Spruce Grouse Habitat Ecology in Maine\u27s Commercially Managed Acadian Forest

Spruce grouse (Falcipennis canadensis) inhabiting the mixed coniferous-deciduous forests of the northeastern United States are at the southern extent of their range. These mixed forests are known collectively as the Acadian forest and represent the transitional zone between the boreal forest to the north and the deciduous northern hardwoods forests to the south. Often assumed to be associated with mature, unharvested forest in this region, few studies have assessed habitat relationships of the species within areas dominated by commercial forest management. We investigated the influence of stand maturity, vertical and horizontal cover, and patchiness on the occupancy and abundance of male spruce grouse during the breeding season (Chapter 1); as well as within stand-scale habitat selection of spruce grouse hens during the brood-rearing season (Chapter 2) in the commercial forests of northcentral Maine. Our study was comprised of six townships that covered 612 km2 within the largest contiguous undeveloped forest in the U.S. Patterns of occupancy and abundance by male spruce grouse were examined by surveying 30 stands during each breeding season (May-June) in 2012-2014. Areas surveyed represented four forest harvest histories including regenerating clearcut (n = 10), pre-commercially thinned (n = 10), selection harvest (n = 4), and mature unharvested conifer (n = 6) stands. We constructed single season occupancy and abundance models with years and stand types considered as groups, while accounting for nuisance variables that could affect survey outcomes (e.g., weather, density of woody vegetation). Probability of detection given occupancy was 0.61, and the probability of occupancy varied by successional stage from 37.4 to 76.8. Across our study area, individual male grouse had a probability of detection of 0.24 and the abundance of male grouse also varied by successional stage from 0.67 to 2.75. Based upon the covariates included in the models, both occurrence and abundance of breeding male spruce grouse were highest in mid-successional, moderately dense, conifer dominated stands that have experienced intensive forestry practices such as clearcutting, herbicide application, and pre-commercial thinning to promote coniferous regeneration. We investigated within stand-scale (i.e., 4th-order selection) habitat selection by female spruce grouse during the brood rearing season (June-October) in 2012-2014 by tracking 30 hens captured in 12 stands, which we equipped with VHF transmitters. We used general linear mixed models to construct resource selection functions to compare use to availability for each hen. Female spruce grouse selected for abundant low vegetation structure ( Spruce-fir forests in the region have declined in recent years and are predicted to decline further under all future climate scenarios. Thus, forms of harvesting and post-harvest treatments that promote moderately dense conifer-dominated regeneration are recommended to maintain spruce grouse presence in commercially managed forests within the Acadian region. Currently, these conditions selected for by spruce grouse occur predominantly in stands with a past history of clearcutting, followed by post-harvest herbicide application and/or pre-commercial thinning. Changing markets, regulations, and other factors have caused the majority of forest harvests to shift towards partial harvest methods in Maine. Given that the extent and size of residual conifer forest patches has declined substantially over the past three decades, opportunities to manage for spruce grouse and other conifer-dominant species in Maine’s commercially managed forests will require future attention and monitoring

Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae

<p>Abstract</p> <p>Background</p> <p>Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise (transitions and transversions, insertions and deletions).</p> <p>Results</p> <p>A nitroreductase- encoding gene was identified in the <it>N. gonorrhoeae </it>FA1090 genome by using a bioinformatic search with the deduced amino acid sequence derived from the <it>Escherichia coli </it>nitroreductase gene, <it>nfsB</it>. Cell extracts from <it>N. gonorrhoeae </it>were shown to possess nitroreductase activity, and activity was shown to be the result of NfsB. Spontaneous nitrofurantoin-resistant mutants arose at a frequency of ~3 × 10^-6- 8 × 10^-8among the various strains tested. The <it>nfsB </it>sequence was amplified from various nitrofurantoin-resistant mutants, and the nature of the mutations determined. Transition, transversion, insertion and deletion mutations were all readily detectable with this reporter gene.</p> <p>Conclusion</p> <p>We found that <it>nfsB </it>is a useful reporter gene for measuring spontaneous mutation frequencies. Furthermore, we found that mutations were more likely to arise in homopolymeric runs rather than as base substitutions.</p

A consensus approach to vertebrate de novo transcriptome assembly from RNA-seq data: assembly of the duck (Anas platyrhynchos) transcriptome

For vertebrate organisms where a reference genome is not available, de novo transcriptome assembly enables a cost effective insight into the identification of tissue specific or differentially expressed genes and variation of the coding part of the genome. However, since there are a number of different tools and parameters that can be used to reconstruct transcripts, it is difficult to determine an optimal method. Here we suggest a pipeline based on (1) assessing the performance of three different assembly tools (2) using both single and multiple k-mer (MK) approaches (3) examining the influence of the number of reads used in the assembly (4) merging assemblies from different tools. We use an example dataset from the vertebrate Anas platyrhynchos domestica (Pekin duck). We find that taking a subset of data enables a robust assembly to be produced by multiple methods without the need for very high memory capacity. The use of reads mapped back to transcripts (RMBT) and CEGMA (Core Eukaryotic Genes Mapping Approach) provides useful metrics to determine the completeness of assembly obtained. For this dataset the use of MK in the assembly generated a more complete assembly as measured by greater number of RMBT and CEGMA score. Merged single k-mer assemblies are generally smaller but consist of longer transcripts, suggesting an assembly consisting of fewer fragmented transcripts. We suggest that the use of a subset of reads during assembly allows the relatively rapid investigation of assembly characteristics and can guide the user to the most appropriate transcriptome for particular downstream use. Transcriptomes generated by the compared assembly methods and the final merged assembly are freely available for download at http://dx.doi.org/10.6084/m9.figshare.1032613

Feline Stem Cell Factor: Isolation and Characterisation of Biological Activity

Cytokines are small proteins produced by many tissue types and have wide ranging effects on the haemopoietic and immune systems. The cloning of human cytokines has facilitated the production of recombinant cytokines in quantities sufficient to enable detailed study of their biological properties. An understanding of the biological effects of these cytokines has led to their introduction as novel therapeutic agents with widespread potential uses, including the treatment of cancer, cytopenias and viral infections. The use of heterologous cytokines in domestic species has been of only limited success, in part due to the variable degree of interspecies conservation. In order to fully realise the potential for cytokines as therapeutic agents and to facilitate further studies of the role of cytokines in diseases of domestic species, the isolation of species specific cytokines is desirable. This thesis describes the approach used to isolate and clone feline stem cell factor (fSCF) and subsequently express the recombinant protein and characterise its biological properties. Stem cell factor is the ligand for the tyrosine kinase receptor encoded by the c-kit gene. It has wide ranging actions on cells of the haemopoietic, reproductive and nervous systems and melanocytes, in particular promoting the survival and development of primitive cells. cDNA clones encoding two isoforms of fSCF were isolated using RT-PCR and their sequences determined. The cDNAs encode a predicted full length fSCF protein of 274 amino-acids and a shorter isoform of 246 amino acids. Feline SCF shows a high degree of homology to the SCFs of other species at both the nucleic acid and protein level. Feline SCF was expressed as a soluble protein using the glutathione S-transferase fusion protein system and purified by affinity, anion exchange and gel filtration chromatography. Murine MC/9 and human TF-1 cells were used to assay fSCF biological activity. The recombinant protein supported the growth of feline granulocyte-macrophage colony forming cells in vitro and in combination with feline phytohaemagglutinin lymphocyte conditioned medium increased colony numbers and sizes were seen. Administration of the recombinant protein to cats produced increases in circulating colony forming cells, induced extramedullary haemopoiesis in the spleens of treated cats and led to increased mast cell numbers at the site of injection. In order to enable assessment of the effects of frSCF upon primitive haemopoietic cells, the production of polyclonal antiserum to CD34 (a transmembrane glycoprotein expressed predominantly on primitive haemopoietic cells) was attempted. Rabbits were used to raise antisera to conserved intracellular epitopes of the CD34 molecule by inoculation with immunogenic peptides. This was of limited success; whilst the antisera recognised the synthetic peptides against which they had been raised, they showed poor affinity for the native protein. These studies provide the basis for further investigations of the potential of this cytokine in the treatment of feline disease, particularly cytopenias associated with neoplasia, chemotherapy or viral disease (e.g. FeLV, FIV) and in the development of peripheral stem cell transplantation. The ability of fSCF to synergise with other cytokines in vitro suggests that it may be combined with other haemopoietic cytokines in vivo to provide more potent haemopoietic stimulation. Furthermore, the recombinant cytokine may be usefully employed to support in vitro growth of haemopoietic cells in this species and so facilitate their study

Differences in influenza virus receptors in chickens and ducks: Implications for interspecies transmission

Avian influenza viruses are considered to be key contributors to the emergence of human influenza pandemics. A major determinant of infection is the presence of virus receptors on susceptible cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid α2,3-galactose (SAα2,3-Gal) linked receptors, whereas human strains bind to sialic acid α2,6-galactose (SAα2,6-Gal) linked receptors. While ducks are the major reservoir for influenza viruses, they are typically resistant to the effects of viral infection, in contrast to the frequently severe disease observed in chickens. In order to understand whether differences in receptors might contribute to this observation, we studied the distribution of influenza receptors in organs of ducks and chickens using lectin histochemistry with linkage specific lectins and receptor binding assays with swine and avian influenza viruses. Although the intestinal epithelial cells of both species expressed only SAα2,3-Gal receptors, we found widespread presence of both SAα2,6-Gal and SAα2,3-Gal receptors in many organs of both chickens and ducks. Co-expression of both receptors may allow infection of cells with both avian and human viruses and so present a route to genetic reassortment. There was a marked difference in the primary receptor type in the trachea of chickens and ducks. In chicken trachea, SAα2,6-Gal was the dominant receptor type whereas in ducks SAα2,3-Gal receptors were most abundant. This suggests that chickens could be more important as an intermediate host for the generation of influenza viruses with increased ability to bind to SAα2,6-Gal receptors and thus greater potential for infection of humans. Chicken tracheal and intestinal epithelial cells also expressed a broader range of SAα2,3-Gal receptors (both β(1-4)GlcNAc and β(1-3)GalNAc subtypes) in contrast to ducks, which suggests that they may be able to support infection with a broader range of avian influenza viruses

Longitudinal study of Asian elephants, Elephas maximus, indicates intermittent shedding of elephant endotheliotropic herpesvirus 1 during pregnancy

Introduction: EEHV-1 is a viral infection of elephants that has been associated with a fatal haemorrhagic syndrome in Asian elephants. Previous studies have suggested that pregnant animals may shed more virus than non-pregnant animals. Methods: This study examined whether pregnancy affected the frequency or magnitude of shedding of elephant endotheliotropic herpesvirus 1 (EEHV1) using Taq man real-time PCR on trunk washes from four female elephants from a UK collection over three time periods between 2011 and 2014. These periods included pregnancies in two animals (period 1 and period 3). Behavioural observations made by keepers were also assessed. Results: During period 1 there was a high degree of social hierarchical instability which led to a hierarchy change, and was associated with aggressive behaviour. Also during period 1 EEHV-1 shedding was of a higher magnitude and frequency than in the latter two time periods. Conclusions: These results suggest that there is no clear relationship between shedding and pregnancy, and that behavioural stressors may be related to an increase in EEHV-1 shedding

A consensus approach to vertebrate de novo transcriptome assembly from RNA-seq data: assembly of the duck (Anas platyrhynchos) transcriptome

For vertebrate organisms where a reference genome is not available, de novo transcriptome assembly enables a cost effective insight into the identification of tissue specific or differentially expressed genes and variation of the coding part of the genome. However, since there are a number of different tools and parameters that can be used to reconstruct transcripts, it is difficult to determine an optimal method. Here we suggest a pipeline based on (1) assessing the performance of three different assembly tools (2) using both single and multiple k -mer (MK) approaches (3) examining the influence of the number of reads used in the assembly (4) merging assemblies from different tools. We use an example dataset from the vertebrate Anas platyrhynchos domestica (Pekin duck). We find that taking a subset of data enables a robust assembly to be produced by multiple methods without the need for very high memory capacity. The use of reads mapped back to transcripts (RMBT) and CEGMA (Core Eukaryotic Genes Mapping Approach) provides useful metrics to determine the completeness of assembly obtained. For this dataset the use of MK in the assembly generated a more complete assembly as measured by greater number of RMBT and CEGMA score. Merged single k -mer assemblies are generally smaller but consist of longer transcripts, suggesting an assembly consisting of fewer fragmented transcripts. We suggest that the use of a subset of reads during assembly allows the relatively rapid investigation of assembly characteristics and can guide the user to the most appropriate transcriptome for particular downstream use. Transcriptomes generated by the compared assembly methods and the final merged assembly are freely available for download at http://dx.doi.org/10.6084/m9.figshare.1032613. © 2014 Moreton, Dunham and Emes

Lipid biophysics and/or soft matter-inspired approach for controlling enveloped virus infectivity

Proven as a natural barrier against viral infection, pulmonary surfactant phospholipids have a biophysical and immunological role within the respiratory system, acting against microorganisms including viruses. Enveloped viruses have, in common, an outer bilayer membrane that forms the underlying structure for viral membrane proteins to function in an optimal way to ensure infectivity. Perturbating the membrane of viruses using exogenous lipids can be envisioned as a generic way to reduce their infectivity. In this context, the potential of exogenous lipids to be used against enveloped virus infectivity would be indicated by the resulting physical stress imposed to the viral membrane, and conical lipids, i.e. lyso-lipids, would be expected to generate stronger biophysical disturbances. We confirm that when treated with lyso-lipids the infectivity three strains of influenza virus (avian H2N3, equine H3N8 or pandemic human influenza H1N1) is reduced by up to 99% in a cell-based model. By contrast, lipids with a similar head group but two aliphatic chains were less effective (reducing infection by only 40–50%). This work opens a new path to merge concepts from different research fields, i.e. ‘soft matter physics' and virology

Associations between small ruminant lentivirus infection and total milk yield and somatic cell count in a dairy sheep flock

BackgroundSmall ruminant lentiviruses (SRLVs) are lentiviruses of sheep and goats, formerly known as maedi–visna (MV) in sheep and caprine encephalitis and arthritis in goats. In sheep, SRLVs commonly cause progressive pneumonia, wasting and indurative mastitis. SRLVs have a long latent period, and chronic production losses are often not recognised until very late. Few studies quantifying the production losses in ewes have been published, and none have been published under UK flock husbandry conditions.MethodsProduction records of milk yield and somatic cell count (SCC) from a dairy flock of 319 milking East Friesian × Lacaune ewes identified as MV infected via routine serological screening for SRLV antibodies were used in multivariable linear regression modelling to estimate the impact of SRLV status on total milk yield and SCC.ResultsMilk yield was reduced in seropositive ewes by 8.1%–9.2% over an entire lactation. SCC counts were not significantly different in SRLV-infected and unifected animals.LimitationsFurther parameters, such as body condition score or clinical mastitis, that were not available may have clarified the underlying cause of milk yield drop.ConclusionsThe study demonstrates substantial production losses in an SRLV-affected flock and highlights the impact of the virus on a farm's economic viability