Major Revisions in Pancrustacean Phylogeny and Evidence of Sensitivity to Taxon Sampling.

The clade Pancrustacea, comprising crustaceans and hexapods, is the most diverse group of animals on earth, containing over 80% of animal species and half of animal biomass. It has been the subject of several recent phylogenomic analyses, yet relationships within Pancrustacea show a notable lack of stability. Here, the phylogeny is estimated with expanded taxon sampling, particularly of malacostracans. We show small changes in taxon sampling have large impacts on phylogenetic estimation. By analyzing identical orthologs between two slightly different taxon sets, we show that the differences in the resulting topologies are due primarily to the effects of taxon sampling on the phylogenetic reconstruction method. We compare trees resulting from our phylogenomic analyses with those from the literature to explore the large tree space of pancrustacean phylogenetic hypotheses and find that statistical topology tests reject the previously published trees in favor of the maximum likelihood trees produced here. Our results reject several clades including Caridoida, Eucarida, Multicrustacea, Vericrustacea, and Syncarida. Notably, we find Copepoda nested within Allotriocarida with high support and recover a novel relationship between decapods, euphausiids, and syncarids that we refer to as the Syneucarida. With denser taxon sampling, we find Stomatopoda sister to this latter clade, which we collectively name Stomatocarida, dividing Malacostraca into three clades: Leptostraca, Peracarida, and Stomatocarida. A new Bayesian divergence time estimation is conducted using 13 vetted fossils. We review our results in the context of other pancrustacean phylogenetic hypotheses and highlight 15 key taxa to sample in future studies.

Consistent Negative Correlations between Parasite Infection and Host Body Condition Across Seasons Suggest Potential Harmful Impacts of Salmincola markewitschi on Wild White-Spotted Charr, Salvelinus leucomaenis.

Assessing the impacts of parasites on wild fish populations is a fundamental and challenging aspect of the study of host-parasite relationships. Salmincola, a genus of ectoparasitic copepods, mainly infects salmonid species. This genus, which is notorious in aquaculture, damages host fishes, but its impacts under natural conditions remain largely unknown or are often considered negligible. In this study, we investigated the potential impacts of mouth-attaching Salmincola markewitschi on white-spotted charr (Salvelinus leucomaenis) through intensive field surveys across four seasons using host body condition as an indicator of harmful effects. The prevalence and parasite abundance were highest in winter and gradually decreased in summer and autumn, which might be due to host breeding and/or wintering aggregations that help parasite transmissions. Despite seasonal differences in prevalence and parasite abundance, consistent negative correlations between parasite abundance and host body condition were observed across all seasons, indicating that the mouth-attaching copepods could reduce the body condition of the host fish. This provides field evidence suggesting that S. markewitschi has a potential negative impact on wild white-spotted charr.

Distribution and bioaccumulation status of polycyclic aromatic hydrocarbons (PAHs) in Veraval coastal waters using copepods as bio-indicators.

The study investigates the pollution characteristics of 16 priority PAHs, accumulated in copepods from a major fishing harbour and its adjacent coastal waters of Veraval, west coast of India. The total PAH accumulation is in the range of 922.16-27,807.49 ng g-1 dw, with the mean concentration of 5776.59 ng g-1 dw. High concentrations of PAHs were present in the copepod samples from inside the harbour. Notably, there was no significant correlation between the lipid content of copepods and the accumulation of PAHs. The molecular diagnostic ratio method (MDR) indicates that the PAH sources are petrogenic in origin, while principal component analysis (PCA) points to petroleum, coal combustion and vehicular emission sources. Total cancerous PAHs (C-PAHs) in the study area dominate by 40% of the total PAHs identified; moreover, the bioaccumulation factor (BAF) is very high in the offshore area, which is also a fishing ground. The global relevance and magnitude of the present study in the Veraval, one of the prime seafood exporting hubs in India, should be dealt with utmost avidity as the accumulation status of PAHs in the zooplankton has never been explored in the Indian coastal waters. Moreover, the current study gives the foremost data on the bioaccumulation status of PAHs in copepods from the tropical waters of India.

A First Record on Microplastic Ingestion by Tropical Estuarine Copepods of Bangladesh.

Microplastics (MPs) pollution is a profound problem around the world yet it's study on the effect on zooplankton including copepods are very limited. The study was conducted between January 2021 and January 2022 in the Lower Meghna Estuary to investigate MPs ingestion in two different family of copepod: Calanoid and Cyclopoid. A method of acid digestion along with Scanning Electron Microscope (SEM) was used to identify MPs ingested by copepods from the conducted area. However, three types of MPs namely fiber, fragment and foam were extracted from this copepod biomass. Fibers represent highest (> 50%) of the ingested MPs from both group of copepod that exceed fragments and foams in all sampling stations. The overall ingestion rate of Calanoid was found higher (0.084 ± 0.002 particles/individual) compared to the Cyclopoid group (0.077 ± 0.001 particles/individual). The results of the study have effectively illustrated that copepod, obtained from multiple sampling sites within the Lower Meghna Estuary, display a propensity to ingest MPs and subsequently endangering the food security of seafood industry.

Differential gene expression and mitonuclear incompatibilities in fast- and slow-developing interpopulation Tigriopus californicus hybrids.

Mitochondrial functions are intimately reliant on proteins and RNAs encoded in both the nuclear and mitochondrial genomes, leading to inter-genomic coevolution within taxa. Hybridization can break apart coevolved mitonuclear genotypes, resulting in decreased mitochondrial performance and reduced fitness. This hybrid breakdown is an important component of outbreeding depression and early-stage reproductive isolation. However, the mechanisms contributing to mitonuclear interactions remain poorly resolved. Here, we scored variation in developmental rate (a proxy for fitness) among reciprocal F2 interpopulation hybrids of the intertidal copepod Tigriopus californicus and used RNA sequencing to assess differences in gene expression between fast- and slow-developing hybrids. In total, differences in expression associated with developmental rate were detected for 2925 genes, whereas only 135 genes were differentially expressed as a result of differences in mitochondrial genotype. Upregulated expression in fast developers was enriched for genes involved in chitin-based cuticle development, oxidation-reduction processes, hydrogen peroxide catabolic processes and mitochondrial respiratory chain complex I. In contrast, upregulation in slow developers was enriched for DNA replication, cell division, DNA damage and DNA repair. Eighty-four nuclear-encoded mitochondrial genes were differentially expressed between fast- and slow-developing copepods, including 12 subunits of the electron transport system (ETS) which all had higher expression in fast developers than in slow developers. Nine of these genes were subunits of ETS complex I. Our results emphasize the major roles that mitonuclear interactions within the ETS, particularly in complex I, play in hybrid breakdown, and resolve strong candidate genes for involvement in mitonuclear interactions.

Beneficial microbes to suppress Vibrio and improve the culture performance of copepod Tigriopus japonicus Mori.

The use of beneficial microbes, i.e., probiotics, to reduce pathogens and promote the performance of the target species is an important management strategy in mariculture. This study aimed to investigate the potential of four microbes, Debaryomyces hansenii, Ruegeria mobilis, Lactobacillus plantarum, and Bacillus subtilis, to suppress Vibrio and increase survival, population growth and digestive enzyme activity (protease, lipase, and amylase) in the harpacticoid copepod, Tigriopus japonicus. Copepod, T. japonicus stock culture with an initial mean density of 50 individual/mL (25 adult male and 25 adult female) was distributed into five treatments (i.e., four experimental and a control, each with four replicates; repeated twice) using 20 beakers (100 mL capacity each). The copepods were fed a mixture of the dinoflagellate Alexandrium tamarense and the diatom Phyaeodactylum tricornutum (3 × 104 cells/mL-1). Each microbe's concentration was adjusted at 108 CFU/mL-1 and applied to the culture condition. D. hansenii, L. plantarum, and B. subtilis all improved the copepods' survival and population growth, likely by including a higher lipase activity (P < 0.05). In contrast, using R. mobilis did not improve the copepod's culture performance compared to control. B. subtilis was the most effective in decreasing the copepod's external and internal Vibrio loading. The probiotic concentrations in the copepod decreased within days during starvation, suggesting that routine re-application of the probiotics would be needed to sustain the microbial populations and the benefits they provide. Our results demonstrated that D. hansenii and B. subtilis are promising probiotics for mass copepod culture as live food for mariculture purposes.

Bioluminescent imaging systems boosting near-infrared signals in mammalian cells.

Bioluminescence (BL) is broadly used as an optical readout in bioassays and molecular imaging. In this study, the near-infrared (NIR) BL imaging systems were developed. The system was harnessed by prototype copepod luciferases, artificial luciferase 30 (ALuc30) and its miniaturized version picALuc, and were characterized with 17 kinds of coelenterazine (CTZ) analogues carrying bulky functional groups or cyanine 5 (Cy5). They were analyzed of BL spectral peaks and enzymatic kinetics, and explained with computational modeling. The results showed that (1) the picALuc-based system surprisingly boosts the BL intensities predominantly in the red and NIR region with its specific CTZ analogues; (2) both ALuc30- and picALuc-based systems develop unique through-bond energy transfer (TBET)-driven spectral bands in the NIR region with a Cy5-conjugated CTZ analogue (Cy5-CTZ); and (3) according to the computational modeling, the miniaturized version, picALuc, has a large binding pocket, which can accommodate CTZ analogues containing bulky functional groups and thus allowing NIR BL. This study is an important addition to the BL imaging toolbox with respect to the development of orthogonal NIR reporter systems applicable to physiological samples, together with the understanding of the BL-emitting chemistry of marine luciferases.

Transcriptomic and targeted immune transcript analyses confirm localized skin immune responses in Atlantic salmon towards the salmon louse.

Atlantic salmon (Salmo salar) are highly susceptible to infestations with the ectoparasite Lepeophtheirus salmonis, the salmon louse. Infestations elicit an immune response in the fish, but the response does not lead to parasite clearance, nor does it protect against subsequent infestations. It is, however, not known why the immune response is not adequate, possibly because the local response directly underneath the louse has been poorly evaluated. The present study describes the transcriptomic response by RNA sequencing of skin at the site of copepodid attachment. Analysing differentially expressed genes, 2864 were higher and 1357 were lower expressed at the louse attachment site compared to uninfested sites in the louse infested fish, while gene expression at uninfested sites were similar to uninfested control fish. The transcriptional patterns of selected immune genes were further detailed in three skin compartments/types: Whole skin, scales only and fin tissue. The elevation of pro-inflammatory cytokines and immune cell marker transcripts observed in whole skin and scale samples were not induced in fin, and a higher cytokine transcript level in scale samples suggest it can be used as a nonlethal sampling method to enhance selective breeding trials. Furthermore, the immune response was followed in both skin and anterior kidney as the infestation developed. Here, newly moulted preadult 1 stage lice induced a higher immune response than chalimi and adult lice. Overall, infestation with salmon louse induce a modest but early immune response with an elevation of mainly innate immune transcripts, with the response primarily localized to the site of attachment.

Mining Lepeophtheirus salmonis RNA-Seq data for qPCR reference genes and their application in Caligus elongatus.

Lepeophtheirus salmonis and Caligus elongatus are two parasitic copepod species posing a significant threat to salmonid aquaculture. Consequently, several gene expression studies are executed each year to gain new knowledge and treatment strategies. Though, to enable accurate gene expression measurements by quantitative real time PCR, stable reference genes are needed. Previous studies have mainly focused on a few genes selected based on their function as housekeeping genes, as these are often stably expressed in various cells and tissues. In the present study, however, RNA-sequencing data from 127 L. salmonis samples from different life stages and diverse environmental conditions were used to identify new candidate reference genes displaying low variation. From this, six genes were selected, and the stability validated by qPCR on samples from different life stages. Since neither a genome nor comprehensive RNA sequencing data are available for C. elongatus, homologous genes to those identified for L. salmonis were identified within a C. elongatus transcriptome assembly and validated by qPCR in different life stages. Overall, the genes eukaryotic translation initiation factor 1A (EIF1A) and serine/threonine-protein phosphatase 1 (PP1) displayed the highest stability in L. salmonis, while the combination of PP1 and ribosomal protein S13 (RPS13) was found to have the highest stability in C. elongatus. These genes are well-suited reference genes for qPCR applications which allow for accurate normalization of target genes.

Strong selective effects of mitochondrial DNA on the nuclear genome.

Oxidative phosphorylation, the primary source of cellular energy in eukaryotes, requires gene products encoded in both the nuclear and mitochondrial genomes. As a result, functional integration between the genomes is essential for efficient adenosine triphosphate (ATP) generation. Although within populations this integration is presumably maintained by coevolution, the importance of mitonuclear coevolution in key biological processes such as speciation and mitochondrial disease has been questioned. In this study, we crossed populations of the intertidal copepod Tigriopus californicus to disrupt putatively coevolved mitonuclear genotypes in reciprocal F2 hybrids. We utilized interindividual variation in developmental rate among these hybrids as a proxy for fitness to assess the strength of selection imposed on the nuclear genome by alternate mitochondrial genotypes. Developmental rate varied among hybrid individuals, and in vitro ATP synthesis rates of mitochondria isolated from high-fitness hybrids were approximately two-fold greater than those of mitochondria isolated from low-fitness individuals. We then used Pool-seq to compare nuclear allele frequencies for high- or low-fitness hybrids. Significant biases for maternal alleles were detected on 5 (of 12) chromosomes in high-fitness individuals of both reciprocal crosses, whereas maternal biases were largely absent in low-fitness individuals. Therefore, the most fit hybrids were those with nuclear alleles that matched their mitochondrial genotype on these chromosomes, suggesting that mitonuclear effects underlie individual-level variation in developmental rate and that intergenomic compatibility is critical for high fitness. We conclude that mitonuclear interactions can have profound impacts on both physiological performance and the evolutionary trajectory of the nuclear genome.

Without a pinch of salt: effect of low salinity on eggs and nauplii of the salmon louse (Lepeophtheirus salmonis).

The salmon louse is an economically important parasite on Atlantic salmon and poses a major threat to aquaculture. Several treatment methods have lost their effect due to resistance development in the lice. A rather new method for combatting sea lice is freshwater treatment where the various life stages of lice are differently affected by this treatment. In this study, we analyzed the effect of freshwater on the egg strings. A 3-h treatment with freshwater had a detrimental effect on the egg strings. First, the water penetrated the string, widening it, then entering the eggs and enlarging them. Finally, the ordered structure of the egg strings collapsed, and no alive animals hatched. Shorter treatments had a lower effectivity, and treatments with brackish water also showed milder effects. The egg strings were found to have a protective effect against low salinities, as hatched nauplii died rapidly under conditions that embryos survived. We also found that embryos react to low salinity on a molecular level by changing gene expression of several genes, when incubated in brackish water. Additionally, the hatching of embryos treated with brackish water was delayed in comparison to seawater controls.

Creation of Artificial Luciferase 60s from Sequential Insights and Their Applications to Bioassays.

In this study, a series of new artificial luciferases (ALucs) was created using sequential insights on missing peptide blocks, which were revealed using the alignment of existing ALuc sequences. Through compensating for the missing peptide blocks in the alignment, 10 sibling sequences were artificially fabricated and named from ALuc55 to ALuc68. The phylogenetic analysis showed that the new ALucs formed an independent branch that was genetically isolated from other natural marine luciferases. The new ALucs successfully survived and luminesced with native coelenterazine (nCTZ) and its analogs in living mammalian cells. The results showed that the bioluminescence (BL) intensities of the ALucs were interestingly proportional to the length of the appended peptide blocks. The computational modeling revealed that the appended peptide blocks created a flexible region near the active site, potentially modulating the enzymatic activities. The new ALucs generated various colors with maximally approximately 90 nm redshifted BL spectra in orange upon reaction with the authors' previously reported 1- and 2-series coelenterazine analogs. The utilities of the new ALucs in bioassays were demonstrated through the construction of single-chain molecular strain probes and protein fragment complementation assay (PCA) probes. The success of this study can guide new insights into how we can engineer and functionalize marine luciferases to expand the toolbox of optical readouts for bioassays and molecular imaging.

Parasitism does not reduce thermal limits in the intermediate host of a bopyrid isopod.

Parasitism has strong effects on community dynamics. Given the detrimental effects parasites have on host health, infection or infestation might be expected to reduce upper thermal limits, increasing the vulnerability of host species to future climate change. Copepods are integral components of aquatic food webs and biogeochemical cycles. They also serve as intermediate hosts in the life cycle of parasitic isopods in the family Bopyridae. As both copepods and isopod parasites play important roles in aquatic communities, it is important to understand how the interaction between parasite and host affects thermal limits in order to better predict how community dynamics may change in a warming climate. Here we examined the effect of infestation by larvae of a bopyrid isopod on the cosmopolitan copepod Acartia tonsa to test the hypothesis that infestation reduces thermal limits. To aid with this work, we developed an affordable, highly portable system for measuring critical thermal maxima of small ectotherms. We also used meta-analysis to summarize the effects of parasitism on critical thermal maxima in a wider range of taxa to help contextualize our findings. Contrary to both our hypothesis and the results of previous studies, we observed no reduction of thermal limits by parasitism in A. tonsa. These results suggest that life history of the host and parasite may interact to determine how parasite infestation affects environmental sensitivity.

Elemental and biochemical nutrient limitation of zooplankton: A meta-analysis.

Primary consumers in aquatic ecosystems are frequently limited by the quality of their food, often expressed as phytoplankton elemental and biochemical composition. However, the effects of these food quality indicators vary across studies, and we lack an integrated understanding of how elemental (e.g. nitrogen, phosphorus) and biochemical (e.g. fatty acid, sterol) limitations interactively influence aquatic food webs. Here, we present the results of a meta-analysis using >100 experimental studies, confirming that limitation by N, P, fatty acids, and sterols all have significant negative effects on zooplankton performance. However, effects varied by grazer response (growth vs. reproduction), specific manipulation, and across taxa. While P limitation had greater effects on zooplankton growth than fatty acids overall, P and fatty acid limitation had equal effects on reproduction. Furthermore, we show that: nutrient co-limitation in zooplankton is strong; effects of essential fatty acid limitation depend on P availability; indirect effects induced by P limitation exceed direct effects of mineral P limitation; and effects of nutrient amendments using laboratory phytoplankton isolates exceed those using natural field communities. Our meta-analysis reconciles contrasting views about the role of various food quality indicators, and their interactions, for zooplankton performance, and provides a mechanistic understanding of trophic transfer in aquatic environments.

Changes in Chemical Composition and Copepod Toxicity during Petroleum Photo-oxidation.

Photoproducts can be formed rapidly in the initial phase of a marine oil spill. However, their toxicity is not well understood. In this study, oil was irradiated, chemically characterized, and tested for toxicity in three copepod species (Acartia tonsa, Temora longicornis, and Calanus finmarchicus). Irradiation led to a depletion of polycyclic aromatic hydrocarbons (PAHs) and n-alkanes in oil residues, along with an enrichment in aromatic and aliphatic oil photoproducts. Target lipid model-based calculations of PAH toxicity units predicted that PAH toxicities were lower in water-accommodated fractions (WAFs) of irradiated oil residues ("irradiated WAFs") than in WAFs of dark-control samples ("dark WAFs"). In contrast, biomimetic extraction (BE) measurements showed increased bioaccumulation potential of dissolved constituents of irradiated WAFs compared to dark WAFs, mainly driven by photoproducts present in irradiated oil. In line with the BE results, copepod mortality increased in irradiated WAFs compared to dark WAFs. However, low copepod toxicities were observed for WAFs produced with photo-oxidized oil slicks collected during the Deepwater Horizon oil spill. The results of this study suggest that while oil photoproducts have the potential to be a significant source of copepod toxicity, dilution and dispersion of these higher solubility products appear to help mitigate their toxicity at sea.

Diel Fluctuation Superimposed on Steady High pCO2 Generates the Most Serious Cadmium Toxicity to Marine Copepods.

Coastal systems experience diel fluctuation of pCO2 and cadmium (Cd) pollution; nevertheless, the effect of fluctuating pCO2 on Cd biotoxicity is poorly known. In this study, we initially performed the isotopically enriched organism bioassay to label Tigriopus japonicus with 113Cd (5 µg/L) to determine the Cd accumulation rate constant (kaccu) under ambient (400 µatm) and steadily (1000 µatm) and fluctuatingly elevated (1000 ± 600 µatm) pCO2 conditions for 48 h. Next, T. japonicus was interactively subjected to the above pCO2 exposures at Cd (control, 5, and 500 µg/L) treatments for 7 d. Biochemical and physiological responses for copepods were analyzed. The results showed that steadily increased pCO2 facilitated Cd bioaccumulation compared to ambient pCO2, and it was more under fluctuating acidification conditions. Despite compensatory reactions (e.g., increased energy production), Cd ultimately induced oxidative damage and apoptosis. Meanwhile, combined treatment exhibited higher toxicity (e.g., increased apoptosis) relative to Cd exposure, and even more if fluctuating acidification was considered. Intriguingly, fluctuating acidification inhibited Cd exclusion in Cd-treated copepods compared to steady acidification, linking to higher Cd kaccu and bioaccumulation. Collectively, CO2-driven acidification could aggravate Cd toxicity, providing a mechanistic understanding of the interaction between seawater acidification and Cd pollution in marine copepods.

Host specificity and the reproductive strategies of parasites.

Environmental stability can have profound impacts on life history trait evolution in organisms, especially with respect to development and reproduction. In theory, free-living species, when subjected to relatively stable and predictable conditions over many generations, should evolve narrow niche breadths and become more specialized. In parasitic organisms, this level of specialization is reflected by their host specificity. Here, we tested how host specificity impacts the reproductive strategies of parasites, a subject seldomly addressed for this group. Through an extensive review of the literature, we collated a worldwide dataset to predict, through Bayesian multilevel modelling, the effect of host specificity on the reproductive strategies of parasitic copepods of fishes or corals. We found that copepods of fishes with low host specificity (generalists) invest more into reproductive output with larger clutch sizes, whereas generalist copepods of corals invest less into reproductive output with smaller clutch sizes. The differences in host turnover rates through an evolutionary timescale could explain the contrasting strategies across species observed here, which should still favour the odds of parasites encountering and infecting a host. Ultimately, the differences found in this study reflect the unique evolutionary history that parasites share both intrinsically and extrinsically with their hosts.

Ceratosomicola oki n. sp., a New Species of Copepod (Cyclopoida: Splanchnotrophidae) Parasitic on the Chromodoridid Nudibranch Glossodoris misakinosibogae Baba, 1988 Off the Oki Islands, Japan, with Microanatomical Observation Using Micro-CT.

A new species of the family Splanchnotrophidae Norman and Scott, 1906 (Cyclopoida) is described based on both sexes collected from off the Oki Islands, the Sea of Japan. Specimens of both sexes of Ceratosomicola oki n. sp. were found in the body cavities of Glossodoris misakinosibogae Baba, 1988 (Nudibranchia: Chromodorididae). The copepod is characterized by the following female characters: the cephalosome with a pair of dorsolateral horn-like processes; the prosome with hemispherical posterolateral lobes on the middle region. Non-destructive, micro-computed tomography (micro-CT) imaging performed on a single specimen of the nudibranch revealed a heavy infection by a total 17 specimens of C. oki n. sp. Almost all individuals of the copepod were attached on the surface of the middle to posterior parts of the visceral sac, forming a dense cluster. The four females bearing developed lateral processes on the prosome faced the anterior end of the visceral sac and positioned the posterior tip of the body under the secondary gills of the host. The males fitted in the gaps between the females' bodies. Further, the distribution and shape of the reproductive organs of both sexes were partially clarified by micro-CT imaging.

Chronic effects of nano and microplastics on reproduction and development of marine copepod Tigriopus japonicus.

This study aimed to examine the impact of chronic (30 days) exposure to polystyrene microplastics (PS-MPs) of different sizes (50 nm and 2 µm) and at different concentrations (0.5 µg/L and 100 mg/L) to marine copepod Tigriopus japonicus. Polystyrene microplastics affected survival rates in size- and concentration-dependent manners. The LC50s values of 50 nm and 2 µm PS-MPs were 0.10 mg/L and 3.92 mg/L, respectively. The developmental time was delayed by 50 nm PS-MPs, and Usp expression was downregulated. Reproduction was negatively affected by 2 µm PS-MPs even at environmentally relevant concentrations; however, the expression of Vtg was not altered. The production rates of reactive oxygen species and nitric oxide also increased after exposure to PS-MPs; but this effect was independent of particle size. The expression levels of Cat and Tnf, genes related to oxidative stress and inflammation, respectively, were upregulated by exposure to PS-MPs, independently of particle size. Meanwhile, the level of oxidative stress in T. japonicus was not significantly affected by PS-MPs at environmentally relevant concentrations. This study suggests that nano-sized PS-MPs are not always more toxic than micro-sized PS-MPs, and that oxidative stress is a key factor in determining the toxic effect on T. japonicus at high concentrations.

Multigenerational study of life history traits, bioaccumulation, and molecular responses of Pseudodiaptomus annandalei to cadmium.

Metal pollution provide a substantial challenge for environmental health. This study investigated the multigeneration effects of cadmium on populations of the copepod species Pseudodiaptomus annandalei, exposed to a sublethal concentration, 40 µg/L of cadmium (Cd), over 10 generations. At the end of each generation, copepod individuals were collected to estimate fecundity, bioaccumulation, and real time qPCR quantification of selected differentially expressed genes to evaluate Cd effects and sex-specific responses of copepods across multiple generations. Our results revealed a sex-specific accumulation of Cd integrating 10 successive generations. The concentration of Cd was significantly higher (p < 0.05) in males than in females. We also observed a generational increase in Cd accumulation. Fecundity increased, with the exception of the first generation, possibly as a compensation for a decrease of copepod population size under Cd exposure. Protein expression of copepods exposed to Cd occurred in a sex-specific manner. Hemerythrin was mostly up-regulated in both copepod sexes exposed to Cd with males having the highest expression levels, while heat shock protein 70 was mostly up-regulated in males and down-regulated in female copepods, both exposed to Cd. Although copepods are known to develop adaptive mechanisms to tolerate toxic chemicals, continuous exposure to metals could lead to the bioaccumulation of metals in their offspring through maternal transfer and direct uptake from the medium over several generations. As a consequence, increased metal concentrations in copepods could result in physiological damage, reducing their fitness, and possibly compromise copepod population structures. This study showed that mortality, life history traits and molecular responses of a copepod species provided important toxicological endpoints and bio-markers for environmental risk assessments. Environmental pressure resulting from continuous exposure to persistent pollutants like Cd, could have evolutionary significance. The tendency for copepods to selectively adapt to a toxic environment through modifications, could increase their chance of survival over a long term.