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Secure land increased farmers’ decision to participate in social groups by 30 percent

Six indices of social capital among the cocoa-based farming households are identified.The density of membership to associations is 0.636, which means that cocoa-based farming households belong to 6 out of 10 associations.Households belong to various associations in order to promote and protect their business interests.The degree of heterogeneity is considerably high in the area of study.This suggests heterogeneous characteristics such as different ethnicities, occupations, religion and neighbourhoods among the cocoa-based farming households.The decision-making value is fairly high in the associations, which implies that most of the cocoa farming household members are actively involved in decision making within the social group they belong.The meeting attendance value is 0.318, which means that the cocoa-based farming households do not attend most of the scheduled statutory meetings.This could be attributed to the fact that the households trekked an average of 0.70 km to designated meeting points.Hence, they only attend meetings whenever important decisions are to be made.The cash contribution value is 0.652, which means that the cash commitment to associations by cocoa-based farming households is relatively high.This shows that cocoa-based farming households are committed to contributing cash to their respective associations.Also, labour contribution has a value of 0.541, which means that the mean labour contribution is 54 days annually.This result compares favourably with Ajani and Tijani.The aggregate social capital, which is the multiplicative value of density of membership, heterogeneity index and decision-making index is 0.568.The result shows that a fairly high level of social capital exists among cocoa farming households in the study area.The factors that influenced the decision to participate in social groups are shown in Table 3.These characteristics include asset, age, years of education, gender, farm size,ebb and flow trays land tenure, loan interest rate, and extension visit.

The negative signs of marginal effects reduce probability of household participation in social groups while positive signs increase the probability of participation.Assets of households significantly affects the probability of participating in social groups.An additional unit of households’ asset decreased the decision to participate in social groups by 10.9 percent.This implies that increase in asset ownership decreases probability to participate in social groups.The age of household head significantly affected the probability of participating in social groups.A year increase in age of household head increased the decision to participate in social groups by 54.8 percent.This could be attributed to the fact that social groups might prefer older farmers to their younger counterparts, because older farmers are more responsible and secured to participate.In addition, due to some cultural beliefs in Africa, younger people might be prevented from participating in social groups.Years of education of household head significantly affected the probability of participating in social groups.An increase in years of education of household head increased the decision to participate in social groups by 7.7 percent.This implies that education gives farmers the ability to access and comprehend information regarding the terms and conditions required to participate in social groups.The gender of household head significantly affected the probability of participating in social groups.A male household head increased the decision to participate in social groups by 17 percent.This could be attributed to the fact that male headed families are willing to take more risk than female headed families.In addition, due to some social-cultural values and norms of Africans, male farmers have more freedom to participate in different social groups compared to the female farmers.The farm size of household significantly affected the probability of participating in social groups.An increase in hectares of farm size increased the decision to participate in social groups by 28 percent.The result implies that farmers with large farms possess the ability and collateral to participate in social groups.Land tenure status of household head significantly affected the probability of participating in social groups.This could serve as a push factor to participate in social groups in order to put resources to optimum use.

Loan interest rate significantly affected the probability of participating in social groups.A one percent increase in interest rate decreased the decision to participate in social groups by 46.6 percent.This implies that high interest rate constitutes a hindrance to loan access.Extension contacts significantly affected the probability of participating in social groups.A contact of household with extension agents increased the decision to borrow by 5.4 percent.This is because extension services could provide farmers with essential information regarding participation in social groups.The results of the factors influencing cocoa-based farming households’ level of participation in social capital groups are presented in Table 3.These include age of household head, years of education, membership in agricultural organization, off farm income, land tenure, interest rate, distance to credit sources, extension visits, decision making, cash contribution, and labour contribution.The age of household head significantly affected level of participation in social groups.A year increase in age of household head increased the level of participation in social groups by 3.164 units.This could be ascribed to the fact that as farmers get older, they become more productive and increase their level of participation in social groups.Years of education of household head significantly affected level of participation in social groups.An increase in years of education of household head increased the level of participation in social groups by 0.662 units.This is because education equips farmers to make informed decisions about their level of participation in social groups.Membership in agricultural organisations significantly affected level of participation in social groups.Being a member of agricultural organisation increased the level of participation in social groups by 2.085 units.This is attributed to the fact that membership in an agricultural organisation increases access to credit.Non-farm income significantly affected level of participation in social groups.An increase in non-farm income of households decreased the level of participation in social groups by 0.041 units.This is because farmers that earn substantial amounts of non-farm income would less likely need external funds.Land tenure significantly affected level of participation in social groups.Households with secure land increased the level of participation in social groups by 1.858 units.This could serve as a push factor to farmers to increase their level of participation in social groups.

Loan interest rate significantly affected level of participation in social groups.A one percent increase in loan interest rate decreased the level of participation in social groups by 1.225 units.High interest rates constitute a hindrance to the level of participation in social groups.Distance to credit source significantly affected level of participation in social groups.A kilometre increase in the distance to credit source decreased the level of participation in social groups by 0.010 units.Farmers who live near the social group’s designated building have a location advantage, which increases their level of participation in social groups.Extension visits significantly affected level of participation in social groups.Households’ contact with extension agents increased the level of participation in social groups by 0.768 units.Extension services provide essential information to farmers regarding participation in social groups.Decision-making significantly affected level of participation in social groups.An increased unit of the decision-making index increased the level of participation in social groups by 1.424 units.This is ascribed to the fact that decision making keeps individuals abreast of the association’s benefits.Cash contribution significantly affected level of participation in social groups.A naira increase in cash contribution increased the level of participation in social groups by 9.923 units.This implies cocoa-based farming households who made adequate financial contributions to social groups have access to substantial amounts of credit compared to households who did not.Labour contribution significantly affected level of participation in social groups.An increase in labour contribution increased the level of participation in social groups by 3.353 units.This implies that cocoa-based farming households who make adequate labour contributions in social groups have access to substantial amounts of credit compared to households who do not.To test for validity of the instrumental variables used in the 3SLS estimation procedure, a correlation analysis between aggregate social capital, farm productivity and food security with the proposed instruments was carried out.The proposed instruments were length of residency, charity donation, membership in religious group,4×8 flood tray and membership in ethnic groups.The results of the correlation analysis are presented in Table 4.The membership in ethnic groups has significant correlations with aggregate social capital, but an insignificant correlation with farm productivity and food security.It also has the highest correlation coefficient with the social capital.This conforms to the findings of Adepoju and Oni.

The basic model is shown in the first column of Table 6.The rationale behind this model is to examine the farm productivity of the households while they are not involved in social capital activities.The Chi2 showed that the econometric modelling is appropriate and correctly specified.Age of household head significantly influenced farm productivity of the cocoabased farming households.This implies that a unit increase in age of household head decreased households’ farm productivity by 0.836 kg/₦.This is attributed to the fact that ability to do farm work and farm output reduces with ageing.Household size significantly influenced farm productivity of the cocoa-based farming households.This implies that a unit increase in household size increased households’ farm productivity by 0.865 kg/₦.This is attributed to the fact that family labour available for farming could increase farm output.The results agree with Atagher.Primary and secondary education significantly influenced households’ farm productivity.The implication of this is that a unit increase in primary and secondary education increased households’ farm productivity by 0.014 and 0.113 kg/₦, respectively.This could be traced to the fact that education empowers farmers to access required skills and to utilise existing resources on the farm to boost their productivity.Farm size significantly influenced farm productivity of the cocoa-based farming households.This implies that a unit increase in the farm size increased households’ farm productivity by 0.214 kg/₦.This is ascribed to the fact that resources on large farms would increase farm productivity.Interest rate significantly influenced farm productivity of the cocoa-based farming households.This implies that a percentage increase in interest rate decreased households’ farm productivity by 0.346 kg/₦.This is because high interest rate discourages farmers from applying for loans and the amount of the loans farmers receive.Correspondingly, this reduces the quantity and quality of farm inputs that the farmer buys and negatively affects productivity farmer.Loan time lag significantly influenced farm productivity of the cocoa-based farming households.This implies that a unit increase in loan time lag decreased households’ farm productivity by 0.660 kg/₦.This is because long loan time lags would delay the procurement of a loan, which implies that farm inputs will not be available to the cocoa farmers at the right time, quantity and quality.This affects productivity of the farmers due to the seasonal nature of agriculture.This model suggests that households’ social-economic characteristics, farm specific and credit variables play a significant role in improving farm productivity.The second column of Table 6 shows the inclusion of six additive forms of social capital variables identified in this study.These include density of membership, decision making, cash contribution, labour contribution, meeting attendance and heterogeneity.The rationale behind the model is to examine the farm productivity of the households while they are involved in social capital activities.This new model has a better farm productivity level as reflected in a Chi2 of 45.34.This suggests that households’ farm productivity improve as members become involved in the affairs of their social groups.This model shows that the effect of social capital on farm productivity can be traced to meeting attendance, decision making, membership density, and cash contribution.This finding is line with the findings of Balogun et al..Meeting attendance significantly influenced farm productivity of the cocoa-based farming households in the study area.This implies that a unit increase in attendance of meetings increased households’ farm productivity by 6.959 kg/₦.This is because farmers who recurrently attended group meetings have access to resources and information to improve their productivity.Decision making significantly influenced farm productivity of the cocoa-based farming households.This implies that a unit increase in active participation in decision of the group decreased households’ farm productivity by 4.824kg/₦.This means that farmers’ involvement in association matters is of no benefit to their farm productivity.Density of membership significantly influenced farm productivity of the cocoa-based farming households.This implies that a unit increase in the number of groups to which a farmer belongs increases productivity by 0.450 kg/₦.This is attributed to the fact that farmers’ commitment in many social groups enhances their access to loans, which can be used to increase their productivity.

Pairwise analyses and significant differences of farming systems were tested with function lsmeans

The significance of the linear model was tested with type III analysis of variance with Kenward-Roger’s method with function anova.OTU data from the amplicon sequencing was normalized using the geometric mean of pairwise ratios method. We performed permutational multivariate analysis of variance using distance matrices with function adonis from vegan 2.5–5 to test the effect of farming system and crop type , and the effect of manure addition, including plants grown in 2018, on microbial community composition. We also conducted 2-D nonmetric multidimensional scaling with stable solution from random starts, axis scaling and species scores with function meta MDS from vegan using the Bray-Curtis dissimilarity index and plotted the NMDS with fitted environmental variables from function envfit in vegan. Fungal and bacterial OTUs indicative for specific rotations were obtained by differential abundance analysis which identified significant groups. Due to the PERMANOVA results we also did additional differential abundance analyses to obtain OTUs indicative for rotation plots that have either received manure or not. The results are presented as paired comparisons between the farming systems for forage and cereal crop rotations, and plots with manure or not, and for the spring and autumn data separately.Bacterial richness, assessed by OTU reads, was significantly higher in the organic rotations compared to the conventional rotations in the autumn data . Furthermore, fungal richness was higher in the organic systems in the autumn data but only significantly for the OMilk rotation. In the spring data bacterial and fungal richness did not differ between farming systems. Neither did bacterial and archaeal 16S rRNA gene copy numbers differ between farming systems in either the spring or autumn data,plastic pots 30 liters whereas fungal ITS copy numbers were higher in the OCer rotation compared to the CCer rotation in the autumn data . The NMDS ordination showed minor differences in bacterial community composition between the farming systems for both crop rotations.

Differences in bacterial community composition, however, were more distinct in the autumn data and higher basal respiration, microbial biomass C and N, as well as extractable C and N variables fitted best with the CMilk rotation. Whereas changes in the fungal community composition between farming systems were more pronounced for both crop rotations and between the seasons . The fungal community composition in the organic rotations were more similar than between the conventional rotations. In the springdata, higher basal respiration rates and microbial biomass C and N variables fitted best with the OMilk rotation. In autumn data, microbial C and N, and extractable C fitted best with the OMilk rotation, and soil pH and basal respiration rates with the OCer rotation, and extractable N with the CMilk rotation. The first PERMANOVA indicated that both farming system and crop type significantly affected the variation in microbial community composition both in the spring and autumn data. However, crop type did not explain the variation in the bacterial community composition in the spring data . Farming system explained 10 and 14% of the variation in bacterial community composition in the spring and autumn data, respectively . Crop type explained 10% of the variation in the autumn bacterial data. In turn, the farming system explained 11 and 14% of the variation in fungal community composition in the spring and autumn data, respectively. Crop type explained even more, 21 and 36% of the variation, in the spring and autumn data, respectively. We also tested the effect of manure in an additional PERMANOVA test, which showed that in addition to farming system manure did not affect the variation in bacterial community composition . However, manure explained additional 14% and 11% of the variation in fungal community composition in the spring and autumn data, respectively. In addition, the impact of manure could not be separated from the impact of crop plant which was grown in all manure fertilized rotation plots in 2018. In the ITS2 based fungal data we could identify on average from 9 to 15 Glomeromycotan AMF OTUs in the spring, and from 12 to 25 in the autumn. In the spring data, the OMilk rotation had the lowest OTU number and the OCer rotation the highest . In the autumn data, the CMilk rotation had the lowest and the OCer rotation the highest AMF OTU number.

The majority of AMF OTUs were shared between the farming systems. PERMANOVA showed that season explained the most variation in the AMF community composition, while crop type and farming system also had an effect. There were large differences in the relative abundances of AMF families between the spring and autumn data of the organic systems . Claroideoglomeraceae was the most abundant family, especially in the autumn data, whereas Acaulosporaceae and Pacisporaceae were not present at all in the CMilk rotation. Archaeosporaceae were abundant in the organic systems and almost absent from the conventional systems. It also seems that Pacisporaceae, while not abundant, is characteristic to the cereal rotations, regardless of the season. The 18S rDNA-based AMF data from composite samples obtained in the autumn identified 38 OTUs with unique genbank accession numbers . Moreover, two Scutellospora species were obtained from the Gigasporaceae family that were not seen in the ITS2 data . Paraglomeraceae, Claroideoglomeracea and Diversisporaceae were the most abundant AMF families based on the 18S data. Organic systems had higher AMF richness; AMF OTU numbers varied from 22 in soil with conventional systems to 35 in soil from OMilk. One out of two Acaulospora sp. and four out of seven Glomus sp. OTUs were only present in the organic systems, with two of them only in the OMilk rotation. Altogether, 19 OTUs were shared between all four rotations. Glomus species were abundant in the OMilk rotation, and similarly to the ITS2 based data, Pacisporaceae was not present in the CMilk rotation, but we could find one Acaulosporaceae OTU present in all rotations, contrary to the ITS2 data. Cereal systems were characteristically associated with Archaeospora trappei, Archaeospora sp, Glomus mosseae, and Pacispora sp. . The more frequent tillage in the conventional system for cereal crops explains partly the lower microbial activity rates and biomasses observed in spring and autumn, a phenomenon which has been observed also by others. Furthermore, the conventional cereal system had bare soil over the winter and is the only treatment without manure addition in the rotation. Thus, the absence of continuous plant cover may have further reduced the microbial biomass C in the spring compared to the respective organic system as earlier reported.

Moreover, the second cut of grass and clover ley was left on the soil surface as a green manure in the organic system for cereal rotation and this may have led to the higher microbial activity and biomass in the following autumn. The lack of chemical agents may also have been reflected in the results since earlier studies have shown negative effects of agrochemicals on soil microbial communities.Higher springtime microbial activity and biomass in the conventional forage crop rotation compared to that of organic cannot be easily explained by the differences in the rotation types as the rotations include various management practices. However, there was 30–40% higher soil P concentration in the conventional system for both cereal and forage crop rotations compared to the organic ones. Indeed, lower water soluble and inorganic P amounts have been reported from organic systems compared to systems receiving synthetic fertilization in a long-term field experiment. Possibly the springtime bacterial community in the conventional forage crop rotation gained competitive advantage from the higher availability of P, since P has been reported to limit bacterial growth in agricultural soils. Later in autumn, the summertime amendments of cow manure would equalize the P availability, and differences between organic and conventional systems for forage crop rotation are no longer detected. The slightly higher autumn pH in the organic system for cereal crop rotation may result from microbial decomposition of fresh plant residues with high N content and mineralization of ammonium which temporarily is known to increase pH. Furthermore, it has been reported that long-term application of manure maintains the soil pH,round plastic pots but inorganic fertilizer decreased it. Consequently, since bacterial growth is known to increase multi-fold with increasing pH, this may explain the increased microbial biomass in the organic system for cereal crop rotation. Alternatively, the increased fungal abundance in the organic system for cereal crop rotation in the autumn indicated that fungi could also be responsible for the higher respiration activity and sequestration of C into their biomass.

Moreover, the organic system for cereal crop rotation had timothy and clover ley as the main crop plant in the sampling year instead of barley as in the respective conventional system. Indeed, higher microbial biomass in production systems including ley grasses have been detected compared to single crop systems only. Our results showed that the farming system induced a clear shift in microbial community composition and that the overall impact of the farming system was about the same magnitude for both bacterial and fungal community composition. Our results are comparable to previous findings that about 10% of variation in microbial communities was explained by the farming practices of conventional and organic systems. Yet, crop type affected fungal community composition in particular, especially in the autumn. A simple explanation would be that changing cultivated plants from barley in the year 2017 to ley in the sampling year 2018 induced a shift in fungal community composition. Furthermore, it is likely that the summertime amendments of synthetic fertilization in the conventional systems have also contributed to the lowered bacterial and fungal richness in the autumn, since the quality of fertilizer is known to impact largely on microbial communities. Thus, the differences in crop rotation, tillage and fertilization practices may all have contributed to the differences in the microbial community but with the current experimental layout we are not able to determine which practices have the strongest impact. Nevertheless, comparisons within the cereal rotations were valuable for indicating the long-term impacts of manure addition and overwintering as bare soil, while comparing the forage crop rotations it was possible to assess the other effects of organic practises beyond manure addition and undersown ley. Moreover, the differences in AMF richness between the farming systems were only moderate and non-existing under the cereal crop rotation. A higher diversity of Acaulospora species, typical to organic systems was also supported by our study. We did not observe Clareideoglomus species to be characteristic to organic systems, instead finding the opposite, which contradicts previous results. Thus, our study supports previous observations which concluded that mycorrhizal diversity is not influenced by the farming system but rather cultivation practices and conditions, and that finding a universal AMF indicator for farming systems is not feasible. Results suggest that the cow manure applied in the forage crop rotation under both the conventional and the organic systems over the years has shaped the bacterial and fungal communities more than any other farming system specific practice, since only a few representatives were typical of either farming system.

This also highlights the commonness of pathogenic fungi in the fields cultivated for fodder and fertilized with manure irrespective of the farming system. On the contrary, AMF richness in the forage crop rotation varied clearly due to the farming system; for instance, Glomus species were indicative in the organic system, while Pacispora sp. was totally missing from the conventional system. Since plants acquire P directly and through their symbiotic AMF, the 40% higher levels of P in the conventional forage crop rotation compared to the respective organic rotation may partly explain the lower AMF richness. However, bacterial representatives were less diverse in the conventional farming system of the cereal rotation compared to the respective organic system. Representative taxa typical of the conventional cereal crop rotation in autumn were affiliated to decomposer and plant-growth promoting and cellulose decomposing bacterial taxa. In contrast, both the spring and autumn data obtained from the organic system for cereal crop rotation revealed a variety of specific taxa with diverse functional roles benefiting soil health. Most of these taxa were also linked to manure fertilization, and included for instance, plant growth-promoting rhizobacteria that include species capable of N-fixation, P solubilization, phytohormone production, and repression of soil-borne plant pathogens, and genera with anti-fungal and antibiotic capability. Furthermore, the spring and autumn data of the organic system for cereal crop rotation contained also season specific taxa, which shared bacterial representatives with similar functional roles.

Scenarios are widely used in research to simulate decision-making environments

Their study found a significant number of behavioural bio-security policies in seven European countries, many of which appeared to match theoretical behavioural change frameworks. However, the most frequent strategies relied on the most basic interventions , and there was little evidence of the systematic use of methods from the behavioural sciences to develop these policies. If this suggests there remains some way to travel before the social sciences are integrated within bio-security policy making, other research continues to highlight the potential value of these approaches. For example, research on the role of information cues reveals that bio-security behaviours can be improved when messages are shown graphically, rather than linguistically or numerically.Drawing on Kahneman and Tversky’s ‘prospect theory’ in which avoiding losses are preferable to accruing gains, Hansson and Lagerkvist show how farmers’ disease management decisions reflect farmers individual assessments of risk. However, when farmers are faced with managing an ongoing disease outbreak, decisions reflect a preference of avoiding losses; gains are only preferred when they seek to prevent future disease outbreaks. Other research has sought to examine how social information and the behaviour of other farmers can influence farmers’ bio-security decisions. Using an experimental simulation, Merrill et al. for instance show that willingness to invest in bio-security decreases when information on environmental disease prevalence is uncertain,stacking pots reflecting an optimism bias that farmers’ herds will not become infected.

Alternatively, when more information is provided about bio-security practices on neighbouring farms, bio-security investment decreases. This work is interesting in that it suggests that social norms of what constitutes ‘bio-security citizenship’ , appropriate conduct or what has been referred to as ‘good farming’ may not be influential in bio-security decision making. Burton suggests that ‘good farming’ refers not only to economic forms of capital, but symbolic cultural capital: the visible demonstration of practical knowledge such as good stockman ship, symbols of appropriate farm maintenance such as clean farmyards and tidy hedgerows, and attributes such as hard work. These symbols are encoded and disseminated within discursive scripts, reinforcing their cultural legitimacy . In this way, good farming acts as a heuristic to provide a strategy to guide, interpret and make decisions in conditions of uncertainty. Other strategies of decision-making are available to farmers, however, and the selection of good farming to guide decisions represents what Sunstein and Ullmann-Margalit refer to as a second-order decision. For Burton and Paragahawewa , the value of the good farmer approach lies in recognising and utilising cultural capital to create more culturally salient agricultural policy. Rather than simply rely on financial payments, they instead recommend the development and incorporation of measures of cultural capital into agricultural policy, and/or restructuring agricultural policy to directly encourage the generation of cultural capital. This may include directly measuring farmers’ ‘skills’ in order to allow them to publicly demonstrate what is valued by the farming community. Whilst Burton and Paragahawewa note that some cultural values might be hard to measure , objectifying cattle purchasing skills may provide a relatively easy way of incorporating the cultural capital of good farming into animal disease management policy. For example, recent research has established a link between farmers’ understandings of good farming and bio-security practices .

In particular, cattle purchasing is likely to be connected to and reflect good farming in a number of ways. Firstly, purchasing cattle risks the introduction and transmission of new diseases to animals within the herd and, for some diseases that can be subsequently transmitted within the local environment, to animals on neighbouring farms. For those farms that need to replace stock, however, different forms of institutional capital – such as certification and ranking schemes – can help provide assurance to the purchaser that they are buying from a good farmer and are running the risk of being labelled a bad farmer by introducing disease into their herd or area. For example, Enticott et al. describe how the number of years a farm has been free from disease effectively establishes a good farming rating that may incentivise improved bio-security when it is required to be displayed at the point of sale. The extent to which these forms of information are a reliable guide to whether the farmer is a ‘good farmer’ may, however, be compromised by farmers’ own spatial understanding of disease transmission and by blaming disease outbreaks on perceived government failings, rather than ‘bad farming’ . Secondly, the avoidance of disease through careful cattle purchasing should allow farmers to display other forms of symbolic cultural capital. An outbreak of bTB, for instance, would lead to a farm’s business being subject to a range of trading restrictions, denying the opportunity to farm with autonomy, which is highly valued by farmers in the farming script of ‘being my own boss’ which symbolises farmers’ success at running their own farm well rather than being told how to farm by government. Indeed, an outbreak of bTB would mean that many farming decisions would be subject to bureaucratic procedures and determined by government officials: farmers would be unable to attend market to sell their cattle. As a result, farms may become over-stocked, and cattle suffer poor welfare. Failing to avoid disease through responsible cattle purchasing therefore compromises farmers’ abilities to display the embodied and practical skills of the good farmer symbolised by good-looking cattle either on show at markets or at pasture. Similarly, participation at livestock markets reflects the significance of the autonomous farmer consistent with good farming. Providing measures of good farming in relation to animal disease may therefore help cattle purchasers identify good farmers, and help them avoid becoming a bad farmer as a consequence of poor cattle purchases. The extent to which such measures can successfully symbolise the good farmer and influence cattle purchasing is explored in the remainder of this paper.

Studies of behavioural influences in disease management reveal two distinct methodological approaches. On the one hand, agricultural economists, drawing on methodologies from behavioural psychology, have conducted experiments to simulate the effects of information provision and financial incentives upon bio-security behaviours. On the other hand, sociological research has sought to conceptualise and describe in-depth farmers’ responses to disease events and policy interventions. Each has their problems. Despite the promise of the experimental approach, research participants are often students responding to hypothetical situations wholly divorced from the practical skills and situational awareness that farmers use to respond to real-life context-dependent situations . By contrast, qualitative analyses of good farming and bio-security, whilst focused on real-world policies and disease incursions, are retrospective and subject to recall and social desirability biases. Rather than adopting one or the other, we seek to develop an innovative mixed-methods approach that allows us to quantitatively and qualitatively assess the value of symbolising good farming to influence farmers’ cattle purchasing decisions to prevent bTB. The following sections firstly provide information on the importance of bTB and the relevance of cattle purchasing before providing a detailed account of our methodological approach. In the United Kingdom, bTB is the UK’s most challenging endemic disease, resulting in the premature death of approximately 35,000 cattle and costing the taxpayer in excess of £100 m every year . Managed by the government, the disease has a complex epidemiology involving transmission by legally protected wildlife, the culling of which for disease control purposes has raised political, social and economic challenges . 2007. Cattle movements have become recognised as an important part of the epidemiology of bTB. Studies have shown how the movement of cattle is one of the most important risk factors in infected herds, whilst movements also translocate disease from areas of high to low prevalence . Whilst infected farms are restricted by law from buying or moving cattle on or off farms, all other farms are free to act as they please. Nevertheless, the limitations of diagnostic tests and their frequency mean that these movements still pose a risk to other farmers.

Indeed, other countries with successful bTB eradication schemes, have governed the movement of all cattle between areas of different epidemiological risk using statutory and/or voluntary policies of ‘risk based trading’ and in doing so identify and provide cultural capital to good farmers. Whilst no such scheme currently exists in the UK for bTB, policy makers view cattle purchasing as an important practice on which to apply the behavioural sciences in order to govern cattle movements through behavioural nudges rather than regulation. To understand the impact of different ways of objectifying good farming,sawtooth greenhouse we devised a novel mixed-methods approach. Avoiding experimental approaches involving non-farmers, our approach involved simulating cattle purchasing with farmers who buy and sell cattle. Many studies within the behavioural sciences involve randomised controlled trials, but this approach was not available and not suitable: we were not able to alter the information provided at the point of sale.The diversity of cattle, buyers and sellers also makes controlling for the effect of a single intervention a significant methodological challenge. Instead, our approach sought to simulate cattle purchasing, whilst also allowing farmers to reflectively deliberate on the reasons for their purchases and the value of different behavioural insights. To do this, we developed cattle purchasing game in Mural – a web-based interactive whiteboard – in which participants moved around a Monopoly-style board . Players progressed around the board by rolling one die. All games were played online via Zoom due to Covid 19 lockdown restrictions. Game play was organised using a “branch and bottleneck” structure. Branches reflect different contextual influences that participants land on at random throughout the game. This allowed us to introduce an element of competitiveness between players: points were awarded for landing on squares that reflected ‘positive’ contexts. No points were awarded for landing on negative blue squares. Red squares were a bTB test: if players landed on these, they were required to roll an even number to pass the bTB test,otherwise they would miss a go. Bottlenecks were cattle purchasing events that all players had to complete at the same time and were located in each corner square of the game board. Once one player reached a corner square, all other players also moved there.

Players were then read a cattle purchasing scenario and asked to make a choice between four adverts.They provide opportunities to elicit attitudes and beliefs about complex and potentially sensitive situations and to examine how people may respond to future events . Scenarios work best when they are based on plausible and familiar situations . Scenarios were therefore developed based on a prior research project on cattle purchasing involving farmers and vets. To ensure the scenarios reflected real-world cattle purchasing opportunities, specific versions were developed for three sectors: dairy, store cattle, and calf-rearing. For each scenario, adverts contained information to symbolise good farming in order to influence purchase choices. Firstly, farmers could use pictures of the animals to derive good farming information. For scenarios 1 and 2, pictures were of cattle in a livestock market, but for scenarios 3 and 4 animals were pictured on farm. Secondly, adverts featured two different conceptual measures of good farming. All adverts contained a logo stating how many years free the herd had been from bTB and the geographical average years free for the area in which the farm was located. Values were set randomly. In this method, good farming is symbolised by longer periods of disease freedom; ‘bad farmers’ would avoid purchasing from farms who had recently had an outbreak for fear of introducing disease. Scenarios 2 and 4 also contained a ‘Good Farmer Rating’ to graphically indicate the percentage of satisfied previous customers for each vendor. The aim of this logo was to convey levels of trust and reputation of the seller, which had seen to be important considerations when purchasing cattle from our previous research, and found in other research by Hidano et al. . Presented as a star rating, the logo was similar to review ratings found on internet shopping sites. Two ratings were set at 95% and two at 70% satisfaction. In addition to these measures of good farming, scenarios 1 and 3 explored the effect of different compensation regimes upon purchase decisions. Two different schemes were presented: two sales adverts stated that the purchaser would receive 50% compensation if the animal ever tested positive for bTB in future.

Especially system functions in arable systems were perceived to be moderately to strongly affected

Enabling conditions in the social domain were e.g. related to rural demographics and/or availability of labor and more horizontal and vertical cooperation and social self-organization . Specifically, in BGArable and RO-Mixed emphasis was put on enabling conditions in the institutional and social domain. In all case studies, interacting thresholds across level and/or domain were observed . More details on the interacting thresholds are presented in the Supplementary Materials 3. Common interactions between critical thresholds occur between field environmental and field-economic, from field-economic to farm economic, from farm-economic to farm-social, from farm-social to farming system-social, and from farming system-social to farm-social . Generally, an environmental issue at field level, for instance, decreasing soil quality , pest diseases , wildlife attacks , or drought is so much of a shock or stress that it leads to yields that are too low to sustain an adequate level of farm income . In a majority of the farming systems, high input prices and decreasing output prices and sales further diminish the farm income. Too low incomes at farm level were in all case studies resulting in reduced attractiveness of farming, farmers quitting or the lack of finding a successor for the farm. In UK-Arable, also reduced farmer happiness due to lack of recognition was mentioned as a reason for quitting a farm. Farmers quitting their farm without having a successor was in multiple farming systems also considered to contribute to a smaller rural population at farming system level . Interestingly,hydroponic nft although socially oriented function indicators and resilience attributes were less often formally included in the discussions, they eventually appeared when explaining how challenges impact the farming system. Having less farms in the farming system was also associated with a lower maintenance of natural resources and a less attractive countryside.

Interactions with critical thresholds in the environmental domain at farm and farming system level were mentioned in a few other case studies. In NL-Arable, at farm level in the environmental domain a narrow rotation in which starch potato is grown every second year was expected to lead to increased pressure of plant parasitic nematodes . In UK-Arable, low income at farm level was expected to lead to declining soil health at field level . In IT-Hazelnut and SE-Poultry, environmental regulations were expected to improve the maintenance of natural resources at farming system level, but also to push farm income levels below a threshold through increased costs . Overall we observed that environmental thresholds certainly feature, but differ in the level at which they play a role and in what direction they evolve. In farming systems for which access to land is an issue , quitting of farmers may also be an opportunity, provided land becomes available on the market for sale or to be leased. In ES-Sheep, quitting of farmers was experienced as a serious issue. In IT-Hazelnut, the retention of young people on the farms was specifically mentioned as something that could support the rural life and vice versa . Both low economic viability at farm level and low attractiveness of farming and a smaller rural population were considered to reduce the access to labor at farm level in BG-Arable, SE-Poultry, PLHorticulture, DE-Arable&Mixed, RO-Mixed, and ES-Sheep. Access to labor in BG-Arable, PL-Horticulture and RO-Mixed was important for the continuation of activities on farms, as lack of labor was expected to push yields below acceptable levels . In BG-Arable lack of labor could be overcome by implementing new technologies, but this would require a labor force with higher levels of education and qualification which is even harder to find. Lack of labor was also expected to push production costs beyond critical thresholds in SE-Poultry and RO-Mixed. Hence, in multiple systems, low economic viability, attractiveness of farming, rural depopulation and low level of services at farming system level, and low access to labor seem to be part of a vicious cycle. Following from Fig. 1, it can be made plausible that after exceeding critical thresholds of challenges, a decline in performance of system’s main function indicators and resilience attributes was expected by workshop participants in most case studies . Across farming systems, the functions “Food production”, “Economic viability”, and the “Natural resources” were in most cases expected to decline moderately or strongly .In ES-Sheep, ongoing decline of function performance was expected to be aggravated.

When discussed in case studies, “Biodiversity & habitat” and “Animal health & welfare” were on average expected to be less impacted compared to other functions. When exceeding critical thresholds of challenges, also a decline in resilience attributes was expected in most case studies, mainly because of a decline in profitability, production being less coupled with local and natural capital, a declining support of rural life and lower levels of self organization . By contrast, participants in BG-Arable and SE-Poultry generally expected improvements in resilience attributes after critical thresholds are exceeded . For instance, infrastructure for innovation was expected to develop positively in BG-Arable and SE-Poultry, while it was expected to develop negatively in other case studies . In the case of BG-Arable, participants expected increased collaboration, leading to innovation, in case the system would collapse. In the case of ES-Sheep, participants expected that the current low profitability of farmers will not allow investment in new infrastructures for innovation. All studied farming systems were perceived to be “close” or “at or beyond” at least one critical threshold for challenges, function indicators or resilience attributes . The actual state of the system may be more or less close to a threshold than the participant’s perception. Obviously, for case studies that are perceived to be “at or beyond” critical thresholds while still continuing business as usual, the actual state must be at a different position than perceived. Still, perceived closeness can be seen as a clear stress signal, indicating that change is needed, expected or even already experienced. An example refers to the ban of crop protection products before alternatives are available. This stress signal could instigate a study about a reasonable time to phase in/ out regulations regarding the use of crop protection products before actually implementing them. Perceptions of being close to or at critical thresholds also indicate that, from the perspective of farming system actors, immediate action is needed to preserve the farming system or guide it in its transition, thus avoiding a situation where sustainability is even lower. Looking at multiple challenges puts individual challenges into perspective.

To give an example, climate change may be a problem causing regime shifts in many socio-ecological systems , but for the studied farming systems this is not the only challenge and often also not perceived to be the most urgent, except for some arable systems . This supports the notion that climate change should be studied in the context of other drivers . At a global level, reducing anthropogenically induced climate change is, of course, urgent and agricultural systems’ contribution to it must be reduced. Some challenges experienced by FS actors, especially farmers, may also be implicitly caused by climate change; for instance changing legislation and high input costs. For most of the farming systems in our study, climate awareness of some stakeholders, such as conventional farmers, is however not likely triggered due to the impact of climate change on their system per se. When deliberated in an appropriate manner with those stakeholders, new legislation in the context of fighting climate change may however have considerably more effect regarding changing stakeholder perceptions. Function indicators for food production and economic viability were often perceived to be close to critical thresholds. This confirms the need to closely monitor economic indicators as is done in the CMEF of the CAP . When discussed, social function indicators were generally perceived to be “not close” or “somewhat close” to a critical threshold, except for ES-sheep where participants experienced that a critical threshold was exceeded . Environmental function indicators were in most cases perceived to be “not close” or “somewhat close” to critical thresholds . Only in arable systems,hydroponic channel environmental functions were experienced “close” or “at or beyond” critical thresholds. This was mainly related to the capacity of soils to deal with an excess or lack of water, often due to climate change. Participants in workshops of arable systems indicated that a lot of effort was already required to maintain rather than to improve the current soil quality. Arable systems, in need for soil improvement to avoid critical thresholds, would benefit from enabling conditions at national and EU level that foster the maintenance of natural resources.

Mitter et al. , based on a mechanistic scenario development approach for EU agriculture, expect improved attention for natural resources only in a scenario following a “sustainability pathway” out of five possible future scenarios. Current conditions and their future development hence do not seem to support a resilient future of arable systems. Overall, perceived closeness to critical economic thresholds could explain the perceived lower importance of social and environmental functions compared to economic and production functions . Defining critical thresholds seemed most difficult for resilience attributes . According to Walker and Salt it is actually impossible to determine critical thresholds for resilience attributes because they all interact. However, function indicators also interact, but were easier to assess for participants. We argue that difficulties in determining critical thresholds are probably more an indication of the perceived redundancy of resilience attributes for system functioning: presence and contribution to resilience was low to moderate according to stakeholders’ perceptions . This could be related to a control rationale , in which keeping a relatively stable environment and improving efficiency is more important than increasing the presence of resilience attributes. It should be noted, however, that participants often could indicate enabling conditions that improve the resilience attributes. This could be an indication that participants are aware of the importance of resilience attributes, but are in need for more concrete, locally adapted indicators that represent the resilience attributes. In any case, suggesting improvements for resilience attributes could be seen as an implicit acknowledgment by participants that building capacities for adaptation or transformation is required. Perceived thresholds may be different than the real threshold. For the systems that are perceived to be “at or beyond” critical thresholds, it is not necessarily too late to adapt in case the real threshold is actually at a different level than the perceived one. The extensive sheep system in Spain was judged to be close to a collapse, but alternative systems and strategies to reach those have been proposed . In IT-Hazelnut, introduction of new machinery in the past has made farming more attractive for the younger generation, thus avoiding depopulation . Further developments in IT-Hazelnut regarding local value chain activities at farming system level rather than farm scale enlargement, are aimed to further stimulate economic viability and the retention of young people in the area . In PL-Horticulture, the case study is relatively close to Poland’s capital where access to land is limited, system actors aim at increasing the economic viability via vertical and horizontal cooperation at farming system level, which keeps re-attracting seasonal laborers from nearby Ukraine, where wages are lower, to the region. The common factor in these examples of adaptation is that resources are needed to implement them. Be it financial, human, social or other forms of resources. The examples above also suggest that coming back to a desired state, even after exceeding a critical threshold, is possible, provided the disturbance causing the exceedance does not last too long , and adaptation strategies are available . The notion of a critical threshold being a combination of magnitude and duration was not discussed much in the workshops but could help to further define critical thresholds. For instance with regard to the number of years the farming system can deal with extreme weather events as was done in NL-Arable. It is worth noting that challenges are perceived to be more often “at or beyond” perceived critical thresholds than function indicators and resilience attributes.

This is not to suggest there is nothing to gain from using some smart farming technologies

Looking more closely at the emergence of smart farming developments, another crucial consideration pertains to the innovation processes that try to generate and integrate new and striking configurations of firms, farmers, and research institutes. New networks of diverse stakeholders are formed; extant networks are re-made, potentially rendering invisible the role of some actors and their interests. In a sense, then, smart farming can resemble the notion of “innovation by withdrawal”,which departs from the view that innovation is “structured around the introduction of a new element, an artefact, a way of operating, a service, and its success is dependent on the number of adopters and the significance of the entities which are articulated with it” . Yet, as demonstrated by the case of no-till farming in France , withdrawal of one element relies on making visible hitherto invisible or overlooked elements , while maintaining problematic practices.To respond, an alternative to focusing on introduction or withdrawal is to recognize that agricultural innovation, like any other practice, is always a topological affair: it is about overseeing and managing configurations of humans and materials and how they flow through a system or across a specific domain, such as a field . Where there are blockages, conduits can be installed to increase flows; where there are leaks, plugs are required. Farmers shift and prod to adjust arrangements of materials or relations with a view to addressing problems, such as falling yields, vulnerabilities to climatic variability, or exposure to viruses. A mechanism of implementing innovations such as those associated with smart farming is to reconfigure topological arrangements. As demonstrated by literature on agricultural biosecurity,farming practice needs to be viewed as occurring against the topological backdrop of an “entangled interplay” , with numerous “contingent intra-actions” occurring across multiple risky “borderlands” .Proximities,distance,nft system connectivities,and modulations of presence/absence figure in the effort to create desired outcomes,with new insertions or removals pursued in efforts to control or steer activities in defined or experimental ways.

Whether the risk is a matter of falling yields or exposure to viruses, it pays to acknowledge the ongoing relationship between farming and innovation through a topological lens; that is, to dwell on farmers as active agents of topological transformation, even if they are rarely acting alone. One way to combine an analytical concern with digital life, corporate interests in establishing a certain type of smart farming, and topological transformations is highlighted by literature on smart farming innovation processes. Consider, for example, how new configurations come into the picture. As highlighted in research on smart farming in Canada, and as referenced by Relf-Eckstein et al.,the Canadian government has recently established innovation ‘superclusters’ to examine and exploit technological opportunities. A recent outcome is an industry-led consortia called Protein Industries Canada, which includes a partnership whereby Lucent BioSciences “will use the hulls of pea and lentil seeds which are a co-product from value-added processing completed by AGT Foods and Ingredients [to create] Soileos: a novel carbon-neutral micro-nutrient fertilizer that uses organic fibre as a carrier to provide micro-nutrients to plants” . As this case suggests, the ‘smart’ in smart farming can involve astute and imaginative arrangements to make new products and chase after profits in novel ways. A similar picture emerges in the Netherlands where a “golden triangle” of agricultural research, industry, and government aims to create “new business ecosystems consisting of focal firms, their suppliers, complementor firms, and customers” . A key feature is the leading role of the Dutch firm Philips, which occupies a prominent position in high-tech urban agriculture , a growing smart farming sector, by “providing the essential technologies, registration of patents, and creation of new business opportunities” . Meanwhile, in the larger and more traditional Dutch agricultural sector, the Food Valley Open Innovation Ecosystem includes “the Wageningen Campus and the planned World Food Centre in Ede” and creates ties between research and development centres run by large firms such as Friesland Campina and Unilever and wider networks of small-to-medium enterprises and startups.

There are 15,000 scientists across Food Valley, with twenty research institutes, 1440 food related, and 70 science related firms . Such configurations of firms, farmers and research institutes will likely create new smart farming products and services and build on Dutch successes in exporting around €9 billion worth of high-tech agrifood innovations, including “energy-efficient greenhouses, precision agricultural systems and new discoveries that make crops more resistant” . Put differently, the topologies of smart farming point toward new forms of “path creation” that involve but also often extend beyond farmers. It is instructive that smart farming today is bound up with efforts to use ‘open innovation’ processes that facilitate co-design or co-innovation between agricultural technology providers, farmers, and others. Such an approach can “blur the boundaries between scientists and agricultural system stakeholders, between agronomists and farmers, and between actors in the agricultural sector and those designing in other sectors” . The virtue of “participatory design processes involving farmers” is that it can yield new tools, such as dashboards , to help farmers understand agro-ecological conditions. There are, however, no guarantees that smart farming developments will yield effective configurations. Indeed, there is significant evidence that smart farming developments are hamstrung not only by the instrumental logics underpinning technology providers but also by ineffective coordination and inadequate arrangements of materials or skills. Consider here the push to develop automated body condition scoring and a soil water outlook tool for Australian dairy farmers. Noting that these versions of smart farming innovation involve “a unique innovation challenge [not least because of] the new knowledge demands for farmers in a highly dynamic, technology-driven environment” , one finding is that the new tools and practices confront limitations in the way agricultural relations are configured with respect to the wider institutional milieu. Making the most of the soil water outlook tool, for example, required but did not receive sufficient input from the Australian Bureau of Meteorology “to help farmers to link the SWO with seasonal climate outlooks” . Then, with regards to automated body condition scoring, the new technology led some farmers to think “maybe we don’t need the [farm] advisor as often,”,with the upshot that “some tools were potentially replacing the skills of advisors”.Yet, because “more remote monitoring of key performance indicator data via online software” can enable farm advisors to make fewer farm visits, smart farming in this context conceivably increases the sense of isolation many farmers already experience .

Elsewhere in Australia, smart farming developments call attention to a different dynamic between farmers and advisors. In some rice farming regions, advisors might be expected to be the “sense makers” who can explain and encourage farmers to adopt new technologies; but in fact one consultant respondent claimed “it’s mainly been the farmers dragging the agronomists along” .At the same time, “insufficient support structures” , for example regarding data compatibility or standards, can hold back adoption and frustrate farmers who are “prepared to use evolving and uncertainty-generating technologies” but find that their knowledge is not effectively tapped. In Canada, ‘broadacre’ smart farming developments occur amid the “critical constraint” of labour shortages and demographic change, but even here “adoption is lower than anticipated” , with one explanation focusing on tensions around what happens to data produced on farms. A problem yet to be overcome is industry self-regulation of data usage and a lack of certainty about the legal ramifications of smart farming. Thus, “[u]ntil clarity is brought to the issue of data, the industry is at risk of losing farmer’s trust and potentially hindering innovation opportunities at the farm level” . Although there are examples from the literature which demonstrate that smart farming innovation involves an ongoing process of trying to reconfigure arrangements of sociotechnical relations, I argue a more accurate and urgent conclusion is to emphasize the ‘mis-configured innovations’ of smart farming. One of the main features of smart farming concerns the limited parameters within which innovations operate. In Canada, for example, an element in smart farming arrangements is models and platforms designed for commodity farmers, not those “farmers working outside of the dominant industrial model” . In effect, “the maps created within those big data platforms developed by industry are made meaningful only if one adheres to a rigid conventional farming strategy of seeding in neat rows separated by areas of soil free of weeds” . A similar result emerges in Australia where observers note that farmers want autosteer technologies, new imagery services, levelling and GPS guidance because “if they’ve got efficient layouts, laser levelled, they’ll make significant water savings and they’ll have reduced labour inputs as well” . Smart farming therefore means that food producers contemplate, “standardizing the environment” in accordance with the commercial imperatives of farmers operating large holdings and using expensive machinery to generate predictable topographies that fit with the new topologies required to make smart farming technologies effective. Built-in biases pervade all algorithmic systems ; the biases in smart farming might only pertain to environments in the first place but they can have broader political-economic effects.

As such, the core problem with the various reconfigurations underpinning smart farming developments is not simply that the absence of one or other action or reform can limit their impact,hydroponic gutter but rather that smart farming innovation processes begin and proceed without adequately conceptualizing the underlying obstacles and limitations confronting food producers today. Technological innovations that reinforce power asymmetries regarding data ownership, for example, or that fail to challenge implicit biases toward certain types of environments, render some interests invisible while reifying specific types of logics, such as narrow measures of economic efficiency. Like any innovation, insertion, or reconfiguration, smart farming entails topological transformation; but problems emerge when the “quieter registers” of smart farming make it possible for “powerful actors to make their presence felt at one remove, to reach into the everyday life of distant others” , for example by dispossessing them of valuable data or establishing algorithmic biases toward standardized farm topographies.It can make sense to use devices or services in new arrangements that create efficiencies or give food producers new access to information that can inform decisions. However, because these developments always by necessity involve reconfiguring arrangements of sociotechnical relations, agricultural innovation processes will continue to introduce new misconfigurations when they pursue discrete solutions to specific problems, rather than integrated developments based on incremental adjustments in information-intensive iterative processes that target systemic or structural change. As insisted upon by scholarship on food sovereignty in critical agrarian studies , the urgent challenge today is to conceptualize a planetary land, agrarian, and food system in which food producers and consumers everywhere are confronted by, but examine ways of overcoming, the same problems of neoliberal capitalism dominated by transnational corporations, authoritarian governance, and climate change. In the shadow of the corporate food regime, producing food in the Netherlands or Canada is bound up with the realities of producing food in India or Kenya. Further, the dynamics of digital life mean smart farming innovations in one place will inform and conceivably move the ‘planetary cognitive ecology’ generally, with unpredictable but connected results playing out elsewhere. The products of smart farming will only reinforce problems if they yield new patents for agricultural technology providers in a place such as Ireland , a widening yield gap between capital- and labour-intensive agrarian systems , or if they increase the likelihood of ‘smart’ food production in one region leading to food dumping in another . Per the vision of a Common Food Policy in the European Union , rather than seeing smart farming developments “reinforcing existing production models, leading to trade-offs between different environmental impacts, or between environmental and social sustainability,” the task is to reorient innovation “towards low-input, diversified agroecological systems” . In the light of these challenges, a sustainable and successful smart farming innovation process requires what we might imagine as the coproduction of ambitious ‘topological repertoires’ that make ongoing assessments of absence, presence, proximity, and reach at the scale of a structure or system and then pursue appropriate technological solutions from the ground up.

Wilcoxon test was used for comparing each treatment to its control in the real-time PCR analyses

All tests were performed using StataSE v12 software.Mushrooms are widely consumed foods whose high contents of bioactive compounds may provide antioxidant, anti-inflammatory, anti-obesity, and antidiabetic properties, among others.Thus, different in vitro and in vivo models have been used to identify and characterized the health benefits of these mushrooms for their potential application as food ingredients. Thereby, Grifola frondosa polysaccharides have been found to exert antioxidant and antidiabetic properties in different animal models . The regulatory pathways of energy homeostasis are highly conserved between C. elegans and mammals, making this nematode a powerful model for exploring the genetic bases of fatty acid synthesis and the regulation of fat storage. Thus, C. elegans has been widely used as a screening model for the identification and evaluation of BACs with healthy properties in the prevention of obesity-related disturbances, together with the characterization of the biological mechanism underlying these effects.

Moreover, thisnematode has been widely used for determining the antioxidant, anti-aging, and life-prolonging properties of BACs present in different food ingredients with beneficial properties in the prevention of aging-related diseases . Previous studies have suggested the anti-obesity properties of G. frondosa. For example, Aoki and colleagues demonstrated that supplementation with 0.4% G. frondosa extract in high fat-induced obese mice for 15 weeks significantly reduced body weight gain and visceral fat accumulation, ameliorated hepatic triglyceride storage, and improved glucose tolerance . They suggested that the anti-obesity and antidiabetic properties of this G. frondosa extract were attributed to its activity as a PPARδ agonist. As mentioned above, our GE represents an important source of different BACs, including beta-glucans, phenolic compounds, PUFAs, and MUFAs. Different studies have reported the lipid-reducing activity of different flavonoids and phenolic acids in C. elegans, including ours . For this reason, we aimed to determine if the combination of the bioactive compounds found in our GE could also affect the lipid homeostasis using the C. elegans model. The intestinal and hypodermal cells of this nematode accumulates lipids in the form of fat droplets, which can be detectable under microscopy using fat-soluble dyes, such as Sudan Black B, Oil red O, and Nile Red .

The quantification of the fluorescence of the fixative Nile Red lipophilic dye has been demonstrated to represent a reliable method to determine the fat content of this nematode, and has been widely used for evaluating the lipid-reducing activity of BACs, with potential uses in the prevention of and therapy for obesity-related diseases . For this experiment, L1 N2 wild-type worms were treated until reaching the L4 stage with and without the GE at the doses of 10 and 20 μg/mL, when nematodes were collected, fixed, and stained with Nile Red . As revealed by the quantification of the fluorescence of the worms , both doses of GE induced a significant reduction in the lipid content of C. elegans, in comparison with untreated control worms . Orlistat-treated worms were used as a positive control of fat reduction. In fact, the reduction induced by the high dose of GE was 18.64%, and a similar result was obtained after Oil Red O staining , confirming this effect on worm fat deposition. Although our extract did not exhibit in vitro genotoxicity in the SOS/umu test, these results could be related to an effect on nematode development. Thus, in order to dismiss this negative effect, we analysed the effect of GE extract treatment from L1 to day 1 of adulthood on worm length, size, and egg laying. No differences were observed in terms of worm length and size between GE-treated and untreated nematodes, suggesting that the lipid-reducing activity of GE is not accompanied by an effect on worm length and size. Furthermore, after 72 h of treatment, both GE and control plates exhibited the presence of both eggs and L1 larvae without differences in the time of appearance. All these results suggest that treatment with our GE from L1 to L4 significantly reduces the C. elegans fat content independently of any effect on worm development. Importantly, the effect of GE on fatty acid synthesis and breakdown was also accompanied by a tendency to higher expression of daf-2 , an ortholog of human IGF1R , INSR , and INSRR . This gene codes for the single receptor protein in the IIS pathway. Moreover, treatment with GE induced a pronounced overexpression of daf-16, the ortholog of human FOXO, which codes for a key transcription factor regulated by the IIS pathway. Daf-16 acts as a nutrient-sensing regulator of energy homeostasis and lipid metabolism .

The significant overexpression of GE on daf-16/foxo was also confirmed at a lower dose of the extract and would suggest that the anti-obesity properties previously observed with GE are mediated by the upregulation of this transcription factor. Finally, GE-treated worms exhibited a significant upregulation of skn-1 , an ortholog of the human NRF2 gene, an important transcription factor of the antioxidant and antiaging responses . No differences were observed in the expression of sod-3. Again, significant upregulation of skn-1 was observed after treatment with a lower dose of GE . SKN-1 activation has been previously shown to be involved in fat metabolism by depleting somatic lipids , so overexpression of this transcription factor by GE might also be involved in the fat-reducing activity observed in our Nile Red and Oil Red O analyses. Our findings demonstrate that our GE extract reduced the C. elegans lipid content when treated from L1 to L4; this effect is mediated by a reduction in the fatty acid biosynthesis and increased oxidation, together with a significant overexpression of the skn-1 and daf-16 transcription factors. In order to further investigate the potential implication of the daf-2/daf-16 and the skn-1/nrf-2 signalling pathways in the anti-obesity properties of the GE extract, we analysed the lipid-reducing activity of GE on C. elegans in a glucose-loaded medium.

Glucose has been used to establish a C. elegans obesity model in various studies , and has been demonstrated to affect both lipid accumulation and oxidative stress responses . Again, we observed that treatment with 20 μg/mL of GE induced a significant reduction in the fat content in comparison with untreated control worms in a glucoseloaded medium . A gene expression analysis performed after this assay demonstrated that, when the medium was supplemented with glucose, no differences were observed in skn-1 gene expression , suggesting that the skn-1 activation previously observed might be involved in the potential antioxidant activity of this extract, more than modulating the lipid accumulation in this model.