Use it and lose it: lipofuscin accumulation in the brain of a coral reef fish

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Gagliano, M, Lema, AK. Depczynski, M & Whalan, SW 2011, 'Use it and lose it: lipofuscin accumulation in the midbrain of a coral reef fish', Journal of Fish Biology, vol. 78, no. 2, pp. 659-666.

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Lipofuscin, an autofluorescent biomarker of physiological wear-and-tear, was concentrated in those areas of a fish's midbrain responsible for visual performance, suggesting a potentially strong link between physiological specialization, ecological adaptation and senescence.

The study of the physiological trade-offs and costs underlying the potential for animal adaptation to changing environments is clearly emerging as a common theme in connection with the impact of global climate change on the biogeography, community composition and food web structures of natural populations (Pörtner & Farrell, 2008). Physiological trade-offs are typically thought to result from the partitioning of finite energetic resources among competing biological functions, where the higher performance of one activity generates negative consequences in other activities (Zera & Harshman, 2001). Costs of living and senescence can be understood within this allocation framework, where the process of ageing inevitably leads to changes in an organism's physiological priorities and hence its subsequent ecological performance. The result is often seen by the trade-off between energetic investments in present day activities (i.e. current reproduction and growth) balanced against an uncertain future (i.e. the possibility of a premature death leading to a reduced lifetime reproductive output).

The cumulative costs of living may be quantified by measuring the aggregation of ‘waste material’ such as lipofuscin that naturally accumulates in animals as a consequence of physiological trade-offs and random damage. Lipofuscin is an intracellular protein aggregation, which forms due to iron-catalyzed oxidation and polymerization of proteins and lipids in the lysosome (Terman & Brunk, 2004). It accumulates over time because it cannot be exocytosed nor be degraded, most likely due to the presence of peptides cross-linked by aldehydes into plastic-like structures (Kikugawa et al., 1989). Because it accumulates over time, lipofuscin has long been known as the autofluorescent ‘age pigment' and given that it is phylogenetically the most widespread cellular manifestation of ageing, it is a recognized hallmark of physiological ageing (Brunk & Terman, 2002). Contrary to chronological ageing which is defined by the passage of time since birth, physiological ageing is characterized by the progressive change in tissues and organs of the body, associated with a decrease in physiological function and increased susceptibility to disease and death (Harman, 2001). Given how obsessed humans are with finding ways to elude the signs of ageing and extend the length of life, it is understandable that so much research effort on physiological ageing and lipofuscin has been largely focused on its underlying causes and concentrated on humans (and relatively few model organisms in laboratory conditions). Nevertheless, exploring variation in physiological ageing within species and among species in nature could not only reveal the influence of biological mechanisms that have evolved in response to variations in the environment to facilitate adaptation (Ricklefs, 2008) but also the extent to which the ageing process itself may be an intrinsic part of this adaptation process (Borup et al., 2008).

Fishes have been identified as offering a tremendous scientific opportunity for gerontological investigations and several small short-lived tropical fish species, such as guppies Poecilia reticulata Peters (Poeciliidae), killifishes (Nothobranchiidae, Rivulidae and Fundulidae) and zebrafish Danio rerio (Hamilton) (Cyprinidae) have indeed been adopted as models and have already facilitated significant progress in the understanding of specific age-related disorders (Gerhard, 2007). Previous studies have examined the accumulation of lipofuscin in these freshwater species (Strauss, 1999; Kishi et al., 2003; Terzibasi et al., 2008), but they have mostly used individuals from laboratory strains maintained under artificial conditions in captivity. These studies have provided invaluable knowledge of the biological processes at play; however, the costs incurred by animals as they experience life within the complexity of their natural environment still need to be explored. Because the behavioural abilities and feats of an organism seem to be governed largely by what it needs in order to pursue its life, this study focused on a common coral reef fish species Thalassoma lunare (L.), which leads a particularly active diurnal life even for a reef fish (Fulton, 2007; Gagliano et al., 2009). It is often observed darting around reef crests and down the front slope, carrying out an array of visually mediated tasks including foraging, predator avoidance and communication (Barry & Hawryshyn, 1999). Does life for such a visually dependent animal come at a cost detectable in those areas of the brain directly orchestrating its visual performance? The accumulation of lipofuscin in the brain of this coral reef fish was examined in relation to its ecology and behaviour in nature. After previous studies have shown that specific parts of the brain may accumulate selectively greater amounts of this pigment, this study specifically examined whether lipofuscin is accumulated uniformly throughout the fish midbrain, where visual and motor control as well as olfactory (Hofmann & Meyer, 1992) and auditory (Bell & Maler, 2005) processing occur.

To do this, a total of 23 individuals of T. lunare (Fig. 1) were collected at Lizard Island in the northern Great Barrier Reef. Fish were captured from shallow reefs using a combination of a monofilament barrier net and hand-nets and immediately transported to the laboratory, where they were humanely sacrificed, their size (standard length, LS) and wet mass recorded. Brains were immediately dissected, fixed with formaldehyde-acetic acid-calcium chloride (FAAC) and stored for subsequent lipofuscin analyses. Fish brains were dehydrated through an automatic tissue processor (Shandon Embedding Centre; www.thermoscientific.com), embedded in paraffin wax and sectioned at 5 µm. To check for autofluorescent lipofuscin-like granules, unstained serial coronal sections (n = 15 slides for each fish brain) were mounted in DPX medium for confocal microscopy and examined using a Leica DM IRE 2 (Leica microsystems; www.leica-microsystems.com) and a ×40 objective (numerical aperture 1·30). Sections were excited with a 488 nm laser and their fluorescence emissions were collected over the spectral range 490–700 nm and followed by spectral unmixing (Fig. 2). The presence of lipofuscin autofluorophores identified by confocal microscopy was then histochemically confirmed by positive staining with Sudan black B (Fig. 3; Lomovasky et al., 2002; Lema, 2008).

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