Sterol Composition of Sponges, Cnidarians, Arthropods, Mollusks, and Echinoderms from the Deep Northwest Atlantic: A Comparison with Shallow Coastal Gulf of Mexico

Triterpenoid biosynthesis is generally anaerobic in bacteria and aerobic in Eukarya. The major class of triterpenoids in bacteria, the hopanoids, is different to that in Eukarya, the lanostanoids, and their 4,4,14-demethylated derivatives, sterols. In the deep sea, the prokaryotic contribution to primary productivity has been suggested to be higher because local environmental conditions prevent classic photosynthetic processes from occurring. Sterols have been used as trophic biomarkers because primary producers have different compositions, and they are incorporated in primary consumer tissues. In the present study, we inferred food supply to deep sea, sponges, cnidarians, mollusks, crustaceans, and echinoderms from euphotic zone production which is driven by phytoplankton eukaryotic autotrophy. Sterol composition was obtained by gas chromatography and mass spectrometry. Moreover, we compared the sterol composition of three phyla (i.e., Porifera, Cnidaria, and Echinodermata) collected between a deep and cold-water region and a shallow tropical area. We hypothesized that the sterol composition of shallow tropical benthic organisms would better reflect their photoautotrophic sources independently of the taxonomy. Shallow tropical sponges and cnidarians from environments showed plant and zooxanthellae sterols in their tissues, while their deep-sea counterparts showed phytoplankton and zooplankton sterols. In contrast, echinoids, a class of echinoderms, the most complex phylum along with hemichordates and chordates (deuterostomes), did not show significant differences in their sterol profile, suggesting that cholesterol synthesis is present in deuterostomes other than chordates.


Introduction
In the deep sea (i.e., below 200 m depth), light and temperature diminish while pressure increases with depth [1]. In addition, both food quantity and quality decrease along the depth gradient [2]. and Echinodermata, the most complex phylum along with those of Hemichordata and Chordata (which include vertebrates), are deuterostomes [23]. Cholesterol biosynthesis pathway (CBP) genes in the basal metazoans were inherited from their last common eukaryotic ancestor and evolutionarily conserved for cholesterol biosynthesis. The genomes of the basal metazoans and deuterostomes retain almost the full set of CBP genes, while Cnidaria and many protostomes have independently experienced multiple massive losses of CBP genes that might have been due the appearance of an exogenous sterol supply and the frequent perturbation of ocean oxygenation [24].
Mar. Drugs 2020, 18, x FOR PEER REVIEW 3 of 21 cholesterol biosynthesis. The genomes of the basal metazoans and deuterostomes retain almost the full set of CBP genes, while Cnidaria and many protostomes have independently experienced multiple massive losses of CBP genes that might have been due the appearance of an exogenous sterol supply and the frequent perturbation of ocean oxygenation [24]. To assess the euphotic contribution from phytodetritus to deep-sea sponges, corals, gastropods, decapods, and echinoids, we used sterols, most of which are absent in prokaryotes, with the exception of modified lanosterol products [21] probably acquired via horizontal gene transfer from eukaryotes [22]. Although photoautotrophs are a rich source of sterols, some have to be altered before they suit metazoan needs, for example, the exchangeable use of cholesterol and stigmasterol [25].
Sterol biosynthesis in invertebrates does not take place or else proceeds at a slow rate; therefore, they should be provided by diet [26]. Certain phytosterols were more efficient in supporting somatic growth of the crustacean Daphnia magna than cholesterol (e.g., fucosterol and brassicasterol), while others were less efficient (e.g., dihydrocholesterol and lathosterol), indicating substantial differences in the assimilation efficiency and further processing within the body for these dietary sterols [27]. Therefore, phytosterols detected in consumers which have no bioconversion capacity can be used as biomarkers.
Here, we evaluated sterol composition of five phyla, Porifera, Cnidaria, Mollusca, Arthropoda, and Echinodermata, from the deep and cold northeast Atlantic Ocean to infer their food sources. We then compared three phyla, Porifera, Cnidaria [28], and Echinoidea, from Echinodermata [29] with published data to investigate if their sterol composition was affected by temperature and depth. We hypothesized that the tissues of shallow, tropical benthic organisms would better represent photoautotrophic sources, To assess the euphotic contribution from phytodetritus to deep-sea sponges, corals, gastropods, decapods, and echinoids, we used sterols, most of which are absent in prokaryotes, with the exception of modified lanosterol products [21] probably acquired via horizontal gene transfer from eukaryotes [22]. Although photoautotrophs are a rich source of sterols, some have to be altered before they suit metazoan needs, for example, the exchangeable use of cholesterol and stigmasterol [25].
Sterol biosynthesis in invertebrates does not take place or else proceeds at a slow rate; therefore, they should be provided by diet [26]. Certain phytosterols were more efficient in supporting somatic growth of the crustacean Daphnia magna than cholesterol (e.g., fucosterol and brassicasterol), while others were less efficient (e.g., dihydrocholesterol and lathosterol), indicating substantial differences in the assimilation efficiency and further processing within the body for these dietary sterols [27]. Therefore, phytosterols detected in consumers which have no bioconversion capacity can be used as biomarkers.
Here, we evaluated sterol composition of five phyla, Porifera, Cnidaria, Mollusca, Arthropoda, and Echinodermata, from the deep and cold northeast Atlantic Ocean to infer their food sources. We then compared three phyla, Porifera, Cnidaria [28], and Echinoidea, from Echinodermata [29] with published data to investigate if their sterol composition was affected by temperature and depth. We hypothesized that the tissues of shallow, tropical benthic organisms would better represent photoautotrophic sources, such as phytoplankton, zooxanthellae, macroalgae, and plants, than tissues in their deep-water counterparts, independently of the phylogenetic relationships (phyla) [30]. To test our hypothesis, we compared sterol profiles of Porifera, Cnidaria, and Echinoidea from two contrasting sites: the Northwest Atlantic continental slope and southwest Gulf of Mexico shallow tropical coral reefs.

Comparison of Sterol Composition of Benthic Invertebrates from the Deep, Cold Sea Northwest Atlantic and Shallow, Tropical Coastal Gulf of Mexico
As hypothesized, we found significant dissimilarity in sterol profiles between shallow tropical and deep cold-water organisms (Figure 2a). In addition, phylogeny had a significant effect on the sterol profile; however, there was a significant interaction effect between depth/temperature and phylogeny ( Figure 2b). Some tropical benthic organisms had significantly more campesterol, gorgosterol, and β-sitosterol from photoautotrophic sources such as zooxanthellae and plants, independent of phylum (t = 8.68, Markov chain probability (p(MC)) = 0.001, Figure 3a). Among them, the cnidarian Montastraea cavernosa had significantly higher proportions of campesterol and gorgosterol from zooxanthellae, while the deep-water cnidarian P. agaricus had a higher proportion of cholesterol, occelasterol, and brassicasterol, thus indicating zooplankton and phytoplankton sources (t = 11.86, p(MC) = 0.001, Figure 3b). Similarly, the sponge Aplysina sp., from the shallow, tropical site, showed high proportions of plant related sterols β-sitosterol and stigmasterol, as well as campesterol and cholesterol. On the other hand, the deep-water sponge T. muricata showed significantly higher proportions of phytoplankton sterols such as 24-methylenecholesterol and episterol (t = 9.43, p(MC) = 0.001, Figure 4a). In contrast to sponges and cnidarians, echinoids did not show significant differences in their sterol profiles (t = 2.09, p(MC) = 0.082, Figure 4b). In quantitative terms, lipids in shallow, tropical benthic organisms were significantly higher than in deep cold-sea organisms. The sponge Aplysina sp. had 15.4 ± 5.4 mg·g −1 wet weight, around fivefold more lipids than T. muricata with 2.6 ± 1.2 mg·g −1 , while the cnidarian M. cavernosa had 7.2 ± 3.6 mg·g −1 , almost twice the content of P. agaricus (4.1 ± 2.2 mg·g −1 ). A similar pattern was detected in echinoids, with Echinometra lucunter having 12.6 ± 1.2 mg·g −1 of lipids and P. placenta having 6.0 ± 3.1 mg·g −1 (Table 3).     Sterol composition of the sponge T. muricata suggests apportionment from phytoplankton, brown algae, and perhaps terrestrial vascular plants (Table 2); however, its δ 13 C at −17.4% ± 4.9% is indicative of phytoplankton sources more than macroalgae or higher plants [31]. Moreover, its δ 15 N of 14.2% ± 0.3% [30] suggests a higher trophic level more related to zooplanktivore and detritivore food habits ( Table 3). The zooplankton and recent detritus diet is more likely because its C:N ratio of 5.4 ± 0.3 is low compared to C:N values as high as 10 with increasing organic matter decomposition, which is more common in old detritus [32].
Similarly, the Cnidaria P. agaricus reflects a phytoplankton supply due the presence of 24-methylenecholesterol and occelasterol characteristic of phytoplankton. They were probably trophically transferred by zooplankton, coinciding with the high proportion of cholesterol [29]. The latter is coincident with a lower trophic level indicated by its δ 15 N mean value of 11.0% ± 1.4% , a wide δ 13 C value of −13.8% ± 4.9% , and a C:N ratio of 6.5 ± 3.1 [30].
The mollusk Buccinum sp. and the arthropod P. tarda had the highest proportion of cholesterol, and a low or zero proportion of the phytosterol β-sitosterol. Cholesterol is probably provided by carnivore and scavenger food habits, since crustaceans with no or limited cholesterol content (<1%) have significantly decreased growth and survival rates in early stages [18]. There is consistency with the lowest C:N ratios of 4.2 ± 0.1 and 3.8 ± 0.04, respectively (Table 3), suggesting high apportionment of nitrogen from proteins characteristic of carnivores.
Lastly, the echinoid P. placenta had a greater variety of sterols including phytoplankton sterols such as occelasterol and zooplankton-related sterols such as 24-nordehydrocholesterol [29], a lower proportion of cholesterol, and a higher C:N ratio of 5.3 ± 0.5, suggesting that they are omnivores. Absence of hopanoids and C stable isotopes values far from metanothropic bacteria with a δ 13 C of −50% to −100% [5] suggest that the principal food supply is eukaryotic photoautotrophic in origin.

Comparison of Sterol Composition of Benthic Invertebrates from the Cold, Deep Northwest Atlantic and Tropical, Shallow Coastal Gulf of Mexico
Small sample sizes were not a statistical issue due to small variation and clear differences among species and sites. Permutational multivariate analysis of variance (PERMANOVA) allows multivariate comparisons of compositional data such as sterol profiles with data with or without a normal distribution of residuals. Here, 996 permutations were performed in the main test, and, in those species with small sample sizes such as sponges, 10 permutations allowed detecting significant differences (p = 0.001). Furthermore, between cnidarians there were 56 permutations allowing detection of significant differences (p = 0.001). In contrast, echinoids with 35 permutations did not show a significant difference (p = 0.082) because sterol profiles of echinoids from shallow and deep sites were quite similar.
Tropical, shallow benthic organisms with a lower complexity such as Porifera and Cnidaria reflected the available food supply in their sterol composition. The most obvious difference was detected in cnidarians because M. cavernosa reflected zooxanthellae sterols such as gorgosterol. Zooxanthellae contribution in deep, cold-water cnidarians is limited by light. In locations with clear water, the reduced light is enough to maintain hermatypic corals deeper than 50 m; for example, in the Atlantic Ocean, Agaricia grahamae grows at 119 m in the Bahamas [33], whereas, in the Red Sea, Leptoseris fragilis grows at 145 m [34]. The deepest record in the Pacific Ocean is for Leptoseris hawaiiensis at 165 m in Johnston Atoll [35]. However, P. agaricus was collected at 1027 m; thus, even a zooplankton supply revealed by 24-methylenecholesterol, occelasterol and brasicasterol is surprising. Similarly, tropical, shallow sponges had higher proportions of macroalgae and vascular plant sterols, in contrast to deep, sea sponges, also reflecting phytoplankton sterols such as 24-methylenecholesterol and episterol.
Echinoids from the tropical, shallow site were herbivores, as their δ 13 C of −10% ± 0.1% from macroalgae and their δ 15 N of 3.3% ± 0.3% [31] suggest a low trophic level (Table 3). In contrast, the echinoids from the cold, deep site had a more depleted δ 13 C of −14.3% ± 0.9% , indicating bentho-pelagic carbon sources, and higher δ 15 N ratios of 12.3% ± 0.3% [30], indicating higher tropic levels, such as those of omnivores or even carnivores (Table 3). However, echinoid sterol profiles did not show significant differences between tropical, shallow and cold, deep sites. There are two possible explanations. The first is that echinoids can select cholesterol rich macroalgae, which could explain why the sea urchin E. lucunter had a high contribution of red algae Galaxaura sp. [31], coincident with the high proportion of cholesterol in Galaxaura sp. [29]. The second is that echinoids can modify phytosterols via dealkylation or synthesize cholesterol similarly to vertebrates among the chordates. The presence of lathosterol, a precursor of cholesterol, in P. placenta suggests that echinoderms can actually synthesize cholesterol. Lathosterol has been recorded in other cold, deep-water echinoderms such as the holoturian Cucumaria japonica [38]. This suggests that the maintenance of the complete set of CBP genes in vertebrates [24] is also maintained in echinoids.
Lastly, as lipids are the storage reservoir of energy in animals [19], the ratio between triacylglycerols and sterols (TAG:ST) can be used as an indicator of nutritional condition in fish larvae [39] and invertebrates [40]. Tropical, shallow sponges, cnidarians, and echinoids showed a significantly higher concentration of lipids and higher TAG:ST ratios, suggesting a better nutritional condition than deep, cold-water invertebrates, even if primary productivity was slightly higher in the Newfoundland offshore with a chlorophyll concentration of 1.24 mg·m −3 [41] in comparison with the Parque Nacional Sistema Arrecifal Veracruzano (PNSAV) with a chlorophyll concentration of 1.0 mg·m −3 [42] in similar seasons. Further investigation comparing essential fatty acids would provide more information about food quality.

Study Area
The geographical location and depth profile of the northwest Atlantic and southwest Gulf of Mexico sites are shown for comparison in Figures 5 and 6. The cold, deep study area was located in zone 3K, which represents the marine area located off the northern coasts of Newfoundland, according to the Northwest Atlantic Fisheries Organization (NAFO) division of the North Atlantic Ocean (Figure 6; more details in [43]). Mean daily bottom temperature during the sampling period at the sampling site ranged between 3.2 and 4.5 • C, with a slight decrease with depth. This region of the Northwest Atlantic is characterized by high productivity levels and is affected by seasonal blooms of large-celled phytoplankton; for instance, in 2012, chlorophyll a ranged from 1.24 mg·m −3 in November to 2.23 mg·m −3 in April [41], and there are strong lateral food inputs [44][45][46]. Due to its well-known productivity, the area has been heavily exploited and fished.  In contrast, the tropical shallow samples were collected in the Veracruz reef system national park (Parque Nacional Sistema Arrecifal Veracruzano; PNSAV), which is located off the State of   In contrast, the tropical shallow samples were collected in the Veracruz reef system national park (Parque Nacional Sistema Arrecifal Veracruzano; PNSAV), which is located off the State of In contrast, the tropical shallow samples were collected in the Veracruz reef system national park (Parque Nacional Sistema Arrecifal Veracruzano; PNSAV), which is located off the State of Veracruz, Mexico, adjacent to the cities of Boca del Rio and Anton Lizardo (19 • 02 24 to 19 • 15 27 north (N), 96 • 12 01 to 95 • 46 46 west (W)). It is part of a larger coral reef system in the Caribbean and Gulf of Mexico. A group of 13 reefs is located near Veracruz and Boca del Rio, and another group of 15 reefs with larger structures is located near Antón Lizardo; the two reefs are separated by the Río Jamapa and delimited to the north by Río La Antigua and to the south by the Río Papaloapan. During the summer, under sustained southward winds, a cyclonic eddy develops off Veracruz Port, which enhances productivity in the area reaching 2.3 ± 1.1 g·m −3 total plankton wet biomass [47] and a chlorophyll concentration of 1 mg·m −3 [42].

Sampling
In the cold, deep site, animals belonging to five phyla were opportunistically collected within 7 days in November-December 2013, during one of the annual multispecies bottom-trawl surveys conducted by Fisheries and Oceans (DFO), Canada. Individuals were sampled onboard the CCGS Teleost, from a total of 23 tows inside a 100 km radius and a depth range of 313 to 1407 m. The gear used to collect the samples (Campelen 1800 shrimp trawl) included a 16.9 m wide net with four panels of polyethylene twine. Once on board, individuals were immediately vacuum-packed and frozen at −20 • C to minimize lipid oxidation and hydrolysis. Individuals were identified to the lowest possible taxonomic level, from direct observation and through photo-identification. A total of 34 deep-sea organisms, belonging to five species from different phyla, were weighed, and samples of 0.7 ± 0.2 g wet weight were collected from each individual and processed for lipid analysis at the Core Research Equipment and Instrument Training Aquatic Research Cluster (CREAIT-ARC) Facility at Memorial University ( Table 1). The following were sampled: pieces of sponges, body walls from cnidarians, foot muscle from gastropods, dorsal abdominal muscle from crustaceans, and wall and tube feet from echinoids.
Sponges, cnidarians, and echinoids sterols from the deep, cold-water site were compared to sterols of sponges and cnidarians [28] and echinoderms [29] from shallow tropical coral reefs. Sponges and echinoderms were collected in September 2007 and coral was collected in October 2009 in the Cabezo and Blanca reefs at depths from 18 to 20 m (Figure 7).
Veracruz, Mexico, adjacent to the cities of Boca del Rio and Anton Lizardo (19°02′24″ to 19°15′27″ north (N), 96°12′01″ to 95°46′46″ west (W)). It is part of a larger coral reef system in the Caribbean and Gulf of Mexico. A group of 13 reefs is located near Veracruz and Boca del Rio, and another group of 15 reefs with larger structures is located near Antón Lizardo; the two reefs are separated by the Río Jamapa and delimited to the north by Río La Antigua and to the south by the Río Papaloapan. During the summer, under sustained southward winds, a cyclonic eddy develops off Veracruz Port, which enhances productivity in the area reaching 2.3 ± 1.1 g·m −3 total plankton wet biomass [47] and a chlorophyll concentration of 1 mg·m −3 [42].

Sampling
In the cold, deep site, animals belonging to five phyla were opportunistically collected within 7 days in November-December 2013, during one of the annual multispecies bottom-trawl surveys conducted by Fisheries and Oceans (DFO), Canada. Individuals were sampled onboard the CCGS Teleost, from a total of 23 tows inside a 100 km radius and a depth range of 313 to 1407 m. The gear used to collect the samples (Campelen 1800 shrimp trawl) included a 16.9 m wide net with four panels of polyethylene twine. Once on board, individuals were immediately vacuum-packed and frozen at −20 °C to minimize lipid oxidation and hydrolysis. Individuals were identified to the lowest possible taxonomic level, from direct observation and through photo-identification. A total of 34 deep-sea organisms, belonging to five species from different phyla, were weighed, and samples of 0.7 ± 0.2 g wet weight were collected from each individual and processed for lipid analysis at the Core Research Equipment and Instrument Training Aquatic Research Cluster (CREAIT-ARC) Facility at Memorial University ( Table 1). The following were sampled: pieces of sponges, body walls from cnidarians, foot muscle from gastropods, dorsal abdominal muscle from crustaceans, and wall and tube feet from echinoids.
Sponges, cnidarians, and echinoids sterols from the deep, cold-water site were compared to sterols of sponges and cnidarians [28] and echinoderms [29] from shallow tropical coral reefs.

Laboratory Analysis
Samples were weighed wet, and around 0.7 ± 0.2 g was immersed in 4 mL of chloroform, sealed under nitrogen gas, and stored in a freezer (−20 • C). Lipids were extracted by adding 2 mL of methanol and 1.8 mL of distilled water, mixed with a vortex, and centrifuged to separate polar and nonpolar phases. The lower phase was recovered in a lipid-cleaned tube; an aliquot was separated into lipid classes by Chromarod thin-layer chromatography (TLC) [48]. Chromarod TLC and Iatroscan flame ionization detection (FID) were used to quantify the lipid classes in the extracts. TLC/FID was performed with Chromarods SIII and an Iatroscan MK VI (Iatron Laboratories, Tokyo, Japan). Concentrations of triacylglycerols (TAG), steryl esters, and free sterols were obtained by interpolation with a calibration curve constructed with five amounts, ranging between 0.5 and 4.0 µg, of the following standards: cholesteryl stearate, triplamitin, and cholesterol (Sigma-Aldrich, Toronto, Canada). The remainder of the extract was separated by a solid-phase extraction column (Strata SI-1 Silica 55 mm, 70 A; Phenomenex, Torrance, CA, USA). Neutral lipid fractions were recovered with 6 mL of chloroform/methanol/formic acid (98:1:0.05); then, galactoglycerolipids were recovered with 6 mL of 100% acetone, and phosphoglycerolipids were recovered with 9 mL of chloroform/methanol (5:4) as a modification of [49]. Quantification of lipids, stable isotopes, and C:N molar ratios for cold, deep samples were obtained from [30], and lipids and stable isotopes of tropical shallow echinoids were obtained from [31]. Stable isotopes and C:N molar ratios were used to assign functional group and possible organic carbon source (Table 3).
Neutral lipid, galactoglycerolipid, and phosphoglycerolipid fractions were transmethyl esterified with Hilditch reagent, and then silylated with bis trimethylsilyl trifluoroacetamide (BSTFA) (Supelco: 3-2024) [49]. Sterols from all samples were recovered in hexane and analyzed in a gas chromatograph coupled to a mass spectrometer (GC-MS (EI): Agilent 6890N GC-5973 MSD (quadrupole) with a DB-5 column 30 m × 0.32 mm × 0.25 µm, Agilent Technologies, Santa Clara, CA, USA). The quadrupole was set at 150 • C, and the mass spectrophotometer ion source was set at 270 • C and 70 eV. The positive ionization voltage on the repeller pushed the positive ions into several electrostatic lenses, producing a positive ionization mode. Peaks were identified by the retention time of standards and mass spectra interpretation. Sterols were identified by reference to [20] and [50] (p. 235). Positions of peaks were useful for identification of sterols with the same molecular ion, and the list provided by [20] [29] (pp. [17][18][19]. No peaks were detected in the galactoglycerolipid or phosphoglycerolipid fractions; therefore, all results refer to the neutral lipid fraction.

Data Analysis
Species differences of individual (univariate) sterols were tested with one-way ANOVA using the Fisher statistic (F). Residual analyses were used to test assumptions of normality and equal variance. The effect of tropical shallow vs. cold deep waters on sponge, cnidarian, and echinoderm sterols was tested with multivariate analyses of sterol profile (>2% in a least one sample) using PRIMER 6.1.16 software and PERMANOVA + 1.0.6 (PRIMER-E, Plymouth, UK). Nonmetric multidimensional scaling was conducted on the basis of the Bray-Curtis similarity coefficient No transformation was used to avoid artificial weighting of sterols that gave only trace contributions to their respective profiles. Similarities among species and depth were investigated using the similarity percentages function (SIMPER), and statistical differences were tested with two-way analysis, for depth and phyla, using permutational multivariate analysis of variance (PERMANOVA), which allows multivariate comparisons with data with or without a normal distribution of residuals. The method of permutation of residuals under a reduced model was used, and significant differences were tested with Markov chain probability p(MC) values because the sample size was n < 30.

Conclusions
In the deep cold sites, gastropods and arthropods had cholesterol as their main sterol. Other extraction systems such as acetic acid and methanol could facilitate finding bioactive sterol metabolites for use against diabetes and oxidative stress-induced inflammatory diseases [51]. Sponges and cnidarians from shallow, tropical environments had plants such as mangrove or seagrass and zooxanthellae sterols, respectively, while cold, deep sponges had 24-ethyl sterols probably synthesized themselves [36], and cnidarians had phytoplankton, zooplankton, and

Data Analysis
Species differences of individual (univariate) sterols were tested with one-way ANOVA using the Fisher statistic (F). Residual analyses were used to test assumptions of normality and equal variance. The effect of tropical shallow vs. cold deep waters on sponge, cnidarian, and echinoderm sterols was tested with multivariate analyses of sterol profile (>2% in a least one sample) using PRIMER 6.1.16 software and PERMANOVA + 1.0.6 (PRIMER-E, Plymouth, UK). Nonmetric multidimensional scaling was conducted on the basis of the Bray-Curtis similarity coefficient.
No transformation was used to avoid artificial weighting of sterols that gave only trace contributions to their respective profiles. Similarities among species and depth were investigated using the similarity percentages function (SIMPER), and statistical differences were tested with two-way analysis, for depth and phyla, using permutational multivariate analysis of variance (PERMANOVA), which allows multivariate comparisons with data with or without a normal distribution of residuals. The method of permutation of residuals under a reduced model was used, and significant differences were tested with Markov chain probability p(MC) values because the sample size was n < 30.

Conclusions
In the deep cold sites, gastropods and arthropods had cholesterol as their main sterol. Other extraction systems such as acetic acid and methanol could facilitate finding bioactive sterol metabolites for use against diabetes and oxidative stress-induced inflammatory diseases [51]. Sponges and cnidarians from shallow, tropical environments had plants such as mangrove or seagrass and zooxanthellae sterols, respectively, while cold, deep sponges had 24-ethyl sterols probably synthesized themselves [36], and cnidarians had phytoplankton, zooplankton, and probably fungal sterols. In contrast, echinoids, belonging to Echinodermata, the most complex phylum along with hemichordates and chordates (deuterostomes), did not show significant differences in their sterol profiles, suggesting that cholesterol synthesis is present in deuterostomes other than chordates. Tropical, shallow sponges, cnidarians, and echinoids showed a significantly higher concentration of lipids, as well as higher TAG:ST ratios, suggesting a better nutritional condition than deep, cold invertebrates. However, other echinoderm groups should be studied such as holothurians which did not have cholesterol as the main sterol, but rather diatomsterol, probably because supercritical fluid extraction was used [38]. Sex was the main factor affecting sterol amount in the gonads (intact, spawned) and gametes of echinoderm A. dufresnii, with differences in their total concentration from intact to spawned states explained by gamete concentrations [52]. As gonads are a good functional food for diabetes mellitus treatment in mice [53], sex and gonad composition of echinoderms should be recorded in future surveys.