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Dinoflagellates and other microalgae can produce a wide spectrum of toxic molecules, which are the main responsible of shellfish poisoning syndromes. During seasonal harmful algal blooms (HABs), many filter-feeding marine invertebrates, including bivalve molluscs, can accumulate phycotoxins at extremely high levels, thus representing a serious threat to human health. Furthermore, HABs also have a severe impact on the aquaculture sector due to the forced prolonged closure of large harvesting areas. Although the targets and mechanism of action of many phycotoxins have been extensively studied on vertebrate model organisms, so far just a little attention has been focused on their effects on marine invertebrates. Here we provide an overview about the molecular response of marine bivalves to phycotoxins, with a particular focus on toxins produced by dinoflagellates. Even though large-scale genomic and proteomic approaches on molluscs are still hindered by the limited molecular knowledge of these organisms, a few studies exploiting the most recent technological advances provide promising perspectives for a better comprehension of the mechanisms involved in shellfish toxicity and for the identification of molecular markers of contamination. [ABSTRACT FROM AUTHOR]

Gene expression studies largely support the understanding of gene-environment interactions in humans and other living organisms but the lack of genomic and genetic information often complicates the analysis of functional responses in non-traditional model species. Nevertheless, the fast advancement of DNA microarray and sequencing technologies now makes global gene expression analysis possible in virtually any species of interest. As regards the Mytilus genus, tens of thousands Expressed Sequence Tags (ESTs) are currently available for M. californianus and M. galloprovincialis, and DNA microarrays have been developed. Among them, Immunochip 1.0 specifically includes 1,820 probes of genes centrally involved or modulated in the innate immune responses of the Mediterranean mussel. This review recalls peculiarities and applications of the existing mussel DNA microarrays and finally summarizes facts concerning a variety of transcript sequences likely involved in the mussel immunity. Beside DNA microarrays, Next Generation Sequencing (NGS) technologies now offer new and broader research perspectives, from the whole transcriptome coverage to the Mytilus genome sequencing. [ABSTRACT FROM AUTHOR]

Background: Genomic Observatories (GOs) are sites of long-term scientific study that undertake regular assessments of the genomic biodiversity. The European Marine Omics Biodiversity Observation Network (EMO BON) is a network of GOs that conduct regular biological community samplings to generate environmental and metagenomic data of microbial communities from designated marine stations around Europe. The development of an effective workflow is essential for the analysis of the EMO BON metagenomic data in a timely and reproducible manner., Findings: Based on the established MGnify resource, we developed metaGOflow. metaGOflow supports the fast inference of taxonomic profiles from GO-derived data based on ribosomal RNA genes and their functional annotation using the raw reads. Thanks to the Research Object Crate packaging, relevant metadata about the sample under study, and the details of the bioinformatics analysis it has been subjected to, are inherited to the data product while its modular implementation allows running the workflow partially. The analysis of 2 EMO BON samples and 1 Tara Oceans sample was performed as a use case., Conclusions: metaGOflow is an efficient and robust workflow that scales to the needs of projects producing big metagenomic data such as EMO BON. It highlights how containerization technologies along with modern workflow languages and metadata package approaches can support the needs of researchers when dealing with ever-increasing volumes of biological data. Despite being initially oriented to address the needs of EMO BON, metaGOflow is a flexible and easy-to-use workflow that can be broadly used for one-sample-at-a-time analysis of shotgun metagenomics data., (© The Author(s) 2023. Published by Oxford University Press GigaScience.)

Background: The European sardine (Sardina pilchardus Walbaum, 1792) is culturally and economically important throughout its distribution. Monitoring studies of sardine populations report an alarming decrease in stocks due to overfishing and environmental change, which has resulted in historically low captures along the Iberian Atlantic coast. Important biological and ecological features such as population diversity, structure, and migratory patterns can be addressed with the development and use of genomics resources., Findings: The genome of a single female individual was sequenced using Illumina HiSeq X Ten 10x Genomics linked reads, generating 113.8 gigabase pairs of data. Three draft genomes were assembled: 2 haploid genomes with a total size of 935 megabase pairs (N50 103 kilobase pairs) each, and a consensus genome of total size 950 megabase pairs (N50 97 kilobase pairs). The genome completeness assessment captured 84% of Actinopterygii Benchmarking Universal Single-Copy Orthologs. To obtain a more complete analysis, the transcriptomes of 11 tissues were sequenced to aid the functional annotation of the genome, resulting in 40,777 genes predicted. Variant calling on nearly half of the haplotype genome resulted in the identification of >2.3 million phased single-nucleotide polymorphisms with heterozygous loci., Conclusions: A draft genome was obtained, despite a high level of sequence repeats and heterozygosity, which are expected genome characteristics of a wild sardine. The reference sardine genome and respective variant data will be a cornerstone resource of ongoing population genomics studies to be integrated into future sardine stock assessment modelling to better manage this valuable resource., (© The Author(s) 2019. Published by Oxford University Press.)

Background: Pistachio (Pistacia vera L.) is one of the most important commercial nut crops worldwide. It is a salt-tolerant and long-lived tree, with the largest cultivation area in Iran. Climate change and subsequent increased soil salt content have adversely affected the pistachio yield in recent years. However, the lack of genomic/global transcriptomic sequences on P. vera impedes comprehensive researches at the molecular level. Hence, whole transcriptome sequencing is required to gain insight into functional genes and pathways in response to salt stress., Results: RNA sequencing of a pooled sample representing 24 different tissues of two pistachio cultivars with contrasting salinity tolerance under control and salt treatment by Illumina Hiseq 2000 platform resulted in 368,953,262 clean 100 bp paired-ends reads (90 Gb). Following creating several assemblies and assessing their quality from multiple perspectives, we found that using the annotation-based metrics together with the length-based parameters allows an improved assessment of the transcriptome assembly quality, compared to the solely use of the length-based parameters. The generated assembly by Trinity was adopted for functional annotation and subsequent analyses. In total, 29,119 contigs annotated against all of five public databases, including NR, UniProt, TAIR10, KOG and InterProScan. Among 279 KEGG pathways supported by our assembly, we further examined the pathways involved in the plant hormone biosynthesis and signaling as well as those to be contributed to secondary metabolite biosynthesis due to their importance under salinity stress. In total, 11,337 SSRs were also identified, which the most abundant being dinucleotide repeats. Besides, 13,097 transcripts as candidate stress-responsive genes were identified. Expression of some of these genes experimentally validated through quantitative real-time PCR (qRT-PCR) that further confirmed the accuracy of the assembly. From this analysis, the contrasting expression pattern of NCED3 and SOS1 genes were observed between salt-sensitive and salt-tolerant cultivars., Conclusion: This study, as the first report on the whole transcriptome survey of P. vera, provides important resources and paves the way for functional and comparative genomic studies on this major tree to discover the salinity tolerance-related markers and stress response mechanisms for breeding of new pistachio cultivars with more salinity tolerance.

Trebouxia is the most common lichen-forming genus of aero-terrestrial green algae and all its species are desiccation tolerant (DT). The molecular bases of this remarkable adaptation are, however, still largely unknown. We applied a transcriptomic approach to a common member of the genus, T. gelatinosa, to investigate the alteration of gene expression occurring after dehydration and subsequent rehydration in comparison to cells kept constantly hydrated. We sequenced, de novo assembled and annotated the transcriptome of axenically cultured T. gelatinosa by using Illumina sequencing technology. We tracked the expression profiles of over 13,000 protein-coding transcripts. During the dehydration/rehydration cycle c. 92 % of the total protein-coding transcripts displayed a stable expression, suggesting that the desiccation tolerance of T. gelatinosa mostly relies on constitutive mechanisms. Dehydration and rehydration affected mainly the gene expression for components of the photosynthetic apparatus, the ROS-scavenging system, Heat Shock Proteins, aquaporins, expansins, and desiccation related proteins (DRPs), which are highly diversified in T. gelatinosa, whereas Late Embryogenesis Abundant Proteins were not affected. Only some of these phenomena were previously observed in other DT green algae, bryophytes and resurrection plants, other traits being distinctive of T. gelatinosa, and perhaps related to its symbiotic lifestyle. Finally, the phylogenetic inference extended to DRPs of other chlorophytes, embryophytes and bacteria clearly pointed out that DRPs of chlorophytes are not orthologous to those of embryophytes: some of them were likely acquired through horizontal gene transfer from extremophile bacteria which live in symbiosis within the lichen thallus.

Synonymous codon usage bias (CUB) is a defined as the non-random usage of codons encoding the same amino acid across different genomes. This phenomenon is common to all organisms and the real weight of the many factors involved in its shaping still remains to be fully determined. So far, relatively little attention has been put in the analysis of CUB in bivalve mollusks due to the limited genomic data available. Taking advantage of the massive sequence data generated from next generation sequencing projects, we explored codon preferences in 64 different species pertaining to the six major evolutionary lineages in Bivalvia. We detected remarkable differences across species, which are only partially dependent on phylogeny. While the intensity of CUB is mild in most organisms, a heterogeneous group of species (including Arcida and Mytilida, among the others) display higher bias and a strong preference for AT-ending codons. We show that the relative strength and direction of mutational bias, selection for translational efficiency and for translational accuracy contribute to the establishment of synonymous codon usage in bivalves. Although many aspects underlying bivalve CUB still remain obscure, we provide for the first time an overview of this phenomenon in this large, commercially and environmentally important, class of marine invertebrates.

We report the identification of a novel gene family (named MgCRP-I) encoding short secreted cysteine-rich peptides in the Mediterranean mussel Mytilus galloprovincialis. These peptides display a highly conserved pre-pro region and a hypervariable mature peptide comprising six invariant cysteine residues arranged in three intramolecular disulfide bridges. Although their cysteine pattern is similar to cysteines-rich neurotoxic peptides of distantly related protostomes such as cone snails and arachnids, the different organization of the disulfide bridges observed in synthetic peptides and phylogenetic analyses revealed MgCRP-I as a novel protein family. Genome- and transcriptome-wide searches for orthologous sequences in other bivalve species indicated the unique presence of this gene family in Mytilus spp. Like many antimicrobial peptides and neurotoxins, MgCRP-I peptides are produced as pre-propeptides, usually have a net positive charge and likely derive from similar evolutionary mechanisms, that is, gene duplication and positive selection within the mature peptide region; however, synthetic MgCRP-I peptides did not display significant toxicity in cultured mammalian cells, insecticidal, antimicrobial, or antifungal activities. The functional role of MgCRP-I peptides in mussel physiology still remains puzzling., (© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The red swamp crayfish (Procambarus clarkii, Girard 1852) is among the most economically important freshwater crustacean species, and it is also considered one of the most aggressive invasive species worldwide. Despite its commercial importance and being one of the most studied crayfish species, its genomic and transcriptomic layout has only been partially studied. Illumina RNA-sequencing was applied to characterize the eyestalk transcriptome and identify its most characterizing genes. A collection of 83,170,732 reads from eyestalks was obtained using Illumina paired-end sequencing technology. A de novo assembly was performed with the Trinity assembly software generating 119,255 contigs (average length of 1,007 bp) and identifying the first sequenced transcriptome in this species. The eyestalk is a major site for the production of neurohormones and controls a variety of physiological functions such as osmotic regulation, molting, epidermal color patterns and reproduction. Hence, its transcriptomic characterization is interesting and potentially instrumental to the elucidation of genes which have not been comprehensively described yet. Moreover, the availability of such a large amount of information supported the characterization of molecular families which have never been described before. The P. clarkii eyestalk transcriptome reported here provides a resource for improving the knowledge of the still incompletely defined neuroendocrinology of this species and represents an important source of data for all the interested carcinologists., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Conversion of one or more amino acids in eukaryotic peptides to the D-enantiomer configuration is catalyzed by specific L/D-peptide isomerases and it is a poorly investigated post-translational modification. No common modified amino acid or specific modified position has been recognized, and mechanisms underlying changes in the peptide function provided by this conversion are not widely studied. The 72 amino acid crustacean hyperglycemic hormone (CHH) in Astacidea crustaceans exhibits a co-existence of two peptide enantiomers with either D- or L-phenylalanine as their third residue. It is a pleiotropic hormone regulating several physiological processes in different target tissues and along different time scales. CHH enantiomers differently affect time courses and intensities of examined processes. The short-term effects of the two isomers on gene expression were examined in the hepatopancreas, gills, hemocytes and muscles of the astacid Pontastacus leptodactylus. Gene expression in muscles and hemocytes was not affected by either of the isomers. Two modes of action for CHH were elucidated in the hepatopancreas and the gills: specific gene induction in both organs by D-CHH, and targeted attenuation caused by both enantiomers in the gills. Consequently, a two-receptor system is proposed for conveying the effect of the two CHH isomers., (© 2014. Published by The Company of Biologists Ltd.)

The rigid crustacean exoskeleton, the cuticle, is composed of the polysaccharide chitin, structural proteins and mineral deposits. It is periodically replaced to enable growth and its construction is an energy-demanding process. Ecdysis, the shedding event of the old cuticle, is preceded by a preparatory phase, termed premolt, in which the present cuticle is partially degraded and a new one is formed underneath it. Procambarus clarkii (Girard 1852), an astacid crustacean, was used here to comprehensively examine the changing patterns of gene expression in the hypodermis underlying the cuticle of the carapace at seven time points along ~14 premolt days. Next generation sequencing was used to construct a multi-tissue P. clarkii transcript sequence assembly for general use in a variety of transcriptomic studies. A reference transcriptome was created here in order to perform digital transcript expression analysis, determining the gene expression profiles in each of the examined premolt stages. The analysis revealed a cascade of sequential expression events of molt-related genes involved in chitin degradation, synthesis and modification, as well as synthesis of collagen and four groups of cuticular structural genes. The new description of major transcriptional events during premolt and the determination of their timing provide temporal markers for future studies of molt progress and regulation. The peaks of the expression of the molt-related genes were preceded by expression peaks of cytoskeletal genes that are hypothesized to be essential for premolt progress through regulating protein synthesis and/or transport, probably by remodeling the cytoskeletal structure., (© 2014. Published by The Company of Biologists Ltd.)

Background: The Mediterranean mussel Mytilus galloprovincialis is marine bivalve with a relevant commercial importance as well as a key sentinel organism for the biomonitoring of environmental pollution. Here we report the RNA sequencing of the mussel digestive gland, performed with the aim: a) to produce a high quality de novo transcriptome assembly, thus improving the genetic and molecular knowledge of this organism b) to provide an initial assessment of the response to paralytic shellfish poisoning (PSP) on a molecular level, in order to identify possible molecular markers of toxin accumulation., Results: The comprehensive de novo assembly and annotation of the transcriptome yielded a collection of 12,079 non-redundant consensus sequences with an average length of 958 bp, with a high percentage of full-length transcripts. The whole-transcriptome gene expression study indicated that the accumulation of paralytic toxins produced by the dinoflagellate Alexandrium minutum over a time span of 5 days scarcely affected gene expression, but the results need further validation with a greater number of biological samples and naturally contaminated specimens., Conclusion: The digestive gland reference transcriptome we produced significantly improves the data collected from previous sequencing efforts and provides a basic resource for expanding functional genomics investigations in M. galloprovincialis. Although not conclusive, the results of the RNA-seq gene expression analysis support the classification of mussels as bivalves refractory to paralytic shellfish poisoning and point out that the identification molecular biomarkers of PSP in the digestive gland of this organism is problematic.

Coelacanths are a critically valuable species to explore the gene changes that took place in the transition from aquatic to terrestrial life. One interesting and biologically relevant feature of the genus Latimeria is ureotelism. However not all urea is excreted from the body; in fact high concentrations are retained in plasma and seem to be involved in osmoregulation. The purine catabolic pathway, which leads to urea production in Latimeria, has progressively lost some steps, reflecting an enzyme loss during diversification of terrestrial species. We report the results of analyses of the liver and testis transcriptomes of the Indonesian coelacanth Latimeria menadoensis and of the genome of Latimeria chalumnae, which has recently been fully sequenced in the framework of the coelacanth genome project. We describe five genes, uricase, 5-hydroxyisourate hydrolase, parahox neighbor B, allantoinase, and allantoicase, each coding for one of the five enzymes involved in urate degradation to urea, and report the identification of a putative second form of 5-hydroxyisourate hydrolase that is characteristic of the genus Latimeria. The present data also highlight the activity of the complete purine pathway in the coelacanth liver and suggest its involvement in the maintenance of high plasma urea concentrations., (© 2013 Wiley Periodicals, Inc.)

The morphological stasis of coelacanths has long suggested a slow evolutionary rate. General genomic stasis might also imply a decrease of transposable elements activity. To evaluate the potential activity of transposable elements (TEs) in "living fossil" species, transcriptomic data of Latimeria chalumnae and its Indonesian congener Latimeria menadoensis were compared through the RNA-sequencing mapping procedures in three different organs (liver, testis, and muscle). The analysis of coelacanth transcriptomes highlights a significant percentage of transcribed TEs in both species. Major contributors are LINE retrotransposons, especially from the CR1 family. Furthermore, some particular elements such as a LF-SINE and a LINE2 sequences seem to be more expressed than other elements. The amount of TEs expressed in testis suggests possible transposition burst in incoming generations. Moreover, significant amount of TEs in liver and muscle transcriptomes were also observed. Analyses of elements displaying marked organ-specific expression gave us the opportunity to highlight exaptation cases, that is, the recruitment of TEs as new cellular genes, but also to identify a new Latimeria-specific family of Short Interspersed Nuclear Elements called CoeG-SINEs. Overall, transcriptome results do not seem to be in line with a slow-evolving genome with poor TE activity., (© 2013 Wiley Periodicals, Inc.)

Background: Latimeria menadoensis is a coelacanth species first identified in 1997 in Indonesia, at 10,000 Km of distance from its African congener. To date, only six specimens have been caught and just a very limited molecular data is available. In the present work we describe the de novo transcriptome assembly obtained from liver and testis samples collected from the fifth specimen ever caught of this species., Results: The deep RNA sequencing performed with Illumina technologies generated 145,435,156 paired-end reads, accounting for ~14 GB of sequence data, which were de novo assembled using a Trinity/CLC combined strategy. The assembly output was processed and filtered producing a set of 66,308 contigs, whose quality was thoroughly assessed. The comparison with the recently sequenced genome of the African congener Latimeria chalumnae and with the available genomic resources of other vertebrates revealed a good reconstruction of full length transcripts and a high coverage of the predicted full coelacanth transcriptome., Conclusion: Given the high genomic affinity between the two coelacanth species, the here described de novo transcriptome assembly can be considered a valuable support tool for the improvement of gene prediction within the genome of L. chalumnae and a valuable resource for investigation of many aspects of tetrapod evolution.

The crustacean Hyperglycemic Hormone (cHH) is a neuropeptide present in many decapods. Two different chiral isomers are simultaneously present in Astacid crayfish and their specific biological functions are still poorly understood. The present study is aimed at better understanding the potentially different effect of each of the isomers on the hepatopancreatic gene expression profile in the crayfish Pontastacus leptodactylus, in the context of short term hyperglycemia. Hence, two different chemically synthesized cHH enantiomers, containing either L- or D-Phe(3), were injected to the circulation of intermolt females following removal of their X organ-Sinus gland complex. The effects triggered by the injection of the two alternate isomers were detected after one hour through measurement of circulating glucose levels. Triggered changes of the transcriptome expression profile in the hepatopancreas were analyzed by RNA-seq. A whole transcriptome shotgun sequence assembly provided the assumedly complete transcriptome of P. leptodactylus hepatopancreas, followed by RNA-seq analysis of changes in the expression level of many genes caused by the application of each of the hormone isomers. Circulating glucose levels were much higher in response to the D-isoform than to the L-isoform injection, one hour from injection. Similarly, the RNA-seq analysis confirmed a stronger effect on gene expression following the administration of D-cHH, while just limited alterations were caused by the L-isomer. These findings demonstrated a more prominent short term effect of the D-cHH on the transcription profile and shed light on the effect of the D-isomer on specific functional gene groups. Another contribution of the study is the construction of a de novo assembly of the hepatopancreas transcriptome, consisting of 39,935 contigs, that dramatically increases the molecular information available for this species and for crustaceans in general, providing an efficient tool for studying gene expression patterns in this organ.

Genes involved in sex determination and differentiation have been identified in mice, humans, chickens, reptiles, amphibians and teleost fishes. However, little is known of their functional conservation, and it is unclear whether there is a common set of genes shared by all vertebrates. Coelacanths, basal Sarcopterygians and unique "living fossils", could help establish an inventory of the ancestral genes involved in these important developmental processes and provide insights into their components. In this study 33 genes from the genome of Latimeria chalumnae and from the liver and testis transcriptomes of Latimeria menadoensis, implicated in sex determination and differentiation, were identified and characterized and their expression levels measured. Interesting findings were obtained for GSDF, previously identified only in teleosts and now characterized for the first time in the sarcopterygian lineage; FGF9, which is not found in teleosts; and DMRT1, whose expression in adult gonads has recently been related to maintenance of sexual identity. The gene repertoire and testis-specific gene expression documented in coelacanths demonstrate a greater similarity to modern fishes and point to unexpected changes in the gene regulatory network governing sexual development.

The discovery of a living coelacanth specimen in 1938 was remarkable, as this lineage of lobe-finned fish was thought to have become extinct 70 million years ago. The modern coelacanth looks remarkably similar to many of its ancient relatives, and its evolutionary proximity to our own fish ancestors provides a glimpse of the fish that first walked on land. Here we report the genome sequence of the African coelacanth, Latimeria chalumnae. Through a phylogenomic analysis, we conclude that the lungfish, and not the coelacanth, is the closest living relative of tetrapods. Coelacanth protein-coding genes are significantly more slowly evolving than those of tetrapods, unlike other genomic features. Analyses of changes in genes and regulatory elements during the vertebrate adaptation to land highlight genes involved in immunity, nitrogen excretion and the development of fins, tail, ear, eye, brain and olfaction. Functional assays of enhancers involved in the fin-to-limb transition and in the emergence of extra-embryonic tissues show the importance of the coelacanth genome as a blueprint for understanding tetrapod evolution.

Antimicrobial peptides (AMPs) play a fundamental role in the innate immunity of invertebrates, preventing the invasion of potential pathogens. Mussels can express a surprising abundance of cysteine-rich AMPs pertaining to the defensin, myticin, mytilin and mytimycin families, particularly in the circulating hemocytes. Based on deep RNA sequencing of Mytilus galloprovincialis, we describe the identification, molecular diversity and constitutive expression in different tissues of five novel transcripts pertaining to the macin family (named mytimacins) and eight novel transcripts pertaining to the big defensins family (named MgBDs). The predicted antimicrobial peptides exhibit a N-terminal signal peptide, a positive net charge and a high content in cysteines, allegedly organized in intra-molecular disulfide bridges. Mytimacins and big defensins therefore represent two novel AMP families of M. galloprovincialis which extend the repertoire of cysteine-rich AMPs in this bivalve mollusk., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The key component of the classical complement pathway C1q is regarded as a major connecting link between innate and acquired immunity due to the highly adaptive binding properties of its trimeric globular domain gC1q. The gC1q domain also characterizes many non-complement proteins involved in a broad range of biological processes including apoptosis, inflammation, cell adhesion and cell differentiation. In molluscs and many other invertebrates lacking of adaptive immunity, C1q domain containing (C1qDC) proteins are abundant, they most probably emerged as lectins and subsequently evolved in a specialized class of pattern recognition molecules through the expanding interaction properties of gC1q. Here we report the identification of 168 C1qDC transcript sequences of Mytilus galloprovincialis. The remarkable abundance of C1qDC transcripts in the Mediterranean mussel suggests an evolutionary strategy of gene duplication, functional diversification and selection of many specific C1qDC variants. A comprehensive transcript sequence survey in Protostomia also revealed that the C1qDC family expansion observed in mussel could have occurred in some specific taxa independently from the events leading to the establishment of a large complement of C1qDC genes in the Chordates lineage., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We generated a panel of monoclonal antibodies (mAbs) selected to recognize components from a Triton X-100 extract of ovine oligodendrocytes. One of these Abs, mAb H, recognizes an O-linked N-acetyl glucosamine residue in a specific conformation and/or environment. mAb H stained, weakly, two bands with Mr x 10(-3) of 209 and 62 in lysates of cultured rat astrocytes, suggesting antigens of low abundance. We have employed immunohistochemistry to investigate the cell and tissue distribution of the mAb H antigen(s). In normal rat and human brains, the sparse reaction products detected were confined, mostly, to fibrous astrocytes. In sharp contrast, when pathological specimens from a variety of brain lesions, including anisomorphic and isomorphic gliosis, were examined, a strong reaction with mAb H was in evidence in all reactive astrocytes, independent of the origin or nature of the lesions. This we interpret as meaning that the gene product(s) recognized by this mAb is (are) upregulated or induced following injury to the brain. Hence, epitope H represents a new addition to the list of molecules that are affected by brain injury. Structural and functional identification of the antigen(s) should shed light on its (their) relevance to the pathophysiology of the disease process.

Fertilized hens' eggs were externally treated, before starting incubation, with a single dose of 2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e., 3.1 mg/egg). Chicks at different developmental stages were examined, extending from 10 day embryos to one day after hatching. Previously, we demonstrated that 2,4-D b.e. produces hypomyelination in chicks born from treated eggs. In search of the causes of this hypomyelination, "myelin markers" such as sulphatides, cerebrosides and 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) activity, as well as protein and nucleic acid contents were determined in the embryonic brains. We have shown in the present study that the chemical alterations occurred even before the period of active myelination, since myelin appears in chicken brain stem and cerebrum approximately after 17 days of incubation, and most of the chemical parameters studied are diminished before that time. The DNA content in brain of treated group is increased from the 14th embryonic day (with a transient diminution at 12th day) to the first hatching day, when compared to the control, suggesting a proliferation of glial cells, possibly oligodendrocytes.

Fertilized hens' eggs were treated externally with 2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e.) (3.1 mg/egg) immediately before starting incubation, and after different times of incubation (5, 10 and 15 days). Controls were treated externally with ether. Hatchability studies demonstrated that fetotoxic effects of 2,4-D b.e. were similar on the 0, 5 and 10 incubation day, but the 15 Day Group improved the hatching percentage. One day after hatching, chicks were decapitated, and CNS tissue was dissected. Myelin markers, as cerebrosides and CNP, were determined in cerebrum, cerebellum, brain stem and spinal cord of the four groups. They were reduced in cerebrum and brain stem of the 0, 5 and 10 Day Groups, but in the 15 Day Group they were in normal levels. Cerebellum presented normal myelin marker contents in each group studied, while spinal cord only presented decreased marker contents in the 5 Day Group. UDP galactose-ceramide galactosyl transferase (EC 2.4.1.45) activity was reduced in whole brain of chicks born from eggs treated preincubation. The results show the importance of time drug application and suggest that the vulnerable period in CNS development includes proliferation and development of myelin forming cells. Among CNS regions, cerebrum and brain stem seem to be the most vulnerable to the toxic action of 2,4-D b.e. in the chick.

The effects of 2,4-dichlorophenoxyacetic butyl ester (2,4-D b.e.) on protein and nucleic acid contents of different tissues of chicks born from externally treated hen's eggs were studied. Residues of 2,4-D b.e. were also determined and quantified by gas liquid chromatography (GLC). A diminished DNA content was observed in skeletal muscle and stomach, an increased one was found in cerebrum and cerebellum, whereas no variations were observed in the other analysed tissues. Protein levels were found decreased in kidney, cerebrum and heart. Besides, the GLC determinations revealed the presence of the 2,4-D b.e. in all the studied tissues finding the highest concentrations in kidney. 2,4-D did not appear to be a significant metabolite of 2,4-D b.e. in this experimental system. The dissimilar effects of 2,4-D b.e. on protein and nucleic acid contents of the studied tissues would be related to a different tissue susceptibility to the drug and/or to drug distribution.

Previous studies have shown that 2,4-Dichlorophenoxyacetic butyl ester (2,4-D b.e.) causes hypomyelination in chicks born from eggs externally treated and alters the myelin chemical composition. In this paper the effect of 2,4-D b.e. on myelin phospholipid and fatty acid composition has been examined. The results of our investigations show significant variations in the phospholipid composition, with the phosphatidyl inositol content increased and sphingomyelin and phosphatidyl ethanolamine contents diminished. The fatty acid pattern of the individual myelin lipids is also significantly altered, with an important reduction of long chain fatty acids and an increase of saturated fatty acids. The observed changes in the chemical composition implicate alterations in the intrinsic properties of this membrane.

Fertilized hen's eggs were painted, before starting incubation, with solutions of commercial 2,4-dichlorophenoxyacetic butyl ester, (0.8 mg, 3.1 mg, 6.3 mg, 9.4 mg and 12.1 mg of pure drug) in ether. Hatchability studies suggest that the fetotoxic effects observed are related to the drug concentration. There are no statistically significant differences between control and treated groups in body weight, cerebral wet, and dry weights and water content. Protein and total lipid levels are dose dependently decreased. Phospholipids are significantly diminished in the 6.3 mg of 2,4-dichlorophenoxyacetic butyl ester group. Gangliosides are increased while cholesterol and glycolipids are diminished; their decrease is inverse to drug concentration. A decrease in glycolipids suggests an alteration of the normal process of myelination.

Fertilized hens' eggs were externally treated with 2,4-dichlorophenoxyacetic (2,4-D) butyl ester (2.4 mg/egg) before starting incubation. The specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) was diminished in the whole brain of animals born from treated eggs, as compared with controls. Myelin was isolated by ultracentrifugation from the central nervous system. Treatment with 2,4-D butyl ester produced a 65% reduction. There were no statistically significant differences in myelin phospholipids, cerebrosides, sulphatides and gangliosides between control and treated animals. Cholesterol was diminished by 12% in the treated group, while cholesterol esters were not detectable in either myelin fraction. Total myelin proteins were also decreased by 40%, without variations in their pattern. There were changes in the following ratios in the myelin of the treated group: lipid/protein, cholesterol/phospholipids, cholesterol/protein, phospholipids/protein. Collectively these findings indicate that 2,4-D butyl ester produced hypomyelination.