This review aimed to capture the key findings that animal models have provided around the role of the alternative pathway and amplification loop (AP/AL) in disease. Animal models, particularly mouse models, have been incredibly useful to define the role of complement and the alternative pathway in health and disease; for instance, the use of cobra venom factor and depletion of C3 provided the initial insight that complement was essential to generate an appropriate adaptive immune response. The development of knockout mice have further underlined the importance of the AP/AL in disease, with the FH knockout mouse paving the way for the first anti‐complement drugs. The impact from the development of FB, properdin, and C3 knockout mice closely follows this in terms of mechanistic understanding in disease. Indeed, our current understanding that complement plays a role in most conditions at one level or another is rooted in many of these in vivo studies. That C3, in particular, has roles beyond the obvious in innate and adaptive immunity, normal physiology, and cellular functions, with or without other recognized AP components, we would argue, only extends the reach of this arm of the complement system. Humanized mouse models also continue to play their part. Here, we argue that the animal models developed over the last few decades have truly helped define the role of the AP/AL in disease. However, we would argue the data generated in animal models is incredibly useful and, when used wisely, answers key questions. Thus, we will begin our section on the AP/AL of complement in animal models by directing you to the ground‐breaking work by Mark Pepys1.2Some important basics worth discussing with respect to small animal models is that the AP of mouse/rat, as in humans, includes C3, FB, FD, properdin, FH & FI, and that these proteins function in a highly analogous way  towards both mC3bBb and (less effectively) hC3bBb.Cfh−/− mice following supplementation with hFH, shows competent inter‐species functional cross‐over.GPI‐DAF−/− mouse‐derived red cells confirmed that mouse DAF could regulate human complement activation .The role of mFH seems to reflect that of hFH;1.3Cfh−/− mouse offered the potential of an animal model to dissect the role of FH in renal disease.C3 glomerulopathy (C3G), encompassing dense deposit disease (DDD) and C3 glomerulonephritis ), describes rare forms of glomerulonephritis  with common clinical features and overlapping histological appearances.Cfh including exon 3 was replaced by the Neor gene).Cfh−/− mice were described as viable and fertile under specific pathogen free conditions. As expected from the studies in pigs and man, loss of murine FH caused almost complete C3 consumption,Cfh+/− (heterozygous) mice also had lower C3 levels, suggesting a direct correlation with FH and intact C3 levels. Cfh−/− mice (6/26) succumbed to suspected renal disease by 8 months of age, with florid C3 and C9 deposition in the glomeruli of the mice, with some evidence of IgG deposits. Thus, Cfh−/− mice appeared to present with many of the salient features of C3G,This challenge was taken up by Matthew Pickering and colleagues in Marina Botto's Laboratories, Imperial College London, using standard gene knockout technology  mice, confirming the causative link between AP/AL and phenotype.To confirm this, the authors backcrossed the Cfi−/− mice were generated using standard knockout technology (deleting exon 4 of the Cfi gene)Cfi−/− mice compared with wildtype or Cfi+/− littermates.Cfi−/− mice was established to be C3b, suggesting full consumption of intact C3, essentially as found in man.Cfh−/− mice will develop a recognizable C3G phenotype.Cfi−/− mouse phenotype was arguable closer to that of the Cfd−/− mice (covered later) than the Cfh−/− mice (where C3 deposition is on the GBM), although all 3 mice exhibit recognizably different renal phenotypes in 8‐month‐old mice.Cfi−/−.Cfh−/− mice was found to be identical to the Cfi−/− mouse, demonstrating that FI was necessary for GBM mediated deposition of C3 and therefore, C3G.Cfh−/− mice is essentially absolute while in the Cfi−/− mice (or Cfi−/−.Cfh−/−or Cfi−/−.Cfh+/−), C3 levels are about 25‐30% normal. Only when Cfh−/− or Cfi−/−.Cfh−/− mice have FI present are iC3b and C3c  fragments generated and detectable in the serum. Furthermore, C3 activation products are rapidly deposited on the GBM; within 72 hour in the case of Cfi−/−.Cfh−/− mice infused with Cfh−/−.C3−/− sera (replete in mouse FI but no C activity).Cfi+/−.Cfh−/− mice have florid C3 deposition on the GBM, underlining the necessity for FI and C3 fragments in the pathogenesis of C3G. The importance of inactivated fragments of C3b in C3G was further explored in an additional double knockout strain—the Itgam−/−.Cfh−/− mice.Itgam−/−.Cfh−/− mice compared with Cfh−/− mice as they aged to 8 months, particularly in animals exposed to greater risk of infection . Bone marrow transfer experiments established that loss of CR3 on white cells drove the renal phenotype. Therefore, while unexpected, signaling via CR3 was protective in Cfh−/− mice and to our mind links back to the importance of the AL/AP in immune complex solubilization and handling as well as the importance of complement breakdown fragments generated by the AL/AP in normal physiological processes. Similar far reaching and complex effects of the AL/AP have also been noted in Cd55−/−  mice,H1 responsesC3−/−, Cfd−/−, and Cfb−/− animals display significantly reduced antigen presenting cell (APC) function and associated T cell responses.In contrast to C3b, iC3b and its metabolites cannot interact with FB or properdin to form a C3 convertase. Studies in man had established that deficiency in FI lead to loss of many complement‐mediated functions,Cr1l) are embryonic lethal.−/− were recovered from 245 births. Thus, control of the AP is critical for embryonic survival beyond 9.5 days of development as Crry is the only complement regulator available to control the AP/AL at these sites. Backcross of Crry−/− mice onto the C3‐deficient background rescued the phenotype and confirmed the role of complement, influx of polymorphonuclear leukocytes and inflammation in the demise of the embryo.In the mouse, Crry fulfils the role of both hDAF and hMCP in control of C3 activation at many surfacesC3−/− used by Molina and colleagues above were developed by standard knockout technology (targeting the promoter and exon 1 C3 gene but with NeoR in the reverse orientation to reduce the risk of a truncated C3 being expressed under the NeoR promoter).−/− mice showed a nearly 2000‐fold increase in bacterial load compared with C3+/− littermates and had much poorer survival profiles. However, these mice appeared to generate a pro‐C3 which may leave any intracellular roles for C3 intact despite complete loss of serum complement activity.−/− mice are distinct from the more commonly used C3 null mouse developed in the laboratories of Mike Carroll  which are available from the Jackson Laboratory, Bar harbour, Maine, US . The C3tm1Crr/J (C3−/−) mice were also developed by standard knockout technology (where the NeoR gene replaced the sequence encoding the arginine rich linker of pro‐C3) and were found to be extensively healthy but had a markedly impaired responses to group B steptococci.−/− mice have helped revolutionize our understanding of the role for complement in an ever‐increasing number of diseases and conditions.The −/−) and Crry failed to correct the phenotype. Further confirmation that the classical pathway played no role in the loss of embryos was noted in mice deficient for C4 and Crry, which were embryonic lethal. Surprisingly, deletion of C5 in the context of Crry‐deficiency also failed to protect embryos from AP/AL activation. Thus, the AP/AL was the only part of the complement system involved in this phenotype.C3−/−.Crry−/− males with C3+/+.Crry+/− females, the authors were able to discern that it was maternal C3 that was responsible for the complement mediated loss of the embryos. It was later shown that properdin deficiency could protect Crry−/− mouse from AP‐mediated embryonic death.Returning to the Crry deficient mouse, it was demonstrated that embryonic lethality had an absolute requirement for the AP/AL,Cfp−/− mice were first developed by Cordula Stover and colleagues, using standard knockout technology (replacing exons 3‐7 with Neor) and deleting the thrombospondin repeats (TSR) with importance for binding to C3b.Cfp−/− sera (in the presence of EGTA) was significantly less than noted in wildtype mice. Using a sepsis model, it was evident that properdin was critical to animal survival and that properdin may interact directly with bacteria to trigger and sustain complement activation. These data fit well with our understanding of properdin as a potential initiator and as the principal positive regulator of the AP/AL. Based on these data, loss of positive regulation of the AP/AL through loss of properdin was predicted to ameliorate C3G but that was not the outcome.Cfp−/−.Cfh−/− mice had very little detectable C3 in their plasma, that is, not significantly different to Cfh−/− mice unlike the essentially normal levels in the Cfp−/− mice. However, C5 plasma levels were strikingly different between Cfp−/−.Cfh−/− (readily detectable) and Cfh−/−  mice, suggesting an important role for properdin in C5 consumption/activation in Cfh−/− mice.m/m) but only a small amount of the mutated FH was detectable in the mouse plasma (presumed a result of an unstable FH mRNA product), insufficient to prevent uncontrolled consumption of C3 and FB. These mice had a renal phenotype akin to the Cfh−/− mice, which again, over time (12 months), developed into a recognizable C3GN. The authors termed this model an “age‐dependent C3 GN”.Crry−/− mice but zymosan mediated AP activation was not significantly disrupted by the lack of properdin. Importantly, these in vivo studies demonstrated that properdin was important for context dependent AP activation rather than necessary for sustained activation of the AL. When this Cfp−/− mouse was backcrossed to the FHm/m mice, as with the previous study,m/m or Cfp−/− mice alone) with all mice dying as a result of renal disease between 8 and 12 weeks.In experiments that paralleled those above and an attempt to develop a mouse model of aHUS, Song's group used gene knock‐in technology to insert a two stop codons at the beginning of CCP19 of mouse FH.cfhloxp/loxp mice (loxP sites engineered 3 prime exon 2 and 5 prime exon 3) had reduced FH plasma levels, suggesting an impact on the expression of this gene across the mouse. This is reminiscent of, although not as marked as, the issues encountered in the generation of the properdin deficient line developed in Song's group above.Cfh−/− and Cfh−/−.Cfp−/− mice. The data again confirmed the need for FH to protect the GBM and indicated that even 20% normal levels was sufficient to protect this particular surface. Another interesting mechanistic insight from this study is that even low levels of FH can restrict AP C5 convertase formation and the consumption of C5. The data also highlighted the role of C5 activation in the formation of thrombotic microangiopathy (TMA) in the kidney and provided a model system that allows analysis of aspects of C3G and aHUS, a phenotype that can be seen in the clinic.The Imperial studies also confirmed differential staining patterns in the kidney in the presence and absence of properdin, suggesting that properdin was facilitating consumption of complement at particular surfaces in the kidney, again demonstrating that properdin is more important in activation of the AP at particular sites than it is in sustaining the AL in the fluid phase.Cfh−/− mice, animals devoid of FH and their FHRs had very little intact C3 in circulation while deletion of all FHRs had no apparent impact of C3 levels. Renal phenotype in 8‐month‐old mice was also established to be comparable between delFH‐FHR and Cfh−/− mice.hCFH and native hCFHR5 genes to the delFH‐FHR strain, partial humanization of the mouse FH locus was achieved. These mice had differential expression of hFH allowing the authors to note the direct correlation with hFH level and that of C3/C5 in the transgenic delFH‐FHR mice. Only hFH‐FHRmut (delFH‐FHR) mice demonstrated pathogenic deposition of C3, properdin and FH/FHRmut protein in the kidney.The FH‐related proteins (and properdin) are such modifiersCFH‐H, see later) but not in mice with low hFH (CFH‐Y) expression.,Humanizing the mouse complement system is now becoming common place due to the advances in gene manipulation technologies but requires careful consideration. One early example was mice humanized with full‐length hFH, which was successful in reversing kidney disease in transgenic mice with expression levels closer to normal mouse FH expression (denoted 1.4Hemolytic uremic syndrome (HUS) is defined as the clinical triad of microangiopathic anemia, thrombocytopenia, and acute renal injury. This can be sub divided into Shiga toxin‐associated HUS (STEC‐HUS) and non‐diarrheal or atypical HUS (aHUS). STEC‐HUS is the most common form, accounting for >90% of cases and is linked to preceding illness with Shiga toxin‐producing bacteria.Cfh−/− mouse to generate a mouse that only expressed the truncated FH protein.Cfh+/− mice, sufficient to prevent accumulation of C3 on the GBM (as noted above for Cfh−/− mice). The Cfh−/−.FHΔ16‐20 mice rapidly succumbed to renal disease characterized by hematuria and TMA, and no evidence of electron dense deposits by EM. In short, the development of this mouse demonstrated that the carboxy terminus of FH is essential to prevent aHUS‐like disease.Cfh+/−.FHΔ16‐20 mice did not develop any disease  also provided support to the concept that additional modifiers were important to precipitate disease in aHUS patients and by extension, that functional deficits in the AP alone were not the full story in aHUS. The Cfh−/−.FHΔ16‐20 mouse is no longer available but additional approaches to explore the AP in aHUS were developed. This includes a mouse developed with a single point mutation in the C terminus of mouse FH  based on a mutation (FH W1183R) identified in aHUS patients.m/m mouseR/R mice (as they were denoted).Crry−/−.DAF−/−.C3−/−; see below)” red cells. FHR/R mice failed to thrive (30% died within 30 weeks postpartum) and had several “clinical” or pathological features consistent with those seen with aHUS patients.R/R mice, with large clot formation in many organs, particularly the liver and brain and about a third of the FHR/R mice developed neurological abnormalities consistent with thrombotic and hemorrhagic stroke,R/R mice, it did not reduce C3 and C9 loading in the kidney.The group at Imperial College London were the first to describe a mouse model of aHUS based on the appreciation that aHUS‐associated defects were predominately in the C terminus of Factor H and distant from the complement regulatory domain in the N terminus (where mutations were more commonly associated with C3G and AMD).R/R mouse, (we) Kate‐smith Jackson et al. focused on C3 mutants associated with aHUS in a large patient cohort. After selecting and testing several patient‐associated mutants using in vitro cell culture analysis, we focused on a C3 D1115N mutation, found in three families world‐wide.D1115N also interacted appropriately with Crry. Homozygote mice  display all the hallmarks of aHUS, that is, acute kidney injury , thrombocytopenia, anemia and thrombotic microangiopathy  associated with significant AP dysregulation . This phenotype can be ameliorated by anti‐C5 mAb, replicating human disease.N/N mice (up to 6 months of age) did not have evidence of extra‐renal disease (unlike the FHR/R mice described above), and therefore, appear more faithful to aHUS in man. We also have begun to tease apart the molecular pathways that drive disease via backcrossing our model onto properdin, C7 and C5aR deficient mice to aid understanding and drug target design. While clearly distinct, our findings share some similarities with those noted for the FHR/R mouse. For instance and most relevant herein, we found properdin deficiency was protective on C3N/N mouse background. Over 6 months, only 2 of 13 C3N/N.Cfp−/− mice succumbed to disease compared with 14 of 15 C3N/N mice. C3N/N.Cfp−/− mice had no evidence of hematuria, proteinuria, or thrombotic microangiopathy . There was no evidence of a dose‐response as C3N/N.Cfp+/− were not protected and succumbed to disease in a similar fashion to properdin sufficient C3N/N . During this study, we also note that C3N/N have low serum levels but significant renal properdin deposits in comparison with wildtype controls. These data potentially indicate that consumption of properdin is a factor in disease progression or that, as with other models, where high kidney C3 deposits are noted, that properdin is sequestered. This makes some sense in that the lack of FH binding to C3 convertase in the model should allow more properdin to bind and remain resident. Properdin deficiency did not appear to alter C3 deposit load in the kidney, again mirroring the FHR/R results, suggesting C3 convertase remains highly active generating significant deposits of C3b. Therefore, given the success of anti‐properdin or properdin deficiency in ameliorating disease in both the C3N/N and FHR/R models of aHUS, where both have defined TP dependency, it seems likely that these effects are related to loss of stability in the AP C5 convertase.Developed in parallel to the FH1.5DAF−/− mice had much poorer renal function during a bilateral renal IRI model than WT (or CD59a−/−); Double DAF/CD59−/− compounded this effect significantly. More severe tubular damage and neutrophil infiltrate was noted in DAF deficient mice, which again was further amplified in the double knockout mouse. Depletion of C3, by CVF, prior to IRI induction in the DAF/CD59 double knockout mice confirmed a role for complement in the renal damage. Furthermore, detailed analysis of C3 deposits confirmed that C3 deposits are routinely seen on tubular cells in healthy mice but that C3 (and C9) deposition in the peritubular capillaries indicated that MAC‐induced microvascular injury was the principal pathogenic mechanism for complement in this model of IRI. Zhou et al illustrated that mice deficient in C3 are protected from IRI and that was not seen in C4 deficient mice.−/− miceCfb−/− mouse to tease apart the role of the AP/AL in pathology, Thurman et al demonstrated that loss of FB significantly protected the kidney from reperfusion injury.The development of DAF (CD55) deficient miceWhile all tissues are not equal, FB deficiency is protective in a model of transient coronary ligation.Cfb−/− mice and those treated with CR2‐FH Local complement production by cells such as hepatocytes, adipocytes, and chondrocytes is well characterized, but in recent years, the hypothesis of intracellular complement has developed and become controversial. This phenomenon was first observed in human T cells,1.8Cfd−/− mice  were healthy and viable with no apparent abnormality noted up to 26 weeks of age (similar to Cfb−/− mice). Cfd−/− animals  showed no difference in body weight or serum concentrations of triglycerides, cholesterol, and free fatty acids.Cfd−/− mice displayed no AP activity in RRBC‐based hemolytic or zymosan C3 deposition assays, despite significantly increased C3 levels compared with wildtype littermates, demonstrating that FD was essential for C3H2OBb formation in mice. Additionally, these experiments underlined the importance of the AL/AP in opsonization of bacteria, with around 66% less opsonization seen in FD deficient sera.Cfd−/− and wildtype animals, when fed a high‐fat diet (HFD).Cfd−/− HFD livers showed decreased hepatic lipid accumulation (independent of HFD‐induced insulin resistance) and reduced expression of genes involved in fatty acid synthesis and the PPARγ/CD36/FATP2 lipid uptake pathway.The association of complement with the field of metabolism is also longstanding; adipose tissue produces a number of complement proteins including C3 and FBCfp−/− mice showing increased body weight, decreased expression of C5a like receptor 2 (C5L2 or C5aR2), decreased insulin sensitivity, obesity‐associated glomerulopathy, and increased hepatic triglyceride.The link between metabolic dysfunction, immunity, and non‐alcoholic fatty liver disease (NAFLD) is gaining greater interest, as NAFLD becomes the predominant global cause of cirrhosis and hepatocellular carcinoma.1.9CFB, CFI, and C3, with AMD has confirmed a role for the AP/AL. Although patients with AMD do exhibit higher serum levels of activation products C3d, Ba, C3a, C5a, SC5b‐9; C3, C4, FB, and FD, allowing for a systemic impact of genetic changes on AMD, local complement activation and the correct regulation of that activated complement are being described as critical to disease progression.Complement activation plays a role in AMD. Initial evidence of the involvement of complement activation in AMD came from the observation that drusen were found to contain various components of the complement system including C3, C5, and C5b‐9.With these findings came an urgency to model complement dysfunction in the mouse. While mice do not possess a macula, it has been proposed that the retinal structures of the mouse eye are similar to those of the peripheral retinas in humans, and that they can exhibit the characteristics of dry AMD.1.10CFH associate so strongly with AMD in manCfh−/− mice for evidence of an AMD phenotype. Analysis of eyes from 2‐year‐old Cfh−/− mice indicated an accumulation of C3 in the neural retinal and a thinning of the Bruch's membrane, associated with disorganization of cellular organelles (mitochondria) in the RPE, providing additional data to support a role for the AP/AL in eye damage in aged mice.Cfh−/− mice revealed evidence of retinal damage,Cfh−/− mice as they age.Cfh−/− mice held in a barrier (specific pathogen free) versus an open environment indicated that the additional stress, and presumably systemic complement activation associated with the open environment, was important for the development of retinal pathology noted in these mice.Cfh−/− mice is therefore considered to represent early AMD .As noted above, the fact that gene polymorphisms in APOE gene) mouse. These animals, when aged and placed on a high‐fat, cholesterol‐enriched (HFC) diet, recapitulate many aspects of the human AMD phenotype, including RPE cell pathology, visual function deficits, and formation of sub‐RPE deposits, including significant deposition of C3.Cfh−/− background (with HFC diet) was reported to generate an aggravated disease while backcross onto the sCrry expressing mice (with HFC diet) ameliorated disease, confirming that in this model of AMD the AP/AL played a distinct role in the eye damage.The role of complement in eye disease was underlined in the human APOE4 knocked‐in  were generated.Cfh+/− were favored, as with advanced age, and use of high‐fat diet, these mice developed sub‐RPE deposit formation which lead to complement activation and ultimately eye pathology which was considered more consistent with AMD.With the understanding that pathological outcomes similar to man were achievable in the eyes of mice that had altered AP/AL components, several studies were undertaken with the aim to tease apart the pathophysiological relevance of the FH Y402H variant. This SCR7 variant is in an area of FH important for “self” cell surface interactions, binding to C‐reactive protein and immune evasion by several pathogens.2Cfb−/− mice in this model.From as early as 1985, we have been aware that components of cigarette smoke can trigger activation of AP/AL in normal human serum (NHS), possibly modifying C3 in such a way to prevent FH from binding;C3−/− animals suggest that systemic and local levels of C3 are critical for progression of inflammatory eye disease.Interestingly, studies with C3−/− mice  at 12 months old, findings that are largely supported above.C3aR−/− (and C3aR−/−C5aR−/−) mice.In other studies, the flip side of the AP/AL was noted, in that mice lacking C3aR were found to develop early onset and progressive retinal degeneration, linking a role in C3a generation to normal retinal function.3CFI sequence (not codon optimized) via a sub‐retinal injection of mouse eyes.One of Professor Peter Lachmann's legacies will of course be his drive to use FI as therapeutic, and with Prof's David Kavanagh and Andrew Lotery was a scientific founder of Gyroscope (now part of Novartis). They aimed to provide precision medicine treatment of patients with AMD via FI gene therapy. One of the supporting studies towards their current clinical trial  was the testing of AAV constructs carrying the human 3.1DAF−/−, Cfh−/−, and C3−/− mice) as described herein, has been/is highly informative but it is rare in man. As noted, point mutations and polymorphisms are sufficient to increase human disease susceptibility; they may have only subtle effects at a molecular level but such variations can have profound and lasting effects on immunity. To us, this fits squarely with Chaos Theory and the Butterfly Effect or as Edward Lorenz put it “For those systems with bounded solutions, it is found that non‐periodic solutions are ordinarily unstable with respect to small modifications, so that slightly differing initial states can evolve into considerably different states”.Deletion of whole molecules/genes (i.e., KJM was a scientific consultant and provided research for Gemini Therapeutics. KJM has also received research funding from Qualasept, Catalyst Biosciences and Idorsia.


We saw considerable overlap in nutrient responses across most bacteria regardless of phyla, with many taxa growing slowly and few taxa that grew quickly. To define plausible life history boundaries based on in situ relative growth rates, we applied Gaussian mixture models to organisms’ joint 18O–13C signatures and found that across experimental replicates, few taxa could consistently be assigned as copiotrophs, despite their potential for fast growth. When life history classifications were assigned based on average relative growth rate at varying taxonomic levels, finer resolutions  were significantly more effective in capturing changes in nutrient response than broad taxonomic resolution . Our results demonstrate the difficulty in generalizing bacterial life history strategies to broad lineages, and even to single organisms across a range of soils and experimental conditions. We conclude that there is a continued need for the direct measurement of microbial communities in soil to advance ecologically realistic frameworks.Study of life history strategies may help predict the performance of microorganisms in nature by organizing the complexity of microbial communities into groups of organisms with similar strategies. Here, we tested the extent that one common application of life history theory, the copiotroph-oligotroph framework, could predict the relative population growth rate of bacterial taxa in soils from four different ecosystems. We measured the change of in situ relative growth rate to added glucose and ammonium using both  The concept of copiotrophy and oligotrophy in microbial communities offers the potential for an organizing principle to describe the complexity of microbial systems. As such, it has been discussed for decades in relation to marine environments  and soilThe copiotroph-oligotroph framework is commonly used for the interpretation of 16S rRNA gene bacterial community data –13. FierIt is also possible that the pressure to select for either copiotrophic or oligotrophic strategies is strong in soils but that the current practice of assignment of bacterial phyla to one strategy or the other is inaccurate Fig.  or impreHere, we analyzed published quantitative stable isotope probing (qSIP) data , 24, 25 0, Null: No bimodality in nutrient response of taxa to nutrient addition , or water with glucose and a nitrogen source . To track growth through isotope assimilation, both 18O-enriched water (97 atom %) and 13C-enriched glucose (99 atom %) were used. In all treatments isotopically heavy samples were paired with matching “light” samples that received water with a natural abundance isotope signatures. For 18O incubations, this design resulted in three soil samples per ecosystem per treatment  while 13C incubations were limited to only C and CN treatments (n = 24). Previous analyses suggest that three replicates is sufficient to detect growth of 10 atom % 18O in microbial DNA with a power of 0.6 and a growth of 5 atom % 18O with a power of 0.3 (12 and 6 atom % respectively for 13C) [To generate experimental data, three replicate soil samples were collected from the top 10 cm of plant-free patches in four ecosystems along the C. Hart Merriam elevation gradient in Northern Arizona. From low to high elevation, these sites are located in the following environments: desert grassland , piñon-pine juniper woodland , ponderosa pine forest , and mixed conifer forest . Soil samples were air-dried for 24 h at room temperature, homogenized, and passed through a 2 mm sieve before being stored at 4 °C for another 24 h. This produced three distinct but homogenous soil samples from each of the four ecosystems that were subject to experimental treatments. Three treatments were applied to bring soils to 70% water-holding capacity: water alone (control), water with glucose  . All soi18O or 13C isotope incorporation into bacterial DNA (excess atom fraction or EAF), the qSIP protocol [18O or 13C isotopes through the method of density fractionation by adding 1 µg of DNA to 2.6 mL of saturated CsCl solution in combination with a gradient buffer  in a 3.3 mL OptiSeal ultracentrifuge tube . The solution was centrifuged to produce a gradient of increasingly labeled (heavier) DNA in an Optima Max bench top ultracentrifuge  with a Beckman TLN-100 rotor  at 18 °C. Each post-incubation sample was thus converted from a continuous gradient into approximately 20 fractions (150 µL) using a modified fraction recovery system (Beckman Coulter). The density of each fraction was measured with a Reichart AR200 digital refractometer . Fractions with densities between 1.640 and 1.735 g cm−3 were retained as densities outside this range generally did not contain DNA. In all retained fractions, DNA was cleaned and purified using isopropanol precipitation and the abundance of bacterial 16S rRNA gene copies was quantified with qPCR using primers specific to bacterial 16S rRNA genes . Triplicate reactions were 8 µL consisting of 0.2 mM of each primer, 0.01 U µL−1 Phusion HotStart II Polymerase , 1× Phusion HF buffer (Thermo Fisher Scientific), 3.0 mM MgCl2, 6% glycerol, and 200 µL of dNTPs. Reactions were performed on a CFX384 Touch Real-Time PCR Detection System  under the following cycling conditions: 95 °C at 1 min and 44 cycles at 95 °C (30 s), 64.5 °C (30 s), and 72 °C (1 min). Separate from qPCR, retained sample-fractions were subject to a similar amplification step of the 16S rRNA gene V4 region  in preparation for sequencing with the same reaction mix but differing cycle conditions – 95 °C for 2 min followed by 15 cycles at 95 °C (30 s), 55 °C (30 s), and 60 °C (4 min). The resulting 16S rRNA gene V4 amplicons were sequenced on a MiSeq sequencing platform . DNA sequence data and sample metadata have been deposited in the NCBI Sequence Read Archive under the project ID PRJNA521534.The procedure of qSIP (quantitative stable isotope probing) is described here but has been applied to these samples as previously published , 21, 25.protocol  was usedprotocol , 32. BriIndependently from previous publications, we processed raw sequence data of forward and reverse reads (FASTQ) within the QIIME2 environment  (release18O and 13C excess atom fraction (EAF) for each bacterial ASV using R version 4.0.3 [https://www.github.com/bramstone/. Negative enrichment values were corrected using previously published methods [n = 3) and less than three density fractions within those two replicates were removed to avoid assigning spurious estimates of isotope enrichment to infrequent taxa. Any ASVs filtered out of one ecosystem-treatment group were allowed to be present in another if they met the frequency threshold. Applying these filtering criteria, we limited our analysis towards 3759 unique bacterial ASVs which accounted for a small proportion of the total diversity but represented 68.0% of all sequence reads, and encompassed most major bacterial groups  and from the 18O EAF of bacteria in CN-amended soils to determine C and N response (Δ18O EAFCN). Because an ASV must have a measurable EAF in both the control and treatment for a valid Δ18O EAF to be calculated, we were only able to resolve the nutrient response for 2044 bacterial ASVs – 1906 in response to C addition and 1427 in response to CN addition.All statistical tests were conducted in R version 4.0.3 . We asse13C and 18O EAF values. To compare both isotopes directly, we divided 18O EAF values by 0.6 based on the estimate that this value (designated as µ) represents the fraction of oxygen atoms in DNA derived from the 18O-water, rather than from 16O within available C sources [18O enrichment but comparatively low 13C enrichment, potentially indicating growth following the depletion of the added glucose, and that were reasonably clustered as oligotrophs in our mixture model.We used Gaussian finite mixture modeling, as implemented by the mclust R package , to dema sources . Two mix18O–13C EAF in a treatment) could predict its growth across replicates . We applied the treatment-level mixture models defined above to the per-taxon isotope values in each replicate, recording when a microorganism’s life history strategy in a replicate agreed with the treatment-level cluster, and when it didn’t. We used exact binomial tests to test whether the number of “successes” (defined as a microorganism being grouped in the same life history category as its treatment-level cluster) was statistically significant. To account for type I error across all individual tests (one per ASV per treatment), we adjusted P values in each treatment using the false-discovery rate (FDR) method [We tested how frequently mixture model clustering of each microorganism’s growth (based on average ) method .18O and 13C EAF values were combined into a single variable using principal components analysis separately for each treatment. Across the C and CN treatments, the first principal component (PC1) was able to explain – respectively – 86% and 91% of joint variation of 18O and 13C EAF values. In all cases, we applied PC1 as the response variable and treated taxonomy and ecosystem as random model terms to limit the potential of pseudo-replication to bias significance values. We used likelihood ratio analysis and Akaike information criterion (AIC) values to compare models where life history strategy was determined based on observed nutrient responses at different taxonomic levels . However, we did observe ASVs with strong positive responses to C and CN addition, despite the prevailing unimodal pattern  while fast-growing  ASVs had mean enrichment values greater than 0.3 . In both the C and CN treatments, most bacterial ASVs (>90%) were clustered into the oligotrophic growth category defined by the mixture model   Fig. .Fig. 3CoPaenibacillus and an unclassified genus within the order Bacillales, Actinobacteria: an unclassified genus within the Micrococcaceae) while the remaining occurred in the oligotrophic cluster.Bacteria generally behaved consistently across replicates, but few ASVs could be identified as soley copiotrophic or oligotrophic with a high degree of statistical confidence. Nevertheless, assignment of bacterial ASVs into different life history clusters, based on groupings from mixture models Fig. , provideL = 12.5, P < 0.001; CN response: L = 10.0, P = 0.0015), providing evidence against hypothesis H0 . This provides strong support for hypothesis H2 , indicating that assumptions about the behavior of any particular taxon cannot be made on the basis of its representative phylum. Fierer et al. note as much in their seminal work [Our results indicate that microbial life history strategies, as currently conceptualized, do not provide a strong predictive framework on the behavior and activity of most microorganisms in the soil environment. Rather, under the conditions of this experiment, microorganisms exhibited a continuous distribution from copiotrophic to oligotrophic strategies as represented from high to low growth rates, with most microorganisms showing low to intermediate growth rates. While this suggests support for our null hypothesis, Hnal work .Copiotrophic and oligotrophic modes are roughly analogous to nutrient acquisition or stress tolerance strategies (respectively) within the yield-acquisition-stress tolerance framework . The ass2. Therefore, while we refute the continued use of categorical assumptions of oligotrophic or copiotrophic life histories for bacterial phyla, our in situ findings suggest that such representations could be useful if made at the genus level  – in agreement with previously reported conclusions [We saw significant improvement in hierarchical linear models of nutrient response when life history strategy was estimated at finer levels of taxonomic resolution  which indicates strong support for hypothesis Hclusions .Despite the best performance of the genus-level models to estimate nutrient responses, we had difficulty confidently characterizing the growth of individual ASVs; few could be consistently labeled as copiotrophs because many grew both quickly and slowly across the replicates in our experiment. Such indeterminancy is partially due to the low sample size of our experiment, but also likely stems from the inherent stochasticity of the soil environment. This context-dependency of bacterial responses  is another argument against categorical application of life history strategy at a broad taxonomic level. For example, it will be difficult to predict the growth of a “copiotroph” if its behavior depends on a complex arrangement of soil characteristics, nutrient availability, and local biotic interactions rather than more relatively static traits such as 16S rRNA gene copy number or genome size.13C-glucose additions to track its utilization specifically. We found high shared variation between 18O and 13C EAF values, suggesting that most microorganisms utilized the added glucose. Further, 18O and 13C EAF values covaried more strongly in the CN treatment, suggesting that N limitation may have limited glucose uptake in the C treatment. Thus, microorganisms with high 18O signatures but low 13C signatures may be those with high N demand who prioritized the decomposition of native soil organic matter to meet their needs. However, the presence of microorganisms with high 18O but low 13C signatures in the CN treatment suggests that N limitation alone may not explain why some microorganisms did not utilize the added glucose. Besides mixing, successional dynamics across the incubation may also explain differences in isotopic signatures of bacteria. Thus, we took this into account in our mixture model specifications such that these microorganisms  were clustered as oligotrophic, based on the possibility that they grew after the depletion of added glucose.The soils used in the current experiment were subject to considerable disturbance including physical disruption, dry-down, and sudden wet-up, inducing a strong pattern of microbial turnover, activity, and respiration from new organic matter made available to the soil community , 45–47. Our results do suggest that – in our aerobic mineral soils at least – the potential for quick growth in response to labile nutrients exists within a small portion of the bacterial community and that this potential seems to be phylum-specific. Among these phyla, however, it is more accurate to understand nutrient response as a continuum rather than a dichotomous classification. If classification is necessary for statistical or narrative purposes, we recommend to restrict life history designations to the family or genus level. These findings (produced by qSIP) were measured by within the context of microbial community interactions which is an important line of inference to better understand microbial trait adaptations. In keeping with other ecological frameworks , stSupplemental InformationBacterial Life History Strategy and TaxonomyExperimental data


We document ontogeny and sex-related isotopic differences indicating distinct diet and habitat use at the time of tooth formation. Adult females have the most distinct isotopic niche, likely feeding on higher trophic level prey in a distinct habitat. This multi-proxy approach characterises an animal’s isotopic niche in greater detail than traditional isotope analysis alone and shows that δ66Znen analysis can highlight intrapopulation dietary variability thereby informing conservation management and, due to good δ66Znen fossil tooth preservation, palaeoecological reconstructions.Trophic ecology and resource use are challenging to discern in migratory marine species, including sharks. However, effective management and conservation strategies depend on understanding these life history details. Here we investigate whether dental enameloid zinc isotope (δ Sex- and ontogeny-related differences in diet and habitat use of endangered sand tiger sharks from Delaware Bay revealed by analysis of shark teeth zinc isotope values. In modern marine food webs, most sharks play the roles of apex and mesopredators, and their removal is likely to result in large-scale, but still uncertain, ecological consequences7. Many shark species experience global population declines, attributed to fishing, habitat degradation, climate change and pollution10. Conservation efforts will depend on a better understanding of extant shark ecology but could also be informed by how ancient species adapted to comparable environmental and climatic pressures in the past.The lineage of the Elasmobranchii originated in the Devonian Period11. Bulk carbon and nitrogen isotope values  analysed in an animal’s organic tissues, including bone and tooth collagen, are the most applied dietary and trophic level proxies14. Generally, both δ13C and δ15N values increase with trophic level due to the preferential loss of the lighter isotopes 12C and 14N in respiration and urea, respectively16. However, δ13C values behave more conservatively, increasing ~1‰, or not at all, per trophic level compared to δ15N values, which increase on average by 3.4‰ per trophic level in most tissues19. Thus, δ13C values are more commonly applied to identify the primary producer(s) in a food web21, whereas δ15N values are used to estimate trophic levels.Stable isotope analysis can be a powerful tool to decipher an animal’s life and migration history, including dietary and habitat preferences. As such, stable isotope studies are becoming more widespread in shark ecology, especially given the difficulty of directly monitoring individuals or populations15N values as a trophic level proxy relies on the assumption of predictable, constant nitrogen isotopic fractionation factors between the diet and the examined consumer tissue. Yet, physiological and environmental factors can also influence diet-tissue fractionation factors shifting them from their predicted values26. In addition, the traditional bulk collagen δ13C and δ15N method used in an archaeological and palaeontological context is not applicable to ‘old’ fossil material  as it is limited by the degree of protein preservation27. In recent years, other non-traditional dietary/trophic isotopic proxies have emerged that can complement the traditional δ15N proxy to provide additional otherwise inaccessible information, as well as act as a substitute in ancient sample material with poor or no collagen preservation34. Among these methods, zinc isotope ratios (66Zn/64Zn expressed as δ66Zn) were recognised as a particularly promising trophic level proxy in mammals35 and fish33. Pristine, i.e., diet-controlled δ66Zn values can be preserved in shark tooth enameloid for millions of years, making it an effective tool to investigate deep-time shark ecology and evolution33.The application of bulk δ37 and undergoes mass-dependent fractionation within an organism40. Zinc intra-organism isotopic fractionation is tissue-specific, depending on the Zn coordination environment40. Heavier Zn is typically concentrated in stiffer bonds, i.e. heavier Zn preferentially binds to ligands with a stronger electronegativity (oxygen>nitrogen>sulphur)41. This preference results in a depletion of 66Zn in organs and muscles relative to the animal’s diet leading to successively lower δ66Zn values as trophic levels increase. For ecological, archaeological, and palaeontological studies, δ66Zn is commonly measured in the mineral phase of skeletal tissues (bioapatite) of bones and teeth35. Notably, there appears to be a predictable offset in δ66Zn values when analysing different bioapatite tissues of the same individual, with bone/tooth dentine having values that are on average 0.2‰ higher compared to tooth enamel/enameloid35. Per trophic level, bioapatite δ66Zn values decrease by approximately 0.4‰ in both marine and terrestrial mammals, although the exact trophic level fractionation factors have yet to be determined experimentally35.Zinc is supplied into vertebrate tissues through diet−1, while deep water usually exhibits higher concentrations, e.g. up to 10 nmol kg−1 in the North Pacific42. Despite this nutrient-like Zn distribution, which is a result of biological uptake, there is no consistent covariation between dissolved Zn concentration and isotopic composition42. The shallow upper ocean exhibits a large δ66Zn variation for dissolved Zn ranging from −1.1 to +0.9‰44. This variability and its causes are still the subject of debate (reviewed by refs. 45). In contrast, dissolved δ66Zn values from deep water are near homogenous at approximately +0.45‰47. Marine Zn sources, mainly stemming from rivers and aeolian dust, are also relatively homogenous with δ66Zn values around +0.33‰48, close to the average upper continental crust value of +0.3‰49.Dissolved zinc is highly depleted in the surface ocean with typical concentrations of 0.01–0.5 nmol kg33. These initial results suggest that, within the same tissues analysed , zinc isotope values have a similar range across geographic locations within the same taxa/trophic levels, implying little variation in marine Zn isotope food web baselines, perhaps even on geological timescales33. However, large-scale marine food web baseline data derived from primary producers are currently non-existent, which limits any detailed discussions of marine Zn isotope baselines at this time.Few studies have investigated zinc isotope variability in marine vertebrates66Zn and δ15N are measured in different components of skeletal tissues, bioapatite and collagen respectively, and are metabolically and environmentally independent of each other. As such, a combined analysis of both trophic level proxies offers the potential of gaining additional ecological information, cross-verification of results and correction for environmental and/or physiological factors influencing either proxy50. The inclusion of δ66Zn may also provide a more direct comparability of marine species trophic ecology between spatially and temporally distinct locations than possible by traditional isotope analysis alone. While it remains unknown for δ66Zn values, both bulk δ13C and δ15N values are known to be strongly affected by marine baseline variability52.Notably, δ66Zn (δ66Znen) values can be used to investigate foraging differences within a single marine predator population. We analyse tooth dentine collagen δ13C and δ15N  values with δ66Znen values of the same tooth from 53 sand tiger shark (Carcharias taurus) individuals that were caught and released in Delaware Bay . Declines in abundance are observed globally for C. taurus53. This species is particularly vulnerable to overexploitation due to slow growth, late maturation, and low reproductive output55. Subsequently, C. taurus is now listed as “Critically Endangered” on the International Union for Conservation of Nature (IUCN) Red List of Threatened Species56. Understanding this species feeding and habitat requirements is essential to facilitate conservation and management efforts.Here, we test, whether enameloid δCarcharias taurus is a large coastal shark that primarily feeds on teleosts and smaller elasmobranchs58. This species is the ideal subject for a study such as this; Carcharias taurus is the only extant member of a genus who’s now extinct members, along with analogue Odontaspididae (e.g. Striatolamia), were globally distributed and abundant throughout the Cenozoic, dating back to the Cretaceous period61. Carcharias is thus a key genus for palaeontological and conservation palaeobiological studies. In addition, while C. taurus is wide-ranging in its distribution, extant populations are isolated and require conservation efforts that are tailored to the population-specific regional ecology62. Carcharias taurus is a migratory species and previous studies suggested differences in the timing and routes of migration between juvenile and adult individuals as well as males versus females within the western Atlantic population63. Yet, little is known about migration, sex, and size-related differences in their foraging ecology. This multi-proxy isotope study investigates the isotopic variability and niches within this population and associated differences in resource use related to sex, ontogeny and migration patterns. Finally, we discuss the implications of our δ66Znen results for ecological and palaeobiological studies investigating intrapopulation dietary variability.15N values from organic tissues to establish trophic levels in marine predators, especially as foraging ecology typically cannot be directly observed. Despite its merits, metabolic and environmental factors may compromise δ15N trophic level interpretations26. In addition, organic substances are commonly not available for fossil studies. Here, we measure δ66Znen, an emerging trophic level indicator preservable in mineralised tissues over geological timescales33, together with bulk dental δ13Ccoll and δ15Ncoll values in western Atlantic C. taurus individuals. All three dietary indicators vary substantially with significant correlations with body size, and, thereby, ontogeny . Collagen δ15N values have a range of 3.6‰, from +13.3 to +16.9‰, with a mean of +14.9 ± 0.8‰ (n  = 53). Enameloid δ66Zn values have a range of 0.64 ‰, from −0.49 to +0.15‰, with a mean of −0.02 ± 0.13‰ (n = 54). All three isotopes show a significant variation in body size, with relationships decreasing from δ13Ccoll to δ66Znen to δ15Ncoll  differ for all four C. taurus groups 55; therefore, the chemical composition of their teeth does not reflect the dietary uptake in late summer Delaware Bay61. Sharks continuously grow their teeth in a conveyor belt system and the timing of tooth formation from below the epithelial tissue to the outermost, functional position was previously quantified as ~260 days for captive Leopard Sharks Triakis semifasciata13. Further, this captive study determined the incorporation rate of dietary nutrients into dentine collagen is 32–83 days13. While the T. semifasciata has different life history traits and evolutionary history from C. taurus, this captive study provides an order of magnitude for the timing delay and formation rate to interpret meaningful ecological data from isotopic results. All C. taurus individuals and their teeth in this study were collected within a 15-day span and we expect the teeth featured here to represent a similar formation timing given each individual was only sampled once, all teeth come from the functional position, from a single population and all teeth within a row form at the same time69. Given the sampling date and timing for a tooth to reach the functional position via the tooth ‘conveyor belt’ system, we estimate a formation date  in December 2011 . In addition, the isotopic incorporation rate of dental collagen means dietary information is integrated into organic carbon and nitrogen from September or November 2011 . The mineralisation rate of enameloid and incorporation rate of dietary zinc is not characterised for sharks, and we expect our calculated timing of organic nutrient incorporation to be a conservative estimate.The Carcharias taurus individuals convene in Delaware Bay in the summer months, but there are variable migratory behaviours during other seasons, likely related to sex and reproductive status. Most movement observations of C. taurus are from acoustic tracking, which is confined to the coastal ocean and embayments where receivers are located to detect tagged animals71. From these studies, we know that generally, C. taurus departs the Delaware Bay and Mid-Atlantic region in September and October71, migrates south in nearshore waters (<15 km from shore), and overwinters off the North Carolina and Florida coast71. The isotope composition from adult males and juveniles of both sexes in this study likely represents integrated dietary signals after leaving Delaware Bay on the route south from nearshore waters off the coasts of Delaware, Maryland, Virginia and North Carolina63.66Znen values spanning from +0.09 to −0.49‰ (n = 13). The expanded isotopic niche of adult males , whereas juveniles with a larger fork length relied more heavily on demersal omnivores. In shelf environments, pelagic phytoplankton has typically lower δ13C values compared to benthic primary producers75. Pelagic prey for juvenile C. taurus sampled in Great South Bay, New York, USA also had lower δ13C and δ15N values compared to demersal prey species74. Thus we tentatively interpret lower and more varied δ13C values in smaller juveniles  as a greater reliance on lower δ13C value pelagic prey than in larger juveniles and adults. However, we cannot exclude the possibility that some variation may also relate to differences in migration timing with ontogeny55, where small juveniles may forage in areas with a different baseline.While δgth Fig. . This re13Ccoll, δ15Ncoll and δ66Znen) and their isotopic niche , these individuals are so distinct in terms of their isotopic composition that this trait is unlikely a sample size artefact alone, rather indicating differences in dietary resources. Other than diet, physiological and/or environmental effects may also contribute to the observed isotopic differences.Four individuals, which are the only adult females analysed, are distinct from the other individuals in terms of their isotopic composition for all three proxies  and hence their isotopic niche  and loggerhead sea turtles (Caretta caretta) document low δ13C variability along the shelf from Cape Hatters and Cape Cod87. Whereas little skate (Leucoraja erinacea) muscle tissue δ13C values document higher values to the northeast of Delaware Bay towards Cape Cod and lower values to the south towards Cape Hatters87. This δ13C spatial pattern in L. erinacea is more consistent with the patterns observed in C. taurus if adult females were foraging NE of Delaware Bay and adult males and juveniles in neritic waters enroute to Cape Hatteras.Baseline δ80, regional isotopic baselines may vary compared to the foraging sites of juveniles and adult males. We are unaware of any documented significant δ13C variability between the area south of Cape Hatteras and the northern neritic waters up to Delaware Bay86 except for some lower values for particulate matter in neritic waters of the Eastern Shore of Virginia85. Due to the novelty of δ66Zn as a marine trophic level proxy, baseline variability remains poorly understood, but previous studies cautiously suggest a potentially more homogenous marine baseline for δ66Zn than for δ13C and δ15N, although this remains to be tested33. Differences in δ13Ccoll, δ15Ncoll and δ66Znen values among sex and ontogenetic stage may also relate to a greater reliance on benthic/demersal prey by large females in lower latitude water. There is also the possibility that these four larger females consumed other elasmobranchs, which are too large to be consumed by smaller C. taurus74.The temporal lag between tooth formation and sampling provides insights into the animal’s past diet, but also complicates ecological inferences where habitat occupation is unknown. For example, if adult females foraged in waters offshore of North Carolina13Ccoll, δ15Ncoll and δ66Znen values documents the distinct ontogenetic isotopic niches and sexual segregation within the Northwest Atlantic C. taurus population more confidently than in the case of a traditional isotope analysis alone. Despite the novelty of zinc isotope compositions as a dietary proxy, and the therewith associated interpretational uncertainties, C. taurus δ66Znen values document ontogenetic and sexual differences in foraging ecology. Therefore, the inclusion of δ66Znen values provides another independent isotope proxy to decipher intraspecific trophic level and niche variability. This is particularly important as bulk δ13Ccoll and δ15Ncoll are likely influenced to some degree also by baseline variation in distinct intraspecific habitats within the population at the time of tooth formation. Notably, δ66Znen is environmentally and metabolically independent from the traditional light isotopes (δ13Ccoll and δ15Ncoll), yet δ66Znen values show the same distinctiveness in terms of the isotopic composition of adult females as observed for the traditional isotopes. Zinc isotope analysis of shark tooth enameloid thus allows to reconstruct not only interspecific dietary differences but also intraspecific ecological variability using both modern and fossil shark teeth33. Indeed, pristine dietary zinc isotope values can be preserved in fossil shark enameloid over millions of years33. As such δ66Zn analysis can enable cross-disciplinary research with an application for modern ecological research questions, including conservation science, and palaeontology, including palaeoecology, as well as the relatively recently named field of conservation palaeobiology, aiming to increase and utilise our understanding of past ecosystems to respond to modern and future conservation challenges88.The multi-isotope proxy approach combining δC. taurus population. Combining δ66Znen values with compound-specific nitrogen isotope analysis of amino acids from dentine collagen (and other tissues) and tagging data could further eliminate uncertainties in baseline variation and allow more detailed reconstructions of habitat resource usage of mobile predators in future studies. Such reconstructions may be particularly valuable when targeting shark teeth, in a multi-proxy approach, across multiple rows with different tooth formation time, potentially allowing a detailed reconstruction of an individual’s migration route and resource uptake along the way14. In any case, zinc isotope analysis is a promising tool, especially combined with other methods to decipher the resource use of extant and extinct mobile marine animals, and as such, has great potential to inform preservation efforts for endangered species, including C. taurus. Finally, δ66Zn analysis has the potential to enable reconstructing population-specific diet variability in extinct species too, especially related to shark ontogeny, as shark tooth size scales allometrically with body size89.Uncertainties remain regarding the movement and habitat use (and therefore isotope baseline) and prey consumption of the imperilled North Atlantic All sharks were caught legally and ethically in Delaware Bay between August 15 and 30, 2012, as part of a joint tagging programme in 2007–2015 between the University of Delaware and Delaware State University with approval from the Delaware Department of Natural Resources and Environmental Control , Delaware State University Institutional Animal Care and Use Committee (IACUC) and the University of Delaware IACUC (1259-2014-0). After sharks were caught, biological data  were recorded, dart tags inserted, and 1–2 teeth in the functional position were extracted (i.e. representing the oldest teeth in the jaw) before sharks were released again. Teeth that were loose were prioritised for sampling. We analysed teeth from 22 females (18 juvenile and 4 adult) and 32 males (19 juvenile and 13 adult) ranging in total length from 143 to 272 cm.13C) and nitrogen (δ15N), dentine collagen was isolated from 53 shark teeth. Powdered dentine samples were collected using a Dremel on low speed with a 300-micron diamond-tipped bit. Samples were demineralised using chilled 0.1 M HCl, then rinsed five times with deionised water, before being freeze-dried overnight90. Samples were weighed to 0.4–0.5 mg in 3 × 5 mm tin capsules. All collagen samples were measured for δ13C and δ15N values using a Costech 4010 Elemental Analyser coupled to a Delta V Plus continuous flow isotope ratio mass spectrometer with a Conflo IV in the Stable Isotope Ecosystem Lab (SIELO) of the University of California Merced. All data were corrected for linearity and drift using a suite of calibrated reference materials: USGS 40 (n = 19), USGS 41a (n = 10) and costech acetanilide (n = 10). Long term standard deviation for the instrument is 0.1‰ for δ 13C and 0.2‰ for δ15N values.To analyse the stable isotopic compositions of organic carbon . All teeth were cleaned by ultrasonication in ultrapure water (Milli-Q water) for 5 min and dried in a drying chamber at 50 °C overnight. Enameloid samples were abraded from the top surface using a dental drill. The enameloid powder were dissolved in closed perfluoroalkoxy vials with 1 ml 1 M HCl on a hotplate for 1 h at 120 °C and then evaporated. The residue was then dissolved in 1 ml 1.5 M HBr and placed in an ultrasonic bath for 30 min. Zinc purification was performed in two steps, following the modified ion exchange method adapted from ref. 91, described in ref. 35, and always included a chemistry blank and reference standard (NIST SRM 1400) to monitor contamination and Zn elution. One ml of AG-1×8 resin (100–200 mesh) was placed in 10 ml hydrophobic interaction columns (Macro-Prep® Methyl HIC). The resin was then cleaned twice with 5 ml 3 % HNO3 followed by 5 ml ultrapure water and subsequently conditioned with 3 ml 1.5 M HBr. Following sample loading, 2 ml HBr were added for matrix residue elution, after which Zn was eluted with 5 ml HNO3. After performing the column chromatography twice, the solution was evaporated for 13 h at 100 °C and the residue re-dissolved in 1 ml 3% HNO3 for analysis.Powdering of samples, zinc purification, and zinc isotope measurements were performed at the Max Planck Institute for Evolutionary Anthropology  following the protocol as described in ref. 92 and Toutain et al. 93. The in-house reference material Zn Alfa Aesar-MPI was used for standard bracketing. All δ66Zn values are expressed relative to the JMC-Lyon standard material (mass-dependent Alfa Aesar-MPI offset of +0.27‰ for δ66Zn94). Zinc concentrations in the respective samples were estimated following a protocol adapted from one used for Sr by ref. 95, applying a regression equation based on the Zn signal intensity (V) of three solutions with known Zn concentrations . The δ66Zn measurement uncertainties were estimated from standard replicate analyses and ranged between ±0.02‰ and ±0.04‰ (2 SD). Samples were typically measured at least twice with mean analytical repeatability of 0.01‰ . Reference material NIST SRM 1400 was prepared and analysed alongside the samples and had δ66Zn values  as reported elsewhere96. Reference materials and samples show a typical Zn mass-dependent isotopic fractionation, i.e. the absence of isobaric interferences, as the δ66Zn vs. δ67Zn and δ66Zn vs. δ68Zn values fall onto lines with slopes close to the theoretic mass fractionation values of 1.5 and 2, respectively  space. NicheROVER uses a probabilistic method to calculate our three-dimensional isotopic niche regions (NR) using a Bayesian framework. Niche overlap was calculated from 1000 Monte Carlo draws with an alpha = 0.95 probability level and is defined as the probability of finding an individual from one group in the NR of another group97.Sharks were sorted into four groups separated by sex  and total length (juvenile/adult). Females and males are considered adults when the total length reaches 220 and 190 cm, respectivelyFurther information on research design is available in the Peer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Despite the change in Helmholtz potential as h+ accumulate we find that a population model can be used to fit the electrocatalytic water oxidation kinetics with a transition between a first and third order regime with respect to hole concentration. Within these two regimes there are no changes in the rate constants for water oxidation, indicating that the rate determining step under these conditions does not involve electron/ion transfer, in-line with it being O–O bond formation.To understand the mechanisms of water oxidation on materials such as hematite it is important that accurate measurements and models of the interfacial fields at the semiconductor liquid junction are developed. Here we demonstrate how electric field induced second harmonic generation (EFISHG) spectroscopy can be used to monitor the electric field across the space-charge and Helmholtz layers in a hematite electrode during water oxidation. We are able to identify the occurrence of Fermi level pinning at specific applied potentials which lead to a change in the Helmholtz potential. Through combined electrochemical and optical measurements we correlate these to the presence of surface trap states and the accumulation of holes (h Electric field induced second harmonic generation spectroscopy is used to monitor the potential drop across the space-charge and Helmholtz layers in a hematite electrode during water oxidation. The low cost, good stability and high abundance of hematite, coupled to its suitable band gap for visible light absorption has led to massive efforts towards engineering more efficient photoelectrodes.1 Mechanistic studies of hematite electrodes, and photoelectrodes aim to elucidate the underlying physico-chemical phenomena controlling water splitting efficiencies which is vital in enabling the design of the next generation of photoelectrodes.Hematite  and a diffuse layer of charges towards the bulk of the electrolyte, with the resulting potential distribution shown in ϕH). Thus, for these electrodes there is a distribution in the electric field along the surface normal direction and understanding how this varies as illumination and/or the potential applied to the electrode is changed is essential if we are to rationalise how a (photo)electrode behaves. But it is challenging to directly monitor these fields under operating (water oxidation) conditions.The electrocatalytic and photoelectrochemical properties of a semiconductor are profoundly affected by the presence of the interfacial electric fields at the semiconductor liquid junction (SCLJ). For hematite, an n-type semiconductor, a depletion of charge carriers within its structure occurs when contacted to an aqueous electrolyte and a positive space charge layer is formed between the bulk of the semiconductor and the electrolyte interface.3 Transient absorption spectroscopy with a UV/vis probe has been used extensively to understand how photogenerated charge carrier dynamics change as the applied potential is altered.4–7 But transient absorption spectroscopy does not directly probe the different fields present. These are instead inferred from the applied potential dependence of the kinetics of the charge carriers. Here we use electric field-induced second harmonic generation (EFISHG) spectroscopy to study a hematite electrode to report on the changes in potential across the Helmholtz and space-charge layers under operating conditions as hole (h+) accumulation occurs at the SCLJ.Capacitance measurements such as Mott–Schottky analyses can be used to study hematite electrodes but they typically require an assumption of the potential distribution across the Helmholtz and space-charge layers. Mott–Schottky analyses are also complicated by the presence of surface and defect states and it has been shown that for hematite, a material where surface states play a key role in determining photoelectrochemical performance, these measurements can give unreliable results.+ accumulation changes the potential distribution across the SCLJ and the subsequent impact of this on water oxidation kinetics is important. It is widely accepted that the slow kinetics of water oxidation, with h+ lifetimes at the SCLJ of milliseconds reported,8–11 limits the efficiency of metal oxide photoanodes. With conventional metallic electrodes the kinetics of electrocatalytic reactions are often interpreted using the Butler–Volmer equation. In this case application of a potential leads to a change in ΔϕH causing a change in the energetics (and hence rate constant) for charge transfer giving rise to an exponential increase in current with applied potential. In contrast for an idealized semiconductor  in a concentrated electrolyte the majority of the potential drop across the SCLJ is expected to occur across the space charge region with minimal change to ΔϕH. The invariance of ΔϕH means that the rate constant for electron transfer can be considered independent of applied potential. Instead of using the Butler–Volmer model well established population models can be used,12,13 where the change in rate of reaction is due to a change in surface electron/hole population. Recently Durrant et al. used a population model (+] to rates of water oxidation (Jwo) for a range of metal oxide electrodes and photoelectrodes including hematite.11,14,15 These experiments indicated that a switch in mechanism occurs on hematite photoelectrodes with the reaction order (α) with respect to [h+] being ∼3 at high [h+] (occurring at ∼1 sun), but ∼1 at low [h+] and that the water oxidation rate constant (kwo) for each pathway is independent of applied potential.15 This behaviour has been interpreted to indicate a change in water oxidation mechanism from occurring at a single Fe centre to a mechanism involving interaction between two adjacent FeIV PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O sites (the proposed surface trapped h+) when the surface coverage of h+ reaches a high enough level. Further studies using transient photocurrent measurements have since also reported a third-order dependence of Jwo on hole density with DFT calculations showing O–O bond formation occurring at sites where three oxo/oxyl groups are found.16Understanding how hon model  to corre2O3,17 which could indicate that minimal change in ΔϕH occurs as [h+] changes. However, as recently highlighted by Zhang and Leng,18 it is surprising that a population model holds for hematite and other oxide photoelectrodes. When the rate of h+ transfer is slow, h+ accumulation causes light-induced Fermi level pinning. Fermi level pinning leads to changes in ΔϕH as the applied potential is changed, which in turn might be expected to change kwo. Supporting the concerns of Zhang and Leng is that in, several studies fitting of Intensity-Modulated Photocurrent Spectroscopy (IMPS) and Photoelectrochemical Impedance Spectroscopy (PEIS) data of hematite electrodes gives kwo values that are dependent on applied potential.19–23 It is therefore clear that to validate the proposed models of electron transfer at the hematite–water interface new measurements that directly address the local electric fields whilst monitoring the h+ population are required.Supporting the use of a population model are measurements of hole activation energies which are constant at a range of applied potentials with α-Feωi induces a polarisation within the material and light is emitted at double this frequency (ωSHG = ω2i).24 The intensity of the SHG (ISHG) signal is proportional to the square of the second order polarisation of the material (P(2)), which in turn is dependent on both the electric field of the incoming photons and the second order susceptibility (χ(2)) of the system under study:Second harmonic generation (SHG) is a second order nonlinear optical process where the electric field of incident light at frequency χ(2) = 0 in centrosymmetric materials and only becomes non-zero at interfaces, where the inversion symmetry is inherently broken. SHG spectroscopy has been used previously to study a variety of catalytic systems at interfaces including monitoring adsorption of gaseous reactants at solid surfaces and even identifying reaction intermediates in liquid phases (when the intermediates have a resonance at the second harmonic wavelength).25,26 This surface-selectivity makes SHG spectroscopy especially useful for studying electrochemical systems. EFISHG is a form of SHG in which the response of a charged interface requires consideration of an additional static electric field(s) (EDC), ISHG through two mechanisms.27 Firstly, by directly interacting with bulk third order susceptibility (χ(3)) which does not usually contribute to the SHG response, but is induced by the additional static field. Thus, the presence of EDC means non-interfacial species will contribute to the second harmonic response, resulting in a change in the SHG intensity. Secondly, the presence of EDC can also change the net orientation of mobile interfacial molecular species, or for a material such as a semiconductor electrode induces a polarization of the lattice, manifesting as a change in χ(2). Thus, the second harmonic polarisation for an electrode held at a potential (ϕ) can be summarised as:Due to inversion symmetry, et al.28,29 However, fewer studies have exploited EFISHG to study the semiconductor electrode|electrolyte interface.30–33 Experiments on a TiO2 electrode34 reported a linear relationship between the applied potential and ISHG at potentials positive of flat band (ϕfb). To rationalise this behaviour, it is important to first consider the relationship between applied potential  and the strength of the electric field across the space charge layer. The Mott–Schottky approximation tells us that for a n-type semiconductor that is held positive of the flat band potential (ϕfb), the electric field varies linearly over the space charge layer and the charge (qSC) is proportional to the square root of ΔϕSC (where ΔϕSC = ϕ − ϕfb), The dependence of the second harmonic polarisation on the interfacial electric field has been used extensively to study the evolution of electric fields at metal electrode|electrolyte interfaces based on the nonlinear electroreflectance studies by Bloembergen 30,34 and it is possible to arrive at In this case the overall charge of the space charge layer is directly related to the magnitude of the electric field ,30,34 anISHG is proportional to the square of PSHG, 2 electrode studied previously the potential drop was primarily across the space charge layer at potentials positive of flat band and that the χ(3) contribution was dominant in the EFISHG response.34 A similar linear dependence of ISHG with applied potential for a n-Si positive of flat-band has also been reported.35 Interestingly, at potentials negative of flat-band a non-linear (parabolic) potential dependence of ISHG occurred, which was rationalised by the electrode becoming more metal like under these conditions leading to ΔϕH changing with applied potential instead of ΔϕSC.28,30 These past studies demonstrate that EFISHG can be used to study the SCLJ and to monitor the distribution of the potential drop, but to the best of our knowledge EFISHG has not been used to study electrodes during water oxidation. SHG has also been used to study the interactions of dissolved ions with hematite.36,37 Here we combine SHG spectroscopy with optical/kinetic measurements of [h+] to follow how the potential drop across the SCLJ varies for a hematite electrode as h+ accumulate at the SCLJ. We show that h+ accumulation leads to changes in ΔϕH but that the water oxidation rate constant is independent of ΔϕH when there is a first or third-order dependence of Jwo on [h+], indicating that under these conditions the rate determining step does not involve electron/ion transfer, in-line with it being O–O bond formation.As 2O3 electrodes on FTO (FTO = F-doped SnO2) glass. Full details of the synthesis and characterisation of the photoelectrodes are found in the Experimental section and ESI .2O3 layers (150 nm thick) that have low surface roughness  in close proximity, 2O3, the electrode itself can act as an optical filter for the (unwanted) underlying interfaces. An incident wavelength of 800 nm was selected for these experiments, generating SHG photons at 400 nm. The absorption coefficient for α-Fe2O3 is reported to be α ∼ 2 × 105 cm−1 (α−1 ∼ 29 nm) at 400 nm.38 Therefore, any SHG from the α-Fe2O3|FTO and FTO|glass interfaces is almost completely absorbed by the hematite layer in these electrodes and it does not contribute to the detected EFISHG response. Photoelectrochemical tests indicate that when the 800 nm laser  is incident on the α-Fe2O3 there is no detectable photocurrent  contribution dominating the EFISHG response  term arising from the space charge layer is leading to the potential dependent ISHG. A full expansion of nd and 3rd order terms can be found in the ESI.†The potential dependence of the SHG response of the α-Fe Fig. S10, therefo Fig. S10 the α-FeFig. S11b shows clresponse .34 Here ISHG with applied electrode potential at >0.6 V+Ag/Ag and between −0.05 and 0.2 V+Ag/Ag may indicate an abrupt change where the majority of potential drop is occurring (i.e. ϕΔHvs. ϕΔSC). Supporting the possibility that a change in potential drop distribution is the Mott–Schottky analysis . Electrochemical measurements  swept to more negative potentials depopulating (reducing) any oxidised trap states, ca. 0.65 V+Ag/Ag was previously assigned to the reduction of h+ that have accumulated at surface trap states,23 supporting our assignment of Femi level pinning and a change in ϕΔH at >0.6 V+Ag/Ag as a result of h+ accumulation. The 2nd broad reduction centred around 0.1 V+Ag/Ag in 23 The good agreement between the potential of these electron/hole trap states and the deviation from the linear rate of change of ISHG leads us to conclude that the lower rate of change of ISHG potential between −0.05 to 0.2 V+Ag/Ag is also due to Fermi-level pinning with the potential drop occurring across the Helmholtz layer, instead of solely over the space-charge layer.To identify if the deviation from the linear rate of change of Jwo) to the [h+]  which is modified by changing the applied potential to the electrode, 15 There they correlated the measured photocurrent for a hematite photoelectrode under strong anodic bias to the optically determined [h+] which was modulated by changing the light intensity. In To understand the impact of the change in potential distribution on the mechanisms and kinetics of water oxidation we have carried out an operando spectroelectrochemistry study of the rate law, and examined its potential dependence, +].15 For both the VIA and PIA experiments there is an approximately first-order dependence of Jwo on hole density (α = 1) at low h+ concentrations, which switches to third order (α = 3) at higher h+ concentrations, st order and 3rd order behaviour occurs at approximately the same hole density in the light and dark  and a Jwo of ∼0.03–0.04 e− s−1 nm−2 (0.4 and 0.6 μA cm−2). This compares well to past PIA studies on planar hematite where a change in reaction order occurred at between ∼0.6 h+ nm−2 and a Jwo ∼ 0.07 e− s−1 nm−2 (∼1 μA cm−2).40 On high aspect ratio photoelectrodes which achieve benchmark photocurrents the change in reaction order to α = 3 occurs at similar surface hole densities (∼1 h+ nm−2) to those seen here and the third-order dependence of Jwo on hole density has been shown to occur at ∼1 sun indicating that it is occurring on many leading photoelectrodes.15 Interestingly here at the highest water oxidation currents studied (Jwo > 0.3 (VIA), Jwo > 0.6 e− s−1 nm−2 (PIA)) in both the dark and light we find that α ≠ 1 or 3 which has not been previously reported. Instead we find that the Jwovs. h+ density data in this region can be reasonably well fitted to a linear function with α ∼ 7–9  which is reasonable given the use of a pseudo reference (Ag wire) and the change in spectroelectrochemical cell. From + accumulation increases at potentials where Jwo has a first or third-order dependence on h+ density (α = 1 or 3 from 0.575 to 0.675 V+Ag/Ag). The EFISHG experiment  oxidation43,44 and DFT calculations on the photoelectrochemical mechanism of water oxidation on hematite that show for low FeIVO coverages (when α = 1) the rate determining step is the interaction of H2O with FeIVO, leading to O–O bond formation.40 At higher coverages (α = 3) the rate determining step is again O–O bond formation, this time due to nucleophilic attack of a water or hydroxide molecule at site where three h+ have accumulated.16,40 In both cases the rate determining step (O–O bond formation) does not involve a formal transfer of a charged species  to or from the solution and the reaction free energy would not be expected to be strongly dependent upon the potential drop across the Helmholtz layer as is seen in our studies. changes . There iperiment  shows thϕH during photohole accumulation, however these are extremely difficult to carry out and to interpret due to the presence (and trapping) of both photogenerated h+ and e−. The demonstration of a common mechanism during electrocatalytic (dark) and photoelectrochemical water oxidation indicates that the rate determining step is also likely to be the same and it is therefore reasonable to conclude that kwo would also be expected to be independent of ΔϕH under conditions where Jwo has a first or third-order dependence on hole density.The good agreement between the PIA and VIA data shows that water oxidation occurs by the same surface mechanisms in the dark and the light on these hematite electrodes. Ideally EFISHG experiments would also be carried out with the electrode under band gap illumination to directly assess the change in ΔJwo on h+ density no longer holds, α ∼ 7 to 9, although there are considerable errors on these linear fits. This behaviour has not previously been reported on hematite. A possible cause could be direct water oxidation due to hole transfer from FTO via pinholes in the α-Fe2O3 layer. But we rule this out as the onset of water oxidation on FTO is significantly positive of that on α-Fe2O3 and Fig. S112O3 has occurred at the highest Jwo measured. It is notable that the potential region where α ≠ 1 or 3 corresponds to that where the [h+] plateaus  at potentials where Jwo has a first or third-order dependence on h+ density. This indicates that the rate determining step in water oxidation on hematite at these lower current densities does not involve a formal transfer of a charge species and instead our results are consistent with O–O formation being the rate determining step. Here we have focused on the behaviour of semiconductor electrodes in the dark but with careful modelling, experiments of photoelectrodes under band gap illumination should be addressable. This combined with the ability to carry out EFISHG with a high spatial resolution (μm) offers an exciting future opportunity to provide a comprehensive understanding of the spatial and potential dependence of the SCLJ during photoelectrochemical water oxidation.In conclusion our study demonstrates the applicability of EFISHG as a probe of the electric fields present at the SCLJ during electrochemical water splitting. Using complimentary UV/vis spectroelectrochemical measurements we have directly correlated changes in [hhttps://doi.org/10.17638/datacat.liverpool.ac.uk/2158.The ESIKhezar H. Saeed: conceptualisation, data curation, formal analysis, investigation, writing – original draft, writing – review & editing. Dora-Alicia Garcia Osario: data curation, formal analysis, investigation, writing – review & editing. Chao Li: data curation, formal analysis, investigation, writing – review & editing. Liam Banerji: data curation, formal analysis, investigation, writing – review & editing. Adrian M. Gardner: conceptualisation, investigation, supervision, writing – review & editing. Alexander J. Cowan: conceptualisation, formal analysis, funding acquisition, investigation, supervision, writing – original draft, writing – review & editing.There are no conflicts of interest to declare.SC-014-D2SC05628C-s001


The triglyceride-glucose (TyG) index is a predictor of cardiovascular diseases; however, to what extent the TyG index is associated with cardiovascular diseases through renal function is unclear. This study aimed to evaluate the complex association of the TyG index and renal function with cardiovascular diseases using a cohort design.This study included participants from the China Health and Retirement Longitudinal Study (CHARLS) free of cardiovascular diseases at baseline. We performed adjusted regression analyses and mediation analyses using Cox models. The TyG index was calculated as Ln [fasting triglyceride (mg/dL) × fasting glucose (mg/dL)/2]. Renal function was defined by the estimated glomerular filtration rate (eGFR).2, those with a higher TyG index and decreased eGFR had the highest risk of cardiovascular diseases . Decreased eGFR significantly mediated 29.6% of the associations between the TyG index and cardiovascular diseases.A total of 6 496 participants were included in this study. The mean age of the participants was 59.6 ± 9.5 years, and 2996 (46.1%) were females. During a maximum follow-up of 7.0 years, 1 996 (30.7%) people developed cardiovascular diseases, including 1 541 (23.7%) cases of heart diseases and 651 (10.0%) cases of stroke. Both the TyG index and eGFR level were significantly associated with cardiovascular diseases. Compared with people with a lower TyG index (median level) and eGFR ≥ 60 ml/minute/1.73 mThe combination of a higher TyG index and lower eGFR level was associated with the highest risk of cardiovascular diseases. Renal function could mediate the association between the TyG index and cardiovascular risk.The online version contains supplementary material available at 10.1186/s12933-023-02055-4. Insulin resistance has been identified as an independent risk factor for the onset of cardiovascular diseases , which hRenal function is another important factor in cardiovascular diseases . StudiesTherefore, this study aimed to investigate the combined association between the TyG index and eGFR level with the onset of cardiovascular diseases given that insulin resistance and renal function are two initial predictors of cardiovascular health. In addition, we tested the hypothesis that the association between the TyG index and cardiovascular diseases is partially mediated by impaired renal function.http://charls.pku.edu.cn/) among Chinese adults aged 45 years or older with four regular biannual surveys between 2011 and 2018. The participants were recruited from both rural and urban areas using a multistage stratified probability proportional-to-size sampling strategy and covered 150 counties or districts of 28 provinces in China. Details of the study design and cohort profile have been previously described . The eGFR level (mL/min/1.73 mCKD-EPI) . ImpaireThe study outcome was any incidence of cardiovascular diseases of coronary heart disease (including heart attack and coronary heart disease) and stroke during the follow-up period (from wave 2 to wave 4). Information on cardiovascular diagnosis history was collected using the standardized question: ‘Have you been told by a doctor that you have been diagnosed with a heart attack, coronary heart disease or stroke?’. The outcomes were assessed by rigorously trained interviewers through standardized questionnaires that are harmonized to international leading aging surveys in the Health and Retirement Study (HRS) and related international aging surveys, including the English Longitudinal Study of Aging (ELSA) and the Survey of Health, Aging and Retirement in Europe (SHARE). Quality control of data recording and checking was conducted to ensure data reliability. Participants who reported heart disease or stroke during the follow-up period were defined as having incident cardiovascular diseases that have been validated and widely used .2 (in meters squared) and grouped into normal weight (BMI < 24.0 kg/m2), overweight (BMI ≥ 24.0 kg/m2) and obesity (BMI ≥ 28.0 kg/m2) according to the overweight and obesity standard for the Asian population [Baseline measurements of age, sex, education level, marital status, residence location, BMI, smoking, and self-reported health conditions (hypertension and diabetes) were included as covariates in the current study. Educational level was categorized as “primary education,” “secondary education,” and “third education.” Marital status included “married” and “others.” Residence location included “urban” and “rural.” Smoking status was defined as “never smoking”, “current smoker” and “former smoker”. BMI was calculated as weight (in kilograms)/height^pulation . Hypertepulation . Diabetepulation . The defht BMI < .0 kg/m2,2 as cutoff points). The dose–response relationship of the TyG index and eGFR level with cardiovascular risk was shown using the restricted cubic spline function using 3 knots at the 10th, 50th, and 90th percentiles. The reference point was set as the median value of varibales among the corresponding populations. We performed multiple sensitivity analyses after additionally adjusting for hs-CRP level (continuous), using 130/80 mmHg to define hypertension, and repeating the analyses using multiple imputed analyses (5 iterations) by Markov chain Monte Carlo. In addition, we reanalyzed the effect of the TyG index and eGFR level on cardiovascular risk using propensity scores of the inverse probability treatment weighting (IPTW) method. The sample weights in the cohort survey procedures were also considered using weighted regression. Furthermore, we performed subgroup analysis under two scenarios. First, we repeated the analysis among those without treatment for diabetes, hypertension or dyslipidemia at baseline. Then, we further excluded those with treatment for diabetes, hypertension or dyslipidemia during follow-up. We also performed analysis stratified by obesity, diabetes, hypertension and metabolic syndrome.Baseline characteristics were described using the mean  for continuous variables and frequency (proportion) for categorical variables. To determine the association between the TyG index, impaired renal function, and the development of cardiovascular diseases, multivariable-adjusted Cox regression models were used to calculate the hazard ratio (HR) with 95% confidence interval (CI), considering the time-to-event framework. Age (continuous), sex, residence , education level , marital status , smoking status , BMI (continuous), hypertension , diabetes , and nonHDL cholesterol (continuous) were included in the adjusted model. The TyG index was grouped into two groups according to the median value. Then, participants were grouped into four categories according to the joint assessment of the TyG index and eGFR value. In addition, the participants were grouped into nine categories according to the joint assessment of the TyG index (using tertile) and eGFR value (60 and 90 ml/minute/1.73 m2) as a mediator (M) and cardiovascular disease onset as the outcome variable (Y). The analysis included four steps: (1) establishing that X is associated with Y (Model Y = βTot X) ; (2) establishing that X is associated with M (Model M = β1 X) (β1 = indirect effect1); (3) determining which part of Y is explained by controlling for X (Model Y = β2 M + βDir X) ; and (4) calculating the proportion of indirect or mediation effect: mediation effect (%) = (β1 × β2/βTol) × 100%. This method has been widely used in previous studies to quantify the mediating effect [We conducted a mediation analysis to assess the direct and indirect associations between the TyG index group and cardiovascular diseases via impaired renal function. In brief, the TyG index group  was used as a predictor variable (X), decreased eGFR . Weighted regression was performed using the ‘survey’ package. Mediation analysis was performed using the ‘mediation’ package, and the IPTW procedure was performed using the ‘PSweight’ package. A two-sided P value < 0.05 was considered statistically significant.A total of 6 496 participants in CHARLS from 2011 to 2018 were included in the final analyses. The mean (SD) age was 59.6 (9.5) years, including 2996 (46.1%) females , followed by those with a lower TyG index and decreased eGFR level and those with a higher TyG and normal eGFR level, as shown in Table In the fully adjusted model, when compared with people with a lower TyG index (less than median level) and eGFR ≥ 60 ml/minute/1.73 m2, the highest HR was still observed among those with the highest TyG and eGFR less than 60 ml/minute/1.73 m2 (Additional file The results remained consistent in multiple sensitivity analyses, as summarized in Fig. Figure Among 6 496 Chinese adults aged 45 years or above followed up to 7.0 years, a higher baseline TyG index and decreased eGFR were significantly associated with a higher risk of cardiovascular diseases. The highest risk of cardiovascular disease onset was observed among those with a higher TyG index and decreased eGFR. The associations persisted even after adjustment for other established cardiovascular risk factors and IPTW procedures. In addition, this study indicated that decreased eGFR partially mediated the relationship between the TyG index and cardiovascular diseases.The positive association between the TyG index and cardiovascular diseases has been widely evaluated in previous studies. The Tehran Lipid and Glucose Study among 7521 Iranian males found that the TyG index is significantly associated with an increased risk of cardiovascular diseases or coronary heart diseases . The KaiIn addition, the association between the TyG index and other established risk factors for cardiovascular diseases has been evaluated among several cohort designs. The Hanzhong Adolescent Hypertension Cohort study found that elevated levels of the baseline TyG index and a higher long-term trajectory of the TyG index were independently associated with increased arterial stiffness , which wThere are possible mechanisms underlining the complex correlation among the TyG index or insulin resistance, renal function, and cardiovascular damage. Generally, insulin resistance plays an important role in metabolic disease-induced chronic kidney disease by causing hyperglycemia and later low-grade inflammation and fibrosis . InsulinSeveral limitations of the current study should be acknowledged. First, owing to the observational nature of the study, we could not confirm the causal association among the TyG index, renal function and cardiovascular risk. Second, although potential cardiac risk factors were adjusted for, we still cannot exclude the possibility of residual or unmeasured confounding given the observational study design of the present analysis. Additionally, HbA1c data were not available in this current analysis, which could affect the definition of diabetes. Clinical information on proteinuria was not available, which could cause bias in the assessment of renal function. Third, the diagnosis of cardiovascular events was self-reported. Medical records were not available in the CHARLS; however, some other large-scale studies, such as the English Longitudinal Study of Aging, found that self-reported incident cardiovascular disease had good agreement with medical records. Fourth, the CHARLS focuses on general Chinese participants aged 45 years and older, and the association found in this study may not be fully generalizable to the general population or disease population. Further studies are needed for validation in other populations and health conditions.In a prospective cohort of Chinese adults, we found that reduced renal function significantly mediated the association between insulin resistance, that is the TyG index, and cardiovascular diseases. The findings recommend the combined assessment of the TyG index and renal function markers to further stratify the risk of cardiovascular diseases. Future research is needed to validate the clinical effect of targeting insulin resistance on renal function and cardiovascular health.Additional file 1: Table S1. Characteristics of 6 496 participants categorized by TyG and eGFR levels. Table S2. Associations of TyG index and eGFR with risk of cardiovascular diseases using sample weighted method. Table S3. Effect of TyG index and eGFR with risk of cardiovascular diseases when analyzed using 3*3 matrix.