De novo transcriptome assemblies of red king crab (Paralithodes camtschaticus) and snow crab (Chionoecetes opilio) molting gland and eyestalk ganglia - Temperature effects on expression of molting and growth regulatory genes in adult red king crab.

Red king crab (Paralithodes camtschaticus) and snow crab (Chionoecetes opilio) are deep-sea crustaceans widely distributed in the North Pacific and Northwest Atlantic Oceans. These giant predators have invaded the Barents Sea over the past decades, and climate-driven temperature changes may influence their distribution and abundance in the sub-Arctic region. Molting and growth in crustaceans are strongly affected by temperature, but the underlying molecular mechanisms are little known, particularly in cold-water species. Here, we describe multiple regulatory factors in the two high-latitude crabs by developing de novo transcriptomes from the molting gland (Y-organ or YO) and eye stalk ganglia (ESG), in addition to the hepatopancreas and claw muscle of red king crab. The Halloween genes encoding the ecdysteroidogenic enzymes were expressed in YO, and the ESG contained multiple neuropeptides, including molt-inhibiting hormone (MIH), crustacean hyperglycemic hormone (CHH), and ion-transport peptide (ITP). Both crabs expressed a diversity of growth-related factors, such as mTOR, AKT, Rheb and AMPKα, and stress-responsive factors, including multiple heat shock proteins (HSPs). Temperature effects on the expression of key regulatory genes were quantified by qPCR in adult red king crab males kept at 4 °C or 10 °C for two weeks during intermolt. The Halloween genes tended to be upregulated in YO at high temperature, while the ecdysteroid receptor and several growth regulators showed tissue-specific responses to elevated temperature. Constitutive and heat-inducible HSPs were expressed in an inverse temperature-dependent manner, suggesting that adult red king crabs can acclimate to increased water temperatures.

Has "The Ceiling" Rendered the Readiness for Interprofessional Learning Scale (RIPLS) Outdated?

PURPOSE: The present study aimed to investigate the preliminary effects of collaborative learning and simulation on readiness to engage in and attitudes toward future interprofessional learning activities. We translated into Norwegian and validated the original Readiness for Interprofessional Learning Scale (RIPLS) (part 1) to measure the efficacy and feasibility of a structured collaborative learning activity (part 2). MATERIALS AND METHODS: Undergraduate social and health care professional students from five Norwegian universities (n = 307) participated in the validation stage of this study (part 1). A Norwegian version of the RIPLS was developed using forward and backward translation. An expert panel discussed discrepancies between the translations and professional concepts. We planned to conduct a principal component analysis to evaluate the structure, reliability, and internal consistency of the Norwegian version of the RIPLS, after investigating the skewness, kurtosis, and range of items included. One hundred fifty students participated in collaborative learning activities; 72 (48%) of these individuals answered the translated RIPLS questionnaire. RESULTS: We found a substantial ceiling effect in the majority of items in the RIPLS, making it difficult to use the instrument as a measure of change. We evaluated the efficacy and feasibility of the collaborative activities based on the changes in the single items that had sufficient univariate normality and ultimately confirmed positive changes in two of these items. CONCLUSION: Norwegian students appear ready for interprofessional learning; however, due to significant ceiling effects, the majority of items in the RIPLS no longer seem suitable for measuring and evaluating the effects of interprofessional learning (part 1). Single-item analysis revealed a potential effect of collaborative learning (part 2). A new questionnaire is needed where readiness is instead understood as self-efficacy in areas such as role awareness and interprofessional communication. Researchers should be aware that even previously validated questionnaires may lose their applicability over time and require revision. Demands for interprofessional learning and practice are continuously evolving, and evaluation methods should be adjusted accordingly.

Low Oxygen Stress During Early Development Influences Regulation of Hypoxia-Response Genes in Farmed Atlantic Salmon (Salmo salar).

Survival and growth of developing salmonids are negatively affected by low oxygen levels within gravel nests in natural streams, and hypoxic stress is often experienced by farmed Atlantic salmon (Salmo salar) within hatcheries. Exposure to hypoxia during early development may have long-lasting effects by altering epigenetic marks and gene expression in oxygen regulatory pathways. Here, we examine the transcriptomic response to low dissolved oxygen (DO) in post-hatch salmon reared continuously in 30%, 60% or 100% DO from fertilization until start of feeding. RNA sequencing revealed multiple differentially expressed genes, including oxygen transporting hemoglobin embryonic α subunit (hbae) and EGLN3 family hypoxia-inducible factor 3 (egln3) which regulates the stability of hypoxia inducible factor 1α (HIF-1α). Both hbae and egln3 displayed expression levels inversely correlated to oxygen concentration, and DNA methylation patterns within the egln3 promoter were negatively associated with the transcript levels. These results suggest that epigenetic processes are influenced by low oxygen levels during early development in Atlantic salmon to upregulate hypoxia-response genes.

Atlantic salmon (Salmo salar) mounts systemic and mucosal stress responses to peracetic acid.

Peracetic acid (PAA), a strong organic peroxide, is considered a relatively sustainable disinfectant in aquaculture because of its broad effectivity against many pathogens at low concentrations and because it degrades spontaneously to harmless residues. The impacts of PAA on fish health must be determined before its use as either a routine disinfectant or chemotherapeutant. Here we investigated the systemic and mucosal stress responses of Atlantic salmon (Salmo salar) to PAA. In experiment 1, salmon were exposed to different nominal concentrations (0, 0.6, and 2.4 ppm) of PAA for 5 min, followed by a re-exposure to the same concentrations for 30 min 2 weeks later. Sampling was performed before exposure to PAA and at 2 h, 48 h, and 2 w after exposures. In experiment 2, fish were subjected to crowding stress prior to PAA exposure at 4.8 ppm for 30 min. The fish were sampled before exposure and 1 h, 4 h, and 2 w after. The two trials were performed in a recirculation system. Both systemic (i.e., plasma cortisol, glucose, lactate, total antioxidant capacity) and mucosal (i.e., expression of antioxidant coding genes in the skin and gills) stress indicators were affected by the treatments at varying levels, and it was apparent that the fish were able to mount a robust response to the physiological demands of PAA exposure. The cortisol levels increased in the early hours after exposure and returned to basal level afterwards. Prior exposure history to PAA did not markedly affect the levels of plasma lactate and glucose when fish were re-exposed to PAA. Crowding stress before PAA treatment, however, did alter some of the stress indicators (i.e., lactate, glucose and expression of antioxidant genes in the gills), suggesting that stress history serves as both a confounding and compounding factor on how stress responses to PAA are mobilised. Nonetheless, the changes were not substantial. Gene expression profile analyses revealed that the antioxidant system was more responsive to PAA in the gills than in the skin. The increased antioxidant capacity in the plasma, particularly at 2.4 ppm and higher, indicates that antioxidants were produced to neutralise the internal redox imbalance resulting from PAA exposure. In conclusion, the results show that salmon were able to mount a robust adaptive response to different PAA doses and exposure times, and a combined exposure to stress and PAA. These results underscore the potential of PAA as a chemotherapeutant for salmon at PAA concentrations commonly applied to control parasitic infestations.

Early Developmental Stress Affects Subsequent Gene Expression Response to an Acute Stress in Atlantic Salmon: An Approach for Creating Robust Fish for Aquaculture?

Stress during early life has potential to program and alter the response to stressful events and metabolism in later life. Repeated short exposure of Atlantic salmon to cold water and air during embryonic (E), post-hatch (PH) or both phases of development (EPH) has been shown to alter the methylome and transcriptome and to affect growth performance during later life compared to untreated controls (CO). The aim of this study was to investigate how the transcriptome of these fish responds to subsequent acute stress at the start feeding stage, and to describe methylation differences that might steer these changes. EPH treated fish showed the strongest down-regulation of corticotropin releasing factor 1, up-regulation of glucocorticoid receptor and 3-oxo-5-alpha-steroid 4-dehydrogenase 2 gene expression and a suppressed cortisol response 3 hr after the acute stress, differences that could influence hormesis and be affecting how EPH fish cope and recover from the stress event. Growth hormone 2 and insulin-like growth factor 1 were more strongly down-regulated following acute stress in EPH treated fish relative to E, PH and CO fish. This indicates switching away from growth toward coping with stress following stressful events in EPH fish. Genes implicated in immune function such as major histocompatibility class 1A, T-cell receptor and toll-like receptor also responded to acute stress differently in EPH treated fish, indicating that repeated stresses during early life may affect robustness. Differential DNA methylation was detected in regions mapping <500 bases from genes differentially responding to acute stress suggesting the involvement of epigenetic mechanisms. Stress treatments applied during early development therefore have potential as a husbandry tool for boosting the productivity of aquaculture by affecting how fish respond to stresses at critical stages of production.

Identification of tomato introgression lines with enhanced susceptibility or resistance to infection by parasitic giant dodder (Cuscuta reflexa).

The parasitic flowering plant genus Cuscuta (dodder) is a parasitic weed that infects many important crops. Once it winds around the shoots of potential host plants and initiates the development of penetration organs, called haustoria, only a few plant species have been shown to deploy effective defense mechanisms to ward off Cuscuta parasitization. However, a notable exception is Solanum lycopersicum (tomato), which exhibits a local hypersensitive reaction when attacked by giant dodder (Cuscuta reflexa). Interestingly, the closely related wild desert tomato, Solanum pennellii, is unable to stop the penetration of its tissue by the C. reflexa haustoria. In this study, we observed that grafting a S. pennellii scion onto the rootstock of the resistant S. lycopersicum did not change the susceptibility phenotype of S. pennellii. This suggests that hormones, or other mobile substances, produced by S. lycopersicum do not induce a defense reaction in the susceptible tissue. Screening of a population of introgression lines harboring chromosome fragments from S. pennellii in the genome of the recurrent parent S. lycopersicum, revealed that most lines exhibit the same defense reaction as shown by the S. lycopersicum parental line. However, several lines showed different responses and exhibited either susceptibility, or cell death that extended considerably beyond the infection site. These lines will be valuable for the future identification of key loci involved in the perception of, and resistance to, C. reflexa and for developing strategies to enhance resistance to infection in crop species.

Impacts of Early Life Stress on the Methylome and Transcriptome of Atlantic Salmon.

Exposure to environmental stressors during early-life stages can change the rate and timing of various developmental processes. Epigenetic marks affecting transcriptional regulation can be altered by such environmental stimuli. To assess how stress might affect the methylome and transcriptome in salmon, fish were treated using cold-shock and air-exposure from the eye-stage until start-feeding. The fish were either stressed prior to hatching (E), post-hatching (PH), pre- and post-hatching (EPH) or not stressed (CO). Assessing transcriptional abundances just prior to start feeding, E and PH individuals were found to have modified the expression of thousands of genes, many with important functions in developmental processes. The EPH individuals however, showed expression similar to those of CO, suggesting an adaptive response to extended periods of stress. The methylome of stressed individuals differed from that of the CO, suggesting the importance of environment in shaping methylation signatures. Through integration of methylation with transcription, we identified bases with potential regulatory functions, some 10s of kb away from the targeted genes. We then followed fish growth for an additional year. Individuals in EPH showed superior growth compared to other treatment groups, highlighting how stress can potentially have long-lasting effects on an organism's ability to adapt to environmental perturbations.

Genetic background and embryonic temperature affect DNA methylation and expression of myogenin and muscle development in Atlantic salmon (Salmo salar).

The development of ectothermic embryos is strongly affected by incubation temperature, and thermal imprinting of body growth and muscle phenotype has been reported in various teleost fishes. The complex epigenetic regulation of muscle development in vertebrates involves DNA methylation of the myogenin promoter. Body growth is a heritable and highly variable trait among fish populations that allows for local adaptations, but also for selective breeding. Here we studied the epigenetic effects of embryonic temperature and genetic background on body growth, muscle cellularity and myogenin expression in farmed Atlantic salmon (Salmo salar). Eggs from salmon families with either high or low estimated breeding values for body growth, referred to as Fast and Slow genotypes, were incubated at 8°C or 4°C until the embryonic 'eyed-stage' followed by rearing at the production temperature of 8°C. Rearing temperature strongly affected the growth rates, and the 8°C fish were about twice as heavy as the 4°C fish in the order Fast8>Slow8>Fast4>Slow4 prior to seawater transfer. Fast8 was the largest fish also at harvest despite strong growth compensation in the low temperature groups. Larval myogenin expression was approximately 4-6 fold higher in the Fast8 group than in the other groups and was associated with relative low DNA methylation levels, but was positively correlated with the expression levels of the DNA methyltransferase genes dnmt1, dnmt3a and dnmt3b. Juvenile Fast8 fish displayed thicker white muscle fibres than Fast4 fish, while Slow 8 and Slow 4 showed no difference in muscle cellularity. The impact of genetic background on the thermal imprinting of body growth and muscle development in Atlantic salmon suggests that epigenetic variation might play a significant role in the local adaptation to fluctuating temperatures over short evolutionary time.

Multiplicity of Buc copies in Atlantic salmon contrasts with loss of the germ cell determinant in primates, rodents and axolotl.

BACKGROUND: The primordial germ cells (PGCs) giving rise to gametes are determined by two different mechanisms in vertebrates. While the germ cell fate in mammals and salamanders is induced by zygotic signals, maternally delivered germ cell determinants specify the PGCs in birds, frogs and teleost fish. Assembly of the germ plasm in the oocyte is organized by the single Buc in zebrafish, named Velo1 in Xenopus, and by Oskar in Drosophila. Secondary loss of oskar in several insect lineages coincides with changes in germline determination strategies, while the presence of buc in mammals suggests functions not associated with germline formation. RESULTS: To clarify the evolutionary history of buc we searched for the gene in genomes available from various chordates. No buc sequence was found in lamprey and chordate invertebrates, while the gene was identified in a conserved syntenic region in elephant shark, spotted gar, teleosts, Comoran coelacanth and most tetrapods. Rodents have probably lost the buc gene, while a premature translation stop was found in primates and in Mexican axolotl lacking germ plasm. In contrast, several buc and buc-like (bucL) paralogs were identified in the teleosts examined, including zebrafish, and the tetraploid genome of Atlantic salmon harbors seven buc and bucL genes. Maternal salmon buc1a, buc2a and buc2b mRNAs were abundant in unfertilized eggs together with dnd and vasa mRNAs. Immunostained salmon Buc1a was restricted to cleavage furrows in 4-cell stage embryos similar to a fluorescent zebrafish Buc construct injected in salmon embryos. Salmon Buc1a and Buc2a localized together with DnD, Vasa and Dazl within the Balbiani body of early oocytes. CONCLUSIONS: Buc probably originated more than 400 million years ago and might have played an ancestral role in assembling germ plasm. Functional redundancy or subfunctionalization of salmon Buc paralogs in germline formation is suggested by the maternally inherited mRNAs of three salmon buc genes, the localized Buc1a in the cleavage furrows and the distribution of Buc1a and Buc2a in the Balbiani body during oogenesis. The extra-ovarian expression of salmon buc genes and the presence of a second zebrafish bucL gene suggest additional functions not related to germ cell specification.

Evolutionary history and adaptive significance of the polymorphic Pan I in migratory and stationary populations of Atlantic cod (Gadus morhua).

The synaptophysin (SYP) family comprises integral membrane proteins involved in vesicle-trafficking events, but the physiological function of several members has been enigmatic for decades. The presynaptic SYP protein controls neurotransmitter release, while SYP-like 2 (SYPL2) contributes to maintain normal Ca(2+)-signaling in the skeletal muscles. The polymorphic pantophysin (Pan I) of Atlantic cod shows strong genetic divergence between stationary and migratory populations, which seem to be adapted to local environmental conditions. We have investigated the functional involvement of Pan I in the different ecotypes by analyzing the 1) phylogeny, 2) spatio-temporal gene expression, 3) structure-function relationship of the Pan I(A) and I(B) protein variants, and 4) linkage to rhodopsin (rho) recently proposed to be associated with different light sensitivities in Icelandic populations of Atlantic cod. We searched for SYP family genes in phylogenetic key species and identified a single syp-related gene in three invertebrate chordates, while four members, Syp, Sypl1, Sypl2 and synaptoporin (Synpr), were found in tetrapods, Comoran coelacanth and spotted gar. Teleost fish were shown to possess duplicated syp, sypl2 and synpr genes of which the sypl2b paralog is identical to Pan I. The ubiquitously expressed cod Pan I codes for a tetra-spanning membrane protein possessing five amino acid substitutions in the first intravesicular loop, but only minor structural differences were shown between the allelic variants. Despite sizable genomic distance (>2.5 Mb) between Pan I and rho, highly significant linkage disequilibrium was found by genotyping shallow and deep water juvenile settlers predominated by the Pan I(A)-rho(A) and Pan I(B)-rho(B) haplotypes, respectively. However, the predicted rhodopsin protein showed no amino acid changes, while multiple polymorphic sites in the upstream region might affect the gene expression and pigment levels in stationary and migratory cod. Alternatively, other strongly linked genes might be responsible for the sharp settling stratification of juveniles and the different vertical behavior patterns of adult Atlantic cod.

Cell wall composition profiling of parasitic giant dodder (Cuscuta reflexa) and its hosts: a priori differences and induced changes.

Host plant penetration is the gateway to survival for holoparasitic Cuscuta and requires host cell wall degradation. Compositional differences of cell walls may explain why some hosts are amenable to such degradation while others can resist infection. Antibody-based techniques for comprehensive profiling of cell wall epitopes and cell wall-modifying enzymes were applied to several susceptible hosts and a resistant host of Cuscuta reflexa and to the parasite itself. Infected tissue of Pelargonium zonale contained high concentrations of de-esterified homogalacturonans in the cell walls, particularly adjacent to the parasite's haustoria. High pectinolytic activity in haustorial extracts and high expression levels of pectate lyase genes suggest that the parasite contributes directly to wall remodeling. Mannan and xylan concentrations were low in P. zonale and in five susceptible tomato introgression lines, but high in the resistant Solanum lycopersicum cv M82, and in C. reflexa itself. Knowledge of the composition of resistant host cell walls and the parasite's own cell walls is useful in developing strategies to prevent infection by parasitic plants.

Gonad specific genes in Atlantic salmon (Salmon salar L.): characterization of tdrd7-2, dazl-2, piwil1 and tdrd1 genes.

Atlantic salmon is a commercially important species. Understanding key processes in their life history, such as germ cell development is essential for further improvements within salmon farming. Since salmonids have undergone an additional whole genome duplication compared to many other fish species, they possess more gene paralogues. Therefore, data on gene expression and function from other species may not apply for salmon. Our aim was to study the spatial and tissue specific expression of genes known from model species to be essential for germ cell development, to identify germ cell specific factors in salmon. Based on homology with other species, selected genes were predicted in the salmon genome assembly. Gene expression was measured by PCR in a variety of juvenile salmon tissues. For genes expressed exclusively in gonads we measured the expression in the same tissues as well as in eggs, embryos and larvae by qPCR. Finally, we revealed the cellular localization of the gonad specific mRNAs by in situ hybridization (ISH). Several of the selected genes (tdrd7, cxcr4b and dazl), were found in more than one copy (indicated by a number following the gene name) in the salmon genome. Expression of tdrd7-2, dazl-2, piwil1 and tdrd1 was detected exclusively in the testis and ovary of juvenile salmon, and transcripts of tdrd7-2, dazl-2 and piwil1 were localized within male and female germ cells. While tdrd7-2, piwil1 and tdrd1 were expressed in unfertilized eggs and all embryo and larval stages measured, dazl-2 was expressed in unfertilized eggs and embryos until the onset of gastrulation. This study shows that several of the genes known from model species to be essential for germ cell development, display paralogues in salmon with dissimilar and similar expression patterns in comparison to other species. Transcripts of tdrd7-2, dazl-2, piwil1 and tdrd1 are detected exclusively in gonads of juveniles and are found among maternal RNA of eggs and subsequent embryos. This information is valuable for further studies aiming at understanding salmon germ cell development.

Cellulase activity screening using pure carboxymethylcellulose: application to soluble cellulolytic samples and to plant tissue prints.

Reliable, rapid and inexpensive detection of cellulolytic enzymes that can be used for a wide variety of biological and environmental samples are currently in high demand. Here, a new cellulase detection protocol is described that circumvents problems observed with popular agar-based methods by exploiting the ability of carboxymethylcellulose (CMC) to form gel-like surfaces on its own. These pure CMC-layers are sensitive to cellulolytic degradation and stainable by Gram's iodine without showing unwelcome reactions with other enzymes. The staining intensity negatively correlates with the enzyme activity and can be used for quantification. Cellulase activities are not obstructed by high sugar contents (e.g., in plant material) which limit the applicability of other quantification methods, making our new method particularly attractive for screening of plant extracts. A useful variant of this new method is its applicability to plant tissue prints for spatial mapping of the cellulolytic activity in a zymogram-like fashion.

Divergent and sex-dimorphic expression of the paralogs of the Sox9-Amh-Cyp19a1 regulatory cascade in developing and adult atlantic cod (Gadus morhua L.).

The factors of the Sox9-Amh-Cyp19a1 cascade play a crucial role in the complex process of sex differentiation in mammals. The involvement of Sox9 and Cyp19a1 paralogs and the single Amh ortholog in sex differentiation and development of the gonads and the brain in Atlantic cod was examined by analyzing bimodal and sex-dimorphic gene expression patterns, respectively, during early stages and in maturing males and females. Expression of sox9a and sox9b were initiated at blastulation, and both paralogs were expressed in chondrogenic tissue in the hatched larvae. The male-specific expression of sox9a in the adult gonads supports a conserved role in testis function, while sox9b was expressed in the maturing testes and ovaries at similar levels. Amh was expressed at low, but variable, levels from late gastrulation prior to the onset of cyp19a1a and cyp19a1b expression. Male-biased amh expression was found in the maturing gonads, but the increased ovarian levels during maturation suggest a role also in females. The larval expression of cyp19a1a and cyp19a1b increased at the expected time of sex differentiation, but showed large individual variation. The ovarian expression of cyp19a1a and amh increased concomitant with increased plasma estradiol levels during vitellogenesis. The testis-specific cyp19a1b expression supports the importance of estrogen in the spermatogenesis, while abundant expression in the male and female brain is probably related to the continuous neurogenesis in fish. These divergent and sex-dimorphic expression patterns of the cod sox9 and cyp19a1 paralogs demonstrate the complexity of the genetic network regulating sexual development in fish.

Sex dimorphic expression of five dmrt genes identified in the Atlantic cod genome. The fish-specific dmrt2b diverged from dmrt2a before the fish whole-genome duplication.

The Doublesex and Mab-3 related transcription factors (Dmrt) are characterised by the zinc finger-like DM domain binding similar DNA sequences, but show different spatio-temporal expression patterns and functions throughout ontogenesis. Dmrt1 is the master regulator of sex determination in very distant metazoans, while Dmrt2 and Dmrt4 are of crucial importance in vertebrate somitogenesis and neurogenesis, respectively. To elucidate the evolutionary divergence of the fish dmrt genes, we examined the expression patterns and the chromosomal synteny of the five dmrt genes identified in the Atlantic cod genome. Quantitative PCR analyses of cod dmrt1, dmrt2a, dmrt3, dmrt4 and dmrt5 revealed distinct expression patterns in the embryo and larvae, and indicated conserved extragonadal functions during early development. Several dmrt genes seem to be involved in the sexual differentiation of gonads and brain, but the sex-dimorphic expression patterns differed substantially between teleosts, suggesting functional switch between Dmrt members. The dmrt2a-dmrt3-dmrt1 cluster was found to be located in a conserved syntenic region, and the flanking genes have become duplicated in teleosts and are closely linked in a paralogous region lacking the dmrt cluster. Similarly, the region containing the fish-specific dmrt2b gene was found to have a paralogous region without a dmrt2b duplicate in a separate linkage group in the teleost genomes. We propose that the teleost segments paralogous to the dmrt2a- and dmrt2b regions, respectively, were formed through the fish-specific whole genome duplication (3R), while dmrt2a and dmrt2b originated from the second round (2R) of whole genome duplication of the ancestral dmrt2. The dmrt2b paralog seems to have been lost in Atlantic cod as in tetrapods and may be a pseudogene in pufferfish, while dmrt2a and dmrt2b have acquired different functions in zebrafish. Contrasting with the retained duplicates of dmrt flanking genes, the massive losses of dmrt duplicates in the vertebrate tetraploidizations suggest that their functions are exquisitely sensitive to gene dosage.

Sexually dimorphic expression of dmrt1 in immature and mature Atlantic cod (Gadus morhua L.).

The Doublesex and Mab-3 related transcription factor 1 (Dmrt1) is implicated in testis development in a variety of vertebrates, including teleost fish. Atlantic cod (Gadusmorhua L.) is a promising cold-water aquaculture species, but early sexual maturation of males in particular is a major problem in today's cod farming. Molecular studies of dmrt1 were initiated to gain knowledge about the regulation of gonad development for the first time in a species of the superorder Paracanthopterygii. The predicted cod Dmrt1 of 310 amino acids contains a highly conserved DM domain, including six Cys residues probably involved in the formation of a double zinc-finger motif for DNA binding. The tissue expression analysis revealed that dmrt1 is expressed exclusively in the gonads, and the signal was localized in the germ cells in both genders by in situ hybridization. Sexually dimorphic expression of dmrt1 was documented by quantitative PCR with the highest mRNA levels in immature males corresponding to the start of spermatogenesis. Although significantly less expressed in the ovary, Dmrt1 might also play a role in oogenesis. Southern blot analysis revealed several DM domain-containing genes in the cod genome, but no sex-linked polymorphism was shown.

The toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival.

Apicomplexa are unicellular eukaryotic pathogens that carry a vestigial algal endosymbiont, the apicoplast. The physiological function of the apicoplast and its integration into parasite metabolism remain poorly understood and at times controversial. We establish that the Toxoplasma apicoplast membrane-localized phosphate translocator (TgAPT) is an essential metabolic link between the endosymbiont and the parasite cytoplasm. TgAPT is required for fatty acid synthesis in the apicoplast, but this may not be its most critical function. Further analyses demonstrate that TgAPT also functions to supply the apicoplast with carbon skeletons for additional pathways and, indirectly, with energy and reduction power. Genetic ablation of the transporter results in rapid death of parasites. The dramatic consequences of loss of its activity suggest that targeting TgAPT could be a viable strategy to identify antiparasitic compounds.

Maternal transfer and transcriptional onset of immune genes during ontogenesis in Atlantic cod.

The immune system in teleosts is not completely developed during embryonic and larval stages and immune competence is assumed to be restricted. This study is the first to address whether immune transcripts are maternally transferred to offspring and when immune genes are transcriptionally active in Atlantic cod (Gadus morhua). In unfertilised eggs, transcripts encoding lysozyme and cathelicidin were found indicating maternal transfer of antibacterial transcripts. Lysozyme activity was also present at this stage suggesting the presence of a functional protein. Transcripts of two other putative antibacterial genes (hepcidin and pentraxin) and antiviral genes (ISG15 and LGP2) were absent in unfertilised eggs. The transcriptional onset of these genes occurred during the gastrula period. Transcripts of the heavy chain constant regions of the immunoglobulin (Ig) D, membrane-associated and secreted form of IgM were absent in unfertilised eggs. Transcription of the heavy chain locus commenced at low levels during the segmentation period indicating the onset of B-cell development. Most innate immune genes showed an increase in transcription around hatch and first feeding, indicating a preparation for increased pathogen exposure at this time. Prior to and during metamorphosis all genes showed a pronounced elevation in transcript levels indicating a further maturation of the immune system during this period.

The ontogeny and extrahepatic expression of complement factor C3 in Atlantic salmon (Salmo salar).

Fish embryos and hatchlings are exposed to pathogens long before maturation of their lymphoid organs. Little is known about defence mechanisms during the earliest stages of life, but innate mechanisms may be essential for survival. The complement system in fish is well developed and represents a major part of innate immunity. Complement factor 3 (C3) is central subsequent to activation of all pathways of the complement system, leading to inflammatory reactions, such as chemotaxis, opsonisation and lysis of pathogens. Hepatocytes represent the major source of C3, but modern molecular biological methods have confirmed that C3 is synthesised at multiple sites. Our main objective was to study the ontogeny of C3 in Atlantic salmon by mapping the commencement of synthesis and localisation of proteins. Eggs, embryos, hatchlings and adult fish were analysed for the presence of C3 mRNA and proteins. From immunohistochemical studies, C3 proteins were detected at several extrahepatic sites, such as the skeletal muscle, developing notochord and chondrocytes of the gill arch. Immunoblotting revealed presence of C3 proteins in the unfertilised egg, but C3 mRNA was only detected after fertilisation by real-time RT-PCR. Taken together, the results implicated the maternal transfer of C3 proteins as well as novel non-immunological functions during development.