Induction of defensive traits in marine plankton—new copepodamide structures

Marine zooplankton release chemical cues, which trigger defenses in unicellular phytoplankton, such as increased toxin production and changes of colony sizes. Here, we identify the structure of two novel alarm cues belonging to the group of copepodamides. Similar to the known copepodamides, one of the compounds described is shown to trigger both amnesic and paralytic shellfish toxin production and chain length shortening in Skeletonema marinoi. In addition, we report the putative structures of another 21 copepodamides, which constitute 28% of the total copepodamides extractable from whole animals, suggesting that the copepodamide concentrations have been underestimated in earlier studies. We introduce a structure‐based nomenclature to handle the increasing number of copepodamides. Analysis of 12 copepod species showed that marine calanoid and freshwater cyclopoid copepods contain copepodamides. The only harpacticoid included in the analysis, Tigriopus californicus, did not appear to produce detectable amounts of copepodamides. Feeding experiments revealed that copepodamide compositions depend on both diet and species‐specific properties. Copepodamides induce both morphological and biochemical defensive traits in phytoplankton and may drive large‐scale trait–mediated effects in marine food webs. The more comprehensive list of copepodamides reported here makes it possible to explore the role of the copepodamide signaling system in the pelagic ecosystem in greater detail.

The free water mass of the oceans, the pelagial, is Earth's largest ecosystem, and its primary producers, phytoplankton, account for half of the planets primary production (Field et al. 1998). The zooplankton community of the upper part of the pelagial is dominated by copepods. In productive areas, they occasionally reach densities of hundreds of individuals per liter (Hamner and Carleton 1979;Ambler et al. 1991) and constitute up to 90% of the zooplankton biomass (Froneman 2001;Pane et al. 2004). Copepods play a key role in the food web as the main vector from the primary producers to higher trophic levels (Hansen et al. 1994;Verity and Smetacek 1996;Stibor et al. 2004).
Copepods exude unique chemical cues into surrounding waters (Selander et al. 2016). Among the exuded compounds are intraspecific and interspecific cues. This includes species, sex, and even life-stage-specific cues (Heuschele and Selander 2014). Exuded compounds also serve as early warnings of predator presence for prey organisms. Prey responses to predator presence include decreased colony size, induction of toxin production, increased bioluminescence, and increased cell wall silicification (Long et al. 2007;Pondaven et al. 2007;Bergkvist et al. 2012;Selander et al. 2015;Lindström et al. 2017). One group of such predator cues is taurine conjugated lipids, copepodamides. Copepodamides trigger paralytic shellfish and amnesic shellfish toxin production in dinoflagellates and diatoms E. Selander et al. unpubl.), increased bioluminescence in Alexandrium tamarense and Lingulodinium polyedra (Lindström et al. 2017), and chain length shortening in chain forming diatoms (E. Selander et al. unpubl.).
Copepodamides are characterized by the scaffold and the fatty acid attached to it. The known copepodamides contain common marine fatty acids such as docosahexaenoic (C22:6), eicosapentaenoic (C20:5), or stearidonic acid (C18:4). Copepods, however, contain many more fatty acids, and it is possible that more copepodamides exist in addition to the ones currently known. Further, copepodamides have only been measured in three species of calanoid copepods, Centropages typicus, Calanus sp., and Pseudocalanus sp. . Here, we report the structure of two novel copepodamides and suggest putative structures for an additional 21 copepodamides. The bioactivity and ecological relevance of one of the newly identified compounds is tested in dose response experiments monitoring morphological and biochemical defensive traits of representatives from the two major marine phytoplankton groups, dinoflagellates and diatoms. Additionally, we report the copepodamide composition of 10 common marine copepods in the NE Atlantic surface waters as well as one harpacticoid and one freshwater species and explore the effect of diets with different fatty acid composition on the copepodamide composition.

Copepodamide extraction and purification
Copepodamides from freeze-dried Calanus finmarchicus (11.32 g) (Calanus AS) were extracted with 200 mL methanol at −20 C for 48 h. The extract was mixed in a blender and sonicated at 40 C and > 20 kHz, for 30 min, and centrifuged for 5 min at 1800 rpm. The supernatant was collected and the procedure repeated with 150 mL methanol. Copepodamides were separated from less polar compounds by liquid-liquid partitioning (Löfgren et al. 2012). The methanol extract was supplemented with 1% aqueous NH 3 + , to a volume of 300 mL and mixed with 300 mL of heptane/methanol (98 : 2). The mixture was incubated on a shaking table for 30 min, transferred to a separation funnel, and 150 mL heptane/methanol (98 : 2) was added. The funnel was shaken vigorously for 3 min. The heptane phase was discarded and the procedure repeated twice with 300 mL portions of heptane/methanol (98 : 2). The methanol phase, containing the copepodamides, was evaporated using a rotary evaporator (Rotavapor R II, Büchi) at 40 C. The extract was resolved in methanol, diluted with milliQ water to 25% methanol (aq), and loaded onto an solid phase extraction (SPE) column (ENVI-18, 19 g, 60 mL; Sigma-Aldrich). Impurities were removed with 70% (v/v) methanol, and the copepodamides eluted in 100% methanol. The copepodamides were further purified by gradient elution on a high-performance liquid chromatography (HPLC; Hitachi ELITE LaChrom L-7100) system with a reversed phase column (KR100, C18, 5 μm, 4.6 × 150 mm; Hichrom) The gradient went from 100% A (methanol : acetonitrile : water; 35 : 35 : 35) to 100% B (2-propanol) over 25 min, 100% B was maintained for 5 min before re-equilibrating in 100% A for 7 min before the next injection. Both eluents were supplemented with 0.2% (v/v) formic acid, the flow rate was 1 mL min −1 . Fractions were collected (Waters Fraction Collector III) at 15 s intervals. Fractions were pooled based on copepodamide content. A final isocratic HPLC clean up step with methanol : acetonitrile : water : isopropanol, 9 : 8 : 3 : 80, with 0.2% (v/v) formic acid was used. Fractions containing significant amounts of pure copepodamides were pooled and stored at −20 C.

Structure verification of novel copepodamides
To separate and identify the hydrolysable fatty acid side chain in position C5 ( Fig. 1) from the copepodamide, we used the alkaline transmethylation modified after Christie (1976). Purified copepodamides were dissolved in 0.2 mL heptane, and 0.5 mL 0.5 mol L −1 sodium methoxide in dry methanol was added before incubation in a sonication bath for 30 min at room temperature. After neutralization with 50 μL acetic acid and 0.8 mL deionized water, the heptane phase was transferred into a new glass vial. Extraction was repeated once with 0.5 mL heptane, and the combined heptane phases were evaporated under N 2 flow at room temperature. The fatty acid methyl esters were dissolved in heptane and analyzed by gas chromatography-mass spectrometry (GC-MS) as described in Selander et al. (2015). To verify the identity of the lysocopepodamide scaffold, we extracted the remaining aqueous phase with chloroform. The chloroform was evaporated and the sample resolved in methanol before analysis on HPLC-MS/MS as described above. The sample was compared to nuclear magnetic resonance (NMR)-verified standards of lysocopepodamide, both individually and coinjected (Supporting Information Fig. S1).

Species-specific copepodamide composition
The marine copepod species Acartia clausi, Centropages hamatus, Centropages typicus, Metridia longa, Oithona sp., Pseudocalanus sp., and Temora longicornis were collected outside the Sven Lovén Center for Marine Sciences, Kristineberg, Sweden. C. finmarchicus, Calanus glacialis, and Calanus hyperboreus were collected from water at Svalbard, Norway, 2014. The intertidal harpacitcoid Tigriopus californicus was purchased from Reefphyto, and the freshwater specie Cyclops sp. was collected in a pond in the Gothenburg botanical garden, Sweden. Three to seven copepods were extracted in 1 mL methanol at −20 C for at least 24 h. The methanol was evaporated at 40 C under N 2 flow and the sample resolved in 40-80 μL methanol before analysis by HPLC-MS/MS as described above. The relative proportions (in %) of the different copepodamides analyzed in each sample were compared by principal component analysis (PCA) using SIMCA (Version 14.1 Umetrics).

Domoic acid analysis
The culture samples consisting of cells suspended in media were acidified with 0.2% (v/v) formic acid and loaded onto SPE columns (Bond Elute C18 LRC 10 mL, 200 mg, 40 μm; Agilent) to collect total (intracellular and extracellular) domoic acid. Columns were desalted with 5 mL milliQ water with 0.2% (v/v) formic acid. About 750 μL aqueous methanol (50%, v/v) was added to the column and left for 1 h, to extract cells. The samples were eluted into HPLC vials and analyzed on an Agilent 1200 LC system coupled to an Agilent 6410 triple quadrupole detector with electrospray interface, injection volume was 10 μL. We used a reversed phase column (Accucore 2.1 × 150 mm, 2.6 μm, Thermo Fisher Scientific) at 45 C and isocratic elution with 25% acetonitrile (aq) with 0.1% (v/v) formic acid. The flow rate was 0.2 mL min −1 . The ion source operated in negative mode at 300 C and 4500 V with a nitrogen gas flow of 5.8 L min −1 at 20 psi. Fragmentor voltage was set at 100 V and collision energy 14 eV. Domoic acid was identified by comparing retention time and diagnostic fragments, precursor ion m/z 312 and product ion m/z 266.2 (Tammilehto et al. 2012), with domoic acid standard solution (National Research Council, Certified Reference Materials Program, Canada).

Gonyautoxin analysis
The samples were transferred to a 1.5 mL reaction tube and centrifuged for 5 min at 16 C, 12,000 × g. The supernatant was discarded and the cell pellets lyophilized. Toxins were extracted in 200 μL acetic acid (0.05 mol L −1 ) by three freeze-thaw cycles. The lysate was centrifuged for 5 min at 12,000 × g, the supernatant transferred into HPLC vials and analyzed on an Agilent 1200 LC system coupled to an Agilent 6410 triple quadrupole detector with electrospray interface, injection volume was 1 μL. We used a reversed phase chromatography (Accucore 2.1 × 150 mm, 2.6 μm; Thermo Fisher Scientific) and isocratic elution with 50% acetonitrile (aq) with 0.1% (v/v) acetic acid, flow rate 0.2 mL min −1 . The electrospray ionization source was operated in positive ionization mode at 4.5 kV and 300 C with a nitrogen gas flow of 10 L min −1 at 35 psi. Fragmentor voltage was set at 104 V and collision energy 23 eV (gonyautoxin 1, 4) or 19 eV (gonyautoxin 2, 3). Gonyautoxins were identified from comparison of retention time and diagnostic fragments, precursor ion m/z 412.0 and product ion m/z 314.1 for gonyautoxin 1 and 4 and precursor ion 396.0 and product ion 298.1 for gonyautoxin 2 and 3 (Dell 'aversano et al. 2005) provided by analysis of standard solutions of gonyautoxin 1,4 and 2,3 (National Research Council, Certified Reference Materials Program, Canada).

Nonlinear regression fits
We performed nonlinear regression fits according to the Michaelis-Menten equation (GraphPad Prism 7, GraphPad Software) with the obtained phytoplankton response variables in relation to the copepodamide concentrations to get information about the maximum induced response: where phytoplankton response refers to toxicity or chain length reduction relative to controls, C denotes copepodamide concentration, R max the maximum induced response, and K m the half saturation constant, that is, the concentration inducing half the maximum induction.

Structure elucidation of novel copepodamides
To search for additional copepodamides, we performed a precursor scan of a polar lipid extract from C. finmarchicus (Fig. 2a). The precursor scan targeting compounds with the diagnostic fragment m/z 430 revealed two prominent peaks with precursor ions (negative mode) m/z 658 and 686 in addition to more than 20 minor constituents (Fig. 2b). GC-MS analysis of fatty acid methyl esters from isolated copepodamides 658 and 686 revealed the fatty acid side chain to be myristic acid (C 14:0) for copepodamide m/z 658 and palmitic acid (C 16:0) for copepodamide m/z 686. MS/MS experiments confirmed that the scaffold part of both compounds is identical to NMR-validated lyso-copepodamide Supporting Information Fig. S1). This confirms the structure of 14:0-copepodamide (C 36 H 68 NO 7 S − ; Fig. 1a) and 16:0-copepodamide (C 38 H 72 NO 7 S − ; Fig. 1b). Accurate mass of the compounds is consistent with the proposed structures (Supporting information 2).
A targeted search for additional copepodamides-and dihydrocopepodamides-containing fatty acids commonly found in copepods (Tiselius et al. 2012) revealed another 21 putative structures of copepodamides listed in Table 1, although we were unable to purify sufficient amounts for full structural elucidation of these. Together with 14:0-and 16:0-copepodamide, the novel copepodamides contribute 28% of total copepodamides in C. finmarchicus extract. We propose a simple structure-oriented nomenclature (Table 1) to name the growing number of copepodamides. The described copepodamides all fall into two groups with either a methyl (-CH 3 ) or a methylene (=CH 2 ) group at position C3 (Fig. 1). We refer to these groups as dihydrocopepodamides (former copepodamides A-C) and copepodamides (former copepodamides D-F). Each group is further characterized by different fatty acids attached to position C5 which is indicated by the prefix (e.g., 22:6-copepodamide, a copepodamide with a methylene group at C3 and docosahexaenoic acid [C 22:6] at C5). The deacylated species are in analogy with other acyl lipids referred to as lyso-dihydrocopepodamide (former G) and lysocopepodamide (former H). The entire group of compounds can collectively be referred to as "copepodamides." Effects of 16:0-copepodamide on phytoplankton We were able to purify sufficient amounts of 16:0copepodamide and performed dose response experiments with three microalgae species to confirm its biological activity. We used established copepod-induced systems in diatoms and dinoflagellates. A. minutum and P. seriata both increase production of toxic secondary metabolites (gonyautoxins and domoic acid, respectively) in response to copepod presence Tammilehto et al. 2015). Skeletonema changes morphology to single celled or shorter chain phenotype when exposed to copepods (Bergkvist et al. 2012). Increased resistance to grazers has been established for defended phenotypes of Alexandrium and Skeletonema, but the grazer deterrent role of domoic acid is still debated (Bergkvist et al. 2012;Selander et al. 2006;E. Selander et al. unpubl.). 18:4-Copepodamide (copepodamide D) was included as a positive control. It has the same molecular backbone as 16:0-copepodamide and induces toxin formation in A. minutum . 16:0-Copepodamide triggered the grazer-induced phenotypes in all three systems (Fig. 3). P. seriata and S. marinoi were sensitive to low amounts of copepodamides and signal perception saturated at 50 pmol L −1 (S. marinoi) and 150 pmol L −1 (P. seriata). In contrast, A. minutum showed no saturation even at high concentrations (up to 24 nmol L −1 ) and is previously shown to do so up to 28 nmol L −1 . However, for the diatoms, the potency of 16:0-copepodamide was lower than that of 18:4-copepodamide as reflected by 63% lower maximum domoic acid increase in P. seriata (Fig. 3b) and 19% lower maximum chain length shortening in S. marinoi (Fig. 3c). For A. minutum, no maximum induced response could be estimated as the copepodamide concentrations tested did not lead to a saturation of the increased toxin production (Fig. 3a), but for the tested concentrations, no difference in potency of 16:0-and 18:4-copepodamide could be observed.
Some species of the same genus have similar copepodamide composition, such as Calanus spp., whereas others like Centropages typicus and Centropages hamatus cluster far apart (Fig. 4a). The freshwater specie Cyclops sp. is not distinctly different from the saltwater species, and has a similar copepodamide signature as Centropages hamatus. The presence of copepodamides in the freshwater copepod suggests that copepodamide signaling may be operating in limnic systems too. Copepodamide composition in Centropages hamatus and C. finmarchicus illustrates the differences in copepodamide profiles from opposite corners of the PCA. Centropages hamatus, is dominated by dihydrocopepodamides (95% of total copepodamides), whereas C. finmarchicus contains only 14% dihydrocopepodamides (Fig. 4b).
Feeding of T. longicornis with different microalgae resulted in a changed composition of copepodamides (Fig. 4c) mirroring the fatty acid content of the algae. Feeding on D. tertiolecta, Rhodomonas sp., and I. galbana resulted in high C18:2-dihydrocopepodamide levels, whereas copepods feeding on N. oculata mainly formed C20:5-dihydrocopepodamide. N. oculata is rich in C20:5 (Roncarati et al. 2004;Ohse et al. 2015) while Rhodomonas algae have a high content of C18:4 and I. galbana is rich in C18:4 and C22:6 (Patil et al. 2007;Dahl et al. 2009;Parrish et al. 2012). The fatty acid composition of D. tertiolecta is typical for chlorophytes, deficient in C20:5 and C22:6 but contain some C18:4 (Veloza et al. 2006;Parrish et al. 2012). The food composition had no effect on which type of copepodamide scaffold was synthesized as both T. longicornis caught in field and the cultured ones only produced dihydrocopepodamides but not copepodamides.

Discussion
We report the structure of two novel copepodamides. Both share the identical molecular scaffold of the known copepodamides but are unique in containing saturated acyl groups. In addition, we identify 21 putative copepodamide structures with acyl groups commonly found in copepod organisms. The new compounds constitute a significant portion of the total Table 1. Composition of copepodamides in a polar lipid extract of C. finmarchicus analyzed by HPLC-MS/MS. The structures of copepodamide A-F are published in Selander et al. (2015), the structures of 14:0-and 16:0-copepodamide are presented in this study, and the remaining copepodamides are not structural elucidated. Copepodamides are now named by the fatty acid residue followed by a suffix reflecting an unsaturated (copepodamide) or saturated (dihydrocopepodamide) scaffold (e.g., 22:6-copepodamide for the former copepodamide D and 22:6-dihydrocopepodamide for the former copepodamide A). Fatty acids marked with * were also identified in taurine-containing lipids in Calanus spp. stage five copepodites in a study of Mayor et al. (2015). copepodamides, which have consequently been underestimated in earlier studies. Ten of the putative structures are likely to be identical to a group of taurine-containing lipids identified in a study of the metabolic response of Calanus spp. to environmental warming and ocean acidification (Mayor et al. 2015) further supporting their presence in calanoid copepods.

Fatty
The inducing effect of copepodamides on domoic acid and gonyautoxin production can facilitate harmful algal bloom formation of both amnesic and paralytic shellfish toxinproducing algae. The grazer deterrent role of harmful algal toxins is debated. Evidence is mainly correlative, where algae producing toxins trigger a wide range of responses in grazers. Some grazers are resistant to toxins and feed seemingly unharmed on harmful algae. Others show decreased feeding rates and rejection of toxic prey (Guisande et al. 2002;Xu et al. 2018) or reduced reproductive success (Dutz 1998;Guisande et al. 2002), altogether leading to less grazing pressure for the algae. Several algal toxins like microcystins, paralytic shellfish toxins, and amnesic shellfish toxins are induced by grazer presence, which supports that they serve as grazer deterrents (Selander et al. 2006;Takabayashi et al. 2006;Jang et al. 2007). Both, domoic acid and paralytic shellfish poison accumulate within the tissue of copepods which play a crucial role by transfer of the toxins to distant trophic levels (Tester et al. 2000;Lincoln et al. 2001). As a consequence, harmful algal blooms sometimes have detrimental effects on, for example, fish and mammals (Todd 1993;Landsberg 2002).
Beside increased toxicity, copepod cues also trigger chain length shortening in Skeletonema, together with a massive transcriptional response (Bergkvist et al. 2012;Amato et al. 2018;E. Selander et al. unpubl.). Copepods have an one order of magnitude lower grazing rate on single cells compared to longer chains, whereas microzooplankton, like ciliates, prefer the smaller units (Bjaerke et al. 2015). By grazing on microzooplankton and large phytoplankton, copepods may act as a switch between two alternate trophic cascades of different food chain lengths and with opposite selective pressure on phytoplankton size distribution (Stibor et al. 2004). Measurements of chain length over time confirm that chain length is negatively correlated to copepod abundance in nature too (Bjaerke et al. 2015). Based on the results reported here and earlier findings-a previous study shows that copepodamides induce bioluminescence in L. polyedra and Alexandrium tamarense (Lindström et al. 2017)-we conclude that copepodamide presence induces a broad range of defensive traits in phylogenetically distant groups which may cause indirect cascading effects in plankton food webs. In particular, copepods and copepodamides will both favor smaller and/or more defended phytoplankton phenotypes.
A full characterization of copepodamides makes it possible to pursue the molecular and ecological mechanisms of indirect effects of copepods in the pelagic ecosystem. For instance, the signal transduction pathway and downstream molecular mechanisms leading to the expression of defensive phenotypes can be studied in controlled laboratory experiments, and plankton communities can be exposed to copepodamides to separate the indirect, copepodamide-driven effects from direct grazing effects.
Dose response experiments comparing copepodamides containing a saturated or unsaturated fatty acid support the importance of the presence of a fatty acyl group at position C3 . Notwithstanding, for the diatoms tested, the potency of the saturated 16:0-copepodamide is lower than for the unsaturated 18:4-copepodamide. This suggests that the identity of the fatty acid side chain is also of some importance for the structure activity relationship and that especially the level of saturation may be important for the activity of the copepodamides. The binding of molecules to proteins is partly driven by entropic terms which mean that molecules with a lower conformational flexibility and less conformational penalty toward the bioactive conformation can bind better than highly flexible molecules (Bostrom et al. 1998;Hoffmann 2000). In comparison to saturated fatty acids, the conformational space of unsaturated fatty acids is smaller, as the double bond introduces a rotational barrier. Assuming that copepodamides are sensed by microalgae via receptor proteins, copepodamides with a higher conformal stability may lead to better binding to the protein and thus stronger biological activity.
However, the response of A. minutum to copepodamides was less sensitive than for the diatoms and there was no difference in the potency of 18:4-and 16:0-copepodamide in toxin induction. Dinoflagellates and diatoms may have evolved different perception or response mechanisms to copepodamides. This is not surprising as dinoflagellates and diatoms are represented in two distinct groups in the eukaryotic tree of life (Burki and Keeling 2014;Worden et al. 2015) and may indicate convergent evolution of copepodamide receptor proteins.
A study monitoring copepodamide concentrations in the field reveals that the average natural concentration of copepodamides in seawater is 0.4 to 2 pmol L −1 . The same study shows that the effective concentration averages < 0.5% of the nominal concentration when administered by coating copepodamides onto culture vessel walls (E. Selander et al. unpubl.). In the present study, saturation in the dose response curves for the diatoms was reached at nominal concentrations of 0.1-0.5 nmol L −1 , resulting in 0.5-2.5 pmol L −1 effective concentrations. Thus, both diatom species respond to copepodamide cues in ecologically relevant concentrations. The lower sensitivity of the dinoflagellate to copepodamides is puzzling, but copepods occasionally reach densities of hundreds per liter (Hamner and Carleton 1979;Ambler et al. 1991) and a single large copepod can exude 120 pmol d −1 . This indicates that even these high concentrations are in a possible range of natural occurring copepodamides. Indeed, copepods do induce toxin production in A. minutum in laboratory experiments, but the lower sensitivity also suggest that there may be additional cues to the copepodamides involved or alternatively that the standing incubations used for A. minutum result in lower effective concentrations than incubation on a rotating plankton wheel used for the diatoms in this study.
Our results indicate that copepods have a specific copepodamide composition, which is linked to the diet and controlled by the amount of available dietary fatty acids. Copepods, although generally omnivorous, are selective feeders on microalgae depending on, for example, size, abundance, or toxicity (Frost 1972;Schultz and Kiørboe 2009;Ray et al. 2016;Xu et al. 2017) and microalgae vary in their fatty acid composition (Roncarati et al. 2004;Patil et al. 2007;Ohse et al. 2015). Additionally, copepods graze on microzooplankton, further differentiating the fatty acid composition of their diet (Tiselius 1989;Calbet and Saiz 2005;Tiselius et al. 2012). Hence, it is conceivably that feeding preferences between different species are mirrored in the copepodamide composition. The specific composition of copepodamides is further influenced by the proportions of dihydrocopepodamides and copepodamides, which seem not to be affected by the diet. It may reflect evolutionary differences in copepodamide biosynthesis among the copepod species tested. This is obviously also the case for the harpacticoid Tigriopus californicus, which does not produce detectable amounts of copepodamides at all. In accordance to the biosynthetic pathway of a group of related compounds in Tetrahymena (Kaya and Sano 1991), it is possible that the two copepodamide scaffolds are synthesized prior to the introduction of the fatty acid residue, resulting in a species-specific distribution of dihydrocopepodamides and copepodamides with a diet-depending variation of the fatty acid side chain.
As the composition of exuded copepodamides of C. finmarchicus (Fig. 4b) is similar to the composition of extractable copepodamides , the specific composition of dihydrocopepodamides and copepodamides observed within different copepod species can have ecological consequences. Dihydrocopepodamides are approximately 10 times more potent inducers of paralytic shellfish toxin production in dinoflagellates than copepodamides . Thus, formation of harmful algae blooms may be more promoted by copepods rich in dihydrocopepodamides such as Acartia clausi, Centropages hamatus, and T. longicornis.
Further, this study represents the first record of copepodamides in freshwater copepods and suggests that copepodamides may act as cue compounds in limnic systems too.
It is currently not known why copepods synthesize copepodamides and why the compounds are released into the water, but kairomones, chemical cues benefitting the receiving organism, are often released involuntary where the compounds original purpose, for example, metabolism, improves overall fitness (Pohnert et al. 2007). One possible hypothesis is that copepodamides serve a function as emulsifiers in the digestive tract of copepods in analogy to taurine-containing lipids found in the gastric juice of crabs and bile salts in other animals (Van Den Oord et al. 1965;Maldonado-Valderrama et al. 2011). Alternatively, Mayor et al. (2015) discuss a speculative role of taurine lipids found in Calanus in the transportation and protection of metabolic substrates from their storage location to the point of oxidative catabolism within mitochondria. Beside this, a variety of possible functions of copepodamides is conceivable and further studies, for example, regarding the localization and biosynthesis of copepodamides are needed to learn more about the function of copepodamides within the copepods.