A method for orienting cryostat sections for three-dimensional reconstructions.

Tissue for cryostat sectioning is embedded together with reference tissue impaled on the needles of a specially constructed trident. To maintain spatial orientation, reference and experimental tissue are frozen in Tissue-Tek O. C. T. compound in the manner usual for enzyme histochemistry using a simply constructed carrier. The trident is removed by heating its handle, thus leaving holes in the reference tissue which in turn are used as reference points to orient successive sections with respect to each other.     

Pleiotropic effects of regulatory variation in tan result in correlation of two pigmentation traits in Drosophila melanogaster

Traits with a common genetic basis frequently display correlated phenotypic responses to selection or environmental conditions. In Drosophila melanogaster, pigmentation of the abdomen and a trident‐shaped region on the thorax are genetically correlated. Here, we used a pooled replicated genomewide association approach (Pool‐GWAS) to identify the genetic basis of variation in thoracic trident pigmentation in two Drosophila melanogaster populations. We confirmed the previously reported large effect of ebony and the association of the cosmopolitan inversion In(3R)Payne. For the first time, we identified tan as another major locus contributing to variation in trident pigmentation. Intriguingly, the regulatory variants of tan that were most strongly associated with female abdominal pigmentation also showed a strong association with trident pigmentation. We validated this common genetic basis in transgenic assays and found qualitatively similar effects on trident and abdominal pigmentation. Further work is required to determine whether this genetic correlation is favoured by natural selection or reflects a neutral by‐product of a shared regulatory architecture.     

Phenotypic variability of thoracic pigmentation in Indian populations of Drosophila melanogaster

Fourteen natural populations of Drosophila melanogaster collected along diverse latitudinal and altitudinal ranges, were analysed for thoracic trident pigmentation at two different breeding temperatures (17 and 25°C) for both sexes. Statistical analyses showed significant intra- as well as interpopulational genetic variations. Clinal patterns for thoracic trident pigmentation along latitude as well as altitude are significantly correlated with thermal amplitude ( T _cv). Crosses between dark (Shimla) and light (Kalka) variants of trident pigmentation produced intermediate phenotypes, and a clear maternal effect was observed in the reciprocal F ₁ crosses. Data suggest that the temperature differences have resulted in phenotypic plasticity of thoracic pigmentation across the Indian subcontinent.     

The molecular genetics of clinal variation: a case study of ebony and thoracic trident pigmentation in Drosophila melanogaster from eastern Australia

Widespread pigmentation diversity coupled with a well‐defined genetic system of melanin synthesis and patterning in Drosophila provides an excellent opportunity to study phenotypes undergoing evolutionary change. Pigmentation variation is highly correlated with different ecological variables and is thought to reflect adaptations to different environments. Several studies have linked candidate genes from Drosophila melanogaster to intra‐population variation and interspecific morphological divergence, but less clearly to variation among populations forming pigmentation clines. We characterized a new thoracic trident pigmentation cline in D. melanogaster populations from eastern Australia, and applied a candidate gene approach to explain the majority of the geographically structured phenotypic variation. More melanized populations from higher latitudes tended to express less ebony than their tropical counterparts, and an independent artificial selection experiment confirmed this association. By partitioning temperature dependent effects, we showed that the genetic differences underlying clinal patterns for trident variation at 25 °C do not explain the patterns observed at 16 °C. Changes in thoracic trident pigmentation could be a common evolutionary response to climatically mediated environmental pressures. On the Australian east coast most of the changes appear to be associated with regulatory divergence of the ebony gene but this depends on temperature.     

银额果蝇自然群体中的mtDNA多态性研究──Ⅱ．银额果蝇的起源和分化

本文以８种限制性内切酶对８个银额果蝇群体进行了ｍｔＤＮＡ的限制性片段长度多态性（ＲＦＬＰ）分析。发现现生银额果蝇种群可以分成三个相对独立的群体,即东部、中部和西部群体。结合其它有关资料,我们推测,银额果蝇可能起源于马来半岛南部和加里曼丹岛一带。起初分成东西两支向北扩散。东支发展成现在的东部群体;西支则在中南半岛北部又分成两个支系;从而形成了现生银额果蝇群体的东部、中部和西部的地理分布模式。     

Selection for abdominal tergite pigmentation and correlated responses in the trident: a case study in Drosophila melanogaster

In Drosophila melanogaster, abdominal tergite pigmentation and the appearance of a trident‐shaped thoracic pattern exhibit similar biogeographical variation and sensitivity to temperature. These pigmentation traits may be under common selection pressure in natural populations or may be genetically correlated. To investigate the nature of this interaction, replicated populations of D. melanogaster were selected for increased or decreased melanization of the abdominal tergites for 40 generations. Selection for abdominal tergite pigmentation leads to correlated changes in trident formation. Although selection was performed only on female flies, male pigmentation also responded to selection. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 287–294.     

A thermal method for the determination of tissue blood flow.

An isothermal flowmeter for the determination of local tissue blood flow is described. Flow is determined by the measurement of the thermal conductivity of the tissue in the vicinity of a heated thermistor maintained at a fixed temperature difference above a reference thermistor. Direct heating of the thermistor is utilized to eliminate the need for specially constructed indirectly heated thermistors. This design results in a device with a voltage output directly proportional to tissue thermal conductivity and to tissue blood flow. The device is shown to be adequate for the qualitative measurement of myocardial blood flow under various situations. Construction is simplified and the size of the circuit reduced by the use of readily available integrated circuits.     

The specificity of interphase FISH translocation probes in formalin fixed paraffin embedded tissue sections is readily assessed using automated staining and scoring of tissue microarrays constructed from murine xenografts

Implementation of interphase fluorescence in situ hybridization (FISH) assays in the clinical laboratory requires validation against established methods. Validation tools in common use include exchange of consecutive sections with another institution that has already established the FISH assay, comparison with conventional banded metaphase cytogenetics, confirmation of specificity using probed normal metaphases, consecutive paraffin sections of a validation set tested by a reference laboratory, and specificity assessment against well characterized cell lines. We have investigated the feasibility of using tissue microarrays (TMA) constructed from murine xenografts as a preliminary specificity-screening tool for validation of interphase FISH assays. Cell lines currently in use for FISH controls are used to generate xenografts in SCID mice which are fixed in formalin and paraffin embedded. A TMA is constructed using duplicate donor cores from the xenograft blocks. Xenografts used represent a wide range of translocations used routinely for formalin fixed paraffin embedded sections evaluated by FISH. Probe cocktails (Abbott-Vysis), for several non-random translocations associated with hematologic neoplasms and soft tissue sarcomas have been used in this manner. On-line deparaffinization, cell conditioning, and prehybridization steps are automated using a staining workstation (Ventana Discovery XT); hybridization and stringency washes are performed manually offline. FISH-probed TMAs are tracked using a Metasystems image scanner and analyzed using classifiers specifically developed for each molecular abnormality. FISH results for each xenograft in the TMA correspond exactly to the genotype previously established for the parent cell line from which the xenograft was prepared. Moderate complexity tissue microarrays constructed from murine xenografts are excellent validation tools for initial assessment of interphase FISH probe specificity.     

Natural variation of ebony gene controlling thoracic pigmentation in Drosophila melanogaster

We identified the causal genetic variation for the difference in the thoracic trident pigmentation intensity between two wild-derived strains of Drosophila melanogaster. It was found to be the difference in expression level of ebony, which codes for an enzyme in the melanin-synthesis pathway and has pleiotropic effects on vision and behavior.     

A new genus and species of demodecid mites from the tongue of a house mouse Mus musculus: description of adult and immature stages with data on parasitism

The study of the parasitofauna of the house mouse Mus musculus (Rodentia: Muridae) Linnaeus is particularly important owing to its multiple relationships with humans – as a cosmopolitan, synanthropic rodent, bred for pets, food for other animals or laboratory animal. This article proposes and describes a new genus and species of the parasitic mite based on adult and immature stages from the house mouse. Glossicodex musculi gen. n., sp. n. is a medium‐sized demodecid mite (adult stages on average 199 µm in length) found in mouse tissue of the tongue. It is characterized by two large, hooked claws on each tarsus of the legs; the legs are relatively massive, consisting of large, non‐overlapping segments. The palps consist of three slender, clearly separated, relatively narrow segments, wherein their coxal segments are also quite narrow and spaced. Also, segments of the palps of larva and nymphs are clearly isolated, and on the terminal segment, trident claws that resemble legs' claws can be found. On the ventral side, in immature stages, triangular scuta, topped with sclerotized spur, can be also observed. Glossicodex musculi was noted in 10.8% of mice with a mean infection intensity of 2.2 parasites per host.     

Divergent enhancer haplotype of ebony on inversion In(3R)Payne associated with pigmentation variation in a tropical population of Drosophila melanogaster

The pattern and intensity of pigmentation have direct impact on individual fitness through various ecological factors. In a Drosophila melanogaster population from southern Japan, thoracic trident pigmentation intensity of most of the strains could be classified into Dark or Light‐type. The expression level variation of the ebony gene correlated well with this phenotype and the allelic differences in expression indicated that the variation is partly due to cis‐regulatory changes. In the ～13 kb gene region, we identified 17 nucleotide sites and 2 indels that were in complete association with the thoracic trident pigmentation intensity. Interestingly, 11 out of 19 sites located within ～0.5 kb of the core epidermis enhancer. These sites had no obvious association with the abdominal pigmentation intensity in the previously analysed African populations from Uganda and Kenya, which suggested that multiple potential mutational pathways in the cis‐regulatory control region of a single gene could lead to similar phenotypic variation within this species. We also found that the Light‐type enhancer haplotype is strongly linked to a cosmopolitan inversion, In(3R)Payne, which is predominant in warmer climatic regions in both hemispheres. The sequence pattern suggested that the strong linkage may be due to selective forces related to thermal adaptation. The inferred selection for lighter pigmentation in the Japanese population is in the opposite direction of the previously reported case of selection for darker individuals in African populations. Nevertheless, both adaptive changes involved cis‐regulatory changes of ebony, which shows that this gene is likely to be a common target of natural selection.     

Solution structure of the ubiquitin-associated domain of human BMSC-UbP and its complex with ubiquitin

Ubiquitin is an important cellular signal that targets proteins for degradation or regulates their functions. The previously identified BMSC-UbP protein derived from bone marrow stromal cells contains a ubiquitin-associated (UBA) domain at the C terminus that has been implicated in linking cellular processes and the ubiquitin system. Here, we report the solution NMR structure of the UBA domain of human BMSC-UbP protein and its complex with ubiquitin. The structure determination was facilitated by using a solubility-enhancement tag (SET) GB1, immunoglobulin G binding domain 1 of Streptococcal protein G. The results show that BMSC-UbP UBA domain is primarily comprised of three alpha-helices with a hydrophobic patch defined by residues within the C terminus of helix-1, loop-1, and helix-3. The M-G-I motif is similar to the M/L-G-F/Y motifs conserved in most UBA domains. Chemical shift perturbation study revealed that the UBA domain binds with the conserved five-stranded beta-sheet of ubiquitin via hydrophobic interactions with the dissociation constant (KD) of approximately 17 microM. The structural model of BMSC-UbP UBA domain complexed with ubiquitin was constructed by chemical shift mapping combined with the program HADDOCK, which is in agreement with the result from mutagenesis studies. In the complex structure, three residues (Met76, Ile78, and Leu99) of BMSC-UbP UBA form a trident anchoring the domain to the hydrophobic concave surface of ubiquitin defined by residues Leu8, Ile44, His68, and Val70. This complex structure may provide clues for BMSC-UbP functions and structural insights into the UBA domains of other ubiquitin-associated proteins that share high sequence homology with BMSC-UbP UBA domain.     

De novo genome assembly of Candida glabrata reveals cell wall protein complement and structure of dispersed tandem repeat arrays

Candida glabratais an opportunistic pathogen in humans, responsible for approximately 20% of disseminated candidiasis. Candida glabrata's ability to adhere to host tissue is mediated by GPI-anchored cell wall proteins (GPI-CWPs); the corresponding genes contain long tandem repeat regions. These repeat regions resulted in assembly errors in the reference genome. Here, we performed a de novo assembly of the C. glabrata type strain CBS138 using long single-molecule real-time reads, with short read sequences (Illumina) for refinement, and constructed telomere-to-telomere assemblies of all 13 chromosomes. Our assembly has excellent agreement overall with the current reference genome, but we made substantial corrections within tandem repeat regions. Specifically, we removed 62 genes of which 45 were scrambled due to misassembly in the reference. We annotated 31 novel ORFs of which 24 ORFs are GPI-CWPs. In addition, we corrected the tandem repeat structure of an additional 21 genes. Our corrections to the genome were substantial, with the length of new genes and tandem repeat corrections amounting to approximately 3.8% of the ORFeome length. As most corrections were within the coding regions of GPI-CWP genes, our genome assembly establishes a high-quality reference set of genes and repeat structures for the functional analysis of these cell surface proteins.     

Quick Staining Method For Frozen Sections

Since the advent and general acceptance of frozen sections in histological and pathological laboratories it has been necessary to devise methods for staining these sections. The usual method is fixing the tissue to a slide by the use of celloidin. This paper is an attempt to describe a permanent, quick method of staining frozen sections without distortion or mechanical tearing of the tissues.     

A method for reconstructing three-dimensional positions of swarming mosquitoes

We developed a new field method for reconstructing the three-dimensional positions of swarming mosquitoes. This method overcame certain inherent difficulties accompanied by conventional stereoscopic methods and is applicable to three-dimensional measurements of other insect species. Firstly, we constructed a probabilistic model for stereoscopy; if mosquitoes and six reference points with known coordinates were photographed simultaneously from two or more perspectives, then from the positions of images of mosquitoes and the reference points on the photographs, 1) the position of each camera with respect to the reference points is estimated; 2) stereo images which correspond to an identical real mosquito are matched; and 3) the spatial positions of the mosquitoes are determined. We automated the processes 1), 2) and 3), developing computer programs based on our model. We then constructed a portable system for three-dimensional measurements of swarming mosquitoes in the field. Initial data that illustrate the application of our method to studying mosquito swarming were presented.     

Testing the long-term stability of marine isoscapes in shelf seas using jellyfish tissues

Maps of the spatial distribution of stable isotope ratios across wide geographic areas (isoscapes) are increasingly used to study mechanisms of nutrient flux, movements of animals, and to improve trophic information derived from stable isotope analyses. Isoscapes are usually constructed from reference samples collected from known geographic positions, a time consuming and costly process. In this study, we test the temporal stability of isoscapes of carbon and nitrogen isotopes across the North Sea over a ten-year period. Using jellyfish tissues as reference organisms, we show that hydrodynamic and biogeochemical processes controlling the distribution of carbon and nitrogen isotope values, and thus that the underlying isoscapes, are temporally stable. Remarkably, broad geographic variations in stable carbon and nitrogen isotope distributions across the North Sea are consistent with previously published variations seen in archaeological cod bones from the ninth to seventeenth centuries, despite dramatic changes in land use in the past 1,000 years. Stable isotope-based studies of trophic interactions or movements in animals with lifespans of a year or more in shelf ecosystems can consequently be referenced to previously published isoscape models, and do not require construction of temporally explicit isotope baseline corrections. Scyphomedusan jellyfish are excellent reference organisms for ecological stable isotope analyses in pelagic ecosystems, due to their widespread distributions, well-defined life histories, and fast tissue growth.     

Using Species Distribution Models For Fungi

Species distribution models (SDMs) are an emerging tool in the study of fungi, and their use is expanding across species and research topics. To summarise progress to date and to highlight important considerations for future users, we review 283 studies that apply SDMs to fungi. We found that macrofungi, lichens, and pathogenic microfungi are most often studied. While many studies only aim to model species response to environmental covariates, the use of SDMs for explicitly predicting fungal occurrence in space and time is growing. Many studies collect fungal occurrence data, but the use of pre-collected records from reference collections and citizen science programs is increasing. Challenges of applying SDMs to fungi include detection and sampling biases, and uncertainties in identification and taxonomy. Further, finding environmental covariates at appropriate spatial and temporal scales is important, as fungi can respond to fine-scale environmental patterns. Fine-scale covariate data can be difficult to gather across space, but we show remote-sensing measurements are viable for fungi SDMs. For those fungi interacting with host species, host information is also important, and can be used as covariates in SDMs. We also highlight that competition among fungi, and dispersal, can affect observed distributions, with the latter particularly prominent for invasive fungi. We show how one can account for these processes in models, when suitable data are available. Finally, we note that environmental DNA records create new opportunities and challenges for future modelling efforts, and discuss the difficulties in predicting invasions and climate change impacts. The application of SDMs to fungi has already provided interesting lessons on how to adapt modelling tools for specific questions, and fungi will continue to be relevant test subjects for further technical development of SDMs.     

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator’s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10¹⁰ viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.     

A Long‐Term Comparison of Hydrology and Plant Community Composition in Constructed Versus Naturally Occurring Vernal Pools

Successful restoration of ephemeral wetlands worldwide is particularly challenging, given the often‐precise relationship between hydrological features and plant community dynamics. Using a long‐term experiment in vernal pool restoration, we compare hydrological and vegetative characteristics of constructed pools with those of adjacent, naturally occurring reference pools. Although constructed and reference pools were similar in maximum water depth and duration of inundation at the beginning of our experiment in 2000, constructed pools were shallower and inundated for shorter periods by 2009. Native vernal pool species were able to establish populations in many constructed pools, and seeding sped their establishment. Comparing seeded plots in constructed pools with unseeded plots in reference pools, we found no significant difference in the cover of seeded species, native species, or exotic species in most years. In recent years, however, native species have declined in both constructed and reference pools. Finally, the cover of native vernal pool species was positively and non‐linearly associated with both water depth and seeding treatment. We conclude that the establishment of appropriate hydrological conditions was necessary, but not sufficient to promote successful performance of vernal pool species in constructed pools. Constructed pools with hydrologic conditions similar to those of reference pools were more likely to support populations of native vernal pool plant species, but only seeded pools were similar to reference pools in abundance of native cover. Most importantly, hydrological conditions in experimental pools have worsened since their construction, which may hamper persistence of native species in this restoration effort.