Flight style in bats as predicted from wing morphometry: the effects of specimen preservation

An as yet unconsidered potential error in studies that predict flight style from morphological measurements of bats is the effect of the specimen type employed. On the basis of the finding that morphological measurements taken from fluid-preserved bat specimens may not yield values equivalent to those taken from the live animal, we compared the values of several variables (lifting surface area, wingspan, mass, aspect ratio, wing loading and minimum power speed) for live and fluid-preserved little brown bats ( Myotis lucifugus ) with the accepted standards for this species given by Norberg & Rayner (1987). Significant differences were detected for lifting surface area, wingspan, mass, aspect ratio and wing loading values taken from live bats and their respective values reported by Norberg & Rayner. Differences between preserved bats and Norberg & Rayner's numbers were limited to lifting surface area and wingspan (extended wing positions only), aspect ratio (all wing positions), and mass (both 70% ethanol- and 45% isopropyl alcohol-preserved specimens). Thus, Norberg & Rayner's values correspond most closely to values obtained from preserved museum specimens, a fact reflecting the source of their data in this instance. This and other limitations involved in attempting to predict the flight style of bats from a few morphological characters are discussed.     

Effects of preservation on pigmentation and length measurements in larval lampreys

The effects of two methods of preservation (fixation and storage in 10% formalin, and fixation in 10% formalin followed by storage in 95% alcohol) on pigmentation and morphometric features used for identification of larval Ichthyomyzon lampreys were analysed. Both short‐term (3 weeks) and long‐term (6 months) studies were conducted using digital analysis of images of fresh and preserved lampreys. Six standard morphometric lengths and 10 areas of pigmentation were analysed. All measurements were significantly affected by preservation. Preservative type affected pigmentation and morphometric characteristics differently, and characters were affected to different degrees. Multiple measurements over time showed that almost all changes occurred within 3 weeks of preservation. Regression equations allowed for accurate correction of preservation effects on morphometric measurements, but the effects on pigmentation levels were less predictable. Effects of preservation on larval lampreys need to be considered when comparing fresh and preserved specimens because they influence critical identification features.     

Accuracy of body mass estimates of formalin-preserved fish – a review

This paper highlights possible effects of physical and chemical mechanisms of formalin fixation and preservation on biological tissue and reviews the consequent potential inaccuracies on estimates of body mass of small fishes fixed and preserved in formalin. Twenty-six papers including 65 independent experiments with 35 species which examine effects of formalin on body mass estimates on small fishes are included. The effect of the formalin on the specimens depends on the salinity of the water used to dilute the commercial formalin (usually 1:9 formalin: water) before being used to fixate and preserve fish. Mean wet body mass of the specimens from the studies using seawater or fresh water diluted formalin deceases by 13% and increases by 7%, respectively, from before to after being immersed in formalin. The same trend is found with condition factor in the few papers that report this parameter. Body length decreases on average by c. 2% in fixated and preserved fish regardless of whether the formalin is diluted in seawater or fresh water.     

Is formalin fixation and ethanol preservation able to influence in geometric morphometric analysis? Fishes as a case study

Geometric morphometric analysis has increased in the recent years, turning into a powerful tool to explore shape and size variation. Several biological studies use specimens that have been through some kind of preservation, mainly formalin preservation, commonly used in biological collections. This study analyzed the effect of preservation in shape on two fish species: Eucinostomus argenteus and Pomadasys corvinaerformis. Twenty-nine individuals of E. argenteus and twenty-five of P. corvinaeformis were collected, photographed twice, preserved in 10 % formalin for 1 week, and then transferred to 70 % ethanol for 83 days. We evaluated three levels of error: (1) error of landmark digitalization, (2) error of taking the picture and storage in JPEG format, and (3) the formalin and ethanol fixation error using Procrustes ANOVA, Discriminant Analysis, and Principal Component Analysis. Significant difference between treatments was observed on both species with Procrustes ANOVA and Discriminant Analysis. In addition, Principal Component Analysis showed a separation between groups of treatment on both species. These results represent the first evidence of preservation effects in studies of geometric morphometrics and show that according to the statistical test utilized, the fixation could affect the shape variations in different ways and could lead the researcher to false results or wrong conclusions. Other methods to explore the shape variation of organisms previously fixed should be tested in order to assess their influence in geometric mophrometric studies.     

Effects of fixation and preservation conditions on immunohistochemical profiles of the skeletal muscle fibers in Japanese macaques

Effects of fixation and preservation conditions of muscle tissues on immunohistochemical profiles are investigated. Samples of the hind limb and epaxial muscles were removed from 4 adult female Japanese macaques (Macaca fuscata) fixated with 10% formalin and preserved in the same solution under different conditions for 6 months to 4 years and 6 months. Sections were stained with indirect immunofluorescence and avidin-biotin peroxidase complex methods using an antibody against fast myosin (Mouse Monoclonal Anti-skeletal Myosin-Fast, clone MY-32, Sigma) as a primary antibody. Clear responses to the antibody were demonstrated in the samples from the specimens fixated by injection or immersion with 10% formalin and preserved in the same solution for 6 months to 1 year and 6 months. Distribution patterns of the fibers reacting to the antibody coincided with that of the fast twitch fibers determined using enzyme-histochemical techniques in these samples. Clear responses to the antibody were not demonstrated in the samples from the specimen repeatedly rinsed in water for gross anatomical dissections during the preservation period. The results of this study warrant applications of immunohistochemical techniques to the study of fiber type composition in muscle samples from specimens fixated with formalin and preserved in the same solution for a long term.     

Rapid microwave fixation--a comparative morphometric study

Summary Tissue blocks taken from healthy human lung tisues, from primary bronchial carcinoma and from mediastinal and hilar lymph nodes were placed in the following solutions. Tris buffer; buffered formalin (0.5%, 1%, 7%); 0.1 mol NaCl; distilled water; DMSO (1%, 10%, 20%); acetone (10%); methanol (50%, 80%, 100%); glutaraldehyde (2.5%), and fixed by use of a commercial microwave oven.Tissue blocks obtained from the same surgical specimens were fixed in 7% buffered formalin for 24 h for comparison. Conventional and microwave-fixed tissue was embedded in paraffin, and stained with haematoxylin and eosin. Fifteen specimens of each group and each solution were examined by light microscopy. Minimum diameter and area of 100 nuclei of each specimen were measured interactively.Histomorphological sections fixed with Tris buffer in a microwave oven revealed best morphological results showing more contrast in chromatin distribution of nuclei and opening of interstitial lung capillaries in comparison to conventional formalin-fixed specimens. No statistically significant differences in area and minimum diameter of nuclei between the different groups were found. Microwave fixation using Tris buffer is a time-saving fixation method at least comparable to conventional formalin fixation. It is not accompanied by hazards to the environment that are unavoidable by formalin fixation.     

Immunoelectron microscopic visualization of neurohypophyseal hormones: evaluation of some tissue preparations and staining procedures.

To obtain optimal electron microscopical localization of vasopressin in the rat neurohypophyses two immunocytochemical staining procedures and several tissue treatments were evaluated. The peroxidase-antiperoxidase technique allowed greater dilution of the first antibody than the indirect method using a commercial peroxidase conjugate. This appeared crucial for the dilution-dependent specificity in the localization of vasopressin. Immersion fixation with formalin gave better results than those obtained with perfusion fixation with glutaraldehyde-paraformaldehyde (resulting in similar preservation of immunoreactivity) and freeze substitution (showing the best preservation of immunoreactivity). However, these latter two tissue fixation methods resulted in less than optimal preservation of general ultrastructure than immersion fixation in formalin alone. Immersion fixation with glutaraldehyde-paraformaldehyde followed by OsO4 improved ultrastructural detail, but immunoreactivity decreased considerably. Fixation with paraformaldehyde-picric acid resulted in poorest preservation of morphologic detail. Immunoreactivity was similar in both Epon 812 and Araldite 6005 embedded tissue.     

Critical steps in tissue processing in histopathology

Histopathological diagnosis using Formalin-Fixed Paraffin Embedded (FFPE) tissues is essential for the prognostic and therapeutic management of cancer patients. Pathologists are being confronted with increasing demands, from both clinicians and patients, to provide immunophenotypic and gene expression data from FFPE tissues to allow the planning of personalized therapeutic regimens. Recent improvements in the protocols for pre-analysis processing of pathological tissues aim to better preserve cellular details and to conserve antigens and nucleic acid sequences. These developments have been recently patented. The international protocol for the transporting of surgical specimens from the surgical theatre to the pathology department is to immerse the specimen in formalin. The alternative method of sealing the specimens into bags under a vacuum and then cooling is a well-accepted and environmentally safe procedure that overcomes the many drawbacks linked to transfer in formalin. Importantly, RNA is notoriously poorly preserved in FFPE tissue. Due to this, successful procedures for the extraction of genetic information from archival tissues have been the object of several studies and patents. Novel molecular approaches for RT-qPCR and gene array analysis on FFPE tissues are presented here. Moreover, a major advance is reported in this study, the observation that tissue fixation in cold conditions allows a much better preservation of nucleic acid sequences.     

Morphometric measurements of dragonfly?wings: the accuracy of pinned,scanned and detached?measurement methods

Large-scale digitization of museum specimens, particularly of insect collections, is becoming commonplace. Imaging increases the accessibility of collections and decreases the need to handle individual, often fragile, specimens. Another potential advantage of digitization is to make it easier to conduct morphometric analyses, but the accuracy of such methods needs to be tested. Here we compare morphometric measurements of scanned images of dragonfly wings to those obtained using other, more traditional, methods. We assume that the destructive method of removing and slide-mounting wings provides the most accurate method of measurement because it eliminates error due to wing curvature. We show that, for dragonfly wings, hand measurements of pinned specimens and digital measurements of scanned images are equally accurate relative to slide-mounted hand measurements. Since destructive slide-mounting is unsuitable for museum collections, and there is a risk of damage when hand measuring fragile pinned specimens, we suggest that the use of scanned images may also be an appropriate method to collect morphometric data from other collected insect species.     

Effects of tissue preservation on murine bone mechanical properties

Murine bone specimens are used extensively in skeletal research to assess the effects of environmental, physiologic and pathologic factors on their mechanical properties. Given the destructive nature of mechanical testing, it is normally performed as a terminal procedure, where specimens must be preserved without affecting their mechanical properties. To this end, we aimed to study the effects of tissue preservation (freezing and formalin fixation) on the elastic and viscoelastic mechanical properties of murine femur and vertebrae. A total of 120 femurs and 180 vertebral bodies (L3–L5) underwent non-destructive cyclic loading to assess their viscoelastic properties followed by mono-cyclic loading to failure to assess their elastic properties. All specimens underwent re-hydration in 0.9% saline for 30 min prior to mechanical testing. Analysis indicated that stiffness, modulus of elasticity, yield load, yield strength, ultimate load and ultimate strength of frozen and formalin-fixed femurs and vertebrae were not different from fresh specimens. Cyclic loading of both femurs and vertebrae indicated that loss, storage and dynamic moduli were not affected by freezing. However, formalin fixation altered their viscoelastic properties. Our findings suggest that freezing and formalin fixation over a 2-week period do not alter the elastic mechanical properties of murine femurs and vertebrae, provided that specimens are re-hydrated for at least half an hour prior to testing. However, formalin fixation weakened the viscoelastic properties of murine bone by reducing its ability to dissipate viscous energy. Future studies should address the long-term effects of both formalin fixation and freezing on the mechanical properties of murine bone.     

Mitochondrial DNA sequence from an extinct population of European vendace, Coregonus albula L

Nucleotide sequence of the mitochondrial DNA control region was determined for the formalin-preserved specimen of the individual European vendace, Coregonus albula, belonging to an extinct population. The specimen has been preserved and stored in formalin for 39 years. A comparison with two previously determined sequences obtained for specimens from other populations of vendace revealed both nucleotide substitutions and site heteroplasmy. The ability to sequence DNA from formalin-preserved museum specimens of rare or extinct taxa provides another sampling strategy for phylogenetic studies on fish.     

Morphometrical and front wing abrasion analysis of a Hungarian cotton bollworm <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> (Lepidoptera: Noctuidae) population

To determine the cotton bollworm migrating population rate in Hungary, we examined the weights and the front wing morphological feautures of trapped moths. We used sex pheromone traps to monitor field populations during the maize vegetation cycle period in 2008. We examined moths trapped at various times, and measured their body mass (m) and morphological features, namely the front wing quotient (fWQ = quotient of length of front wing/width of front wing), modified wing loading (WL = weight of moth/surface of front wing), and the relative thorax size (RTS = width of thorax/width of head). The data were analysed by Student t-test, anterior wing abrasion and darkness were analysed by a Adobe Photoshop 7.0 software. The Hungarian appearance of three cottom bollworm generations in 2008 was also observed. Based on the examined morphological features we found regularity in body mass, front wing quotient and modified wing loading changes during the flight period. The specimens trapped in the first and third part of the flight period had lower body mass, larger wing surface, longer wings and more favourable modified wing loading than the specimens trapped in the middle of the flight period. The abrasion and colour of the anterior wings of cotton bollworms were concordant to morphometric investigations. The abrasion in darker spots E1 and E3 clearly showed a more intensive usage of the wings in case of specimens trapped at the beginning and at the end of the flight period.     

Spatial and temporal continuity of kangaroo rat populations shown by sequencing mitochondrial DNA from museum specimens

Summary The advent of direct sequencing via the polymerase chain reaction (PCR) has opened up the possibility of molecular studies on museum specimens. Here we analyze genetic variation in populations over time by applying PCR to DNA extracted from museum specimens sampled from populations of one species over the last 78 years. Included in this study were 43 museum specimens of the Panamint kangaroo ratDipodomys panamintinus from localities representing each of three geographically distinct subspecies. These specimens were originally collected and prepared as dried skins in 1911, 1917, or 1937. For each specimen, a 225-bp segment of the mitochondrial genome was sequenced. These mitochondrial DNA sequences were compared to those of 63 specimens collected at the same localities in 1988. The three subspecies were nearly completely distinct. Only 2 of the 106 individuals shared mitochondrial types between subspecies. For all three localities, the diversity levels were maintained between the two temporal samples. The concordance observed between the two temporally separate phylogenies supports the use of museum specimens for phylogenetic inference. This study demonstrates the accuracy and routine nature of the use of museum specimens in the analysis of mitochondrial sequence variation in natural populations and, importantly, that a temporal aspect can now be added to such studies.     

Changes in body dimensions of larval Chaoborus in ethanol and formalin

The effects of fixation and preservation in formalin and ethanolon various body size measurements of the planktonic midge, Chaoborus,were determined. The magnitude of the observed change variedwith the species and the fixative/preservative used. Failureto consider changes in body dimensions induced by fixation andpreservation may significantly bias the estimation of body weightfrom the length of preserved specimens.     

Effects of freezing on white perch Morone americana (Gmelin, 1789): implications for multivariate morphometrics

This study tested the hypothesis that duration of freezing differentially affects whole‐body morphometrics of a derived teleost. Whole‐body morphometrics are frequently analyzed to test hypotheses of different species, or stocks within a species, of fishes. Specimens used for morphometric analyses are typically fixed or preserved prior to analysis, yet little research has been done on how fixation or preservation methods or duration of preservation of specimens might affect outcomes of multivariate statistical analyses of differences in shape. To determine whether whole‐body morphometrics changed as a result of freezing, 23 whole‐body morphometrics of age‐1 white perch (Morone americana) from western Lake Erie (n = 211) were analyzed immediately after capture, after being held on ice overnight, and after freezing for 100 or 200 days. Discriminant function analysis revealed that all four groups differed significantly from one another (P < 0.0001). The first canonical axis reflected long‐axis morphometrics, where there was a clear pattern of positive translation along this axis with duration of preservation. Re‐classification analysis demonstrated fish were typically assigned to their original preservation class except for fish frozen 100 days, which assigned mostly to frozen 200 days. Morphometric comparisons using frozen fish must be done on fish frozen for identical periods of time to avoid biases related to the length of time they were frozen. Similar experiments should be conducted on other species and also using formalin‐ and alcohol‐preserved specimens.     

Handling specimens in liquid preservative: adding and removing the liquid paraffin overlayer

Adding a overlayer of liquid paraffin to formalin and subsequently removing it with oil-absorbent sheets in order to observe the specimen are very useful procedures when handling specimens preserved in formalin.     

Museum metabarcoding: A novel method revealing gut helminth communities of small mammals across space and time

Natural history collections spanning multiple decades provide fundamental historical baselines to measure and understand changing biodiversity. New technologies such as next generation DNA sequencing have considerably increased the potential of museum specimens to address significant questions regarding the impact of environmental changes on host and parasite/pathogen dynamics. We developed a new technique to identify intestinal helminth parasites and applied it to shrews (Eulipotyphla: Soricidae) because they are ubiquitous, occupy diverse habitats, and host a diverse and abundant parasite fauna. Notably, we included museum specimens preserved in various ways to explore the efficacy of using metabarcoding analyses that may enable identification of helminth symbiont communities from historical archives. We successfully sequenced the parasite communities (using 12S mtDNA, 16S mtDNA, 28S rDNA) of 23 whole gastrointestinal tracts. All gastrointestinal tracts were obtained from the Museum of Southwestern Biology, USA, and from recent field collections, varying both in time since fixation (ranging from 4?months to 16?years) and preservation method (70% or 95% ethanol stored at room temperature, or flash frozen in liquid nitrogen and stored at ?80?°C). Our proof of concept demonstrates the feasibility of applying next generation DNA sequencing techniques to authoritatively identify the parasite/pathogen communities within whole gastrointestinal tracts from museum specimens of varying age and fixation, and the value of future preservation of host-associated whole gastrointestinal tracts in public research archives. This powerful approach facilitates future comparative examinations of the distributions and interactions among multiple associated groups of organisms through time and space.     

Gut wash,body soak,blender and heat-fixation: approaches to the effective collection,fixation and preservation of trematodes of fishes

Advice is offered on some effective methods for collecting and preserving trematodes from fishes for taxonomy and systematics. Emphasis is placed on obtaining high-quality specimens that have reliable data and that are amenable to study by both morphological and molecular approaches. We emphasise the importance of the freshness of the host specimen, the reliability of its provenance and the labelling of the specimens. For the collecting itself, we recommend a ‘gut-wash’ approach for gastro-intestinal species and specific searches for atypical taxa such as didymozoids, aporocotylids, Saturnius Manter, 1969 and transversotrematids. For metacercariae, we recommend a ‘blender’ approach to release parasites from host tissues. For fixation, we argue in favour of heat-killing in fluid at close to boiling temperature. We recommend against flattening as a routine procedure for collecting specimens for morphology. Preservation for morphological study is best in formalin or alcohol, and alcohol works well for molecular samples. The importance of reliable labelling and the deposition of specimens in museums is emphasised.     

Ultrastructural observations on tissues processed by a quick-freezing,rapid-drying method: Comparison with conventional specimen preparation

A quick-freeze, rapid-dry method for processing unfixed tissue for electron microscopy has been developed. The technique employs freezing on a cryogenchilled metal surface and drying in a cryosorption vacuum apparatus that allows osmium-vapor fixation and epoxy-resin embedment under high vacuum. Liver, kidney, bone marrow, and monolayer cultures of ventricular myocytes were selected as tissue specimens representing a wide range of physical properties, to demonstrate the practical aspects of achieving good ultrastructural morphology by freeze drying. A comparison was made between freeze drying and conventional processing using aldehyde fixation and alcohol dehydration. The preservation of cellular ultrastructure achieved by freeze drying allowed the identification of specific cell types within each specimen. Membranous organelles were well preserved, surrounded by cytoplasmic ground substance devoid of ice crystal damage. Electron-dense material was observed within the rough endoplasmic reticulum and Golgi cisternae and vesicles of frozen-dried, but not conventionally processed cells. This suggests the preservation by freeze drying of cytoplasmic components otherwise extracted from the cell by solvent exposure.