Reproductive performance of Peregrine falcons relative to the use of organochlorine pesticides, 1946-2021.

Populations of some fish- and meat-eating birds suffered dramatic declines globally following the introduction of organochlorine pesticides during the late 1940s and 1950s. It has been hypothesised that these population declines during the 1950s-1970s were largely driven by a combination of reproductive failure due to eggshell-thinning, egg breakage and embryonic death attributable to DDT and its metabolites, and to enhanced mortality attributable to the more toxic cyclodiene compounds such as aldrin and dieldrin. Using 75 years (1946-2021) of Peregrine falcon (Falco peregrinus) monitoring data (315 unique nest-sites monitored for 6110 nest-years), we studied the breeding performance of a resident Peregrine population in southern Scotland relative to the spatiotemporal pattern of organochlorine pesticide use. We show that (i) Peregrine breeding success and measures of breeding performance increased substantially following the reduction in, and subsequently a complete ban on, the use of organochlorine pesticides; (ii) improvements in Peregrine breeding performance were more dramatic in southeastern Scotland where agriculture was the predominant land use than in southwestern Scotland where there was less arable and more forested land; (iii) Peregrines nesting closer to the coast generally had higher fledging success (that is, a higher proportion of clutches that produced at least one fledgeling) than those nesting inland farther away from the coast; (iv) low temperatures and excessive rain in May negatively affected Peregrine fledging success; and (v) Peregrine abundance increased in parallel with improvements in reproductive performance following the reduction and then complete ban on the use of organochlorine pesticides in the UK. However, recovery was gradual and occurred over four decades, and rate of recovery varied among measures of reproductive performance (egg, nestling and fledgeling production). Our results suggest that the temporal pattern of organochlorine pesticide use strongly influenced Peregrine reproductive parameters but that the pattern of influence differed regionally. Overall results are consistent with the hypothesis that reproductive failure caused by organochlorine pesticides was an important driver of the decline in the south Scottish Peregrine population, and that improvements in all measures of breeding performance following a reduction and eventual ban on organochlorine use facilitated the observed increase in this population.

Loss of adenomatous polyposis coli function renders intestinal epithelial cells resistant to the cytokine IL-22.

Interleukin-22 (IL-22) is a critical immune defence cytokine that maintains intestinal homeostasis and promotes wound healing and tissue regeneration, which can support the growth of colorectal tumours. Mutations in the adenomatous polyposis coli gene (Apc) are a major driver of familial colorectal cancers (CRCs). How IL-22 contributes to APC-mediated tumorigenesis is poorly understood. To investigate IL-22 signalling in wild-type (WT) and APC-mutant cells, we performed RNA sequencing (RNAseq) of IL-22-treated murine small intestinal epithelial organoids. In WT epithelia, antimicrobial defence and cellular stress response pathways were most strongly induced by IL-22. Surprisingly, although IL-22 activates signal transducer and activator of transcription 3 (STAT3) in APC-mutant cells, STAT3 target genes were not induced. Our analyses revealed that ApcMin/Min cells are resistant to IL-22 due to reduced expression of the IL-22 receptor, and increased expression of inhibitors of STAT3, particularly histone deacetylases (HDACs). We further show that IL-22 increases DNA damage and genomic instability, which can accelerate cellular transition from heterozygosity (ApcMin/+) to homozygosity (ApcMin/Min) to drive tumour formation. Our data reveal an unexpected role for IL-22 in promoting early tumorigenesis while excluding a function for IL-22 in transformed epithelial cells.

Interkinetic nuclear migration and basal tethering facilitates post-mitotic daughter separation in intestinal organoids.

Homeostasis of renewing tissues requires balanced proliferation, differentiation and movement. This is particularly important in the intestinal epithelium where lineage tracing suggests that stochastic differentiation choices are intricately coupled to the position of a cell relative to a niche. To determine how position is achieved, we followed proliferating cells in intestinal organoids and discovered that the behaviour of mitotic sisters predicted long-term positioning. We found that, normally, 70% of sisters remain neighbours, while 30% lose contact and separate after cytokinesis. These post-mitotic placements predict longer term differences in positions assumed by sisters: adjacent sisters reach similar positions over time; in a pair of separating sisters, one remains close to its birthplace while the other is displaced upward. Computationally modelling crypt dynamics confirmed that post-mitotic separation leads to sisters reaching different compartments. We show that interkinetic nuclear migration, cell size and asymmetric tethering by a process extending from the basal side of cells contribute to separations. These processes are altered in adenomatous polyposis coli (Apc) mutant epithelia where separation is lost. We conclude that post-mitotic placement contributes to stochastic niche exit and, when defective, supports the clonal expansion of Apc mutant cells.

Paneth Cell-Rich Regions Separated by a Cluster of Lgr5+ Cells Initiate Crypt Fission in the Intestinal Stem Cell Niche.

The crypts of the intestinal epithelium house the stem cells that ensure the continual renewal of the epithelial cells that line the intestinal tract. Crypt number increases by a process called crypt fission, the division of a single crypt into two daughter crypts. Fission drives normal tissue growth and maintenance. Correspondingly, it becomes less frequent in adulthood. Importantly, fission is reactivated to drive adenoma growth. The mechanisms governing fission are poorly understood. However, only by knowing how normal fission operates can cancer-associated changes be elucidated. We studied normal fission in tissue in three dimensions using high-resolution imaging and used intestinal organoids to identify underlying mechanisms. We discovered that both the number and relative position of Paneth cells and Lgr5+ cells are important for fission. Furthermore, the higher stiffness and increased adhesion of Paneth cells are involved in determining the site of fission. Formation of a cluster of Lgr5+ cells between at least two Paneth-cell-rich domains establishes the site for the upward invagination that initiates fission.

Dispersal: a matter of scale.

Population density around the natal site is often invoked as an explanation for variation in dispersal distance, with the expectation that competition for limiting resources, coupled with increased intra-specific aggression at high densities, should drive changes in dispersal distances. However, tests of the density-dependent dispersal hypothesis in long-lived vertebrates have yielded mixed results. Furthermore, conclusions from dispersal studies may depend on the spatial and temporal scales at which density and dispersal patterns are examined, yet multi-scale studies of dispersal are rare. Here, we present the findings of a long-term study examining factors influencing natal dispersal distances for the non-migratory population of Peregrine Falcons (Falco peregrinus) in the British Isles across distinct spatial and temporal scales. Our smallest scale study included Peregrines ringed as nestlings and subsequently recaptured alive in south Scotland-north England, an area that was intensively studied during the time periods 1974-1982 and 2002-2016. Second, we examined dispersal patterns of birds ringed as nestlings in south Scotland-north England, but subsequently recaptured alive or recovered dead anywhere in the British Isles. Finally, we examined the natal dispersal patterns for Peregrines ringed and recaptured or recovered anywhere in the British Isles from 1964 to 2016. Consistent with prior findings, females dispersed farther than males across all scales. However, the patterns of dispersal were strongly scale dependent. Specifically, we found a lack of a discernible relationship between index of density and dispersal distance in the limited study area, but when region-wide recaptures and recoveries were included in the analyses, a negative relationship was revealed. Our results suggest that conclusions of dispersal studies may be scale dependent, highlighting the importance of spatial and temporal scales in examining and interpreting the relationship between population density and dispersal patterns.

Acoustic Sensing and Ultrasonic Drug Delivery in Multimodal Theranostic Capsule Endoscopy.

Video capsule endoscopy (VCE) is now a clinically accepted diagnostic modality in which miniaturized technology, an on-board power supply and wireless telemetry stand as technological foundations for other capsule endoscopy (CE) devices. However, VCE does not provide therapeutic functionality, and research towards therapeutic CE (TCE) has been limited. In this paper, a route towards viable TCE is proposed, based on multiple CE devices including important acoustic sensing and drug delivery components. In this approach, an initial multimodal diagnostic device with high-frequency quantitative microultrasound that complements video imaging allows surface and subsurface visualization and computer-assisted diagnosis. Using focused ultrasound (US) to mark sites of pathology with exogenous fluorescent agents permits follow-up with another device to provide therapy. This is based on an US-mediated targeted drug delivery system with fluorescence imaging guidance. An additional device may then be utilized for treatment verification and monitoring, exploiting the minimally invasive nature of CE. While such a theranostic patient pathway for gastrointestinal treatment is presently incomplete, the description in this paper of previous research and work under way to realize further components for the proposed pathway suggests it is feasible and provides a framework around which to structure further work.

Dichlorodiphenyldichloroethylene exposure reduces r-GCS via suppressed Nrf2 in HepG2 cells.

p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE), the major isomer of persistent 1,1-Bis(4-chlorophenyl)-2,2,2-trichloroethane metabolite, is highly associated with the risk of liver cancer. γ-glutamyl-cysteine synthetase (γ-GCS), which is the rate-limiting enzyme of glutathione (GSH) biosynthesis and an important scavenger of reactive oxygen species (ROS), is considered as a potential therapeutic target for many cancers. However, the association between the body burden of p,p'-DDE and γ-GCS has not been fully established. Here, we indicated that low doses of p,p'-DDE exposure promoted the proliferation and decreased γ-GCS activity of HepG2 cells in a dose- and time-dependent manner. In addition, p,p'-DDE elevated ROS content and attenuated glutathione peroxidase activity. This was accompanied with inhibitions of NF-E2-related factor 2 (Nrf2) at the mRNA and protein levels. ROS inhibitor supplement could significantly reverse these effects. Moreover, the addition of the proteasome inhibitor, MG132, strongly reversed the p,p'-DDE-reduced Nrf2 expression and γ-GCS activity. Consistently, GSH content was in line with the alteration of γ-GCS. Collectively, the results indicate that p,p'-DDE treatment downregulates γ-GCS activity in HepG2 cells by inducing ROS-mediated Nrf2 loss.

Expression, purification and renaturation of truncated human integrin ß1 from inclusion bodies of Escherichia coli.

Integrins are a family of transmembrane receptors and among their members, integrin ß1 is one of the best known. It plays a very important role in cell adhesion/migration and in cancer metastasis. Preparation of integrin ß1 has a great potential value especially in studies focused on its function. To this end, recombinant plasmids were constructed containing DNA segments representing 454 amino acids of the N-terminal of integrin ß1. The recombinant plasmid was transformed into Escherichiacoli BL21 (DE3) cells and after induction by isopropyl-ß-D-thiogalactopyranoside (IPTG), the recombinant protein (molecular weight: 53 kD) was expressed, mainly in the form of inclusion bodies. The inclusion bodies were solubilized by 8M urea solution then purified by nickel affinity chromatography. The recombinant protein was renatured by a stepwise dialysis and finally dissolved in phosphate buffered saline. The final yield was approximately 5.4 mg/L of culture and the purity of the renatured recombinant protein was greater than 98% as assessed by SDS-PAGE. The integrity of the protein was shown by Western blot using monoclonal antibodies against his-tag and integrin ß1. Its secondary structure was verified as native by circular dichroism spectra and the bioactivity of the recombinant protein was displayed through the conformation switch under Mn(2+) stimulation.

Demography of population recovery: survival and fidelity of peregrine falcons at various stages of population recovery.

Factors influencing vital demographic rates and population dynamics can vary across phases of population growth. We studied factors influencing survival and fidelity of peregrine falcons in south Scotland-north England at two stages of population growth: when the population was recovering from pesticide-related declines and density was low, and when it had largely recovered from pesticide effects and density was high. Fidelity was higher for: adults and subadults than for juveniles, females than for males, and juveniles and adults during the low-density than during the high-density study period. Survival was age specific, with lower survival for juveniles than for older birds (juveniles, 0.600 ± SE 0.063; subadults, 0.811 ± 0.058; adults, 0.810 ± 0.034). Furthermore, there was some evidence that survival was generally lower for all age classes during the low-density period than during the high-density period, possibly due to a chronic, persistent effect of organochlorine pesticides as the population recovered. Evidence for a density-dependent effect on survival was weak, but a negative effect of density on fidelity of juveniles (dispersing age class) during the recovery phase suggests density-dependent dispersal when the population was increasing. Our results show how population density can influence demographic parameters differently and how such influences can vary across phases of population growth.

The microtubule poison vinorelbine kills cells independently of mitotic arrest and targets cells lacking the APC tumour suppressor more effectively.

Colorectal cancers commonly carry truncation mutations in the adenomatous polyposis coli (APC) gene. The APC protein contributes to the stabilization of microtubules. Consistently, microtubules in cells lacking APC depolymerize more readily in response to microtubule-destabilizing drugs. This raises the possibility that such agents are suitable for treatment of APC-deficient cancers. However, APC-deficient cells have a compromised spindle assembly checkpoint, which renders them less sensitive to killing by microtubule poisons whose toxicity relies on the induction of prolonged mitotic arrest. Here, we describe the novel discovery that the clinically used microtubule-depolymerizing drug vinorelbine (Navelbine) kills APC-deficient cells in culture and in intestinal tissue more effectively than it kills wild-type cells. This is due to the ability of vinorelbine to kill cells in interphase independently of mitotic arrest. Consistent with a role for p53 in cell death in interphase, depletion of p53 renders cells less sensitive to vinorelbine, but only in the presence of wild-type APC. The pro-apoptotic protein BIM (also known as BCL2L11) is recruited to mitochondria in response to vinorelbine, where it can inhibit the anti-apoptotic protein BCL2, suggesting that BIM mediates vinorelbine-induced cell death. This recruitment of BIM is enhanced in cells lacking APC. Consistently, BIM depletion dampens the selective effect of vinorelbine on these cells. Our findings reveal that vinorelbine is a potential therapeutic agent for colorectal cancer, but they also illustrate the importance of the APC tumour suppressor status when predicting therapeutic efficacy.

Direct contacts with colon cancer cells regulate the differentiation of bone marrow mesenchymal stem cells into tumor associated fibroblasts.

Tumor-stroma interactions are referred to as essential events in tumor progression. There has been growing attention that bone marrow-derived mesenchymal stem cells (BMSCs) can travel to tumor stroma, where they differentiate into tumor-associated fibroblast (TAF)-like cells, a predominant tumor-promoting stromal cell. However, little is definitively known about the contributors for this transition. Here, using an in vitro direct co-culture model of colon cancer cells and BMSCs, we identify that colon cancer cells can induce adjoining BMSCs to exhibit the typical characteristic of TAFs, with increased expression of α-smooth muscle actin (α-SMA). Importantly, the present data also reveals that activated Notch signaling mediates transformation of BMSCs to TAFs through the downstream TGF-ß/Smad signaling pathway.

Genetic dissection of differential signaling threshold requirements for the Wnt/beta-catenin pathway in vivo.

Contributions of null and hypomorphic alleles of Apc in mice produce both developmental and pathophysiological phenotypes. To ascribe the resulting genotype-to-phenotype relationship unambiguously to the Wnt/beta-catenin pathway, we challenged the allele combinations by genetically restricting intracellular beta-catenin expression in the corresponding compound mutant mice. Subsequent evaluation of the extent of resulting Tcf4-reporter activity in mouse embryo fibroblasts enabled genetic measurement of Wnt/beta-catenin signaling in the form of an allelic series of mouse mutants. Different permissive Wnt signaling thresholds appear to be required for the embryonic development of head structures, adult intestinal polyposis, hepatocellular carcinomas, liver zonation, and the development of natural killer cells. Furthermore, we identify a homozygous Apc allele combination with Wnt/beta-catenin signaling capacity similar to that in the germline of the Apc(min) mice, where somatic Apc loss-of-heterozygosity triggers intestinal polyposis, to distinguish whether co-morbidities in Apc(min) mice arise independently of intestinal tumorigenesis. Together, the present genotype-phenotype analysis suggests tissue-specific response levels for the Wnt/beta-catenin pathway that regulate both physiological and pathophysiological conditions.

Microtubule assembly by the Apc protein is regulated by importin-beta--RanGTP.

Mutations in the tumour suppressor Adenomatous polyposis coli (Apc) initiate most sporadic colorectal cancers. Apc is implicated in regulating microtubule (MT) dynamics in interphase and mitosis. However, little is known about the underlying mechanism or regulation of this Apc function. We identified importin-beta as a binding partner of Apc that regulates its effect on MTs. Apc binds importin-beta in vitro and in Xenopus egg extracts, and RanGTP inhibits this interaction. The armadillo-like repeat domain of importin-beta binds to the middle of Apc, where it can compete with beta-catenin. In addition, two independent sites in the C terminus of Apc bind the N-terminal region of importin-beta. Binding to importin-beta reduces the ability of Apc to assemble and bundle MTs in vitro and to promote assembly of microtubule asters in Xenopus egg extracts, but does not affect the binding of Apc to MTs or to EB1. Depletion of Apc decreases the formation of cold-stable spindles in Xenopus egg extracts. Importantly, the ability of purified Apc to rescue this phenotype was reduced when it was constitutively bound to importin-beta. Thus, importin-beta binds to Apc and negatively regulates the MT-assembly and spindle-promoting activity of Apc in a Ran-regulatable manner.

Loss of APC induces polyploidy as a result of a combination of defects in mitosis and apoptosis.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene initiate a majority of colorectal cancers. Acquisition of chromosomal instability is an early event in these tumors. We provide evidence that the loss of APC leads to a partial loss of interkinetochore tension at metaphase and alters mitotic progression. Furthermore, we show that inhibition of APC in U2OS cells compromises the mitotic spindle checkpoint. This is accompanied by a decrease in the association of the checkpoint proteins Bub1 and BubR1 with kinetochores. Additionally, APC depletion reduced apoptosis. As expected from this combination of defects, tetraploidy and polyploidy are consequences of APC inhibition in vitro and in vivo. The removal of APC produced the same defects in HCT116 cells that have constitutively active beta-catenin. These data show that the loss of APC immediately induces chromosomal instability as a result of a combination of mitotic and apoptotic defects. We suggest that these defects amplify each other to increase the incidence of tetra- and polyploidy in early stages of tumorigenesis.

The confounding effects of source isotopic heterogeneity on consumer-diet and tissue-tissue stable isotope relationships.

Stable isotope analysis of consumer tissues document patterns of resource use because data are linearly related to isotope compositions of their source(s) (i.e., food, water, etc.). Deviations in parameters estimated for these relationships can arise from variations in consumer tissue-diet spacing (Δ(TS)) and the level of isotopic heterogeneity in the source(s). We present a set of simple hypotheses that distinguish between the effects of Δ(TS) and source isotope heterogeneity. The latter may arise via mixed diets, during tissue turnover, or by isotopic routing of dietary components. We apply these concepts to stable carbon and nitrogen isotope relationships between gut contents and body tissues of large mammal herbivores from mixed C(3)/C(4) South African savannas and test predictions based on the compound- and/or time-specific data archived within each material. Predicted effects of source isotope heterogeneity are readily detected in carbon isotope relationships between materials representing different time periods or comprising bulk versus protein-only diet components. Differences in Δ(TS) of carbon isotopes across mammal herbivore species with very different feeding niches (and diet isotope compositions) are likely to be small or non-existent in these habitats. Variations in Δ(TS) estimated for nitrogen isotopes are much greater, leading to inconsistencies that cannot be explained by diet or trophic level effects alone. The effects of source heterogeneity on isotopic relationships generate numerical artefacts that have been misinterpreted as variations in Δ(TS). We caution against generalized application of hypotheses based on assumptions of source isotopic homogeneity, even for single diets commonly used in laboratory studies. More careful consideration of how heterogeneity affects consumer-diet relationships is needed for many field and laboratory systems.

A Learning-Based Microultrasound System for the Detection of Inflammation of the Gastrointestinal Tract.

Inflammation of the gastrointestinal (GI) tract accompanies several diseases, including Crohn's disease. Currently, video capsule endoscopy and deep bowel enteroscopy are the main means for direct visualisation of the bowel surface. However, the use of optical imaging limits visualisation to the luminal surface only, which makes early-stage diagnosis difficult. In this study, we propose a learning enabled microultrasound ( µ US) system that aims to classify inflamed and non-inflamed bowel tissues. µ US images of the caecum, small bowel and colon were obtained from mice treated with agents to induce inflammation. Those images were then used to train three deep learning networks and to provide a ground truth of inflammation status. The classification accuracy was evaluated using 10-fold evaluation and additional B-scan images. Our deep learning approach allowed robust differentiation between healthy tissue and tissue with early signs of inflammation that is not detectable by current endoscopic methods or by human inspection of the µ US images. The methods may be a foundation for future early GI disease diagnosis and enhanced management with computer-aided imaging.

Ultrasound mediated delivery of quantum dots from a proof of concept capsule endoscope to the gastrointestinal wall.

Biologic drugs, defined as therapeutic agents produced from or containing components of a living organism, are of growing importance to the pharmaceutical industry. Though oral delivery of medicine is convenient, biologics require invasive injections because of their poor bioavailability via oral routes. Delivery of biologics to the small intestine using electronic delivery with devices that are similar to capsule endoscopes is a promising means of overcoming this limitation and does not require reformulation of the therapeutic agent. The efficacy of such capsule devices for drug delivery could be further improved by increasing the permeability of the intestinal tract lining with an integrated ultrasound transducer to increase uptake. This paper describes a novel proof of concept capsule device capable of electronic application of focused ultrasound and delivery of therapeutic agents. Fluorescent markers, which were chosen as a model drug, were used to demonstrate in vivo delivery in the porcine small intestine with this capsule. We show that the fluorescent markers can penetrate the mucus layer of the small intestine at low acoustic powers when combining microbubbles with focused ultrasound during in vivo experiments using porcine models. This study illustrates how such a device could be potentially used for gastrointestinal drug delivery and the challenges to be overcome before focused ultrasound and microbubbles could be used with this device for the oral delivery of biologic therapeutics.

Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability.

Most sporadic colorectal tumors carry truncation mutations in the adenomatous polyposis coli (APC) gene. The APC protein is involved in many processes that govern gut tissue. In addition to its involvement in the regulation of beta-catenin, APC is a cytoskeletal regulator with direct and indirect effects on microtubules. Cancer-related truncation mutations lack direct and indirect binding sites for microtubules in APC, suggesting that loss of this function contributes to defects in APC-mutant cells. In this study, we show that loss of APC results in disappearance of cellular protrusions and decreased cell migration. These changes are accompanied by a decrease in overall microtubule stability and also by a decrease in posttranslationally modified microtubules in the cell periphery particularly the migrating edge. Consistent with the ability of APC to affect cell shape, the overexpression of APC in cells can induce cellular protrusions. These data demonstrate that cell migration and microtubule stability are linked to APC status, thereby revealing a weakness in APC-deficient cells with potential therapeutic implications.

Small populations and offspring sex-ratio deviations in eagles.

Stochastic variation of sex ratio has long been appreciated as a potential factor driving small populations to extinction, but it is not the only source of sex-ratio bias in small populations. We examined whether some consequences of sex allocation could affect extinction risk in small populations of size-dimorphic birds such as eagles. We report variations in sex ratio at fledging from a long-term study of a declining population of Spanish Imperial Eagles (Aquila adalberti). Nestling sex-ratio deviation apparently was mediated by age of breeders, whereas territory quality had no obvious effect. Adult-adult pairs produced the same proportion of both sexes in high- or low-density situations, but pairs with at least one member in nonadult plumage class produced more males. As the population declined over a period of years, the proportion of breeders with immature plumage increased; consequently, the proportion of fledgling males increased. However, when population density was high, the proportion of breeders with immature plumage decreased and more female offspring were produced. This relationship between population density, composition of breeder age, and fledgling sex ratios allowed us to make predictions of extinction risk due to nonstochastic deviations of sex ratio in small, declining populations. In the study population, on the basis of the Vortex simulation results, an estimated reduction of 42.5% in predicted mean time to extinction was attributed solely to biased sex ratio.

The role of age of first breeding in modeling raptor reintroductions.

The present biodiversity crisis has led to an increasing number of reintroduction programs, and this conservation method is likely to be increasingly used in the future, especially in the face of climate change. Many fundamental questions in population ecology are focused on the mechanisms through which populations escape extinction.Population viability analysis (PVA) is the most common procedure for analyzing extinction risk. In the use of PVA to model the trajectories of reintroduced populations, demographic values are sometimes taken from other existing wild populations or even from individuals in captivity.Density dependence in productivity is usually considered in viability models, but density-dependent variation in age of first breeding is usually ignored. Nevertheless, age of first breeding has a buffering effect on population fluctuations and in consequence on population persistence.We simulated the viability of Spanish Imperial Eagle (Aquila adalberti) and Osprey (Pandion haliaetus) populations using data from established and reintroduced populations in southern Spain.Our results show that reduction in the age of first breeding is critical in the success of reintroductions of such long-lived birds. Additionally, increases in productivity allow populations to growth at maximum rate. However, without considering variation in age of breeding, and the associated increasing overall productivity, reintroduced populations seem nonviable.To ignore density dependence in age of breeding in PVA means that we are seriously limiting the potential of the model population to respond to fluctuations in density, thereby reducing its resilience and viability. Variation in age of first breeding is an important factor that must be considered and included in any simulation model involving long-lived birds with deferred maturity.