Assessing the Patterns of Evolution in Anuran Vocal Sexual Signals

Many animal species have evolved signalling traits to mediate various intra-specific interactions. Signals are particularly important for inter-sexual selection, where females use male signalling traits to select mates. Female preferences are therefore a major selective force in the evolution of these male signals, and these preferences can facilitate rapid changes in these traits in an evolutionary timeframe. This introduction of high levels of variation in inter-sexual signals may overshadow any phylogenetic patterns present. Such shadowing effects, however, should be dependant on the characteristics of traits (e.g. morphological, physiological and behavioural). Using male advertisement calls from 72 species of anuran amphibians, we tested the levels of phylogenetic signal present for a variety of call features in relation to trait types, and for calls as whole units using phylogenetic principal components analysis. We found that most call features displayed some level of phylogenetic autocorrelation (or signal), with traits that are dependent on morphology having much stronger phylogenetic signals than those based on behaviour. In addition, when calls were analysed as whole units, closely related species were found to be similar to each other, indicating that phylogenetic patterns had not been cancelled out by selection via female preferences. We suggest that signal functions, such as indicating male quality (e.g. mediated by body size) to potential mates, may place constraints on the amount of variation that can be introduced by female preferences. More research, particularly studies on other taxa, will be required to elucidate whether the patterns found in anurans are general across the animal kingdom.     

Absence of selection against aneuploid mouse sperm at fertilization.

Is there selection against aneuploid sperm during spermatogenesis and fertilization? To address this question, we used male mice doubly heterozygous for the Robertsonian (Rb) translocations Rb(6. 16)24Lub and Rb(16.17)7Bnr, which produce high levels of sperm aneuploid for chromosome 16, the mouse counterpart of human chromosome 21. The frequencies of aneuploid male gametes before and after fertilization were compared by analyzing approximately 500 meiosis II spermatocytes and approximately 500 first-cleavage zygotes using fluorescence in situ hybridization with a DNA painting probe mixture containing three biotin-labeled probes specific for chromosomes 8, 16, and 17 plus a digoxigenin-labeled probe specific for chromosome Y. Hyperhaploidy for chromosome 16 occurred in 20.0% of spermatocytes and in 21.8% of zygotes. Hypohaploidy for chromosome 16 occurred in 17.0% and 16.7% of spermatocytes and zygotes, respectively. In addition, there was no preferential association between chromosome 16 aneuploidy and either of the sex chromosomes, nor was there an elevation in aneuploidy for chromosomes not involved in the Rb translocations. These findings provide direct evidence that there is no selection against aneuploid sperm during spermiogenesis, fertilization, and the first cell cycle of zygotic development.     

Monoclonal antibody against a melanosomal protein in melanotic and amelanotic human melanoma cells

BALB/c mice were immunized with tyrosinase, partially purified in two stages from a human melanoma cell line. A hybridoma was obtained which produced monoclonal antibody (MoAb 1C11) reactive with 8/10 melanoma cell lines and 10/10 primary cultures of human melanocytes, neval cells, and melanomas. Immunoreactivity correlated to a certain extent with tyrosinase activity but not with melanin content. No crossreactivity was obtained with neuroblastoma, medulloblastoma, fibroblasts, keratinocytes, lymphoid cells, or murine melanomas. Purification of the antigen directly from cell lysates with a MoAb 1C11 CNBr-Sepharose affinity column gave a green-brown protein of 56 kDa with no detectable tyrosinase activity. This protein was therefore different from 60 kDa active tyrosinase, identified by enzyme activity and Western blotting with a MoAb derived previously (MoAb 5C12). Unlike 5C12, 1C11 reactivity was not destroyed by pretreatment of the antigen with periodate. Immunogold labelling showed that the 1C11-reactive antigen was associated with melanosomes, and there was close correlation between 5C12 and 1C11 reactivity in resistance to trypsin and in staining various melanocytic cell populations. MoAb 1C11 may therefore recognise a polypeptide epitope in a molecule closely linked to melanin biosynthesis.     

In silico exploration of the effects of host genotype and nutrition on the genetic parameters of lambs challenged with gastrointestinal parasites

An in silico mathematical model was used to explore the effect of, and the interaction between, (i) nutrition, (ii) genotype for growth and (iii) genotype for resistance, on the estimates of genetic parameters for resistance and performance in a population of lambs trickle-challenged daily with 3,000 L3s of Teladorsagia circumcincta. A previously published model for nematode infections in sheep was developed to include heritable variation in sheep growth traits, as well as in immunologically controlled traits such as establishment of incoming larvae, mortality of the adult worms and fecundity of the adult female worms. The simulated population comprised 10,000 lambs, these being the offspring of 250 sires mated to 5,000 dams. The model assumed the lambs to be parasitologically naïve at weaning (2 months of age), at which point the trickle challenge commenced and the model was updated daily until slaughter (at 6 months of age). Dietary treatments included a good and a poor quality feed, offered ad libitum. Two genotypes for growth were assumed: (i) fast and (ii) slow growing. Three genotypes for resistance were used: (i) benchmark, (ii) susceptible and (iii) resistant, differing in their ability to cope with nematode infections. Genetic parameters for output traits, including growth rate, food intake, worm burden and faecal egg count were estimated using a linear mixed model, fitting sire as a random effect to capture genetic effects. Heritabilities and correlations were found to change over time. In general, the heritabilities of immunity traits increased over time, whereas genetic correlations between production and immunity traits became weaker. Diet had a significant effect on the means and the estimated correlations of output traits, while genotypes for growth and resistance had smaller effects. These results suggest that discrepancies between published genetic parameters for nematode resistance may be a function of environmental factors rather than differences in host genotype.     

Crossmodal session rating of perceived exertion response at low and moderate intensities

Session rating of perceived exertion (SRPE) permits global effort estimations after an exercise bout and has shown promise for evaluating training load. However, factors mediating SRPE are not well understood. The purpose of this study was to compare SRPE between cycling and treadmill exercise at low and moderate intensities. In a counterbalanced order, male subjects (n = 7) completed a VO2max trial on a cycle ergometer and a motor-driven treadmill. Then, participants completed trials at 50 and 75% mode-specific VO2max on a cycle ergometer (BK75, BK50) and a treadmill (TM75, TM50) to achieve ～ 400-kcal energy expenditure per trial. Acute RPE (i.e., during exercise) at 5 minutes, midway, and test termination were recorded with SRPE (20-minutes postexercise) expressed as overall (SRPEO), legs (SRPEL), and breathing also recorded were heart rate (HR) and change in rectal temperature (ΔTrec). Significance was accepted at p ≤ 0.05. Repeated-measures analysis of variance revealed significantly greater SRPE for higher intensities within each mode. Crossmodal comparisons also show a higher SRPE at moderate (75% VO2max) intensities [SRPEO] = BK75: 7.6 ± 1.0, TM75: 6.9 ± 1.3) vs. lower (50% VO2max) intensities (BK50: 4.6 ± 1.4, TM50: 4.6 ± 1.1). Within modes, SRPE corresponded well with ΔTrec and HR. Acute RPE was linked with intensity and drifted upward across time. Results indicated that overall and differentiated SRPEs are magnified with exercise intensity with the corresponding disruption in internal environment potentially mediating subjective responses. From a practical application standpoint, SRPE provides a subjective assessment for immediate evaluation of daily training. Results indicate that, when using SRPE to monitor training, consideration should be given to responses across differing exercise modes.     

A novel SINE family occurs frequently in both genomic DNA and transcribed sequences in ixodid ticks of the arthropod sub-phylum Chelicerata

Reassociation kinetics and flow cytometry data indicate that ixodid tick genomes are large, relative to most arthropods, containing>or=10(9) base pairs. The molecular basis for this is unknown. We have identified a novel small interspersed element with features of a tRNA-derived SINE, designated Ruka, in genomic sequences of Rhipicephalus appendiculatus and Boophilus (Rhipicephalus) microplus ticks. The SINE was also identified in expressed sequence tag (EST) databases derived from several tissues in four species of ixodid ticks, namely R. appendiculatus, B. (R.) microplus, Amblyomma variegatum and also the more distantly related Ixodes scapularis. Secondary structure predictions indicated that Ruka could adopt a tRNA structure that was, atypically, most similar to a serine tRNA. By extrapolation the frequency of occurrence in the randomly selected BAC clone sequences is consistent with approximately 65,000 copies of Ruka in the R. appendiculatus genome. Real time PCR analyses on genomic DNA indicate copy numbers for specific Ruka subsets between 5800 and 38,000. Several putative conserved Ruka insertion sites were identified in EST sequences of three ixodid tick species based on the flanking sequences associated with the SINEs, indicating that some Ruka transpositions probably occurred prior to speciation within the metastriate division of the Ixodidae. The data strongly suggest that Class I transposable elements form a significant component of tick genomes and may partially account for the large genome sizes observed.     

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein^1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.     

Combining otolith chemistry and telemetry to assess diadromous migration in pinkeye mullet, <Emphasis Type="Italic">Trachystoma petardi</Emphasis> (Actinopterygii,Mugiliformes)

Little is currently known regarding microbial community structure, and the environmental factors influencing it, within the anchialine ecosystem, defined as near-shore, land-locked water bodies with subsurface connections to the ocean and groundwater aquifer. The Hawaiian Archipelago is home to numerous anchialine habitats, with some on the islands of Maui and Hawaii harboring unique, laminated orange cyanobacterial–bacterial crusts that independently assembled in relatively young basalt fields. Here, benthic and water column bacterial and micro-eukaryotic communities from nine anchialine habitats on Oahu, Maui, and Hawaii were surveyed using high-throughput amplicon sequencing of the V6 (Bacteria-specific) and V9 (Eukarya-biased) hypervariable regions of the 16S- and 18S-rDNA genes, respectively. While benthic communities from habitats with cyanobacterial–bacterial crusts were more similar to each other than to ones lacking it on the same island, each habitat had distinct benthic and water column microbial communities. Analyses of the survey data in the context of environmental factors identified salinity, site, aquifer, and watershed as having the highest explanatory power for the observed variation in microbial diversity and community structure, with lesser drivers being annual rainfall, longitude, ammonium, and dissolved organic carbon. Our results epitomize the abiotic and biotic uniqueness characteristic of individual habitats comprising the Hawaiian anchialine ecosystem.     

Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.     

Further investigation of simian immunodeficiency virus Vif function in human cells

Primate lentivirus Vif proteins function by suppressing the antiviral activity of the cell-encoded apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) proteins APOBEC3G and APOBEC3F. It has been hypothesized that species-specific susceptibilities of APOBEC proteins to Vif proteins may help govern the transmission of primate lentiviruses to new host species. Consistent with this view and with previous results, we report that the Vif proteins of several diverse simian immunodeficiency viruses (SIVs) that are not known to infect humans are not effective inhibitors of human APOBEC3G or APOBEC3F when assessed in transient-transfection experiments. Unexpectedly, this lack of SIV Vif function did not prevent the replication of two vif-deficient SIVs (SIVtan and SIVmnd1; isolated from tantalus monkeys and mandrills, respectively) in a human T-cell line, HUT78, that expresses both APOBEC 3G and APOBEC3F, a finding which demonstrates that some SIVs are partially resistant to the antiretroviral effects of these enzymes irrespective of Vif function. Additional virus replication studies also revealed that the Vif protein of SIVtan is, in fact, active in human T cells, as it substantially enhanced the replication of its cognate virus and human immunodeficiency virus type 1. In sum, we now consider it improbable that species-specific restrictions to SIV Vif function can explain the lack of human infection with certain SIVs. Instead, our data reveal that the species-specific modulation of Vif function is more complex than previously envisioned and that additional (as-yet-unidentified) viral or host factors may be involved in regulating this dynamic interaction between host and pathogen.