Biogerontology

, Volume 11, Issue 6, pp 643–669

Suitability of the clonal marbled crayfish for biogerontological research: a review and perspective, with remarks on some further crustaceans

Authors

    • Department of ZoologyUniversity of Heidelberg
Review Article

DOI: 10.1007/s10522-010-9291-6

Cite this article as:
Vogt, G. Biogerontology (2010) 11: 643. doi:10.1007/s10522-010-9291-6

Abstract

This article examines the suitability of the parthenogenetic marbled crayfish for research on ageing and longevity. The marbled crayfish is an emerging laboratory model for development, epigenetics and toxicology that produces up to 400 genetically identical siblings per batch. It is easily cultured, has an adult size of 4–9 cm, a generation time of 6–7 months and a life span of 2–3 years. Experimental data and biological peculiarities like isogenicity, direct development, indeterminate growth, high regeneration capacity and negligible senescence suggest that the marbled crayfish is particularly suitable to investigate the dependency of ageing and longevity from non-genetic factors such as stochastic developmental variation, allocation of metabolic resources, damage and repair, caloric restriction and social stress. It is also well applicable to examine alterations of the epigenetic code with increasing age and to identify mechanisms that keep stem cells active until old age. As a representative of the sparsely investigated crustaceans and of animals with indeterminate growth and extended brood care the marbled crayfish may even contribute to evolutionary theories of ageing and longevity. Some relatives are recommended as substitutes for investigation of topics, for which the marbled crayfish is less suitable like genetics of ageing and achievement of life spans of decades under conditions of low food and low temperature. Research on ageing in the marbled crayfish and its relatives is of practical relevance for crustacean fisheries and aquaculture and may offer starting points for the development of novel anti-ageing interventions in humans.

Keywords

Marbled crayfishCrustaceaNegligible senescenceAllocation of resourcesEpigeneticsStem cellsSocial stress

Introduction

Laboratory experiments with animal model organisms have significantly contributed to the understanding of the biology of ageing and longevity. Such experiments, supplemented by observations from wild populations, pursue the following goals: (1) to compile a catalogue of ageing phenomena for the animal kingdom, (2) to compare ageing and longevity among and within animal clades, (3) to investigate the biological processes of ageing in detail, and (4) to learn how to achieve healthy old age and improved longevity in humans. The mouse Mus musculus, the rat Rattus norvegicus and the rhesus monkey Macaca mulatta are the most popular models of biogerontology due to their close phylogenetic relationship to humans and their long history as laboratory animals (Conn 2006; Enns et al. 2008; Bizon and Woods 2009; Masoro 2009a; Osorio et al. 2009). Among the invertebrates, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster are the most intensely studied ageing models, mainly because of short life cycles and well investigated development and genetics (Johnson 2008; Kennedy 2008; Austad 2009; Partridge 2009). The invertebrate models have proven particularly powerful for research on genetics of ageing, investigation of conserved metabolic pathways modulating longevity, and drug discovery in ageing (Martin et al. 1996; Lithgow et al. 2005; Kuningas et al. 2008; Masoro 2009a). They even turned out to be suitable for the examination of some age-related human diseases (Link 2001; Augustin and Partridge 2009).

In the last decade, several further species were publicised as promising model candidates to compensate for shortcomings of the classical ageing models. Among the candidates suggested are mammals (Brunet-Rossinni and Austad 2004; Buffenstein 2005), birds (Holmes and Ottinger 2003), fish (Gerhard 2007) and species from various invertebrate phyla (Keller and Jemielity 2006; Ebert 2008; Hengherr et al. 2008; Austad 2009; Mouton et al. 2009; Philipp and Abele 2010). Vertebrate candidates have the advantage of closer biological similarity to humans whilst invertebrates have the advantage of easier culture, lower costs and fewer restrictions by animal protection and welfare acts. The introduction of a new laboratory model for ageing research makes sense if the candidate belongs to a poorly investigated animal group (comparative ageing research) or if it possesses biological characteristics that allow investigation of basic ageing mechanisms from a new perspective (Austad and Podlutsky 2006; Holmes and Kristan 2008; Austad 2009).

For instance, the mole rat Heterocephalus glaber, the longest-lived rodent known, and the killifish Nothobranchius furzeri, the shortest-lived vertebrate may be exploitable for life span manipulation studies (Terzibasi et al. 2007; Buffenstein 2008). The zebra fish Danio rerio, a common laboratory model in development and genetics, may serve for investigation of the molecular biology of ageing with the help of well established molecular tools and transgenic lineages (Gerhard 2007). The ocean quahog Arctica islandica, which can reach an age of 400 years, could serve as a prototype for negligible senescence and highly effective anti-oxidative strategies (Philipp and Abele 2010). The honey bee Apis mellifera is of interest due to its unique diversity in life span, which varies from a few weeks to more than 2 years, depending on the social environment (Münch et al. 2008). Bdelloid rotifers and tardigrades may be useful because they can extend their life spans for weeks or even years by anhydrobiosis, either ignoring or counting the time spent in the cryptobiotic state (Hengherr et al. 2008). And the hydrozoan Turritopsis nutricula is expected to contribute a new mechanism to ageing research because it can reverse development resulting in morph rejuvenation (Piraino et al. 2004).

Among the animal groups that have been largely ignored by biogerontologist are the decapod crustaceans (Finch 1990; Animal Ageing and Longevity Database AnAge, http://genomics.senescence.info/species/), although they provide many ecologically and economically important taxa and have been used for laboratory experiments for more than a century (Martin and Davis 2001; Vogt 2002). The Decapoda comprise approximately 10,000 species including shrimps, prawns, lobsters, freshwater crayfish, sea spiders and crabs. Morphologically, they are highly diverse but the histology and cytology of their internal organs is rather uniform. Decapods have maximum adult sizes of 1 cm to 4 m and life spans between 1 and 100 years (Martin and Davis 2001). Most of them are characterized by indeterminate growth (Hartnoll 1982), high regeneration capacity (Hopkins et al. 1999) and pronounced resistance to tumour formation (Vogt 2008a). Indeterminate growth refers to growth that continues in the adult life period in contrast to determinate growth that stops once a genetically pre-determined final stage has formed. So far, research on ageing in Decapoda focussed almost exclusively on the analysis of life histories and age structures in wild populations in order to estimate their ecological dynamics and fishery potential (Belchier et al. 1998).

This paper examines the suitability of the marbled crayfish, an emerging laboratory model for development, epigenetics and toxicology (Vogt 2007, 2008b), for biogerontological research. It reviews published and unpublished findings from 10 years of investigation and gives a perspective for future studies. The article starts with a description of the biological peculiarities of the marbled crayfish qualifying for ageing research, continues with an analysis of its potential for non-genetic and genetic topics of biogerontology and closes with evolutionary considerations. In some chapters, I have included observations and data that are not yet statistically firm in order to stimulate research in these directions and to facilitate planning of future experiments. Where appropriate, I also refer to ageing phenomena in relatives of the marbled crayfish, particularly other freshwater crayfish and the closely related lobsters.

Biology of the marbled crayfish and peculiarities qualifying for ageing research

The marbled crayfish (Fig. 1) or Marmorkrebs (Decapoda, Astacida) was discovered in the mid 1990s in the German aquarium trade (Scholtz et al. 2003; Vogt et al. 2004). It is the first of more than 10,000 decapod crustaceans that was shown to produce high amounts of genetically identical offspring by obligatory parthenogenesis (Martin et al. 2007; Vogt et al. 2008). Males are unknown. Mainly due to its clonal nature and easy culture the marbled crayfish was introduced as a laboratory animal in the year 2000 by myself and, almost simultaneously, by colleagues at the universities of Ulm and Berlin. Presently, it is used by more than a dozen laboratories in Europe and the United States for morphofunctional, cytological, developmental, neurobiological, epigenetic, behavioural, chronobiological and toxicological research. Its basic biology is rather well investigated (Vogt et al. 2004; Seitz et al. 2005; Alwes and Scholtz 2006; Rieger and Harzsch 2008; Vogt 2008b, c; Farca Luna et al. 2009, 2010; Kawai et al. 2009). Just recently it has been shown that the marbled crayfish is a parthenogenetic form of the slough crayfish Procambarus fallax (Hagen 1870; Martin et al. 2010). This cambarid species is native to Florida and south Georgia and occurs in wetland, stream, ditch and pond habitats (Dorn and Volin 2009). Wild marbled crayfish have not been found as yet in Florida and Georgia, their presumed site of origin, but thriving outdoor populations have developed in tropical Madagascar as a result of deliberate release in about 2003 (Jones et al. 2009).
https://static-content.springer.com/image/art%3A10.1007%2Fs10522-010-9291-6/MediaObjects/10522_2010_9291_Fig1_HTML.jpg
Fig. 1

Brooding marbled crayfish. Despite the clonal nature of this species each specimen is recognized throughout life by an individual marmoration pattern. The eggs and first three juvenile stages develop under the maternal pleon (pl) but can also be raised in vitro. Black arrow denotes stage-2 juvenile on pleopod. Arrowheads indicate ‘total length’ from tip of rostrum (ro) to end of tail fan (tf), a standard measure of body size in crayfish. White arrow, mouthparts; a1, 1st antenna; a2, 2nd antenna; ca, carapace; p1–p3, pereopods 1–3 (from Vogt and Tolley 2004). Inset: egg with embryo at 70% development surrounded by yolk (from Vogt et al. 2004)

The life cycle of the marbled crayfish can be subdivided into embryonic, juvenile, adolescent and adult phases. The embryonic period starts with spawning of the eggs and ends 17–28 days later with hatching of the first juvenile stage. The juvenile phase includes approximately seven stages, which are characterized by a spotted pigmentation pattern. The eggs and the first three juvenile stages are carried on the maternal pleopods and protected, fanned and cleaned by the mother (Vogt and Tolley 2004; Vogt 2008c). Stage 1 and stage 2 juveniles are permanently on the pleopods thriving on their yolk reserves (Fig. 1) whilst stage 3 juveniles become free lancing and start to feed. The juvenile stages are followed by seven to ten adolescent stages, which become increasingly marmorated and display clearly visible female sexual characteristics (Vogt et al. 2004). The adult phase spans from first spawning to death and includes several alternating growth and reproduction phases. Despite genetic identity, marbled crayfish are individually recognized by their marmoration pattern (Fig. 1), which is principally maintained throughout life (Vogt et al. 2008).

Adult marbled crayfish usually measure 4–8 cm in total length (tip of rostrum to end of tail fan, Fig. 1), having weights of 1.5–15 g. The largest specimen of my laboratory colony had reached a length of 8.8 cm and a weight of 23.5 g. Under tropical outdoor conditions the maximum length measured was 10.7 cm (Jones et al. 2009). The maximum individual life span, defined as the time period from hatching to death, recorded so far in my laboratory was 1610 days (Fig. 2). However, most adults died between day 400 and 1000 of life. The mean life span of 49 reproducing adults was 718.8 ± 221.6 days (Fig. 2) and the mean life span of the most long-lived 10% was 1154 ± 264.2 days. The maximum number of reproduction phases per female was seven. First time spawners usually had ages between 150 and 250 days, in an exceptional case 524 days. The latest spawning ever recorded occurred at day 1530 of life (Fig. 2). The highest number of eggs and juveniles per batch were 589 and 427, respectively. The maximum total number of offspring produced per female exceeded 800.
https://static-content.springer.com/image/art%3A10.1007%2Fs10522-010-9291-6/MediaObjects/10522_2010_9291_Fig2_HTML.gif
Fig. 2

Mortality curve of adult marbled crayfish. Black dots denote age at death of 49 reproducing specimens. Individuals were genetically identical and reared under similar water and feeding conditions either communally or individually. Life span varied between 312 and 1610 days. Earliest spawning occurred at day 157 and latest spawning at day 1530. Note rather constant mortality between 450 and 950 days. Forty-two specimens died during moulting and seven specimens died of unknown causes

Marbled crayfish can be kept either individually or communally in simple housing systems at room temperature and can tolerate broad ranges of environmental conditions for longer periods of time (Vogt et al. 2004; Seitz et al. 2005; Vogt 2007, 2008c; Jimenez and Faulkes 2010). They are omnivorous and can be fed with a single pellet food (TetraWafer Mix) from their first feeding stage to death, which is very exceptional in both vertebrates and invertebrates (Vogt et al. 2004; Vogt 2008c). The pellets are obviously a wholly adequate diet because I have kept marbled crayfish on this food for eight generations without loss of vitality. Clutch sizes in captivity are comparable to those of wild population (Jones et al. 2009). The maximal age cannot be compared between our laboratory colony and wild populations because analyses of the age structure of wild populations are not available as yet. Our simple housing and feeding system facilitates performance of highly standardized experiments with the marbled crayfish. Moreover, the use of pellets as the only food source and tap water as the only water source minimizes the risk of introducing toxicants and infectious diseases during long-term experiments. Tap water has the additional advantage that it is delivered in a constantly high quality and permanently controlled by the municipal water supplier.

Marbled crayfish show a broad spectrum of behaviours like other freshwater crayfish and establish dominance hierarchies even among genetically identical batch-mates, starting approximately from juvenile stage 7, the first life stage with sclerotized claws suitable for agonistic fighting (Vogt et al. 2008; Farca Luna et al. 2009). Dominance is usually size dependent, and the intensity of agonistic interaction varies with space and shelter. The behavioural spectrum of the marbled crayfish is similar to that of the noble crayfish Astacus astacus, the ethogram of which comprises 66 behavioural elements belonging to 12 functional cycles (Lundberg 2004).

Rearing of marbled crayfish under optimized laboratory conditions in the absence of predators and diseases allows specimens to grow to their biological age limit, which should facilitate detection of senescence related dysfunctions. In a heterogeneous population of several age classes, mortality is usually rather high in juveniles and early adolescents and low in adults, depending on stocking density, abundance of shelter and availability of food. In juveniles, the primary cause of death is cannibalism probably followed by moulting disturbances. Their survival rate can be considerable enhanced by grouping specimens of the same size at low density. For instance, in two such trials with 26 and 37 individuals per 600 cm2, survival rate was 61.5 and 56.2% in the 88 days following brood care, which is the most delicate period of life. In smaller groups of size-matched adults, mortality can be zero for more than a year at best (Fig. 3, B1–B8). The primary cause of death in adults is moulting as shown for a sample of 49 specimens: 85.7% of them died during ecdysis or in the first 2 days thereafter and 14.3% died of unknown causes. The causes of death are rather well known for humans but remain obscure in most animals including the invertebrate models Drosophila melanogaster and Caenorhabditis elegans (Vermeulen and Loeschcke 2007).
https://static-content.springer.com/image/art%3A10.1007%2Fs10522-010-9291-6/MediaObjects/10522_2010_9291_Fig3_HTML.gif
Fig. 3

Relationships between life span and growth and life span and reproduction in marbled crayfish. Specimens A and B were the founders of two laboratory lineages indicated by white and black dots, respectively. A1 and B1–B8 were F1-progeny, B31 and A11–A14 were F2-progeny, and B31-1 was F3-progeny. Female B and batch-mates B1–B8 were communally housed in a 60 × 30 × 30 cm aquarium at room temperature and fed daily ad libitum with TetraMin Wafer Mix pellets. The same holds for A11–A14. Specimens A, A1, B31 and B31-1 were individually reared in 30 × 20 × 20 cm aquaria for most of their adolescent and adult life. Water and feeding conditions were similar to communal rearing. Rectangular frames indicate specimens with regeneration of multiple appendages. Asterisk denotes most heavily damaged individual (still alive at end of experiment). The data indicate that life span varies considerably among isogenic batch-mates even in the same environment (B1–B8, A11–A14), that there is no simple relationship between life span and investments in growth and reproduction (compare relative position of individuals in left and right panel), that individual rearing promotes life span extension (A, A1, B31, B31-1), and that damage and repair does not necessarily impair longevity (B31, B31-1). The specimens depicted died of unsuccessful moulting with the exception of B2, B7 and B8, which were sacrificed for determination of global DNA methylation

Easy culture, individual recognisability, high fertility, breeding at any time of the year, easy accessibility of all life stages including embryos (Fig. 1, inset), high tolerance to physical manipulation and adaptability to a wide spectrum of environmental and nutritional conditions qualify the marbled crayfish as a general laboratory animal. Special advantages for research on ageing and longevity are genetically identical offspring, direct development, indeterminate growth, lack of resting stages, alternation of growth and reproduction phases, high regeneration capacity, broad behavioural spectrum, DNA methylation throughout life, and possession of several functionally diverse stem cell systems. Moreover, marbled crayfish have well developed circulatory, hormone and immune systems (Vogt 2002; Gherardi et al. 2010), which in mammals are primary targets of age-related alterations.

The generation time of the marbled crayfish of 6–7 months is longer than in the model organisms Caenorhabditis elegans, Drosophila melanogaster, Mus musculus and Rattus norvegicus but within the range of Danio rerio. The same holds for its maximal life span, which is longer than in worm and fruit fly, comparable to mouse and rat, but shorter than in zebrafish. Many researchers prefer small and short-lived ageing models like the fly and the worm because they can be followed for their entire life and across generations within a reasonable amount of time for a reasonable cost. Such models have proven invaluable for several aspects of ageing as noted above. However, short-lived and tiny ageing models also have some shortcomings, among them the impossibility of individual biochemical and physiological studies (Johnson 2003; Austad 2009), which caused some researchers to advocate for the additional use of larger and longer-lived models in ageing research (Holmes 2004; Buffenstein 2005; Philipp and Abele 2010).

The marbled crayfish may be a good compromise between small, short-lived models and larger, long-lived model candidates because it is small enough to be mass-cultured and large enough to allow individual physiological and biochemical analyses. It also allows individual longitudinal studies by taking blood samples or biopsies or by the analysis of exuviae of subsequent life stages, which provide an excellent archive of morphological traits inclusive of external sense organs. The life span of the marbled crayfish is not too long to complete experiments in reasonable periods of time but long enough to follow in detail time consuming processes like regeneration of damaged body parts, production of diseases by low chronic doses of toxicants, or alteration of the epigenetic code with age.

Suitability of the marbled crayfish for research on non-genetic aspects of ageing and longevity

This section deals with aspects of ageing that are mainly determined by non-genetic parameters. It includes highly topical issues like the relationship between senescence and stem cell function and the alteration of the epigenome with increasing age.

Stochastic components of ageing and longevity

Ageing and longevity is considerably influenced by stochastic developmental variation (intrinsic chance variation), which is generated by processes like gene expression, intracellular signalling, reaction–diffusion-like patterning mechanisms, and self-reinforcing circuitries involving behaviour, metabolism and neuroendocrine control (Finch and Kirkwood 2000; Kirkwood et al. 2005; Rea et al. 2005; Vogt et al. 2008). In the marbled crayfish, stochastic developmental variation was shown to be produced in all life stages and to cause variation in a broad range of morphological characters and life history traits including longevity (Vogt et al. 2008). For instance, the life spans of eight communally reared batch-mates varied between 437 and 910 days (mean ± SD: 621 ± 132.5 days), although they were exposed to the same environmental conditions at any time of their life and fed ad libitum with a single pellet food (Fig. 3, specimens B1–B8). In another group of four communally reared batch-mates, life span varied between 618 and 861 days (mean ± SD: 731 ± 125.1 days) (Fig. 3, specimens A11–A14). Batch-mates of the marbled crayfish appear particularly suitable to determine the influence of stochastic developmental variation on ageing and longevity because they are isogenic and share the same developmental history.

Allocation of metabolic resources between maintenance, growth and reproduction

The allocation of metabolic resources between maintenance, growth and reproduction is among the key topics of ageing research and is the mainstay of the ‘disposable soma theory’ of ageing (Kirkwood 2008; Jasienska 2009). Extensive utilization of metabolic resources for growth and reproduction is thought to result in shorter life span because of inadequate maintenance and tissue repair. Animals with indeterminate growth like the marbled crayfish, which have no conclusive adult size, experience a life history trade-off in resource allocation between maintenance, reproduction and growth until the end of their life (Heino and Kaitala 1999). This is in contrast to animals with determinate growth like the ageing models Mus musculus, Drosophilamelanogaster and Caenorhabditiselegans that must allocate their resources only between maintenance and reproduction once they have reached their adult size. In the marbled crayfish, growth is not a continuous process like in mammals but occurs step-wise by moulting. In adults, each growth phase is usually followed by a reproduction phase, at which moulting and growth are inhibited. Therefore, this species appears well suitable to investigate the relationship between allocation of metabolic resources and longevity in depth.

Our marbled crayfish moulted and reproduced up to seven times in their adult period of life (Fig. 3). They produced approximately 50–500 polylecithal eggs per female, depending on size of the spawner. These eggs had a mean diameter of 1.6 mm and were filled with triglycerides and vitellin, the energetic and nutritional basis for the development of the embryo and the first three juvenile stages (Vogt and Tolley 2004). Due to the synthesis of special high molecular weight molecules like vitellin, a lipo-glyco-carotino-protein, allocation of metabolic resources towards egg production is not only a quantitative but also a qualitative issue. In our colony, the clutch sizes varied between 3.5 and 11.9% of the mother’s body weight (mean ± SD: 7.41 ± 2.35) (Fig. 4), indicating that a considerable proportion of the metabolic resources is shifted towards egg production. This effort is higher than in humans, where the average birth weight of the offspring is 5.2% of the mother’s weight. Usually, breeding females lose some weight because they either completely cease or drastically reduce feeding in the 30–40 days lasting brooding period but spent energy for their basic metabolism and fanning of the eggs and juveniles.
https://static-content.springer.com/image/art%3A10.1007%2Fs10522-010-9291-6/MediaObjects/10522_2010_9291_Fig4_HTML.gif
Fig. 4

Relationships between clutch size, body size and age in marbled crayfish. Black dots indicate specimens used for determination of length-weight relationship only. White dots, rectangles and asterisks denote specimens with known length, weight, clutch size and age at spawning. Rectangles and asterisks indicate early (c1) and late (c3, c4, c7) clutches of females B1 and A1. Clutch size is given in percent of body weight of spawner (% bw) and age is given in days (d). The graph illustrates that investment in reproduction varies among individuals but, in average, remains in the same order of magnitude with increasing body size and age

First experiments on the allocation of metabolic resources in two batches (B1–B8, A11–A14) revealed that there is no clear correlation between growth and/or reproduction and longevity in the marbled crayfish (Fig. 3). Instead, the picture is complicated showing variation in all directions. However, the specimen with the shortest life span (B1) showed the fastest growth in the adolescent period, the earliest onset of reproduction, and the shortest time intervals between subsequent reproduction phases suggesting ‘exhaustion’ by rapid growth and intense reproduction (Vogt 2009). The pronounced variation in allocation strategies among batch-mates may be related to differences in basic metabolism and feeding behaviour, which seems to diverge with increasing age despite genetic identity of the sibs (Vogt et al. 2008).

In our experiments we determined the organismal or overall response to get a first idea on the allocation of metabolic resources in the marbled crayfish. In future research, this approach could be supplemented by estimation of energetic and metabolic parameters. Such studies were already made with other decapod species. For instance, in the Chinese midden crab Eriocheir sinensis Jiang et al. (2009) analysed the transfer of nutrients from the hepatopancreas, the main storage organ of energy and nutrients, to the reproductive organ by transcriptom analysis. They identified seventeen genes relevant to the control of nutrition mechanisms and eleven genes involved in regulation of reproduction. Baeza and Fernández (2002) determined the energetic cost of breeding in the crab Cancer setosus and measured doubling of the metabolic rate in females carrying late embryos.

It has to be noted that in our experiment food was available in excess at any time of life, which is usually not the case under natural conditions. In the wild, the animals have to adapt their allocation strategy to the environmental challenges. Therefore, experiments on the relationship of metabolic allocation and longevity under conditions of limited food may reveal quite different results. Under conditions of prolonged starvation, crayfish and other decapods reduce or cease growth and reproduction. In an early phase of famine they live on the reserves of the hepatopancreas. Later on, they catabolize structural components of the musculature and other tissues and re-absorb vitellogenic oocytes in the ovary (Vogt et al. 1985).

Regeneration and longevity

Decapod crustaceans are traditional models for studying regeneration, particularly regeneration of the appendages (Skinner and Cook 1991). The repair of internal organs is less well examined. Like most decapods, marbled crayfish can autotomize and regenerate their limbs from the first juvenile stage to death. Autotomy or self-amputation is a natural reflex and occurs at a pre-formed fracture plane, which, in the pereopods, is located in the basis-ischium segment. High and enduring mechanical stress on a limb causes the crayfish to actively abandon the distal part of this limb by a specific mode of action of the proximal limb musculature. The fracture plane is immediately closed by a pre-existing membrane, minimizing blood loss. Regeneration of the limb is then initiated by the formation of a blastema of mitotically active cells, which produce different tissues like epidermis, connective tissue, musculature and nervous tissue (Hopkins et al. 1999).

In the marbled crayfish, regeneration of lost appendages apparently does not shorten life span as long as the disabled specimen is protected from predation and cannibalism and fed ad libitum. For instance, specimen B31 had lost one cheliped and 3 of 8 walking legs at the age of 235 days but recovered fully in an individual housing system throughout the next two moults. Despite its severe damages, it lived for 1610 days, the highest age ever recorded in my laboratory population. However, at a given time both growth and frequency of spawning were lower than in undamaged specimen A1, although both were reared individually under very similar environmental and nutritional conditions (Fig. 3). For example, at day 850 of life, B31 had a weight of 8.7 g and 3 spawnings compared to 16.6 g and 5 spawnings in A1. The same tendency was shown by specimen B31-1, which had lost both chelipeds and 6 walking legs at the age of 402 days. At day 850 of life it had a weight of 14.3 g but only 2 spawnings, suggesting that regeneration in the marbled crayfish requires a considerable proportion of the metabolic resources at cost of growth and/or reproduction as shown earlier for other animals (Maginnis 2006).

Reproductive and mechanical senescence

In many animals, the last period of life is characterized by senescence, the age-related decline in structural and functional status (Finch 1990; Kirkwood 1996). Senescence can be measured at the organismic, organ, cellular or biochemical level and can affect behaviour, reproduction, mechanical structures and soft tissues. Whether senescence is rapid like in salmon, flies and nematodes, gradual like in placental mammals and humans or negligible like in reptiles, molluscs and echinoderms is apparently dependent on the phylogeny and life strategy of a species or higher taxon (Finch 1990; Grotewiel et al. 2005; Abele et al. 2008; Ebert 2008; Finch 2009). As a rule of thumb, animals with determinate growth usually show a marked decline of structure and function with age, whereas animals with indeterminate growth do not show such features. Therefore, negligible senescence is the most likely ageing feature to be expected for the marbled crayfish.

Reproductive senescence or the decline of breeding success with age is well documented for a variety of vertebrates and invertebrates (Grotewiel et al. 2005; Kirkwood 2008; Sharp and Clutton-Brock 2010). In our marbled crayfish, earliest spawning was noted at day 157 of life and latest spawning at day 1530, which is close to the recorded maximal age of 1610 days (Fig. 2). In general, clutch sizes are positively correlated with body size, which itself is roughly correlated with age, but there is considerable variation among individuals (Seitz et al. 2005; Jones et al. 2009). In our marbled crayfish, there was no marked age-related difference in reproductive investment, which becomes particularly obvious by comparison of spawners younger than 6 months to spawners older than 2 years, which had mean clutch sizes of 7.45 and 7.97%, respectively (Fig. 4). The absence of reproductive senescence in the marbled crayfish was corroborated by examination of successive clutches of individual females. For example, specimens A1 and B1 had clutch sizes of 4.8 and 6.3% at their first spawning and 6.9 and 6.5% at their last spawning, respectively (Fig. 4). There is apparently also no decrease in the viability of the eggs with increasing age of the mother because the highest number of juveniles per clutch (427) was produced by a 3-year old female in its last reproduction phase.

Mechanical senescence is an important component of ageing in animals with body parts that are subject to mechanical wear and tear but are not replaced. Examples are most teeth of mammals or the wings of insects (Finch 1990). Particularly in grass eating mammals like ruminants and horses this type of senescence can be the primary cause of death. Marbled crayfish escape from mechanical senescence by moulting, which occurs up to 25 times during lifetime. Moulted are the exoskeleton (Fig. 5a), various types of sensory setae (Fig. 5b–d), the masticatory structures around the mouth and in the stomach (Fig. 5e, f), filtering structures in the stomach (Fig. 5f, g) and various grooming structures (Fig. 5h) that keep the gills and other body parts clean. In early juveniles, the intermoult period lasts a few days, extending to weeks in the adolescents and months in the adults.
https://static-content.springer.com/image/art%3A10.1007%2Fs10522-010-9291-6/MediaObjects/10522_2010_9291_Fig5_HTML.jpg
Fig. 5

Escape from mechanical senescence and concurrent enhancement of sensory capacity by moulting in marbled crayfish and relatives. a Marbled crayfish juvenile shedding its old exoskeleton (arrow) (from Vogt 2008c). b Olfactory aesthetascs (arrow) on 1st antenna of marbled crayfish. These sensory units are replaced and increased in number at each moult. c Gustatory corrugated setae (arrow) on 2nd pereiopod of marbled crayfish, being also replaced and augmented by moulting. d Statocyst of marbled crayfish showing sensory setae (arrowheads) and parts of the statolith (arrow). The setae are replaced by moulting and the statolith by active insertion of sand particles that are clued together. e Cutting edges of freshly moulted mouthparts of marbled crayfish juvenile showing unworn masticatory (arrowheads) and sensory (arrow) structures. m, mandible; m1, 1st maxilla; m2, 2nd maxilla; mp, 1st maxilliped (from Vogt 2008c). f Gastric mill in cardiac stomach of spiny-cheek crayfish Orconectes limosus, consisting of medial tooth at roof of stomach (mt), lateral teeth (lt) and accessory teeth (at). Although located in the interior of the animal, this masticatory apparatus is moulted together with the primary filters (pf) at the bottom of the stomach (from Vogt 2002). g Secondary filter in pyloric stomach of noble crayfish Astacus astacus consisting of numerous filter tubes (ft) covered by filter setae (arrow). These filter tubes are renewed and increased in number at each moult (from Vogt 2002). h Grooming setae from gill chamber of freshly moulted marbled crayfish showing unworn cleaning shovels (arrow). Inset: serrated cleaning shovel in detail

Some mechanical structures of the marbled crayfish are not only replaced and adapted to the new size but are also increased in number by moulting. For instance, the total number of olfactory aesthetascs on the first antennae (Figs. 1, 5b) was augmented from 10 in stage 2 juveniles, the first stage of their appearance, to 154 in an adult of 4.7 cm total length. The gustatory corrugated setae on the pereiopods 1–3 (Figs. 1, 5c) were increased from 74 to 556 in the same specimen. The aesthetascs were only slightly enlarged with age, whereas the corrugated setae were three times bigger in the adult than in stage-2 juveniles. Each of the newly added aesthetascs includes approximately 100 olfactory receptor neurons, as shown for the yabby crayfish Cherax destructor (Sandeman and Sandeman 2003). The axons of these neurons grow towards the olfactory lobe of the brain to be integrated into the existing neuronal network, resulting in an enlargement of the olfactory lobe and an alteration of its wiring pattern. These changes are thought to enhance the efficiency of the olfactory system and may even enhance the cognitive capacity of the brain with increasing age.

Our observations suggest that moulting in the marbled crayfish serves multiple anti-ageing purposes. Most evident are the replacement and thickening of the calcified exoskeleton, which protects to some degree against attacks of predators and conspecifics, and the renewal of the food processing and cleaning structures. Less obvious are the enhancement of the sensory capacity by moulting and the contribution of moulting to maintenance of the immune system. The cuticle is not only a mechanical barrier against the invasion of pathogens and parasites but also includes components of the proPO-system, the major immune defence system of crayfish, providing a first line of defence (Cerenius et al. 2008). However, the prevention of mechanical senescence by moulting also has its price, which includes the energetic and metabolic costs of the new cuticle plus the energetic cost for fuelling the basic metabolism in the five to 6 days around ecdysis, when feeding is inhibited. The cuticle of the marbled crayfish accounts for 20–25% of the body weight, and its synthesis requires special proteins and high amounts of chitin and calcium carbonate.

Senescence of soft tissues: stem cell function, anti-oxidative defence and cellular waste treatment

Structural and functional decline of the soft tissues with age is much more difficult to analyse than reproductive and mechanical senescence and requires thorough knowledge of the organs of interest. Since the dynamics of tissues of crayfish and shrimps under normal and pathological conditions was at the centre of my research for more than 20 years (see references in Vogt 1999, 2002, 2008a; Gherardi et al. 2010), this requirement for examining age-related tissular alteration in the marbled crayfish was fulfilled. Light and electron microscopic investigations of the hepatopancreas, heart and ovary as the central organs of metabolism, circulation and reproduction, respectively, revealed that senescence of the soft tissues is negligible in the marbled crayfish. The hepatopancreas cells (Fig. 6a) and the myocardiocytes (Fig. 6c) of a 3-year old specimen showed similar features as in early adults, and the ovary of the same specimen included primary and secondary vitellogenic oocytes typical of a re-maturing ovary (Fig. 6d). Since loss of stem cell integrity, oxidative damage and lysosomal dysfunction are among the main causes of tissular senescence (Passos et al. 2007; Rossi et al. 2008; Hwang et al. 2009) I will discuss in the following what is known on these mechanisms in the marbled crayfish and other decapod crustaceans.
https://static-content.springer.com/image/art%3A10.1007%2Fs10522-010-9291-6/MediaObjects/10522_2010_9291_Fig6_HTML.jpg
Fig. 6

Negligible senescence and stem cell integrity in major tissues of marbled crayfish and relatives. a Detail of hepatopancreatic R-cell in 3-year old marbled crayfish. Ultrastructural features like lipid globule (lg), mitochondria (m) and rough endoplasmic reticulum (arrow) are similar to those of younger adults. b Stem cell niche at blind end of hepatopancreas tubule of adult marbled crayfish showing numerous E-cells (arrowhead) and some mitotic stages (arrow). hs, haemolymph space; lu, lumen (from Vogt 2008b). c Section of heart of 3-year old marbled crayfish showing small satellite cell (sc, arrowheads) and large myocardiocyte with nucleus (n), myofibrils (mf), mitochondria (asterisk) and glycogen fields (gl). d Re-maturing ovary of 3-year old marbled crayfish showing numerous pre-vitellogenic (arrowheads) and vitellogenic (arrow) oocytes, resembling first-time maturing females. e Germarium in re-maturing ovary showing oogonia (o) enclosed in meshwork of matrix cells (arrow) and collagen fibres (arrowhead). Inset: dividing oogonium. f Neurogenic system in deutocerebrum of juvenile red swamp crayfish Procambarus clarkii showing neurogenic niche (nn), migratory stream (arrow) and lateral proliferation area (lp). BrdU labelled progenitor cells (arrowheads) originate in neurogenic niche and migrate to proliferation area where they divide and differentiate into neurons. This neurogenic system remains active throughout life (from Song et al. 2007)

The marbled crayfish and its relatives harbour a variety of interesting stem cell systems with different activity patterns (Fig. 6b, c, e, f). Some of them are continuously active over longer periods of time, for instance during embryonic development or limb regeneration. Others are cyclically active for short periods only like the satellite cells in the skeletal and heart musculature (Fig. 6c) and the E-cells in the hepatopancreas (Fig. 6b). The satellite cells are activated by moulting (Martynova 1993) and the E-cells by feeding (Vogt and Quinitio 1994). Highly proliferative stem cells are also found in the ovary (Fig. 6e), the haematopoietic tissue and some areas of the brain (Fig. 6f) (Zhang et al. 2009). The haematopoietic tissue can be cultured and investigated in vitro (Fig. 8c) (Söderhäll et al. 2005). Regulation of the activity of these stem cells must be very robust because tumour formation is unknown in the marbled crayfish and extremely rare in other decapod crustaceans (Vogt 2008a). Due to their easy accessibility and robustness the stem cells of the marbled crayfish appear well suitable to study the molecular mechanisms that keep stem cells running until old age without having deleterious side effects.

Among the molecular mechanisms that cause stem cell related decline of tissues in mammals is loss of the replicative capacity of stem or progenitor cells by telomere shortening (Jiang et al. 2007). Telomere shortening can be counteracted by activation of telomerase, a reverse transcriptase, which adds new repeats to the ends of the chromosomes. In humans, most adult tissues lack telomerase activity, whereas it is expressed in embryonic tissues, stem cells and tumour cells (Jiang et al. 2007; Song et al. 2009). In contrast, in the smaller and much shorter-lived mice telomerase is expressed in several somatic tissues, for instance the liver, and the telomeres are not shortened with cell division. These striking differences among the two determinately growing mammals were explained by disparities in life history and body mass (Gorbunova and Seluanov 2009). Animals with indeterminate growth are thought to express telomerase in their tissues also in the adult stages as was shown for the rainbow trout Oncorhynchus mykiss and the American lobster Homarus americanus (Klapper et al. 1998). In the lobster, a rather close relative of the marbled crayfish, telomerase activity was present at different ages and in all organs examined, being highest in the hepatopancreas, the organ with the fastest cellular turnover in decapod crustaceans.

The hepatopancreas is a particularly interesting model for investigation of stem cells and their role in tissue regeneration due to its unique polar architecture and rapid cellular turnover. The hepatopancreas serves for the absorption of nutrients, synthesis of digestive enzymes, storage of reserves, and detoxification of xenobiotics (Vogt 2002; Gherardi et al. 2010). In adults, it is composed of more than a hundred blindly ending tubules that fuse together to form collecting ducts that finally terminate in the antechamber of the stomach (Vogt 1994). This tubular system displays a distinct polarity because its stem cells are confined to the blind ends of the tubules (Fig. 6b). These so-called E-cells divide in a late phase of each digestive cycle and give rise to three mature cell types, R-cells, F-cells and B-cells, which exert different functions and remain post-mitotic until cell death. Mitotic pulses of the E-cells push the older epithelial cells from the embryonic zone downstream, resulting in the establishment of a distinct age gradient along the tubules (Fig. 7b) and the collecting ducts.
https://static-content.springer.com/image/art%3A10.1007%2Fs10522-010-9291-6/MediaObjects/10522_2010_9291_Fig7_HTML.jpg
Fig. 7

Cellular waste treatment in selected organs of decapod crustaceans. a Electron micrograph of residual bodies in aged hepatopancreas cell of noble crayfish Astacus astacus including membranous (arrow) and electron dense, lipofuscin-like (arrowhead) material (from Vogt 1994). b Increase and enlargement of metal-loaded residual bodies (arrows) from embryonic zone (ez) to mature zone (mz) in hepatopancreas tubule of copper treated giant tiger prawn Penaeus monodon, demonstrating intracellular persistence of residual bodies until cell death. Arrowhead, tubular lumen; dz, differentiation zone; hs, haemolymph space (from Vogt and Quinitio 1994). c Apocrine secretion (arrowheads) of metal-loaded residual body (rb) from antennal gland cell of lead treated Penaeus monodon, indicating continuous removal of residual bodies from this cell type. lu, lumen; n nucleus (from Vogt and Quinitio 1994). d, e Augmentation and enlargement of lipofuscin-loaded residual bodies (arrows) with age in terminal medulla of eyestalk of European lobster Homarus gammarus. Arrowheads denote nuclei of neurons. Shown are a 4.7-year old specimen (d; from Sheehy et al. 1996) and a ca. 60-year old individual (e; from Sheehy 2002)

In the marbled crayfish, the E-cells are thought to undergo more than 1000 divisions in their life-time, resembling closely the stem cells in the small intestine of mouse. In the con-generic crayfish Procambarus acutus, mitotic pulses of the E-cells were shown to completely regenerate the hepatopancreas in about one to two weeks (Davis and Burnett 1964). The hepatopancreas tubules of the marbled crayfish are big enough to allow specific sampling of mitotic and post-mitotic areas, which would facilitate comparison of telomerase activity and telomere length in both tissue compartments. Such investigations might help to understand in more detail the role of telomeres and telomerase on cell ageing in situ, particularly since telomerase was shown to also have functions different from elongation of the telomeres (Artandi 2008).

Another widespread mechanism of tissue ageing is long-term stress caused by reactive oxygen species. These molecules are generated mainly in the mitochondria as common by-products of vital oxidative enzyme complexes. At high levels they can exert damaging effects on the cells resulting in tissular senescence. Such toxic reactions are at the centre of the ‘free radical theory of ageing’ (Cadenas and Davies 2000; Dowling and Simmons 2009). Long-lived animal species with negligible senescence were shown to have particularly effective anti-oxidative strategies comprising detoxifying enzymes and free radical scavengers (Abele et al. 2008; Pérez et al. 2009). Decapod crustaceans possess the universal detoxifying enzymes superoxide dismutase, catalase and glutathione peroxidase and the free radical scavenger glutathion. In addition, they produce astaxanthin, one of the best free radical scavengers and inhibitors of lipid peroxidation in the animal kingdom, which was occasionally appraised as a ‘super vitamin E’ (Sagi et al. 1995; Naguib 2000; Beytut et al. 2009).

In decapod crustaceans, astaxanthin is produced in the hepatopancreas from dietary carotenoids and is delivered to other organs, where it occurs in quite different concentrations. In the red claw crayfish Cherax quadricarinatus, it was shown to be present in relatively high amounts in the hepatopancreas, the cuticle and vitellogenic oocytes either in free or esterified form (Sagi et al. 1995). The same holds for ß-carotene, which is also a potent free radical scavenger. In the hepatopancreas, astaxanthin has probably anti-oxidative functions. In the cuticle, it is a constituent of the pigments and is assumed to be photo-protective. The astaxanthin in the oocytes may serve as a reservoir for anti-oxidants and pigment components for the embryos and first juvenile stages. In the giant tiger prawn Penaeus monodon, the anti-oxidative capability and hepatopancreatic function was greatly enhanced by dietary astaxanthin, resulting in a higher resistance to ammonia stress (Pan et al. 2003). Since astaxanthin was also proven to be effective against salinity stress, thermal stress and oxygen depletion in cultured shrimp it probably contributes to maintenance of tissue structure until old age and may therefore be a valuable target organ of more intense research on anti-oxidative strategies.

In virtually all animal clades including decapod crustaceans, defective cell organelles are degraded in lysosomal compartments, the autophagosomes, usually resulting in myelinated or electron dense residual bodies (Fig. 7a). Proper functioning of this system seems to be another mechanism to avoid cell senescence (Terman and Brunk 2006; Yen and Klionsky 2008; Rajawat et al. 2009). The lysosomal system is obviously uniform throughout the animal kingdom regarding its basic components but differs even between tissues of the same species with respect to the fate of the residual bodies (Vogt and Quinitio 1994). In the marbled crayfish and some other decapods the residual bodies were shown to accumulate in the hepatopancreas cells with increasing age (Fig. 7a, b), which may have no detrimental consequences due to the short life time of these cells. In contrast, in the antennal gland, the main organ of excretion and osmoregulation, residual bodies are continuously discharged by apocrine secretion (Fig. 7c), avoiding accumulation of cellular waste with age. A different picture was found in some areas of the decapod brain, for instance in the terminal medulla of the eyestalk. There, cellular waste was accumulated throughout life and deposited in lipofuscin granules. In the crayfish Cherax quadricarinatus and the European lobster Homarus gammarus, the area fraction of these granules increased linearly with age (Fig. 7d, e), making them a reliable age indicator (Sheehy et al. 1996; Sheehy 2002). At present, it is controversially discussed whether extensive lipofuscin accumulation in human or animal tissues is neutral or detrimental (Fonseca et al. 2005; Gray and Woulfe 2005; Jung et al. 2007).

The results obtained so far with the marbled crayfish and its relatives suggest that, unlike in mammals, insects and nematodes, there is no prolonged period of reproductive, mechanical and tissular senescence prior to death. The only sign of senescence found so far in freshwater crayfish is the deposition of lipofuscin in some areas of the brain. This fits well to the observation, that in the protected environment of a laboratory the main cause of death in adult marbled crayfish is unsuccessful moulting. This fatal incident occurs in all age classes and can therefore not be considered as an age-related phenomenon. There is presumably also no marked behavioural senescence in the marbled crayfish because, at first view, movement and feeding behaviour seemed to be quite similar in adults of increasing age.

Life span extension by caloric restriction and low temperature

There is now a large body of evidence on life span extension in animals by caloric restriction or low temperature (Yen et al. 2004; Masoro 2005). Earlier, this effect was explained by the ‘rate of living theory of ageing’, which states that longevity is inversely related to the metabolic rate of an animal. Today, this theory is largely rejected and replaced by an intense debate on other possible mechanisms including reduction of endogenously produced damaging molecules, enhancement of protective and repair mechanisms, increased resistance against environmental stressors, adaptation of the neuroendocrine system or hormesis (López-Torres et al. 2002; Houthoofd and Vanfleteren 2006; Min et al. 2008; Masoro 2009b). The response to caloric restriction is obviously not uniform in the animal kingdom and is thought to be dependent on the phylogeny and life history of a species or clade (Mockett et al. 2006; Phelan and Rose 2006; Shanley and Kirkwood 2006). For instance, species naturally adapted to regular periods of famine might respond differently to caloric restriction than unadapted species. Crayfish are among the clades that are used to starve for certain periods of time because they cease feeding during moulting and breeding (Reynolds 2002).

Reduction of the caloric intake can be achieved either by reducing the energetic content of the food or by temporal starvation. We have not yet performed experiments in these fields but the natural adaptation of the marbled crayfish to periodical starvation and its ability to feed on pellets makes this species an interesting research candidate. Moult related starvation occurs up to 25 times in its life time and lasts approximately 1–2 days in juveniles and 5–6 days in adults. Breeding related famine occurs up to 7 times and usually lasts from spawning to the appearance of stage 3-juveniles, which corresponds to 30–40 days (Vogt et al. 2004; Vogt 2008c; Vogt et al. 2008). Pellets enable a much better control of the caloric intake than other food sources and can be modified with respect to their caloric and biochemical content. The hormone system, the immune defence system, and the antioxidant system of crayfish are relatively well studied (Vogt 2002, Cerenius et al. 2008; Beytut et al. 2009; Gherardi et al. 2010), which would allow testing of some of the hypotheses listed above for life span extension by caloric restriction.

A particularly exciting topic that could be investigated with the marbled crayfish is the relationship between temporal starvation, stem cell activity and longevity. There is experimental evidence from crayfish and shrimp that the E-cells in the hepatopancreas, which are normally activated at the end of each digestive cycle to renew parts of the organ, remain quiescent during starvation (Vogt and Quinitio 1994), resulting in sparing of the stem cells. Validation of this idea with the marbled crayfish would possibly reveal a new mechanism for the explanation of the life-extending effect of caloric restriction.

Life span extension at low temperature has been shown for different poikilothermic animals like fish, insects and nematodes (Liu and Walford 1972; Miquel et al. 1976; Van Voorhies and Ward 1999). For example, wild-typ Caenorhabditis elegans raised at 10°C lived four times longer than their 25°C counterparts. Temperature related life span extension was often explained by reduction of the metabolism, inspired by the Arrhenius equation on the temperature dependency of the rate of chemical reactions. However, even invertebrates do not reduce their metabolism linearly with lowering of the temperature but display numerous compensatory physiological adaptations. For example, in the lobster Homarus gammarus cardiac output remains relatively constant from 20 to 2°C, as the temperature dependencies of some elements in the cardiac system are compensated for by others (Worden et al. 2006). Another temperature adapting mechanism is shown by the narrow-clawed crayfish Astacus leptodactylus that can change the transport capacity of its oxygen carrier haemocyanin via alteration of gene expression for the subunit types of its quarterny structure (Decker and Föll 2000).

The marbled crayfish seems principally suitable to investigate the relationship between temperature reduction and life extension because it can be exposed for longer periods of time to a broad range of temperatures (for details see section on environmental stress). In juveniles and adolescents, optimal survival was obtained at 20°C but optimal growth at 25°C (Seitz et al. 2005). Unfortunately, these temperature experiments were not performed long enough to see effects on longevity. Likewise, it is not yet known, which deviation from the optimal rearing temperature would exert a positive and potentially life extending stress and which would be detrimental and life shortening. Enhancement of protective mechanisms by mild temperature stress has been suggested as an explanation for life extension at lowered temperature (Rikke and Johnson 2004). This hypothesis could be tested with the marbled crayfish because, due to its large size, longitudinal measurement of protection indices like blood cell number or the concentration of anti-oxidative enzymes is principally feasible.

The decapod crustaceans also offer some long-lived species that are naturally adapted to food shortage or low temperature or both. These come from caves, a habitat with constantly low temperature (~10°C) and irregular food supply, the deep sea and arctic and antarctic waters. For instance, the troglobitic crayfish Orconectes australis and Procambarus erythrops, a species con-generic to the marbled crayfish, have maximal life spans of 50 years and >16 years, respectively, which is a factor of 5–10 higher than in their epigean relatives (Huryn et al. 2008). Cave dwelling decapods are well adapted to the periodic food supply as shown for Troglocaris anophthalmus that can starve for more than 27 months thriving on its reserves in the hepatopancreas (Vogt and Štrus 1999). The maximal life span of this shrimp is not yet known but is estimated to be >10 years. Atyaephyra desmarestii, its closest epigean relative, has a life span of 12–18 month only. The abundant circum-antarctic shrimp Notocrangon antarctica lives in the Weddell Sea in a very narrow temperature range of 0.4°C to −1.88°C and reaches an age of roughly 10 years, which is much longer than the 2–3 years of its relatives in the shallow waters of lower latitudes (Bluhm and Brey 2001).

The longest-lived decapod is probably the lobster, a temperate to cold water species that spends parts of its adult life in deeper waters of 50 and more meters. The European lobster Homarus gammarus has a maximal life span of 72 ± 9 years as determined by the lipofuscin method (Sheehy et al. 1999) and the American lobster Homarus americanus is even estimated to reach 100 years and more (Martin and Davis 2001). Such long-lived decapods are unsuitable for lifelong laboratory experiments but may be suitable to study anti-oxidative enzymes or tissue structures in wild caught specimens of different ages or to obtain cells and genes for laboratory experiments as suggested by Austad (2001) for slowly ageing organisms.

Influence of environmental and social stress on longevity

Under natural conditions, individual ageing and the age structure of a population are significantly influenced by predators, diseases, scarcity of shelters, harsh environmental conditions, food shortage and high population density (Finch and Seeman 1999; Kirkwood and Austad 2000; Mangel 2008). Stress is not necessarily detrimental and can even have life extending effects as shown for caloric restriction and low temperature. Apparently, it depends on the metabolic cost of the stress response whether stress is neutral or negative for longevity (Vermeulen and Loeschcke 2007). In poikilothermic animals with indeterminate growth like freshwater crayfish the environment is thought to be particularly influential on life history parameters including longevity (Sebens 1987).

The marbled crayfish appears well suitable to investigate the influence of environmental and social stress on ageing and longevity because it can be maintained in a broad range of abiotic and social environments. It can be kept individually or communally in either small or large aquaria at low or high temperature and abundance or scarcity of food and shelter. In a first study on the influence of temperature on growth and survival, Seitz et al. (2005) have kept juveniles and adolescents for several weeks at temperatures between 30 and 8°C. Adults were shown to even survive the German winter outdoors in ice covered vessels (Pfeiffer 2005). There are also some data available on the influence of environmental toxicants on the development and survival of the marbled crayfish. Exposure of the eggs to 17α-methyl testosterone resulted in decreased hatching rate and increased mortality and teratogenic effects in juveniles (Vogt 2007). The temperature and testosterone experiments revealed that environmental factors can indeed affect the ageing structure in marbled crayfish populations via alteration of the survival rate. However, none of these experiments was run long enough to estimate effects on longevity of the survivors.

Understanding of the relationship between social stress and longevity is relevant for all organisms that live in high population densities, including farm animals, aquaculture species and humans (Cheng and Muir 2004; Sterlemann et al. 2010). Social stress can become effective under conditions of high population density, scarcity of resources or social rank fighting. Presently, we have only few data on this topic but since the four oldest specimens of our laboratory colony were reared individually for most of their lifetime (Fig. 3) social stress is thought to reduce life expectancy in the marbled crayfish. Social stress in the marbled crayfish can be experimentally varied by manipulating food and shelter, stocking density or the size spectrum within a group, facilitating more refined experiments on this topic in future.

Since marbled crayfish establish dominance hierarchies even among genetically identical batch-mates they might also be suitable to examine the relationship of dominance and subordination on longevity. In social animal groups, longevity of the dominant can either be prolonged or shortened, depending on the social organization and the degree of stress that is exerted on the dominants or subordinates (von Holst et al. 1999; Sapolsky 2005). The social hierarchy of the marbled crayfish is special in as far as there are no males, which are the most aggressive specimens in crayfish populations. In the marbled crayfish, the biggest female is usually the dominant. This rank can be lost to another rapidly growing group member when growth of the dominant is stopped by reproduction.

Two observations on this topic might be worth to be mentioned here. The first addresses the relationship between social status and growth and survival. In aquaria without shelter juvenile batch-mates of the marbled crayfish established particularly pronounced social hierarchies that included one dominant, one subdominant and three subordinates (Vogt et al. 2008). Interestingly, the dominants grew much faster than the subordinates, although all specimens had unlimited access to the food and fed regularly. This phenomenon was probably caused by self-reinforcing circuitries including behaviour, metabolism and neuroendocrine control. Unfortunately, this experiment was not run long enough to determine longevity of the different social ranks but may provide ideas for future experiments in this direction. The second observation addresses the relationship between social status and survival of newborn neurons in the olfactory lobe of the crayfish brain that are continuously produced by stem cells as explained above. In Procambarus clarkii, a species con-generic to the marbled crayfish, survival and further development of the precursor cells into neurons was higher in dominants than in subordinates (Song et al. 2007), demonstrating that social rank can influence ageing of the brain.

Age-related diseases and stem cell diseases

The age structure of an animal population and individual longevity are considerably influenced by diseases. Two types of diseases are particularly relevant for biogerontology, namely diseases that emerge in an advanced age and stem cell diseases that impair tissue regeneration. For humans, there are numerous infectious and non-infectious stem cell diseases known, particularly from the haematopoietic tissue (Corey et al. 2007; Ozden et al. 2007; Banerjee et al. 2010). Typical age-related diseases are Alzheimer's disease, arthritis, diabetes, osteoporosis and cancer (Rattan 2006; Rossi et al. 2008; Anisimov et al. 2009). Many of the age-related diseases are caused by environmental and endogenously generated toxicants causing macromolecular damage and physiological dysregulation. Therefore, well functioning detoxification pathways are major longevity assurance mechanisms. In order to improve the quality of human life in old age a better understanding of both pathophysiology and detoxification mechanisms are urgently required (Rattan 2006; Zimniak 2008; Campisi et al. 2009). The latter could at least partly be studied in longer-lived animals that do not develop age-related diseases.

Age-related diseases have not yet been found in the marbled crayfish or its relatives, although more than 200 infectious, nutritional and environmental diseases have been described for the decapod crustaceans (references in Vogt 1999; 2008a; 2009). Likewise, age-related cancer, and tumour formation in general, is very rare in the Decapoda (Vogt 2008a), although some species like lobsters can grow nearly as old as humans. Such a scarcity of age-related diseases and tumours was also found in vertebrates with indeterminate growth and high regeneration capacity such as the axolotl (Roy and Gatien 2008) and is apparently related to the preservation of stem cell integrity throughout life. Crayfish have stem cell systems that are active until old age as discussed above but also have effective protection and detoxification mechanisms involving anti-oxidative enzymes, free radical scavengers, metallothioneins and cytochrome P450 (James and Boyle 1998; Leignel et al. 2008; Beytut et al. 2009). Moreover, they possess powerful immune response mechanisms including melanization and encapsulation of infected and pathologically transformed tissue areas (Cerenius et al. 2008; Gherardi et al. 2010).

In crayfish and shrimps there are at least three stem cell diseases known. The penaeid rod-shaped DNA virus (PRDV) was detected in the heart of the kuruma prawn Penaeus japonicus in both the satellite cells (Fig. 8a) and the post-mitotic cells. The satellite cells even seemed to be the primary target of the virus (Miyazaki et al. 2008). In the hepatopancreas of Penaeus monodon, fading of the nuclei and subsequent lysis was observed in the E-cells and their descendants after feeding of a compound feed that included leaf meal of the leguminous shrub Leucaena leucocephala (Fig. 8b) (Vogt 1990). This cytopathology was related to the non-protein amino acid mimosine, which occurs in rather high amounts in the Leucaena leaves. And the white spot syndrome virus (WSSV), one of the most disastrous disease agents in shrimp aquaculture, was shown to infect and destroy haematopoietic stem cells of the signal crayfish Pacifastacus leniusculus in vivo and in vitro (Fig. 8c) (Jiravanichpaisal et al. 2006). All of these stem cell diseases resulted in high mortalities, probably as a result of tissue necrosis accompanied by failure of tissue regeneration.
https://static-content.springer.com/image/art%3A10.1007%2Fs10522-010-9291-6/MediaObjects/10522_2010_9291_Fig8_HTML.jpg
Fig. 8

Stem cell diseases in crayfish and shrimp. a Penaeid rod-shaped DNA virus (PRDV) infection of satellite cell in heart of kuruma prawn Penaeus japonicus. Black arrow denotes virus particles in nucleus (n) of satellite cell (marked by arrowheads). The nucleus of the neighbouring myocardium cell (white arrow) is not infected. m, mitochondrium, mf, myofibril (from Miyazaki et al. 2008). b Mimosine-induced pathological decondensation of chromatin in nucleus (asterisk) and nucleolus (arrowhead) of E-cell progeny in hepatopancreas of giant tiger prawn Penaeus monodon. Arrow denotes normal nucleolus. n, nucleus of healthy cell (from Vogt 1990). cWhite spot syndrome virus (WSSV) infection (arrows) of cultured haematopoietic stem and progenitor cells from signal crayfish Pacifastacus leniusculus, visualized by in situ hybridization technique (from Jiravanichpaisal et al. 2006)

Marbled crayfish can be cultured under disease and toxicant free laboratory conditions as explained in the biological properties section. Therefore, they provide good material to test how ageing and longevity is modulated by diseases and chronic sublethal concentrations of xenobiotics. Moreover, disease free marbled crayfish could be utilized to estimate the metabolic cost of immune defence reactions and its effects on longevity. This aim could be achieved by eliciting immune responses either by infections with crayfish parasites of low pathogenicity (Vogt 1999) or by injection of a non-pathological immune stimulant and subsequent measurement of the energy expenditure (Martin et al. 2002).

Epigenetic drift during ageing and epigenetic intervention

There is increasing evidence that phenotypic characters and life history traits including longevity are shaped not only by the genetic code but also by the epigenetic code, which can be altered in response to environmental and intrinsic influences (Jaenisch and Bird 2003; Fraga and Esteller 2007; Calvanese et al. 2009; Martin 2009). The epigenetic code includes methylation of cytosines, histone modifications, DNA-binding proteins and microRNAs and can, unlike the genetic code, be erased and rewritten during life-time. Alteration of the epigenetic code can result in activation and silencing of genes or “dimmer switching” of gene expression and can affect susceptibility to diseases, ageing and longevity (Dolinoy et al. 2007; Van Vliet et al. 2007; Liang et al. 2009). Fraga et al. (2005) demonstrated that in human monozygotic twins the epigenetic code is similar in the first years of life but becomes diverse with increasing age (epigenetic drift).

DNA methylation is one of the best investigated epigenetic mechanisms (Fraga and Esteller 2007). However, not all of the established ageing models are suitable to investigate alterations of DNA methylation throughout life because Caenorhabditis elegans lacks methylated DNA at all (Bird 2002) and Drosophila melanogaster possesses methylated DNA only in the early stages of embryonic development (Lyko et al. 2006). The marbled crayfish, in contrast, has methylated DNA in both the juvenile and adult life stages, corresponding to approximately half of the value of humans (Schiewek et al. 2007; Vogt et al. 2008). Measurement of global 5-methyl cytosine methylation in two communally reared batches of the marbled crayfish revealed variations among batch-mates, tissue and age (Vogt et al. 2008). In a group of four 188-days old adolescents with total lengths of 3.2–4.2 cm DNA methylation amounted to 1.86 ± 0.088% in the hepatopancreas and 2.01 ± 0.060% in the abdominal musculature. These values were higher than in a group of three 626-days old adults of 6.2–7.0 cm total length, which had methylation values of 1.65 ± 0.130% in the hepatopancreas and 1.84 ± 0.076% in the musculature, suggesting that DNA methylation is slightly reduced with age. Global loss of DNA methylation is apparently also typical of ageing humans and other vertebrates, although individual genes can get hypermethylated at higher age (Richardson 2003; Fraga and Esteller 2007; Bollati et al. 2009).

Our first results on DNA methylation in the marbled crayfish suggest that this species may be well suitable to contribute to the emerging field of ageing epigenetics. Alterations of global DNA methylation during lifetime could be determined from blood or limb muscle samples using a refined analytical technique to precisely measure low methylation levels in small sample sizes (Schiewek et al. 2007). Age-related changes of the genome-wide DNA methylation patterns in various tissues could be determined by restriction landmark genomic scanning even without knowing particular genes (Thompson et al. 2010). The marbled crayfish could also be used to study the influence of epigenetic interventions on ageing and longevity, for instance by feeding methyl depleted and methyl enriched diets or by inhibition of methyltransferases (Cooney et al. 2002; Rattan and Singh 2009; Kuck et al. 2010).

Suitability of the marbled crayfish for research on genetic aspects of ageing and longevity

Knowledge on genetics of ageing and longevity has much increased in the last decade (Finch and Ruvkun 2001; Braeckman and Vanfleteren 2007; Bartke 2008; Kuningas et al. 2008; Paaby and Schmidt 2009). The key genes and molecular pathways identified so far encode for metabolism, maintenance and repair mechanisms and are often evolutionarily conserved. At present, the marbled crayfish is not yet ready to be used for investigation of genetic aspects of ageing and longevity because of poor knowledge on genetics in crayfish. However, due to the availability of rapid sequencing techniques and other molecular genetics tools closure of this gap is just a question of time and effort. The genome size of the marbled crayfish is expected to be similar to that of the con-generic Procambarus clarkii, which has a C-value of 4.46 pg (http://www.genomesize.com/). Such a genome size would be closer to the average of animals than the small genomes of Caenorhabditis elegans (0.10 pg) and Drosophila melanogaster (0.16 pg).

At present, there is only one crustacean genome fully sequenced, namely that of the water flea Daphnia pulex (Crustacea, Branchiopoda) (http://wfleabase.org; McTaggart et al. 2009). This species is a short-lived monitor organism in ecotoxicology and has occasionally been used for ageing research as well (Dudycha 2003). A genome sequencing project for the first decapod crustacean, the commercially most valuable Pacific white shrimp Litopenaeus vannamei, is on the way and the necessary EST databases, linkage maps or BAC libraries are rapidly increasing (Stillman et al. 2008; Koyama et al. 2010). Moreover, techniques for the identification of genes and their products are more and more adopted for shrimp and crayfish (Shechter et al. 2007; Robalino et al. 2009). For the short term, the gap of knowledge on genetics of ageing in decapod crustaceans could be bridged by searching for ageing genes on the basis of Drosophila data (Helfand and Rogina 2003; Kennedy 2008; Paaby and Schmidt 2009). Since the Crustacea and Hexapoda are taxonomical sister groups (Giribet et al. 2005) many orthologous genes are expected to be shared by the marbled crayfish and the fly. This idea is corroborated by analysis of the genome of the water flea, which showed closest gene homology to insects but also many homologies to mouse (http://wfleabase.org).

Another promising approach would be the production of transgenic lineages in the marbled crayfish. The transfer of genes into the ovary and spawned eggs by microinjection, particle gun bombardment, electroporation and transfection was already successfully accomplished in the crayfish Procambarus clarkii and the shrimp Litopenaeus vannamei (Sarmasik et al. 2001; Sun et al. 2005) and could easily be adopted for the marbled crayfish. There is even an example that an introduced vertebrate growth hormone gene was physiologically effective in the acceptor shrimp (Arenal et al. 2008). With respect to genetic engineering of ageing, the parthenogenetic marbled crayfish would have an advantage over gonochoristic species since an introduced transgene would be propagated in pure form across generations.

Suitability of the marbled crayfish and other crustaceans for research on evolutionary aspects of ageing and longevity

Ageing and longevity vary very much among the higher metazoan taxa. Some hydrozoans, for instance, are virtually immortal because they can reverse development and rejuvenate their tissues. Fishes, reptiles and crustaceans show no or little functional senescence, whereas mammals and insects develop a pronounced decline of vitality in their post-reproductive life period. However, even within the same animal group, there can be great differences, depending on the life strategies of the species. For example, within the mammals ageing and longevity differs markedly between mice, which are short-lived r-strategists, and primates that are long-lived K-strategists. Therefore, understanding of the evolution of ageing, or testing and refining of the evolutionary theories of ageing (Rose 1991; Kirkwood 2002, 2008; Hughes and Reynolds 2005), requires information from all of the major animal groups and the most representative life histories.

One of the hot topics of the evolutionary biology of ageing is the relationship of senescence and growth formats. As a rule of thumb, determinately growing animals like most mammals and insects are usually characterized by reproductive, mechanical and tissular senescence, lack of telomerase in the post-mitotic tissues and the occurrence of age-related diseases. Therefore, they are well suitable to investigate tissue degeneration with age and development of age-related diseases inclusive of cancer (Rossi et al. 2008; Salomon and Jackson 2008; Anisimov et al. 2009). Indeterminately growing animals like most fish, reptiles, bivalves, echinoderms and decapod crustaceans usually display negligible senescence, have telomerase in their somatic tissues and do not develop age-related diseases (Klapper et al. 1998; Miller 2001; Abele et al. 2008; Ebert 2008; Vogt 2008a). They are thus suitable to study the molecular mechanisms that prevent tissue deterioration and age-related diseases and keep stem cells running until old age. Maximal insight into the ageing process is expected by comparison of determinately and indeterminately growing species, particularly if these come from the same phylogenetic clade like mammals and fish in the vertebrates and insects and crustaceans in the arthropods.

Of great value are probably also those animals, which deviate from the rule of thumb given above. For instance, mice express telomerase in some somatic tissues and mole rats show negligible senescence despite determinate growth (Buffenstein 2008; Gorbunova and Seluanov 2009). And the water flea Daphnia magna shows reproductive and tissular senescence in its final period of life despite indeterminate growth (Schulze-Röbbecke 1951). In the Decapoda, a small minority of the species deviate from the indeterminate growth format having either a fixed number of adult instars like the spider crab Maja squinado or variable numbers of adult instars like the brachyuran crab Carcinus maenas, depending on conditions (Hartnoll 1982). Both species are easily available and can be cultured. In Maja squinado growth is stopped after 2–3 years with a final moult. Thereafter, life continues for another 2–5 years and includes several reproduction cycles. In this period of life, wear and tear of the exoskeleton becomes increasingly obvious indicating mechanical senescence. It would be interesting to see if there is also senescence of the soft tissues in this determinately growing decapod and if stem cell activity ceases after the final moult.

So far, the Crustacea have largely been neglected in evolutionary considerations on ageing and longevity (Rose 1991; Kirkwood 2002) although they are placed fourth among the Metazoa in terms of species diversity (52.000 species) and first with respect to morphological diversity (Martin and Davis 2001). The differences in both maximum size of the adults and life span in the crustaceans are close to a factor of one thousand, which is very exceptional. The use of short-lived ‘lower crustaceans’ such as Copepoda, Cladocera and Anostraca was occasionally propagated for studying the evolutionary biology of ageing but only rarely implemented (Reznick 1993; Dudycha 2000, 2003). The use of the longer-lived Decapoda was probably impeded by difficulties in age estimation because decapods have no growth ring-bearing structures like bivalves or fishes. However, this shortcoming is no problem in the laboratory where the age of specimens is usually known. Ageing of decapods in the wild is meanwhile possible by the lipofuscin based ageing technique, which proved to be the highly reliable in crayfish, shrimps and lobsters (Sheehy 2002), and by newly developed long-term marking methods (Buřič et al. 2008).

Marbled crayfish may contribute to the evolutionary biology of ageing as a representative of the decapod crustaceans and of animals with indeterminate growth, high regeneration capacity and extended brood care (K-strategist). They reach their essential life span, the time point required to ensure the continuation of generations, in about 7 months already but usually live on and reproduce for another 8–24 months. Decapod species with other biological features are available to refine the ageing picture in the Decapoda. For instance, atyid shrimps of the genus Caridina are shorter-lived (≤1 year) than the marbled crayfish and clawed lobsters of the genus Homarus are much longer-lived (>70 years) (Sheehy et al. 1999). Penaeid shrimps lay up to 500.000 eggs per spawning and release the eggs directly into the water (r-strategists), lobsters and crabs have clutch sizes of many thousand eggs and care for the eggs only, whilst freshwater crayfish have maximal egg numbers of a few hundred and care for the eggs and the first juvenile stages (K-strategists) (Vogt and Tolley 2004).

Within the Crustacea, the water fleas Daphnia pulex and Daphnia magna may serve as antipodes to the marbled crayfish. Daphnia species are arguably among the best understood organisms and serve as model organisms in ecotoxicology, environmental genomics and evolutionary biology. They can reproduce by obligate parthenogenesis like the marbled crayfish, depending on strain and culture condition, and also show indeterminate growth and extended brood care. However, they measure only 3 mm as adults, have a generation time of 5–10 days and a life span of 40–50 days at 25°C (Dudycha 2000). Despite indeterminate growth, they show clear signs of reproductive and tissular senescence in the last 15% of life, starting with depletion of the energy reserves in the fat body and continuing with damages of the alimentary canal (Schulze-Röbbecke 1951).

Conclusion

For some time there was an intense debate whether invertebrate ageing models, which bear little or no resemblance to the ageing biology of humans, can be useful for understanding or even postponing human ageing (Austad and Podlutsky 2006). At present, there is little doubt that the invertebrate models Drosophila melanogaster and Caenorhabditis elegans have proven invaluable for ageing research, particularly with respect to genetics of ageing (Partridge 2009). Another occasionally addressed question is whether there is a role for further invertebrate models in ageing research (Austad 2009). Introduction of a new invertebrate ageing model makes only sense, if it displays ageing phenomena that are not yet covered by the existing models. This is clearly the case in the marbled crayfish, arguing for a more intense use of this crustacean in ageing research. Features that are not covered by the established invertebrate models are direct development, suitable size for individual biochemical analyses and longitudinal studies, negligible senescence, high regenerative capacity, intense stem cell activity in the adult life stages and methylation of the DNA throughout life.

The results obtained so far with the marbled crayfish extend our knowledge on ageing and longevity in the poorly investigated Decapoda and thus add to the catalogue of ageing phenomena in the animal kingdom. Some of the recorded phenomena are clade specific, for example the escape from mechanical senescence by moulting, but others have a more general character and appear well suitable to contribute to the understanding of basic issues of ageing. Because of its clonal nature and eurytopicity, the marbled crayfish is particularly promising to investigate non-genetic aspects of ageing such as stochastic developmental variation, allocation of metabolic resources, environmental and social stress, caloric restriction and regeneration. However, in future, genetically manipulated lineages may be useful for research on genetics of ageing as well. The marbled crayfish has also a good potential for studying highly topical ageing issues such as age-related epigenetic drift and maintenance of adult stem cell function until old age. Last but not least, it may contribute to mechanistic and evolutionary theories of ageing, representing the speciose decapod crustaceans and animals with indeterminate growth, high regeneration capacity, social hierarchy and extensive brood care.

Ageing research in the marbled crayfish and its relatives is not only of academic interest but also of practical relevance. In crustacean fisheries, knowledge on ageing and longevity is required to correctly estimate the fishery potential of wild populations. In aquaculture, it is helpful to optimize grow-out and reproduction strategies of highly valuable species and strains. Some of the ageing phenomena recorded for the marbled crayfish and its relatives even seem to have a good potential for the development of anti-ageing interventions in humans. One promising approach is the use of astaxanthin as a highly effective scavenger of free radicals and inhibitor of lipid peroxidation, which already has been the subject of some studies (Hussein et al. 2006). Another approach may be inferred from maintenance of stem cell integrity until old age in the virtual absence of cancer. Understanding of the underlying molecular mechanisms may help to develop a therapy that improves tissue regeneration and diminishes tumour formation in elderly people.

Copyright information

© Springer Science+Business Media B.V. 2010