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Influence of diatoms on copepod reproduction. III. Consequences of abnormal oocyte maturation on reproductive factors in Calanus helgolandicus
Abstract
Observations of gonads and oocyte development stages (OS) have been achieved in Calanus helgolandicus females fed different algal diets and starved in filtered sea water under laboratory conditions during 8days. The effects
of 20 diets on egg production rates (EPR), hatching success (HS) and proportion of abnormal larvae (AL, development stages
N1-2) were examined. With the control diet Prorocentrum minimum EPR and HS values were high, while AL was very low, coinciding with intact cell structures in oogonia (OO) and normal OS
(OS1-OS4). With the other diets, oocyte maturation, EPR, HS and AL patterns were partially or totally impaired. Decrease of
EPR coincided with the arrest of OS3 maturation and oocyte degradations, characterised by cell fragmentation, presence of
apoptotic bodies in the OS3, degradation of cytoplasm in OS3 and OS4 and consequently the arrest of OS4 formation. These degradations
were reversible when females were fed a favourable diet. Results reflect the presence of two distinct inhibitory mechanisms.
Inhibition mechanism (1) impairs egg production. It was induced by starvation or by several species belonging to Bacillariophyceae
(Chaetoceros calcitrans, Guinardia delicatula, Guinardia
striata, Odontella regia, Rhizosolenia setigera, Stephanopyxis turris, Thalassiosira pseudonana) and mixed-diatom assemblages (collected in the field) and to the prymnesiophyte Pavlova lutherii. Remarkably other diatoms like Navicula sp., Nitzschia sp., Skeletonema costatum and Thalassiosira rotula did not induce mechanism (1) Inhibitory mechanism (2) affected exclusively HS and AL and was triggered by species independent
of the production of polyunsaturated aldehydes (PUA), which are supposed to have adverse impacts on HS and larval development.
... These compounds are not constitutively present in the cells but are only produced when the cell is damaged as would occur during grazing (Pohnert, 2000). Owing to the teratogenic nature of diatom oxylipins, the mechanism of chemical defense in diatoms functions through induction of apoptosis which can occur in maturing oocytes (Poulet et al., 2007) or during embryo development (Romano et al., 2003) and in newly hatched nauplii (Ianora et al., 2004; Poulet et al., 2003). The toxicity of oxylipins, and more specifically of PUAs, has been demonstrated in classical feeding experiments using diatoms as copepod food (Ianora et al., 2003; Poulet et al., 2007) or in in vitro tests by incubating embryos and adults in known concentrations of pure molecules dissolved into seawater (Ianora et al., 1999; Romano et al., 2003; Adolph et al., 2004; Caldwell et al., 2005; Taylor et al., 2007). ...
... Owing to the teratogenic nature of diatom oxylipins, the mechanism of chemical defense in diatoms functions through induction of apoptosis which can occur in maturing oocytes (Poulet et al., 2007) or during embryo development (Romano et al., 2003) and in newly hatched nauplii (Ianora et al., 2004; Poulet et al., 2003). The toxicity of oxylipins, and more specifically of PUAs, has been demonstrated in classical feeding experiments using diatoms as copepod food (Ianora et al., 2003; Poulet et al., 2007) or in in vitro tests by incubating embryos and adults in known concentrations of pure molecules dissolved into seawater (Ianora et al., 1999; Romano et al., 2003; Adolph et al., 2004; Caldwell et al., 2005; Taylor et al., 2007). Recently, Ianora et al. used the dinoflagellate Prorocentrum minimum, which does not produce oxylipins, as a live carrier of 2-trans,4-trans-decadienal (henceforth called decadienal or DD), which has been widely used as a model aldehyde to study the deleterious effects of these compounds on marine invertebrates (Ianora et al., 2004). ...
... Another interesting finding here is that when the percentage of egg viability for C. helgolandicus was below 25% and adult survival was less than 50% (Fig.·4B,D), gonad tissues of some females appeared positively stained by TUNEL (Fig.·6A), suggesting that they were undergoing apoptosis. The apoptotic region corresponded to oocyte 3-stage producers (OS3) (Poulet et al., 2007). Follicle chambers with apoptotic body-like spots were similar to structures found in histological sections by these authors (Fig.·6B). ...
... While PUAs can explain several instances of poor copepod reproduction, their negative impact is not universal. Several field and laboratory assays gave no evidence of adverse PUA effects [9][10][11][12][13] while others did (reviewed in [5]). Higher molecular weight oxylipins and other toxins might provide additional explanations for observed teratogenic effects of diatoms on copepods [4,5]. ...
... Nutritional inadequacies of some diatom species [14] and poor nutrient uptake due to rapid gut passage time [15] were also discussed. Most reports fuelling the debate about PUA toxicity focus on observations of the outcome of feeding experiments, while very few mechanistic studies address the origin of these findings and the mode of action of PUAs [9,16]. Data is especially lacking on how the metabolites are delivered to and distributed in feeding copepods. ...
... This accumulation supports the activity of PUAs as antiproliferative metabolites. The targeted delivery of these reactive metabolites to the reproductive organs explains observation that diatom rich diets cause oocyte degradations characterised by cell fragmentation, presence of apoptotic bodies and degradation of cytoplasm in Calanus helgolandicus [9]. In that study histological and cytological observations on gonads and oocyte development stages in C. helgolandicus were performed and several mechanisms as well as other factors besides PUA content of the diet were postulated to cause adverse effects in copepod oocyte maturation. ...
Polyunsaturated aldehydes (PUAs) are released by several diatom species during predation. Besides other attributed activities, these oxylipins can interfere with the reproduction of copepods, important predators of diatoms. While intensive research has been carried out to document the effects of PUAs on copepod reproduction, little is known about the underlying mechanistic aspects of PUA action. Especially PUA uptake and accumulation in copepods has not been addressed to date. To investigate how PUAs are taken up and interfere with the reproduction in copepods we developed a fluorescent probe containing the α,β,γ,δ-unsaturated aldehyde structure element that is essential for the activity of PUAs as well as a set of control probes. We developed incubation and monitoring procedures for adult females of the calanoid copepod Acartia tonsa and show that the PUA derived fluorescent molecular probe selectively accumulates in the gonads of this copepod. In contrast, a saturated aldehyde derived probe of an inactive parent molecule was enriched in the lipid sac. This leads to a model for PUAs' teratogenic mode of action involving accumulation and covalent interaction with nucleophilic moieties in the copepod reproductive tissue. The teratogenic effect of PUAs can therefore be explained by a selective targeting of the molecules into the reproductive tissue of the herbivores, while more lipophilic but otherwise strongly related structures end up in lipid bodies.
... Buttino et al. (2008) found that diatom PUAs could produce the early signs of apoptosis in female gonad tissue. They suggest that eggs from Reduced hatching, abnormal nauplii Poulet et al. (1995) Rhizosolenia setigera L English Channel Reduced fecundity Poulet et al. (2006) Reduced fecundity, increased hatching Poulet et al. (2007a) No negative effect Wichard et al. (2008) (2004) Reduced hatching Ban et al. (1997) Chaudron et al. (1996) Pohnert et al. (2002) Reduced fecundity & hatching Poulet et al. (1994) Apoptosis & necrosis in nauplii Poulet et al. (2003) Increased fecundity & hatching Poulet et al. (2006) Gulf of Naples Apoptosis in eggs Romano et al. (2003) English Channel No negative effect Wichard et al. (2008) Thalassiosira weissflogii L English Channel Reduced hatching Ban et al. (1997) Chaudron et al. (1996) Increased fecundity Poulet et al. (2006) Centropages hamatus Avoided when feeding, reduced naupliar survival Koski et al. (2008) Chaetoceros decipiens L North Sea Avoided when feeding, reduced naupliar survival Koski et al. (2008) Chaetoceros socialis L North Sea Avoided when feeding, reduced naupliar survival Koski et al. (2008) Dunaliella tertiolecta L North Sea Reduced ingestion & fecundity Arendt et al. (2005) Leptocylindrus danicus L North Sea Reduced fecundity & hatching Dutz et al. (2008) No negative effect Koski et al. (2008) Phaeodactylum tricornutum L English Channel Reduced fecundity & hatching Ban et al. (1997) Skeletonema costatum L North Sea Reduced fecundity & hatching Dutz et al. (2008) No negative effect Koski et al. (2008) Thalassiosira pseudonana L North Sea Avoided when feeding, reduced naupliar survival Koski et al. (2008) Thalassiosira rotula L North Sea Reduced hatching Dutz et al. (2008) No negative effect Koski et al. (2008) Reduced naupliar survival Koski et al. (2008) Thalassiosira weissflogii L English Channel Reduced fecundity & hatching Ban et al. (1997) L North Sea High egg production & hatching Arendt et al. (2005) Reduced hatching Dutz et al. (2008) No negative effect Koski et al. (2008) these females were probably laid but died soon afterwards producing a negative effect on hatching success rather than egg production. However, Poulet et al. (2007a) showed that reduced fecundity, when seen, is caused by the arrest of oocyte maturation and oocyte degradations, similar to those seen in starved animals, and effects were reversible once females were fed a favourable diet. ...
... They suggest that eggs from Reduced hatching, abnormal nauplii Poulet et al. (1995) Rhizosolenia setigera L English Channel Reduced fecundity Poulet et al. (2006) Reduced fecundity, increased hatching Poulet et al. (2007a) No negative effect Wichard et al. (2008) (2004) Reduced hatching Ban et al. (1997) Chaudron et al. (1996) Pohnert et al. (2002) Reduced fecundity & hatching Poulet et al. (1994) Apoptosis & necrosis in nauplii Poulet et al. (2003) Increased fecundity & hatching Poulet et al. (2006) Gulf of Naples Apoptosis in eggs Romano et al. (2003) English Channel No negative effect Wichard et al. (2008) Thalassiosira weissflogii L English Channel Reduced hatching Ban et al. (1997) Chaudron et al. (1996) Increased fecundity Poulet et al. (2006) Centropages hamatus Avoided when feeding, reduced naupliar survival Koski et al. (2008) Chaetoceros decipiens L North Sea Avoided when feeding, reduced naupliar survival Koski et al. (2008) Chaetoceros socialis L North Sea Avoided when feeding, reduced naupliar survival Koski et al. (2008) Dunaliella tertiolecta L North Sea Reduced ingestion & fecundity Arendt et al. (2005) Leptocylindrus danicus L North Sea Reduced fecundity & hatching Dutz et al. (2008) No negative effect Koski et al. (2008) Phaeodactylum tricornutum L English Channel Reduced fecundity & hatching Ban et al. (1997) Skeletonema costatum L North Sea Reduced fecundity & hatching Dutz et al. (2008) No negative effect Koski et al. (2008) Thalassiosira pseudonana L North Sea Avoided when feeding, reduced naupliar survival Koski et al. (2008) Thalassiosira rotula L North Sea Reduced hatching Dutz et al. (2008) No negative effect Koski et al. (2008) Reduced naupliar survival Koski et al. (2008) Thalassiosira weissflogii L English Channel Reduced fecundity & hatching Ban et al. (1997) L North Sea High egg production & hatching Arendt et al. (2005) Reduced hatching Dutz et al. (2008) No negative effect Koski et al. (2008) these females were probably laid but died soon afterwards producing a negative effect on hatching success rather than egg production. However, Poulet et al. (2007a) showed that reduced fecundity, when seen, is caused by the arrest of oocyte maturation and oocyte degradations, similar to those seen in starved animals, and effects were reversible once females were fed a favourable diet. Carotenuto et al. (2002) found that early stage Temora stylifera (Dana, 1849) nauplii raised on diatom diets had high mortality rates and failed to complete development, and field studies have also shown reduced naupliar survival following peaks of PUA producing Thalassiosira spp Pierson et al., 2005). ...
... The differences in growth rate in this case were not related to PUA ingestion, and in some cases lack of development was caused by the inability to feed on diatoms due to size or morphology. Poulet et al. (2007a) and Wichard et al. (2008) also found that decreased egg viability and increased production of abnormal nauplii were not related to PUA production of the diatoms, but recent studies have shown that other oxylipins can also affect copepod reproduction (Fontana et al., 2007b;Ianora et al., 2008). Tang & Dam (2001) found that exudates from 'suspect' diatom cultures had no negative effect on Acartia tonsa egg viability leading to the conclusion that toxins must be released through grazing cell damage, and therefore are diet-induced maternal effects rather than external effects. ...
Many planktonic algae produce biotoxins that can affect grazer feeding, mortality and recruitment rates, or alter swimming behaviour and therefore predation risk. This report reviews the literature on the effects of algal biotoxins (excluding cyanobacteria) on pelagic copepod biology, focussing on copepod species that are important in Scottish waters. Marine Scotland Science currently holds more than 10 years inshore coastal monitoring data on the occurrence of toxic phytoplankton and corresponding zooplankton species from the Stonehaven and Loch Ewe monitoring sites, and these data are summarised. The relevant biotoxins that could influence copepod population dynamics in Scottish waters are identified as poly-unsaturated aldehydes from diatoms, and toxins associated with paralytic shellfish poisoning (Alexandrium spp.), diarrhetic shellfish poisoning (Dinophysis spp.) and amnesic shellfish poisoning (Pseudo-nitzschia spp.). There is potential for these Scottish field data and further experimental studies to provide insights into the way that interspecies relationships and energy flow in food webs can be affected by the production, transfer and effects of algal biotoxins.
... We have monitored PUA content of the diets, nutritional parameters/indicators specified as polyunsaturated fatty acids, the DHA/EPA [22:6 x3/20:5 x3] and C/N ratios as well as environmental parameters (chlorophyll a, water-temperature). The work belongs to a series of extended field experiments from spring to fall in 2003 and 2004 ( Poulet et al., 2006Poulet et al., , 2007a). We used a sensitive in situ derivatisation approach for determination and quantification of PUAs in phytoplankton ( Wichard et al., 2005b). ...
... As the dinoflagellate Prorocentrum minimum (PM) supports high egg production and hatching success ( Ban et al., 1997;Poulet et al., 2006), this diet was used as a control to prove that the selected copepods were healthy and able to spawn viable eggs and that the experimental conditions allowed optimum reproduction. In order to survey the fertility of the collected copepods, 4-5 females were randomly collected for histology preparation and observation of the oocyte maturation (see Poulet et al., 2007a). ...
... Some diatom species impair HS but not EPR and vice versa, hence it was concluded that more than one factor has to be taken into account to explain the role of maternal diets. This multi factor dependence of EPR and HS was also confirmed by other laboratory studies ( Lacoste et al., 2001;Ceballos and Ianora, 2003;Poulet et al., 2007a). Nevertheless in our field-survey egg production and hatching success are coupled ( Fig. 2), indicating that these two reproductive factors might be influenced by the same parameters in the coastal waters off Roscoff in 2004. ...
We set up a survey involving comprehensive chemical analyses of phytoplankton and the simultaneous monitoring of the reproductive response of Calanus helgolandicus. Hatching success and larvae development of C. helgolandicus were monitored weekly with female specimens sampled along with phytoplankton in the coastal waters off Roscoff (Western English Channel) from March/April to October in 2003 and 2004. Polyunsaturated aldehyde (PUA) production and nutritional parameters of phytoplankton [i.e. polyunsaturated fatty acids (PUFAs), particulate organic carbon (POC) and nitrogen (PON)] were measured. The hatching success varied between 0% and 80% during the two spring–summer spawning seasons. Neither the nutritional parameters measured nor polyunsaturated aldehydes could be found responsible for the frequently observed hatching failure. However, egg production and hatching rates were strongly positively correlated, suggesting a functional link between these reproductive parameters. PUA production in phytoplankton was mainly determined by Thalassiosira rotula. The occurrence of this diatom species might be used as a marker of PUAs in the study area. Laboratory feeding experiments showed that single species diets of freshly isolated diatoms (T. rotula, Guinardia striata, Guinardia delicatula and Rhizosolenia setigera) caused high or low egg viability and naupliar survival of C. helgolandicus independent of PUA production and PUFA content. The impact of PUA on the hatching success of Calanus among different ecosystems is discussed. The combined approach of feeding experiments and chemical analysis supports the idea that parameters other than PUAs and PUFAs have to be taken into account in order to explain the highly variable hatching rates observed in this environment.
... The use of aldehydes as defensive compounds in terrestrial systems and by macroalgae is well known (Cavill and Hinterberger 1960 ;Kubo et al. 1976 ;Camazine et al. 1983 ;Morimoto et al. 2002 ;Schnitzler et al. 2001) . PUA are often semiochemically active in several different ways, for example antimicrobial, fertilization inhibiting, embryotoxic, sperm motility inhibiting, larval toxic, grazer toxic, and feeding deterrent (Caldwell et al. 2002(Caldwell et al. , 2004aPoulet et al. 2003Poulet et al. , 2007Tosti et al. 2003 ;Adolph et al. 2004 ;Lewis et al. 2004) . Bioactivity is directed at cellular and molecular targets such as microtubule and microfilament stability, DNA replication, and the activity of the G2-M promoting complex cyclin B-Cdk1, which are all crucial during embryonic development Hansen et al. 2004a) . ...
... PUA toxic to developmental stages of a number of aquatic invertebrate species, including copepods, sea urchins, polychaetes, and ascidians. Reported symptoms of PUA exposure include gamete infertility and dysfunction, fertilization failure, and abnormal morphological development, indicating that the timing and/or level of exposure may result in subtle, yet deleterious departures from normal reproductive patterns (e.g., Miralto et al. 1999 ;Ianora et al. 2004 ;Adolph et al. 2004 ;Poulet et al. 2007) . Given the importance of oxylipins in reproduction and development, exposure of invertebrates to altered environmental titers at particular ontogenetic stages may therefore disrupt highly sensitive endocrine processes. ...
... Therefore, gonadal accumulation is feasible in theory, despite the reactivity of PUA resulting in significant losses before reaching the target organs. PUA will likely form DNA adducts within animal tissues (Loureiro et al. 2000) , the significance of this within aquatic grazers is unknown but may be related to observed patterns of apoptosis in gonadal, embryonic, and larval tissue Poulet et al. 2003Poulet et al. , 2007 . At the present time, this remains purely theoretical, since it is extremely difficult to demonstrate, and a number of protocol issues need to be addressed before this mechanism can be systematically examined (cf. ...
On the one hand, in marine systems, a long history of research established the roles of PUFA derivatives as pheromones and
in grazer defense (Sects. 4.3 and 4.4). For a long time, this work was directed largely toward littoral interactions involving
seaweeds (notably phaeophytes and rhodophytes). On the other hand, in freshwater systems, the nutritive value of algal PUFA
in food webs has been recognized for some time, but their potential role(s) in chemical defense has been only acknowledged
recently; and was similarly first convincingly elucidated in littoral communities (Jüttner 2001). Planktonic and littoral algal PUFA derivatives have also long been notorious as potent taste-odor agents in freshwater
supplies, and even more extensively characterized in the food and flavor/perfume industries as sources of rancid, oily, fishy,
cucumber, fruity, and floral flavors in both essential oils and spoiled lipid-rich food products (Watson et al. 2000, 2001; Watson 2003).
... There was also a clear effect on adult survivorship with mortality linked with apoptotic events in the somatic tissues. Interestingly, Poulet et al. [98] described two separate oogenic syndromes in C. helgolandicus as a result of diatom-feeding, although in this case the indicative species were not those associated with PUA-production. Syndrome one was characterised by reduced egg production whereas egg hatching was unaffected. ...
... Also the overall oocyte diameter increased. Similar observations were reported by Poulet et al. [98]. Using morphological criteria, the oocytes were determined to have undergone necrotic cell death as opposed to apoptosis; however additional diagnostic tests were not performed to confirm this. ...
... Necrosis is linked to pathological inflammatory conditions following exposure to extreme physical, biological or chemical conditions. Cell death in adults, gonads, early embryonic stages and larvae in response to decadienal exposure has been described previously [5,69,75,98], however in these cases, cell death was determined to have followed an apoptotic pathway. A combination of both apoptosis and necrosis was described by Poulet et al. [106] in copepod nauplii spawned from mothers feeding on a PUA-producing diatom strain. ...
Diatoms are one of the main primary producers in aquatic ecosystems and occupy a vital link in the transfer of photosynthetically-fixed carbon through aquatic food webs. Diatoms produce an array of biologically-active metabolites, many of which have been attributed as a form of chemical defence and may offer potential as candidate marine drugs. Of considerable interest are molecules belonging to the oxylipin family which are broadly disruptive to reproductive and developmental processes. The range of reproductive impacts includes; oocyte maturation; sperm motility; fertilization; embryogenesis and larval competence. Much of the observed bioactivity may be ascribed to disruption of intracellular calcium signalling, induction of cytoskeletal instability and promotion of apoptotic pathways. From an ecological perspective, the primary interest in diatom-oxylipins is in relation to the potential impact on energy flow in planktonic systems whereby the reproductive success of copepods (the main grazers of diatoms) is compromised. Much data exists providing evidence for and against diatom reproductive effects; however detailed knowledge of the physiological and molecular processes involved remains poor. This paper provides a review of the current state of knowledge of the mechanistic impacts of diatom-oxylipins on marine invertebrate reproduction and development.
... The effective concentrations of DD, often used as a model of PUA, range between 0.05 and 30 lg ml )1 . In copepods, PUAs have been shown to induce apoptosis in maturing oocytes (Poulet et al., 2007a), during embryo development and in newly hatched nauplii (Ianora et al., 2004). The consequences are a reduced hatching success and the induction of malformations in larval stages, leading to low survival rates. ...
... Nevertheless, the link between PUAs and reproductive failure is not very clear. The effective role of PUAs has been challenged by recent studies that failed to correlate PUA production and reproductive failure in the field (Poulet et al., , 2007aWichard et al., 2008) and in the laboratory (Dutz et al., 2008). ...
... Interestingly, although data on freshwater micro-algae are scarce, they seem to be less sensitive to PUAs than do marine micro-algae (Adolph et al., 2004;Leflaive et al., 2008). For all the phototrophic organisms tested, the observed effects of PUAs were similar to apoptosis, as described for animals (Ianora et al., 2004;Poulet et al., 2007a). This is shown by cell shrinkage, chromatin condensation and the degradation of nuclear DNA to nucleosomal size fragments. ...
Chemicals produced by aquatic organisms, and especially micro-organisms, have received increasing attention in the last decade for their role in shaping interactions and communities. Several cases emphasize the fact that chemical signals or defence may modulate interspecific interactions. Notably, it has been shown that diatoms, unicellular algae and key primary producers in aquatic ecosystems produce a wide range of bioactive metabolites. Among these compounds, polyunsaturated short-chain aldehydes in vitro strongly impair the reproduction of various potential grazers. In the field, the relationship between aldehyde production and reproductive failure in copepods remains unclear. Recent studies have suggested that these putative defence compounds may also be involved in intercellular communication and in interactions with competitors. Potential effects of the aldehyde precursors on various organisms have also been described. This review presents an overview of various results obtained in the last decade that could help us to understand the role of polyunsaturated aldehydes and their precursors in the ecology of diatoms. It is focused on the dichotomy between freshwater and marine environments. Indeed, most of the results on anti-proliferative aldehydes concern marine planktonic diatoms, whereas they are also known to be produced by benthic and freshwater species.
... These compounds are not constitutively present in the cells but are only produced when the cell is damaged as would occur during grazing (Pohnert, 2000). Owing to the teratogenic nature of diatom oxylipins, the mechanism of chemical defense in diatoms functions through induction of apoptosis which can occur in maturing oocytes (Poulet et al., 2007) or during embryo development (Romano et al., 2003) and in newly hatched nauplii Poulet et al., 2003). The toxicity of oxylipins, and more specifically of PUAs, has been demonstrated in classical feeding experiments using diatoms as copepod food Poulet et al., 2007) or in in vitro tests by incubating embryos and adults in known concentrations of pure molecules dissolved into seawater Romano et al., 2003;Adolph et al., 2004;Caldwell et al., 2005;Taylor et al., 2007). ...
... Owing to the teratogenic nature of diatom oxylipins, the mechanism of chemical defense in diatoms functions through induction of apoptosis which can occur in maturing oocytes (Poulet et al., 2007) or during embryo development (Romano et al., 2003) and in newly hatched nauplii Poulet et al., 2003). The toxicity of oxylipins, and more specifically of PUAs, has been demonstrated in classical feeding experiments using diatoms as copepod food Poulet et al., 2007) or in in vitro tests by incubating embryos and adults in known concentrations of pure molecules dissolved into seawater Romano et al., 2003;Adolph et al., 2004;Caldwell et al., 2005;Taylor et al., 2007). ...
... Another interesting finding here is that when the percentage of egg viability for C. helgolandicus was below 25% and adult survival was less than 50% (Fig.·4B,D), gonad tissues of some females appeared positively stained by TUNEL (Fig.·6A), suggesting that they were undergoing apoptosis. The apoptotic region corresponded to oocyte 3-stage producers (OS3) (Poulet et al., 2007). Follicle chambers with apoptotic body-like spots were similar to structures found in histological sections by these authors (Fig.·6B). ...
In the last decade, there has been an increased awareness that secondary metabolites produced by marine diatoms negatively impact the reproductive success of their principal predators, the copepods. Several oxylipins, products of the enzymatic oxidation of fatty acids, are produced when these unicellular algae are damaged, as occurs during grazing. In the past, the dinoflagellate Prorocentrum minimum, which does not produce the oxylipin 2-trans,4-trans-decadienal (DD), has been used as a live carrier to calculate daily ingestion rates of this molecule by copepod crustaceans. However, since the interaction between oxylipins and live carriers is unknown, the question as to how much and for how long ingestion of these molecules affects copepod reproduction remains a critical point to understanding the functional role of such compounds at sea. In the investigation presented here we used giant liposomes ( approximately 7 mum) as a delivery system for the oxylipin DD, prepared in the same size range as copepod food and containing known amounts of DD. The aim of this work was to relate the ingestion of DD to the reproductive failure of the copepods Temora stylifera and Calanus helgolandicus. Liposomes were very stable over time and after 10 days of feeding, liposomes encapsulating DD reduced egg hatching success and female survival with a concomitant appearance of apoptosis in both copepod embryos and female tissues. Concentrations of DD inducing blockage were one order of magnitude lower that those used in classical feeding experiments demonstrating that liposomes are a useful tool to quantitatively analyze the impact of toxins on copepods.
... Among the five microalgae tested in this study, I. galbana is known as an excellent food for marine planktonic copepods due to its appropriate size for ingestion, ease of digestion because of its thin cell wall, and favorable nutritional profile for copepods (Li et al., 2006;Zheng, 2011). On the other hand, diatoms generally are not considered as ideal diets for marine planktonic copepods because their cell wall contains silicon, some species produce toxic secondary metabolites, and some species are deficient in essential nutrients for copepods (Malej & Harris, 1993;Carotenuto et al., 2002;Li et al., 2006;Poulet et al., 2007;Taylor et al., 2007;Zheng, 2011). It is generally believed that dinoflagellates are a nutritional food source for marine copepods and conducive to their growth and development. ...
... Previous studies have indicated that the diatom S. costatum can produce unsaturated aldehydes as a secondary metabolite, which could have severe negative impacts on naupliar survival of pelagic calanoid copepods. Poulet et al. (2007) reported that when S. costatum was fed to the pelagic calanoid copepod Calanus helgolandicus (Claus), abnormal nauplii that were not viable were produced. Similarly, Carotenuto et al. (2002) found that when S. costatum was fed at 0.6 lgC ml -1 to newly hatched first-stage nauplii (N I ) of another calanoid copepod T. stylifera, none of the nauplii were able to develop to copepodite stage and all died within 8 days. ...
It has been reported that certain microalgae have the ability to cause various negative effects on pelagic calanoid copepods. However, whether such pelagic microalgae have similar negative effects on benthic copepods has had little attention. The results of the present study indicated that both benthic naupliar and copepodite stages of the harpacticoid copepod Tigriopus japonicus could effectively utilize five species of pelagic microalgae to complete their development. The effects of different concentrations of various microalgae species on naupliar survival were significant; however, no significant effects on copepodite survival were detected. When fed with two diatoms Chaetoceros muelleri and Skeletonema costatum as well as the dinoflagellate Prorocentrum micans and the chrysophyte Isochrysis galbana at high concentrations (≥8.50 μgC ml−1), the survival of both naupliar and copepodite stages of T. japonicus was more than 90%. However, when the diatom Nitzschia closterium f. minutissima was fed at the high concentrations of 8.50 μgC ml−1, the naupliar survival was 76.3 ± 3.2 %. Interestingly, the negative effects of N. closterium f. minutissima as a diet in the naupliar stage did not affect the survival of the copepodites, which may be explained by increased mobility of copepodites.
... The present results provide evidence that 2-trans,4-transdecadienal negatively affects survival, development and reproduction in the copepod Pseudodiaptomus annandalei in a dose-dependent manner. Previous studies have reported that exposure of embryos and larvae to PUA can severely impact development, growth and fitness of other copepod species (Miralto et al., 1999;Caldwell et al., 2002;Carotenuto et al., 2002;Ianora et al., 2004Ianora et al., , 2011Vargas et al., 2006;Poulet et al., 2007;Kâ et al., 2014). Decadienal has also been shown to potentially impact other marine organisms (Romano et al., , 2010Caldwell et al., 2005;Tosti et al., 2003). ...
... Diatom PUA have also been shown to impair copepod egg hatching success (Miralto et al., 1999), with induction of apoptosis in both somatic and gonadal tissues of females (Buttino et al., 2008) and naupliar stages Kâ et al., 2014). Teratogenic impacts (birth defects and hatching of malformed larvae) have also been recorded when females are fed with certain diatom diets (Poulet et al., 1995(Poulet et al., , 2007Uye, 1996;Ianora et al., 2011). In our study, 45-93% reduction was observed in nauplii production with increasing concentrations of decadienal. ...
Partially treated or untreated domestic (including livestock slurry) and industrial waste waters are discharged into the ambient environment rendering harmful effects on human and environment. Poor local air quality caused by slurry and flushing of Livestock may also cause community health risks besides releasing greenhouse gases. Therefore we need to explore a system in which individual households or a cluster of homes in cities can also treat their domestic wastewater on-site in a de-centralized manner to mitigate negative impacts on environment. For sustainability the key attribute of the natural system of self-regulating and self-supporting need to be exploited. Benthic invertebrate and zooplankton play important role in self-purification in reducing microbial load of waste water. Similar benefits are expected when wastewater is filtered through a medium containing large numbers of earthworms and bacterivorus zooplankton. We set up a biofiltration project (vermifiltration+ bacterivorus zooplankton) to process the wastewater (urban sewage and slurry from cowshed) for water recycling and management of bio solids. Vermifiltration reorded an average removal of TSS, TDS, COD and BOD from waste water as 90.3%, 88.98%, 67.47% and 86.44% respectively, whereas, the holistic biofilter enhanced the efficiency and recorded 95.3%, 92.8%, 87.47% and 96% respectively. Water collected from the holobiofilter recorded an order of magnitude lower bacterial count than the vermifilter treated water. Our results suggest that vermifiltration along with the application of bacterivorous zooplankton is more economical and effective technique of wastewater treatment. This is Decentralized onsite application of this technique advisable. Biofiltered water is microbe free, odorfree and adequately disinfected to be recycled for , cowshed flushing, farm irrigation and gardens.
... The present results provide evidence that 2-trans,4-transdecadienal negatively affects survival, development and reproduction in the copepod Pseudodiaptomus annandalei in a dose-dependent manner. Previous studies have reported that exposure of embryos and larvae to PUA can severely impact development, growth and fitness of other copepod species (Miralto et al., 1999;Caldwell et al., 2002;Carotenuto et al., 2002;Ianora et al., 2004Ianora et al., , 2011Vargas et al., 2006;Poulet et al., 2007;Kâ et al., 2014). Decadienal has also been shown to potentially impact other marine organisms (Romano et al., , 2010Caldwell et al., 2005;Tosti et al., 2003). ...
... Diatom PUA have also been shown to impair copepod egg hatching success (Miralto et al., 1999), with induction of apoptosis in both somatic and gonadal tissues of females (Buttino et al., 2008) and naupliar stages Kâ et al., 2014). Teratogenic impacts (birth defects and hatching of malformed larvae) have also been recorded when females are fed with certain diatom diets (Poulet et al., 1995(Poulet et al., , 2007Uye, 1996;Ianora et al., 2011). In our study, 45-93% reduction was observed in nauplii production with increasing concentrations of decadienal. ...
... The present results provide evidence that 2-trans,4-transdecadienal negatively affects survival, development and reproduction in the copepod Pseudodiaptomus annandalei in a dose-dependent manner. Previous studies have reported that exposure of embryos and larvae to PUA can severely impact development, growth and fitness of other copepod species (Miralto et al., 1999;Caldwell et al., 2002;Carotenuto et al., 2002;Ianora et al., 2004Ianora et al., , 2011Vargas et al., 2006;Poulet et al., 2007;Kâ et al., 2014). Decadienal has also been shown to potentially impact other marine organisms (Romano et al., , 2010Caldwell et al., 2005;Tosti et al., 2003). ...
... Diatom PUA have also been shown to impair copepod egg hatching success (Miralto et al., 1999), with induction of apoptosis in both somatic and gonadal tissues of females (Buttino et al., 2008) and naupliar stages Kâ et al., 2014). Teratogenic impacts (birth defects and hatching of malformed larvae) have also been recorded when females are fed with certain diatom diets (Poulet et al., 1995(Poulet et al., , 2007Uye, 1996;Ianora et al., 2011). In our study, 45-93% reduction was observed in nauplii production with increasing concentrations of decadienal. ...
Primarily, diatoms have been considered to support higher trophic level production in marine ecology theory. However, the progressive evidence of detrimental effects of diatoms on marine organisms from the last two decades has challenged the pelagic food web theory. Diatoms, unicellular algae and major primary producers in aquatic ecosystems produce a wide range of bioactive metabolites. Among these compounds, polyunsaturated aldehydes (PUAs) strongly impair the reproduction and developmental processes including gametogenesis, gamete functionality, fertilization, embryonic mitosis, and larval fitness and competence of various potential grazers. PUAs are well-known to impair with diatom cells themselves inhibit the growth of other phytoplankton and inhibit or stimulate pathogenic bacteria. 2-trans, 4-trans decadienal, due to its higher bioactivity has been studied extensively to inhibit the embryonic development, causes detrimental influence on the egg viability, hatching success, larval fitness and survival in copepods. The influence of decadienal on the reproductive performance of two most dominant estuarine copepods Pseudodiaptomus annandalei and Apocyclops royi were tested in the laboratory in Taiwan. A range of concentrations varied from 0.75 µM to 6 µM was used for the experiment. The algae, Isochrysis galbana was given as a food source to each treatment. The negative impact of decadienal was found on nauplii production, number of egg bearing females per day and survival of cohort females. Both copepod species responded differentially to the same diatom toxin. Nauplii number produced by per females was significantly higher with the algae Isochrysis galbana (control- without decadienal) compared to decadienal applied medium. Decadienal caused continuous mortality of cohort female in a dose dependent manner.
From an ecological prospective, our results indicate potential impact of PUAs in energy flow in planktonic systems during higher abundance of diatom and from an aquaculture prospective, the present results suggest for avoiding the use of PUAs producing diatoms in the cultivation of these copepod species in aquaculture industry.
Keywords: diatom, polyunsaturated aldehydes, decadienal, estuarine copepod, reproduction.
... However, upon cell damage several species produce cytotoxic compounds such as poly-unsaturated aldehydes (PUAs) and other oxylipins deriving from the oxidative metabolism of fatty acids (d 'Ippolito et al., 2004;Fontana et al., 2007;Pohnert, 2000). Oxylipins and PUAs in particular impair several reproductive and developmental processes in copepods, such as gametogenesis (Poulet et al., 2007a), embryogenesis (Ianora et al., 1995), egg viability , larval development (Carotenuto et al., 2002) and sex differentiation (Carotenuto et al., 2011). They also induce malformations (teratogenesis) and apoptosis in newly hatched nauplii (Ianora et al., 2004). ...
... Thus, our observed higher expression of TCP-1 in Calanus helgolandicus after feeding on Skeletonema marinoi might be related to the need of repairing misfolded or degraded cytoskeleton proteins induced by the diatom diet. Similarly, up-regulation of RNF-121 could be associated to a possible deleterious effect of this diatom on C. helgolandicus oocyte development (Poulet et al., 2007a). This protein belongs to the zinc-finger protein class, which are involved in numerous cellular mechanisms, such as regulation of gene transcription, protein synthesis, cytoskeleton organization, protein folding and chromatin remodeling (Laity et al., 2001). ...
Diatoms dominate productive regions in the oceans and have traditionally been regarded as sustaining the marine food chain to top consumers and fisheries. However, many of these unicellular algae produce cytotoxic oxylipins that impair reproductive and developmental processes in their main grazers, crustacean copepods. The molecular mode of action of diatoms and diatom oxylipins on copepods is still unclear. In the present study we generated two Expressed Sequence Tags (ESTs) libraries of the copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi and the cryptophyte Rhodomonas baltica as a control, using suppression subtractive hybridization (SSH). Our aim was to investigate differences in the transcriptome between females fed toxic and non-toxic food and identify differentially expressed genes and biological processes targeted by this diatom. We produced 947 high quality ESTs from both libraries, 475 of which were functionally annotated and deposited in GenBank. Clustering and assembling of ESTs resulted in 376 unique transcripts, 200 of which were functionally annotated. Functional enirchment analysis between the two SSH libraries showed that ESTs belonging to biological processes such as response to stimuli, signal transduction, and protein folding were significantly over-expressed in the S. marinoi-fed C. helgolandicus compared to R. baltica-fed C. helgolandicus library. These findings were confirmed by RT-qPCR analysis. In summary, 2 days of feeding on S. marinoi activated a generalized Cellular Stress Response (CSR) in C. helgolandicus, by over-expressing genes of molecular chaperones and signal transduction pathways that protect the copepod from the immediate effects of the diatom diet. Our results provide insights into the response of copepods to a harmful diatom diet at the transcriptome level, supporting the hypothesis that diatom oxylipins elicit a stress response in the receiving organism. They also increase the genomic resources for this copepod species, whose importance could become ever more relevant for pelagic ecosystem functioning in European waters due to global warming.
... Recent studies add a physiological explanation concerning low assimilation eYciency of essential compounds in copepod guts ( Dutz et al. 2008), a depletion of polyunsaturated fatty acids (especially eicosapentaenoic acid EPA) in certain diatoms caused by the PUA production ( Wichard et al. 2007) and compounds inducing oxidative stress ( Fontana et al. 2007) to the list of suggested mechanisms behind the negative eVects of diatoms. Since it seems, at present, likely that neither PUA production nor direct nutritional limitations can fully explain the diatom eVects ( Poulet et al. 2006Poulet et al. , 2007Dutz et al. 2008;Wichard et al. 2008), the actual mechanism(s) remain, however, unknown. Similarly, Weld studies on copepod-diatom interactions present controversial results, and the Weld relevance of diatom eVects is under debate. ...
... Copepod-diatom interactions are both species and strain-speciWc ( Pohnert et al. 2002), and changes in algae growth conditions can have large consequences for nutritional quality ( Klein Breteler et al. 2005) and also change the PUA production ( Ribalet et al. 2007). In addition, maternal diets have potentially a large eVect on the development of the new generation, which has been shown thoroughly with diatom diets ( Ianora et al. 2004; Table 6) and starvation ( Poulet et al. 2003Poulet et al. , 2007). The possible explanations for the large variability between experiments could therefore be (1) use of diVerent algae strains with diVerent properties, (2) changes in algae condition aVecting PUA production, nutritional quality or both or (3) maternal eVects complicating the nauplii response to the diet. ...
We measured development, growth and juvenile mortality of the common copepod Temora longicornis on 11 different monospecific diatom diets in order to estimate (1) how common the negative effects of diatoms are on the development of this copepod and (2) whether the arrested development is connected to deleterious polyunsaturated aldehydes
(PUA) or food nutritional quality. Four diatom species (Thalassiosira weissflogii, Thalassiosira rotula CCMP1647, Leptocylindricus danicus CCPM469 and Skeletonema costatum CCMP1281) supported complete development, whereas development failed in or before metamorphosis on seven diatom species/strains (Chaetoceros affinis CCMP158, C. decipiens CCMP173, C. socialis, T. rotula CCMP1018, Thalassiosira pseudonana CCMP1010 and CCMP1335). However, four out of these seven species were not ingested by nauplii, either due to morphology (Chaetoceros spp.) or large size (T. pseudonana CCMP1010). The growth rate did not correlate with the ingestion rate of PUA, neither with ingestion of food mineral (nitrogen) nor with biochemical (polyunsaturated fatty acids, sterols) components. We show that, although some diatoms are of inferior food quality, this is unlikely to be connected to toxicity or due to a direct limitation by a single food nutritional compound.
... The response observed with the toxic cyanobacterium was similar to that reported by Lacoste et al. (2001) and by Poulet et al. (2007) on C. helgolandicus feeding on a variety of food diets or kept under starvation. Degradation of cytoplasm and cell fragmentation in OS3, and lack of OS4 observed in A. tonsa fed N. spumigena resemble these cell abnormalities described for starved C. helgolandicus females and those fed a variety of EPR deleterious food (Poulet et al., 2007). ...
... The response observed with the toxic cyanobacterium was similar to that reported by Lacoste et al. (2001) and by Poulet et al. (2007) on C. helgolandicus feeding on a variety of food diets or kept under starvation. Degradation of cytoplasm and cell fragmentation in OS3, and lack of OS4 observed in A. tonsa fed N. spumigena resemble these cell abnormalities described for starved C. helgolandicus females and those fed a variety of EPR deleterious food (Poulet et al., 2007). Those abnormalities were seen in conjunction with very low or decreasing EPR. ...
Organisms present a series of cellular mechanisms to avoid the effects of toxic compounds. Such mechanisms include the increase in activity of detoxification enzymes [e.g., 7-ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST)], which could explain the low retention of ingested toxins generally observed in copepods. In addition, decreasing gross growth efficiency (GGE) of copepods with increasing concentration of toxic diets could be caused either by a high expenditure coping with toxins (e.g., increase in the activity of detoxification enzymes) or by a deterioration of reproductive tissues. To assess the effect of toxic phytoplankton on the activity of detoxification enzymes and on oocyte maturation of Acartia tonsa and Temora longicornis, feeding and egg production experiments were carried out with a variety of toxic diets and an adequate non-toxic food control (Rhodomonas spp.) all provided as single species diets. Toxic diets included the nodularin-producing cyanobacterium Nodularia spumigena, the dinoflagellates Alexandrium minutum, and A. tamarense, which contained Paralytic Shellfish Poisoning (PSP) toxins, the dinoflagellate Prorocentrum lima with Diarrhetic Shellfish Poisoning (DSP) toxins and the haptophyte Prymnesium parvum, which produces ichtyotoxins with haemolytic activity. Feeding on toxic diets was lower than on Rhodomonas spp., except for A. minutum and A. tamarense. In addition, toxic diets negatively affected reproduction in both copepod species with the production of oocytes and oocyte development impaired with A. minutum and N. spumigena. While the negative effect of N. spumigena seemed to be connected to gonad atresia likely caused by severe food limitation (starvation), the negative effect of A. minutum could have been either caused by a direct effect of saxitoxins or nutritional inadequacy on oocyte production. We could not detect EROD activity in the copepods, while the activity of GST was generally higher with the non-toxic food control and positively related to the feeding and egestion rates, suggesting relation to feeding conditions rather than to exposure to toxic diets. No relationship was found between GGE and GST activity. Our results refute the hypothesis that toxic diets, provided at ecologically relevant levels, would induce cellular mechanisms in copepods regarding GST activity. GST activity thus seems to play no role in detoxification of copepods confronted with toxic phytoplankton. Toxin detoxification and its cost for copepods still remain an open question.
... There have been several recent studies that failed to find a correlation between reduced hatching success and PUA production. Poulet et al. (2007a) studied the influence of diatoms on egg production and hatching success in Calanus hegolandicus fed 20 different algal diets in the laboratory. They reported that egg production was impaired by several diatom diets (Chaetoceros calcitrans, Guinardia delicatula, G. striata, Odontella regia, Rhizosolenia setigera, Stephanopyxis turris, Thalassiosira pseudonana) and mixed diatom assemblages collected in the field, as well as by the prymnesiophyte Pavlova lutherii. ...
... They found that hatching success was generally [80% in the copepods Temora stylifera and Centropages typicus, but survivorship of the first naupliar stage was very low, with mean values of 12% in T. stylifera and 67% in C. typicus. Furthermore, in T. stylifera, on nearly 40% of the sampling dates not a single nauplius 1 survived to moult to the second larval stage, similar to the findings of Halsband-Lenk et al. (2005) and Poulet et al. (2007a). Laboratory experiments showed that maternal feeding on Isochrysis galbana or Prorocentrum minimum for 7 days did not enhance naupliar survival in either copepod species, indicating that the negative effects of maternal diets did not disappear after feeding on a high quality food. ...
Traditionally, diatoms have been regarded as providing the bulk of the food that sustains the marine food chain and important fisheries. However, this view was challenged almost two decades ago on the basis of laboratory and field studies showing that when copepods, the principal predators of diatoms, feed on certain diatom diets, they produce abnormal eggs that either fail to develop to hatching or hatch into malformed (i.e. teratogenic) nauplii that die soon afterwards. Over the years, many explanations have been advanced to explain the causes for reproductive failure in copepods and other marine and freshwater invertebrates including diatom toxicity, or nutritional deficiency and poor assimilation of essential compounds in the animal gut. Here we review the literature concerning the first possibility, that diatoms produce cytotoxic compounds responsible for growth inhibition and teratogenic activity, potentially sabotaging future generations of grazers by inducing poor recruitment. The cytotoxic compounds responsible for these effects are short chain polyunsaturated aldehydes (PUAs) and other oxygenated fatty acid degradation products such as hydroxides, oxo-acids, and epoxyalcohols (collectively termed oxylipins) that are cleaved from fatty acid precursors by enzymes activated within seconds after crushing of cells. Such toxins are suggested to have multiple simultaneous functions in that they not only deter herbivore feeding but some also act as allelopathic agents against other phytoplankton cells, thereby affecting the growth of competitors, and also signalling population-level cell death and termination of blooms, with possible consequences for food web structure and community composition. Some oxylipins also play a role in driving marine bacterial community diversity, with neutral, positive or negative interactions depending on the species, thereby shaping the structure of bacterial communities during diatom blooms. Several reviews have already been published on diatom-grazer interactions so this paper does not attempt to provide a comprehensive overview, but rather to consider some of the more recent findings in this field. We also consider the role of diatom oxylipins in mediating physiological and ecological processes in the plankton and the multiple simultaneous functions of these secondary metabolites.
... wounding, grazing, or competition for nutrients) (Dittami et al., 2010;Ribalet et al., 2014), and can persist in the water even after phytoplankton bloom decline (Bartual and Ortega, 2013). Experimental works demonstrated that PUAs can induce changes in microzooplankton growth dynamic and community structure (Franzè et al., 2018), have a teratogenic effect on copepods (Romano et al., 2010) and inhibitory effects on the reproduction and development of marine invertebrates, thus they are considered an anti-grazer defense (Miralto et al., 1999;Poulet et al., 2007;Lauritano et al., 2012;Brugnano et al., 2016). Field studies reported differential effects of PUAs on the bacterial community in terms of growth and metabolic activity (Balestra et al., 2011;Paul et al., 2012) and on ciliates (Lavrentyev et al., 2015). ...
Chemical interactions between macroalgae and other organisms play an important role in determining species compositions and dominance patterns, and can explain the widespread success of some species in establishing their predominant populations in a specific coastal area. Allelopathy could act as a self-regulatory strategy of the algal community, being not only a succession regulator but also an active mechanism maintaining the species diversity especially in a delimited environment, such as the benthic ecosystem. Polyunsaturated aldehydes (PUAs) are among the most studied allelopathic compounds and are commonly released into the aquatic environment by different phytoplankton species in response to environmental stressors (e.g. wounding, grazing, or competition for nutrients). Diatom-released PUAs were observed to affect phytoplankton community dynamics and structure, and showed inhibitory effects on the reproduction and development of marine invertebrates. As for macroalgae, there are only a few reports that attest to the production of PUAs, and mostly refer to Ulva spp. In this study, the production of PUAs by several Mediterranean macroalgae was investigated at different sampling times, aiming at providing the first evidence of potential allelochemical activity. Results highlighted the potential production by macroalgae of a variety of aldehydes, among which some have never reported so far. Some species (i.e. D. polypodioides and U. cf. rigida) were found to produce higher PUAs amounts than others, and even a wider variety of structures (e.g. length of the carbon chain); these species might exert strong effects on epiphytic species or other organisms of the benthic community, especially considering the differential sensitivities of the various taxa. A high dPUA concentration (order of μM) potentially due to the release of PUAs by algal species was found, and might affect the population dynamics of the epiphytic organisms (e.g. microalgae, meiofauna), of grazers, as well as of the microbial community.
... Its role is still not fully known, although it has been suggested that altered expression of this gene in Daphnia magna females exposed to ibuprofen, was associated with failed oogenesis, abnormal oocytes possibly being re-absorbed and blocked embryogenesis [68]. It is therefore possible that reduced expression of VMO1 genes could play a role in the arrested oocyte maturation and oocyte degradation previously observed in C. helgolandicus females feeding on oxylipin-producing diatoms [69]. ...
Diatoms are the dominant component of the marine phytoplankton. Several diatoms produce secondary metabolites, namely oxylipins, with teratogenic effects on their main predators, crustacean copepods. Our study reports the de novo assembled transcriptome of the calanoid copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi. Differential expression analysis was also performed between copepod females exposed to the diatom and the control flagellate Prorocentrum minimum, which does not produce oxylipins. Our results showed that transcripts involved in carbohydrate, amino acid, folate and methionine metabolism, embryogenesis, and response to stimulus were differentially expressed in the two conditions. Expression of 27 selected genes belonging to these functional categories was also analyzed by RT-qPCR in C. helgolandicus females exposed to a mixed solution of the oxylipins heptadienal and octadienal at the concentration of 10 µM, 15 µM, and 20 µM. The results confirmed differential expression analysis, with up-regulation of genes involved in stress response and down-regulation of genes associated with folate and methionine metabolism, embryogenesis, and signaling. Overall, we offer new insights on the mechanism of action of oxylipins on maternally-induced embryo abnormality. Our results may also help identify biomarker genes associated with diatom-related reproductive failure in the natural copepod population at sea.
... As PUAs can play important roles in the interaction and regulation of algae-herbivore relationships, they have become a common parameter, determined along with other metadata in many studies. Being produced on cell damage of microalgae (Pohnert 2000;) PUAs have been made responsible for numerous effects on the grazer reproductive success such as the inhibition of copepod egg hatching or the action as causative agents for abnormal larval morphology (Miralto et al. 1999;Ianora et al. 2004;Poulet et al. 2007). However, the ecological relevance of these findings is still under discussion (Wichard et al. 2008). ...
Polyunsaturated aldehydes (PUAs) are a group of microalgal metabolites that have attracted a lot of attention due to their biological activity. Determination of PUAs has become an important routine procedure in plankton and biofilm investigations, especially those that deal with chemically mediated interactions. Here we introduce a fast and direct derivatization free method that allows quantifying PUAs in the nanomolar range, sufficient to undertake the analysis from cultures and field samples. The sample preparation requires one simple filtration step and the initiation of PUA formation by cell disruption. After centrifugation the samples are ready for measurement without any further handling. Within one chromatographic run this method additionally allows us to monitor the formation of the polar oxylipins arising from the cleavage of precursor fatty acids. The robust method is based on analyte separation and detection using ultra high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (UHPLC-APCI MS) and enables high throughput investigations by employing an analysis time of only 5 min. Our protocol thus provides an alternative and extension to existing PUA determinations based on gas chromatography-mass spectrometry (GC-MS) with shorter run times and without any chemical derivatization. It also enables researchers with widely available LC-MS analytical platforms to monitor PUAs. Additionally, non-volatile oxylipins such as ω-oxo-acids and related compounds can be elucidated and monitored.
... Diatoms, the major components of the marine microalgae, are assumed to be the principal food source for herbivorous copepods (Mann 1993). The effects of various diatoms (such as S. costatum, Thalassiosira rotula, T. weissflogii, P. tricornutum, Chaetoceros calcitrans) on the reproductive success, development, ingestion and growth rate of copepods vary greatly due to species-specific influences (Abu-Rezq, Yule & Teng 1997;Carotenuto, Ianora, Buttino, Romano & Miralto 2002;Ceballos & Ianora 2003;Poulet, Cueff, Wichard, Marchetti, Dancie & Pohnert 2007), but there are conflicting comments. Some researchers considered that diatoms have deleterious effects on development, egg production and hatching success of copepods because of polyunsaturated aldehydes (PUA) (Miralto, Barone, Romano, Poulet, Ianora, Russo, Buttino, Mazzarella, Laabir, Cabrini & Giacobbe 1999;Carotenuto et al. 2002;Ianora, Miralto, Poulet, Carotenuto, Buttino, Romano, Casotti, Pohnert, Wichard, Colucci-D'Amato, Terrazzano & Smetacek 2004). ...
We investigated the ingestion, fecundity and population growth of Harpacticus sp. fed on monodiet or mixed diets to evaluate the effects of different algae on Harpacticus sp. Harpacticus sp. fed on diatoms (Skeletonema costatum and Chaetoceros curvisetus) had higher ingestion and pellet production. Time to attain 100% in proportions of gravid females differed, with quickest to slowest: S. costatum, C. curvisetus, Gymnodinium sp. and Heterosigma akashiwo, with the exception of Prymnesium parvum (≤16.67%). S. costatum or C. curvisetus produced higher populations than the other three diets, supported complete development to adulthood, and resulted in doubling copepod population within four days, while no population growth occurred for the other three diets. Mixed-diet experiments showed that egg production and gross growth efficiencies reduced significantly when fed on H. akashiwo, Gymnodinium sp. or P. parvum mixed with Isochrysis galbana, in comparison with 100% I. galbana. Thus, S. costatum and C. curvisetus were beneficial foods while the other three diets were potentially toxic for Harpacticus sp. The data in this article provide further recognition of nutrient deficiency or toxicity of different algae on copepods.
... It is reported that diatoms such as Thalassiosira, Skeletonema, Odontella, Chaetoceros, Navicula, Nitzschia, among others, together with some dinofl agellates (Dinophyceae) such as Prorocentrum micans, Gymnodinium sanguineum, and Lingulodinium polyedra (formerly Gonyaulax polyedra) induce low hatching and low fertilization success in copepods, even in high egg production. Those that could hatch were presented with some deformities, expressing marked morphological asymmetry which resulted in their death a few days after (Ianora and Poulet 1993, Ianora et al. 1999, Lee et al. 1999, Caldwell et al. 2002, Poulet et al. 2007). These morphological development alterations were proposed to be the result of compounds with some kind of mitosis inhibition effect that could accumulate in the reproductive organs of the copepods that were fed with a diatom or dinofl agellate rich diet and somehow were transferred to the eggs. ...
... It is reported that diatoms such as Thalassiosira, Skeletonema, Odontella, Chaetoceros, Navicula, Nitzschia, among others, together with some dinofl agellates (Dinophyceae) such as Prorocentrum micans, Gymnodinium sanguineum, and Lingulodinium polyedra (formerly Gonyaulax polyedra) induce low hatching and low fertilization success in copepods, even in high egg production. Those that could hatch were presented with some deformities, expressing marked morphological asymmetry which resulted in their death a few days after (Ianora and Poulet 1993, Ianora et al. 1999, Lee et al. 1999, Caldwell et al. 2002, Poulet et al. 2007). These morphological development alterations were proposed to be the result of compounds with some kind of mitosis inhibition effect that could accumulate in the reproductive organs of the copepods that were fed with a diatom or dinofl agellate rich diet and somehow were transferred to the eggs. ...
... To date most studies have focused on the effects of PUAs on copepod reproduction in laboratory feeding experiments (Ban et al., 1997;Carotenuto et al., 2011;Poulet et al., 2007;Turner et al., 2001), generally confirming the toxicity of PUA-producing diatoms. On the other hand, field studies on the impact of PUAs on copepod reproduction have yielded highly-variable results. ...
... Miralto et al. (1999) demonstrated that inhibitory PUas blocked embryogenesis in copepods and sea urchins, although other authors proposed alternative roles for aldehydes (Dutz et al. 2008). Several mechanisms have been suggested to explain these negative effects (Poulet et al. 2007). ...
Plants evolve the production of wound-activated compounds (WACs) to reduce grazing pressure. In addition, several plant-produced WACs are recognized by various invertebrates, playing the role of infochemicals. Due to co-evolutionary processes, some invertebrates recognize plant infochemicals and use them to identify possible prey, detect the presence of predators or identify algae containing various classes of toxic metabolites. Different metabolites present in the same algae can play the role of toxins, infochemicals or both simultaneously. We investigated the infochemical activity of compounds extracted from three diatoms epiphytes of the seagrass Posidonia oceanica, by conducting choice experiments on invertebrates living in the same community or in close proximity. Furthermore, the specific toxicity of the extracts obtained from the same algae was tested on sea urchin embryos using a standard bioassay procedure, to detect the presence
of toxins. The comparison of the two effects demonstrated that invertebrates are subjected to diatom wound activated toxicants when these algae are not associated with their own habitat, but they are able to recognize volatile infochemicals derived from diatoms associated with their habitats. The specific toxicity of WACs was shown to be inversely correlated to the perceptive ability of invertebrates towards volatile compounds liberated by the same algae. Hence, when the recognition of specific algae by a given invertebrate species evolves, their detrimental effects on the receiving organism may be lost.
... Essential fatty acids in Skeletonema were found to deplete strongly after cell disruption resulting in imbalance of important fatty acids. Our results are consistent with studies showing higher egg production on dinoflagellate than on diatom diets (Niehoff, 2004;Poulet et al., 2007). In addition, natural spring bloom dominated by the dinoflagellate Gymnodinium corollarium, which co-occurs and is isomorphic with S. hangoei, supports higher egg production than phytoplankton composition dominated by S. marinoi (Vehmaa et al., subm. ...
Mild winters are modifying the plankton spring bloom composition so that diatoms are decreasing and dinoflagellates increasing. We used two common spring bloom phytoplankton species, a diatom and a dinoflagellate to study the effects of changing bloom composition on the reproduction of the calanoid copepod Acartia bifilosa Giesbrecht, a dominant species in the northern Baltic Sea. Egg production was significantly higher when copepods were fed with Scrippsiella hangoei (Schiller) Larsen dinoflagellates or a mixture of Scrippsiella and Skeletonema marinoi Sarno and Zingone diatoms than when they were provided with Skeletonema only. This effect was observed despite the fact that the Skeletonema strain did not produce polyunsaturated aldehydes (PUAs) and its nutritional quality was high according to polyunsaturated fatty acid (PUFAs) and sterol measurements, and moderate according to mineral (C:N and C:P) measurements. When offered mixtures of Skeletonema and Scrippsiella, copepods ingested both, even when the other one was rare. This indicates potential positive effect of multispecies diets not verified in this study. Here we show that increasing dinoflagellate:diatom ratio might have a positive effect on copepod reproduction. (C) 2011 Elsevier B.V. All rights reserved.
... C. helgolandicus is among the most widely studied copepod species when investigating the influence of temperature and food on development, growth, egg production and hatching success (Bonnet et al., 2009;Cook et al., 2007;Guisande and Harris, 1995;Jónasdóttir et al., 2005;Kang et al., 2000;Pond et al., 1996;Rey-Rassat et al., 2002a, 2002b. This species has also been the subject of several experimental and field studies focused on the negative effects of diatom secondary metabolites deriving from the oxidative metabolism of fatty acids (oxylipins), on hatching success and naupliar fitness Ianora and Miralto, 2010;Ianora et al., 2004;Irigoien et al., 2000a;Laabir et al., 1995;Miralto et al., 2003;Poulet et al., 1995Poulet et al., , 2006Poulet et al., , 2007Wichard et al., 2008). These experiments have traditionally been conducted on wild-caught specimens kept for a few days or weeks in small volume containers, but so far, the species has never been reared for multiple generations. ...
We tested the amenability of the calanoid copepod Calanus helgolandicus to be mass cultivated in a large volume system. The copepod was reared on two different occasions (during 2009 and 2010), for at least four generations and six months with a mixed flagellate diet of Isochrysis galbana, Rhodomonas baltica and Prorocentrum minimum, or a mixed diet of I. galbana, R. baltica and the diatom Thalassiosira weissflogii. The generation time of C. helgolandicus from nauplius to adult was 21 days, with relative duration of naupliar and copepodid phases of 7 and 14 days, respectively. Population growth rates when the second generation of adults developed and reproduced in the tank, were ~2-times higher (0.067 d−1 vs. 0.038 d−1) and abundances increased 6-times more (40-fold vs. 6.7-fold) during 2010 than 2009. This higher production was related to the addition of T. weissflogii to the mixed R. baltica+I. galbana diet at low concentrations (0.17 mg C l−1) and for short periods (b2 months), which induced higher egg production rate and percentage of spawning females and lower copepodid mortality in the population. However, supplementation of T. weissflogii at higher concentrations (0.40 mg C l−1) or longer feeding times (>3 months), reduced copepod egg hatching success, and hence population growth of C. helgolandicus. In conclusion, our study shows that by using an appropriate mixed diatom assemblage for short periods, it is possible to obtain healthy individuals of all larval stages of C. helgolandicus for biological, toxicological and gene expression studies year-round.
... A negative effect of PUA on the reproduction success of herbivorous copepods was proposed by several authors based on evidence from feeding experiments [2], application of pure PUA [3] or PUA encapsulating liposomes [4] (reviewed in [5,6]). While some evidence for the action of PUA was found in the field [7], several investigations failed to correlate diatom abundance with reproduction failure of copepods [8,9] as well as PUA production with lower egg hatching success10111213. Additionally, modeling studies suggest that PUA-based chemical defense against herbivory is not advantageous for a diatom population, since it cannot be considered as an evolutionary driving force [14]. ...
Several marine and freshwater diatoms produce polyunsaturated aldehydes (PUA) in wound-activated processes. These metabolites are also released by intact diatom cells during algal blooms. Due to their activity in laboratory experiments, PUA are considered as potential mediators of diatom-bacteria interactions. Here, we tested the hypothesis that PUA mediate such processes in a close-to-field mesocosm experiment. Natural plankton communities enriched with Skeletonema marinoi strains that differ in their PUA production, a plankton control, and a plankton control supplemented with PUA at natural and elevated concentrations were observed. We monitored bacterial and viral abundance as well as bacterial community composition and did not observe any influence of PUA on these parameters even at elevated concentrations. We rather detected an alternation of the bacterial diversity over time and differences between the two S. marinoi strains, indicating unique dynamic bacterial communities in these algal blooms. These results suggest that factors other than PUA are of significance for interactions between diatoms and bacteria.
... In an effort to optimise the feeding regime of cultured P. canaliculus larvae we started with this simple two species model. However, the diatom C. 'calcitrans' may also have detrimental side effects upon the grazer's health (Poulet et al., 2007). Harmful effects may be attributable to metabolites (Webb and Chu, 1982; Rico-Villa et al., 2006), mechanical interference (Chauvaud et al., 1998) or pathogenic bacteria associated with the algal cells or culture medium (Torkildsen et al., 2005); in each case deleterious effects are likely to be influenced by conditions within the diatom culture environment, particularly as cells age or become resource-limited (Enright et al., 1986b). ...
The diatom Chaetoceros calcitrans forma pumilum is an important dietary component for cultured Greenshell™ mussel larvae, Perna canaliculus. However concerns surrounding potentially deleterious side effects have motivated a series of experimental trials to optimise the safe usage of this alga. Bioassays involved raising veliger larvae in the purpose-built Cawthron Ultra-Density Larval rearing (CUDL) system; an array of 2.5-L tanks were stocked with 2-day-old veligers (200 larvae mL−1) supplied with inflowing water dosed with sufficient microalgae to maintain a fixed concentration of cells, after compensating for ingestion. The nutritional role of C. ‘calcitrans’ was examined by adjusting its cellular fraction in the feed environment. Diets of 0, 5, 66, 95 and 100% C. ‘calcitrans’ were each offered to six replicate rearing tanks, using Isochrysis aff. galbana (T-Iso clone) to maintain a total of 40 cells μL−1 in the larval cultures. The 66% C. ‘calcitrans’ diet was also offered at 3, 20, 60 and 120 cells µL−1 (n = 6). Higher C. ‘calcitrans’ fractions sustained faster growth, with 95% and 100% treatments producing 23-day-old pediveligers of 240 ± 15 μm and 228 ± 5 μm mean shell length, respectively; compared to 212 ± 7 μm in the 66% treatment (40 cells μL−1). However, high C. ‘calcitrans’ treatments were more volatile, had higher mortality and greater predisposition towards population crashes. Similarly, reduced survival was observed in the high feed treatment of 120 cells µL−1, reflected in a final pediveliger yield of 29 ± 6%, compared to 47 ± 3% at 60 cells µL−1 and 36 ± 7% at 40 cells µL−1. The 5% C. ‘calcitrans’ and 20 cells µL−1 treatments showed signs of nutrient limitation, while larvae fed 0% C. ‘calcitrans’ or a total of 3 cells µL−1 starved, failing to reach metamorphosis. A standard diet consisting of 66% C. ‘calcitrans’ and 34% I. aff. galbana maintained at 40 cells μL−1 was used to test the hypothesis that culture age increased deleterious side effects associated with feeding C. ‘calcitrans’. The final stage of batch culture, using 20-L nylon carboys inoculated with 1010C. ‘calcitrans’ cells was allowed to age for 2, 3, 4, 5 or 6 days before being fed to larvae. After 21 days eating 2-day-old C. ‘calcitrans’ larvae reached a mean shell length of 236 μm, were eating 35,000 cells larva−1 day−1, and had a survival rate of 59 ± 3%; in contrast, larvae eating 6-day-old cells only reached 214 µm, eating 17,000 cells larva−1 day−1, with an overall survival of 46 ± 7%. While C. ‘calcitrans’ is valuable in the larval culture of P. canaliculus and many other bivalves, appropriate feeding protocols are needed when using this diatom.
... Dutz et al., 2008;Flynn and Irigoien, 2009). PUAs are geno-and cytotoxic (Adolph et al., 2004;Caldwell et al., 2011;Hansen et al., 2004) and, depending on the degree of exposure, toxic endpoints may be manifested as a variety of impacts including parental sterility/infertility, abortive embryogenesis, impaired hatching success, reduced larval fitness and mortality of neonates (Caldwell, 2009;Caldwell et al., 2002;Ceballos and Ianora, 2003;Ianora et al., 2004;Poulet et al., 2007b). ...
Numerous species of diatoms liberate oxylipins including polyunsaturated aldehydes (PUAs) in response to cellular damage such as may occur during grazing. PUAs are cyto- and genotoxic and negatively disrupt reproductive processes in copepods, their principal grazers, although experimental evidence would suggest that the grazer response may be species specific. The reproduction of the benthic harpacticoid copepod Tisbe holothuriae was compared over two generations. Copepods were reared using four diet treatments: PUA-producing diatom strains Skeletonema marinoi (Adriatic Sea Isolate FE6) and Melosira nummuloides (CCAP 1048/6); and non-PUA-producing diatom strains Phaeodactylum tricornutum (CCAP 1052/A) and Skeletonema marinoi (Seasalter (Walney) Ltd). Life tables were generated for each treatment using measured reproductive parameters and the net reproductive rate (R0) calculated. No significant differences were observed between the individual reproductive parameters of T. holothuriae fed PUA-producing diatoms compared to those fed non-PUA producing diatoms although diets of P. tricornutum resulted in some decreases in individual reproductive parameters in the second generation. There were no significant differences in the R0 values between the four tested diets. These observations indicate that T. holothuriae exhibits a tolerant of known PUA-producing diatom diets that has not been similarly demonstrated in pelagic calanoid copepods. Harpacticoid copepods may have a greater capacity to detoxify diatom oxylipins than their planktonic calanoid counterparts.
... The lack of feeding on these species by C. sinicus may indicate that they are nutritionally insufficient or toxic (Delgado and Alcaraz, 1999;Albuquerque et al., 2005;Dam and Colin, 2005). However, C. helgolandicus and Temora stylifera exhibited high egg production and hatching rates when feed on P. minimum compared with diatoms (Laabir et al., 1999;Turner et al., 2001;Poulet et al., 2007). These results suggest that feeding interactions between copepods and dinoflagellates may be species-specific. ...
Shipboard incubations were conducted in spring (April) and autumn (October/November) 2006 to measure the feeding and egg production
rates (EPR) of Calanus sinicus in the Yellow Sea, China. The ingestion rate (2.08–11.46 and 0.26–3.70 μg C female−1 day−1 in spring and autumn, respectively) was positively correlated with microplankton carbon concentrations. In the northern part
of the Yellow Sea, feeding on microplankton easily covers the respiratory and production requirements, whereas in the southern
part in spring and in the frontal zone in autumn, C. sinicus must ingest alternative food sources. Low ingestion rates, no
egg production and the dominance of the fifth copepodite (CV) stage indicated that C. sinicus was in quiescence inside the
Yellow Sea Cold Bottom Water (YSCBW) area in autumn. Calanus sinicus ingested ciliates preferentially over other components
of the microplankton. The EPR (0.16–12.6 eggs female−1 day−1 in spring and 11.4 eggs female−1 day−1 at only one station in autumn) increased with ciliate standing stock. Gross growth efficiency (GGE) was 13.4% (3–39%) in
spring, which was correlated with the proportion of ciliates in the diet. These results indicate that ciliates have higher
nutrient quality than other food items, but the low GGE indicates that the diet of C. sinicus is nutritionally incomplete.
... pungens, Rhizosolenia setigera, and T. nitzschioides) have so far not been reported harmful for A. tonsa or other Acartia spp. However, three of the species have been reported to be toxic for other copepods: Chaetoceros curvisetus for Temora stylifera (Koski et al. 2008), N. acicularis for Calanus chilensis (Poulet et al. 2007b), and R. setigera for Calanus helgolandicus (Poulet Oecologia (2009) 159:207–215 213 et al. 2007a). Other species of Chaetoceros have been found toxic for A. tonsa and other Acartia spp. ...
The feeding selectivity and the growth and reproductive success of the copepod Acartia tonsa have been studied in mesocosms fertilized at different Si:N ratios (0-1.75:1) and, therefore, at different compositions of the phytoplankton communities. Phytoplankton composition showed a strong response to nutrient ratios, with diatoms comprising >90% at Si:N ratios >1:1 of total biomass as opposed to <20% at the lowest ratio. A. tonsa strongly preferred feeding on motile prey (flagellates and ciliates) to feeding on diatoms. Nevertheless, diatoms comprised a substantial part of the diet at the highest Si:N ratios. A. tonsa egg production and the final (after 4 weeks) abundance of adults and copepodites showed no response to Si:N ratios while nauplii production slightly increased with Si:N ratios. It is concluded that the frequently reported deleterious effect of diatoms on copepod reproduction is rather unusual when copepods are confronted with a naturally diverse phytoplankton assemblage instead of clonal cultures in the laboratory.
We hypothesize that algae with different cell composition are differently perceived by their predators and consequently subjected to selective grazing. Five populations of the diatom Phaeodactylum tricornutum that differed for organic and elemental composition, but were otherwise identical, were generated by acclimation to distinct growth regimes. The different populations were then mixed in pairs and subjected to predation by either the rotifer Brachionus plicatilis or the copepod Acartia tonsa. The presence of rotifers had no impact on the ratio between any two algal populations. The presence of copepods, instead, affected the ratio between algae previously acclimated to a medium containing 1 mM NH4+ and algae acclimated to 0.5 mM NO3‐ and to either a lower irradiance or a higher CO2 concentration. We discuss the possible reason for the influence of different nutritional histories on the vulnerability of algae to predators. The differential impact of grazers on the growth of algae with different nutritional history may result from direct selective grazing (i.e., grazers can detect algae with the most palatable cell composition), alone or combined to an asymmetric utilization of the nutrients regenerated after predation by co‐existing algal populations. Our results strongly suggest that the nutritional history of algae can influence the relationships between phytoplankton and grazers and hint at the possibility that algal cell composition is potentially subject to natural selection, because responsible, to some extent, of the survival of algae to their predators. This article is protected by copyright. All rights reserved.
Diatoms, ubiquitous primary producers in marine systems, synthesize oxylipins, which impair copepod fitness through a maternal effect. While oxylipins do not directly affect the adults, these chemicals alter basic cellular and developmental processes of copepod embryos, thereby negatively affecting egg hatching and naupliar survival. Inspecting the effects of oxylipins on copepod reproductive success is extremely challenging, because wide variations in their synthesis potentials among diatom genera, species, populations and strains have been detected. In parallel, distinct copepod species and populations can be differently sensitive to oxylipin-producing diatoms. Lately, application of chemical and molecular methods allowed to finely characterize the oxylipin profiles of single diatom strains and to highlight detoxification responses of copepods through the relative expression of selected genes of interest. Thus far, integrative experimental approaches encompassing physiological, chemical and molecular data were mostly applied to laboratory setups, whereas few in situ experiments were presented in this perspective. Moreover, only a restricted number of papers tested the effects of oxylipins on the plankton community as a whole. This review synthesizes latest advances in conceptual and methodological approaches in diatom–copepod interactions and aims at discussing possible re-shaping of the plankton community in response to oxylipin-producing diatom occurrence.
Two diatoms, Chaetoceros muelleri and Nitzschia closterium f. minutissima, were used for rearing nauplii to adults of Tigriopus japonicus, Acartia pacifica, and Pseudodiaptomus annandalei at different concentrations. The survival rates to adults and activities of superoxide dismutase (SOD) and glutathione‐S‐transferase (GST) of the adult copepods were measured. The results showed that when fed on N. closterium f. minutissima, A. pacifica was unable to complete naupliar development; while SOD and GST activities of P. annandalei fed N. closterium f. minutissima were significantly higher than those fed the control algae Isochrysis galbana (Prymnesiod), suggesting this diatom species is harmful to these calanoid copepods. The survival rates of T. japonicus were very high when fed N. closterium f. minutissima, indicating that T. japonicus has high acceptance to diatoms. To evaluate whether calanoid copepod had the capacity to acclimatize to diatoms, P. annandalei was cultured for four generations on the two diatoms at 1.7 μgC/ml and survival rates as well as SOD and GST activities were determined for each generation. It was shown that starting at the second generation, P. annandalei exhibited adaption to the diatoms with improved survival. When fed on N. closterium f. minutissima, both SOD and GST activities of P. annandalei showed a trend of decreasing with the progress of generation, whereas no significant difference was found among four generations on C. muelleri. This study demonstrates that whether a diatom species is considered harmful to copepods, which is largely species specific, and diatom concentration is an important influencing factor. Additionally, the capacity of copepod adaption to diatoms over time should not be neglected.
Variations of egg production rate (EPR), hatching success (HS), production of abnormal larvae (AL) and histology of gonads have been investigated with Calanus chilensis females sampled weekly, from late November to December 2004, at a station located in the coastal zone off Dichato (Chile), at time diatom concentration in phytoplankton bloom was high. Weekly EPR estimate in nature did not change significantly during this period. It remained close to normal values (25–40 eggs/female/day), whereas HS was constantly low and high proportions of AL were observed. In parallel, bioassays revealed that EPR was strongly depressed by artificially enriched diets, corresponding to natural diatom assemblages (NDA) occurring in the field, while abnormal HS and AL values could not be improved. Ingestion of diatoms by females was estimated by faecal pellet production rates and SEM examination of diatom remains in pellet samples. Low HS and the high amounts of abnormal larvae were not reversible when females were offered a favourable food, the dinoflagellate Prorocentrum minimum (PM). Minor cell degradations were observed in gonads of females fed NDA diets. Results showed that the two major reproductive factors HS and AL in C. chilensis could be significantly impaired by diets in the field or diatom enriched diets.
A unialgal diet of a non-neurotoxic strain of the dinoflagellate Alexandrium tamarense strongly modified egg production and hatching success in the copepod Temora stylifera, even though grazing rates were relatively constant over time. Both exponential and stationary cultures of A. tamarense reduced egg production and hatching success, but the effect of stationary cultures on hatching success was dramatic, with egg viability dropping to 0 % after 24 h of feeding. HPLC analyses revealed that the A. tamarense clone was non-neurotoxic, with a mean toxin content of about 0.005 fmol per cell, indicating that adverse effects were not due to saxitoxins or neosaxitoxins. 1H-NMR analyses also revealed that diatom-derived PUSCAs (polyunsaturated short-chain aldehydes) were not responsible for reduced hatching rates, since these compounds were absent in A. tamarense as compared to the diatom Skeletonema costatum. Extracts of A. tamarense and the diatom-derived PUSCA 2-trans-4-trans-decadienal were also assessed in terms of biological effects on sea urchin embryo cell divisions. A. tamarense did not show anti-mitotic properties, as extracts did not block first-cell cleavage compared to decadienal. However, A. tamarense extracts did block fertilization success when sea urchin oocytes were first incubated for 30 min in extracts and then fertilized, as opposed to S. costatum extracts, which did not affect normal elevation of the fertilization membrane. This is the first report that dinoflagellates produce antiproliferative compounds, other than PUSCAs, that can potentially reduce copepod egg production and hatching success.
The factors controlling the temporal dynamics of egg production rate (EPR) in Temora longicornis were investigated during a 3 yr study. T longicornis produced eggs all year round, with an annual maximum (EPRmax) between 27 and 48 eggs female(-1) d(-1) during spring/summer and minimum of 1.4 eggs female(-1) d(-1) in autumn/winter. The highest EPR values were measured when copepods were heaviest and in situ chlorophyll a (chl) increased above similar to 1 to 3.5 mu g l(-1), irrespective of ambient temperature, EPR was positively related to copepod body weight (BW) and chl, whereas it was negatively related to suspended particulate matter (SPM) and temperature. Stepwise multivariate analysis showed that copepod BW, chl and SPM explained most of the variability associated with fecundity, outweighing the effect of temperature. Moreover, the lower EPRmax measured in spring 1996, as compared to 1997 and 1998, suggests that food nutritional quality was probably another important factor affecting copepod fecundity. Since BW was inversely related to temperature, by controlling copepod BW, temperature indirectly determined the maximum potential reproductive output of T longicornis. Acting on at least in Situ physical parameters like temperature and SPM, climate change could represent an important modulator of secondary production in this and other copepod species.
We determined the egg production efficiency (EPE: egg production/ingestion) and egg viability of the copepod Acartia tonsa Dana under different heterotrophic and autotrophic diets. EPE was estimated in adult females either as the slope of the linear relationship between specific egg production (EPR) and ingestion rates, or as the quotient: EPR/ingestion rate. The diets, offered in mono-culture, were the heterotrophic ciliates Strombidium sulcatum or Mesodinium pulex, the heterotrophic dinoflagellate Gymnodinium dominans, the autotrophic cryptophyte Rhodomonas salina and the autotrophic dinoflagellate Gymnodinium sanguineum. The diets were also analyzed for fatty acid contents and composition, relationships with EPE and reproductive success were determined. Clear differences were found in the fatty acid contents and the composition of the different diets offered, but these differences did not correspond with variability in EPE. However, egg viability was correlated with ingestion of certain prey essential fatty acids; interestingly, our data do not show that ciliates and heterotrophic dinoflagellates are nutritionally superior prey for marine copepods, contrary to general expectations.
Fecundity of the copepod Calanus helgolandicus was monitored at a coastal station in the western English Channel during January to September 1994 and varied seasonally from 3 to 33 eggs female(-1) d(-1) (mean 17 eggs female(-1) d(-1)). Particulate fatty acid levels, particularly in the <20 mu m size fraction, were more highly correlated with fecundity than either chi a, particulate carbon or nitrogen, and this reflected the size and species composition of the microplankton. Diatoms, dinoflagellates and ciliates all sustained high fecundity. The mean volume of eggs produced by C. helgolandicus was inversely related to both food availability and fecundity, with increased numbers of smaller eggs produced during periods of high food availability. Female C. helgolandicus with the highest body nitrogen and carbon produced eggs with the highest carbon and nitrogen contents, although these eggs were also smaller. Hatching viability was consistently high with a seasonal mean of 83% and both viability and the incidence of deformed nauplii were negatively correlated with the carbon and nitrogen content of females and eggs. Hatching viability was not correlated with the abundance of diatoms, but was closely correlated with the levels of 18:2(n-6) and 20:4(n-6) fatty acids in both the environment and in the eggs. Particulate concentrations of the polyunsaturated fatty acids 20:5(n-3) and 22:6(n-3), although abundant in the environment and therefore good indicators of food availability, were not correlated with either egg viability or naupliar survival. The levels of saturated and monounsaturated fatty acids in the eggs were important factors determining naupliar survival under starvation conditions. Although food availability limited fecundity, female recruitment was possibly limited by predation pressure, particularly during the late summer. This study highlights the value of seasonal and nutritional based studies in understanding variability in zooplankton fecundity.
Evidence is presented on the harmful impact of a diatom (Thalassiosira rotula) on the reproductive biology of the copepod Calanus helgolandicus. When adult females were fed the diatom, both total egg production and hatching success were significantly lower than with the dinoflagellate Prorocentrum minimum. Embryonic development in the copepod was arrested when eggs were exposed to diatom but not dinoflagellate extracts from these same species of phytoplankton, Depending on the age of the eggs before exposure, cell division was blocked either prior to fusion of male and female pronuclei, or during mitosis. Embryos underwent strikingly abnormal development. The structural anomalies included a dark brown, opaque outer membrane, globular cytoplasm, blockage of pronuclei, or dispersed chromatin scattered in the egg matrix of non-hatched eggs either spawned by naturally occurring females, spawned by females fed with diatoms, or in embryos incubated in diatom extracts. The production by several species of diatoms of inhibitory compound(s) detrimental to the development and survival of their principal grazers may have major implications on secondary production and on the flow of energy in the marine food web.
Calanus helgolandicus females were incubated for 6 to 25 d in single and mixed diets of the diatom Coscinodiscus curvatulus and the dinoflagellate Gymnodinium sanguineum using 2 incubation protocols, and fecundity, hatching success, egg cannibalism and faecal pellet production were measured. Both fecundity and hatching success were significantly reduced by single or mixed diets containing high concentrations of diatoms. The deleterious effect of the diatom was diminished when females were fed diets containing low diatom concentration and also when egg cannibalism represented ca > 20% of the total daily egg production. Egg cannibalism was higher with the wheel-incubation method than with the vial-incubation method. The results suggest that diatom inhibition of copepod reproduction can be reduced in various ways, including decreasing the diatom concentration, switching from diatom to dinoflagellate diets, increasing the diversity of food items and also by egg cannibalism (eggs are of high nutrient value).
A method for determining Calanus egg production rates from preserved, net-tow samples is proposed. In the sea off Nova Scotia, in situ egg production rates (eggs per female per day) of Calanus finmarchicus are significantly related to an index of gonadal development in preserved females. This relationship could be used in combination with data on female abundance to estimate daily production of eggs in the water column. The method is illustrated with data from a transect across Browns Bank.
Traditionally, diatoms have been associated with productive pelagic food chains that lead, through suspension-feeding planktonic copepods, to top consumers and important fisheries. Here, 15 laboratories located worldwide in 12 different countries and representing a variety of marine, estuarine and freshwater environments present strong evidence that diatom diets are in fact inferior for copepod reproduction. When fed to females of 16 copepod species, all but 1 of the 17 diatoms examined significantly reduced egg production rates or egg viability compared to non-diatom controls. These effects are hypothesized to influence copepod recruitment patterns and the flow of energy in marine food webs.
Egg production rate (EPR) and subsequent egg-hatching success of Temora longicornis from the southern Gulf of St. Lawrence were measured in the laboratory and at sea during 2 cruises in late June and early July of 2000 and 2001. A quantitative, functional Ivlev relationship between EPR, food concentration (µg C l-1) and temperature was fit to the laboratory results. This relationship can serve as a template for expressing environmental control of T. longicornis egg production in models of the species population dynamics. Among the findings was a very low EPR at high temperature (18°C) when food was limiting (<200 µg C l-1). The EPR of T. longicornis in the southern Gulf of St. Lawrence in early summer varied as a function of chlorophyll a concentration (integrated 0 to 50 m), and corresponded well with the functional laboratory relationship using a carbon/chlorophyll a conversion factor of 80. Mean hatching success in the laboratory was 42.8% for experimental temperatures <14°C, but declined dramatically to 7.5% at temperatures over 14°C. No relationship between hatching success and food concentration was observed. In the southern Gulf of St. Lawrence in late June, hatching success varied between 0 and 56% for mean temperatures between 3.3 and 10.9°C (median 6.6°C). While the presence of resting eggs at this time of year is the most probable explanation of our observations of low hatching success, other factors that may inhibit egg hatching, including methodology, insufficient fertilization and food quality, may also be implicated. We conclude that for T. longicornis, EPR is food-limited in early summer in this region. Comparison with other studies indicated that environmental controls on fecundity of T. longicornis may differ among regions where this species is the dominant copepod.
A unialgal diet of a non-neurotoxic strain of the dinoflagellate Alexandrium tamarense strongly modified egg production and hatching success in the copepod Temora stylifera, even though grazing rates were relatively constant over time. Both exponential and stationary cultures of A. tamarense reduced egg production and hatching success, but the effect of stationary cultures on hatching success was dramatic, with egg viability dropping to 0% after 24 h of feeding. HPLC analyses revealed that the A. tamarense clone was non-neurotoxic, with a mean toxin content of about 0.005 fmol per cell, indicating that adverse effects were not due to saxitoxins or neosaxitoxins. H-1-NMR analyses also revealed that diatom-derived PUSCAs (polyunsaturated short-chain aldehydes) were not responsible for reduced hatching rates, since these compounds were absent in A. tamarense as compared to, the diatom Skeletonema costatum. Extracts of A. tamarense and the diatom-derived PUSCA 2-trans-4-trans-decadienal were also assessed in terms of biological effects on sea urchin embryo cell divisions. A. tamarense did not show anti-mitotic properties, as extracts did not block first-cell cleavage compared to decadienal. However, A. tamarense extracts did block fertilization success when sea urchin oocytes were first incubated for 30 min in extracts and then fertilized, as opposed to S. costatum extracts, which did not affect normal elevation of the fertilization membrane. This is the first report that dinoflagellates produce antiproliferative compounds, other than PUSCAs, that can potentially reduce copepod egg production and hatching success.
Field evidence is presented showing that hatching success in the copepods Acartia clausi and Calanus helgolandicus was greatly modified during 2 major late-winter diatom blooms in the North Adriatic (Mediterranean) Sea in 1997 and 1998 compared to post-bloom conditions in early summer of 1997. In February of both years, diatoms represented >90% of the microplankton at all depths of inshore stations, and from 40 to 95% at different depths of offshore stations. The diatom blooms differed between the 2 yr in that Skeletonema costatum (66.3%) and Pseudo-nitzschia delicatissima (23.2%) dominated in 1997, whereas the following February the bloom was almost entirely composed of P. delicatissima (80.6%). In June 1997, diatoms were much less abundant, even though they still comprised about 50% of the algal population at some of the more coastal stations; the diatom species composition in this period had shifted to a dominance of Chaetoceros decipiens and Dactlyliosolen fragilissimus. The population structure and vertical distribution of the copepods A. clausi and C. helgolandicus were examined to determine the effects of these diatom blooms on population recruitment rates. A. clausi was the most abundant copepod sampled in June, constituting 47.7% of total copepod numbers, whereas low abundances were recorded in February 1997 (1.6%) and 1998 (1.8%). C. helgolandicus numbers were also low in winter 1998 (0.82% of total copepod numbers). Egg production rates (EPR) for A. clausi were highest during the blooms, with values of 10.6 and 18.4 eggs female(-1) d(-1) in 1997 and 1998 respectively, compared to 6.2 eggs female(-1) d(-1) in June 1997. However the most dramatic differences between bloom and post-bloom conditions were in terms of egg hatching viability, with mean values of 15.4 and 16.1% in February 1997 and 1998 respectively, as opposed to 89.1% in June. Fecal pellet numbers for females were highest in winter, indicating active feeding on diatoms. Total egg production rate (TEP, the product of EPR and total number of females m(-3)) was much greater in June (863 eggs m(-3) d(-1)) than in February 1997 (17.6 eggs m(-3) d(-1)) and 1998 (140 eggs m(-3) d(-1)) due to the higher number of females in early summer. In turn, naupliar recruitment rates (TNR) were extremely high in June (790.2 nauplii m(-3) d(-1)) as opposed to February 1997 (2.8 nauplii m(-3) d(-1)) and February 1998 (15.6 nauplii m(-3) d(-1)). EPR for C. helgolandicus in February 1998 was low (9.7), with only 6.5% hatching viability. Values for TEP (54 eggs m(-3) d(-1)) and TNR (4.7 nauplii m(-3) d(-1)) were lower than for A. clausi. We conclude that the main factor responsible for reduced population recruitment rates of both species in winter was low hatching viability and that the bulk of annual recruitment in the North Adriatic Sea occurred in post-bloom conditions, since the mixed protistan community that succeeded the bloom was probably a better food source for copepod embryogenesis. The results are discussed in relation to laboratory findings on the toxic effects of diatoms on copepod reproduction.
Diatoms are small eukaryotic plants with over 10,000 known species, constituting one of the major components of the phytoplankton in freshwater and marine environments. Traditionally, they have been regarded as beneficial to the growth and survival of marine organisms, and to the transfer of organic material through the marine food chain to top consumers and important fisheries. From 1993 onwards, however, evidence has accumulated that has progressively challenged the classic view that diatoms are good and harmless food items for copepods, the dominant constituent of the zooplankton, which sustain the production and growth of larval fish. Laboratory results in recent years have shown that some diatoms potentially reduce copepod egg viability up to 100%; at times, egg production rates are adversely affected as well. Thus, while diatoms may provide a source of energy for copepod larval growth. they often reduce fecundity or hatching Success or both. These results constitute the paradox of diatom-copepod interactions in the pelagic food web. This biological model is new and has no equivalent in marine plant-herbivore systems, since most of the known negative plant-animal interactions have been related to repellent or poisoning processes, but never to reproductive failure. This paper reviews the literature on the inhibitory effects of diatoms on the reproductive biology of copepods and focuses on the present status and problems regarding diatom-copepod interactions.
Experiments were conducted over 10 to 20 d periods to study the grazing and reproductive success of the copepod Temora sWera fed on unialgal cultures of the diatom Thalassiosira rotula (THA) or the dinoflagellate Prorocentrum minimum (PRO), as well as mixtures of THA and PRO (MIX experiments) and alternating diets of THA and PRO switched daily (SWITCH experiments). Adult females ate both THA and PRO, and while rates of feeding on the 2 diets were similar in terms of carbon ingestion, egg production was generally higher on the diatom diet. In contrast, copepod egg-hatching success was low on the diatom diet, declining rapidly after 2 d from > 80 to 0% by Day 17. The diminution in hatching success was slower when females were fed MIX or SWITCH diets, but nonetheless diminished to 0 and < 25% by the end of the experiment, depending on the incubation method. Only in the case of the PRO diet was egg viability high and stable with time (87 to 96%), regardless of whether female and male couples were incubated as individual couples in crystallizing dishes or as triplicate couples in rotating bottles. However, in most other cases, the incubation method (crystallizing dishes vs rotating bottles) had very strong effects on egg and fecal pellet production, and hatching success. Higher egg production rates were generally obtained when females were incubated in crystallizing dishes, whatever the diet, although fecal pellet, production rates were significantly higher in the rotating bottle experiments in most cases. Egg-hatching success was also strongly affected by incubation method, with generally higher hatching rates in the rotating bottles. This was probably due to the fragility of non-viable eggs, which were more easily destroyed by mechanical disturbance in rotating bottle experiments. The results support the recent discovery that reproductive failure in copepods can be due to deleterious antimitotic compounds present in some diatoms that arrest normal embryonic division. Reduction in egg viability was not only visible when females were fed unialgal diatom diets, but also when they were fed mixed diets. However, on mixed diets there was a 'dilution effect' in that hatching was reduced by approximately half, and this took about twice as long to occur. The evolutionary advantages for diatoms in producing antimitotic compounds are discussed, as well as questions of why copepods feed on diatoms with impunity, even though some diatoms are detrimental to copepod reproductive success.
The productive regions of the ocean are characterized by seasonal blooms of phytoplankton which are generally dominated by diatoms. This algal class has, therefore, traditionally been regarded as providing the bulk of the food that sustains the marine food chain to top consumers and important fisheries. However, this beneficial role has recently been questioned on the basis of laboratory studies showing that although dominant zooplankton grazers such as copepods feed extensively on diatoms, the hatching success of eggs thus produced is seriously impaired. Here we present evidence from the field showing that the hatching success of wild copepods feeding on a diatom-dominated bloom is also heavily compromised, with only 12% of the eggs hatching compared with 90% in post-bloom conditions. We report on the structure of the three aldehydes isolated from diatoms that are responsible for this biological activity, and show that these compounds arrest embryonic development in copepod and sea urchin bioassays and have antiproliferative and apoptotic effects on human carcinoma cells.
Abnormal embryonic development is described for the copepod Calanus helgolandicus. Anomalies followed ingestion of diatoms by females which led to erroneous synchronization between nuclear division and cytokinesis, with malformation of the cellular membrane between daughter cells during mitosis. Similar types of cellular anomalies and morphological deformities, reported for the first time in the embryonic and naupliar stages of C. helgolandicus, were observed both in the laboratory and in situ. The ecological importance of this process as a factor controlling mortality and recruitment of copepods in the ocean is discussed.
Traditionally, diatoms have been associated w ~ t h productive pelagic food chains that lead, through suspension-feeding planktonic copepods, to top consumers and important fisheries Here, 15 laborator~es located ivorldw~de in 12 d~ffer-ent countries and representing a variety of marine, estuarine and freshwater environments present strong evidence that diatom diets are in fact inferior for copepod reproduction When fed to females of 16 copepod species, all but 1 of the 17 diatoms e x a m ~ n e d significantly reduced egg production rates or egg viability compared to non-diatom controls These effects are hypothesized to influence copepod recruitment patterns and the flow of energy in marine food webs.
Clutch size and hatching success in the marine calanoid copepod Pseudocalanus newmani were examined using two diatom species, Chaetoceros gracilis and Phaeodactylum tricornutum, and two non-diatom species, Pavlova sp. and Heterocapsa triquetra, as food. Both clutch size and hatch ing success varied with food algae after the 3rd clutch. The clutch size was largest with C. gracilis, in termediate with H. triquetra and Pavlova sp., and smallest with Ph. tricornutum. Hatching success was significantly lower with the diatom diets than with the non-diatom ones. With a mixed diet of C. gracilis and Pavlova sp., the clutch size was similar to that with C. gracilis or Pavlova sp. alone, while the hatching success rate was slightly lower than that with Pavlova sp. but higher than that with C. gracilis. Eggs exposed to high concentrations of a C. gracilis extract at 7X107cells ml"1 failed to hatch, but those at the same concentration of Pavlova sp. extract successfully hatched at more than 89%. These results suggest the presence of an anti-mitotic substance in these diatoms. The low hatching success on diatom diets might be due to the presence of as yet unidentified embryogenesis-inhibitory-compounds. Furthermore, both the diatom diets included a less amount of 18:3g;3, 18:4g)3 and 22:6co3 (DHA) fatty acids than the two non-diatom ones. The clutch size was related to neither the inhibitory compounds nor any fatty acids. Probably, nutritional components other than fatty acids in the diets play an important role in determining clutch size. Additionally, feeding the mixed diets of diatom and non-diatom species is considered to reduce the diatom effect on the cope-pods through lowering the amount of diatoms ingested, as P. newmani utilized equally diatom and non-diatom species in its diet.
Test compounds including natural hormones, endocrine disrupters, environmentally occurringcompounds, and reference compounds were tested for acute toxicity and inhibitory effect on larval development in the copepod Acartia tonsa. Three compounds, 17α-ethinylestradiol, p-octylphenol, and tamoxifen, known for their differing effects on the vertebrate estrogen system, were potent inhibitors of naupliar development. Other estrogens, 17β-estradiol, estrone, and bisphenol A, had little potency. Testosterone and progesterone did not inhibit development, but the antiandrogen flutamide had inhibitory effect. Juvenile hormone III was a potent inhibitor, as was expected based on the literature, whereas 20-hydroxyecdysone had no effect. 3,4-Dichloroaniline was inhibitory on development, whereas other control compounds, potassium dichromate and 3,5-dichlorophenol, did not inhibit development. Six of the 17 test compounds had 50% lethal concentration to 50% effective concentration (EC50) ratios higher than 10. The results suggest that naupliar development, as a parameter, is able to detect hormonal disrupters in addition to other chemicals that have other specific modes of action.
The effect of the chemical composition of food on the reproductive success of the copepods Acartia tonsa Dana and A. hudsonica Pinhey was studied in the laboratory. Laboratory-reared individuals were fed one of three monoalgal diets at different stages of growth: the diatom Thalassiosira weissflogii, the flagellate Rhodomonas lens and the dinoflagellte Prorocentrum minimum. The diet was analyzed for carbon, nitrogen, protein, carbohydrate and fatty acid content. Reproductive success was measured as eggs female-1 day-1 (E
r) and as the hatching success of the eggs. The E
r of Acartia spp. was correlated with protein and specific fatty acids [16:17 (negative), 20:53, 22:63, and 18:0 (positive)] and, especially, the fatty acid composition of the algae expressed as the 3:6 and 20:22 fatty acid ratios. The youngest diatom cultures and exponentially-growing flagellates displayed the highest E
r; the lowest E
r was recorded for females fed the senescent diatom cultures. The development time of eggs was affected by the age of the phytoplankton culture fed to the female. Hatching success of eggs decreased with the age of the algal culture, but no correlation was found with the meansured chemical components of the food.
Oogenesis and gonad development ofCalamus finmarchicus during the winter spring transition in a fjord in Northern Norway were studied. A combination of observations on whole animals
(macroscopic), taking advantage of their transparency, and histological sections was used to establish a system of gonad maturation
stages, to describe the spawning cycle and to estimate egg production rates from preserved samples. During their development,
oocytes change their shape and size, the morphology of the nucleus, and the appearance of the ooplasm. Four oocyte development
stages were identified and related to a macroscopic system of four gonad developmental stages (GS) that can be applied to
whole stained animals. During gonad development, the macroscopic morphological modifications of the gonads are mainly manifested
in an increase in number and size of the oocytes and their distribution in diverticula and oviducts. The maturation processes
during the spawning cycle (the period between egg depositions) were described from histological sections of females preserved
at regular intervals after egg deposition. The macroscopic GS did not vary during the spawning cycle. This GS was a highly
significant indicator for females, which spawned within 24 h after collection at 5°C. Another histological indicator for the
last third of the spawning cycle is the appearance of distinct chromosomes, which, however, are only seen in histological
sections. For the prediction of egg production rate of aCalanus population, in addition to the number of females spawning, knowledge of clutch size and the interval of clutch deposition
is required. Clutch size was determined by counting the number of the oocyte stages to be released during the next spawning
event in serial sections of the gonads. There was no significant difference in clutches laid by the females during 5 days
before preservation. From the observations it seems that GS 4 predicts females that are ready to spawn within the minimum
spawning interval characterisitic for a given temperature. If this holds true, it should be possible to predict in situ egg
production rates solely from preserved samples.
Dinoflagellates, which comprise an important part of the phytoplankton in the neritic region of the Southern California Bight, are known to be grazed byCalanus pacificus; rates of ingestion, development and survival of nauplii are influenced by the food quality of dinoflagellates. We have examined the effects of dinoflagellate food quality on reproduction ofC. pacificus females sampled in La Jolly Bay (32N; 117W) between 6 April and 4 May 1988. Four sets of experiments were conducted in which copepods were fed five different species:Gymnodinium splendens, Gyrodinium resplendens., G. dorsum, Exuviella mariae lebourae andGymnodinium simplex. These species were selected on the criterion of their success in supporting the growth of nauplii in previous rearing experiments. The experimental culture concentration was maintained at 100g C 1–1, near that of the natural seston. As indices of successful reproduction, we measured the percentage of spawning females and the number of eggs laid per female per day. Laboratory results were compared to stages of ovarian maturation of wild females. Four stages of maturation were determined using histological and microscopic observations.Gymnodinium splendens, Exuviella mariae lebourae, andG. simplex yielded clearly inferior results: few females were able to lay eggs repetitively over the first few days of confinement.Gyrodinium resplendens and, above all,G. dorsum yielded the best results. The stage of sexual maturation in situ appears to influence the rhythmicity of egg-laying events. In the presence of high-quality food, females are able to maintain high reproductive rates by sustaining the maturation of successive batches of eggs; good food quality appears to assure this secondary vitellogenesis and the rapid turnover of ovocytes.
The main reproductive period of herbivorous Antarctic copepods is coupled to the phytoplankton bloom in spring, while omnivorous or carnivorous species apparently reproduce year round. However, our knowledge of the reproduction during autumn and winter is limited. Therefore this study during a cruise with RV "Polarstern" aimed to measure reproductive activity in two dominant copepod species, Metridia gerlachei and Ctenocalanus citer, in late autumn (April/May). For this purpose, gonad development stages were determined from both species, taken with Bongo net in the eastern Bellingshausen Sea (Antarctic Peninsula region), and related to the ambient feeding conditions represented as chlorophyll a concentration. In addition, experiments were conducted with M. gerlachei to study the influence of feeding and starvation on gonad development. Gonad maturity of M. gerlachei differed considerably between stations and decreased over time. The correlation of chlorophyll a and the proportion of mature females, though significant, was weak and highly variable. In experiments, the gonads of feeding females were in better condition than those of their starving counterparts, showing that reproductive activity in M. gerlachei was related to ambient feeding conditions. Mature females of C. citer were found at each station (17-67%). The gonad stage composition was rather similar at all stations, showing no clear temporal and spatial trend. In this species, the proportion of mature females was not related to the chlorophyll a concentration, indicating either that the ambient phytoplankton stock was sufficient to fuel maturation or that other food sources were used. Our study shows that both species are reproducing during the austral autumn, indicating that their reproduction is partially decoupled from the spring phytoplankton bloom.
Egg production and hatching success of the copepod Temora longicornis were measured in laboratory experiments and in the field (North Sea). In the laboratory, ingestion of four algal species (Thalassiosira weissflogii, Phaeocystis globosa, Isochrysis sp. and Dunaliella tertiolecta) was followed and the content of fatty acids in the algae was determined. The two food types (T. weissflogii and Isochrysis sp.) that provided the highest ingestion of carbon and long chain fatty acids also resulted in the highest egg production rate (E
r) and hatching success (H%). In contrast, D. tertiolecta led to both low ingestion of carbon and long chain fatty acids, resulting in low reproductive success. There was a positive relationship between the amount of eicosapentaenoic fatty acid [20:5(n-3), EPA] ingested and E
r and H%, and of the ratio between docosahexaenoic and eicosapentaenoic fatty acid [22:6(n-3)/20:5(n-3), DHA/EPA] in the ingested food and H%. In the field, chlorophyll a and specific fatty acids were measured and protists were enumerated, in order to investigate the link between these factors and the reproductive success of T. longicornis. Hatching time was found to be related to temperature and exceeded 120 h at 6°C. No relationship was found between chlorophyll a and reproductive success (E
r or H%). E
r correlated with the concentration of diatoms and ciliates, which were the dominating protists in early spring, indicating that food quantity was the limiting factor for E
r. As in the laboratory experiments, H% was dependent on the fatty acid DHA and the ratio of DHA/EPA, which indicates that the quality of eggs (H%) is linked to the quality of food.
Vital fluorescent probes have routinely been used to distinguish viable from non-viable embryos in various veterinary and aquaculture studies. Here, we present new protocols to rapidly detect embryo viability in the copepod Calanus helgolandicus using three of these probes, fluorescein diacetate (FDA), SYTOX green and 7-aminoactinomycinD (7-AAD), and the confocal laser scanning microscope. The percentage of fluorescent-FDA embryos and non-fluorescent SYTOX green and 7-AAD embryos were compared with the percentage of hatched unstained embryos and with the percentage of embryos that had been stained, washed, and allowed to hatch. Results showed that all three dyes accurately predicted embryo viability and could be used to rapidly calculate C. helgolandicus egg-hatching success. We also tested the possible applications of SYTOX green in egg-production/egg-hatching assays in which the dinoflagellate Prorocentrum minimum or the diatom Skeletonema costatum are used to investigate for the possible negative impact of diatoms on embryo viability. Other possible applications for fluorescence methods in studies on the reproductive biology of zooplankton, and in particular of copepods, are discussed.
In recent years a negative influence of diatom-derived ,,,-unsaturated aldehydes (PUA) on the reproductive success of copepods and invertebrates has been suggested. Since adverse chemical properties of diatoms would question the traditional view of the marine food web, this defense mechanism has been investigated in detail, but the PUA-release by test organisms has only been determined in a few cases. The observed effects were nevertheless frequently discussed from a general point of view often leading to contradictory conclusions. We have examined the PUA-production of 50 diatom species (71 isolates) in order to provide a basis for the interpretation of laboratory and field results on the influence of diatom food on the reproductive success of their consumers. PUA-production is species and strain dependent. Thirty-six percent of the investigated species (38% of the cultivated isolates) release ,,,-unsaturated aldehydes upon cell disruption in concentrations from 0.01 to 9.8fmol per cell. Thalassiosira rotula and Thalassiosira pacifica, major spring-bloom forming diatoms isolated from Roscoff (Bretagne, English Channel, France) and Puget Sound (Washington, USA) were among the PUA-producing strains.
The effects of several food items on larvae production and survival of the mysid Mysidopsis almyra were compared. A total of six diets were used. The diets were: 1) phytoplankton (Isochrysis galbana), 2) an artificial diet (Liqualife®, Cargill, Minneapolis, MN), 3) a mixed diet composed of both zooplankton (mostly copepods) and phytoplankton, 4) 750 mg g-1 of HUFA enriched Artemia nauplii and 250 mg g-1 of the artificial diet, 5) newly hatched Artemia nauplii (24-hour incubation at 28 °C) and 6) newly hatched Artemia nauplii enriched with HUFA (SELCO®, INVE Inc., Ghent, Belgium) for 12 hours. Mysids fed HUFA enriched Artemia nauplii (diet 6) had the highest production and survival rates, although not significant (P > 0.05), compared to diets 3, 4 and 5, while the phytoplankton and the artificial diet had significantly lower production and survival rates (p > 0.05).
Cholesterol is required for animal growth, yet it cannot be biosynthesized de novo by crustaceans. Dietary sterols are thus necessary for the growth and reproduction of copepods. Sterols vary widely in both composition and concentration in phytoplankton. To explore the potential for sterol limitation of copepod growth, I did experiments with adult copepods feeding on diatoms with and without cholesterol supplementation. Cholesterol was added to the diet by (1) homogenizing the insoluble cholesterol with an ultrasonic disintegrator or (2) preparing gelatin/ acacia microcapsules with and without cholesterol using triolein as a carrier. Egg-production rates were measured after 2-4 d on the experimental diets, except in one experiment, in which an 18-d feeding period was used. Egg- production rates increased 1.5-2-fold for Acartia hudsonica, Acartia tonsa, and Calanus finamrchicuswhen the diatom Thalassiosira weissflogii was supplemented with 5-20 m gL 21 cholesterol; supplementation at 1 m gL 21 had no effect. Nearly three-fold higher rates of egg production were observed with C. finmarchicusafter feeding for 18 d on the supplemented diet. Cholesterol had a positive effect on egg-production rates when added by either method. Egg hatch rates for A. hudsonica were also higher when they were fed T. weissflogii supplemented with cholesterol (91% hatched) than when they were fed an unsupplemented diet (40% hatched). Egg production of Centropages hamatus feeding on T. weissflogii was unaffected by supplementation. Egg-production rates of A. hudsonica were unaffected when Chaetoceros affinis, Phaeodactylum tricornutum, or Thalassiosira rotulawere supplemented. High- er egg-production rates in supplemented treatments were caused by a decrease in the frequency of small clutches during spawning events rather than an increase in maximum clutch size. These experiments demonstrate the potential for limitation of egg production by dietary sterols when copepods feed exclusively on diatoms.
Conflicting hypotheses have been formulated regarding the origin of wood mouse (Apodemus sylvaticus) populations in northern Africa. In this study, the mtDNA restriction patterns of mice (n = 28) collected in Tunisia and Morocco are compared with those of representatives from southern Europe (n = 102). The neighbour-joining tree confirms the existence of the three lineages previously found in the Mediterranean area: western, Tyrrhenian-Balkan, and Sicilian. The western group is isolated from the two others, with bootstrap values of 89 and 95%. Northern African patterns are included in the western group. Their variability is low, the same pattern being shared by five Tunisian and all Moroccan animals (n = 18), caught either in the north of the country (Cap Spartel) or in the south (Marrakech). This implies that northern African wood mouse populations have a southwestern European origin and that their presence in the region is probably recent, which corresponds to both paleontological data and the hypothesis of anthropogenic introduction.
Gonad morphology and maturation of the dominant Arctic copepod species Calanus glacialis and C. hyperboreus were studied using a combination of observations on whole animals (“whole-body analysis”) and histological sections to describe basic reproductive processes. The comparison with C. finmarchicus shows that the internal organisation of the gonads is the same in the Calanus species: from the dorsally laying ovary, two anterior diverticula extent into the head region and two posterior diverticula extend into the abdomen. In the diverticula, the oocytes increase in size and development stage in a dorso–ventral direction. There were no physiological differences between the oocytes in the diverticula and oviducts in any of the three species. During gonad development, the morphological modifications of the gonads mainly refer to an increase in number, developmental stage and size of the oocytes both in diverticula and oviducts. During spawning the most ventral layer of oocytes is released, and then the adjacent dorsal layer begins final maturation. Thus, the final maturation processes occur simultaneously as it is essential to produce clutches.
In Hemidiaptomus ingens, fertilization occurs at the time of oocyte spawning in the ovisac. Fertilization induces the reactivation of the oocytes, arrested in meiotic prophase I, and above all, the development of two successive membrane envelopes arising before the formation of the eggshells. At once, within 25 min after spawning, a first cortical reaction induces the development of the fertilization envelope. This envelope is composed of a thin amorphous coat delaminated from the plasma membrane and of a material exocytosized from cortical vesicles which coalesces with the coat. This process results in the formation of a perivitelline space between the fertilization envelope and the plasma membrane. Subsequently, a new envelope appears from 25 to 60–90 min after spawning according to the same process, i.e., the association of a coat arising from a second delamination of the plasma membrane and of a material issued from the exocytosis of another type of cortical vesicles. This second envelope divides the perivitelline space into two compartments, an outer compartment or primary shell space, and an inner one or secondary shell space. An outer multi-layered shell begins to develop in the inner compartment within 1 h after spawning from exogenous material which is presumably produced by specialized shell glands located in the last segment of the prosome. This shell reaches its maximal thickness 48 h after spawning. Finally, when the outer shell is completed, an inner shell arises from embryos.
Estimates of production rate and viability of eggs are reported for Remora styIzj2ra collected in the Bay of Naples (Italy). Complementary observations on rates of fecal pellet and spcrmatophore production and female longevity were also conducted during the incubations. In situ egg viability was highly variable during the 2 yr of observation, with mean monthly values of ~80% of total egg production. Under experimental food-saturated conditions, the nature and hatching success of eggs were modified depending on type of food. With a dinoflagellate diet (Prorocentrum minimum), the responses of copepods were characterized by maximum production of viable eggs, high female longevity in the presence or absence of males, and low rates of fecal pellet production. The opposite was true with a diatom diet (Thalassiosira rot&z), except fecal pellets were produced at a higher rate, showing indirectly that this algae was neither properly assimilated nor transformed into production. Total and mean daily egg production were more or less the same with the two diets, indicating that under poorer food conditions copepods can maximize total egg production but not viability.
Within a cohort of young copepods, the individual size or growth rate becomes increasingly diversified during the development. The longer the development, the greater the resulting anatomical or functional diversity, since the differences between fast growing and slow growing individuals increase with time. Different factors may contribute to this variability, such as genetic diversity and metabolic differences in the ability to utilize food resources.
Yolk formation in the oocytes of the free-living, marine copepod, Labidocera aestiva (order Calanoida) involves both autosynthetic and heterosynthetic processes. Three morphologically distinct forms of endogenous yolk are produced in the early vitellogenic stages. Type 1 yolk spheres are formed by the accumulation and fusion of dense granules within vesicular and lamellar cisternae of endoplasmic reticulum. A granular form of type 1 yolk, in which the dense granules within the cisternae of endoplasmic reticulum do not fuse, appears to be synthesized by the combined activity of endoplasmic reticulum and Golgi complexes. Type 2 yolk bodies subsequently appear in the ooplasm but their formation could not be attributed to any particular oocytic organelle.
In the advanced stages of vitellogenesis, a single narrow layer of follicle cells becomes more developed and forms extensive interdigitations with the oocytes. Extra-oocytic yolk precursors appear to pass from the hemolymph into the follicle cells and subsequently into the oocytes via micropinocytosis. Pinocytotic vesicles fuse in the cortical ooplasm to form heterosynthetically derived type 3 yolk bodies.
In the present work, we questioned the natural or artifactual origin of the large amounts of free fatty acids recovered in lipids from the diatom Skeletonema costatum. Using rapid cell harvest and a very drastic method to extract the lipids, we demonstrated that these lipid patterns rich in free fatty acids were relevant to the degradation of complex lipids. A revised lipid composition of S. costatum is given which differs from those previously described by the absence of free fatty acids, while the proportion of polar lipids is substantially increased. Membrane lipids are mostly represented and storage lipids are less abundant, since we analysed the cells during their exponential growth phase. However, the fatty acid composition is in agreement with previous data. Polyunsaturated fatty acids (PUFA) consist essentially of 20:5 ω3, 16:3 ω4 and 16:4 ω1, while 16:1 ω7 is the main monousaturated FA. This confirms that, with regard to its lipid composition, the diatom, S. costatum, provides a good diet for molluscs, particularly through its high proportion of ω3 PUFA. Using this extraction procedure, sterols were found to be identical to previous results but a new compound, a 18:11 fatty alcohol, was detected. The consequences of this reassessement on the lipid compositions of diatoms so far published and their application to the lipid diet of mollusc larvae in aquaculture are discussed.
The effects of several food items on larvae production and survival ofthe mysid Mysidopsis almyra were compared. A total of sixdiets were used. The diets were: 1) phytoplankton (Isochrysisgalbana), 2) an artificial diet (Liqualife, Cargill,Minneapolis, MN), 3) a mixed diet composed of both zooplankton (mostlycopepods)and phytoplankton, 4) 750 mg g–1 of HUFA enrichedArtemia nauplii and 250 mgg–1 of the artificial diet, 5) newly hatchedArtemia nauplii (24-hour incubation at 28C) and 6) newly hatched Artemia naupliienriched with HUFA (SELCO, INVE Inc., Ghent, Belgium) for 12 hours. Mysidsfed HUFA enriched Artemia nauplii (diet 6) had the highestproduction and survival rates, although not significant (P > 0.05), comparedto diets 3, 4 and 5, while the phytoplankton and the artificial diet hadsignificantly lower production and survival rates (p > 0.05).
The Biological Station on Helgoland has one of the longest data series for phytoplankton species composition in the world. Since 1962, phytoplankton has been counted to a species level on a daily basis (weekdays). One of the main reasons why this data set has rarely been used is that it has never been subjected to proper quality control or inventoried properly. This paper describes the quality control which we have now carried out on this data set and also represents the first inventory of the data and its meta-information. A comparison of the electronic database where the data is archived and the original recorded lists from the different persons dealing with the samples was an integral part of the evaluation. Apart from recording inconsistencies in species identification, we also recorded and corrected the many differences found between the electronic and paper data. The evaluation was carried out in a direct comparative manner to discern taxonomic discrepancies, and in order to find random errors in the transfer of data from paper to computer random checks of the data were carried out. This paper serves to illustrate the level of quality control required when dealing with archived phytoplankton data and illustrates the typical problems encountered.
Two double-labelling methods, Tunel+propidium iodide and AnnexinV-FITC+propidium iodide, have been tested to diagnose cell degradation processes in N1–N2 nauplius stages produced by spawning females of the copepod Calanus helgolandicus fed either non-toxic (PRO: control) or toxic (TR1) diets under laboratory conditions. Observation of labelled samples with a confocal laser-scanning microscope revealed that the maternal-food effect, following absorption of the toxic diet, induced cell apoptosis and necrosis in 80–100% of the offspring, which sooner or later died. Similar cell damages were also observed in 18–50% of the nauplii produced by females incubated in filtered seawater. Such cell degradation processes and high mortality were not observed in nauplii produced by females fed the non-toxic diet. Our protocols allowed detection of cell degradations before death of unhealthy larvae, preferentially with a confocal microscope or a conventional visible-fluorescent-light microscope.
Feeding, egg production, hatching success and early naupliar development of Calanus finmarchicus were measured in three north Norwegian fjords during a spring bloom dominated by diatoms and the haptophyte Phaeocystis pouchetii. Majority of the copepod diet consisted of diatoms, mainly Thalassiosira spp. and Chaetoceros spp., with clearance rates up to 10mlind−1h−1 for individual algae species/groups. Egg production rates were high, ranging from ca 40 up to 90 eggsf−1d−1, with a hatching success of 70–85%, and fast naupliar development through the first non-feeding stages. There was no correlation
between the egg or nauplii production and diatom abundance, but the hatching success was slightly negatively correlated with
diatom biomass. However, the overall high reproductive rates suggested that the main food items were not harmful for C. finmarchicus reproduction in the area, although direct chemical measurements were not conducted. The high population egg production (>1,20,000
eggsm−2d−1) indicated that a large part of the annual reproduction took place during the investigation, which stresses the importance
of diatom-dominated spring phytoplankton bloom for population recruitment of C. finmarchicus in these northern ecosystems.
Gonad maturation processes were studied in Pseudocalanus spp. females from Georges Bank and Cape Cod Bay (northwest Atlantic) using a combination of morphological analysis and experiments. For light microscopy of the oocytes, females of different maturation stages were preserved immediately after capture. The maturation processes during the spawning cycle were described from observations of live females which were exposed to feeding and starvation at two temperatures, 8 and 15C, for 12days. The gonad morphology of these females was examined in 24h intervals, and spawning events were recorded. Both light microscopy and whole animal observation revealed that oocytes during maturation change in shape and size, in the morphology of the nucleus, and in the appearance of the ooplasm. Due to these modifications of oocyte morphology and due to oocyte migration, the morphology of the gonads changed distinctly during a spawning cycle. Five oocyte development stages were identified by light microscopy and related to a macroscopic system of four gonad development stages, that can be applied to whole animals and allows the identification of females ready to spawn. The experiment showed that food and temperature had strong effects on gonad maturation processes. High proportions of mature females were found when food was available, whereas the proportion of immature females increased shortly after exposure to starvation. Compared to 15C, gonad maturation at 8C was prolonged and thus spawning frequency was lower. The final maturation processes at food saturated conditions were slower than the embryonic development, and no indication was found that mature oocytes are stored in the diverticula waiting to be released. The duration of the interspawning interval would thus be determined by the duration of final oocyte maturation which is dependent on both temperature and food supply.
Calanus finmarchicus, one of the dominant copepods species of the North Atlantic, often encounters low food concentration or quality during the reproductive period; however, our knowledge on the effect of these conditions on reproduction processes is scarce. The present study combines experiments with histological observations to describe the response of C. finmarchicus to limited food focussing on (1) oocyte maturation processes, (2) gonad morphology, (3) egg production rates as a function of spawning frequency and clutch size, and (4) the fuelling of egg production by measuring carbon and nitrogen content of the females. In the laboratory, C. finmarchicus females were exposed to 0, 10, 50, 150 or >300 μg C l−1 for several days. To account for food quality and season, reproductive activity was compared in April and July 1999 between females feeding on diatoms or dinoflagellates. The effect of feeding history was studied in February with females fed and starving prior to the experiment. Feeding conditions had severe effects on oocyte maturation process. Hence, egg production varied significantly with food concentration and quality, season and feeding history due to variation in both clutch size and spawning frequency. Clutch size differed by a factor of 2–4 between food limited and well fed females, and is thus an important parameter for modelling egg production. Changes in clutch size were related to changes of the number of maturing oocytes in the females gonads indicating that the latter can be used to precise the prediction of egg production from preserved samples. The proportion of females carrying at least some mature oocytes was relatively high at low food availability. Apparently, these females used internal body reserves as the carbon and nitrogen content decreased significantly under these conditions. These results indicate that C. finmarchicus embarks on the strategy to enable reproduction in all or many females of a population at low rates when feeding conditions are unfavourable.
Failure of female reproductive capacity in the copepod Calanus helgolandicus was related to number and combination of the phytoplankton species in the diets. The maternal food effects were detectable at different levels: fecundity, oogenesis and hatching. Fecundity and hatching were normal with two single (ca. Isochrysis galbana and Prorocentrum minimum) and one mixed (Phaeodactylum tricornutum+Dunaliella tertiolecta+Pavlova lutherii+I. galbana+P. minimum) diets. With the single P. lutherii diet, fecundity decreased, but hatching remained optimal. The daily egg production and hatching rates decreased significantly in females fed the other single P. tricornutum, D. tertiolecta and mixed (P. tricornutum+D. tertiolecta+P. lutherii+I. galbana) diets, or starved. The fecundity decrease coincided with gonad atresia, which was reversible when P. tricornutum and P. lutherii diets were replaced by P. minimum diet. It was irreversible when D. tertiolecta was replaced by P. minimum, leading to female sterilization expressed by the deterioration of OS3 and OS2 oocytes, as a function of the feeding duration. We assume that atresia of female gonads was caused by the limitation of essential nutrients in food, such as fatty acids, which induced catabolism and recycling of yolk reserves and thus, maintenance of gonad integrity and low spawning rates. With the D. tertiolecta diet, abnormally high increase of ornithine concentrations in eggs showed that the ornithine metabolism and polyamine pathway were affected during oogenesis, leading atresia of oocytes to be deeply disturbed and followed up by necrosis of the gonads.
Copepod secondary production has traditionally been linked to the spring diatom bloom in temperate and high latitudes, but laboratory studies have recently challenged this view and have shown either reduced fecundity or viability of offspring when copepods were fed high concentrations of – mostly unialgal – diatoms. However, field evidence that diatoms affect copepod reproduction is still scarce. We analyzed the reproductive response of a common, small calanoid copepod of the boreal Pacific, Pseudocalanus newmani, to spring diatom blooms in Dabob Bay, a semi-enclosed fjord of Puget Sound, Washington, USA. Abundance patterns, egg production rates, egg hatching success, and naupliar viability of the egg-carrying copepod were examined between February and early May in the years 2002–2004. The population underwent strong variations in abundance during both years, with high abundance of all stages from February to mid-March, but dramatically decreasing individual numbers later in spring. A recovery to higher numbers occurred in July.
Four batches of Pecten maximus (L.) larvae were grown under hatchery conditions and fed the monospecific diets Pavlova lutheri Droop, Isochrysis aff. galbana Green (clone T-iso; termed Tahitian Isochrysis), Chaetoceros calcitrans Takano and Dunaliella tertiolecta Butcher which were selected on their very different polyunsaturated fatty acid (PUFA) profiles. Neutral and polar lipid PUFA compositions of larvae were influenced by diets. The constant level of (n-3) PUFA in neutral lipids of larvae although very different in the diet indicated that neutral lipid composition was partially controlled by the larval metabolism. Similarly, the stability of total saturated, monounsaturated and PUFA levels (25, 15 and 55% of total fatty acids), as well as total (n-3) PUFA level and the preferential incorporation of the 22:6(n-3) in polar lipids clearly indicated a metabolic regulation of the fatty acid composition. The 20:4(n-6) and 22:5(n-6) acids were also preferentially incorporated. The accumulation of 18:3(n-3) or 20:5(n-3) with depletion of 22:6(n-3) in neutral and polar lipids of larvae fed diets with high levels of the first two fatty acids but little or no 22:6(n-3), indicated that elongation-desaturation of dietary fatty acids was too low in P. maximus larvae to maintain high 22:6(n-3) levels in polar lipids. The selective incorporation of dietary long chain PUFA into neutral and polar lipids by acyltransferases may be the main mechanism controlling the fatty acid composition of larvae. These results indicate that P. maximus larvae have essential requirements for long chain (n-3) and (n-6) PUFA, similar to many other marine species.