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    Complex networks of marine heatwaves reveal abrupt transitions in the global ocean

    SST data
    I used the National Oceanic and Atmospheric Administration (NOAA) daily optimum interpolation SST gridded dataset V2.0 to identify MHWs in the period 1 January 1982 to 31 December 20184,28. The dataset is a blend of observations from satellites, ships and buoys and includes bias adjustment of satellite and ship observations to compensate for platform differences and sensor biases. Remotely sensed SSTs were obtained through the Advanced Very High Resolution Radiometer and interpolated daily onto a 0.25° × 0.25° spatial grid globally. Data were provided by the NOAA/OAR/ESRL PSD, Boulder, Colorado, USA, from their Web site at https://www.esrl.noaa.gov/psd/ accessed in January 2019.
    I also obtained data from simulation models implemented in the fifth phase of the Coupled Model Intercomparison Project (CMIP5). I used the first ensemble member r1i1p1 of 12 coupled Earth System Models that allowed the analysis of variation in daily SSTs in all scenarios: CNMR-CM5, GFDL-CM3, GFDL-ESM2G, GFDLESM2M, IPSL-CM5A-LR, IPSL-CM5A-MR, MPI-ESM-LR, MPI-ESM-MR, MIROC5, MIROC-ESM, MRI-CGCM3, MIROC-ESM-CHEM. A climatology (the statistical properties of the timeseries, including the mean, variance, seasonal cycle and quantiles; see section “Identifying marine heatwaves” below for derivation) was obtained from historical simulations over the period 1861–2005 and used to identify MHWs for the historical scenario and for simulated SSTs over the period 2006–2100 following high and low emission scenarios (RCP 8.5 and RCP 2.6, respectively; RCP, representative concentration pathway). All the analyses on simulated data were implemented on a multi-model ensemble obtained by averaging the twelve models, unless otherwise indicated. For comparisons with the simulated SSTs, the satellite 0.25° × 0.25° data were regridded by averaging daily onto a regular 1° × 1° grid. The climatology for the satellite MHWs was derived from the whole observational period (1982–2018).
    Whether a fixed climatology is appropriate instead of using shifting baselines to define MHWs is a matter of debate13,40. Here, the historical scenario provides a common reference to gauge shifts in the spatiotemporal dynamics of projected MHWs under high (RCP 8.5) and low (RCP 2.6) emission scenarios.
    Identifying marine heatwaves
    I identified MHWs from daily observed and simulated SST timeseries within each 1° × 1° cell following Hobday et al.27, who define a MHW as an anomalously warm water event with daily SSTs exceeding the seasonally varying 90th percentile (climatological threshold) for at least 5 consecutive days. The climatological mean and threshold were computed for each calendar day within a 11-day window centered on the focal day across all years within the climatological period. The mean and threshold were further smoothed by applying a 31-day moving average. Two events with a break of less than 3 days were considered the same MHW. I then derived characteristic metrics of MHWs, including duration, intensity and frequency and linked them to network properties (see below, Network analysis). Only SST timeseries with less than 10% of missing data were used in the analysis. I used the R package heatwaveR to identify marine heatwaves from SSTs41.
    Topological data analysis and the Mapper workflow
    Topological Data Analysis (TDA) is a collection of statistical methods based on topology, the field of mathematics that deals with the study of shapes, to find structure in complex datasets24. The Mapper algorithm is one tool of TDA that allows reducing high-dimensional data into a combinatorial object that encapsulates the original topological and geometric information of the data, such that points close to each other are more similar than distant points. The combinatorial object, also called a shape graph, is indeed a network with nodes and edges. The statistical properties of the TDA-based Mapper algorithm and how it relates to other non-linear dimensionality reduction techniques have been discussed in Ref.22. Here, I briefly summarize the five key steps involved in a Mapper analysis (Fig. 1). The first step of MAPPER consists of collapsing a raster stack of spatiotemporal data of MHWs into a binary 2D matrix where rows are timeframes (days) and columns are 1° × 1° cells arranged sequentially to represent the occurrence of MHWs across the global ocean. The first column of the matrix corresponds to the upper-left pixel of the raster centered at 89.5°N and − 180°W and the subsequent 364 columns represent adjacent pixels within the same latitude. Column 366 is centered at 88.5°N and − 180°W and so on, with the last column of the matrix corresponding to the lower-right pixel at − 89.5°S and 180°E. Although this scheme would result in matrices with 64,800 columns (360 × 180), I used reduced matrices in computations by excluding pixels on land or where missing SST values prevented the identification of MHWs. The final size of the matrices used in the analysis is (rows × columns) 13,514 × 42,365 for observed SSTs and 52,960 × 41,968, 34,675 × 41,074 and 34,675 × 41,482 for simulated SSTs under the historical, RCP 2.6 and RCP 8.5 scenarios, respectively.
    The second step of Mapper involves dimensionality reduction or filtering. I used the Uniform Manifold Approximation and Projection dimensionality reduction (UMAP) algorithm to perform nonlinear dimensionality reduction42. This algorithm is similar to t-distributed Stochastic Neighbor Embedding (tSNE), which is widely used in machine learning43. The advantage of UMAP is that it has superior run time performance compared to tSNE, while retaining the ability to preserve the local structure of the original high-dimensional space after projection into the low-dimensional space.
    The third step of Mapper consists of dividing the output range generated by the filtering process into overlapping bins. The number of bins and the amount of overlap are determined by the resolution (R) and gain (G) parameters, respectively. I used an optimization procedure to objectivity identify the combination of parameters R and G that best localized timeframes with similar cumulative intensity of MHWs nearby in the network (see “Parameter search and sensitivity analysis”). This procedure selected R = 24 and G = 45 for observed SSTs and for the RCP 8.5 scenario, R = 22 and G = 45 for the historical scenario and R = 12 and G = 25 for the RCP 2.6 scenario.
    The fourth step of Mapper consists of partial clustering of timeframes within bins. Although Mapper is flexible and can accommodate different clustering methods and distance functions, I employed single-linkage clustering with Euclidean distance25. It is worth noting that this approach does not involve averaging of timeframes within clusters, so the original information is preserved in a compressed representation of the data.
    The fifth and final step involves the generation of the network graph from the low dimensional compressed representation of the data. Clusters become nodes in the network and nodes become connected if they share one or more timeframes. I implemented the TDA-based Mapper algorithm using a parallelized version of function mapper2D in the R package TDAmapper44.
    Network analysis
    I employed two widely used measures of network topology, modularity and node degree, to compare the structure of the four MHW networks. Modularity describes the strength of division of a network into communities—i.e. cohesive groups of nodes that have dense connections among them and that are only sparsely connected with nodes in other groups. High modularity indicates the presence of distinct regimes of spatiotemporal dynamics of MHWs. As a second measure of network structure, I used mean node degree—where the degree of a node is the number of edges that are incident to that node. High mean node degree indicates that many nodes share one or more timeframes and depicts similar spatiotemporal patterns of MHWs within those nodes. In contrast, low node degree indicates the occurrence of many isolated nodes with few timeframes in common and more isolated MHWs. I computed modularity and node degree with functions ‘modularity’ and ‘degree’ in the R package igraph45.
    To provide significance tests for the observed measures of network topology and to evaluate if they originated simply from non-stationarity properties in the original data, I run two null models based on surrogate data for each of the four networks. Surrogate data can be obtained through the Fourier transform of the original timeseries, shuffling the phases and applying the inverse transform to generate the surrogate series46. Phase randomization preserves the power spectrum, autocorrelation function and other linear properties of the data, but not the amplitude distribution. To address this potential drawback, I generated surrogate timeseries via the Theiler’s Amplitude Adjusted Fourier Transform (AAFT) using function ‘surrogate’ in the R package fractal, which also preserves the amplitude distribution of the original timeseries47. Using this approach, I applied two schemes of randomization—one employing a random sequence for each timeseries and one employing the same sequence for all timeseries. Randomizing using a fixed sequence for all timeseries (constant phase) randomizes the nonlinear properties of the data while preserving linear properties, such as the linear cross-correlation function. The randomization scheme based on random sequences (random phase) also disrupts linear relationships in the data. A significant departure of the observed statistic from the null model under constant phase randomization allows rejecting the null hypothesis that the observed time series is a monotonic nonlinear transformation of a Gaussian process. A significant departure from the null model under random phase allows rejecting also the null hypothesis that the original data come from a linear Gaussian process. To assess significance, 1000 randomizations were performed for each network under each scheme of random and constant phase and a two-tailed test was performed at α = 0.025 to account for multiple testing (Bonferroni correction).
    To quantify temporal transitions of MHWs, I estimated node degree of the temporal connectivity matrix (TCM) obtained from each network. The degree for each node in the TCM was estimated by counting the number of non-zero edges connected to that node22. Temporal fluctuations in node degree were benchmarked against the confidence intervals of the random phase null model, estimated as twice the standard deviation of the null distribution. A Generalized Additive Model (GAM) smooth function was fitted to node degree data to visualize temporal trends. The timing of collapse of node degree for the historical scenario was estimated as the year when the smooth curve intersected the upper confidence limit of the null model. To provide a measure of uncertainty, I obtained analogous estimates of the year of collapse by repeating the whole analysis for each of the twelve ESMs separately and computing the median and the bootstrap standard error (n = 1000) of these estimates. A similar analysis was done on the RCP 8.5 data to estimate the year when node degree increased again and diverged significantly from the null distribution. To determine the duration of the different period of connectivity identified in the TCMs, I used the change point algorithm implemented in function cpt.mean of package changepoint48.
    Parameter search and sensitivity analysis
    To objectively identify parameters R and G (resolution and gain) as part of the binning process in the Mapper algorithm, I used an optimization procedure that best localized timeframes with similar patterns of MHWs nearby in the network. Localization can be done for any of the properties of MHWs. I used cumulative intensity as the localization criterion since it was a good proxy for other properties of MHWs, such as duration (r = 0.88, p  More

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    Radiolysis generates a complex organosynthetic chemical network

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    Author Correction: Clustered versus catastrophic global vertebrate declines

    Affiliations

    Department of Biology, McGill University, Montreal, Quebec, Canada
    Brian Leung & Anna L. Hargreaves

    Bieler School of Environment, McGill University, Montreal, Quebec, Canada
    Brian Leung

    Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada
    Dan A. Greenberg

    School of Biology and Ecology, University of Maine, Orono, ME, USA
    Brian McGill

    Mitchell Center for Sustainability Solutions, University of Maine, Orono, ME, USA
    Brian McGill

    Centre for Biological Diversity, University of St Andrews, St Andrews, UK
    Maria Dornelas

    Indicators and Assessments Unit, Institute of Zoology, Zoological Society of London, London, UK
    Robin Freeman

    Authors
    Brian Leung

    Anna L. Hargreaves

    Dan A. Greenberg

    Brian McGill

    Maria Dornelas

    Robin Freeman

    Corresponding author
    Correspondence to Brian Leung. More

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    Analysis of global human gut metagenomes shows that metabolic resilience potential for short-chain fatty acid production is strongly influenced by lifestyle

    Our results are consistent with a non-industrial gut harboring a more resilient ecology with respect to SCFA production, while the industrial gut ecology would be vulnerable to disruption of such pathways, yet the pattern is complex and nuanced. The increased gene abundance in non-industrial populations and overall ratio of acetate:butyrate:propionate generally agrees with previous studies of SCFAs5,12. Similarly, the higher genus-level diversity of bacteria encoding acetate, compared to the other SCFAs, is expected and matches studies that have documented the taxa that encode different SCFAs13,17,19. The overall high richness, high diversity at Hill numbers 1 and 2, and high Gini-Simpson indices found in non-industrial populations at the genus level indicates a highly diverse and evenly distributed production of SCFAs. From an ecological perspective, uneven production of SCFA dominated by a few bacteria in industrial gut microbiomes means lower functional diversity and less redundancy, which ultimately leads to an expectation of decreased resilience. In other words, this study finds that industrial gut microbiomes are at a higher risk of reduced SCFA production because SCFA synthesis is dominated by only a few genera. Given the lower resilience, factors that disrupt the gut ecology are expected to have a more extreme consequence to those living an industrial lifestyle.
    While there is an overall trend of increased genus-level functional diversity and redundancy for SCFA production in non-industrial populations, variation exists when examining the SCFAs and populations individually. At the genus-level, the pastoral and rural agricultural populations have increased richness of genera encoding genes involved in acetate and butyrate synthesis, while there is similarity across the different lifestyles for genus richness for propionate encoding taxa. Although hunter-gatherers have similar, or lower, genus richness as industrial populations, they have significantly higher diversity at Hill number orders 1 and 2 and Gini-Simpson indices for butyrate and propionate. Additionally, the pastoralists have a generally similar profile to the industrial populations for acetate and propionate Hill number diversity, as well as similarity to the industrial populations in species PD, which may be linked to this pastoralist group having a diet similar to some industrial populations; namely, a diet high in dairy and red meat consumption, coupled with few dietary sources of plant-derived fibers23. This paints a complex picture. Non-industrial populations have a high diversity of genera encoding butyrate synthesis, and butyrate production is spread more evenly across genera in non-industrial populations than in industrial populations. Hunter-gatherers and rural agriculturalists have significantly greater evenness of propionate production, even though they have fewer number of total genera encoding this SCFA. Finally, the richness and evenness of genera encoding acetate is similar between industrial and non-industrial populations. Ecologically, we would expect the industrial populations to be less resilient for production of butyrate and propionate when faced with a shift in taxonomic composition, while non-industrial populations may be only marginally more resilient for acetate production compared to industrial populations. Intriguingly, SCFA relative abundance does not appear to correlate to resilience profile. Acetate and butyrate are significantly more abundant in non-industrial populations but only butyrate shows much stronger resilience profile for non-industrial populations. Additionally, propionate is slightly more abundant in industrial populations, although not significantly, yet our results indicate greater resilience in non-industrial groups for propionate production. This indicates that measuring only total gene, and/or molar, abundance is not enough to make statements about metabolic processes in the human microbiome; rather, ecological approaches are necessary to understand diversity in functional potential of the human microbiome.
    The increased species-level alpha diversity in industrial populations initially runs counter to the genus-level results but the genus and species level results ultimately yield similar interpretations after accounting for ecology and ascertainment bias, as discussed below. The substantially higher species richness in industrial populations is striking; however, the differences in PD between industrial and non-industrial populations are not nearly as extreme. This means that the high species richness in the industrial populations is driven by species that are closely phylogenetically related. Indeed, we observed SCFA producing genera found at high abundance in industrial populations (Bacteroides and Clostridium) to have up to nine species encoding SCFAs, while highly abundant non-industrial genera only have one or two species. Therefore, what first appears to indicate high species-level ecological resilience in SCFA production in the industrial populations is actually the result of closely related species performing the same function. It follows that closely related species may be prone to changes in abundance or even elimination after certain types of ecosystem shift events. For example, narrow-spectrum antibiotics33 and exposure to various xenobiotic compounds that lead to variable bacterial metabolic responses34 are events that can affect a limited range of bacteria and lead to shifts in microbial abundance and metabolic activity. While this result has ecological implications, it is also likely the result of historical trends of microbiology research. Bacterial taxa at high abundance in non-industrial gut microbiomes have not been a focus of microbiological isolation and species identification until recently; therefore, we expect more species to be identified from non-industrial gut microbiomes in the future35. Additionally, classification of bacteria into distinct genera and species is undergoing a revolution in the genomic era36 meaning that the high number of species classified to Bacteroides and Clostridium may ultimately be reclassified to different genera. Nevertheless, the fact that we observe a large jump in species richness, but only a minor increase in species PD, in the industrial gut microbiomes suggests that the high industrial species richness is driven by closely related species and therefore, results in the same interpretation as the genus richness results: diversity is high in non-industrial populations.
    Ascertainment bias extends to the databases used to identify taxa and genes: fewer genes were identified in non-industrial populations and a smaller proportion of these genes can be linked back to bacteria at every taxonomic level, in non-industrial gut microbiomes. In some cases, such as butyrate synthesis genes, less than 10% of genes are identified to species for non-industrial populations, while over 50% of such identifications were possible for industrial populations. A decreased ability to identify the genus and species encoding SCFA synthesis genes in non-industrial populations means that the ecological metrics underestimate the true ecological diversity of these genes. Moreover, the drop-off in classification from the genus to the species level was significantly greater in non-industrial populations compared to industrial populations. This drop-off means a much lesser ability to identify species compared to genera in non-industrial populations, which helps explain why species diversity was substantially lower in non-industrial populations. Nevertheless, the statistically significant differences observed at the genus-level send a strong signal of the high functional diversity, and potential resilience, of SCFA synthesis genes in non-industrial gut microbiomes.
    The metagenome-wide poor performance in terms of gene identification and classifying SCFA genes to genera and species indicates a bias in reference databases that underrepresents diversity in non-industrial gut microbiomes, which is unsurprising. Bias is expected because the vast majority of human gut microbiome studies have used samples from industrial populations. There is an immense challenge in including non-industrial communities in biomedical research, including recruiting research participants, sustaining longitudinal sampling, building culturally appropriate community relationships, and even securing transport of samples35. This has resulted in comparatively few metagenomic studies of human gut microbiomes from non-industrial settings35. Nevertheless, our data demonstrate the extent of this bias and how it can hinder more in-depth study of human gut microbiome health. Given this sizable ascertainment bias favored industrial populations, the non-industrial populations are likely even more diverse, more resilient, than our databases can sufficiently characterize, making our genus-level results even stronger. Without a serious investment to include such populations, the characterization of microbiomes will remain naive to the ecological breadth of the core, healthy, human gut. Imagine studying forest ecology, with only city parks at your disposal. This has been, overwhelmingly, the analogous practice of human microbiome research.
    The relative lack of microbiome studies with non-industrial populations means an underrepresentation of not only metagenomic data and genome annotation but also fewer opportunities for cultivation and validation of novel species of bacteria. This ultimately leads to an inequality in the depth to which researchers can describe microbiome samples from non-industrial communities, compared to industrial microbiomes, as diverse groups of novel taxa may be grouped into a single group of “unknown” or “unclassified” bacteria35. Similarly, an incomplete picture of microbial functional potential means that genes may be misidentified or even unannotated completely. Unknown taxa and misidentified genes may be playing key roles in ecological and metabolic processes but researchers are unable to confidently identify them, let alone make statements about their importance in a microbial ecology35. Recent human gut microbiome metagenome studies from diverse populations will undoubtedly improve database representation but the number of studies and metagenomic samples from non-industrial populations still pales in comparison to industrial gut microbiomes26,35,37,38.
    Limitations in annotating the full extent of microbial diversity impacts health research. Recently proposed ‘Microbiota Insufficiency Syndrome (MIS)’2 postulates that, while the microbiome has adapted to industrialization, these adaptations are maladaptive to human health. The decreased phylogenetic diversity and loss of specific taxa (e.g. Prevotellaceae, Succinivibrionaceae, and Spirochaetaceae) observed in industrial gut microbiomes may contribute to the increase in non-communicable chronic diseases found at higher prevalence in industrial populations. The root cause of MIS in industrial populations is undoubtedly multifactorial; however, diet is suggested to play a major role2. This syndrome is compelling and we postulate that this insufficiency precisely rests on the stability of functional capacity. Our findings of decreased resilience in industrial populations, as well as species-level diversity driven by a few closely related species, fits in well with MIS. Low resilience in SCFA production may ultimately manifest itself as altered colonocyte function and/or autoimmune disruptions (both symptoms of MIS) due to a decrease in SCFA bioavailability after a group SCFA-producing bacteria were wiped-out during an ecological shift, such as antibiotic or xenobiotic exposure. Similar to MIS, diet is likely to play an important role in SCFA resilience. The non-industrial populations studied in this paper consume much more fiber than industrial populations, on average3,5,14,25,26, and microbial fermentation of dietary fibers is a major source of SCFAs in the human digestive tract39. A diet poor in dietary fiber means less substrate for microbial fermentation and therefore less SCFA production and also higher competition for that fiber, potentially resulting in competitive exclusion and less microbial diversity. Nevertheless, if we are unable to fully characterize and annotate non-industrial gut microbiomes then we will be unable to paint a complete picture of MIS. Currently, we have confidence that there is a wealth of undiscovered resilience in non-industrial gut microbiomes. Once we describe the extent of this diversity/resilience, through increased sampling and focus on partnerships with research institutes in industrializing countries, we will have a more complete picture of MIS and possibly develop therapeutic approaches to combat non-communicable chronic diseases related to the human gut microbiome.
    Improved sampling, metabolic profiling, and annotation will not only improve our understanding of SCFA resilience, but it will also permit more detailed picture microbiome wide resilience. Our work shows the value of focusing on specific SCFA genes, due to their importance in human biology and previously reported variation in SCFA molar abundance between industrial and non-industrial populations31,32; however, future work will undoubtedly add to our findings. One avenue for future work is through analyzing SCFA molar concentrations in fecal samples in a longitudinal setting and comparing these results to predicted SCFA resilience from metagenome panels. Unlike genomic data, where we can infer about SCFA production potential via taxonomic diversity, one-time measures of fecal SCFA molar concentrations will not inform about future resilience because SCFA molar concentrations carry no information about which taxa produce each SCFA. Longitudinal SCFA concentration and metagenomic data from non-industrial populations, or animal models, is necessary to inform about SCFA resilience and production in diverse lifestyles. Another avenue for future work is to focus resilience analysis on other microbiome functions of interest, such as resilience of antibiotic resistance genes and amino acid biosynthetic pathways. These valuable studies would be valuable for comparing microbiome resilience dynamics for different functions, with the caveat that there is sufficient genomic annotation data to yield interpretable results.
    Lack of sample diversity is not unique to human microbiome research, as human genetics research has been grappling with this very issue for decades. In 2009, 96% of individuals included in human genome-wide association studies (GWAS) claimed European ancestry, as compared to 78% in 201940. Thus, while there have been improvements, GWAS clearly fail to reflect the breadth of human diversity. Incorporating diverse populations in human genome and microbiome research has the potential to greatly benefit the scientific community’s understanding of human biology and develop treatments that are based on human diversity rather than European-ancestry genetics and microbiomes. A key component of increasing representation in genetics and microbiome studies is that these studies are designed as partnerships with minority and/or indigenous communities in a manner that builds both trust between the community and researchers, as well as facilitates the ability for the sample donors to exercise their rights on how data are treated and shared41. More

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    A shift towards early-age desexing of cats under veterinary care in Australia

    This is the first large scale analysis of feline desexing practices in Australia using outcomes documented in the patient medical record. The findings complement those of previous studies that used survey data to analyse the attitudes and opinions of veterinary professionals and owners to desexing31,34,38. The prevalence of desexing among cats in Australia, found to be 83.6%, confirms that desexing rates in Australia are among the highest reported internationally. Survey-based studies have reported that approximately 90% of cats in Australia are desexed, compared with 80% in the USA, and 43% in Italy39,40,41,42,43,44,45. A recent EPR-based study conducted in the UK reported the prevalence of feline desexing as 77%46. While population-wide analyses of desexing status provide a useful snapshot of practices in a region, most do not consider reproductive history.
    A clear shift over time towards desexing cats at a younger age was evident here. EAD was 1.76 times more likely to have been carried out among desexed cats born between 2010 to 2017, than in those born between 1995 and 2009. This move towards earlier desexing was apparent in all age groups studied. Despite this trend, EAD had been carried out in only 21.5% of desexed females in the recent period. In fact, only 59.8% of females had been desexed by 6 months of age, which is the traditional recommendation and the most common recommendation reported by vets in Australia31,47. Despite a move towards earlier desexing, opportunities to control reproduction by prepubertal desexing are still being lost.
    For an individual female cat, desexing at 6 months or later may be of little consequence, since they may not yet have reached puberty or had access to a mate. A recent survey of cat owners in Australia and New Zealand however found that 66% of cats had outdoor access45. From a population control perspective, eliminating the possibility of pregnancy by adopting EAD as standard has merit. The body of scientific evidence generated specifically to address the short-term and long-term safety of EAD overwhelmingly validates this practice4,17,22,23,24,25,26,27,28,29.
    The impact that tighter control of reproduction among owned cats would have on shelter and stray populations is not yet clear. Populations of owned cats (completely reliant on humans) feral cats (living independently of humans) and stray cats (intermediate relationship with humans) do not exist in isolation48. Anthropogenic factors, including the provision of food, abandonment, and failure to curb reproduction, influence cat abundance and movement through these populations. Modelling population dynamics in owned, unowned (stray and feral) and shelter-housed cats holds promise to inform cat management strategies in the future49.
    In multivariable models, for cats born 2010–2017, sex, breed, state and socioeconomic indices were all significantly associated with both desexing status and age at surgery. Females were less likely than males to be desexed and, among desexed cats, females were less likely than males to have been desexed at ≤ 4 months, supporting future measures to promote EAD in female cats. The reasons for this difference were not investigated but, conceivably, it may be due to higher fees for desexing females at some practices, or a greater awareness of spraying and roaming behaviours in males than pregancies in young female cats.
    Not surprisingly purebred cats were less likely to be desexed than mixed breeds. In contrast, the finding that purebred cats were 2.7 times less likely to undergo EAD was unexpected because breeders commonly request EAD so that progeny for the pet market can be sold without delay50. It is plausible that this result reflects a greater demand in Australia for EAD from the charity and shelter sector, where mixed breed cats predominate, than from breeders. In line with this possibility, recent surveys found 70–80% of cats in Australia and New Zealand are of mixed breed and acquired from shelters, veterinary clinics, friends and as strays40,45,51. The higher odds of EAD in males than females was even greater among purebred cats, a result that may have been influenced by the practice of retaining more entire females than males for breeding.
    The breeding season in Australia and New Zealand extends year round with peaks of kittening in spring and summer inferred from shelter admissions9,52,53. Cats born in winter had the lowest odds of being desexed in each age group. One explanation for this finding is that promotion of desexing by veterinary practices and welfare groups is less likely in winter because fewer kittens are born. This seasonal difference is certainly seen in the UK, where the RSPCA conducts desexing campaigns in Autumn to prevent the peak of spring litters54.
    State or territory influenced both whether a cat was desexed, and the odds of EAD. Compared with cats in New South Wales (NSW), those in Victoria (VIC) and South Australia (SA) were more likely, and those in Queensland (QLD) less likely to be desexed. Again, compared with NSW, the odds of being desexed at ≤ 4 months were 1.45 greater for cats in VIC and 1.5–2.3 times less for those in QLD, SA and ACT. Desexing is handled inconsistently between Australia’s states and territories. Mandatory desexing legislation exists in ACT (by 3 months of age) and in SA, Tasmania (TAS), WA (by 6 months of age), with some exceptions. No legal requirement to desex cats exists at state level in VIC, QLD, NSW or Northern Territory (NT), although desexing is indirectly incentivized in NSW (by 6 months of age) and VIC (by 3 months of age) where registration is mandatory, and reduced registration fees are applied for desexed cats. No consistent relationship between our findings and state legislation related to desexing cats was identified. In fact, in ACT, where desexing of pet cats at 3 months of age has been a legal requirement since 2007, the second lowest odds of EAD were identified. Most veterinarians practicing in ACT (90%), surveyed 10 years after the legislation was introduced, gave recommendations inconsistent with the legislation and 35% were unaware that desexing by 3 months was mandatory in the ACT34. Whether and how legislation might be an appropriate tool to influence reproduction in owned cats and, indirectly, overpopulation should be further investigated.
    Socioeconomic conditions influenced both whether a cat was desexed or not, and the age at desexing. Entire cats were more common in remote, low income and disadvantaged areas. This finding is concerning, given that outdoor access was more likely in non-urban than urban areas in a study of households in Australia and New Zealand45, implying more opportunity to find a mate. In addition, stray cat density correlated positively with socioeconomic deprivation in a New Zealand-based study employing geographically weighted regression analyses55. Together, these findings support the promotion of desexing campaigns in non-urban areas.
    Economic indicators such as household income influenced whether a cat was desexed; the odds of being desexed were around 1.2 times greater in the highest compared to the lowest income areas. A similar, but more dramatic effect was reported in a study conducted in the USA where the prevalence of desexing increased from 51.4% to 96.2% as household income increased43. Among desexed cats, EAD was least likely in low income areas, but highest in the most socio-economically disadvantaged areas. Although this might seem paradoxical, IRSD is based on broader indicators of disadvantage than income alone. A UK study, similarly, identified that EAD was most likely in the most deprived regions, and that chances of being desexed by 6 months were more likely in higher income areas56. Possible explanations for these observations include the preferential targeting of areas of greatest disadvantage, rather than those with fewer economic resources, by discount desexing programs promoting EAD, or preferential sourcing of kittens in disadvantaged areas from organizations that routinely practice EAD, such as shelters.
    There are limitations to our study that should be considered when interpreting the results. Cats that were either not registered with a veterinary practice, or were registered with a practice that did not contribute to VCA during the study periods were not studied. Therefore actual desexing prevalences are almost certainly lower than the estimates reported here. The study population represents cats that are accessible for desexing and is expected to comprise cats kept as pets, for breeding, owned by shelters, semi-owned cats and others. Provenance and lifestyle were not investigated because we chose not to collect data from the examination text field in VCA because of its low positive predictive value57, and because these data are inconsistently recorded. This precluded the analysis of other variables that may have been related to desexing outcomes such access to outdoors and the number and species of pets. Data collection was not uniform across Australia and variations in sample size, for example between states, may have affected our results. Also it is possible that data for the same cat presenting at more than one practice could be counted more than once, although a previous study using VCA found that  More

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