Stable isotope analyses reveal major nutritional deficiencies in captive vs. field juvenile individuals of Pinna nobilis

Highlights

• Disease outbreaks in Pinna nobilis have forced dietary studies for captive breeding.

• Captive individuals feed phytoplankton gel/sediment diets were enriched in δ¹³C and δ¹⁵N.

• These patterns suggest the use of own N and C pools due to severe nutritional stress.

• Field individuals showed a 34.9% contribution from the zooplankton fraction.

• Zooplankton consumption was experimentally confirmed using Artemia nauplii.

Abstract

The pen shell, Pinna nobilis L. is critically endangered by the spread of a haplosporidan parasite. Stable isotopes have been shown an association with dietary assimilation, trophic level, and body condition, and can provide valuable information for ex situ conservation and breeding. In this context, the aim of this study was to investigate the nutritional status of individuals using δ¹³C and δ¹⁵N patterns across pen shell body tissues as tracers of elemental incorporation from treatment diets based on commercial gels and living phytoplankton and/or sediment. Further comparisons were also conducted with field animals and diets to better understand nutritional needs. Captive individuals, were enriched in δ¹³C and δ¹⁵N (~18.9 and 1.3‰, respectively), suggesting severe nutritional stress and utilization of own N and C pools, especially muscle. The mixing model for field individuals evidenced a large contribution from the zooplankton fraction (34.9%), which was further confirmed by experimental feeding with Artemia nauplii.

Introduction

The fan mussel, Pinna nobilis, is a critically endangered bivalve mollusk endemic to the Mediterranean Sea (Kersting et al., 2019). During the fall of 2016, populations started to be seriously affected by the spread of a parasitic disease caused by the protozoan Haplosporidium pinnae (Catanese et al., 2018), alone or in combination with a new species of Mycobacteria (Carella et al., 2019, 2020). Mass mortality events close to 100% of the individuals were first observed in Spanish waters during the fall of 2016, and then spread to other Mediterranean countries including France, Italy, Morocco, Tunisia, Turkey, Cyprus, Malta, and Greece, putting the future of species in jeopardy in less than three years (García-March et al., 2020). Given the threatening scenario for the species, the Spanish government implemented a rescue project for 215 individuals from the remaining populations, and 100 adult pen shells from the Alfacs Bay in the Ebro Delta were kept at IRTA facilities. However, pen shells appeared to be stressed under captive conditions and were also prone to mortality by emerging bacterial pathogens such as Vibrio mediterranei thus preventing successful breeding (Prado et al., 2020a; Andree et al., 2021; Cabanellas–Reboredo et al., 2019). Also, larval cultures using field individuals have so far failed to obtain viable seed, apparently for similar reasons related to bacterial vulnerability (Trigos et al., 2018), thus hindering the species production under captivity conditions. Yet, bivalve aquaculture has provided effective solutions for the rehabilitation of other endangered bivalve populations subjected to overexploitation (Loor et al., 2016; Lodeiros et al., 2016), pointing that further research is needed to establish adequate protocols for pen shell maintenance and growth.

In particular, the proper formulation of diets is generally indicated as a major bottleneck for rearing bivalves in captivity (FAO, 2006). A balanced diet as close as possible to field feeding patterns may reinforce the efficiency of the immune system in cultured bivalves (Hégaret et al., 2004). For instance, distinctive growth patterns and mortality rates by V. mediterranei have been observed for juvenile pen shells fed different combinations of phytoplankton gels (1–3 species) and an in situ produced diet based on three microalgae species, suggesting that diet quality can mediate disease resistance (Prado et al., 2020b). Similarly, the failure of larval cultures of P. nobilis (Trigos et al., 2018) due to an association between disease susceptibility and unbalanced diet formulation cannot be discarded. P. nobilis has been indicated to ingest a diversity of phytoplankton species (up to 68 different taxa), along with large quantities of detrital material (up to 95%), and some micro and mesozooplankton species (Davenport et al., 2011; Morton and Puljas, 2019), although the assimilation of the different fractions has not been assessed and dietary contributions are inferred from presence within stomach contents. For logistical reasons, most larval hatcheries including attempts conducted with P. nobilis (Trigos et al., 2018), only use 2–3 species of microalgae (at least one Haptophyceae and one Bacillariophyceae; Robert and Gérard, 1999) to feed the bivalves without a clear knowledge of their dietary requirements (Muller-Feuga et al., 2003). Besides, the finding of an adequate formulation could become even more challenging for species such as P. nobilis that may use resources other than phytoplankton.

The determination of stable isotope abundances is an important tool for assessing dietary contributions and element assimilation by consumers over long periods of time (months and years; Hobson and Clark, 1992). Isotopic signatures of potential dietary resources can be used to determine their relative contributions to the mixed signature of the animal using mass balance equations (Phillips and Gregg, 2003), but their application relies on the existence of distinctive signatures among diet sources (DeNiro and Epstein, 1978), and on the availability of accurate estimates of consumer-diet fractionation (Prado et al., 2012). Organic matter contents in phytoplankton and zooplankton fractions, and in sediment samples are expected to have locally distinctive isotopic signatures (e.g., Prado et al., 2014a), that may allow the determination of the relative contribution of these broad resources to pen shell diets in situ. Besides, experimental evaluation of isotopic signatures under captivity conditions may also provide useful information about the efficiency of elemental assimilation (C and N) under each dietary condition (Prado et al., 2012), thus aiding dietary formulation. In regard to fractionation, bivalves are usually considered to be slightly enriched in δ¹³C by 0–1‰ (i.e. Δ¹³C) and to resemble the value of their diet (McCutchan et al., 2003; Post et al., 2007; Marín Leal et al., 2008). For Δ¹⁵N (i.e. the increase in δ¹⁵N between consumers and their diets), bivalves have been reported lower enrichment values than the usually applied 3.4‰ (Kwak and Zedler, 1997, Peterson et al., 1985). Raikow and Hamilton (2001) examined available data on marine bivalves from Minagawa and Wada (1984) and Fry (1988) and suggested that bivalves tend to be only 1.7‰ enriched in δ¹⁵N relative to suspended food resources. Furthermore, Post et al. (2007) indicated a Δ¹⁵N value of only 0.4 for freshwater mussels, and Deudero et al. (2009) consistently found negative values of Δ¹⁵N (from −0.11 to −2.5‰) and Δ¹³C (0.27 to −2.29‰) across tissues of Mytilus galloprovincialis from an oligotrophic Mediterranean environment.

The population of P. nobilis in the Alfacs Bay has been indicated as one of the largest in the Mediterranean within an estimate of over 90,000 individuals (Prado et al., 2014b). Unfortunately, the area was infected by H. pinnae in July 2018, although most of the inner bay remains unaffected (Prado et al. in press). A few months earlier, in late January 2018, abundant juvenile recruitment was observed on an emerged sand-bar (less than 10 cm water depth) adjacent to the area of adult collection for the adult rescue project implemented by the Spanish government (Prado et al., 2020a). These juveniles, which would have died in the following months due to desiccation in the sand bar, provided a suitable source of individuals for field and dietary experimentation at the IRTA aquaculture facilities (see also Prado et al., 2020b). In this context, the objectives of this study were twofold: (1) to assess the adequacy of artificial diets for the species maintenance using stable isotope analysis across P. nobilis tissues, and when possible, determining the dietary contributions of each diet item; and (2) to evaluate the contributions of the three potential natural resources (i.e., the phytoplankton, and zooplankton fractions and the detrital material) to the diets of field individuals. Besides, a feeding experiment at different concentrations of newly hatched Artemia nauplii was conducted to account for possible discrepancies between ingestion and assimilation of zooplankton.