genetic variation as their primary tumours was shown to be false. After a tumour is implanted within a mouse there is a dramatic shift in the genetic landscape of the PDX tumours with a median of 12.3% (range, 0-58.8%) within 4 passages (Figure 2). This is a reduction of total genetic alterations in PDX models as compared with cell lines.

However, given the labelling “early, medium, and late” of cell line passaging, the actual amount of passaging would be much greater than 4 even for “early”. This could account for some of the increase of genetic alterations when compared with PDX models.

Next, when PDX’s were compared to CLDX’s, it is shown that PDX has a much higher change in the genetic landscape. This does not necessarily mean that CLDX’s better represent the primary tumour, unfortunately. Certain aspects of the CLDX model, such as they are xenografted from well-established cell lines. Thus, before being xenografted each CLDX would have already undertaken large changes within their genetic landscapes, causing less observed changes when passaged within mice.

Figure 2 – Mosaic of Model-acquired genetic alterations per model type. Credit: Uri, et al (2017)

Overall this does not look good for PDX’s – It does not look good for any of the three models.

The change in environment from a human to a mouse does not seem to be spared from specific selection pressures causing genomic alterations. Thus, PDX models diverge from their primary tumours after being xenografted and do not accurately represent the primary tumour. PDX’s also do not necessarily represent the primary tumour better than cell lines of CLDX’s.

What is even more concerning for current cancer research is their next finding.

The PDX models can lose signature chromosomal aberrations through passaging – large gains or losses of chromosomes that are believed to cause the specific cancer type. We see an example of this is in figure 3, the hallmark chromosomal gains of breast cancer chromosomes 1q and 8q are lost in PDX models.

Figure 3

Figure 3 –  gain and losses of signature chromosomal aberrations within breast cancer PDX – Modified Figure 4 b  Uri, et al (2017)

This could have a huge therapeutic impact.

Most therapies target aspects like these signature alterations. If the signature alterations are no longer present in the models, it is highly likely that the targets of therapies are altered too. Potentially leading to many therapies having been discarded due to the failure of the model to accurately represent the primary tumour and not due to the therapy being ineffective.

Think of all the wasted time and research grants.

Most scientists already knew the flaws of working with cancer cells line and CLDX models but assumed the same flaws would not be present with tumours grown in mice. This assumption clearly was incorrect. Uri et al, 2017 has created a clear divide within cancer research where, after the publication, PDX’s (once thought infallible) are now subject to the same criticism subjected to cell lines and cell line derived xenografts. The search for cancers next model top model needs to continue.

I’m sorry to say.

Patient-derived Xenografts you are no longer in the running to be cancers next top model.

giphy-2

America’s Next Top Model elimination Credit: https://giphy.com/gifs/girl-fangirling-antm-eanhZUqf1PaIo

Topic Paper; Uri et al. (2017) Patient-derived xenografts undergo mouse-specific tumor evolution. Nature Genetics, 49 (11) 1567-1575. doi:10.1038/ng.3967

Addition Reading

Cell line evolution’s effects on Drug Response: Uri, et al. (2018)Genetic and transcriptional evolution alters cancer cell line drug response, Nature, 560, 325-330. doi.org/10.1038/s41586-018-0409-3

Organ on a chip – new human drug model: Zhang, et al. (2018) Advances in organ-on-a-chip engineeringNature Reviews Materials3(8), 257. https://doi.org/10.1038/

Further PDX Info: Tentler, et al. (2012) Patient-derived tumour xenografts as models for oncology drug development. Nat. Rev. Clin. Oncol. 9, 338-350. http://www.nature.com/doifinder/10.1038/nrclinonc.2012.61

Clinical relevance of Cancer Cell Lines: Gillet et al (2013). The clinical relevance of cancer cell lines. J. Natl. Cancer Inst. 105, 452–458. doi-org.ezproxy.massey.ac.nz/10.1093/jnci/djt007

 

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Finding Sequels

I’ve always, always, always loved animals. As a kid, I wanted to be a vet (“also an actor,” thought this 30-something genetics student, “kids are weird”). My Mum worked at a Safari Park in the UK, which didn’t help, as I grew up in a house of darling dogs, cute cats, silly snakes and lovable lizards. My own, personal, private zoo, and I loved it. Of course, as a kid I’ve also always, always, always, always loved Disney movies, particularly of the Pixar variety. Finding Nemo, then, holds an especially dear, squishy, jellyfish-shaped place in my heart.

If you don’t know anything about Finding Nemo , then you probably haven’t lived, but besides that our story begins in Australia’s Great Barrier Reef: 2000+ kilometers of astounding ecological diversity; 1,500+ species of fish;  3000 species of mollusks; 350 species of hard corals, and never-mind all the soft corals, clams, sponges, etc (Gutierrez, 2014). Despite being a computer animated film, every shade and hue you see in Finding Nemo is no exaggeration about what’s really out there, ready to astound you.

Coral

Why, if you ain’t the bluest thing I ever did see. (c) 2004 Richard Ling

Finding Nemo released in 2003. I was around 15-years-old. Even now, however, I remember a thought I had while snug in those big cinema-chairs, holding the obligatory empty bucket of popcorn, eviscerated before the film even began:

“How much of it still looks like that?”

Yes, kids are weird, but I wasn’t an especially jaded 15-year-old. I’m sure a lot of people were aware of things like coral bleaching, but being the son of a conservationist perhaps made it more present in my mind. I’m sure a lot of people are still aware of bleaching events, because it’s still a thing. Climate change, among other things, contributes to these bleaching events, and refers to when the corals lose those fantastic colours. “Bleaching”, it turns out, is quite literal.

Keppelbleaching

Corresponding healthy (background) and bleached (foreground) corals. (c) CC BY 3.0

Coral bleaching boils down to an endosymbiotitic relations between coral polyps (which we’ll refer to as corals for the sake of simplicity), and their micro-algae Symbiodinium. Corals are immobile or “sessile” creatures often referred to as “reef builders”: they pump out the calcium-carbonate responsible for these hard coral reefs forming. The micro-algae are photosynthetic (hopefully not surprising anybody) and can live inside the corals, exchanging photosynthates (sugary treats from photosynthesis) for some of the corals’ own inorganic molecules (ammonium, nitrate, etc).

Increasing water temperatures, as a result of climate change, causes stress on both the corals and the algae (I empathize, being English makes me about as thermally intolerant as you can imagine). The corals may expel their algae as a short term survival tactic, reducing their burden as a host. Alternatively, the algae my decide to jump ship, making things easier for themselves. Sadly as the corals are sessile, they rely on their symbiotic relationships for up to 90% of their nutritional needs (Falkowski et al, 1984). Prolonged bleaching events means the corals eventually to starve, leading to a slight case of death. While coral reefs can recover, climate change has resulted in these recovery periods becoming shorter and shorter, with many organisms unable to keep up (Hoegh-Guldberg and Bruno, 2010).

Disney is going to end up having a hard time Finding Sequels. Aha.

As mentioned, the thermal tolerance of the algae, as well as the corals, contribute to these bleaching events (Pandolfi et al, 2011). As the algae themselves have much shorter life-spans (days, Wilkerson, 1988) than the corals (years, Babcock, 1991), perhaps the algae can evolve their thermal tolerance quicker than the corals? If this so, then perhaps the algae’s increased thermal tolerance can help prevent the corals becoming bleached?

coralbleaching-large

Your handy-dandy guide to coral bleaching. (c) National Oceanic and Atmospheric Administration

Experimental evolution can be thought of as a kind of indirect artificial selection: as opposed to picking and choosing which organisms can breed based on a shared desirable trait, we instead change the environment that our organisms inhabit, favouring the traits we wish to develop. In terms of Symbiodinium, this would involve growing a strain at an elevated temperature and hoping to observe positive growth (ie growth that is equal to or better than growth observed at a more common temperature). If positive growth is observed, we can try to grow a sample of this strain at an even higher temperature, and so on and so on.

This is precisely what Chakravarti and van Oppen tried to achieve in their recent 2017 study. They took 5 genetically distinct Symbiodinium strains and attempted to grow them at 30°C, a temperature that Symbiodinium are known to tolerate in the literature. If “positive growth” was observed for these strains (explained below), then a sample of those strains were then grown at 31°C, and so on and, indeed, so on. This process was repeated over the course of a year.

fmars-05-00227-g001

Chakravarti and van Oppen’s simplified experimental design. Blue indicates the control temperature (27), red the hotter experimental temperatures (30, 31, etc). The yellow colour refers to fresh media the strains were transplanted into at the corresponding elevated temperature, to compare the growth of the wild -type (WT) and experimental strains (SS).

“Positive growth” in this study was assessed in terms of “growth rate” and “photosynthetic efficiency”. Growth rate compared the initial cell density with cell density at a later time, as well as the cell doubling or generation time at a given  experimental temperature. This makes intuitive sense: if an experimental strain’s rates of growth are positive after prolonged exposure to an elevated temperature (where the corresponding wild-type may struggle), it suggests a stable, adaptive change for the experimental strains’ thermal tolerance.

“Photosynthetic efficiency” was assessed using common plant stress measurements : the maximum and effective quantum yield. Maximum quantum yield assesses how well our plants can photosynthesize after being “dark-adapted”, whereas the effective quantum yield assesses photosynthesis when light is available at a steady state.

Without going too much into the biochemistry of it all, photosynthesis involves the movement of electrons, excited by light energy, or “photons”. Electron movement is facilitated by “reaction centres” that carry the electrons. A dark-adapted plant should have more available reaction centers, as there’s been no light available to excite electrons for them to be carried by the reaction centres. When light is available at a steady state, however, there’s a constantly back-and-forth between reaction centers picking up and dropping off excited electrons. A stressed plant will have fewer available reaction centres, resulting in reduced maximum and effective quantum yields.

The figure below from another study (Roth, Goericke, and Deheyn, 2012) helps us visualize the effect of both heat and cold on the photophysiology (ie photosynthetic efficiency) of the coral Acropora yongei.

Figure 3

Here, we see the stress caused by both cold (downward arrows) and heat (upward arrows) have detrimental effects on the effective and maximum quantum yields of the corals, compared to the control temperature (white circles). Heat is shown as being more deleterious over time.

(We also see the effects of maximum excitation pressure of photosystem II, Qm, which from what I can understand involves the availability of some reaction centers in response to environmental factors such as heat and light, Grey et al, 1996).

This again makes intuitive sense: if a strain, after prolonged exprosure to a higher temperature, can photosynthesis better than its wild-type at the same temperature, it also suggests a stable adaptive change has taken place.

It is important to remember that, while Charkravarti and van Oppen’s claim to have observed stable adaptive changes in their experimental strains (a great start), it’s certainly not the end of the line. Experimental evolution, as with experiments as a whole, involves controlling a number of variables. Controlling variables means it’s easier to understand the relationship between our results (stable adaptive change) and the experimental conditions we did change (temperature).

Only two traits (growth rate and photosynthetic efficiency) were assessed in this experiment. In a real world setting, these two traits will not be the only ones that matter. Chakravarti and van Oppen discuss in their paper future experiments that need to be considered. Do these evolved strains contribute to the corals during heat stress? Being photosynthetically efficient in vivo is a great start, as many species of coral are “broadcast spawners“. Adapted strains could be released during a spawning period, surviving elevated temperatures ex hospite before being taken up by the coral spawn. Being photosynthetically efficient, however, doesn’t mean the strains release photosynthates when in hospite (Stat, Morris, and Gates, 2008). We also need to ask if these evolved strains can and will infect the corals and be retained by them (Gabay, Weis, and Davy, 2018).

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“Finding Spawn” would have been a very different movie. (c) Jamie Craggs.

What about where these evolved strains exist on the symbiotic spectrum of parasitism to mutualism (Baker, 2018)? Studies have shown that an algae may be more or less beneficial to a coral depending on the coral’s life stage, such as one strain may be more symbiotic for juveniles (Suzuki et al, 2013), but more parasitic in mature corals (Stat, Morris, and Gates, 2008). Corals also rarely host only one strain of Symbiodinium, and so we ask how these corals perform with a single evolved strain compared to being given a “cocktail” of thermally tolerant strains.

What about the thermal tolerance of the corals themselves? Some recent papers by Liew et al and Yong et al have suggested bringing about epigenetic changes in the corals by using “coral nurseries”. It is thought that growing corals in nurseries at elevated temperatures (rather than relying on sporadic, “natural” temperature increases) may cause epigenetic changes that would increase expression of thermal tolerance genes. These young corals would then better react to haphazard temperature changes, rather than hoping these’s enough time between bleaching events for the corals to develop these changes more naturally. Yong et al go on to say that these epigenetic changes also causes changes in transcription that responds to algal symbiosis of the corals.

It’s all exciting stuff, as we consider the myriad of ways in which we might be able to help. Charkravarti and van Oppen have used experimental evolution here as a tool to improve the thermal tolerance of the endosymbiont Symbiodinium, claiming to have observed stable adaptive change in 3 of their 5 experimental strains. I believe combining some of the approaches discussed here, and other incredibly clever ways, is the best hope we have to aid these diverse ecological systems. I hope these efforts bare fruit. I hope there’s a time where I can watch Finding Nemo and no longer think: How much of it still looks like that?

“I hope”.

(Different movie, I know. Thanks for reading!)

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Antibiotics in Agriculture: We Can’t Afford It.

NZ nature

A snapshot from our Massey University Albany campus.

I am fairly certain my housemate saved my life that day. It was the first week of graduate school and she found me delirious, dehydrated and with a temperature above 40°C. Overconfident, I babbled about having the flu but being fine, the futon had hurt my back and that I just needed some water. It turned out I had a progressed kidney infection. That is how I came to start my graduate course in bacterial evolution; alongside courses of antibiotics, prescribed to combat the bacteria ravaging my internal organs.

We often take antibiotics for granted. But every day there are more and more people for whom antibiotics don’t work. Hundreds of thousands now die each year from bacterial infections that can no longer be treated with these life saving drugs. A recent report estimated that by the year 2050, more people will die of antibiotic resistant infections each year than from cancer (O’Neill 2016).

That is a truly scary prospect. We may still be able to avoid this fate but it will take real changes in the way we use these precious medicines. We have over used antibiotics in a deeply irresponsible way and it is time for us to reassess our values. You and I, lawmakers, doctors and farmers must all carefully consider the sacrifices that we are willing to make to save human lives. What is a life worth?

For global antibiotic awareness week this year I want to focus on antibiotics in agriculture. This is not what we generally think of first when we discuss this issue but it should be. According to the Food and Drug Administration, in the US, 80% of all antibiotics are used in agriculture (2009 FDA report).

How can that be? For reasons that are still not clear to science when low doses of antibiotics are given to the farm animals they increase the amount of meat that they put on their bones (2012 Review). This use of antibiotics is called “growth promotion” and it is no longer allowed in Europe or New Zealand. For those places where it is still permitted it is a huge boon for farmers who are looking for ways of producing cheap meat. Bigger animals mean bigger profits for individuals and corporations who are trying to feed the world meat products.

Not long ago the New Zealand Veterinary Association (NZVA) vowed “by 2030 New Zealand will not need antibiotics for the maintenance of animal health and wellness.” In the same report the NZVA also proudly declared that New Zealand is already the third lowest user of antibiotics for animals in the OECD (Organisation for Economic Co-operation and Development) (Hillerton) (Fig. 1). This sort of statistic is enough to swell the heart of any red blooded Kiwi soul. We are proud of our beautiful rolling hills and our happy grass fed sheep and cattle. “Of course we are doing well,” we sigh contentedly, “we are clean and green and so naturally we are miles ahead of places like the US” (ranked 28/30).

Antibiotics ag

Figure 1. Antimicrobial use in humans and agriculture by mg/Kg biomass in OECD countries, 2012 (Hillerton).

Unfortunately, as with many a national myth, this one does not hold up to closer inspection. The first tip off is the metric that is being used by the NZVA; Antibiotic use in milligrams per kilogram of biomass (mg/Kg). To be clear, the NZVA did not invent this reporting measurement, they are simply following a very cleverly opaque convention. By reporting the mg/Kg of biomass we benefit simply by being a country that has a lot of large animals to put in the denominator, a single chicken weighs a little less than 2 kg on average but a single cow weighs 160 times that. Any country in the OECD with a high proportion of cattle and sheep is going to have a lower reported mg/Kg, even if every chicken and pig in the nation is being force fed unnecessary antibiotics in it’s feed each day. As it happens, Cattle and Sheep, which are generally (but not always) free range in NZ, make up 98% of the weight of the animals that we raise for food production each year (Fig. 2).

Bio mass NZ

Figure 2. Animal biomass in New Zealand broken down by animal type.

Poultry and Pigs combined are 2% of the animal weight we put in our denominator but these two groups consume 34% or 22,000 kgs of antibiotics sold for agriculture each year. That is phenomenal to me. Ultimately, we may look good by this measure but we should not pat ourselves on the back for simply having a lot of cattle. We are still pouring huge amounts of antibiotics directly into the feed of many of the poultry and pigs here in NZ.

Why do we do it? This use of antibiotics is called “prophylactic use”. Essentially, we deliver antibiotics to these animals in their water or feed in order to prevent them from getting sick. This is common practice when animals are being kept in less than ideal conditions. Stressed, over crowded, close quartering of small animals is cheaper for farmers but these are also ideal conditions for infections to spread. Constant prophylactic antibiotic administration is one way of keeping these animals healthy.

“Who cares?” you may say, “I am no battery hen!”

Antibiotics Res spreads

Figure 3. Antibiotic use in one environment can affect antibiotic resistance rates in many other environments (adapted from Andersson and Hughes 2014).

I was recently involved in the drafting of the most recent white paper on ‘Antimicrobial Resistance – Implications for New Zealanders’ from the Royal Society of New Zealand. This is an excellent source of NZ relevantdata on this topic and I can recommend it. Two important points are that antibiotics that go into agricultural production are not isolated there and they don’t simply go away. They assert pressure on bacteria in the soil to become more resistant to those antibiotics (Fig. 3). If these are pathogens we can become infected when we ingest food that has been treated with antibiotics. The second point raised by the RSNZ report is that the rates of antibiotic resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), carbapenemase-producing Enterobacteriaceae and beta-lactamase resistant Enterobacteriaceae in New Zealand are already on the rise.

According to a 2015 study published in the Proceedings of the National Academy of Science, New Zealand is using more antibiotics in food animals per Km2 than many other OECD countries (Fig. 4). This means that we are running the same risks in terms of the affect of these antibiotics on the bacteria that infect us as any of our less favourably ranked OECD colleagues. This also means we still need to do better!

Select Counties Use-01

Figure 4. Antibiotic use in animals by land or per person in select OECD countries.

None of us wants our friends or loved ones to be the next person in hospital who will be told that the antibiotics aren’t working because the bacteria are already resistant to everything we have. It’s a nightmare scenario. What are we willing to give up to avoid the post-antibiotic era?

-I encourage lawmakers in New Zealand to take antibiotic use seriously and regulate agricultural use. This should include labelling antibiotic use in the foods we see in the supermarket.

-I implore farmers to reconsider their practices if they are using antibiotics for anything other than saving lives. Let us strive towards practices that will eliminate such use.

-As a consumer, ask yourself what cost we are all really paying for the cheap meat we find in the supermarket. Can you afford it at this price?

 

 

 

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The life of a pea aphid, and why you don’t want to be him

Aphids live pretty simple lives, suck up some plant juices, find a mate, and make some babies…sounds quite chilled, right? Wrong. In fact, the life of a pea aphid or Acyrthosiphon pisum, is a constant battle against predators and parasites. In fact, if you were to compare aphid life to anything, it would be Ridley Scott’s Alien movies. These films were pretty gory (one of the reasons I illustrated this post with the Shrek reference), and I always wondered where he came up with the idea of baby aliens bursting out of people’s chests. Well I can I quit my wondering, because parasitoid wasps or Aphidius ervidid it first.

Picture1

Aliens bursting out of chests is something you only see in movies, right…?

Now before you join team aphid, you have to realise that aphids are actually pests of multiple crops, and cause problems by spreading devastating plant viruses. And those parasitoid wasps you thought were the bad guys? Farmers actually use them as biological control agents to keep down aphid populations. Isn’t that the plot twist of the 21st century!

Now for those of you who are still on team aphid (who likes vegetables anyway), fear not, these little guys have someone defending them. Some lucky aphids harbour a symbiont, the Enterobacteriaceae, Hamiltonella defensa. However, unlike the Captain America that I have portrayed them as, these bacteria require something in return (so much for superhero). The aphid provides the bacteria with nutrients, and the bacteria provides toxins which act to prevent the development of parasitoid wasp larvae. Or specifically, the bacteriophage H. defensa harbours produces the toxins. Is three a crowd? Obviously not to the pea aphid!

Now the question that is addressed in the study I’m reviewing for you here is, is it all really worth it? Does harbouring this symbiont really save these pea aphids from the doom of being eaten alive by wasp larvae? Think I’m just being pessimistic? You may just change your mind after reading what they discovered in this article.

In this study, the first experiment they performed determined what level of defence H. defensa provided to the aphids. This involved growing parasitoid wasps on two aphid populations, one harbouring the symbiont, the other not, and counting how many stung aphids survived (ie. didn’t hatching any wasp larvae). They found that:

143/200 aphids harbouring H. defensa survived

33/400 aphids not harbouring H. defensa survived

So that symbiont bacteria really does make a difference to aphid survival! What we can also see from this data is that aphids also have an innate (or natural) immunity, evident from the fact that while 143 of the non-symbiont-harbouring aphids hatched wasp larvae and died, there were still 57 that survived.

The second experiment they performed was an evolution experiment that monitored components of fitness from parasitoids grown on symbiont-harbouring or non-symbiont-harbouring aphids over 10 generations.

Experiment 2

Experiment 2: Measuring fitness components of parasitoid wasps during evolution

Now fitness in the science world doesn’t mean the most muscular or the fastest organism, instead it means ability to survive (guess you can tell your friends you’re fit now). In this experiment, the fitness components measured were; effective parasitism rate (of the number of aphid stung, how many hatched wasp larvae), and the size of the left tibia of the wasps that emerge (used as a determinate of wasp size).

Experiment 2 results

Components of fitness measured from experiment 2. Modified from Dion, Zele, Simon, & Outreman, 2011.

It was found that after just 4 generations, the parasitism rate of wasps grown on the symbiont-harbouring population had increased to equal that of the wasps grown on the non-symbiont-harbouring population. Did H. defensa take a holiday? Nope, that’s the power of evolution kids. Just like you evolved to eating cereal out of a pot because your flat mates never clean the dishes, these parasitoid wasps can evolve against the symbiont-mediated resistance in their aphid prey. Basically, there is massive selection pressure on these wasps, either their young survive, or they don’t. Therefore, only the wasps best adapted that carry the most useful genes (possibly acting to de-toxify that symbiont toxin), will survive to contribute to the next generation.

The other fitness component measured was the left tibia of surviving wasps, which estimates the body size. The researchers wanted to know if the wasps that emerged from the aphids harbouring the symbiont got smaller, suggesting that there had been a trade-off. Essentially, some of the nutrients wasps would normally use for growth are siphoned off (or traded) for the production of proteins that protect the wasp larvae from toxins. And indeed, these results were found in the experiment. After 10 generations, the wasps grown on the symbiont-harbouring population got smaller. Therefore, while these parasitoid wasps are getting the better of the only defence these aphids have, at least they’re looking a little bit starved in the process. Totally makes up for their babies hatching out of aphid larvae and eating them alive…

Interestingly, a further study by these researchers found that a trade-off occurs in the aphids as well, from their symbiont-mediated resistance. Aphids that harboured the symbiont suffered higher predation by ladybugs because they didn’t use as many defensive behaviours (Polin, Simon, Outreman, 2014). The researchers called this an “evolutionary cost”, and suggested that symbiosis might not be as beneficial as it’s talked up to be. Guess that’s even more bad news for the pea aphid!

Experiment 3

Experiment 3: Determining if selection on symbiont-harbouring aphids increases survival rate of wasps

The last experiment they performed really hits hard the shortcomings of H. defensa. For this experiment, they used the same wasp populations that were used in experiment 2 and exposed them to two different H. defensa-harbouring aphid clones (aphids can reproduce asexually essentially cloning themselves, these aphids are the same species but different clones). They then measured the effective parasitism rate (how often a stung aphid hatches wasp larvae), of the two different wasp populations and compared this to the effective parasitism rate they calculated from experiment 1, before evolution. This experiment will determine if selection of symbiont-mediated resistance will help these wasps survive on new symbiont-harbouring aphids.

Experiment 3 results

Effective parasitism rates before and after evolution to the symbiont-harbouring aphid. Modified from Dion, Zele, Simon, & Outreman, 2011.

And would you look at that, in both clones the wasps that had been grown on a symbiont-harbouring aphid population had a higher effective parasitism rate after evolution. Therefore, was it all worth it? Well, carrying the symbiont H. defensa did provide protection to the aphids, however it doesn’t last for long in these laboratory conditions. I did tell you this story doesn’t have a happy ending! However, this study does illustrate the power of evolution and that in nature, there will always be predators and prey, that’s life. No symbiont will offer complete protection, which is good news for farmers (and you if you like vegetables)!

Now you might be wondering what the practical uses of this study are. As I mentioned earlier, farmers are using these wasps to manage aphid populations. When they were first introduced in North America, the native parasitoid wasp populations were displaced and only one remained to compete with A. ervi (Schellhorn, Kuhman, Olson, Ives, 2002). This shows how important it is that we understand the biological systems we are manipulating and the implications that could occur. The use of parasitoid wasps as biological control agents disrupts the ecological system in crops, and the evolution and adaption of these organisms must be studied to anticipate and manage any changes that could occur.

It is becoming ever more prevalent that nature is a complex system and when meddled with, can result in disaster. I’m sure you’ve all heard about “super bugs” that are becoming resistant to every antibiotic, how is this different to pests becoming resistant to their parasitoids? There are also stories about messing with the food chain, such as this interesting story about shark finning. Science and evolution is a way to understand how to manipulate nature to our advantage, while preventing any catastrophes. Essentially, we need to be aware about what could happen if we introduce something new into the ecological system or take something away. Therefore this study is just one small part of the bigger picture, which is to understand and safely manipulate this biological system.

However, all this still doesn’t help the poor pea aphids.  At least they helped invent a block buster movie enterprise! Someone should really let them know they aren’t suffering for nothing…

 

References:

Dion, E., Zele, F., Simon, J., & Outreman, Y. (2011). Rapid evolution of parasitoids when faced with the symbiont-mediated resistance of their hosts. Journal of Evolutionary Biology 24, 741-750. doi: 10.1111/j.1420-9101.2010.02207.x

Polin, S., Simon, J., Outreman, Y. (2014). An ecological cost associated with protective symbionts of aphids. Ecology and Evolution 4(6), 836-840. doi: 10.1002/ece3.991

Schellhorn, N. A., Kuhman, T. R., Olson, A. C., & Ives, A. R. (2002). Competition between Native and Introduced Parasitoids of Aphids: Nontarget Effects and Biological Control. Ecology, 83(10), 2745-2757. doi:10.2307/3072012

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Taking sibling rivalry to a whole new level.

Picture it. You are a larva, freshly hatched from an egg, and squirming around with just one goal. Survive. How best to survive? Well, you need food pronto; you’ve literally never been hungrier in your life!! You survey your options, in addition to the flour surrounding you there are a bunch of lush round eggs, some full siblings, some half siblings, and some of no relation at all. While flour alone may be sufficient for survival, cannibalising some eggs would provide you with an edge over the other larvae.
So now you have to choose. Will you become a cannibal, bettering your chances of survival? Does your relatedness to the egg affect the degree of cannibalism?
Are you… confused?

beetle

Adult Confused Flour Beetle.

In 1980, Michael J. Wade decided to study the tendency of larvae from the coolest named beetle, the confused flour beetle (Tribolium confusm), to cannibalise eggs. He used it as a platform to investigate the conditions required for the evolutionary theory of kin selection.

Kin selection is defined as:

“Natural Selection in which an apparently disadvantageous characteristic increases in the population due to increased survival of individuals genetically related to those possessing the characteristic” 

So here, larvae eat eggs due to a genetically inherited behaviour which you think would be bad for the population but since larvae that ate the eggs thrive, the behaviour has stuck around and even increased in the population.

In 1980 this theory was poorly defined, while it was agreed that the relatedness of individuals is important, there were many differing opinions to whether the breeding structure was important leading Wade to his big research question.

However, firstly I had a question of my own: Why bother developing evolutionary theories, they require such specific conditions that it does not seem to be realistic or applicable in nature?

It had really stumped me as it made the whole study of evolution seem rather redundant. Thankfully I have come up with an answer which is actually pretty simple, the experiments we all conduct give information on the present. Organisms in the present time, how they act and react, right at this moment. Developing evolutionary theories and taking the time to carefully refine them is all we have, it is the very best we can do to take what we see happening right now and use it, not only opening up the mystery of what occurred in ancient populations as species fought to survive but to also predict what will happen to populations in the future.

Now sense of importance restored, back to Wade and his big research question!

What is the role of population breeding structure in the evolution of social behaviour?

To test this Wade designed 3 different population structures which tested both the effect of the relatedness of individuals and the breeding structure on the evolution of the egg cannibalism behaviour.

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Experimental process of population structure one.

Population Structure One is shown above. 5 larvae from each of the selected 15 mating pairs were put into separate vials; in both groups 45 fully related eggs (R=50) were offered over time and all beetles were allowed to mature then counted to determine the degree of cannibalism. If no cannibalism occurred, you would have 50 adults in each vial (5 larvae + 45 eggs). The mature beetles are then subjected to different breeding structures, either random or within-group mating. Larvae were again collected from 15 mating pairs and the experiment was repeated for 8 generations.
Population Structure Two is the same only half related eggs (R=25) were offered.
Population Structure Three is also the same but with non-related eggs (R=00) offered.

What Wade found…

results1

Raw results with no significant trends. (Wade, 1980)

At first glance the results seemed to be a bust with no trends in the rate of cannibalism for either breeding structure, there are fluctuations, but they are attributed to an uncontrollable aspect of environmental variation.

But don’t despair!

results2

Adjusted results revealing the within-group mating trend. (Wade, 1980)

When Wade plotted the difference between the average cannibalism rate when eggs were related and when the eggs were not related, effectively normalising the results, theoretical expectations as predicted by the kin selection model were met!!

There was a lower cannibalism rate when larvae and egg are closely related, but only in the within-group breeding structure. Population structure did not seem to affect the cannibalism rate when random breeding occurs.

It turns out this all comes down to the variance of the genetic basis for this behaviour within the groups. It is either preserved (within-group mating) or destroyed (random mating).
Variance is essential for the ability of a behaviour to become pronounced, so it is no surprise that when random mating occurs there are no behavioural trends.
Furthermore, he concludes that population genetic models which assume random mating of individuals are inappropriate to the study of evolution of these traits.

To simply answer Wade’s question, population breeding structure DOES plays a critical role in determining how a social behaviour evolve and should be considered when studying the kin selection theory!

Let’s now take a moment to appreciate the irony that clarity is provided by none other than the confused flour beetle!
Maybe, just maybe, this wee beetle is not as confused as first suggested…

To read the full paper by Michael J Wade click here.

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A comfort zone is a beautiful place, but nothing ever grows there

Experimental evolution is not a subject that I would describe as being within my comfort zone. This may explain the interesting experience I have had with this topic and why my appreciation for experimental evolution has grown considerably.

In many ways, lack of growth within one’s comfort zone is a nice analogy for the evolutionary process. Changes in environment push populations outside of their comfort zones creating the selection pressure required to drive the evolutionary process. Take the highly diverse beak shapes of Darwin’s finches for example, changes in the environment altered food availability. This pushed some members of the population outside of their comfort zone, increasing selection pressure which drives evolution.

beak-shape-image

Different beak shapes in finches are specialized for different types of food. Changes in food availability can push birds with the wrong beak shape out of their comfort zone and drive selection towards the best suited beak shape for the available food.

The concept of evolution as proposed by Darwin, first shook the world over 150 years ago, since then science and technology advancements have continued to drastically change the world we live in. Despite this our understanding of evolution remains incomplete, we are constantly revealing new insights into the evolutionary processes which have happened and which are continuing to happen around us. Interestingly it is now possible to observe evolution in real time, and in real organisms through studies designed with microbes and specific environmental parameters. This field of research has been dubbed “Experimental Evolution” and the findings being produced are both interesting and relevant to a wide range of biological fields.

A recent paper in this field which really sparked my interest was the article by Vogwill et al on population bottlenecks released earlier this year.

A population bottleneck is the biological term used to describe a dramatic reduction in population size. Bottlenecks occur frequently, and in a variety of situations both biological and in the physical world around us. For example, a bottleneck occurs when traffic builds around areas where many cars from many roads are trying to move onto the same road. All cars are used for transport, but they vary greatly in shape, size and colour.  If at any one time only 5 cars were able to enter the road and all 5 of them were vans, we would suddenly have a “population” of cars which was all vans. When a species experiences a population bottleneck a similar situation to the 5 cars occurs. Only a few members of the species survive the bottleneck and the genetic composition of the future population is derived only from the surviving member’s genes.

bottleneck-image

Bottlenecks can occur in a variety of settings. As described above we can now see a “population” of 5 vans. 

The study by Vogwill et al set out to investigate the effects of population bottlenecks on the rate and mechanisms of adaptation to the environmental conditions.

Using a simple experimental design, of artificially bottlenecking a Pseudomonas fluorescens Pf0-1 bacterial population each day, the group was able to study the effect of different bottleneck intensities on the bacteria’s ability to adapt. In order to produce a selection pressure, the bacteria were exposed to antibiotics at a constant concentration throughout the experiment. Each day the researches took a small proportion of the bacterial population and transferred them to a “fresh” environment consisting of fresh, plentiful nutrient supply, oxygen and the antibiotic selection pressure. After 110 total generations of bacterial growth, genetic analysis was carried out on members of the surviving populations to determine which genes were being modified and how.  They found that in the Pseudomonas strains mutations which allowed survival arose most often in two specific genes (rpoB and cpxA).  Both genes are known to be involved in the acquisition of antibiotic resistance, but rpoB is more specific to the antibiotics used in this study than cpxA. Further to this, of the three different bottleneck intensities (weak, medium and strong) used in this study, rpoB was mutated at higher frequencies in both the weak and strong bottleneck conditions. The intermediate bottleneck condition lead to the most variation in gene mutation with both genes being favoured.  The researchers explained this finding by looking at the effect mutations in each gene had on the absolute fitness of the population. They suggested that under strong bottleneck conditions the higher fitness caused by the rpoB mutations was necessary to survive. However in the weak bottleneck populations both  rpoB and cpxA mutations competed with each other and the greater fitness of rpoB lead to the observed increase in frequency. This study wasn’t perfect for a range of reasons, and the authors outlined a variety of ways in which their work could be improved and built upon. They did however highlight some interesting observations on the trends evolution seems to follow when experiencing a bottleneck.

experiment-image

Experimental design used in this study to investigate the effects of three different population bottleneck intensities.

Despite this the paper got me thinking about how we can use experimental evolution studies to further understand not only how bacteria evolve in laboratory conditions but how diseases evolve and mutate.

My first and true love when it comes to genetics is cancer, I never cease to be amazed at the mechanisms the disease develops for surviving despite natural and medical attempts to remove it. Cancer can be described as “the uncontrollable growth of cells within the body”, basically cells are being rapidly replicated, not unlike the bacteria in the bottleneck study.

Perhaps then information from studies like this can provide insight into the patterns of genetic changes seen in cancer cells during treatment. The drugs used to treat cancer can create a bottleneck for the disease, most of the cancer cells are die but those that survive will be the few cells that are best able to adapt and respond to changing environments. So while it sounds great to hear that 90% of the tumor is gone, knowing that the 10% that survived are the sneakiest, most adaptable cells of the cancer isn’t exactly the kind of news most patients are hoping to hear. Experimental evolution studies may hold the key to understanding and ultimately treating the sneaky, adaptable cancer cells causing painful and expensive treatments to continue and in some cases relapse.

Despite the validity of some of the results from the bottleneck study being a little bit questionable, this paper definitely succeed in expanding my comfort zone and making a fan of experimental evolution out of me!

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One Guppy to Rule Them All.

Trying to find a paper on experimental evolution wasn’t an easy task for me. I found papers that I thought were interesting, but were lacking in experimental evolutionary methods and others that bored me to death. Personally guppies are what come to mind when I think about natural selection and evolution – being pretty and shiny is just an added bonus!

Natural selection: “the process whereby organisms better adapted to their environment tend to survive and produce more offspring. The theory of its action was first fully expounded by Charles Darwin, and it is now regarded as be the main process that brings about evolution” – wikipedia

Endler (1980) used guppies to look at two aspects of natural selection in both natural and artificial environments. He has been quite a prominent fixture in evolutionary mechanisms within guppies and provides an example of natural selection with the simplicity needed to grasp this topic by following colour patterns and spot sizes in these guppy populations. Guppies are common additions to household aquariums, but would generally be overlooked in terms of their importance in understanding evolution.

This experiment, which utilized the vast expanses and populations of guppies found in Northern Trinidad and Venezuela, demonstrates the impact of natural and sexual selection in these guppy populations. Its a simple, yet comprehensive, study that is still relevant today. Some websites have taken the fundamentals from this experiment and delivered in it a way that is fun and easy to understand. With that in mind, lets take a look at what Endler’s (1980) study entailed.

This is an awesome website that summarizes the experiments outlined in this paper!

 Greenhouse Experiment

greenhouse1

Artificial pools for greenhouse experiment

Ten artificial pools were set up that mimicked natural guppy environments. Two control
pools with no predators, 4 pools with the weak predators (Rivulus hartii) and 4 pools
with the dangerous predators (Crenichichla atla) were used for these experiments. For full details of the methods used in this study have a look at the paper by Endler (1980)

timeline

Timeline for greenhouse experiments

Endler (1978)  had previously observed that predators influence male
color patterns
 and with this knowledge was able to predict the results from this experiment. Dangerous predators (Crenichichla) result in guppies with dull colors and smaller spots while weak predators (Rivulus hartii) result in guppies with brighter colour patterns and larger spots (Endler, 1978). Only male guppies exhibit these elaborate colour patterns –  indicating an element of sexual selection, more on this later.

The figures that follow (Endler, 1980) support these previous observations (Endler, 197
8) and demonstrate the impact predation has on natural selection.

Figure 1

Endler, 1980

Figure 2

Endler, 1980

Figure 3

Endler, 1980

The colour pattern and spot sizes increased in the presence of weaker predators and decreased in the presence of dangerous predators (Fig. 1-3). Blue and iridescent (shiny) spots were seen to almost diminish in the presence of dangerous predators (Fig. 2 & 3). It was later suggested that blue and iridescent spots were highly reflective and eye catching to predators, so a decrease in the presence of dangerous predators is concurrent with this view as something yummy and shiny is easier to see when you’re looking for it, similar to spotting a donut with sprinkles as you walk past a bakery window.

With these findings in mind they then compared the results they obtained from the field experiments to see if similar results were drawn.

Field Experiment

feild1

Aripo Introduction site for natural experimental population

The field experiment used an isolated portion of a stream (Aripo I), with only the presence of the weak predator (Rivulus hartii), to relocate guppies from a high predation area (c) to a low predation area (x). In this experiment they expected to see a shift in colouration and spot size from that seen in high predation environments (dull colours and small spots) to low predation environments (colourful guppies with large spots). After two years the guppies in the Aripo Introduced population had phenotypes similar to that of guppies found in low predation environments (r), more colourful and displaying larger spots. This deviates from the high predation phenotype that was initially exhibited prior to translocation indicating natural selection within this population.

Figure 4

Endler, 1980

Habitual Background

The second parameter considered in this study was the influence gravel colour and size had on colour patterns and spot sizes in the guppies. If you think about it, for guppies in the presence of dangerous predators the better you are at blending into your surroundings the more likely you are to evade the lunch menu. With this in mind, colour patterns and spot sizes were observed and scored to see what influence this had on natural selection. It was observed that the overall body sizes were proportionate to the gravel backgrounds they inhabited and the colour proportions in the gravel background. This was more obvious in the greenhouse pools as the artificial gravel colours were brighter in some pools than others and this reflected in the results (Fig. 2-4).

Sexual Selection

Sexual selection is a concept that is prevalent in any social standing. People (and animals) choose a mate based on their ideal preferences. Generally, it can be considered what society deems as an appropriate fit but this ingrained preference goes a little deeper than that, even if we aren’t consciously aware of it. A study by Godin and Dugatkin (1996) looked at female mating preferences in guppies and identified their tendencies to mate with brightly coloured males. Surely this isnt based entirely on aesthetically pleasing characteristics? Godin and Dugatkin (1996) identified that males with brighter colours were more likely to approach predators in both the absence and presence of females. This resonates in many social situation where a male tries to portray his masculinity by challenging someone larger than him in order to woo a mate. This boldness ultimately leads to these characteristics  being passed onto their sons and thus increasing genetic fitness of both parents. The protective element is always desirable as males exhibiting these characteristics are able to fend for and protect their families from danger and thus contribute, genetically, to the next generation.

I enjoyed looking at this paper again. I was first introduced to Endler and his guppies in my undergrad degree, where we were given a computer simulation that was based on this experiment. At the time I didn’t appreciate the intricacies of it, clicking aimlessly until it was time for lunch, but the concept stuck with me. It’s quite a cool concept to consider when you’re thinking about natural selection, as it is not only the predation element that is operating in this study but also sexual selection.

I had been watching a program on tv about fertility issues some couples face and later discussed this with my husband. With the addition of modern medicine and the ability to over come issues that would have initially stalled reproduction in some individuals, will this ultimately lead to an increase in the susceptibility of future generations? This can be applied to many evolutionary concepts where adaptation culls the weak and selects for the strong. If we are able to alter this selection process, what does that mean for the future? Will we see increasing amounts of people succumbing to illness? Is this changing the natural course of evolution? These are questions that I often think about and by looking at the current population its easy to see the shift. This is a deviation from what guppies can tell us at this point in time but simply put, only the strongest survive.

 

References

  1. Godin, J.-G.J. & L.A. Dugatkin, Female Mating Preference for Bold Males in the Guppy, Poecilia reticulata. Proceedings of the National Academy of Sciences of the United States of America, 1996. 93(19): p. 10262-10267.
  2. Endler, J.A., A Predator’s View of Animal Color Patterns, in Evolutionary Biology, M.K. Hecht, W.C. Steere, and B. Wallace, Editors. 1978, Springer US: Boston, MA. p. 319-364.
  3. Endler, J.A., Natural Selection on Color Patterns in Poecilia reticulata. Evolution, 1980. 34(1): p. 76-91.
  4. Amy E. Deacon, Hideyasu Shimadzu, Maria Dornelas, Indar W. Ramnarine & Anne E. Magurran. From species to communities: the signature of recreational use on a tropical river ecosystem, 2015. Ecology and Evolution 5 (23): 5561–5572. DOI:10.1002/ece3.1800. (Feature Image).
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