Stevia as a natural additive on gut health and cecal microbiota in broilers.

Stevia mash (SM), leaves of Stevia rebaudiana Bertoni plant, is an additive used in poultry that enhances growth and health. Objective: to determine the effect of 1 % SM on productive parameters, gut health, and the cecal microbiome in broilers between the first 15 and 21 days old. One hundred sixty male, 1-day-old broilers (48.5 ± 2.5 g) were divided into Control (C) without SM and Treated (T) with 1 % SM on diet, during 15/21 days. Each subgroup had eight broilers/five repetitions/treatment. At day 15 or 21, all broilers were dissected, Fabricius Bursa and Gut removed and processed for histomorphometry, followed by Villi Height/Crypt Deep (VH/CD) ratio. Conversion Index (CI) was determined. The V3-V4 region of 16S rRNA gene was amplified from DNA obtained from pooled cecal contents and sequenced on Illumina Miseq PE 2 × 250 platform. Sequence processing and taxonomic assignments were performed using the SHAMAN pipeline. Both T groups have better VH/CD Ratios than C groups (p ≤ 0.05). In guts, increased plasmatic and goblet cells number and thicker mucus layer were found in T15 and T21. All groups received SM showed early immunological maturity in Fabricius Bursa. IC was similar between all treatments. Faecalibacterium, Ruminococcus torques group, and Bacteroides were the major genera modulated by SM addition. At 15 and 21 days old, SM exerts a impact on diversity and evenness of the cecal microbiome.  Conclusion: SM (1 %) produced early immunologic maturity on Fabricius Bursa, increased intestinal functionality, and modified the microbiota, increasing beneficial microbial genera and microbial diversity.

Systematic assessment of nymphs of Diesingiinae (Pentastomida: Sebekidae) parasitizing Palloceros harpagos (Cyprinodontiformes: Poeciliidae) from Parque Nacional Iguazú, Argentina: filling gaps in an incomplete phylogenetic framework.

During a search for parasites in fishes from Iguazu River, Argentina, a nymph of pentastomid was found encysted on mesenteries of Phalloceros harpagos. The 28S rDNA and COI mt-DNA were used to compare with the sequences deposited in the GenBank. Pentastomid nymphs belong to the subfamily Diesingiinae (Sebekidae) for having chloride cell pores distributed in a single row per annulus; also, the hooks and rows of spines of our material differ to other genera. Present specimens are most likely Diesingia sp., having 63-74 annuli, a U shaped oral cadre with fibers closing anteriorly and a peg like extension of the oral cadre. The 28S rDNA analysis places our samples into a sister group of Alofia sp., but the COI mt-DNA situate them on the base of the clade. In conclusion, our pentastomid positively belongs to Diesingia sp., however, indisputable assignation requires a reliable description of the nymph, or the availability of sequences linking nymphs and adults, which even could provide evidence of a new hitherto undescribed genus. Aditionally, the systematic position of Sebekia oxycephala previously described by P. harpagos is discussed on the basis of our results, allowing us to suggest a re-assignation of such specimens to the genus Diesingia.

How many species of genus Lernaeopoda Blainville, 1822 (Siphonostomatoida: Lernaeopodidae) are there in the southwestern Atlantic?

The family Lernaeopodidae includes 14 genera parasitizing elasmobranchs. Fourteen species of this family have been cited from Argentina, four of which were found on chondrichthyans. Schroederichthys bivius Mller and Henle and Galeorhinus galeus (Linnaeus) from Argentina harbored parasitic copepods of the genus Lernaeopoda. The objective of this study was to identify the species using an integrative approach. The morphology was examined by Optical Microscopy and Scanning Electron Microscopy and the molecular analysis was based on partial sequences of the COI mtDNA gene. Despite differences in the antenna, antennule, mandible, maxilliped and maxillae among the specimens, the morphological and molecular analyses revealed that they belonged to Lernaeopoda bivia Leigh-Sharpe, 1930. The species so far reported for Argentina are L. bivia and L. galei Kryer, 1837, which are distinguished by the size (less and greater than 7 mm, respectively). Here, we report partial sequences of the COI mtDNA gene of L. bivia for the first time, obtained from eleven specimens attached to the mouth, fins, anal slit and claspers of the two shark hosts. The COI mtDNA gene tree shows that the Lernaeopoda group forms a sister clade with Pseudocharopinus bicaudatus (Kryer, 1837), while the genus Pseudocharopinus does not appear to be a natural group. We propose that the material described from Argentinean waters as L. galei was misidentified and actually belongs to L. bivia. The wide variability within the specimens of L. bivia emphasizes the importance of using an integrative approach to revise the taxonomy of the Lernaeopoda species from all over the world.

New insights on Pomphorhynchus sphaericus Gil de Pertierra, Spatz et Doma, 1996 (Acanthocephala: Pomphorhynchidae).

The finding of Pomphorhynchus sphaericus in new localities from La Plata River allowed the reevaluation of the species using a taxonomic integrative approach. The newly found specimens in Pimelodus maculatus from Samborombon Bay differ from P. sphaericus by the roots of hooks 1-6 which not form a wide sheet split into 2 apophysis, the slender, separated and equatorial testicles, the position of the cement glands, the shape of the proboscis, the shape and length of lemnisci, and the eggs size. Despite the notorious observed morphological differences, the COI mtDNA analysis confirmed that Pomphorhynchus individuals are the same conspecific, and showed that there is a high phenotypical plasticity in this species. Pomphorhynchus sphaericus is the first South American species analyzed to a DNA level (COI mtDNA, ITS, and 18S rDNA genes). The molecular analysis relates P. sphaericus to P. bulbocolli and P. purhepechus.

Molecular data reveal hidden diversity of the genus Clinostomum (Digenea, Clinostomidae) in Argentina, with the description of a new species from Ardea cocoi (Ardeidae).

The genus Clinostomum has been recently a subject of a large number of molecular phylogenetic studies that have uncovered a larger species diversity than we thought. In Argentina, only two nominal species have been reported, namely C. detruncatum and C. marginatum. Three putative species represented by metacercariae were recently molecularly diagnosed, and there are at least two additional metacercarial morphotypes diagnosed on morphological grounds. Here, we molecularly characterized specimens of Clinostomum sampled from freshwater fishes and fish-eating birds from Argentina through mtDNA cytochrome c oxidase subunit I (COI). Unexpectedly, the phylogenetic analysis uncovered three new additional genetic lineages, two of them corresponding to metacercarial stages and another matching COI sequences of C. heluans Braun, 1899, being considered conspecific, whereas the others still require formal description. Additionally, we add a new host species for a lineage molecularly diagnosed in a previous study. The adult specimens recovered from A. cocoi in Buenos Aires Province represented a new species clearly distinguished from the two species previously reported in Argentina. Clinostomum detruncatum is distinguished from the new species by possessing a characteristic tenoidean uterus, and testes located more posteriorly. Clinostomum marginatum is morphologically similar but differs from the new species in having rounded ovary, posterior testis lobated, and lateral cirrus-sac and displacing the anterior testis, and vitelline follicles not extending beyond the caeca end. Our study raises the number of Clinostomum species in Argentina up to 10. We describe the new species herein.

Morphological and molecular identification of hymenolepidid cestodes in children and synanthropic rodents from rural Mexico.

Hymenolepidid cestodes of synanthropic rodents represent a risk for public health. In order to describe the occurrence of hymenolepidids in children and the role of rodents as a potential source of infection, we conducted a morphological and molecular survey on cestodes in two rural villages from Yucatan, Mexico. One hundred and thirty-five stool samples from children (64 from Paraíso and 71 from Xkalakdzonot), 233 Mus musculus (159 from Paraíso and 74 from Xkalakdzonot) and 125 Rattus rattus (7 from Paraíso and 118 from Xkalakdzonot) were analyzed for the presence of cestodes. Three hymenolepidid species were identified morphologically: Hymenolepis nana in 7.8% of children from Paraíso, Hymenolepis microstoma in 4.4% of M. musculus from Paraíso and Hymenolepis diminuta in 15.3% of R. rattus from Xkalakdzonot. The molecular characterization and phylogenetic analysis based on mitochondrial cytochrome c subunit 1 (CO1) gene and ribosomal internal transcribed spacer 1 (ITS1) region, confirmed the identity of the three cestodes isolated from Yucatan. Phylogeny of the CO1 gene identified intraspecific genetic differences within H. nana ranging from 0 to 5%, in H. microstoma from 0 to 0.4%, and in H. diminuta ranged from 0 to 6.5% which suggests, the presence of complex species within H. nana and H. diminuta infecting humans and rodents, as reported by other authors. Based on the morphological and molecular results, and the epidemiological evidence, infections with H. nana suggest a non-zoonotic transmission; however, the presence of H. microstoma and H. diminuta in synanthropic rodents serve as a possible source for human infection.

Hidden diversity in waterfall environments: The genus Acrorbis (Gastropoda: Planorbidae) from the Upper-Paraná Atlantic Forest.

High-energy freshwater environments such as rapids and waterfalls in the Upper-Paraná Atlantic Forest are home to highly endemic minute freshwater snails of the genus Acrorbis. Only one species, Acrorbis petricola, is currently included within this genus, whose geographical distribution is restricted to three known populations, one in Brazil and the other two in Argentina. Because of habitat specificity and limited geographical distribution, the species is considered vulnerable in Argentina and endangered in Brazil. In this work, we identify five new populations of A. petricola in southern Upper-Paraná Atlantic Forest, exclusively found on waterfalls from the Misiones Province, Argentina. Based on these populations and on specimens of one of the two historical populations from the Misiones Province, we explored the morphological features of shells and reproductive system of specimens from each location and provide the first molecular data on the species. We used DNA sequences from cytochrome c oxidase subunit I (COI) and 16S-rRNA genes to investigate the molecular diversity, genetic distances and genealogical relationships among populations. We verified the existence of intra- and interpopulation morphological variability, with the greatest variation being found in spire, spiral sculpture, penis sheath, flagella, prostatic diverticula and bursa copulatrix. We found interpopulation genetic diversity, with no intrapopulation variation, and identified six geographically structured genetic lineages with maximum genetic distances of up to 2.3%. Different combinations of morphological characters with the same genetic background within each locality were observed. The finding of new populations genetically differentiated not only broadens the known distribution of the species, but also illustrates that waterfall environments in the Atlantic Forest harbour a hidden diversity of Acrorbis that still remains to be discovered. This scenario suggests a complex evolutionary history that needs to be unveiled and taken into account for future development of conservation strategies in this endemic genus.

Identification of two species of Binema Travassos 1925 (Oxyurida: Travassosinematidae) and Cameronia arecoensis Marchissio and Miralles 1987 (Oxyurida: Thelastomatidae) based on morphological and 18S rRNA partial sequence.

Oxyurid nematodes parasitizing the mole cricket Neoscapteriscus vicinus were isolated in the framework of sampling fields of mole-crickets from the pampean region, in Argentina. In this work, molecular characterization of the 18S rRNA partial sequence of nematodes belonging to the families Thelastomatidae (Cameronia arecoensis Marchissio and Miralles 1987) and Travassosinematidae (Binema korsakowi Sergiev 1923 and Binema klossae, Marchissio and Miralles 1993) were carried out. This is the first world report of sequences belonging to B. klossae and C. arecoensis and first Argentinian report of B. korsakowi sequence. Also, morphological and morphometric features of B. klossae, B. korsakowi and C. arecoensis from Argentinian populations are reported.

Natural occurrence in Argentina of a new fungal pathogen of cockroaches, Metarhizium argentinense sp. nov.

The aim of this study was to search for entomopathogenic fungi that infect wild cockroaches in forest ecosystems in two protected natural areas of Argentina. Two isolates of Metarhizium argentinense were obtained and identified from wild cockroaches (Blaberidae: Epilamprinae) through the use of morphological characteristics and molecular phylogenetic analyses. This novel species was found in Argentina and is a member of the Metarhizium flavoviride species complex. Phylogenetic analyses, based on sequence similarity analysis using internal transcribed spacer (ITS) and a set of four protein-coding marker sequences (EF1A, RPB1, RPB2 and BTUB), supported the status of this fungus as a new species. In addition, we tested the biological activity of the new species through assays against Blattella germanica nymphs and found that the two evaluated isolates were pathogenic. However, isolate CEP424 was more virulent and caused a confirmed mortality of 76 % with a median lethal time of 7.2 d. This study reports the southernmost worldwide location of a Metarhizium species that infects cockroaches and will help expand the knowledge of the biodiversity of pathogenic fungi of Argentine cockroaches.

Geometric morphometric on a new species of Trichodinidae. A tool to discriminate trichodinid species combined with traditional morphology and molecular analysis.

This study reports a new Trichodinidae, Trichodina bellottii n. sp., parasitizing the pearly fish Austrolebias bellottii from Buenos Aires, Argentina. Its small subunit ribosomal ribonucleic acid (SSU rDNA) was sequenced for the first time. Based on the results from morphological identification, SSU rDNA sequencing, and Elliptical Fourier analysis, the new species was identified and compared with similar species. Phylogenetic analysis revealed that the genetic distances among the new species and similar species reached interspecific levels, furthermore, the phylogenetic study also validated the identification of T. bellottii n. sp. and its placement in the genus Trichodina. To be able to quantitatively describe the differences in shapes with similar species, this study used the elliptic Fourier analysis by first time in this genus.

Biological characterization of Aedes albopictus (Diptera: Culicidae) in Argentina: implications for arbovirus transmission.

Aedes albopictus (Diptera: Culicidae) is an invasive mosquito, native to Asia, that has expanded its range worldwide. It is considered to be a public health threat as it is a competent vector of viruses of medical importance, including dengue, chikungunya, and Zika. Despite its medical importance there is almost no information on biologically important traits of Ae. albopictus in Argentina. We studied life cycle traits, demographic parameters and analyzed the competence of this mosquito as a virus vector. In addition, we determined the prevalence of Wolbachia strains in Ae. albopictus as a first approach to investigate the potential role of this bacteria in modulating vector competence for arboviruses. We observed low hatch rates of eggs, which led to a negative growth rate. We found that Ae. albopictus individuals were infected with Wolbachia in the F1 but while standard superinfection with wAlbA and wAlbB types was found in 66.7% of the females, 16.7% of the females and 62.5% of the males were single-infected with the wAlbB strain. Finally, despite high levels of infection and dissemination, particularly for chikungunya virus, Ae. albopictus from subtropical Argentina were found to be relatively inefficient vectors for transmission of both chikungunya and dengue viruses.

Mutation in a D-alanine-D-alanine ligase of Azospirillum brasilense Cd results in an overproduction of exopolysaccharides and a decreased tolerance to saline stress.

Bacteria of the genus Azospirillum are free-living nitrogen-fixing, rhizobacteria that are found in close association with plant roots, where they exert beneficial effects on plant growth and yield in many crops of agronomic importance. Unlike other bacteria, little is known about the genetics and biochemistry of exopolysaccharides in Azospirillum brasilense. In an attempt to characterize genes associated with exopolysaccharides production, we generated an A. brasilense Cd Tn5 mutant that showed exopolysaccharides overproduction, decreased tolerance to saline conditions, altered cell morphology, and increased sensitivity to detergents. Genetic characterization showed that the Tn5 was inserted within a ddlB gene encoding for a d-alanine-d-alanine ligase, and located upstream of the ftsQAZ gene cluster responsible for cell division in different bacteria. Heterologous complementation of the ddlB Tn5 mutant restored the exopolysaccharides production to wild-type levels and the ability to grow in the presence of detergents, but not the morphology and growth characteristics of the wild-type bacteria, suggesting a polar effect of Tn5 on the fts genes. This result and the construction of a nonpolar ddlB mutant provide solid evidence of the presence of transcriptional coupling between a gene associated with peptidoglycan biosynthesis and the fts genes required to control cell division.