Isotopic discrimination of stable isotopes of nitrogen (δ15N) and carbon (δ13C) in a host-specific holocephalan tapeworm.

During the past decade, parasites have been considered important components of their ecosystems since they can modify food-web structures and functioning. One constraint to the inclusion of parasites in food-web models is the scarcity of available information on their feeding habits and host-parasite relationships. The stable isotope approach is suggested as a useful methodology to determine the trophic position and feeding habits of parasites. However, the isotopic approach is limited by the lack of information on the isotopic discrimination (ID) values of parasites, which is pivotal to avoiding the biased interpretation of isotopic results. In the present study we aimed to provide the first ID values of δ(15)N and δ(13)C between the gyrocotylidean tapeworm Gyrocotyle urna and its definitive host, the holocephalan Chimaera monstrosa. We also test the effect of host body size (body length and body mass) and sex of the host on the ID values. Finally, we illustrate how the trophic relationships of the fish host C. monstrosa and the tapeworm G. urna could vary relative to ID values. Similar to other studies with parasites, the ID values of the parasite-host system were negative for both isotopic values of N (Δδ(15)N = - 3.33 ± 0.63‰) and C (Δδ(13)C = - 1.32 ± 0.65‰), independent of the sex and size of the host. By comparing the specific ID obtained here with ID from other studies, we illustrate the importance of using specific ID in parasite-host systems to avoid potential errors in the interpretation of the results when surrogate values from similar systems or organisms are used.

Automatic cell counting in vivo in the larval nervous system of Drosophila.

Identification and counting of cells is necessary to test biological hypotheses, for instance of nervous system formation, disease, degeneration, injury and regeneration, but manual counting is time consuming, tedious, and subject to bias. The fruit fly Drosophila is a widely used model organism to analyse gene function, and most research is carried out in the intact animal or in whole organs, rather than in cell culture. Inferences on gene function require that cell counts are known from these sample types. Image processing and pattern recognition techniques are appropriate tools to automate cell counting. However, counting cells in Drosophila is a complex task: variations in immunohistochemical markers and developmental stages result in images of very different properties, rendering it challenging to identify true cells. Here, we present a technique for counting automatically larval glial cells in three dimensions, from confocal microscopy serial optical sections. Local outlier thresholding and domes are combined to find the cells. Shape descriptors extracted from a data set are used to characterize cells and avoid oversegmentation. Morphological operators are employed to divide cells that could otherwise be missed. The method is accurate and very fast, and treats all samples equally and objectively, rendering all data comparable across specimens. Our method is also applicable to identify cells labelled with other nuclear markers and in sections of mouse tissues.

Gliatrophic and gliatropic roles of PVF/PVR signaling during axon guidance.

Evidence of molecular and functional homology between vertebrate and Drosophila glia is limited, restricting the power of Drosophila as a model system to unravel the molecular basis of glial function. Like in vertebrates, in the Drosophila central nervous system glial cells are produced in excess and surplus glia are eliminated by apoptosis adjusting final glial number to axons. The underlying molecular mechanisms are largely unknown, as the only gliatrophic pathway known to date in flies is the EGFR and its ligands. The PDGFR signaling pathway plays a major role in regulating oligodendrocyte migration and number in vertebrates. Here, we show that the Drosophila PDGFR/VEGFR homologue PVR is required in midline glia during axon guidance for glial survival and migration, ultimately enabling axonal enwrapment. The midline glia migrate aided by the VUM and the MP1 midline neurons--sources of PVF ligands--and concomitantly interactions with neurons maintain midline glia survival. Upon loss of function for PVF/PVR signaling midline glia apoptosis increases, and gain of function induces supernumerary midline glia. Midline glial cells are displaced towards ectopic sources of PVF ligands. PVR signaling promotes midline glia survival through AKT and ERK pathways. This work shows that the PVR/PDGFR pathway plays conserved gliatrophic and gliatropic roles in subsets of glial cells in flies and vertebrates.

Automatic identification of Mycobacterium tuberculosis by Gaussian mixture models.

Tuberculosis and other kinds of mycobacteriosis are serious illnesses for which early diagnosis is critical for disease control. Sputum sample analysis is a common manual technique employed for bacillus detection but current sample-analysis techniques are time-consuming, very tedious, subject to poor specificity and require highly trained personnel. Image-processing and pattern-recognition techniques are appropriate tools for improving the manual screening of samples. Here we present a new technique for sputum image analysis that combines invariant shape features and chromatic channel thresholding. Some feature descriptors were extracted from an edited bacillus data set to characterize their shape. They were statistically represented by using a Gaussian mixture model representation and a minimal error Bayesian classification procedure was employed for the last identification stage. This technique constitutes a step towards automating the process and providing a high specificity.

Ecological, morphological and phylogenetic correlates of interspecific variation in plasma carotenoid concentration in birds.

Carotenoids are important as pigments for bright coloration of animals, and as physiologically active compounds with a wide array of health-related benefits. However, the causes of variation in carotenoid acquisition and physiology among species are poorly known. We measured the concentration of carotenoids in the blood of 80 wild bird species differing in diet, body size and the extent of carotenoid-based traits. Preliminary analyses showed that diet significantly explains interspecific variability in plasma carotenoids. However, dietary influences were apparently overridden by phylogenetic relationships among species, which explained most (65%) of this variability. This phylogenetic effect could be due partly to its covariation with diet, but may also be caused by interspecific differences in carotenoid absorption from food to the blood stream, mediated, for example by endothelial carriers or gut parasites. Carotenoid concentrations also decreased with body size (which may be explained by the allometric relationship between ingestion rate and body mass), and correlated positively with the extent of carotenoid-dependent coloration of plumage and bare parts. Therefore, the acquisition of carotenoids from the diet and their use for both health and display functions seem to be constrained by ecological and physiological aspects linked to the phylogeny and size of the species.

Offspring body condition and immunocompetence are negatively affected by high breeding densities in a colonial seabird: a multiscale approach.

Why avian colonies vary in size and how food competition among nearby colonies affects offspring quality are still not completely understood. We simultaneously examined the effects of four scales of breeding density on two measures of offspring viability (body condition and T-cell-mediated immunity) in the colonial Magellanic penguin. Body condition of fledglings was inversely correlated with breeding density within 100 m(2) of nests, and decreased with increasing numbers of breeding pairs competing within the parental foraging ranges (100 km), probably as a result of density-dependent food depletion. The T-cell-mediated immune response was positively correlated with body condition, reflecting, to some extent, the previous breeding-density effects, and was negatively correlated with colony size, which may be related to social stress. However, given the effect of protein intake on cell immunity, this result could also indicate a thus far neglected cost of coloniality, namely the consumption of low-protein food to compensate for the depletion of optimal prey. These results were not influenced by other traits, nor by the current exposure of birds to parasites and diseases, as measured by serological variables. Since body condition and the T-cell-mediated immune response of fledgling birds are indicators of their survival and recruitment prospects, the costs we have identified can explain variability in colony size in relation to food competition with surrounding colonies, as well as the skewed distribution toward small colonies in this species.

Genetics, local environment and health as factors influencing plasma carotenoids in wild American kestrels (Falco sparverius).

Carotenoids are important as pigments for bright coloration of animals, and as physiologically active compounds with a wide array of health-related functions. Carotenoid-dependent coloration may have evolved as a signal to conspecifics; however, factors that may limit availability of carotenoids are poorly known. We investigated how the acquisition of carotenoids may be constrained by availability in the environment, diet, genetic make-up and health status of wild American kestrels (Falco sparverius). Plasma concentrations of siblings at the time of fledging showed a high degree of resemblance; however, a cross-fostering experiment revealed that variance was largely explained by nest of rearing, rather than nest of origin, thus indicating a low genetic component. A multivariate analysis of attributes of nestlings (sex, size, plasma proteins, immune function), parental reproduction (laying date, clutch size) and rearing conditions (brood size, size hierarchy, nestling mortality) showed only a small significant effect of leucocyte differentials on carotenoid concentrations of nestlings. A strong environmental effect on plasma carotenoids was demonstrated by levels of adult kestrels being correlated within mated pairs, and having a significant association with the abundance of voles, the primary prey species, per territory.

The T-cell-mediated immune response and return rate of fledgling American kestrels are positively correlated with parental clutch size.

Life-history theory predicts that parents face a trade-off between the number and viability of the progeny they produce. We found evidence for an apparent trade-off in a free-living population of American kestrels (Falco sparverius), as larger clutches produced more but lighter fledglings. However, while the body mass of fledglings has traditionally been used as a measure of survival prospect, offspring immunocompetence should also play an important role. We thus measured the T-cell-mediated immune response of fledgling kestrels in relation to brood traits and nest-rearing conditions through a cross-fostering experiment. The immune response was positively correlated with the body condition of fledglings, but was also higher in those hatched from five-egg than four-egg clutches. These results were not influenced by other brood traits, nor by current exposure to stressors and infectious agents, as measured by serological variables. Such ability to resist pathogens may account for why the probability of offspring returning to the study area in subsequent years, when controlling for brood size, was higher for five-egg than four-egg clutches. These results suggest an optimal clutch size through maternal effects on offspring immunocompetence rather than a trade-off between the number and quality of the offspring.

Diet explains interpopulation variation of plasma carotenoids and skin pigmentation in nestling white storks.

Carotenoids have a dietary origin in birds, but mechanisms by which they are absorbed in the gut, transported in the blood, metabolized at various sites, and deposited in the integument remain poorly understood. Variation in both plasma carotenoid levels and external color may reflect different access to dietary carotenoids or individual physiological differences in the uptake and deposition of carotenoids. We compared total plasma carotenoid concentration in nestling white storks (Ciconia ciconia) from 11 Spanish colonies in two consecutive years. The main food item in one of the colonies was the red swamp crayfish (Procambarus clarkii), a recently introduced species. Storks in the remaining colonies ate a variety of foods but no crayfish. Total plasma carotenoid levels in the colony where crayfish were consumed were about five times higher than in any other colony. These differences were maintained after controlling for the significant interyear variability, as well as for sex, age, and body mass of birds. Skin pigmentation also differed, being intensely orange in storks that consumed crayfish but white (unpigmented) in the remaining individuals. With thin-layer chromatography (TLC) and electronic absorption spectroscopy, astaxanthin was confirmed as the major carotenoid in crayfish as well as in the plasma, skin, and body fat of crayfish-eating storks, whereas lutein was the main carotenoid in plasma samples from the other colonies. These results indicate that a newly available carotenoid in the environment, astaxanthin, can be absorbed in large quantities from the gut and be transported in the blood before deposition in different tissues.

Environmental and genetic variation in T-cell-mediated immune response of fledgling American kestrels.

We investigated genetic and environmental components of variance in avian T-cell-mediated immune response (CMI) through a cross-fostering experiment conducted on wild American kestrels (Falco sparverius). CMI was evaluated in vivo by an experimental challenge with phytohaemagglutinin, a T-cell mitogen, injected intradermally in fledglings. Additionally, we assessed two measures of nutritional condition (body mass and circulating plasma proteins) which could influence the variance components of CMI. A two-way nested ANOVA indicated that CMI of fledgling kestrels was explained more by the nest where the bird was reared (33% of the explained variance) than by the nest of origin (12%). Body mass was explained equally by familial and environmental components, while plasma proteins were only related to the rearing environment. CMI of fledglings was not related to their circulating plasma proteins, but was positively correlated with their body mass. Fledgling body mass seemed to be influenced by pre-hatching or post-hatching maternal effects prior to manipulation since resemblance in body mass of sibships at the age of manipulation was high (h 2≤0.58), and body mass at this age predicted body mass at fledging. Therefore, pre-manipulation parental effects on body mass, such as investment in egg size, could have inflated the familial effects on body mass of fledglings and then on its correlated CMI. When controlling for body mass, most of the variation in CMI of fledglings was explained by the nest where the bird was reared (36.6%), while the variance explained by the nest of origin (4%) was not significant. This means that environmental influences are major determinants of offspring CMI. The low proportion of variance explained by the familial component may have been due to the high correlation of CMI to fitness.

Habitat, world geographic range, and embryonic development of hosts explain the prevalence of avian hematozoa at small spatial and phylogenetic scales.

The factors explaining interspecific differences in prevalences of blood parasites in birds are poorly known. We simultaneously assessed 20 social, ecological, life history, and sampling-related variables that could influence hemoparasite prevalences among diurnal birds of prey in Spain. Our results show that multiple factors are responsible for the studied host-parasite association. We confirmed for the first time that prevalence is inversely correlated to the embryonic development period, and thus probably to immune performance, even among closely related birds. Macrohabitat features related to vector availability are also important, prevalences being higher in species breeding in forested habitats. Finally, prevalence is positively correlated with the host's world geographic range. We hypothesize that larger geographic ranges offered more opportunities for host-vector-hemoparasite associations to become established. The results from our multivariate analyses differ from those obtained through univariate ones, showing that all potential factors should be assessed jointly when testing any ecological or evolutionary hypothesis dealing with parasites.

A comparison of spectrophotometry and color charts for evaluating total plasma carotenoids in wild birds.

The study of the role of carotenoids on the physiology and evolutionary ecology of birds demands methods for their quantification in the bloodstream. We compared color-chart scores of plasma hue with the actual concentration of plasma carotenoids obtained by spectrophotometry in 356 wild birds from 26 species. Repeatability of chart scores between three independent observers was high. However, color scores did not correlate with the spectrophotometric results in interspecific analyses. Within species (n = 3), one showed no relationship and two showed weak but significant positive correlations. Hemoglobin, and probably other substances, may mask the color of carotenoids, making the accurate use of color charts difficult. Spectrophotometry should be the method of choice as it permits precise quantifications of total plasma carotenoids and objective comparisons among studies.