ABSTRACT
Coordinated spawning of marine animals releases millions of planktonic eggs into the environment, known as egg boons. Eggs are rich in essential fatty acids and may be an important lipid subsidy to egg consumers. Our aim was to validate the application of fatty acid and stable isotope tracers of egg consumption to potential egg consumers and to confirm egg consumption by the selected species. We conducted feeding experiments with ctenophores, crustaceans and fishes. We fed these animals a common diet of Artemia or a commercial feed (Otohime) and simulated egg boons for half of them by intermittently supplementing the common diet with red drum (Sciaenops ocellatus) eggs for 10–94 days. Controls did not receive eggs. Fatty acid profiles of consumers fed eggs were significantly different from those of controls 24 h after the last egg-feeding event. Consumers took on fatty acid characteristics of eggs. In fishes and ctenophores, fatty acid markers of egg consumption did not persist 2–5 days after the last egg-feeding event, but markers of egg consumption persisted in crustaceans for at least 5–10 days. Additionally, consumption of eggs, which had high values of δ15N, led to δ15N enrichment in crustaceans and a fish. We conclude that fatty acids and nitrogen stable isotope can be used as biomarkers of recent egg consumption in marine animals, validating their use for assessing exploitation of egg boons in nature.
INTRODUCTION
Acquisition of nutrients through feeding has profound effects on all biological processes, scaling from cellular to ecosystem levels. Nutritional qualities of food resources regulate individual survival, growth and fecundity, with upscaling effects on population and community dynamics (Atkinson et al., 2017; Van de Vaal et al., 2018; Guo et al., 2021; Danger et al., 2022). In the past, stoichiometric research has predominantly focused on limiting macronutrients, such as carbon, nitrogen and phosphorus, and their influences on consumer growth and other ecological processes (Van de Vaal et al., 2018). Beyond standard stoichiometry, there is growing recognition that micronutrients, such as essential fatty acids (EFAs), can limit the trophic transfer of macronutrients (Müller-Navarra et al., 2000) and consumer performance when not present in sufficient amounts in food resources (Gladyshev et al., 2009; Ruess and Müller-Navarra, 2019; Závorka et al., 2019). Examining the transfer of these micronutrients through food webs can reveal important trophic linkages and elucidate how consumer fitness and ecosystem functions are affected by changes in bioavailability of those nutrients.
EFAs cannot be synthesized de novo by animals or are synthesized in quantities well below their physiological requirements. Thus, animals must acquire these micronutrients through dietary sources (Sargent et al., 1999; Tocher, 2003; Bell and Sargent, 2003; Parrish, 2009; Glencross, 2009; Ruess and Müller-Navarra, 2019; Flecker et al., 2019). Marine ecosystems are uniquely important sources of omega-3 polyunsaturated fatty acids (n-3 PUFAs) for consumers across ecosystems (Gladyshev et al., 2013; Twining et al., 2020). Aquatic animals require n-3 PUFA, such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) (Carlson and Neuringer, 1999) for a broad range of physiological, cognitive and life history traits (McKenzie, 2001; Hulbert, 2008; Pilecky et al., 2021). PUFAs are produced at the base of the food web and are selectively retained by consumers at higher trophic levels. PUFAs are especially concentrated in eggs of marine fishes, and superabundances of eggs released in temporally and spatially discrete patches create pulsed nutritional resources for egg consumers, called ‘egg boons’ (Fuiman et al., 2015). Generally, 90% or more of marine planktonic eggs enter the food web before they hatch (Fuiman et al., 2015; Nielsen et al., 2019), and consumers of eggs can be at a higher or lower trophic level than the adults that produce the eggs (Heyman et al., 2001; Plirú et al., 2012; Taylor and Dunn, 2017). Therefore, egg boons have been suggested to constitute an important trophic pathway in marine ecosystems through which animals repackage PUFAs and redistribute them counter to the main direction of trophic flow (i.e. to smaller animals at lower trophic levels; Fuiman et al., 2015).
Predation on fish eggs by various aquatic consumers has been traced using direct observation, gut content analysis and DNA-based diet analysis (Purcell, 2003; Webster et al., 2015; Taylor and Dunn, 2017; Lutz et al., 2020). These techniques can provide insights into egg consumption as long as remnants remain in the gut, but they are not well suited for identifying egg consumption after absorption and gut clearance (Hayden et al., 2014; Nielsen et al., 2017). Biomarkers, such as fatty acids (FAs) and stable isotopes (SIs), are increasingly being used in combination to identify food resources and understand trophic linkages (Peterson and Fry, 1987; Dalsgaard et al., 2003). These techniques provide a time-integrated measure of diet, and are based on the principle that a consumer's diet is reflected in the patterns of FAs and SIs of their tissue (Hooker et al., 2001; Galloway and Budge, 2020; Guerrero et al., 2021).
Major dietary items often have characteristic FA compositions that can be used to trace the contribution of a dietary item to a consumer's diet (Dalsgaard et al., 2003). However, FA markers can be ambiguous with regard to a consumer's trophic level (Dalsgaard et al., 2003; Kelly and Scheibling, 2012). The ratio of the two stable isotopes of nitrogen (δ15N) increases predictably with trophic position (3–4‰ per trophic step) and is thus used to infer the trophic position of a consumer (Minagawa and Wada, 1984). The ratio of the two stable isotopes of carbon (δ13C) provides information about carbon sources at the base of the food web (Peterson and Fry, 1987). However, δ13C and δ15N values of dietary resources frequently overlap, making their contribution to consumer diets difficult to estimate using SIs alone (Newell et al., 1995; Twining et al., 2020). Additionally, incorporation of dietary FAs and SIs into consumer tissues is affected by metabolic processes, such as catabolism and anabolism, which occur during digestion, assimilation and excretion (Peterson and Fry, 1987; Taipale et al., 2011). Hence, using FAs and SIs in combination has greater potential than use of a single technique to discriminate between potential food sources contributing to a consumer's diet. However, because a consumer's metabolism can modify FAs and SIs, controlled feeding experiments that assess transfer and integration of dietary FAs and SIs are necessary before these biomarkers can be applied to field-collected samples (Galloway and Budge, 2020; Schaub et al., 2021). Such validation studies are especially needed to advance the application of FAs and SIs as tracers of trophic connections.
The aim of the present study was to validate, through laboratory experiments, the application of FA and SI (δ13C and δ15N) tracers to detect consumption of eggs of the teleost red drum (Sciaenops ocellatus) by potential egg consumers occupying lower trophic levels than adult red drum, and to confirm that the selected species consume red drum eggs. We also assessed the duration for which FA biomarkers of egg consumption persisted in those egg consumers. We found that in a controlled setting, a combination of FAs and SI of nitrogen can be used to trace recent fish egg consumption in several species of egg consumers.
MATERIALS AND METHODS
All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Texas at Austin (AUP-2019-00306 and AUP-2021-00248). We conducted feeding experiments to quantify effects of egg consumption on consumer FA profiles and SI ratios in a controlled setting. Red drum eggs for laboratory experiments were collected from broodstock maintained at the Fisheries and Mariculture Laboratory (FAML; henceforth FAML eggs) of the University of Texas Marine Science Institute (UTMSI) in Port Aransas and the Texas Parks and Wildlife Department (TPWD; henceforth TPWD eggs) CCA Marine Development Center in Corpus Christi, TX, USA. The broodstock at the FAML were fed shrimp, squid and fish, and those at the TPWD were fed shrimp, squid, fish and beef liver. Spawning was induced by manipulating temperature and photoperiod regimes. Captive-spawned eggs were used because it was not feasible to collect sufficient quantities of eggs from the field for the feeding experiments.
Mnemiopsis leidyi A. Agassiz 1865 (warty comb jelly; 4.0–8.0 cm total length) and juvenile male Callinectes similis Williams 1966 (lesser blue crab) (2.5–5.5 cm carapace length) were collected in March–April 2022, and Beroe ovata Bruguière 1789 (brown comb jelly; 3.0–4.5 cm total length) was collected in November 2022, using a plankton net (50 cm diameter, 500 µm mesh) from Aransas Pass inlet at the FAML pier in Port Aransas (27.8396°N, 97.0726°W). Non-gravid Palaemon pugio (Holthuis 1949) (daggerblade grass shrimp) was collected using a plankton net from Corpus Christi Bay (27.8035°N, 97.0898°W) in Port Aransas in July 2021. Juvenile Opisthonema oglinum (Lesueur 1818) (Atlantic thread herring) and Lagodon rhomboides (Linnaeus 1766) (pinfish) (8.0–12.0 cm total length for both fishes) were collected using a seine (6.4 m wide by 1.2 m high with 5 mm square mesh) from Aransas Pass inlet at Port Aransas (27.8396°N, 97.0726°W) in August 2021 and Redfish Bay at Aransas Pass (27.8611°N, 97.07632°W) in May 2022, respectively.
The experimental protocol (i.e. length of egg-feeding event and experimental duration) was different for each species because of species-specific differences in tissue FA and SI turnover, as determined from a series of preliminary experiments and previously published studies (Antonio and Richoux, 2016; Schaub et al., 2021; Mohan et al., 2016; Thomas et al., 2020). The live animals of each species were divided into two treatments (control and experimental). Both treatments were fed a common diet of either live Artemia sp. nauplii (enriched with Alga-Mac 3050; Aquafauna Bio-Marine, Inc., Hawthrone, CA, USA) or commercial fish food, Otohime (EP1, Reed Mariculture, Inc., Campbell, CA, USA) during the acclimation period of 10–45 days (Fig. 1). After acclimation (day 0), both treatments received a common diet of Artemia or Otohime, and the diet of experimental treatments was supplemented with red drum eggs for a period of 10–94 days following the protocol provided in Fig. 1. Controls did not receive eggs. Each taxon was provided with eggs from one source (i.e. either TPWD or FAML; Supplementary Materials and Methods) throughout the experiment. For each study species, three to eight replicate tanks were sampled at the end of acclimation (day 0), and 24 h and 2–5 days after the last day the experimental treatment received eggs (Fig. 1).
Mnemiopsis leidyi, B. ovata and C. similis were held in rectangular tanks (26.7 cm long×16.5 cm wide×16.5 cm deep), and P. pugio and fishes were held in circular tanks (106.7 cm in diameter, 43.2 cm deep) with recirculating filtered water. Within each rectangular tank, individuals of C. similis were held separately in round plastic containers (12 cm in diameter, 6.4 cm deep) with perforated lids to prevent aggressive contact. For the same reason, individuals of L. rhomboides were kept in separate perforated cylindrical enclosures (30 cm in diameter, 45 cm high) within each circular tank. Excess food and solid waste were siphoned daily from all tanks, and complete water changes were performed in rectangular tanks every 2–4 days. Environmental conditions were measured daily and were constant throughout the experiment (temperature: 21–24°C, salinity: 28–35 ppt, and photoperiod: 12 h light and 12 h dark).
Invertebrates removed from both treatments on sampling days (Fig. 1) were kept in clean sea water overnight to evacuate their guts and were killed the following morning. For taxa with low dry mass (i.e. ctenophores), 3–4 individuals from each tank were pooled to make a replicate. A single individual per tank of C. similis, and three individuals of P. pugio (subsamples, n=3) per tank were removed at each sampling day (Fig. 1). On each sampling day, one fish per tank was removed and immediately euthanized with a lethal dose (300 mg l−1) of tricaine methanesulfonate (MS-222). Euthanized fish were placed on ice while sampling the liver and a fillet of dorsal white muscle tissue (DM). Subsamples (n=5–21) of diets provided to both treatments (i.e. FAML eggs, TPWD eggs, Artemia and Otohime) were collected throughout the experiments for each taxon. All samples were rinsed twice in distilled water and frozen at −80°C until analysis.
Invertebrates were analyzed whole, except for C. similis, for which the exoskeleton was excluded. Fish DM, a standard tissue for SI analyses (Busst et al., 2015), was analyzed for carbon (δ13C) and nitrogen (δ15N) isotopes. Fatty fishes (e.g. clupeids) generally accumulate FAs primarily in muscle tissues, whereas lean fishes (e.g. gadids and flatfishes) store relatively more FAs in their liver (Ando et al., 1993; Copeman and Parrish, 2004; Nanton et al., 2007; Zeng et al., 2010; Budge et al., 2011; Guil-Guerrero et al., 2011; Mohan et al., 2016). We analyzed both DM and liver tissues for FAs in O. oglinum (clupeid) and L. rhomboides (sparid) to determine whether both species stored FAs from egg consumption in these tissues.
Samples were lyophilized, homogenized and weighed. Fatty acid profiles for all samples were obtained following established procedures (Faulk and Holt, 2005). Lipids were cold-extracted from each sample by homogenizing in a chloroform–methanol solution (2:1 volume:volume). Fatty acid methyl esters (FAMEs) were prepared by saponification in 0.5 mol l−1 potassium hydroxide, followed by 14% boron trifluoride in methanol. FAMEs were analyzed using a Shimadzu GC-2014 gas chromatograph (GC) with a flame ionization detector (GC-FID; Shimadzu Scientific Instruments, Columbia, MD, USA). Individual FAs were identified by comparison to commercial FAME standards (marine PUFA no. 3, Bacterial and Supelco 37 component FAMEs mix; Sigma-Aldrich, St Louis, MO, USA). FA profiles expressed each of the identified FAs as a percentage of total FAs. Thirty-four FAs were measured on every sample.
Another known subsample (0.5–0.7 mg of crustacean and fishes, 4.9–5 .2 mg of M. leidyi, 1.0–1.2 mg of B. ovata) of lyophilized and homogenized tissue was weighed into a tin capsule at the UTMSI Core Isotope Facility. A Thermo Fisher Scientific Flash Elemental Analyzer-Isolink coupled to a Thermo Fisher Scientific Delta V Plus Isotope Ratio Mass Spectrometry (Thermo Fisher Scientific, Waltham, MA, USA) in continuous-flow (Helium) mode was used to determine carbon and nitrogen isotopic compositions. Casein was used as a protein standard. Nitrogen was expressed (with analytical precision within ±0.2‰) relative to atmospheric nitrogen, and carbon relative to Vienna Pee Dee Belemnite. Isotope ratios are expressed in the δ unit notation as per the following equation: δX=[(Rsample/Rstandard)−1]×1000, where X represents 13C or 15N, and R is 13C/12C or 15N/14N ratio. For tissue samples with C/N ratios>3.5 (Post et al. 2007), δ13C values were corrected prior to analyses for lipid content using the following formula: δ13Cnormalized=δ13Cuntreated−3.32+0.99×C:N, where δ13Cnormalized and δ13Cuntreated are the lipid corrected and uncorrected δ13C values, respectively.
Data analysis
Data were analyzed separately for each taxonomic group, and for fish liver and DM tissues. For animals collected 24 h after the experimental treatments received eggs, a correspondence analysis (CA) (Graeve and Greenacre, 2020) was performed on proportions (% total FAs) of 34 FAs to summarize the patterns of variation in FA profiles receiving different dietary treatments (control or experimental). Differences in FA composition of control and experimental treatments were assessed using permutational multivariate analysis of variance (PERMANOVA; adonis function, VEGAN package, R). Differences in FA composition of common diets (i.e. Otohime, Artemia) and red drum eggs were also assessed using PERMANOVA. Differences in the means of individual FA among the dietary treatments (control or experimental), and among the diets (i.e. Otohime, Artemia and red drum eggs) were tested using a Student's t-test. A Wilcoxon rank sum test was used when the assumptions of normality and homoscedasticity were violated. P-values obtained from t-tests or Wilcoxon tests were adjusted using a Benjamini–Hochberg false discovery rate correction. Fatty acids that were significantly higher (or lower) in red drum eggs and in the experimental animals receiving eggs were selected as markers of red drum egg consumption (Supplementary Materials and Methods). The persistence of selected markers of red drum egg consumption in animals collected 2–5 days after the last egg feeding event was analyzed using a t-test or a Wilcoxon rank sum test.
For P. pugio, repeated measures ANOVAs (RM-ANOVA) were used to assess differences in FA profiles of subsamples (n=3) collected from three tanks for each treatment (control and experimental). If no significant differences were detected, subsamples were treated as separate samples, making a total of 9 samples (3 subsamples×3 tanks) for each treatment type. Friedman's test was conducted if the assumptions of normality, homoscedasticity and sphericity were not met. For the DM of O. oglinum, RM-ANOVAs were conducted for each treatment to assess whether there were differences among FA profiles of animals collected on days 39 and 91 (24 h after the experimental animals received eggs). If no significant differences were detected, data for those days were pooled for each treatment. Differences in the means of δ13Cnormalized and δ15N among the dietary treatments and among the diets were tested using a t-test or a Wilcoxon rank sum test. Significance for all tests was inferred at P≤0.05. All statistics and graphics were performed using R v.4.2.1.
RESULTS
FA analyses
The common diet of Artemia or Otohime had significantly different FA composition from that of red drum eggs (PERMANOVA P<0.001) (Table S2). Of the 34 FAs, 20–29 differed significantly among the diets (Table S2).
Differences in the FA profiles of control and experimental treatments were clearly represented by the CA axes for all species. The effect of the diet treatment was on the first CA axis (CA1) for ctenophores and crustaceans (Fig. 2) but on CA1 and CA2 for fishes (Fig. 3). The first two axes explained approximately 84% of the variance, with CA1 explaining 61.5–78.8% and CA2 explaining 5.3–15.3% of the total variance.
Ctenophores
FA composition of controls was significantly different from that of experimental treatments for M. leidyi on day 6 (PERMANOVA pseudo-F1,12=19.4, P=0.0008) (Fig. 2A), and for B. ovata on day 8 (PERMANOVA pseudo-F1,10=8.8, P=0.003) (Fig. 2B). Out of 34 FAs, 17 FAs in M. leidyi and 11 FAs in B. ovata differed significantly between the control and experimental treatments (adjusted P≤0.05) 24 h after the last egg-feeding event (Fig. 2A,B). Of these, 13 FAs in M. leidyi and 5 FAs in B. ovata changed (increased or decreased) in the same direction as FAs in red drum eggs (Table 1) and were selected as markers of red drum egg consumption in these animals (Table S1). For M. leidyi 4 days after the last egg feeding event, none of the 13 FAs persisted as a marker in egg consumers. However, 22:6n–3 decreased and a-15:0 increased significantly from controls, possibly due to individual variation in FA profiles (P≤0.02) (Fig. 4A). For B. ovata, five of the FA markers did not persist in egg consumers 2 days after the last egg feeding event (P>0.05) (Fig. 4B).
Crustaceans
For P. pugio, three individuals sampled from each of the three control and experimental tanks were not significantly different from each other on sampling days 32 (10 days after the last egg-feeding event) and 48 (24 h after the last egg-feeding event) (RM-ANOVA or Friedman test P>0.05). So, individuals were treated as replicates, making 9 (3×3) replicates for each treatment on both days.
FA composition of experimental animals differed significantly from that of the controls for P. pugio on day 48 (PERMANOVA pseudo-F1,16=38.6, P=0.0001) (Fig. 2C), and for C. similis on day 23 (PERMANOVA pseudo-F1,10=23.7, P=0.002) and day 49 (PERMANOVA pseudo-F1,9 =26.5, P=0.002) (Fig. 2D). Out of 34 FAs, 23 FAs in P. pugio and 18 FAs in C. similis differed significantly between the control and experimental treatments (adjusted P≤0.05) (Fig. 2C,D). Sixteen FAs in P. pugio and 15 FAs in C. similis changed in the same direction as FAs in red drum eggs (Table 1) and were selected as markers of red drum egg consumption in these animals (Table S1).
Ten days after the last egg-feeding event (day 32), 13 markers of egg consumption persisted in P. pugio (P≤0.05), while three markers, 22:5n-3, i-15:0 and i-17:0, did not persist (Fig. 5A).
Five days after the last egg feeding event, 11 markers of egg consumption persisted in C. similis. However, four markers, 15:0, 16:3n-4, 17:0 and 18:1n-9, did not persist (Fig. 5B).
Fishes
Lagodon rhomboides
Liver and DM FA compositions of control and experimental animals differed significantly on day 27 (liver PERMANOVA pseudo-F1,14=6.5, P=0.002; DM PERMANOVA pseudo-F1,14=10.3, P=0.002) and day 57 (liver PERMANOVA pseudo-F1,14=18.1, P=0.0003; DM PERMANOVA pseudo-F1,14=6.9, P=0.008) (Fig. 3A,B). For the liver tissue, eight FAs on day 27 and 16 FAs on day 57 were significantly different between the treatments (adjusted P≤0.05) (Fig. 3A, Table 1). Seven FAs on day 27 and an additional four on day 57 changed in the same direction as FAs in red drum eggs (Table 1) and were selected as markers of red drum egg consumption (Table S1). For the DM tissue, 17 FAs differed significantly from the controls on day 27 (Fig. 3B). Fourteen FAs changed in the same direction as FAs in red drum eggs and were selected as markers of red drum egg consumption (Table S1). However, on day 57, only six DM FAs differed significantly from the controls, and five changed in the same direction as FAs in red drum eggs and were selected as markers of red drum egg consumption (adjusted P≤0.05) (Table 1). Three to five days after the egg feeding events (i.e. days 30, 60 and 62), three FA markers of egg consumption (17:0, 20:5n-3, 22:4n-6) persisted in the liver, and four (18:1n-7, 18:2n-6, 20:3n-6, 20:5n-3) persisted in the DM (P≤0.002) (Fig. 6A,B).
Opisthonema oglinum
Liver and DM FA composition of experimental animals differed significantly from that of the controls on day 39 (DM PERMANOVA pseudo-F1,7=6.8, P=0.023; liver PERMANOVA pseudo-F1,7=5.5, P=0.006). However, on day 91, only DM FA composition differed significantly from that of the controls (DM PERMANOVA pseudo-F1,8=7.6, P=0.015; liver PERMANOVA pseudo-F1,8=0.3, P=0.6) (Fig. 3C). Therefore, DM was chosen as the appropriate tissue for selecting FA markers of egg consumption in O. oglinum. Approximately 11 DM FAs were marginally non-significant (adjusted P≤0.1) from the controls on days 39 and 91. Failure to obtain significant differences for individual FA when their P-values were adjusted might be due to small sample size (N=5). Therefore, FA profiles of each treatment were pooled for days 39 and 91 as FA profiles of each treatment on day 39 did not differ significantly from those on day 91 (Friedman or RM-ANOVA, adjusted P>0.05). The FA composition of pooled data differed significantly from that of the controls (PERMANOVA pseudo-F1,17=14.6, P=0.0002). Out of 34 FAs, 14 FAs were significantly different from the controls (adjusted P≤0.05) (Fig. 3C). Of those, 11 FAs changed in the same direction as FAs in red drum eggs and were selected as markers of red drum egg consumption (Table 1; Table S1). Three days after the last egg-feeding event, only one marker of egg consumption (18:2n-6) persisted (P=0.04) (Fig. 6C).
Bulk stable isotope analyses
TPWD eggs (13.6±0.08‰) and FAML eggs (13.1±0.1‰) (δ15N: mean±s.e.m.), had significantly more enriched δ15N values than Otohime (11.2±0.2‰) or Artemia (11.2±0.1‰) (P<0.001) (Fig. 7A). For P. pugio, the treatment that received eggs had significantly more enriched δ15N (12.0±0.1‰) compared with the controls (11.1±0.2‰) on day 48 (t-test, t14=−3.7, P<0.003) (Fig. 7B). Similarly, C. similis that received eggs had significantly more enriched δ15N (day 23: 13.0±0.2‰, day 49: 13.5±0.04‰) compared with the controls (day 23: 11.7±0.1‰, day 49: 12.1±0.1‰) on day 23 (t-test, t9=5.83, P<0.001) and day 49 (Wilcox test, z=30, P=0.007) (Fig. 7B). For L. rhomboides, δ15N of the experimental treatments (13.9±0.04‰) did not differ from that of the controls (14.0±0.09‰) on day 27, but was significantly more enriched (14.0±0.07‰) compared with controls (13.7±0.06‰) on day 57 (t-test, t14=3.25, P<0.01) (Fig. 7B). For M. leidyi, B. ovata and O. oglinum, δ15N values did not differ from those of the controls (P>0.05).
TPWD eggs and FAML eggs had significantly different δ13Cnormalized values from those of Otohime or Artemia (P<0.03) (Table 2, Fig. 7A). For all study species except L. rhomboides, δ13Cnormalized did not differ among treatments (P>0.05) (Fig. 7C, Table 2). For L. rhomboides, δ13Cnormalized of the experimental treatments was slightly but significantly more enriched compared with the controls on day 27 (Table 2). However, δ13Cnormalized of the experimental treatments did not differ from that of the controls on day 57 (Fig. 7C, Table 2).
DISCUSSION
Our study tested whether FAs and SIs could be used as biomarkers to confirm that the eggs of large marine fishes are consumed by a variety of taxa at lower trophic levels, at least in laboratory feeding experiments. This study is the first to experimentally identify biomarkers of fish egg consumption across taxonomic groups. To the best of our knowledge, this is also the first study to show that Beroe ovata could be a potential consumer of fish eggs in the wild. Beroe is generally thought to be a predator of other ctenophores (Swanberg, 1974; Haddock, 2007). But biomarkers indicated egg consumption in our experiments, which we confirmed by direct observation of B. ovata ingesting eggs. All egg consumers in this study took on FA characteristics of the eggs. This observation supports the idea that certain FAs are transferred conservatively (Dalsgaard et al., 2003) from eggs to egg consumers, validating their use as markers for identifying red drum egg consumption. The egg consumers took on FA characteristics of the eggs within a few days (ctenophores) or weeks (crustaceans and fishes), indicating that FAs could potentially be used effectively to trace the trophic transfer of these ephemeral nutritional resources in marine food webs. Other studies have reported similar findings where FA profiles in marine invertebrates and fishes changed within days or weeks after a complete diet change (McLeod et al., 2013; Copeman et al., 2013; Thomas et al., 2020).
Suites of FAs identified as potential markers of red drum egg consumption were species specific (Table 1), indicating differences in egg FA turnover in each species of egg consumer (Dalsgaard et al., 2003). Each species of egg consumer either retained certain FAs when red drum eggs had higher levels than Artemia or Otohime, or lost certain FAs when they were lower than the control diet. Species-specific differences among egg consumers in egg FA uptake and retention imply differences in nutritional processing, which are accompanied by differences in functional traits acting on organismal performance (Kirk et al., 1999; Graeve et al., 2005; Schälicke et al., 2020). Docosahexaenoic acid (DHA, 22:6n-3), an EFA that cannot be synthesized de novo by most marine animals, was highly retained by all egg consumers. Efficient retention of DHA underpins its importance in maintaining normal physiological functioning in all study organisms (Kainz et al., 2004; Strandberg et al., 2015). Further, retention of 20- and 22-carbon PUFAs from red drum eggs (Table 1; i.e. 20:4n-3, 20:5n-3, 22:5n-3, 22:6n-3, 20:4n-6, 22:4n-6, 22:5n-6) indicates that study organisms might have limited abilities to synthesize these PUFAs, and might be dependent on dietary sources for their supply in estuarine ecosystems. Indeed, most marine fishes and some marine invertebrates have limited ability to biosynthesize EFAs de novo and thus rely on a dietary supply of 20:5n-3 (EPA), DHA, or closely related 18-carbon precursors (Bell et al., 2007; Bell and Tocher, 2009; Nyunoya et al., 2021; but see Kabeya et al., 2018).
In fishes and ctenophores, FA markers of egg consumption did not persist 2–5 days after the last egg-feeding event (Figs 4 and 6). However, most FA markers of egg consumption persisted in crustaceans, 5–10 days after the last egg-feeding event (Fig. 5). This suggests that FA markers of egg consumption are metabolized or bio-converted more slowly, or are incorporated and retained in tissues for a longer duration in epibenthic crustaceans in comparison to pelagic ctenophores and fishes. Epibenthic crustaceans, P. pugio and C. similis, may have limited or occasional access to planktonic fish eggs, when these animals move into the water column or when dead eggs sink to the bottom. On acquisition of planktonic eggs, epibenthic species might be incorporating physiologically important FAs from the eggs into their lipid stores, or into non-storage tissues for growth, reproduction and survival (Thomas et al., 2020; Taipale et al., 2021). Pelagic animals, in contrast, lost FA markers of egg consumption within a short period of time, indicating that these FAs might not be limiting in their habitat. Further, some gelatinous zooplankton, such as M. leidyi, have limited abilities to store dietary lipids (Deibel et al., 1992; Lucas, 1994; Lee et al., 2006) and likely catabolize the dietary FAs for energy to fuel rapid somatic growth or gamete production (Deibel et al., 1992; Schaub et al., 2021).
The dorsal muscle of O. oglinum was indicative of egg consumption throughout the experiment, lending support to the general idea that fatty fishes accumulate dietary FAs in their muscle tissue (Ando et al., 1993; Copeman and Parrish, 2004; Nanton et al., 2007; Guil-Guerrero et al., 2011; Mohan et al., 2016). However, in L. rhomboides, which is thought of as a lean fish (Darcy, 1985), the DM had more FA markers of egg consumption than the liver after receiving eggs for a brief period of time (sporadically over 27 days). But, when L. rhomboides received eggs for a longer duration (sporadically over 57 days), their liver FA signature was more similar to the FA profile of fish eggs compared with their DM FA. This indicates that tissue FA composition of L. rhomboides is affected by the span of time over which the animal receives a certain diet. Other studies have also reported susceptibility of dietary FAs to rapid compositional changes and the magnitude of this change being dependent on the dietary FA composition, days of feeding, tissue type and fish growth (Budge et al., 2011; Copeman et al., 2013; Mohan et al., 2016). The longer time required for dietary signals of egg consumption to appear in the liver may be due to the higher amount of lipid in the liver compared with the DM, and longer amounts of time required to dilute this larger pool of endogenous lipid with lipid from the consumption of eggs (Budge et al., 2011). Therefore, both liver and muscle should be analyzed when studying FA markers of egg consumption in L. rhomboides and possibly other lean fish species.
Red drum eggs (TPWD and FAML) had more enriched δ15N and significantly different δ13Cnormalized values than the common diet of Artemia or Otohime (Fig. 7A). More enriched δ15N values were also observed for three study species (i.e. L. rhomboides, C. similis and P. pugio) that received a diet of red drum eggs (Fig. 7B), indicating nitrogen isotopic turnover in response to egg consumption in these species. However, except for L. rhomboides on day 27, differences in δ13Cnormalized were not observed for these study species (Table 2 and Fig. 7C). Slower turnover of δ13C compared with δ15N for fish and crustaceans has also been reported in previous studies (Barton et al., 2019; Madigan et al., 2021; Viozzi et al., 2021). While the exact mechanism underlying the differential turnover rates of δ13C and δ15N is unclear, it may be due to δ13C values representing longer periods of past resource use because of slow accrual of new biomass (growth) and slow elemental turnover within a tissue resulting in slower catabolic tissue replacement or catabolic turnover (Vander Zanden et al., 2015; Barton et al., 2019; Madigan et al., 2021; Viozzi et al., 2021). Enrichment of δ15N and differences in δ13Cnormalized were not observed for ctenophores or for O. oglinum, which suggests that the experimental duration for which these animals were fed eggs (6–21 days; Fig. 1) may not be long enough for measurable carbon and nitrogen isotopic turnover to occur in their body tissue. The absence of δ15N enrichment in ctenophores and O. oglinum could also be due to excretion of ammonia as a waste product (i.e. ammonotelic). Ammonotelic animals are thought to have lower δ15N enrichment than ureotelic or uricotelic animals (Vanderklift and Ponsard, 2003). However, the other three study species that showed δ15N enrichment were also ammonotelic. Therefore, it is more likely that the absence of δ15N enrichment in ctenophores and O. oglinum is more related to the number of days that these animals received eggs than to nitrogen excretion mode. Experiments with ctenophores were not conducted for a longer duration, as shrinkage was observed in these animals when kept on a monotypic diet of eggs for more than 1 week.
Our study demonstrated that in a controlled setting, a combination of FAs and SI of nitrogen can be used to trace recent fish egg consumption in several species of egg consumers. All egg consumers took on FA characteristics of eggs. However, δ15N values reflective of egg consumption were detected in only three of the six egg consumers. This indicates faster turnover of FAs in the tissue of egg consumers in comparison to nitrogen SI. Similar findings were reported by Mohan et al. (2016), where turnover of FAs in tissues of juvenile Atlantic croaker occurred within a few weeks after a dietary shift compared with months for SIs.
The species of egg consumers selected for this study were smaller and occupied lower trophic levels than the adult red drum that produced the eggs. This highlights the possible existence of counter-gradient flow of nutrients in marine food webs, from larger to smaller organisms, as proposed by Fuiman et al. (2015). The presence of counter-gradient flow and the importance of egg boons to marine food webs needs to be confirmed through field sampling, using the biomarkers identified in this study.
Acknowledgements
We would like to thank Cynthia K. Faulk, Leigh S. Walsh, Rene Lopez and Grace Walsh for their valuable assistance in the field and lab. We thank Dr Ryan Hladyniuk and Patricia Garlough at the UTMSI Core Isotope Facility for their help with bulk isotope analyses. A special thanks to the staff at the Texas Parks and Wildlife Department CCA Marine Development Center for providing red drum eggs for lab experiments.
Footnotes
Author contributions
Conceptualization: P.N., L.A.F.; Methodology: P.N., C.M.M., L.A.F.; Validation: P.N., L.A.F.; Formal analysis: P.N., L.A.F.; Investigation: P.N., C.M.M.; Resources: L.A.F.; Data curation: P.N., C.M.M.; Writing - original draft: P.N.; Writing - review & editing: P.N., C.M.M., L.A.F.; Visualization: P.N.; Supervision: L.A.F.; Project administration: P.N., L.A.F.; Funding acquisition: P.N., L.A.F.
Funding
This work was funded by the National Science Foundation Biological Oceanography program (Award number: OCE-2023618).
Data availability
Data are available from BCO-DMO: www.bco-dmo.org/project/817943
References
Competing interests
The authors declare no competing or financial interests.