Acinetobacter species in the Great Fish, Keiskamma and Tyhume rivers in the Eastern Cape Province, South Africa. Water samples and values of the environmental factors were taken from the rivers for 12 months. The density of presumptive Acinetobacter species was estimated from the culture of water samples on a CHROMagar selective medium, while the Acinetobacter-specific recA gene was targeted for the identification of Acinetobacter species using PCR assay. The multivariate relationship between seasons and changes in variables was created using PCA, while the effect of seasonal shifts in the environmental variables on the density of Acinetobacter species was evaluated using correlation test and topological graphs. Positive association patterns were observed between the seasons, environmental factors and the bacterial density in the rivers. In addition, temperature, TBS, TSS and BOD tended to influence the bacterial density more than other physicochemical factors in the rivers across the seasons. Of the total 1107 presumptive Acinetobacter species, 844 were confirmed as Acinetobacter species. Therefore, these findings suggested that the rivers contain Acinetobacter species that could be useful for basic and applied study in ecology or biotechnology, while their clinical relevance in causing diseases cannot be underestimated.Certain environmental variables are responsible for the survival of microorganisms in aquatic environments. The influence of these environmental factors in each season  of the year can be used to track changes in a microbial population in freshwater resources. In this study, we assessed the effect of seasonal shifts in environmental variables including temperature, pH, total dissolved solids (TDS), total suspended solids (TSS), biochemical oxygen demand (BOD) and turbidity (TBS) among others on the density of  Escherichia coli in rivers have been reported to be very sensitive to the changes in physicochemical parameters because of pollution [Microbial communities in freshwater resources are extremely diverse ,2 and thollution .Acinetobacter species have been implicated in wound infection [Acinetobacter species have shown ecological and economic importance such as in bioremediation of industrial effluents [Some nfection , bloodstnfection  and meninfection . Additioffluents , degradaffluents  productiffluents ,17,18 anffluents .Acinetobacter species. One of the characteristics of the Acinetobacter species is the ability to survive in adverse environmental conditions [Seasonal changes faced by the microbial communities inhabiting freshwater resources provide an opportunity to appraise the extent to which variation in the aquatic physicochemical variables affect the density and survival of the nditions ,21. The Acinetobacter species in the three major rivers in the Eastern Cape Province of South Africa. Understanding the response of Acinetobacter species in freshwater resources to changes in the environment during different seasons could serve as a tool to monitor the stress response of the microorganism when exposed to a sudden shift in environmental conditions. Therefore, given that some Acinetobacter species are potential waterborne pathogens [Acinetobacter species in the freshwater resources.Thus, the uncertainty that characterizes the shifts in weather conditions in South Africa was considered for the evaluation of the survival of the athogens  and someathogens , this stWater samples were collected from three rivers, namely the Great Fish, Keiskamma and Tyhume rivers in the Eastern Cape Province, South Africa, between April 2017, and March 2018. The Great Fish River is located in the Chris Hani District Municipality in the Eastern Cape Province and it is one of the major rivers used for irrigation and livestock farming in South Africa. This river is prone to agricultural and municipal runoffs and functions as the receiving stream of effluents from various wastewater treatment plants (WWTPs), particularly those located in urban residential districts such as Craddock. The Keiskamma and Tyhume rivers are located in the Amathole District Municipality in the Eastern Cape Province and are disclosed to different anthropogenic activities such as livestock drinking and irrigation agriculture in the rural and urban communities along the river courses. In summation, these rivers receive effluents from wastewater treatment plants (WWTPs) situated close to their banks. The river sampled and the corresponding sampling points are presented in Water samples were collected aseptically in sterile 1 L glass bottles from the different sampling points along the river courses by midstream dipping of sample bottles at 25–30 cm down the water column, with the mouth tilting against the flow of the river. Between April 2017 and March 2018, 180 water samples were collected of which 30, 45, 30 and 75 water samples were collected in autumn, winter, spring and summer respectively. All the samples were labelled properly and transported to Applied and Environmental Microbiology Research Group (AEMREG) laboratory in an ice chest and processed within 6 h of collection.3), total suspended solids (TSS (mg/dm3) and dissolved oxygen  were measured on-site using a multi-parameter meter . Turbidity (TBS) (NTU) was determined using a turbidimeter . The five-day biochemical oxygen demand  of the samples was determined using a biochemical oxygen demand meter  [The physicochemical parameters such as temperature (°C), pH, electrical conductivity , salinity (PSU), total dissolved solids  .Acinetobacter species in the water samples was determined by the membrane filtration technique [Acinetobacter species selective medium, CHROMagar Acinetobacter plus selective supplement , which was prepared according to the manufacturer’s instructions. The plates were incubated at 37 °C for 24 h and all colonies of microorganisms on plates were taken as bacterial density, while colonies showing red, a typical Acinetobacter species appearance on the CHROMagar medium were counted as presumptive Acinetobacter species (CFU/100 mL). The pure colonies of presumptive Acinetobacter species were stored in glycerol stocks at −80 °C for use.Bacterial density and presumptive echnique . Three vechnique . These mrecA gene by using Acinetobacter genus-specific primers for the identification of Acinetobacter species according to [Acinetobacter species in the initial presumptive Acinetobacter species density was confirmed.DNA extraction from the bacterial isolates was carried out using the direct boiling method according to . Polymerrding to . Thus, tH-test was employed for the statistical analysis because the months that make the seasons are not parametric, while multiple comparison post hoc was used to determine the significant values at p < 0.05. Principal component analysis (PCA) (Biplots) was employed to define the seasonal changes in the physicochemical variables in each river using PAST . The correlation between the physicochemical variables and the density of presumptive Acinetobacter species in the three rivers across the four seasons was measured by correlation test in Statistica software version 13. Coefficient of correlation (r) between physicochemical variables and presumptive Acinetobacter species was determined using Pearson’s correlation , while further validation of the relationship between physicochemical variables and presumptive Acinetobacter species was performed using Kohonen analysis (p < 0.05) trial version of XLstat2018 .Seasonal changes in the physicochemical variables in the rivers were evaluated using Kruskal–Wallis ANOVA (H-test). The Kruskal–Wallis The physicochemical variables of the freshwater resources were recorded within the four seasons of the year in South Africa. p < 0.05) variation in pH across the seasons in the Great Fish , Keiskamma  and Tyhume  rivers.The pH values of the water samples collected from the Great Fish river remained within the alkaline range (8.0–8.2), while the Keiskamma and Tyhume rivers were relatively neutral (7.2–7.9). However, there was significant (p < 0.05) increase in temperature, compared to other seasons when evaluated in the Great Fish , Keiskamma  and Tyhume  rivers.The temperature increased from winter to summer (winter<autumn<spring<summer), and the Great Fish was the hottest among the rivers studied. Summer came with a significant (p < 0.05) lower during summer compared to other seasons. In both the Great Fish and Keiskamma rivers, EC during the autumn, winter and spring was not significantly different (p < 0.05) from each other, but there was a significant decrease (p < 0.05) in EC during the summertime. The EC in Tyhume was similarly low in summer, but no statistical difference (p < 0.05) was observed. However, there was significant shift in EC in Great Fish  and Keiskamma  but not in Tyhume  across seasons. Comparable to EC, TDS was significantly lower (p < 0.05) in summer compared to other seasons in all the rivers evaluated in this study. Nonetheless, the change in TDS was statistically significant across season in Great Fish , Keiskamma  but not in Tyhume .An average EC and TDS of the water samples from the three rivers were significantly  and Keiskamma  compared to Tyhume .Additionally, there was substantial change in salinity in each of the river across the seasons in both Great Fish (p < 0.05) than other seasons. However, change in TSS was statistically significant across the season in Great Fish , Keiskamma  but not in Tyhume . Similarly, while TBS increased from spring to summer (spring<winter<autumn<summer) in all the rivers, there was significant (p < 0.05) increase in TBS in the summertime compared to autumn, winter and spring. However, TBS in the Great Fish  was statistically significant compared to both Keiskamma  and Tyhume  rivers in all the seasons.TSS increased from spring to summer (spring<autumn<winter<summer). TSS in summer was significantly higher (p < 0.0%) in DO between autumn, winter and spring in each of the rivers. However, DO in the summertime was lower than autumn, winter and spring in each of the rivers. The seasonal change in DO was statistically significant in the Great Fish , Keiskamma  and Tyhume  rivers in all the seasons.Additionally, there was no significant change (p < 0.05) in the values across the seasons. In the Keiskamma River, BOD was notably high in spring compared to autumn and winter, but no significant difference (p < 0.05) between the BOD was recorded in the spring and summer seasons. In the Tyhume river, BOD in spring was similarly high compared to the other seasons studied. Notwithstanding, there was statistical significance of BOD in the Great Fish , Keiskamma  and Tyhume  rivers in all the seasons.BOD in the Great Fish river was highest in winter even though there was no statistical difference  in the Great Fish, Keiskamma and Tyhume rivers respectively. However, for a convenient interpretation of the analysis, variables with loadings (coefficients) as indicated below were retained to establish the influence of each variable on the component. Biplot of PC1 versus PC2 in each of Great Fish, Keiskamma and Tyhume rivers are presented in Of the nine principal components of the analysis, the first two components (PC1 and PC2) were chosen for further analysis in all the rivers using the scree plot principle of eigenvalues. The two PCs explain 70.11%, 66.63% and 64.90% of the observed variation (r = 0.41), TDS (r = 0.41), salinity (r = 0.41) and DO (r = 0.36); and negative association with temperature (r = −0.35), TSS (r = −0.35) and TBS (r = −0.35). Both pH (r = 0.05) and BOD (r = -0.05) had a weak positive and negative correlation with PC1. PC2 (15.95%) had a large positive correlation with BOD (r = 0.66) and temperature (r = 0.36) and negative correlation with DO (r = −0.38), while association with other parameters was weak at ≥95% confidence level.In the Great Fish river, PC1 explains 54.17% of the observed variability. PC1 had large positive associations with EC (r = 0.46), TDS (r = 0.45) and salinity (r = 0.46), while pH (r = −0.16), temperature (r = −0.26), TSS (r = −0.28) and TBS (r = −0.29) had weak negative associations. However, DO (r = 0.30) and BOD (r = 0.18) had a weak positive association with PC1. PC2 (20.23%) had a large positive correlation with TSS (r = 0.48) and TBS (r = 0.47) and large negative correlation with pH (r = −0.45) and temperature (r = −0.47) at ≥95% confidence level.Similarly, PC1 in the Keiskamma river accounts for 46.4% of the observed variability showing large positive associations with EC (r = 0.46), TDS (r = 0.44), and salinity (r = 0.46), while pH (r = −0.17), temperature (r = −0.30), TSS (r = −0.28) and TBS (r = −0.29) had a weak negative association with PC1. PC2 (21.09%) had a large positive correlation TSS (r = 0.57) and TBS (r = 0.56) at ≥95% confidence level. Correlation between DO and PC1 was r = 0.32, while both PC1 and PC2 had a negative association with temperature , BOD  and pH .PC1 in the Tyhume river accounts for 43.81% of the observed variability with large positive associations with EC .he plate , differeAcinetobacter species recovered during the autumn, winter, spring and summer was 1602 ± 257.38, 684.89 ± 114.91, 2312.67 ± 477.36 and 11655.33 ± 608.70 (CFU/100 mL) in the Great Fish River. Presumptive Acinetobacter species in Keiskamma were 460.97 ± 118, 1775.78 ± 223.15, 2577.67 ± 1006.99 and 10040.53 ± 598.48 (CFU/100 mL), while 4096 ± 805.45, 830 ± 111.14, 9216.67 ± 689.74 and 10331.07 ± 862.83 (CFU/100 mL) were recorded in Tyhume. In all the rivers, there was significant (p < 0.05) recovery of presumptive Acinetobacter species during the summer season compared to other seasons in this study. However, in the Tyhume River, the density of presumptive Acinetobacter species recovered during spring and summer seasons were significantly higher (p < 0.05) than autumn and winter (Acinetobacter species recovered during the winter and autumn was significantly lower (p < 0.05) than the spring and summer seasons in the Great Fish river, recovery of the Acinetobacter species in the Keiskamma river during the summer seasons were significantly higher (p < 0.05) than other seasons. Nonetheless, a substantial number of Acinetobacter species were recovered at sampling point 2 (Keiskamma river) during the winter and spring compared to other sampling sites and autumn  of presumptive d winter C. Additid autumn B.Acinetobacter species in more details, we examined whether both the physicochemical variables and the density have any correlation using correlation test and topological graphs . r) and statistical significance  of the correlation test, while To further evaluate the effect of changes in the environmental variables on the density of Acinetobacter species in the river varied significantly  as seasonal change affected physicochemical variables , TSS , TBS  and BOD  were responsible for the bacterial density in the Great Fish River. In the Keiskamma River, pH  temperature , TSS , TBS  and BOD  were the driving factors for the density of Acinetobacter species. While all these factors influenced the density of Acinetobacter species in Tyhume, DO did not correlate with the density. Further consideration of PCA in Acinetobacter species in the rivers.The density of the presumptive ariables . The posAcinetobacter species in the Great Fish, Keiskamma and Tyhume rivers respectively. SOMs and codes plots of the physicochemical variables showing significant (p < 0.05) effect on the density of Acinetobacter species in each of the rivers are captured in a rectangle and circular shapes respectively.Comparable with the correlation/association test, Kohonen self-organizing maps (SOMs) and codes plots were used to connect the bacterial counts and physicochemical variables in the various rivers studied. The average distance to the closest codebook vector was made with 100 iterations (training progress) for uniformity among the rivers. Acinetobacter species.The SOMs visually indicated that temperature, TSS, TBS and BOD are responsible for the bacterial counts in the Great Fish River, meanwhile, pH, temperature, TSS and TBS were positively correlated with the bacterial density in the Keiskamma River. Likewise, in the Tyhume River, both pH and DO did not have any relationship with the density of presumptive Acinetobacter species in the river across the season. As such, code plots indicated that the bacterial density in Tyhume River correlates with all the physicochemical variables except DO. Although, correlation-association test showed the relationships between the physicochemical variables and bacteria counts. However, both SOMs and codes plots showed better pieces of evidence about the levels of the relationship between the bacterial density and physicochemical variables.Even so, depending on the magnitude of each variable in the weight vector, the codes plot further confirmed the effect of each physicochemical variable on the density of presumptive Acinetobacter species on CHROMagar) was 1107 presumptive Acinetobacter species of which 370, 309 and 428 were isolated from Great Fish, Kieskamma and Tyhume respectively.As shown in Acinetobacter-specific recA gene  and Codes plots  were advanced to measuring the effect of physicochemical variables and density of  factors ,28. HoweAcinetobacter species within the freshwater resources, even though the relationship between seasonal shifts in physicochemical variables and the density of presumptive Acinetobacter species were river-dependent. Anthropogenic activities  were identified using the molecular confirmatory test of by targeting recA gene [recA gene as a reliable method for identification of Acinetobacter [From the forging, bacteria belonging to the genus ecA gene ,48, whictobacter .Acinetobacter species has created an opportunity to detect statistical significance in the relationship between the density of presumptive Acinetobacter species and physicochemical variables in the Great Fish, Keiskamma and Tyhume rivers. From the various analyses in this study, temperature, TSS, TBS and BOD were the main but not the only factors that were influencing the density of Acinetobacter species in the various rivers. Overall, seasonal changes in the identified variables tended to control the quantity of presumptive Acinetobacter species recovered from each river. The response of Acinetobacter species to the shifts in environmental conditions is a semblance of a model to predict the nature of Acinetobacter species in the freshwater resources. As such, this study revealed that the three rivers that were investigated contain Acinetobacter species that might be useful for basic and applied study in ecology, microbiology and biotechnology, while their clinical relevance in causing diseases cannot be overemphasized. Thus, these rivers deserve further evaluation using advanced methods such as metagenomics.This study of the effect of seasonal shifts in environmental factors on the survival of


Many observers worry that growing numbers of international institutions with overlapping functions undermine governance effectiveness via duplication, inconsistency and conflict. Such pessimistic assessments may undervalue the mechanisms available to states and other political agents to reduce conflictual overlap and enhance inter-institutional synergy. Drawing on historical data I examine how states can mitigate conflict within Global Governance Complexes (GGCs) by dissolving or merging existing institutions or by re-configuring their mandates. I further explore how “order in complexity” can emerge through bottom-up processes of adaptation in lieu of state-led reform. My analysis supports three theoretical claims: (1) states frequently refashion governance complexes “top-down” in order to reduce conflictual overlap; (2) “top-down” restructuring and “bottom-up” adaptation present alternative mechanisms for ordering relations among component institutions of GGCs; (3) these twin mechanisms ensure that GGCs tend to (re)produce elements of order over time–albeit often temporarily. Rather than evolving towards ever-greater fragmentation and disorder, complex governance systems thus tend to fluctuate between greater or lesser integration and (dis)order. International agreements and international law have expanded rapidly over the past century with the result that many areas of global politics are today co-governed by multiple institutions whose membership and competences overlap. The resulting “fragmentation” of global governance has been widely depicted as a source of rule inconsistency, duplication, and jurisdictional conflict , which I define as a system of diverse rule sets and agents that assert overlapping authority claims to govern a specific global policy issue. I have three main goals.First, I seek to draw a conceptual distinction between “ordered” and “disordered” governance complexes according to the degree of synergy and coordination among separate component institutions.Second, I theorize two mechanisms for ordering governance complexes through either “deliberate restructuring” or “spontaneous adaptation.” Deliberate restructuring entails the use by collective state principals of treaty-reform or the creation of cross-cutting institutions tasked with managing complexity. Spontenous adaptation refers to positive feedback processes in complex social systems which reinforce certain behaviours so that ongoing interactions among separate institutions tend—over time—to produce elements of order through mutual accommodation by individual institutions Anderson : 219. ThThird, I explain the prevalence of order versus disorder in GGCs as a function of institutional actors’ incentives and capacities for either top-down restructuring or bottom-up adaptation, which in turn reflect the alignment of state preferences and available organizational resources for adaptation.The empirical part of the article examines processes of ordering in the GGC for international civil aviation from 1910 to 2010—as proof of concept., Aggarwal I am not the first to observe that ongoing interactions among overlapping institutions can lead to stable divisions of labour through functional specialization or other forms of decentralized adaptation order. A central claim of this article is that processes of top-down restructuring and bottom-up adaptation tend—over time—to (re)produce elements of both local and system-wide order in GGCs. Nevertheless, “order” in open complex systems remains precarious as existing task-divisions and exchange relationships are frequently upset by exogenous shocks . Thus, GGCs tend to evolve through a pattern of “punctuated equilibrium” Krasner  in whichThe article is organized as follows. Section My empirical analysis shows that both top-down restructuring and bottom-up adaptation are common mechanisms for ordering relationships among institutions. It also illustrates that order in the absence of centralized authority remains precarious in that existing exchange relationships are frequently upset by exogenous shocks which disrupt established patterns of interaction. Despite recurrent shocks, however, the GGC for civil aviation demonstrates a tendency to reestablish elements of order over time. Its evolution thus conforms to a cyclical pattern of punctuated equilibrium in which periodic shocks trigger growing conflict and disorder, only to be followed by periods of (re)consolidation and stability until a new crisis hits. In the concluding section I consider the implications of these findings for the wider study of institutional complexity in global governance.institutions as rules . While fully compatible with the concept of a regime-complex, the notion of a global governance complex—a system of diverse rule sets and agents that assert overlapping authority claims to govern a specific global policy issue—highlights the diversity of rules  and agents (public and private) involved in global governance  builds closely on Raustiala and Victor’s  concept A second contrast between GGCs and existing notions of regime complexes concerns the relationships between component rule-sets and agents. Raustiala and Victor cite the absence of hierarchy as a constitutive feature of an RC , GGCs as a whole often sit at the nexus of several distinct issue-areas order from the notion of complexity.In this section I consider what constitutes order in complex governance systems, not synonymous with disorder or chaos. Simply put, “order” is an arrangement of things in relation to each other according to a particular, recognizable method or pattern. It refers to the nature of relations between a system’s components rather than their number or diversity. A governance system consisting of a handful of like institutions with limited ties between them will score low on complexity but may nevertheless be disordered if relationships are unpredictable and in constant flux. By contrast, a more complex system consisting of numerous diverse institutions with multiple ties between them may exhibit elements of order if institutional relationships are transparent and stable order in international governance complexes was made by Biermann et al. :20 who dregimes and “loosely integrated” regime complexes . In a GGC featuring diverse institutions and agents, what amounts to effectiveness for one may represent failure for another. Thus, from the perspective of component institutions, the effectiveness of a GGC is largely in the eyes of the beholder. Effectiveness at the level of the system as a whole is equally difficult to measure: effective compared to what? Nevertheless, we may assume that conflicting rules and governance techniques which result in frequent contestation of established routines and exchange relationships (i) incur higher transaction costs, and (ii) are indicators that a system is failing to generate satisfactory outcomes in the eyes of many participants. Conversely, coordination of overlapping authority claims through mutual accommodation or overt collaboration are indicators of general “buy-in” to joint governance processes. Thus, an ordered GGC may be seen to signify general satisfaction with collective governance processes which may in turn be regarded as a proxy for effectiveness.Unlike their component institutions GGCs as a whole are not purposefully constituted but evolve through interactions among dispersed institutions and agents. This does not imply that institutional complexity is always unintentional. States may purposefully engineer institutional overlap to enable forum shopping  may also suffer negative consequences from institutional complexity. Growing institutional density may trigger competition for scarce resources like state funding or expert staff. Jurisdictional overlap can also limit international organizations’ ability to induce policy change, as opportunities for forum shopping allow states and other target actors to evade commitments . According to ECOWAS member states, at whose behest the merger took place, “the driving force behind WAHO’s creation was the incongruence of the agendas that were being pursued by the two existing intergovernmental health organisations in the sub-region”.4These brief examples are far from unique. Since 1900 hundreds of formal inter-governmental organizatoins (IGOs) have “disappeared” from the international system, being either dissolved or replaced by their founding states, merged with other organizations, or simply left to die  is to create cross-cutting institutions tasked with coordinating the activities of separate governing bodies. Previous studies have pointed to the creation of “super-institutions” tasked with promoting governance coherence order relations among IGOs. However, states may also engage in restructuring of mixed public-private organizations, private for-profit IOs and NGOs via “orchestration”  is that they exhibit “emergent behaviour”, meaning that undirected interactions between component elements generate distinctive behaviour at the level of the system as a whole. Starting in the 1980s, economists and organization scholars have drawn on theories of CAS in biology and physics to suggest that complex socio-economic systems may have capacity for spontaneous “self-organization” through “coLeaning on such ideas, some observers have suggested that ongoing interactions between overlapping international institutions may gradually produce a stable division-of-labour as institutions adjust to one-another by developing increasingly separate tasks Young :10. But One bureaucratic response to growing institutional overlap might be to modify or reduce the scope of institutional activities to avoid direct competition with peers; a strategy of “avoidance”  in ordering GGCs? When should we rather expect bottom-up adaptation? State incentives to order GGCs depend firstly on the nature of interdependencies between existing institutions. Whereas overlapping institutions may be mutually undermining due to incompatible rules or excessive transaction costs, co-governance by diverse actors may also increase flexibility and widen technical and political knowledge thereby reducing policy uncertainty and increasing problem-solving capacity  resource scarcity may set in, constraining further expansion and fuelling competition  mainly comprise government-led institutions with low autonomy, leaving less scope for bottom-up adaptation. Undoubtedly, a comparative research-design featuring systematic comparison to other domains such as international trade or human rights would offer additional insights. Yet such a design would take me beyond the limits of a single article. Instead I focus on exploring within-case variation and process-tracing to demonstrate the plausibility of my theory.Observationally, I expect exogenous shocks to lead to growing institutional density and resulting resource competition. These are my main independent variables at the system level. I then look for evidence of top-down restructuring and bottom-up adaptation. My framework predicts top-down restructuring when governments are relatively united in their desire for institutional change. Given a failure of top-down ordering, a more gradual process of bottom-up adaptation is expected; firstly via competitive task-differentiation and more gradually via growing cooperation. Importantly, the level of task-differentiation/cooperation is a property of the system as a whole, rather than of any single institution or pair of institutions. However, I assume that system-level changes are driven by underlying changes in the behavior of individual units. I therefore consider how individual institutions adapt to complexity through specialization or cooperation. The behavior of institutional agents is ultimately what is “ordered” through processes of adaptation. Despite the profound cross-border implications of international civil aviation there has never been a single, integrated regime governing international civil aviation. Since the early 1900s international aviation has been regulated by an array of overlapping bilateral and multilateral agreements implemented by IGOs, NGOs, individual government agencies, and private commercial actors. The complexity of international aviation governance is partly dictated by the complexity of air travel itself. For international flights to operate smoothly, numerous legal regimes must co-function, including those governing radio and telecommunications, meteorological services, and customs. International aviation touches directly on major economic and social issue-areas such as commerce, tourism, and immigration. At the same time, the speed and reach of commercial flights give rise to overlap and potential jurisdictional conflict vis-à-vis international regimes governing public health, environmental protection, and security. As a result, decisions affecting international air travel are made in multiple venues, many of which do not focus directly on aviation.par excellence.The complexity of international aviation governance is, however, not merely a function of its technical aspects. Functions at the heart of aviation governance, such as the granting of overflight and landing rights, licensing of airlines, fixing of international routes and fares, and standardization of safety procedures are all made by multiple separate institutions with overlapping functions and membership. Despite periodic attempts to integrate these functions into a single regime, international aviation governance has remained strongly fragmented. The system is thus a complex governance system Since the early twentieth century, the GGC for international aviation has undergone several changes that can be described as fundamental restructurings of its institutional architecture. Along with examining the roots of complexity, this section focuses on how the system has evolved over time; expanding and contracting in terms of the number, diversity and density of component institutions, and alternating between periods of greater and lesser (dis)order.The first international flight was accomplished in 1909 by French pilot, Louis Bleriot, crossing the Channel to England. The following year, France hosted the first international aviation conference to agree basic principles of international air law. This conference exposed a rift between opposing institutional designs. On one side stood the ideal of international “freedom of the air” supported by Germany and France; on the other, the model of sovereign control of national airspace, favoured by Britain Jönsson :146–47. Attempts at intergovernmental cooperation were renewed after World War I. Among the chief concerns of state representatives to the Paris Peace Conference in 1919 were military uses of civilian aircraft technology and the need to maintain strong communication and transport ties with overseas colonies Jönsson . The ConIn addition to confirming the principle of unreserved “air-sovereignty”, the 1919 Paris Conference introduced a geographical divide in international aviation governance. The Paris Convention was ratified by the British Empire, seven European air-faring states (excluding Germany), and Japan. The United States and the Soviet Union declined to join, preferring to develop their own regional agreements Jönsson :277–78. Washington’s rejection of ICAN echoed wider US resistance to the League of Nations under whose direction ICAN was placedDuring the 1920s, several international conferences were convened to negotiate a centralized global aviation regime. All ended in deadlock (ICAO-History). Instead, growing demand for regulation led to a rapid proliferation of task-specific bodies. During the 1920s and 1930s, a cascade of new institutions emerged alongside ICAN to address technical issues arising from expanding air-traffic; from the allocation of routes, to radio-communications, weather forecasts, and operation of signals and lights (ICAO-History). These institutions included standing international aviation conferences among West and Central European states and sub-regional conferences such as the Mediterranean Aeronautical Conference and the Aeronautical Conference of the Baltic States and the Balkans. Many existing IGOs like the International Office of Public Hygiene, the International Meteorological Organization, and the International Radiotelegraph Union also placed aeronautical issues on their agendas (ibid). A limited measure of information-exchange and coordination of technical standards was provided by the International Technical Committee of Experts in Air Law, founded in 1925, which counted members from both PACCA and ICAN Ide . All othAlongside the proliferation of public institutions, numerous private bodies emerged to address aeronautical matters. The most prominent was the International Air Traffic Association (IATA) founded in 1919 by representatives of major airlines to harmonize international operations. Other private institutions included the non-profit Aircraft International Register 1926), the Aerospace Medical Association (1929), and the International Committee for the Study of Sanitary Aviation (1934). This growing assemblage of private institutions filled regulatory gaps left by states’ inability to agree a centralized regime to govern aviation safety and to allocate international routes and carrier capacities. Thus, overall, institutional density and overlap was rapidly growing . Among the issues that could not be effectively addressed by any regional institution was the liabilities of private air carriers operating in international airspace. If an international airliner caused death or injury to passengers, or surface damage to third parties, who would be liable? How could one protect passengers’ rights while also shielding national airlines from crippling compensation claims from third parties? (Wilberforce ICAN.Org). ICAN’s Director also attended the many regional aviation conferences (ICAN.org). Among private institutions, the International Committee for the Study of Sanitary Aviation created in 1934 introduced an element of coordination between the International Aeronautical Federation, The Permanent Committee of International Congresses of Sanitary Aviation, and The League of Red Cross Societies to develop joint guidelines for aviation health, and to liaise jointly with the public International Office for Public Hygiene.13As predicted, failure by governments to restructure an increasingly complex governance system was gradually compensated by bottom-up adaptation (Hypothesis 3). The parallel operation of a large number of overlapping aviation institutions increased operational costs due to complicated information-exchange and widespread duplication. Often, the same technical and legal experts were represented in different aviation institutions, doing parallel work.The fact that adaptation was achieved mainly through informal coordination rather than task-differentiation or more permanent institutionalized cooperation may be explained by a relatively resource rich environment. During this period more and more states developed capacities for international air transport. Continuous market-growth combined with rapid technological progress which required constant updates to existing protocols and procedures meant that individual institutions could expand their functions and membership without coming into direct conflict, while reducing governance costs and increasing effectiveness through decentralized coordination. Given relatively abundant resources, institutional relations thus developed in broadly synergistic ways with individual institutions supplying overlapping but mutually reinforcing tasks, and parallel processes of standard development facilitating communal learning through regular information-exchange.This harmonious state of affairs ended during World War II due to two exogenous shocks. First, the war effort led to the creation of new international air routes and bases—from New York to Paris, from Africa to the Arctic—unleashing an international race to expand commercial air-travel , which was tasked with ensuring “maximum uniformity in regulations and standards of civil aviation” by “recommending certain practices that member countries should follow” (Art. 44).Renewed failure to agree an integrated international aviation regime led to further institutional proliferation (Hypothesis 2). In the decades following the Chicago Conference more than 1000 bilateral accords were made exchanging commercial air rights - their content varying depending on the relative bargaining strength of the parties . Nevertheless, limited top-down adjustments were made. The move to a purely market-based system rendered redundant many existing intergovernmental aviation organizations. As no state saw an interest in continuing to fund these obsolete organizations doing away with them was relatively easy (Hypothesis 2). In May 1947 the International Technical Committee of Experts in Air Law was dissolved by member states, its functions transferred to ICAO along with those of the International Legal Committee on Aviation . Between the two overarching governance bodies—ICAO and IATA—an increasingly stable of division of labor emerged. While ICAO promoted multilateral cooperation to standardize navigation practices, IATA facilitated cooperation among airlines (many still state-owned) on scheduling, reservation and communication systems, handling of passengers, baggage, and cargo . The consolidation of a system of “free but controlled competition” Jönsson  ushered During the 1960s and 1970s a series of shocks hit the global airline industry. First, the birth of jet airliners and the introduction of national carriers in many developing countries led to a rapid expansion of transport capacity which far outstripped consumer demand  have created potential for regulatory conflict by placing air travel within the scope of two overlapping domains of international law: the Chicago Convention, and the WHO’s International Health Regulations (IHRs) . In 2006 representatives from ICAO, the WHO, IATA, the Airports Council International and the UN World Tourism Organization founded the Collaborative Arrangement for the Prevention and Management of Public Health Events in Civil Aviation (CAPSCA) to keep emergency guidelines for states up to date. Focused on facilitating safe air-transport during global health emergencies, CAPSCA “performs the important function of ‘overlap manager’ between different domains of law” (ÓCuinn and Switzer The GGC for civil aviation has evolved through stages characterized by varying levels of (dis)order. During a first stage (1900–1943) rapid technological developments combined with conflicting state interests triggered rapid proliferation of overlapping institutions. During this period, a resource-rich environment facilitated a combination of individual task-expansion and informal decentralized coordination. Despite growing institutional density, the system thus remained relatively ordered as separate institutions supplied overlapping but compatible functions. During a second period (1944–72) growing institutional fragmentation prompted limited top-down restructuring, followed by gradual bottom-up adaptation through growing task-differentiation and resource-pooling. A third stage (1973–1990) was marked by severe economic shocks to the aviation industry, leading to increasing market competition and growing disorder in respect to the governance of international routes, frequencies and fares. Since the 1990s, growing overlap between the functions of ICAO and IATA and institutions in adjacent governance domains like public health and security has been addressed mainly through bottom-up adaptation, ensuring broadly synergistic policy outcomes.Overall, the evolution of the GGC illustrates that institutional density and disorder often increase in the wake of exogenous shocks which upset existing exchange relationship. Between shocks, however, state principals have (sometimes) succeeded in rationalizing cooperation by terminating or reforming redundant institutions, while institutional agents have sought to improve their individual fitness through coordination and/or resource-exchange thereby providing an element of bottom-up ordering of the system as a whole. The GGC has thus evolved through a pattern of punctuated equilibrium in which periodic shocks have resulted in growing disorder, followed by periods of (re)consolidation via top-down restructuring and bottom-up adaptation (Hypothesis 5).Many observers have argued that a proliferation of international agreements and international law in recent decades is causing a deleterious fragmentation of global institutional architectures. There can be little doubt that the international system has become increasingly “crowded” in terms of the sheer number of institutions in play  as the result of weak intergovernmental governance mechanisms in this domain which have their origin in the early post-war period. The history of international aviation governance thus vividly illustrates how the emergence of new rules and practices is shaped and constrained by existing institutional frameworks , leading to different institutional trajectories. Exploring variation in the historical evolution of GGCs may thus help us to distinguish between systemic causes of growing complexity and proximate causes that lead to institutional proliferation in specific instances.At the same time as it invites comparison between different domains of global governance, my analysis also highlights the promise of studying the evolution of


Journal of General Virology in February 2019 has been retracted following findings reported by a reader of the journal. The immunoblots shown in Figs 3a, b, 4a, b and 6 are believed to have been manipulated and to contain duplicated images.The article ‘Host protein CD63 promotes viral RNA replication by interacting with human astrovirus non-structural protein nsP1a/4’ which was published in the Journal of General Virology has attempted to contact the authors but to date has not received a response. All authors have been notified of this decision.


This study investigates genetic and environmental variation in starch content and characteristics of 14 French bread cultivars. Understanding the impact of these factors on wheat quality is important for processors and especially bakers to maintain and meet the requirements of industrial specifications. Different traits were evaluated: starch content, distribution of starch granules, percentage of amylose and amylopectin and their molecular characteristics . Genetic, environment and their interaction had significant effects on all parameters. The relative magnitude of variance attributed to growth conditions, for most traits, was substantially higher (21% to 95%) than that attributed to either genotype (2% to 73%) or G × E interaction (2% to 17%). The largest environmental contribution (95%) to total variance was found for starch dispersity. The highest genetic influence was found for the percentage of A-type starch granules. G × E interaction had relatively little influence (≈7%) on total phenotypic variance. All molecular characteristics were much more influenced by environment than the respective percentages of amylose and amylopectin were. This huge difference in variance between factors obviously revealed the importance of the effect of growing conditions on characteristics of cultivars. GroD-glucopyranosyl units with a degree of polymerization (DP) in the range 500–6000 glucose residues. A fraction of the AML molecules are slightly branched by α--linkages [5 to 3 × 106 glucose units. The formation of AML is mainly due to the granule bound starch synthase (GBSS) coded by the waxy genes [Starch is found in the form of starch granules trapped in a protein matrix. Three classes of granule can be distinguished: type A granules with a diameter greater than 15 µm and a lenticular shape, type B granules between 5 and 15 µm with a spherical shape and type C granules which are smaller than 5 µm ,8. For plinkages ,10. The linkages ,12,13 anxy genes ,15 namedxy genes ,17. GBSSxy genes ,19. AMP xy genes ,21,22. Txy genes ,25,26,27Considering the current and projected environmental impacts , it is essential to better understand these phenomena to implement new breeding strategies for sustainable quality. In this study, 14 winter wheat cultivars were grown under field conditions in locations widely scattered across France and Europe, and flours were subjected to a comprehensive analysis of wheat starch traits. The main objective was to assess the effects of genetic (G), environment (E) and G × E interaction on the starch parameters. In addition, the present study offers a comprehensive discussion on variations of starch biosynthesis.In the frame of the collaborative research project established between Arvalis, UniLaSalle and private European plant breeding companies working in France, multilocal trials were carried out during two different growing seasons (2015 and 2016) . Fourteen winter wheat cultivars were grown in ten locations in the European areas, providing a very large contrast of growing environments .-Stability of their known bread-making quality. Some of them are considered stable and some varieties are recognized to be less stable.-The allelic form at the GluD1 locus considering the weight of this gene on the final rheological quality.-Earliness to heading stage which may allow adaptation to very wide geographical locations exhibiting different pedo-climatic conditions.Wheat cultivars were chosen as function of:Ten experimental sites were identified by the breeders based on the quality of the results regularly observed at these locations, with the aim of maximizing the sources of variability in the expression of breadmaking quality.Experimental trials were conducted in the field, including randomized plots with three replicates. Conventional agronomic practices, with mineral and fungicide treatments, were used to achieve optimal grain yield without any water irrigation.-Classically adapted sites  consisted of locations with a low frequency of abiotic stress where regularly stable breadmaking quality is obtained in the control cultivars.-Classically invalidated sites  with episodes of heat and hydric stress leading to regularly variable breadmaking values in the control cultivars.-Sites with restrictive climatic conditions, particularly end-of-cycle temperatures from European networks (EU sites).Three kinds of locations were chosen:w/v) SDS, 1% (w/v) dithiothreitol (DTT), 10% (v/v) glycerol) at room temperature. At each washing step, starch granules were disrupted with sonication for 20 s at a power setting of 20% using a stepped microtip probe (6 mm diameter) . Granules were then washed three times for 5 to 10 min with cold water, once with cold acetone and finally air-dried at room temperature overnight. Each purification step was followed by centrifugation at 3500× g for 5 min. The extractions were conducted in triplicate.Starch was extracted using the method developed previously with some technical modifications . Wheat g® glass tube. The samples were wetted with 0.2 mL of an 80% (v/v) aqueous ethanol solution for dispersion and stirred on a vortex mixer. Then, 3 µL of thermostable α-Amylase prepared in 3-Morpholinopropanesulfonic acid (MOPS) solution buffer was added to the slurry and thoroughly and vigorously stirred on a vortex mixer. To start enzymatic digestion, samples were incubated at 100 °C for 6 min with stirring every 2 min. The tubes were then placed in a water bath at 50 °C and 4 mL of sodium acetate solution  was added. Amyloglucosidase preparation (0.1 mL) was introduced and samples were stirred on a vortex mixer and incubated for 30 min at 50 °C. After incubation, the samples were transferred to 50 mL tubes and the volume was then adjusted with 0.7 mL of distilled water. The samples were mixed vigorously and centrifuged at 3000 rpm for 10 min at 20 °C. The diluted solutions (1 mL) were transferred in triplicate to Pyrex® glass tubes. A solution (3 mL) of glucose oxidase peroxidase 4-aminoantipyrine (GOPOD) reagent was added to tubes and incubated for 20 min at 50 °C. Glucose controls consisted of 0.1 mL of glucose standard solution, while reagent Blank Solutions consisted of 0.1 mL of distilled water. The absorbance was read against the reagent blank at 510 nm for each tube including that containing the glucose control. The starch content was expressed as a percentage of flour dry weight (DW).The total starch content of whole flour was calculated using a Total Starch Amyloglucosidase/α-Amylase Assay Kit . Wheat flours (100 mg) were weighed, in triplicate, into a 20 mL PyrexThe size distribution of the purified starch granules was measured with a Mastersizer 2000 laser particle size analyzer  following the protocol previously reported . Each stv/v) and heated for 60 min at 110 °C. Gelatinized starch was precipitated using 5 mL of absolute ethanol and then centrifuged at 20,000× g for 20 min at 20 °C. Supernatants were discarded, while pellets were kept and mixed with 4 mL of NaOH solution (20 mM) and transferred into 10 mL pressurized vessels . Solubilization was conducted using microwave heating at variable power  for 8 min at 135 °C. Solubilized starchy polymers were then filtered through 5 µm cellulose nitrate syringe filters  and 100 µL was injected into the A4F system. Pullulan standard polymers (110 to 800 kDa) were used for initial operational checking of the whole Eclipse™ A4F system. A4F analysis was accomplished using an Eclipse3 F System  combined with a multi-angle light scattering (MALS) detector  and an Optilab® T-rEX™ refractive index (RI) detector, . The light scattering detector was calibrated using toluene as a standard. Constant calibration of the RI detector was calculated using sodium chloride at different concentrations and the temperature was set at 35 °C. The short separation channel (195 mm in length) had a trapezoidal geometry. The thickness of the spacer was 0.35 mm. The accumulation wall was constituted of regenerated cellulose ultrafiltration membrane with a cut-off of 10 kDa (Superon). An Agilent HPLC 1200 Series  was used in tandem with the A4F system. The eluent was ultra-pure deionized water with 0.02% NaN3 (w/v) added as a preservative. The mobile phase was degassed and filtered through a 0.1 µm inline filter  before entering the A4F channel.Starch was solubilized according to the method established previously . About 1Separation of AML and AMP was achieved following the protocol reported in our previous work . AML andThe polymer characteristics of AML and AMP measured using A4F, were: weight-average molar mass Mw (expressed in g/mol); number-average molar mass Mn (in g/mol); polydispersity Mw/Mn; and gyration radius R (in nm).® software. Statistical significance was accepted at p < 0.05, p < 0.01, p < 0.001 and p < 0.0001.All statistical analyses  were performed using StatgraphicsOverall means of starch content, starch granule distribution and molecular features of the 14 wheat cultivars grown in the 20 growing conditions (Year × Location) are shown in Starch content for all wheat samples varied from 54% to 69.48% with a mean value of 61.10%, showing a low coefficient of variation (CV) of about 3.66%. The granule content distribution showed higher levels for A-type granules with a mean of 75%, followed by B-type granules, with an overall mean of 21%, then C-type granules with an overall mean of 4%. Percentage volume of A granules which represents the highest fraction, showed the lowest CV (~4%) amongst total granule distribution. This variability could be related to the low variability observed for total starch content. The widest range of granule distribution was observed for B-type granules with a CV of ~14% followed by C-type granules with CV of ~11.76 . These rSeparation of AML and AMP showed mass fraction proportions, respectively, of 30% and 70% . These r6 to 1.4 × 106 g/mol with a mean value of 0.4 × 106 g/mol. The MWD has not been widely examined for wheat starch; however, it is known that the Mw of AML varied from 105 to 106 g/mol [6 to 58.4 × 106 g/mol, with a mean of 23.4 × 106 g/mol. Molar masses were reasonably consistent with those reported for AML and AMP from several cereals including barley, triticale and wheat [7 to 109 g/mol [5 to 4 × 105 g/mol for AML and from 1 × 108 to 4 × 108 g/mol for AMP [Characterization of the MWD of grain storage starch polymers using A4F was here performed for the first time in a research program studying genotypic and environmental effects on bread wheat. Mw, Mn, Mw/Mn and R for AML and AMP and Mw/Mn means and CV are shown in 06 g/mol . AMP Mw nd wheat . In the 09 g/mol ,36,37,38 for AMP .AML and AMP were moderately to highly dispersed since AML showed an overall mean Mw/Mn value of 1.65 while AMP exhibited a value of 4.41. As a result, starch was extremely dispersed, showing an overall mean value of 24.68. The results indicate very strong heterogeneity of the lengths of the starchy chains formed during grain filling.Values of the AMP gyration radius, Rw, ranged from 53.2 to 141.9 nm. It showed an overall mean of 95.61 nm, twice that of AML (48.92 nm). Values agreed reasonably with those found for AML and AMP in different species . The higOn the other hand, all molecular attributes showed wide dispersity among the 14 cultivars and the 20 growing conditions, with CV values ranging from 13.17% to 34.04% for AMP gyration radius and AML Mn, respectively. The highest CVs were obtained for the molecular weights of the AML and AMP chains. These results, mainly those related to molar masses, imply that the molar masses had a higher level of useful genetic variability among all starch attributes studied. Greater CVs for starch attributes could indicate greater potential for favorable advances in wheat breeding.The relative contribution of cultivar (G), environment and G × E interaction to the variation observed in starch, starch granule distribution and the molecular properties of starch polymers is reported in p < 0.0001) source of variation for starch content, starch granule distribution and for all molecular features of its components  calculations showed very wide ranges in all parameter means studied except for starch whole flour content, percentage of A-type granules and percentage of AMP mass fractions (Environment CV (CVractions . The mosp < 0.0001) for starch content, granule distribution, AML% and AMP%  for all features (6 g/mol at Orsenville (France) in the 2015 crop season, to 0.52 × 106 g/mol at Champigny (France) in the 2015 crop season. The same results were obtained for Mw which varied between 0.25 × 106 g/mol and 0.69 × 106 g/mol. It should be noted that the latter is one of the atypical locations. In addition, the Mn of AMP oscillated from 3.673 × 106 to 8.15 × 106 g/mol at Estrées Saint Denis (France) and Navarre (Spain), respectively, in the 2015 crop season. The Mw ranged from 18.64 × 106 g/mol in an atypical area, Échemines (France) in the 2015 crop season, to 30.51 × 106 g/mol at Navarre (Spain) in the same crop season. To verify our choice of locations used in our study and to detect possible effects exerted by groups of locations, we processed the data according to the characteristics of the locations. Thus, we have formed three groups of places as indicated in the materials and methods section. The means and the results of the Duncan analysis are shown in For the first time, the molecular features of starch polymers were tested for the influence of environment. Without exception, the results showed significant environmental impacts . Typical locations in France showed higher values for molecular features  for AML and AMP than those found in atypical locations. In-depth analyses will be carried out to understand and determine the exact source of these differences. We will be able to determine the weight of each factor  using advanced statistical analyses (PLS for example).In the current study, the results show that environment was the major source of variation for biosynthesis of starch and its components .E/CVG ratios clearly show the relative importance of the growing conditions on all molecular parameters except for starch content and starch granules , corresponding to endosperm differentiation or cell division phases . Starch Reported RNA-Seq results highlighted 166 homologs of starch biosynthesis-related genes expressed during the development of wheat grain endosperm ; 74 seleAny punctual and severe environmental event during the starch deposition phases would have variable effects depending on the stages of wheat grain development and on the sensitivity of starch biosynthesis-related genes and their regulators. Thus, AGP, SS, GBSS and SBE are reported to be the most sensitive to abiotic stress such as high temperature and drought ,43,80,81Variations in the transcripts, translation and enzyme activity could explain the genotypic and environmental variations observed in our study. Changes in SS activity could explain variations of starch content since SSs have been shown to be the most sensitive to drought and largely explain the starch content in grain wheat . In wheaIn addition, activity of AGP could also explain variations in starch content as it is involved in the most limiting step of starch biosynthesis by providing glucose molecules in the form of ADG-glucose. AGP activity declines drastically under water stress and leads to premature cessation of starch accumulation ,85. It hGBSS is considered to be less sensitive to elevated temperature while it is significantly affected by drought ,84,87. ASBE activity seems to be sensitive to heat stress in maize and rice ,89. The For all of the French bread wheat parameters tested, environmental growing conditions were considerably more important than variations associated with genetic factor, being excluded to starch content and granule distribution. This obviously reveals the importance of growing conditions on starch components and related variables. As expected, the relative contribution of the G × E interaction to total variation was substantially lower than that of either genetic or environment.Our results point clearly to the presence of genetic mechanisms controlling starch polymerization and structuration  that can be considered by breeders in future wheat breeding programs, since some variations of these traits have already been related to technological differences between cultivars. However, genetic and enzymatic mechanisms were influenced by growing environment. In order to maintain a product of uniform and expected quality, the challenge for the common wheat industry is to manage as much as possible the impacts of the changing growing environment on wheat quality by stabilizing not only protein-related parameters, but also starch-related characteristics.


Symbiochlorum hainanensis by culturing it under two temperature settings (26.0 and 32.0°C) crossed with two pH levels (8.16 and 7.81). The results showed that the growth of S. hainanensis was positively affected by H, A, and the combined treatment (AH). However, no superimposition effect of H and A on the growth of S. hainanensis was observed under AH. The analysis of chlorophyll fluorescence, pigment content, and subcellular morphology indicated that the chloroplast morphogenesis (enlargement) along with the increase of chlorophyll fluorescence and pigment content of S. hainanensis might be a universal mechanism for promoting the growth of S. hainanensis. Transcriptomic profiles revealed the effect of elevated temperature on the response of S. hainanensis to acidification involved in the down-regulation of photosynthesis- and carbohydrate metabolism-related genes but not the up-regulation of genes related to antioxidant and ubiquitination processes. Overall, this study firstly reports the growth, morphology, and molecular response of the thermo-tolerant alga S. hainanensis to future climate changes, suggesting the predominance of S. hainanensis in its associated corals and/or coral reefs in the future.Ocean acidification and warming affect the growth and predominance of algae. However, the effects of ocean acidification and warming on the growth and gene transcription of thermo-tolerant algae are poorly understood. Here we determined the effects of elevated temperature (H) and acidification (A) on a recently discovered coral-associated thermo-tolerant alga  Tely 26°C , which i at 35°C . Hence, S. hainanensis (CCTCC M2018096), isolated from coral species Porites lutea in the tropical reef regions of the SCS  and washed three times with sterile phosphate-buffered saline. Then, cell pellets were re-suspended in autoclaved artificial seawater medium and used for further experiments.Cultures of  the SCS , 2019, wS. hainanensis were acclimated to four conditions: (1) pCO2 level 1,000 μatm (pH = 7.81), 26°C , (2) pCO2 level 395 μatm (pH = 8.16), 32°C , (3) pCO2 level 1,000 μatm (pH = 7.81), 32°C , and (4) pCO2 level 395 μatm (pH = 8.16), 26°C . To achieve the different pCO2/pH conditions, 1,000-ml Erlenmeyer flasks with 500 ml autoclaved artificial seawater medium were bubbled with sterilized air containing either ambient  or elevated . To achieve the different temperature conditions, the Erlenmeyer flasks were incubated in water baths , H (n = 3), AH (n = 3), and C (n = 3) conditions was monitored every 2 days by optical density (OD) value at 750 nm with a UV spectrophotometer  at 750 nm. The growth rate was calculated based on the cell concentration variations of S. hainanensis at exponential phase (day 2 to day 24). The dry cell weight in the mid-exponential phase under the A (n = 3), H (n = 3), AH (n = 3), and C (n = 3) conditions was measured by filtering the algal suspension through a pre-dried and pre-weighted 0.45-μm cellulose nitrate membrane filter  and drying in an oven at 80°C for 24 h.The cell concentration of S. hainanensis under the A (n = 3), H (n = 3), AH (n = 3), and C (n = 3) conditions was measured using a Turner fluorometer with the in vivo module . Algal cells in the mid-exponential phase under the A (n = 3), H (n = 3), AH (n = 3), and C (n = 3) conditions were sampled for chlorophyll a, chlorophyll b, and total carotenoids measurements according to the acetone-based method  were determined based on scanning electron and transmission electron microscopy observations according to our previous study (Cells of us study .n = 3), H (n = 3), AH (n = 3), and C (n = 3) conditions were harvested in the mid-exponential phase by centrifugation at 5,000 rpm for 5 min at 4°C for total RNA extraction and RNA-seq. In detail, cell pellets under different conditions were snap-frozen in liquid nitrogen and stored at −80°C before RNA extraction. Total RNA was extracted from algal cells as previously described , H (n = 3), AH (n = 3), and C (n = 3) conditions were processed using Trimmomatic v0.33 for trimming adapters as well as low-quality bases from the ends of the reads . Bray–Curtis dissimilarity-based principal coordinate analysis (PCoA) pictures of differentially transcribed genes were drawn using Primer-e2 for comparing differentially expressed genes under different conditions.The platform . Additioplatform . The cluplatform . Differeplatform . Fold chn = 3), H (n = 3), AH (n = 3), and C (n = 3) conditions was measured using quantitative PCR (qPCR). The list of genes and primers is available in the electronic To validate the RNA-seq results, the expression level of 17 differentially transcribed genes under the A (P < 0.01) in the OD value, dry cell weight, chlorophyll fluorescence, and pigment content under the A (n = 3), H (n = 3), AH (n = 3), and C (n = 3) conditions were tested by ANOVA using the stats package in R software (P < 0.01) differences in the Bray–Curtis distances of differentially transcribed genes under the A (n = 3), H (n = 3), AH (n = 3), and C (n = 3) conditions were determined by permutational multivariate analysis of variance (MANOVA) in Primer-e (see text footnote 2). Significant (P < 0.01) differences in qPCR-measured genes under the A (n = 3), H (n = 3), AH (n = 3), and C (n = 3) conditions were calculated using the pair-wise fixed reallocation randomization test in REST.All results are presented in the text as mean ± standard error. Significant differences , acidification , and the combined treatment  compared with the control . The growth rate of S. hainanensis increased 1.05-fold but decreased 1.04-fold in the combined treatment compared with individual elevated temperature  and acidification , respectively , and the shape of the chloroplast changed . More starch granules under acidification and warming conditions were observed (B2–B4). Moreover, the sporangium of S. hainanensis was observed under acidification condition (C1–C4).The features of the cell morphology of cumented . The celA total of 12 transcriptomic sequencing libraries were generated for the four conditions  with three biological replicates. These libraries were sequenced with an Illumina platform with an output of 88.36 G clean reads (an average of 7.36 G reads per sample) . The cleS. hainanensis was significantly different under the C, H, A, and AH conditions (P < 0.001). The PCoA analysis showed the following order of the effect of different conditions on gene transcription: A-VS-C > AH-VS-H > H-VS-C. Among the detected genes, 688 genes were significantly differentially transcribed, and more than half of these genes had unknown functions  protein, and atpA and atpB involved in photosynthetic ATP synthesis were down-regulated more than 1–15-fold by the combined treatment (AH) (lci and cah1 genes encoding low CO2-inducible protein and soluble carbonic anhydrase related to carbon acquisition by CO2 concentrating mechanism (CCM) were significantly down-regulated more than fourfold by individual acidification and the combined treatment  , while gent (AH) , 6. In areatment .S. hainanensis , cell wall polysaccharide synthesis, and β oxidation of fatty acids to be significantly up-regulated more than threefold. However, the transcription of those genes was down-regulated approximately 1–17-fold by the combined treatment (AH).Acidification and elevated temperature had different effects on the genes related to the carbohydrate metabolism of nanensis . Almost ftsZ, which is involved in chloroplast division, was significantly up-regulated by A, H, and AH compared with the control (C).Elevated temperature (H) also had a minimal effect on the transcription of genes involved in the cell and life cycles. However, almost all of these genes were up-regulated by individual acidification (A-VS-C), while most of them were down-regulated by the combined treatment (AH-VS-H) . Genes e3– transporter and nitrate reductase involved in nitrogen metabolism were down-regulated over threefold under elevated temperature (H-VS-C). Under the combined treatment (AH), genes involved in nitrogen and phosphate metabolism were up-regulated over threefold relative to the acidification treatment (A). In addition, the gene encoding transferrin involved in Fe3+ transporter showed a higher transcription under the control (C) and elevated temperature (H) conditions than under the other conditions, and it was significantly down-regulated approximately fivefold by acidification (A) and the combined treatment (AH).The transcription of genes related to nutrient transport and metabolism is illustrated in Genes encoding glutathione S-transferase, catalase isozyme, UvrB/uvrC motif, heat shock 22-kDa protein, and chaperones were differentially transcribed under single acidification (A) or elevated temperature condition (H) . SpecifiS. hainanensis, a recently discovered coral-associated thermo-tolerant alga, to future ocean acidification and warming conditions by culturing it under two temperature settings (∼26.0 and ∼32.0°C) crossed with two pH levels (∼8.16 and ∼7.81) in a 28-day lab-scale experiment. We found that this thermo-tolerant alga exhibited a positive growth response to individual acidification, elevated temperature, and the combined treatment. We observed a no-superimposition effect of individual acidification and elevated temperature on the growth of S. hainanensis compared with the combined treatment of acidification and elevated temperature. Interestingly, our present data indicated that the chloroplast enlargement (possibly controlled by the ftsZ gene) along with the increase of chlorophyll fluorescence and pigment content might be a universal mechanism for the stimulative growth of S. hainanensis under ocean acidification and warming conditions, implying the predominance of S. hainanensis in its associated corals and/or coral reef areas in the future. This study provides novel insights into the growth, morphology, and molecular-level responses of thermo-tolerant algae to ocean warming and acidification conditions.In this study, we mainly explored the growth, morphology, and molecular level response of S. hainanensis to ocean acidification reported in this study is consistent with the previous studies in several species of cyanobacteria, diatoms, and dinoflagellates  caused by the increasing partial pressure of CO2 (pCO2) in seawater under acidification (2-inducible protein (lci) and soluble carbonic anhydrase (cah1) associated with CCMs were significantly down-regulated by acidification. Although the genes encoding RubisCO for CO2 fixation were not differentially transcribed in S. hainanensis under acidification, one gene encoding RubisCO LSMT was significantly up-regulated. RubisCO LSMT was reported to exhibit methyltransferase activity toward RubisCO , which might contribute to the enhanced growth of S. hainanensis as well. Similar results were also reported in higher plants under elevated CO2 or stresses  were down-regulated by acidification. According to one previous study of diatom  to acidification is that the increasing pCO2 (carbon resource) and the concomitantly decreasing pH (acidification) separately have different effects on the growth and photosynthesis. Interestingly, we observed that the chlorophyll fluorescence and pigment content of S. hainanensis significantly increased under acidification. Concomitantly, enlargement of chloroplast and significant up-regulation of one key gene ftsZ (encoding cell division protein homolog 1) for controlling chloroplast division and/or morphogenesis  was disrupted and inhibited at 32 and 35°C, respectively (Durusdinium spp.) associated with corals, both net growth and negative effect of photosynthesis at 32°C were reported   . In addiSynechococcus was promoted by the combined treatment, whereas elevated temperature and acidification had no effects on Prochlorococcus . Meanwhile, we observed no superimposition effect on the growth of S. hainanensis in response to acidification and elevated temperature alone, compared to the combined treatment.Laboratory studies have shown that the effects of the combined treatment of elevated temperature and acidification on the growth of marine algae are species specific . For exarococcus . Similarletonema , but it etoceros . The groreatment . Our preS. hainanensis, a superimposition effect of the individual elevated temperature (H) and acidification (A) on the gene transcription of S. hainanensis was observed under combined treatment (AH). Meanwhile, our present transcriptomic data revealed a balanced strategy used by S. hainanensis for maintaining moderate growth under the combined treatment of elevated temperature and acidification (AH). For example, the transcription of genes related to photosynthesis, CO2 biofixation, and carbohydrate metabolism was inhibited, but genes related to antioxidant and ubiquitination processes were promoted under combined treatment (AH) compared with individual elevated temperature (H) and acidification (A).For transcriptional profiles, the effects of individual acidification and warming conditions on the response of marine algae and terrestrial plant have been widely reported . To our S. hainanensis, a recently discovered coral-associated thermo-tolerant alga, to future ocean acidification and warming conditions. The promoted growth of S. hainanensis under these conditions suggests the strong acclimation of this alga to future ocean environmental changes. An antagonistic effect on the growth of S. hainanensis was observed between elevated temperature and acidification, which are involved in the balance of gene transcription related to basic metabolism and stress responses. The present data revealed that chloroplast morphogenesis (possibly controlled by ftsZ gene) along with increasing chlorophyll fluorescence and pigment content might serve as a universal mechanism for promoting the growth of this alga under both acidification and warming conditions. This alga is also ecologically important because it is highly abundant in corals in the SCS, and the optimum growth temperature of 32°C for S. hainanensis isolated from bleached corals in the SCS is higher than those of the symbiotic Cladocopium spp. and Durusdinium spp. . The present study provides novel and valuable data on the growth of S. hainanensis and improves the knowledge of the molecular functions underpinning the growth response of thermo-tolerant algae to future ocean acidification and warming. This study also lays a foundation for a further evaluation of the distribution and the ecological significance of S. hainanensis in coral–algae symbionts and/or coral reef areas.This study mainly describes the growth, morphology, and molecular response of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/SG and ZL conceived and designed the experiments. SG and YX performed the cultivation experiment and sampling. SG performed the molecular and bioinformatic analyses. SG, XJ, and ZL wrote the manuscript. All the authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition to being a marker of cardiovascular (CV) aging, aortic stiffening has been shown to be independently associated with increased CV risk (directly and/or indirectly due to stiffness-gradient attenuation). Arterial stiffness determines the rate at which the pulse pressure wave propagates . Thus, propagated PWV  was proposed as an arterial stiffness index. Presently, aortic PWV is considered a gold-standard for non-invasive stiffness evaluation. The limitations ascribed to PWV have hampered its use in clinical practice. To overcome the limitations, different approaches and parameters have been proposed . In turn, it has been proposed to determine PWV considering blood pressure (BP) levels (β-PWV), so as to evaluate intrinsic arterial stiffness. It is unknown whether the different approaches used to assess PWV or β-PWV are equivalent and there are few data regarding age- and sex-related reference intervals (RIs) for regional and local PWV, β-PWV and PWV ratio. Aims: (1) to evaluate agreement between data from different stiffness indexes, (2) to determine the need for sex-specific RIs, and (3) to define RIs for PWV, β-PWV and PWV ratio in a cohort of healthy children, adolescents and adults. Methods: 3619 subjects (3–90 y) were included, 1289 were healthy and non-exposed to CV risk factors. Carotid-femoral (cfPWV) and carotid-radial (crPWV) PWV were measured (SphygmoCor System (SCOR)) and PWV ratio (cfPWV/crPWV) was quantified. Local aortic PWV was obtained directly from carotid waves  and indirectly  from radial  and brachial ) recordings. β-PWV was assessed by means of cardio-ankle brachial (CAVI) and BP-corrected CAVI (CAVIo) indexes. Analyses were done before and after adjustment for BP. Data agreement was analyzed . Mean and standard deviation (age- and sex-related) equations were obtained for PWV parameters . Results: The methods and parameters used to assess aortic stiffness showed different association levels. Stiffness data were not equivalent but showed systematic and proportional errors. The need for sex-specific RIs depended on the parameter and/or age considered. RIs were defined for all the studied parameters. The study provides the largest data set related to agreement and RIs for stiffness parameters obtained in a single population. The arterial wall thickens and stiffens with aging. Such aging-related structural and functional changes are not distributed homogeneously but predominate in the aorta and central elastic arteries ,2. On thArterial stiffness determines the rate at which the pulse pressure wave propagates, that is, the pulse wave velocity (PWV). Thus, propagated PWV (obtained as the distance traveled by the pulse wave divided by the transit time) was proposed as an arterial stiffness index. Furthermore, up to now, PWV ) is considered a gold standard for non-invasive arterial stiffness evaluation ,4. AlthoThe detrimental impact of arterial stiffness on the CV system would be mediated by a direct effect of the increased stiffness and/or by the attenuation of the center-to-periphery stiffness gradient . In this regard, the age-related reduction in the stiffness gradient is associated with adverse clinical outcomes . Then, aCurrently, different devices, approaches and parameters are used to non-invasively assess arterial stiffness. It is unknown whether the different methods used to quantify PWV provide equivalent information and/or if it is possible to define equations to convert data obtained with a given approach into data corresponding to another one. Additionally, there is little information regarding age and/or sex-related reference intervals (RIs) for regional and local PWV, β-PWV (or CAVI) and PWV ratio obtained at the same time in a large healthy population including children, adolescents and adults.This work’s aims were: (1) to evaluate agreement between PWV data obtained with different approaches, (2) to determine the need for sex-specific RIs, and (3) to define RIs for PWV, β-PWV and PWV ratio in a cohort of healthy children, adolescents and adults from South America.The study was carried out in the context of the Centro Universitario de Investigación, Innovación y Diagnóstico Arterial (CUiiDARTE) project ,11,12,132; <18 y: z-BMI ≥ 2.0). The cut-off values used to define the healthy sub-population were chosen, whenever possible, to be similar to those used to indicate increased risk in clinical guidelines   estimated from the regression for mean cfPWV_Real_SCOR curve, and likewise from the regression for SD curve. Continuing the example, FPs with powers [2 [Briefly, fitting FPs age-specific MV and SD regression curves for the different variables  were defined using an iterative procedure . Then, age-specific equations were obtained for the different parameters. For instance, the cfPWV_Real_SCOR MV equation would be cfPWV_Real_SCOR MV = a + b*Ageowers [2 . Residuap < 0.05 was considered statistically significant.The minimum sample size required was 377 . Like inSimple bivariate and concordance correlation data are shown in Likewise, regardless of whether it was considered aoPWV, CAVI or CAVIo, (i) the levels of association were always significant , and (ii) in all cases, CCC showed low (or even non-existent) levels of agreement between data . CCC valCAVI data showed significant systematic differences. On the other hand, with the exception of CAVI_aoPWV_Radial_SCOR and CAVI_aoPWV_Carotid_SCOR, they also showed proportional differences. In turn, CAVIo data showed both, systematic and proportional errors .Jointly analyzing the findings, it could be said that the methods used to assess PWV, CAVI or CAVIo were not equivalent.There was no association between arterial stiffness  and atherosclerotic plaque presence  .However, when age and sex interaction was considered, there were: (i) parameters that required sex-specific RIs from certain ages , (ii) parameters for which sex-specific RIs were necessary in all ages  and (iii) parameters that did not require sex-specific RIs  , Part 1.p > 0.05), while for older ages, cfPWV was gradually higher in males. About this: (i) at the age of ~23 y (23.15 y), cfPWV values in males were 0.1373 m/s higher than in females, (ii) at ~40 y (39.43 y), the difference was 0.2789 m/s, (iii) at the age of ~60 y (59.78 y), male values were 0.456 m/s higher than those in females whereas (iv) at ~80 y (80.13 y), the difference was 0.633 m/s. Similarly, from 29 years of age (28.9 y), the crPWV_SCOR levels were gradually higher in males than in females   , Part 2.ctively) , Part 3.The work’s main findings can be summarized as follows:First, for methods and parameters used to assess aortic stiffness , their CAVI and CAVIo, showed different association levels (some non-significant). In no case was the association very strong (r ≥ 0.80) .For PWV, CAVI and CAVIo, the highest levels of association were obtained for cfPWV_Real_SCOR and aoPWV_Brachial_MOG , whereas the lowest levels were obtained for aoPWV_Carotid_SCOR and aoPWV_Brachial_MOG . Regardless of whether or not there was agreement  on absolute levels, there were methods with no association, so it would not be possible to arrive at similar conclusions from similar trends in their variations .p < 0.0001; mean error/SD: 0.40 ± 2.23 m/s) for the association between aoPWV_Brachial_MOG and cfPWV_Real_SCOR [n = 100, r = 0.46; p < 0.0001; mean error/SD: 0.28 ± 2.94 m/s); (ii) PulsePen ETT System ; (iii) PulsePen ET System  [n = 102) the authors found differences in the associations between methods used to assess aortic stiffness . On the contrary, in the work of Salvi et al., methods that evaluated propagated PWV  showed Pearson Coefficients > 0.8 [Recently, Salvi et al. obtained an r = 0.64  . In agrets > 0.8 .On the other hand, when BP dependence was considered (CAVI and CAVIo), there was a reduction in the levels of association . In all Second, approaches and parameters used to assess aortic stiffness  were not equivalent but showed systematic and proportional errors .Mean differences (absolute mean errors) were between 0.13 and 1.5 m/s . In turnSalvi et al. found a mean error equal to 0.40 ± 2.23 m/s (mean ± SD) when analyzing aoPWV_Brachial_MOG and cfPWV_Real_SCOR data. In addition, the authors found a negative proportional bias . The finPWV data showed low (the lowest) differences when aoPWV_Radial_SCOR and aoPWV_Carotid_SCOR were compared. Statistically significant mean and proportional errors were observed when they were compared to cfPWV_Real_SCOR .Third, the need for sex-specific arterial stiffness RIs relied on the approach used to assess stiffness and/or on the age considered. In this respect, according to the measurement system, sex-specific RIs were not necessary, necessary regardless of age or only needed after a certain age .The relationship between sex and arterial stiffening is controversial and at least two issues are still discussed: (i) whether there are sex-related differences in mean arterial stiffness as a function of age, and (ii) whether there are sex-related differences in the rate of age-associated stiffness increase. In this work, we found that both issues may be closely related. The confusion or controversy regarding them could depend on the site of recording and/or on the approach (and device) used to assess arterial stiffness.p = 0.055). In addition, none of the variables showed a significant interaction between age and sex. Unfortunately, the internal algorithms that allow quantifying aoPWV by means of the different approaches are unknown, which does not allow adequate analysis of factors that could explain or be associated with the findings .The aoPWV_Brachial_MOG values were systematically higher in males than in females , Part C.For propagated PWV (cPWV and crPWV), there were no significant sex-related differences in childhood, but thereafter these became gradually larger . This is in agreement with previous data published by our group regarding local carotid stiffness (carotid elastic modulus) in children and adolescents . The finAs was discussed the need for RIs separated by sex varied depending on the approach used to assess arterial stiffness. In all cases, we opted for defining RIs for the population as well as for males and females separately.Fourth, this work’s main result is the definition of population-based reference values for PWV, CAVI and CAVIo indexes, obtained in the same subjects, with different non-invasive approaches. The definition of RIs is an important step when considering the introduction of PWV, CAVI and CAVIo indexes as a tool for the detection of subclinical target organ damage in the general population . To our In biomechanical terms, while the impact of aortic stiffness on the myocardium is explained by increased cardiac afterload and reduced perfusion, peripheral target organ damage is best explained by the arterial stiffness gradient hypothesis . As a reIn this work, RIs for stiffness parameters were obtained working with a large population of healthy subjects, selected taking into account inclusion and exclusion criteria previously used by our and other groups. Tables and Figures showing age-related (year by year) profiles for the different parameters  are included in Both cfPWV and crPWVcurves showed the expected behavior. About this, cfPWV evidenced the expected curvilinear relationship between age and aortic stiffness , in whicFinally, the different approaches used to assess aortic stiffness did not show similar age-related profiles . While cOur results should be interpreted within the context of this work’s strengths and limitations. As the study is a cross-sectional one, it provides no data on longitudinal age-related changes in cfPWV, crPWV or aoPWV (and hence in CAVI and CAVIo). Since no outcome data were considered, cut-off points  were not defined based on CV risk but on value distribution in the RIs group. Whether or not the reference values should be used as cutoff values for diagnosis and treatment is not known. In this work, the concept of arterial stiffness is mainly presented as a “static process”, rather than the composite of (i) fixed  and (ii) variable  activation-related arterial stiffness (viscoelasticity) modulation). Two issues should be considered in this regard. First, the increases in arterial stiffness with increasing age do not necessarily reflect “concrete-like” changes, but rather a variable and less known increase in stiffness associated with increases in VSM tone  ,55. ThisMethods and parameters used to assess aortic stiffness  and their CAVI and CAVIo, showed different association levels, which in no case were very strong (r ≥ 0.80). Data from different approaches were not equivalent but showed systematic and proportional errors.The need to define sex-specific RIs relied on the approach used to assess the stiffness and/or on the age considered. In this work, population-based reference values for PWV, stiffness gradient, CAVI and CAVIo indexes were defined; a key step when considering the introduction of the indexes as a tool for the detection of subclinical target organ damage.