Using aquatic animals as partners to increase yield and maintain soil nitrogen in the paddy ecosystems

  1. Liang Guo
  2. Lufeng Zhao
  3. Junlong Ye
  4. Zijun Ji
  5. Jian-Jun Tang
  6. Keyu Bai
  7. Sijun Zheng
  8. Liangliang Hu  Is a corresponding author
  9. Xin Chen  Is a corresponding author
  1. College of Life Sciences, Zhejiang University, China
  2. Bioversity International, Italy
  3. Yunnan Academy of Agricultural Sciences, China

Abstract

Whether species coculture can overcome the shortcomings of crop monoculture requires additional study. Here, we show how aquatic animals (i.e. carp, crabs, and softshell turtles) benefit paddy ecosystems when cocultured with rice. Three separate field experiments and three separate mesocosm experiments were conducted. Each experiment included a rice monoculture (RM) treatment and a rice-aquatic animal (RA) coculture treatment; RA included feed addition for aquatic animals. In the field experiments, rice yield was higher with RA than with RM, and RA also produced aquatic animal yields that averaged 0.52–2.57 t ha-1. Compared to their corresponding RMs, the three RAs had significantly higher apparent nitrogen (N)-use efficiency and lower weed infestation, while soil N contents were stable over time. Dietary reconstruction analysis based on 13C and 15N showed that 16.0–50.2% of aquatic animal foods were from naturally occurring organisms in the rice fields. Stable-isotope-labeling (13C) in the field experiments indicated that the organic matter decomposition rate was greater with RA than with RM. Isotope 15N labeling in the mesocosm experiments indicated that rice used 13.0–35.1% of the aquatic animal feed-N. All these results suggest that rice-aquatic animal coculture increases food production, increases N-use efficiency, and maintains soil N content by reducing weeds and promoting decomposition and complementary N use. Our study supports the view that adding species to monocultures may enhance agroecosystem functions.

Editor's evaluation

This is a well conducted experimental study in which rice monocultures are compared with rice-aquatic animal co-cultures at multiple sites over multiple years. Co-culture increases plant yield and adds animal yield, but requires extra input of animal feed. Overall co-culture benefits yield and sustainability.

https://doi.org/10.7554/eLife.73869.sa0

eLife digest

Monoculture, where only one type of crop is grown to the exclusion of any other organism, is a pillar of modern agriculture. Yet this narrow focus disregards how complex inter-species interactions can increase crop yield and biodiversity while decreasing the need for fertilizers or pesticides. For example, many farmers across Asia introduce carps, crabs, turtles or other freshwater grazers into their rice paddies. This coculture approach yields promising results but remains poorly understood. In particular, it is unclear how these animals’ behaviours and biological processes benefit the ecosystem.

To examine these questions, Guo, Zhao et al. conducted three separate four-year field experiments; they compared rice plots inhabited by either carp, mitten crabs or Chinese softshell turtles with fields where these organisms were not present.

With animals, the rice paddies had less weeds, better crop yields and steady levels of nitrogen (a natural fertiliser) in their soil. These ecosystems could breakdown organic matter faster, use it better and had a reduced need for added fertilizer. While animal feed was provided in the areas that were studied, carp, crabs and turtles obtained up to half their food from the field itself, eating weeds, algae and pests and therefore reducing competition for the crops.

This work helps to understand the importance of species interactions, showing that diversifying monocultures may boost yields and make agriculture more sustainable.

Introduction

Biological simplification and reliance on chemicals have increased the concern with the low levels of biodiversity in modern, intensive agriculture (Li et al., 2007; Smith et al., 2008; Kremen et al., 2012; Ren et al., 2014; Brooker et al., 2021). In natural ecosystems, experiments have shown that increases in species number can increase ecosystem productivity and stability (Hector et al., 1999; Tilman et al., 2006; Loreau and de Mazancourt, 2013; Tilman et al., 2014; van der Plas, 2019). These positive effects of species diversity on ecosystem functioning are mainly explained by niche partitioning, facilitation, and complementary resource use (Cardinale et al., 2002; Isbell et al., 2009; Cardinale, 2011; Brooker et al., 2021).

Interspecific facilitation (which occurs when one species makes conditions more favorable for another species) or complementary resource use are common in terrestrial, marine, and wetland ecosystems (Bruno et al., 2003; Brooker et al., 2007; He et al., 2013; Bulleri et al., 2015; Wright et al., 2017). Plants can make the local environment more favorable for their co-existing partners by reducing thermal, drought, and salt stress (Gómez-Aparicio et al., 2004; Gómez‐Aparicio et al., 2008; Pretzsch et al., 2013; Anthelme et al., 2014); by increasing nutrient availability Li et al., 2007; by removing competitors or deterring predators (Callaway et al., 2005; Gómez‐Aparicio et al., 2008; Flory et al., 2014); and by stimulating beneficial soil microorganisms (Hortal et al., 2013; Rodríguez‐Echeverría et al., 2015). Animals can also enhance plant growth and population development by improving the soil environment (Daleo et al., 2007; Booth et al., 2019; by removing competitors Cushman et al., 2011); or by facilitating dispersal of fruits and seeds (Bronstein et al., 2006; Carlo et al., 2014).

Facilitative interactions have recently been successfully applied to forest restoration in arid areas (Gómez-Aparicio et al., 2004); to the establishment of plant communities in salty marshes or on beaches (Bruno, 2000); and to coral reef restoration (Abelson, 2006). Researchers have also proposed that facilitation or resource complementarity between species may increase the sustainability of agricultural production (Ren et al., 2014; Brooker et al., 2021). Although an increase in species richness in a natural plant community is expected to result in an increase in plant mass, an increase in species richness in agriculture may not always lead to increases in yield due to competition for light or nutrients (Omer et al., 2007). Understanding how species may or may not benefit is therefore critical for using species diversity in agriculture.

Intercropping systems (e.g. the interplanting of corn or wheat with a legume) or the planting of cover crops are examples of the successful use of crop diversity in agriculture. In legume-based cropping systems, legume crops provide intercropped non-legume crops with symbiotically fixed nitrogen (N) (Li et al., 2007; Tsialtas et al., 2018) and with increased phosphorus (P) availability due to the lowering of soil pH by N2-fixing bacteria (Li et al., 2007); these effects increase the yield of the intercropped species (Li et al., 2007). Other intercropping systems can increase the diversity of soil microorganisms, natural enemies, and pollinators (Cardinale et al., 2003; Kremen et al., 2007; Letourneau et al., 2011; Norris et al., 2018). Using diverse cover crops can also help reduce soil erosion and greenhouse gas emissions (Kaye and Quemada, 2017).

Because paddy fields provide a shallow water habitat suitable for some aquatic animals (e.g. carp, crabs, and softshell turtles), the coculturing of rice with aquatic animals has been practiced in many countries (e.g. Bangladesh, China, Egypt, India, Indonesia, Myanmar, Malaysia, the Philippines, Thailand, and Vietnam) (Halwart and Gupta, 2004; Frei and Becker, 2005b; Ahmed and Garnett, 2011). Several rice-aquatic animal coculture systems (e.g. rice-carp, rice-crab, and rice-turtle) have been developed (Hu et al., 2016). Field surveys and experiments have shown that these coculture systems can increase rice yields and soil fertility while reducing the need for fertilizers and pesticides compared to rice monoculture (Xie et al., 2011; Hu et al., 2016; Zhang et al., 2016; Guo et al., 2020). Why coculturing these aquatic animals with rice can reduce the application of fertilizers and pesticides, however, is poorly understood. Understanding how aquatic animals contribute to the reductions in fertilizer and pesticide application in coculture systems would help the development of sustainable rice production.

Animal behaviors (e.g. moving and grazing) are important drivers of ecosystem processes (e.g. carbon and nutrient cycling, and energy flux) (Vanni et al., 2006; Schmitz et al., 2018; McInturf et al., 2019). In wetland or aquatic ecosystems, grazing, ‘muddying’, and burrowing by aquatic animals have important roles in nutrient cycling (Vanni, 2002; Thrush et al., 2006; Devlin et al., 2015; Atkinson et al., 2021). For paddy ecosystems in which rice and aquatic animals coexist, understanding whether and how the behavior of aquatic animals affects ecosystem processes and functions could help researchers predict the effects of a coculture system of rice and an aquatic animal, and could also help improve the coculture system.

In this study, we conducted three field experiments and three mesocosm experiments to determine how coculture with aquatic animals benefits a rice paddy ecosystem in terms of productivity, nutrient-use efficiency, and the stability of soil N content. Because some fresh water animals (e.g. carp, crabs, crayfish, and softshell turtles) that are cultured in fish ponds or in paddy fields are omnivores and may use weeds, algae, and phytoplankton as food, we expected that the coculture of these aquatic animals would increase rice yield by reducing competitors of rice. We also expected that the cocultured aquatic animals would promote organic matter decomposition because of their feeding activity and would thereby promote nutrient recycling in the paddy ecosystem. Feed is often applied in the form of pellets to increase the growth of aquatic animals in coculture systems (Hu et al., 2016; Guo et al., 2020), and significant percentages of the N in the feed is often unconsumed and unassimilated by the animals. We therefore expected that this unconsumed and unassimilated feed-N could be used by rice plants, resulting in higher N-use efficiency and a more stable soil N content in a coculture system than in a rice monoculture.

Results

Yield, soil N content, and N-use efficiency in the field experiments

We conducted three 4-year-long field experiments: one with rice-carp, one with rice-crabs, and one with rice-turtles. We found that rice yield was significantly higher in the RA treatment (the treatment with the coculture of rice and an aquatic animal) than in the RM treatment (the treatment with rice monoculture) in the rice-carp experiment (F1,10=7.828, p = 0.019), the rice-crab experiment (F1,10=5.957, p = 0.035), and the rice-turtle experiment (F1,10=12.472, p = 0.005) (Figure 1a). Compared to the corresponding monoculture, average rice yield over the 4 years in the RA treatment was 9.13% ± 3.11% higher for rice-carp, 12.05% ± 1.16 higher for rice-crabs, and 8.69% ± 1.74 higher for rice-turtles. During the experimental period, the average annual aquatic animal yield (in t ha–1) was 0.85 for rice-carp, 0.56 for rice-crab, and 2.66 for rice-turtle systems (Figure 1a).

Yields of rice and aquatic animals (a), soil nitrogen content (b), and apparent N-use efficiency (c) in the field experiments.

In (a), rice yields are indicated by symbols and lines, and aquatic animal yields are indicated by bars. Values are means ± SE (n = 6).

Averaged across all 4 years, total soil N content was not significantly different in the RA vs. the RM treatment in all three experiments (F1,10=0.294 and p = 0.687 for the rice-carp experiment; F1,10=1.325 and p = 0.154 for the rice-crab experiment; and F1,10=0.236 and p = 0.345 for the rice-turtle experiment) (Figure 1b). At the end of the experiments, total soil N contents had not changed relative to initial values in the RA treatment of the rice-carp (t5 = −0.533, p = 0.631), rice-crab (t5 = 0.213, p = 0.842), and rice-turtle systems (t5 = −1.279, p = 0.259) (Appendix 1—figure 1).

Compared to the RM treatment, the RA treatment received extra N from fish feed (Appendix 2—table 1). Data from the 4 years of the experiments showed that apparent N-use efficiency (ANUE) was higher in the RA treatment than in the RM treatment for the rice-crab system (F1,10=9.557, p = 0.011) and the rice-turtle system (F1,10=7.302, p = 0.022) but not for the rice-carp system (F1,10=0.209, p = 0.657) (Figure 1c).

Weed biomass, food sources, and decomposition in the field experiments

Weed biomass was significantly lower in the RA treatment than in the RM treatment in the rice-carp experiment (F1,10=513.456, p = 0.000), the rice-crab experiment (F1,10=538.032, p = 0.000), and the rice-turtle experiment (F1,10=557.659, p = 0.000) (Figure 2). In all three experiments, weed biomass significantly decreased over time in the RA treatment (p < 0.05) but not in the RM treatment (p > 0.05).

Weed biomass in the field experiments.

No herbicides were used in the experiment. Values are means ± SE (n = 6).

Food source analysis showed that 50.2%, 34.9%, and 16.0% of the carp, crab, and turtle foods, respectively, were from the field environment rather than from applied feed (Figure 3). The main non-feed food sources for the aquatic animals in the rice fields included weeds, macro-algae, phytoplankton, zooplankton, and zoobenthos (Figure 3).

Diet components of aquatic animals as determined by dual stable isotopes (δ13C and δ15N), and the contribution of food sources to the aquatic animal diet in the field experiments.

In each of the three plots in the figure, the white zone represents the proportion of food that aquatic animals (i.e. carp, crabs, or turtles) obtained from feed, and the grey zone represents the percentage of food that aquatic animals obtained from the rice field. The values in the rectangles to the right indicate the rice field food components as percentages of the total food obtained by the aquatic animals. POM: particulate organic matter.

Determination of the stable isotope (13C) content in maize leaves indicated that the percentage remaining in maize litter tubes at 40 days after the beginning (DAB) of the experiment was lower in the RA treatment than in the RM treatment in the rice-turtle experiment (F1,10 = 23.353, p = 0.001) (Figure 4) but did not significantly differ between RM and RA treatments in the rice-carp experiment (F1,10 = 0.076, p = 0.788) or the rice-crab experiment (F1,10 = 1.092, p = 0.321) (Figure 4). At 80 DAB, however, the decomposition rate was higher in the RA treatment than in the RM treatment in all three experiments (for rice-carp: F1,10 = 11.432, p = 0.007; for rice-crab: F1,10=15.572, p = 0.003; for rice-turtle: F1,10 = 14.349, p = 0.004) (Figure 4).

Organic matter decomposition in the field experiments at 40 and 80 days after the beginning (DAB) of the experiment.

A higher percentage of 13C remaining indicates slower decomposition. Values are means ± SE (n = 6). An asterisk indicates a significant difference between RM (rice monoculture) and RA (rice-aquatic animal coculture) at p < 0.05; ns indicates that the difference was not statistically significant.

Complementary utilization of feed-N by aquatic animals and rice in the mesocosm experiments

The δ15N percentage in the rice plant biomass was significantly higher in the RA treatment than in the RM treatment in all three mesocosm experiments (for rice-carp: F1,10 = 1278, p = 0.000; for rice-crab: F1,10 = 210.320, p = 0.000; for rice-turtle: F1,10 = 91.572, p = 0.000) (Appendix 1—figure 2). The results from the mesocosm experiments also indicated that rice used from 13.02% to 35.13% of the feed-15N (Figure 5a). The N in feed that was not consumed by aquatic animals represented 9.61–30.65% of the rice biomass-N in the RA treatments (Figure 5b).

The fate of feed-N as determined by 15 N labeling in the mesocosm experiments.

(a) Percentages of feed-N in rice plants, aquatic animals, and the environment (e.g., soil and water). (b) Total N in rice biomass at the end of the experiments. Values are means ± SE (n = 6).

N accumulation in the mesocosm experiments

The δ15N content in the soil in the mesocosm experiments did not significantly differ at the beginning vs. the end for the RM treatments (for rice-carp: t10 = 0.131, p = 0.449, n = 6; for rice-crab: t10 = 0.115, p = 0. 455, n = 6; for rice-turtle: t10 = 0.523, p = 0.623, n = 6), but was significantly higher at the end than at the beginning for the RA treatments (for rice-carp: t10 = 2.178, p = 0.027, n = 6; for rice-crab: t10 = 2.153, p = 0.028, n = 6; for rice-turtle: t10 = 3.292, p = 0.004, n = 6) (Figure 6a). The total N concentration in the soil was also significantly higher at the end than at the beginning of the experiments for the RA treatments (for rice-carp: t10 = 2.765, p = 0.009, n = 6; for rice crab: t10 = 3.204, p = 0.005, n = 6; for rice-turtle: t10 = 2.519, p = 0.015, n = 6) but not for the RM treatments (for rice-carp: t10 = 0.477, p = 0.322, n = 6; for rice-crab: t10 = 1.774, p = 0.053, n = 6; for rice-turtle: t10 = 0.132, p = 0.449, n = 6) (Figure 6b).

Soil δ15N content and total soil N content at the beginning vs the end of the mesocosm experiments.

(a) δ15N value in soil at the beginning and end of the mesocosm experiments. (b) Total N in soil at the beginning and end of the mesocosm experiments. Values are means ± SE (n = 6). An asterisk indicates a significant difference between the before and after values for each treatment in each rice-aquatic system at p < 0.05; ns indicates that the difference was not statistically significant.

Discussion

Researchers have been investigating whether the use of species interactions can overcome the limitations of monocultures, which depend on high fertilizer and pesticide input and which fail to take advantage of the possible beneficial effects of species interactions (Kremen et al., 2012; Ren et al., 2014; Brooker et al., 2021). Intercropping is an important and successful way to use biodiversity in agriculture (Li et al., 2007; Brooker et al., 2015; Brooker et al., 2021). The current study provides another example of exploiting positive species interactions in agriculture. In this case, the interactions involve the use of an aquatic animal (i.e. carp, crabs, or turtles) as a partner crop for rice with the goals of stabilizing soil N and increasing the productivity and N-use efficiency of paddy ecosystems.

The role of animals in ecosystems has been increasingly recognized (Vanni, 2002; Dirzo et al., 2014; Schmitz et al., 2018). Animals can mediate carbon exchange between ecosystems, can mediate organic matter transformation within ecosystems, and can also drive nutrient cycling (Schmitz et al., 2013; Schmitz et al., 2018; Vanni, 2002; McInturf et al., 2019). In many aquatic ecosystems, aquatic animals can also significantly affect the plant community, primary productivity, and nutrient availability (Attayde and Hansson, 2001; Vanni, 2002; Vanni et al., 2006). In this study, coculturing with aquatic animals (i.e. carp, crabs, and turtles) increased rice yield and N-use efficiency, and helped maintain soil N compared to the corresponding rice monoculture.

The aquatic animals had two important roles in these cocultured paddy ecosystems. One role involved competition, that is, aquatic animals reduced competitors (i.e. weeds) of rice plants and thereby enhanced rice yield. Some freshwater animals (e.g. carp, crabs, and crayfish) are omnivores that may consume some living organisms (e.g. weeds, algae, and phytoplankton) that compete with rice plants for nutrients. Carp and crabs, for example, can greatly reduce weeds in rice-carp systems (Frei and Becker, 2005b; Xie et al., 2011) and in rice-crab systems (Lv et al., 2011). Our field experiments also showed a reduction (45.3–51.9%) of weeds in the plots with carp, crabs, or turtles compared to the monoculture plots without herbicide use (Figure 2). Our dietary reconstruction based on stable-isotope data of δ13C and δ15N showed that carp and crabs obtained 34.8–50.2% of their total food from the rice field, including weeds, macro-algae, and phytoplankton (Figure 3); these results provide indirect evidence that carp and crabs reduced competitors of rice. Although some freshwater animals (e.g. turtles in our study) do not prefer to feed on weeds and other vegetative food sources, their activities disturb the paddy soil and thereby inhibit weed germination and growth (Hu et al., 2016; He, 2017).

A second role of aquatic animals concerned N, that is, aquatic animals increased the recycling of N in these cocultured paddy ecosystems. Many studies have shown that grazers can accelerate nutrient cycling in natural grassland and freshwater ecosystems by increasing nutrient availability in soil and nutrient-use efficiency of plants (McNaughton et al., 1997; Atkinson et al., 2017). In our study, the carp, crabs, and turtles obtained 50.2, 34.8, and 16.0%, respectively, of their food from the field rather than from the feed although sufficient feed was applied in our experiment to support the aquatic animals (Figure 3). Similar to the effects of grazers in the natural ecosystems, the aquatic animals (carp, crabs, and turtles) in the paddy ecosystem foraged on weeds, algae, phytoplankton, zooplankton, and benthic invertebrates that used N directly from the paddy field. Once these food source organisms are ingested, the aquatic animals convert them into biomass, feces, and excretions. Because ammonia represents from 75% to 85% of the N in aquatic animal excretions (Chakraborty and Chakraborty, 1998), the excretions can be directly utilized by rice plants. The aquatic animal feces may release nutrients once they are decomposed, or they may be stored in the form of soil organic matter. Thus, the promotion of N cycling by aquatic animals apparently explains, at least in part, why N-use efficiency of rice was higher and soil N content was more stable in the rice-aquatic animal coculture plots than in the monoculture plots (Figure 1).

In addition to reducing competition and increasing nutrient availability for rice plants via grazing, aquatic animals apparently increased nutrient availability for rice plants by enhancing organic matter decomposition. The percentage of maize leaves (added to the plots in ‘litter tubes’) that remained after 80 days (as indicated by the percentage of 13C remaining) was significantly lower in the three RA treatments than in the RM treatment (Figure 4), indicating that carp, crabs, and turtles promoted organic matter decomposition in the field. Nutrients (e.g. N and P) in the organic matter (e.g. unconsumed feed, aquatic animal feces, and leaf litter) may be released by decomposition and then used by rice plants or other organisms in the field. Our tracing of feed-15N demonstrated that 13.0–35.1% of the feed-N was found in the rice plants (Figure 5a). These results suggested that the N in unconsumed or unassimilated feed was released via decomposition and was then used by the rice plants.

Unlike traditional rice-fish coculture systems in which no fish feed is applied (Xie et al., 2011), the current coculture systems, like those described in this study, often include the application of feed in order to obtain high aquatic animal yields (Hu et al., 2016). Whether such feed affects rice yield and soil fertility was also assessed in our study. The mesocosm experiments showed that 1.27 ± 0.09 g m–2 (rice-carp), 0.36 ± 0.09 g m–2 (rice-crab), and 1.12 ± 0.10 g.m–2 (rice-turtle) of feed-15N accumulated in the rice plant biomass (grain and straw) (Figure 5b). The mesocosm experiments also showed that feed-15N accumulated in the soil (Figure 6). These results suggest that N use by cocultured rice and animals can be complementary and can increase N-use efficiency. The results also suggest that the unconsumed or unassimilated feed can function as a fertilizer for rice and can thereby increase the rice yield and the N content in the soil. In addition to N, phosphorus (P) also entered rice-animal coculture systems via feed in our study (Appendix 2—table 1). Like N, P is important for rice growth and yield (Ahmed et al., 2017). In our 4-year field experiments, the level of soil total P was similar at the end vs. the beginning under both rice monocultures and rice-animal cocultures (Appendix 1—figure 3), but rice yields were higher and more P was removed with the harvested products with coculture than with monoculture (Appendix 2—table 2). It follows that the P input via feed may contribute to the rice yield increase and the maintenance of soil P in the coculture systems.

The current results increase our understanding of how agricultural systems can use species diversity to increase sustainability. Planting diverse wild or crop species in field margins has been found to improve the management of crop pests and their natural enemies (Bianchi et al., 2006; Tschumi et al., 2016). Overyielding often occurs in intercropping systems when the coexisting crops benefit each other or when one benefits the others (Snapp et al., 2010; Kremen et al., 2012; Li et al., 2014; Ren et al., 2014; Brooker et al., 2021). In our study, the rice plants and the three aquatic animals (i.e. carp, crabs, and turtles) have a similar growing period and have similar water and temperature requirements, making it possible to develop a rice–aquatic animal partnership. Although carp, crabs, and turtles differ in biological traits and feeding activities, they play similar roles in increasing rice yield and N-use efficiency, and in stabilizing soil N.

Rice paddies provide food for half of the world’s population (Gross and Zhao, 2014; Edzesi et al., 2016), and also provide other ecosystem services, including groundwater recharge, flood control, water purification, and the conservation of biodiversity, landscapes, and human cultures (Bouman et al., 2007; Natuhara, 2013; Wang et al., 2018). Modern rice farming currently faces the great challenge of how to increase yield while minimizing negative environmental effects (Mueller et al., 2012; Chen et al., 2014). Our study suggests that this challenge can be at least partially met by adding certain species of aquatic animals to rice monocultures. The resulting cocultures could produce more food (rice grain and fish) with less fertilizer and pesticides than rice monocultures. In our field experiment, an average annual aquatic animal yield ranging from 0.52 to 2.57 t ha–1 was produced from the rice fields (Figure 1), suggesting that local farmers can obtain more income from their paddy fields. Moreover, the prices for grain and aquatic animal products from these cocultures were higher than from the local rice monoculture (Hu et al., 2016). Although costs of the cocultures are higher than the costs of monoculture because of the feed input and increased labor required for the management of two species, net income was still higher for cocultures than for monocultures because of the higher prices of the products and the reduced use of fertilizers and pesticides (Hu et al., 2016; Wang et al., 2018). Over the last two decades in China, the increased income has greatly increased farmer enthusiasm for the rice–aquatic animal cocultures (Tang et al., 2020).

While our current study and other previous studies have shown the positive effects of rice-aquatic animal coculture on rice production, farmer income, and soil N, possible negative effects resulting from the input of feed and the increased decomposition rate should be considered. These potential negative effects include eutrophication and increased carbon emission. Previous studies, however, found that rice-aquatic animal coculture would not cause serious N eutrophication in the field when the target aquatic animal yields were set below the following thresholds: 2.11 ± 0.22 t ha–1 for rice-carp, 0.66 ± 0.08 t ha–1 for rice-crab, and 3.62 ± 0.25 t ha–1 for rice-turtle coculture systems (Hu et al., 2013; Zhang et al., 2016; Hu et al., 2016). The effects of rice-aquatic animal coculture on carbon emission (e.g. CH4 emission) varied among reports (Ding et al., 2020; Sun et al., 2021). Some experiments indicated that CH4 emissions were lower in rice–aquatic animal coculture systems than in rice monocultures (Yuan et al., 2009; Sun et al., 2021), while other experiments indicated that CH4 emissions were higher in rice–aquatic animal coculture systems than in rice monocultures (Frei and Becker, 2005a; Wang et al., 2019). These differences in CH4 emission could be caused by differences in aquatic animals, natural environments, and field management (Ding et al., 2020; Dai et al., 2022). In our study, the aquatic animals increased organic matter decomposition in the paddy field (Figure 4), suggesting that the release of nutrients but also of CO2 would be higher with coculture than with monoculture.

In our previous survey, the three typical types of coculture (i.e. rice-carp, rice-crab, and rice-turtle) were compared in terms of financial returns for the farmers. Financial returns were higher for rice-turtle systems (which is the most common type of coculture in south China) than for rice-carp and rice-crab systems (Hu et al., 2016; Wang et al., 2018). In addition to these three types of coculture, other types (e.g. rice-prawn/crayfish, rice-frogs, and rice-ducks) are also practiced in China and in other Asian countries (Hu et al., 2015; Ahmed and Turchini, 2021). In some areas, farmers apply feed to obtain higher animal yield from the cocultures, and the main components of the feed are residues of soybean after the oil has been extracted. Given the millions of hectares of rice fields with suitable conditions for rice-aquatic animal coculture, the cropland available for soybean production and the integration of rice-aquatic animal coculture with soybean production should be taken into account before substantial increases in coculture area are promoted.

Materials and methods

The rice–aquatic animal coculture systems in the study

Request a detailed protocol

Three coculture systems, namely rice-carp, rice-crab, and rice-turtle (Figure 7), were studied. These coculture systems have been developed and adapted to different rice-growing areas in China (Hu et al., 2016). The rice-carp coculture system, for example, has a long history and is widely practiced in south China (Xie et al., 2011; Wang et al., 2018). In recent decades, the rice-crab coculture system has been rapidly developing in northeastern China (Xu et al., 2019), and the rice-turtle coculture system has been rapidly developing in south China (Zhang et al., 2017). In these systems, the aquatic animals (i.e. carp, crabs, and softshell turtles, Figure 7) are partnered with rice plants during the whole rice-growing period (130–150 days) and are harvested every year when the rice matures. These three aquatic animals are economically important, and are usually cultured in fish ponds or paddy fields by local farmers. The meat of all these species is a popular food of the local people. To increase the growth and quality of aquatic animals in coculture systems, farmers often apply feed in the form of pellets (Appendix 2—table 1).

Illustration of the field experiments.

The rice plants and carp (A), crab (B), and turtle (C) were photographed in the corresponding experimental plots (D, E, and F), which were arranged in completely randomized blocks in three rice-planting areas in Qingtian County (G), Panshan County (H), and Deqing County (I), respectively. The red circle in A indicates a rice hill. Photos of A, B and C were taken by Lufeng Zhao, Zhiming Li and Genlian Wang, respectively.

The common carp (Cyprinus carpio) has adapted to paddy habitats and is predominantly used for rice-carp coculture (Figure 7A, Xie et al., 2011; Wang et al., 2018). The growing season for rice-carp systems is usually from late May to early October (ca. 125 days). Carp fry (ca. 40 g each) are often released into the rice field 1 week after rice is transplanted. The carp, which do not feed on rice plants, live in the paddy field until the rice plants are mature, at which time the carp are harvested. In the current study, pellet feed (5.37% N) was applied each day at ca. 6:30 am. The daily amount of feed was initially set as 4% of the fish fresh weight and was increased by 3% every 10 days afterwards. By carp harvest, the total inputs of the feed and feed-N for RA plots were 1.46 t ha–1 and 78.41 kg ha–1, respectively (Appendix 2—table 1). After they are harvested from paddy fields, the carp are directly used as food or are temporary cultured in fish ponds until they are used as food.

The Chinese mitten crab (Eriocheir sinensis Milne-Edwards) is used in the rice-crab system and is also well adapted to the paddy environment (Figure 7B, Xu et al., 2019). In the rice-crab system, the growing season is from middle May to middle October (ca. 150 days). The juvenile crabs (ca. 1.15 g each) are released into rice fields 1 week after rice is transplanted. The juvenile crabs do not feed on rice plants and ‘live’ together with rice plants until harvest. The crabs molt several times during their time in the paddy field. In the current study, pellet feed (4.52% N) was applied once each day at ca. 6:30 am. The daily amount of feed was initially set as 3–5% of the crab fresh weight and was increased by 3% every 10 days afterwards. By crab harvest, the total inputs of the feed and feed-N for RA plots were 1.78 t ha–1 and 80.46 kg ha–1, respectively (Appendix 2—table 1). After they are harvested from the paddy fields, the crabs are used as food or are placed in ponds and are used as a source of crabs for the next year.

The Chinese softshell turtle (Pelodiscus sinensis) is often cultured in paddy fields by local farmers in southeastern China (Figure 7C). The turtles are omnivores but prefer animal diets (He, 2017). In this rice-turtle system, the growing season is from middle June to early November (ca. 140 days). The baby turtles (ca. 150 g each) are released into the field 1 week after rice is transplanted. The turtles remain with the rice plants for the whole rice-growth period. In the current study, pellet feed (6.51% N) was applied twice per day at ca. 7:00 am and 5:00 pm throughout the coculture period. The daily amount of feed was initially set as 0.5%–1.0% of the turtle fresh weight but was increased as the turtles grew. By turtle harvest, the total inputs of the feed and feed-N for RA plots were 1.62 t ha–1 and 105.46 kg ha–1, respectively. (Appendix 2—table 1). After they are harvested from paddy fields, the turtles are used as food or are temporarily cultured in fish ponds until they are used as food.

Ethics statement

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In the following experiments, the samples of all aquatic animals (i.e. carp, crabs, and softshell turtles) were collected and measured in accordance with the approved guidelines of the Zhejiang University Experimental Animal Management Committee (reference number SYXK(Zhe)2018–0016). Details on the handling of animal samples were described in the Methods section.

Field experiments

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Each of three field experiments (one each for rice-carp, rice-crab, and rice-turtle systems) was conducted for 4 years (2017–2020) at a site (one system per site) where the particular system was widely practiced. The three sites are described in the supporting information (see Appendix 3).

All three experiments used a completely randomized block design with two treatments: rice monoculture (RM) and rice-aquatic animal coculture (RA) with feed addition for aquatic animal (Appendix 2—table 1). Each treatment at each site was represented by six replicate plots with a size of 0.01 ha per plot for rice-carp and rice-crabs, and 80 m2 per plot for rice-turtles. No chemicals were used to control weeds, pests, or diseases during the experiments. The plot size and detailed experimental procedures are described in Appendix 4.

In 2018, the stable-isotope 13C labeling method was used to determine the organic matter decomposition rate in the three field experiments (Cheng et al., 2012). ‘Litter tubes’ containing maize (Zea mays L.) leaves enriched with 13C (δ13C –13.6‰) were used as described in Appendix 5. The percentage of 13C remaining in the litter tubes after 40 and 80 days represented the decomposition rate.

We used a stable isotope (13C and 15N) technique to determine how the aquatic animals (i.e. carp, crabs, or turtles) used food resources (e.g. weeds, zoobenthos, zooplankton, and phytoplankton) in the field (Michener and Schell, 1994). During the rice growing period in 2019, we collected living organisms that were consumed by the aquatic animals in each of the three experiments (Caut et al., 2009; Guo et al., 2016) (see Appendix 6). Before and after the experiments, we also collected muscles from the aquatic animals (carp, crabs, or turtles) (see Appendix 7).

We ground all dried samples of food sources and aquatic animals and analyzed their isotopic δ value and content (13C and 15N). The δ value was calculated as ([Rsample /Rstandard]−1) × 1000, where R represents 13C:12C or 15N:14N (Peterson and Fry, 1987). Dietary contributions of input feed and potential food sources from the rice field were analyzed by stable isotopic dietary reconstruction with the R package ‘siar’ (Phillips and Gregg, 2003; Phillips et al., 2005; Parnell and Jackson, 2011; R Core Team, 2021). The discrimination factors of 13C and 15N for carp, crabs, and turtles in dietary reconstruction were previously determined (see Appendix 8).

Three weeks before rice was harvested in each experimental year (from 2017 to 2020), 5 quadrats (1 m2) were randomly placed in each plot to evaluate weed infestation. For each quadrat, the aboveground dry weed biomass was measured.

In each experimental year (from 2017 to 2020), rice and aquatic animals were harvested from the whole experimental plots when rice plants matured. Rice yield was determined by manually harvesting entire plots. Rice grain was air-dried and weighed. Rice yield was estimated as tons of air-dried grain per ha. One week before rice was harvested, aquatic animals were collected from entire plots by driving them into the corner of field as the water was drained. Yield was expressed as tons of fresh aquatic animal biomass per ha.

At harvest in each year of the experiments, samples of rice plants and aquatic animals were collected for determining N content. Five hills of rice were collected in each plot. The separated grain and straw were oven-dried at <65℃ to a constant weight. The aquatic animal samples were kept in water for 24 hr to permit the emptying of intestinal contents. The clean aquatic animal samples were oven-dried at 105℃ to a constant weight. Both rice and aquatic animal samples were ground with a ball mill (RETSCHMM 400, Germany). The N content in rice straw and grain and in the aquatic animals was measured by the Kjeldahl method (Bremmer and Mulvaney, 1982).

Every experimental year, soil samples (0–20 cm depth) were collected immediately after harvest from each plot. All soil samples were air dried. Soil organic matter (SOM) content was determined by the K2Cr2O7 oxidation method, and total nitrogen (N) content was determined by the Kjeldahl method (Lu, 1999).

We used the data collected in 2018 to estimate apparent N-use efficiency (ANUE) by calculating percentage of the input N that was used by rice and aquatic animals (Moll et al., 1982; Mayer et al., 2015; Zhang et al., 2017) as follows:

(1) ANUE %=NyNs×100

where Ny is the total amount of N contained in the grain and straw of rice plants, and in the aquatic animals that were removed from the paddy system, and Ns is the total input of fertilizer-N and feed-N. Ny was determined by multiplying the biomass of rice (grain and straw) and aquatic animals by the percentage of N in rice and aquatic animals. We assumed that the natural N input (e.g. N fixed by bacteria, N in the irrigation water, and atmospheric N deposition) was similar between RM and RA plots, and natural N input was therefore not included in our estimations of ANUE.

Statistical analysis was conducted using the GLM in SPSS (V.20.0, RRID: SCR_002865). All data were subjected to a homogeneity test. If the data did not meet the assumptions of normality and homogeneity, they were log-transformed before analysis. For each field experiment, ANOVAs with a split-plot design (i.e. treatment RM and RA as the main plots and experimental years as the sub-plots) were performed on rice yields, total soil N content, ANUE, and weed biomass. For RM or RA plots, total N in the soil at the beginning and end of the experiment were compared by using paired t-tests (SPSS V.20.0, RRID: SCR_002865).

Mesocosm experiments

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To determine whether unconsumed and unassimilated feed-N is used by aquatic animals in rice-carp, rice-crab, or rice-turtle systems, we conducted three independent mesocosm experiments (one for each kind of system) at the Experimental Station of Zhejiang University in Deqing County, Zhejiang Province (30°33′N, 119°32′E.). The mesocosm experiments were conducted in 2019. The fate of feed-N was traced by using stable-isotope 15N-labeled feed in each mesocosm experiment.

For rice-carp and rice-crab experiments, each mesocosm was a cylindrical, 1017 L plastic stock tank. Intact soil (300 L) from a corresponding rice field was added to each mesocosm to a depth of 30 cm. The mesocosms were placed in a rice paddy field (the corresponding fields that were used for the three field experiments) so that their bottoms were 20 cm below the soil surface, and the top of each microcosm was above the water line. For the rice-turtle system, 2 m × 2 m plots were established in a rice paddy field. Each plot had an independent water inlet and outlet and was separated from water mixing by concrete ridges.

Each of the three mesocosm experiments had a completely randomized block design with six replicate blocks. The treatments were rice monoculture (RM) and rice-aquatic animals coculture (RA). For the RM and RA, varieties of rice and aquatic animal species were the same as in the field experiment. The detailed procedures for each mesocosm experiment are provided in Appendix 9.

Because soybean is the major raw ingredient of feeds for the three aquatic animals (carp, crabs, and turtles), we first labeled soybean powder with 15N (see Appendix 10). We then mixed the labeled soybean powder with the general feed of carp, crabs, and turtles as indicated in Appendix 2—table 3.

At harvest, samples of rice plants, aquatic animals (carp, crabs, and turtles), and soil were collected from each mesocosm (see Appendix 11). The 15N content in all samples of rice plants, aquatic animals, and soil was quantified with a ThermoFinnigan DELTA Plus continuous flow isotope ratio mass spectrometer.

We calculated the contribution of feed-N to total rice biomass-N with a linear mixing model (Phillips and Gregg, 2003):

(2) δ15NRM×a+δ15Nfeed×b=δ15NRA
(3) a+b=1
(4) b=(δ15NRAδ15NRM)/(δ15Nfeedδ15NRM)

where a is the contribution of soil N to rice total biomass-N; b is the contribution of feed-N to rice total biomass-N; δ15NRM is the δ15N value of the rice plants in the RM treatment; δ15Nfeed is the δ15N value of 15N-labeled feed; and δ15NRAS is the δ15N value of rice plants in the RA treatment. For RM or RA mesocosms, δ15N and total N in the soil at the beginning and end of the experiment were compared by using one-tailed t-tests under the assumption that δ15N and total N in the soil would increase after the experiments (SPSS V.20.0, RRID: SCR_002865).

Appendix 1

Appendix 1—figure 1
Total soil N content in rice monoculture (RM) and coculture treatments (RA) before and after the field experiments.

RM: rice monoculture; RA: rice coculture with an aquatic animal. Values are means ± SE (n = 6), ns indicates no significant difference between RM and RA (p > 0.05).

Appendix 1—figure 2
δ15N of rice shoots in rice monoculture (RM) and coculture treatments (RA) in the mesocosm experiments.

RM: rice monoculture; RA: rice coculture with an aquatic animal. Values are means ± SE (n = 6), * indicates a significant difference between RM and RA (p < 0.05).

Appendix 1—figure 3
Total soil P content in rice monoculture (RM) and coculture treatments (RA) before and after the field experiments.

RM: rice monoculture; RA: rice coculture with an aquatic animal. The P contents in soil were analyzed with a San++ Continuous Flow Analyzer (Skalar, Netherlands) after the air-dried soil samples were and digested by the K2SO4-CuSO4-Se method (Lu, 1999). For RM or RA plots, total P in the soil at the beginning and end of the experiment were compared by using paired t-tests (SPSS V.20.0, RRID: SCR_002865). Values are means ± SE (n = 6), ns indicates no significant difference between RM and RA (p > 0.05).

Appendix 2

Appendix 2—table 1
Input of N and P into rice-animal coculture systems via feed and fertilizer application.
SystemTreatmentNitrogen input (kg ha–1)Phosphorus input (kg ha–1)
FeedFertilizer*FeedFertilizer
Base fertilizerTop-dress fertilizerTotalBase fertilizerTop-dress fertilizerTotal
Rice-carpRM---82.5029.40111.90---36.025.3141.33
RA78.4182.50---82.5019.6336.02---36.02
Rice-crabRM---112.544.94157.44---49.1211.7960.91
RA80.46112.5---112.5020.6249.12---49.12
Rice-turtleRM---82.544.94127.44---36.0211.7947.81
RA105.4682.5---82.5036.6136.02---36.02
  1. *

    The total amount of feed-N and - P input were determined by multiplying the amount of feed by the percentage of N and P in feed that were analyzed with a San++ Continuous Flow Analyzer (Skalar, Netherlands) (Lu, 1999).

Appendix 2—table 2
The total P removed with the harvested products under rice monoculture and coculture in the field experiment.

Values are means ± SE (n = 6)

SystemTreatmentRice grain*(kg ha–1)Rice straw*(kg ha–1)Aquatic animal*(kg ha–1)Total(kg ha–1)
Rice-carpRM18.53 ± 0.925.49 ± 0.25---24.02 ± 1.15
RA21.68 ± 0.506.44 ± 0.327.45 ± 0.9535.57 ± 0.96
Rice-crabRM26.38 ± 1.538.60 ± 0.67---34.98 ± 1.87
RA34.74 ± 2.1913.67 ± 1.041.07 ± 0.1549.48 ± 2.79
Rice-turtleRM19.57 ± 0.7212.64 ± 0.44---32.21 ± 0.42
RA24.19 ± 1.1717.19 ± 0.6920.66 ± 1.4762.04 ± 2.60
  1. *

    The total amount of P contained in the grain and straw of rice plants, and in the aquatic animals were determined by multiplying the biomass of rice (grain and straw) and aquatic animals by the percentage of P in rice and aquatic animals that were analyzed with a San++ Continuous Flow Analyzer (Skalar, Netherlands) (Lu, 1999).

Appendix 2—table 3
Properties of original feed and 15N-labeled feed for the three field experiments.

The original feed (in which most N was supplied by soybean powder) was supplemented with 15N-labeled soybean powder to obtain 15N-labeled feed.

ExperimentOriginal feed15N-labeled soybean powder15N-labeled feed% of labeled soybean powder in the 15N-labeled feed
N%δ15NN%δ15NN%δ15N
Rice-carp5.581.736.611,5395.2564933
Rice-turtle8.218.416.984457.5412830
Rice-crab5.941.196.984457.2714533

Appendix 3

Sites of the field experiments

The rice-carp (common carp, Cypinius carpia) field experiment was conducted in Qingtian County (120°16′02″E, 28°01′24″N), Southeast Zhejiang Province. The rice-carp system in that hilly and mountainous county has a history of more than 1000 years and exclusively cultures an indigenous breed of common carp, which was used for our experiment. The experimental site has a subtropical monsoon climate (mean annual temperature: 17.5 °C, mean annual rainfall: 1432 mm) and an acidic sandy loam soil (total organic matter: 30.72–32.92 g kg–1, total N: 2.09–2.79 g kg–1, pH: 5.4).

The rice-crab (Chinese mitten crabs, Eriocheir sinensis) field experiment was conducted in Panshan County, Liaoning Province (41°9′24″N, 122°15′1″E). Because of its proximity to the Bohai Sea, this province provides natural habitats for the breeding and growing of Chinese mitten crabs. The experimental site has a monsoon continental climate (mean annual rainfall: 620 mm, mean annual temperature: 9.2℃) and a loamy soil (total organic matter: 23.08–29.53 g kg–1, total N: 1.42–1.66 g kg–1, pH: 8.2).

The rice-turtle (soft-shelled turtle, Pelodiscus sinensis) field experiment was conducted in Deqing County, Northern Zhejiang Province (120°7'30"E, 30°37'42"N). The soft-shelled turtle used for the experiment is indigenous breed to that flat, open county. The experimental site has a subtropical monsoon climate (mean annual rainfall: 1379 mm, mean annual temperature: 14℃) and a sandy loam soil (total organic matter: 30.72–32.92 g kg–1, total N: 2.09–2.79 g kg–1, pH: 5.4).

Appendix 4

Field experiment details

For the rice-carp field experiment, we randomly assigned one RM plot and one RA plot (0.01 ha per plot) to each of six blocks. We also separated field plots by 50-cm-high concrete brick walls. Independent water inlets and outlets were setup for each plot to prevent interference between plots. Rice (ZhongZheYou No. 8) was transplanted 4 weeks after germination, with a spacing of 30 cm × 30 cm (one seedling per hill). Two days before rice transplanting, a compound fertilizer (15% N, 15% P2O5, 15% k2O) used as basal fertilizer was broadcast at the rate of 550 kg ha–1 for both treatments. No top-dress fertilizer was applied for RA, but urea (46% N) at the rate of 37.5 kg ha–1 and compound fertilizer (15% N, 15% P2O5, 15% K2O) at the rate of 81 kg ha–1 were used as top-dress fertilizer for RM during the rice growth period (Appendix 2—table 1). No pesticide was applied for both treatments. During the rice growth period, all plots were continuously flooded at about 20 cm depth. Six days after rice transplanting, we released 60 carp fry (ca. 40 g each, purchased from a local farmer) into each RA plot. Pellet feed (5.37% N) was added each day at ca. 6:30 am to feed the carp fry. The daily amount of feed was initially set as 4% of the fish fresh weight and was increased by 3% every 10 days afterwards. By carp harvest, the total inputs of the feed and feed-N for RA plots were 1.46 t ha–1 and 78.41 kg ha–1, respectively.

For the rice-crab experiment, we randomly assigned one RM plot and one RA plot (0.01 ha per plot) to each of six blocks. We also separated the plots with 50-cm-high PVC boards. Independent water inlets and outlets were setup for each plot to prevent interference between plots. Rice plants (Yanfeng No. 47) were transplanted into each plot 4 weeks after germination at a spacing of 30 cm × 30 cm (one seedling per hill). Two days before rice transplanting, a compound fertilizer (15% N, 15% P2O5, 15% k2O) used as basal fertilizer was broadcast at the rate of 750 kg·ha–1 for both treatments. No top-dress fertilizer was applied for RA, but urea (46% N) at the rate of 39 kg ha–1 and compound fertilizer (15% N, 15% P2O5, 15% K2O) at the rate of 180 kg ha–1 were used as top-dress fertilizer for RM during the rice growth period (Appendix 2—table 1). No pesticide was applied for both treatments. One week after rice transplanting, 375 g of juvenile crabs (ca. 1.15 g each) were released into each RA plot. During the experiment, pellet feed (4.52% N) was applied once each day at ca. 6:30 am. The daily amount of feed was initially set as 3%–5% of the crab fresh weight and was increased by 3% every 10 days afterwards. By crab harvest, the total inputs of the feed and feed-N for RA plots were 1.78 t ha–1 and 80.46 kg·ha–1, respectively.

For the rice-turtle experiment, we randomly assigned one RM plot and one RA plot (8 m × 10 m per plot) to each of six blocks. The plots were separated by 1.5-m-high concrete ridges. Independent water inlets and outlets were setup for each plot to prevent interference between plots. Rice (Qing-Xi No. 8) was transplanted into each plot 4 weeks after germination, at a spacing of 30 cm × 30 cm (one seedling per hill). Two days before rice transplanting, a compound fertilizer (15% N, 15% P2O5, 15% k2O) used as basal fertilizer was broadcast at the rate of 550 kg ha–1 for both treatments. No top-dress fertilizer was applied for RA, but urea (46% N) at the rate of 39 kg ha–1 and compound fertilizer (15% N, 15% P2O5, 15% K2O) at the rate of 180 kg ha–1 were used as top-dress fertilizer for RM during the rice growth period (Appendix 2—table 1). No pesticide was applied for both treatments. One week after rice transplanting, 8 young turtles (ca. 150 g each) were released into each RA plot. Pellet feed (6.51% N) was applied twice per day at about 7:00 am and 5:00 pm throughout the experiment. The daily amount of feed was initially set as 0.5%–1.0% of the turtle fresh weight but was increased as the turtles grew. By turtle harvest, the total inputs of the feed and feed-N for RA plots were 1.62 t ha–1 and 105.46 kg ha–1, respectively.

Appendix 5

Method for testing organic matter decomposition rate

As a C4 plant, maize (Zea mays) is naturally 13C-enriched as compared to C3 plants like rice. We therefore used maize tissue to determine the decomposition rate of organic matter in our experiment. Maize was planted in a loam soil and was harvested just before flowering. Maize leaves were dried and cut into pieces ( < 1.0 cm2). About 0.5 g of the leaf segments, mixed with 50 g of sands, was added to a tube (10 cm long, 5 cm in diameter), the ends of which were covered with a 20 μm mesh; this resulted in a “litter tube”, which was analogous to a litter bag. The initial δ13C contents in the litter tubes are listed in Appendix 5—table 1.

Appendix 5—table 1
Initial δ13C contents in materials used to test the decomposition rate in the three field experiments.
ExperimentC (%)δ13C13C (mg)
rice-carp0.41–16.212.18
rice-crab0.44–16.242.39
rice-turtle0.43–15.622.33

In each plot, we randomly buried 20 tubes at 0–10 cm soil depth in the plant row and harvested them after 40 and 80 days. On each sampling date, we retrieved five tubes from each plot. The material (sample) in the tubes was air-dried and ground (RETSCHMM 400, Germany). Carbon content and the ratio of 13C to 12C were determined using a continuous flow isotope ratio mass spectrometer (CF-IRMS, ThermoFinnigan DELTA Plus, Waltham, MA, USA). The 15C remaining was expressed as a percentage of 15C in the initial materials.

Appendix 6

Method for sample collection of aquatic animal food sources

In 2019, we collected all possible aquatic animal food sources in the field, including weeds, algae, azollaceae, phytoplankton, zooplankton, zoobenthos, and particulate organic matter (POM). Weeds, algae, and azollaceae were collected, rinsed, and oven-dried. Zoobenthos collected from 0 to 20 cm topsoil were placed in distilled water for 48 hr. Zooplankton were collected from field water with a 0.064 mm nylon net. To collect phytoplankton and POM, field water was first passed through the nylon net; the phytoplankton and POM were then collected on glass fiber filters (GF/C, Whatman, pre-combusted at 450℃) via vacuum-filtration in the laboratory. All samples were oven dried at 50℃ and stored until analyzed.

Appendix 7

Method of aquatic animal sample collection

Before and after the experiments in 2018, carp, crabs, or turtles were randomly sampled and stored at –20℃. Muscles were peeled from every animal sample, oven dried at 50℃, and separated into two sub-samples. One sub-sample was directly used for 15N analysis, and the other was used for 13C analysis after being pretreated with 2:1 chloroform and methanol for lipid removal.

Appendix 8

Determination of the discrimination factors of 13C or 15N

In the dietary reconstruction, the discrimination factor (△) is the difference in δ13C or δ15N values between food sources and food consumers (Peterson and Fry, 1987). In our study, △15N and △13C were 2.73‰ and 1.71‰, respectively, for carp (Hu et al., 2015), and were 2.75‰ and 0.75‰, respectively, for crabs and turtles (Caut et al., 2009).

Appendix 9

Mesocosm experiments

For the rice-carp mesocosm experiment, five hills of rice plants (one seedling per hill) were planted in each mesocosm. One week after rice transplanting, five fish fry (ca. 10 g each) were added to each mesocosm. A water level of 25 cm was maintained during the experiment, with pH 6.0–6.5, NO2- < 0.01 mg L–1, and NH4+ < 0.2 mg L–1. No fertilizer was applied. At 4 days after the fry were introduced and on every day of the experiment thereafter, the 15N-labeled feed was applied twice per day (at 7:00 and 17:00). Throughout the experiment, the total amount of feed applied was 126 g m–2.

For the rice-crab mesocosm experiment, four hills of rice plants (three rice seedlings per hill) were transplanted into each mesocosm. One week after rice transplanting, 50 juvenile crabs (ca. 1.22 g each) were released into each mesocosm. A water level of 25 cm was maintained during the experiment, with pH 8.0–8.5, NO2- < 0.1 mg L–1, and HN4+ < 0.5 mg L–1. At 3 days after crab release and on every day of the experiment thereafter, the 15N-labeled feed was applied once per day at 18:00. Throughout the experiment, the total amount of feed applied was 124 g m–2.

For the rice-turtle mesocosm experiment, four hills of rice plants (three rice seedlings per hill) were transplanted into each mesocosm. One week after rice transplanting, 14 young turtles (ca. 3.5 g each) were released into each mesocosm. A water level of 30 cm was maintained during the experiment, with pH 8.0–8.5, NO2- < 0.1 mg L–1, and NH4+ < 0.5 mg L–1. Four days after turtle release and on every day of the experiment thereafter, the 15N-labeled feed was applied twice each day (at 6:00 and 16:00). Throughout the experiment, the total quantity of feed applied was 130 g m–2.

Appendix 10

Method of obtaining soybean powder labeled with15N

Soybean plants were planted in pots in a green house. On 3 days during the growing period, 15N-enriched (NH4)2SO4 (99.7% 15N) was added to the soil at the rate of 5 mg N kg–1 soil. After they were harvested, soybeans were oven-dried at 65℃ and ground into powder, which was used as the 15N labeling ingredient in the 15N-enriched feed.

Appendix 11

Sample collecting and preparing for rice, aquatic animals, and soil

At harvest, the aboveground portions of rice plants on one hill from each mesocosm were separated into grain and straw and were oven-dried at 65 °C to a constant weight. Samples of carp, crabs, or turtles were randomly collected from each mesocosm. These aquatic animal samples were placed in clean water for 48 hr to empty their guts and were stored at –80 °C after being freeze-dried. Soil samples (0–20 cm depth) were collected from each mesocosm before and after the experiment and were air-dried.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1–6 and Appendix 1—figure 1–3.

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    Research of Agricultural Modernization 39:875–882.

Decision letter

  1. Bernhard Schmid
    Reviewing Editor; University of Zurich, Zurich, Switzerland
  2. Detlef Weigel
    Senior Editor; Max Planck Institute for Developmental Biology, Tübingen, Germany
  3. Bernhard Schmid
    Reviewer; University of Zurich, Zurich, Switzerland

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Decision letter after peer review:

Thank you for submitting your article "Using aquatic animals as partners to increase yield and soil nitrogen in the paddy ecosystem" for consideration by eLife. Your article has been reviewed by 4 peer reviewers, including Bernhard Schmid as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Detlef Weigel as the Senior Editor.

Essential Revisions:

1) Statistical analysis: it seems this was done correctly, e.g using years as subplots and not as independent measurements. However, this should be explained in more detail by presenting nominator and denominator degrees of freedom for F-tests and number of replicates in figures or legends.

2) Report amount of N (and P?) input via animal feed and arriving in soil. Discuss this in relation to other N in system and potential consequences of added P.

3) Discuss broader consequences of rice-animal co-culture in terms of economic costs/benefits to farmer, potential side effects on land used to produce animal feed and on nutrient and carbon cycle (including greenhouse-gas emissions).

4) Explain in more detail how animals were treated, grew (one or several years), affected rice plants and were "harvested" (every year all animals?).

5) It would be nice to include pictures of some experimental plots in the display material. Some graphs could be improved by extending y-axes only as far as data points occur.

Reviewer #1:

This is a well conducted experimental study in which rice monocultures are compared with rice-aquatic animal co-cultures over multiple years. Co-culture increases plant yield and adds animal yield, but requires extra input of animal feed. Overall co-culture seems to benefit yield and sustainability.

It would be interesting to discuss the broader consequences of the experimental results for application, e.g. potential costs of the additional animal feed (and additional labor?) required for the co-culture, farmer income, other ecosystem services including eutrophication and greenhouse-gas emissions.

The following are points that should be addressed in revising the manuscript:

1) Statistical analysis: it seems this was done correctly, e.g using years as subplots and not as independent measurements. However, this should be explained in more detail by presenting nominator and denominator degrees of freedom for F-tests and number of replicates in figures or legends.

2) Report amount of N (and P?) input via animal feed and arriving in soil. Discuss this in relation to other N in system and potential consequences of added P.

3) Discuss broader consequences of rice-animal co-culture in terms of economic costs/benefits to farmer, potential side effects on land used to produce animal feed and on nutrient and carbon cycle (including greenhouse-gas emissions).

4) Explain in more detail how animals were treated, grew (one or several years), affected rice plants and were "harvested" (every year all animals?).

5) It would be nice to include pictures of some experimental plots in the display material. Some graphs could be improved by extending y-axes only as far as data points occur.

Reviewer #2:

This manuscript reports interesting results from a rigorous set of experiments, each with six true replicates of each treatment randomized to experimental units. The authors aimed to compare rice monocultures to co-cultures of rice and an aquatic animal (carp, crab, or turtle). The data analyses appear adequate, though I do request the degrees of freedom for F-tests to double check that the repeated measures analysis was properly implemented. The findings are of widespread importance, given that rice is one of the most widely consumed crops worldwide. Increasing the yield of rice through co-culture may reduce the demand of land for rice production, leaving more land for non-human species.

I have two main suggestions for the authors, which I hope will help further improve their manuscript. First, it is necessary to clarify in visible places, including the Abstract, that feed was added in co-culture. This confounds, to some extent, the extra nutrient inputs in the feed with the comparisons between monocultures and co-cultures. It might also help to include further discussion of the extent to which the increased rice yield is likely explained by each of these two things: the animals or the nutrient inputs in the feed. The diet analyses help get at this and are another strength of this study.

Second, it would help to also briefly discuss the broader implications of these results for land use and climate change. Currently, the manuscript presents only the positive outcomes. Would changing from monocultures to co-cultures likely lead to more carbon emissions? The decomposition results suggest that this should be further investigated. Would changing from rice monocultures to co-cultures likely lead to more demand for cropland, given that soybean feed source for the aquatic animals? These implications of the results deserve a bit more attention in the Discussion section.

Title

I recommend removing 'soil nitrogen' from the title, given the non-significant results shown in Figure 1b.

Abstract

L44: here or elsewhere in the Abstract, please include a few words to let readers know that there were also feed inputs.

Introduction

L115: It would help to use a different word here, rather than 'functions' because some of the previous studies, including those in the preceding sentence, considered ecosystem functions. Also, I tend to find it more compelling when studies explain why a knowledge gap needs to be filled, rather than stating that it exists. Many things are unknown. Why should we know more about this particular knowledge gap?

L129-131: This argument seems sound if these animals do not eat the rice plants. Do these aquatic animals eat any parts of the rice plant?

Results

L147: Here and throughout, please include the numerator and denominator degrees of freedom for the F-tests. This will help readers check that the split-plot-in-time repeated measures analysis was correctly implemented.

Figure 1: Note typo at top of Figure one: carb should be crab, I believe

Figure 1: Yield should have units of mass per unit area per unit time.

The diet analyses really strengthen this study. Nicely done.

Discussion:

L288, see also Dirzo et al., 2014 Science

L294-301: Here and elsewhere, it would help to have further discussion of the implications of the results. Note that this enhanced decomposition would not only contribute to the benefit of N recycling, but would also affect the C cycle in a potentially undesirable manner.

L314-316: I am not sure that I fully understand the proposed mechanism here. Is this interpretation similar to the arguments made by McNaughton et al., (1997 Science)? It would help readers to add a bit more explanation here.

L333-334: here and in other visible places, such as the abstract, I believe it is necessary to also note that feed was added.

Materials and methods:

L338: Somewhere early on in the Methods section, please clarify how much feed was added and in what form(s).

L341-352: It would help to clarify here how each of these animal species is used, given that the production of their biomass is presented as a co-benefit alongside increased rice yield. For example, is the meat of all these species consumed by people for food?

L364-365: Do farmers typically apply pesticides? If so, then it would be interesting to include a sentence in the Discussion clarifying how the weed suppression by the animals compares, on a percentage basis, to weed suppression by pesticides.

L411: Shouldn't this percentage also be multiplied by the percentage of N in rice and aquatic animals that came from the fertilizer/feed?

L414: Please clarify how much (mass per unit area or per experimental uint) N was added as fertilizer and feed.

L443-446: This is helpful information about the feed, but more information is needed (e.g., how much N was added in the feed?) and it should be presented much earlier, as requested in comments above.

Appendix 2

L793-801: I see this helpful information here. Perhaps just move or repeat this information above, where requested in the Methods section. It will be difficult for some readers to find these important details in this appendix.

Reviewer #3:

Agricultural intensification has increased yields but has also negatively affected the environment. Food production needs a comprehensive overhaul in terms of sustainability, with better approaches to producing safe and healthy food, while leaving little footprint. Globally, chemical fertilizers, herbicides and pesticides are rolled back as part of our commitment to the Sustainable Development Goals (SDG) and the urgent need for a greener renaissance in sustainability (SDG 2, 12 and 13).

The coupling of two agricultural systems (i.e., integrated agriculture-aquaculture or integrated crop-livestock) have been proposed as one of the important ways that reduce the negative effects of food production on the environment: using lesser chemical fertilizers, no herbicide was needed for weed suppression, uneaten animal feed serving as nutrients for rice, no eutrophication, etc. While the idea of harnessing co-cultivation of plant-animal system for food production is great and beneficial to the environment, the availability of large-scale and long-term experimental data is lacking. Thus, this manuscript is an important contribution to the literature on integrated agriculture and aquaculture. The science in the manuscript is well thought out and executed and the manuscript is well written. The central research question – can yields in rice and aquatic animals be maintained in coupled rice-"animal" ("animal" refers to carps, crabs and turtles, collectively) systems, is timely.

The authors described their long-term and integrated agriculture-aquaculture experimental systems and the results of the multi-year study highlighted a 'solution' to the problem of historical environmental impacts caused by our current and common intensive food production systems (mainly monocultural).

The results suggested that the coupling of rice with aquatic animal production could achieve synergies between food production systems while reducing their associated environmental impacts per unit of production.

Going beyond the science, to help policy makers to better appreciate the co-cultivation strategy, a mention of the comparative financial returns (for the farmers) should perhaps be provided by the authors in their China-wide research? Such analyses (financial returns from selling rice in monocultures, selling rice and aquatic animals together) would strengthen the case for supporting various options: the crab-rice versus fish-rice co-cultivation systems in certain localities. Perhaps in certain situations (e.g. seasonality, lack of farm labour, brackish water issues), the other aquatic animals (prawns/amphibians/ducks) could be the preferred approach?

It is interesting to note that in China, rice yields were similar or higher using the rice-"animal" co-cultivation systems than under monocultures of rice. To help the readers, I would recommend that some photos for the coupled rice-"animal" ("animal" refers to carps, crabs and turtles, collectively) systems be included as a figure in this paper. The alternate is to provide some illustration to highlight the coupled rice-"animal" systems.

With the strong focus of this paper on N, which appeared to be an important (or dominant driver) factor, we still cannot rule out the plausible influence of animal manures releasing additional phosphorus (P) to drive rice growth. Thus, is there some baseline data about the dynamics in the soils and irrigation waters among the research sites? Or from past literature about P dynamics in relation to the present or absence of crabs/fishes/turtles, within a certain level of N? Current plant physiological papers, especially those involving rice growth and yield had frequently mentioned the dual roles of N and P? Some comments from the authors about P would be appropriate with reference to soil fertility and health

Other salient observations, suggestions and notes:

Importantly and across all the three co-cultivation systems, there was no requirement for weed control via herbicides. This salient finding could be included in the proposed figure,

The paper is well written and organised in a logical fashion in line with the given guidelines. If possible, some proof-reading is needed to further improve the English language.

The literature cited are relevant and updated. Appropriate statistical methods were used by the authors to analyse the data.

The sample size, field survey approaches, apparatus utilised and chemical analyses (especially stable isotopes using the IRMS, Isotope Ratio Mass Spec) were appropriate.

Reviewer #4:

This study by Guo et al., is set in the framework of a highly interesting system: the coculture of different species in agricultural production. In this case, not different plant species are grown together (as typically done in intercropping) but aquatic animals (carp, crabs and turtles) are cultured together with rice. Using three multi-year field experiments (one experiment for each combination between one animal species and rice plants) and three corresponding field mesocosm experiments, the authors found that generally, rice yield and apparent nitrogen-use efficiency were higher and weed infestation of the rice paddies lower in coculture with animals, compared with rice monocultures. These results are attributed to the additional feed-N unused by the animals that was shown to be taken up by plants, as well as likely feeding on insect pests and weeds by the animals and higher decomposition (also measured in the experiment) in the coculture plots. Overall, these results provide support for multispecies systems in rice production. Additionally, animal yield was measured in this study and adds to the increased economic advantage of such a system compared with rice monocultures.

This study is interesting for both, ecologists with an interest in biodiversity effects and food webs, and for agronomists aiming to increase yield while supporting agricultural diversity and limiting synthetic inputs at the same time. It is a very valuable applied study that underlines the value of diversity in agriculture. I appreciated the interesting combination of organisms across trophic levels in this culture. I also found it fascinating that this old cultural form of agriculture has a strong relevance for today's production schemes which aim for low input of synthetic fertilizers and pesticides and high organismal diversity in agricultural areas.

The experimental design of the study is clear and simple. It has relatively low power (n=6 for each treatment) but the experiments were done across several years, increasing the applicability of the results. The authors measured many relevant variables (e.g. nitrogen use efficiency) and even use C and N labelling to follow the fate of different elements in the system.

The results of the study are well supported and indicate that the coculture with animals indeed has many advantages. However, I missed a discussion of caveats and potential disadvantages of this coculture method. As far as I can see, the authors did not determine whether rice plants were destroyed or fed upon by the animals. Furthermore, coculture may pose logistical challenges such as a higher effort in management or harvest of two species that may increase costs to farmers and may make them reluctant to use this method. In addition, total nitrogen that entered the system was different between treatments (because no animal feed was used in the rice monocultures, I think), so this additional input may account for differences in yield, even if no animals are used.

I have a few major points regarding potential improvements to the analysis and presentation of the data.

Statistical analysis: I struggled to understand how the data was analysed. Was year nested within plot? Or was the experiment completely restarted every year and the data points across years can be considered independent replicates (I think they cannot, see also the temporal development in Figure 1,2)? I was also confused at times about the sample sizes behind the figures and when averages across years or replicates were used. Please be more specific here (e.g. state n in legends or on figure panels or in the text). Generally, many tests are being used where one model (testing several explanatory variables, e.g. RA/RM, time and their interaction) would likely have been the better solution.

Ethical considerations: The manuscript contains an ethics statement. However, there are few methodological details regarding the treatment of animals in the study. For example, I assume that the animals were killed in some way before drying/freezing at -20{degree sign}? How were they "harvested"?

Methodological details: I missed some details on the general method for non-experts of this culture technique. Most importantly: In this system, is harvest of the aquatic animals done every year? How long is the usual growing season? Do the animals grow as fast as rice? Even the turtles? Is the amount of fertilizer added in the experiment (550-750kg/ha) similar to what would normally be added to regular rice monocultures?

https://doi.org/10.7554/eLife.73869.sa1

Author response

Essential revisions:

1) Statistical analysis: it seems this was done correctly, e.g using years as subplots and not as independent measurements. However, this should be explained in more detail by presenting nominator and denominator degrees of freedom for F-tests and number of replicates in figures or legends.

Thank you very much for the comments.

(1) In our field experiments, each treatment rice monoculture (RM) or rice- animal coculture (RA) was conducted in same experimental plot every year during the 4 experimental year. We thus used years as subplots and not as independent measurements in the statistical analysis.

(2) We checked all the results of the statistical analysis. The nominator and denominator degrees of freedom for F-tests or T-tests have been provided in text. The number of replicates have been provided in legends. Please see line 151-152, line 159-169, line 177-179, line 204-209, line 221-222, line 234-243, line and Figure 1-2, Figure 4-6 of legends.

2) Report amount of N (and P?) input via animal feed and arriving in soil. Discuss this in relation to other N in system and potential consequences of added P.

Thank you very much for the comments. In the revision, the amount of N and P input via animal feed were applied in Appendix 2- table 1. Soil P before and after experiment in both RM and RA treatment were also provided in Appendix 1- figure 3. Because P is not our focus in this study, we did not present the data of P in the main text. But we had a brief discussion on the potential effects of feed- P on yield increase and soil P maintenance in RA compared to RM. Please see line 335-343.

3) Discuss broader consequences of rice-animal co-culture in terms of economic costs/benefits to farmer, potential side effects on land used to produce animal feed and on nutrient and carbon cycle (including greenhouse-gas emissions).

Thanks. We accepted all of these comments.

(1) Basing on this current study, our previous survey and other reported studies, we had a discussion on the differences of the economic costs/benefits between rice monoculture and rice-animal coculture, and a discussion on economic benefits that the local farmers obtain from the rice-animal cocultures. Please see line 363-375.

(2) As to the potential side effects of the rice-animal co-cultures, possible negative effects (including eutrophication, greenhouse-gas emissions) resulting from the input of feed and the increased decomposition rate were discussed in the revision. Please see line 376-395.

(3) As to the animal feed that main components are residues of soybean after the oil has been extracted. Because soybean is major oil crop in China and in other Asian countries, using residues of soybean in rice-animal coculture can achieve the recycling of resources in agriculture. Thus the integration of rice-aquatic animal coculture with soybean production would help develop sustainable rice-animal coculture. A brief discussion was provided in the revision. Please see line 396-408.

4) Explain in more detail how animals were treated, grew (one or several years), affected rice plants and were "harvested" (every year all animals?).

Thanks, we accepted all of these comments.

(1) In this study, the animals (carp, crab and softshell turtles) were released into paddy field one week after rice seedlings (15-20cm height) were transplanted. The animals (carp, crab and softshell turtles) did not affect (eat or knock down) the rice plants because the rice plants have established when the carp fry or juvenile crabs or baby turtles were released into paddy field each year.

(2) The animals (carp, crab and softshell turtles) were cultured with rice plants during the whole rice-growing period (130-150 days), and were harvested every experimental year when the rice matures.

The detail information of the animals in the experiment were provided in the Method section. Please see line 425-461, and Appendix 4. Also see Figure 7.

5) It would be nice to include pictures of some experimental plots in the display material. Some graphs could be improved by extending y-axes only as far as data points occur.

We thank this great comment. Pictures of the animals and experimental plots were provided in Figure 7.

Reviewer #1:

This is a well conducted experimental study in which rice monocultures are compared with rice-aquatic animal co-cultures over multiple years. Co-culture increases plant yield and adds animal yield, but requires extra input of animal feed. Overall co-culture seems to benefit yield and sustainability.

It would be interesting to discuss the broader consequences of the experimental results for application, e.g. potential costs of the additional animal feed (and additional labor?) required for the co-culture, farmer income, other ecosystem services including eutrophication and greenhouse-gas emissions.

Thanks for these valuable comments.

(1) Basing on this current study, our previous survey and other reported studies, we had a discussion on the differences of the economic costs/benefits between rice monoculture and rice-animal coculture, and a discussion on economic benefits that the local farmers obtain from the rice-animal cocultures. Please see line 363-375.

(2) Other possible effects of the rice-animal co-cultures (including eutrophication, greenhouse-gas emissions) were also discussed in the revision. Please see line 376-395.

The following are points that should be addressed in revising the manuscript:

1) Statistical analysis: it seems this was done correctly, e.g using years as subplots and not as independent measurements. However, this should be explained in more detail by presenting nominator and denominator degrees of freedom for F-tests and number of replicates in figures or legends.

Thanks, we accepted all of these comments.

(1) In our field experiments, each treatment rice monoculture (RM) or rice- animal coculture (RA) was conducted in same experimental plot every year during the 4 experimental year. We thus used years as subplots and not as independent measurements in the statistical analysis.

(2) We checked all the results of the statistical analysis. The nominator and denominator degrees of freedom for F-tests or T-tests have been provided in text. The number of replicates have been provided in legends. Please see line 151-152, line 159-169, line 177-179, line 204-209, line 221-222, line 234-243, line and Figure 1-2, Figure 4-6 of legends.

2) Report amount of N (and P?) input via animal feed and arriving in soil. Discuss this in relation to other N in system and potential consequences of added P.

Thanks, we accepted all of these comments.

In the revision, the amount of N and P input via animal feed were applied in Appendix 2- table 1. Soil P before and after experiment in both RM and RA treatment were also provided in Appendix 1- figure 3. Because P is not our focus in this study, we did not present the data of P in the main text. But we had a brief discussion on the potential effects of feed- P on yield increase and soil P maintenance in RA compared to RM. Please see line 335-343.

3) Discuss broader consequences of rice-animal co-culture in terms of economic costs/benefits to farmer, potential side effects on land used to produce animal feed and on nutrient and carbon cycle (including greenhouse-gas emissions).

Thanks, we accepted all these comments. Combining with the comments of other reviewers, we have majorly revised the Discussion section.

(1) Basing on this current study, our previous survey and other reported studies, we had a discussion on the differences of the economic costs/benefits between rice monoculture and rice-animal coculture, and a discussion on economic benefits that the local farmers obtain from the rice-animal cocultures. Please see line 363-375.

(2) As to the potential side effects of the rice-animal co-cultures, possible negative effects (including eutrophication, greenhouse-gas emissions) resulting from the input of feed and the increased decomposition rate were discussed in the revision. Please see line 376-395.

(3) As to the animal feed that main components are residues of soybean after the oil has been extracted. Because soybean is major oil crop in China and in other Asian countries, using residues of soybean in rice-animal coculture can achieve the recycling of resources in agriculture. Thus the integration of rice-aquatic animal coculture with soybean production would help develop sustainable rice-animal coculture. A brief discussion was provided in the revision. Please see line 396-408.

4) Explain in more detail how animals were treated, grew (one or several years), affected rice plants and were "harvested" (every year all animals?).

Thanks, we accepted all of these comments.

(1) In this study, the animals (carp, crab and softshell turtles) were released into paddy field one week after rice seedlings (15-20cm height) were transplanted. The animals (carp, crab and softshell turtles) did not affect (eat or knock down) the rice plants because the rice plants have established when the carp fry or juvenile crabs or baby turtles were released into paddy field each year.

(2) The animals (carp, crab and softshell turtles) were cultured with rice plants during the whole rice-growing period (130-150 days), and were harvested every experimental year when the rice matures.

The detail information of the animals in the experiment were provided in the Method section. Please see line 425-461, and in Appendix 4. Also see Figure 7.

5) It would be nice to include pictures of some experimental plots in the display material. Some graphs could be improved by extending y-axes only as far as data points occur.

We thank this great comment. Pictures of the animals and experimental plots were provided in Figure 7. We also revised the graphs to improve the quality.

Reviewer #2:

This manuscript reports interesting results from a rigorous set of experiments, each with six true replicates of each treatment randomized to experimental units. The authors aimed to compare rice monocultures to co-cultures of rice and an aquatic animal (carp, crab, or turtle). The data analyses appear adequate, though I do request the degrees of freedom for F-tests to double check that the repeated measures analysis was properly implemented. The findings are of widespread importance, given that rice is one of the most widely consumed crops worldwide. Increasing the yield of rice through co-culture may reduce the demand of land for rice production, leaving more land for non-human species.

Thank you very much for the comments. We accepted the comments about the degrees of freedom for F-tests and the repeated measures analysis.

(1) We have checked and confirmed that using "the split-plot-in-time" repeated measures analysis in our study was statistically proper because each treatment rice monoculture (RM) or rice- animal coculture (RA) was conducted in same experimental plot every year across the 4 experimental years. in our study.

(2) We checked all the results of the statistical analysis. The nominator and denominator degrees of freedom for F-tests or T-tests have been provided in text. The number of replicates have been provided in legends. Please see line 151-152, line 159-169, line 177-179, line 204-209, line 221-222, line 234-243, line and Figure 1-2, Figure 4-6 of legends.

I have two main suggestions for the authors, which I hope will help further improve their manuscript. First, it is necessary to clarify in visible places, including the Abstract, that feed was added in co-culture. This confounds, to some extent, the extra nutrient inputs in the feed with the comparisons between monocultures and co-cultures. It might also help to include further discussion of the extent to which the increased rice yield is likely explained by each of these two things: the animals or the nutrient inputs in the feed. The diet analyses help get at this and are another strength of this study.

Thanks. We accepted all these comments.

(1) The information of feed addition in rice-aquatic animal (RA) treatments were provided in abstract (line 44-45) and in Introduction (line 138-144).

(2) The amount of N and P input in the RA systems via feed was provided in Appendix 2- table 1. The detailed information of feed use were also provided in Method section and Appendix 4.

(3) Combining the comments of other reviewers, we have reconstructed the Discussion section. In the revision, we separately discussed the effects of animals and feed input in rice-aquatic animal coculture systems.

The aquatic animals had two important roles in these cocultured paddy ecosystems. One role involved competition, i.e., aquatic animals reduced competitors (i.e., weeds) of rice plants and thereby enhanced rice yield. A second role of aquatic animals concerned N, i.e., aquatic animals increased the recycling of N in these cocultured paddy ecosystems. Detailed discussion indicate in line 266-323.

Unlike traditional rice-fish coculture systems in which no fish feed is applied, the current coculture systems, like those described in this study, often include the application of feed in order to obtain high aquatic animal yields. The potential effects of nutrients (i.e. N and P) via feed input yield increases and soil N and P maintenance in RA compared to RM. Detailed discussion indicate in line 324-343

Second, it would help to also briefly discuss the broader implications of these results for land use and climate change. Currently, the manuscript presents only the positive outcomes. Would changing from monocultures to co-cultures likely lead to more carbon emissions? The decomposition results suggest that this should be further investigated. Would changing from rice monocultures to co-cultures likely lead to more demand for cropland, given that soybean feed source for the aquatic animals? These implications of the results deserve a bit more attention in the Discussion section.

Thank you very much for the comments. Combining with the comments of other reviewers, we have majorly revised the Discussion section.

(1) Basing on this current study, our previous survey and other reported studies, we had a discussion on the differences of the economic costs/benefits between rice monoculture and rice-animal coculture, and a discussion on economic benefits that the local farmers obtain from the rice-animal cocultures. Please see line 363-375.

(2) As to the potential side effects of the rice-animal co-cultures, it would be some possible negative effects (including eutrophication, greenhouse-gas emissions) resulting from the input of feed and the increased decomposition rate. The detailed discussion indicate in line 376-395.

(3) As to the animal feed, the main components of the feed are residues of soybean after the oil has been extracted. Because soybean is major oil crop in China and in other Asian countries, using residues of soybean in rice-animal coculture can achieve the recycling of resources in agriculture. Thus the integration of rice-aquatic animal coculture with soybean production would help develop sustainable rice-animal cocultures. A brief discussion was provided in the revision. Please see line 396-408.

Title

I recommend removing 'soil nitrogen' from the title, given the non-significant results shown in Figure 1b.

Thank you very much for the comments. Yes, no significant difference of soil N between RA and RM (Figure 1), and between before and after field experiment (Figure S1) were found. These results suggested that rice yield increased while soil N did not change in RA compared to RM.

We thus changes title to "Using aquatic animals as partners to increase yield and maintain soil nitrogen in the paddy ecosystem".

Abstract

L44: here or elsewhere in the Abstract, please include a few words to let readers know that there were also feed inputs.

Thanks, we accepted this comment. The information of feed addition in rice-aquatic animal (RA) treatments were provided in abstract and main text ( Introduction, Result and Method sections). We also provided the information of the amount of N and P input in the RA systems via feed in Appendix 2- table 1. Please see line 44-45, line 138-144, line 165-166; Appendix 4.

Introduction

L115: It would help to use a different word here, rather than 'functions' because some of the previous studies, including those in the preceding sentence, considered ecosystem functions. Also, I tend to find it more compelling when studies explain why a knowledge gap needs to be filled, rather than stating that it exists. Many things are unknown. Why should we know more about this particular knowledge gap?

Thank you very much for these good comments. In the revision, we changed the sentences as "Why coculturing these aquatic animals with rice can reduce the application of fertilizers and pesticides, however, is poorly understood. Understanding how aquatic animals contribute to the reductions in fertilizer and pesticide application in coculture systems would help the development of sustainable rice production.". Please see line 114-118.

L129-131: This argument seems sound if these animals do not eat the rice plants. Do these aquatic animals eat any parts of the rice plant?

Yes, the animals (carp, crab and turtle) used in our study do not eat the rice plants.

In this study, the animals (carp, crab and softshell turtles) were released into paddy field one week after rice seedlings (15-20cm height) were transplanted. The animals (carp, crab and softshell turtles) did not eat or knock down the rice plants because the rice plants have established when the carp fry or juvenile crabs or baby turtles were released into paddy field each year. In the revision, the detail information of the animals were provided in the Method section. Please see line 425-461, and Appendix 4. Also see Figure 7.

Results

L147: Here and throughout, please include the numerator and denominator degrees of freedom for the F-tests. This will help readers check that the split-plot-in-time repeated measures analysis was correctly implemented.

Thanks, we accepted this comment.

The nominator and denominator degrees of freedom for F-tests or T-tests have been provided in text. The number of replicates have been provided in legends. Please see line 151-152, line 159-169, line 177-179, line 204-209, line 221-222, line 234-243, line and Figure 1-2, Figure 4-6 of legends.

Figure 1: Note typo at top of Figure one: carb should be crab, I believe

Thank you very much. The correction has been done.

Figure 1: Yield should have units of mass per unit area per unit time.

Thank you very much. The correction has been done.

The diet analyses really strengthen this study. Nicely done.

Thank you very much.

Discussion

L288, see also Dirzo et al., 2014 Science

Thank you very much. We have read this nice paper by Dirzo et al., 2014 in Science and added it as a reference. Please see line 266-267.

L294-301; Here and elsewhere, it would help to have further discussion of the implications of the results. Note that this enhanced decomposition would not only contribute to the benefit of N recycling, but would also affect the C cycle in a potentially undesirable manner.

Thanks, we accepted these comments. Combining with the comments of other reviewers, we have majorly revised the Discussion section.

We have a brief discussion on the possible negative effects (including eutrophication, greenhouse-gas emissions) resulting from the input of feed and the increased decomposition rate. The detailed discussion indicate in line 376-395.

L314-316: I am not sure that I fully understand the proposed mechanism here. Is this interpretation similar to the arguments made by McNaughton et al., (1997 Science)? It would help readers to add a bit more explanation here.

Thank you very much for these great comments. Yes, the mechanism that the aquatic animals increased N-use efficiency and maintained soil N in our study was something similar to the effects of grazers in natural ecosystem as reported by McNaughton et al., (1997, Science). In the revision, we gave more explanation on this mechanism. Please see line 292-310.

L333-334: here and in other visible places, such as the abstract, I believe it is necessary to also note that feed was added.

Thanks, we accepted this comment. The information of feed addition in rice-aquatic animal (RA) treatments were provided in abstract and main text ( Introduction, Result and Method sections). We also provided the information of the amount of N and P input in the RA systems via feed in Appendix 2- table 1. Please see line 44-45, line 138-144, line 165-166; Appendix 4.

Materials and methods

L338: Somewhere early on in the Methods section, please clarify how much feed was added and in what form(s).

Thanks, we accepted this comment.

In the revision, the use of feed, and the information of amount and form of fee used in rice-carp, rice-crab and rice-turtle were provided in Introduction, Result and Method section. Please see line 44-45, line 138-144, line 165-166, line 245-261 and Appendix 2- table 1.

L341-352: It would help to clarify here how each of these animal species is used, given that the production of their biomass is presented as a co-benefit alongside increased rice yield. For example, is the meat of all these species consumed by people for food?

Thanks for these good comments. Yes, These three aquatic animals are economically important, and are usually cultured in fish ponds or paddy fields by local farmers. The meat of all these species is a popular food of the local people.

In the revision, we have rewritten this paragraph and described each of the animal species used in the coculture systems, including their economic uses, biological traits and the interactions with rice plants. Please see line 410-461.

L364-365: Do farmers typically apply pesticides? If so, then it would be interesting to include a sentence in the Discussion clarifying how the weed suppression by the animals compares, on a percentage basis, to weed suppression by pesticides.

Yes, farmers typically apply pesticides and herbicides to control insect pests and weeds in the rice monoculture. In the revision, we had a discussion on how many percentage of weeds be reduced by the animals compared to by pesticides. Please see line 283-284.

L411: Shouldn't this percentage also be multiplied by the percentage of N in rice and aquatic animals that came from the fertilizer/feed?

Yes, the total output Ny was obtained by multiplying the biomass of rice (grain and straw) and aquatic animals by the percentage of N in rice and aquatic animals that came from the fertilizer, feed and soil. We have provided this information in the revision. Please see line 535-538.

L414: Please clarify how much (mass per unit area or per experimental uint) N was added as fertilizer and feed.

Thanks, we accepted this comment. The information of fertilizer-N and feed-N used in each experimental plot were provided in the Appendix 2- table 1.

L443-446: This is helpful information about the feed, but more information is needed (e.g., how much N was added in the feed?) and it should be presented much earlier, as requested in comments above.

Thanks, we accepted this comment. In revision, the information of feed containing N were mentioned in the Result section and also provided in the Method section. Please see line 165-166, and line 425-461.

Appendix 2

L793-801: I see this helpful information here. Perhaps just move or repeat this information above, where requested in the Methods section. It will be difficult for some readers to find these important details in this appendix.

Thanks, we accepted this comment. In the revision, the information of feed application in the Appendix were repeated in the Methods section. please see line 430-436, line 443-447, and line 455-461.

Reviewer #3:

Agricultural intensification has increased yields but has also negatively affected the environment. Food production needs a comprehensive overhaul in terms of sustainability, with better approaches to producing safe and healthy food, while leaving little footprint. Globally, chemical fertilizers, herbicides and pesticides are rolled back as part of our commitment to the Sustainable Development Goals (SDG) and the urgent need for a greener renaissance in sustainability (SDG 2, 12 and 13).

The coupling of two agricultural systems (i.e., integrated agriculture-aquaculture or integrated crop-livestock) have been proposed as one of the important ways that reduce the negative effects of food production on the environment: using lesser chemical fertilizers, no herbicide was needed for weed suppression, uneaten animal feed serving as nutrients for rice, no eutrophication, etc. While the idea of harnessing co-cultivation of plant-animal system for food production is great and beneficial to the environment, the availability of large-scale and long-term experimental data is lacking. Thus, this manuscript is an important contribution to the literature on integrated agriculture and aquaculture. The science in the manuscript is well thought out and executed and the manuscript is well written. The central research question – can yields in rice and aquatic animals be maintained in coupled rice-"animal" ("animal" refers to carps, crabs and turtles, collectively) systems, is timely.

The authors described their long-term and integrated agriculture-aquaculture experimental systems and the results of the multi-year study highlighted a 'solution' to the problem of historical environmental impacts caused by our current and common intensive food production systems (mainly monocultural).

The results suggested that the coupling of rice with aquatic animal production could achieve synergies between food production systems while reducing their associated environmental impacts per unit of production.

Going beyond the science, to help policy makers to better appreciate the co-cultivation strategy, a mention of the comparative financial returns (for the farmers) should perhaps be provided by the authors in their China-wide research? Such analyses (financial returns from selling rice in monocultures, selling rice and aquatic animals together) would strengthen the case for supporting various options: the crab-rice versus fish-rice co-cultivation systems in certain localities. Perhaps in certain situations (e.g. seasonality, lack of farm labour, brackish water issues), the other aquatic animals (prawns/amphibians/ducks) could be the preferred approach?

Thanks, we accepted all these comments. Combining with the comments of other reviewers, we have reconstructed the Discussion section.-

(1) A discussion on the comparative financial returns (e.g. economic costs/benefits. product price) to farmer of the rice-animal co-cultures was provided in the revision. Please see line 356-375.

(2) We have a brief discussion on the differences of financial return among the types of cocultures (rice-carp, rice-crab and rice turtle) in the revision. We also discussed the potential application of other types of cocultures (prawns/amphibians/ducks). Please see line 396-402.

It is interesting to note that in China, rice yields were similar or higher using the rice-"animal" co-cultivation systems than under monocultures of rice. To help the readers, I would recommend that some photos for the coupled rice-"animal" ("animal" refers to carps, crabs and turtles, collectively) systems be included as a figure in this paper. The alternate is to provide some illustration to highlight the coupled rice-"animal" systems.

Thank you very much for these great comments. In the revision, we grouped the photos of the animals and experimental plots for the coupled rice-"animal" in Figure 7.

With the strong focus of this paper on N, which appeared to be an important (or dominant driver) factor, we still cannot rule out the plausible influence of animal manures releasing additional phosphorus (P) to drive rice growth. Thus, is there some baseline data about the dynamics in the soils and irrigation waters among the research sites? Or from past literature about P dynamics in relation to the present or absence of crabs/fishes/turtles, within a certain level of N? Current plant physiological papers, especially those involving rice growth and yield had frequently mentioned the dual roles of N and P? Some comments from the authors about P would be appropriate with reference to soil fertility and health

Thank you very much for these great comments.

In the revision, the amount of N and P input via animal feed were applied in Appendix 2- table 1. Soil P before and after experiment in both RM and RA treatment were also provided in Appendix 1- figure 3. Because P is not our focus in this study, we did not present the data of P in the main text. But we had a brief discussion on the potential effects of feed- P on yield increase and soil P maintenance in RA compared to RM. Please see line 335-343.

Other salient observations, suggestions and notes:

Importantly and across all the three co-cultivation systems, there was no requirement for weed control via herbicides. This salient finding could be included in the proposed figure,

Thanks, we accepted this comment. In the revision, the information of no herbicides use in the three co-cultivation systems was provided in the legend of Figure 2.

The paper is well written and organised in a logical fashion in line with the given guidelines. If possible, some proof-reading is needed to further improve the English language.

Thanks, we have asked Dr. Bruce Jaffee who is a retire professor from University of California at Davis to help to improve the English language.

The literature cited are relevant and updated. Appropriate statistical methods were used by the authors to analyse the data.

Thank you very much for the comments.

The sample size, field survey approaches, apparatus utilised and chemical analyses (especially stable isotopes using the IRMS, Isotope Ratio Mass Spec) were appropriate.

Thank you very much for the comments.

Reviewer #4:

This study by Guo et al., is set in the framework of a highly interesting system: the coculture of different species in agricultural production. In this case, not different plant species are grown together (as typically done in intercropping) but aquatic animals (carp, crabs and turtles) are cultured together with rice. Using three multi-year field experiments (one experiment for each combination between one animal species and rice plants) and three corresponding field mesocosm experiments, the authors found that generally, rice yield and apparent nitrogen-use efficiency were higher and weed infestation of the rice paddies lower in coculture with animals, compared with rice monocultures. These results are attributed to the additional feed-N unused by the animals that was shown to be taken up by plants, as well as likely feeding on insect pests and weeds by the animals and higher decomposition (also measured in the experiment) in the coculture plots. Overall, these results provide support for multispecies systems in rice production. Additionally, animal yield was measured in this study and adds to the increased economic advantage of such a system compared with rice monocultures.

This study is interesting for both, ecologists with an interest in biodiversity effects and food webs, and for agronomists aiming to increase yield while supporting agricultural diversity and limiting synthetic inputs at the same time. It is a very valuable applied study that underlines the value of diversity in agriculture. I appreciated the interesting combination of organisms across trophic levels in this culture. I also found it fascinating that this old cultural form of agriculture has a strong relevance for today's production schemes which aim for low input of synthetic fertilizers and pesticides and high organismal diversity in agricultural areas.

The experimental design of the study is clear and simple. It has relatively low power (n=6 for each treatment) but the experiments were done across several years, increasing the applicability of the results. The authors measured many relevant variables (e.g. nitrogen use efficiency) and even use C and N labelling to follow the fate of different elements in the system.

The results of the study are well supported and indicate that the coculture with animals indeed has many advantages. However, I missed a discussion of caveats and potential disadvantages of this coculture method. As far as I can see, the authors did not determine whether rice plants were destroyed or fed upon by the animals. Furthermore, coculture may pose logistical challenges such as a higher effort in management or harvest of two species that may increase costs to farmers and may make them reluctant to use this method. In addition, total nitrogen that entered the system was different between treatments (because no animal feed was used in the rice monocultures, I think), so this additional input may account for differences in yield, even if no animals are used.

Thank you very much for the comments.

(1) We accepted the comments that the manuscript lacks a discussion of caveats and potential disadvantages of the coculture method. Combining with the similar comments of other reviewers we reconstructed the Discussion section, and had a discussion on possible negative effects (including eutrophication, greenhouse-gas emissions) resulting from the input of feed and the increased decomposition rate. Please see line 376-395.

2) As to the "---rice plants were destroyed or fed upon by the animals", the three animals (carp, crab and turtle) do not feed on rice plants because rice seedlings have grown up (about 40 days old, 15-20 cm height) when the animals are released into paddy field. The young animals do eat the hard stems of rice plants. This information has provided in Method section. Please see line 425-461.

3) As to the fee-N input, yes, rice plants used some of fee-N in the coculture compared to rice monoculture. Thus, we have a discussion on how complementary use of feed-N between rice plants and animal can contribute N use efficiency in coculture systems. We also discuss potential effect of other nutrients (e.g. P) in the feed on rice yield and soil fertility. Please see line 324-343.

I have a few major points regarding potential improvements to the analysis and presentation of the data.

Thank you very much.

Statistical analysis: I struggled to understand how the data was analysed. Was year nested within plot? Or was the experiment completely restarted every year and the data points across years can be considered independent replicates (I think they cannot, see also the temporal development in Figure 1,2)? I was also confused at times about the sample sizes behind the figures and when averages across years or replicates were used. Please be more specific here (e.g. state n in legends or on figure panels or in the text). Generally, many tests are being used where one model (testing several explanatory variables, e.g. RA/RM, time and their interaction) would likely have been the better solution.

Thank you very much for the comments.

(1) Statistical analysis: yes, the experimental year nested within plot. In our study, the same treatment (RM or RA) in each experimental plot continued across 4 years, not completely restarted every year. We thus used years as subplots and not as independent measurements in the statistical analysis.

(2) We accepted the comments that more specific explanations should be provided in legends or on figure panels or in the text. We checked all the results of the statistical analysis. The nominator and denominator degrees of freedom for F-tests or T-tests have been provided in text. The number of replicates have been provided in legends. Please see line 151-152, line 159-169, line 177-179, line 204-209, line 221-222, line 234-243, line and Figure 1-2, Figure 4-6 of legends.

(3) For the field experiment, we used a repeated measure design with four years and two culture types (RM vs RA). In our statistical analysis, we actually used repeated-measure ANOVA and focused on the effect of RM and RA, but we did not care about the effect of time or the interaction between experimental year and culture type. We thus only presented the effect of culture type in our text.

Ethical considerations: The manuscript contains an ethics statement. However, there are few methodological details regarding the treatment of animals in the study. For example, I assume that the animals were killed in some way before drying/freezing at -20{degree sign}? How were they "harvested"?

Thank you very much for the comments. In the revision, detail methods of the animal sampling and harvesting in the field, and methods of animal sample handling in lab were provided in. Method section and Appendix 7.

Methodological details: I missed some details on the general method for non-experts of this culture technique. Most importantly: In this system, is harvest of the aquatic animals done every year? How long is the usual growing season? Do the animals grow as fast as rice? Even the turtles? Is the amount of fertilizer added in the experiment (550-750kg/ha) similar to what would normally be added to regular rice monocultures?

Thank you very much for the comments.

(1) In our study, the aquatic animals were harvested every year. The growing season for rice-carp was from late May to early October (around 125days), for rice-crab was from middle May to middle October (around 150 days), for rice-turtle was from middle June to early November (around 140 days). In the revision, these information have provided in Method section. Please see line 425-461

(2) The coculture systems, both rice plants and aquatic animals grow as the time passed on. But growth rate of rice plants and aquatic animals was not the same. These information have provided in Method section.

3) Yes, to test the effects of animals, we designed the amount of fertilizer added in the experiment were similar to regular rice monocultures.

https://doi.org/10.7554/eLife.73869.sa2

Article and author information

Author details

  1. Liang Guo

    College of Life Sciences, Zhejiang University, Hangzhou, China
    Contribution
    Formal analysis, Investigation, Methodology, Software, Writing – original draft
    Contributed equally with
    Lufeng Zhao
    Competing interests
    No competing interests declared
  2. Lufeng Zhao

    College of Life Sciences, Zhejiang University, Hangzhou, China
    Contribution
    Formal analysis, Investigation, Methodology, Software
    Contributed equally with
    Liang Guo
    Competing interests
    No competing interests declared
  3. Junlong Ye

    College of Life Sciences, Zhejiang University, Hangzhou, China
    Contribution
    Formal analysis, Investigation, Methodology, Software
    Competing interests
    No competing interests declared
  4. Zijun Ji

    College of Life Sciences, Zhejiang University, Hangzhou, China
    Contribution
    Formal analysis, Investigation, Methodology, Software
    Competing interests
    No competing interests declared
  5. Jian-Jun Tang

    College of Life Sciences, Zhejiang University, Hangzhou, China
    Contribution
    Formal analysis, Investigation, Methodology, Resources
    Competing interests
    No competing interests declared
  6. Keyu Bai

    Bioversity International, Maccarese, Italy
    Contribution
    Formal analysis, Investigation, Methodology, Software
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3644-0844
  7. Sijun Zheng

    1. Bioversity International, Maccarese, Italy
    2. Yunnan Academy of Agricultural Sciences, Kunming, China
    Contribution
    Formal analysis, Investigation, Methodology, Software
    Competing interests
    No competing interests declared
  8. Liangliang Hu

    College of Life Sciences, Zhejiang University, Hangzhou, China
    Contribution
    Formal analysis, Investigation, Methodology, Software
    For correspondence
    zjuhull@126.com
    Competing interests
    No competing interests declared
  9. Xin Chen

    College of Life Sciences, Zhejiang University, Hangzhou, China
    Contribution
    Conceptualization, Methodology, Supervision, Validation, Writing – original draft, Writing - review and editing
    For correspondence
    chen-tang@zju.edu.cn
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6622-1074

Funding

Natural Science Foundation of China (U21A20184)

  • Xin Chen

Science and Technology Program of Zhejiang Province (2022C02058)

  • Xin Chen

Science and Technology Program of Zhejiang Province (LGN22C030002)

  • Liangliang Hu

Natural Science Foundation of China (31661143001)

  • Xin Chen

Natural Science Foundation of China (31770481)

  • Xin Chen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Minfang Wu, Yongqing Yu, and Genlian Wang for their assistance with the field experiments.

Ethics

In the following experiments, the samples of all aquatic animals (i.e., carp, crabs, and softshell turtles) were collected and measured in accordance with the approved guidelines of the Zhejiang University Experimental Animal Management Committee. Details on the handling of animal samples were described in the Methods section.

Senior Editor

  1. Detlef Weigel, Max Planck Institute for Developmental Biology, Tübingen, Germany

Reviewing Editor

  1. Bernhard Schmid, University of Zurich, Zurich, Switzerland

Reviewer

  1. Bernhard Schmid, University of Zurich, Zurich, Switzerland

Publication history

  1. Received: September 14, 2021
  2. Accepted: January 13, 2022
  3. Version of Record published: February 22, 2022 (version 1)

Copyright

© 2022, Guo et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Liang Guo
  2. Lufeng Zhao
  3. Junlong Ye
  4. Zijun Ji
  5. Jian-Jun Tang
  6. Keyu Bai
  7. Sijun Zheng
  8. Liangliang Hu
  9. Xin Chen
(2022)
Using aquatic animals as partners to increase yield and maintain soil nitrogen in the paddy ecosystems
eLife 11:e73869.
https://doi.org/10.7554/eLife.73869

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