Search

Rice blast, caused by Magnaporthe oryzae, is a destructive disease threatening the production of staple foods worldwide. Quinone outside inhibitors (QoIs) are a group of chemicals exhibiting excellent activity against a majority of plant pathogens, with the disadvantage that pathogens can easily develop resistance to QoIs.Here, we investigated the activity of picoxystrobin against M. oryzae, which showed a great inhibitory effect on 100 strains of M. oryzae with half-maximal effective concentrations (ECOur findings suggested that M. oryzae had a mid to high risk of resistance to picoxystrobin. Considering this, we should be vigilant to the resistance risk and apply picoxystrobin sensibly in the field. © 2022 Society of Chemical Industry.

Methadone metabolism and clearance are determined principally by polymorphic cytochrome P4502B6 (CYP2B6). Some CYP2B6 allelic variants affect methadone metabolism in vitro and disposition in vivo. We assessed methadone metabolism by CYP2B6 minor variants in vitro. We also assessed the influence of CYP2B6 variants, and P450 oxidoreductase (POR) and CYP2C19 variants, on methadone clearance in surgical patients in vivo.CYP2B6 and P450 oxidoreductase variants were coexpressed with cytochrome bIn vitro, CYP2B6.4 was more active than wild-type CYP2B6.1. CYPs 2B6.5, 2B6.6, 2B6.7, 2B6.9, 2B6.17, 2B6.19 and 2B6.26 were less active. CYPs 2B6.16 and 2B6.18 were inactive. CYP2B6.1 expressed with POR variants POR.28, POR.5 and P228L had lower rates of methadone metabolism than wild-type reductase. In vivo, methadone clinical clearance decreased linearly with the number of CYP2B6 slow metabolizer alleles, but was not different in CYP2C19 slow or rapid metabolizer phenotypes, or in carriers of the POR*28 allele.Several CYP2B6 and POR variants were slow metabolizers of methadone in vitro. Polymorphisms in CYP2B6, but not CYP2C19 or P450 reductase, affected methadone clearance in vivo. CYP2B6 polymorphisms 516GT and 983TC code for canonical loss of function variants and should be assessed when considering genetic influences on clinical methadone disposition. These complementary translational in vitro and in vivo results inform on pharmacogenetic variability affecting methadone disposition in patients.

Litchi downy blight, caused by Phytophthora litchii, is one of the most important diseases of litchi. Ametoctradin, as the only QioI (quinone inside and outside inhibitor) fungicide, has been registered in China in 2019. However, the ametoctradin-resistance risk and molecular basis in Phytophthora litchii have not been reported.In this study, the sensitivity profile of 144 Phytophthora litchii strains to ametoctradin was determined, with a mean median effective concentration (ECThese results suggest that Phytophthora litchii has a medium to high resistance risk to ametoctradin in the laboratory. Two changes, S33L and D228N, in PlCyt b are likely to be associated with the observed ametoctradin resistance. © 2022 Society of Chemical Industry.

We investigated the controversial origin of domestic sheep (Ovis aries) using large samples of contemporary and ancient domestic individuals and their closest wild relatives: the Asiatic mouflon (Ovis gmelini), the urial (Ovis vignei) and the argali (Ovis ammon). A phylogeny based on mitochondrial DNA, including 213 new cytochrome-b sequences of wild Ovism confirmed that O. gmelini is the maternal ancestor of sheep and precluded mtDNA contributions from O. vignei (and O. gmelini × O. vignei hybrids) to domestic lineages. We also produced 54 new control region sequences showing shared haplogroups (A, B, C and E) between domestic sheep and wild O. gmelini which localized the domestication center in eastern Anatolia and central Zagros, excluding regions further east where exclusively wild haplogroups were found. This overlaps with the geographic distribution of O. gmelini gmelini, further suggesting that the maternal origin of domestic sheep derives from this subspecies. Additionally, we produced 57 new CR sequences of Neolithic sheep remains from a large area covering Anatolia to Europe, showing the early presence of at least three mitochondrial haplogroups (A, B and D) in Western colonization routes. This confirmed that sheep domestication was a large-scale process that captured diverse maternal lineages (haplogroups).

We recently proposed a diffusible reactive (oxygen) species (DRS/DROS) based function for cytochrome b complexes (CBC) and quinones (Q)/quinols (QH

Silonia silondia is a commercially important fish distributed in Asian countries, which is under threat due to overexploitation. This study focuses on the morphological analysis and genetic variation of S. silondia individuals, through truss network and sequencing of two mitochondrial regions, respectively, from six wild populations of the Ganga and Mahanadi river systems in India. A total of 38 haplotypes was observed by analysing combined mitochondrial genes (cytochrome b + ATPase 6/8) in 247 individuals of S. silondia collected from six populations. Average haplotype and nucleotide diversities were 0.8508 and 0.00231, respectively. Genetic structure analysis showed the predominant cause of genetic variation to be within populations. The two clades were observed among the haplotypes and time of divergence from their most probable ancestor was estimated to be around 0.3949 mya. Analysis of combined mitochondrial genes in six populations of S. silondia resulted into three management units or genetic stocks. The truss network analysis was carried out by interconnecting 12 landmarks from digital images of specimens to identify phenotypic stocks. Sixty-five truss morphometric variables were analysed for geometric shape variation which revealed morphological divergence in River Son specimens. The present study presents molecular markers and genetic diversity data which can be critical input for conservation and management of differentiated populations and future monitoring of the genetic bottleneck. The morphological shape analysis clearly shows that variation in the insertion of adipose fin is an important parameter influencing the morphological discrimination.

Background Atovaquone and azithromycin (A&A) with supportive care improve survival rates in cats with cytauxzoonosis. Resistance to atovaquone via parasite cytochrome b gene (cytb) mutations occurs in other Apicomplexan protozoans but is not described in Cytauxzoon felis. Objective To serially characterize the C. felis cytb sequences from a cat that remained persistently infected after A&A treatment. Animal A cat with naturally occurring C. felis infection. Methods Case report of the anemic cat persistently infected with C. felis before, during and after A&A treatment. Cytauxzoon felis cytb genes were amplified and sequenced before, during and after A&A treatment. Results Cytauxzoon felis was detected before, during and after A&A treatment including samples collected 570 days after treatment. After A&A treatment, the cat's anemia improved slightly. Cytb sequencing revealed only wild-type cytb methionine (M128) in samples collected before treatment. In samples collected after treatment, the cytb coded for isoleucine (M128I) and valine (M128I) at 2- and 4-months after treatment. These M128I and M128V mutations persisted even after a repeat treatment course with a higher dose atovaquone combined with the standard dose of azithromycin. Conclusions and clinical importance This report documents C. felis atovaquone resistance associated with M128 cytb mutations. This study suggests parasites with mutations of cytb M128 can be selected and impart resistance to A&A treatment even with higher atovaquone dosing.

The swamp buffalo (Bubalus carabanensis) is mainly bred for meat, transport and rice cultivation in China and Southeast Asian countries. In the current study, we investigated the genetic diversity, maternal origin and phylogenetic relationship of swamp buffalo by analyzing 1,786 mitochondrial cytochrome b (cytb) sequences from China, Vietnam, Laos, Thailand, India and Bangladesh. Our results indicated that the swamp buffalo can be divided into two major lineages (SA and SB with the sublineages) and three rare lineages (SC, SD and SE), which showed strong geographic differentiation. The SA1 lineage represented a major domestication event, which involved population expansion. Regions III and V showed higher haplotype diversity than the other regions, indicating that the regions of Southwest China and IndoChina are potential domestication centers for the swamp buffalo. In addition, the swamp buffalo showed a closer phylogenetic relationship to tamaraw. In conclusion, our findings revealed a high level of genetic diversity and the phylogenetic pattern of the swamp buffalo.

Background and aims: The progression of nonalcoholic fatty liver disease (NAFLD) into severe histological forms (steatohepatitis – NASH) is paralleled by the occurrence of complex molecular processes. Mitochondrial dysfunction is a hallmark feature of advanced disease. Mitochondrially encoded cytochrome B (cytochrome b, MT-CYB), a member of the oxidative phosphorylation system, is a key component of the respirasome supercomplex. Here, we hypothesized that NAFLD severity is associated with liver tissue cytochrome b mutations and damaged mitochondrial DNA (mtDNA). Methods: We included 252 liver specimens of NAFLD patients – in whom histological disease ranged from mild to severe – which were linked to clinical and biochemical information. Tissue molecular explorations included MT-CYB sequencing and analysis of differential mtDNA damage. Profiling of circulating Krebs cycle metabolites and global liver transcriptome was performed in a subsample of patients. Tissue levels of 4-hydroxynonenal – a product of lipid peroxidation and 8-hydroxy-2’-deoxyguanosine, a marker of oxidative damage – were measured. Results: Compared to simple steatosis, NASH is associated with a higher level of MT-CYB variance, 12.1 vs. 15.6 substitutions per 103 bp (P = 5.5e-10). The burden of variants was associated with increased levels of 2-hydroxyglutarate, branched-chain amino acids, and glutamate, and changes in the global liver transcriptome. Liver mtDNA damage was associated with advanced disease and inflammation. NAFLD severity was associated with increased tissue levels of DNA oxidative adducts and lipid peroxyl radicals. Conclusion: NASH is associated with genetic alterations of the liver cellular respirasome, including high cytochrome b variation and mtDNA damage, which may result in broad cellular effects. Fil: Pirola, Carlos José. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina Fil: Garaycoechea, Martin Enrique. Gobierno de la Provincia de Buenos Aires. Hospital El Cruce Doctor Nestor Carlos Kirchner. Centro de Medicina Traslacional.; Argentina Fil: Flichman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas en Retrovirus y Sida. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas en Retrovirus y Sida; Argentina Fil: Castaño, Gustavo Osvaldo. Gobierno de la Ciudad de Buenos Aires. Hospital "Dr. Abel Zubizarreta"; Argentina Fil: Sookoian, Silvia Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; Argentina

In this study, population genetic and demographic parameters were inferred using sequence data from 151 individuals of Anguilla mossambica originating from continental south and south-east Africa and Madagascar. The analyses were based on a 532 bp segment of the mitochondrial cytochrome b gene. The absence of genetic structuring was observed thereby supporting the hypothesis of panmixia for the endemic A. mossambica. The overall nucleotide diversity π = 0.002 and the haplotype diversity reached h = 0.691. Significant negative values from several tests of neutrality and mismatch analysis pointed to fluctuating historical population sizes. Bayesian averaging resulted in higher support for population growth models vs. a constant population-size model. Population decline and subsequent growth most likely predated the last glacial and were probably related to extended periods of extreme drought followed by wetter and more stable hydroclimate between 150 and 75,000 years before present (kBP). According to this scenario the female effective population size has increased since 110 kBP by c. two orders of magnitude to a recent level of about 650,000 (219,317-2,292,000).

BACKGROUNDIn spider mites, mutations in the mitochondrial cytochrome b Qo pocket have been reported to confer resistance to the Qo inhibitors bifenazate and acequinocyl. In this study, we surveyed populations of the two‐spotted spider mite Tetranychus urticae for mutations in cytochrome b, linked newly discovered mutations with resistance and assessed potential pleiotropic fitness costs.RESULTSWe identified two novel mutations in the Qo site: G132A (equivalent to G143A in fungi resistant to strobilurins) and G126S + A133T (previously reported to cause bifenazate and acequinocyl resistance in Panonychus citri ). Two T. urticae strains carrying G132A were highly resistant to bifenazate but not acequinocyl, whereas a strain with G126S + A133T displayed high levels of acequinocyl resistance, but only moderate levels of bifenazate resistance. Bifenazate and acequinocyl resistance were inherited maternally, providing strong evidence for the involvement of these mutations in the resistance phenotype. Near isogenic lines carrying G132A revealed several fitness penalties in T. urticae ; a lower net reproductive rate (R0), intrinsic rate of increase (rm) and finite rate of increase (LM); a higher doubling time (DT); and a more male‐biased sex ratio.CONCLUSIONSSeveral lines of evidence were provided to support the causal role of newly discovered cytochrome b mutations in bifenazate and acequinocyl resistance. Because of the fitness costs associated with the G132A mutation, resistant T. urticae populations might be less competitive in a bifenazate‐free environment, offering opportunities for resistance management. © 2019 Society of Chemical Industry

Shads of the genus Alosa are essential to commercial fisheries across North America and Europe, but in some areas their species boundaries remain controversial. Traditional morphology-based taxonomy of Alosa spp. has relied heavily on the number of gill rakers and body proportions, but these can be highly variable. We use mitochondrial (mt)DNA (coI and cytb) and genome-wide single nucleotide polymorphisms (SNP) along with morphological characters to assess differentiation among endemic Ponto-Caspian shads in the Sea of Azov. Morphological species assignments based on gill-raker number were not congruent with genetic lineages shown by mtDNA and SNPs. Iterative analysis revealed that genetic lineages were associated with sampling location and several other morphometric traits (caudal peduncle depth, pre-anal length and head length). Phylogenetic analysis of the genus placed Ponto-Caspian Alosa spp. in the same evolutionary lineage as endangered Alosa spp. endemic to Greece, highlighting the importance of these findings to conservation management. We conclude that gill-raker number is not reliable for delimiting species of Alosa. This taxonomic uncertainty should be addressed by examining type material to provide a robust integrative classification for these commercially important fishes.

Background Babesiosis caused by Babesia gibsoni is recognized throughout the world and can be difficult to treat. Resistance to atovaquone is associated with mutations in the B. gibsoni mitochondrial genome, specifically the M128 position of cytochrome b (cytb). The prevalence of cytb mutations in North America has not been reported. Hypothesis/objectives The objective of our study was to describe the prevalence of cytb M128 mutations in B. gibsoni in canine blood samples submitted to a US veterinary diagnostic laboratory. A secondary objective was to determine whether or not some dogs had wild-type cytb in our initial samples then had M128 mutations detected in follow-up samples. Animals One-Hundred seventy-four dogs that tested positive for the presence of B. gibsoni between 2012 and 2017. Methods Case series of consecutive samples submitted to a veterinary diagnostic laboratory. Partial B. gibsoni cytb genes were amplified by polymerase chain reaction and screened for the presence of mutations at the M128 position. Results The overall prevalence of M128 mutants was 3.5% (6/173 dogs) in the initial samples. The incidence of new cytb mutants in dogs that tested positive for B. gibsoni, which then had follow-up testing, was 12.1% (5/41). Conclusions and Clinic Importance: Our study reaffirms that B. gibsoni infection is widespread and most commonly detected in American Staffordshire Terrier/American Pit Bull Terrier dogs (128/174, 74% of the infected dogs in our study). The prevalence of cytb mutations does not warrant pretreatment genotyping.

The recently developed environmental DNA (eDNA) analysis has been used to estimate the distribution of aquatic vertebrates by using mitochondrial DNA (mtDNA) as a genetic marker. However, mtDNA markers have certain drawbacks such as variable copy number and maternal inheritance. In this study, we investigated the potential of using nuclear DNA (ncDNA) as a more reliable genetic marker for eDNA analysis by using common carp (Cyprinus carpio). We measured the copy numbers of cytochrome b (CytB) gene region of mtDNA and internal transcribed spacer 1 (ITS1) region of ribosomal DNA of ncDNA in various carp tissues and then compared the detectability of these markers in eDNA samples. In the DNA extracted from the brain and gill tissues and intestinal contents, CytB was detected at 95.1 ± 10.7 (mean ± 1 standard error), 29.7 ± 1.59 and 24.0 ± 4.33 copies per cell, respectively, and ITS1 was detected at 1760 ± 343, 2880 ± 503 and 1910 ± 352 copies per cell, respectively. In the eDNA samples from mesocosm, pond and lake water, the copy numbers of ITS1 were about 160, 300 and 150 times higher than those of CytB, respectively. The minimum volume of pond water required for quantification was 33 and 100 mL for ITS1 and CytB, respectively. These results suggested that ITS1 is a more sensitive genetic marker for eDNA studies of C. carpio.

Organisms continuously release DNA into their environments via shed cells, excreta, gametes and decaying material. Analysis of this 'environmental DNA' (eDNA) is revolutionizing biodiversity monitoring. eDNA outperforms many established survey methods for targeted detection of single species, but few studies have investigated how well eDNA reflects whole communities of organisms in natural environments. We investigated whether eDNA can recover accurate qualitative and quantitative information about fish communities in large lakes, by comparison to the most comprehensive long-term gill-net data set available in the UK. Seventy-eight 2L water samples were collected along depth profile transects, gill-net sites and from the shoreline in three large, deep lakes (Windermere, Bassenthwaite Lake and Derwent Water) in the English Lake District. Water samples were assayed by eDNA metabarcoding of the mitochondrial 12S and cytochrome b regions. Fourteen of the 16 species historically recorded in Windermere were detected using eDNA, compared to four species in the most recent gill-net survey, demonstrating eDNA is extremely sensitive for detecting species. A key question for biodiversity monitoring is whether eDNA can accurately estimate abundance. To test this, we used the number of sequence reads per species and the proportion of sampling sites in which a species was detected with eDNA (i.e. site occupancy) as proxies for abundance. eDNA abundance data consistently correlated with rank abundance estimates from established surveys. These results demonstrate that eDNA metabarcoding can describe fish communities in large lakes, both qualitatively and quantitatively, and has great potential as a complementary tool to established monitoring methods.

International audience; The amount of iron in the diet directly influences the composition of the microbiota. Inversely, the effects of the microbiota on iron homeostasis have been little studied. So, we investigate whether the microbiota itself may alter host iron sensing. Duodenal cytochrome b and divalent metal transporter 1, involved in apical iron uptake, are 8- and 10-fold, respectively, more abundant in the duodenum of germ-free (GF) mice than in mice colonized with a microbiota. In contrast, the luminal exporter ferroportin is 2-fold less abundant in GF. The overall signature of microbiota on iron-related proteins is similar in the colon. The colonization does not modify systemic parameters as plasma transferrin saturation (20%), plasma ferritin (150 ng/L), and liver (85 µg/g) iron load. Commensal organisms (Bacteroides thetaiotaomicron VPI-5482 and Faecalibacterium prausnitzii A2-165) and a probiotic strain (Streptococcus thermophilus LMD-9) led to up to 12-fold induction of ferritin in colon. Our data suggest that the intestinal cells of GF mice are depleted of iron and that following colonization, the epithelial cells favor iron storage. This study is the first to demonstrate that gut microbes induce a specific iron-related protein signature, highlighting new aspects of the crosstalk between the microbiota and the intestinal epithelium.

Hybridization is an important evolutionary process, with ecological and behavioural factors influencing gene exchange between hybrids and parent species. Patterns of hybridization in anemonefishes may result from living in highly specialized habitats and breeding status regulated by size-based hierarchal social groups. Here, morphological, ecological and genetic analyses in Kimbe Bay, Papua New Guinea, examine the hybrid status of Amphiprion leucokranos, a nominal species and presumed hybrid between Amphiprion sandaracinos and Amphiprion chrysopterus. We test the hypothesis that habitat use and relative size differences of the parent species and hybrids determine the patterns of gene exchange. There is strong evidence that A. leucokranos is a hybrid of smaller A. sandaracinos and larger A. chrysopterus, where A. chrysopterus is exclusively the mother to each hybrid, based on mtDNA cytochrome b and multiple nDNA microsatellite loci. Overlap in habitat, depth and host anemone use was found, with hybrids intermediate to parents and cohabitation in over 25% of anemones sampled. Hybrids, intermediate in body size, colour and pattern, were classified 55% of the time as morphologically first-generation hybrids relative to parents, whereas 45% of hybrids were more A. sandaracinos-like, suggesting backcrossing. Unidirectional introgression of A. chrysopterus mtDNA into A. sandaracinos via hybrid backcrosses was found, with larger female hybrids and small male A. sandaracinos mating. Potential nDNA introgression was also evident through distinct intermediate hybrid genotypes penetrating both parent species. Findings support the hypothesis that anemonefish hierarchical behaviour, habitat use and species-specific size differences determine how hybrids form and the evolutionary consequences of hybridization.

Summary 1. Patterns of diversity and turnover in macroorganism communities can often be predicted from differences in habitat, phylogenetic relationships among species and the geographical scale of comparisons. In this study, we asked whether these factors also predict diversity and turnover in parasite communities. 2. We studied communities of avian malaria in two sympatric, ecologically similar, congeneric host species at three different sites. We asked whether parasite prevalence and community structure varied with host population, host phylogeography or geographical distance. 3. We used PCR to screen birds for infections and then used Bayesian methods to determine phylogenetic relationships among malaria strains. Metrics of both community and phylogenetic beta diversity were used to examine patterns of malaria strain turnover between host populations, and partial Mantel tests were used determine the correlation between malaria beta diversity and geographical distance. Finally, we developed microsatellite markers to describe the genetic structure of host populations and assess the relationship between host phylogeography and parasite beta diversity. 4. We found that different genera of malaria parasites infect the two hosts at different rates. Within hosts, parasite communities in one population were phylogenetically clustered, but there was otherwise no correlation between metrics of parasite beta diversity and geographical or genetic distance between host populations. Patterns of parasite turnover among host populations are consistent with malaria transmission occurring in the winter rather than on the breeding grounds. 5. Our results indicate greater turnover in parasite communities between different hosts than between different study sites. Differences in host species, as well as transmission location and vector ecology, seem to be more important in structuring malaria communities than the distance–decay relationships frequently found in macroorganisms. Determining the factors affecting parasite community diversity and turnover has wide-ranging implications for understanding the selective pressures shaping host ecology and ecosystem structure. This study shows that metrics of community and phylogenetic beta diversity can be useful tools for disentangling the ecological and evolutionary processes that underlie geographical variation in parasite communities.

The cornerstone of fisheries management relies on a solid taxonomic base and an understanding of how animals can be grouped into coherent management units. Surprisingly, little is known about the basic biology and ecology of opah (Lampris guttatus), a globally distributed species that is commercially exploited and regionally common in the North Pacific. Recent efforts to collect life history data on this species uncovered evidence of two North Pacific morphotypes. Sequencing of the mitochondrial cytochrome c oxidase I gene (655 bp) for these morphotypes and other specimens collected worldwide (n = 480) produced five strongly diverged and well-supported clades. Additional sequence data from the cytochrome b gene (1141 bp) as well as the nuclear recombination activating gene 1 (1323 bp) corroborated these results, suggesting these five clades probably represent separate species. Our conclusion that opah is a complex of five separate species has implications for management and indicates a need to gather additional data on these poorly understood fishes.

Scope Hyperglycemia is a hallmark of diabetes mellitus but slighter increases of blood glucose levels are observed also during ageing. Using the Caenorhabditis elegans mev-1 mutant, we identified molecular mechanisms underlying the protection from glucose toxicity by the polyphenol quercetin. Methods and results We fed C. elegans mev-1 mutants on a liquid medium supplemented with 10 mM glucose, which resulted in a reduced survival at 37°C. The polyphenol quercetin (1 μM) was able to prevent glucose-induced lifespan reduction completely. RNA interference revealed that the sirtuin SIR-2.1, the nuclear hormone receptor DAF-12, and its putative co-activator MDT-15 were critical for the quercetin effects. Moreover, RNA interference for key factors of proteostasis reduced survival, which was not further affected by glucose or quercetin, suggesting that those proteins are a target for both substances. Besides unfolded protein response, proper functionality of the proteasome was shown to be crucial for the survival enhancing effects of quercetin and the polyphenol was finally demonstrated to activate proteasomal degradation. Conclusion Our studies demonstrate that lowest concentrations of quercetin prevent a glucose-induced reduction of survival. SIR-2.1, DAF-12, and MDT-15 were identified as targets that activate unfolded protein response and proteasomal degradation to limit the accumulation of functionally restricted proteins.

Dinoflagellates in the genus Symbiodinium are ubiquitous in shallow marine habitats where they commonly exist in symbiosis with cnidarians. Attempts to culture them often retrieve isolates that may not be symbiotic, but instead exist as free-living species. In particular, cultures of Symbiodinium clade E obtained from temperate environments were recently shown to feed phagotrophically on bacteria and microalgae. Genetic, behavioral, and morphological evidence indicate that strains of clade E obtained from the northwestern, southwestern, and northeastern temperate Pacific Ocean as well as the Mediterranean Sea constitute a single species: Symbiodinium voratum n. sp. Chloroplast ribosomal 23S and mitochondrial cytochrome b nucleotide sequences were the same for all isolates. The D1/D2 domains of nuclear ribosomal DNA were identical among Western Pacific strains, but single nucleotide substitutions differentiated isolates from California (USA) and Spain. Phylogenetic analyses demonstrated that S. voratum is well-separated evolutionarily from other Symbiodinium spp. The motile, or mastigote, cells from different cultures were morphologically similar when observed using light, scanning, and transmission electron microscopy; and the first complete Kofoidian plate formula for a Symbiodinium sp. was characterized. As the largest of known Symbiodinium spp., the average coccoid cell diameters measured among cultured isolates ranged between 12.2 (± 0.2 SE) and 13.3 (± 0.2 SE) μm. Unique among species in the genus, a high proportion (approximately 10-20%) of cells remain motile in culture during the dark cycle. Although S. voratum occurs on surfaces of various substrates and is potentially common in the plankton of coastal areas, it may be incapable of forming stable mutualistic symbioses.

Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi-aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P. bivittatus from water-borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P. bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P. bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P. bivittatus-specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P. bivittatus DNA was consistently detectable up to 96 h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P. bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P. bivittatus. This study shows P. bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.

The theory of classical and cryptic Pleistocene refugia is based mainly on historical changes in temperature, and the refugia are usually defined within a latitudinal gradient. However, the gradient of oceanic-continental climate (i.e. longitudinal) was also significantly variable during glacial cycles with important biotic consequences. Range-wide phylogeography of the European ground squirrel (EGS) was used to interpret the evolutionary and palaeogeographical history of the species in Europe and to shed light on its glacial-interglacial dynamic. The EGS is a steppe-inhabiting species and the westernmost member of the genus in the Palaearctic region. We have analysed 915 specimens throughout the present natural range by employing mitochondrial DNA sequences (cytochrome b gene) and 12 nuclear microsatellite markers. The reconstructed phylogeography divides the species into two main geographical groups, with deep substructuring within both groups. Bulgaria is the centre of the ancestral area, and it also has the highest genetic diversity within the species. The northernmost group of the EGS survived in the southern part of Pannonia throughout several glacial-interglacial cycles. Animals from this population probably repeatedly colonized areas further to the north and west during the glacial periods, while in the interglacial periods, the EGS distribution contracted back to this Pannonian refugium. The EGS thus represents a species with a glacial expansion/interglacial contraction palaeogeographical dynamics, and the Pannonian and southeastern Balkanian steppes are supported as cryptic refugia of continental climate during Pleistocene interglacials.

There is growing interest in broad-scale biodiversity assessments that can serve as benchmarks for identifying ecological change. Genetic tools have been used for such assessments for decades, but spatial sampling considerations have largely been ignored. Here, we demonstrate how intensive sampling efforts across a large geographical scale can influence identification of taxonomic units. We used sequences of mtDNA cytochrome c oxidase subunit 1 and cytochrome b, analysed with maximum parsimony networks, maximum-likelihood trees and genetic distance thresholds, as indicators of biodiversity and species identity among the taxonomically challenging fishes of the genus Cottus in the northern Rocky Mountains, USA. Analyses of concatenated sequences from fish collected in all major watersheds of this area revealed eight groups with species-level differences that were also geographically circumscribed. Only two of these groups, however, were assigned to recognized species, and these two assignments resulted in intraspecific genetic variation (>2.0%) regarded as atypical for individual species. An incomplete inventory of individuals from throughout the geographical ranges of many species represented in public databases, as well as sample misidentification and a poorly developed taxonomy, may have hampered species assignment and discovery. We suspect that genetic assessments based on spatially robust sampling designs will reveal previously unrecognized biodiversity in many other taxa.

Iron (Fe) deficiency is endemic worldwide. Little data are available regarding acute effects of dietary protein on intestinal Fe absorption. The current study evaluated the short-term effects of increasing dietary protein on Fe absorption and expression of genes involved in Fe homeostasis. Sprague Dawley rats (24, female) were randomly assigned to custom-formulated isocaloric diets containing 40, 20 (control), or 5% protein (as percentage of total kilocalories) for 7 d. Whole-body Fe balance studies demonstrated that Fe retention was greater in the 40% group than in the 5% group (30.8 vs. 7.3%; P0.01). In a separate study utilizing stable iron isotopes, the 40% group absorbed 30% of ingested Fe, while the 20% group absorbed 18% (P=0.005). Whole-genome profiling revealed that increasing dietary protein from 5 to 40% increased duodenal transcript expression of divalent metal transporter 1 (DMT1) 3.2-fold, duodenal cytochrome b (Dcytb) 1.8-fold, and transferrin receptor (TfR) 1.8-fold. Consistent with these findings, DMT1 transcript expression was 4-fold higher in RNA prepared from duodenal mucosa in the 40% group compared to the 20% group (P0.001). These data suggest that increasing dietary protein increases intestinal Fe absorption in part by up-regulating DMT1, Dcytb, and TfR.

Biogeographic patterns displayed by obligate freshwater organisms are intimately related to the nature and extent of connectivity between suitable habitats. Two of the more significant barriers to freshwater connections are seawater and major drainage divides. South-eastern Australia provides a contrast between these barriers as it has discrete areas that are likely influenced to a greater or lesser extent by each barrier type. We use continental shelf width as a proxy for the potential degree of river coalescence during low sea levels. Our specific hypothesis is that the degree of phylogeographic divergence between coastal river basins should correspond to the continental shelf width of each region. This predicts that genetic divergences between river basins should be lowest in regions with a wider continental shelf and that regions with similar continental shelf width should have similar genetic divergences. Pygmy perches (Nannoperca australis and Nannoperca 'flindersi') in south-eastern Australia provide an ideal opportunity to test these biogeographic hypotheses. Phylogeographic patterns were examined based on range-wide sampling of 82 populations for cytochrome b and 23 polymorphic allozyme loci. Our results recovered only limited support for our continental shelf width hypothesis, although patterns within Bass clade were largely congruent with reconstructed low sea-level drainage patterns. In addition, we identified several instances of drainage divide crossings, typically associated with low elevational differences. Our results demonstrate high levels of genetic heterogeneity with important conservation implications, especially for declining populations in the Murray-Darling Basin and a highly restricted disjunct population in Ansons River, Tasmania.

Species identification through noninvasive sampling is increasingly used in animal conservation genetics, given that it obviates the need to handle free-living individuals. Noninvasive sampling is particularly valuable for elusive and small species such as rodents. Although rodents are not usually assumed to be the most obvious target for conservation, of the 21 species or near-species present in Iberia, three are considered endangered and declining, while several others are poorly studied. Here, we develop a genetic tool for identifying all rodent species in Iberia by noninvasive genetic sampling. To achieve this purpose, we selected one mitochondrial gene [cytochrome b (cyt-b)] and one nuclear gene [interphotoreceptor retinoid-binding protein (IRBP)], which we first sequenced using tissue samples. Both genes allow for the phylogenetic distinction of all species except the sibling species Microtus lusitanicus and Microtus duodecimcostatus. Overall, cyt-b showed higher resolution than IRBP, revealing a clear barcoding gap. To allow these markers to be applied to noninvasive samples, we selected a short highly diagnostic fragment from each gene, which we used to obtain sequences from faeces and bones from owl pellets. Amplification success for the cyt-b and IRBP fragment was 85% and 43% in faecal and 88% and 64% in owl-pellet DNA extractions, respectively. The method allows the unambiguous identification of the great majority of Iberian rodent species from noninvasive samples, with application in studies of distribution, spatial ecology and population dynamics, and for conservation.

In this study, the phylogenetic relationships among 13 pangasiids and six schilbids of Thailand were reconstructed based on the almost complete mitochondrial cytochrome b (cyt b), 12S rRNA, tRNA-Val and 16S rRNA, as well as the partial nuclear recombination-activating gene 1 (rag1) sequences by using the maximum likelihood and the Bayesian inference methods of phylogenetic reconstruction. The reconstructed phylogeny based on the concatenated sequence data set recovered Pangasiidae and Schilbidae as reciprocally monophyletic groups. Within Pangasiidae, four major clades were recovered, which according to the cyt b genetic distances can be categorized into four genera: Pangasius, Pseudolais, Helicophagus and Pangasianodon. The genus Pangasianodon was strongly supported as the most basal taxon within pangasiids, whereas Pseudolais + Helicophagus were recovered as a sister group of Pangasius. Within the latter, the giant pangasius Pangasius sanitwongsei was recovered as a sister group of the spot pangasius Pangasius larnaudii, Pangasius krempfi as a sister group of Pangasius nasutus + Pangasius conchophilus and Pangasius polyuranodon as a sister species of Pangasius macronema. Other internal phylogenetic relationships, however, were unresolved. Within Schilbidae, Pseudeutropius was supported as the most basal lineage. Eutropiichthys was recovered as a sister group of Clupisoma. The enigmatic Clupisoma sinense was recognized as more closely related to Laides longibarbis than to Clupisoma prateri. Thus, based on the cyt b genetic distances, a recategorization of C. sinense to the genus Laides is suggested. On the basis of a relaxed clock fossil calibration, the divergence of pangasiids and schilbids was estimated to have occurred 14·93 million years before present (b.p.) during the Miocene epoch. The separation between Pangasiidae and Schilbidae took place c. 13·12 Mb.p. during the early middle Miocene. The estimated divergence time of pangasiids is similar to the age of the calibrated fossil, Cetopangasius chaetobranchus, which was discovered in north-central Thailand. This suggests that the oldest pangasiid ancestor diverged into diverse genera in the area.

Cutaneous leishmaniasis (CL) is a parasitic infection and occurs in tropical and subtropical regions worldwide and in the region of the Mediterranean Sea. The diagnosis is based on the clinical appearance and biopsy findings that may be supplemented with polymerase chain reaction (PCR). In this study 20 cases were selected if (i) a histopathological diagnosis of granulomatous dermatitis was made, (ii) CL was taken into consideration or (iii) the diagnosis was CL. PCR analysis with primers specific for leishmania was performed on archived histological specimens and was positive in 6 of the 20 cases. In two cases both the clinical and histopathological diagnosis concurred with CL. In the remaining four cases a clinical diagnosis other than CL was made. In two of these cases the histopathology showed granulomatous dermatitis, and detection of parasites led to consideration of CL. In the last two cases leishmaniasis was not taken into consideration by clinicians or pathologists. Our study shows that CL may occur more often than anticipated in Norway, but clinicians do not consider the diagnosis as often as they should. Pathologists may also fail to diagnose or suggest CL especially when parasites are not visualized in the histopathological specimen.

Sequence-based species identification relies on the extent and integrity of sequence data available in online databases such as GenBank. When identifying species from a sample of unknown origin, partial DNA sequences obtained from the sample are aligned against existing sequences in databases. When the sequence from the matching species is not present in the database, high-scoring alignments with closely related sequences might produce unreliable results on species identity. For species identification in mammals, the cytochrome b (cyt b) gene has been identified to be highly informative; thus, large amounts of reference sequence data from the cyt b gene are much needed. To enhance availability of cyt b gene sequence data on a large number of mammalian species in GenBank and other such publicly accessible online databases, we identified a primer pair for complete cyt b gene sequencing in mammals. Using this primer pair, we successfully PCR amplified and sequenced the complete cyt b gene from 40 of 44 mammalian species representing 10 orders of mammals. We submitted 40 complete, correctly annotated, cyt b protein coding sequences to GenBank. To our knowledge, this is the first single primer pair to amplify the complete cyt b gene in a broad range of mammalian species. This primer pair can be used for the addition of new cyt b gene sequences and to enhance data available on species represented in GenBank. The availability of novel and complete gene sequences as high-quality reference data can improve the reliability of sequence-based species identification.

One mtDNA gene (cytochrome b), one nuclear DNA fragment, five microsatellites and a suite of morphological characters were evaluated in samples of Rutilus spp. from Skadar, Ohrid and Prespa Lakes. Both genetic and phenotypic data supported two sympatric taxa in Lake Skadar, whereby Prespa and Ohrid Lakes revealed only a single taxon each. One of the taxa from Lake Skadar was similar to samples from Lake Prespa, whereas the second taxon was the most divergent in the data set. The estimated time to the most recent common ancestor of these two sympatric taxa in Lake Skadar was between 125 000 and 500 000 years. The data did not support existing taxonomic schemes for Rutilus in these lakes. This study poses the following working hypothesis: (1) Rutilus prespensis lives both in Lake Prespa and Lake Skadar and therefore is not endemic to Lake Prespa, (2) Rutilus ohridanus lives in Lake Ohrid only and therefore could be considered an endemic if its species status is retained and (3) a third recently described taxon (Rutilus albus) sympatric to R. prespensis lives in Lake Skadar.

Variability in cytochrome b (cytb) in European populations of bream Abramis brama was assessed. The cytb gene was found to be strongly conserved in A. brama relative to other cyprinid taxa. This limits the usefulness of this marker in examining geographical genetic structure in this species and raises interesting questions as to the recent evolutionary history of the species.

Crested gibbons, genus Nomascus, are endemic to the Indochinese bioregion and occur only in Vietnam, Laos, Cambodia, and southern China. However, knowledge about the number of species to be recognized and their exact geographical distributions is still limited. To further elucidate the evolutionary history of crested gibbon species and to settle their distribution ranges, we analyzed the complete mitochondrial cytochrome b gene from 79 crested gibbon individuals from known locations. Based on our findings, crested gibbons should be classified into seven species. Within N. concolor, we recognize two subspecies, N. concolor concolor and N. concolor lu. Phylogenetic reconstructions indicate that the northernmost species, N. hainanus, N. nasutus, and N. concolor branched off first, suggesting that the genus originated in the north and successively migrated to the south. The most recent splits within Nomascus occurred between N. leucogenys and N. siki, and between Nomascus sp. and N. gabriellae. Based on our data, the currently postulated distributions of the latter four species have to be revised. Our study shows that molecular methods are a useful tool to elucidate phylogenetic relationships among crested gibbons and to determine species boundaries. Am. J. Primatol. 72:1047–1054, 2010. © 2010 Wiley-Liss, Inc.

Studies on feeding habits based on faeces dissecting are imperative to understand the natural history of Tibetan foxes and their functions in the transmission of a lethal zoonotic parasite, Echinococcus multilocularis. However, Tibetan foxes and red foxes live sympatrically on the Tibetan plateau, China. Therefore, the faeces of Tibetan foxes must be distinguished from those of red foxes. We established a diagnostic method to distinguish the faeces of the two species by amplifying a portion of the mitochondrial cytochrome b gene (cytb) and digesting with the restriction enzymes BamHI and SspI, to produce specific diagnostic banding patterns. This PCR-RFLP assay enabled rapid, accurate and easily performed identification and differentiation of the two species.

Haehnel et al. synthesized 399 different artificial cytochrome b (aCb) models. They consist of a template-assisted four-helix bundle with one embedded heme group. Their redox potentials were measured and cover the range from -148 to -89 mV. No crystal structures of these aCb are available. Therefore, we use the chemical composition and general structural principles to generate atomic coordinates of 31 of these aCb mutants, which are chosen to cover the whole interval of redox potentials. We start by modeling the coordinates of one aCb from scratch. Its structure remains stable after energy minimization and during molecular dynamics simulation over 2 ns. Based on this structure, coordinates of the other 30 aCb mutants are modeled. The calculated redox potentials for these 31 aCb agree within 10 mV with the experimental values in terms of root mean square deviation. Analysis of the dependence of heme redox potential on protein environment shows that the shifts in redox potentials relative to the model systems in water are due to the low-dielectric medium of the protein and the protonation states of the heme propionic acid groups, which are influenced by the surrounding amino acids. Alternatively, we perform a blind prediction of the same redox potentials using an empirical approach based on a linear scoring function and reach a similar accuracy. Both methods are useful to understand and predict heme redox potentials. Based on the modeled structure we can understand the detailed structural differences between aCb mutants that give rise to shifts in heme redox potential. On the other hand, one can explore the correlation between sequence variations and aCb redox potentials more directly and on much larger scale using the empirical prediction scheme, which--thanks to its simplicity--is much faster.

The establishment and partial characterization of a continuous cell line from the dorsal fin of red sea bream, Pagrus major, are described. The cell line, designated RSBF-2, has been subcultured for more than 100 passages since its initiation in November 2000. It was optimally maintained at 28 degrees C in Leibovitz L-15 medium with 10% foetal bovine serum. Propagation of RSBF-2 cells was serum dependent and exhibited low plating efficiency (

Of the 5000 fish species on coral reefs, corals dominate the diet of just 41 species. Most (61%) belong to a single family, the butterflyfishes (Chaetodontidae). We examine the evolutionary origins of chaetodontid corallivory using a new molecular phylogeny incorporating all 11 genera. A 1759-bp sequence of nuclear (S7I1 and ETS2) and mitochondrial (cytochrome b) data yielded a fully resolved tree with strong support for all major nodes. A chronogram, constructed using Bayesian inference with multiple parametric priors, and recent ecological data reveal that corallivory has arisen at least five times over a period of 12 Ma, from 15.7 to 3 Ma. A move onto coral reefs in the Miocene foreshadowed rapid cladogenesis within Chaetodon and the origins of corallivory, coinciding with a global reorganization of coral reefs and the expansion of fast-growing corals. This historical association underpins the sensitivity of specific butterflyfish clades to global coral decline.

Rivers can act as both islands of mesic refugia for terrestrial organisms during times of aridification and barriers to gene flow, though evidence for long-term isolation by rivers is mixed. Understanding the extent to which riverine barrier effects can be heightened for populations trapped in mesic refugia can help explain maintenance and generation of diversity in the face of Pleistocene climate change. Herein, we implement phylogenetic and population genetic approaches to investigate the phylogeographic structure and history of the ground skink, Scincella lateralis, using mtDNA and eight nuclear loci. We then test several predictions of a river- refugia model of diversification. We recover 14 well-resolved mtDNA lineages distributed east―west along the Gulf Coast with a subset of lineages extending northward. In contrast, ncDNA exhibits limited phylogenetic structure or congruence among loci. However, multilocus population structure is broadly congruent with mtDNA patterns and suggests that deep coalescence rather than differential gene flow is responsible for mtDNA―ncDNA discordance. The observed patterns suggest that most lineages originated from population vicariance due to riverine barriers strengthened during the Plio―Pleistocene by a climate-induced coastal distribution. Diversification due to rivers is likely a special case, contingent upon other environmental or biological factors that reinforce riverine barrier effects.

Mitochondria encoded Cytochrome B (CYTB) gene mutations were reported in tumors of different anatomic origin but the functional significance of these mutations are not well studied. Earlier, we found a 7-amino acid deletion mutation in the CYTB gene in a primary bladder cancer patient. In the present study, we overexpressed this 7-amino acid deletion mutation of CYTB gene in SV-40 transformed human uroepithelial HUC-1 cells. The nuclear transcribed mitochondrial CYTB (mtCYTB) was targeted into the mitochondria and generated increased copies of mitochondria and mitochondrial COX-I protein in the transfected HUC-1 cells. The pro-apoptotic protein Bax largely remained confined to the cytoplasm of the mtCYTB transfected HUC-1 cells without release of Cytochrome C. The downstream apoptotic proteins PARP also remained uncleaved along with increased Lamin B1 in the mtCYTB transfected cells. Our results demonstrate that forced overexpression of mtCYTB in transformed human uroepithelial HUC-1 cells triggered mitochondrial proliferation and induction of an anti-apoptotic signaling cascade favoring sustained cellular growth. Coding mitochondrial DNA mutations appear to have significant functional contribution in tumor progression.

The host-feeding patterns of Anopheles quadrimaculatus Say and Anopheles punctipennis (Say) were examined in order to evaluate their potential contributions to the transmission of eastern equine encephalitis virus (EEEv) and other arboviruses in the northeastern U.S.A. Engorged mosquitoes of the two species were collected from EEEv foci in central New York (NY) and throughout New Jersey (NJ), and their bloodmeals were identified using a polymerase chain reaction (PCR)-based assay and sequencing portions of the mitochondrial cytochrome b gene. Analysis of 131 An. quadrimaculatus and 107 An. punctipennis from NY revealed that 97.7% and 97.2%, respectively, had acquired blood solely from mammalian hosts. Similarly, examination of 288 An. quadrimaculatus and 127 An. punctipennis from NJ showed 100% and 96.0%, respectively, contained mammalian-derived bloodmeals. Mosquitoes containing mixed bloodmeals from both avian and mammalian hosts were detected in 1.6% of An. quadrimaculatus from NY, and 2.8% and 4.0% of An. punctipennis from NY and NJ, respectively. White-tailed deer (Odocoileus virginianus) constituted the most common vertebrate host for these anopheline mosquitoes, accounting for 85.8-97.7% of all bloodmeals identified. The predominance of white-tailed deer as a source of bloodmeals supports enzootic amplification of deer-associated arboviruses in this region, including Jamestown Canyon, Cache Valley and Potosi viruses. One horse- and two human-derived bloodmeals were also detected in An. quadrimaculatus collected in NJ. Limited avian-derived bloodmeals were detected from mourning dove (Zenaida macroura), sharp-shinned hawk (Accipiter striatus) and house finch (Carpodacus mexicanus), mostly in mixed bloodmeals. Occasional feeding on avian hosts suggests that these mosquitoes may participate as epizootic-epidemic bridge vectors of EEEv from viraemic birds to mammalian hosts of concern, including horses and humans. An isolate of EEEv was recovered from the head and thorax of an An. punctipennis mosquito collected in NY.

A seahorse specimen from Banco Açores (Azores Archipelago) was identified using morphological and molecular genetic data as Hippocampus erectus. This specimen represents the first record of H. erectus in the eastern Atlantic Ocean, well outside its reported range, and may provide evidence of long-distance translocation in what are assumed to be relatively sedentary fish.

The aim of this work was to investigate the possible origin of local Brazilian pig breeds through Cytochrome b (MT-CYB) mitochondrial analyses. The results indicated that the main local pig breeds descended from two different European maternal lineages, both Iberian varieties. The haplotype relationship analysis showed that Monteiro, Nilo, Piau and Tatu breeds share haplotypes only with Iberian varieties, while the Moura breed presented a different maternal lineage. The Moura appears to share a high frequency of haplotypes with the Black Hairy Iberian variety and Hungarian Mangalica breed.

Whether microevolution on small islands differs from that on larger landmasses is a key question in biology with substantial implications for species conservation. However, due to the difficulties faced in producing adequately replicated samples and in controlling for confounding variables, prior attempts to examine evolutionary questions relating to habitat area and population size have produced equivocal results. Here we show, using experimental design criteria that reduce the potential for such confounding, that bird species on larger landmasses have higher rates of molecular evolution. The study involves a global dataset of 48 independent contrasts for the cytochrome b gene encompassing all possible paired sister species comparisons (from seven orders and 17 families) that were available at the time of dataset assembly. A more rapid evolutionary tempo in larger areas has important ramifications for biodiversity conservation because it indicates a new imperative, beyond that of simply maintaining preexisting genetic diversity, for securing large areas for threatened species. This result suggests that the trend of confining species to limited refugia is likely to be slowing the tempo of microevolution. That effect might constrain the potential for adaptive shifts in response to changing environments such as those associated with global warming.

South East Asian countries are known for illegal poaching and trade of crocodiles clandestinely, to be used in skin, medicinal, and cosmetic industries. Besides crocodiles being listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora, India has its Wildlife Protection Act, 1972 for conservation of crocodile species. Hitherto, lack of any rapid and reliable technique for examinations of crocodile-based crime exhibits such as skin, bones, etc. has been a major problem for an effective promulgation of law on illegal trade. DNA-based identification of species using PCR-RFLP technique for an apt identification of all the three Indian crocodile species namely, Crocodylus porosus, Crocodylus palustris and Gavialis gangeticus is presented here. A 628 bp segment of cytochrome b gene was amplified using novel primers followed by restriction digestion with three enzymes i.e., HaeIII, MboI, and MwoI, separately and in combination. The technique has produced a species-specific pattern for identifying the three crocodile species individually, which fulfills the requirement for its forensic application. It is expected that the technique will prove handy in identification of all the three Indian crocodile species and strengthen conservation efforts.

Guanaco (Lama guanicoe) is a protected and widely distributed ungulate in South America. A poacher, after killing guanacos in Valle Chacabuco, Chilean Patagonia, transported and stored the meat. Samples were retrieved by local police but the suspect argued that the meat was from a horse. Mitochondrial cytochrome b gene (774 pb), 15 loci microsatellites, and SRY gene were used to identify the species, number of animals and their population origin, and the sex of the animals, respectively. Analysis revealed that the samples came from a female (absence of SRY gene) Patagonian guanaco (assignment probability between 0.0075 and 0.0282), and clearly distinguishing it from sympatric ungulates (E-value = 0). Based on the evidence obtained in the field in addition to forensic data, the suspect was convicted of poaching and illegally carrying fire arms. This is the first report of molecular tools being used in forensic investigations of Chilean wildlife indicating its promising future application in guanaco management and conservation.

The colonisation history and genetic structure of the common vole (Microtus arvalis) was investigated in the region of the Alps by analysing the mitochondrial cytochrome b gene (mtDNA) and 19 microsatellite loci (nucDNA) for 137 voles from 52 localities. mtDNA data provided a much refined distribution of three highly divergent evolutionary lineages in the region compared to previous studies. Although high mountain ranges are widely accepted to be barriers for colonisation processes for many organisms and especially small terrestrial mammals, our phylogeographic analyses showed clear evidence of four transalpine colonisation events by the common vole. Individual-based phylogenetic analyses of nucDNA and two alternative Bayesian-clustering approaches revealed a deep genetic structure analogous to mtDNA. Incongruence between nucDNA and mtDNA at the individual level was restricted to the regions of contact between the lineages. mtDNA patterns and strong female philopatry in M. arvalis suggest that the crossings of the Alps occurred during the colonisation of the region when it was free from ice after the last glaciation. nucDNA patterns suggest that some of the transalpine elements of this phylogeographic pattern were subsequently eroded by male-biased gene flow. We conclude that the combination of phylogeography and landscape genetics at the individual level can provide very detailed insights into colonisation events and may even allow differentiation between historical and more recent processes.

Wohlfahrtia magnifica (Schiner) (Diptera: Sarcophagidae) is a major cause of traumatic myiasis in livestock in Central and Eastern Europe and in countries bordering the Mediterranean. The present study explored the utility of external body characters, genitalia characters and mitochondrial DNA characters for identification of this and related species in the subfamily Paramacronychiinae. Sequence analyses of the 3' terminal 273 bp of the mitochondrial cytochrome b gene revealed two lineages of W. magnifica, one from Spain and France and the other from the rest of Eurasia, differing by only two base pairs. Phylogenetic analysis of cytochrome b showed that W. magnifica and Wohlfahrtia vigil Walker were sister species; this conclusion was not contradicted by a phylogenetic analysis of the morphological characters. Based on cytochrome b, the genetic distance between specimens of W. vigil from Europe and North America was sufficiently large to justify the recognition of more than one species. A new species, Wohlfahrtia monegrosensis, from northern Spain, was described, based on morphology and cytochrome b. A unique combination of external body characters of males or females were diagnostic for W. magnifica, the W. vigil group and Wohlfahrtia bella, but only the genitalia characters were diagnostic for all nine species studied.

Summary. Restriction endonuclease fragment patterns of mitochondrial DNA (mtDNA) in sheep were analysed with 11 enzymes. Four breeds (Merinolandschaf, Rhoenschaf, Schwarzkoepfiges Fleischschaf and Skudde) of domestic sheep and European Mouflon were examined. A restriction map with 28 cleavage sites of seven enzymes was established. KpnI and PstI do not cut ovine mtDNA. Two EcoRI fragments of Merinolandschaf, Rhoenschaf and Mouflon each were cloned and partially sequenced. Intraspecific nucleotide sequence differences within 1.101 kb ranged from 0.09 to 0.27%. Hybridization analysis with a fragment of porcine mtDNA along with sequencing data from cloned fragments was used for orientation of the restriction map along the bovine sequence. Ovine mtDNA sequences encompassing parts of the Cyt.b-, ND5-, CoIII- and ATPase6 genes were compared with the corresponding sequences of the bovine mtDNA.

Supramolecular protein polymers: When a heme moiety was introduced to the surface of an apo-cytochrome b(562)(H63C) mutant, supramolecular polymers formed through noncovalent heme-heme pocket interactions. The incorporation of a heme triad as a pivot molecule in the protein polymer further led to a two-dimensional protein network structure, which was visualized by tapping-mode atomic force microscopy (see picture).

Primer pairs were designed and protocols developed to selectively amplify segments of vertebrate mitochondrial cytochrome oxidase subunit 1 (COI) and cytochrome b (Cyt b) mtDNA from the bloodmeals of mosquitoes (Diptera: Culicidae). The protocols use two pairs of nested COI primers and one pair of Cyt b primers to amplify short segments of DNA. Resultant sequences are then compared with sequences in GenBank, using the BLAST function, for putative host identification. Vertebrate DNA was amplified from 88% of our sample of 162 wild-caught, blood-fed mosquitoes from Oregon, U.S.A. and GenBank BLAST searches putatively identified 98% of the amplified sequences, including one amphibian, seven mammalian and 14 avian species. Criteria and caveats for putative identification of bloodmeals are discussed.