Evaluation of the ¹³⁷Ba mass-marking technique and potential effects in the early life history stages of Sepioteuthis lessoniana

Additional keywords: barium isotopes, hatchling size, statolith chemistry.

Cephalopods are crucial in commercial fisheries (Hunsicker et al. 2010; Jereb and Roper 2010) and are a vital component in marine food webs as a dietary source for numerous marine organisms (Clarke 1996; Klages 1996). The growth and population dynamics of cephalopods respond to environmental conditions, such as food abundance, temperature and water (Jackson and Moltschaniwskyj 2002; Forsythe 2004). Similar to most marine organisms, the degree of larval dispersal and distributional range of cephalopods determine connectivity of the population and are related to conservation of the ecosystem (Swearer et al. 1999; Cowen and Sponaugle 2009). Several methods for investigating dispersal patterns and migratory behaviours of marine animals in their early life history stages have been developed, but are not always applicable. For example, external tags may harm organisms and increase mortality rates (Sauer et al. 2000; Replinger and Wood 2007; Barry et al. 2011). In addition, the large tag size is impossible to use on squid paralarvae or juveniles (Nagasawa et al. 1993; Semmens et al. 2007). Although alternative biomarkers, like parasite communities and molecular markers, are not restricted by larval size, these methods do not provide detailed information on larval dispersal and movement patterns (Bower and Margolis 1991; Buresch et al. 2006). Traditional marking methods clearly have their limitations, and advanced marking techniques for early life history stages are required.

Recently, enriched stable isotope-marking techniques have been used, such as injecting enriched stable isotopes into mature females (Thorrold et al. 2006; Almany et al. 2007) or immersing offspring in water with enriched stable isotopes (Munro et al. 2008; Smith and Whitledge 2011; Woodcock et al. 2011a). Marked individuals with unique isotopic signatures in their biogenic carbonates are distinguishable from natural populations (Munro et al. 2008; Smith and Whitledge 2011). Stable isotopes of barium and strontium are commonly used in the marking experiments. Both elements have similarities in their ionic radius to Ca²⁺ and will likely be a substitute for Ca²⁺ in biogenic carbonates (Speer 1983). It is easier to perform this technique with barium for marine organisms because barium concentrations are low in natural seawater (varying in the range 0.007–15 ppm in seawater and fresh water; Bernat et al. 1972; Kresse et al. 2007). In addition, marking by feeding Ba-enriched dietary items has been suggested as a more effective method in marine systems (Woodcock and Walther 2014). The ¹³⁷Ba isotope is stable in lower abundance (11.23%) and is not the major barium isotope (71.1% for ¹³⁸Ba; Rosman and Taylor 1998). Because enrichment with ¹³⁷Ba in calcified structures is greater than environmental levels, the mark is easily detected and shows a difference from natural seawater signatures (Thorrold et al. 2006). Therefore, ¹³⁷Ba mass-marking techniques are likely suitable for tracking larval dispersal and movement patterns of cephalopods in the natural environment.

To date, only two studies have evaluated the enriched stable isotope mass-marking technique in cephalopod early life stages (Pecl et al. 2010; Payne et al. 2011). Maternal injection of stable isotopes in cephalopods may not mark offspring because there is difficulty in determining the oocyte maturity stage in cephalopod ovaries (Pecl et al. 2010). By contrast, Payne et al. (2011) marked Sepia apama hatchlings by immersing their spawning eggs in solutions spiked with different ¹³⁷Ba concentrations (0.3 and 1 ppb) and for different durations (2, 5 and 8 days). These authors demonstrated the potential of using stable isotope-marking techniques to assess the population dynamics of cephalopods in the field (Payne et al. 2011). However, the efficacy of marking techniques varies among species. Thus, which method produces high-quality marks with lower costs in target species must be assessed (Warren-Myers et al. 2018).

Compared with fluorescent dyes (e.g. alizarin complexone), techniques for mass marking with enriched stable isotopes are usually seen as non-toxic to experimental offspring (Williamson et al. 2009; Woodcock et al. 2011a; Warren-Myers et al. 2018). Yet, there is evidence to suggest that enriched stable isotope mass-marking techniques may affect hatchling size (Williamson et al. 2009; Starrs et al. 2014a, 2014b). The size at hatch is crucially related to swimming and foraging abilities, which subsequently affect survival rate and reproduction (Sogard 1997). The effect of marking method on the hatchling size of cephalopod species is unclear. Moreover, the process of enriched stable isotope marking may alter physiological regulation, which may cause erroneous interpretations of cephalopod behaviours (de Vries et al. 2005). Such effects need to be carefully evaluated and considered in enriched stable isotope mass-marking experiments.

The present study used the method of immersing eggs of Sepioteuthis lessoniana with the enriched isotope of ¹³⁷Ba and aimed to evaluate the optimum spiked concentration and immersion duration for successful marking of their statoliths. In addition, we examined hatchling size (mantle length (ML) and bodyweight) and growth condition factor (Fulton’s condition factor K) after marking, in addition to analysing elemental concentrations (²⁴Mg, ²⁵Mn, ⁶³Cu, ⁶⁴Zn, ⁸⁸Sr and ²⁰⁸Pb) in hatchling statoliths. Changes to statolith chemistry may be induced by physiological regulation and varying accretion rates of calcium carbonate (Hamer and Jenkins 2007; Sturrock et al. 2015). This study looked at the potential effects of the enriched stable isotope mass-marking technique on cephalopod size at hatch and statolith chemistry.

Bigfin reef squid S. lessoniana are widely distributed in the neritic waters of the Indo-Pacific Ocean (Okutani 2015). They spawn almost throughout the entire year, with the embryo development period being ~23–26 days when the temperature is ~25°C (Segawa 1987). S. lessoniana egg capsules were collected by SCUBA diving from artificial bamboo reefs (depth ~20–25 m) at Wanghaixiang Bay in northern Taiwan in August 2015. The egg capsules were put in an opaque plastic bucket with natural seawater and immediately transported (<2 h) to the aquaculture station of the National Museum of Marine Science and Technology (Keelung, Taiwan). Before the experiments, all the egg capsules were suspended on nylon threads in a 200-L tank for initial acclimation. Natural seawater was collected from Wanghaixiang Bay and pumped through a filter bed to supply the rearing system. During the experiment, the seawater temperature was maintained at a mean (±s.d.) temperature of 25 ± 1°C, salinity was maintained at 34.1–34.7 PSU and experiments were conducted under a 12-h light–dark cycle.

In all, 150 eggs with visible embryos at 23–25 developmental stages, which were classified according to Segawa (1987), were randomly selected for each group and reared in a 10-L tank. There were nine experimental groups in total: three ¹³⁷Ba spike concentrations (0.2, 0.5 and 1 ppm) and three immersion durations (1, 3 and 7 days). These groups were compared against a control group with no spiking. Different ¹³⁷Ba concentrations were prepared by dissolving ¹³⁷Ba-enriched BaCO₃ (≥91% ¹³⁷Ba and 8% ¹³⁸Ba; Trace Sciences International, Richmond Hill, ON, Canada) in ultrapure water. For groups immersed for >1 day, half the rearing seawater was replaced daily and extra ¹³⁷Ba spike was added to maintain the concentration of the ¹³⁷Ba spike. After immersion, eggs were returned to the natural seawater until they hatched. The ML (mm) and bodyweight (mg) of the hatchlings were measured. Individuals were then killed by exposure to a high concentration of ethyl alcohol and their statoliths extracted. The experimental procedures followed the Guidelines for the Care and Welfare of Cephalopods in Research – A Consensus Based on an Initiative by CephRes, FELASA and the Boyd Group (Fiorito et al. 2015). The growth condition factor of hatchlings was estimated based on Fulton’s condition factor K, calculated as follows (Ricker 1975):

Statoliths were extracted under a stereomicroscope (SteREO Discovery, V12; Carl Ziess Microscopy GmbH, Jena, Germany), cleaned ultrasonically with 70% hydrogen peroxide to remove adhering tissue, rinsed three times in ultrapure water, placed into acid-washed Eppendorf microcentrifuge tubes and oven dried overnight. The statoliths were then transferred to 1.5-mL acid-washed high-density polyethylene vials and weighed on a microbalance to the nearest 10 μg. Individual pairs of statoliths were dissolved in 0.5 mL of 0.3 M ultrapure nitric acid. Solutions were analysed using inductively coupled plasma–mass spectrometry (ICP-MS; ELEMENT XR ICP-MS; Thermo Scientific, Bremen, Germany) at the Institute of Earth Science, Academia Sinica, Taipei, Taiwan. Nine isotopes (²⁵Mg, ⁴³Ca, ⁵⁵Mn, ⁸⁸Sr, ¹³⁷Ba, ¹³⁸Ba and ²⁰⁸Pb) were analysed in a low-resolution mode and two isotopes (⁶³Cu and ⁶⁴Zn) were evaluated in a medium-resolution mode. Element concentration is shown as a ratio relative to the concentration of calcium (mean element (Me) : Ca ratio). The carbonate (otolith)-certified reference material FEBS-1 (National Research Council, Ottawa, ON, Canada) was used to determine the Me : Ca ratio of samples and analysed every fifth sample to instrument drift. In regard to the matrix effect, statolith solutions in various calcium concentrations (0.5, 1, 5, 25 and 50 ppm) were tested and the Me : Ca ratios of every sample were normalised at the same level of matrix concentration. The relative standard deviations of the Me : Ca ratio measurements of FEBS-1 were lower than 4% for most elements expect Mn : Ca (Mg : Ca 3.37%; Mn : Ca 5.16%; Sr : Ca 1.79%; Ba : Ca 3.44%; Pb : Ca 2.46%; Cu : Ca 1.85%; Zn : Ca 3.06%), and the percentage accuracy of the Me : Ca ratios was better for Mg : Ca, Sr : Ca, Ba : Ca and Pb : Ca (1.04, 0.36; 0.60 and 1.21% respectively) than for Mn : Ca, Cu : Ca and Zn : Ca (7.20, 7.91 and 6.76% respectively). However, Mn concentrations detected were close to the background level and were excluded from further analyses.

All statistical analyses were performed using SPSS (ver. 20, IBM Corp., Armonk, NY, USA), as described below. A Shapiro–Wilk was used to assess the normality of the data, and Ba stable isotope ratios were found to be non-normally distributed. Therefore, a non-parametric Scheirer Ray Hare extension of the Kruskal–Wallis test was used to examine the effects of spiked concentration and immersion duration on ¹³⁸Ba : ¹³⁷Ba ratios. In addition, the effect of ¹³⁷Ba spikes on the size and condition of marked hatchlings were analysed by two-way analysis of variance (ANOVA). If significant differences were detected, Tukey’s post hoc test was used to evaluate the difference between groups. For statolith chemistry, a forward stepwise canonical discriminant analysis was used to evaluate variations in element composition (Mg : Ca, Sr : Ca, Zn : Ca, Cu : Ca and Pb : Ca) among the control and all treatment groups, and cross-validation was further conducted to assess the percentage of successful classifications. In addition, Spearman’s ρ test was used to assess correlations between barium stable isotopes (¹³⁷Ba : Ca and ¹³⁸Ba : Ca) and other trace elements.

The ¹³⁷Ba spike was successfully marked in statoliths because ¹³⁸Ba : ¹³⁷Ba values decreased with increasing spike concentration or immersion duration. The mean (±s.d.) ¹³⁸Ba : ¹³⁷Ba ratio in statoliths in the control group was 6.28 ± 0.17, which decreased to 3.50 ± 0.22 after 7 days of immersion in 1-ppm ¹³⁷Ba-spiked solution (Fig. 1). Significant interactions were found between immersion duration and the concentration of the ¹³⁷Ba spike on ¹³⁸Ba : ¹³⁷Ba ratios in hatchling statoliths (Scheirer–Ray–Hare extension of the Kruskal–Wallis test, d.f. = 9, SS = 166601.9, H = 34.577, P < 0.001), so separate Dunn’s tests were used to compare the mean ¹³⁸Ba : ¹³⁷Ba ratios within groups. Overall, 7 days of immersion produced significantly lower mean ¹³⁸Ba : ¹³⁷Ba ratios than 1 day immersion for the same spiked concentration (Z > 4.057, P < 0.001), and the mean ¹³⁸Ba : ¹³⁷Ba ratios of the 1-ppm treatment were significantly lower than those of the 0.2-ppm treatment for the same immersion duration (Z > 3.510, P < 0.01). Longer immersion durations (3 and 7 days) with higher spiked concentrations (0.5 and 1 ppm) produced significantly lower ¹³⁸Ba : ¹³⁷Ba ratios than seen in the control group (Z > 4.564, P < 0.001). An additional significant difference was detected between 3- and 1-day immersions in the 0.2-ppm ¹³⁷Ba-spiked group (Z > 3.510, P = 0.015).

Fig. 1.  Mean (±s.d.) Ba isotope ratios in statoliths of hatchlings immersed in water with different concentrations of ¹³⁷Ba spike, namely 0.2 ppm (light grey bars), 0.5 ppm (dark grey bars) and 1 ppm (black bars), for 1, 3 and 7 days. The percentage of mark success for each group is shown below the columns. The dashed line indicates the natural ¹³⁸Ba/¹³⁷Ba ratio.

Following the criteria of Payne et al. (2011), the critical value of successfully marked squid was set at 5.78, which was the mean ratio of the control group minus 3 s.d. for ¹³⁸Ba : ¹³⁷Ba. A successfully marked statolith was defined as a ¹³⁸Ba : ¹³⁷Ba ratio in the hatchling statolith that was lower than this value. Higher spiked concentrations and longer immersion duration both increased the success rate of statolith marking (Fig. 1). For example, no mark was found after 1 day of immersion with a 0.2-ppm ¹³⁷Ba spike, but the success rate increased to 40% after 3 days of immersion with the same concentration. In total, 100% of squid were successfully marked after 3 days of immersion with the 0.5- and 1-ppm concentrations and after 7 days of immersion with all concentrations.

All eggs hatched 1–5 days after marking. The mean ML of the hatchlings in each group ranged from 5.54 to 5.99 mm, the mean bodyweight ranged from 24.4 to 31.3 mg and mean Fulton’s condition factor K ranged from 12.98 to 16.43 (Fig. 2). No interaction between spike concentration and immersion duration was found for ML (F = 0.795, P = 0.622), bodyweight (F = 1.162, P = 0.321) or Fulton’s condition factor K (F = 0.821, P = 0.597) of hatchlings. The spiked concentration of ¹³⁷Ba significantly affected ML (F = 5.789, P = 0.001) and bodyweight (F = 6.687, P < 0.001) of hatchlings, but not Fulton’s condition factor K (F = 2.530, P = 0.058). Hatchlings exposed to spike concentrations of 0.2 and 1 ppm were significantly longer than those in the control group (Tukey’s honest significant difference (HSD), P = 0.001 and 0.008 respectively; Fig. 2a). The bodyweight of hatchlings in the control group was significantly lower than that of hatchlings in all spiked groups (P < 0.01, Fig. 2b). Conversely, the ML (F = 5.190, P = 0.002), bodyweight (F = 8.222, P < 0.001) and Fulton’s condition factor K (F = 3.214, P = 0.024) of hatchlings differed significantly among immersion duration treatments. Individuals in most immersion duration groups had a larger size in terms of ML and bodyweight than those in the control group, except for bodyweight observed after 1 day immersion (P = 0.063). In addition, there was a significant difference in Fulton’s condition factor K between 1 and 3 days of immersion (P = 0.013; Fig. 2c).

Fig. 2.  Mantle length, bodyweight and Fulton’s condition factor K of hatchlings immersed in water with different concentrations of ¹³⁷Ba spike, namely 0.2 ppm (light grey bars), 0.5 ppm (dark grey bars) and 1 ppm (black bars), for 1, 3 and 7 days and the control group. The boxes show the interquartile range, with the median value indicated by the horizontal line; whiskers show the range. Circles indicate outliers in each experimental group.

According to canonical discriminant analysis, hatchling statolith element composition did not show a clear pattern of discrimination between the control and all experimental groups (Fig. 3). The variations explained by Functions 1 and 2 were 53.9 and 24.0% respectively. Cu primarily contributed to Function 1 and Zn contributed to Function 2 (Table 1). The cross-validated classification success for all hatchlings was 24.7%, and ranged from 0% (7 days of immersion with 0.2 ppm of ¹³⁷Ba) to 53.3% (7 days of immersion with 1 ppm of ¹³⁷Ba) (Appendix 1).

Fig. 3.  Forward stepwise canonical discriminant analysis using Mg, Sr, Zn, Cu and Pb in the statoliths of hatchlings among control and all experimental groups immersed in water containing different concentrations of ¹³⁷Ba spike (0.2, 0.5 and 1 ppm) for 1, 3 and 7 days.

Table 1.  Structure matrix coefficients for Discriminant Function (DF) 1 and DF2 for each mean element : Ca ratio used in canonical discriminant analysis for hatchling statoliths among the control and experimental groups
Main elements contributing are in bold

Although statoliths were enriched with ¹³⁷Ba, their elemental : Ca ratios (Cu : Ca, Zn : Ca and Pb : Ca) positively correlated to ¹³⁸Ba but not to ¹³⁷Ba (Table 2; Fig. 4). The regressions of ¹³⁸Ba : Ca with Cu : Ca, Zn : Ca and Pb : Ca were significant (P < 0.01), with determination coefficients (R²) of 0.865, 0.741 and 0.248 respectively.

Table 2.  Summary of Spearman’s ρ test between Ba stable isotopes and trace elements in the statoliths of hatchlings
Significant correlations (P < 0.05) are in bold

Fig. 4.  Linear regressions between mean element : Ca ratios and ¹³⁸Ba : Ca in the statoliths of hatchlings. Symbols indicate different treatment durations (circle, control; square, 1 day; diamond, 3 days; triangle, 7 days) and difference ¹³⁷Ba spike concentrations (white, 0.2 ppm; grey, 0.5 ppm; black, 1 ppm). Error bars indicate the s.d.

Because of its crucial role in marine ecosystems and being a highly attractive fishery target, effective ecological monitoring and resource management of S. lessoniana are needed. In particular, larval dispersal patterns and demographic population connectivity have significant effects on marine organism resources (Cowen et al. 2000; Thorrold et al. 2001; Jones et al. 2005; Cowen and Sponaugle 2009). There are many factors influencing the success rate of mass marking (e.g. spike concentration or developmental stage; Payne et al. 2011; Woodcock and Walther 2014). Consistent achievement of 100% mark success is a vital goal for any mass-marking technique (Warren-Myers et al. 2018). For fish larvae or eggs, concentrations of ≥0.1 ppm of ¹³⁷Ba have been used to achieve 100% mark success by immersion (Woodcock et al. 2011a, 2011b; de Braux et al. 2014, Warren-Myers et al. 2015). However, the eggs of many cephalopod species (e.g. myopsid squid and sepioidea cuttlefish) are coated with encapsulation substances (i.e. a capsule) that are effective barriers against metal uptake into the embryo (Rosa et al. 2015). Therefore, the present study examined higher ¹³⁷Ba spike concentrations to mark large numbers of S. lessoniana hatchlings and found that 100% mark success was achieved steadily after 3 days of immersion with concentrations >0.5 ppm of the enriched barium stable isotope.

This study revealed that 7 days of immersion with lower spike concentrations could also achieve 100% mark success, indicating that immersion duration is a critical factor for marking S. lessoniana statolith through egg immersion. This may be because the perivitelline fluid, which is in the capsule and encasing the embryo, is conducive to ambient seawater influx and swells gradually during the late development stage (Cronin and Seymour 2000). Extension of immersion during egg swelling results in the uptake of the spiked water, decreasing the ¹³⁸Ba : ¹³⁷Ba ratio within eggs. A similar effect of immersion duration on Ba stable isotope ratios in otoliths of fish species has been reported (Munro et al. 2008; de Braux et al. 2014). Yet, this is inconsistent with the results reported for S. apama by Payne et al. (2011), who found a significant interaction between the concentration of enriched ¹³⁷Ba and immersion duration, but no significant differences among immersion durations for the lower concentration tested (0.3 ppb). Species and physiological differences may explain these different results. For example, egg swelling time varies according to embryo development period, thus the longer embryo development of S. apama (3–5 months; Hall and Fowler 2003) would dilute the contribution of immersion time to the ¹³⁸Ba : ¹³⁷Ba ratios in S. apama statoliths. Moreover, the low enriched ¹³⁷Ba concentration may need a longer time of immersion, and the effect of immersion duration would become significant. In the study of Payne et al. (2011), extension of immersion duration from 2 to 8 days did decrease ¹³⁸Ba : ¹³⁷Ba ratios for the higher-concentration (1 ppb) treatment group. Therefore, determining the appropriate concentration and corresponding time of immersion before using this technique on a species of interest is important, because life history characteristics (e.g. developmental stage) and habitats (e.g. seawater or fresh water) may affect the effectiveness and the costs for mass marking.

The ML of hatchlings in this study was consistent with that reported by Lee et al. (1994), who continuously cultured S. lessoniana through three successive generations and whose hatchlings averaged 5.3 mm ML, ranging from 3.5 to 6.4 mm ML. The bodyweight of hatchlings in past studies varies, from a range of 4.3–12.0 mg (mean 8.2 mg; Lee et al. 1994) to 50 mg (Segawa 1987); the bodyweight of hatchlings in the present study fell between values published in the literature. In the present study, ¹³⁷Ba mass marking slightly increased the ML and bodyweight of marked hatchlings in some of the experimental groups. Larger hatchling size may benefit from an increased attack speed (Sugimoto and Ikeda 2013) and a reduction in the distance required to capture prey accurately (Chen et al. 1996). In addition, hatchling size is linked to vulnerability to predators (Blaxter 1986; Sogard 1997), so that larger size hatchlings would have a greater survival rate in the early life history stages. Moreover, the growth condition (K) is related to embryo development and environmental variables, and individuals in a better condition (K) have higher survivorship and greater growth rate (Bolger and Connolly 1989). However, the K values of hatchlings in the present study only differed significantly between two immersion duration groups, indicating that ¹³⁷Ba mass marking did not affect hatchling growth condition. Previous experimental results of the effects of transgenerational marking (i.e. injection method) on the condition of larval fish were species specific. Positive (Starrs et al. 2014a, 2014b), negative (Williamson et al. 2009) and no significant effects (Zitek et al. 2013; Warren-Myers et al. 2015) on size at hatch, yolk sac area, oil globule area and eyeball diameter were found among species. The findings of the present study provide additional information on cephalopod species marked using the immersion method. As noted by Starrs et al. (2014b), the effects of such mass marking with stable isotopes on hatchling morphology require additional research, as does the roles of barium during the developmental of S. lessoniana embryos.

We found different element compositions of statoliths in hatchlings that were related to size at hatch, and significant correlations were found between Me : Ca ratios (Cu : Ca, Zn : Ca and Pb : Ca) and ¹³⁸Ba : Ca. The effects on element composition of statoliths are not often mentioned in the literature when marking cephalopod offspring with enriched stable isotopes. Element uptake in cephalopod statoliths is presumably similar to the observations in fish otoliths (Gillanders et al. 2013) and is primarily associated with environmental changes, such as water chemistry composition (Arkhipkin et al. 2004) and ambient temperature (Ikeda et al. 2002; Zumholz et al. 2007). However, in this study the rearing seawater was maintained at consistent conditions and egg capsules in the same cluster were used to eliminate any possible affects from the maternal yolk (e.g. Lloyd et al. 2008). The difference in growth rate between control and experimental groups was a potential explanation for variations in element incorporation. Growth rate has been confirmed to be negatively correlated with the elemental partition coefficient in otoliths of teleost species (Walther et al. 2010). A faster growth rate could result in more calcium-binding proteins, altering relative ion concentrations in the calcifying fluid (Kalish 1989). Therefore, trace elements such as Cu and Zn have a greater likelihood of being associated with organic matrix protein (Miller et al. 2006). In addition, a fast growth rate usually occurs with higher calcium carbonate accretion rate (Ikeda et al. 1999), which raises the pH of the calcifying fluid and reduces trace element concentrations in the endolymph, resulting in a negative relationship between the Me : Ca ratio and accretion rate (Sinclair 2005; Sinclair and Risk 2006; Hamer and Jenkins 2007). The lower hatchling size in the control group could simultaneously lead to elevated patterns of Me : Ca in statoliths. Although the effects of growth rate on the element composition of cephalopod statoliths have not been adequately clarified, the physiological processes do significantly affect the microchemistry of biogenic carbonates. We emphasise that the mechanisms of trace element incorporation into statoliths should be carefully considered to avoid confounding environmental signatures with artificial marking.

Understanding the population connectivity and migration of cephalopods is critical in developing approaches for the resource management and conservation of marine ecosystems. Stable isotope mass-marking techniques can be successfully used in fishes. This study demonstrated unique signatures in S. lessoniana statoliths with 100% marking success after 3 days of immersion in ¹³⁷Ba and provides a method to unravel the questions regarding dispersal mechanisms and movement patterns in cephalopods. However, we also found potential effects of stable isotope mass marking on offspring size at hatch that are consistent with those reported by an increasing number of studies. The effects on embryo development and growth may induce variations in element composition in statoliths, probably reflecting physiological processes and statolith accretion, which affect statolith chemistry and may subsequently affect the accuracy of interpreting an individual’s environmental history. The present study defined the successful marking conditions for use with stable isotope-marking techniques on S. lessoniana and their potential effects on cephalopod statoliths. We highlight that the effects of this technique need to be taken into consideration in field applications. Additional research investigating the relationships among multiple elements and physiological responses to enriched stable isotope incorporation will advance our knowledge for the application of these techniques to wild cephalopods.

Chia-Hui Wang declares that she is a guest editor of the Otoliths Symposium special issue for Marine and Freshwater Research but took no part in the review and acceptance of this manuscript. The authors declare that they have no further conflicts of interest.

This study was supported by the Ministry of Science and Technology, Taiwan (grant number 106-2611-M-019-004).