While the use of biochemical biomarkers in marine food-web studies has a long and successful tradition in shallow-water ecosystems, starting from the 1970s with the use of stable isotopes (McConnaughey and McRoy, 1979) and lipids (Lee et al., 1971), their application in deep-water environments is relatively new (e.g., Iken et al., 2001; Polunin et al., 2001; Howell et al., 2003). Undoubtedly, technological advances made over the past few decades have allowed the exploration of ever deeper ecosystems with more refined techniques. Iken et al. (2001) were among the first to provide a comprehensive analysis of a deep-sea food web, which was sampled at a depth of ∼ 4840 m at the Porcupine Abyssal Plain (PAP, northeast Atlantic), by using bulk stable N and C isotope ratios (δ15N and δ13C, respectively) as trophic markers. In the same year, Polunin et al. (2001) used the same approach to study the trophic relationships of a slope megafaunal assemblage collected off the Balearic Islands (western Mediterranean). Since these first two investigations, several others have been carried out across different oceanic regions and climes, such as the Canadian Arctic (Iken et al., 2005), the Arabian Sea (Jeffreys et al., 2009), and the Sea of Japan (Kharlamenko et al., 2013). Furthermore, over the past decade, it has become evident that the simultaneous use of different trophic markers (e.g., δ15N, δ13C, and fatty acids, FAs) and techniques (e.g., bulk or compound specific isotope analysis, as well as FAs, gut content, and morphometric analyses) provides a more complete picture of trophic structure and dynamics. Indeed, while the first investigations relied on a single method (Iken et al., 2001; Polunin et al., 2001; Howell et al., 2003), the latest trend in deep-sea food-web studies favors an integrative approach, which maximizes the efficiency of each technique, while increasing the resolution of the investigation (e.g., Stowasser et al., 2009; Parzanini et al., 2017).

For the first time since the implementation of trophic markers in studies of deep-sea food webs, this review synthesizes current knowledge in this growing field of research, mainly focusing on heterotrophic ecosystems (i.e., relying on photosynthetic primary production). In addition, it provides a preliminary overview of large-scale geographic trends from the analysis of isotopic and FA data for macro- and megafauna, along with guidance for future investigations. In particular, the present contribution (i) briefly defines various trophic biomarkers and their respective advantages; (ii) describes deep-sea food webs, based on examples from the literature; (iii) lists the sources of variation among the different studies to highlight pitfalls and gaps; and (iv) provides a preliminary quantitative analysis across studies by using relevant data sets.

The analysis of gut contents was among the first techniques (together with in situ observation of feeding behaviors) applied in trophic ecology and food-web studies in aquatic systems (Gartner et al., 1997; Michener and Kaufman, 2007). Subsequently, other methods were developed as alternative or supplementary means of studying diet and feeding behaviors within the same ecosystems. Among them, the use of biochemical markers as trophic tracers rapidly grew in popularity in food-web ecology since it is relatively simple and should overcome many of the issues ascribed to gut content analysis (Michener and Kaufman, 2007). In this regard, Table 1 lists strengths and drawbacks of gut content analysis and of the two most popular biochemical techniques, i.e., bulk stable isotope and FA analyses. For instance, bulk stable isotope and FA analyses may, theoretically, be performed on any species, regardless of feeding mode and food sources, whereas gut content analysis can only be applied to those organisms characterized by a sufficiently large and full stomach. Except in cases where individuals are too small and have to be analyzed whole, biochemical analyses are typically conducted on target tissues (e.g., muscle) that provide long-term dietary data and reduce intra-individual variability (Table 1). In addition, the use of biochemical tracers requires shorter processing times than gut content analysis. Thanks to this integrative approach and faster output, the application of food-web tracers has been particularly helpful in deep-sea studies, which are often plagued by financial and logistical constraints. Furthermore, due to its relative ease of use, it has favored the analysis of wider sets of taxa/feeding guilds, primary producers included, rather than focusing on one or a few focal groups. However, the interpretation of isotopic and FA data is complex, and both techniques require dedicated and sophisticated instrumentation (e.g., gas chromatograph, mass spectrometer) and knowledge of intrinsic sources of variations (see Sect. 1.4). Although each method needs a sufficient sample size, only gut content analysis may provide direct and clear taxonomic evidence of the diet (Table 1). Therefore, as stated above, the latest trend in trophic ecology advocates a multifaceted approach, on the understanding that each technique may offer unique and valuable data.

The principle behind the use of food-web tracers is that the biochemical signature of consumers reflects that of their diet. Among them, δ15N and δ13C are the most popular. While the former is used to study trophic positions and dietary sources, with an enrichment factor of 2 ‰–4 ‰ between a consumer and its food (Minagawa and Wada, 1984), the latter undergoes little fractionation (< 1 ‰) and, therefore, is used to distinguish primary food sources (McConnaughey and McRoy, 1979). For further details, refer to Sulzman (2007) and Michener and Kaufman (2007), who have provided extensive reviews on the chemistry behind stable isotopes and their use as food-web tracers, respectively. In addition, sterols, FAs, and amino acids, which are important constituents of lipids (for the first two) and proteins (for the last), have successfully been used to study trophic relationships and dietary sources in deep-water systems (Howell et al., 2003; Drazen et al., 2008a, b). Their use is based on the principle that certain FAs and amino acids are considered essential for animals, being required for optimal fitness. However, most species cannot synthesize these essential compounds de novo and, therefore, they must gain them through their diet. Indeed, only primary producers and a few consumers possess the enzymatic apparatus to synthesize essential FAs and amino acids de novo. Conversely, a few taxa are unable to synthesize sterols de novo, which are critical for them; therefore, they have to acquire these essential sterols through diet (Martin-Creuzburg and Von Elert, 2009). Because sterols, FAs, and amino acids undergo little or no alteration when consumed, it is possible to detect dietary sources within the consumers' tissues (Parrish et al., 2000). The isotopic signature of amino acids can also be used to study trophic position through compound specific analysis (δ15N), as some of these acids show trophic enrichment (Bradley et al., 2015). Detailed information about FA analysis was outside the scope of this study, and is provided by Parrish (2009) and Iverson (2009), whereas the use of sterols as food-web tracers was outlined in Martin-Creuzburg and Von Elert (2009) and Parrish et al. (2000). McClelland and Montoya (2002) and Larsen et al. (2009) discuss the use of amino acids as trophic biomarkers.

As there is no photosynthetically derived primary production in the deep sea, deep-water ecosystems are mostly heterotrophic (Gage, 2003), and may hence largely rely on particulate organic matter (POM) that passively sinks from the surface waters as a primary source of nutrients (Hudson et al., 2004). Nonetheless, food can also be actively transported down by those animals that carry out vertical diel migrations through the water column (Trueman et al., 2014); it can also be provided by the occasional fall of large animal carcasses (Smith and Baco, 2003), and/or by lateral inputs, from inland and shelf areas towards abyssal offshore regions (Pfannkuche, 2005). Although most of the deep-water ecosystems are heterotrophic, a few, such as hydrothermal vents and cold seeps, are fueled by chemical energy (e.g., methane, hydrogen sulfide) and rely on chemosynthetic microorganisms for the production of organic matter. Each of these primary food sources has a specific isotopic composition and biochemical signature, resulting from a combination of chemical and physical processes reflective of its origin. By knowing the composition of the food source(s) that fuel(s) a given food web, it is possible to reconstruct its trophic structure and dynamics. Conversely, by measuring the signatures of the food-web components, it is possible to assess food sources on which they rely. For instance, Iken et al. (2001) showed that phytodetritus was the primary energy input of the deep-sea benthic community at PAP, and also defined two different trophic pathways: a pelagic and isotopically lighter one in which sinking POM and small pelagic prey constituted the main food sources, and a benthic and more isotopically enriched trophic pathway, fueled by degraded sedimented POM. In fact, once POM settles on the seafloor, it undergoes continuous degradation by microbes and is reworked through bioturbation and feeding activities, thus leading to a more isotopically enriched material relative to the sinking one (Iken et al., 2001). Depending on the primary food source they relied on, benthic organisms at PAP were thus characterized by either lower or higher values of δ15N. Similar scenarios of dual trophic pathways characterizing benthic systems were also found by Iken et al. (2005) in the Canadian Arctic, Drazen et al. (2008b) in the North Pacific, Reid et al. (2012) within the benthic community sampled on the mid-Atlantic Ridge, Valls et al. (2014) in the western Mediterranean, and Parzanini et al. (2017) in the northwest Atlantic. Moreover, Kharlamenko et al. (2013) used both stable isotopes and FAs to study the dietary sources of benthic invertebrates collected along the continental slope (500–1600 m depth) in the Sea of Japan. The authors recognized different trophic pathways (i.e., planktonic, benthic, microbial) and dietary sources by using biochemical tracers, and they proposed a strong link with the primary production of the surface waters, as the FA composition of the deep-sea echinoderms and mollusks was similar to that of the shallow-water counterparts.

As POM sinks through the water column, its δ15N increases, reflecting the preferential assimilation of the lighter isotope, 14N, by microbes; in particular, a gradient in POM δ15N has been detected with depth, where POM at greater depths is more enriched (Altabet et al., 1999). For this reason, Mintenbeck et al. (2007) carried out a study in the high-Antarctic Weddell Sea to assess whether this gradient was reflected in the isotopic signature of POM consumers sampled at 50–1600 m. In this regard, only those organisms feeding directly on sinking POM (e.g., suspension feeders) showed increasing values of δ15N with depth, whereas the increase was less evident for the deposit feeders (Mintenbeck et al., 2007). Similar results for suspension feeders were obtained by Bergmann et al. (2009), who analyzed a benthic food web sampled at the deep-water observatory HAUSGARTEN, west of Svalbard (Arctic), between 1300 and 5600 m depth. Conversely, deposit feeders exhibited a negative trend along the bathymetric gradient in terms of δ15N, and predator/scavengers were not affected. In another study, Sherwood et al. (2008) did not detect any relationships with depth in the δ15N values measured from cold-water corals collected on a slope environment in the northwest Atlantic. Among the explanations suggested for these inconsistencies and differences among feeding groups, Mintenbeck et al. (2007) and Sherwood et al. (2008) included feeding preferences with respect to the size and sinking velocity of POM. According to these authors, only those organisms feeding on small particles of sinking POM should reflect a bathymetric gradient in δ15N. In fact, small-sized particles sink at a lower velocity and, therefore, experience high rates of degradation, with more evident changes in δ15N (Mintenbeck et al., 2007). Based on these findings, depth-stratified sampling should ideally be conducted when studying a system characterized by a bathymetric gradient, as it would prevent biases in the interpretation of the isotopic data.

Deep-water systems are generally characterized by a limited food supply, as the quantity of food being transferred from the surface to the bottom diminishes with increasing depth (Gage, 2003). In addition, in temperate areas, food arrives as intermittent pulses, following the spring and late summer blooms of primary (and secondary) productivity. For this reason, deep-water benthic communities can only rely on fresh, high-quality phytodetritus within short temporal windows following algal blooms; whereas reworked and resuspended POM fuels these communities for the rest of the year (Lampitt, 1985). Deep-sea benthic organisms have hence developed adaptations and strategies to increase their feeding success and minimize competition for food, including trophic niche expansion and specialization. In this regard, certain benthic taxa (e.g., pennatulacean corals, hexactinellid sponges) and/or feeding groups (e.g., suspension and deposit feeders) at PAP showed vertical extension of their trophic niches (i.e., omnivory), which, according to Iken et al. (2001), was most likely driven by a strong competition for food. In other words, some species belonging to the same taxon or feeding guild shared similar food sources (i.e., exhibiting similar δ13C values), but they were located at different trophic levels (i.e., exhibiting a wide range of δ15N). Similarly, Jeffreys et al. (2009) reported trophic niche expansion among and within feeding guilds sampled between 140 and 1400 m depth, at the Pakistan margin (Arabian Sea). Pennatulacean corals and other sestonivorous cnidarians, for example, displayed the greatest niche expansion; they fed on not only POM, but also small invertebrates (e.g., zooplankton). Moreover, ophiuroids, which are typically selective deposit feeders, switched to an omnivorous diet under food-limited conditions (Jeffreys et al., 2009). Apart from trophic niche expansion, Iken et al. (2001) proposed that specialization on certain food items represented another adaptation developed by benthic organisms at PAP to mitigate competition for food. Holothuroid echinoderms, for instance, were thought to accomplish food specialization through a combination of different factors involving changes in morphology, mobility, and digestive abilities (Iken et al., 2001). Further examples of trophic niche segregation and food partitioning, as strategies to minimize competition, were also reported for deep-sea demersal fishes in the northwest Mediterranean Sea (Papiol et al., 2013) and for asteroid echinoderms in the northwest Atlantic (Gale et al., 2013). Howell et al. (2003) detected trophic niche expansion across different species of deep-sea asteroids (1053–4840 m) by analyzing their FA composition. In particular, multivariate analysis of FA proportions discriminated three different feeding guilds among the asteroids analyzed, including mud ingesters, predators and scavengers, and suspension feeders.

When comparing studies relying on biochemical analysis, there are numerous sources of variation, which may influence results and findings and also prevent the detection of similarities and general trends. However, their importance may depend on the scale of the investigation (i.e., local, regional, or global). In this section, the main sources of variation are illustrated and explained by type (Table 2).

Comparisons among multiple groups of deep-sea organisms were run through t tests and one-way analysis of variance (ANOVA). In particular, isotopic (i.e., δ15N, δ13C) and FA (i.e., ARA, EPA, and DHA) data were compared across organisms from different latitudes (i.e., tropical, temperate, and polar), habitats (i.e., pelagic, demersal, benthic), and collection depths (i.e., mesopelagic, bathypelagic, meso-bathypelagic, abyssopelagic, bathyal, bathyal-abyssal, abyssal, and hadal) to detect any significant differences. When the normality assumption was violated, Mann–Whitney rank sum test, Kruskal–Wallis one-way ANOVA on ranks, and Dunn's method pairwise comparisons were performed instead. In addition, multivariate statistics, i.e., principal coordinate analysis (PCO) and permutational MANOVA (PERMANOVA), were used to study the variability in the isotopic and FA composition of deep-water organisms across different latitudes, habitats, collection depths, and phyla. In addition, a distance-based linear model (DistLM) was run to assess which of these four factors contributed the most to such a variability. PCO, PERMANOVA, and DistLM were run on resemblance matrices, based on Euclidean distance for the isotopic data, and Bray–Curtis for the FA data. Data were not normalized or transformed prior to analysis. Univariate statistics were conducted using Sigmaplot 12.5, while PCO, PERMANOVA, and DistLM were run through Primer 7.0 with the add-on package PERMANOVA+ (Clarke and Gorley, 2006).

Analyses revealed both latitudinal and depth-related trends for isotopic and essential FA composition. In particular, mean values (± SD) of δ15N and δ13C were significantly lower in deep-sea fauna sampled at high latitudes than in that collected at low latitudes (δ15N, ANOVA on ranks, H = 35.6, p ≤ 0.001; δ13C, ANOVA on ranks, H = 277.9, p ≤ 0.001; Fig. 2). Conversely, no difference was detected across latitudes in terms of ARA, but mean proportions (± SD) of EPA and DHA were significantly greater at polar latitudes than at temperate and tropical areas (EPA, ANOVA on ranks, H = 11.4, p=0.003; DHA, ANOVA on ranks, H = 63.6, p ≤ 0.001; Fig. 3). Similarly, PERMANOVA detected significant differences across latitudes in terms of both stable isotopes (pseudo-F = 81.4, p(perm) = 0.0001) and essential FAs (pseudo-F = 11.0, p(perm) = 0.0001).

When deep-water species were analyzed separately according to their habitat, the same latitudinal trend in the isotopic composition was shown for deep-water benthic species (δ15N, ANOVA on ranks, H = 40.5, p ≤ 0.001; δ13C, ANOVA on ranks, H = 171.2, p ≤ 0.001), whereas, for demersal and pelagic species, only the δ13C ratios were significantly lower at higher latitudes (ANOVA on ranks, H = 105.7, p ≤ 0.001, for demersal species; ANOVA on ranks, H = 11.5, p=0.003, for pelagic species). PERMANOVA showed that the isotopic composition of deep-sea animals was indeed statistically different across the three habitats (pseudo-F = 112.6, p(perm) = 0.0001), and benthic and demersal species had higher stable N and C isotope ratios than the pelagic counterparts (p<0.05). Conversely, only benthic and pelagic species revealed a latitudinal gradient in their essential FA composition (EPA, ANOVA on ranks, H = 12.1, p=0.002; DHA, ANOVA on ranks, H = 43.6, p ≤ 0.001, for benthic species; EPA, ANOVA, H = 6.4, p=0.011, for pelagic taxa). In this regard, pelagic, demersal, and benthic taxa had a different essential FA composition (ARA, ANOVA on ranks, H = 39.7, p ≤ 0.001; EPA, ANOVA on ranks, H = 12.5, p=0.002; DHA, ANOVA on ranks, H = 76.9, p ≤ 0.001; pseudo-F = 19.7, p(perm) = 0.0001). Benthic species had the highest proportions of ARA and EPA (p<0.05), while demersal species had the highest levels of DHA, although similar to those of pelagic species.

While mean values of both stable N and C isotope ratios significantly increased with depth for benthic and demersal species (δ15N, ANOVA on ranks, H = 63.9, p ≤ 0.001; δ13C, ANOVA on ranks, H = 126.2, p ≤ 0.001), only δ13C ratios showed the same trend in pelagic taxa (ANOVA on ranks, H = 125.5, p ≤ 0.001). Proportions of EPA significantly decreased along the bathymetric gradient for pelagic taxa (ANOVA on ranks, H = 12.3, p=0.002), and levels of ARA were significantly higher at abyssal depths for benthic and demersal species (ANOVA on ranks, H = 39.7, p ≤ 0.001). In addition, levels of δ15N, δ13C, and ARA increased for benthic and demersal organisms with increasing depth (δ15N, ANOVA on ranks, H = 84.7, p ≤ 0.001; δ13C, ANOVA on ranks, H = 105.0, p ≤ 0.001; ARA, ANOVA on ranks, H =  22.8, p ≤ 0.001). PERMANOVA revealed significant differences in the isotopic (pseudo-F = 74.6, p(perm) = 0.0001) and essential FA composition (pseudo-F = 8.6, p(perm) = 0.0001) across collection depths.

Among the four variables considered (i.e., latitude, habitat, collection depth, and phylum), analyses revealed that “habitat” and “phylum” were the most important factors influencing the variability of the stable isotope (respectively 12 % and 9 %; DistLM, adjusted R2=0.4) and FA (respectively 8 % and 11 %; DistLM, adjusted R2=0.3) composition of deep-water organisms (Fig. 4).

The present analysis shows, for the first time, the existence of (a) latitudinal trends in both stable isotope and essential FA composition of deep-sea organisms, with decreasing δ13C ratios and increasing ω3 LC-PUFAs towards the poles, and (b) global bathymetric trends in the isotopic composition of deep-water fauna for which mean levels of δ15N, δ13C, and ARA increased with increasing depth. In addition, it provides further evidence of the link, across latitudes and depth, between surface primary production of the surface waters and the deep-water consumers. The present findings generally align with reports of decreasing values of δ13C in surface-water plankton and POM towards the polar regions, in both the Southern Hemisphere and Northern Hemisphere (Sackett et al., 1965; Rau et al., 1982; Francois et al., 1993), as well as of increasing POM isotopic ratios along a bathymetric gradient (Altabet et al., 1999). They also agree with Colombo et al. (2016), who noticed that proportions of ω3 LC-PUFAs were higher in marine organisms from polar and temperate regions in comparison to tropical regions, and with Parzanini et al. (2018a), who detected increasing proportions of ARA along a slope area in the deep northwest Atlantic.

Water temperature, in combination with other abiotic (e.g., oceanographic and biogeochemical processes, nutrient supply) and biological factors (e.g., species metabolism, taxonomic composition of deep-water communities, microbial remineralization processes), seems to play a role in these trends (Rau et al., 1982; Francois et al., 1993; Altabet et al., 1999; Colombo et al., 2016). In particular, water temperature influences isotopic fractionation processes and, typically, higher fractionation is associated with lower temperatures (Sackett et al., 1965). High fractionation rates are also linked to the pronounced denitrification activities characterizing oligotrophic areas, such as observed in some areas of the tropics (Hetherington et al., 2017). This may explain the higher δ15N ratios of the deep-sea organisms from the tropical latitudes analyzed in this study. Furthermore, water temperature affects membrane fluidity, and lower temperatures decrease the fluidity of cell membrane (Parrish, 2013; Colombo et al., 2016). Thus, in order to maintain normal membrane function and condition, i.e., health, ectotherms may counteract variations in water temperature by readjusting their FA composition (Cossins and Lee, 1985; Parrish, 2013). For example, larger proportions of long-chain unsaturated FAs (e.g., ARA, EPA) within the lipid bilayer help increase membrane fluidity (Parrish 2013), as these molecules are characterized by a higher flexibility (DeLong and Yayanos, 1985; Colombo et al., 2016).

Trends in the isotopic and FA composition of deep-sea organisms were also seen along a depth gradient. As a proxy for water temperature as well as nutrient supply, depth may influence biochemical composition of marine consumers (Parzanini et al., 2018a, b). POM becomes more isotopically enriched while sinking to deeper depth due to microbial degradation (Altabet et al., 1999). Thus, the isotopic composition of deep-water organisms which feed on POM may vary accordingly (Mintenbeck et al., 2007). In the present analysis, levels of ARA were globally higher at deeper depths, similar to the study by Parzanini et al. (2018a), which may be due to (i) a higher reliance of deeper-dwelling organisms on the benthic-detrital trophic pathway, and/or (ii) the need to maintain membrane fluidity at low temperatures via increasing the unsaturation levels of membrane phospholipids.

Finding latitudinal trends in the biochemical composition of deep-water organisms that mirror results from shallow depths provides further evidence of the link between the two systems, in that deep-sea benthic communities rely on POM sinking from the surface water as a primary food source (Gage, 2003; Hudson et al., 2004). Close dependence of deep-sea food webs on near-surface processes raises important concerns. According to the latest climate estimates, both air and water temperatures have been rising, and continue to increase, and seawater pH has already dropped by 0.1 units due to large CO2 emissions and is expected to decrease further (IPCC, 2014). Furthermore, models predict that increasing surface water temperature will favor stratification, while reducing vertical mixing as well as enhancing variability in the transport of primary production and energy (i.e., carbon) transport to the deep sea (Smith et al., 2009; Jones et al., 2014; Sweetman et al., 2017). At the same time, deep-water benthic biomass is expected to decrease due to the increasing variability in the food supply, which may in turn affect health and functioning of benthic ecosystems, as well as global biogeochemical cycles (Jones et al., 2014). Hixson and Arts (2016) showed that the FA composition of the six most common fresh- and saltwater phytoplankton species responded to temperature and, specifically, that their ω3 PUFA levels decreased with increasing temperature. Not only do ω3 PUFAs, such as EPA and DHA, play an important role in the response to temperature variations in aquatic systems, but they are also essential nutrients and are highly required by aquatic organisms for optimal growth and health (Parrish, 2009). A case in point, Rossoll et al. (2012) showed experimentally that growth and reproduction of the copepod Acartia tonsa were severely compromised by the alteration of FA content and composition of its primary food source, the diatom Thalassiosira pseudonana, exposed to high CO2 levels. The present investigation, therefore, suggests that changes in amounts and composition of surface production could also result in changes in essential nutrients and biomarkers in deep-sea benthic organisms that feed on it, with possible cascading effects throughout deep-water food webs. Such variations may alter nutrient intake of deep-sea benthic organisms, as well as trophodynamics; and they may also influence species' abilities to cope with deep cold waters.