Nicotinamide Adenine Dinucleotide Dehydrogenase Subunit Research Articles

Morphological and molecular data establishes Clinostomum dolichorchum n. sp. (Digenea: Clinostomidae) in the great blue heron Ardea herodias L. and American bullfrog Rana catesbeiana Shaw.

Clinostomum is a cosmopolitan genus of trematodes that infect piscivorous birds, freshwater molluscs, freshwater fish and amphibians. Herein, a novel species of Clinostomum is described based on morphological and molecular data from an adult in the oral cavity of the great blue heron Ardea herodias and metacercariae collected from the gills and skin of American bullfrog tadpoles Rana catesbeiana. The novel species shares similar qualitative and quantitative morphological features with a congener, Clinostomum marginatum, which has overlap in host and geographic distribution. The most notable morphological difference when compared to C. marginatum is the greater posterior testis length of the novel species. Molecular data resolved similarities with morphological comparisons to nominal species and supports the establishment of a novel species. Molecular data include partial small ribosomal subunit (18S rRNA gene), ribosomal internal transcribed spacer regions (ITS1, 5.8S rRNA gene, and ITS2), partial large ribosomal subunit (28S rRNA gene), cytochrome c oxidase subunit 1 gene (cox1), and nicotinamide adenine dinucleotide dehydrogenase subunit 1 gene (nad1) sequences. Phylogenetic analyses place the novel species in a sister clade to C. marginatum. Morphological and molecular data, combined with phylogenetic analyses support the establishment of Clinostomum dolichorchum n. sp.

The completed mitochondrial genomes of Globodera vulgaris reveals new insights into the genus Globodera phylogeny

Due to the highly conserved structure, animal mitochondrial genome (mtDNA) is widely used in classification, evolution, phylogeny, population genetic structure and other fields. We reported on the five circle multipartite mtDNAs of a newly described species of Globodera, Globodera vulgaris (Gv) from potatoes in China. The results showed that the mtDNA of Gv was obtained through second- and third-generation sequencing, with a total length of 42,995 bp. It contained 12 protein-coding genes, two rRNA genes and 17 tRNA genes, which were distributed in different subgenomic circles. Comparison of the differences in mtDNA among Gv, G. rostochiensis, G. pallida and G. ellingtonae showed that the size and arrangement of the genes in the mtDNA of the genus Globodera were variable and not conserved. The codon usage bias of the mitochondrial protein-coding gene of Gv showed that Gv might have originated from locally and more primitive group of existing Globodera. Based on the cytochrome c oxidase subunits I genes (COX1) and the nicotinamide adenine dinucleotide dehydrogenase subunits I genes (ND1), and the results showed that Gv was clustered with Globodera spp. according to the COX1 and ND1 in scmtDNA-V, while Gv was clustered with Meloidogyne spp. according to ND1 in scmtDNA-III. The results of this study provided a new basis for understanding the multipartite structure of mtDNA as a phylogenetic and taxonomic feature of the genus Globodera. The number of subgenomic circles is a diagnostic feature of species and the arrangement order and size of mitochondrial protein-coding genes also have important application value in species identification within the genus.

FIRST REPORTS OF LIGULA INTESTINALIS AND A SCHISTOCEPHALUS SP. INFECTING SMALL-BODIED FISH IN NEW BRUNSWICK, CANADA.

Morphological characteristics and DNA sequencing were used to identify plerocercoids of a Schistocephalus sp. infecting slimy sculpin (Cottus cognatus) from northern New Brunswick and plerocercoids of Ligula intestinalis infecting blacknose dace (Rhinichthys atratulus) in Fundy National Park (FNP, New Brunswick). To our knowledge, no previous publications documented either cestode from New Brunswick, Canada. Blacknose dace represent a new host record for L. intestinalis. Identifications were made based on the presence or absence of segmentation and sequencing partial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1; mitochondrial DNA) and/or partial cytochrome c oxidase subunit 1 (COI; mitochondrial DNA). Plerocercoids from blacknose dace in FNP were identified as Ligula intestinalis based on >99% nucleotide identity with COI for this species in the NCBI GenBank database. Plerocercoids in slimy sculpin from northern New Brunswick were identified as a Schistocephalus sp. based on high nucleotide identity with congenerics in the NCBI GenBank database. The absence of GenBank entries with sufficient high percent identity to our specimens, and potential species hybrids in this genus, prevents species-level identification of Schistocephalus sp. plerocercoids currently. The absence of previous documentation of these cestodes might reflect recent environmental change promoting the transmission of these parasites that can modulate host fish behavior, induce sterility of host fishes, and contribute to epizootics.

Cystic echinococcosis in donkeys in eastern Africa.

Cystic echinococcosis (CE) is endemic in humans and domestic animals in eastern Africa. All the species of the Echinococcus granulosus sensu lato complex have been reported in this region except for E. equinus, possibly due to the small number of studies involving equids. This study reports the frequency of different Echinococcus species in donkeys from eastern Africa. A total of 5961 donkeys were examined during meat inspection in 3 slaughterhouses in Kenya. Identification of Echinococcus spp. was achieved through polymerase chain reaction-restriction fragment-length polymorphism and sequencing of the mitochondrial nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 gene. The prevalence of CE was 5.7% (337/5961). The 263 genotyped cysts belonged to E. equinus (n = 163), E. granulosus sensu stricto (n = 70), E. canadensis (G6/7) (n = 26) and E. ortleppi (n = 4). One donkey harboured a metacestode of Spirometra theileri. All E. equinus cases, except 2, originated from southern Ethiopia, whereas the other species were more evenly distributed across the study area. Most of the cysts belonging to E. equinus were fertile (111/163), while those of the other species were non-fertile. This is the first report of Echinococcus spp. in donkeys from sub-Saharan Africa and the first confirmation of E. equinus in East Africa. The frequent fertility of E. equinus cysts in donkeys affirms their suitability as intermediate hosts of this species, while low frequency and cyst fertility suggest a marginal role of donkeys in the transmission of E. granulosus s. s., E. canadensis (G6/7) and E. ortleppi.

Genetic variation of sequences of partial mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (pnad1) and pnad5 genes of Toxascaris leonina isolates from foxes and dogs in Jiuquan City of Gansu Province

To investigate the genetic variations of Toxascaris leonina isolates from different hosts in Jiuquan City, Gansu Province. The mitochondrial sequences of partial mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 (pnad1) and pnad5 of eleven T. leonina isolates from domestic dogs, foxes and pet dogs in Jiuquan City, Gansu Province, were amplified using PCR, and the amplification product was sequenced. The genetic variations of pnad1 and pnad5 genes in T. leonina isolates were analyzed. The sequences of T. leonina pnad1 and pnad5 genes measured 530 bp and 550 bp in size, respectively. The nucleotide sequence homology was 99.4% to 100.0% for T. leonina pnad1 gene and 99.5% to 99.8% for T. leonina pnad5 gene, and the sequences of T. leonina pnad1 and pnad5 genes shared 99.2% to 99.9% and 99.1% to 99.9% with corresponding sequences of known T. leonina isolates. In addition, there were 19 and 24 polymorphic sites detected in the sequences of T. leonina pnad1 and pnad5 genes, with 10 and 9 haplotypes, haplotype diversity of 0.982 and 0.964 and nucleotide diversity of 0.039 4 and 0.034 2, respectively. Phylogenetic analysis based on pnad1 and pnad5 gene sequences showed that the eleven T. leonina isolates and known T. leonina isolates were clustered into the same branch with a random distribution, which were close to the branch where Toxocara canis was clustered, and far from the branch where other Ascaris species were clustered. There is a minor genetic variation in pnad1 and pnad5 genes of T. leonina isolates from different hosts in Jiuquan City, Gansu Province, and the pnad1 gene is more suitable as a molecular marker than pnad5 gene for analysis of genetic variations in T. leonina.

First record of Patagifer bilobus (Rudolphi, 1819) Dietz, 1909 (Digenea: Echinostomatidae), with a morphological and molecular characterization from two threskiornithid species in Mexico.

Patagifer Dietz, 1909 is a small genus of echinostomatids, with 12 recognized species, mostly parasitising threskiornithid birds, distributed worldwide. In the current research, adult specimens of the type species, Patagifer bilobus (Rudolphi, 1819) Dietz, 1909 from the white faced ibis (Plegadis chihi) and white ibis (Eudocimus albus) were re-described, providing new metrical data for the number of head collar spines. Those specimens were recorded from eight localities in Mexico and compared morphologically with specimens previously identified as Patagifer lamothei. A total of 19 specimens identified as P. bilobus including two hologenophores were sequenced with three molecular markers: domains D1-D3 of the large subunit (LSU), the internal transcribed spacer (ITS1, ITS2) plus 5.8S from the nuclear rDNA, and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) from mitochondrial DNA. The new sequences were aligned with other sequences of Patagifer spp., downloaded from GenBank. Phylogenetic trees inferred from each data set, placed all the specimens in a clade, confirming that the isolates belonged to the same species. The morphological examination of specimens previously identified as P. lamothei by Ortega-Olivares MP, Hernández-Mena DI, Pérez-Ponce de León G, García-Varela M (2011) Helminths of the white ibis,Eudocimus albus (Aves Therskiornithidae) in Mexico. (Zootaxa 3088, 15-26. 10.11646/zootaxa.3088.1.2) and in combination with molecular data confirms that those specimens should be reassigned to P. bilobus. In addition, this is the first study in P. bilobus using an integrative taxonomy approach.

Population Genetic Structure and Diversity of Metaphire remanens (Oligochaeta: Megascolecidae) Based on Mitochondrial DNA Analysis, with a Note on a New Species of Metaphire remanens sp. nov.

Metaphire remanens sp. nov. is widely distributed throughout Hunan Province, China. We sequenced the mitochondrial DNA to investigate its population genetic structure and genetic diversity, including cytochrome c oxidase subunit I, cytochrome c oxidase subunit II, 12S ribosomal (r)RNA, 16S rRNA, and nicotinamide adenine dinucleotide dehydrogenase subunit 1, derived from 39 individuals from seven geographic locations in Hunan Province. The genetic diversity indices showed that populations of M. remanens have a strong genetic structure and obvious dispersal histories. M. remanens did not experience population expansion, except in Xiangtan City. This may be because of its evolution toward parthenogenesis. The divergence time estimates indicated that M. remanens originated at 19.2055 Ma and then generated two main lineages at 1.7334 Ma (Quaternary glaciation). These results indicate that glaciation, geographic isolation, and dispersal ability are significant factors that influence the differentiation and dispersal of M. remanens. In this study, we describe Metaphire remanens sp. nov. in morphology.

Molecular identification and genetic-polymorphism analysis of Fasciola flukes in Dali Prefecture, Yunnan Province, China.

This study aimed to identify species of Fasciola flukes in Dali Prefecture (Yunnan Province, China) and analyze their genetic diversity. Fasciola flukes (n=122) were collected from cattle livers in a farmers' market in Xiaguan Town, Dali Prefecture. Nucleotide sequences of ribosomal internal transcribed spacer (ITS) as well as nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) and mitochondrial cytochrome c oxidase subunit 1 (CO1) were amplified, sequenced, and subjected to homology analysis. The heterozygosity ratios of different ITS alleles were determined using the peak-height ratio of heterozygous loci. Multiplex PCR analysis of the nuclear protein coding gene, phosphoenolpyruvate carboxykinase (pepck), was used to identify Fasciola species. Multiple ND1 sequence alignments enabled further genetic diversity analysis of regional Fasciola flukes. Seven ITS sequences belonged to F. hepatica and 115 belonged to Fh/Fg heterozygous flukes. Sequencing analysis of heterozygous flukes revealed 11 heterozygous loci with double peaks, with significantly variable ratios among individuals. ND1 and CO1 results indicated that one specimen was identical to F. hepatica, while 121 specimens were identical to F. gigantica or contained one variable site. Multiplex PCR results for pepck showed that double bands for F. hepatica and F. gigantica were amplified from Dali Fasciola specimens; hence, they were all heterozygous. By combining ITS, ND1, and CO1 sequences with multiplex pepck PCR results, all 122 specimens were identified as Fh/Fg heterozygous Fasciola flukes. Our experimental results preliminarily confirmed a high degree of Fh/Fg heterozygosity among Fasciola flukes in the Dali area. Selecting multiple molecular markers for concurrent analysis will provide more comprehensive and accurate genetic information.

Host-induced phenotypic plasticity in Saccocoelioides lamothei Aguirre-Macedo and Violante-González, 2008 (Digenea: Haploporidae) a parasite of freshwater, brackish and marine fishes from Middle America.

Saccocoelioides is a genus of trematodes associated with fishes from the Americas. In the current research, morphologically distinct specimens of Saccocoelioides spp. were collected from six countries in Middle America. Specimens were sequenced using three molecular markers, the domains D1-D3 of the large subunit (LSU) from the nuclear rDNA, the cytochrome c oxidase subunit 1 (cox1) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) from mitochondrial DNA. A total of 74 new sequences were compared and aligned with other sequences available in GenBank. Maximum likelihood and Bayesian inference analyses were inferred from the LSU and cox1 datasets, revealing unequivocally that all the specimens correspond to S. lamothei. A haplotype network was built with 119 sequences of the nad1 gene. The network detected 57 distinct haplotypes divided into three haplogroups. To explore morphological differences among samples of S. lamothei, 17 morphological features were measured from 53 specimens from three fish families: Eleotridae, Mugilidae and Gobiidae. Principal component analysis yielded three main polygons that corresponded with each family analysed, suggesting host-induced phenotypic plasticity. The current evidence suggests that S. lamothei infects at least five fish families along the Pacific coasts of Mexico, Guatemala, El Salvador, Honduras, Nicaragua and Costa Rica.

Morphology and Molecular Identification of Echinostoma revolutum and Echinostoma macrorchis in Freshwater Snails and Experimental Hamsters in Upper Northern Thailand.

Echinostome metacercariae were investigated in freshwater snails from 26 districts in 7 provinces of upper northern Thailand. The species identification was carried out based on the morphologies of the metacercariae and adult flukes harvested from experimental hamsters, and on nucleotide sequences of internal transcribed spacer 2 (ITS2) and nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) genes. Twenty-four out of 26 districts were found to be infected with echinostome metacercariae in freshwater snails with the prevalence of 40.4%. The metacercariae were found in all 6 species of snails, including Filopaludina martensi martensi (21.9%), Filopaludina doliaris (50.8%), F. sumatrensis polygramma (61.3%), Bithynia siamensis siamensis (14.5%), Bithynia pulchella (38.0%), and Anenthome helena (4.9%). The echinostome metacercariae found in these snails were identified as Echinostoma revolutum (37-collar-spined) and Echinostoma macrorchis (45-collar-spined) morphologically and molecularly. The 2-week-old adult flukes of E. revolutum revealed unique features of the cirrus sac extending to middle of the ventral sucker and smooth testes. E. macrorchis adults revealed the cirrus sac close to the right lateral margin of the ventral sucker and 2 large and elliptical testes with slight indentations and pointed posterior end of the posterior testis. The ITS2 and nad1 sequences confirmed the species identification of E. revolutum, and the sequences of E. macrorchis have been deposited for the first time in GenBank. The presence of the life cycle of E. macrorchis is a new record in Thailand and the snail F. doliaris as their second intermediate host seems to be new among the literature.

XIAP Protects Retinal Ganglion Cells in the Mutant ND4 Mouse Model of Leber Hereditary Optic Neuropathy.

PurposeLeber hereditary optic neuropathy (LHON) is a genetic form of vision loss that occurs primarily owing to mutations in the nicotinamide adenine dinucleotide dehydrogenase (ND) subunits that make up complex I of the electron transport chain. LHON mutations result in the apoptotic death of retinal ganglion cells. We tested the hypothesis that gene therapy with the X-linked inhibitor of apoptosis (XIAP) would prevent retinal ganglion cell apoptosis and reduce disease progression in a vector-induced mouse model of LHON that carries the ND4 mutation.MethodsAdeno-associated virus (AAV) encoding full length hemagglutinin-tagged XIAP (AAV2.HA-XIAP) or green fluorescent protein (AAV2.GFP) was injected into the vitreous of DBA/1J mice. Two weeks later, the LHON phenotype was induced by AAV delivery of mutant ND4 (AAV2.mND4FLAG) to the vitreous. Retinal function was assessed by pattern electroretinography. Optic nerves were harvested at 4 months, and the effects of XIAP therapy on nerve fiber layer and optic nerve integrity were evaluated using immunohistochemistry, transmission electron microscopy and magnetic resonance imaging.ResultsDuring LHON disease progression, retinal ganglion cell axons are lost. Apoptotic cell bodies are seen in the nuclei of astrocytes or oligodendrocytes in the optic nerve, and there is thinning of the optic nerve and the nerve fiber layer of the retina. At 4 months after disease onset, XIAP gene therapy protects the nerve fiber layer and optic nerve architecture by preserving axon health. XIAP also decreases nuclear fragmentation in resident astrocytes or oligodendrocytes and decreases glial cell infiltration.ConclusionsXIAP therapy improves optic nerve health and delays disease progression in LHON.

Mitochondrial transport mediates survival of retinal ganglion cells in affected LHON patients.

The mutations in the genes encoding the subunits of complex I of the mitochondrial electron transport chain are the most common cause of Leber's hereditary optic neuropathy (LHON), a maternal hereditary disease characterized by retinal ganglion cell (RGC) degeneration. The characteristics of incomplete penetrance indicate that nuclear genetic and environmental factors also determine phenotypic expression of LHON. Therefore, further understanding of the role of mutant mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit proteins and nuclear genetic factors/environmental effects in the etiology of LHON is needed. In this study, we generated human-induced pluripotent stem cells (hiPSCs) from healthy control, unaffected LHON mutation carrier, and affected LHON patient. hiPSC-derived RGCs were used to study the differences between affected and unaffected carriers of mitochondrial DNA point mutation m.11778G > A in the MT-ND4 gene. We found that both mutated cell lines were characterized by increase in reactive oxygen species production, however, only affected cell line had increased levels of apoptotic cells. We found a significant increase in retrograde mitochondria and a decrease in stationary mitochondria in the affected RGC axons. In addition, the messenger RNA and protein levels of KIF5A in the LHON-affected RGCs were significantly reduced. Antioxidant N-acetyl-L-cysteine could restore the expression of KIF5A and the normal pattern of mitochondrial movement in the affected RGCs. To conclude, we found essential differences in the mutually dependent processes of oxidative stress, mitochondrial transport and apoptosis between two LHON-specific mutation carrier RGC cell lines, asymptomatic carrier and disease-affected, and identified KIF5A as a central modulator of these differences.

First report of pre-Hispanic Fasciola hepatica from South America revealed by ancient DNA.

It is generally assumed that the digenean human liver fluke, Fasciola hepatica, gained entry to South America during the 15th century upon arrival of Europeans and their livestock. Nonetheless in Patagonia, Argentina, digenean eggs similar to F. hepatica have been observed in deer coprolites dating back to 2300 years B.P. The main objective of our present study was to identify and characterize these eggs using an ancient DNA (aDNA) study. Eggs were isolated and used for aDNA extraction, amplification and sequencing of partial regions from the cytochrome c oxidase subunit 1 and the nicotinamide adenine dinucleotide dehydrogenase subunit 1 mitochondrial genes. Also, phylogenetic trees were constructed using Bayesian and maximum likelihood. Our results confirm the presence of F. hepatica in South America from at least 2300 years B.P. This is the first report and the first aDNA study of this trematode in South America prior to the arrival of the European cattle in the 15th century. The present work contributes to the study of phylogenetic and palaeobiogeographical aspects of F. hepatica and its settlement across America.

Lutein upregulates the PGC-1α, NRF1, and TFAM expression by AMPK activation and downregulates ROS to maintain mtDNA integrity and mitochondrial biogenesis in hyperglycemic ARPE-19 cells and rat retina.

Hyperglycemia (HG) affects cellular organelle including mitochondrion in retina that diminishes mitochondrial biogenesis by downregulation of nuclear transcription factors peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1α) and mitochondrial transcription factor A (TFAM). Mitochondrial dysfunction has been linked to diabetic retinopathy (DR). Carotenoids reported to modulate mitochondrial biogenesis in HG. Aim of the study was to explore the role of lutein, oxidized lutein (purified upon UV oxidation of lutein) and drug metformin, on mitochondrial biogenesis in HG-induced ARPE-19 cells and rat retina. Results showed higher uptake of lutein and oxidized lutein in ARPE-19 cells and rat retina of HG group than the control groups. Further, lutein and oxidized lutein augmented the AMPK phosphorylation and activation of mitochondrion signaling molecule TFAM (protein expression) and mRNA expression of PGC-1α, TFAM, and nuclear respiratory factor 1 (responsible for mitochondria biogenesis) along with lowered reactive oxygen species in HG compared with control and metformin groups. Higher mRNA expression of nicotinamide adenine dinucleotide dehydrogenase subunits mt-ND1, mt-ND4, mt-ND6, and cytochrome C that aid maintenance of mtDNA integrity was also evidenced. To conclude, lutein and oxidized lutein found to upsurge mitochondrial biogenesis in ARPE-19 cells and rat retina under HG, which may be due to upregulation of AMPK phosphorylation. Finally, lutein and oxidized lutein may provide a therapeutic basis to ameliorate HG-induced DR.

Metabolic surgery reduces increased urinary mitochondrial DNA copy number in obese patients

At present, obesity has become an independent risk factor for chronic kidney disease through its direct or indirect effects. Urine mitochondrial DNA (mtDNA) is a new marker of mitochondrial damage in various kidney diseases, but the differences in urinary mtDNA between obese and healthy subjects and the effect of obesity intervention on urinary mtDNA copy number are unknown. In January 2019, The Journal of Clinical Endocrinology & Metabolism published an article Bariatric surgery reduces elevated urinary mitochondrial DNA copy number in obese patients [Lee H, Oh S, Yang W, et al. J Clin Endocrinol Metab, 2019, 104(6): 2257-2266], with the permission of the original journal, we translate the article into Chinses. This article studied the difference of urinary mtDNA levels between obese and healthy subjects and the changes of urinary mtDNA after metabolic surgery intervention. The results revealed that obesity was associated with the increase of urinary nicotinamide adenine dinucleotide dehydrogenase subunit 1 (mtND-1) copy number, and urinary mtDNA copy number was elevated in obese patients. Metabolic surgery reduced urinary mtND-1 copy number in obese patients. This study shows that metabolic surgery can alleviate mitochondrial damage in kidney cells in obese patients. Key words: Metabolic surgery; Obesity; Urinary mitochondrial DNA

Development of a quantitative polymerase chain reaction assay and environmental DNA sampling methods for Giant Gartersnake (Thamnophis gigas).

The Giant Gartersnake (Thamnophis gigas) is a low density visually evasive species with a low detection probability based on standard field survey methods (e.g., traps, visual census). Habitat loss has resulted in extirpations or serious declines for T. gigas populations throughout the southern two thirds of its historic range. Uncertainty regarding its current distribution and occupancy present management challenges for the species. Enhancing survey sensitivity through development of environmental DNA sampling (eDNA) methods would improve compliance monitoring under the Endangered Species Act, recovery planning for T. gigas, and evaluation of California’s Central Valley tule marsh habitat on which this species depends. To address these needs, we designed and validated diagnostic quantitative Polymerase Chain Reaction (qPCR) assays for identifying portions of the Cytochrome B (CytB) and the Nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4 (ND4) genes of the T. gigas mitochondrial genome. The designed ND4 qPCR assay was not specific to T. gigas DNA and amplified DNA from a closely related and spatially co-occurring Thamnophis species (T.s. fitchi). The CytB T. gigas qPCR assay proved specific to a species level with a sensitivity that reliably detected T. gigas DNA at a concentration of 2.0x10-5 ng μL-1. To assess detection range, coordinated field sampling was conducted at aquatic sites with an observed and documented population of T. gigas. The T. gigas qPCR assay reliably detected DNA from samples taken 300m downstream from the known source. We then used environmental eDNA sampling and qPCR analysis to augment unsuccessful trap surveys in the southern range of T. gigas and detected DNA in 28 of the 52 locations sampled, confirming that T. gigas was still present at some sites where physical trapping failed to identify presence. QPCR-based DNA detection coupled with eDNA sampling methods provides an effective means to obtain critical population metrics from this otherwise cryptic, federally protected and hard to study organism, offering great promise for elucidating patterns of occupancy with greater efficiency and at far less cost than trapping methods, particularly where detection probabilities are low.

S-Nitrosoglutathione Reductase Is Essential for Protecting the Female Heart From Ischemia-Reperfusion Injury.

Protein S-nitros(yl)ation (SNO) has been implicated as an essential mediator of nitric oxide-dependent cardioprotection. Compared with males, female hearts exhibit higher baseline levels of protein SNO and associated with this, reduced susceptibility to myocardial ischemia-reperfusion injury. Female hearts also exhibit enhanced S-nitrosoglutathione reductase (GSNO-R) activity, which would typically favor decreased SNO levels as GSNO-R mediates SNO catabolism. Because female hearts exhibit higher SNO levels, we hypothesized that GSNO-R is an essential component of sex-dependent cardioprotection in females. Male and female wild-type mouse hearts were subjected to ex vivo ischemia-reperfusion injury with or without GSNO-R inhibition (N6022). Control female hearts exhibited enhanced functional recovery and decreased infarct size versus control males. Interestingly, GSNO-R inhibition reversed this sex disparity, significantly reducing injury in male hearts, and exacerbating injury in females. Similar results were obtained with male and female GSNO-R-/- hearts using ex vivo and in vivo models of ischemia-reperfusion injury. Assessment of SNO levels using SNO-resin assisted capture revealed an increase in total SNO levels with GSNO-R inhibition in males, whereas total SNO levels remained unchanged in females. However, we found that although GSNO-R inhibition significantly increased SNO at the cardioprotective Cys39 residue of nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 3 in males, SNO-NADH dehydrogenase subunit 3 levels were surprisingly reduced in N6022-treated female hearts. Because GSNO-R also acts as a formaldehyde dehydrogenase, we examined postischemic formaldehyde levels and found that they were nearly 2-fold higher in N6022-treated female hearts compared with nontreated hearts. Importantly, the mitochondrial aldehyde dehydrogenase 2 activator, Alda-1, rescued the phenotype in GSNO-R-/- female hearts, significantly reducing infarct size. These striking findings point to GSNO-R as a critical sex-dependent mediator of myocardial protein SNO and formaldehyde levels and further suggest that different therapeutic strategies may be required to combat ischemic heart disease in males and females.

Clinostomum poteae n. sp. (Digenea: Clinostomidae), in the trachea of a double-crested cormorant Phalacrocorax auritus Lesson, 1831 and molecular data linking the life-cycle stages of Clinostomum album Rosser, Alberson, Woodyard, Cunningham, Pote & Griffin, 2017 in Mississippi, USA.

Clinostomum spp. (Digenea: Clinostomidae) are a group of trematodes commonly found in the buccal cavity and oesophagus of a variety of piscivorous birds. The metacercariae, colloquially known as "yellow grubs," have been reported from a diverse group of freshwater fishes worldwide. In the catfish farming region of the southeastern USA, piscivorous birds present a continuous challenge for aquaculturists in the form of fish depredation and the introduction of trematodes into these static, earthen pond systems. Clinostomum spp. are commonly encountered in farm-raised catfish. While generally considered pests of minimal importance, heavy infections can result in unmarketable fillets. Of the piscivorous birds that frequent catfish aquaculture operations in the southeastern US, the double-crested cormorant (Phalacrocorax auritus Lesson) is one of the most damaging, although reports of Clinostomum spp. from P. auritus are limited. In this study, adult trematodes morphologically consistent with Clinostomum sp. were found in the trachea of a double-crested cormorant captured in Lowndes Co., Mississippi, USA. These specimens differed from other recognised Clinostomum spp. in several key morphological characters. Moreover, sequence data of mitochondrial cytochrome c oxidase subunit 1 gene (cox1), nicotinamide adenine dinucleotide dehydrogenase subunit 1 gene (nad1) and ribosomal internal transcribed spacer (ITS) regions did not match any known Clinostomum sp. for which sequence data are available. While genetically similar to C. marginatum and C. album Rosser, Alberson, Woodyard, Cunningham, Pote & Griffin, 2017 reported from the great egret Ardea alba L. in Mississippi, these adult clinostomids were larger in size and limited to the trachea, whereas both C. marginatum Rudolphi, 1819 and C. album are found in the oral cavity and esophagus. Given these distinct morphological and molecular characters we propose a new member of the genus, known hereafter as Clinostomum poteae n. sp. Additionally, larval stages in the life-cycle of C. album are morphologically and molecularly identified for the first time from ramshorn snails Planorbella trivolvis Say and fathead minnows Pimephales promelas Rafinesque.

A new species of fan-throated lizard of the genus Sitana Cuvier, 1829 from coastal Kerala, southern India.

We here describe Sitana attenboroughii sp. nov., a new species of fan-throated lizard of the genus Sitana Cuvier, 1829 from coastal Kerala in southern India. The new species morphologically is closer to Sitana visiri Deepak, 2016 (in Deepak et al. 2016a), however, differs in having higher numbers of ventral scales and a comparatively short but richly colored dewlap. Genetically the new species shows affinity to Sitana marudhamneydhal Deepak, Khandekar, Varma Chaitanya, 2016 from which it differs in an uncorrected pairwise sequence divergence of 2.2% for a fragment of mitochondrial Nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 2 gene.

Exploring the diversity of Diplostomum (Digenea: Diplostomidae) in fishes from the River Danube using mitochondrial DNA barcodes

BackgroundMetacercariae of Diplostomum are important fish pathogens, but reliable data on their diversity in natural fish populations are virtually lacking. This study was conducted to explore the species diversity and host-parasite association patterns of Diplostomum spp. in a large riverine system in Europe, using molecular and morphological data.MethodsTwenty-eight species of fish of nine families were sampled in the River Danube at Nyergesújfalu in Hungary in 2012 and Štúrovo in Slovakia in 2015. Isolates of Diplostomum spp. were characterised morphologically and molecularly. Partial sequences of the ‘barcode’ region of the cytochrome c oxidase subunit 1 (cox1) and complete sequences of the nicotinamide adenine dinucleotide dehydrogenase subunit 3 (nad3) mitochondrial genes were amplified for 76 and 30 isolates, respectively. The partial cox1 sequences were used for molecular identification of the isolates and an assessment of haplotype diversity and possible host-associated structuring of the most prevalent parasite species. New primers were designed for amplification of the mitochondrial nad3 gene.ResultsOnly lens-infecting Diplostomum spp. were recovered in 16 fish species of five families. Barcoding of representative isolates provided molecular identification for three species/species-level genetic lineages, D. spathaceum, D. pseudospathaceum and ‘D. mergi Lineage 2’, and three single isolates potentially representing distinct species. Molecular data helped to elucidate partially the life-cycle of ‘D. mergi Lineage 2’. Many of the haplotypes of D. spathaceum (16 in total), D. pseudospathaceum (15 in total) and ‘D. mergi Lineage 2’ (7 in total) were shared by a number of fish hosts and there was no indication of genetic structuring associated with the second intermediate host. The most frequent Diplostomum spp. exhibited a low host-specificity, predominantly infecting a wide range of cyprinid fishes, but also species of distant fish families such as the Acipenseridae, Lotidae, Percidae and Siluridae. The nad3 gene exhibited distinctly higher levels of interspecific divergence in comparison with the cox1 gene.ConclusionsThis first exploration of the species diversity and host ranges of Diplostomum spp., in natural fish populations in the River Danube, provided novel molecular, morphological and host-use data which will advance further ecological studies on the distribution and host ranges of these important fish parasites in Europe. Our results also indicate that the nad3 gene is a good candidate marker for multi-gene approaches to systematic estimates within the genus.