Determining the distribution of melanin in river lamprey (Lampetra fluviatilis) using histological sections

Posted by lcb14 at Feb 06, 2018 12:10 PM |
PhD Researcher Chris Nedza was awarded the Whitaker Award in Geology last year to assist his studies into melanin in river lamprey. Here he writes about how this funding has helped his research...
Determining the distribution of melanin in river lamprey (Lampetra fluviatilis) using histological sections

Figure 1. Haematoxylin and eosin stained histological section through the head region of a pristine (day 0) lamprey showing several anatomical features. arc, arcualia; ey, eye; my, myomeres; nc, notochord; olf, olfactory organ; Sk, skin; rpe, retinal pigm

Understanding the distribution of pigmented anatomy in fossilised animals is crucial to correctly interpreting their physiology, ecology and development. Do the dark stripes on a fossil fish represent colour patters used for camouflage and communication, or something else entirely? To answer this question one has to study the pigment melanin, the same substance that gives colour to your hair and skin. Vast amounts of research has been conducted that looks at the sub-cellular structures that produce this pigment – melanosomes – in relation to the colour patterning and ecology of feathered dinosaurs. However, little research has focused on this pigment in aquatic vertebrates and to what extent the decay and fossilisation processes have upon its distribution. It was the aim of this John Whitaker Award funded project to determine the distribution of melanin throughout the tissues of the extant river lamprey (Lampetra fluviatilis) using stained histological sections. Doing so would allow a greater understanding of the development and evolution of important pigmented tissues in a ‘primitive’ vertebrate and allow accurate comparisons to be drawn with fossil lamprey.

Samples of adult lamprey were kindly donated by Marine Nutrition in Grimsby, transported live to Leicester before being euthanized with an overdose of tricaine methanesulfonate (MS-222) and placed in decay vessels. Individual lamprey were destructively sampled at several intervals over a two week period. Samples were fixed in 10% formalin for a minimum of two days before being taken to the histology facility within the university to be sectioned. Sections were taken through anatomy located towards the anterior region of the organism. Staining of the sections using haematoxylin and eosin (H+E) helps to reveal important tissues and structures (Figure 1) including proteins (stained red), cell nuclei (stained purple) and melanin (stained black). Sections were imaged by using a Zeiss AxioImager.M2m with an x5 objective to create a montage of the whole slide.

Analysis of the sections through the anterior anatomy of the lamprey revealed several striking details (Figure 2). Melanin can be seen in a variety of tissues including the skin, eyes, olfactory organ, notochord, gills and sporadically between different tissues within the body cavity. Furthermore, tissue degradation is evident from day 2 of decay onwards, with the breakdown of tissue structure and integrity increasing as decay progresses. From day 4 onwards structural collapse of the body is noticeable, particularly in the gill region. By day 11 decay has progressed to a point where identification of specific organs and structures becomes problematic due to tissue loss, distortion and the movement of pigment throughout the organism. Over increasing time, and therefore decay, the intensity of colouration from the haematoxylin and eosin staining diminishes. This could be attributed to the breakdown of proteins and nucleic acids during decay that would normally be highlighted by the staining process.

Figure 2. Stained (H+E) histological sections of the anterior region of L. fluviatilis during various stages of decay.
Figure 2. Stained (H+E) histological sections of the anterior region of L. fluviatilis during various stages of decay. A-D show areas of sectioning. (A) anterior most region of the head with notable eyes and olfactory organ (B) section through the eyes, olfactory organ and the inception of the notochord (C) section through the gills and digestive tract (D) posterior most section through the liver and heart. All sections contain skin.

Whilst the original aim of this project was to reconstruct the pigmented anatomy in three dimensions using the histological sections, it transpired that this wasn’t feasible or likely to be as informative as the sections themselves. Therefore, the sections funded by the John Whitaker Award were used to provide a basis for a study of the distribution and morphology of melanin and melanosomes in extant river lamprey. The results from this study were presented as a poster at the annual Palaeontological Association meeting in London this year, which won the Council Poster prize. Similarly, I submitted several images of the lamprey sections to the university Images of Research competition, where they won the People’s Choice award (Figure 3).

Figure 3. ‘A Slice of Fish’. Images of Research People’s Choice award winning images.
Figure 3. ‘A Slice of Fish’. Images of Research People’s Choice award winning images. Colour negatives of lamprey histological sections showing the distribution of melanin, stained white, throughout internal tissues: eyes (middle), the gills (left) and gonads (right).

The funding received from the John Whitaker Award has enabled me to conduct the first comprehensive study of the distribution of melanin throughout an evolutionarily important, extant vertebrate. The data obtained from this project will be invaluable to future work which will aim to further our understanding of the timing and development of key evolutionary events and ecological traits within early vertebrates, alongside serving as a starting point for a larger database focused on mapping the distribution of pigmented anatomy within extant aquatic vertebrates.

Chris is currently studying for his PhD in the School of Geography, Geology and the Environment - "Pigmented and organically preserved soft-bodied fossils – using taphonomic mode to interpret anatomy"

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