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.

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.

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.

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.

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).

La symbiose est l'un des principaux moteurs de l'évolution. Le génotype du symbiote est capable d'altérer le phénotype de l'hôte, et vice-versa : c'est le « phénotype étendu ». Dans ce contexte, les endosymbioses à Wolbachia sont remarquables. Cette bactérie intracellulaire est un parasite de la reproduction capable d'induire la féminisation des mâles génétiques ou l'incompatibilité cytoplasmique chez ses hôtes crustacés isopodes terrestres. Actuellement, aucun mécanisme moléculaire régissant ces effets n'est connu. Dans le but d'identifier des gènes impliqués dans la féminisation, nous avons utilisé une approche intégrative qui combine à la fois des analyses génomiques, d'expression de gènes et phénotypiques. Nous avons tout d'abord analysé l'évolution moléculaire de la voie de la recombinaison homologue dans les génomes de Wolbachia, source importante de plasticité génomique pouvant être liée à la diversité des phénotypes. Ensuite, afin d'effectuer des études comparatives qui augmenteraient considérablement la compréhension des mécanismes de la féminisation, nous avons établi un système où la souche féminisante wVulC féminise deux hôtes isopodes (hôte naturel : Armadillidum vulgare : hôte hétérologue : Cylisticus convexus) présentant un timing différent de la différenciation sexuelle. En effet, l'effet féminisant étant supposé avoir lieu avant ou pendant la différenciation sexuelle, il est important de distinguer l'effet de Wolbachia dû à la différenciation sexuelle de celui dû au développement. Enfin, une approche par gènes candidats (du séquençage de génome bactérien à l'analyse comparative d'expression de gènes bactériens durant le développement de l'hôte) a permis de déterminer une liste réduite de 29 gènes (parmi les 1885 gènes de wVulC) dont la probabilité qu'ils soient impliqués dans la féminisation est élevée. Le rôle potentiel de ces gènes candidats comme effecteurs supposés de la féminisation induite par wVulC est ensuite discuté. Ce travail contribue grandement à l'identification de facteurs potentiels d'endosymbiotes qui ont un impact évolutif sur la détermination du sexe de leurs hôtes.