My Favorite System

The long-lived clam Arctica islandica, a new model species for ageing research

Ridgway I, Richardson, C.A., Scourse, J.D., Wanamaker, A.D. Jr., & Butler, P.

School of Ocean Sciences, College of Natural Sciences, Bangor University

Introduction

The Ocean Quahog, Arctica islandica (Linnaeus 1767), is the oldest non-colonial animal known to science, attaining an age in excess of 400 years [1]. Funded by Research into Ageing^TM we seek to establish A. islandica as a new model ageing species.

Arctica islandica

Also known as the Iceland Cyprina, Ocean Quahog and Mahogany Clam, A. islandica is a long-lived suspension feeding bivalve mollusc. It lives burrowed into the top 5cm of sand and muddy substrates (figure 1) in suitable habitats around the shelf seas of North European and North American continents, ranging from the Bay of Biscay in the south to the sub-Arctic waters off Iceland, commonly at depths between 25 & 80 m. The species is dioecious (separate sexes), with larval development taking between 30 to 60 days depending on seawater temperature, and sexual maturity is reached at between 7 and 13 years of age. 

Figure 1.  View of Arctica islandica buried just below and also on the sediment surface.

The shells of A. islandica contain an ontogenetic record of shell growth in the form of wide annual summer growth increments separated by narrow growth lines.  Counting the number of lines gives an accurate estimate of the age of the clams and measurement of increment width an estimate of inter-annual growth rate (see figure 2). The discovery of what appeared to be an extremely old, long-lived A. islandica clam in the 1980s, far older than was thought previously possible, was the stimulus to investigate the periodicity of the increments. On the basis of seasonal changes in stable oxygen isotope profiles [2] and from field mark-recapture experiments [3] it was shown that the increments were deposited annually. Specimens with ages of between 100 and 200 years old are documented in the literature with dead collected northern North Sea and Icelandic specimens attaining ages of 268 and 374 years old respectively [7, 4]. However it was not until relatively recently that the documented maximum lifespan of A. islandica increased dramatically, from 268 years in 2003 [5, 7] to 374 years in 2005 [4], with the latest oldest reported specimen being at least 405 years old [1].  It is still to be demonstrated if these exceptionally old clams demonstrate evidence of physiological ageing, except for the accumulation of age pigment [6].

The anatomy, behaviour, physiology and more recently the ecology of A. islandica have been extensively studied because of their commercial importance along the American east coast; 150,000 tonnes are collected globally each year, principally by hydraulic clam dredges. This exceptionally long-lived clam has been termed the ‘tree of the sea' as the growth increment series measured from the shells could be used retrospectively to reconstruct marine environmental change. The School of Ocean Sciences, Bangor University is a world leader in the field and has been working toward constructing a 1000-year master chronology for the marine environment using the growth increment series in A. islandica shells [1, 7].

Figure 2. Idealized preparation of an A. islandica shell for sclerochronological studies (from Scourse et al., 2006). (A) External valve face, showing line of section through the left valve used to generate acetate peel replicas. (B) Acetate peel replica cross-section of valve showing annual growth band increments along shell margin (arrows) and within the hinge plate. The axis of growth through the hinge plate used to generate increment data is indicated by the dashed white line. (C) Numbered annual growth bands are measured from the earliest growth bands to the most recent along the axis of growth. Juvenile early bands are wide and reflect the ontogenetic growth curve of the individual. (D) Narrow senescent late bands from the outer part of the hinge plate axis.

What is known about ageing in A. islandica?

Despite interest in this clam's longevity and the measurement of growth increment series, little research into how this species has apparently managed to defy the onset of the ageing processes has been conducted. Earlier studies into the responses to stress with age were undertaken from a bio‑monitoring/eco‑toxicology angle, however in the past 5 years there has been an increasing number of papers documenting age associated trends in bivalves, normally focussing on the ‘free radical theory of ageing'. To date there has only been one publication on ageing in A. islandica which documented accumulation of lipofuscin with age, but showed no accumulation of protein carbonyl in the gill tissues [6]. Comparative biology of ageing in bivalves has demonstrated that better preservation of mitochondrial and antioxidant enzyme activities and the avoidance of waste accumulation may have enabled longer-lived individuals to live longer [8] than shorter lived species.  Shorter-lived bivalve species show a more pronounced decrease in mitochondrial function with ontogeny [9] and long-lived cold water dwelling species possess antioxidant activity that is higher than in shorter-lived species [10].

Why study Arctica islandica?

Significant advances in our understanding of the processes involved in ageing have been made using classical model organisms of biogerontological research (e.g. yeasts, fruit flies, nematodes and rodents). Despite the advantages of these organisms, where their basic biology and genome are well known, and their short life spans enable cheap and quick longitudinal studies and experimental manipulations to be conducted, they have been primarily chosen for convenience, rather than for specific features pertinent to human ageing [11]. Long-lived organisms may be more appropriate models to compare with human ageing as the most intensively used animal models used in biogerontological research lack the very trait needed to emulate longevity, i.e. the ability to live long and therefore they have poorly developed defences against the destructive processes of ageing [12].

This alternative approach investigates the nature of exceptionally effective defences against the destructive ageing processes that biological evolution has designed. If evolution has produced a model of successful resistance to the damage of ageing, it might be possible to learn from the investigation of that model [12]. Despite being identified as a potential area of study for ageing research over 70 years ago, the study of long-lived animals has advanced only recently. Comparative ageing research based on the longest-lived non-colonial animal known, Arctica islandica offers a unique opportunity to remedy this situation. The term "negligible senescence" has been coined by Caleb Finch at USC to describe very slow or negligible ageing [13]. He listed several animals with this characteristic, including vertebrates and invertebrates and specifically named A. islandica as a potential organism.

Of the organisms identified by Finch [13] as having slow or negligible ageing it is believed that A. islandica is the most suitable for establishment as a long-lived model organism for biogerontological research. All other organisms identified (e.g. sturgeons, bowhead whales, turtles, rockfish and possibly lobsters) are either aggressive, too large to maintain in captivity in aquaria or prohibitively expensive to study. These logistical problems associated with documenting exceptionally long-lived species have resulted in few ageing studies specifically focusing on long-lived animals.  A. islandica offers a unique opportunity to study a long-lived organism as it is possible to collect large numbers of individuals and to maintain them in laboratory aquaria where they are relatively cheap to grow and study. Clams are anatomically simple, but have a germ line/soma distinction, unlike hydra - another marine species documented with extreme longevity.

What do we aim to do?

Through a 12 month programme of research we aim to provide the basis for future ageing research in A. islandica. This is a collaborative project between marine biologists at Bangor University and biogerontologists from Brighton University, Cardiff University and University College London. A large thrust of the research will be to ascertain when the species demonstrates signs of physiological ageing; loss of organ structure/function, incidence of neoplasms, and decline in cell proliferation rates. Through field sampling and interrogation of a 10,000 A. islandica shell archive housed in the School of Ocean Sciences it is anticipated that accurate estimations of the species maximum lifespan potential (MLSP) can be obtained for discreet populations. Current estimations are based on single specimen finds rather than demographic analysis of the populations. Before research into A. islandica can proceed a few key questions need to be answered: 1) do they exhibit signs of physiological ageing? 2) Can we establish cell cultures from A. islandica tissues 3) Are there age associated changes in fecundity? and finally 4) what is the MLSP of the species?

Knowledge of the ageing process in this clam will be obtained from studies of age-related changes in the rate of cellular proliferation, the ability of the cells to culture and cellular resistance to stress and histopathology of organ structures. Establishing cell cultures from marine invertebrates has been problematic, but in the last 5 years there has been some success. In vitro applications are alternative and exceptionally important tools for animal experimentation, for biotechnological applications and pathological investigations [14]. The use of cell cultures also optimises the use of animals and allows ethical testing of resistance to stress.

Conclusion

In order for A. islandica to be established as a model for ageing research there is a need for more demographic data on wild clam populations and for some basic research to be conducted into its biology and cellular biology, and how these factors change with age. Holmes [15] concluded that the adoption of new and unusual animals for research exploring basic ageing processes need not be difficult as long as it involves careful collaborations between biogerontologists and zoologists well versed in evolutionary principles and judicious application of the comparative method. We believe this Research into Ageing^TM funded project, is a prime example of the kind of collaboration which will enhance our understanding of the ageing processes in invertebrates.

Figure 1.  View of Arctica islandica buried just below and also on the sediment surface.

Figure 2. Idealized preparation of an A. islandica shell for sclerochronological studies (from Scourse et al., 2006). (A) External valve face, showing line of section through the left valve used to generate acetate peel replicas. (B) Acetate peel replica cross-section of valve showing annual growth band increments along shell margin (arrows) and within the hinge plate. The axis of growth through the hinge plate used to generate increment data is indicated by the dashed white line. (C) Numbered annual growth bands are measured from the earliest growth bands to the most recent along the axis of growth. Juvenile early bands are wide and reflect the ontogenetic growth curve of the individual. (D) Narrow senescent late bands from the outer part of the hinge plate axis.

Francis RG Amrit  BSc (Hons), MSc

Elizabeth K Marsh  BSc (Hons)

Molecular Pathobiology

School of Biosciences

University of Birmingham

Introduction

The idea of spending the day looking down a microscope at a culture of worms is something that greatly amuses our friends; little do they know!  The organism we are referring to, the nematode Caenorhabditis elegans, which we employ as a model in our laboratory has truly revolutionised biological thinking.

The animal was first adapted as a laboratory model by Sydney Brenner over 40 years ago.  He selected this particular soil nematode to be a model system for a number of reasons, which are clear to all C. elegans biologists, indeed the practical advantages of the animals to the laboratory are endless (Brenner, 1974). 

Firstly, they have a short generation time of four days and a lifespan of approximately three weeks under laboratory conditions.  During this period the animals progress through four larval stages to the 1mm-long adult.  This generation time can be regulated by varying the incubation temperature, similar to regulating the reproduction of a bacterial culture!  However, there is an alternative to the L3 stage called the dauer stage.  Animals enter this state upon crowding or lack of food.  Here they are highly resistant to abiotic as well as to pathogenic stress, and they do not age (Riddle et al, 1997).

Figure 1 shows the nematode is transparent and stocks are easy to maintain: animals are cultivated in petri dishes on a modified agar substrate seeded with an Escherichia coli mutant as a food source.   

    (a)         

       (b)              

Figure 1(a) : A growing C. elegans culture: the population is dominated by self-fertilising hermaphrodites with a rare occurrence of males. 1(b) : A male worm with a distinctive sword shaped tail.

So, why do we study this animal?  Well, this simple multicellular organism shares a number of biological features and pathways with higher vertebrates.  Stringent studies on this animal therefore point towards key genes and pathways in these higher vertebrates and, having been identified, can be investigated further saving both time and money. 

In this way Brenner and his colleagues initially used C. elegans as an organism in which to study animal development and behaviour.  During this work, the nervous system was re-constructed, the entire somatic cell lineage was mapped out and the process of programmed cell death was characterised (Riddle et al, 1997).  Brenner was co-awarded the Nobel Prize for Medicine in 2002, along with Bob Horvitz and John Sulston, for these studies. The worm has since been awarded a second Nobel Prize in 2006 for the discovery of RNA interference (Fire et al, 1998).

Since then, C. elegans has been used as a model to study a wide-range of biological issues.  The organism has been so extensively studied that there is a vast amount of genotypic and phenotypic information available, and the model is genetically tractable.  The worm was the first eukaryotic organism to be sequenced (C. elegans Sequencing Consortium, 1998) and this project formed the basis of the human sequencing program which followed shortly afterwards.  The wealth of information that is accessible through online resources search as WormBase (http://www.wormbase.org/), has again contributed to the success of C. elegans as a model organism. 

In our laboratory we use C. elegans as a whole-organism approach to study the links between gender, immunity and abiotic stress and the subsequent impact these factors have on animal lifespan.

Ageing and Immunity in C. elegans

Ageing which is ironically, an age-old problem in biology is characterised by widespread degenerative changes and an increase in incidence of various age related pathologies or diseases, which include conditions such as cancer, diabetes and stroke. Rather than treating ageing to be an uncontrollable entity, a new wave of research using C. elegans as a model organism has set off trying to understand the mechanics of ageing and to look at it from a different perspective by treating ageing as a problem or a disease.

Calorie restriction was one of the first techniques that employed physiological means to slower the rate of ageing with the first experiments done in the late 1950's where lab mice were successfully made to live for about 40% longer using calorie restriction. But, recently genetic studies in this area have shown that there are molecular mechanisms that are conserved evolutionarily which govern ageing. By intervening with these genetically, it has been shown that the lifespan, immunity and stress resistance (pathogenic invasion, heat etc.) of model organisms like worms (Kenyon et al, 1993), flies (Clancy et al, 2001) and mice (Holzenberger et al, 2003) can be altered and are likely to have a similar effect on vertebrates such as humans. Both these mechanism ultimately result in a better immune and stress response, which also influences lifespan in a positive way.

Single gene mutations that increase lifespan, in some cases doubling it, were first identified in C. elegans in 1993 (Kenyon et al, 1993). Initially the idea of lifespan extension was controversial. It was a radical notion to many scientists who considered ageing as an uncontrolled process of deterioration that isn't controlled by gene regulation. But since the long lived nematode strain was proven to be a result of a mutation in the hormone controlled pathway of molecular signals, there have been over 70 genes identified in the past two decades that lengthen lifespan which is a testament of acceptance and tremendous progress in this field (Johnson, 2003).

Figure 2: A diagram depicting the various possible ways and techniques that can be employed to increase longevity.

The innate immune system which is evolutionarily conserved                                                                                                                                                                                                                             among all organisms responds immediately upon invasion by pathogens and also contributes towards the initiation of the acquired immune system that is unique to vertebrates. This appears to play the major role in immunity as a whole (Schulenburg et al, 2004). The highly similar features of the innate immune system across organisms suggest that it has a common origin and has been conserved over millions of years' of evolution. Non-vertebrates such as C. elegans can therefore be used to understand the immune system in higher vertebrates (Hasshoff et al, 2007; Schulenburg et al, 2004), which has already been shown by numerous studies on nematode bacterial interaction (Mahajan-Miklos et al, 1999). Molecular studies have helped in the elucidation of hormonal pathways controlling genes involved in antimicrobial actions and stress response that have been documented in C. elegans and these pathways appear to be at work in mammals as well. Mutations in similar hormonal driven pathways have resulted in lifespan extension in other organisms such as fruit flies and mice raising the prospect that this could slow ageing or enable to age with better health in humans too.

There are several known pathways that contribute towards the innate immunity of C. elegans  (Garsin et al, 2003; Kim et al, 2002; Mallo et al, 2002; Nicholas and Hodgkin, 2004; Schulenburg et al, 2004). Of these the evolutionarily conserved insulin like/IGF-1 signalling pathway is the most studied with it being shown to regulate several characters such as longevity and metabolism (Paradis and Ruvkun, 1998). This pathway is part of a global endocrine system that is triggered by hormones that resemble the hormone insulin in humans and controls whether animals grow reproductively or arrest at the dauer diapause stage (Finch and Ruvkun, 2001). Single gene mutations in this pathway have been shown to result in a large increase in mean lifespan of about 250 to 300% of the wild type depending on the mutation. The significance of such studies is the possibility to zoom in on a few similar genes as promising targets for drugs in humans that could hence result in a similar outcome.   

In 1997 (Kimura et al, 1997) the DNA sequence for the longevity causing mutation (DAF-2) was identified and to everyone's surprise this protein resembled the human cell surface proteins or receptors that respond to insulin and another hormone called insulin like Growth Factor (IGF-1). Also the downstream component DAF-16 (another longevity causing mutation) was identified and it turned out to encode a DNA binding protein that controls expression of its downstream target genes.

In our lab, we study two essential downstream components of the insulin like/IGF-1 signalling pathway, which we propose to be key determinants of immunity, stress resistance and ageing (May, 2007). One of them is the DAF-16 transcription factor which is considered as the "molecular link" that controls the transcription of a whole array of approximately a few hundred downstream genes. In addition we study the antimicrobial lysozyme 7 gene which is one of the many genes transcriptionally controlled by DAF-16.

In nematodes both lifespan and stress resistance have been shown to have gender specific and species specific variability hence suggesting that there is a common molecular mechanism or a "molecular link" which we propose as DAF-16. By establishing this "molecular link" we also plan to address the problem of post reproductive ageing as, if immunity is increased then that would also increase lifespan even after the animal has reproduced which cannot be an evolutionarily selected trait.

Worm research on ageing and immunity has had tremendous success in the recent past with outcomes that have great potential applications in humans.  Though there are a lot of questions left to answer, the resounding impact of the IGF-1 signalling pathway research, due to its evolutionary conservation across organisms is reassuring.

References

A. Fire, S.Q.X., M.K. Montgomery, S.A. Kostas, S. E. Driver, C.C. Mello 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 391:808-811.

Brenner, S. 1974. The Genetics of Caenorhabditis elegans. Genetics. 77.

C. elegans Sequencing Consortium, 1998. Genome sequence of the nematode C. elegans: a platform for investigating biology. Science. 282:2012-2018.

David J. Clancy, D.G., 1* Lawrence G. Harshman,2, H.S. Sean Oldham, 3 Ernst Hafen,3 Sally J. Leevers,4,5, and L. Partridge1. 2001. Extension of Life-Span by Loss of CHICO, a Drosophila Insulin Receptor Substrate Protein. Science. 292,:104.

Donald L. Riddle, T.B., Barbara J. Meyer and James R. Priess  1997. C. elegans II. Cold Spring Harbor Laboratory Press.

Finch, C.E., and G. Ruvkun. 2001. The Genetics of Aging. Annu. Rev. Genomics Hum. Genet. 2:435-462.

Garsin, D.A., J.M. Villanueva, J. Begun, D.H. Kim, C.D. Sifri, S.B. Calderwood, G. Ruvkun, and F.M. Ausubel. 2003. Long-Lived C. elegans daf-2 Mutants Are Resistant to Bacterial Pathogens. Science. 300:1921-.

Hasshoff, M., C. Bohnisch, D. Tonn, B. Hasert, and H. Schulenburg. 2007. The role of Caenorhabditis elegans insulin-like signaling in the behavioral avoidance of pathogenic Bacillus thuringiensis. FASEB J. 21:1801-1812.

Holzenberger, M., J. Dupont, B. Ducos, P. Leneuve, A. Geloen, P.C. Even, P. Cervera, and Y. Le Bouc. 2003. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature. 421:182-187.

Johnson, T.E. 2003. Advantages and disadvantages of Caenorhabditis elegans for aging research. Experimental Gerontology. 38:1329-1332.

Kenyon, C., J. Chang, E. Gensch, A. Rudner, and R. Tabtiang. 1993. A C. elegans mutant that lives twice as long as wild type. Nature. 366:461-464.

Kim, D.H., R. Feinbaum, G. Alloing, F.E. Emerson, D.A. Garsin, H. Inoue, M. Tanaka-Hino, N. Hisamoto, K. Matsumoto, M.-W. Tan, and F.M. Ausubel. 2002. A Conserved p38 MAP Kinase Pathway in Caenorhabditis elegans Innate Immunity. Science. 297:623-626.

Kimura, K.D., H.A. Tissenbaum, Y. Liu, and G. Ruvkun. 1997. daf-2, an Insulin Receptor-Like Gene That Regulates Longevity and Diapause in Caenorhabditis elegans. Science. 277:942-946.

Mahajan-Miklos, S., M.-W. Tan, L.G. Rahme, and F.M. Ausubel. 1999. Molecular Mechanisms of Bacterial Virulence Elucidated Using a Pseudomonas aeruginosa- Caenorhabditis elegans Pathogenesis Model. Cell. 96:47-56.

Mallo, G.V., C.L. Kurz, C. Couillault, N. Pujol, S. Granjeaud, Y. Kohara, and J.J. Ewbank. 2002. Inducible Antibacterial Defense System in C. elegans. Current Biology. 12:1209-1214.

May, R.C. 2007. Gender, Immunity and the regulation of longevity. Willey Periodicals, BioEssays. 29:795-802.

Nicholas, H.R., and J. Hodgkin. 2004. The ERK MAP Kinase Cascade Mediates Tail Swelling and a Protective Response to Rectal Infection in C. elegans. Current Biology. 14:1256-1261.

Paradis, S., and G. Ruvkun. 1998. Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes Dev. 12:2488-2498.

Schulenburg, H., C. Leopold Kurz, and J.J. Ewbank. 2004. Evolution of the innate immune system: the worm perspective. Immunological Reviews. 198:36-58.