Metagenomic Analysis of the Bacterial and Fungal Community Associated to the Rhizosphere of Tabebuia chrysantha and T. billbergii.

The rhizosphere of plants contains a diversity of microorganisms, some of which play an important role in the growth and development of the host plant. In this work, the diversity of fungi and bacteria associated to the rhizosphere of Tabebuia chrysantha and T. billbergii plants was analyzed. The molecular identification was performed by sequencing the ITS and 16S rDNA for fungi and bacteria, respectively. The analysis of the rDNA sequences of the rhizosphere of T. billergii showed that for domain Eukaria, the most abundant phyla were Glomeromycota (56%) and Ascomycota (39%), and for domain Bacteria, the phylum Firmicutes (19.17%) was the most abundant followed by Actinobacteria (14.90%) and Proteobacteria (8.94%). In the rhizosphere of T. chrysantha the most abundant phylum of Eukaria was Ascomycota (98%), and for Bacteria the most representative phyla were Proteobacteria (18.61%) and Actinobacteria (11.93%). A diversity of genera and species of fungi and bacteria was observed, to be more significant in T. chrysantha than T. billbergii. The taxonomic assignment of metagenomic sequences revealed a homology associated with genomic sequences of 546 bacteria and 147 fungi in T. chrysantha and 154 bacteria and 122 fungi in T. billbergii.

Genomic characterization and probiotic potential of lactic acid bacteria isolated from feces of guinea pig (Cavia porcellus).

Background: Presently, there exists a growing interest in mitigating the utilization of antibiotics in response to the challenges emanating from their usage in livestock. A viable alternative strategy encompasses the introduction of live microorganisms recognized as probiotics, exerting advantageous impacts on the immune system and nutritional aspects of the host animals. Native lactic acid bacteria, inherently possessing specific properties and adaptive capabilities tailored to each animal, are deemed optimal contenders for probiotic advancement. Aim: In the current investigation, microorganisms exhibiting probiotic potential were isolated, characterized, and identified from the fecal samples of guinea pigs (Cavia porcellus) belonging to the Peruvian breed. Methods: The lactic acid bacteria isolated on Man, Rogosa, and Sharpe agar underwent Gram staining, catalase testing, proteolytic, amylolytic, and cellulolytic activity assays, low pH tolerance assessment, hemolytic evaluation, antagonism against Salmonella sp., determination of autoaggregation and coaggregation capacity, and genotypic characterization through sequencing of the 16S rRNA gene. Results: A total of 33 lactic acid bacteria were isolated from the feces of 30 guinea pigs, also 10 isolates were selected based on Gram staining and catalase testing. All strains exhibited proteolytic activity, while only one demonstrated amylolytic capability, and none displayed cellulase activity. These bacteria showed higher tolerance to pH 5.0 and, to a lesser extent, to pH 4.0. Furthermore, they exhibited antagonistic activity against Salmonella sp. Only two bacteria demonstrated hemolytic activity, and were subsequently excluded from further evaluations. Subsequent assessments revealed autoaggregation capacities ranging from 4.55% to 23.19%, with a lesser degree of coaggregation with Salmonella sp. ranging from 3.53% to 8.94% for the remaining eight bacterial isolates. Based on these comprehensive tests, five bacteria with notable probiotic potential were identified by molecular assays as Leuconostoc citreum, Enterococcus gallinarum, Exiguobacterium sp., and Lactococcus lactis. Conclusion: The identified bacteria stand out as promising probiotic candidates, deserving further assessment in Peruvian breed guinea pigs. This exploration aims to enhance production outcomes while mitigating the adverse effects induced by pathogenic microorganisms.

MALDI mass spectrometry-based identification of antifungal molecules from endophytic Bacillus strains with biocontrol potential of Lasiodiplodia theobromae, a grapevine trunk pathogen in Peru.

Lasiodiplodia theobromae, a grapevine trunk pathogen, is becoming a significant threat to vineyards worldwide. In Peru, it is responsible for Botryosphaeria dieback in many grapevine-growing areas and it has spread rapidly due to its high transmissibility; hence, control measures are urgent. It is known that some endophytic bacteria are strong inhibitors of phytopathogens because they produce a wide range of antimicrobial molecules. However, studies of antimicrobial features from endophytic bacteria are limited to traditional confrontation methods. In this study, a MALDI mass spectrometry-based approach was performed to identify and characterize the antifungal molecules from Bacillus velezensis M1 and Bacillus amyloliquefaciens M2 grapevine endophytic strains. Solid medium antagonism assays were performed confronting B. velezensis M1 - L. theobromae and B. amyloliquefaciens M2 - L. theobromae for antifungal lipopeptides identification. By a MALDI TOF MS it was possible identify mass spectra for fengycin, iturin and surfactin protoned isoforms. Masses spectrums for mycobacillin and mycosubtilin were also identified. Using MALDI Imaging MS we were able to visualize and relate lipopeptides mass spectra of fengycin (1463.9 m/z) and mycobacillin (1529.6 m/z) in the interaction zone during confrontations. The presence of lipopeptides-synthesis genes was confirmed by PCR. Liquid medium antagonism assays were performed for a proteomic analysis during the confrontation of B. velezensis M1 - L. theobromae. Different peptide sequences corresponding to many antifungal proteins and enzymes were identified by MALDI TOF MS/MS. Oxalate decarboxylase bacisubin and flagellin, reported as antifungal proteins, were identified at 99 % identity through peptide mapping. MALDI mass spectrometry-based identification of antifungal molecules would allow the early selection of endophytic bacteria with antifungal features. This omics tool could lead to measures for prevention of grapevine diseases and other economically important crops in Peru.

Metagenomic analysis of the intestinal microbiome in goats on cactus and Salicornia-based diets.

Background: The Peruvian coast is characterized by its arid and saline soils, the cactus being an alternative for arid soils and Salicornia for saline soils. Therefore, it is necessary to develop nutrition based on the intestinal microbiota in goats. Aim: To identify the intestinal microbiota in goats through a metagenomic analysis. Methods: In this study, goats and kids were randomly selected and fed cacti and Salicornia as potential forage species compared to native grass to study the changes in the microbiota using massive sequencing using the 16S rRNA gene as a marker. Results: The sequencing results showed the taxonomic levels of Bacteroidetes and Firmicutes at the phylum level as the most abundant in creole goats' microbiome, varying from 18% to 36% and 47% to 66%, respectively. At the genus level, variants of the genus Ruminococcaceae stand out, related to cellulose degradation, as the most dominant in all samples, followed by Christensenellaceae, Rikenellaceae, and Prevotellaceae. Also, the genus Akkermansia appeared in greater abundance in kids fed with cactus, being necessary for being related to the intestinal mucosa's health and avoiding the adhesion of pathogens to the intestinal epithelium. Conclusion: These microbiota changes based on diets with high fiber content are necessary to understand the adaptation of this species to favorable dietary changes.

Species-informative SNP markers for characterising freshwater prawns of genus Macrobrachium in Cameroon.

Single Nucleotide Polymorphisms (SNPs) are now popular for a myriad of applications in animal and plant species including, ancestry assignment, conservation genetics, breeding, and traceability of animal products. The objective of this study was to develop a customized cost-effective SNP panel for genetic characterisation of Macrobrachium species in Cameroon. The SNPs identified in a previous characterization study were screened as viable candidates for the reduced panel. Starting from a full set of 1,814 SNPs, a total of 72 core SNPs were chosen using conventional approaches: allele frequency differentials, minor allele frequency profiles, and Wright's Fst statistics. The discriminatory power of reduced set of informative SNPs were then tested using the admixture analysis, principal component analysis, and discriminant analysis of principal components. The panel of prioritised SNP markers (i.e., N = 72 SNPs) distinguished Macrobrachium species with 100% accuracy. However, large sample size is needed to identify more informative SNPs for discriminating genetically closely related species, including M. macrobrachion versus M. vollenhovenii and M. sollaudii versus M. dux. Overall, the findings in this study show that we can accurately characterise Macrobrachium using a small set of core SNPs which could be useful for this economically important species in Cameroon. Given the results obtained in this study, a larger independent validation sample set will be needed to confirm the discriminative capacity of this SNP panel for wider commercial and research applications.

Colonisation and mass rearing: learning from others.

Mosquitoes, just as other insects produced for the sterile insect technique (SIT), are subjected to several unnatural processes including laboratory colonisation and large-scale factory production. After these processes, sterile male mosquitoes must perform the natural task of locating and mating with wild females. Therefore, the colonisation and production processes must preserve characters necessary for these functions. Fortunately, in contrast to natural selection which favours a suite of characteristics that improve overall fitness, colonisation and production practices for SIT strive to maximize only the few qualities that are necessary to effectively control populations. However, there is considerable uncertainty about some of the appropriate characteristics due to the lack of data. Development of biological products for other applications suggest that it is possible to identify and modify competitiveness characteristics in order to produce competitive mass produced sterile mosquitoes. This goal has been pursued--and sometimes achieved--by mosquito colonisation, production, and studies that have linked these characteristics to field performance. Parallels are drawn to studies in other insect SIT programmes and aquaculture which serve as vital technical reference points for mass-production of mosquitoes, most of whose development occurs--and characteristics of which are determined--in an aquatic environment. Poorly understood areas that require further study are numerous: diet, mass handling and genetic and physiological factors that influence mating competitiveness. Compromises in such traits due to demands to increase numbers or reduce costs, should be carefully considered in light of the desired field performance.

Morphological and molecular characterization of freshwater prawn of genus Macrobrachium in the coastal area of Cameroon.

Macrobrachium (Bate, 1868) is a large and cosmopolitan crustacean genus of high economic importance worldwide. We investigated the morphological and molecular identification of freshwater prawns of the genus Macrobrachium in South, South West, and Littoral regions of Cameroon. A total of 1,566 specimens were examined morphologically using a key described by Konan (Diversité morphologique et génétique des crevettes des genres Atya Leach, 1816 et Macrobrachium Bate, 1868 de Côte d'Ivoire, 2009, Université d'Abobo Adjamé, Côte d'Ivoire), leading to the identification of seven species of Macrobrachium: M. vollenhovenii (Herklots, 1857); M. macrobrachion (Herklots, 1851); M. sollaudii (De Man, 1912); M. dux (Lenz, 1910); M. chevalieri (Roux, 1935); M. felicinum (Holthuis, 1949); and an undescribed Macrobrachium species M. sp. To validate the genetic basis of the identified species, 94 individuals representing the species were selected and subjected to genetic characterization using 1,814 DArT markers. The admixture analysis revealed four groups: M. vollenhovenii and M. macrobrachion; M. chevalieri; M. felicinum and M. sp; and M. dux and M. sollaudii. But, the principal component analysis (PCA) separated M. sp and M. felicinum to create additional group (i.e., five groups). Based on these findings, M. vollenhovenii and M. macrobrachion may be conspecific, as well as M. dux and M. sollaudii, while M. felicinum and M. sp seems to be different species, suggesting a potential conflict between the morphological identification key and the genetic basis underlying speciation and species allocation for Macrobrachium. These results are valuable in informing breeding design and genetic resource conservation programs for Macrobrachium in Africa.

Diseño y evaluación de la expresión de una potencial vacuna de ADN contra el virus de la Tilapia de Lago (TiLV)

El Virus de la Tilapia del Lago (TiLV), es un patógeno causante de mortalidades masivas tanto en poblaciones de tilapias cultivadas y silvestres alrededor del mundo. El desarrollo de una vacuna efectiva contra este patógeno emergente es imperativo para prevenir pérdidas económicas. En este trabajo se diseñó y evaluó un vector de expresión como una potencial vacuna de ADN contra este virus. Inicialmente, se realizó un análisis de enhebramiento para predecir las estructuras tridimensionales y las funciones de las proteínas del TiLV. Se encontraron homologías estructurales entre las proteínas correspondientes al segmento genómico 1 y al segmento genómico 4 del TiLV, con las proteínas de ARN polimerasa dependiente de ARN del virus de la influenza B (56%) y la proteína neuraminidasa que pertenece a la cápside del virus de la influenza A (12%), respectivamente. Se insertó el producto de PCR del gen neuraminidasa viral en el vector plasmídico de expresión pCMV. Finalmente, se inyectó el constructo plasmídico en juveniles de la tilapia del Nilo Oreochromis niloticus y se midió su expresión mediante RT-PCR en tiempo real a las 8h, 16h, 24h, 72h después de la segunda inyección inmunizante. Se logró detectar expresión génica en los cuatro tiempos evaluados, con mayor expresión a las 16 horas post inyección. Estos resultados constituyen el primer paso para el desarrollo de una vacuna efectiva para la protección de los stocks de tilapias alrededor del mundo.

Tilapia Lake Virus (TiLV) is a pathogen that causes massive mortalities in both cultured and wild tilapia populations around the world. The development of an effective vaccine against this emerging pathogen is imperative to prevent economic losses. In this work an expression vector was designed and evaluated as a potential DNA vaccine against this virus. Initially, a threading analysis was done to predict the threedimensional structures and functions of the TiLV proteins. Structural homologies were found between the TiLV proteins corresponding to the genomic segment 1 and the genomic segment 4, with the RNA-dependent RNA polymerase proteins of the influenza B virus (56%) and the neuraminidase protein belonging to the influenza A virus capsid (12%), respectively. The PCR product of the viral neuraminidase gene was inserted into the expression plasmid vector pCMV. Finally, the plasmid construct was injected into juveniles of the Nile tilapia Oreochromis niloticus and its expression was measured by real time RT-PCR at 8h, 16h, 24h, and 72h after the second immunizing injection. It was possible to detect gene expression in the four evaluated times and greater expression at 16 hours post injection. These results are the first step in the development of an effective vaccine for the protection of tilapia stocks around the world.

Respuesta fisiológica y molecular de Anadara tuberculosa (Arcoida: Arcidae) al estrés de salinidad / Physiological and molecular response of Anadara tuberculosa (Arcoida: Arcidae) to salinity stress

Resumen: La concha negra Anadara tuberculosa es una especie emblemática del ecosistema manglar que está actualmente en condición vulnerable. El desarrollo de su acuicultura requiere identificar biomarcadores moleculares, en particular asociados al estrés por salinidad en mira al inicio de programas de mejoramiento genético. Se recolectaron ejemplares de Anadara tuberculosa del manglar colindante a la Bahía de Puerto Pizarro (Tumbes, Perú) entre enero 2015 y febrero 2016. Estos individuos fueron sometidos a condiciones de estrés hipo-osmótico (extremo: 5 y 10 ppt); (moderado: 15 y 25 ppt) y sin estrés (grupo control: 33 ppt) por 16 días después de haber sido separados en grupos de diez animales y por triplicado. La presencia de biomarcadores del estrés por salinidad fue evaluada a nivel genético con la detección por PCR de 19 genes reportados como actores claves de la osmorregulación en bivalvos como ostras y mejillones y a nivel proteomico con la secuenciación de péptidos expresados en tejidos de animales expuestos a diferentes salinidades por espectrometría de doble masa. Ninguno de los marcadores genéticos probados pudo ser amplificado por PCR lo que sugiere que A. tuberculosa presente diferencias genéticas significativas en comparación con otros moluscos. El análisis proteómico realizado por MALDI TOF/TOF a nivel de tejido branquial de A. tuberculosa permitió identificar 26 péptidos expresados de formas presenciales y diferenciales a las diferentes salinidades evaluadas, resaltando posibles marcadores como la HSP70 y una proteína transmembrana de transporte de cloruro que están relacionadas con la adaptación a la salinidad. Estas secuencias aminoacídicas permitirán diseñar iniciadores nucleotidicos específicos a A. tuberculosa para la puesta en marcha de futuras investigaciones en ecofisiología de este importante recurso.

Abstract: The pustulose ark A. tuberculosa is an emblematic species of mangrove ecosystem that is currently in a vulnerable condition. The development of its aquaculture, to begin with genetic breeding programs, requires the identification of molecular biomarkers, particularly those associated with salinity stress. With this purpose, specimens of A. tuberculosa were collected from the adjacent mangroves of Puerto Pizarro bay (Tumbes, Perú), from January 2015 to February 2016. Different assays (groups of ten animals in triplicate) were undertaken in separated periods of 16 days: hypo-osmotic stress (extreme: 5, 10 ppt); (Moderate: 15, 25 ppt) and no stress (control group: 33 ppt). The presence of salinity stress biomarkers was assessed at the genetic level throughout PCR detection of 19 genes reported to be key actors in osmoregulation, and at the proteomic level with the sequencing of peptides (tandem mass spectrometry MALDI TOF/TOF), expressed in ark tissues exposed to different salinities. None of the tested genetic markers could be amplified by PCR, suggesting that A. tuberculosa has significant genetic differences compared to other mollusks. Proteomic analysis by mass spectrometry on A. tuberculosa gill tissue, allowed to identify 26 peptides expressed in presential and differential forms at different salinities, highlighting possible markers such as HSP70 and trans-membrane chloride channel transportation protein, to be related with salinity adaptation. These amino acid sequences will allow the design of target specific primers for A. tuberculosa, to implement future research in ecophysiology of this important fishery resource. Rev. Biol. Trop. 65 (3): 1142-1151. Epub 2017 September 01.