Category Archives: Lab Methods

Gaining Genetic and Epigenetic Data from a Single Established Next-Generation Sequencing Approach

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Post provided by Marco Crotti

How organisms adapt to the environment they live in is a key question in evolutionary biology. Genetic variation, i.e. how individuals within populations differ from each other in terms of their DNA, is an essential element in the process of adaptation. It can arise through different mechanisms, including DNA mutations, genetic drift, and recombination.

Example of how genetic drift can occur over generations via random sampling (i.e. random mating) in a population. (Picture credit: Gringer).

Differences in DNA sequences between individuals can results in differences in the expression of genes. This can therefore determine the organism’s capacity to grow, develop, and react to environmental stimuli. However, a growing body of literature reveals that there are other ways organisms can change the way they interact with the world without mutations in the DNA sequence.

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A Surgical Approach to Dissection of an Exotic Animal

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Post provided by Aaron T Irving, Justin HJ Ng and Lin-fa Wang

An Australian black flying fox – missing an ear, but fit for release.

Bats. They’re amazing creatures. Long-lived (with relevance to their body size), echolocating (for microbats and some megabats), metabolically-resilient (apparently resilient to most virus infections) flying mammals (with heart beats up to 1200 bpm for hours during flight). There are 1,411 species of this incredible creature. But very little is known about their physiology and unique biological traits. And detailed evolutionary analysis has only just begun.

The problem is, they’re an ‘exotic’ animal (wildlife that most people do not come into contact with). Being a long-lived animal producing minimal offspring (most only have one baby per year), they’re not suited to the kind of experimental studies we do with other animals like mice. Unavoidably, some aspects of biology require the use of tissues and cells. These samples can be used for sequencing, genomics, molecular evolution studies, detailed transcriptomic analysis, functional experiments with specific cell types and much more. Some methodology is beginning to be published – such as capture techniques and wing punch/genomic isolation – but there’s been an absence of protocols for the processing of bats. This is essential for the field to maximise the potential application of each individual and for minimising non-essential specimen collection.

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The Self-Preserving eDNA Filter: How It Works and Why You Should Use It

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Researchers at Washington State University and Smith-Root recently invented an environmental DNA (eDNA) filter housing that automatically preserves captured eDNA by desiccation. This eliminates the need for filter handling in the field and/or liquid DNA preservatives. The new material is also biodegradable, helping to reduce long-lasting plastic waste associated with eDNA sampling.

This video explains their new innovation in the field of eDNA sampling technology:

To find out more about the self-preserving eDNA filter, read the full, Open Access Methods in Ecology and Evolution article ‘A self‐preserving, partially biodegradable eDNA filter‘
(No Subscription Required).

If you’re using interesting new field techniques like this, why not submit a Practical Tools manuscript about them? You can find out more about Practical Tools manuscripts here.

Stereo DOV: A Non-Invasive, Non-Destructive Way to Study Fish Populations

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It’s more important than ever for us to have accurate information to help marine conservation efforts. Jordan Goetze and his colleagues have provided the first comprehensive guide for researchers using diver operated stereo-video methods (or stereo-DOVs) to survey fish assemblages and their associated habitat.

But what is Stereo DOV? What makes it a better method than the traditional UVC (Underwater Visual Census) method? And when should you use it? Find out in this video:

To find out more about stereo DOVs, read the full Methods in Ecology and Evolution article ‘A field and video analysis guide for diver operated stereo‐video‘
(No Subscription Required).

If you’re using interesting new field techniques like this, why not submit a Practical Tools manuscript about them? You can find out more about Practical Tools manuscripts here.

Quantifying Animal Movement from Videos

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Quantifying animal movement is central to research spanning a variety of topics. It’s an important area of study for behavioural ecologists, evolutionary biologists, ecotoxicologists and many more. There are a lot of ways to track animals, but they’re often difficult, especially for people who don’t have a strong background in programming.

Vivek Hari Sridhar, Dominique G. Roche and Simon Gingins have developed a new, simple software to help with this though: Tracktor. This package provides researchers with a free, efficient, markerless video-based tracking solution to analyse animal movement of single individuals and groups.

Vivek and Simon explain the features and strengths of Tracktor in this new video:

Read the full Methods in Ecology and Evolution article ‘Tracktor: Image‐based automated tracking of animal movement and behaviour‘
(No Subscription Required).

Download and start using Tracktor via GitHub.

How to Assemble, Fill and Clean Metapopulation Microcosm Plates: Two Video Tutorials

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Metapopulation Microcosm Plates (MMP) are devices which resemble 96-well microtiter plates in size and shape, but with corridors connecting the wells in any configuration desired. They can be used to culture microbial metapopulations or metacommunities with up to 96 habitat patches.

In these two video tutorials, Helen Kurkjian explains how you can assemble, fill and clean MMPs in your lab.

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Scales, Slime and Dragon(fly) Wings? Investigating the Surfaces of Organisms

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Post provided by Dylan Wainwright

Our recent Methods in Ecology and Evolution paper – ‘Imaging biological surface topography in situ and in vivo‘ – shows how to use gel-based profilometry to image various biological surfaces. To start you need to press a gel into a surface of interest. The bottom surface of the gel is coated in a paint to create an impression of the surface that has standard optical properties (not clear, shiny, or coloured). Then lights are shone on the gel at different angles and photographs are taken at six different lighting angles. These photographs allow us to study the surface in incredible detail. The following images give more information on how we can do this and the benefits of it.

Our first picture shows the peduncle and tail of a yellow perch (Perca flavescens) being pressed into a gel. We use a gel-based profilometry system manufactured by GelSight Inc. (http://www.gelsight.com/). Image: Dylan Wainwright.
The six greyscale photographs in this image are of the scales from the Hawaiian dascyllus (Dascyllus albisella). Each image has a different lighting angle and all six will be used to reconstruct the surface topography on this patch of scales. Imaging a surface is as fast as positioning the specimen and taking six photographs. No specimen preparation is required – this method can be done on clear, shiny, wet, and slimy surfaces! Images: Dylan Wainwright and the Freshwater and Marine Image Bank.
In this picture you can see the surface topography of Dascyllus albisella, reconstructed from the six greyscale images in the previous image. This image captures the lateral line, visible at the top of the image as a row of scales connected by a canal. Heights on this surface are shown as colours: the warmer the colours (oranges and reds), the higher the heights. The height range of this surface is just over 200 microns – the highest parts of the surface are over 200 microns higher than the lowest . Images: Dylan Wainwright and the Freshwater and Marine Image Bank.
Each reconstructed surface is made up of over 18 million three-dimensional points (x, y, and z). This allows for a substantial amount of digital zoom with the ability to still recover surface features. Above is an enlarged view of the posterior margin of a scale from Dascyllus albisella from the same image as the previous two slides. The posterior margin of this scale is made of ctenii, which are small interlocking spines that are present on the scales of many species of fish. Those at the margin are the longest and newest, with older ctenii becoming shortened and serving as a scaffold to interlock with newer ones. Images: Dylan Wainwright and the Freshwater and Marine Image Bank.
The three-dimensional topography data recovered by gel-based profilometry can help you make unique observations on the surface texture of biological surfaces, such as the armor-like ganoid scales of Polypterus endlicheri (see ‘Materials design principles of ancient fish armour’ by Bruet et al. http://go.nature.com/2ivXi8I for more information on poylpterus armor). Using software for surface analysis, height profile lines can be generated (shown above), along with a variety of roughness and surface measurements (not shown). This topographic data is crucial for understanding how biological surfaces interact with their environments. Images: Dylan Wainwright and George Albert Boulenger.
With gel-based profilometry, you can tune the gel properties to match even very soft surfaces, such as the epidermis and mucus that covers the scales of live fish. Above, we show a bluegill (Lepomis macrochirus) that was imaged with and without mucus. Without mucus, many surface details of scales are obvious, such as the concentric growth lines of each scale, the lateral line, and clear margins made of spiny ctenii. When mucus is present, the surface details are obscured. Below each image we provide tables of common surface parameters including root-mean-square roughness (Sq – http://bit.ly/2Amhpeb), kurtosis (Sku – http://bit.ly/2zUY8ne), and skew (Ssk – http://bit.ly/2zUY8ne). Roughness is much lower on the surface with mucus, demonstrating its smoothing effect. This smoothing effect and the material properties of mucus will likely affect the swimming performance of this fish, and these results show how useful this technique can be for exploring surfaces of live animals. Images: Dylan Wainwright and the Freshwater and Marine Image Bank.
Gel-based profilometry is non-invasive and only needs pressure to be applied to the surface of interest to get the image. Above is the surface topography of the back of a human hand. The pores are evident as small blue regions with low elevation. Long flexible structures like hairs will be pressed flat by the sampling gel, as seen in the hairs above. Image: Dylan Wainwright.
You can see the surface of a Boston fern (Nephrolepis exaltata) above. This image was taken at high magnification and then cropped to a 1 mm by 1 mm square. Stomata with guard cells are visible on the surface of the leaf as ring-shaped cells. Images: Dylan Wainwright and Marija Gajić (http://bit.ly/2AxryHp).
This is the forewing of a dragonfly. The wing venation pattern is obvious using this technique, and small spines are present on many of the veins, especially the distal veins towards the wing tip. We produced this image without any special preparation of the subject and without damaging these delicate wings. Images: Dylan Wainwright and Wellcome Library, London (http://bit.ly/2AkcT1J).
The above image shows a dorsal patch of skin from the Chinese crocodile lizard (Shinisaurus crocodilurus). This lizard is an endangered semiaquatic species with skin similar in appearance to a crocodiles (as its name suggests). Gel-based profilometry provides a non-destructive way of investigating the skin morphology of this species using museum specimens. Images: Dylan Wainwright and spacebirdy (CC-BY-SA-3.0) (http://bit.ly/2jCtvb4).
Above we have both a greyscale image and a height map from the hand of a Sulawesi lined gliding lizard (Draco splinotus). For two or one-dimensional measurements, greyscale images can be valuable because of their high contrast. Gel-based profilometry produces grayscale images at a range of sizes, comparable to low to medium magnification scanning electron microscopy. Images: Dylan Wainwright and A.S.Kono (http://bit.ly/2BCFY6W).
The denticles from the lateral flank of a leopard shark (Triakis semifasciata) were imaged and you can see the topographic reconstruction above. Denticles have been shown to increase swimming performance and understanding their surface topography is crucial for connecting the form of shark denticles to hydrodynamic function (see ‘The hydrodynamic function of shark skin and two biomimetic applications’ by Oeffner and Lauder, for example). Images: Dylan Wainwright and Tom Hilton (http://bit.ly/2BpW3vv).
This image shows the skin texture of the white marlin. Although most fish only have one type of bony structure in their skin (scales), white marlin have two. The first are larger, teardrop shaped scales with forked ends that are embedded in the dermis – they’re visible as larger impressions above. The second bony structure present on white marline skin are smaller peaks that are attached to the skin surface and look like small grains in the images above. Understanding these structures is an important step to understanding the function of marlin skin and the reasons behind these modifications (for more information on these scales see ‘Comparative morphology of the scales of roundscale spearfish Tetrapturus georgii and white marlin Kajikia albida’ by Loose et al. – http://bit.ly/2Bq5UBM). Images: Dylan Wainwright and public domain image.

To find out more, read our Methods in Ecology and Evolution article ‘Imaging biological surface topography in situ and in vivo’. And to find out more about Dylan’s research, visit his website.

Animation Meets Biology: Shedding New Light on Animal Behaviour

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Below is a press release about the Methods paper ‘Integrating evolutionary biology with digital arts to quantify ecological constraints on vision-based behaviour‘ taken from the La Trobe University.

Ctenophorus fionni (Peninsula Dragon), male push up display - Copyright Jose Ramos, La Trobe University

Ctenophorus fionni (Peninsula Dragon), male push up display. © Jose Ramos, La Trobe University

Many animals rely on movement to find prey and avoid predators. Movement is also an essential component of the territorial displays of lizards, comprising tail, limb, head and whole-body movements.

For the first time, digital animation has been used as a research tool to examine how the effectiveness of a lizard’s territorial display varies across ecological environments and conditions. The new research was published today in the journal Methods in Ecology and Evolution.

A team from La Trobe University’s School of Life Sciences, led by Dr Richard Peters, worked with academics from Monash University’s Faculty of IT to create, using 3D animation, a series of varied environmental settings and weather conditions, comprising different plant environments and wind conditions, to quantify how lizard displays are affected by this variation. Continue reading →

Imperfect Pathogen Detection: What to Do When Sampling and Diagnostic Tests Produce Inaccurate Results

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Post Provided by Graziella DiRenzo

A salamander having its skin swabbed to test for Bsal infection.

A salamander having its skin swabbed to test for Bsal infection.

Imagine you’re at the doctor’s office. You’re waiting to hear back on a critical test result. With recent emerging infectious diseases in human populations, you are worried you may be infected after a sampling trip to a remote field site. The doctor walks in. You sit nervously, sensing a slight tremble in your left leg. The doctor confidently declares, “Well, your tests results came back negative.” In that moment, you let out a sigh of relief, the kind you feel throughout your body. Then, thoughts start flooding your mind. You wonder– what are the rates of false negatives associated with the test? How sensitive is the diagnostic test to low levels of infection? The doctor didn’t sample all of your blood, so how can they be sure I’m not infected? Is the doctor’s conclusion right?

 Now, let’s say I’m the doctor and my patient is an amphibian. I don’t have an office where the amphibian can come in and listen to me explain the diagnosis or the progression of disease − BUT I do regularly test amphibians in the wild for a fatal fungal pathogen, known as Batrachochytrium dendrobatidis (commonly known as Bd). Diseases like Bd are among the leading causes of the approximately one-third of amphibian species that are threatened, near threatened, or vulnerable to extinction. To test for Bd, and the recently emerged sister taxon Batrachochytrium salamandrivorans (hereafter referred to as: Bsal), disease ecologists rely on non-invasive skin swabs. Continue reading →

Reptile DNA Sexing: Easier Than You Ever Thought

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Post provided by Lukáš Kratochvíl and Michail Rovatsos

The sand lizard (Lacerta agilis).

The sand lizard (Lacerta agilis).

Many researchers, breeders and hobbyists need to know sex of their animals. Sometimes it’s easy – in sexually dimorphic species you only have to look. In other species or juveniles it’s often not so straightforward though. And it’s often impossible – but sometimes essential – in embryos or in tissue samples. Determining sex from DNA is the most practical option, or sometimes even the only possibility, in these cases.

Molecular sexing is routinely used in mammals and birds, but until now it has only been available for a handful of reptile species. Many people didn’t believe that this situation would improve considerably any time soon. But why? Continue reading →