Mitochondrial and ribosomal markers in the identification of nematodes of clinical and veterinary importance.

BACKGROUND: Nematodes of the Ascarididae, Ancylostomatidae and Onchocercidae families are parasites of human and veterinary importance causing infections with high prevalence worldwide. Molecular tools have significantly improved the diagnosis of these helminthiases, but the selection of genetic markers for PCR or metabarcoding purposes is often challenging because of the resolution these may show. METHODS: Nuclear 18S rRNA, internal transcribed spacers 1 (ITS-1) and 2 (ITS-2), mitochondrial gene cytochrome oxidase 1 (cox1) and mitochondrial rRNA genes 12S and 16S loci were studied for 30 species of the mentioned families. Accordingly, their phylogenetic interspecies resolution, pairwise nucleotide p-distances and sequence availability in GenBank were analyzed. RESULTS: The 18S rRNA showed the least interspecies resolution since separate species of the Ascaris, Mansonella, Toxocara or Ancylostoma genus were intermixed in phylogenetic trees as opposed to the ITS-1, ITS-2, cox1, 12S and 16S loci. Moreover, pairwise nucleotide p-distances were significantly different in the 18S compared to the other loci, with an average of 99.1 ± 0.1%, 99.8 ± 0.1% and 98.8 ± 0.9% for the Ascarididae, Ancylostomatidae and Onchocercidae families, respectively. However, ITS-1 and ITS-2 average pairwise nucleotide p-distances in the three families ranged from 72.7% to 87.3%, and the cox1, 12S and 16S ranged from 86.4% to 90.4%. Additionally, 2491 cox1 sequences were retrieved from the 30 analyzed species in GenBank, whereas 212, 1082, 994, 428 and 143 sequences could be obtained from the 18S, ITS-1, ITS-2, 12S and 16S markers, respectively. CONCLUSIONS: The use of the cox1 gene is recommended because of the high interspecies resolution and the large number of sequences available in databases. Importantly, confirmation of the identity of an unknown specimen should always be complemented with the careful morphological examination of worms and the analysis of other markers used for specific parasitic groups.

Mitochondrial and nuclear gene sequencing confirms the presence of the invasive sea anemone Diadumene lineata (Verrill, 1869) (Cnidaria: Actiniaria) in Argentina.

Background: Diadumene lineata is one of the most widespread sea anemone species worldwide. Although this species has been reported a few times on the Argentine coast since 2004, its identification has traditionally been based on external morphological characteristics, and in most cases no voucher specimens are available to support previous records. Methods: In this study, we obtained DNA sequences of two mitochondrial markers (12S and 16S) and two nuclear markers (18S and 28S) from specimens of D. lineata collected in two locations on the Argentine coast separated by almost 800 km. Additionally, we conducted an analysis of the morphology, as well as the types and size ranges of cnidae, using specimens collected at three different locations along the Argentine coast. Furthermore, since introduced populations of D. lineata are presumably ephemeral and only reproduce asexually outside their native range, we examined the internal anatomy of representatives from the Argentine coast for gametogenic tissue as an indication of whether they might be capable of sexual reproduction. Results: DNA data support our morphological identification, including cnidae analyses, of the specimens as D. lineata. Furthermore, all specimens examined were determined to be sterile. Discussion: Genetic sequence comparisons, phylogenetic reconstruction, and cnidae data support the identification of individuals of D. lineata from Mar Chiquita and Garipe Beach, confirming the presence of the species on the Argentine coast using both morphological and molecular tools. The absence of fertile specimens suggests that each sampled population is likely reproducing only by asexual reproduction and possibly composed of clones. The presence of an additional category of longer p-mastigophores B2a in the actinopharynx and filaments, as well as holotrichs in the column, is also reported. Conclusions: For the first time, we have confirmed the presence of D. lineata in Argentina through molecular data. Additionally, our findings indicate that the analyzed specimens are sterile, suggesting that this species is not engaging in sexual reproduction in the studied localities. It is crucial to continue monitoring the populations of D. lineata along the Argentine coast to assess whether they establish sexual reproduction, expand their distribution range or disappear, or potentially cause any harm to local species or alterations in benthic communities.

Expression of mitochondrial dynamic markers in adenoid cystic carcinoma.

BACKGROUND: Mitochondrial fission and fusion processes are known as mitochondrial dynamics and the occurrence of imbalances in the mitochondrial activity is related to the pathogenesis of many human cancers. However, the importance of mitochondrial dynamics in malignant salivary gland tumours remains unknown. Therefore, we aimed to investigate its prognostic significance in adenoid cystic carcinoma. METHODS: Fifty-seven formalin-fixed paraffin-embedded cases were retrieved and disposed in tissue microarray. Histological sections were submitted to immunohistochemical reactions against AMT, DRP1, FIS1, MFN1, MFN2 and OPA1 proteins. Clinical data were retrieved from the patients' medical files, including specific and disease-free survival data. RESULTS: It was observed that 50.9% of the cases were strongly positive for AMT and DRP1, and 49.1%, 21.1%, 22.8% and 24.6% strongly positive for FIS1, MFN1, MFN2 and OPA1, respectively. Reactions were observed in both epithelial and myoepithelial components of the tumour. The higher expression of MFN2 was associated with solid microscopic pattern (p = 0.016). DRP1 overexpression showed a trend towards a shorter overall survival (p = 0.054), while negative/weak OPA1 showed a trend towards a lower disease-free survival (p = 0.051) in the univariate analysis, but no mitochondrial marker represented an independent prognostic determinant under multivariate analysis. CONCLUSION: In conclusion, mitochondrial dynamics markers do not seem to carry a prognostic significance for adenoid cystic carcinoma patients, but these proteins may play an important role in its pathogenesis.

Revisiting the genetic variability of Brazilian peridomestic populations of the Chagas disease vector Triatoma sordida () (Hemiptera, Triatominae).

Triatoma sordida is an endemic species to South America, currently considered the species most frequently found in the peridomestic environment in Brazil. This triatomine has a wide ecological tolerability that allows it to inhabit several ecotopes and use different food sources. Although the species is considered predominantly peridomestic, it is also capable of colonizing households and forming numerous intra-household colonies, leading to its inclusion among the priorities for triatomine control campaigns in Brazil. All Brazilian populations of T. sordida are considered as T. sordida sensu stricto by chromosomal analyses (which highlights their epidemiological importance), although molecular studies that characterize the genetic diversity of these populations are scarce, being restricted only to the region of Minas Gerais. Thus, several populations of this vector distributed in the states of Bahia, Goiás, Mato Grosso do Sul, and Minas Gerais were analyzed using mitochondrial markers (cyt b and nd1). Low nucleotide diversity, high haplotypic diversity, low genetic distance, and high FST value were observed, as well as the formation of a monophyletic clade of the Brazilian populations of T. sordida, which confirms that this species has low genetic variability, with all specimens in Brazil grouped in T. sordida sensu stricto. In addition to the genetic and evolutionary importance for the knowledge of the biology of these vectors, these results are important from an epidemiological point of view, thus being able to direct vector control programs.

NAD+ precursor increases aerobic performance in mice.

PURPOSE: Nicotinamide riboside (NR) acts as a potent NAD+ precursor and improves mitochondrial oxidative capacity and mitochondrial biogenesis in several organisms. However, the effects of NR supplementation on aerobic performance remain unclear. Here, we evaluated the effects of NR supplementation on the muscle metabolism and aerobic capacity of sedentary and trained mice. METHODS: Male C57BL/6 J mice were supplemented with NR (400 mg/Kg/day) over 5 and 10 weeks. The training protocol consisted of 5 weeks of treadmill aerobic exercise, for 60 min a day, 5 days a week. Bioinformatic and physiological assays were combined with biochemical and molecular assays to evaluate the experimental groups. RESULTS: NR supplementation by itself did not change the aerobic performance, even though 5 weeks of NR supplementation increased NAD+ levels in the skeletal muscle. However, combining NR supplementation and aerobic training increased the aerobic performance compared to the trained group. This was accompanied by an increased protein content of NMNAT3, the rate-limiting enzyme for NAD + biosynthesis and mitochondrial proteins, including MTCO1 and ATP5a. Interestingly, the transcriptomic analysis using a large panel of isogenic strains of BXD mice confirmed that the Nmnat3 gene in the skeletal muscle is correlated with several mitochondrial markers and with different phenotypes related to physical exercise. Finally, NR supplementation during aerobic training markedly increased the amount of type I fibers in the skeletal muscle. CONCLUSION: Taken together, our results indicate that NR may be an interesting strategy to improve mitochondrial metabolism and aerobic capacity.

Expression of mitochondrial dynamics markers during melanoma progression: Comparative study of head and neck cutaneous and mucosal melanomas.

BACKGROUND: Head and neck mucosal melanomas (MMs) are rare tumors with adverse outcomes and poorer prognoses than their more common cutaneous counterparts (cutaneous melanomas-CMs). Few studies have compared the expression of mitochondrial dynamic markers in these tumors. This study aimed to assess the correlations of mitochondrial markers with melanoma progression and their potential as predictors of lymph node involvement and distant metastasis. METHODS: Immunohistochemistry against anti-mitochondrial (AMT), dynamin-related protein 1 (DRP1), mitochondrial fission protein 1 (FIS1), mitofusin-1 (MFN1), and mitofusin-2 (MFN2) antibodies was performed in 112 cases of head and neck CM and MM. A Cox regression multivariate model was used to assess the correlation of AMT, FIS1, and MFN2 expressions considering the risk for nodal and distant metastasis. RESULTS: All markers studied presented higher staining in tumor cells than normal adjacent tissues. Higher mitochondrial content was observed in MM than in CM, and it was significantly associated with nodal metastasis in oral melanomas. Both FIS1 and DRP1 expressions were related to advanced Clark's levels in CM, and they were overexpressed in oral melanomas. Moreover, increased immunoexpression of MFN2 was significantly associated with a higher risk of metastasis in CM, and it was also overexpressed in sinonasal melanomas. CONCLUSIONS: Our results suggest that mitochondrial fission and fusion processes can play an important role during multiple stages of tumorigenesis and the development of nodal and distant metastasis in cutaneous and mucosal melanomas.

Genetic diversity, population structure and rickettsias in Amblyomma ovale in areas of epidemiological interest for spotted fever in Brazil.

Amblyomma ovale (Ixodida: Ixodidae) Koch, 1844 is widely-reported in the neotropical region and is the main vector in the epidemic cycle of Rickettsia parkeri strain Atlantic rainforest, a bioagent of a milder variety of spotted fever (SF). Because species with wide geographical distributions are known to exhibit variations that influence their vectorial capacity, the present study aimed to analyze genetic diversity and rickettsia infection of A. ovale collected during the investigation and surveillance of SF cases in the Cerrado and Atlantic rainforest (ARF) Brazilian biomes. Samples had their DNA extracted, amplified and sequenced for 16S rDNA, 12S rDNA, cytochrome oxidase subunit II and D-loop markers for tick analyses, as well as the gltA, htrA, ompA and ompB genes for rickettsia detection. Between 11 and 33 A. ovale haplotypes were identified, all of them exclusive to areas within individual analyzed biome areas. The A. ovale populations appeared to be structured, with Cluster I restricted to Cerrado + ARF isolated in Caatinga and Cluster II to ARF continuous area. Rickettsia bellii, R. parkeri strain Atlantic rainforest (first report for Goiás state, Cerrado), Rickettsia asemboensis (first record in A. ovale for Brazil) and Rickettsia felis (first detection in this ixodid) were identified. A. ovale clusters were not associated with rickettsia types.

Genetic Pattern and Demographic History of Salminus brasiliensis: Population Expansion in the Pantanal Region during the Pleistocene.

Pleistocene climate changes were major historical events that impacted South American biodiversity. Although the effects of such changes are well-documented for several biomes, it is poorly known how these climate shifts affected the biodiversity of the Pantanal floodplain. Fish are one of the most diverse groups in the Pantanal floodplains and can be taken as a suitable biological model for reconstructing paleoenvironmental scenarios. To identify the effects of Pleistocene climate changes on Pantanal's ichthyofauna, we used genetic data from multiple populations of a top-predator long-distance migratory fish, Salminus brasiliensis. We specifically investigated whether Pleistocene climate changes affected the demography of this species. If this was the case, we expected to find changes in population size over time. Thus, we assessed the genetic diversity of S. brasiliensis to trace the demographic history of nine populations from the Upper Paraguay basin, which includes the Pantanal floodplain, that form a single genetic group, employing approximate Bayesian computation (ABC) to test five scenarios: constant population, old expansion, old decline, old bottleneck following by recent expansion, and old expansion following by recent decline. Based on two mitochondrial DNA markers, our inferences from ABC analysis, the results of Bayesian skyline plot, the implications of star-like networks, and the patterns of genetic diversity (high haplotype diversity and low-to-moderate nucleotide diversity) indicated a sudden population expansion. ABC allowed us to make strong quantitative inferences about the demographic history of S. brasiliensis. We estimated a small ancestral population size that underwent a drastic fivefold expansion, probably associated with the colonization of newly formed habitats. The estimated time of this expansion was consistent with a humid and warm phase as inferred by speleothem growth phases and travertine records during Pleistocene interglacial periods. The strong concordance between our genetic inferences and this historical data could represent the first genetic record of a humid and warm phase in the Pantanal in the period since the Last Interglacial to 40 ka.

A new parrot taxon from the Yucatán Peninsula, Mexico-its position within genus Amazona based on morphology and molecular phylogeny.

Parrots (Psittaciformes) are a diverse group of birds which need urgent protection. However, many taxa from this order have an unresolved status, which makes their conservation difficult. One species-rich parrot genus is Amazona, which is widely distributed in the New World. Here we describe a new Amazona form, which is endemic to the Yucatán Peninsula. This parrot is clearly separable from other Amazona species in eleven morphometric characters as well as call and behavior. The clear differences in these features imply that the parrot most likely represents a new species. In contrast to this, the phylogenetic tree based on mitochondrial markers shows that this parrot groups with strong support within A. albifrons from Central America, which would suggest that it is a subspecies of A. albifrons. However, taken together tree topology tests and morphometric analyses, we can conclude that the new parrot represents a recently evolving species, whose taxonomic status should be further confirmed. This lineage diverged from its closest relative about 120,000 years ago and was subjected to accelerated morphological and behavioral changes like some other representatives of the genus Amazona. Our phylogenies, which are so far the most comprehensive for Amazona taxa enabled us to consider the most feasible scenarios about parrot colonization of the Greater and Lesser Antilles and Central America from South America mainland. The molecular dating of these migrations and diversification rate were correlated with climatic and geological events in the last five million years, giving an interesting insight into Amazon parrot phylogeography and their evolution in general.

Self-fertilization, long-distance flash invasion and biogeography shape the population structure of Pseudosuccinea columella at the worldwide scale.

Population genetic studies are efficient for inferring the invasion history based on a comparison of native and invasive populations, especially when conducted at species scale. An expected outcome in invasive populations is variability loss, and this is especially true in self-fertilizing species. We here focus on the self-fertilizing Pseudosuccinea columella, an invasive hermaphroditic freshwater snail that has greatly expanded its geographic distribution and that acts as intermediate host of Fasciola hepatica, the causative agent of human and veterinary fasciolosis. We evaluated the distribution of genetic diversity at the largest geographic scale analysed to date in this species by surveying 80 populations collected during 16 years from 14 countries, using eight nuclear microsatellites and two mitochondrial genes. As expected, populations from North America, the putative origin area, were strongly structured by selfing and history and harboured much more genetic variability than invasive populations. We found high selfing rates (when it was possible to infer it), none-to-low genetic variability and strong population structure in most invasive populations. Strikingly, we found a unique genotype/haplotype in populations from eight invaded regions sampled all over the world. Moreover, snail populations resistant to infection by the parasite are genetically distinct from susceptible populations. Our results are compatible with repeated introductions in South America and flash worldwide invasion by this unique genotype/haplotype. Our study illustrates the population genetic consequences of biological invasion in a highly selfing species at very large geographic scale. We discuss how such a large-scale flash invasion may affect the spread of fasciolosis.

Molecular analysis of phylogeographic subspecies in three Ponto-Caspian sturgeon species.

Sturgeons (Order Acipenseriformes) represent an extremely valuable natural resource that is now facing depletion. In the current study we evaluate if the traditional classification in subspecies of Acipenser gueldenstaedtii, Acipenser stellatus and Huso huso, endemic to Ponto-Caspian region is sustained by molecular analysis and if these represent Evolutionary Significant Units (ESUs) that should be managed separately in conservation programs. To examine the classification of taxonomic entities we sequenced a fragment of the mitochondrial control region in case of three sturgeon species that inhabit the North-western of Black Sea and migrate for reproduction in the Lower Danube. Beside these sequences, we used previously published sequences from sturgeon individuals sampled in the Black Sea, Azov Sea and Caspian Sea. We determined the genetic diversity and genetic differentiation, conducted a Population Aggregation Analysis (PAA) and inferred an intraspecific molecular phylogeny and haplotype network. The results indicated a low level of genetic differentiation between the geographically designated subspecies and did not support a significant divergence or reciprocal monophyly between them. Our results confirm previous genetic studies with smaller samples sizes, but additional analyses including nuclear markers should be conducted for proper recommendations aiming at the development of conservation programs.

Low genetic diversity in Wolbachia-Infected Culex quinquefasciatus (Diptera: Culicidae) from Brazil and Argentina / Baixa diversidade genética em Culex quinquefasciatus (Diptera: Culicidae) infectado por Wolbachia do Brasil e Argentina

Culex quinquefasciatus is a vector of human pathogens, including filarial nematodes and several viruses. Although its epidemiological relevance is known to vary across geographical regions, an understanding of its population genetic structure is still incipient. In light of this, we evaluated the genetic diversity of Cx. quinquefasciatus and Cx. pipiens x Cx. quinquefasciatus hybrids collected from nine localities in Brazil and one site in Argentina. We used mitochondrial genes cox1 and nd4, along with the coxA and wsp genes of the maternally-inherited Wolbachia endosymbiont. The nd4 fragment was invariant between samples, whilst cox1 exhibited four haplotypes that separated two types of Cx. quinquefasciatus, one clustered in southern Brazil. Low sequence diversity was generally observed, being discussed. Both Brazilian and Argentinian mosquitoes were infected with a single Wolbachia strain. As reported in previous studies with these populations, cox1 and nd4 diversity is not congruent with the population structure revealed by nuclear markers or alar morphology. Future Cx. quinquefasciatus research should, if possible, evaluate mtDNA diversity in light of other markers.

Culex quinquefasciatus é vetor de patógenos humanos, incluindo nematódeos filarídeos e vários vírus. Embora a sua relevância epidemiológica varie entre as diferentes regiões geográficas, o conhecimento da estrutura genética da população é ainda incipiente. Em vista disso, foram avaliados os níveis de diversidade genética de Cx. quinquefasciatus e de híbridos Cx. quinquefasciatus x Cx. pipiens de nove cidades do Brasil e em La Plata, na Argentina. Para os testes foram utilizados fragmentos dos genes mitocondriais cox1 e nd4, juntamente com coxA e wsp do endossimbionte Wolbachia, herdado maternalmente. O fragmento nd4 não apresentou variação entre as amostras, e o cox1 exibiu quatro haplótipos que separaram dois tipos de Cx. quinquefasciatus, com um deles agrupado no sul do Brasil. Os dados de sequência mostraram baixa diversidade, sendo esta discutida. Ambas as amostras de mosquitos brasileiros e argentinos estão infectados com uma única cepa de Wolbachia. A diversidade apresentada por nd4 e cox1 não é congruente com a estrutura da população revelada por marcadores nucleares e morfologia alar de estudos anteriores com estas mesmas populações. Pesquisas com Cx. quinquefasciatus devem, se possível, avaliar a diversidade por DNA mitocondrial na luz de outros marcadores.