Varying aerobic capacity in relation to breeding stage and reproductive success in giant petrels (Macronectes spp.).

Reproduction, and parental care in particular, are among the most energy-demanding activities within the annual cycle of adult birds. Parents that cannot meet the metabolic demands and other physiological costs of raising offspring may opt to abandon chicks in favour of self-maintenance and future reproduction. Recent work examining reproductive trade-offs in birds revealed an important role of oxygen carrying capacity in mediating variation in parental effort. This study explores the aerobic factors underlying the success or failure of parental care in two closely-related petrel species during their breeding season on Bird Island, South Georgia: northern giant petrels (Macronectes halli) and southern giant petrels (M. giganteus). Failed breeders of both sexes and species had significantly lower hematocrit levels (by 5.48 ± 0.64%) than successful breeders, and reticulocyte counts also tended to be lower in failed males, consistent with the hypothesis that parental care and workload depend on aerobic capacity. We discuss these results in relation to differences in the foraging ecology of both species and sexes.

Unravelling the relative roles of top-down and bottom-up forces driving population change in an oceanic predator.

In the open ocean ecosystem, climate and anthropogenic changes have driven biological change at both ends of the food chain. Understanding how the population dynamics of pelagic predators are simultaneously influenced by nutrient-driven processes acting from the "bottom-up" and predator-driven processes acting from the "top-down" is therefore considered an urgent task. Using a state-space demographic model, we evaluated the population trajectory of an oceanic predator, the Macaroni Penguin (Eudyptes chrysolophus), and numerically assessed the relative importance of bottom-up and top-down drivers acting through different demographic rates. The population trajectory was considerably more sensitive to changes in top-down control of survival compared to bottom-up control of survival or productivity. This study integrates a unique set of demographic and covariate data and highlights the benefits of using a single estimation framework to examine the links between covariates, demographic rates and population dynamics.

Longitudinal bio-logging reveals interplay between extrinsic and intrinsic carry-over effects in a long-lived vertebrate.

Carry-over effects have major implications for individual fitness and population and evolutionary dynamics. The strength of these effects is dependent on an individual's intrinsic performance and the environmental conditions it experiences. However, understanding the relative importance of environmental and intrinsic effects underpinning seasonal interactions has proved extremely challenging, since they covary. A powerful approach is longitudinal measurement of individuals across a range of conditions, whereby each animal is effectively acting as its own control. We related time spent foraging during the nonbreeding period to subsequent breeding performance in European Shags Phalacrocorax aristotelis. By following individuals for up to six years, we could test simultaneously for extrinsic and intrinsic effects using random regression modeling. We detected significant annual and among-individual variation in daily foraging time during the late winter, and clear variation among individuals in the quadratic relationship between foraging time and date. Shorter foraging times were associated with earlier and more successful breeding, driven by differences among years and individuals, with no evidence of individual variation in the slope of these relationships. That both environmental and intrinsic variation shape carry-over effects has important implications for population responses to environmental change.

Fringe boundaries coincide with Notch-dependent patterning centres in mammals and alter Notch-dependent development in Drosophila.

In both vertebrate and invertebrate development, cells are often programmed to adopt fates distinct from their neighbors. Genetic analyses in Drosophila melanogaster have highlighted the importance of cell surface and secreted proteins in these cell fate decisions. Homologues of these proteins have been identified and shown to play similar roles in vertebrate development. Fringe, a novel signalling protein, has been shown to induce wing margin formation in Drosophila. Fringe shares significant sequence homology and predicted secondary structure similarity with bacterial glycosyltransferases. Thus fringe may control wing development by altering glycosylation of cell surface and/or secreted molecules. Recently, two fringe genes were isolated from Xenopus laevis. We report here the cloning and characterization of three murine fringe genes (lunatic fringe, manic fringe and radical fringe). We find in several tissues that fringe expression boundaries coincide with Notch-dependent patterning centres and with Notch-ligand expression boundaries. Ectopic expression of murine manic fringe or radical fringe in Drosophila results in phenotypes that resemble those seen in Notch mutants.

Variation in blood mercury concentrations in brown skuas (Stercorarius antarcticus) is related to trophic ecology but not breeding success or adult body condition.

Mercury is a pervasive environmental contaminant that can negatively impact seabirds. Here, we measure total mercury (THg) concentrations in red blood cells (RBCs) from breeding brown skuas (Stercorarius antarcticus) (n = 49) at Esperanza/Hope Bay, Antarctic Peninsula. The aims of this study were to: (i) analyse RBCs THg concentrations in relation to sex, year and stable isotope values of carbon (δ13C) and nitrogen (δ15N); and (ii) examine correlations between THg, body condition and breeding success. RBC THg concentrations were positively correlated with δ15N, which is a proxy of trophic position, and hence likely reflects the biomagnification process. Levels of Hg contamination differed between our study years, which is likely related to changes in diet and distribution. RBC THg concentrations were not related to body condition or breeding success, suggesting that Hg contamination is currently not a major conservation concern for this population.

A quantitative assay for the progenitors of bone marrow-associated lymphocytes.

A cell transfer assay system was developed to study the precursors of bone marrow-associated (B) lymphocytes in the adult mouse. The rationale of the assay is to inject into irradiated mice a cell suspension depleted of B lymphocytes, to wait a period of time to let precursor cells differentiate to B lymphocytes, then to correlate the number of B cells present in the recipient mice with the number of precursor cells injected. The assay as described was shown to be linear in the range of 10(5)-3 x 10(6) fractionated bone marrow cells. Kinetic studies indicated that precursor cells start producing detectable numbers of B cells within 3 days after transplantation; B cell activity then increases with a doubling time of 24 hr. Physical characterization of that precursor cell has shown that it is lighter and sediments faster than small lymphocytes. Precursor cells were found in bone marrow and spleen but could not be detected in peripheral lymph nodes. Results of physical analysis also indicate that the precursors of B lymphocytes described here may not be pluripotent stem cells for the immune system.

Restriction of specificity in the precursors of bone marrow-associated lymphocytes.

Previous work has shown that the immediate precursor of B lymphocytes (PB cell) has many properties that distinguish it from both B lymphoctes and hemopoietic stem cells. Size, density, tissue distribution, and sensitivity to cytotoxic antisera differ for each type of cell. The work described here was designed to study three aspects of the differentiation of PB cells. First, since PB cells probably have immunoglobulin surface receptors, fluorescein-labeled anti-immunoglobulin antiserum was used in an attempt to investigate directly the physical properties of PB cells. The use of this labeled antiserum revealed a population of cells with properties similar to the PB cells defined by the functional assays. Second, the differentiative potential of PB cells was studied by comparing the size of the total population of PB cells, as determined with fluorescein-labeled anti-immunoglobulin antiserum, to the size of the population of PB cells responding in a functional assay with a specific antigen. The cells responding in the functional assay represent only 0.1% of the total population of PB cells. This observation suggests that PB cells are not pluripotent stem cells of the immune system. Finally, the kinetics of the differentiation of PB cells to B lymphocytes was studied. The differentiation to mature lymphocytes involves at least one intermediate stage in which cells larger than mature B cells are active in a functional assay for B cells. These large B cells are present in irradiated mice soon after transplantation of PB cells, but by 20 days the majority of the B cells are typical small lymphocytes.

Inhibition of the immune response by 7S antibody mechanism and site of action.

A cell culture system was used to investigate the mechanism of action of the feedback inhibition caused by specific 7S antibody. It was found that preincubation of spleen cells with specific 7S antibody led to a marked reduction in the in vitro response of the treated spleen cells to the antigen used to prepare the antibody. The inhibition was not caused by a carry-over of free antibody nor by the release of 7S antibody from the cells. Rather, the preincubation appeared to specifically inactivate one of the cells required for initiation of an in vitro response. Since the suppression could be reversed by addition of untreated cells, it was possible to characterize the properties of the reconstituting cell. This cell is identified as the nonlymphoid accessory cell (A cell) by several criteria. (a) Suppression can be demonstrated only in assay systems requiring functional A cells. (b) The most active sources for reconstitution are also good sources for A cells. (c) The sedimentation velocity of the reconstituting cell is identical with that for A cells. (d) Like A cells, the reconstituting cell is resistant to high doses of ionizing radiation. (e) The reconstituting ability is not affected by anti-theta antibody. Of the three cells required for the initiation of an immune response, A cells, bone marrow-derived cells, and thymus-derived cells, the data are only compatible with the reconstituting cell being an A cell. Additional experiments suggest that the Fc portion of 7S antibody binds to the surface of A cells. Thus, fluorescein isothiocyanate-labeled 7S antibody binds specifically to cells with properties similar to those described above, and F(ab')(2) fragments, lacking Fc portion, are unable to cause immunosuppression when they are preincubated with spleen cells. It is possible that this binding is related to the specific suppression caused by 7S antibody molecules.

Quantitative studies on the precursors of cytotoxic lymphocytes. III. The lineage of memory cells.

In the course of a mixed lymphocyte culture, memory cells are produced which can give rise to a large secondary cytotoxic lymphocyte response on reexposure to the sensitizing alloantigen. We have studied the lineage of these memory cells using a clonal assay for precursors of cytotoxic T lymphocytes (CLP). Our data provide conclusive evidence that individual CLP, upon stimulation with alloantigens, gives rise to clones which contain memory cells of the same specificity as the CLP. Only half of the clones that responded in the primary stimulation could be reactivated upon exposure to the original priming alloantigen.

The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems.

The precise relationship between the stem cells for the lymphoid system and those for the blood-forming system is unclear. While it is generally assumed that the hemopoietic stem cell, the spleen colony-forming unit (CFU-S), is also the stem cell for the lymphoid system, there is little evidence for this hypothesis. To investigate the stem cells in these two systems, we irradiated bone marrow cells to induce unique chromosome aberrations in the stem cell population and injected them at limiting dilution into stem cell-deficient recipients. Several months (between 3 and 11) were allowed for the injected cells to repopulate the hemopoietic system. At that time, the bone marrow, spleen, and thymus were examined for a high frequency of cells having the same unique chromosome aberration. The presence of such markers shows that the marker was induced in a cell with extensive proliferative capacity, i.e., a stem cell. In addition, the splenic lymphocytes were stimulated with phytohemagglutinin (PHA) or lipopolysaccharide (LPS) to search for unique chromosomes in dividing T and B cells, respectively. Finally, bone marrow cells were injected into secondary irradiated recipients to determine if the marker occurred in CFU-S and to determine whether or not the same tissue distributions of marked cells could be propogated by bone marrow cells in a second recipient. After examination of 28 primary recipients, it was possible to identify three unique patterns of stem cell regeneration. In one set of mice, a unique chromosome marker was observed in CFU-S and in PHA- and LPS-stimulated cultures. These mice provide direct evidence for a pluripotent stem cell in bone marrow. In addition, two restricted stem cells were identified by this analysis. In three recipients, abnormal karyotypes were found only in myeloid cells and not in B and T lymphocytes. These mice presumably received a marked stem cell restricted to differentiate only into myeloid progeny. In three other recipients, chromosome aberrations were found only in PHA-stimulated cells; CFU-S and cells from LPS cultures did not have cells with the unique chromosome. This pattern suggests that bone marrow contains cells committed to differentiation only into T lymphocytes. For each of the three types of stem cells, secondary recipients had the same cellular distribution of marked cells as the primary recipients. This observation provides further evidence that unique markers can be induced in both pluripotent and restricted stem cells.

Requirement for non-T cells in the generation of cytotoxic T lymphocytes in vitro. I. Use of nude mice as source of non-T cells.

The ability of small numbers of LN cells to produce cytotoxic lymphocytes on in vitro culture with allogeneic stimulator cells is greatly augmented by the addition of spleen cells from athymic nude mice. The possibility that the synergism is a result of improved culture conditions or a "feeder effect" is excluded. All cytotoxic cells found in these cultures are shown to be T cells and to arise from precursors contained in the LN-cell component. The nude spleen cell component appears to be providing a required non-T cell which has been lost from the LN component through dilution. Synergism between the two components can occur whether they are syngeneic or allogeneic provided that both can recognize the same alloantigens in the stimulator population.

In vivo requirement for a radiation-resistant cells in the immune response to sheep erythrocytes.

Experiments have been done to establish whether the radiation-resistant or A cell has a specific function in the initiation of an immune response in mice to sheep erythrocytes (SRBC). All previous demonstrations using accessory (A) cells have involved in vitro assays and are possibly explainable as tissue culture artifacts. If A cells are essential, it should be possible to demonstrate their requirement in vivo. Therefore we first established such conditions. Two methods were found for creating an A-cell deficiency in vivo: (a) A cells disappear gradually from the spleens of irradiated mice, presumably by migration since A-cell function was shown not to be decreased by irradiation. If 3 days elapse between irradiation and transplantation of mixtures of bone marrow and thymus cells (which provide B and T but few A cells), the usual synergistic response does not occur. Addition of large numbers of freshly irradiated spleen cells to the mixture of bone marrow and thymus completely restores the immune response. (b) Injection of 10(10) horse erythrocytes into mice suppresses A-cell activity in these mice 24 hr later; a much reduced response to SRBC is obtained when they are given at this time. The response can be partially restored if irradiated spleen cells are given with the SRBC. This observation formed the basis for a quantitative in vivo assay for A cells in which the magnitude of restoration by various suspensions of irradiated cells was used to estimate the A-cell activity of that suspension. A quantitative in vitro assay for A cells was also developed. It was essential for this assay that the total cell number, B-cell number, and T-cell number be kept constant and that only the number of A cells be allowed to vary. Only under these conditions was the response a linear function of the number of A cells added. If the in vivo and in vitro assays are detecting the same class of radiation-resistant cells, the physical properties of the cells active in each assay should be identical. Spleen cells were separated on the basis of both density and sedimentation velocity. Fractions from both separation methods were tested for their content of A cells using both the in vivo and in vitro assays. The density and sedimentation profiles of A cells were similar in both assays. The demonstration that a radiation-resistant cell is required in vivo and that this cell has properties identical to the radiation-resistant cell required in vitro indicates that this cell (the A cell) is directly involved in the initiation of an immune response to erythrocyte antigens.

An expanded population of natural killer cells in mice with severe combined immunodeficiency (SCID) lack rearrangement and expression of T cell receptor genes.

Mice with severe combined immunodeficiency syndrome (SCID) exhibit an impairment in both T and B cell maturation, whereas myelopoiesis remains unaffected. We report here that spleens from SCID mice have undergone phenotypic expansion of cells bearing the NK-2 and asialo GM1 markers (70-80%) characteristic of NK cells and this expansion is accompanied by a 3-4-fold enrichment in NK cytolytic activity over their normal C.B-17 littermates. Furthermore, the NK cells from SCID mice do not rearrange or express T cell receptor alpha or beta genes, or a third T cell rearranging gene, gamma. These findings suggest that (a) T cell receptors are not necessary for NK-mediated cytolysis, and (b) either NK cells constitute an entirely distinct lineage or NK cell function is acquired in pre-T cells prior to the expression of T cell receptor genes.

High frequency of normal DJH joints in B cell progenitors in severe combined immunodeficiency mice.

The severe combined immunodeficiency (scid) mouse has a defective V(D)J recombinase activity that results in arrested lymphoid development at the pro-B cell stage in the B lineage. The defect is not absolute and scid mice do attempt gene rearrangement. Indeed, approximately 15% of all scid mice develop detectable levels of oligoclonal serum immunoglobulin and T cell activity. To gain more insight into the scid defect and its effect on V(D)J rearrangement, we analyzed DJH recombination in scid bone marrow. We determined that DJH structures are present in scid bone marrow and occur at a frequency only 10-100 times less than C.B-17+/+. The scid DJH repertoire is limited and resembles fetal liver DJH junctions, with few N insertions and predominant usage of reading frame 1. Moreover, 70% of the DJH structures were potentially productive, indicating that normal V(D)J recombinants should be arising continually.

Speciation and phylogeography of giant petrels Macronectes.

We examine global phylogeography of the two forms of giant petrel Macronectes spp. Although previously considered to be a single taxon, and despite debate over the status of some populations and the existence of minimal genetic data (one mitochondrial cytochrome b sequence per form), the current consensus based on morphology is that there are two species, Northern Giant Petrel M. halli and Southern Giant Petrel M. giganteus. This study examined genetic variation at cytochrome b as well as six microsatellite loci in giant petrels from 22 islands, representing most island groups at which the two species breed. Both markers support separate species status, although sequence divergence in cytochrome b was only 0.42% (corrected). Divergence was estimated to have occurred approximately 0.2mya, but with some colonies apparently separated for longer (up to 0.5 my). Three clades were found within giant petrels, which separated approximately 0.7mya, with the Southern Giant Petrel paraphyletic to a monophyletic Northern Giant Petrel. There was evidence of past fragmentation during the Pleistocene, with subsequent secondary contact within Southern Giant Petrels. The analysis also suggested a period of past population expansion that corresponded roughly to the timing of speciation and the separation of an ancestral giant petrel population from the fulmar Fulmarus clade.

Migration and stopover in a small pelagic seabird, the Manx shearwater Puffinus puffinus: insights from machine learning.

The migratory movements of seabirds (especially smaller species) remain poorly understood, despite their role as harvesters of marine ecosystems on a global scale and their potential as indicators of ocean health. Here we report a successful attempt, using miniature archival light loggers (geolocators), to elucidate the migratory behaviour of the Manx shearwater Puffinus puffinus, a small (400 g) Northern Hemisphere breeding procellariform that undertakes a trans-equatorial, trans-Atlantic migration. We provide details of over-wintering areas, of previously unobserved marine stopover behaviour, and the long-distance movements of females during their pre-laying exodus. Using salt-water immersion data from a subset of loggers, we introduce a method of behaviour classification based on Bayesian machine learning techniques. We used both supervised and unsupervised machine learning to classify each bird's daily activity based on simple properties of the immersion data. We show that robust activity states emerge, characteristic of summer feeding, winter feeding and active migration. These can be used to classify probable behaviour throughout the annual cycle, highlighting the likely functional significance of stopovers as refuelling stages.

Identification of germline and somatic mutations affecting the retinoblastoma gene.

Retinoblastoma (RB) is a malignant tumor of developing retina that arises when abnormalities resulting in loss of function affect both alleles of the gene at the retinoblastoma locus (RB1) on chromosome 13q. The majority of RB tumors do not show gross alterations in a 4.7-kb fragment (4.7R), which is a candidate RB1 gene. To search for more subtle mutations, the ribonuclease protection method was used to analyze 4.7R messenger RNA from RB tumors. Five of 11 RB tumors, which exhibit normal 4.7R DNA and normal-sized RNA transcripts, showed abnormal ribonuclease cleavage patterns. Three of the five mutations affected the same region of the messenger RNA, consistent with an effect on splicing involving an as yet unidentified 5' exon. The high frequency of mutations in 4.7R supports the identification of 4.7R as the RB1 gene. However, the unusual nature of some of the abnormalities of 4.7 R alleles indicates that the accepted sequence of genetic events involved in the genesis of RB may require reevaluation.

A model of human acute lymphoblastic leukemia in immune-deficient SCID mice.

A human acute lymphoblastic leukemia (ALL) cell line that was transplanted into immune-deficient SCID mice proliferated in the hematopoietic tissues, invaded various organs, and led to the death of the mice. The distribution of leukemic cells in SCID mice was similar to the course of the disease in children. A-1 cells marked with a retrovirus vector showed clonal evolution after the transplant. SCID mice that were injected with bone marrow from three patients with non-T ALL had leukemic cells in their bone marrow and spleen. This in vivo model of human leukemia is an approach to understanding leukemic growth and progression and is a novel system for testing new treatment strategies.

Genetic origin of mutations predisposing to retinoblastoma.

Retinoblastoma is one of several human tumors to which predisposition can be inherited. Molecular genetic analysis of several nonheritable cases has led to the hypothesis that this tumor develops after the occurrence of specific mitotic events involving human chromosome 13. These events reveal initial predisposing recessive mutations. Evidence is presented that similar chromosomal events occur in tumors from heritable cases. The chromosome 13 found in the tumors was the one carrying the predisposing germline mutation and not the homolog containing the wild-type allele at the Rb-1 locus. These results suggest a new approach for identifying recessive mutant genes that lead to cancer and a conceptual basis for accurate prenatal predictions of cancer predisposition.

Quantifying individual specialization using tracking data: a case study on two species of albatrosses.

Many predictive models of spatial and temporal distribution (e.g. in response to climate change or species introductions) assume that species have one environmental niche that applies to all individuals. However, there is growing evidence that individuals can have environmental preferences that are narrower than the species niche. Such individual specialization has mainly been studied in terms of dietary niches, but a recent increase in the availability of individual movement data opens the possibility of extending these analyses to specialisation in environmental preferences. Yet, no study to date on individual specialisation has considered the environmental niche in its multidimensionality. Here we propose a new method for quantifying individual specialisation in multiple dimensions simultaneously. We compare the hypervolumes in n-dimensional environmental niche space of each individual against that of the population, testing for significant differences against a null model. The same method can be applied to a 2-dimensional geographic space to test for site fidelity. We applied this method to test for individual environmental specialisation (across three dimensions: sea surface temperature, eddy kinetic energy, depth) and for site fidelity among satellite-tracked black-browed albatrosses (Thalassarche melanophris) and grey-headed albatrosses (Thalassarche chrysostoma), during chick-rearing at South Georgia. We found evidence for site fidelity in both species and of environmental specialisation among individual grey-headed but not black-browed albatrosses. Specialisation can affect the resilience of populations affected by natural and anthropogenic changes in the environment, and hence has implications for population dynamics and conservation.