Several strains, one disease: experimental investigation of Vibrio aestuarianus infection parameters in the Pacific oyster, Crassostrea gigas.

This study investigated oyster infection dynamics by different strains of Vibrio aestuarianus isolated before and after the apparent re-emergence of this pathogen observed in France in 2011. We conducted experiments to compare minimal infective dose, lethal dose 50 and bacterial shedding for six V. aestuarianus strains. Whatever the strain used, mortality was induced in juvenile oysters by intramuscular injection and reached 90-100% of mortality within 5 days. Moreover, bacterial shedding was comparable among strains and reached its maximum after 20 h (≈10 EXP5 bacteria/mL/animal). Similarly, our first estimations of lethal dose 50 were comparable among strains (minimal infective dose around 0.4 × 10EXP5 bacteria/mL and LD50 around 10EXP5 bacteria/mL) by using seawater containing freshly shed bacteria. These results indicate that, at least with these criteria, despite V. aestuarianus strains genetic diversity, the disease process is similar. The strains isolated after the apparent re-emergence of the bacteria in 2011, do not present a more acute virulence phenotype than the reference strains isolated between 2002 and 2007. Finally, our study provides original and noteworthy data indicating that infected oysters shed bacteria at a level above the threshold of LD50 a few days before they die, meaning that infection is expected to spread in a susceptible population.

Genomics investigation of the potentially invasive firefly Photinus signaticollis Blanchard 1845: Complete mitochondrial genome, multigene phylogenies and obtention of the luciferase and luciferin-regenerating genes.

A genomic investigation of the potentially invasive firefly Photinus signaticollis Blanchard1845 has been performed and led to the obtention of its complete 16,411 bp long mitochondrial genome. The mitogenome encodes 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes. With other species of the Photinus complex it shares several premature terminations of some protein-coding genes and also an overlap between cox1 and tRNA-Tyr. By data-mining, the complete luciferase and luciferin-regenerating genes were also identified from the contigs file and compared with existing data, in addition to WG and CAD, two genes used in pioneering phylogenetic studies on fireflies. Three maximum likelihood phylogenies were derived from all these data. The multigene phylogeny based on all mitochondrial protein-coding genes strongly associates P. signaticollis with Photinus pyralis Linnaeus, 1758 and the lantern-less daily "winter firefly", Photinus corruscus Linnaeus, 1767. A second phylogeny based on concatenated sequences of the cox1, WG and CAD genes positions P. signaticollis as a sister clade to a large cluster of species containing the 7 sub-groups previously evidenced among the North American species of the Photinus complex. A third phylogeny based on the amino-acid sequence of the luciferase protein associates P. signaticollis to Photinus scintillans. The analysis presented here will most certainly help to come to a better understanding of the very complex inter-relationships in the very large Photinus genus.

The mitochondrial genome of the bioluminescent fish Malacosteusniger Ayres, 1848 (Stomiidae, Actinopterygii) is large and complex, and contains an inverted-repeat structure.

We determined the complete mitogenome sequence of the bioluminescent fish Malacosteusniger using long-read sequencing technologies. The 21,263 bp mitogenome features a complex structure with two copies of a 1198-bp inverted-repeat and a region of 2616-bp containing alternating copies of 16 and 26 bp repeat elements. Whole mitogenome phylogenies inferred from both nucleotide and amino-acid datasets place M.niger among Melanostomiinae. The need for additional complete mitogenome sequences from the subfamily Malacosteinae is discussed.

Quick Spreading of Populations of an Exotic Firefly throughout Spain and Their Recent Arrival in the French Pyrenees.

In August 2018, a firefly (Coleoptera: Lampyridae) of American origin was observed in several localities in Girona (Catalonia, Spain) and was described as Photinus immigrans by Zaragoza-Caballero and Vinolas, 2018. Here, we show that this species dispersed very quickly throughout northeastern Spain and was, in 2020, observed in the French Pyrenees. The animal's quick progress is documented, and part of its biology is described (dispersion speed, land use, phenology, identification of all life stages). An additional population was localized in Extremadura, and its special status is discussed. We were able to determine its Argentinian-Uruguayan origin and propose, therefore, to consider Photinus immigrans as a synonym of Photinus signaticollis (Blanchard, 1846) (=Photinus immigrans Zaragoza-Caballero and Viñolas, 2018, syn. nov.). Our data clearly show that at least the Catalan and French populations are spreading very quickly and are able to settle permanently if adequate ecosystems are found. The species is highly expansive and may well be invasive; our citizen science platforms are ideally suited to monitor their progress throughout Spain and France. This is important for avoiding future ecological problems with diverse native faunas, such as glow-worms, fireflies and earthworms. If no ways are found to stop the species' progression, the animals will quite probably invade substantial areas of France, Spain and the rest of Europe in the years to come.

Easier detection of invertebrate "identification-key characters" with light of different wavelengths.

The marine α-taxonomist often encounters two problems. Firstly, the "environmental dirt" that is frequently present on the specimens and secondly the difficulty in distinguishing key-features due to the uniform colours which fixed animals often adopt.Here we show that illuminating animals with deep-blue or ultraviolet light instead of the normal white-light abrogates both difficulties; dirt disappears and important details become clearly visible. This light regime has also two other advantages. It allows easy detection of very small, normally invisible, animals (0.1 µm range). And as these light wavelengths can induce fluorescence, new identification markers may be discovered by this approach.

Pathogenic Vibrio harveyi, in contrast to non-pathogenic strains, intervenes with the p38 MAPK pathway to avoid an abalone haemocyte immune response.

Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone epidemics associated with massive mortalities in France, Japan, and Australia. The aim of this study was the understanding of a possible role of the p38 MAPK in abalone haemocyte responses towards this bacterium. First, the pathogenicity of different V. harveyi strains was compared in both immersion and injection trials, and clear differences were detected. The three strains, ORM4, 04/092, and 05/053, all isolated from moribund abalone, induced up to 80% mortalities in immersion or injection challenges (LD(50) (ORM4) = 2.5 x 10(2) CFU animal(-1)). The two strains, LMG 4044T and LMG 7890 were non-pathogenic towards abalone in immersion trials, and needed very high numbers for killing by intramuscular injections (LD(50) = 8.9 x 10(4) and 1.6 x 10(5) CFU animal(-1), respectively). To start unraveling the mechanism explaining these differences, the p38-MAPK, a keyplayer in antimicrobial immune response, was studied. The non-pathogenic strain, LMG 7890 can be eliminated by abalone haemocytes and induces haemocyte phagocytosis and high ROS production. With different concentrations of a p38-specific inhibitor, SB203580, p38 implication was shown. This inhibitor reduced phagocytosis and ROS induction leading to LMG 7890 proliferation. In the case of the pathogenic ORM4 which can not be eliminated by abalone haemocytes, no phagocytosis and ROS production was induced, and a retarded p38 activation was observed. Taken together, our results suggest that p38 MAPK modulation may be one of the ways of virulent V. harveyi to attack its host and escape abalone immune response.

Construction of a stable GFP-tagged Vibrio harveyi strain for bacterial dynamics analysis of abalone infection.

Vibrio harveyi is a bacterial marine pathogen that can cause fatal disease in a large range of vertebrates and invertebrates, including the commercially important marine gastropod, Haliotis tuberculata. Since 1997, strains of this bacterium have regularly been causing high mortalities in farmed and wild abalone populations. The way in which the pathogen enters into abalone and the disease transmission mechanisms are thus far unknown. Therefore, a pathogenic strain, ORM4, was green fluorescent protein-tagged and validated both for its growth characteristics and for its virulence as a genuine model for abalone disease. The strain allows V. harveyi quantification by flow cytometry in seawater and in abalone haemolymph as well as the in situ detection of the parasite inside abalone tissues.

Diversity of cytosolic HSP70 Heat Shock Protein from decapods and their phylogenetic placement within Arthropoda.

The 70kDa heat shock proteins (HSP70) are considered the most conserved members of the HSP family. These proteins are primordial to the cell, because of their implications in many cellular pathways (e. g., development, immunity) and also because they minimize the effects of multiple stresses (e. g., temperature, pollutants, salinity, radiations). In the cytosol, two ubiquitous HSP70s with either a constitutive (HSC70) or an inducible (HSP70) expression pattern are found in all metazoan species, encoded by 5 or 6 genes (Drosophila melanogaster or yeast and human respectively). The cytosolic HSP70 protein family is considered a major actor in environmental adaptation, and widely used in ecology as an important biomarker of environmental stress. Nevertheless, the diversity of cytosolic HSP70 remains unclear amongst the Athropoda phylum, especially within decapods. Using 122 new and 311 available sequences, we carried out analyses of the overall cytosolic HSP70 diversity in arthropods (with a focus on decapods) and inferred molecular phylogenies. Overall structural and phylogenetic analyses showed a surprisingly high diversity in cytosolic HSP70 and revealed the existence of several unrecognised groups. All crustacean HSP70 sequences present signature motifs and molecular weights characteristic of non-organellar HSP70, with multiple specific substitutions in the protein sequence. The cytosolic HSP70 family in arthropods appears to be constituted of at least three distinct groups (annotated as A, B and C), which comprise several subdivisions, including both constitutive and inducible forms. Group A is constituted by several classes of Arthropods, while group B and C seem to be specific to Malacostraca and Hexapoda/Chelicerata, respectively. The HSP70 organization appeared much more complex than previously suggested, and far beyond a simple differentiation according to their expression pattern (HSC70 versus HSP70). This study proposes a new classification of cytosolic HSP70 and an evolutionary model of the distinct forms amongst the Arthropoda phylum. The observed differences between HSP70 groups will probably have to be linked to distinct interactions with co-chaperones or other co-factors.

Establishment of stable GFP-tagged Vibrio aestuarianus strains for the analysis of bacterial infection-dynamics in the Pacific oyster, Crassostrea gigas.

Several marine pathogens are thought to be implicated in the summer mortality phenomenon that strikes the Pacific oyster stocks (Crassostrea gigas) in Europe since more than a decade. Although, since 2008, a herpes virus variant (microvar) is considered the main responsible for juvenile mortalities, the role of several associated bacteria is less clear. One of these, Vibrio aestuarianus, has often been detected in moribund oysters, and laboratory challenges proved its involvement in oyster death. However, the mechanisms by which this pathogen enters the oyster and transmits in-between specimens or collaborates with other pathogens remain thus far almost unknown. To establish genuine model strains, which allow the detection of the bacteria during the first hours of infection, both a highly pathogenic (02/41) and a weakly pathogenic strain (01/308) were transformed with green fluorescent protein-expression vectors. The clones obtained were compared to the parental strains for their growth characteristics, basic metabolism, antibiotic-resistance and virulence. The 02/41 derivative was in all aspects indistinguishable from the parental strain. In contrast, in the 01/308 strain GFP expression led to a significant increase of virulence pointing to the dangers of GFP-tagging. The 02/41 GFP strain allows easy quantification by flow cytometry in both seawater and oyster haemolymph, and most importantly, its in situ detection will permit discerning the bacterium's routes inside the oyster tissues.

PML/RARA oxidation and arsenic binding initiate the antileukemia response of As2O3.

As(2)O(3) cures acute promyelocytic leukemia (APL) by initiating PML/RARA oncoprotein degradation, through sumoylation of its PML moiety. However, how As(2)O(3) initiates PML sumoylation has remained largely unexplained. As(2)O(3) binds vicinal cysteines and increases reactive oxygen species (ROS) production. We demonstrate that upon As(2)O(3) exposure, PML undergoes ROS-initiated intermolecular disulfide formation and binds arsenic directly. Disulfide-linked PML or PML/RARA multimers form nuclear matrix-associated nuclear bodies (NBs), become sumoylated and are degraded. Hematopoietic progenitors transformed by an As(2)O(3)-binding PML/RARA mutant exhibit defective As(2)O(3) response. Conversely, nonarsenical oxidants elicit PML/RARA multimerization, NB-association, degradation, and leukemia response in vivo, but do not affect PLZF/RARA-driven APLs. Thus, PML oxidation regulates NB-biogenesis, while oxidation-enforced PML/RARA multimerization and direct arsenic-binding cooperate to enforce APL's exquisite As(2)O(3) sensitivity.

Summer immune depression associated with increased susceptibility of the European abalone, Haliotis tuberculata to Vibrio harveyi infection.

Haliotis tuberculata mortality outbreaks have occurred in France since 1998 and were attributed to a pathogenic Vibrio harveyi. These mortalities were recorded in September, a month with abalone reproduction and characterised by high seawater temperatures. The importance of gonadal maturation and temperature increase on abalone immunity and susceptibility to V. harveyi infection needed to be clarified. Therefore, an immune survey analyzing a large panel of parameters was performed from June to September 2007 on abalone from the Bay of Brest. The data obtained were put in relation with abalone reproductive status and its susceptibility to V. harveyi. Most parameters showed clear patterns from early to late summer and during gametogenesis, phagocytosis and phenoloxidase activity were reduced, whereas basal reactive oxygen species production and agglutination titres were significantly increased. Total haemocyte counts went up after the partial spawning event at the end of June, and cell complexity diminished. Using a Principal Component Analysis, the "haemolymph profile" was shown to decrease in parallel with spawning and gonadal maturation processes, and reached a minimum just after total spawning. A significant correlation between this "haemolymph profile" and disease susceptibility allowed us to establish for the first time in abalone, a clear concordance between maturation and spawning processes, immune status and abalone susceptibility to V. harveyi.

Morphologic, cytometric and functional characterisation of abalone (Haliotis tuberculata) haemocytes.

This work presents the first detailed microscopic and functional analysis of the haemocytes of an abalone; the European Haliotis tuberculata. It is shown that in contrast to the situation in bivalves, only very few basophilic "granulocytes" could be found and exclusively with a histological stain. Neither flow cytometry, phase contrast observation nor transmission electron microscopy were able to detect any granular cells. The large majority of cells was constituted of "hyalinocytes", which could be sorted by flow cytometry, for the first time, into small (blast-like) and large cells. This permits a detailed analysis of haemocytes and especially of the lowly represented blast-like cells. The differences in haemolymph cell composition between bivalves and gastropods is reviewed in depth and discussed in view of the new data we present. Most of the abalone haemocytes analysed harbour many vacuoles, large glycogen deposits, lipid inclusions and acidic compartments. However, although the number of these "inclusions" was rather variable in between individual hyalinocytes, these experiments did not allow to discern subpopulations using these criteria, and the population appears more as a "differentiation continuum". Haemocytes adhere very rapidly and are immunologically active as they quickly phagocytose latex beads and zymozan particles. This study is the first step towards understanding the H. tuberculata immune system by adapting new tools to gastropods and in providing a first detailed morpho-functional study of their haemocytes.

Roscovitine targets, protein kinases and pyridoxal kinase.

(R)-Roscovitine (CYC202) is often referred to as a "selective inhibitor of cyclin-dependent kinases." Besides its use as a biological tool in cell cycle, neuronal functions, and apoptosis studies, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, viral infections, and glomerulonephritis. We have investigated the selectivity of (R)-roscovitine using three different methods: 1) testing on a wide panel of purified kinases that, along with previously published data, now reaches 151 kinases; 2) identifying roscovitine-binding proteins from various tissue and cell types following their affinity chromatography purification on immobilized roscovitine; 3) investigating the effects of roscovitine on cells deprived of one of its targets, CDK2. Altogether, the results show that (R)-roscovitine is rather selective for CDKs, in fact most kinases are not affected. However, it binds an unexpected, non-protein kinase target, pyridoxal kinase, the enzyme responsible for phosphorylation and activation of vitamin B6. These results could help in interpreting the cellular actions of (R)-roscovitine but also in guiding the synthesis of more selective roscovitine analogs.

Hypoxia enhances human B19 erythrovirus gene expression in primary erythroid cells.

Human B19 erythrovirus replicates in erythroid progenitors present in bone marrow and fetal tissues where partial oxygen tension is low. Here we show that infected human primary erythroid progenitor cells exposed to hypoxia (1% O2) in vitro increase viral capsid protein synthesis, virus replication, and virus production. Hypoxia-inducible factor-1 (HIF-1), the main transcription factor involved in the cellular response to reduced oxygenation, is shown to bind an HIF binding site (HBS) located in the distal part of the B19 promoter region, but the precise mechanism involved in the oxygen-sensitive upregulation of viral gene expression remains to be elucidated.