Managed wildlife breeding faces high costs and genetic diversity challenges associated with caring for small populations. Biobanking (freezing of sex cells and tissues for use in assisted breeding) and associated reproductive technologies could help alleviate these issues in koala captive management by enhancing retention of genetic diversity in captive-bred animals and lowering program costs through reductions in the size of the required live captive colonies. Australia’s zoos and wildlife hospitals provide rare opportunities to refine and cost-effectively integrate these tools into conservation outcomes for koalas due to extensive already-existing infrastructure, technical expertise, and captive animals.Phascolarctos cinereus). Managed breeding programs held across zoo-based networks typically face high economic costs and can be at risk of adverse genetic effects typical of unavoidably small captive colonies. Emerging evidence suggests that biobanking and associated assisted reproductive technologies could address these economic and genetic challenges. We present a modelled scenario, supported by detailed costings, where these technologies are optimized and could be integrated into conservation breeding programs of koalas across the established zoo and wildlife hospital network. Genetic and economic modelling comparing closed captive koala populations suggest that supplementing them with cryopreserved founder sperm using artificial insemination or intracytoplasmic sperm injection could substantially reduce inbreeding, lower the required colony sizes of conservation breeding programs, and greatly reduce program costs. Ambitious genetic retention targets  could be possible within realistic cost frameworks, with output koalas suited for wild release. Integrating biobanking into the zoo and wildlife hospital network presents a cost-effective and financially feasible model for the uptake of these tools due to the technical and research expertise, captive koala colonies, and ex situ facilities that already exist across these networks.Zoo and wildlife hospital networks are set to become a vital component of Australia’s contemporary efforts to conserve the iconic and imperiled koala ( The continued decline of species in the wild typically drives an increased reliance on captive management, particularly on breed-for-release programs . DespiteBiobanking and assisted reproductive technologies have been advocated as potential technological solutions to address challenges of high costs and genetic diversity in captive programs. We define biobanking as a term encompassing methods for the frozen storage of sex cells and tissues , These models also revealed the potential to meet or exceed long-standing and challenging genetic diversity targets (90% heterozygosity retention for 100 years ) in captMustella nigripes) in North America. In this case, a captive program was developed with the last remaining 18 wild ferrets which over time experienced reproductive complications due to inbreeding [in situ conservation efforts , has resulted in the release of thousands of ferrets back to wild habitats [Despite clear potential benefits of these technologies ,4,18, thbreeding ,22,23,24breeding . The prohabitats ,22. It ihabitats .Phascolarctos cinereus), the world’s most iconic marsupial [https://amp.abc.net.au/article/12941370; accessed on 17 March 2022).The koala . Furthermore, historic advances have been made through the successful use of koalas as a research model for assisted reproduction [Although conventional strategies are allowing for the maintenance of optimal genetic diversity in existing koala captive populations , the limoduction  . Alloduction .This study aims to explore a reality where these technologies are optimized and could be integrated into koala captive management. We examine the potential of using biobanked koala sperm and assisted reproductive technologies to enhance the cost-effectiveness and genetic benefits possible if these technologies were to become a practical reality for koala captive management. We model the potential benefits of integrating biobanking and assisted reproductive technologies  into the captive management of koalas by adapting the available modelling systems described above ,4 to theP. cinereus) is currently listed as ‘vulnerable’ under the International Union for the Conservation of Nature (IUCN) Red List [http://www.environment.gov.au/cgi-bin/sprat/public/spratlookupspecies.pl?name=Phascolarctos+cinereus&searchtype=Sciname; accessed on 17 March 2022). We selected the koala as a case study due to the availability of primary data required for modelling; particularly detailed captive holding costs  with or without the use of biobanking and assisted reproductive technologies. We modelled a range of 100-year heterozygosity targets, including 90% of source population heterozygosity, which was first proposed in [N) and program costs required to meet the described genetic retention targets under: (1) closed captive breeding conditions ; and (2) in hypothetical captive colonies where assisted reproductive technologies  are used to backcross the colony to founder male koalas using biobanked sperm every generation , up to 100 years (14 generations) after the program is established. We have modelled 100-year captive breeding scenarios where live colony sizes of koalas reflect the potential numbers required to maintain different proportions of source population heterozygosity . In l koalas . Economiex situ holding cost per koala (C) is given by Equation (1) below.H represents the economic holding cost per individual koala (given in I represents variable on-site infrastructure and facilities costs. Year 1 economic cP) for any non-backcrossed conventional captive koala population and for populations integrating biobanking and assisted reproductive technologies (bcP) are given by Equations (2) and (3) below.C is Year 1 economic ex situ holding cost per koala, as given by Equation (1), B represents biobanking costs  and heterozygosity (tH/H0) in Equation (4) [tH/H0) at each generation (t) for which tF was estimated. F0 for founders was assumed to be 0.The predicted rate of loss of heterozygosity from koala populations was derived from the relationship between the inbreeding coefficient (tion (4) . This altF  from eN (effective population size) and t−F1 . This allows sequential determination of tF for any generation (t) up to the number required to reach 100-year heterozygosity benchmarks .The increase in inbreeding between generations in captive koala colonies without backcrossing was determined from the relationship in Equation (5)  (p. 271)eN represents the effective number of male koala founders and t is the number of backcross generations. For detailed derivation of Equation (6) see [We employed an iterative process ,4, usingWe provide detailed costings across a mapped network of zoos as well as wildlife hospital and rehabilitation centers which could provide nodes for the collection of founder sperm from wild koala populations or act as source populations for assisted reproductive research. The approach taken to construct this network is provided below. B) described above and presented in We conducted a comprehensive online search to select zoos and wildlife hospitals (hereafter referred to as “nodes”) in New South Wales, Queensland, Victoria, and South Australia. Selection criteria for inclusion of nodes in the network were: (1) specialize in capture, care and/or treatment of koalas ; and (2) sufficient on-site infrastructure to hold koalas and facilitate collection and temporary or long-term storage of sperm in additional frozen storage infrastructure that could be held on-site or that may benefit from the integration of assisted reproduction for genetic management . OrganieN = 67; N = 223) in captive koalas would require significant Year-1 start-up costs of >A$5.2 million, followed by >A$73 million in 100-year costs to maintain residual heterozygosity of 0.9004  and 99%  of source population heterozygosity without assisted reproductive technologies would require further increased Year 1 start-up costs of >A$10.8 million and >A$54.9 million respectively, as well as increased total 100-year program costs of >A$149.4 million and >$A760.2 million respectively  requires >5-fold reduced start-up costs of A$904k, and >6-fold reduced total 100-year program costs of A$10.6 million (N = 33) retention target, start-up costs and total 100-year program costs are reduced >8-fold and >9-fold to A$1.2 million and A$16 million respectively (N = 167) retention target  to use assisted reproductive technologies such as artificial insemination or intracytoplasmic sperm injection to backcross live captive female koalas to founder males using frozen sperm would allow substantial reductions in required colony census numbers and total program costs for all genetic retention targets across 100 years of captive management . Backcro million . Cost re million . The sam million . Despite million . In the ectively . Start-un target .n = 5), Queensland (n = 7), Victoria (n = 2), and South Australia (n = 2) for management of unique and independent koala regions as informed by understanding of population and genetic differentiation across jurisdictions  cWe recognize that koalas are held in captivity for a number of other reasons  and the cost savings presented here apply to the specific case study of a closed breed-for-release program with or without integration of assisted reproduction. If these technologies were a practical reality, an already established network of 16 wildlife hospitals and zoos could act as vital collection points for koala founder sperm, with some institutions having appropriate infrastructure and staff support for temporary or permanent storage and use of samples after thawing . With adWe have presented an assessment of the existing infrastructure across a potential National node network for the integration of biobanking and/or assisted reproductive technologies to support koala conservation . This stThe influence of genetic issues and on-going loss of genetic diversity in wild koala populations suggest a need for novel tools to maximize the adaptability and resilience of the koala to prevent further decline and ultimately extinction ,50. Habihttps://www.smh.com.au/environment/conservation/new-genetic-tools-to-stop-koalas-other-species-going-down-the-drain-20210211-p571kh.html; accessed on 17 March 2022). This research will provide a diagnostic tool to develop a genetic baseline for extant and declining koala populations; determine rates of reproduction and disease prevalence; evaluate population size and adaptive potential; identify genetic variance in populations; and may ultimately identify priority koala populations for conservation action [These genetic diversity challenges and their potential to arise have driven a policy and funding focus towards the genetic management of wild koala populations. One step to address this has been significant investment in recent years, including $A1 million in research investment for the frozen storage and genomic sequencing of non-living tissue samples collected from wild koalas . If the approach advocated in the present study is optimized and becomes a practical reality, it has the potential to provide a mechanism to address genetic issues in wild and captive koala populations. We advocate for the imperative and neglected development of practical tools to actually retain living reproductive koala cells and tissues , and integrate genetic material into wild populations through reintroduction and genetic rescue ,59. Reduin situ decline of these populations and their unique genetic material; and (3) cost-effectively hold viable colony sizes long enough to meet globally accepted genetic diversity targets .The substantial network of infrastructure  supportiRecover and make use of genetic material from koalas that can no longer contribute to the population ;Proactively capture the valuable genetic material from important or imperiled wild koala populations and produce genetically fit koalas for release to the wild;Slow the rate of inbreeding by reintroducing founder genomes through frozen-thawed spermatozoa  comprehensive assisted breeding programs integrating assisted reproduction where the backcrossing to captive colonies modelled here is feasible  in the koala, there is still much to learn regarding the reliable cryopreservation of the koala sperm cell (reviewed in ) and theChlamydia or the recovery of sperm from recently post-mortem koalas), live and frozen genome banks both offer an exciting new paradigm for koala conservation and genetic management. The use of chilled semen, while not a long-term solution, nevertheless still provides a useful model in establishing workable operational logistics in preparation for a biobank based on a frozen repository. These ideas have led to the development of a pilot study known as the live koala genome bank paradigm ,60, whicConducting this applied research across multi-million-dollar programs will require conservation practitioners to leverage large research and development budgets. For example, the optimized backcross protocol for the iconic black-footed ferret required an estimated US $4.2 million in dedicated applied research funding . The rout/HoH retention; N of around 100 may be closer to a realistic minimum. For example, Species Survival Plan Programs had an average N of 137 [Biobanking and assisted reproduction are unique tools, in that they could future-proof populations through capturing the genetics of key populations and long-dead individuals of which can be re-introduced into at risk populations using optimized assisted breeding. These reproductive tools are an essential addition to the koala conservation toolbox that require significant funding support from policymakers. Biobanking and assisted reproduction are proactive tools that could offer genetic management of populations through the production of individual koalas suited for wild release, and with the frequent collection and frozen storage of koala spermatozoa would act as an insurance policy against local extinction in the event of stochastic events. For high value banked genetic resources from koalas, some risk management strategies would be recommended, such as storage of replicated samples in multiple locations as proposed across our node network  the successful wild release of 4300 ferrets across 29 sites in North America . However16 the suWe have modelled scenarios where biobanking and assisted reproductive technologies are optimized and are a practical reality for integration into koala conservation breeding programs. Our genetic and economic modelling which compares closed captive koala populations suggest that supplementing with cryopreserved founder sperm using artificial insemination or intracytoplasmic sperm injection could substantially reduce inbreeding, lower required colony sizes of conservation breeding programs, and greatly reduce program costs. We have shown that ambitious genetic retention targets  could be possible within realistic cost frameworks with output koalas suited for wild release. Our results provide clear evidence for policymakers and funding bodies to support basic and applied research that supports the optimization and then the integration of biobanking and assisted reproductive technologies as vital tools to support koala conservation. Biobanking and assisted reproduction, once developed for the koala, could function as cost-effective and financially feasible conservation tools for applied genetic management in koala breed-for-release programs across Australia. Importantly through the reduced colony sizes and significant reduction in the required investment to maintain source population heterozygosity under a biobanking framework, could free valuable conservation funding. These cost savings could support the generation of captive programs for other at-risk species, and the on-ground actions, such as habitat restoration efforts, and monitoring of individuals, required to ensure optimal outcomes after release from these innovative potential programs.


Suillus luteus, Boletus edulis, Cantharellus cibarius, and Rozites caperata with LAB strains (Lacticaseibacillus casei LUHS210 and Liquorilactobacillus uvarum LUHS245) and to evaluate the influence of this technology on colour characteristics, pH, mould/yeast count, liking, emotional response, volatile compound (VC) profile, and the formation of biogenic amines (BA). Additionally, ultrasonication or prolonged thermal treatment were applied before fermentation. The LUHS245 strain showed better preservation properties in the case of fungal inhibition; however, prolonged thermal treatment and/or ultrasound pre-treatment ensure safer fermentation. Mushroom species and type of pre-treatment had a significant effect on colour coordinates and pH (p ≤ 0.0001). A greater variety of VC was identified in pre-treated and fermented samples. Significant differences were found between the emotions induced in consumers. The lowest sum of BA was found in thermally pre-treated and fermented R. caperata, while the highest was in ultrasonicated and fermented B. edulis. Finally, despite good overall acceptability, it is important to select appropriate LAB strains for the fermentation of edible mushrooms to ensure their safety in the case of BA formation.There is scarce data on the influence of fermentation with lactic acid bacteria (LAB) on the quality and safety of edible mushrooms. The aim of this study was to ferment  Lacticaseibacillus casei LUHS210 and Liquorilactobacillus uvarum LUHS245 used in this study were isolated from spontaneous rye sourdough and, as our previous studies showed, these LAB strains can be effective antimicrobial agents in food preservation [Wild edible mushrooms have been a part of the human nutrition culture for many years because of their sensory characteristics, good nutritional and functional characteristics, and attractive culinary characteristics . Mushrooervation . Howeverervation . In fermervation ,15. A smervation . Precurservation . TherefoSuillus luteus, Boletus edulis, Cantharellus cibarius, and Rozites caperata. Preserving these mushrooms for an extended period of time is needed because they grow only in summer and autumn for a short time and have specific beneficial characteristics for human nutrition. S. luteus possess high antioxidant activity, but their extracts have low antimicrobial activity [B. edulis mushrooms are a good source of bioactive molecules, including polyunsaturated fatty acids and phenolic compounds [C. cibarius are rich in potassium and vitamins D and C [R. caperata compounds are characterised by their antiviral activity [Lacticaseibacillus casei LUHS210 and Liquorilactobacillus uvarum LUHS245 strains for the fermentation of edible mushrooms  and to evaluate the influence of the technology used on the colour characteristics, pH, mould/yeast count, liking, emotional response, volatile compound profile, and formation of biogenic amines. Additionally, ultrasonication or prolonged thermal treatment were applied before fermentation.Commercially used preservation techniques for mushrooms still have some limitations in terms of product quality and economic efficiency. Therefore, further improvement in the quality of preserved mushrooms by applying a sustainable biological approach could gain more attention. The popularity of fermented foods is increasing due to their beneficial health-related attributes and attractive sensory properties. As studies on mushroom fermentation with LAB are scarce, there is a need to analyse the influence of this preservation method on the quality and safety of mushrooms. Therefore, in this study, we hypothesised that the use of selected LAB strains for the biopreservation of wild edible mushrooms could lead to better sensory properties of the final product because of the formation of different volatile compounds, and the safest LAB strain could be selected in accordance with the lowest concentration of BA. For this study, some of the most popular edible wild mushrooms in Central-Eastern Europe were selected: activity . B. edulompounds . C. ciba D and C  and show D and C . R. capeactivity . TherefoS. luteus, B. edulis, C. cibarius, and R. caperata mushrooms were collected in the Varenos Forests (Eastern Lithuania) in September of 2021. Images of the mushrooms are given in L. casei LUHS210 and L. uvarum LUHS245 strains were incubated separately and multiplied in De Man, Rogosa, and Sharpe (MRS) broth culture medium  at 30 °C under anaerobic conditions. Boiled mushrooms (100 g) were inoculated with 3 mL of LAB multiplied in MRS (average cell concentration 9.0 log10 CFU/mL) followed by fermentation for 7 days at 22 ± 2 °C. The principal scheme of the first stage of the experiment is shown in Selected LAB strains were previously isolated from spontaneously fermented cereal, identified and characterised by Bartkiene et al. . These LS. luteus mushrooms, they were not used for subsequent experiments.Further, mushroom samples were visually evaluated to determine which treatment led to the lowest intensity of visible mould colonies on samples’ surfaces. Then, the most effective LAB strain was selected for further treatment. Because of the dark unacceptable colour of The parameters of thermal treatment and ultrasonication before fermentation were optimised. Ultrasonication was performed at a 37 kHz frequency, using a 100 W power level. The equipment employed was an ultrasonication processor . Each sample was processed for 30 min. The principal scheme of the second stage of the experiment is shown in Before measuring the mushrooms’ colour characteristics and pH, samples were homogenised using a blender.The colour coordinates  were assessed using a CIELAB system .The pH was measured using a pH electrode . The dry matter of the samples was evaluated with a saccharometer .10 of colony-forming units per gram (log10 CFU/g).Yeasts and fungi in edible mushrooms were counted on chloramphenicol agar  after incubation at 25 °C for 5 days. All experiments were carried out in triplicate, and the number of microorganisms was expressed as log®-5MS capillary column  was used for analysis. The temperature gradient was programmed from a start at 35 °C (5 min hold) to 200 °C (10 °C/min) up to 280 °C . The VC were identified according to mass spectra libraries .The VC in edible mushrooms were analysed by gas chromatography–mass spectrometry (GC-MS); we used a method with our own developed modification. A solid phase micro-extraction (SPME) device with Stableflex™ fibre coated with a 50 µm PDMS-DVB-Carboxen™ layer  was used for analysis. Before the experiment, mushroom samples were homogenised using a blender. For headspace extraction, 1 g of homogenised mushrooms were added to the 20 mL extraction vial which was sealed with polytetrafluoroethylene septa and thermostatted at 60 °C for 15 min. The fibre was then exposed to the headspace of the vial for 10 min and desorbed in the injector liner for 2 min (splitless injection mode). Prepared samples were analysed with a GCMS-QP2010 gas chromatograph  and a mass spectrometer. The following conditions were used for analysis: injector temperature 250 °C, ion source temperature 220 °C, and interface temperature 280 °C. Helium  was used as a carrier gas at a flow rate of 0.97 mL/min. The RxiThe overall acceptability of the mushroom samples was established by 20 judges, using a 10-point scale ranging from 0 (‘dislike extremely’) to 10 (‘like extremely’) . SamplesSample preparation and determination of BA in mushroom samples were performed according to the method of Ben-Gigirey et al. , with sop ≤ 0.05.Results were expressed as the mean ± standard deviation (SD). Thermal treatment, ultrasonication, and fermentation of mushrooms were performed once; all analyses were performed in triplicate. Results were analysed using the statistical package SPSS for Windows V15.0 . A linear Pearson’s correlation was used to quantify the strength of the relationship between pH, mould/yeast count, biogenic amines, volatile compounds, overall acceptability, and emotional response. The correlation coefficients were calculated using the statistical package SPSS for Windows V15.0 . Results were recognised as statistically significant at L. uvarum LUHS245 was selected for the subsequent experiment. Prolonged thermal treatment and ultrasonication pre-treatment were suggested to avoid the moulding of mushrooms. S. luteus was not used for subsequent experiments because of its dark unacceptable colour.Images of the fermented mushrooms are given in L. uvarum LUHS245 are given in Images of the pre-treated (boiled and ultrasonicated) mushrooms fermented with L. uvarum LUHS245 were used.For the subsequent experiments, both thermal treatment and ultrasonication of mushrooms, and fermentation with L. casei LUHS210 and L. uvarum LUHS245, originally isolated from fermented cereals, showed good antifungal activity against Aspergillus nidulans, Penicillium oxalicum, Penicillium funiculosum, Fusarium poae, and Fusarium graminearum [L. uvarum LUHS245 was used for the subsequent experiment according to the intensity of visible mould colonies on the surface of the samples.Food spoilage caused by fungi represents a big concern not just for economic losses, but also for its risk to human health. Some of the compounds excreted by LAB possess antifungal properties, so various LAB are used for food preservation. In this study, minearum . In mostminearum , there aB. edulis samples, showed higher redness (a*) and yellowness (b*) than those of ultrasonicated samples . However, higher coordinates of lightness (L*) were found for the ultrasonicated B. edulis and R. caperata samples than for thermally treated samples . Ultrasonicated Cantharellus and R. caperata samples showed higher a* and b* coordinates than those of thermally treated samples of the same species . There were no significant differences in the pH of thermally treated and ultrasonicated unfermented mushrooms of the same species; the highest pH was found in Cantharellus mushrooms . Comparing the colour coordinates of thermally treated and ultrasonicated fermented mushrooms, ultrasonicated fermented B. edulis and R. caperata showed higher L*, and ultrasonicated fermented Cantharellus and R. caperata showed higher a* and b* coordinates than those of thermally treated and fermented samples of the same species . While comparing the pH of thermally treated and ultrasonicated fermented samples, a significantly lower pH was found in thermally treated and fermented R. caperata samples than in ultrasonicated and fermented ones. However, no significant differences were established between other mushroom samples. Multivariate analysis of variance showed that mushroom species, thermal treatment and/or ultrasonication, and the interaction of these factors had a significant effect on the samples’ colour coordinates and pH (p ≤ 0.0001). In comparing the mould and yeast count in thermally treated and ultrasonicated unfermented mushrooms of the same species, a significantly lower mould and yeast count was found in thermally treated unfermented R. caperata samples; however, no significant differences were established between thermally treated and ultrasonicated fermented mushroom samples of the same species. When comparing thermally treated and ultrasonicated mushrooms with fresh samples, a lower mould and yeast count were established in treated samples in all cases, and a significant moderate positive correlation  was found between the samples’ pH and mould and yeast count.The colour characteristics and pH of the edible mushrooms are given in B. edulis and R. caperata found in this study. Thermal treatment could also lead to the loss of desirable food compounds, including colour pigments [B. edulis samples.The colour of mushrooms is one of the most important indicators of their quality, affecting consumers’ purchase intentions. The darkening of the mushroom surface is mainly related to microbial contamination, enzymatic activity, or mechanical injuries . Lactic pigments . Thermalpigments . In our The production of organic acids by LAB causes a reduction in pH and reflects the progress of fermentation. It is known that fermented product with a pH of 4.0–4.1 is durable . In the B. edulis VC profile (61.9%). The other most abundant VCs were 3-octanol and 1-octanol in B. edulis, oct-(2E)-enal and (E)-2-octen-1-ol in C. cibarius, and benzaldehyde in R. caperata.The whole VC profile of the untreated and treated mushrooms is given in N-heterocyclic compounds cause individual odours between different mushroom species [B. edulis.Mushroom flavour is mainly formed by the aliphatic components, such as 1-octen-3-ol, 2-octen1-ol, 3-octanol, 1-octanol, and 3-octanone . Compoun species . The uns species . Zhuang  species  found thB. edulis, a higher 1-octen-3-ol and (E)-2-octen-1-ol content was found in ultrasonicated samples , and no 3-octanol was established in ultrasonicated B. edulis. Comparing the VC in thermally treated and ultrasonicated unfermented C. cibarius samples, the content of the main VC (1-octen-3-ol and oct-(2E)-enal) remained similar, but the hexanal content in thermally treated C. cibarius was 1.5 times higher than that in ultrasonicated samples. The content of the main VC in R. caperata  was similar in thermally treated and ultrasonicated samples .Comparing the VC profile of fresh mushrooms with thermally treated and ultrasonicated ones, a greater variety of VC was identified in treated mushrooms . CompariUsually, the high temperature elicits a significant decrease in total volatile compounds because most of these compounds are thermosensitive . Selli eC. cibarius in which the main VC was oct-(2E)-enal). Considering the influence of the treatment before fermentation on the 1-octen-3-ol content in mushrooms, various tendencies were established: in thermally treated and fermented B. edulis, the 1-octen-3-ol content was, on average, 2.0 times higher than in ultrasonicated fermented samples; however, in C. cibarius and R. caperata, a higher 1-octen-3-ol content was established in ultrasonicated and fermented samples . Other VC which accounted for more than 5% of the VC profile were: (E)-2-octen-1-ol in thermally treated and fermented B. edulis; 3-methyl-1-butanol, 3-octanone, and 3-octanol in ultrasonicated and fermented B. edulis; acetoin, 3-methyl-1-butanol, 3-octanone, and 2-pentylfuran in thermally treated and fermented C. cibarius; 3-octanone, 3-octanol, oct-(2E)-enal, and 1-octanol in ultrasonicated and fermented C. cibarius; hexanal, 2-heptenal, and oct-(2E)-enal in thermally treated and fermented R. caperata; and 3-methyl-1-butanol, ethyl tiglate, benzaldehyde, 3-octanone, and hexanoic acid ethyl ester in ultrasonicated and fermented R. caperata.The greatest variety of VC was shown in thermally treated and/or ultrasonicated fermented samples . In mostC. cibarius, in which the main VC was oct-(2E)-enal). Considering the influence of the treatment before fermentation on the 1-octen-3-ol content in mushrooms, various tendencies were established: in thermally treated and fermented B. edulis, the 1-octen-3-ol content was, on average, 2.0 times higher than in ultrasonicated fermented samples; however, in C. cibarius and R. caperata, a higher 1-octen-3-ol content was established in ultrasonicated and fermented samples . Other VC which accounted for more than 5% of the VC profile were: (E)-2-octen-1-ol in thermally treated and fermented B. edulis; 3-methyl-1-butanol, 3-octanone, and 3-octanol in ultrasonicated and fermented B. edulis; acetoin, 3-methyl-1-butanol, 3-octanone, and 2-pentylfuran in thermally treated and fermented C. cibarius; 3-octanone, 3-octanol, oct-(2E)-enal, and 1-octanol in ultrasonicated and fermented Cantharellus; hexanal, 2-heptenal, and oct-(2E)-enal in thermally treated and fermented R. caperata; and 3-methyl-1-butanol, ethyl tiglate, benzaldehyde, 3-octanone, and hexanoic acid ethyl ester in ultrasonicated and fermented R. caperata.In most of the fermented mushroom samples, 1-octen-3-ol was the main VC  [The odour of benzaldehyde is described as almond, fruity, powdery, nutty, and benzaldehyde-like, and the taste is sweet, oily, almond, cherry, nutty, and woody; the odour of hexanal is green, fatty, leafy, vegetative, fruity, and clean with a woody nuance, and the taste is green, woody, vegetative, apple, grassy, citrus, and orange with a fresh, lingering aftertaste; the odour of 3-methyl-1-butanol is fusel, alcoholic, pungent, ethereal, cognac, fruity, banana, and molasses, and the taste is fusel, fermented, fruity, banana, ethereal, and cognac; 3-octanone, used as a flavour and fragrance ingredient ,47 and cophilus) ; its odoophilus) .Lactobacillus, produce unique flavour profiles in fermented foods [In the present study, the increased variety of VC in fermented mushrooms is related to the fact that most LAB strains, as well as ed foods . VC proded foods . Biochemed foods .The overall acceptability and emotions induced in consumers by edible mushrooms are given in B. edulis samples , and a moderate positive correlation was found between the emotion ‘neutral’ and samples’ pH . The emotion ‘happy’ was expressed most strongly for ultrasonicated and fermented B. edulis samples. Other samples induced significantly weaker expression of the emotion ‘happy’, and a moderate positive correlation was found between the emotion ‘happy’ and samples’ yellowness (b*) . Ultrasonicated and fermented B. edulis samples also induced the highest intensity of the emotions ‘sad’, ‘angry’, ‘surprised’, and ‘contempt’, and a moderate positive correlation was found between the emotion ‘angry’ and samples’ pH . The strongest expression of the emotion ‘scared’ was established for thermally treated R. caperata samples. This perhaps could be explained by these mushrooms looking similar to toxic ones.However, significant differences were found between the emotions induced in judges. The emotion ‘neutral’ was expressed most strongly for the thermally treated unfermented samples of all species and for ultrasonicated and fermented p = 0.027); however, significant correlations were found between the emotion ‘happy’ and hexanal, benzaldehyde, 3-octanone, octanal, 3-ethyl-2-methyl-1,3-hexadiene, oct-(2E)-enal, 6-methyl-hept-2-en-4-ol, and nonanal . It can be concluded that an increase in the content of benzaldehyde, 3-octanone, octanal, and nonanal reduced the intensity of the ‘happy’ emotion expression in consumers. In contrast, increasing the content of hexanal, 3-ethyl-2-methyl-1,3-hexadiene, oct-(2E)-enal, and 6-methyl-hept-2-en-4-ol led to the stronger expression of the emotion ‘happy’.Correlations between the VC and overall acceptability of thermally treated and ultrasonicated unfermented and fermented mushrooms, as well as between VC and the emotion ‘happy’, are given in p = 0.0001; r = 0.999, p = 0.0001; r = 0.819, p = 0.0001; r = 0.622, p = 0.006; r = 0.999, p = 0.0001; r = 0.999, p = 0.0001, respectively). An increase in the percentage of benzaldehyde, oct-(2E)-enal, (E)-2-octen-1-ol, and 2-nonanone in the VC profile of fermented mushrooms reduced the intensity of the ‘happy’ emotion expression .Significant positive correlations were also established between the emotion ‘happy’ induced by fermented mushrooms and 3-methyl-1-butanol, 2,3-butanediol, 3-octanone, 3-octanol, 2-iodo-3-methyl-butane, and N-hexyl-1-hexanamine . Putrescine was found in B. edulis and C. cibarius . In addition, cadaverine was established in fresh C. cibarius (14.6 mg/kg). Comparing the sum of BA in fresh mushrooms, the highest was established B. edulis; that in C. cibarius and R. caperata was 7.3 and 7.2 times lower, respectively.The content of separate BA a,b and tB. edulis . As in fresh samples, phenylethylamine and spermidine were established in all thermally treated and ultrasonicated mushrooms. Additionally, putrescine was found in both thermally treated and ultrasonicated B. edulis and C. cibarius. However, the sum of BA in thermally treated and ultrasonicated samples was lower in treated samples of B. edulis and C. cibarius than in fresh mushrooms: 2.2 and 1.2 times lower, respectively, in thermally treated samples, and 1.9 and 1.2 times lower, respectively, in ultrasonicated samples.In comparing thermally treated and ultrasonicated unfermented mushroom groups, tryptamine was found in thermally treated and ultrasonicated B. edulis (75.5 mg/kg), and cadaverine was established in both thermally treated and ultrasonicated fermented B. edulis samples . Ultrasonication before fermentation, in comparison with thermal treatment before fermentation, increased the sum of BA in B. edulis and R. caperata samples . In contrast, ultrasonication before fermentation reduced the sum of BA in C. cibarius samples .When comparing thermally treated and ultrasonicated fermented mushroom groups, tryptamine was found in ultrasonicated and fermented p ≤ 0.0001). This could be explained by the fact that moulds and yeasts could also contribute to BA production [Microbial activities could lead to amino acid decarboxylation which induces BA formation. . The amooduction . Howeveroduction . Certainoduction . The rouoduction . The redoduction , which moduction .B. edulis, were in this range. Toxicological effects and various health disorders  are related to the high concentration of BA consumed by humans [The total BA content in food should not exceed 750–900 mg/kg . Accordiy humans . These ey humans . HoweverB. edulis, C. cibarius, R. caperata, or even other fermented mushrooms have not been analysed for the content of BA. Studies on BA in mushrooms are quite scarce and focus on raw or processed  mushrooms, but the reported concentrations of BA significantly differ among species ,61,62. SL. casei LUHS210 and L. uvarum LUHS245 were evaluated. As pre-treatment, ultrasonication or prolonged thermal treatment were applied before fermentation. Pre-treatment significantly affected physicochemical properties, while the greatest variety of volatile compounds was obtained after pre-treatment and fermentation of tested mushrooms. The emotional response was a more sensitive approach to measuring the perception of the tested mushrooms. All ultrasonicated and fermented B. edulis samples elicited the expression ‘happy’. The lowest sum of BA was found in thermally pre-treated and fermented R. caperata, while the highest one was in ultrasonicated and fermented B. edulis. Finally, it can be stated that despite good overall acceptability, it is important to select appropriate LAB strains for the fermentation of edible mushrooms to ensure their safety in the case of BA formation.Although lactic acid fermentation is traditionally used to preserve mushrooms, studies in this field are still scarce. Therefore, there is a need to analyse the impact of this biological approach on the quality and safety of mushrooms. In the present study, the quality and safety traits of edible mushrooms fermented with


Smart city construction plays an important role in environmental governance and public health. Based on the panel data of 216 prefecture-level cities across China during the period 2009–2018, this study uses the multi-time difference-in-differences method to evaluate the haze reduction effect of smart city construction. The estimated results demonstrate that the construction of smart cities can reduce haze pollution in pilot cities significantly. The main conduction mechanisms are the technical effects and the environmental regulatory effects that promote the reduction of corporate emissions. The heterogeneity analyses show that the haze reduction effect of smart city construction is more evident in southern cities, inland cities and resource-efficient cities. In addition, the benefit analyses show that smart cities can reduce the carbon emission intensity and promote economic growth. These results provide empirical support for accelerating the construction of a new type of smart city and building a new type of people-oriented urbanization. AlthougSmart city construction provides a new way to solve various urban diseases caused by rapid industrialization and urban expansion and promote sustainable urban development. In 1992, Singapore took the lead in proposing the “Smart Island Project” as a prototype of the “Smart City” concept . In 2008The Chinese government is also keeping pace with advanced city construction around the world. The Department of Information Industry of Guangdong Province signed a memorandum of cooperation with IBM in 2009 to strengthen the field of information technology and information services to promote the construction of smart cities, marking the official opening of the construction of smart cities in China, thereby opening the practical exploration of the construction of smart cities with Chinese characteristics . The MinHowever, the question whether or not such new urbanization measures are conducive to improving environmental quality and promoting green and sustainable urban development urgently needs to be answered. Thus, this paper focuses on the impact of smart city construction on haze pollution by taking the smart city pilot policy as an entry point. Does the construction of smart cities help reduce the level of haze pollution in pilot cities? If the answer is yes, what are the mechanisms that influence haze pollution? Are there regional heterogeneities in their haze pollution control effects? Can smart city construction achieve collaborative governance of haze and carbon emissions under the dual challenges of air pollution and climate change? Are the governance effects achieved at the expense of economic benefits? All these issues deserve to be discussed further.Existing literature reviews mainly focus on two aspects: the first is about the influencing factors of haze pollution. Many scholars have examined the factors influencing haze pollution from multiple perspectives and have achieved many results. Fiscal decentralization, industrial agglomeration, energy prices, transportation infrastructure, and urbanization are all important factors that influence haze pollution ,19,20,212.5 from Dalhousie University and converts it into panel data of prefecture-level municipalities, which overcomes the barrier of discontinuity in the available data and overcomes the possible endogeneity problems of using the DID method. (3) Based on mechanism analysis, this study not only explores the haze reduction mechanism of smart city construction, but also finds that smart city construction can reduce carbon emission intensity and promote economic growth in pilot cities, which provides a new path to effectively solve urban diseases and promote high-quality urban development.By combining the above literature, many useful results have been obtained concerning the research on the policy effects of smart city construction, but there are a few studies in the literature directly discussing the effects of smart city construction on haze pollution, which provides a research opportunity for this paper. As such, this study takes the panel data of 216 prefecture-level cities in China from 2009 to 2018 as research sample and uses the multi-time difference-in-differences (DID) method to examine the impact of smart city construction on haze pollution. The contributions of this study include: (1) Existing studies focus on the economic effects of smart cities, where limited attention is paid to environmental effects, especially haze management effects. This study contributes to the literature by focusing on the effect of smart city construction on haze reduction, which may improve urban governance and residents’ happiness. This study contributes to the literature by focusing on the effect of smart city construction on haze reduction, which may improve urban governance and residents’ happiness. (2) In terms of data processing and method selection, this paper takes the raster PMFundamentally, smart city construction is a systematic and innovative project of reform, which is the centralized embodiment of scientific and technological innovation, organizational innovation, and resource-allocation innovation. In theory, this innovation effect is primarily reflected in haze control through smart environmental protection, technology, and haze-control technology, as well as smart transportation and smart buildings.First, smart environmental protection requires a total of six modern information technologies, including comprehensive use of the Internet of Things, cloud computing, intelligent GIS, integrated remote-sensing monitoring, massive data mining, and environmental model simulation technology, to provide comprehensive solutions for environmental-quality-monitoring systems, environmental early warning and forecasting systems, environmental emergency management systems, and other areas of environmental protection. This enables environmental policymakers and air pollution controllers to accurately grasp trends in environmental quality and thus provide data support for haze-reduction actions. Second, technological innovation is an important means for haze management. In the process of smart city construction, the green technology for haze control has been fully utilized, such as the “multi-scale spraying and haze removal technology” used in Chengdu City and the haze removal tower built in Xi’an City. Third, the development of intelligent transportation in the construction of smart cities also indirectly contributes to haze control. The planning of the shortest travel path or the best route in the intelligent transportation system helps the public to choose the best driving route, thus effectively alleviating traffic congestion and reducing pollutant emissions. Fourth, smart city construction is conducive to the promotion of smart buildings, which use energy-saving materials and technologies in the planning, design, construction, and use process, thereby alleviating haze pollution.Based on the above understanding, the first hypothesis is proposed as follows: Hypothesis 1.Smart city construction is beneficial for reducing the level of haze pollution in pilot cities.At the same time, possible transmission routes are discussed as follows:The first is the technology effect. Technological innovation is an important driving force for green development and important support for fighting the battle of pollution prevention and control, thereby promoting ecological civilization construction and high-quality development. The construction of smart cities is conducive to the promotion of research and development of green technology and its application, and the technological progress it offers can effectively improve capabilities for pollution prevention and control. For example, the intelligent environmental protection big data system can observe and analyze the air pollution situation in real time, such that environmental managers can use the system to effectively manage pollution sources and polluting industries, thereby reducing haze pollution levels.Second are the structural effects. Smart city construction reduces haze pollution by promoting development of smart city industry and upgrading of industrial structure. Unlike the traditional development model, the smart city is a new development model supported by a new generation of information technology, which has generated a huge demand for emerging wisdom technologies and promoted the formation and development of the smart city industry and its associated industries. The development of the smart city industry is conducive to accelerating the upgrading of urban industrial structures and the construction of a modern urban industrial system, thus creating a smart city ecological industrial chain. Among them, characteristics of low pollution, low energy consumption, low emissions, high-tech, and high industrial added value of the smart city industry will reduce haze pollution.Third is the environmental regulation effect. Environmental regulation, as an important element of social regulation, is an important tool for improving haze pollution. Distinct from general pollution control policies, smart cities can strengthen environmental regulations by strengthening environmental monitoring systems, thereby reducing haze pollution. On the one hand, the integrated environmental monitoring system of smart cities can be used to monitor the pollutant emission status of enterprises in an all-around and all-time manner and restrict the illegal emission behavior of relevant enterprises. On the other hand, in the process of smart city construction, the public has access to more comprehensive pollution emission data, which enhances the public awareness regarding environmental protection, thereby enhancing the intensity of public-participation-based environmental regulation.Considering the above factors, we present the second hypothesis:Hypothesis 2.Smart city construction affects haze pollution levels in three ways, through technological, structural, and environmental regulation effects.In 2012, 2013, and 2014, construction of smart cities in China was introduced in batches through promotion of pilot projects which were expected to inevitably ensure operational experience and help form long-term operational mechanisms. However, the overall knowledge is still in its infancy.The smart city pilot policy in China is a good “quasi-natural experiment” in that the selection of the pilot is not randomly assigned. Therefore, this paper uses the multi-time difference-in-differences method, which is one of the methods in the quasi-natural experiment, to estimate its impact on haze control. Research subjects were identified by the smart city pilot list announced by the Ministry of Housing and Construction, and the prefecture-level cities that have implemented the smart city pilot were selected as the test group samples, while the remaining prefecture-level cities that were not approved for smart city construction were considered as the control group sample. A prefecture-level city is one of China’s administrative divisions, with the same administrative status as a region, autonomous prefecture, or union, under the jurisdiction of a province or autonomous region. The following criterion were used to construct the sample: (1) Exclude prefecture-level cities that are not fully piloted; that is, those that implement smart city construction only in certain districts or counties within the city. The reason is that some of the pilot units of smart cities are implemented in county-level areas, e.g., Chaoyang District, Beijing, and Heihe County, Jinzhou City. Setting these cities with partial regional implementation of pilots as pilot cities would lead to an overestimation of the estimated results. (2) Exclude newly established prefecture-level cities within the study interval (2009 to 2018), such as Bijie and Tongren. During the study period of this paper’s sample, these cities experienced administrative divisional adjustments. For example, in 2011, the State Council approved the abolition of the Bijie district to establish the prefecture-level Bijie city; on 22 October 2011, the Tongren district was abolished to establish the prefecture-level Tongren city. Additionally, there is a large number of missing statistics for these cities before their elevation to non-prefecture-level cities. (3) Exclude prefecture-level cities with serious data deficiencies, such as Lhasa. Finally, the dataset of 91 cities in the experimental group and 125 in the control group was obtained. Among the samples in the experimental group, there were 35, 33, and 23 prefecture-level cities that implemented smart city construction in 2012, 2013, and 2014, respectively. The model was set as follows:The study herein focuses on assessing the haze control effect of smart city construction while considering the possible endogeneity problems caused by omitted variables, and following Sun et al., a series of control variables are also included . The var(1)2.5 concentrations to measure the haze pollution level [10, PM2.5, and other haze-related data, which means that the sample interval 2009–2018 contains incomplete data. Therefore, the continuous PM2.5 data published by the Atmospheric Composition Analysis Group of Dalhousie University in Canada were used in this study from 2000 to 2018 [Haze pollution level Following Chen and Xiao, this study used annual average PMon level . Since 2 to 2018 . However(2)2 emission and CO2 emission intensity. The DMSP and VIIRS night-light data were used to estimate the CO2 emissions and the CO2 emission intensity of the sample cities, which were, respectively, used as explained variables for regression in the other benefit analysis section. The data come from the study of Chen et al. [COn et al. .(3)Smart City pilot policy. Whether a city is a smart city pilot will be measured by a dummy variable. The dummy variables were assigned according to the smart city pilot list announced by the Ministry of Housing and Construction. If a city was identified as a smart city pilot, the dummy variable was assigned a value of 1 for the current year and subsequent years; otherwise, it was assigned a value of 0. The values of this variable were manually compiled.(4)Control Variables were the level of economic development, expressed as the logarithm of the real GDP per capita of each prefecture-level city, which also incorporates the squared term of the level of economic development to test the existence of the Kuznets curve hypothesis; climate condition, measured by the logarithm of average annual precipitation; government size, measured by the logarithm of government fiscal spending as a share of GDP; the level of financial development, measured by the logarithm of the loan balance of financial institutions as a share of GDP at the end of the year; the level of greening, expressed as the logarithm of green space per capita in prefecture-level municipalities; the level of openness to the outside world, measured by the logarithm of the proportion of the actual use of foreign investment in GDP in that year. Basic data were obtained from the China City Statistical Yearbook and the China Regional Statistical Yearbook for each year. Descriptive statistics of the main variables are shown in 2.5 concentration in the pilot city compared to the control group. In total, the estimated coefficients of the baseline model capture the average treatment effect over seven years, which implies that the smart city pilot policy contributed to a reduction in PM2.5 concentrations of about 0.181 ug/m3 per year. The annual average PM2.5 concentration in the control group cities was 42.6 micrograms per cubic meter. The smart city construction resulted in a 2.98% reduction in the annual average PM2.5 concentration in the pilot cities.2.5 is changing at 13.906 (the U-shaped inflection point is at 13.906), and the maximum value of the logarithm of per capita GDP in the sample of this paper is 12.655. This indicates that the relationship between economic development and haze pollution is already in the second half of the inverted U-shaped curve, which is inconsistent with the findings of Shao et al. [The estimated coefficients of the control variables show some interesting results. The estimated coefficients of actual GDP per capita and its squared term are −35.677 and 1.283, respectively, both of which are significantly negative at the 1% level in o et al. . Climatikth year the smart city pilot policy was implemented. Specifically, k < 0 indicates the kth year before the pilot implementation; k = 0 indicates the year of smart city pilot implementation; k > 0, it indicates the kth year after pilot implementation. By the end of 2018, the first and last batches of smart city pilots were implemented in 2012 and 2014, respectively, and the sample interval in this study is from 2009 to 2018. As such, this study covered the first five and last six years of pilot implementation. The first year prior to the establishment of the smart city is taken as the base period, so k < 0, it means that the cities in the control and experimental groups satisfy the parallel trend hypothesis in terms of haze pollution levels.A basic condition which must be satisfied for the policy-effect assessment using the DID method is that without the intervention of the smart city construction policy, the urban haze pollution levels in the control and treatment groups should have a common trend of change; that is, a parallel trend assumption needs to be satisfied. Following the method of Beck et al., the dynamic DID model is used to perform a parallel trend test . The spek < 0, and both fluctuate around zero, indicating that the treatment and control groups satisfy the parallel trend hypothesis. When The effect of urban characteristics that do not vary over time on haze pollution was controlled by adding the urban fixed effects above, but some of the omitted variables vary over time, which affects the accuracy of the estimation results. Therefore, to test whether the baseline estimation results were affected by omitted variables, such as that in Chetty et al., this paper constructs DID variables based on a randomly generated dummy list of smart city pilots and conducts 500 baseline regressions; the specific regression results and their distributions are shown in The most ideal situation using the difference-in-difference method is that pilot and non-pilot cities are randomly selected. In reality, however, the determination of the list of smart city pilots is not random. According to the “Notice on National Smart City Pilot Work” issued by the Ministry of Housing and Construction, the declaration of smart city pilot work requires the inclusion of smart city construction work in the local National Five-Year Plan or relevant special plans, the completion of the preparation of the outline of the smart city development plan, and the existence of a clear funding plan and guaranteed channels. These conditions can reflect the inherent differences between cities, which have an impact on urban haze pollution levels with time trends, leading to bias in the estimation results. Following Lu et al., the stochastic selection problem of pilot cities was mitigated by including, in the benchmark equation, an interaction term between the benchmark factors affecting the selection of pilot cities and the linear trend over time . The speAmong them, Other environmental policies pursued during the sample period of this study may have confounded the baseline estimation results. Therefore, this study compiled the large environmental policies implemented since 2009 at the city level, specifically the Low-Carbon-City Pilot Policy implemented in 2010, the Special Emission Limits for Air Pollutants Policy, and the Air Pollution Prevention and Control Action Plan implemented in 2013. Referring to the way the smart city policy variables are set up, cross-terms were constructed based on the corresponding list of cities and the time point of policy implementation, which were added to the baseline regression equation, thus controlling the effects of these three environmental policies on the estimation results. The corresponding estimated coefficients are reported in Column (2) of (1) Special samples and outliers were excluded. First, considering that the large-scale haze control actions pioneered in the Beijing–Tianjin–Hebei region since 2013 may interfere with the results, all prefecture-level cities in the Beijing–Tianjin–Hebei region were excluded from the regression, and the regression results are shown in column (1) of (2) The explanatory variables were treated with a one-period lag. Considering the lagged effect of smart city construction on haze pollution, a lagged one-period treatment was applied to the smart city variable. To avoid errors in the joint cubic equation, all explanatory variables were lagged by one period, as shown by Shen and Jin . The reg(3) Following the method in Fan and Tian, time-counterfactual tests were conducted by changing the time of policy implementation . The spe2 emissions for the following reasons. First, SO2 pollution is an important factor in haze pollution, and also a key environmental indicator concerned by Chinese governments. China set up acid-rain-control zones or sulfur dioxide pollution-control zones to control sulfur dioxide pollution as early as 1998, and further included sulfur dioxide emissions as a binding indicator in the national economic and social development plan from 2006. Therefore, the changes in SO2 emissions could reflect the intensity of environmental regulation by local governments to a certain extent. In addition, the data of sulfur dioxide emissions of each prefecture-level city are published in the China City Statistical Yearbook since 2003, which brings convenience to this study. Second, although the emission of particulates is an important source of PM2.5 pollution, no official data on particulate matter emissions at the city level have been published in China to date. As such, the specific mechanism of haze pollution reduction by smart city construction can be tested according to Equations (4)–(6). This is true specifically if The results of the above-mentioned types of tests show that the smart city construction significantly reduces the level of haze pollution. Therefore, what are the specific conduction mechanisms? According to the theoretical analysis, the smart city construction may affect haze pollution in three ways: technological innovation effect, industrial structure effect, and environmental regulation effect. Herein, following the method of Baron and Kenny, causal stepwise regression was used to identify the three aforementioned possible transmission pathways . The speThe results of the mechanism analysis are listed in The average effect of smart city construction on haze pollution was analyzed above. However, the analysis based on the overall sample may have masked the differences that existed between regions. In particular, there are large differences in geographic location, resource endowment, population, and economic agglomeration among regions in China, resulting in very different levels of haze pollution from city to city. To this end, this study further tests whether smart city construction has a heterogeneous effect on haze pollution. First, considering that winter heating is an important factor of haze pollution, the sample cities were divided into northern and southern cities for analysis. Columns (1) and (2) in To cope with the dual challenges of environmental pollution and climate change, China proposed the collaborative governance idea of “pollution reduction and carbon reduction”. Therefore, this study investigates whether the smart city construction has the same inhibiting effect on carbon emissions in addition to the haze governance effect; that is, whether the smart city pilot can realize the collaborative governance of haze pollution and carbon emissions. For this reason, this study adds carbon dioxide emissions and carbon intensity as explanatory variables for regression, and the regression results are shown in columns (1) and (2) of 2.5 concentration. Secondly, the technical and environmental regulation effects are the main pathways in smart city construction for reducing the level of haze pollution. Thirdly, the impact of smart city construction on haze pollution levels is influenced by factors such as the geographical location of the city and resource endowment, and its haze reduction effect is more obvious in southern, inland cities and resource-efficient cities. Fourthly, smart city construction reduces haze pollution while reducing carbon emission intensity and promoting regional GDP, which demonstrates that smart city construction can not only achieve collaborative governance of haze and carbon emissions, but also considers economic growth at the same time.Taking the smart city pilot policy as the entry point, this study assesses the haze governance effect of smart city construction using a multi-time DID method based on a panel of 216 prefecture-level cities across China from 2009 to 2018. The following results can be obtained: firstly, the smart city construction can significantly reduce the level of urban haze pollution. Specifically, it can achieve a 2.98% reduction in the annual average PMThe policy implications of this study are as follows. First, although the direct goal of smart city construction in China is to improve the level of urban governance and service capacity, green development in the form of intelligent environmental protection, energy-saving, and other forms throughout the entire process of smart city construction was also improved. The empirical results of this study also show that smart city construction can reduce the level of haze pollution in pilot cities; as such, improving the level of smartness can be an alternative path to reduce urban air pollution and improve the quality of healthy life. Second, smart city construction reduces the level of haze pollution by promoting technological innovation and improving the intensity of environmental regulation rather than adjusting the industrial structure. Thus, the future construction of smart cities should be optimized to adjust to the urban industrial system, to strengthen the construction and improvement of new industrial chains, support the development of new industries with high technology and low energy consumption, improve the degree of integration and application of new-generation information technology in the traditional industries of the city, and promote the transformation of industries into environmentally friendly ones. Third, the haze-reduction effect of smart cities is more significant in southern and inland cities; on the one hand, the smartness of heat and other energy systems remains to be improved, while on the other hand, the technological innovation level of industrial development in inland cities remains to be improved with the development of smart cities. Fourth, under the new round of technological revolution and industrial change, green development requires the construction of new smart cities not only based on digital intelligent technologies such as the Internet of Things and cloud computing, but also by adhering to focusing on the human perspective to promote the realization of its diversified goals, combined with the development of local advantages and characteristics to improve the quality of cities.2.5 have large variance in a year. This may have some influence on the model results. However, it is difficult to calculate the variance of PM2.5 concentrations due to the unavailability of daily mean or monthly mean PM2.5 concentration data from 2000–2018. This issue will be further investigated if higher-frequency data are available. Second, haze pollution is spatially metastatic and mobile, so spatial factors can be further incorporated in the future to select a suitable method to analyze the spatial effects of smart-city pilot policy implementation in the mechanism analysis section. Third, in addition to the smart city pilots in this paper, the construction of emerging smart cities has been proposed in recent years, and the policy effects of their construction can be evaluated in future studies. Finally, this paper measures the overall effect of the pilot construction of smart cities on haze management. This may be considered further by studying the threshold effect of the different level of urban smartness on haze reduction, exploring the specific threshold values that produce the effect during policy implementation.Based on the research in this paper, there are also some limitations, and the following aspects can be expanded. First, haze pollution shows obvious cyclical characteristics. In China, haze pollution is concentrated in a November–March outbreak, which makes PM


Eimeria tenella are limited. In this study, the coding sequence of E. tenella rhoptry protein 35 (EtROP35) was cloned, and then its localization, expression in parasite, potential role within invasion, and protective efficacy were investigated. Sequence analysis and subcellular localization revealed that EtROP35 is a rhoptry protein of E. tenella. Sporozoite invasion-blocking assay and protective efficacy indicated that EtROP35 might be involved in the parasite invasion process and may be a potential vaccine candidate against E. tenella.Rhoptry proteins (ROPs) of phylum Apicomplexa parasites are important secretory virulence factors as well as candidate vaccines. However, studies on ROPs of Eimeria tenella ROPs are not well studied. This study identified and characterized a novel E. tenella ROP (EtROP35), which showed the highest levels among 28 putative ROPs in previous sporozoite and merozoite transcriptomes. Sequence analysis showed that EtROP35 contains an N-terminal secretory signal and a protein kinase domain including eight conserved ROP35-subfamily motifs. Subsequent experiments confirmed that it is a secretory protein. Subcellular localization revealed it localized at the apical end of the sporozoites and merozoites, which was consistent with the ROPs of other Apicomplexan parasites. To further understand the biological meaning of EtROP35, expression levels in different developmental stages and sporozoite invasion-blocking assay were investigated. EtROP35 showed significantly higher levels in sporozoites (6.23-fold) and merozoites (7.00-fold) than sporulated oocysts. Sporozoite invasion-blocking assay revealed that anti-EtROP35 polyclonal antibody significantly reduced the sporozoite invasion rate, suggesting it might participate in host cell invasion and be a viable choice as a vaccine candidate. The immunological protective assays showed that EtROP35 could induce a high level of serum IgY and higher mean body weight gain, and lower cecum lesion score and oocysts excretion than the challenged control group. These data indicated that EtROP35 had good immunogenicity and may be a promising vaccine candidate against E. tenella.Rhoptry proteins (ROPs) of Apicomplexa are crucial secreted virulence factors and sources of vaccine candidates. To date,  E. tenella is a highly destructive and virulent pathogen threatening global poultry industry [Toxoplasma, Plasmodium, Eimeria, Babesia, Neospora and Cryptosporidium. As an obligate intracellular apicomplexan protozoan parasite, the infection and intracellular development of E. tenella rely on three unique apical secretory organelles: rhoptries, micronemes, and dense granules. Rhoptries discharging ROPs are club-shaped organelles divided into two distinct substructures (posterior bulb and anterior neck) [industry . It beloor neck) ,3. ROPs or neck) ,5,6.E. tenella genome, 28 are likely to be ROPs [E. tenella ROPs have been reported, including EtROP1, Et-ROPK-Eten5-A, EtROP17 and EtROP30 [Toxoplasma gondii [T. gondii have been widely studied since tachyzoite can be continuously propagated in vitro and a set of powerful genetic manipulation and functional analysis tools can be applied, some ROPs  have been proved to be key virulence factors and control targets. In contrast, studies on E. tenella ROPs were limited, possibly due to in-vitro culturing and genetic manipulation difficulties.Due to the presence of protein kinase domains, ROPs are specific members of eukaryotic protein kinases (ePK) . Among t EtROP30 ,10,11,12a gondii . Et-ROPKT. gondii rhoptry protein 35 (TgROP35) in the evolutionary relationship analysis. TgROP35 strongly stimulates the host immune response and is considered a promising drug target and vaccine candidate antigen [E. tenella were evaluated.A previous RNA-Seq analysis of sporozoite and merozoite transcriptomes found the highest concentration of EtROP35 among 28 putative ROPs . This ob antigen . Here, EE. tenella separated from Shandong (SD-01 strain) was propagated in our laboratory [boratory . Unsporuboratory . The DonE. tenella sporozoite cDNA by PCR . Bioinformatic analyses were performed using ExPASy . All EtROP35 homology protein sequences from Eimeria species were downloaded from the NCBI database : E. tenella (XP_013228816.1), E. necatrix (XP_013434354.1), E. brunetti (CDJ51900.1), E. acervulina (XP_013247055.1), E. maxima (XP_013335700.1), E. praecox (CDI74752.1), and E. mitis (XP_013358067.1).The coding sequence of EtROP35 gene was cloned from E. coli BL21 carrying a pET32a-EtROP35 plasmid and purified by His-tag Protein Purification . The anti-EtROP35 polyclonal antibody was generated to localize the parasites using indirect immunofluorescence assay (IFA) following a previous method [The recombinant EtROP35 protein was expressed in s method . BrieflyAccording to a previous method , sporozoE. tenella were detected by employing qRT-PCR using SYBR Green I   with the 18S rRNA as the standard reference gene . The translation levels of EtROP35 were detected by western blot using the anti-EtROP35 polyclonal antibody (1:1000). Parasites of four stages were ground in liquid nitrogen and the protein lysates were prepared in RIPA buffer including protease inhibitor . The ImageJ 1.8.0 software  was used to measure Grey values.The transcription levels of EtROP35 in four stages  of 5 sporozoites per well of a 24-well plate. After culturing for 12 h, the uninvaded free sporozoites were gathered and counted. The inhibition rates were calculated from the percentage of the uninvaded sporozoites.The sporozoite invasion-blocking assay was conducted based on the observation that sporozoites invaded DF-1 (chicken embryo fibroblast cell line) cells. The sporozoites were pretreated with anti-EtROP35 serum and mouse serum at 37 °C for 2 h with multiple dilutions, separately. DF-1 cells were infected with 3 × 10E. tenella sporulated oocysts were orally ingested in the EtROP35 group and the challenged control group (8000/chicken), while the unchallenged group were ingested PBS. The cecal lesion scores of the chickens (n = 5) from each group were recorded using a continuous number from 0 (none) to 4 (severe) at 5 days post-infection (dpi); the grading criteria were in line with the previous description [Three groups (20 chickens/group) were used to evaluate the immune protection of EtROP35.Chickens were immunized in the neck region subcutaneously with 200 µL/chicken 100 µg) purified His-EtROP35 protein in complete Freund’s adjuvant at 7-day age in EtROP35 group. The challenged and unchallenged control groups received injections of complete Freund’s adjuvant emulsified in PBS. A second immunization was given at the same dose in incomplete Freund’s adjuvant at the following week. One week later, fresh cription . Bodywei0 µg puri450) was recorded by an automated microplate reader .The serum antibody levels (five chickens/group) were detected by ELISA at 0 and 7 dpi following reported methods . Brieflyt-test. The statistical significance level was expressed as p < 0.05.Statistical analysis was performed using GraphPad Prism8 . Data were analyzed by the two-tailed E. tenella were divided into ten subfamilies by evolutionary adaptation and into active and inactive enzymatic ROPs based on the presence or not of the “catalytic triad” [253, D344, and D362) essential for catalysis . Bioinformatics analysis confirmed the presence of a 25-aa N-terminal secretory signal sequence and an ePK domain (197–60 aa) A. From ac triad” . Accordiatalysis B, indicaia mitis C. EtROP3complexa D.E. tenella sporozoites and merozoites (E. tenella.Recombinant His-tag EtROP35 protein was expressed and purified by His bind purification Kit (TransGen Biotech), we prepared anti-EtROP35 polyclonal antibody to recognize EtROP35. The polyclonal antibody could recognize the recombinant EtROP35 protein A. A uniqrozoites D; this op < 0.01) compared to that in the unsporulated oocysts by 3.07-, 8.91-, and 10.90-fold, respectively (p > 0.05).Results of the qRT-PCR showed that transcription level of EtROP35 was higher in the sporulated oocysts, sporozoites, and merozoites (ectively A. MerozoE. tenella were detected through Western blot (p < 0.001) compared to that in the sporulated oocysts by 6.23- and 7.00-fold (p < 0.05).The translation levels of EtROP35 during the different developmental phases of ern blot B. EtROP3.00-fold C. MerozoE. tenella sporozoite invasion of host cells.The sporozoite invasion-blocking assay was performed to measure the impact of EtROP35 on the host cell invasion capacity of the sporozoites. Pretreatment with anti-EtROP35 polyclonal antibody significantly reduced the sporozoite invasion of the host cells . The invE. tenella recognized the recombinant EtROP35 protein . IgY levels were substantially higher at 7 dpi than at 0 dpi in the EtROP35-immunized group (p < 0.05) and the challenged group (p < 0.001). These results indicated that EtROP35 induced a high-level serum antibody response for E. tenella infection.Levels of IgY in serum against the EtROP35 at 0 and 7 dpi were detected by ELISA B. Levelsp < 0.0001) in the body weight gain (85.4 ± 5.3 g) compared to the challenged control group (56.2 ± 6.1 g) (p < 0.0001). The mean cecum lesion score (1.58 ± 0.17) was significantly lower in the EtROP35-immunized group of chickens (p < 0.001) compared to that in the challenged control group (2.48 ± 0.21) (p < 0.0001) between the EtROP35-immunized group and the challenged control group  C, with t ± 0.21) D. The nuol group E, while E. tenella (EtROP35) was identified and characterized, showing features consistent with other ROPs of Apicomplexan members. EtROP35 was expressed at relatively higher levels during the parasite invasion stages (sporozoites and merozoites) and might participate in host cell invasion. Furthermore, EtROP35 exhibited good immunogenicity and emerged as a promising vaccine candidate.In the current study, a novel ROP of E. tenella.Protein transport and secretion mechanisms are evolutionarily conserved in Apicomplexan protozoans . MoreoveE. tenella, the reported proteins associated with host cell invasion are mainly microneme proteins [Host cell invasion is crucial for Apicomplexan parasite infection and intracellular parasitism. Studies have reported the association of ROPs with host cell invasion . Neverthproteins ,26, and T. gondii and Plasmodium falciparum have emerged as hopeful vaccine candidates and achieved exciting results [T. gondii RH strain [T. gondii [E. tenella, Et-ROPK-Eten5-A showed significant effects as a vaccine and was superior to Et-SAG, Et-SAG13, and Et-GRA12 [Eimeria. They are immunologically vulnerable and functionally vital for parasite infection. The membrane or secreted protein that enhanced expression in both stages is more conducive for an ideal vaccine [E. tenella.As critical secretory virulence factors, ROPs are considered to be sources of potential vaccine candidates. Many ROPs from  results ,28. The  results . Mice imH strain . TgROP16. gondii . In E. tEt-GRA12 . EtROP17Et-GRA12 ,12. In a vaccine . In the E. tenella with consistent common ROP characteristics of sequence features, secretion, and localization. In addition, EtROP35 showed higher relative expression during the invasion stages and was associated with host cell invasion. It displayed good immunogenicity and could be a promising vaccine candidate against this parasite.This study identified EtROP35 as a novel ROP of


We studied the evolution of genetic variance in the dairy sheep breed Manech Tête Rousse, which has been selected for increasingly complex objectives, including, in this order, milk yield, milk contents, scrapie resistance, and somatic cell score. We estimated base population genetic variance and genetic variance by sex and per year of birth from 1981 to 2014. The data consisted of 1,842,295 milk yield records (from 1978 to 2017) and a pedigree including 530,572 females (96% of them with records) and 3,798 artificial insemination males. As a measure of drift, we computed average relationships for each cohort from which we derived expected reduction of variance due to increased relationships. The difference between observed and expected reductions in genetic variances is the reduction in genetic variance due to selection. Average relationships increased steadily but slowly in both sexes. For females, genetic variance reduced with time until a plateau was reached at around 90% of the initial genetic variance. The reduction due to relationships (roughly 3% cumulated in 30 yr) was smaller than that due to selection (roughly 10% across the last years). A smaller loss due to selection was seen in recent years, possibly due to a change in selection objectives. These results agree well with theoretical expectations. The pattern of the evolution of genetic variance in males was similar to that for females but with a stronger reduction because of strong selection of AI males at birth. We conclude that the reductions in genetic variation due to selection and drift agree with expectations, and none of the reductions are very strong in this population because of control of inbreeding and smooth changes in selection objectives over time. LD) among QTL, also known as the Bulmer effect. In an infinitesimal model and in the short term, the reduction of genetic variance is due mostly to negative covariance between QTL, whereas directed changes in allele frequencies have a small impact  reduces genetic variation as animals become increasingly related. This reduction is well known and can be understood as drift . It actsl impact . TypicalReduction of genetic variance affects genetic gain and its prediction. The genetic gain is often predicted based on base population parameters, without accounting for the Bulmer effect. The reduction in genetic variance also decreases heritability, thus affecting accuracy of selection . The BulRecent introduction of genomic selection has renewed interest in the Bulmer effect . van GreMTR) sheep, a breed that extensively uses AI .Dairy sheep is an interesting species in which to study the Bulmer effect. In France, the cooperative schemes have strategies at the breed level to handle inbreeding. These schemes also have clearly defined and consensual selection objectives at each time period. This is opposite to dairy cattle where different AI studs may propose different portfolios of bulls, and breeding objectives and strategies may differ among actors. Also, in dairy sheep, the populations are large enough  for accurate inferences of the evolution of Bulmer effect. The objective of this work was to estimate the trajectory of genetic variance over years, and the reduction of genetic variance due to coancestry and Bulmer effect, for the trait yearly milk yield in Manech Tête Rousse  and pedigree of MTR (roughly with the same time span as the milk records). A total of 1,842,295 records of milk yield and 540,999 individuals were included in the pedigree . The average generation interval was approximately 4 yr, so there are about 10 generations. There is no formal use of optimal contribution selection in this population, but matings among cousins are avoided, so recent inbreeding is avoided. Data were precorrected for heterogeneity of variances using the method of Genetic evaluation was by pedigree BLUP animal model with permanent environment effect to account for repeated measurements. The linear model included contemporary group , age, lactation number, month of lambing, and interval lambing to first milk recording. Random effects were animal and permanent environment. Because there are approximately 20% missing sires in the pedigree, the model included 13 unknown parent groups every 3 yr.t (1981 to 2014). We computed the variance of the samples of EBV for each of these 34 × 2 = 68 groups. These variances were, in turn, samples from the posterior distributions of genetic variances of males (m) at time tf) at time tWe followed the method presented in t ist are inbred (which increases the variance) and related (which decreases the variance). The reasoning extends to separate sexes by computing separate averagesThe expected genetic variance as a function of average inbreeding+F¯t-A¯t). This exNBUPGF90 .The difference between the observed genetic varianceThe genetic variance at equilibrium was calculated using the program SelAction 2.2 , modelin2) 499.2 ± 5.2, permanent environmental variance was 409.3 ± 3.6, and residual variance was 857.3 ± 1.2. The estimated heritability (h2) was 0.28 ± 0.003. This h2 value is very similar to a previous estimate  in 2008 and increased in the following years, as for females. The reduction in genetic variance for AI males was stronger than that for females. As mentioned previously, the trend for males included not only reduction due to drift and Bulmer effect, but also (and importantly), reduction due to strong preselection of males at birth based on parent average EBV. In contrast, in the female population, there is very little preselection pressure at birth.In The increase in coancestry explains only a small proportion of the reduction of genetic variance in MTR, whereas the remaining was attributable to the Bulmer effect  and to the Bulmer effect and selection at birth . The reduction due to the Bulmer effect (and selection) seemed to stabilize in the last decade. However, it is not possible to say whether this plateau was due to stabilization of the Bulmer effect (as predicted by theory) or to changes in the breeding objective (as happens in this breeding scheme).Finally, the results for the theoretical reduction in genetic variance computed with SelAction are as follows. A scheme selecting for milk yield (breeding objective at the beginning of the scheme) has, at equilibrium, a 15% reduction due to Bulmer effect, whereas a scheme selecting for milk yield, composition, and SCS has an 8% loss for milk yield. These numbers agree with our estimations of an approximate 10% loss due to the Bulmer effect in females. The cumulated loss due to increased coancestry was approximately 3% in both cases.Our results have interest on their own. To our knowledge, there are few estimates of the reduction of genetic variance with real data , and nonIn conclusion, for milk yield in MTR dairy sheep, there has been a steady reduction of genetic variance due to drift (roughly 3% in 30 yr) and reduction due to selection (roughly 10% in 30 yr). The loss due to selection reached an asymptotic value due either to the nature of the Bulmer effect or to the change of selection objectives. These results highlight that there is a need to check the evolution of genetic variability and that common strategies diversifying selection objectives and controlling inbreeding result in small losses of genetic variation.


Sterna hirundo). Using 28 years of data comprising approximately 6000 pedigreed individuals, we find that additive genetic variances in the zero‐inflated and Poisson components of lifetime fitness were effectively zero but estimated with high uncertainty. Similarly, additive genetic variances in adult annual reproductive success and survival did not differ from zero but were again associated with high uncertainty. Simulations suggested that we would be able to detect additive genetic variances as low as 0.05 for the zero‐inflated component of fitness but not for the Poisson component, for which adequate statistical power would require approximately two more decades (four tern generations) of data collection. As such, our study suggests heritable variance in common tern fitness to be rather low if not zero, shows how studying the quantitative genetics of fitness in natural populations remains challenging, and highlights the importance of maintaining long‐term individual‐based studies of natural populations.Additive genetic variance in fitness is a prerequisite for adaptive evolution, as a trait must be genetically correlated with fitness to evolve. Despite its relevance, additive genetic variance in fitness has not often been estimated in nature. Here, we investigate additive genetic variance in lifetime and annual fitness components in common terns ( Fisher's Fundamental Theorem of Natural Selection postulates that “the rate of increase in fitness of any organism at any time is equal to its genetic variance in fitness at that time” Fisher . As suchConsiderable debate has surrounded the question of whether additive genetic variation in fitness is expected to be low   in New Zealand had negligible additive genetic variance in lifetime fitness , while Wolak et al. (Melospiza melodia) of Mandarte Island in Canada harbored substantial additive genetic variance in female and male fitness .Data constraints might partially explain the paucity of studies testing for the heritability of fitness in the wild and the heterogeneity among estimates of additive genetic variance, although steadily growing datasets collected from long‐term study populations gradually alleviate the problem . The common tern is a Nearctic and Palearctic colonially breeding, serially monogamous and migratory seabird. The study colony is located in northern Germany; common terns from this colony spend their winters in western Africa and return to the breeding colony in early spring to breed or prospect potential breeding locations . The Banter See colony consists of six concrete islands, each of which is surrounded by a 60‐cm wall. Walls are equipped with 44 elevated platforms, each containing an antenna that reads transponder codes. The individual‐based study at the Banter See was initiated in 1992, when 101 adult birds were caught and marked with individually numbered subcutaneously injected transponders. Since 1992, all locally hatched birds have been similarly marked with a transponder shortly before fledging, and the presence and reproductive performance of marked individuals have been monitored following a standard protocol (Becker and Wendeln and were younger than 11 years old because (i) they are known to not be, or cannot yet be assumed to be, dead, and (ii) lifetime fitness of individuals older than 10 years and those who died showed a high correlation (r > 0.8) in our dataset. Hence, we included birds that completed their life histories (n = 5836), as well as birds that were still alive but older than 10 years (n = 163), to avoid introducing a cohort truncation bias by nonrandomly removing longer‐lived birds . Additionally, we performed data simulations to investigate (i) whether we can effectively detect “small but substantial” additive genetic variances in fitness  and year of observation . As fixed effects, we modeled the trait intercept and age (continuous trait ranging from 1 to 23 years), as fledgling production is known to linearly increase with age  and year of observation . As fixed effects, we modeled the trait intercept and age (continuous trait ranging from 1 to 23 years), as AAS is known to linearly decrease with age . Narrow‐sense heritabilities (h2) were conditional on the variance explained by fixed effects and were estimated as the proportion of the total phenotypic variance explained by the additive genetic variance. Evolvabilities (IA) were estimated by dividing the additive genetic variance by the squared population mean , population size actually varied dramatically across years and did not decline Fig. , partialsmall but substantial signal for the zero‐inflated component of lifetime fitness: we generated a zero‐inflated component of fitness with an additive genetic variance of 0.01 and found that the average posterior mode was similar to the simulated value of VA , our simulations showed that we would be able to detect those values . The lower 95% CI always exceeded a value of 0.0001 in both simulated cases  and found that the lower 95% CI limit was on average zero in all cases , with nonzero lower 95% CI in both cases , although statistical power differed between the two components of lifetime fitness: we would have power to detect slightly larger signals (additive genetic variances of at least 0.05) for the zero‐inflated, but not Poisson, component of fitness. As such, our work demonstrated that estimating additive genetic variance in fitness is very difficult in wild populations, partly due to the expected low values of genetic variation in fitness in locally adapted populations but also to the challenges associated with collecting sufficient phenotypic and pedigreed data.The most direct measure of the adaptive potential of a population is its standing additive genetic variance in fitness Fisher . Here, wA data‐scale = 0.004, 95% CI = 0 ‐ 0.008, Table A data‐scale ∼ 0, 95% CI = 1.4×10−11 ‐ 0.0038, de Villemereuil et al. −10 ‐ 5.7). Our posterior mode estimate was overall larger , while the Poisson process generating the observed fitness distribution mostly captures adult lifetime reproductive success. If we compare our nominally zero additive genetic variance in the zero‐inflated component of lifetime fitness Table  with estAdult lifetime reproductive success is the sum of annual reproductive events across the life of an individual and hence can be decomposed into annual reproductive success and adult annual survival. Our quantitative genetic analyses of these two annual fitness components revealed a lack of substantial additive genetic variance for both Table . This fiEstimating quantitative genetic parameters with precision is a data‐hungry endeavor. Researchers are therefore faced with the challenge of collecting hard‐to‐quantify lifetime fitness data from an unbiased sample of the population  that comprises a sufficiently large number of individuals of known relatedness Burt . In addiIn addition to the difficulty of estimating the heritability of fitness with precision, our knowledge of the genetic architecture of fitness components is limited. Extending our genomic understanding of fitness variation in wild populations will provide important insights into how genetic variation underpinning fitness may be maintained and, overall, will help to better predict the evolutionary dynamics of natural populations (Merilä and Sheldon A better understanding of the genetic architecture of fitness will also provide added benefits, as, for instance, it would allow a deeper understanding of the genetic underpinnings of complex traits such as fitness, which might be subjected to different pleiotropic effects Mackay . For insOur quantitative genetic study of fitness in a wild population of common terns reported low to zero estimates of additive genetic variance in lifetime and annual fitness components, which were associated with high uncertainty. Those analyses, however, were overshadowed by a lack of statistical power to detect additive genetic variation in fitness more accurately and precisely. The continuation of long‐term individual‐based studies should be safeguarded (also see Clutton‐Brock and Sheldon M.M. conceived the study with input from S.B. and A.C. M.M. designed and conducted the analyses and wrote the manuscript. S.B. manages the tern data and collated the dataset. All authors contributed to editing the final paper.The authors declare no conflict of interest.https://doi.org/10.5061/dryad.8kprr4xqj (Moiron et al. Data have been archived in the Dryad Digital Repository: Associate Editor: G. MarroigHandling Editor: A. G. McAdamSupplementary Table S1. Model of lifetime fitness with parental effects.Supplementary Table S2. Model of lifetime fitness with brood effects.Supplementary Table S3. Models of AAS and ARS without age.Supplementary Text S1. Pedigree information.Supplementary Text S2. Prior specifications.Supplementary Text S3 and Figs. S1‐S4. Data simulations.Supplementary Text S4 and Fig. S5 Data simulations with increased pedigree depth.Supplementary Fig. S6. Temporal variation in population sizeClick here for additional data file.