Structural and functional characterization of an egg-laying hormone signaling system in a lophotrochozoan - The pacific oyster (Crassostrea gigas).

The egg-laying hormones (ELHs) of gastropod mollusks were characterized more than forty years ago. Yet, they have remained little explored in other mollusks. To gain insights into the functionality of the ELH signaling system in a bivalve mollusk - the oyster Crassostrea gigas, this study investigates the processing of its ELH precursor (Cragi-ELH) by mass spectrometry. Some of the ELH mature peptides identified in this study were subsequently investigated by nuclear magnetic resonance and shown to adopt an extended alpha-helix structure in a micellar medium mimicking the plasma membrane. To further characterize the ELH signaling system in C. gigas, a G protein-coupled receptor phylogenetically related to ecdysozoan diuretic hormone DH44 and corticotropin-releasing hormone (CRH) receptors named Cragi-ELHR was also characterized functionally and shown to be specifically activated by the two predicted mature ELH peptides and their N-terminal fragments. Both Cragi-ELH and Cragi-ELHR encoding genes were mostly expressed in the visceral ganglia (VG). Cragi-ELH expression was significantly increased in the VG of both fully mature male and female oysters at the spawning stage. When the oysters were submitted to a nutritional or hyposaline stress, no change in the expression of the ligand or receptor genes was recorded, except for Cragi-ELHR only during a mild acclimation episode to brackish water. These results suggest a role of Cragi-ELH signaling in the regulation of reproduction but not in mediating the stress response in our experimental conditions.

A proteomic study of resistance to Brown Ring disease in the Manila clam, Ruditapes philippinarum.

Marine mollusk aquaculture has more than doubled over the past twenty years, accounting for over 15% of total aquaculture production in 2016. Infectious disease is one of the main limiting factors to the development of mollusk aquaculture, and the difficulties inherent to combating pathogens through antibiotic therapies or disinfection have led to extensive research on host defense mechanisms and host-pathogen relationships. It has become increasingly clear that characterizing the functional profiles of response to a disease is an essential step in understanding resistance mechanisms and moving towards more effective disease control. The Manila clam, Ruditapes philippinarum, is a main cultured bivalve species of economic importance which is affected by Brown Ring disease (BRD), an infection induced by the bacterium Vibrio tapetis. In this study, juvenile Manila clams were subjected to a 28-day controlled challenge with Vibrio tapetis, and visual and molecular diagnoses were carried out to distinguish two extreme phenotypes within the experimental clams: uninfected ("RES", resistant) and infected ("DIS", diseased) post-challenge. Total protein extractions were carried out for resistant and diseased clams, and proteins were identified using LC-MS/MS. Protein sequences were matched against a reference transcriptome of the Manila clam, and protein intensities based on label-free quantification were compared to reveal 49 significantly accumulated proteins in resistant and diseased clams. Proteins with known roles in pathogen recognition, lysosome trafficking, and various aspects of the energy metabolism were more abundant in diseased clams, whereas those with roles in redox homeostasis and protein recycling were more abundant in resistant clams. Overall, the comparison of the proteomic profiles of resistant and diseased clams after a month-long controlled challenge to induce the onset of Brown Ring disease suggests that redox homeostasis and maintenance of protein structure by chaperone proteins may play important and interrelated roles in resistance to infection by Vibrio tapetis in the Manila clam.

Behavior of Antimicrobial Peptide K4 in a Marine Environment.

K4 is a de novo peptide with antibacterial activity on human pathogens. It has a short sequence (14 amino acids), with a cationic N-terminal moiety and an amphipathic ɑ-helix structure. The present paper demonstrates its activity on Vibrio bacteria in a marine environment. It was found non-toxic on marine organisms including Artemia salina, Dicentrarchus labrax, and Magallana gigas at different developmental stages, but influenced the growth of unicellular organisms like microalgae, depending on the algal strain and on K4 concentration. Furthermore, an original approach coupling liquid chromatography (RP-HPLC) and mass spectrometry (MS/MS) allowed us to monitor the degradation time course of the peptide for the first time in conditions close to a hatchery environment, i.e., in the presence of oyster spat. We detected truncated forms over time, and the full K4 was gradually no longer found in these filter-feeder oysters. Finally, using an automated optical density meter, we monitored the growth of several aquatic bacteria identified as pathogenic on animals. K4 had a bactericidal effect on Aeromonas salmonicida and Vibrio splendidus LGP32 at concentrations below 45 µg mL-1. Our results show that K4 could be an environment-friendly alternative to antibiotics, non-toxic to several marine organisms. The use of K4 would be particularly useful to decrease the bacterial load associated with food intake in the early developmental stages of marine animals reared in hatcheries.

Ethoxylated adjuvants of glyphosate-based herbicides are active principles of human cell toxicity.

Pesticides are always used in formulations as mixtures of an active principle with adjuvants. Glyphosate, the active ingredient of the major pesticide in the world, is an herbicide supposed to be specific on plant metabolism. Its adjuvants are generally considered as inert diluents. Since side effects for all these compounds have been claimed, we studied potential active principles for toxicity on human cells for 9 glyphosate-based formulations. For this we detailed their compositions and toxicities, and as controls we used a major adjuvant (the polyethoxylated tallowamine POE-15), glyphosate alone, and a total formulation without glyphosate. This was performed after 24h exposures on hepatic (HepG2), embryonic (HEK293) and placental (JEG3) cell lines. We measured mitochondrial activities, membrane degradations, and caspases 3/7 activities. The compositions in adjuvants were analyzed by mass spectrometry. Here we demonstrate that all formulations are more toxic than glyphosate, and we separated experimentally three groups of formulations differentially toxic according to their concentrations in ethoxylated adjuvants. Among them, POE-15 clearly appears to be the most toxic principle against human cells, even if others are not excluded. It begins to be active with negative dose-dependent effects on cellular respiration and membrane integrity between 1 and 3ppm, at environmental/occupational doses. We demonstrate in addition that POE-15 induces necrosis when its first micellization process occurs, by contrast to glyphosate which is known to promote endocrine disrupting effects after entering cells. Altogether, these results challenge the establishment of guidance values such as the acceptable daily intake of glyphosate, when these are mostly based on a long term in vivo test of glyphosate alone. Since pesticides are always used with adjuvants that could change their toxicity, the necessity to assess their whole formulations as mixtures becomes obvious. This challenges the concept of active principle of pesticides for non-target species.