Age-related changes in fatty acid profile and locomotor activity rhythms in Nothobranchius korthausae

Experimental Gerontology 46 (2011) 970–978

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Age-related changes in fatty acid profile and locomotor activity rhythms in Nothobranchius korthausae A. Lucas-Sánchez ⁎, P.F. Almaida-Pagán, J.A. Madrid, J. de Costa, P. Mendiola Department of Physiology, Faculty of Biology, University of Murcia, 30100 Murcia, Spain

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Article history: Received 15 December 2010 Received in revised form 25 July 2011 Accepted 18 August 2011 Available online 28 August 2011 Section Editor: Andrzej Bartke Keywords: Nothobranchius korthausae Ageing Fatty acids Locomotor activity Circadian rhythms

a b s t r a c t The life cycle of Nothobranchius korthausae, a Cyprinodontiformes fish, was studied in our laboratory to characterise the ageing process. Some morphological changes, such as spine curvature, skin colour, and fin and eye appearance are described. Growth and survival curves reflected a fast life cycle with rapid initial growth until 4 weeks of age, after which the fish grew more slowly before reaching their final size in week 40. Senescence onset was established at week 48 with a decrease in spawn size and viability and a general decline in the animal's appearance (weight and colouration losses, caudal fin degradation, and cataractogenesis). The fatty acid composition changed with age, with high unsaturation in the adult stage as reflected by a high peroxidation index, a condition that is associated with high susceptibility to oxidative damage if elevated reactive oxygen species (ROS) production occurs. Senescent fish had an increase in monounsaturated fatty acid proportions and a lower peroxidation index (226.5 ± 19.7 in adults versus 120.2 ± 19.1 in senescent fish, P b 0.05). The circadian system, as reflected by locomotor activity rhythms, showed noticeable changes with age. Twenty-four-week-old fish (adults) had a robust diurnal rhythm that showed a decrease in total activity, an increase in rhythm fragmentation, and a fall in amplitude and regularity with age. Changes were clearly reflected in the Circadian Function Index variations (0.56, 0.47 and 0.25 at 24, 48 and 72 weeks of age, respectively). In conclusion, N. korthausae appears to be a species with appropriate characteristics for ageing studies because it manifests clear signs of progressive ageing. Comparing species of Nothobranchius genus with different lifespans may be useful for increasing our understanding of the ageing process. © 2011 Elsevier Inc. All rights reserved.

1. Introduction As in any other research field concerning human biology, ageing research needs animal models to throw light on the basic mechanisms involved. However, vertebrate models with short life cycles are scarce. This has led to the potential for different fish species to be explored (Gerhard, 2007), including the so-called killifish (Cyprinodontiformes order). This group includes some small fish that are characterised by their colonisation of seasonal ponds through the production of desiccation-resistant eggs (Jubb, 1982). Several studies have shown that annual fish have a defined lifespan in captivity that can be modulated by water temperature and other factors. They also manifest age-related histological degeneration in many tissues including an increased incidence of tumours, making

⁎ Corresponding author at: Department of Physiology, Faculty of Biology, University of Murcia, Campus de Espinardo, 30100 Murcia, Spain. Tel.: + 34 868 884 931; fax: + 34 868 883 963. E-mail address: [email protected] (A. Lucas-Sánchez). 0531-5565/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2011.08.009

them good candidates for use as vertebrate models for ageing studies (Walford and Liu, 1965; Walford et al., 1969; Liu and Walford, 1970, 1975; Markofsky and Perlmutter, 1972; Markofsky, 1976; Markofsky and Milstoc, 1979a,b; Cooper et al., 1983; Genade et al., 2005; Di Cicco et al., 2011). To date, several studies have been performed in various species of Nothobranchius, especially N. furzeri and N. rachovii. In these studies, different biomarkers have been used to characterise the ageing process, with an emphasis on animal growth and survival curves (Valdesalici and Cellerino, 2003; Valenzano et al., 2006a,b; Terzibasi et al., 2009; Hsu and Chiu, 2009), the quantification of macroscopic morphological factors, such as body mass loss, the cessation of animal growth, spine curvature and colouration loss with age (Valenzano et al., 2006b; Terzibasi et al., 2009); the analysis of microscopic factors, such as age-related pigment deposition (lipofuscin, fluoro-Jade B) (Valenzano et al., 2006b; Terzibasi et al., 2007, 2009; Hsu et al., 2008; Hsu and Chiu, 2009); genetic factors, such as the shortening of chromosome telomeres (Hsu et al., 2008; Hartmann et al., 2009); and the use of behavioural criteria based on the quantification of the decline in cognitive ability with age (Valenzano and Cellerino, 2006; Valenzano et al., 2006a; Terzibasi et al., 2007, 2009).

A. Lucas-Sánchez et al. / Experimental Gerontology 46 (2011) 970–978

Ageing seems to be the result of a complex chain of events in which different factors are involved; the cause–effect relationship between these factors has yet to be established. According to the free radical theory of ageing, reactive oxygen species (ROS) generated as a result of normal cell metabolism play a pivotal role in the ageing process by causing cumulative damage to cell macromolecules (lipids, proteins and DNA), leading to progressive dysfunction with age (Harman, 1956). The oxidative status, a result of the balance between oxidant production and defence and repair systems, has been studied by measuring the activity of antioxidant enzymes or the protein and lipid oxidative damage caused by ROS during the life cycle of fish species proposed as ageing models (Hsu et al., 2008; Hsu and Chiu, 2009). Amongst different types of macromolecule damage, lipid peroxidation is quantitatively the most important oxidative process in tissues; in addition, according to the membrane pacemaker theory, the lipid profile of animals is the key factor that determines oxidative stress susceptibility (Hulbert, 2005). This theory, an extension of the oxidative stress theory, proposes that animal species with highly unsaturated lipid profiles (especially in their membranes) have an increased metabolic rate that is associated with high ROS production and an increased susceptibility to great oxidative damage. Although the relationship between the lipid profile and lifespan has been established in many vertebrates (Hulbert et al., 2007; Sanz et al., 2006), age-related fatty acid profile changes have barely been explored (Castelluccio et al., 1994). The metabolic and morphological age-related changes described above involve a deterioration in the ability of animals to cope with environmental changes (Weinert, 2000). The 24 hour light–dark cycle caused by the rotation of the earth has resulted in the evolution of the circadian system. The circadian system is composed of a network of hierarchically organised structures responsible for the generation of circadian rhythms and their synchronisation with the environment. This timekeeping system is essential for anticipating periodic changes in the environment and for keeping a precise internal temporal order between different physiological and behavioural rhythms. Biological rhythms are driven by a central pacemaker located in the central nervous system and a set of peripheral oscillators located in most tissues and organs. Under natural environmental conditions, the circadian system is reset everyday by periodic environmental cues (called synchronisers or zeitgebers) such as light– dark and temperature cycles or feeding time. During the life cycle of an organism the circadian system suffers several changes, particularly during ontogeny and senescence (Weinert and Schuh, 1988; Turek et al., 1995; Weinert, 2000, 2005). If the circadian system develops normally, a long period will pass until it begins to deteriorate. For this reason, circadian rhythms might be used as markers of the progressive impairment of the functionality of an organism and as ageing indicators (Mailloux et al., 1999). One of the most important biological rhythms of an animal is spontaneous locomotor activity, a behavioural activity that is repeated over 24 hour cycles and is one of the output components of the circadian system. Locomotor activity has been used for circadian function analysis and shows impairment with age, as described in humans (Van Someren et al., 1999), rodents (Kondratov, 2007) and zebrafish (Zhdanova et al., 2008). The main objective of this study was to characterise age-related phenotypes in Nothobranchius korthausae. This species has the advantage of an intermediate lifespan compared to other species used as models in ageing studies and so potentially allows for long-term studies to be performed at various stages in the ageing process. However, the lifespan of N. korthausae is not as long as in zebrafish (4 years), allowing for longitudinal studies. We analyse two new criteria to determine ageing. One is related to oxidative stress susceptibility and, therefore, lifespan, through changes in fatty acid composition. The other is circadian system function, whose impairment with age makes it a potential ageing marker.

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2. Materials and methods 2.1. Animals and husbandry N. korthausae (Meiken 1973; Actinopterygii, Cyprinodontiformes, Nothobranchiidae) adults of the Mafia Island population (TAN 02–05) were obtained from a local commercial supplier (Marine Pond Garden, Murcia) and acclimated to the Fish Chronobiology Laboratory at the University of Murcia facilities. Twenty to 25 fish with 1:2 (male:female) ratios were kept in 70 L tanks equipped with a recirculating fresh water system (4 L/h flow) and biological and mechanical filtration. The water parameters were controlled to conform to the following values: water hardness b6°dKH, NO3− b 0.1 mg/L, NO2− b 0.1 mg/L, NH3 b 0.5 mg/L, pH= 7.4, and temperature = 26 ± 2 °C. The photoperiod was set at 12 L:12D (lights on at 8:00 am). To obtain spawns, breeders were separated in 25 L tanks in groups of 2 males and 4 females (reproductive unit, RU), using coconut fibre as spawning substratum. After two weeks, containers with coconut fibre were removed and examined for egg production. Substrates with eggs were partially desiccated and then stored in plastic bags and incubated at 26 ± 2 °C and 12 L:12D photoperiod. They were inspected fortnightly; when the golden iris was evident, a sign of completely developed embryos, hatching was induced by immersion in aerated fresh water (18 °C). Larvae were fed newly hatched nauplii from Artemia for 15–20 days. Fish were then fed frozen bloodworms (Chiromonus, Frozen Fishfood, Holland) provided once a day (10:00 am), ad libitum, until the end of their lives. As described in Section 2.2.4, bloodworm proximate composition (AOAC, 1997) and the fatty acid profiles of A. salina nauplii and bloodworms were analysed (Supplementary Tables 1 and 2). The data reported in this paper correspond to three different populations (n = 58, 27, and 32) obtained from the initial adults. This experimental procedure agrees with current Spanish law on animal experiments, and the experimental protocol was approved by the Bioethics Committee of the University of Murcia. 2.2. Age determination 2.2.1. Growth and survival Body length (BL) was measured weekly by placing fish individually into a small container with a very low water level so that the fish was laid flat on the bottom over a millimetric scale (1 mm). Fish were measured from the tip of the snout to the tip of the tail. The instantaneous growth rate (IGR: Δlength/Δtime) was computed. BL data were fitted to the Von Bertalanffy growth function (VBGF), used as a model of fish growth (Moreau, 1987). VBGF is determined by the formula L = L∞ [1−e −k(t–to)], which has three main parameters: K (growth constant), L (asymptotic length), and t0 (age at length = 0). To make this adjustment, it is necessary to estimate the L∞ value with Taylor's method, assuming that the oldest fish typically reach 95% of the asymptotic length (L∞ = Lmax/0.95) (Taylor, 1962; Pauly, 1980). The time to reach Lmax (tmax) can be estimated as a function of the growth constant K, as tmax ≈ 3/k. The number of dead fish was recorded daily to obtain the survival curve of the species by the Kaplan–Meier method. 2.2.2. Morphological changes Different morphological changes were observed during N. korthausae ageing: spine curvature, colouration loss (especially in males), shape distortion of the caudal fin, loss of body mass and the increase in the vertical diameter of the eye. Photographs were made to record these observations. Eye diameter was measured by digital analysis (Leica EZ4 D Microscope, ×8 magnification). 2.2.3. Age groups Three main age groups of N. korthausae were established: undifferentiated (0–4 weeks), adult (5–48 weeks), and senescent (more

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than 48 weeks). The adult stage began when secondary sexual colouration appeared and included two secondary stages: a first fastgrowth stage (from 5 to 18 weeks) and a second slow-growth stage (from 19 to 48 weeks). The senescent stage began when any of the changes described in Section 2.2.2 were observed. 2.2.4. Fatty acid profile To characterise changes in the lipid composition of N. korthausae during their life cycle, the fatty acid profiles of whole animals (males and females) were obtained at three different stages: undifferentiated (2 weeks old), adult (20 weeks old) and senescent (more than 64 weeks old). For this analysis, six fish were sampled at each stage. Undifferentiated and adult fish were euthanised by exposure to the anaesthetic MS222 (200 mg/L), whilst senescent fish were obtained by natural death (maximum time post-mortem of 6 h). Analyses were performed to verify that no differences existed between euthanised and naturally-dead fish. The total amount of lipids in samples was determined gravimetrically after extraction, essentially as described by Folch et al. (1957). The total lipid extracts were subjected to acid-catalysed transmethylation using the method described by Christie (2003). The obtained fatty acid methyl-esters (FAME) were separated and quantified by gas–liquid chromatography using a Hewlett–Packard 5890 gas chromatograph equipped with a capillary column (SPTH-2560, SUPELCO, 100 m × 0.25 mm I.D., 0.20 μm thick). Peaks were identified by comparing their retention times with appropriate FAME standards (purchased from Sigma Chemical Company, St. Louis, MO, USA). Concentrations of individual fatty acids were expressed as percentages of total fatty acids. Various parameters of interest, such as the sum of saturated fatty acids, monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), highly unsaturated fatty acids (HUFA, fatty acids of 20 or more carbon atoms and 3 or more double bonds) and the peroxidation index (PI, as a susceptibility index) were calculated. 2.2.5. Locomotor activity rhythms The locomotor activity of adult and senescent N. korthausae fish was monitored to characterise circadian system function and, therefore, the state of temporal organisation of the fish in relation to ageing. For this purpose, three groups of fish were established (n = 6, males and females), representing three different ages in the life cycle of N. korthausae: 24 weeks (the middle of the adult phase), 48 weeks (early senescent phase) and 72 weeks (advanced senescence). Each group was kept in a tank (30 L) equipped with two photoelectric sensors (model E3S-AD62, Omron Corporation, Japan), one sensor 5 cm below the surface and the other 5 cm from the bottom of the tank. When the fish swam in front of the sensor, the interruption of the infrared light beam was recorded as one output signal (count). The number of counts was continuously collected every 10 min with a data acquisition system (Electronic Services of the University of Murcia, Spain). Total activity was calculated as the sum of the two photoelectric sensors in each tank. All data were processed with software specific for time series analyses (El Temps, A. Díez-Noguera, Universitat de Barcelona, 1999). In this way, we obtained double-plotted actograms of the locomotor activity and performed two different analyses: a) the Rayleigh test, which measures the acrophase regularity or the time of day with the maximum activity, and b) Fourier analysis, which fits the data to different sinusoidal functions, estimating the relationship between the power (percentage of variability explained) of the first harmonic (fit to a 24-hour sinusoidal wave) and the sum of all harmonics analysed (up to the 6th harmonic, corresponding to sinusoidal waves of less than 24 h; Circadian/ Ultradian Ratio). The Circadian Function Index (CFI), which offers an approach for understanding the general functioning of the circadian system (Ortiz-Tudela et al., 2010), was also obtained. This index integrates three non-parametric indices: a) intradaily

variability (IV), which depends on the endogenous character of the circadian rhythms and illustrates any fragmentation of the rhythms, b) interdaily stability (IS), which quantifies rhythm stability over different days and is more dependent on external factors, and c) relative amplitude (RA), which results from internal and external influences (Van Someren et al., 1999). 2.3. Statistical analysis The body lengths and mortality data of N. korthausae at different phases of their life cycle are presented as the mean ± standard deviation (SD). Statistical comparisons of the BL and the instantaneous growth rate (IGR) of males and females in the adult stage were performed by using a Student's t-test for independent samples. The significance of the BL data adjusted to the VBGF was obtained by an Ftest. The fatty acid composition of whole animals is represented as the mean ± SD. Comparisons between values from the different life cycle stages were made using a one-way ANOVA, and a Tukey-b post hoc test for multiple comparisons. In all tests, P values below 0.05 were considered to be statistically different. All of the statistical analyses were performed using SPSS software (SPSS Inc., Chicago, IL, v. 15.0). 3. Results 3.1. Life cycle of N. korthausae in captivity Fig. 1 represents the different stages of the N. korthausae life cycle. Because embryos are transparent, it was possible to follow their development. For embryo monitoring, three stages were defined: an initial stage (I), when the embryo was still undifferentiated and had a shiny appearance along with a lipid drop in one of the poles; an intermediate stage (II), when the embryo still had a shiny appearance but also showed the first cell clusters; and a final stage (III), when the embryo had lost its shiny appearance and looked fully differentiated, with a clear golden iris in its eyes. Embryonic development was completed over a period of 30–60 days. After hatching, N. korthausae larvae had an average BL of 4 mm. The specimens grew very quickly in the first four weeks of development (IGR: 1.9 per week), reaching a BL of 11.6 ± 1.3 mm (Fig. 2). A high mortality rate (32.60 ± 13.82%) was observed during the first four weeks, mainly due to development defects. After these four weeks, sexual dimorphism became evident, an event currently considered to be the beginning of the adult stage (Fig. 1). Both males and females showed a high growth rate until week 18, males reaching a BL of 36.5 ± 1.3 mm and females a BL of 29.7 ± 4.0 mm. These gender size differences were consolidated by 11–13 weeks, when the growth rate of males was significantly higher than that of females (2.3 mm compared to 0.6 mm per week; P = 0.023, Fig. 2). After week 19, growth slowed and there were no differences in the growth rate between males and females (0.3 mm per week for both sexes). Finally, growth finished at the end of the adult stage (week 39 for females and week 43 for males) when males reached 43.8 ± 1.3 mm and females 37.7 ± 0.6 mm in length (P = 0.001). When the BL data were fitted to the VBGF (P b 0.05, Fig. 2), K values of 0.07 and 0.08 mm per week were obtained for males and females, respectively, providing a corresponding estimated tmax of 43.5 or 39.0 weeks. In this case, tmax represents the time spent to reach the final length. No differences in mortality between males and females were observed, and the data for both were combined. Between 5 and 22 weeks some animals died due to residual developmental malformations and to aggression between individuals (especially by dominant males). Fish mortality stabilised at week 23, and almost all individuals reached the senescent stage (Fig. 3).

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3 Fig. 1. Life cycle of Nothobranchius korthausae in captivity. Different embryonic development stages (I, II and III): undifferentiated stage (1); adult stage (2), with marked sexual dimorphism; senescent stage (3).

Week 48 was considered to be the beginning of the senescent stage for N. korthausae in captivity. Around this age there was a decrease in the number and size of spawns and in their viability (week 20: 165 eggs/RU, 47% viability; week 28: 130 eggs/RU, 28.4% viability; week 40: 108 eggs/RU, 5.6% viability; week 52: 48 eggs/ RU, 0% viability). In the senescent stage, growth was null; survival 50

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3.2. Morphological age-related changes Several changes in morphology were observed throughout the life cycle of N. korthausae. During the senescent stage, body deformation consisted of marked spine curvature (Fig. 1), body mass and colouration loss (more obvious in males than in females), and progressive deterioration of the fins, especially the caudal fin (Fig. 4).

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Age (weeks) Fig. 2. Body length (BL) growth of Nothobranchius korthausae in captivity. Data are represented as the mean ± SD in males (n = 4) and females (n = 4) during their successive life cycle stages: undifferentiated stage (1), fast-growth adult stage (2a), slow-growth adult stage (2b) and senescent stage (3). Solid lines represent the growth curves for males and females (BL) fitted to the Von Bertalanffy growth function (VBGF).

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The eye diameter of N. korthausae increased significantly until the end of their life cycle, even when animals stopped growing in the senescent stage (Fig. 5). Moreover, some senescent fish had visible signs of lens opacity or cataracts. 3.3. Lipid profile Table 1 presents the fatty acid composition of whole N. korthausae (males and females) in different stages of their life cycle (undifferentiated, adult and senescent). Data from males and females were combined as they did not differ in fatty acid composition. In each stage, unsaturated fatty acids were higher (N60%) than saturated ones. Total saturated fatty acids were unaffected by age, whilst MUFA and PUFA varied significantly (P b 0.05). In the transition from the undifferentiated stage to the adult stage there was a significant decrease in the percentage of MUFA, whilst PUFA were not affected. In the senescent stage, MUFA increased, whilst both PUFA and HUFA decreased significantly. The percentage of individual fatty acids also varied significantly (P b 0.05) in the three stages studied. With regard to saturated fatty acids, the percentage of palmitic acid significantly increased with age. Similar changes were observed in the case of oleic acid, the principal MUFA. Significant levels of both docosapentaenoic acid and docosahexaenoic acid (DHA) were recorded in the undifferentiated stage, despite their absence from the diet. In the adult stage, their relative amounts changed substantially; whilst the docosapentaenoic acid percentage fell sharply, that of DHA increased significantly. Both fatty acids showed a significant decline in senescence. Oxidative susceptibility, reflected by the PI, increased in adult fish compared with the levels observed in undifferentiated fish; in senescent animals the PI fell sharply. 3.4. Locomotor activity rhythms The data obtained from monitoring locomotor activity in N. korthausae of 24, 48 and 72 weeks of age are represented in Fig. 6 as the mean waveforms, double-plotted actograms and Rayleigh tests. The results from the analysis of these data are presented in Table 2. As can be seen from the mean waveforms and the actograms, the activity pattern of 24-week-old N. korthausae was typically diurnal (82.6% of total activity happened during the photophase), with a robust, stable and regular rhythm (Fig. 6A and B). The maximum daily activity occurred around 2–4 h after the lights were turned on, coinciding with feeding time (Fig. 6A) and with a marked daily regularity (Fig. 6C), Furthermore, fish showed an increase in activity prior to feeding called feeding anticipatory activity (FAA; Fig. 6A). The 48week-old fish also had a diurnal rhythm (79.4% of activity concentrated in photophase) with a regular acrophase, whilst total mean activity (mesor) decreased and rhythm fragmentation increased (see IV in Table 2). FAA was maintained in 48-week-old fish. Finally, an impairment in locomotor activity rhythm was observed in 72-week-old animals. The total mean activity (mesor) decreased drastically, and only 57.2% of activity occurred during the photophase, matched by an increase in fragmentation (increased IV) and an irregular acrophase (Fig. 6C). In addition, the Circadian/Ultradian Ratio decreased, whilst FAA disappeared. The impairment of locomotor activity rhythms during the senescent stage in N. korthausae was highlighted

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by a marked decline in total mean activity (mesor), IS, RA and CFI (Table 2). 4. Discussion The life cycle of N. korthausae in captivity is very similar to that observed in other species of the same genus. Embryonic development follows the stages described for annual fish (Wourms, 1972). BL growth curves revealed an accelerated and finite growth, with sexual maturity reached at 4 weeks and final length reached around week 40. From this week onwards, a fall in the reproductive capacity, reflected by the number of fish spawns as well as by their viability, was observed, agreeing with observations made in the American annual fish Austrofundulus limnaeus (Podrabsky, 1999), which shows the same pattern although with differences in the timing; and with the numerous amateur breeders of Nothobranchius also mentioned by Genade et al. (2005). The decrease in the number and size of spawns agrees well with the age-related changes in gonads observed in N. furzeri (Di Cicco et al., 2011). N. korthausae showed reproductive senescence and had an extended post-reproductive lifespan. This is in contrast to other long-lived fish species that maintain reproductive capacity their whole lives (Reznick et al., 2002). The VBGF has been widely used as a model for fish growth (Moreau, 1987). Our results indicate that, under our laboratory conditions, the ageing process of N. korthausae did not begin until after the fish exceeded their life expectancy in nature. In any case, the VBGF model is useful for comparing different species or different treatments within the same species, as has been shown for zebrafish in da Rosa et al. (2010). N. korthausae exhibited a longer captive lifespan (maximum = 81 weeks) than other species from the same genus, such as the N. furzeri GRZ strain (13 weeks) (Valdesalici and Cellerino, 2003) and N. rachovii (37 weeks) (Herrera and Jagadeeswaran, 2004). Certain questions arise when comparing data from fish belonging to the same genus and having similar features, such as body size, but displaying different lifespans (Table 3): for example, what mechanisms are involved in differences in ageing rate? In this respect, N. korthausae would be a useful species to study. It would also therefore seem to be an appropriate species for studies involving relatively long-term experimental protocols. It has been proposed that the different lifespans observed in the genus Nothobranchius are related to the rainfall patterns in the original habitat (Genade et al., 2005). The habitat of N. furzeri is very dry (veldts of Zimbabwe), and the water bodies stand for a short period of time. The N. guentheri habitat (Zanzibar) has two wet seasons and might not dry out every year (www.worldweather.org, Table 3). The habitat of N. korthausae (Mafia Island, Tanzania) is similar to that of N. guentheri. Based on the University of East Anglia 0.5 × 0.5° 1961–1990 Monthly Climatology (New et al., 2000), the climate of Mafia Island can be classified as tropical (Aw in Koppen classification). Temperatures are mild, varying from a maximum temperature of 28 to 31° to a minimum of 19 to 22° throughout the year. The total annual precipitation is around 1800 mm and falls mainly from November to May (rainy and hot season). Due to this, the pools are likely not to dry up every year. Therefore, the habitat characteristics agree well with the higher maximal life expectancy observed in the laboratory for both the N. korthausae and N. guentheri species (Table 3).

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Fig. 4. Caudal fin appearance of Nothobranchius korthausae: 24 weeks (A), 40 weeks (B), 48 weeks (C) and 72 weeks (D) (×8 magnification).

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Weeks Fig. 5. Eye growth during the life cycle of Nothobranchius korthausae. Pictures show the eye of the animals at 12 weeks (A), 24 weeks (B), 48 weeks (C) and 72 weeks (D), when cataracts develop in some specimens (×8 magnification). The vertical line indicates the age at which length growth stops: growth stage (1), non-growth stage (2). Data are represented as the mean ± SD for males (n = 4) and females (n = 4). Statistical analysis was performed in the non-growth stage (2). The different superscripts indicate significant differences obtained with a Tukey-b post hoc for multiple comparisons test (P b 0.05).

The ageing phenotype of N. korthausae displayed marked morphological changes, including spine curvature, emaciation, loss of colouration and fin deterioration, as observed in other species of the genus (Genade et al., 2005; Terzibasi et al., 2008), zebrafish (Gerhard et al., 2002) and medaka (Hatakeyama et al., 2008). In addition, the external diameter of the eye continued to increase even after BL growth ceased (an observation that has not been described previously), and older fish had symptoms of cataractogenesis. All of these morphological changes could be used as ageing markers in this or related species. The lipidic prolife of N. korthausae varies significantly with age. In the three studied stages of its life cycle (undifferentiated, adult and senescent), significant amounts of the most unsaturated HUFAs docosapentaenoic acid (22:5n−3) and DHA (22:6n−3) were measured. The sum of both docosapentaenoic acid and DHA represents over 20% of the total fatty acid content of the animal in the undifferentiated and adult stages (28.84 and 23.64%, respectively). This observation points to the ability of this species to convert diet fatty acids into ones with a higher number of double bonds and longer chains from the undifferentiated stage onwards, as has been observed in the fry of other fish species (Sargent et al., 2002). Moreover, these results illustrate the high unsaturation level of lipids in N. korthausae, which has been suggested to play an important role in lifespan determination (Sanz et al., 2006; Hulbert et al., 2007). It has been observed that animal species with very polyunsaturated membranes have high metabolic rates, which prompts the high production of reactive oxygen species (ROS). The damage done to the constitutive organism biomolecules, particularly lipids composed of fatty acids with a high number of double bonds, is considerable and quickly accumulates, leading to accelerated ageing. These facts agree with the data obtained in N. rachovii by Hsu et al. (2008), in which lipidic peroxidation and protein oxidation increased with age. The susceptibility to lipid oxidative damage can be estimated by calculating the PI from the fatty acid composition (Hulbert et al., 2007). In this study, the Peroxidation Index decreased significantly in the senescent stage. This is probably due to general impairment of the lipids, represented by a substantial decrease in HUFA levels and an increase in the relative

amount of monounsaturated fatty acids such as oleic acid (18:1n −9, OA) and other saturated fatty acids like palmitic acid (16:0), both important elements of fish cellular membranes. Lipid profile changes with age have been observed in the hearts of rats, including a decline in the unsaturation index and DHA (Castelluccio et al., 1994), and in Daphnia, with a decrease in total HUFA (Barata et al., 2005). In the case of membrane phospholipids, a continual remodelling that replaces peroxidised fatty acids with fresh PUFA from the triglyceride pool has been proposed (Girón-Calle et al., 1997). Changes in fish locomotor activity rhythms are also considered to be a potential ageing marker. Different parameters can be measured by tracking fish movements, allowing for an accurate characterisation of their circadian system status. One of these parameters is the mesor (mean value of a rhythm, in this case, mean locomotor activity). A decrease in this parameter might reflect ageing; such a decrease was observed in N. korthausae at 48 weeks and in 72-week-old fish. An age-dependent reduction in total activity was described in N. furzeri (Valenzano et al., 2006a) and zebrafish (Zhdanova et al., 2008). However, the most significant characteristics of circadian system impairment are a decrease in rhythm amplitude and an increase in rhythm fragmentation (Weinert and Schuh, 1988; Scarbrough et al., 1997). Both symptoms were observed in 72-week-old N. korthausae. An age-related reduction in the daily amplitude of activity has also been described in zebrafish (Zhdanova et al., 2008). The main functions of the circadian system are the internal temporal order of the animal and the ability to anticipate the environmental changes that are repeated on a daily basis. Such anticipation determines the proper use of these events; for example, when feeding time is at the same hour every day. Feeding anticipatory activity (FAA) was observed in 24- and 48-week-old animals but not in 72week-old animals. FAA has been observed in other species, such as goldfish, which also showed an increase in digestive enzyme activity prior to feeding (Vera et al., 2007). Therefore, the loss of FAA could be another marker of age-related circadian system impairment. The non-parametric indices of circadian system analysis Intradaily Variability (IV), Interdaily Stability (IS) and Relative Amplitude (RA), have been used to identify differences between healthy and

A. Lucas-Sánchez et al. / Experimental Gerontology 46 (2011) 970–978

Fatty acid⁎

Undifferentiated

Adult

Senescent

14:0 15:0 16:0 18:0 20:0 21:0 22:0 23:0 24:0 ∑ Saturated 15:1n−5 16:1n−7 17:1n−7 18:1n−9 18:1n−7 20:1n−9 24:1 n−9 ∑ Monounsaturated 18:2n−6 18:3n−6 18:3n−3 18:4n−3 20:2n−6 20:3n−6 20:4n−6 20:5n−3 22:4n−6 22:5n−3 22:6n−3 ∑ Polyunsaturated ∑ n−3 ∑ n−6 ∑ HUFA PI

0.68 ± 0.36a 0.06 ± 0.15a 9.58 ± 1.30a 7.73 ± 1.08a 4.99 ± 0.41b 4.46 ± 0.38 3.30 ± 1.62b 2.30 ± 1.83b 1.74 ± 1.91b 34.85 ± 2.09 1.48 ± 0.78b 4.03 ± 0.31a 4.39 ± 0.25c 5.98 ± 0.75a 2.34 ± 2.14a – 10.60 ± 2.45c 29.46 ± 2.98b 1.34 ± 0.18a 0.06 ± 0.14a – – – 0.31 ± 0.48 2.67 ± 2.41 – – 25.36 ± 2.06c 3.48 ± 0.41a 35.69 ± 3.31b 28.84 ± 2.34b 4.37 ± 2.28a 31.83 ± 2.22b 196.00 ± 12.04b

2.00 ± 0.77b 0.54 ± 0.11b 18.57 ± 2.09b 15.28 ± 3.41b 0.80 ± 0.16a – 0.76 ± 0.16a – 1.07 ± 0.31ª.b 39.02 ± 5.86 – 4.99 ± 1.79a 0.25 ± 0.16a 10.25 ± 1.37b 3.68 ± 0.58a 0.57 ± 0.25 0.62 ± 0.16b 20.73 ± 2.93a 6.16 ± 0.44b 0.36 ± 0.22b 1.44 ± 0.82b 0.47 ± 0.50 0.95 ± 0.99b 0.28 ± 0.25 3.47 ± 0.73 1.85 ± 0.69 0.77 ± 0.37b 2.93 ± 1.00b 20.71 ± 1.62c 40.25 ± 4.30b 28.05 ± 3.29b 11.99 ± 1.53b 30.65 ± 2.50b 226.51 ± 19.65c

3.23 ± 0.37c 0.82 ± 0.09c 24.00 ± 2.97c 7.13 ± 0.78a 0.46 ± 0.07a – 0.28 ± 0.11a 0.17 ± 0.01a – 36.08 ± 2.46 0.20 ± 0.17a 14.10 ± 1.53b 0.55 ± 0.10b 14.89 ± 1.81c 6.58 ± 1.06b 0.45 ± 0.46 0.26 ± 0.11a 37.51 ± 1.78c 6.55 ± 1.54b 1.49 ± 0.41b 0.58 ± 0.18a 0.17 ± 0.07 0.24 ± 0.07ª.b 0.50 ± 0.14 3.95 ± 0.71 1.29 ± 0.49 1.68 ± 0.65c 0.71 ± 0.15a 8.96 ± 1.71b 26.41 ± 2.24a 11.82 ± 2.12a 14.41 ± 1.49b 17.20 ± 3.37a 120.17 ± 19.14a

A

50 45

24-week-old

40

48-week-old

35

72-week-old

30 25 20 15 10 5 0

2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 18:00 19:00 20:00 21:00 22:00 23:00 0:00 1:00

Table 1 Fatty acid profile of N. korthausae from three different stages of their life-cycle (% of total fatty acids).

Counts/10min

976

Hour of the day

B

24-week-old

48-week-old

72-week-old

C

Undifferentiated: 2 weeks old. Adults: 20 weeks old. Senescent: more than 64 weeks old. Data represent the mean ± standard deviation (n = 6). Values on the same line with different superscripts are significantly different and were obtained with a Tukey-b post hoc for multiple comparisons test (P b 0.05). Peroxidation Index, PI = 0.025 × (% monoenoics)+ 1 × (% dienoics) + 2 × (% trienoics)+ 4 × (% tetraenoics) + 6 × (% pentaenoics) + 8 × (% hexaenoics). ⁎ Some minor fatty acids (b 0.1 g/100 g of fatty acids) are not shown.

unhealthy humans (Van Someren et al., 1999; Skjerve et al., 2004; Dowling et al., 2008) and rodents (Lax et al., 2011). We decided to apply this analysis to fish locomotor activity for the first time to test its applicability for characterising variations in circadian system status with age. IS is related to the capability of the individual to synchronise with different zeitgebers (mainly the light–dark cycle). Thus, the low values obtained for this index for fish in the senescent stage could be due to the impairment of input pathways, such as the visual system, or the central biological clock. It has been suggested that ocular alterations may have this effect (Drouyer et al., 2008; Lax et al., 2011) and may also increase rhythm fragmentation, as reflected by the high IV values recorded. Age-dependent retina degeneration associated to oxidative stress has been described in zebrafish (Kishi et al., 2008). Another visual alteration, cataractogenesis, has been attributed to age-related changes in the lipid profile induced by an oxidative status imbalance in salmon (Toivonen et al., 2004). In the present work, cataractogenesis was observed in senescent N. korthausae individuals; its development could be a reason for synchronisation to be lost and fragmentation increased. However, fish also have non-visual photoreceptors as input pathways (deep-brain, pineal, dermal cells) and peripheral oscillators in tissues such as the heart and kidney that can be light-entrained in vitro (Whitmore et al., 1998, 2000; Peirson et al., 2009). All these form a complex network of central and peripheral clocks that are well-coordinated to maintain physiological

Fig. 6. Locomotor activity rhythms of Nothobranchius korthausae in different stages of their life cycle. The mean waveform for 24-, 48- and 72-week-old fish (6 animals per group) is represented as the mean ± SD (A). The bar above the mean waveforms represents the photoperiod. The broken line indicates feeding time. Fig. 6(B) represents the double-plotted actograms and (C) the Rayleigh Test for 24-, 48- and 72-week-old fish (6 animals per group). In both, the dark band represents the scotophase. The direction of the Rayleigh Test module indicates the acrophase, and its length is a rhythm stability value.

Table 2 Locomotor activity rhythm of N. korthausae in three different stages of their life cycle.

Mesor Diurnal Activity (DA) Nocturnal Activity (NA) Rayleigh Test (r) Circadian/Ultradian Ratio Interdaily Stability (IS) Intradaily Variability (IV) Relative Amplitude (RA) Circadian Function Index (CFI)

24-week-old

48-week-old

72-week-old

21.8 36.0 ± 5.1 [82.6] 7.6 ± 3.0 [17.4] 0.98 86.95 0.36 0.80 0.72 0.56

6.3 10.0 ± 5.0 [79.4] 2.6 ± 1.7 [20.6] 0.94 59.62 0.28 1.33 0.81 0.47

3.6 4.2 ± 1.8 [57.2] 3.2 ± 1.2 [42.8] 0.47 31.77 0.16 1.51 0.34 0.25

Mesor, DA and NA are presented in counts/10 min (mean and mean ± standard deviation, respectively) and percentage between brackets. r, IS, RA and CFI range values are between 0 and 1. IV between 0 and 2. Circadian/Ultradian Ratio = % of variance explained by the first harmonic/Σ % variance explained by harmonics 1 to 6. CFI = (IS + (2 − IV)/2 + RA) / 3.

A. Lucas-Sánchez et al. / Experimental Gerontology 46 (2011) 970–978

977

Table 3 Habitat characteristics and lifespan of species of genus Nothobranchius. Species

Annual rainfall

Habitat type

Mean lifespan (weeks)

Maximum lifespan (weeks)

References

N. N. N. N. N. N.

326 mma 400 mmb 1000 mmb 1200 mmb 846 mma 1877 mmc

Semi-arid Semi-arid Sub-humid Humid Humid Humid

9 20 24 34 64 57

13 26 32 37 112 81

Valdesalici and Cellerino (2003) Terzibasi et al. (2008) Terzibasi et al. (2008) Herrera and Jagadeeswaran (2004) Markofsky and Perlmutter (1972) Present work

a b c

furzeri (GRZ strain) fuzeri (MZM-04/10 strain) furzeri (MZM-04/03 strain) rachovii guentheri korthausae

Data obtained from www.worldweather.org. Data obtained from Polacik et al. (2011). Data obtained from New et al. (2000).

integrity. But if central clock activity (the pineal gland in fish) is altered with ageing (Reiter, 1995; Zhdanova and Reebs, 2006) and melatonin circadian signals decrease, then peripheral clocks can oscillate independently, leading to a fall in coordination that may alter the integrity of physiological functions. The CFI, which integrates IS, IV and RA, is a recently-proposed highly accurate index of the overall circadian system status (OrtizTudela et al., 2010). CFI values decreased with age (0.56 at 24weeks-old, 0.47 at 48 weeks old and 0.25 at 72 weeks old) in the present work, pointing once again to circadian system impairment during N. korthausae ageing. Twenty-four-week-old fish showed a robust, typically diurnal rhythm that deteriorated with age and lost regularity and amplitude with a marked fragmentation of locomotor activity. Something similar has been described in mammals (Weinert and Schuh, 1988; Scarbrough et al., 1997). Thus, these indices (IS, IV, RA, CFI) act as good markers for characterising ageing in animal models and for testing the effect of treatments that affect the circadian system. Taking all the discussed data into consideration, the loss in locomotor activity may be explained by the accumulated damage to cell macromolecules with age, in which lipids play an important role. The damage to any element in the circadian system will be reflected in alterations in both the genesis and maintenance of rhythms. Moreover, the oxidative status of the cell may in itself be a key factor in the circadian activity of the central system oscillators (Rutter et al., 2001; Cardona, 2004). Thus, variations in cellular oxidative status generated by increased ROS production could be the direct cause for alterations in the locomotor activity rhythm. Although important, the role of fatty acids (especially DHA) in behavioural processes is still unclear. In a review of the role of omega-3 fatty acids in rodent behaviour, Fedorova and Salem (2006) suggested that DHA is strongly retained by the adult mammalian brain where it seems to have many important physiological functions. The presence of HUFA in cell membranes ensures fluidity and a high speed of neuronal transmission (Levant et al., 2004). In rats, it has been observed that an n−3 PUFA and DHA imbalance in the brain produces variations in locomotor activity (Enslen et al., 1991; Levant et al., 2006; Vancassel et al., 2007). The decrease in n−3 PUFA observed in the senescent stage of N. korthausae is accompanied by a decrease in total locomotor activity (from 21.8 to 3.6 counts/10 min), but more research is needed to reveal the nature of the link between the two observations. In summary, the phenotypic manifestations of ageing in N. korthausae were tracked at different levels using a multitude of markers (morphological, behavioural, and biochemical). Morphological manifestations of ageing such as an increased incidence of unrepaired tail damage, emaciation, and reduction in fertility were easily recognisable. Lipid profiles and locomotor activity rhythms also changed in a manner consistent with ageing, and thus constitute potential ageing biomarkers. N. korthausae was found to have a lifespan longer than that of other species in the genus, such as N. furzeri. In addition, N. korthausae can be easily maintained and reproduced in the laboratory.

At a general level, the genus Nothobranchius contains species with very different lifespans. Further research is necessary to understand the multiple mechanisms involved in ageing and their cause–effect relationships; as vertebrate models for ageing studies, Nothobranchius species may be useful in this effort. Supplementary materials related to this article can be found online at doi:10.1016/j.exger.2011.08.009.

Acknowledgements This project was funded by the Seneca Foundation (12005/PI/09), the Instituto de Salud Carlos III (RETICEF, RD06/0013/0019) and the Ministerio de Educación y Ciencia (BFU2007-60658/BFI). Thanks to José Ramón Tauste, aquarium fish expert and to Antonio Martinez for helping with the data analysis. Thanks also to Juan Pedro Montávez for assistance with researching climatology data about Mafia Island.

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