Sticklebacks From ‘trash fish’ to supermodel: the rise and rise of the threespined stickleback in evolution and ecology Sticklebacks are often described as ‘trash ish’ in managed isheries where they are often removed in large numbers to reduce competition with target species. However, in the journal Nature, Gibson (2005) described the three-spined stickleback as a ‘supermodel’ for evolutionary biology. “Y ou’re joking, right? You can’t be serious!” – the usual response upon hearing of an international conference devoted to sticklebacks. Why all the fuss about these seemingly insigniicant little ish, generally considered of interest only to children with hand nets and jam jars? Sure, many biolo- gists will be aware of Nico Tinbergen’s Nobel prize-winning experimental behavioural research, much of which was carried out on sticklebacks, but that was over 50 years ago. Iain Barber and Swati Nettleship University of Leicester, UK Title image: Three-spined stickleback (Gasterosteus aculeatus) Close-up of male. Photo: Winfried Wisniewski/FLPA Biologist Vol 57 No 1 February 2010 15 Sticklebacks What could we still wish to know about the humble ‘prickleback’, ‘baggie minnow’ or ‘red doctor’ (some of the many vernacular names for the three-spined stickleback, Gasterosteus aculeatus; Figure 1), and how can they inform modern biology? Well, since the irst international stickleback conference in 1984, which focussed almost solely on behavioural studies, the way that biologists use sticklebacks in research has developed considerably, and the number of ields that have embraced the utility of this little ish has exploded. As a result, the ish has experienced an astounding rise in its proile and is now irmly established as a biological ‘supermodel’ (Gibson, 2005). In July 2009, twenty-ive years after the irst stickleback meeting, over 100 delegates from 16 countries attended the sixth meeting of the now triennial International Conference on Stickleback Behaviour and Evolution at the University of Leicester (Figure 2). With symposium themes including ‘Adaptation and phylogeny’, ‘Behaviour’, ‘Evolution and development’, ‘Responses to anthropogenic disturbances’, ‘Paleobiology’ and ‘Photoperiodism’, the far-reaching applications of this small, ubiquitous ish begin to become apparent. In this article we hope to explain the reasons for the popularity of the stickleback in research, and highlight some of the most fascinating recent advances. Invaders from the sea The sticklebacks (Gasterosteidae) are a family of small teleost ishes with ive genera and up to 16 species currently recognised on Fish- Figure 1. Main photograph: a male three-spined stickleback, Gasterosteus aculeatus, resplendent in full breeding coloration, tends his nest. Inset: a gravid female threespined stickleback displays the ‘head up’ posture, a characteristic pre-spawning signal (Photos: Iain Barber) 16 Biologist Vol 57 No 1 February 2010 Figure 2. Main photograph: Over 100 delegates attended the 6th International Conference on Stickleback Behaviour and Evolution at the University of Leicester in July 2009 (Photo: Paul J B Hart). Inset: the conference logo. Base (www.ishbase.org). In the UK there are three recognised species: the three-spined stickleback Gasterosteus aculeatus and the nine-spined stickleback Pungitius pungitius can be found in freshwater, saltwater or brackish waters, whereas the ifteen-spined stickleback Spinachia spinachia is purely marine. North America boasts three more species; the brook stickleback Culaea inconstans, the four-spined stickleback Apeltes quadracus and the black-spotted stickleback Gasterosteus wheatlandi, with the remaining species being described from Europe and Asia (Wootton, 1976). Of these species, it is the three-spined stickleback that has risen to prominence as a research model (Östlund -Nilsson et al, 2006). In a nutshell, the great utility of threespined sticklebacks as models in biological research hinges on two key attributes; their suitability for laboratory study and their extensive intra-speciic variation. Sticklebacks can be bred and reared to adult size in modestlysized aquaria that nonetheless permit their full behavioural repertoire to be exhibited, greatly facilitating their use as experimental models in behavioural research. However, the value of the three-spined stickleback as a model species in evolution and ecology lies in the natural intra-speciic variability of freshwater populations (Bell and Foster, 1994). Many readers will cherish childhood memories of catching sticklebacks in streams and ponds (Figure 3). However the ocean is the ancestral environment of three-spined sticklebacks, and large populations of purely marine three-spined sticklebacks (which tend to be larger and more heavily armoured than freshwater counterparts) still exist in the coastal waters of the Northern hemisphere. Inland populations in Northern Europe were only founded 8,000-10,000 years ago, when marine forms invaded newly created freshwater habitats as glacial ice sheets retreated. Their Sticklebacks Figure 3. A ‘typical’ British stickleback lake: Llyn Frongoch in mid-Wales (Photo: Phil Bennett). Inset: a good catch of three-spined sticklebacks sampled using a wire mesh minnow trap. (Photo: Iain Barber) subsequent evolution in response to selection pressures – different in each newly colonised habitat – has driven an extraordinary adaptive radiation (Figure 4). As a result, freshwater three-spined sticklebacks show remarkable variation in almost every conceivable trait, from jaw morphology to anti-predator behaviour. This extraordinary diversity provides evolutionary biologists with a rich testing ground for examining the genetic and ecological basis of trait adaptations, and the Darwinian itness consequences of phenotypic variation. populations remain enigmatic, a number of factors are likely to have been important. In fresh water ecosystems, piscivorous ish – against which armour is an effective deterrent – are at least partly replaced by birds and Figure 4. An example of an adaptive radiation of sticklebacks from the Moray Firth region of Scotland. (a) Marine three-spined stickleback from coastal salt marsh. (b) Freshwater sticklebacks from inland lochs around the Moray Firth. Note the general smaller size and less bulky appearance of freshwater forms, and the diversity among them of traits such as tail stalk length, body depth, spine length, head and eye size and jaw morphology. (Photos: S A Arnott and I Barber). Quick-change artists One of the most striking aspects of stickleback morphology is the extensive external armour that some ish exhibit. Unusually for teleost ish, sticklebacks lack scales; instead, bony lateral plates – and the spines that give them their name – protect their bodies. The dorsal and ventral spines are linked by a bony ascending process that effectively completes a ‘pectoral girdle’ around the central part of the ish’s body, providing protection against many predators. The marine types are more or less uniform in appearance across the globe, with a full complement of 30-35 plates running the entire length of their body and well-developed spines (see Figure 5a). Freshwater populations, however, are far more variable, with both the number of lateral plates and the development of spines often being considerably reduced (Figure 5b). Although the precise evolutionary mechanisms favouring armour loss in freshwater Biologist Vol 57 No 1 February 2010 17 Sticklebacks Figure 5. Cleared and stained specimens of (a) marine and (b) freshwater three-spined stickleback, showing the characteristic reduction in lateral plate number, dorsal and pelvic spine size and altered head morphology of the derived freshwater form. (Photos: Iain Barber) aquatic invertebrates as major predators of sticklebacks, reducing the anti-predator value of armour. Furthermore, in contrast to the open habitats occupied by marine populations, freshwater habitats are typically more structurally complex and extensively vegetated, and so restrictive armour may have been abandoned in favour of the greater manoeuvrability and escape performance conferred by plate loss. Freshwaters also offer a reduced bioavailability of calcium ions, meaning that the energetic costs of ossiication may be dramatically increased when ish invade freshwaters from the sea. Recent work also suggests that pleiotropic effects of alleles for high lateral plate number lead to reduced growth, placing heavily armoured individuals at a disadvantage Figure 6. Fossil Miocene Gasterosteus doryssus recovered from a shale quarry in Nevada, USA. The three dorsal spines and a pelvic spine are clearly visible (arrows). (Photo: Iain Barber). 18 Biologist Vol 57 No 1 February 2010 in freshwater populations. Temporal changes in anti-predator morphology are evident from a remarkable series of fossil Miocene sticklebacks (Gasterosteus doryssus) recovered from shale deposits in Nevada. Well-preserved fossil sticklebacks are abundant in the rock, laid down around 10 million years ago (Figure 6). Morphological and stratigraphic analysis of this fossil series has revealed that, over a period of 100,000 years, both the number of dorsal spines and the extent of pectoral girdle development changed substantially, suggesting evolved responses to changes in predation pressure. However, population level changes in stickleback morphology can occur much more rapidly than this, and two recent examples provide rare evidence of ‘evolution in action’. In the Aleutian Islands, coastal lagoons formed following tectonic uplift during the great Alaska earthquake of 1964 have since become freshwater lakes, and marine sticklebacks trapped in these bodies now exhibit freshwater-like morphology (Gelmond et al, 2009). Similarly, when fully plated ish of marine origin colonised an Alaskan lake previously cleared of resident sticklebacks, freshwater-like armour and foraging morphology evolved after just 12 years – i.e. about six generations (Bell et al, 2004). The speed of these morphological changes suggests that despite their phenotypic uniformity, marine populations retain suficient genetic diversity to diverge rapidly. This inbuilt ‘evolvability’ may be one of the main reasons why sticklebacks have been so successful in repeatedly colonising freshwater habitats from marine environments, and the basis of this ability now forms a major focus of research. Do sticklebacks have personalities? Much of what we know about aggression, Sticklebacks learning, territoriality and courtship in animals has been informed by pioneering studies of sticklebacks by Tinbergen’s group, and sticklebacks are now used routinely as a model to address the most pressing questions in behavioural biology. Over the past few years, stickleback studies have contributed signiicantly to our understanding of the role of animal ‘personality’ in evolutionary ecology. Those who spend time watching animals know intuitively that individuals often differ consistently and predictably in their behaviour; for example, aggressive individuals may also be the ones that are quick to explore novel objects or habitat types. Variation in contextually different behaviours can therefore be related in a behavioural ‘syndrome’, and there has been a realisation among behavioural scientists that the existence of these syndromes – and variation in their structure – is likely to have important implications for species ecology and evolution. This has sparked an upsurge in research activity, and stickleback studies have been at the forefront. Interestingly the personality of sticklebacks also appears to have been shaped by the presence of predators. Sticklebacks from stillwater populations on the island of Anglesey in Wales exhibited a range of behavioural syndromes, but the often documented syndrome between exploratory behaviour, activity and aggression was only found among individuals from populations inhabiting ponds with a history of evolutionary interaction with ish-eating predators (Dingemanse et al, 2007). When predation pressure is relaxed, it seems that a wider range of behavioural strategies appears possible. Host-parasite interactions Sticklebacks will eat almost anything, and in turn almost everything eats them. This gives sticklebacks a pivotal role in aquatic food webs, and as a result they are often infected with a wide variety of endoparasites. Add to this the diversity of aquatic habitat types occupied by three-spined sticklebacks, as well as their wide geographical spread, and it is perhaps not surprising that a recent review listed over 150 species of stickleback parasites. One of these parasites has been particularly well studied. In its adult form, Schistocephalus solidus is a tapeworm living in the intestine of ish-eating birds, but the larval stage undergoes most of its growth in the body cavity of a three-spined stickleback. Individual ish can harbour a number of these worms and in some cases their combined mass can exceed that of the ish! Infected ish exhibit a range of changes that appear to increase the likelihood that they will end up as a meal for a ish-eating bird – exactly what is needed for the parasite to complete its life cycle. For example, infected ish show markedly reduced escape behaviour in response to overhead stimuli, and in some populations infection is associated with the complete loss of dorsal pigment, rendering the ish ghostly-white and highly conspicuous to aerial predators (Figure 7). The stickleback-Schistocephalus system has become important in parasitology because both ish and parasites can be bred under lab conditions, making experimental infections of previously naïve ish possible (Barber and Scharsack, 2010). This has allowed recent studies to demonstrate that the level of allelic diversity in the major histocompatibility complex (the MHC, a region of the genome known to play an important role in the immune system) is associated with differential protection against infection. What is more, experimental trials have shown that female sticklebacks can ‘sniff out’ males that have complementary MHC alleles to their own and show odour-based preferences for males that would provide maximum genetic protection for Figure 7. Plerocercoid larvae of the tapeworm Schistocephlaus solidus are large and important parasites of threespined sticklebacks. (a) Four plerocercoids recovered from the body cavity of a heavily-infected ish. (b) Loss of skin pigment among infected sticklebacks from Wolf Lake, Alaska, USA. (Photos: Iain Barber). Biologist Vol 57 No 1 February 2010 19 Sticklebacks Figure 8. How many biologists have been enthused and inluenced by childhood experiences such as catching their irst stickleback? (Photos: Iain Barber.) their own offspring against parasites (Milinski, 2006). Sticklebacks in a changing world For many years, ish have served as aquatic ‘canaries’ in ecotoxicological studies designed to provide safe release guidelines for harmful chemicals and industrial efluents. However, none of the traditional model species – fathead minnows, medaka and zebraish – is a European native, so extrapolating the results of laboratory studies to natural exposure situations in the UK is challenging. Furthermore, none of these species has known genetic sex-determination systems, creating problems when trying to assess the effects of endocrine disrupting chemical pollutants on sexual development. As a result the three-spined stickleback, which has well understood genetic sex determination, is beginning to rival traditional species as the model of choice in ecotoxicological studies in Europe. Its wide environmental tolerances mean that the effects of both marine and freshwater pollutants can be assessed under a range of biotic and abiotic environmental conditions. Sticklebacks also have complex yet well understood reproductive behaviours that may be more sensitive to disruption by pollutants, and readily-assayed physiological endpoints of both male and female sexual maturation (the nesting glue spiggin and the egg yolk precursor protein vitellogenin respectively). Sticklebacks are also making their mark as model organisms for studying the ecological effects of eutrophication. In recent years a large body of work by researchers in Finland has shown that nutrient enrichment of the Baltic Sea is having a signiicant impact on many 20 Biologist Vol 57 No 1 February 2010 aspects of stickleback reproductive behaviour. The increased algal growth associated with eutrophication alters social interactions between males and allows a higher proportion of males to defend territories and construct nests, including parasitised individuals and those in poor condition that would otherwise not be able to nest. Females looking for a male to mate with are also less discriminating of male quality in eutrophic conditions, perhaps because of reduced sensory capacity or because of increased costs associated with mate sampling. Normal processes of sexual selection resulting from male-male competition and female mate choice are therefore relaxed in eutrophic conditions, and this may have signiicant implications for individual itness and the long-term viability of populations (Wong et al, 2007). The genomic revolution In 2001, a signiicant milestone in stickleback research was reached when researchers at Stanford University published a genome-wide linkage map for the three-spined stickleback (Peichel et al, 2001). This was followed, in 2006, by the publication of the full genome sequence (www.ensembl.org/Gasterosteus_ aculeatus/index.html). This has opened the loodgates of research into the genetic basis of intra-speciic diversity. Most of the stickleback genome has a base coverage of approximately 11x, providing an invaluable tool for identifying and evaluating candidate genes for phenotypic traits, particularly when used in combination with other sequenced teleost genomes (i.e. medaka and zebraish). It may come as a surprise to learn that advances in the understanding of stickleback genetics even have the potential to inform medical research on human health and disease, yet a number of genes have recently been discovered that control analogous developmental processes in sticklebacks and humans. For example, the gene Pitx1 controls pelvic structure variation in sticklebacks and is also implicated in hind limb development in mice and humans, the gene Eda encodes the signalling molecule ectodermal dysplasin and is responsible for lateral plate number in sticklebacks and the development of hair and teeth in human embryos and the Kit ligand gene, which contributes to the natural variation in stickleback pigmentation, also has a signiicant effect on human skin colour (Miller et al, 2007, Shapiro et al, 2006). Clearly there is enormous potential for studying how the expression of these genes is regulated in the stickleback, with considerable potential to inform human biomedical sciences. Sticklebacks Where next for the stickleback model? Making predictions about the likely direction of research is notoriously dificult, but the fact that the next international meeting will be held at the renowned Fred Hutchinson Cancer Research centre in Seattle shows how far sticklebacks have come in the past 25 years, and perhaps hints at the kind of questions that they may help tackle in the future. The use of next-generation, whole-genome sequencing technologies now provides a realistic and cost-effective technique for scoring tens of thousands of genetic markers in hundreds of individuals in just a few days to identify genes under active selection. This powerful approach means that by the time the stickleback research community meet again in Seattle in 2012, we will no doubt be able to link a large number of phenotypic traits to genes, and it seems likely there will be an increased interest from biomedical as well as life scientists. However, whilst many of the most groundbreaking advances will arise from the use of cutting edge technology, the key to the popularity of the stickleback model lies in its fascinating natural history. Finally, we must never underestimate the importance of sticklebacks and other native wildlife in sparking a lifetime of interest in biology (Figure 8). References Barber I and Scharsack J P (2010) The three-spined stickleback – Schistocephalus solidus system: an experimental model for investigating host-parasite interactions in ish. Parasitology, in press. Bell M A, Aguirre W E and Buck N J (2004) Twelve years of contemporary armor evolution in a threespine stickleback population. Evolution, 58, 814-824. Bell M A and Foster S A (Eds) (1994) The Evolutionary Biology of the Threespine Stickleback, Oxford, UK, Oxford University Press. Dingemanse N J, Wright J, Kazem A J N, Thomas D K, Hickling R and Dawnay N (2007) Behavioural syndromes differ predictably between 12 populations of stickleback. Journal of Animal Ecology, 76, 1128-1138. Gelmond O, von Hippel F A and Christy M S (2009) Rapid ecological speciation in three-spined stickleback Gasterosteus aculeatus from Middleton Island, Alaska: the roles of selection and geographic isolation. Journal of Fish Biology, 75, 2037 – 2051. Gibson G (2005) The synthesis and evolution of a supermodel. Science, 307, 1890-1891. Milinski, M. 2006. The major histocompatibility complex, sexual selection, and mate choice. Annual Review of Ecology Evolution and Systematics, 37, 159-186. Miller C T, Beleza S, Pollen A A, Schluter D, Kittles R A, Shriver M D and Kingsley D M (2007) cis-regulatory changes in kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans. Cell, 131, 1179-1189. Östlund-Nilsson S, Mayer I and Huntingford F A (2006) Biology of the three-spined stickleback, Boca Raton, FL, CRC Press. Peichel C L, Nereng K S, Ohgi K A, Cole B L E, Colosimo P F, Buerkle C A, Schulter D and Kingsley D M (2001) The genetic architecture of divergence between threespine stickleback species. Nature, 414, 901-905. Shapiro M D, Bell M A and Kingsley D M (2006) Parallel genetic origins of pelvic reduction in vertebrates. Proceedings of the National Academy of Sciences of the United States of America, 103, 13753-13758. Wong B B M, Candolin U and Lindstrom K (2007) Environmental deterioration compromises socially enforced signals of male quality in three-spined sticklebacks. American Naturalist, 170, 184-189. Wootton R J (1976) The biology of the sticklebacks. London, Academic Press. Iain Barber is Senior Lecturer in Animal Biology at the University of Leicester. Email: ib50@le.ac.uk Swati Nettleship is a NERC-funded PhD student in the Department of Biology at the University of Leicester. Society of Biology Photo competition Ready, aim, inspire! The UN has declared 2010 ‘International Year of Biodiversity’. To mark and celebrate this, the Society of Biology is organising a photographic competition with a top prize of £1,000. Visit www.societyofbiology.org for full details, including deadlines and rules. Photo: Bill Parry Biologist Vol 57 No 1 February 2010 21