ICES Journal of Marine Science (2011), 68(10), 2053–2058. doi:10.1093/icesjms/fsr144 Telomere length analysis in crustacean species: Metapenaeus macleayi, Sagmariasus verreauxi, and Jasus edwardsii Rosamond M. Godwin1*, Stewart Frusher2, Steven S. Montgomery3, and Jennifer Ovenden1 1Molecular Fisheries Laboratory, Agri-Science Queensland, Department of Employment, Economic Development and Innovation, Level 3 Ritchie Building (64A C-Wing), Research Road, The University of Queensland, Brisbane 4067, Australia 2Tasmanian Aquaculture and Fisheries Institute, University of Tasmania, Private Bag 49, Hobart 7001, Australia 3Industry and Investment NSW, Cronulla Fisheries Research Centre of Excellence, PO Box 21, Cronulla, NSW 2230 Australia *Corresponding Author: tel: +61 7 3346 6518; fax: +61 7 3346 6501; e-mail: [email protected] Downloaded from https://academic.oup.com/icesjms/article/68/10/2053/611301 by guest on 29 September 2021 Godwin, R. M., Frusher, S., Montgomery, S. S., and Ovenden, J. 2011. Telomere length analysis in crustacean species: Metapenaeus macleayi, Sagmariasus verreauxi, and Jasus edwardsii. – ICES Journal of Marine Science, 68: 2053–2058. Received 1 March 2011; accepted 20 June 2011; advance access publication 15 September 2011. Estimates of age and growth in crustaceans have been historically problematic and presented significant challenges to researchers. Current techniques of age determination provide valuable data, but also suffer from disadvantages. Telomeric DNA has been proposed as an age biomarker because it shortens with age in some species. In this study, the feasibility of using telomere length (TL) to estimate age was examined in the school prawn Metapenaeus macleayi and the spiny lobsters Sagmariasus verreauxi and Jasus edwardsii. Carapace length (CL) was used as a surrogate for age, and terminal restriction fragment assays were used to test the relationship between TL and size. Degradation of telomeric DNA with time during storage significantly influenced TL estimates, particularly for M. macleayi. TLs obtained from species in this study were 10–20 kb. No relationship between CL and TL was detected for any of the test species, and TL did not differ between male and female M. macleayi. TLs of J. edwardsii pueruli were unexpectedly shorter than those of J. edwardsii adults. The suitability of TL as an age biomarker in crustaceans may be limited, but further research is needed to elucidate telomere dynamics in these species with their different life histories and lifespans. Keywords: age biomarker, age estimation, Australia, crustacean, telomere length, TRF. Introduction In many vertebrate somatic cells, telomeres shorten with each Accurate assessments of age are required for understanding mitotic cell division over the animal’s life as a result of incomplete growth, maturity, reproduction, longevity, and mortality of replication of the DNA (Watson, 1972). This shortening has been animals in a wild population (Campana, 2001). They are basic correlated with age in mammals and some birds (Haussmann and considerations in stock assessment models for sustainable manage- Vleck, 2002; Haussmann et al., 2003; Vleck et al., 2003; Nakagawa ment of fisheries. et al., 2004), as well as in fish (Hatakeyama et al., 2008; Hartmann Estimating age and growth of crustaceans has been historically et al., 2009). It is also correlated with age in oysters and shell size in problematic, presenting significant challenges for researchers. abalone (RMG, unpublished data). Therefore, telomere length Methods used to estimate age or growth include tag-recapture, (TL) has been suggested as a potential age biomarker in animals size frequency distributions, size or weight measurements, and where it is difficult to determine age using conventional lipofuscin accumulation. Such methods provide valuable data methods (von Zglinicki and Martin-Ruiz, 2005; Haussmann and but can be inaccurate, subject to biases, costly, time-consuming, Mauck, 2008). The telomere repeat sequence of crustaceans is and deleterious to the animal (reviewed by Wahle and Fogarty, (TTAGG)n, which is common in many arthropod species 2006). Direct measures of size or weight are only weakly correlated (Klapper et al., 1998; Sahara et al., 1999; Lang et al., 2004; with age in crustaceans (Zheng et al., 1995; Uglem et al., 2005). Vı´tkova´ et al., 2005; Elmore et al., 2008). Detailed studies of TL Lipofuscin pigments, which accumulate with age in crustacean in crustaceans, however, are yet to be conducted. neural tissue, have yielded limited success, and the accuracy of In this study, the relationship between TL and age (as indicated the method is still under debate (Harvey et al., 2008; Sheehy, by size) is examined in three crustacean species with different life- 2008); accumulation of lipofuscin is not independent of environ- spans and life-history characteristics. The test species were school mental effects, particularly temperature (Wahle and Fogarty, prawns, Metapenaeus macleayi, and two species of spiny lobster, 2006). Therefore, the development of an alternative, low-cost, Sagmariasus verreauxi (formerly Jasus verreauxi) and Jasus non-lethal method to estimate age in crustaceans reliably would edwardsii. All three are commercially important and common in be a major improvement for fisheries management. Australian waters. School prawns are short lived (lifespan 12–18 Telomeres are the structures of highly repeated DNA sequences months; Montgomery et al., 2010) and endemic to estuarine and and their associated proteins that protect the ends of the linear inshore waters from southern Queensland to eastern Victoria. chromosomes from degradation and fusion (Blackburn, 1991). Juvenile and subadult prawns inhabit estuaries, generally near # The State of Queensland (through the Department of Employment, Economic Development and Innovation), 2011. 2054 R. M. Godwin et al. beds of seagrass. Adults are found mainly in marine waters, but 3-year-old lobsters were originally caught off Port Hacking as also in small numbers in estuaries (Ruello, 1977; Coles and recently settled pueruli. These small lobsters were then grown in Greenwood, 1983). Sagmariasus verreauxi and J. edwardsii captivity for some 3 years before sampling. The sampled J. edwardsii have relatively long lifespans, 30 years for S. verreauxi comprised two broad size classes, pueruli (CL unknown) and adults (Montgomery et al., 2009), and potentially longer for J. edwardsii. (CL 80–120 mm). Sagmariasus verreauxi is distributed in warmer waters along the Lobsters were transported to the laboratory alive and eutha- east coast of Australia and the North Island of New Zealand nased in ice slurry. Sampled tissues (Table 1) were dissected (Montgomery et al., 2009), and J. edwardsii widely around from the animals, placed in tubes, immediately snap-frozen in southern mainland Australia, Tasmania, and New Zealand liquid nitrogen, and then transferred to a 2808C freezer for long- (Booth, 2007). term storage. Lobsters such as S. verreauxi and J. edwardsii exhibit indetermi- nate growth patterns (Wahle and Fogarty, 2006), and despite its Downloaded from https://academic.oup.com/icesjms/article/68/10/2053/611301 by guest on 29 September 2021 shorter life, M. macleayi is also likely to exhibit indeterminate Extraction of total genomic DNA and TRF assays growth. Hence, the three species grow continuously throughout Genomic DNA of high molecular weight was extracted from their life and theoretically have no upper size limit. frozen tissue using standard methods (Sambrook and Russell, The aim of this study was to determine whether TL was suitable 2001). Extracted crustacean DNA appeared to be susceptible to as an age biomarker in crustaceans. If suitable, it could provide a degradation, so to minimize the co-purification of contaminating molecular method of age determination that would allow age to be material, a smaller quantity of fresh tissue was used during cell estimated from small samples of tissue, a non-lethal and cheaper lysis. Hence, 50 mg of tissue was digested per millilitre of digestion alternative to conventional methods of collecting age data. The buffer (10 mM Tris, pH 8.0, 5.0 mM EDTA, pH 8.0, 0.5% SDS, strategy was to compare TLs in at least two divergent age/size –1 200 mgml proteinase K). Different combinations of tempera- cohorts per species and to add data from other cohorts if ture and incubation period were optimal for cell lysis and digestion differences were indicated. TL can be estimated from very small of fresh tissue for each species (Table 2). Proteins and other con- quantities of tissue using PCR methods (Cawthorn, 2002), but taminants were removed by salt precipitation in 5 M potassium in the absence of any available sequence data for these test acetate and phenol/chloroform extractions. species, a terminal restriction fragment (TRF) assay was used in The resulting DNA was dissolved in TE (Tris–EDTA) buffer, this initial study. Our hypothesis was that there would be an pH 8.0, with 1 mM dithiothreitol added to minimize degradation inverse relationship between TL and age, as has been observed in and oxidation of the telomeric DNA. All DNA samples were stored many vertebrate species. at 48C. The quality and the quantity of genomic DNA were assessed by gel electrophoresis before each assay. High-quality Material and methods DNA samples contained molecular weight fragments .23 kb Sampling and tissue storage and a minimal presence of low molecular weight fragments Size (as indicated by carapace