Prevalence and intensity of a Rickettsiales-like organism in cultured pleasure oyster, Crassostrea corteziensis, from Nayarit, Mexico.

Fastidious endosymbiotic Rickettsiales-like organisms (RLOs) have been observed in the digestive diverticula of the cultured pleasure oyster (Crassostrea corteziensis) from Nayarit, Mexico since 2007. In a few mollusk species, these bacteria have been associated with mortality events and production losses. The type of relationship between the RLOs and the pleasure oyster is largely unknown and further investigations are needed to determine if these bacteria warrant management concern in C. corteziensis. In this study, the morphological characteristics of the RLOs were studied by histology and SEM, and the taxonomic affiliations of the bacteria were evaluated by 16S rRNA amplicon sequencing. In addition, the prevalence and intensity of the RLOs was recorded from 2007 to 2017 by histology. The RLOs were observed inside circular basophilic cytoplasmic membrane bound vacuoles (MBVs) that had an average length and width of 15.70 ± 15.24 µm and 15.42 ± 14.95 µm respectively. Apart from cellular hypertrophy, no tissue alterations were observed in the areas adjacent to the RLOs. Individual bacteria within the MBVs were coccoid in shape with an average length of 0.65 ± 0.12 µm and an average width of 0.38 ± 0.09 µm. The bacterial microbiota of a selected number of samples (one sample without RLOs and two samples with RLOs) showed the presence of intracellular parasite OTUs corresponding to the families Rickettsiaceae and Anaplasmataceae, suggesting that the RLOs from the pleasure oyster is associated with the order Rickettsiales. A mean prevalence of 5 % was observed throughout the study period and the majority of the organisms (89 %) presented low intensity of Grade 1 (30-61 RLOs) of the MBVs. A higher prevalence of the RLOs was observed during warmer months. The lack of tissue alterations, the low prevalence and the low intensity of the MBVs suggest that the RLOs from C. corteziensis is a commensal endosymbiont that presents little risk for oyster production in Nayarit, México. However, regular monitoring is needed to detect if any variation in this relationship occurs, mainly in a scenario where extreme environmental fluctuations may occur.

The emerging pathogen Enterocytozoon hepatopenaei drives a degenerative cyclic pattern in the hepatopancreas microbiome of the shrimp (Penaeus vannamei).

The microsporidian Enterocytozoon hepatopenaei (EHP) is an emerging pathogen that causes substantial economic losses in shrimp (Penaeus spp.) aquaculture worldwide. To prevent diseases in shrimp, the manipulation of the gut microbiota has been suggested. However, prior knowledge of the host-microbiome is necessary. We assessed the modulation of the microbiome (bacteria/fungi) and its predicted functions over the course of disease progression in shrimp experimentally challenged with EHP for 30 days using high throughput 16S rRNA and ITS amplicon sequencing. Infection grade was assessed for the first time by quantitative digital histopathology. According to the infection intensity, three disease-stages (early/developmental/late) were registered. During the early-stage, EHP was not consistently detected, and a high diversity of potentially beneficial microorganisms related to nutrient assimilation were found. In the development-stage, most of the shrimp start to register a high infection intensity related to a decrease in beneficial microorganisms and an increase in opportunistic/pathogenic fungi. During late-stage, animals displayed different infection intensities, showed a displacement of beneficial microorganisms by opportunistic/pathogenic bacteria and fungi related to pathogen infection processes and depletion of energetic reserves. The degenerative cyclic pattern of EHP infection and its effects on beneficial microorganisms and beneficial functions of the shrimp hepatopancreas microbiome are discussed.

Microbiome analysis from formalin-fixed paraffin-embedded tissues: Current challenges and future perspectives.

Formalin-fixed paraffin-embedded (FFPE) tissues stored in thousands of human and animal pathology laboratories around the globe represent mines of stored genetic information. In recent years, the use of FFPE tissues as a viable source of DNA for diverse genetic studies has attracted attention for interrogating microbiomes from this sample type. These studies have proven that 16S rRNA amplicon sequencing-based microbiome studies are possible from FFPE samples but present some particular challenges. In this review, we summarize all aspects of microbiome studies from FFPE tissues including the challenges associated with working highly degraded DNA, best practices for reducing environmental contamination, and we propose solutions to address these issues. Finally, we discuss how the combination of FFPE microbiome studies and Laser Capture Microdissection and/or Laser Microdissection could enable to determine the spatial heterogeneity underlying complex bacterial communities.

Transcriptome analysis of Catarina scallop (Argopecten ventricosus) juveniles treated with highly-diluted immunomodulatory compounds reveals activation of non-self-recognition system.

Marine bivalve hatchery productivity is continuously challenged by apparition and propagation of new diseases, mainly those related to vibriosis. Disinfectants and antibiotics are frequently overused to prevent pathogen presence, generating a potential negative impact on the environment. Recently, the use of highly diluted compounds with immunostimulant properties in marine organisms has been trailed successfully to activate the self-protection mechanisms of marine bivalves. Despite their potential as immunostimulants, little is known about their way of action. To understand their effect, a comparative transcriptomic analysis was performed with Argopecten ventricosus juveniles. The experimental design consisted of four treatments formulated from pathogenic Vibrio lysates at two dilutions: [(T1) Vibrio parahaemolyticus and Vibrio alginolyticus 1D; (T2) V. parahaemolyticus and V. alginolyticus 7C]; minerals [(T3) PhA+SiT 7C], scorpion venom [(T4) ViT 31C]; and one control (C1) hydro-alcoholic solution (ethanol 1%). The RNA sequencing (RNAseq) analysis showed a higher modulation of differentially expressed genes (DEG) in mantle tissue compared to gill tissue. The scallops that showed a higher number of DEG related to immune response in mantle tissue corresponded to T1 (V. parahaemolyticus and V. alginolyticus lysate) and T3 (Silicea terra® - Phosphoric acid®). The transcriptome analysis allowed understanding some interactions between A. ventricosus juveniles and highly-diluted treatments.

Efecto de medicamentos homeopáticos sobre crecimiento, supervivencia y microbiota gastrointestinal, en juveniles del pectínido Argopecten ventricosus / Effect of homeopathic medicines on growth, survival and gastrointestinal microbiota of juvenile scallop Argopecten ventricosus

RESUMEN Objetivo. Estudiar el efecto de medicamentos homeopáticos sobre el crecimiento, supervivencia y microbiota del tracto gastrointestinal (TGI) de almeja Catarina Argopecten ventricosus. Materiales y Métodos. Se aplicaron cinco tratamientos homeopáticos derivados de bacterias [(T1) ViP-ViA 1D, (T2) ViP-ViA 7C], minerales [(T3) AcF-MsS 1D, (T4) PhA-SiT 7C], o venenos [(T5) ViT 31C] y tres controles: (C1) etanol diluido 1:99, (C2) etanol dinamizado 1C y (C3) agua destilada. La microbiota se determinó secuenciando la región V3-V5 del gen 16S rRNA. Resultados. El mayor crecimiento en longitud de la concha correspondió a T1 (117 µm d-1) y T2 (108 µm d-1), la mayor supervivencia a T3 y T5 y el mejor resultado global a T3. Las curvas de rarefacción de los grupos tratados y controles mostraron una clara separación. Se encontraron diferencias significativas (p≤0.05) entre filos (Proteobacteria > Actinobacteria > Firmicutes > Bacterloidetes> Chloroflexi y para los Géneros: Symbiobacterium > Microbacterium > Methylobacillus > Bacillus > Paenibacillus > Burkholderia > Nostoc > Methylobacterium > Leucobacter). El género Symbiobacterium fue dominante (p≤0.05) para T5, respecto a todos los tratamientos y grupos controles. La especie Microbacterium maritypicum (Actinobacteria) mostró la mayor abundancia relativa (p≤0.05) en T1 y T3 y Symbiobacterium toebii (Firmicutes) en T5 y T2 (p≤0.05), ambas con respecto al inicio del estudio T0. Conclusiones. Se presenta por primera vez la composición de la microbiota del TGI de A. ventricosus y la aplicabilidad potencial de la homeopatía para mejorar el rendimiento productivo y modular la microbiota gastrointestinal de la especie.

ABSTRACT Objective. To study the effect of homeopathic medicines on growth, survival and gastrointestinal (GIT) microbiota of Catarina scallop Argopecten ventricosus. Materials and methods. Five homeopathic (HOM) treatments derived from bacteria [(T1) ViP-ViA 1D, (T2) ViP-ViA 7C], minerals [(T3) AcF-MsS 1D, (T4) PhA-SiT 7C] or venoms [T5) ViT 31C] and three controls: [(C1) diluted ethanol 1:99, (C2) diluted/succussed ethanol 1C and (C3) distilled water] were evaluated (21 days) in triplicate. Microbiota was analysed by sequencing the V3-V5 region of the 16S rRNA genes. Results. The best growth in shell-length corresponded to T1 (117 µm d-1) and T2 (108 µm d-1) and the highest survival to T3 and T5, stating T3 as the best HOM-treatment. A clear separation was found in rarefaction curves of HOM-treated against un-treated control scallops. Significant differences (p≤0.05) were found for Phyla (Proteobacteria> Actinobacteria> Firmicutes> Bacterloidetes>Chloroflexi and for Genera: Symbiobacterium> Microbacterium> Methylobacillus> Bacillus> Paenibacillus> Burkholderia> Nostoc> Methylobacterium> Leucobacter). The genus Symbiobacterium was dominant in T5, finding significant differences (p≤0.05) with respect to all treatments. At species level, Microbacterium maritypicum (Actinobacteria) showed a greater relative abundance (p≤0.05) in T1 and T3 and Symbiobacterium toebii (Firmicutes) was also significantly higher (p≤0.05) in abundance in T5 and T2, both against initial T0. Conclusions. This study showed for a first time, the composition of GIT microbiota in A. ventricosus and focused on the potential applicability of homeopathy to improve overall performance and modulate the GIT microbiota of the species.