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We report on a dense bloom (∼1.80 × 10 5 cells mL −1 ) of the marine dinoflagellate species Amphidinium carterae (Genotype 2) in a shallow, small intermittently open coastal lagoon in south eastern Australia. This bloom co-occurred with the deaths of >300 individuals of three different species of fish. The opening of the lagoon to the ocean, as well as localized high nutrient levels, preceded the observations of very high cell numbers. A. carterae is usually benthic and sediment-dwelling, but temporarily became abundant throughout the water column in this shallow (<2 m) sandy habitat. Histopathological results showed that the Anguilla reinhardtii individuals examined had damage to epithelial and gill epithelial cells. An analysis of the bloom water indicated the presence of a compound with a retention time and UV spectra similar to Luteophanol A, a compound known from a strain of Amphidinium . Assays with a fish gill cell line were conducted using a purified compound from cells concentrated from the bloom, and was found to cause a loss of 87% in cell viability in 6 h. The fish deaths were likely due to the low dissolved oxygen levels in the water and/or the presence of Luteophanol A-like compounds released during the bloom. [ABSTRACT FROM AUTHOR]

Ichthyotoxic Karlodinium veneficum has become a persistent problem in the eutrophic Swan River Estuary (SRE) near Perth, Western Australia. Karlotoxin (KmTx) concentrations and K. veneficum were sampled from March to July 2005, spanning a bloom confirmed by microscopy and genetics (ITS sequence), and a fish kill coincident with end of the bloom. The objective of this study was to investigate K. veneficum cell and toxin dynamics, and water quality conditions, leading up to the bloom and fish kill in this estuarine system. Abundance of K. veneficum increased as diatom abundance decreased over a 3-month period (Jan–Mar) preceding the bloom. Low freshwater flow to the SRE characterized the bloom initiation period, while elevated seasonal flows altered water quality and preceded the end of the bloom and fish kill. The bloom of K. veneficum was localized over a bottom layer of hypoxic water in a stratified water column. Low nitrate levels, DIN:DIP (mol) near unity, and particulate C:N:P of K. veneficum -rich water samples were consistent with nitrogen limitation of phytoplankton. A KmTx 2 congener was present in the concentration range 0–1052 ng KmTx mL −1 , levels that were sufficient to kill larval fish in the laboratory within 4 h. A KmTx cell quota of 2.8 pg KmTx cell −1 was estimated for the bloom, which is moderately high for the species. Gill histopathology of fish from this fish kill showed signs of damage similar to those caused by KmTx in the lab. Results from this study suggest that conditions in the SRE, including elevated K. veneficum abundance and KmTx cell quotas, as well as hypoxia in the upper SRE, likely contribute to seasonal fish kills observed in this system. [ABSTRACT FROM AUTHOR]

Juvenile sablefish were fed a low taurine, basal feed with seven graded levels of supplemental taurine to determine taurine requirements for growth and feed efficiency. The basal feed was plant based, formulated primarily with soy and corn proteins with a minimal (9%) amount of fishmeal. The unsupplemented, basal feed contained 0.14% taurine. Experimental feeds were supplemented with 0, 0.25, 0.50, 1.0, 1.5, 3.0, and 6.0% taurine. Using the five parameter, saturated kinetic model (5 SKM), peak weight gain was predicted at 1.5% dietary taurine. Optimum weight gain, as defined as the region of the 5 SKM curve corresponding to at least 95% of peak, was predicted between 0.4% and 5.8% dietary taurine. Peak feed efficiency was predicted at 1.1% dietary taurine with optimum weight gain predicted between 0.4% and 4.2%. Whole body and muscle tissue protein and lipid content were not affected by taurine supplementation. Tissue taurine content increased asymptotically with increasing dietary taurine supplementation. Whole body tissue became saturated at 0.25 ± 0.02% taurine, expressed on a wet weight basis. Muscle tissue became saturated at 0.34 ± 0.02% taurine. Results from this study should increase the performance of alternative, plant based feeds formulated for sablefish and enable regulatory agencies better estimate the potential human exposure to taurine from the consumption of sablefish receiving these feeds. [ABSTRACT FROM AUTHOR]

Abstract: This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7–40% from log phase growth to late log – early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20–31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60–90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with MTX as the primary hemolytic compound produced by Gambierdiscus species. While the results from inhibition studies require validation by LC–MS analysis, the available data strongly suggest differences in hemolytic activity observed in this study reflect maitotoxicity. [Copyright &y& Elsevier]

Increasing petroleum costs and climate change have resulted in microalgae receiving attention as potential biofuel producers. Little information is available on the diversity and functions of bacterial communities associated with biofuel-producing algae. A potential biofuel-producing microalgal strain, Nannochioropsis oceanica IMET1, was grown in Permian groundwater. Changes in the bacterial community structure at three temperatures were monitored by two culture-independent methods, and culturable bacteria were characterized. After 9 days of incubation, N. oceanica IMET1 began to aggregate and precipitate in cultures grown at 30°C, whereas cells remained uniformly distributed at 15°C and 25°C. The bacterial communities in cultures at 30°C changed markedly. Some bacteria isolated only at 30°C were tested for their potential for aggregating microalgae. A novel bacterium designated HW001 showed a remarkable ability to aggregate N. oceanica IMET1, causing microalgal cells to aggregate after 3 days of incubation, while the total lipid content of the microalgal cells was not affected. Direct interaction of HW001 and N. oceanica is necessary for aggregation. HW001 can also aggregate the microalgae N. oceanica CT-1, Tetraselmis suecica, and T. chuii as well as the cyanobacterium Synechococcus WH8007. 16S rRNA gene sequence comparisons indicated the great novelty of this strain, which exhibited only 89% sequence similarity with any previously cultured bacteria. Specific primers targeted to HW001 revealed that the Strain originated from the Permian groundwater. This study of the bacterial communities associated with potential biofuel-producing microalgae addresses a little-investigated area of microalgal biofuel research and provides a novel approach to harvest biofuel-producing microalgae by using the novel bacterium strain HW001. [ABSTRACT FROM AUTHOR]

Fogel and Hastings first hypothesized the existence of voltage-gated proton channels in 1972 in bioluminescent dinoflagellates, where they were thought to trigger the flash by activating luciferase. Proton channel genes were subsequently identified in human, mouse, and Ciona intestinalis, but their existence in dinoflagellates remained unconfirmed. We identified a candidate proton channel gene from a Karlodinium veneficum cDNA library based on homology with known proton channel genes. K. veneficum is a predatory, nonbioluminescent dinoflagellate that produces toxins responsible for fish kills worldwide. Patch clamp studies on the heterologously expressed gene confirm that it codes for a genuine voltage-gated proton channel, kHV1: it is proton-specific and activated by depolarization, its gH-V relationship shifts with changes in external or internal pH, and mutation of the selectivity filter (which we identify as Asp51) results in loss of proton-specific conduction. Indirect evidence suggests that kHV1 is monomeric, unlike other proton channels. Furthermore, kHV1 differs from all known proton channels in activating well negative to the Nernst potential for protons, EH. This unique voltage dependence makes the dinoflagellate proton channel ideally suited to mediate the proton influx postulated to trigger bioluminescence. In contrast to vertebrate proton channels, whose main function is acid extrusion, we propose that proton channels in dinoflagellates have fundamentally different functions of signaling and excitability. [ABSTRACT FROM AUTHOR]

We set out to determine whether probiotic addition would improve larval development in the false percula clownfish Amphiprion ocellaris and to determine what molecular responses could be observed in the larvae following probiotic exposure. We supplied the probiotic bacterial strain Lactobacillus rhamnosus IMC 501 to clownfish larvae from the first day posthatch simultaneously by live prey and with addition to rearing water (group 2) and exclusively by live prey (group 3). We observed twofold higher body weight in both clownfish larvae and juveniles when probiotics were supplied via live prey and added to the rearing water. In addition, development was accelerated with metamorphosis occurring 3 days earlier in fingerlings treated with probiotic. Alteration in molecular biomarkers supported the faster growth observation. There was significantly increased gene expression of factors involved in growth and development (insulin-like growth factors I and II, myostatin, peroxisome proliferator-activated receptors α and β, vitamin D receptor a, and retinoic acid receptor γ) when probiotics were delivered via live prey and added to the rearing water. Moreover, probiotic treatment lessened the severity of the general stress response as exhibited by lower levels of glucocorticoid receptor and 70-kDa heat shock protein gene expression. Furthermore, an improvement of skeletal head development was observed, with a 10-20% reduction in deformities for juveniles treated with probiotic. All data suggest a potent effect on development resulting from the administration of lactic acid bacteria to larval clownfish, and this study provides a preliminary molecular entry path into the investigation of mechanisms responsible for probiotic enhancement in fish development. [ABSTRACT FROM AUTHOR]

Abstract: Reduced fishery harvests and increased consumer demand for seafood have precipitated an increase in intensive fish farming, predominantly in coastal and open ocean net-pens. However, as currently practiced, aquaculture is widely viewed as detrimental to the environment and typical operations are vulnerable to environmental influences, including pollution and endemic diseases. Here we report the development of a land-based, marine recirculating aquaculture system that is fully contained, with virtually no environmental impact as a result of highly efficient biological waste treatment and water recycling. Over 99% of the water volume was recycled daily by integrating aerobic nitrification to eliminate toxic ammonia and, for the first time, simultaneous, anaerobic denitrification and anaerobic ammonium oxidation, to convert ammonia and nitrate to nitrogen gas. Hydrogen sulfide generated by the separated endogenous organic solids was used as an electron source for nitrate reduction via autotrophic denitrification and the remaining organic solids were converted to methane and carbon dioxide. System viability was validated by growing gilthead seabream (Sparus aurata) from 61 g to 412 g for a total of 1.7 tons in a record 131 days with 99% fish survival. Ammonia nitrite and nitrate did not exceed an average daily concentration of 0.8 mg/l, 0.2 mg/l and 150 mg/l, respectively. Food conversion values were 16% lower than recorded levels for net-pen aquaculture and saltwater usage of less than 16 l/every kg of fish produced. The system is site-independent, biosecure, devoid of environmental contaminants and is not restricted to a single species. [Copyright &y& Elsevier]

Abstract: The parasitic dinoflagellate Amoebophrya sp. ex Karlodinium veneficum was used to test two hypotheses: (1) infection of cells decreases with increasing host toxicity and (2) parasitism causes the catabolism of host toxin. To test the first hypothesis, host strains differing in toxin content were inoculated with dinospores of Amoebophrya sp. derived from infected cultures of toxic and non-toxic K. veneficum, with resulting infections assessed following 24-h incubations. Contrary to expectations, infection of K. veneficum by Amoebophrya sp. was positively correlated with host toxicity. To examine the second hypothesis, synchronous infection with >80% of cells being parasitized was induced using a toxic strain of K. veneficum, and total toxin concentration (intracellular plus extracellular levels of KmTX1) was followed over the 3-day infection cycle. Toxin content ml−1 increased with growth of K. veneficum in uninfected control cultures, but declined in infected cultures as the parasite completed its life cycle. On a cellular basis, toxin content of infected and uninfected cultures differed little during the experiment, suggesting that the parasite does not actively catabolise host toxin. Rather, infection appears to promote degradation of toxins via death of host cells and subsequent bacterial activity. Results indicate that Amoebophrya sp. ex K. veneficum has greater potential to impact toxic strains relative to non-toxic host strains in natural systems. Thus, Amoebophrya sp. ex. K. veneficum may limit the occurrence of toxic K. veneficum blooms in marine and estuarine environments, while simultaneously functioning as a pathway for dissipation of host toxin. [Copyright &y& Elsevier]

• Spatiotemporal distribution of marine and freshwater phycotoxins. • Eight phycotoxins were detected, with high prevalence of co-occurrence. • First report of azaspiracids on the east coast of the U.S.; producer is unknown. • Ubiquitous spatiotemporal distribution of okadaic acid and dinophysistoxin-1. • Broad spatial distribution of domoic acid, but only low levels detected. Harmful algal blooms (HABs), varying in intensity and causative species, have historically occurred throughout the Chesapeake Bay, U.S.; however, phycotoxin data are sparse. The spatiotemporal distribution of phycotoxins was investigated using solid-phase adsorption toxin tracking (SPATT) across 12 shallow, nearshore sites within the lower Chesapeake Bay and Virginia's coastal bays over one year (2017-2018). Eight toxins, azaspiracid-1 (AZA1), azaspiracid-2 (AZA2), microcystin-LR (MC-LR), domoic acid (DA), okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), and goniodomin A (GDA) were detected in SPATT extracts. Temporally, phycotoxins were always present in the region, with at least one phycotoxin group (i.e., consisting of OA and DTX1) detected at every time point. Co-occurrence of phycotoxins was also common; two or more toxin groups were observed in 76% of the samples analyzed. Toxin maximums: 0.03 ng AZA2/g resin/day, 0.25 ng DA/g resin/day, 15 ng DTX1/g resin/day, 61 ng OA/g resin/day, 72 ng PTX2/g resin/day, and 102,050 ng GDA/g resin/day were seasonal, with peaks occurring in summer and fall. Spatially, the southern tributary and coastal bay regions harbored the highest amount of total phycotoxins on SPATT over the year, and the former contained the greatest diversity of phycotoxins. The novel detection of AZAs in the region, before a causative species has been identified, supports the use of SPATT as an explorative tool in respect to emerging threats. The lack of karlotoxin in SPATT extracts, but detection of Karlodinium veneficum by microscopy, however, emphasizes that this tool should be considered complementary to, but not a replacement for, more traditional HAB management and monitoring methods. [ABSTRACT FROM AUTHOR]

In 1957 Abbott and Ballantine described a highly toxic activity from a dinoflagellate isolated from the English Channel in 1949 by Mary Park. From a culture maintained at Plymouth Laboratory since 1950, we have been able to isolate two toxic molecules (abbotoxin and 59-E-Chloro-abbotoxin), determine the planar structures by analysis of HRMS and 1D and 2D NMR spectra, and found them to be karlotoxin (KmTx) congeners. Both toxins kill larval zebrafish with symptoms identical to those described by Abbot and Ballantine for gobies (Gobius virescens). Using surface plasma resonance the sterol binding specificity of karlotoxins is shown to require desmethyl sterols. Our results with black lipid membranes indicate that karlotoxin forms large-conductance channels in the lipid membrane, which are characterized by large ionic conductance, poor ionic selectivity, and a complex gating behavior that exhibits strong voltage dependence and multiple gating patterns. In addition, we show that KmTx 2 pore formation is a highly targeted mechanism involving sterol-specificity. This is the first report of the functional properties of the membrane pores formed by karlotoxins and is consistent with the initial observations of Abbott and Ballantine from 1957., (© 2024. The Author(s).)

Dinoflagellates are one of the largest groups of marine microalgae and exhibit diverse trophic strategies. Some dinoflagellates can produce secondary metabolites that are known to be toxic, which can lead to ecologically harmful blooms. Amphidinium carterae is one species of dinoflagellate that produces toxic compounds and is used as a model for dinoflagellate studies. The impact of the microbiome on A. carterae growth and metabolite synthesis is not yet fully understood, nor is the impact of bacterial data on sequencing and assembly. An antibiotic cocktail was previously shown to eliminate 16S amplification from the dinoflagellate culture. Even with drastically reduced bacterial numbers during antibiotic treatment, bacterial sequences were still present. In this experiment, we used novel Nanopore long-read sequencing techniques on A. carterae cultures to assemble 15 full bacterial genomes ranging from 2.9 to 6.0 Mb and found that the use of antibiotics decreased the percentage of reads mapping back to bacteria. We also identified shifts in the microbiome composition and identified a potentially deleterious bacterial species arising in the absence of the antibiotic treatment. Multiple antibiotic resistance genes were identified, as well as evidence that the bacterial population does not contribute to toxic secondary metabolite synthesis.

The toxic dinoflagellate Karlodinium veneficum forms fish killing blooms in temperate estuaries worldwide. These blooms have variable toxicity which may be related to bloom stage and in situ growth rates of the constituent K. veneficum cells. Measurement of in situ growth rates is challenging and methods such as the mitotic index technique require knowledge of the dynamics of cell division. In order to better understand these dynamics, we determined the duration of cell division (t d ) in four geographically distinct laboratory strains of K. veneficum at three different environmentally relevant temperatures. The results demonstrated that the t d value for each strain, growing at strain-specific optimal temperatures, was 1.6 ± 0.1 h. This value corresponded to a range of growth rates from 0.17 ± 0.08 d -1 to 0.62 ± 0.07 d -1 . Equivalent values of t d spread across four geographically distinct laboratory strains and a nearly fourfold range of growth rates implies that 1.6 h represents the t d value of K. veneficum. Additionally, temperature conditions yielding this value for t d and the highest growth rates varied among strains, indicating cold-adapted (Norway), warm-adapted (Florida, USA), and eurythermally-adapted (Maryland, USA) strains. These differences have been apparently retained in culture over many years, indicating a conserved genetic basis that suggests distinct thermal ecotypes of the morphospecies K. veneficum. This knowledge together with the first estimate of t d for K. veneficum will be useful in future field studies aimed at correlating bloom toxicity with in situ growth rate using the mitotic index technique., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Dinoflagellates are unicellular organisms that are implicated in harmful algal blooms (HABs) caused by potent toxins that are produced through polyketide synthase (PKS) pathways. However, the exact mechanisms of toxin synthesis are unknown due to a lack of genomic segregation of fat, toxins, and other PKS-based pathways. To better understand the underlying mechanisms, the actions and expression of the PKS proteins were investigated using the toxic dinoflagellate Amphidinium carterae as a model. Cerulenin, a known ketosynthase inhibitor, was shown to reduce acetate incorporation into all fat classes with the toxins amphidinol and sulpho-amphidinol. The mass spectrometry analysis of cerulenin-reacted synthetic peptides derived from ketosynthase domains of A. carterae multimodular PKS transcripts demonstrated a strong covalent bond that could be localized using collision-induced dissociation. One multi-modular PKS sequence present in all dinoflagellates surveyed to date was found to lack an AT domain in toxin-producing species, indicating trans -acting domains, and was shown by Western blotting to be post-transcriptionally processed. These results demonstrate how toxin synthesis in dinoflagellates can be differentiated from fat synthesis despite common underlying pathway.

Oysters ( Crassostrea virginica ) were screened for 12 phycotoxins over two years in nearshore waters to collect baseline phycotoxin data and to determine prevalence of phycotoxin co-occurrence in the commercially and ecologically-relevant species. Trace to low concentrations of azaspiracid-1 and -2 (AZA1, AZA2), domoic acid (DA), okadaic acid (OA), and dinophysistoxin-1 (DTX1) were detected, orders of magnitude below seafood safety action levels. Microcystins (MCs), MC-RR and MC-YR, were also found in oysters (maximum: 7.12 μg MC-RR/kg shellfish meat wet weight), warranting consideration of developing action levels for freshwater phycotoxins in marine shellfish. Oysters contained phycotoxins that impair shellfish health: karlotoxin1-1 and 1-3 (KmTx1-1, KmTx1-3), goniodomin A (GDA), and pectenotoxin-2 (PTX2). Co-occurrence of phycotoxins in oysters was common (54%, n = 81). AZAs and DA co-occurred most frequently of the phycotoxins investigated that are a concern for human health (n = 13) and PTX2 and KmTxs co-occurred most frequently amongst the phycotoxins of concern for shellfish health (n = 9). Various harmful algal bloom (HAB) monitoring methods and tools were assessed for their effectiveness at indicating levels of phycotoxins in oysters. These included co-deployed solid phase adsorption toxin tracking (SPATT) devices, toxin levels in particulate organic matter (POM, >1.5 μm) and whole water samples and cell concentrations from water samples as determined by microscopy and quantitative real-time PCR (qPCR). The dominant phycotoxin varied between SPATTs and all other phycotoxin sample types, and out of the 11 phycotoxins detected in oysters, only four and seven were detected in POM and whole water respectively, indicating phycotoxin profile mismatch between ecosystem compartments. Nevertheless, there were correlations between DA in oysters and whole water (simple linear regression [LR]: R 2  = 0.6, p < 0.0001, n = 40), and PTX2 in oysters and SPATTs (LR: R 2  = 0.3, p = 0.001, n = 36), providing additional monitoring tools for these phycotoxins, but oyster samples remain the best overall indicators of seafood safety., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Dinoflagellates are unicellular protists that display unusual nuclear features such as large genomes, condensed chromosomes and multiple gene copies organized as tandem gene arrays. Genetic regulation is believed to be controlled at the translational rather than transcriptional level. An important player in this process is initiation factor eIF4E which binds the 7-methylguanosine cap structure (m7G) at the 5'-end of mRNA. Transcriptome analysis of eleven dinoflagellate species has established that each species encodes between eight to fifteen eIF4E family members. Determining the role of eIF4E family members in gene expression requires a method of knocking down their expression. In other eukaryotes this can be accomplished using translational blocking morpholinos that bind to complementary strands of RNA, therefore inhibiting the mRNA processing. Previously, unmodified morpholinos lacked the ability to pass through cell membranes, however peptide-based reagents have been used to deliver substances into the cytosol of cells by an endocytosis-mediated process without damaging the cell membrane. We have successfully delivered fluorescently-tagged morpholinos to the cytosol of Amphidinium carterae by using a specific cell penetrating peptide with the goal to target an eIF4e-1a sequence to inhibit translation. Specific eIF4e knockdown success (up to 42%) has been characterized via microscopy and western blot analysis.

Many dinoflagellate species make toxins in a myriad of different molecular configurations but the underlying chemistry in all cases is presumably via modular synthases, primarily polyketide synthases. In many organisms modular synthases occur as discrete synthetic genes or domains within a gene that act in coordination thus forming a module that produces a particular fragment of a natural product. The modules usually occur in tandem as gene clusters with a syntenic arrangement that is often predictive of the resultant structure. Dinoflagellate genomes however are notoriously complex with individual genes present in many tandem repeats and very few synthetic modules occurring as gene clusters, unlike what has been seen in bacteria and fungi. However, modular synthesis in all organisms requires a free thiol group that acts as a carrier for sequential synthesis called a thiolation domain. We scanned 47 dinoflagellate transcriptomes for 23 modular synthase domain models and compared their abundance among 10 orders of dinoflagellates as well as their co-occurrence with thiolation domains. The total count of domain types was quite large with over thirty-thousand identified, 29 000 of which were in the core dinoflagellates. Although there were no specific trends in domain abundance associated with types of toxins, there were readily observable lineage specific differences. The Gymnodiniales, makers of long polyketide toxins such as brevetoxin and karlotoxin had a high relative abundance of thiolation domains as well as multiple thiolation domains within a single transcript. Orders such as the Gonyaulacales, makers of small polyketides such as spirolides, had fewer thiolation domains but a relative increase in the number of acyl transferases. Unique to the core dinoflagellates, however, were thiolation domains occurring alongside tetratricopeptide repeats that facilitate protein-protein interactions, especially hexa and hepta-repeats, that may explain the scaffolding required for synthetic complexes capable of making large toxins. Clustering analysis for each type of domain was also used to discern possible origins of duplication for the multitude of single domain transcripts. Single domain transcripts frequently clustered with synonymous domains from multi-domain transcripts such as the BurA and ZmaK like genes as well as the multi-ketosynthase genes, sometimes with a large degree of apparent gene duplication, while fatty acid synthesis genes formed distinct clusters. Surprisingly the acyl-transferases and ketoreductases involved in fatty acid synthesis (FabD and FabG, respectively) were found in very large clusters indicating an unprecedented degree of gene duplication for these genes. These results demonstrate a complex evolutionary history of core dinoflagellate modular synthases with domain specific duplications throughout the lineage as well as clues to how large protein complexes can be assembled to synthesize the largest natural products known., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

Dietary insufficiencies have been well documented to decrease growth rates and survival (and therefore overall production) in fish aquaculture. By contrast, the effects of dietary insufficiencies on the sensory biology of cultured fish remains largely unstudied. Diets based solely on plant protein sources could have advantages over fish-based diets because of the cost and ecological effects of the latter, but plant proteins lack the amino acid taurine. Adequate levels of taurine are, however, necessary for the development of a fully functional visual system in mammals. As part of ongoing studies to determine the suitability of plant-based diets, we investigated the effects of normal and reduced taurine dietary levels on retinal anatomy and function in European sea bass (Dicentrarchus labrax). We could not demonstrate any effects of dietary taurine level on retinal anatomy, nor the functional properties of luminous sensitivity and temporal resolution (measured as flicker fusion frequency). We did, however, find an effect on spectral sensitivity. The peak of spectral sensitivity of individuals fed a 5% taurine diet was rightward shifted (i.e., towards longer wavelengths) relative to that of fish fed a 0% or 1.5% taurine diet. This difference in in spectral sensitivity was due to a relatively lower level of middle wavelength pigment (maximum absorbance .500 nm) in fish fed a 5% taurine diet. Changes in spectral sensitivity resulting from diets containing different taurine levels are unlikely to be detrimental to fish destined for market, but could be in fishes that are being reared for stock enhancement programs., Competing Interests: The authors have declared that no competing interests exist.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the detection and quantitation of karlotoxins in the selected reaction monitoring (SRM) mode. This novel method was based upon the analysis of purified karlotoxins (KcTx-1, KmTx-2, 44-oxo-KmTx-2, KmTx-5), one amphidinol (AM-18), and unpurified extracts of bulk cultures of the marine dinoflagellate Karlodinium veneficum strain CCMP2936 from Delaware (Eastern USA), which produces KmTx-1 and KmTx-3. The limit of detection of the SRM method for KmTx-2 was determined as 2.5 ng on-column. Collision induced dissociation (CID) spectra of all putative karlotoxins were recorded to present fragmentation patterns of each compound for their unambiguous identification. Bulk cultures of K. veneficum strain K10 isolated from an embayment of the Ebro Delta, NW Mediterranean, yielded five previously unreported putative karlotoxins with molecular masses 1280, 1298, 1332, 1356, and 1400 Da, and similar fragments to KmTx-5. Analysis of several isolates of K. veneficum from the Ebro Delta revealed small-scale diversity in the karlotoxin spectrum in that one isolate from Fangar Bay produced KmTx-5, whereas the five putative novel karlotoxins were found among several isolates from nearby, but hydrographically distinct Alfacs Bay. Application of this LC-MS/MS method represents an incremental advance in the determination of putative karlotoxins, particularly in the absence of a complete spectrum of purified analytical standards of known specific potency., Competing Interests: The authors declare no conflict of interest.

Uptake of polychlorinated biphenyls (PCBs) by fish is controlled by the bioavailability of ingested PCBs in the gut and the freely dissolved concentration in the water moving across the gills. The prediction of bioaccumulation in fish relies on models that account for these exposure routes; however, these models typically do not account for incidental ingestion of sediment by fish, which is not well studied. The literature values for the PCB assimilation efficiency in the gut have been reported for compounds in food matrices and not associated with sediment particles. It is also unclear how mitigation strategies that alter PCB bioavailability in sediments affect predictions made by the bioaccumulation models when sediment ingestion is involved. To test the bioavailability of PCBs from treated and untreated sediments, dietary assimilation efficiencies were measured for 16 PCB congeners in mummichogs (Fundulus heteroclitus) that were fed 4 experimental diets. Diets consisted of PCB-spiked earthworms, spiked untreated sediment mixed with earthworms, spiked activated carbon-treated sediment mixed with earthworms, and spiked activated carbon mixed with earthworms. Assimilation efficiencies were determined by calculating the ratio of PCB mass in the fish tissue to the PCB mass in the food after a pulse feeding experiment. Assimilation efficiencies of PCBs associated with earthworm diet were similar to the values reported in the literature. Fish that were fed the PCB-spiked untreated sediment and activated carbon particles exhibited the highest and lowest assimilation efficiencies, respectively, over a wide K OW range. Assimilation efficiencies of sediment-bound PCBs were significantly reduced (31-93% reduction for different congeners) after amendment with activated carbon. The present study indicates that assimilation of PCBs can be reduced by sorption to black carbon. Environ Toxicol Chem 2017;36:3480-3488. © 2017 SETAC., (© 2017 SETAC.)

Most dinoflagellates in culture are bacterized, complicating the quantification of protein synthesis, as well as the analysis of its regulation. In bacterized cultures of Amphidinium carterae Hulbert, up to 80% of protein synthetic activity appears to be predominantly bacterial based on responses to inhibitors of protein synthesis. To circumvent this, axenic cultures of A. carterae were obtained and shown to respond to inhibitors of protein synthesis in a manner characteristic of eukaryotes. However, these responses changed with time in culture correlating with the reappearance of bacteria. Here we show that culture with kanamycin (50 μg/mL), carbenicillin (100 μg/mL), and streptomycin sulfate (50 μg/mL) (KCS), but not 100 units/mL of penicillin and streptomycin (PS), prevents the reappearance of bacteria and allows A. carterae protein synthesis to be quantified without the contribution of an associated bacterial community. We demonstrate that A. carterae can grow in the absence of a bacterial community. Furthermore, maintenance in KCS does not inhibit the growth of A. carterae cultures but slightly extends the growth phase and allows accumulation to somewhat higher saturation densities. We also show that cultures of A. carterae maintained in KCS respond to the eukaryotic protein synthesis inhibitors cycloheximide, emetine, and harringtonine. Establishment of these culture conditions will facilitate our ability to use polysome fractionation and ribosome profiling to study mRNA recruitment. Furthermore, this study shows that a simple and fast appraisal of the presence of a bacterial community in A. carterae cultures can be made by comparing responses to cycloheximide and chloramphenicol rather than depending on lengthier culture-based assessments.

The UAG termination codon is generally recognized as the least efficient and least frequently used of the three universal stop codons. This is substantiated by numerous studies in an array of organisms. We present here evidence of a translational readthrough of a mutant nonsense UAG codon in the transcript from the cysteine sulfinic acid decarboxylase ( csad ) gene (ENSDARG00000026348) in zebrafish. The csad gene encodes the terminal enzyme in the taurine biosynthetic pathway. Taurine is a critical amino acid for all animals, playing several essential roles throughout the body, including modulation of the immune system. The sa9430 zebrafish strain (ZDB-ALT-130411-5055) has a point mutation leading to a premature stop codon (UAG) 20 amino acids 5' of the normal stop codon, UGA. Data from immunoblotting, enzyme activity assays, and mass spectrometry provide evidence that the mutant is making a CSAD protein identical to that of the wild-type (XP&#95;009295318.1) in terms of size, activity, and amino acid sequence. UAG readthrough has been described in several species, but this is the first presentation of a case in fish. Also presented are the first data substantiating the ability of a fish CSAD to utilize cysteic acid, an alternative to the standard substrate cysteine sulfinic acid, to produce taurine., Competing Interests: The authors declare no conflict of interest.

Dinoflagellates make up a diverse array of fatty acids and polyketides. A necessary precursor for their synthesis is malonyl-CoA formed by carboxylating acetyl CoA using the enzyme acetyl-CoA carboxylase (ACC). To date, information on dinoflagellate ACC is limited. Through transcriptome analysis in Amphidinium carterae, we found three full-length homomeric type ACC sequences; no heteromeric type ACC sequences were found. We assigned the putative cellular location for these ACCs based on transit peptide predictions. Using streptavidin Western blotting along with mass spectrometry proteomics, we validated the presence of ACC proteins. Additional bands showing other biotinylated proteins were also observed. Transcript abundance for these ACCs follow the global pattern of expression for dinoflagellate mRNA messages over a diel cycle. This is one of the few descriptions at the transcriptomic and protein level of ACCs in dinoflagellates. This work provides insight into the enzymes which make the CoA precursors needed for fatty acid and toxin synthesis in dinoflagellates., Competing Interests: The authors declare no conflict of interest.

Although taurine has been shown to play multiple important physiological roles in teleosts, little is known about the molecular mechanisms underlying dietary requirements. Cell lines can provide useful tools for deciphering biosynthetic pathways and their regulation. However, culture media and sera contain variable taurine levels. To provide a useful cell line for the investigation of taurine homeostasis, an adult zebrafish liver cell line (ZFL) has been adapted to a taurine-free medium by gradual accommodation to a commercially available synthetic medium, UltraMEM™-ITES. Here we show that ZFL cells are able to synthesize taurine and be maintained in medium without taurine. This has allowed for the investigation of the effects of taurine supplementation on cell growth, cellular amino acid pools, as well as the expression of the taurine biosynthetic pathway and taurine transporter genes in a defined fish cell type. After taurine supplementation, cellular taurine levels increase but hypotaurine levels stay constant, suggesting little suppression of taurine biosynthesis. Cellular methionine levels do not change after taurine addition, consistent with maintenance of taurine biosynthesis. The addition of taurine to cells grown in taurine-free medium has little effect on transcript levels of the biosynthetic pathway genes for cysteine dioxygenase (CDO), cysteine sulfinate decarboxylase (CSAD), or cysteamine dioxygenase (ADO). In contrast, supplementation with taurine causes a 30% reduction in transcript levels of the taurine transporter, TauT. This experimental approach can be tailored for the development of cell lines from aquaculture species for the elucidation of their taurine biosynthetic capacity., Competing Interests: The authors declare no conflict of interest.

The dinoflagellate Prorocentrum donghaiense is a dominant harmful algal bloom (HAB) species on the East China Sea (ECS) coast. The co-occurrence of Karlodinium veneficum with P. donghaiense is often observed and can later develop into dense blooms. However, the role of K. veneficum in P. donghaiense population dynamics is unknown. In the current study, three K. veneficum (GM1, GM2, and GM3) strains were isolated from the ECS with one (GM1) from a mixed, dense bloom of P. donghaiense and other HAB species. All three isolates had identical ITS sequences that were concordant with the species designation. Unique karlotoxin congeners were isolated from one strain (GM2). The sterol compositions of P. donghaiense and K. veneficum were consistent with sensitivity to karlotoxin in the former and insensitivity in the latter. Additional experimentation showed that: (1)in monocultures, higher growth rate of P. donghaiense than K. veneficum is observed in nutrient-enriched and nutrient-depleted media. In co-cultures, the growth of P. donghaiense is inhibited; (2) feeding on P. donghaiense by K. veneficum is clearly demonstrated by fluorescent dye tracking; and (3) the isolated karlotoxin is lethal to P. donghaiense in a concentration-dependent manner. From these studies we propose that K. veneficum may play a negative role in P. donghaiense bloom maintenance and that P. donghaiense may in turn be a bloom initiator as a prey item for K. veneficum .

Dinoflagellates are unusual eukaryotes with large genomes and a reduced role for transcriptional regulation compared to other eukaryotes. The mRNA in dinoflagellates is trans -spliced with a 5'-spliced-leader sequence, yielding a 22-nucleotide 5'-sequence with a methylated nucleotide cap. Since the control of gene expression is primarily post-transcriptional, this study focuses on mRNA recruitment as a means for regulating gene expression and specifically on the diversity of eIF4E family members. Three novel clades related to the cap binding initiation factor eIF4E have been recognized in alveolates that are distinct from the three metazoan classes of eIF4E. We have analyzed the characteristics of five of the fourteen eIF4E family members from Karlodinium veneficum , four from clade 1 and one from clade 2. Members of each clade all bear the distinctive features of a cap-binding protein. We examined their ability to interact with the cap analogue, m 7 GTP using an in vitro bead-binding assay. We show that recombinant eIF4E-1 family members are able to bind the cap analogue m 7 GTP, but eIF4E-2b binds poorly. Overall, the eIF4E-1 family members may be serving as general cap-binding translation initiation factors, while the eIF4E-2 (and perhaps eIF4E-3) family members may serve a regulatory role in gene expression.

Karlotoxins, polyketide derivatives produced by the dinoflagellate Karlodinium veneficum, are associated with fish kills in temperate estuaries world-wide. In this study, the acute effects of 3 pure karlotoxin analogs (KmTx 1, KmTx 3 and KmTx 2) have been examined in mice. Transient lethargy and increased respiratory rates were observed soon after dosing with the karlotoxins by intraperitoneal injection, but no deaths were recorded in animals dosed with KmTx 2 at up to 500 μg/kg or with KmTx 1 or KmTx 3 at up to 4000 μg/kg. Animals dosed intraperitoneally with KmTx 1 and KmTx 3 at 4000 μg/kg showed a pronounced decrease in food and water intake, lasting 3-4 days after dosing, accompanied by a significant decrease in body weight. After this time, the lost body weight was regained and the behavior and appearance of the mice remained normal throughout the following 10-day observation period. No effects were seen in mice dosed orally with KmTx 1 or KmTx 3 at a dose of 4000 μg/kg. It is concluded that contamination of seafood if it were to occur with these karlotoxins is unlikely to pose a major risk of acute intoxication in consumers., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We examined growth performance and the lipid content in juvenile cobia, Rachycentron canadum, fed a taurine supplemented (1.5 %), plant protein based diet with two fish oil replacements. The first fish oil replacement was a thraustochytrid meal (TM + SOY) plus soybean oil (~9 % CL) and the second was a canola oil supplemented with the essential fatty acids (EFA) docosahexaenoic acid (DHA) and arachidonic acid (ARA) (~8 % CL). The diet using the thraustochytrid meal plus soybean oil performed equivalently to the fish oil diet; both resulting in significantly higher growth rates, lower feed conversion ratios, and higher survival than the supplemented canola oil diet, even though all three diets were similar in overall energy and met known protein and lipid requirements for cobia. The poor performance of the canola oil diet was attributed to insufficient addition of EFA in the supplemented canola oil source. Increasing levels of EFA in the supplemented canola oil above 0.5 g EFA kg(-1) would likely improve results with cobia. When fish fed either of the fish oil replacement diets were switched to the fish oil control diet, fatty acid profiles of the fillets were observed to transition toward that of the fish oil diet and could be predicted based on a standard dilution model. Based on these findings, a formulated diet for cobia can be produced without fish products providing 100 % survivorship, specific growth rates greater than 2.45 and feed conversion ratios less than 1.5, as long as taurine is added and EFA levels are above 0.5 g EFA kg(-1).

The Nannochloropsis genus contains oleaginous microalgae that have served as model systems for developing renewable biodiesel. Recent genomic and transcriptomic studies on Nannochloropsis species have provided insights into the regulation of lipid production in response to nitrogen stress. Previous studies have focused on the responses of Nannochloropsis species to short-term nitrogen stress, but the effect of long-term nitrogen deprivation remains largely unknown. In this study, physiological and proteomic approaches were combined to understand the mechanisms by which Nannochloropsis oceanica IMET1 is able to endure long-term nitrate deprivation and its ability to recover homeostasis when nitrogen is amended. Changes of the proteome during chronic nitrogen starvation espoused the physiological changes observed, and there was a general trend toward recycling nitrogen and storage of lipids. This was evidenced by a global down-regulation of protein expression, a retained expression of proteins involved in glycolysis and the synthesis of fatty acids, as well as an up-regulation of enzymes used in nitrogen scavenging and protein turnover. Also, lipid accumulation and autophagy of plastids may play a key role in maintaining cell vitality. Following the addition of nitrogen, there were proteomic changes and metabolic changes observed within 24 h, which resulted in a return of the culture to steady state within 4 d. These results demonstrate the ability of N. oceanica IMET1 to recover from long periods of nitrate deprivation without apparent detriment to the culture and provide proteomic markers for genetic modification.

Hematodinium spp. infections have been reported from blue crabs Callinectes sapidus in high-salinity waters of the USA from New Jersey to Texas. Recently, H. perezi (genotype III) has been proposed as the parasite species and genotype infecting blue crabs from Virginia; however, it is unknown whether this same genotype is present in blue crabs from other locations. To address this question, we collected 317 blue crabs from Massachusetts, Virginia, Georgia, Florida, Louisiana, and Texas to test for the presence of H. perezi (III) using a specific PCR assay targeting the first internal transcribed spacer (ITS1) region of the ribosomal RNA gene complex. To examine the genetic variation within H. perezi (III), ITS1 region sequences from the parasite in blue crabs from multiple locations were compared to each other and to those of H. perezi (III) found in alternate hosts from Virginia. In total, 34 distinct ITS1 sequence variants of the parasite were identified from blue crabs alone, and 38 distinct variants were identified when alternate hosts were included. However, a single ITS1 sequence variant appeared in all geographic regions and hosts, and also in blue crabs sampled from a previous study. The high similarity among all the ITS1 region sequences examined (>98%) and the observation of a single variant found throughout a large geographic range, strongly suggests that a single species and genotype of Hematodinium, specifically H. perezi (III), infects blue crabs from Virginia to Texas and multiple alternate host species in Virginia.

Understanding phenotypic differentiation among populations of wide-ranging species remains at the core of life-history research, because adaptation to local environmental conditions is expected. For example, when energy resources influence offspring fitness (as in oviparous ectotherms), the egg and hatchling environments are expected to influence selection by acting on the amount of energy allocated to offspring. Here we identify population variation in egg mass, length, width, and volume from diamondback terrapin Malaclemys terrapin eggs collected in Rhode Island (RI), Maryland (MD), and South Carolina (SC). Egg size (mean volume: 7.6, 8.1, and 9.1 cc in RI, MD, and SC, respectively) and clutch size (mean no. eggs: 16.1, 12.2, and 6.0 in RI, MD, and SC, respectively) differed among populations, which indicated that females produce larger clutches with smaller eggs at high latitudes and smaller clutches of larger eggs at lower latitudes. Lipid analyses indicated that eggs from SC contained yolks with a higher proportion of nonpolar lipids than did eggs from MD or RI (mean percentage of nonpolar lipids: 22.3%, 22.5%, and 31.8% in RI, MD, and SC, respectively). Thus, female terrapins in SC are laying larger eggs with increased lipid content to provide more energy for the developing embryo. Interestingly, total triacylglycerol (energetic lipid) was greater in southern populations but occurred in higher proportions in northern populations (total triacylglycerol: 88.0%, 85.4%, and 81.9% in RI, MD, and SC, respectively). This variation in triacylglycerol levels demonstrates the necessity for quantifying each lipid component. These data indicate a difference in reproductive strategy by which females in northern populations invest in higher fecundity with less energetic resources per offspring, whereas females in southern populations invest in larger eggs with considerably greater energy reserves.

The greatest diversity of eukaryotic species is within the microbial eukaryotes, the protists, with plants and fungi/metazoa representing just two of the estimated seventy five lineages of eukaryotes. Protists are a diverse group characterized by unusual genome features and a wide range of genome sizes from 8.2 Mb in the apicomplexan parasite Babesia bovis to 112,000-220,050 Mb in the dinoflagellate Prorocentrum micans. Protists possess numerous cellular, molecular and biochemical traits not observed in "text-book" model organisms. These features challenge some of the concepts and assumptions about the regulation of gene expression in eukaryotes. Like multicellular eukaryotes, many protists encode multiple eIF4Es, but few functional studies have been undertaken except in parasitic species. An earlier phylogenetic analysis of protist eIF4Es indicated that they cannot be grouped within the three classes that describe eIF4E family members from multicellular organisms. Many more protist sequences are now available from which three clades can be recognized that are distinct from the plant/fungi/metazoan classes. Understanding of the protist eIF4Es will be facilitated as more sequences become available particularly for the under-represented opisthokonts and amoebozoa. Similarly, a better understanding of eIF4Es within each clade will develop as more functional studies of protist eIF4Es are completed.

Toxins produced by the harmful algal bloom (HAB) forming, mixotrophic dinoflagellate Karlodinium veneficum have long been associated with fish kills. To date, the perceived ecological role for toxins has been relief from grazing pressures. Here, we demonstrate that karlotoxins also serve as a predation instrument. Using high-speed holographic microscopy, we measure the swimming behavior of several toxic and nontoxic strains of K. veneficum and their prey, Storeatula major, within dense suspensions. The selected strains produce toxins with varying potency and dosages, including a nontoxic one. Results clearly show that mixing the prey with the predatory, toxic strains causes prey immobilization at rates that are consistent with the karlotoxins' potency and dosage. Even prey cells that continue swimming slow down after exposure to toxic predators. The swimming characteristics of predators vary substantially in pure suspensions, as quantified by their velocity, radii of helical trajectories, and direction of helical rotation. When mixed with prey, all toxic strains that are involved in predation slow down. Furthermore, they substantially reduced their predominantly vertical migration, presumably to remain in the vicinity of their prey. Conversely, the nontoxic control strain does not alter its swimming and does not affect prey behavior. In separate experiments, we show that exposing prey to exogenous toxins also causes prey immobilization at rates consistent with potency. Clearly, the toxic predatory strains use karlotoxins as a means of stunning their prey, before ingesting it. These findings add a substantiated critical understanding for why some HAB species produce such complex toxin molecules.

The shallow depth of field of conventional microscopy hampers analyses of 3D swimming behavior of fast dinoflagellates, whose motility influences macroassemblages of these cells into often-observed dense "blooms." The present analysis of cinematic digital holographic microscopy data enables simultaneous tracking and characterization of swimming of thousands of cells within dense suspensions. We focus on Karlodinium veneficum and Pfiesteria piscicida, mixotrophic and heterotrophic dinoflagellates, respectively, and their preys. Nearest-neighbor distance analysis shows that predator and prey cells are randomly distributed relative to themselves, but, in mixed culture, each predator clusters around its respective prey. Both dinoflagellate species exhibit complex highly variable swimming behavior as characterized by radius and pitch of helical swimming trajectories and by translational and angular velocity. K. veneficum moves in both left- and right-hand helices, whereas P. piscicida swims only in right-hand helices. When presented with its prey (Storeatula major), the slower K. veneficum reduces its velocity, radius, and pitch but increases its angular velocity, changes that reduce its hydrodynamic signature while still scanning its environment as "a spinning antenna." Conversely, the faster P. piscicida increases its speed, radius, and angular velocity but slightly reduces its pitch when exposed to prey (Rhodomonas sp.), suggesting the preferred predation tactics of an "active hunter."

We examined seasonal and age-related variation in digestive organ sizes and enzyme activities in female western sandpipers (Calidris mauri) refueling at a coastal stopover site in southern British Columbia. Adult sandpipers exhibited seasonal variation in pancreatic and intestinal enzyme activities but not in digestive system or organ sizes. Spring migrants had 22% higher total and 67% higher standardized pancreatic lipase activities but 37% lower total pancreatic amylase activity than fall migrants, which suggests that the spring diet was enriched with lipids but low in glycogen. Spring migrants also had 47% higher total intestinal maltase activity as well as 56% higher standardized maltase and 13% higher standardized aminopeptidase-N activities. Spring migrants had higher total enzymic capacity than fall migrants, due primarily to higher total lipase and maltase activities. During fall migration, the juvenile's digestive system was 10% larger than the adult's, and it was composed differently: juveniles had a 16% larger small intestine but a 27% smaller proventriculus. The juvenile's larger digestive system was associated with lower total enzymic capacity than the adult's due to 20% lower total chitinase and 23% lower total lipase activities. These results suggest that juvenile western sandpipers may process food differently from adults and/or have a lower-quality diet.

Plasma triacylglycerol, phospholipid, cholesterol, cholesterol esters, fatty acids, and total lipids were measured in 30 female and 20 male desert tortoises (Gopherus agassizii) during the annual reproductive cycle in the eastern Mojave desert, Nevada. Blood samples were collected at monthly intervals from April to October. All lipid fractions, with the exception of free fatty acids, were significantly higher in female plasma than in male plasma in all months of the year. In contrast, free fatty acids were higher in male plasma than in female plasma in all months. The seasonal pattern in estradiol secretion mirrored that of triacylglycerol, phospholipid, cholesterol, and total lipid, all of which showed a significant correlation with the hormone. Estradiol and the vitellogenesis-associated lipids were all significantly higher in August, September, October, and April than in June. The seasonal variation in cholesterol ester levels in females did not correlate with any of the reproductive events and did not appear to be involved in yolk precursor formation. Total lipid in males showed a negative correlation with testosterone and spermatogenesis. Individual fatty acids in the June and August samples (at the highest and lowest estradiol levels) were compared in male and female plasma. The percent of C18:3n3, C18:2n6, C18:1n9, C20:5n3, and C22:5 were significantly higher in the June female plasma sample than in the August sample. Docosahexanoic (C22:6n3) acid was barely detectable in female plasma in either month., (Copyright 2002 Wiley-Liss, Inc.)