What Was Old Is New Again: The Pennate Diatom Haslea ostrearia (Gaillon) Simonsen in the Multi-Omic Age.

The marine pennate diatom Haslea ostrearia has long been known for its characteristic blue pigment marennine, which is responsible for the greening of invertebrate gills, a natural phenomenon of great importance for the oyster industry. For two centuries, this taxon was considered unique; however, the recent description of a new blue Haslea species revealed unsuspected biodiversity. Marennine-like pigments are natural blue dyes that display various biological activities-e.g., antibacterial, antioxidant and antiproliferative-with a great potential for applications in the food, feed, cosmetic and health industries. Regarding fundamental prospects, researchers use model organisms as standards to study cellular and physiological processes in other organisms, and there is a growing and crucial need for more, new and unconventional model organisms to better correspond to the diversity of the tree of life. The present work, thus, advocates for establishing H. ostrearia as a new model organism by presenting its pros and cons-i.e., the interesting aspects of this peculiar diatom (representative of benthic-epiphytic phytoplankton, with original behavior and chemodiversity, controlled sexual reproduction, fundamental and applied-oriented importance, reference genome, and transcriptome will soon be available); it will also present the difficulties encountered before this becomes a reality as it is for other diatom models (the genetics of the species in its infancy, the transformation feasibility to be explored, the routine methods needed to cryopreserve strains of interest).

Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes.

Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 (CCR1), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1-edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1-edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes.

Complex Relationships between the Blue Pigment Marennine and Marine Bacteria of the Genus Vibrio.

Marennine, the water-soluble blue pigment produced by the marine diatom Haslea ostrearia, is known to display antibacterial activities. Previous studies have demonstrated a prophylactic effect of marennine on bivalve larvae challenged with a pathogenic Vibrio splendidus, suggesting that the blue Haslea is a good candidate for applications in aquaculture as a source of a natural antimicrobial agent. Indeed, the genus Vibrio is ubiquitous in aquaculture ecosystems, and regular events of pathogenic invasion cause some of the biggest losses worldwide. To better characterize the effects of marennine on Vibrios, a panel of 30 Vibrio strains belonging to 10 different species was tested, including bivalve pathogenic species (e.g., Vibrio crassostreae and Vibrio harveyi). Vibrio strains were first exposed to 10 and 25 µg mL-1 of Blue Water (BW), a concentrated culture supernatant of H. ostrearia containing marennine. This screening evidenced a great diversity in responses, from growth stimulation to a total inhibition, at both the interspecific or intraspecific level. In a second series of experiments, 10 Vibrio strains were exposed to BW at concentrations ranging from 5 to 80 µg mL-1. The highest concentrations of BW did not systematically result in the highest growth inhibition as hormetic responses-opposite effects regarding the concentration-were occasionally evidenced. The relationships between marennine and Vibrio strains appear more complex than expected and justify further study-in particular, on the mechanisms of action-before considering applications as a natural prophylactic or antibiotic agent in aquaculture.

Metabolite Quantification by Fourier Transform Infrared Spectroscopy in Diatoms: Proof of Concept on Phaeodactylum tricornutum.

Diatoms are feedstock for the production of sustainable biocommodities, including biofuel. The biochemical characterization of newly isolated or genetically modified strains is seminal to identify the strains that display interesting features for both research and industrial applications. Biochemical quantification of organic macromolecules cellular quotas are time-consuming methodologies which often require large amount of biological sample. Vibrational spectroscopy is an essential tool applied in several fields of research. A Fourier transform infrared (FTIR) microscopy-based imaging protocol was developed for the simultaneous cellular quota quantification of lipids, carbohydrates, and proteins of the diatom Phaeodactylum tricornutum. The low amount of sample required for the quantification allows the high throughput quantification on small volume cultures. A proof of concept was performed (1) on nitrogen-starved experimental cultures and (2) on three different P. tricornutum wild-type strains. The results are supported by the observation in situ of lipid droplets by confocal and brightfield microscopy. The results show that major differences exist in the regulation of lipid metabolism between ecotypes of P. tricornutum.

Screening of Helicoverpa armigera Mobilome Revealed Transposable Element Insertions in Insecticide Resistance Genes.

The cotton bollworm Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is an important pest of many crops that has developed resistance to almost all groups of insecticides used for its management. Insecticide resistance was often related to Transposable Element (TE) insertions near specific genes. In the present study, we deeply retrieve and annotate TEs in the H. armigera genome using the Pipeline to Retrieve and Annotate Transposable Elements, PiRATE. The results have shown that the TE library consists of 8521 sequences representing 236,132 TE copies, including 3133 Full-Length Copies (FLC), covering 12.86% of the H. armigera genome. These TEs were classified as 46.71% Class I and 53.29% Class II elements. Among Class I elements, Short and Long Interspersed Nuclear Elements (SINEs and LINEs) are the main families, representing 21.13% and 19.49% of the total TEs, respectively. Long Terminal Repeat (LTR) and Dictyostelium transposable element (DIRS) are less represented, with 5.55% and 0.53%, respectively. Class II elements are mainly Miniature Inverted Transposable Elements (MITEs) (49.11%), then Terminal Inverted Repeats (TIRs) (4.09%). Superfamilies of Class II elements, i.e., Transib, P elements, CACTA, Mutator, PIF-harbinger, Helitron, Maverick, Crypton and Merlin, were less represented, accounting for only 1.96% of total TEs. In addition, we highlighted TE insertions in insecticide resistance genes and we successfully identified nine TE insertions belonging to RTE, R2, CACTA, Mariner and hAT superfamilies. These insertions are hosted in genes encoding cytochrome P450 (CyP450), glutathione S-transferase (GST), and ATP-binding cassette (ABC) transporter belonging to the G and C1 family members. These insertions could therefore be involved in insecticide resistance observed in this pest.

Investigating the Molecular Genetic Basis of Cytoplasmic Sex Determination Caused by Wolbachia Endosymbionts in Terrestrial Isopods.

In animals, sexual differences between males and females are usually determined by sex chromosomes. Alternatively, sex may also be determined by vertically transmitted intracellular microbial endosymbionts. The best known cytoplasmic sex manipulative endosymbiont is Wolbachia which can, for instance, feminize genetic males into phenotypic females in the terrestrial isopod Armadillidium vulgare. However, the molecular genetic basis of cytoplasmic sex determination is unknown. To identify candidate genes of feminization induced by Wolbachia strain wVulC from A. vulgare, we sequenced the genome of Wolbachia strain wCon from Cylisticus convexus, the most closely related known Wolbachia strain to wVulC that does not induce feminization, and compared it to the wVulC genome. Then, we performed gene expression profiling of the 216 resulting wVulC candidate genes throughout host developmental stages in A. vulgare and the heterologous host C. convexus. We identified a set of 35 feminization candidate genes showing differential expression during host sexual development. Interestingly, 27 of the 35 genes are present in the f element, which is a piece of a feminizing Wolbachia genome horizontally transferred into the nuclear genome of A. vulgare and involved in female sex determination. Assuming that the molecular genetic basis of feminization by Wolbachia and the f element is the same, the 27 genes are candidates for acting as master sex determination genes in A. vulgare females carrying the f element.

Feminization of the Isopod Cylisticus convexus after Transinfection of the wVulC Wolbachia Strain of Armadillidium vulgare.

Reproductive parasites such as Wolbachia are able to manipulate the reproduction of their hosts by inducing parthenogenesis, male-killing, cytoplasmic incompatibility or feminization of genetic males. Despite extensive studies, no underlying molecular mechanism has been described to date. The goal of this study was to establish a system with a single Wolbachia strain that feminizes two different isopod species to enable comparative analyses aimed at elucidating the genetic basis of feminization. It was previously suggested that Wolbachia wVulC, which naturally induces feminization in Armadillidium vulgare, induces the development of female secondary sexual characters in transinfected Cylisticus convexus adult males. However, this does not demonstrate that wVulC induces feminization in C. convexus since feminization is the conversion of genetic males into functional females that occurs during development. Nevertheless, it suggests that C. convexus may represent a feminization model suitable for further development. Knowledge about C. convexus sexual differentiation is also essential for comparative analyses, as feminization is thought to take place just before or during sexual differentiation. Consequently, we first described gonad morphological differentiation of C. convexus and compared it with that of A. vulgare. Then, wVulC was injected into male and female C. convexus adult individuals. The feminizing effect was demonstrated by the combined appearance of female secondary sexual characters in transinfected adult males, as well as the presence of intersexes and female biases in progenies in which wVulC was vertically transmitted from transinfected mothers. The establishment of a new model of feminization of a Wolbachia strain in a heterologous host constitutes a useful tool towards the understanding of the molecular mechanism of feminization.

Multiple Conserved Heteroplasmic Sites in tRNA Genes in the Mitochondrial Genomes of Terrestrial Isopods (Oniscidea).

Mitochondrial genome structure and organization are relatively conserved among metazoans. However, in many isopods, especially the terrestrial isopods (Oniscidea), the mitochondrial genome consists of both ∼14-kb linear monomers and ∼28-kb circular dimers. This unusual organization is associated with an ancient and conserved constitutive heteroplasmic site. This heteroplasmy affects the anticodon of a tRNA gene, allowing this single locus to function as a "dual" tRNA gene for two different amino acids. Here, we further explore the evolution of these unusual mitochondrial genomes by assembling complete mitochondrial sequences for two additional Oniscidean species, Trachelipus rathkei and Cylisticus convexus. Strikingly, we find evidence of two additional heteroplasmic sites that also alter tRNA anticodons, creating additional dual tRNA genes, and that are conserved across both species. These results suggest that the unique linear/circular organization of isopods' mitochondrial genomes may facilitate the evolution of stable mitochondrial heteroplasmies, and, conversely, once such heteroplasmies have evolved, they constrain the multimeric structure of the mitochondrial genome in these species. Finally, we outline some possible future research directions to identify the factors influencing mitochondrial genome evolution in this group.

Signs of neutralization in a redundant gene involved in homologous recombination in Wolbachia endosymbionts.

Genomic reduction in bacterial endosymbionts occurs through large genomic deletions and long-term accumulation of mutations. The latter process involves successive steps including gene neutralization, pseudogenization, and gradual erosion until complete loss. Although many examples of pseudogenes at various levels of degradation have been reported, neutralization cases are scarce because of the transient nature of the process. Gene neutralization may occur due to relaxation of selection in nonessential genes, for example, those involved in redundant functions. Here, we report an example of gene neutralization in the homologous recombination (HR) pathway of Wolbachia, a bacterial endosymbiont of arthropods and nematodes. The HR pathway is often depleted in endosymbiont genomes, but it is apparently intact in some Wolbachia strains. Analysis of 12 major HR genes showed that they have been globally under strong purifying selection during the evolution of Wolbachia strains hosted by arthropods, supporting the evolutionary importance of the HR pathway for these Wolbachia genomes. However, we detected signs of recent neutralization of the ruvA gene in a subset of Wolbachia strains, which might be related to an ancestral, clade-specific amino acid change that impaired DNA-binding activity. Strikingly, RuvA is part of the RuvAB complex involved in branch migration, whose function overlaps with the RecG helicase. Although ruvA is experiencing neutralization, recG is under strong purifying selection. Thus, our high phylogenetic resolution suggests that we identified a rare example of targeted neutralization of a gene involved in a redundant function in an endosymbiont genome.