Age determination method using neurolipofuscin: a reply to Buesa

Estimates of age of some types of animals such as fish and bivalves are possible by analysing hard structures such as otoliths, shells and vertebrae, which are retained throughout life and show agerelated changes. However, for animals such as crustaceans and other invertebrates that have no hard structures that persist throughout their lives, alternativemethods for determining age have emerged. One alternative method, and the one that we used in our 2009 paper in the New Zealand Journal of Marine and Freshwater Research (Matthews et al. 2009) and other work on the Caribbean spiny lobsterPanulirus argus (Maxwell et al. 2007, 2009, 2013), is histological quantification of lipofuscin in soft tissues. Lipofuscin is amixture of oxidised, cross-linked proteins that accumulate post-mitotically in cells of diverse types. They are associated with the lifespan of single post-mitotic cells, and thus also with the lifespan of the animal. Their rate of accumulation is correlated with metabolic activity, and thus they are an indicator of metabolic age more than chronological age. Lipofuscin has been used tomeasure the age inmany species, including humans, other vertebrates and also many invertebrates (e.g. Brunk & Terman 2002; Lomovasky et al. 2002; Porta 2002; Jung et al. 2007; Doubleday & Semmens 2011). Lipofuscin has been applied to the study of crustaceans. Beginning in the late 1980s, Matt Sheehy and colleagues pioneered using histologically measured neurolipofuscin for aging various species of crustaceans, including crayfish, clawed lobsters and spiny lobsters (Sheehy 1989, 1990a, 1992; Sheehy et al. 1996, 1998; de Kerros et al. 1995; Vila et al. 2000; Fonseca et al. 2003, 2005a,b). Other researchers have also used it for aging other crustacean species, including crayfish, American lobster, shrimp, ghost shrimp, amphipods and mantis shrimp (Wahle et al. 1996; Belchier et al. 1998; Bluhm&Brey 2001;Bluhm et al. 2001a,b;Kodama et al. 2006). In these studies, neurolipofuscin content has consistently been found to be more highly correlatedwith age than is size to age, becausewhile growth rates may vary with age, the free-radicalgenerating respiratory catabolism and lipofuscin accumulation continue. A central tenet of lipofuscin-age research is that lipofuscin accumulates predominately relative to chronological age and temperature, not somatic growth. In his critique of our work, Buesa concludes that ‘neurolipofuscin cannot be used to determine age structure and growth rates of the Caribbean spiny lobster Panulirus argus’. He bases this statement in large part on his assertion that the population of lobsters used to determine the ageneurolipofuscin content relationship in our study was not in the same physiological condition as our experimental animals. According to Buesa, our laboratory animals must have had a higher energy intake and lower energy expenditure than field animals, thus having a higher metabolic rate and different relationship between age and neurolipofuscin content compared to field animals. Buesa’s concerns that food availability may influence neurolipofuscin are not new and were directly