Impacts of UVB provision and dietary calcium content on serum vitamin D3 , growth rates, skeletal structure and coloration in captive oriental fire-bellied toads (Bombina orientalis).

Many amphibian species are dependent on ex situ conservation interventions for their long-term persistence. However, projects have been jeopardised by husbandry issues involving poor calcium metabolism and nutritional metabolic bone disease (NMBD). Healthy calcium metabolism requires appropriate dietary calcium content and access to vitamin D3 . In many animals, vitamin D3 can be photobiosynthesised in skin exposed to UVB radiation, as well as extracted from the diet, but the extent of vitamin D3 photobiosynthesis in amphibians is poorly known. Additionally, prey insects for captive amphibians are deficient in calcium and calcium content must be artificially increased, but the effects of different levels of augmentation and their interaction with UVB exposure are also little understood. We fed captive fire-bellied toads (Bombina orientalis) with crickets augmented to contain 5% and 10% calcium and housed them with and without UVB exposure. Despite additional dietary vitamin D3 supplementation, we found that toads exposed to UVB radiation exhibited significantly higher serum vitamin D3 levels, indicating that this species may partly rely on photobiosynthesis sources of vitamin D3 . These data are the first to show a direct link between UVB exposure and serum vitamin D3 in an amphibian. We found significant positive effects of UVB exposure and 10% dietary calcium content on skeletal structure, as well as complex interactions between treatments. We also found UVB radiation exposure resulted in more rapid natural coloration acquisition. Together, this indicates that standard calcium plus vitamin D3 supplementation methods may not fully substitute for UVB exposure and for increased feeder insect calcium content. This may have implications for the success of ex situ amphibian conservation, as well as for the welfare of captive amphibians in general. Our data lend support for the provision of UVB radiation for captive, basking amphibians.

Can carotenoids mediate the potentially harmful effects of ultraviolet light in Silurana (Xenopus) tropicalis larvae?

Amphibians have recently experienced unprecedented declines in the wild, the causes of which are often difficult to mitigate. This has increased the importance of ex situ conservation; however, long-term maintenance and breeding of amphibians in captivity often has limited success. In vertebrates, vitamin D is required for calcium homeostasis and is produced endogenously in skin exposed to ultraviolet (UV) light; however, UV light can be harmful to amphibians in some instances. Carotenoids are dietary pigments that may provide protection from UV light. The aim of this study was to assess the protective capability of carotenoids against the potentially harmful effects of UV light in Silurana (=Xenopus) tropicalis larvae raised in an enhanced or limited UV environment. Tadpole survival and the size and developmental stage reached by the end of the study period were measured. Carotenoids had a significantly positive effect on developmental rate in both UV-limited and UV-enhanced environments. Larvae in an enhanced UV environment were significantly larger than those raised under a limited UV environment, irrespective of diet. Carotenoid-fed larvae tended to have increased survival in relation to those raised without carotenoids, but only in a limited UV environment. Carotenoids appear to provide little protection against UV light in this case. The role of carotenoids in amphibian health has not previously been studied. We show that carotenoid availability significantly influences development and may increase survival in S. (X.) tropicalis larvae. This finding may have important implications for recommendations made on the nutrition of amphibians in captivity.

Plant genotype mediates the effects of nutrients on aphids.

Soil nutrients, and factors which influence their concentrations and bioavailability, form a basic component of bottom-up control of ecosystem processes, including plant-herbivore interactions. Increased nutrient levels are linked, through plant defence theory, with increased levels of herbivore susceptibility. The focal point of many ecological experiments examining this link is at the species level, where the response of single species is the average of many different genotypes. Here, we focus on the genetic basis of indirect ecological interactions. We investigated the effects of nutrient concentration on the population growth of an aphid herbivore across multiple genotypes of barley in relation to plant growth rate. In general, both aphid population size and plant growth rate increased with nutrient concentration. However, they were both dependent on the interaction between nutrient concentration and barley genotype. Our data raise the testable possibility of differential defense responses between genotypes of barley, for example divergent, fixed and inducible defences against aphids. We provide evidence that the indirect effects of soil nutrients on aphid population size are mediated by the genetics of the host plant.

Tritrophic effects on survival and development in a Coccidophagous coccinellid.

Chilocorus nigritus is currently considered one of the most successful biological control agents of armoured scale insects. However, establishment of this beetle in crop pest situations has not always been successful and there are still gaps in our knowledge of its ecology and behaviour. The research involved an examination of tritrophic effects on the survival and development of this common diaspid predator. The effect of a forced change in host plant on the developmental time of the juvenile stages was also examined. The prey and host plants used were the armoured scales Aspidiotus nerii Bouché Homoptera: Diaspididae and Abgrallaspis cyanophylli (Signoret) Homoptera: Diaspididae, on potatoes (Solanum tuberosum L.) and Butternut squashes (Cucurbita moschata Duchesne ex Lamarck). C. nigritus eggs were incubated on four treatments of scales on potatoes or squashes for ten days, half the second instar larvae were then switched to the same scales on the other host plant. Daily observations were made during development to adult emergence. C. nigritus larvae survived and completed development on two species of diaspid scales and the two host plants examined with varying levels of success. Larvae were able to switch from feeding on A. nerii on potatoes to A. nerii on squashes or A. cyanophylli on potatoes to A. cyanophylli on squashes and vice versa with little or no deleterious effects when compared to those beetles reared on one prey and host plant throughout. There were significant differences in survival of larvae reared to the adult stage on both A. nerii and A. cyanophylli on potatoes when compared to larvae reared on these scales on squashes. Squash appears to be a less desirable and potato a more favourable host plant for survival and development. The results have implications for rearing programmes, and the release and establishment of C. nigritus, in fields and glasshouses where scale pests may be present on a variety of host plants, or may be on host plants different to those used in the insectary.

Acromegaly due to a somatroph adenoma in a dog.

A 10-year-old uncastrated male Dalmatian dog was referred for gait abnormalities consisting of chronic progressive stiffness and rigidity. Other symptoms were polyphagia associated with weight gain, polyuria and polydipsia, excessive panting, and an inspiratory stridor. The owner had noticed progressive thickening of the skin and enlargement of the tongue over the last 3 years. Physical examination revealed thickening of the skin, redundant skin folds, and enlargement of the tongue. The only remarkable abnormalities found on routine laboratory examination were mild anaemia and an increased serum fructosamine concentration. Circulating concentrations of total thyroxine, free thyroxine, and cTSH, and the results of an ACTH stimulation test were all within reference ranges. The basal serum growth hormone (GH) concentration was markedly elevated (23microg/l) and did not decrease during a glucose tolerance test or after somatostatin administration. The serum insulin-like growth factor-1 concentration was also markedly elevated (1254microg/l). Basal serum insulin concentration was high (95mU/l) and insulin concentrations increased considerably after glucose loading, consistent with insulin resistance. Abdominal ultrasonography showed no abnormalities. Survey radiographs of the vertebral column showed severe spondylosis deformans extending from the cervical to the lumbosacral spine. CT scanning of the skull showed an enlarged pituitary gland with normal enhancement pattern. On post-mortem examination, the entire vertebral column appeared as a single and inflexible structure due to the presence of multiple fused osteophytes. The pituitary gland contained an acidophilic adenoma that immunostained positively for GH (and negatively for ACTH and alpha-MSH). In conclusion, this Dalmatian dog with acromegaly and insulin resistance represents the first case of GH hypersecretion proven to be due to a somatotroph adenoma.

Multivariate survival analysis of histological parameters and clinical presentation in canine cutaneous mast cell tumours.

The Patnaik histological grade is a good method for the prediction of long-term mast cell tumour behaviour but it is influenced by subjective inter-observer variations and intratumoral heterogeneity. The present study evaluated each of the histopathological parameters used to formulate Patnaik's grade in terms of prognosis and tested whether they have a different prognostic sensitivity, thereby disclosing which could be considered more useful in the prediction of tumour recurrence and patient survival. Clinical presentation (single or multiple tumours) was also considered as possible prognostic factor. The results demonstrated that individual histological criteria together with multiple presentation may be of value in predicting the outcome of mast cell tumours. Among these, invasiveness (beta1.85; standard error 1.15) and the number of mitotic figures (beta3.01; standard error 1.18) showed high prognostic significance (Cox proportional hazard regression for censored data; chi-squared = 15.52, degree of freedom = 6, p = 0.016) and could serve as reliable prognostic indicators avoiding more subjective parameters such as cellular differentiation, nuclear morphology and tumoural pattern.

A pan-neotropical analysis of hunting preferences.

Hunting in the neotropics is a widespread form of resource extraction. However, there is increasing concern that current activities are leading to the decline and extirpation of vulnerable species; particulary ateline primates, large ungulates (such as tapirs and white-lipped peccaries) and large birds such as curassows. Hunting patterns are expected to be a product of two principal influences: the value of return for a given amount of effort invested into hunting, and cultural factors that determine the prestige and usefulness of prey. Previous work has suggested that hunting profiles change in a predictable way over time, becoming more diverse and more dependent on smaller bodied species as preferred, large-bodied prey become scarcer. In this paper, we evaluate the hunting profiles of 78 neotropical communities in Central and South America. We investigate the uniformity of species preferences, whether communities that are geographically closer have similar hunting profiles, and whether the age and size of settlements can be used to predict the type and diversity of species targeted. We found that there was only a weak correlation between the structure of communities' hunting profiles and their geographical proximity. Neither a community's size nor age was a good predictor of the shape and structure of its hunting profile. Our data suggest that either the availability of prey or the cultural influences dictating the value of different species can change rapidly over small distances, and that older and larger settlements do not impact prey species distributions in a predictable way.

Granulocyte-macrophage colony-stimulating factor and fluconazole enhance anti-cryptococcal activity of monocytes from AIDS patients.

OBJECTIVE: To investigate the effect of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF) and fluconazole on anti-cryptococcal activity of monocytes from AIDS patients and normal subjects. DESIGN: The effect of GM-CSF and fluconazole on fungistatic and fungicidal activity of monocytes was studied in an in vitro system. METHODS: Monocytes were treated in vitro with hrGM-CSF and fluconazole or either agent alone for 24 or 48 h, and fungistatic and fungicidal activity was evaluated in a colony-forming unit inhibition assay. CD11b/CD18 expression in monocytes was measured by flow cytometry analysis. Superoxide anion generation by peripheral blood monocytes was measured in the presence of pre-opsonized zymosan. RESULTS: Defective antifungal capacity of monocytes from AIDS patients was observed. GM-CSF treatment of monocytes from AIDS patients increased fungistatic activity, and the combination of hrGM-CSF and fluconazole resulted in fungicidal activity. The mechanisms involved in the GM-CSF-mediated effect appeared to be mediated by (i) enhancement of phagocytic activity, (ii) increase of superoxide anion generation, and (iii) upregulation of CD11b/CD18 expression on the monocyte surface. CONCLUSIONS: Our data highlight the effect of GM-CSF on anti-cryptococcal activity of human monocytes and show a synergistic effect of GM-CSF with fluconazole, suggesting a new therapeutic strategy in the treatment of cryptococcosis.

Lifetime selection on adult body size and components of body size in a waterstrider: opposing selection and maintenance of sexual size dimorphism.

Sexual size dimorphism (SSD), the difference in body size between males and females, is common in almost all taxa of animals and is generally assumed to be adaptive. Although sexual selection and fecundity selection alone have often been invoked to explain the evolution of SSD, more recent views indicate that the sexes must experience different lifetime selection pressures for SSD to evolve and be maintained. We estimated selection acting on male and female adult body size (total length) and components of body size in the waterstrider Aquarius remigis during three phases of life history. Opposing selection pressures for overall body size occurred in separate episodes of fitness for females in both years and for males in one year. Specific components of body size were often the targets of the selection on overall body size. When net adult fitness was estimated by combining each individual's fitnesses from all episodes, we found stabilizing selection in both sexes. In addition, the net optimum overall body size of males was smaller than that of females. However, even when components of body size had experienced opposing selection pressures in individual episodes, no components appeared to be under lifetime stabilizing selection. This is the first evidence that contemporary selection in a natural population acts to maintain female size larger than male size, the most common pattern of SSD in nature.

The spatial structure of the physical environment.

There is substantial environmental variance at small spatial scales (1 m or less) in both natural and disturbed environments. We have investigated the spatial structure of physical variables at larger scales (up to 106 m). We analysed surveys of edaphic properties of Wisconsin forest soils, of the water chemistry of lakes in Ontario and Labrador, and of temperature and precipitation in northeastern North America. We found no clear indication that the variance among sites approaches some maximal value as the distance between them increases. We suggest instead that the variance of the physical environment tends to increase continually with distance. The slope of the log-log regression of variance on distance provides a means of comparing the heterogeneity of different environments with respect to a given factor, or of comparing different factors within a given environment. This slope provides a useful measure of environmental structure that can be related to the biodiversity or plasticity of native organisms.

Cell proliferation patterns in canine infundibular keratinizing acanthoma and well differentiated squamous cell carcinoma of the skin.

The aim of the present study was to investigate if the evaluation of cell proliferation of the well differentiated squamous cell carcinoma (WDSCC) and infundibular keratinizing acanthoma (IKA) could be useful in the differential diagnosis between these two tumours. Eighteen IKAs and ten WDSCCs were selected for this study. Two different methods were used to assess the activity of cell proliferation: MIB1 immunohistochemical detection and AgNOR proteins silver staining. The quantification of proliferative parameters was performed by means of an image analyzer and expressed as MIB1 index and AgNOR area (MNORA). Both MIBI immunohistochemical and AgNOR histochemical patterns were different in WDSCC and IKA; moreover analysis of variance showed a significant difference for both parameters employed (MIB1 index, MNORA) between WDSCC and IKA (P<0.003 for MIB1 index; P<0.0001 for AgNOR area). The results show that canine WDSCC and IKA have a different proliferative behaviour and the assessment of cell proliferation can be considered as a useful adjunctive tool to the histopathological investigation in the differential diagnosis of these tumours.

Assessment of fibromyxoma cell proliferation in a tench (Tinca tinca L.) by the silver staining nucleolar organizer region (AgNOR) technique.

A previously described fibromyxoma in a tench was examined for AgNOR proteins and mitotic index to evaluate the cell proliferation of the different tissue areas forming the tumorous mass. The different areas were: myxoid (myxoid cells in a mucous ground substance with few reticular fibres), collagenous (fibroblast-like cells and collagenous fibres) and mixed areas (intermediate cytological and histological features). A significant (P < 0.01) difference in cell proliferative activity between the three different areas was found by the AgNOR technique and confirmed by the mitotic index. The myxoid areas appeared to be the true proliferative compartment of the tumour, whereas the collagenous areas probably played a structural role, related to low cellular proliferative activity. The AgNOR technique appeared to be a convenient method for the rapid evaluation of cell proliferation in fish tissues.

Prognostic value of intratumoral vessel density in cutaneous mast cell tumors of the dog.

The biological behaviour of canine cutaneous mast cell tumours (MCTs) is highly variable. The aim of this study was to investigate whether intratumoral microvessel density (IMVD), as an indicator of angiogenesis, was predictive of biological behaviour in MCTs of canine skin, and to relate this parameter to histological grade and other histomorphological features. IMVD was evaluated by immunohistochemical labelling of the endothelial marker factor VIII (Von Willebrand's factor)-related antigen in 32 primary cutaneous MCTs. Computer image analysis was used to measure IMVD, expressed as the number of microvessels per square millimetre. Cases were assigned to low and high vessel density groups, the median value (14.1) being used as the "cut-off". Kaplan-Meier-estimated survival curves of the groups revealed that IMVD was significantly associated with survival time (P=0.046). One-way analysis of variance, performed to compare the IMVD values of tumours of different histological grades, showed a significant difference between grades I and III (P=0.0002). There was a significant difference in IMVD between moderately invasive and highly invasive tumours (P=0.002) and between tumours with different numbers of mitoses (P=0.0046). In multivariate analysis, performed to test the combined influence on survival of each parameter, only the mitotic index and IMVD were independent prognostic variables (P=0.00019). The relation of angiogenesis to biological behaviour and histological parameters of malignancy of cutaneous MCTs was consistent with previous findings in human and animal tumours.

Detection of proliferating cell nuclear antigen (PCNA) in canine and feline mammary tumours.

Quantitation of immunohistochemical staining of the proliferating cell nuclear antigen (PCNA, clone PC10) by image analysis was performed on benign and malignant mammary tumours of dogs and cats. Scoring of the slides was carried out by image analysis to assess the percentage of labelled nuclei (expressed as a ratio of areas). Either the strongly labelled nuclei (SP-PCNA index), or all of the stained nuclei (TP-PCNA index) were counted as positive to determine the growth fraction and its correlation with the histopathological classification and nuclear grade (degree of nuclear differentiation, considered a morphological correlate of tumour aggressiveness). A significant difference in the values of PCNA indices was seen between benign and malignant growths (P < 0.0001, dog; P < 0.05, cat). Neither of the PCNA indices showed correlation with nuclear grade in dogs (P = 0.14 for SP-PCNA index and P = 0.31 for TP-PCNA index) or cats (P = 0.09 for SP-PCNA index and P = 0.07 for TP-PCNA index). A significant difference in the number of mitoses, expressed as mitotic index, was seen between benign and malignant growths in the dog (P < 0.01) but not in the cat (P = 0.078). Good correlation of mitotic index with nuclear grade was revealed in canine malignant growths (P < 0.05), but in feline malignant tumours such correlation (P < 0.05) was shown only when the values of intermediate plus typical forms were compared with the data for atypical forms. It is concluded that quantitation of PCNA-positive nuclear area by image analysis provides an objective method for assessing proliferative activity in benign and malignant mammary tumours of dogs and cats.

Laminin expression in testicular tumours of the dog.

The expression of laminin was studied to determine the distribution pattern of basement membranes (BMs) in normal testes and in a series of 40 canine testicular tumours (seminomas, Leydig and Sertoli cell tumours). BM was always present around seminiferous tubules and blood vessels in normal testes and in seminomas and Sertoli cell tumours of the intratubular type without invasion. BM changes (fragmentation or loss, or both) were usually found in invasive neoplasms which retained their tubular structure; disruption or absence was observed in tumours, with a diffuse pattern. The BM was never expressed in Leydig cell tumours, except around vessels, irrespective of their histological growth pattern (cystic-vascular, pseudoadenomatous, diffuse). An attempt was made to relate the degree of BM modification to proliferative monoclonal antibodies and mitotic index. In parallel with the progressive loss of BM an increase in proliferative activity occurred, indicating that BM changes are additional useful prognostic indicators in testicular tumours of the dog.

Proliferative activity assessed by anti-PCNA and Ki67 monoclonal antibodies in canine testicular tumours.

The recent availability of monoclonal antibodies raised against cell cycle nuclear antigens makes possible, by means of immunohistochemical techniques, an easy and quick method of evaluating tumour kinetic activity, in addition to older methods such as measurement of the mitotic index. Some of these antibodies can be used on formalin-fixed paraffin wax-embedded samples, thus allowing the use of archival material. In the present study the proliferative activity of testicular tumours of the dog (seminomas and Sertoli and Leydig cell tumours) was investigated with two monoclonal antibodies to proliferating cell nuclear antigen (PCNA) clone PC10, and Ki67 clone MIB1. The former recognizes a formalin-resistant epitope of PCNA, and MIB1 the same antigen as Ki67 in formalin-fixed, paraffin wax-embedded sections after incubation in a microwave oven. Three parameters of proliferative activity were considered: PCNA and Ki67 indices (percentage of nuclear area positive to PCNA and to Ki67), and mitotic index (number of mitoses per 1000 cells). The PCNA index and Ki67 index revealed a good correlation in linear regression analysis (P < 0.001) as did the mitotic index (P < 0.01). None of the parameters considered revealed a significant difference in proliferative activity of the three types of tumour (P > 0.05-Spearman test), but in both seminomas and Sertoli cell tumours the progression from tubular to diffuse pattern paralleled an increase in growth fraction. It is interesting that some seminomas of the diffuse type, often considered on histological grounds to be the most malignant, showed the highest values of the above-mentioned parameters.

Evaluating mitotic activity in canine and feline solid tumors: standardizing the parameter.

Three different methods for evaluating mitotic activity (mitotic count, mitoses/area, mitotic index) were applied to different types of canine and feline solid tumors to determine the method that is most objective and correlates best with other parameters of cell proliferation. Mitotic activity was evaluated on toluidine blue stained histological sections. Slides stained with histochemical (AgNOR proteins) and immunohistochemical (MIB1, PCNA) markers of cell proliferation were available for each case. Quantitation of mitotic activity and cell proliferation parameters was performed with an image analyzer. Mitotic activity assessment was compared with cell proliferation indices and its ability to discriminate tumors grouped on histologically based criteria including the histological type, malignant or benign characteristics, and grade. A significant correlation by linear regression analysis with other parameters assessing cell proliferation revealed that mitotic index correlated 1000% and mitoses/area and mitotic count correlated 40% of the time. In discriminating the proliferative activity of tumors grouped by histological criteria, mitotic index and mitotic count revealed 1000% concordance with the other parameters of cell proliferation, while mitoses/areas showed 80% concordance.

Cell proliferation and apoptosis in the pathogenesis of oesophagogastric lesions in pigs.

Recent studies have stressed the importance of epithelial hyperproliferation in the pathogenesis of early lesions (parakeratotic hyperkeratosis) of the porcine gastric pars oesophagea (PO). In this study, immunohistochemical staining with Ki67 (clone MIB1) and AgNOR proteins silver staining were used to evaluate, by means of image analysis, cell proliferation in normal and parakeratotic (parakeratotic hyperkeratosis) epithelia of the PO. Apoptotic activity was also assessed with the TUNEL assay and compared with cell proliferative parameters. Early lesions of the PO were characterised by a significant increase in epithelial proliferative activity while there was no difference in the apoptotic activity between normal and parakeratotic epithelia. Our data confirm the hyperproliferative nature of epithelial changes preceding degeneration and erosion/ulcer of the PO and suggest that an underlying feature of gastric ulcers in pigs is an imbalance between cell proliferation and programmed cell death.

Quantification of nucleolar organiser regions in canine perianal gland tumours.

The proliferative activity in 28 canine perianal gland tumours was examined by the quantitative evaluation of the nucleolar organiser regions associated with argyrophil proteins (AgNORs). These regions were stained with a specific silver stain and quantified directly by light microscopy and by computerised image analysis. The relationships between some of the parameters used (the number of AgNORs per nucleus, the area of AgNORs per nucleus and the ratio of the area of AgNORs to the area of the nucleus) were also examined in relation to the histopathological grades of the tumours. All the parameters showed a high correspondence with the tumours' proliferative activity, and the ratio of the area of AgNORs to the area of the nucleus was the most significantly correlated with the tumours' histological patterns.

Hypertrophy of intestinal smooth muscle in cats.

Pathological findings of four cats with severe and diffuse smooth muscle hypertrophy of the small intestine (MHSI) are reported and compared to those of five cats with segmental MHSI secondary to neoplastic obstruction and four controls. Histology demonstrated a constant association between idiopathic MHSI and submucosal fibrosis and chronic lymphoplasmacytic enteritis. Morphometry (gut diameter, thickness and area of muscular layers, number and density of smooth muscle nuclei) and MIB-1-immunolabelling showed that the thickness increase was mostly due to hypertrophy, but hyperplasia was also evident. Microbiology from ileal content samples was performed in two cats with primary MHSI, and Campylobacter spp. were isolated, which were also demonstrated by immunohistochemistry and ultrastructure. The association of chronic enteritis with idiopathic MHSI suggests that factors released in intestinal inflammation may also act as hypertrophy stimuli for smooth muscle cells.