Toxoplasma gondii, is essential to protect the health of vulnerable animals and humans. As high-level predators and scavengers, foxes represent a potentially sensitive indicator of the circulation of T. gondii in environments where humans co-exist. The objectives of our research were to compare serological and molecular assays to detect T. gondii, generate baseline data on T. gondii antibody and tissue prevalence in foxes in northern Canada, and compare regional seroprevalence in foxes with that in people from recently published surveys across northern Canada.In changing northern ecosystems, understanding the mechanisms of transmission of zoonotic pathogens, including the coccidian parasite Vulpes vulpes/Vulpes lagopus, n = 749) were collected by local trappers from the eastern (Labrador and Québec) and western Canadian Arctic  during the winters of 2015–2019. Antibodies in heart fluid were detected using a commercial enzyme-linked immunosorbent assay. Toxoplasma gondii DNA was detected in hearts and brains using a magnetic capture DNA extraction and real-time PCR assay.Fox carcasses  compared to younger foxes (22%). More males (36%) than females (31%) were positive for antibodies to T. gondii. Tissue prevalence in foxes from western Nunavik (51%) was higher than in eastern Nunavik (19%). At the Canadian scale, T. gondii exposure was lower in western Inuit regions (13%) compared to eastern Inuit regions (39%), possibly because of regional differences in fox diet and/or environment. Exposure to T. gondii decreased at higher latitude and in foxes having moderate to little fat. Higher mean infection intensity was observed in Arctic foxes compared to red foxes. Fox and human seroprevalence showed similar trends across Inuit regions of Canada, but were less correlated in the eastern sub-Arctic, which may reflect regional differences in human dietary preferences.Antibodies against T. gondii in foxes in northern Canada and shows that foxes serve as a good sentinel species for environmental circulation and, in some regions, human exposure to this parasite in the Arctic.Our study sheds new light on the current status of The online version contains supplementary material available at 10.1186/s13071-022-05229-3. Toxoplasma gondii is found in a vast array of ecosystems and can infect virtually all warm-blooded vertebrates. As approximately one third of the global human population has been exposed, T. gondii is one of the most successful parasites in the world [The apicomplexan parasite he world , 2. Thishe world . While tT. gondii have been reported in people, with seroprevalence ranging from 8% in the western Arctic [In humans, a higher seroprevalence is found in Latin America, parts of Eastern/Central Europe, the Middle East, parts of southeast Asia, and Africa . This prn Arctic  to 40–60T. gondii varies regionally from 27 to 56% [T. gondii has been reported in various northern wildlife species, including birds, ungulates, and carnivores [T. gondii, sharing transmission dynamics with humans.Globally, exposure to the parasite in human populations increases along a north-to-south gradient, but seroprevalence in some Inuit communities in Canada’s North is much higher than in other parts of North America. In Nunavik, northern QC, exposure to 7 to 56% , signifi7 to 56% , 11. Wil7 to 56% . Neverth7 to 56% . Game mernivores ; wild caT. gondii has also been documented in foxes in some parts of the Canadian Arctic. For example, Bachand et al. [95%: 40–44) in people from this region [95%: 28–50) in Nunavut foxes compared to an exposure of 32% (CI95%: 29–36) in Inuit populations from Nunavut [T. gondii circulation in Arctic ecosystems [Animals can act as ideal sentinel hosts for select pathogens if they possess the following features: adequate availability (population stability), measurable response , earlier response than sympatric wild species or humans, high levels of exposure, and ideally do not serve as a direct source of human exposure , 16. Arcd et al.  reporteds region . Similars region  found a osystems .The current study has a much broader geographic scale and larger sample size than these previous surveillance studies, allowing, for the first time, a high level view of the suitability of foxes as indicators of environmental transmission , 18. In Vulpes lagopus) are affected by environmental change, such as increased competition with other carnivores like the red fox (Vulpes vulpes) moving onto the tundra [T. gondii and its hosts. Warmer, wetter, and more extreme climatic events (with the largest increases in temperature occurring in the western Canadian Arctic) will likely increase exposure rates to T. gondii in the Canadian North [T. gondii in suitable sentinel species, like foxes, will also allow us to detect changing risks due to climate change.Although the fox population in Canada appears to be stable, elsewhere in the world, Arctic foxes  [T. gondii circulating in wild mammals. In North American wildlife, type 12 strain is considered to be a common genotype [T. gondii can reveal regional transmission dynamics, spatial patterns of T. gondii in relation to the genetic structure of hosts, and differences in virulence in animals and people [The methods used to screen for ng lived . Less cong lived . Bioassaor cats) . Commercor cats) . Magnetior cats)  and has or cats) , 29. Morgenotype , 31, folgenotype . Moleculd people –36.T. gondii and associated risk factors in harvested foxes across northern Canada. Finally, our third objective was to compare regional seroprevalence for antibodies to T. gondii in harvested foxes (current study) and people (other studies) from different regions in northern Canada to determine if foxes are good sentinels of environmental transmission and human risk of exposure to this zoonotic parasite.As serological tests are rarely optimized for wildlife, our first objective was to determine cutoff values for two serological tests (ELISA and IFAT), followed by comparison of these serological results with detection of DNA of the parasite in tissues using MC-qPCR. Our second objective was to determine antibody and tissue prevalence of Harvested foxes were sampled from across northern Canada (> 4000 km), including all four Inuit regions of Canada: Inuvialuit Settlement Region (ISR) , Nunavut , Nunavik (QC), and Nunatsiavut (NL) Fig. . These fn = 548, Arctic: n = 171, unknown: n = 30) were harvested by local trappers and collaborators during regular, licensed fur-trapping activities during the winters of 2015–2019. Carcasses were kept frozen at − 20 °C (in a freezer) or left outside in an enclosed shelter during winter , until shipped for necropsy at the Department of Environment and Natural Resources in Inuvik (NT), the Western College of Veterinary Medicine in Saskatoon (SK), the Churchill Northern Studies Centre (MB), the Faculté de Médecine Vétérinaire in Saint-Hyacinthe (QC), the Nunavik Research Center in Kuujjuaq (QC), and the wildlife division office at the Department of Fisheries, Forestry and Agriculture in Goose Bay (NL). As predilection sites for T. gondii in animals [Foxes . Kappa values of ≤ 0.40, 0.40–0.60, 0.61–0.80, and ≥ 0.81 represented poor to fair, fair to moderate, moderate to substantial, and substantial to almost perfect agreement, respectively [As commercially available ELISA and IFAT assays were not validated in foxes, we evaluated sample dilutions for ELISA and IFAT. Samples from nine foxes known to be positive and negative on magnetic capture were used to determine cutoff values: three negative animals on MC-qPCR (Group N), three low positives , and three high positives . The ELISA was performed at four dilutions: no dilution (ND), 1:2 (recommended as per manufacturer’s instruction), 1:4, and 1:8. The IFAT was performed at four dilutions , and the results were graded from 1 + to 3 + based on fluorescence intensity. The slides were screened by two blinded readers, and the results were compared. The between-reader repeatability of the IFAT was assessed using a weighted kappa value (ectively . All samT. gondii were detected in heart fluid (1:2 dilution) using the commercially available ID Screen® Toxoplasmosis Indirect Multi-species kit . The ELISA was performed as per manufacturer’s instructions. The optical density (OD) was recorded at 450 nm using an ELISA microplate reader . Samples presenting an S/P % ≤ 40% were considered negative. Samples ≥ 70% were considered positive. If the S/P % was between 40 and 70%, the test result was considered doubtful and repeated. All samples were tested in duplicate. Positive and negative controls provided by the manufacturers were used in each batch. In addition, positive and negative serum samples from naturally exposed foxes were used as reference controls [Neospora caninum (bovine) and Hammondia hammondi (feline) were provided by the Centre for Food-borne and Animal Parasitology (CFAP) in Saskatoon. The ELISA relative sensitivity and specificity using magnetic capture PCR as a reference test were 94% and 100%, respectively [Antibodies to controls . To checectively .T. gondii antibodies. Tachyzoites with only apical or no staining were recorded as negative. If discontinuous peripheral staining on tachyzoites was observed, the result was considered doubtful and repeated. All samples were tested in duplicate. Positive and negative serum samples from naturally exposed foxes were used as reference controls, previously confirmed by IFAT [N. caninum  (bovine) and H. hammondi (feline) were provided by the Centre for Food-borne and Animal Parasitology (CFAP) in Saskatoon. The IFAT relative sensitivity and specificity using magnetic capture PCR as a reference test were 94% and 100%, respectively [The IFAT was performed on heart fluid (1:50 dilution) using the commercially available antigen-coated Teflon-masked slides from VMRD following manufacturer’s instructions . Anti-canine IgG antibodies conjugated to fluorescein isothiocyanate  were applied on slides. Slides were viewed under an Olympus DP70 fluorescence microscope at 40× objective. A complete staining around the tachyzoites was considered positive for  by IFAT . To checectively .To determine the best-performing serological test, heart fluid from 158 randomly chosen foxes  were tested (in duplicate) by ELISA and IFAT, and results were compared using magnetic capture PCR as a reference test.T. gondii in Inuit from Inuvialuit (NT), Nunavut (NU), Nunavik (QC), Nunatsiavut (NL), and Cree from James Bay (QC) to overall exposure in harvested foxes from the same regions. The human surveys used commercial  and homemade immunoenzymatic assays (ELISAs) to detect IgG antibodies against T. gondii at the National Reference Centre for Parasitology  for Inuit communities and the National Microbiology Laboratory  for Cree communities.We used data from the International Polar Year Inuit Health Survey in 2007–2008, the Nunavik Health Survey in 2017, and Sampasa-Kanyinga et al.  to compaT. gondii [T. gondii tachyzoite-stock (VEG type III) used for spiking was 2.5 × 106/ml. A 10-times dilution series was made in ultrapure water to obtain 2.5 × 105 and 2.5 × 104/ml. For positive controls, 100 µl of these dilutions was added to 50 g of beef samples, resulting in samples spiked with 2500 and 25,000 tachyzoites. Cell-cultured tachyzoites of T. gondii were obtained from CFAP, Saskatoon. For each sample, a back-up of tissue lysate (50 ml) was kept at − 20 °C. The extracted DNA was stored at 4 °C if used in the following 3 days or at − 20 °C until further use.Magnetic capture (MC)-qPCR was used to determine infection status and quantify infection intensity (parasite burden) based on detection of DNA of . gondii , 32. DNA. gondii . Each ru. gondii sequence within the 529 repeat-element was performed using the Bio-Rad CFX 96 DNA thermal cycler , as per Bachand et al. [A real-time PCR using the Tox 9F (5′-aggagagata tcaggactgtag-3′) and Tox 11R (5′-gcgtcgtctc gtctagatcg-3′) primers for the detection of the 188 bp Td et al. . A reactd et al. . The intn = 113, Cq value < 30) was thawed at room temperature, centrifuged at 3500 rpm for 15 min, and 250 µl transferred into a 1.5-ml Eppendeorf tube. DNA was extracted using the High Pure PCR Template Preparation kit . Protocol followed manufacturer’s instructions except the first step was skipped, as the lysate already contained proteinase K, and 75 µl was used for elution in the final step to increase DNA amount. All samples were extracted in duplicate and treated with 1 µl of RNAse A Solution . Frozen aliquots of template DNA (50 µl) were sent within 24 h to the Department of Microbiology, University of Tennessee, Knoxville, TN, USA, for further genetic characterization. A Mn-PCR–RFLP method employing ten genetic markers  from strongly positive samples on MC-qPCR (nd Apico ) was usek) was used to determine the level of agreement between the two tests. After being retested, all doubtful results from serological tests were considered negative. Analyses were performed using IBM SPSS .Proportion of positive results was compared between the ELISA and IFAT, IFAT and MC-qPCR, and ELISA and MC-qPCR, using McNemar’s chi-square tests for paired data. If not significantly different, the kappa coefficient (A Spearman's rank-order correlation coefficient was performed to determine the covariation between antibody concentration (S/P%) and Cq values obtained from ELISA and MC-qPCR, respectively.T. gondii prevalence (exposure and infection). Foxes with missing data were not included in the regression, nor were individuals of unknown species. We checked that no group was under-represented and that groups were distributed similarly in each category. We checked for multicollinearity between explanatory variables by using the variance inflation factor (VIF)  may vary yearly. However, we could not include year as a fixed effect in our model as it was dependent on region (and thus latitude). We first produced a mixed-effect model including year as a random effect [Seroprevalence, tissue prevalence, and their 95% confidence intervals (CI) were calculated from the proportion of positive results using EpiTools epidemiological calculators . We usedm effect  and compT. gondii, we used a generalized linear model (linear regression) in IBM SPSS . We found no outliers (Cook’s distance was < 0.5) and no heteroskedasticity [T. gondii  was not normally distributed, and data were thus log 10 transformed . Foxes with missing data were also excluded from the model. An omnibus test was performed to evaluate the model coefficients  and tissue prevalence was 27% (CI95%: 24–31). Prevalence and number of samples per species, sex, age, geographic location, and BCI are shown in Table Overall, seroprevalence was 36%  and one in Group LP (ID W2015-218) (Table T. gondii were detected in 53% (83/158) of foxes using ELISA and in 46% (72/158) using IFAT. There was excellent agreement for the between-reader repeatability of the IFAT (k = 0.88). There was no statistical difference between serological and molecular results using the McNemar chi-square test . There was substantial agreement for ELISA (k = 0.82) and only moderate agreement for IFAT (k = 0.61) compared to MC-qPCR. The relative sensitivity and specificity of ELISA were 94% and 89%, respectively, compared to 76% and 85% for IFAT. ELISA was therefore used for subsequent analyses.Antibodies to 2 = 45.6, df = 1, p < 0.001, n = 728). There was substantial agreement between the two tests (k = 0.75).Seventy foxes had positive serology and negative molecular tests, nine were serologically negative but tissue positive, 190 were positive on both, and 459 were negative on both. There was no statistical difference between serological and molecular results using the McNemar chi-square test (χp < 0.001). As Cq values are inversely related to DNA quantity, this means higher antibody levels were associated with higher amounts of DNA.A moderate, negative, statistically significant correlation was found between ELISA antibody concentration (S/P%) and Cq values on MC-qPCR  of foxes using ELISA . Seropositivity increased from young , to mature , to older  foxes. More males  than females  were positive for antibodies to T. gondii. In general, seroprevalence was highest in foxes in eastern Hudson Bay and southern QC and lowest in the western Canadian Arctic . Finally, antibodies were detected more frequently in foxes with abundant fat  than moderate  and little fat . The results are presented in Table Antibodies to T. gondii antibodies were three times  higher in male than female foxes. Mature and old foxes had significantly higher exposure to T. gondii than young foxes—two times  and seven times , respectively. The probability of being exposed to T. gondii decreased at higher latitude as well as for foxes having moderate to little fat  in the Inuvialuit region, 32%  in Nunavut, 37%  in eastern Nunavik, 56%  in western Nunavik, 11%  in Nunatsiavut, and 9%  in James Bay. Regional human seroprevalences were compared to regional fox seroprevalences in the present study in Fig. According to previous studies , 9, 19, T. gondii was detected in 27% (CI95%: 24–31) of foxes using MC-qPCR. Older foxes  were more frequently infected than mature  and young  foxes. A trend in location similar to seroprevalence was also observed for detection of T. gondii DNA in foxes. The results are presented in Table DNA of T. gondii DNA. The odds of presence of T. gondii DNA were four times  higher in male than female foxes. Old foxes had three times higher odds of being positive for DNA of T. gondii in tissues  than young foxes. The probability of being infected with T. gondii decreased at higher latitude as well as for male red foxes and foxes having moderate fat . Mean (SE and CI95%) TEG among different classes of risk factors is shown in Table T. gondii in foxes are provided in Table Mean infection intensity was 8879 TEG , using MC-qPCR as a reference test. IFAT has subjective endpoint criteria based on visual inspection, which can lead to bias when reporting seroprevalence. ELISA results are quantitative and therefore more objective and had higher relative sensitivity and specificity than IFAT in the current study. For these reasons, we used ELISA as our primary serological test.After determining optimal dilutions and conducting our pilot study on heart fluid, we detected better agreement with MC-qPCR using ELISA compared to IFAT. In contrast, Sharma et al.  reportedT. gondii in 36% of the foxes using ELISA, whereas we detected DNA of T. gondii in 27% of foxes. Sixty-seven foxes were positive on ELISA but negative on MC-qPCR. This was not unexpected, as seroprevalence is often higher than tissue prevalence [k = 0.75), using both serological and molecular tests concurrently maximized detection of T. gondii in foxes in the current study.We detected antibodies to evalence , 50, 51.evalence . On the evalence , 53. EveT. gondii in food sources of Arctic foxes, as they may be less exposed to oocysts, and possibly due to higher level of carnivorous specialization in the Arctic versus red fox [T. gondii exposure [The overall detection of antibodies was higher in red foxes compared to Arctic foxes, but this was not significant when both species were present at the same latitude. The species effect in our model is likely hidden by the latitude effect  are present in Ungava Bay, but not Hudson Bay. A similar pattern is seen in people, with a seroprevalence of 36% in Ungava Bay and 56% in Hudson Bay [T. gondii for marine animals are thought to be oocysts washed into the sea through water run-off and transported by marine currents [Higher seroprevalence and tissue prevalence were observed in foxes from Hudson Bay (65% and 51%) versus Ungava Bay (29% and 19%) in Nunavik, northern QC. This difference was unexpected, as lynx  and Nunavut (32%) compared to the more southern region of Nunavik [T. gondii seroprevalence is much lower. Caribou meat is the most consumed country food in ISR and Nunatsiavut [T. gondii in tissues [Our overall geographical findings indicate widespread exposure to and infection with  regions . For insn Alaska . Prevale Nunavik . However Nunavik  and 9% [ Nunavik , respect Nunavik . Indeed,e in ISR % and Nun Nunavik , but notatsiavut , 68, whi tissues .T. gondii present in foxes across the Canadian Arctic would provide valuable information about the population structure and transmission of this parasite; for example, if strains in foxes were identical to those in marine mammal species or felids in sub-Arctic regions. However, as we found, recovering sufficient DNA of T. gondii from naturally infected wildlife can be challenging, as they have low tissue burden and therefore yield low levels of DNA [T. gondii circulating in foxes in northern Canada.Determining the genotypes/strains of s of DNA . In futuT. gondii ecology still remains uncertain in northern regions, but may be more severe in the western North American Arctic, where the parasite is not currently as successful as in the Eastern Arctic, and the magnitude of rapid and directional climate change is more pronounced than almost anywhere in the globe. The western Canadian Arctic  is currently experiencing the largest increases in warming and precipitation due to climate change, which could bring increases in survival and infectivity of oocysts in the future [T. gondii may expand via aquatic environments and migratory wildlife in these ecosystems. The presence of fish and marine invertebrate species in new areas that were previously too cool for their survival represents a risk for increased spread, since they can accumulate oocysts in their gills and filtration organs [The effect of climate change on e future . As a ren organs . As welln organs . IncreasT. gondii in a future of climate change. Foxes are widespread across northern Canada, year-round residents, and exposed at similar rates and routes as humans in most of the Canadian Arctic. While the western Arctic may be most vulnerable, the higher prevalence in the eastern Canadian Arctic warrants continued study to monitor prevalence of T. gondii in wildlife species that are consumed in northern communities. Future research could investigate additional predictors of T. gondii presence and prevalence in foxes, such as diet . The geographic scale of the present study speaks to significant community engagement from trappers and territorial, provincial, and indigenous governments. Synergizing sentinel animal surveillance with human disease surveillance is critical to identifying and addressing the potential human and animal health risks associated with altered transmission of T. gondii in a rapidly changing Arctic.This study supports foxes as appropriate sentinels for potential human exposure and transmission of Additional file 1: Fig. S1. Density of data for each species (Arctic fox in blue and red fox in yellow) compared to latitude in decimal coordinates indicating strong relationships (VIF = 146)Additional file 2: Table S1. a Model selection results for hypotheses of risk factors influencing Toxoplasma gondii infection intensity in foxes in Canada. b Model selection results for hypotheses including “BCI^lat” interaction term and other possible risk factors influencing Toxoplasma gondii infection intensity in foxes in Canada.


Ficus, Moraceae) and pollinator wasps  is a classic example of cospeciation. However, examples of phylogenetic incongruencies between figs and their pollinators suggest that pollinators may speciate by host shifting. To investigate the mechanism of speciation by host shifting, we examined the phylogenetic relationships and population genetic structures of six closely related fig species and their pollinators from southern China and Taiwan-Ryukyu islands using various molecular markers. The results revealed 1) an extraordinary case of pollinator sharing, in which five distinct fig species share a single pollinator species in southern China; 2) two types of copollination, namely, sympatric copollination by pollinator duplication or pollinator migration, and allopatric copollination by host migration and new pollinator acquisition; 3) fig species from southern China have colonized Taiwan repeatedly and one of these events has been followed by host shifting, reestablishment of host specificity, and pollinator speciation, in order. Based on our results, we propose a model for pollinator speciation by host shifting in which the reestablishment of host-specificity plays a central role in the speciation process. These findings provide important insights into understanding the mechanisms underlying pollinator speciation and host specificity in obligate pollination mutualism.The obligate pollination mutualism between figs ( Phylogenetic and population genetic analyses reveal mechanisms of pollinator sharing, copollination and speciation by host-shift in fig-wasp mutualism. Obligate pollination mutualism is an extreme example of plant-insect interactions, and one of the classic examples of such interactions is the association between figs  and their wasp pollinators 8. Figs depend on wasps for pollination and the wasps depend on figs to complete their life cycle by providing sites for oviposition, larval hatching, growth, development and reproduction. Indeed, all of these stages in the life history of wasps take place within the closed inflorescence (syconium) of a fig9. While it may seem that obligate pollination mutualism would limit the speciation of hosts and pollinators, Ficus is one of the most widespread and speciose plant genera in tropical and subtropical regions, with more than 750 described species12. It has been estimated that approximately the same number of wasp species pollinate figs, and of these, 319 species have been described to date12. Investigation of speciation mechanisms associated with fig-wasp mutualism may help us to understand the high levels of species diversity in both groups.Interactions between angiosperms and insect pollinators are believed to have contributed to the considerable species diversity that is evident in both groups13, since it has been considered that this form of mutualism is characterized by high host-specificity, i.e., the “one to one” rule, where one fig species is pollinated by only one species of wasp14. However, recent studies have demonstrated numerous instances of co-pollination, i.e., where one fig species is associated with more than one pollinator species, as well as some examples of pollinator sharing, i.e., where one pollinator species is associated with more than one fig species26. The most significant implication of these findings is that numerous pollinator species and some fig species have not arisen by strict cospeciation13. Moreover, while molecular phylogenetic analyses broadly support the co-cladogenesis in figs and pollinator wasps at higher taxonomic levels28, the phylogenies at the species level of figs and wasps do not match perfectly; indeed, the existence of such incongruencies32 suggests that mechanisms other than strict cospeciation also play a significant role in the species diversity13. In response to these reports, it has been suggested that pollinator wasps may speciate by host shifting between closely related figs, or by duplication 13. However, little is known about the mechanisms underlying these speciation processes; for example, which ecological conditions favour host shifting or duplication, and which speciation mechanism, sympatric or allopatric, is employed during host shifting and duplication. Cook and Segar13 proposed that the duplication mechanism of pollinator speciation comprised three main steps: first, fig and wasp populations split into geographically and genetically isolated sub-populations; second, divergence occurs between the sub-populations, but proceeds more rapidly in wasps than in figs; third, on secondary contact between the sub-populations, the figs, but not the wasps, can interbreed which results in the co-occurrence of the two wasps (co-pollinators). Under this scenario, geographic isolation appears to be the key factor underlying the duplication in pollinators. In this study, we hypothesize that host shifting acts as a trigger that leads to the sympatric speciation of pollinators through the reestablishment of host specificity. The main aim of this study was to find support for this hypothesis by examining the phylogenetic relationships between figs and their pollinators in areas where host shifting and host-specificity reestablishment are expected to occur.The obligate mutualism between figs and pollinator wasps has long provided an ideal framework for addressing questions related to coevolution and cospeciation between insects and plants12. Some of the fig species in this region have spread northward to mainland China, Taiwan, and Japan, including the Ryukyu Islands. For species in highly species-specific mutualistic relationships, long-distance island colonization may disrupt their mutualistic relationships due to the need for synchronous co-dispersal of both partners. However, in cases where only one of the partners in a mutualistic relationship colonize an island, the possibility exists that new mutualistic relationships can be established if suitable partners can be found34. It is expected that such colonization events have occurred repeatedly during the northward range expansion of fig-wasp mutualism to Taiwan and the Ryukyu Islands, which represent the northernmost boundary of the Ficus distribution range. In addition, molecular phylogenetic analyses have suggested that co-pollinator and pollinator sharing, which possibly arose by host shifting, have arisen in dioecious figs in Taiwan24. Consequently, the figs and pollinator wasps distributed throughout the Taiwan-Ryukyu islands appear to be well suited to elucidating how pollinator sharing, co-pollination, and host-shift speciation arise in fig-wasp mutualism.Most fig species (ca. 511 species) are distributed in the Indo-Australasian region, with the centre of Malesia being a hotspot of species richness (359 species)36. Therefore, as materials for this study, we selected six very closely related fig species from the F. erecta complex, which comprises ~17 species37 that are distributed in southern China and/or Taiwan-Ryukyu islands. We clarified the population genetic structure and detailed phylogenetic relationships among the six fig species and their pollinator wasps using various molecular data, with extensive sampling from the aforementioned distribution areas , and glyceraldehyde 3-phosphate dehydrogenase (g3pdh) are often used in phylogenetic analyses of fig plants37. However, due to the limited phylogenetic information that can be obtained from these markers at the species level, it is difficult to clarify the phylogenetic relationships among closely related figs. Therefore, in this study, we used multiplexed ISSR genotyping by sequencing (MIG-seq) data to analyze the phylogenetic relationships among six closely related fig species, because the MIG-seq data are expected to provide finer taxonomic resolution for these fig species. The average number of raw and high quality reads obtained from 60 fig individuals belonging to eight species was 1,133,084  and 491,420 , respectively. De novo assembly yielded 80,138 loci, of which 5284 were polymorphic. After removing the non-phylogenetically informative sites, the final data set for phylogenetic analyses produced 2198 variable nucleotide sites. The maximum likelihood (ML) and Bayesian inference (BI) trees  population only and the other comprised the Fujian (FJ), Taiwan (TW) and Yaeyama Islands (YY) populations  regions of the nuclear rRNA gene, F. erecta, F. formosana, and F. vaccinioides  and Hong Kong (HK). However, MIG-seq  showed that the Taiwan-Ryukyu populations of F. erecta are more closely related to the FJ population than the GD and HK populations. This inconsistency suggests that there may be some dispersal of pollen, but not seeds, of F. erecta between the FJ and TW populations (Discussion).In plants, both seed and pollen dispersal promote gene flow between different populations. To confirm whether there is any differential dispersal of seeds and pollen, plastid DNA was analyzed from a total of 418 individuals belonging to populations of the three fig species, es Table . Clusterseq Fig.  and micrF. erecta, F. formosana, F. abelii, F. pyriformis and F. variolosa) in southern China were all identified as belonging to the same species, Blastophaga silvestriana. To clarify whether there was any host specificity between B. silvestriana and the five fig species, we analyzed the ITS sequences of fig pollen carried by foundresses  and fig leaves . Phylogenetic analysis showed that the ITS sequence of the pollen carried by a foundress (C18-040mA) collected from inside the receptive syconia of F. pyriformis was identical to that of F. abelii, but that it differed from F. pyriformis  collected from inside the receptive syconia of F. formosana had identical ITS sequences to those of F. formosana , contained wasps collected from continental populations of the five fig species; all of these wasps were genetically indistinguishable and were morphologically identified as B. silvestriana. The pollinator wasps associated with F. vaccinioides were separated into Clade 2 and Clade 3, which correspond to the southern TW population (including the LY population) and the eastern TW population , respectively  and F. formosana (TW). Clade 5 contained the pollinator wasps from all of the Japanese populations of fig F. erecta. Interestingly, the pollinator wasps associated with F. erecta and F. formosana in the TW population were grouped into a single clade (Clade 4); together with the Japanese populations (Clade 5), the wasps in Clade 4 were split from one of the two clades of pollinator wasps associated with F. vaccinioides  es Table  and 5. CCOI-COII sequences, could be attributed to mitochondrial introgression. Therefore, to corroborate the results of the mtDNA sequence analyses, phylogenetic analysis was performed based on nuclear 28 S rDNA and ITS1 sequences. Given the marked differences in the ITS1 sequences between Blastophaga and the outgroup species, these sequences could not be aligned correctly. As a result, phylogenetic analysis was performed separately for the 28 S rDNA sequences with outgroups, and the ITS1 sequences without outgroups. The pollinator wasps in Clade 1 and those in Clades 2–5  and the other pollinator wasp species (Clades 2–5), but small sequence differences among Clades 2–5. Further, the phylogenetic tree shows that the B. silvestriana wasps from the five fig species were genetically indistinguishable 38 and Clade 3 as B. sp., in this study.The phylogenetic clades of ies Fig. , and 8. COI-COII and 28 S rDNA sequences from pollinator wasps representing the clades shown in Fig. Blastophaga species (pollinator of F. ischnopoda), and ten species belonging to seven genera as outgroups. BEAST analysis , the population genetic structure was analyzed using nine microsatellite loci (SSR: simple sequence repeats) with a total of 447 individuals representing all of the populations of the three fig species . We then performed separate analyses using samples from each group, and the Evanno’s ΔK showed peaks at K = 2 for Group 1 and at K = 4 for Group 2, with the individuals from Group 1 and Group 2 classified into two and four separate clusters, respectively , although some individuals from TW were genetically similar to individuals from MT and FJ . The phylogenetic relationships among the genetic clusters (K = 6) showed that the GD population of F. erecta was largely separated from other F. erecta populations, which were closely related to each other .To investigate the genetic differentiation among the geographical populations of the three fig species  , between the TW and MT/FJ populations  of F. erecta, and between the TW and GD/HK populations of F. formosana . Some genetic differentiation was observed between the YN and IS/IR populations  and between the FJ and MT populations   were clearly separated from other populations of B. nipponica and “B. taiwanensis”  from a total of 263 individuals representing all populations of the pollinators 5 loci frers Fig. . Interesers Fig. , althougis” Fig. . These ris” Fig. .FST analysis (Supplementary Table B. yeni/B. sp. (TW) and the other populations. Two Ryukyu populations of B. nipponica (IS and YN) also differed markedly from the other populations. Moderate levels of genetic differentiation were observed between the TW and MT/FJ populations of B. nipponica, and a slight, but significant, genetic difference was also observed between B. nipponica and “B. taiwanensis” in the TW population  are all closely related, but their morphologies are extremely variable37. The molecular analyses based on MIG-seq and SSR data showed that these fig species were clustered into distinct groups . The range of sequence divergence in the COI gene at the species level is typically considered to be 0.8 ± 0.6%35 and less than 1.4%23. Compared to these previous findings, the sequence differences within Clade 1 were considered to be at the intraspecific level. The phylogenetic analysis based on nuclear ITS1 sequences corroborated the results obtained from the COI-COII sequence analyses   B. nipponica diverged from the pollinator lineage, B. sp.  From the sampling conducted as part of this study, B. nipponica is not widely distributed in continental China, and seems to be limited to the Fujian region. In addition, if the dispersal of pollinator wasps was bidirectional, then it would be expected that the continental species, B. silvestriana, would occur in Taiwan. However, there are no records of B. silvestriana in Taiwan or Japan12, and we have never collected individuals of B. silvestriana from either of these regions; however, a failure to collect specimens is not equivalent to absence. It is possible that unidirectional dispersal may have been caused by typhoons because fig-pollinating wasps are small insects (~2 mm in body length) and are unable to disperse long distances under their own power. They actively fly above the canopy and then rely on the wind to disperse them passively over long distances43. On average, about four typhoons a year pass through Taiwan and reach the Fujian region of Mainland China , and it seems highly likely that these typhoons transport pollinator wasps from Taiwan to the Fujian region.Through the comparisons, we identified three examples of co-pollination. First, the Fujian (FJ) population of an Figs.  and 6d, sp. Fig.  or the cF. vaccinioides, is divided into two genetically distinct clades  and promote their geographic divergence. However, no genetic differentiation has been observed between the host figs of these two clades , which were co-split from the F. formosana and B. silvestriana lineage, respectively  ~5.12 Ma  inhabited the ancient Taiwan region on the continent, and these taxa became established after Taiwan split from the continent  through host shifting of the pollinators associated with F. vaccinioides  extended to the Ryukyu Islands and the Kanto region of Japan, currently the northernmost boundary of the genus Ficus  that were carrying pollen dispersed from Taiwan to the Fujian area, as discussed above, resulting in hybridization of the figs between the two areas through the dispersal of pollen, but not seeds. As a result, the nuclear DNA of the FJ population became more similar to that of the TW population  in the FJ region.Given the similarities in the plastid DNA of ted Fig.  and 2, wion Fig. , while pion Fig. . In contF. formosana, a slight (but significant) genetic difference was detected between the TW and GD/HK populations  some individuals of the pollinator wasp B. sp., host-shifted to F. erecta from their original host F. vaccinioides; (3) these pollinator wasps then became specialized and became associated with F. erecta; and (4) the pollinators then differentiated from B. sp. and speciated to become B. nipponica  Theica Fig. b and e. F. formosana also migrated to Taiwan from mainland China, but later than F. erecta  by restricting pollen flow; however, as discussed above, there are other factors that prevent hybridization between host figs. Consequently, testing whether the species-specificity between F. formosana and “B. taiwanensis” has been established will be an important future challenge. Such analysis will provide more direct evidence for whether “B. taiwanensis” is in the process of speciation.Similarly, cta Fig. . This waata Fig. . Hybridi49 presented genomic evidence of prevalent hybridization between fig species, implying that host-shifting events might have occurred frequently over the evolutionary history of fig-wasp pollination mutualism. Our study provides important findings for understanding the outcomes of pollinator host-shifting between fig species. After host-shifting, pollinator sharing may continue for some time between the two host species. Two scenarios are considered for pollinator sharing24. One is that pollinator sharing is maintained, increasing the chances of hybridization between the two host fig species. The other scenario is that the host-shifted pollinator wasps adapt to become associated with the new host species, leading to genetic differentiation and pollinator speciation. The findings of the present study provide evidence to support the second scenario, and suggest that fig-wasp pollination mutualism may exert an evolutionary force or selection pressure through factors such as differences in floral scents and/or syconium morphology, that may facilitate the establishment of host-specificity in pollinators. Such an evolutionary force may be central to understanding the maintenance of host-pollinator specificity and the speciation mechanisms found in fig-wasp pollination mutualism.The findings of the present study show that under certain circumstances, the species-specific relationships between figs and pollinators can be disrupted and reestablished through the acquisition of new pollinators through host shifting. Such host shifting could potentially lead to co-pollination or pollinator sharing, and pollinator speciation by reestablishment of host-specificity. A recent studyF. erecta, F. formosana, F. vaccinioides, F. abelii, F. pyriformis, and F. variolosa) and their pollinator wasp species were examined in this study. As mentioned in the Introduction, these fig species were considered to be well suited for this study because they are distributed in the Taiwan-Ryukyu islands and/or the adjacent continent (mainland China), and are most closely related with each other in the F. erecta complex, which consists of 17 species37. According to the Flora of China (www.eFloras.org), the approximate distributions of these figs and their pollinator wasps are as follows: F. erecta: S. China, Taiwan, Ryukyu Islands and W. Japan, Vietnam, and Cheju Island (Korea); F. formosana: S. China, Taiwan, and N. Vietnam; F. vaccinioides: endemic to S. Taiwan; F. abelii: S. China, part of S. Asia, and part of S.E. Asia; F. pyriformis: S. China and Vietnam; F. variolosa: S. China, Laos and Vietnam. We sampled these fig species and their pollinator wasps from S. China (Fujian and Guangdong), Hong Kong, Taiwan, Lanyu Island, Matsu Islands, Ryukyu Islands, and W. Japan  were collected from male fig trees and kept in a 30 ml plastic tube for collection of wasps. Pollinator wasps that emerged from the syconia were preserved in 70–80% and 99.5% ethanol solution for morphological examination and DNA analyses, respectively. Some pollinator wasps were collected from inside the receptive syconia of fig trees. These wasps are generally called foundresses, which entered the receptive syconia for pollination and/or to lay eggs. To avoid using pollinator wasps from close relatives, in this study, we used wasp samples from syconia that were collected from different fig trees where possible. The sample numbers of figs and pollinator wasps collected from each population of each species are shown in Table F. erecta, F. formosana, F. abelii, F. pyriformis and F. variolosa) in S. China and Hong Kong were morphologically indistinguishable, we treated them as Blastophaga silvestriana according to Grandi50 and Hill39. In the Fujian population, two pollinator wasp species, B. silvestriana and B. nipponica, which are morphologically distinguishable, were collected from one fig species, F. erecta; even from the same individual fig tree. In the Taiwan population, it is difficult to discriminate between the two pollinator species associated with F. erecta and F. formosana by morphology. We therefore tentatively treated the pollinator wasps from F. erecta as B. nipponica, and those from F. formosana as “B. taiwanensis”38.Since the pollinator wasps reared from the five fig species  dried with silica gel using a DNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. The DNA samples were finally eluted in a 200 μL AE buffer (Qiagen). These DNA samples were used as templates for PCR amplification of plastid DNA, ITS, MIG-seq, and SSR sequences  according to the manufacturer’s instructions. The DNA samples were finally eluted in 100 μL AE buffer (Qiagen) and used as a template for PCR amplification of the nuclear 28 S rDNA, ITS1, 51 were used for phylogenetic analysis. A total of 60 fig samples representing the eight fig species (including two outgroup species) were used for the MIG-seq analysis  were quantified on a Synergy H1 plate reader (BioTek) using QuantiFluor dsDNA System (Promega), and the quality of the product was determined on an Agilent 2100 bioanalyzer using a High Sensitivity DNA kit (Agilent Technologies). The libraries from each sample were pooled in equimolar concentrations. The mixed libraries were then purified and fragments in the size range of 200–1000 bp in the purified library were isolated using AMPure XP beads (Beckman Coulter). The final PCR product libraries were sequenced (2 × 76 bp) on an Illumina NextSeq 500 system using a NextSeq 500/550 High Output Kit v2.5 (Illumina).To investigate the phylogenetic relationships among the six closely related fig species, genome-wide SNPs detected by the MIG-seq techniquehttp://hannonlab.cshl.edu/fastx_toolkit/). Low-quality nucleotide sites  and low-quality reads (sequence length under 50 bp) were removed from raw reads using Sickle v1.33 (https://github.com/najoshi/sickle). For phylogenetic analyses, the cleaned reads were unified to 50 bp in length. De novo assembly and SNP detection were conducted using Stacks v2.252. The minimum depth option for creating a stack was set to 5, and default settings for all other options were used. Stacks v2.2 was also used to select phylogenetically informative SNP sites for phylogenetic analysis by removing sites in which there were no SNP differences between samples, such as single SNPs.The adaptor and primer were removed in the sequenced reads using FASTX trimmer  , and pollen DNA samples from three foundresses (one from F. pyriformis and two from F. formosana)  or KOD-Plus (TOYOBO) DNA polymerase in 25 μL reaction mixtures containing 1 μL (figs) or 2 μL (pollinator wasps) of template DNA and following the procedures outlined in previous studiesCOI-COII sequences of a total of 317 wasp individuals of four Blastophaga species were amplified together with 12 outgroup wasps belonging to 11 species   and Exonuclease I (Takara), and directly sequenced with an ABI 3130xl Genetic Analyser  using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer’s instructions.54 with a GTR + Γ model, and bootstrapping was performed with 1000 replicates. The pgsumtree v2.0 (https://github.com/astanabe/Phylogears) was used to output the tree (Newick file). BI analysis was performed using MrBayes v3.2.7a55 under the GTR + I + Γ model with two separate runs each of four chains for 1,000,000 generations. The trees were sampled every 1000 generations. Convergence of runs was assessed with Tracer v1.7.156 and the first 25% of trees were discarded as burn-in, by default.A final SNP alignment dataset containing 2198 variable nucleotide sites of MIG-seq data obtained from the 60 fig samples was used for phylogenetic analysis. ML analysis was carried out using RAxML v8.2.957. The obtained sequences were aligned using MAFFT v7.310 with L-INS-i option58. The alignment (3753 bp) of plastid DNA sequences was used to construct a statistical-parsimony network using TCS v.1.2159. The ITS sequences alignment (671 bp) was used for phylogenetic analyses with ML and BI methods. ML analysis was performed on MEGA X60 under the T92 model (the best-fit model selected by MEGA X) with 1000 bootstrap replicates and complete deletion of gaps. BI analyses were conducted as described above using MrBayes55 under the GTR + I + Γ model.The plastid DNA and ITS sequences were assembled and manually edited using the GAP4 programme included in the STADEN v2.0.0b9 packageCOI-COII regions were assembled and manually corrected using the GAP457, followed by alignment using MAFFT with L-INS-i58. The sequence alignments were then subjected to phylogenetic analysis with ML and BI methods. For ML analysis, the model selection for each dataset was performed on MEGA X60. The obtained best-fit models were TN93 + I + Γ for COI-COII, TN93 + Γ for 28 S rDNA and HKY for ITS1. ML trees were constructed in MEGA X with 1000 bootstrap replicates and complete deletion of gaps. BI analyses were conducted using MrBayes55 under the GTR + I + Γ model. To analyze the sequence differences in the COI-COII dataset, an NJ tree was also constructed using uncorrected pairwise p-distances. Outgroups in three separate genera were used for the analysis of the 28 S rDNA sequence data, and two species, Eupristina verticillata and Platyscapa sp., which are much more closely related to Blastophaga, were used for the phylogenetic analysis of COI-COII sequences28.As described above, all sequences of the 28 S rDNA, ITS1, and 61 using representative COI-COII and 28 S rDNA sequences from pollinator wasps representing each clade in Fig. COI-COII and 28 S rDNA sequences were set. Each model employed a strict clock model, a birth-death tree prior, and analytical population size integration. Following Cruaud et al.28, the following three calibration points were used to estimate the divergence time of pollinator wasps: split between Blastophaga and Wiebesia (~40.5 Ma [54.7 − 27.6]), split between Platyscapa and Eupristina + Pegoscapus (~43.5 Ma [55.5 − 32.7]), and the crown of Agaonidae (~75.1 Ma [94.9 − 56.2]) . Convergence of runs was assessed with Tracer56 and trees were generated with the burn-in removed (10%) using TreeAnnotator v2.1.2 (BEAST 2 package). The resulting species tree was visualized with FigTree v1.4.4 (https://github.com/rambaut/figtree).A dated coalescent-based species tree was inferred using StarBEAST 2, which is implemented in BEAST version 2.6.462, FinsM5, FinsQ563, FM1-2764, and FereACOI  in 10 μL reaction mixtures containing 1 μL of template DNA. The temperature conditions used for the PCR are shown in Supplementary Table F. erecta, F. formosana, and F. vaccinioides  was simulated using 20 iterations. The number of potential clusters was assessed with K = 1–14 for the SSR data. The most likely K of the dataset was obtained by estimating ΔK value71 using STRUCTURE_HARVESTER v0.6.9472. CLUMPP73 was used to align and visualize the results of multiple runs. Arlequin68 was used for population genetic analysis with default settings. The pairwise FST values among the populations were estimated using the distance method (pairwise differences) and the FSTP values were based on 110 permutations (default setting). In the Arlequin analysis, populations  containing fewer than 20 individuals were removed from the SSR dataset to avoid obtaining biased results.Nine SSR loci were used: Frac241, Frub061, Frub391, Frub416, Frac22274  was estimated by STRUCTURE_HARVESTER72. Arlequin68 was used to analyze pairwise FST values between populations.Fifteen SSR loci were used for pollinator wasps: Ega01, Ega03, Ega06, Ega07, Ega08, Ega09, Ega10, Ega11, Hga04, Hga09, Hga10, Eca15, Eca22, Hca07, and Hca1975 was used to delimit species based on the phylogenetic tree inferred using the COI-COII sequences  with default settings75. The delimitation results were inferred by ML and the highest Bayesian supported solution.A PTP model77, a software programme for phylogenetic tree reconciliation under the duplication-transfer-loss (DTL) model, was used to determine whether the pairs of figs and their pollinator wasps coevolved with each other by comparing their phylogenetic relationships. The input tree topologies (Supplementary Fig. B. silvestriana wasps associated with the five fig species are genetically indistinguishable (Fig. eMPRess GUI Version 1.0ble Fig. . The anaFurther information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Dietary organic selenium (Se) is commonly utilized to increase formation of selenoproteins, including the major antioxidant protein, glutathione peroxidase (GPx). Inorganic Se salts, such as sodium selenite, are also incorporated into selenoproteins, and there is evidence that nanoelemental Se added to the diet may also be effective. We conducted two trials, the first investigated inorganic Se (selenite), organic Se (L-selenomethionine) and nanoelemental Se, in conventional mice. Their bioavailability and effectiveness to increase GPx activity were examined. The second trial focused on determining the mechanism by which dietary Se is incorporated into tissue, utilising both conventional and germ-free (GF) mice. Mice were fed a diet with minimal Se, 0.018 parts per million (ppm), and diets with Se supplementation, to achieve 0.07, 0.15, 0.3 and 1.7 ppm Se, for 5 weeks . Mass spectrometry, Western blotting and enzymatic assays were used to investigate bioavailability, protein levels and GPx activity in fresh frozen tissue  from the Se fed animals. Inorganic, organic and nanoelemental Se were all effectively incorporated into tissues. The high Se diet (1.7 ppm) resulted in the highest Se levels in all tissues and plasma, independent of the Se source. Interestingly, despite being ~11 to ~25 times less concentrated than the high Se, the lower Se diets  resulted in comparably high Se levels in liver, ileum and plasma for all Se sources. GPx protein levels and enzyme activity were significantly increased by each diet, relative to control. We hypothesised that bacteria may be a vector for the conversion of nanoelemental Se, perhaps in exchange for S in sulphate metabolising bacteria. We therefore investigated Se incorporation from low sulphate diets and in GF mice. All forms of selenium were bioavailable and similarly significantly increased the antioxidant capability of GPx in the intestine and liver of GF mice and mice with sulphate free diets. Se from nanoelemental Se resulted in similar tissue levels to inorganic and organic sources in germ free mice. Thus, endogenous mechanisms, not dependent on bacteria, reduce nanoelemental Se to the metabolite selenide that is then converted to selenophosphate, synthesised to selenocysteine, and incorporated into selenoproteins. In particular, the similar efficacy of nanoelemental Se in comparison to organic Se in both trials is important in the view of the currently limited cheap sources of Se. This proteins ,6. SeMet β-lyase . On the pathways . H2Se isenosugar . Anotherenosugar , but its+VI, selenate [2Se, that could be readily incorporated into selenoproteins [The production of nanoelemental forms of Se can be achieved by biological reduction in the oxyanions, selenite and selenite, or by chemical means ,13,14. Eselenate  and sulpselenate  could feproteins . The purThe study utilized C57Bl/6 mice from the Animal Resources Centre (ARC)  and the Walter and Eliza Hall Institute  germfree mouse facility . Ethics was approved by the University of Melbourne and Florey Institute Animal Ethics Committees.2SeO3, Sigma, Sydney, Australia), l-selenomethionine  and nanoelemental Se, as pure red amorphous elemental Se nanoparticles in an aqueous suspension. Nanoelemental Se was produced in the laboratory by reducing selenite (Se+IV) with reduced glutathione (GSH) in the presence of bovine serum albumin (BSA). For this, 2.4 mL of 25 mM sodium selenite in water was added to 9.6 mL water containing 73.8 mg reduced glutathione and 480 mg BSA. Sodium hydroxide (1M) was added dropwise until red coloration appeared. After this reaction, the nanoparticles were cleaned from residues of the reaction by repeated (5×) ultrafiltration  and washing with nanopure water. After several washing steps, ultrafiltration and LC-ICP-MS was used to ensure as previously described to ensure that no residual selenite was present. A small aliquot (2–3 µL) of the washed suspension was dried on a silicon wafer before sputtering using Au−Pd. Particles sizes were then determined by scanning electron microscopy (SEM) on a Supra 40 V system  using an in-lens and a secondary electron detector as previously described [The Se formulations that were investigated were sodium selenite (Naescribed . The Se Selenium levels in prepared foods were determined by mass spectrometry using a Gas Chromatography Mass Spectrometer (GC-MS) at the National Measurement Institute .n = 14).Weaned C57Bl/6 mice, 4 weeks old, were placed on the prepared diets detailed below . Concentrations were chosen based on the current Food and Drug Administration (FDA) requirements of complete feed for livestock to not exceed 0.3 ppm  (1), 2019), with two diets below required threshold and one well above, at 1.7 ppm, added to determine if excessive inclusion would increase GPx activity or result in detectable toxicity. The source Se was made up to the desired concentration in distilled water and was added fresh each day to powdered Speciality Feeds low Se (0.018 ppm Se) diet  and mixed thoroughly. The low Se diet (SF16-014) was based on the standard AIN (American Institute of Nutrition) 93 rodent diet, but with no added selenium and with 30 g/100 g Torula yeast used instead of casein as the protein source to further reduce selenium. Extra vitamins were added to diets to account for expected losses during irradiation. The complete list of ingredients added to the diet can be found below . Diets w2 SO4. Total Se present for the diets  was 0.3 ppm. Total sulphur (S) present was 5600 ppm (5.6 mg/kg), primarily derived from sulphur-containing amino acids . Mice were placed on the organic sulphur diets (5600 ppm) with or without potassium sulphate (K2SO4) added to yield 0.6 g inorganic sulphur/kg, i.e., 600 ppm S. Se formulations were mixed with the organic S or the K2SO4 supplemented diet and irradiated at 25 kGy . Groups of eight mice  were fed diets with or without added potassium sulphate in the control (low Se) or Se supplemented diets (n = 64).For sulphate exclusion experiments, the base AIN93 rodent diet contained no added Se or sulphate, in the form of Kn = 36). Three mice for each different Se diet formulation were placed on the diet with no added Se (n = 12).Germ-free (GF) C57Bl/6 mice and matched specific pathogen free mouse (SPF) mice (4 weeks old) were placed on the low Se diet (0.018 ppm Se) or a 0.3 ppm Se enriched diet  for 5 weeks. Diets were irradiated at 50 kGy . Six mice  were in each Se enriched group  in either GF isolators or a SPF mouse unit in the same facility . For harvesting tissues, mice were anesthetised with a mixture of xylazine  and ketamine hydrochloride  diluted in sterile PBS, given subcutaneously. Terminal blood was collected from the anesthetized mice by cardiac puncture and the mice were then killed by decapitation for collection of tissue. Ileum, liver, skeletal muscle and fecal samples were taken for Se and enzyme determination. Wet weight was recorded and samples were placed in an Eppendorf tube and snap frozen in liquid nitrogen for Se determination by mass spectrometry; ileum and liver were snap frozen for determination of glutathione peroxidase (GPx) protein levels by Western blot, and GPx activity by functional assay. The terminal blood was collected in microvette EDTA tubes  and spun down (850 g for 10 min at 4 °C) and plasma was collected. The blood plasma was aliquotted into samples for determining GPx activity, Western blot and Se concentrations. Animals were weighed every day for the first 5 days then at longer intervals . Weight gains were not different between groups, consistent with other studies ,24.g for 15 min at 4 °C to extract protein. Total protein concentration of the liver and ileum lysates, as well as the plasma samples, were assayed with the Quick Start Bradford Protein Assay Kit .Snap frozen liver and ileum samples were sonicated in 5 mL ice cold buffer  per 1 g of tissue, followed by centrifugation at 10,000× To measure GPx activity, lysates were diluted to the following protein concentrations, in order to fall within the dynamic range of the assay, liver, 0.3 mg/mL; ileum, 3.5 mg/mL and plasma, 10 mg/mL. Extracts were assayed in duplicate using the Glutathione Peroxidase Assay Kit . Bovine erythrocyte GPx was used as a positive control for the assay (see manufacturer’s datasheet). Results are expressed as nMol/min GPx activity per mg total protein (nMol/min/mg).For Western blot analysis, the same liver and ileum lysates, as well as the plasma samples were diluted to 1 μg/μL in Laemmli sample buffer (BioRad) with 50 mM DTT and boiled for 10 min. Then, 20 μL of each was then loaded onto 4–15% Mini-PROTEAN TGX Stain-Free Protein Gels (BioRad) and electrophoresed at 100 V with 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3 running buffer. Precision Plus Protein All Blue Prestained Protein Standard (BioRad) was used for molecular weight determination. Gels were then placed in a ChemiDoc MP imaging system (BioRad) and exposed to UV light. Gels were then transferred to 0.2 μm nitrocellulose membranes using the Trans-Blot Turbo semi-dry transfer system and transfer packs (BioRad). The Stain-Free total protein labelling on the blot was then imaged, followed by air drying for 1 h, then blocking for 1 h in Odyssey blocking solution (Li-Cor). Primary antibodies see  were dilt-tests comparing ranks (Mann–Whitney test) were performed for comparison of ceca weights between GF and SPF mice. Statistical significance is designated as p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001 ****.All data are presented as mean ± SEM. One-way ANOVAs were performed on the datasets with a post hoc Tukey’s multiple comparisons test. We compared between Se groups in bioavailability experiments using a general ANOVA with a post hoc Tukey’s test  is provided in  were not significantly different . However, we found that the levels of GPx activity between mice on a nominally sulphate free diet and a sulphate enriched diet . The ceca of the GF mice were significantly enlarged F,G, whicp < 0.001) in GF than in SPF mice for the nanoelemental Se diets  selenoproteins. This could feasibly be through bacterial conversion [0 and Se+IV were incorporated into selenoproteins (Se−Ⅱ) with comparable high efficiencies, suggesting a common metabolic pathway  lead to incorporation of Se into ileum, liver and muscle A–E. Consnversion . We testnversion . These e pathway .−Ⅱ in order to incorporate selenite Se+ IV or elemental Se (Se0) into proteins (2Se (Se−Ⅱ) [−Ⅱ) that can be incorporated into selenocysteine by trans-selenation [+II) diglutathione (reaction 1). Selenodiglutathione (GS-Se-SG) is a highly efficient oxidant of reduced thiols, such as thioredoxin, and is a substrate for mammalian thioredoxin reductase. The overall reaction yielding H2Se is depicted below [Selenite and elemental Se both need to be reduced to Seproteins , via thee (Se−Ⅱ) ,29. In ced below ,31  to elemental Se as follows: .0 is known to readily react with numerous sulphur compounds of varying oxidation states, ranging from oxidized, e.g., sulphite (S +IV), through more reduced thiosulfate (S+Ⅱ) forms to the most reduced S compounds . The generalized thiol reaction yields selenium in fully reduced form (H2Se):This reaction represents a possible pathway linking selenite to elemental Se that could be further reduced to selenide . Se0 is In mammals, redox homeostasis is maintained by corresponding reductases. The oxidized disulfide form of glutathione (GSSG) for instance, is reduced, with NADPH consumption, to GSH by glutathione reductase. Hydrogen selenide is highly reactive  and is prone to oxidation to elemental Se, causing to some degree the generation of reactive oxygen species (simplified) according to:2Se; or trans-selenation in the case of selenomethionine) and enzymatic activity (eliminating ROS generated) [In addition, generation of reactive oxygen species (ROS) has also been observed upon chemical reaction of GSH and selenite  .Our work in vivo implies that after nanoelemental Se enters the small intestine it is reduced, perhaps in multiple steps, and incorporated into selenoproteins, which is consistent with earlier studies demonstrating increases in GPx phospholipid hydroperoxide, and thioredoxin reductase activity in response to dietary nanoelemental Se . If absoAn interesting observation was that elemental Se was absorbed as efficiently as selenite, but was closer to selenomethionine in its metabolic effect (increase in GPx activity). This confirms an earlier observation of similarly increased GPx activity in mice fed nanoparticle Se compared to selenomethionine . This isGiven its effective uptake, low toxicity  and low The current study shows that increased GPx protein levels and activity are very similar whether Se is delivered in nanoelemental form or as an organic form. Its absorption and metabolism is not dependent on bacterial conversion.


Schizophrenia is associated with elevated levels of circulating C-reactive protein (CRP) and other inflammatory markers, but it is unclear whether these associations extend to psychotic symptoms occurring in adolescence in the general population. A symptom-based approach may provide important clues for apparent trans-diagnostic effect of inflammation, which is also associated with depression and other psychiatric disorders.Based on data from 2421 participants from the Avon Longitudinal Study of Parents and Children birth cohort, we examined associations of serum CRP levels assessed around age 16 with ten positive and ten negative symptoms of psychosis assessed using questionnaires around age 17, using both individual symptoms and symptom dimension scores as outcomes. Regression models were adjusted for sex, body mass index, depressive symptoms, substance use, and other potential confounders.r=0.51; P < 0.001) than positive symptoms . The associations of CRP with positive and negative symptom dimension scores were similar. At individual symptom level, after adjusting for potential confounders including depressive symptoms, CRP was associated with auditory hallucinations  and anhedonia .Most prevalent positive symptoms were paranoid ideation (4.8%), visual (4.3%) and auditory (3.5%) hallucinations. Negative symptoms were more strongly correlated with concurrent depressive symptoms (Inflammation is associated with sub-clinical psychotic symptoms in young people in general population. Association of CRP with symptoms commonly shared between mood and psychotic disorders, such as auditory hallucinations and anhedonia, could be one explanation for the apparent trans-diagnostic effect of inflammation. •We examined psychotic symptoms in adolescents from a population-based birth cohort.•Common positive symptoms were paranoid ideation, visual and auditory hallucinations.•Negative symptoms were correlated with depressive and positive symptoms.•CRP was similarly associated with positive and negative symptom dimension scores.•CRP was specifically associated with auditory hallucinations and anhedonia. Meta-anPsychotic disorders consist of diverse symptoms, some of which are shared by other disorders. For instance, auditory hallucinations can occur in psychosis, depression and anxiety disorders . AnhedonWe present a longitudinal study of serum CRP concentration assessed around age 16 years and subsequent self-reported positive and negative symptoms of psychosis assessed around age 17 years in the Avon Longitudinal Study of Parents and Children (ALSPAC), a general population-representative birth cohort from the United Kingdom. We hypothesised that elevated CRP levels would be associated with specific psychotic symptoms commonly shared by other disorders. We used individual symptoms and factors scores for positive and negative symptom dimensions as our outcomes. Mood symptoms often co-occur with psychotic symptoms  and are 22.1http://www.bristol.ac.uk/alspac), and the sample characteristics and methodology have been described previously . The risk set for the current study comprised 3488 participants with data on serum CRP levels around age 16 years. All analysis was based on maximum available sample (see Tables). The analysis for association of CRP with positive and negative symptoms comprised 2421 and 2419 participants, respectively. Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees.The ALSPAC birth cohort initially recruited 14541 pregnant women resident in study catchment areas in Southwest England who had expected delivery dates between April 1, 1991 and December 31, 1992, resulting in 14062 live births. Additional children and parents were subsequently recruited into the cohort. Since age 7 years, the children attended annual clinical assessments during which they participated in various face-to-face interviews and physical tests. Detailed information about the ALSPAC cohort can be found on the study website . Participants fasted overnight before attending the clinic if seen in the morning, or at least for 6 h if seen in the afternoon. Blood samples were immediately spun, frozen and stored at −80 °C, which were analysed within 3–9 months of blood sampling with no freeze-thaw cycles in between. High sensitivity CRP (hsCRP) was measured by automated particle-enhanced immunoturbidimetric assay . No other inflammatory makers were measured. Among all 3488 participants, serum CRP level ranged from 0.07 to 72.55 mg/L; 62 participants (1.77%) had CRP levels ≥10 mg/L. Based on the American Heart Association and the US Centers for Disease Control and Prevention guidelines on the use of hsCRP levels in epidemiological studies , hsCRP w2.3hallucinations ; delusions ; thought interference . For each question, participants were given three options: definitely yes; may be; no, never. Cases were defined as participants reporting to have definitely experienced a psychotic symptom. The other two groups  were merged and used as control. This is because due to lack of cross-questioning, as in an interview, we are less confident whether those reporting “may be” in the questionnaire indeed experienced a positive symptom.Participants completed a questionnaire at average age 16.6 years (SD = 0.23), which included 10 questions about positive symptoms of psychosis asking whether they had experienced these symptoms at least once since 15th birthday . The que2.4Negative symptoms were assessed using a questionnaire, which included 10 questions based on items from the validated Community Assessment of Psychic Experiences (CAPE) questionnaire , adminisAccording to empirical research  and the 2.5Ethnicity and father's occupation were recorded during pregnancy; sex was recorded at birth; body mass index (BMI) was recorded around blood collection for CRP assay; age (in months) was recorded at the time of assessment of psychotic symptoms. Father's occupation was coded in six categories as per the UK Office of National Statistics classification system: I, II, III non-manual, III manual, IV, V . Depressive symptoms were assessed at the time of assessment of psychotic symptoms using the validated short mood and feelings questionnaire (SMFQ) . The SMFSmoking, alcohol, cannabis and other drug use were recorded using a questionnaire administered alongside assessment of psychotic symptoms. Number of cigarettes/roll ups smoked per day was coded as categorical variable: do not smoke; 1–5; 6–10; >10 cigarettes. Frequency of alcohol use was coded as never; monthly or less frequently; two to four times a month; twice a week or more frequently. The amount of cannabis used in past three months was coded as nil; <8th of an ounce; >8th of an ounce. Use of any other drugs since 15th birthday was coded as a single binary variable. Other drugs included inhaling/sniffing gas, solvent, aerosol, glue/poppers ; stimulants , hallucinogens ; heroin, ketamine, anabolic steroids.2.62.6.1Distributions of covariates were compared among groups with low, medium and high levels of CRP at baseline using one-way analysis of variance for continuous variables  and Chi-squared test for categorical variables . We tested correlation of total negative symptom score with any positive symptoms (binary) using point bi-serial correlation, and with total SMFQ score using Spearman's correlation.2.6.2First, we carried out exploratory factor analysis (EFA) based on 10 positive and 10 negative symptoms to test whether these symptoms represent one, two or three underlying factors. The two-factor solution representing positive and negative symptoms was chosen as optimum based on the interpretability of factors, and model fit indices (comparative fit index (CFI), Tucker-Lewis index (TLI), and Root Mean Square Error of Approximation (RMSEA)) obtained using MPlus software . Then, w2.6.3We used logistic regression to calculate odds ratio (OR) and 95% confidence intervals (CI) for common, individual symptoms  for participants with medium and high, compared with low, CRP levels. ORs were also calculated for any hallucinations, delusions and thought interference (separately). All regression models were adjusted for age, sex, BMI, father's occupation, ethnicity, total SMFQ score, smoking, alcohol, cannabis and other drug use.2.6.4We used logistic regression to examine association between CRP  and anhedonia, asociality and alogia, so the ORs represent increase in risk of each symptom per SD increase in CRP. We used linear regression for the other negative symptom subtypes and two major domain scores as these were coded as continuous variables. Regression models were adjusted for all confounders mentioned above.2.6.5We re-examined the association between CRP and auditory hallucinations after excluding participants who had reported to experience any positive symptoms only in the context of substance use (within 24 h of using cannabis or other drugs), illness (fever) or sleep . We also re-examined this association after excluding participants who had previously been assessed to have any positive symptoms at age 12 years  to minim2.6.6P-values for unadjusted ORs for specific positive/negative symptoms were corrected using Holm-Bonferroni method  had high CRP levels (>3 mg/L). CRP associated with BMI and tobacco, cannabis and other drug use, but not with sociodemographic factors or total SMFQ score .Table 1B3.2Around age 17 out of total 5126 participants assessed, 682 (13.3%) reported at least one positive symptom . Paranoi3.3=0.51; P < 0.001; N = 4981) than positive symptoms .The median (IQR) for total negative symptom score was 0 (0–2). Total negative symptom score was more strongly correlated with total SMFQ score (Spearman's rho3.4r=0.44 (P < 0.05). The association of CRP with positive  and negative  symptom dimension scores was similar  . CRP was3.6P=0.003. Evidence for this association remained after adjusting for current mood (P=0.007), and all potential confounders (P=0.023) (P = 0.042), but not with expressive deficit ; P=0.023) . The ass deficit .Table 3A3.7Evidence for association between CRP levels and auditory hallucinations remained after excluding participants who experienced positive symptoms in the context of cannabis/other drug use, illness or sleep; adjusted OR = 2.49 . Evidence for this association remained after excluding participants who had reported any psychotic symptom previously at age 12 years; adjusted OR = 4.52 . Evidence for association between CRP and auditory hallucinations also remained after excluding participants with CRP>10 mg/L at baseline; adjusted OR = 2.82 ; please see 3.8P-values for unadjusted ORs for auditory hallucinations (P=0.042) and anhedonia (P=0.015) remained significant , an enzyme that breaks down tryptophan along the kynurenine pathway, leading to increased levels of kynurenic acid and quinolinic acid . KynurenStrengths of the work include use of a longitudinal, general population-based sample, data on positive and negative symptoms, and a number of relevant confounders including depressive symptoms and substance misuse. We used two different analytic approaches, regression and factor analysis, which yielded consistent results. A key limitation of the study is the use of self-reported symptoms, as lack of scrutiny from an interviewer and misinterpretation of questions by participants could contribute to measurement error especially for negative symptoms. To minimize such error, we recoded individual positive and negative symptoms into binary variables to ensure that only symptoms reported as definitely present or often/always present were coded as ‘yes’. The prevalence of these symptoms, such as hallucinations in our sample, were not overly high, suggesting that participants did not endorse symptoms wholesale. Majority of participants had attended a face-to-face interview for psychotic symptoms previously at age 12 with a trained assessor , so theyThe prevalence of any positive symptom in this adolescent sample (13.3%) is comparable to that in other studies. According to a systematic review of population-based studies, the median prevalence of questionnaire-assessed positive symptoms in childhood/adolescence is around 9%  . StudiesThe length of follow-up for this longitudinal study was approximately one year; CRP was measured around age 16 and psychotic symptoms were assessed around age 17. However, the questionnaire elicited occurrence of positive symptoms anytime since 15th birthday, so the possibility that some participants might have experienced these symptoms before CRP measurement cannot be ruled out. It is noteworthy that evidence for association between CRP and auditory hallucinations persisted even after excluding participants who had reported any psychotic symptoms previously at age 12.Other limitations include use of one off measurement for CRP and lack of data on other inflammatory markers. However, CRP is a reliable marker of low-grade inflammation that has been used extensively in the literature. Anhedonia, asociality and alogia were measured using a single question, so in future studies should use more detailed assessments for these symptoms. Number of cases for some of the rare symptoms, such as thought interference, were relatively small, so in future studies with larger samples are required.Mechanisms for how inflammation cause auditory hallucinations require further investigation. Childhood abuse/maltreatment is associated with increased risk of auditory hallucinations, psychotic disorders  and withAssociations of CRP with positive and negative symptom factor scores were similar. However, composite scores can sometimes mask heterogeneity of association at individual symptom level in the case of heterogeneous syndromes like psychosis or depression, as evident from our data. A symptom-based approach may help to provide a better understanding of the role of inflammation in psychiatric disorders. For instance, our findings suggest that association of CRP with symptoms commonly shared between mood and psychotic disorders, such as auditory hallucinations and anhedonia, could be one explanation for the apparent trans-diagnostic effect of inflammation. Using a symptom-based approach, emerging evidence provides some clarity regarding potential role of inflammation in depression. Findings from the Dutch NESDA cohort , UK ALSPA clearer understanding of inflammation-related phenotypes could inform clinical trials of anti-inflammatory drugs for depression and schizophrenia. Elevated inflammatory markers at baseline predict poor antipsychotic response in first episode psychosis . Anti-inIn summary, we provide evidence that previously reported association of inflammation with psychotic disorders extend to sub-clinical manifestations of psychotic symptoms in young people. Our findings also suggest that association of CRP with symptoms commonly shared between mood and psychotic disorders, such as auditory hallucinations and anhedonia, could be one explanation for the apparent trans-diagnostic effect of inflammation. Replication of these findings in other samples particularly in patients with psychosis are required.Wellcome Trust, UK (Grant ref: 102215/2/13/2); and University of Bristol. Dr Khandaker acknowledges grant support from the: Wellcome Trust, UK (201486/Z/16/Z); MRC, UK (MC_PC_17213); and MQ: Transforming Mental Health (MQDS17/40). Professor Jones acknowledges grant support from the: Wellcome Trust, UK (095844/Z/11/Z & 088869/Z/09/Z); National Institute for Health Research (NIHR), UK . Prof Dantzer acknowledges grant support from the: National Institute of Health (NIH), USA ; and Cancer Center Support Grant (P30 CA016672). The funding agencies had no role in the design, conduct, and analysis of this study or writing of this manuscript.Core support for the ALSPAC birth cohort is provided by the: United Kingdom (UK) Medical Research Council (MRC); GMK designed the study, carried out data analysis and drafted the manuscript. JS contributed to study design, data analysis and revised the manuscript. SZ, GL, RD, and PBJ contributed to study design and revised the manuscript.The authors have no competing financial interests in relation to the work described. Prof Jones received a honorarium that he donated to his department, from Roche (UK), for taking part in an advisory board to advise on education about schizophrenia for psychiatrists. Prof Dantzer has received honorarium from Danone Nutricia Research unrelated to this study.


The kidney is the main site for the removal of chromogranin A (CgA). Previous studies have found that patients with renal impairment displayed elevated concentrations of CgA in plasma and that CgA concentrations reflect a deterioration of renal function. In this study, we aimed to estimate serum CgA levels and to evaluate the role of serum CgA in the early diagnosis of diabetic nephropathy (DN).n = 121), microalbuminuria (n = 73), or macroalbuminuria (n = 25) groups based on their urine albumin to creatinine ratios (UACRs). The degree of DN is reflected by UACR. A control group consisted of 45 healthy subjects. The serum CgA levels were measured by ELISA, and other key parameters were assayed.A total of 219 patients with type 2 diabetes mellitus (T2DM) were included in this cross-sectional study. These patients were classified into normoalbuminuria (P < 0.05). The levels of serum CgA increased gradually with the degree of DN (P < 0.001). Serum CgA levels showed a moderate-intensity positive correlation with UACRs (P < 0.001). A cutoff level of 3.46 ng/ml CgA showed 69.86% sensitivity and 66.12% specificity to detect DN in the early stage.Serum CgA levels were higher in patients with T2DM than in control subjects, and a statistically significant difference among the studied subgroups regarding CgA was found (The levels of serum CgA increased gradually with the degree of DN and can be used as a biomarker in the early detection of DN. Diabetes is one of the fastest growing health challenges of the twenty-first century. According to recent estimates from the International Diabetes Federation (IDF), 463 million adults are currently living with diabetes. The IDF estimates that there will be 578 million adults with diabetes by 2030 and 700 million by 2045. Globally, 11.3% of deaths are due to diabetes , the microalbuminuria group [4.83  ng/mL] and the macroalbuminuria group [6.35  ng/mL] regarding serum CgA levels (P < 0.001). A nonparametric Jonckheere-Terpstra test showed that CgA increased gradually with the degree of DN . The results are shown in Fig. A comparison of serum CgA levels among subgroups of patients divided according to UACR and healthy subjects was performed using the Kruskal–Wallis test. The serum CgA levels were higher in patients with T2DM  than in healthy subjects [1.89  ng/mL] . Weak correlations were found between serum CgA levels and FBG, HbA1c, sCr, BUN, eGFR, and HDL-C , and there was no correlation between serum CgA levels and age, SBP, DBP, sUA, TC, TG, or LDL-C in all subjects. There was a moderate-intensity positive correlation between serum CgA levels and UACRs in patients with T2DM (P < 0.001). Weak correlations were found between serum CgA levels and known diabetes duration, DBP, HbA1c, sCr, BUN, and eGFR , but there was no correlation between serum CgA levels and age, SBP, FBG, sUA, TC, TG, LDL-C, or HDL-C in patients with T2DM. The correlation coefficients between serum CgA levels and clinical and laboratory data are presented in Table The Spearman rank correlation test demonstrated that there was a moderate-intensity positive correlation between serum CgA levels and known diabetes durations and UACRs in all subjects (P < 0.001) and BUN  were independently associated with the occurrence of early DN when adjusted for age, sex, known diabetes duration, SBP, DBP, FBG, HbAlc, TC, TG, HDL-C, LDL-C, sUA, BUN, sCr and CgA. (Enter).We performed multivariate logistic regression analyses in patients with T2DM between the normoalbuminuria group and the microalbuminuria group. As shown in Table P < 0.001) in the differentiation of T2DM patients with early DN , patients with DN with microalbuminuria (n = 8), and patients with macroalbuminuria (n = 42). It is noteworthy that in their study, the P value of the difference between patients with normoalbuminuria and DN was 0.07. The failure to reach a significant difference may be due to the small sample size. Moreover, other studies have found that patients with renal impairment display elevated concentrations of CgA in plasma and that CgA concentrations reflect a deterioration of renal function [We found for the first time that serum CgA increased gradually with the degree of DN and that serum CgA levels were associated with the occurrence of microalbuminuria. The multivariate logistic regression analyses in patients with T2DM between the normoalbuminuria group and the microalbuminuria group revealed that CgA levels and not eGFR were independently associated with microalbuminuria, which suggests that CgA reflects progression of renal damage even at an early stage without apparent reduction of kidney function, and thus might represent a sensitive marker of DN. One study showed that serum CgA was a poor indicator of DN since patients with T2DM and a reduced glomerular filtration rate failed to show any significant increase in serum CgA . In theifunction , 36. CgAfunction . The assfunction .ROC analysis showed the diagnostic accuracy of serum CgA in differentiating between T2DM patients with early DN and those without DN. Using a cutoff of 3.46 ng/mL for serum CgA, we found a sensitivity of 69.86%, a specificity of 66.12%, and a diagnostic accuracy of 71.4% in predicting early DN.The in vivo isolated cleavage products of CgA in humans include vasostatin-1 and vasostatin-2, pancreastatin (PST), WE-14, cateslytin, and catestatin (CST). All of these products have significant and specific biological effects. Recent studies suggest that WE-14, CST and PST contribute to the development of different diabetes mellitus forms . At the The findings of this study are limited by the sample size and the cross-sectional design of the study; therefore, the direction of causality cannot be determined from these results. Moreover, the selected T2DM patients did not undergo renal biopsy. Although we formulated strict standards for excluding other patients with renal dysfunction caused by nondiabetic diseases, it cannot be ruled out that there could be patients with renal diseases other than DN selected into groups. Additional prospective studies are needed to examine associations between CgA and early DN risk.The results of this study show that serum CgA levels were higher in patients with T2DM than in healthy subjects and increased gradually with the degree of DN. CgA could also be used as a biomarker in the early detection of DN.