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Effects of fertiliser applications on survival and recruitment of the apple snail, Pomacea canaliculata (Lamarck)
Crop Protection 64 (2014) 78e87
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Crop Protection journal homepage: www.elsevier.com/locate/cropro
Effects of fertiliser applications on survival and recruitment of the apple snail, Pomacea canaliculata (Lamarck) Alexander M. Stuart, Alvaro Nogues Palenzuela, Carmencita C. Bernal, Angelee Fame Ramal, Finbarr G. Horgan* Crop and Environmental Sciences Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 January 2014 Received in revised form 28 May 2014 Accepted 30 May 2014 Available online
Since its introduction to Asia in the 1980s, the golden apple snail, Pomacea canaliculata (Lamarck), has represented a major constraint to the profitability of rice (Oryza sativa L.) farming by damaging rice seedlings during crop establishment. This study describes a series of experiments designed to determine the effects of nitrogenous fertilisers on snail fitness. We examined the possibility of a two-phase model of snail response to nitrogen, whereby fertilisers initially increase snail mortality through toxicity, but once assimilated into the rice ecosystem, eventually favour snail reproduction and survival. In experimental arenas, fertiliser had lethal effects: Complete fertiliser (14:14:14), urea, ammonium sulphate and organic fertilisers were associated with snail mortality, generally affecting adult snails more than juvenile snails, and with greater effects when applied to saturated soil that was subsequently flooded (as opposed to direct application to flooded soil). Snail mortality was found to decline considerably when snails were added to arenas one day after fertiliser application e this occurred in arenas with soil and water, but not in arenas with water only, suggesting that soil can reduce the toxic effects of fertilisers. In a field experiment, snail numbers declined in both fertilised and non-fertilised plots at the time of crop establishment. Numbers increased in all plots after rice tillering, with significantly more recruitment in plots with high nitrogen. Although the responses were generally weak in the field experiment, they did support the two-phase model. The consequences of fertiliser applications for snail management and ecosystem health are discussed. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Ammonium sulphate Benthic organisms Crop establishment Invasive species Nitrogen Urea
1. Introduction Pressures to increase food production to meet a growing world population and global food demands have led to an increasing use of chemical and organic inputs in agriculture; particularly nitrogenbased chemical fertilisers (FAO, 2008). The global demand for fertilisers is forecast to grow annually by 1.9%, from around 180 million tonnes in 2012 to around 194 million tonnes by 2016 (FAO, 2012). The use of nitrogen in agriculture has been linked to sustaining agricultural productivity (Stewart et al., 2005) and due to limitations in land availability, increasing fertiliser use on existing cropland is considered the most likely path farmers will take to increase production (FAO, 2008). However, high fertiliser use has a number of negative effects on ecosystems, such as altering plant community composition (Vitousek, 1994; Tilman and Lehman, 2001);
* Corresponding author. Tel.: þ63 2 580 5600x2708. E-mail address: [email protected] (F.G. Horgan). http://dx.doi.org/10.1016/j.cropro.2014.05.020 0261-2194/© 2014 Elsevier Ltd. All rights reserved.
increasing greenhouse gas (nitrous oxide) emissions; and contaminating groundwater, rivers, lakes and coastal waters, causing eutrophication (Zhu and Chen, 2002; Galloway et al., 2003). Rice (Oryza sativa L.) is the only major cereal crop that is grown in standing water, thus the use of nitrogen fertilisers during rice production is likely to have major consequences for the aquatic flora and fauna that inhabit rice ecosystems. Many aquatic organisms are susceptible to water-borne chemicals, including fertilisers, and to changes in floodwater chemistry as a result of chemical inputs (Simpson et al., 1994a). However, few studies have examined the effects of nitrogen fertilisers on aquatic organisms in rice ecosystems. Aquatic biomass, in particular green algae, followed by populations of ostracods and chironomid as well as mosquito larvae have been shown to increase following inorganic nitrogen fertiliser applications in rice fields (Simpson et al., 1994a,b; Sunish and Reuben, 2002; Mutero et al., 2004). Simpson et al. (1994a) indicated that benthic molluscan communities change over the course of rice crop development and that this may be associated with changing water chemistry resulting from fertiliser use.
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Furthermore, De La Cruz et al. (2001) demonstrated that inorganic nitrogen fertilisers cause mortality of golden apple snails (Pomacea canaliculata [Lamarck]) within a few days of application during land preparation for rice crops. Applications of nitrogen fertilisers from land preparation to within 14 days of crop establishment, followed by one to two further applications during the rice cropping season, are common practices in rice fields in South-East Asia (Buresh et al., 2007; Palis et al., 2007). The addition of nitrogen to the rice crop initially increases photosynthesis levels and pH and reduces concentrations of dissolved oxygen in the floodwater (Mikkelsen et al., 1978; Simpson et al., 1994a). However, assimilation of nitrogen also increases plant vigor, which benefits the crop, and increases primary productivity in the aquatic environment. This includes new growth of macrophytes and algae. Increasing plant and algal productivity can also benefit aquatic herbivores by increasing food quality and availability (Horgan et al., 2014). For aquatic herbivores, nitrogen fertilisers can therefore have both negative effects through toxicity and by altering the physio-chemical properties of the water, and positive effects by increasing the production (biomass) and quality of aquatic vegetation. Because of the dynamic nature of nitrogen in aquatic agro-ecosystems like rice, including sudden peaks during application and gradual declines post-application, interactions between nitrogen fertilisers and aquatic herbivores will be complex. The present study examines the effects of fertilisers on the survival of P. canaliculata (Lamarck) in rice systems. P. canaliculata was introduced to much of Asia, including the Philippines, during the 1980s and is considered a major pest of rice during early crop stages, but a beneficial herbivore of weeds during later crop stages (Joshi et al., 2006; Hidaka et al., 2007; Horgan et al., 2014). Previous field experiments by De La Cruz et al. (2001) indicated a reduction in rice damage from snails (missing hills) as a result of basal applications of inorganic nitrogen fertilisers, but the effects of compound fertilisers on snail mortality were largely inconclusive. Furthermore, as plants are affected by fertiliser treatments, i.e., increased growth rates, increased tolerance, and an increased capacity to compensate for damage, field experiments that monitor missing hills or other forms of damage to the crop do not clearly differentiate between the direct effects of snails on the plants and indirect effects mediated through snail mortality. Therefore, we conducted a series of controlled experiments to examine the direct effects of fertilisers on snail mortality. Furthermore, we addressed a two-phase model of snail responses to fertilisers, whereby mortality is increased soon after nitrogen application, but where survival and fitness is eventually enhanced once the excess nitrogen has been assimilated into plant tissues, creating a more favorable environment for herbivore growth and development.
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et al., 1999; Yusa et al., 2006). Shell height (also known as shell length; Youens and Burks, 2007) was measured from the tip of the apex to the lower margin of the aperture (Tanaka et al., 1999; Yoshida et al., 2009). 2.2. Experiment 1 e fertiliser (type) induced mortality The effects on snail mortality caused by four different fertilisers were examined. The fertilisers tested were ammonium sulphate [21-0-0 NPK], complete fertiliser [14-14-14 NPK], urea [46-0-0], and organic fertiliser (chicken manure) [2.6-3.0-3.0]. All treatments were replicated six times and a replicate set of control pots without fertiliser was maintained. In addition, we examined two flooding treatments (flooded, delayed flooding), and two sizes of snails (juvenile, adult) were tested. The fertiliser applications and flooding regimes were designed to simulate two contrasting farmer practices. One hundred and twenty pots (20.5 cm diameter) were filled with 10 cm of dry garden soil followed by the addition of water to a depth of 2 cm above the soil surface. In half the pots (60), the water was allowed to settle with 2 cm of standing water (flooded treatment). In the remaining pots, the water was drained over a 48 h period so that the soil was completely saturated but lacked standing water (delayed flooding treatment). Each fertiliser was added, based on the weight of soil in the pots, to contribute the equivalent of 80 kg/ha of nitrogen. Fertiliser was added directly to the pots by sprinkling over the water in the flooded pots and directly onto the saturated soil in the delayed flooding pots. Snails of two sizes (10e15 mm; 20e25 mm) were added immediately to the pots. Ten snails were added to each pot, maintaining snail size as a treatment. Thus, each pot either received ten juvenile snails or ten adult snails. After 2 h, water was added to the delayed flooded pots to a level of 2 cm. After a further 22 h, water was added to all pots to a level of 5 cm to prevent the snails from aestivating. Pots were interspersed on a greenhouse bench as a completely randomized design. The bench was under shade netting to prevent direct sunlight reaching the pots and to prevent unusually high temperatures. A fan was also used during the hottest daytime periods. Snails were provided with an ample supply of fresh lettuce throughout the experiment. Each pot was covered with a fine mesh lid to prevent snails from escaping. The greenhouse temperature during the experiment ranged from 24.4 to 36.3 C. Daylight hours were from about 0600 h to 1800 h. Daily pH readings, taken from treated pots without snails, ranged from 6.96 to 8.59 with no apparent effects of any of the treatment regimes. Snail mortality was monitored daily for the first five days, with an extra observation after seven days. Where snails appeared inactive, they were gently prodded to evaluate condition (dead or alive). Dead snails were removed from the pots daily.
2. Materials and methods 2.3. Experiment 2 e fertiliser (level) induced mortality 2.1. Study species The P. canaliculata in our experiments were collected from lowland irrigated rice fields at the International Rice Research ~ os, Institute Experimental Station (henceforth IRRI), Los Ban Philippines (14110 N, 121150 E). Although several Pomacea species have been introduced into Asia (and the Philippines), recent molecular analysis has indicated P. canaliculata as the most common and perhaps only apple snail species present in the Philippines (Hayes et al., 2008). Using published primers (Cooke et al., 2012), we verified P. canaliculata as the species occurring at our field sites. We separated adult and juvenile snails based on shell height, roughly classifying those with a shell height of 10e15 mm as juveniles and with a shell height of 20e25 mm as adults (Tanaka
The effects of three different application rates of nitrogen fertiliser on snail mortality were examined. In addition, we tested two snail introduction times in relation to fertiliser application times (snails added at the same time as fertiliser application, snails added 24 h after fertiliser application), two flooding treatments (drained, non-drained) and two sizes of snail (juvenile, adult). All treatment combinations were replicated six times. The water regimes were designed to simulate contrasting farmer practices and the delayed introduction of snails was designed to simulate snails moving into areas treated with fertiliser. One hundred and forty-four pots (20.5 cm diameter) were filled with 10 cm of homogenized paddy soil (with all gastropods removed by hand), water was added and allowed to settle in all the
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pots to a depth of 2 cm of standing water. Pots received three nitrogen treatments (ammonium sulphate equivalent to 0, 40 and 80 kg/ha nitrogen). The fertiliser was added directly to the pots, as a single application, by sprinkling over the standing water. Ten snails of each size category (10e15 mm or 20e25 mm) were added to the pots, maintaining snail size as a treatment, with either ten juvenile snails or ten adult snails added per pot. In half of the pots (72), fertiliser was applied 24 h before snails were added. In the remaining pots, fertiliser was applied immediately before snails were added. In half of each group of pots (36 2), the water level was increased to 5 cm 24 h after adding fertiliser (non-drained treatment) to prevent snails from aestivating. In the other half of each group, pots were gradually drained of standing water (by percolation through the soil) 24 h after fertiliser applications and maintained without water for five days, after which time water was added again to a depth of 5 cm (drained treatment). All pots were interspersed on a greenhouse bench under shade netting as a completely randomized design. The greenhouse temperature during the experiment ranged from 25.4 to 36.1 C. Daylight hours were from about 0600 h to 1800 h. Snails were provided with an ample supply of fresh lettuce throughout the experiment. Each pot was covered with a fine mesh lid to prevent snails escaping. Snail mortality was monitored over a 14-day period; one day after their introduction into pots and every other day thereafter. In pots that were drained, snails were monitored one day after their introduction into the pots, one day after the drained pots were re-flooded and every other day thereafter. Final observations were made after 14 days where snails had been added immediately and after 13 days where the snails had been added one day after nitrogen application. 2.4. Experiment 3 e substrate influence on fertiliser induced mortality The effects of soil substrate on nitrogen fertiliser induced snail mortality were examined. Following the protocol described for Experiment 2, 48 pots were prepared, 24 with soil and 24 without soil. Water was added to a depth of 2 cm and all pots received nitrogen treatments (as above) to an equivalent of 80 kg/ha. Ten juvenile snails (10e15 mm) each were added to half the pots and ten adult snails (20e25 mm) each to the remaining pots; these were each divided into two groups of pots with snails added immediately after fertiliser application to half the pots and snails added after 24 h to the remaining pots. 2.5. Experiment 4 e field study During the 2013 dry season at IRRI, snails were sampled from a group of 18 rice plots (400 m2 plot size). Each plot had been transplanted with two rice varieties, IR62 and IR64, as two subplots. Variety had no apparent effect on snail life-histories within plots (Horgan, unpublished). Plots were treated with three nitrogen (ammonium sulphate) regimes (six plots each at 0, 60, and 150 kg/ ha), each with independent irrigation and drainage systems to avoid fertiliser spread and mixture. Each plot received 0, 20 or 50 kg nitrogen/ha per application on three dates over the rice cropping season. These included a basal application to recently rotavated plots prior to final soil preparation and rice transplanting, a second application during the rice tillering stage, and a third application at the rice flowering stage. During basal application, 47 kg of solophos and 34 kg of muriate of potash were also added to the nitrogen treated plots. To sample snails, five 1 1 m quadrats were randomly placed in each plot and visible snails were collected manually. In addition, five circular quadrats, using an open-bottomed bucket
(27 cm diameter; 0.053 m2), were randomly placed in each plot and the soil (to a depth of 5 cm below the soil surface) and water within the bucket were collected and passed through a sieve (17 cm diameter, 2 mm mesh) to extract all snails. Snails were sampled two days before transplanting (one day prior to the basal nitrogen application and final soil preparation), eight days after transplanting (DAT) when fields were first flooded after transplanting, and during subsequent flooding events at the tillering stage of the rice crop (20 and 27 DAT). Further samples were taken every two to three weeks thereafter until the fields were drained for harvest. Sampled snails were measured before being returned to their original plots. 2.6. Statistical analyses 2.6.1. Experiment 1 e fertiliser (type) induced mortality To investigate the effects of fertiliser type (including the control), snail size, and time of flooding relative to fertiliser application on cumulative snail mortality at the end of the experiment (after seven days), univariate GLM was performed using ranktransformed data. Post-hoc multiple comparisons were conducted using Tukey tests. To investigate the effects of fertiliser type (excluding the control) on daily snail mortality rates over time (Days 1, 2, 3, 4, 5, 7), a repeated measures GLM with a compound symmetry variance-covariance structure was performed. The main factors entered into the model were fertiliser type, snail size and time of flooding. Data was rank-transformed. The control was excluded from this analysis of mortality rate over time as no snail mortality was observed in the control treatments and at the end of the experiment accumulated mortality was found to be significantly higher among all pesticides treatment when compared to the control. The repeated measures analysis therefore examined patterns in mortality among the fertilizer treatments only. 2.6.2. Experiment 2 e fertiliser (level) induced mortality To analyse the effects of nitrogen level, time of snail introduction relative to nitrogen application, drainage and snail size on daily mortality rates over time (on days 1, 6e7, and 13e14 after snail addition to pots), a repeated measures GLM with a compound symmetry variance-covariance structure was performed using rank-transformed data. Post-hoc multiple comparisons were conducted using Tukey tests. 2.6.3. Experiment 3 e substrate influence on fertiliser induced mortality To investigate the effects of soil (versus without soil) on daily mortality, a repeated measures GLM with a compound symmetry variance-covariance structure was performed using ranktransformed data for 1, 3 and 5 days after fertiliser treatment. The main factors entered into the model were substrate (soil, no soil), time of snail introduction and snail size. 2.6.4. Experiment 4 e field study To analyze differences in snail abundance in the field between nitrogen levels and different crop stages, a repeated measures GLM with a compound symmetry variance-covariance structure was performed using log-transformed data. Post-hoc multiple comparisons were conducted using Tukey tests. Univariate GLM was used to analyse differences in mean shell height between nitrogen levels and crop stages from snails collected from the circular quadrats. All analyses were conducted using SPSS (version 18).
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3. Results 3.1. Experiment 1 e fertiliser (type) induced mortality Snail mortality at the end of the experiment was affected by fertiliser type/treatment and snail size (Table 1; Fig. 1), with the highest mortality occurring among adults. Post-hoc analysis revealed a significant difference (P < 0.05) in snail mortality between the control and all four fertiliser types, but no significant difference between the four fertilisers tested (Table 1; Fig. 1). There was higher adult mortality in pots that received either complete fertiliser or urea, but similar levels of mortality between adults and juveniles in the other treatments resulting in a significant fertiliser*snail size interaction (Table 1). There was also a significant fertiliser type*flooding treatment interaction (Table 1) because mortalities were similar between flooded and delayed flooding treatments with organic fertilizer and ammonium sulphate, but delaying flooding increased mortality due to complete and urea fertilisers (Fig. 1). The control pots were removed from the repeated measure analysis because no snails had died in the control pots. Among the remaining pots, adult snails had higher fertiliser-induced mortality than juveniles and mortality was higher where flooding had been delayed (Table 2; Fig. 2). As with the cumulative data (above), there was a significant fertilizer*snail size interaction (Table 2). Mortality increased over the course of the experiment, with mortality levels generally diverging as the experiment progressed: this resulted in significant time*fertiliser type and time*snail size interactions (Table 2; Fig. 2): After application of ammonium sulphate and complete fertiliser, snails (particularly adults) rapidly died e the highest mortality generally occurring within the first two days (Table 2, Fig. 2). In comparison, the lethal response to urea and organic fertiliser was slightly delayed, with higher levels of mortality observed at least three days after fertiliser application. The mortality of adults and juveniles in pots treated with organic manure were similar throughout the experiments (Fig. 2).
time*drainage*fertiliser timing and drainage*snail size*fertiliser timing interactions (Table 3; Fig. 3).
3.2. Experiment 2 e fertiliser (level) induced mortality Snail mortality increased over the course of the experiment and was positively related to nitrogen levels resulting in significant nitrogen level and time*nitrogen level effects (Table 3, Fig. 3). Tukey tests indicated significant increases in mortality with each increase in nitrogen level (P 0.05). Mortality was also higher when snails were added to the pots on the same day as nitrogen fertiliser. Mortality of adults was higher than mortality of juveniles when snails were added one day after fertiliser, resulting in significant 3and 4-way interaction terms (Table 3, Fig. 3). In general, the lowest mortality occurred in pots that had been drained and where snails were added one day after the fertiliser. This produced significant
Table 1 Results of univariate GLM of the effects of fertiliser type, snail size and flooding treatment on cumulative snail mortality following a 14-day observation period (see also Fig. 1).
Fertiliser type/treatment Snail size Flooding treatment Fertiliser*snail size Fertiliser*flooding treatment Snail size*flooding treatment Fertiliser type*snail size*flooding treatment Error
Fig. 1. The mean proportion of adult and juvenile Pomacea canaliculata that had died during a 7-day observation period following application of ammonium sulphate, complete fertiliser, organic manure and urea in delayed flooded (A) and flooded (B) arenas.
df
F
P
4 1 1 4 4 1 4 100
8.933 44.392 0.967 6.516 3.427 0.131 0.034
<0.001 <0.001 0.328 <0.001 0.011 0.718 0.998
3.3. Experiment 3 e substrate influence on fertiliser induced mortality Snail mortality gradually increased over time (Table 4). Snail mortality was lower in pots that contained soil compared to pots without soil and with water only (Table 4, Fig. 4). Adding snails to the pots one day after the fertiliser was added also reduced mortality (Table 4, Fig. 4). Mortality levels were similar on day 1 of the experiment, but diverged as the experiment progressed increasing to higher levels when snails, particularly adults, were added on the same day as the fertiliser and producing a significant time*snail size*fertiliser timing interaction (Table 4, Fig. 4). 3.4. Experiment 4 e field study Across all treatments, snail abundance fluctuated throughout the course of crop development (Table 5, Fig. 5A); abundance declined following the basal application of fertiliser and final soil preparation until the tillering stage of the rice crop and then tended to increase until the end of the rice cropping season. Tukey tests indicated a higher abundance of snails only during landpreparation with no significant differences among later crop stages (P 0.05). Although, both the decline in abundance during the early crop stages and the increase in abundance during the later crop stages were greater in the high nitrogen plots, the effect of
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Table 2 Results from repeated measures GLM of the effects of fertiliser type, snail size and flooding treatment on snail mortality (see also Fig. 2). df Between-subjects effects Fertiliser type Snail size Flooding treatment Fertiliser*snail size Fertiliser*flooding treatment Snail size*flooding treatment Fertiliser type*snail size*flooding treatment Error Within-subjects effects Time Time*fertiliser type Time*snail size Time*flooding treatment Time*fertiliser type*snail size Time*flooding treatment*snail size Time*fertiliser type*flooding treatment Time*fertiliser type*snail size*flooding treatment Error
F
P
3 1 1 3 3 1 3 80
1.809 30.342 4.305 3.092 1.373 2.358 0.522
0.152 <0.001 0.041 0.032 0.257 0.129 0.668
5 15 5 5 15 5 15 15 400
5.855 6.311 6.234 0.933 6.136 0.866 1.233 0.824
<0.001 <0.001 <0.001 0.460 <0.001 0.504 0.244 0.651
nitrogen level and the crop stage*nitrogen interaction were not statistically significant (Table 5). Over the rice cropping season, snail shell height ranged from 4.1 mm to 36.1 mm (Fig. 5B). Mean snail shell height fluctuated over the course of crop development (F6,255 ¼ 5.505, P < 0.001). There
was no significant effect of nitrogen level (F2,255 ¼ 0.582, P ¼ 0.560). However, there was a significant crop stage*nitrogen interaction (F12,255 ¼ 1.817, P ¼ 0.046) due to a large increase in juvenile snails (10 mm in shell height) in nitrogen-treated plots towards the end of the season (Fig. 5B). 4. Discussion Our study has shown that nitrogen fertilisers have lethal effects on apple snails. This confirms findings by De La Cruz et al. (2001) that nitrogen pulses in irrigated rice ecosystems initially cause a decrease in P. canaliculata survival. In our laboratory studies we found 10e60% mortality of adult snails when confined in the presence of fertiliser and in the field we found larger declines in snail numbers (18%) in fertilised plots compared to control plots. Complete fertiliser and urea appeared to be more toxic to snails compared to ammonium sulphate and organic fertiliser. However, the lethal response of adults to fertiliser was faster following application of ammonium sulphate and complete fertiliser than with urea and organic fertiliser. In one of three experiments conducted by De La Cruz et al. (2001), complete fertiliser was shown to cause a significant reduction in snail damage to rice when compared to urea. That experiment was conducted during only three days. However, our results indicate that the toxic affect of urea may be greater after 3e5 days. Our findings also demonstrate that the flooding conditions before and after fertiliser application play an important role in the
Fig. 2. The mean proportion of adult (A,C,E,G) and juvenile (B,D,F,H) Pomacea canaliculata that died over time following application of ammonium sulphate (A,B), complete fertiliser (C,D), organic manure (E,F) and urea (G,H) in flooded (open circles) and delayed flooded (closed circles) arenas, respectively.
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Table 3 Results of repeated measures GLM of the effects of nitrogen fertiliser level, drainage, snail size and timing of fertiliser application relative to snail introduction (fertiliser timing) on snail mortality (see also Fig. 3). df Between-subjects effects N2 level Drainage Snail size Fertiliser timing N2 level*drainage N2 level*snail size N2 level*fertiliser timing Snail size*fertiliser timing Drainage*snail size Drainage*fertiliser timing N2 level*drainage*snail size N2 level*drainage*fertiliser timing N2 level*snail size*fertiliser timing Drainage*snail size*fertiliser timing Error
2 1 1 1 2 2 2 1 1 1 2 2 2 1 122
F 13.739 0.104 0.261 36.715 0.017 0.192 1.256 1.226 0.260 0.401 1.886 1.803 0.273 4.995
P <0.001 0.748 0.610 <0.001 0.983 0.826 0.288 0.270 0.611 0.528 0.156 0.169 0.761 0.027
lethal impact of nitrogen fertiliser on P. canaliculata. Snails are more likely to survive if the water is drained or lost through percolation 24 h after fertiliser application, as often occurs after basal application during land preparation. Snails also survived better when the soil was flooded at the time of fertiliser application. In our experiments, snail mortality was greater when flooding was delayed, i.e. nitrogen applied to saturated soil which was subsequently
Within-subjects effects Time Time*N2 level Time*drainage Time*snail size Time*fertiliser timing Time*N2 level*drainage Time*N2 level*snail size Time*drainage*snail size Time*N2 level*fertiliser timing Time*drainage*fertiliser timing Time*snail size*fertiliser timing Time*N2 level*drainage*snail size Time*N2 level*drainage*fertiliser timing Time*N2 level*size*fertiliser timing Time*drainage*snail size*fertiliser timing Error
df
F
P
2 4 2 2 2 4 4 2 4 2 2 4 4 4 2 244
0.083 4.206 2.584 0.411 2.149 1.044 1.689 0.286 4.294 0.199 3.276 1.750 0.191 3.341 0.637
0.920 0.003 0.078 0.663 0.119 0.385 0.153 0.752 0.002 0.820 0.039 0.140 0.943 0.011 0.530
flooded and maintained in flooded conditions for several days, a practice recommended in rice cultivation to reduce losses from ammonia volatilization and/or nitrificationedenitrification (Smith and Diday, 2003; Norman et al., 2009). For many species of aquatic organisms, juveniles are usually more sensitive to toxicants than adults (Tchounwou et al., 1991; Mohammed, 2013). However, contrary to what might be
Fig. 3. The mean proportion of adult and juvenile Pomacea canaliculata that died over time following application of three levels of ammonium sulphate either immediately before snails were added (A, C) or 24 h before snails were added (B, D) to arenas that were continuously flooded (A, B, respectively) or were drained for a 5-day period (C, D, respectively).
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Table 4 Results of repeated measures GLM of the effects of substrate, snail size and timing of fertiliser application relative to snail introduction (fertiliser timing) on snail mortality (see also Fig. 4). df
F
P
Between-subjects effects Substrate Snail size Fertiliser timing Substrate*snail size Substrate*fertiliser timing Snail size*fertiliser timing Substrate*snail size*fertiliser timing Error
1 1 1 1 1 1 1 33
50.34 0.6 7.925 0.932 1.022 0.047 0.069
<0.001 0.808 0.008 0.341 0.319 0.829 0.795
Within-subjects effects Time Time*substrate Time*snail size Time*fertiliser timing Time*substrate*snail size Time*substrate*fertiliser timing Time*snail size*fertiliser timing Time*substrate*snail size*fertiliser timing Error
2 2 2 2 2 2 2 2 66
6.289 0.99 0.837 1.056 1.32 1.664 3.616 2.69
0.003 0.377 0.438 0.354 0.274 0.197 0.032 0.075
expected, our findings showed adult P. canaliculata to be more sensitive to nitrogen fertiliser than juvenile snails. These findings are similar to a study by Borlongan et al. (1998) in which tobacco dust caused higher mortality in larger and older brackish water pond snails (Cerithidea cingulata [Gmelin]) compared to smaller
Fig. 4. The mean proportion of adult and juvenile Pomacea canaliculata that died over time in arenas with soil and without soil following application of ammonium sulphate either immediately before snails were added (A) or 24 h before snails were added (B).
individuals. We found the lethal effects to also depend on the concentration of fertiliser: With ammonium sulphate, higher concentrations led to a higher mortality in snails. The same effects have been found in a previous laboratory study investigating the toxicity of ammonium sulphate and urea on Helisoma trivolvis (Say) and Biomphalaria havanensis, (Pfeiffer) two freshwater snails that inhabit rice fields in Cameroon (Tchounwou et al., 1991). Snail mortality may also have been underestimated in the study of Tchounwou et al. (1991) since their experiments were restricted to the first 48 h after fertiliser application. Snail mortality was reduced in our study by draining water for a period after the application of fertiliser or by adding snails to experimental arenas one day after the fertilizer was added. This indicates that the toxicity effects are diluted or diminished over time, through elutriation, volatilization, and/or neutralization. In an experiment where we removed soil from the arenas, we found the mortality of adults to decline slightly when snails were added one day later, indicating a possible dilution effect. However, in arenas with soil, mortality of both adults and juveniles declined considerably when added one day later, suggesting that soil plays a large role in neutralizing the toxic effects of fertilisers. This has consequences for the management of apple snails, as draining the field during the early stages of the rice crop is recommended to reduce snail movement and therefore reduce damage to seedlings (Litsinger and Estano, 1993). Our results indicate that a better management option might be to maintain fields flooded with a low water depth (i.e., 2 cm) following fertiliser application. This would limit the movement of larger snails, which are more likely to damage rice than smaller snails, but still expose the snails to the toxic effects of the fertilisers. Snails that move into treated rice fields after fertiliser application are less likely to be affected; however, the movement of large snails between rice fields is apparently rare (Stuart, unpublished data). We have described the gross responses of snails to fertiliser types and concentrations, but little is known of the mechanisms involved. Simpson et al. (1994c) have suggested a number of explanations for declining gastropod numbers in rice fields in the Philippines treated with fertilisers. These included indirect responses such as interspecific competition due to an increase in competitive invertebrates, changes in floodwater chemistry, such as lower dissolved oxygen caused by an increase in photosynthetic aquatic biomass, or because of a shift in balance from nitrogenfixing algae to non-nitrogen-fixing algae. However, the rapid lethal effects of some fertiliser types and the lower mortality of snails that were introduced one day after fertiliser treatments in our experiments suggest that P. canaliculata mortality is more likely the result of a direct response to nitrogen fertilizers and not due to increasing oxygen demands in the floodwater. Furthermore, under low oxygen conditions, Pomacea spp. use their siphons to breath air (Santos et al., 1987). Hydrolysis or ammonia volatilization following fertiliser application (Overrein and Moe, 1967; Mikklesen et al., 1978) could possibly have toxic effects on snails. However, there was no major change in the pH levels of water following fertiliser application in our experiments. Direct consumption of fertiliser by snails is another possible cause of snail mortality. Higher mortality of adults compared to juveniles in arenas applied with urea and complete fertiliser suggests that mortality is likely determined by the total amount of fertiliser consumed. Observations by De La Cruz et al. (2001) of intensified mucus secretion by snails after fertiliser applications are similar to observations of toxicity from molluscicides (Horgan, pers. obvs.), further suggesting that the lethal effects of fertilisers are likely due to poisoning after ingestion. Furthermore, if the toxic effects worked through the skin, we would expect higher mortality in juveniles due to higher surface area to body ratios in small snails, but we found smaller snails to better survive
A.M. Stuart et al. / Crop Protection 64 (2014) 78e87 Table 5 Results of repeated measures GLM of the effects of nitrogen level on snail abundance over different crop stages of a dry season rice crop (see also Fig. 5A). df
F
Between-subjects effects N2 level Error
P
2 15
0.559
0.583
Within-subjects effects Crop stage Crop stage*N2 level Error
6 12 90
5.209 1.017
<0.001 0.440
fertiliser applications. Information on the mechanisms of fertiliserinduced mortality of apple snails is still lacking, but would represent a convenient model for assessing the effects of fertilisers on benthic organisms in general.
85
In the field experiment we found reduced (but not significantly so) snail numbers at the start of the crop in plots with higher nitrogen. These effects were likely masked by the overall decrease in abundance across all treatments as a result of harrowing activities during final soil preparation, a management practice known to cause mortality in snails (Wada, 2004), and possibly as a result of intermittent flooding and draining during the early crop stages to reduce snail damage. Numbers increased after tillering, mainly due to recruitment, and this led to a significant change in snail size in the plots, suggesting the plots with higher nitrogen were more favourable for snail reproduction. This is likely to be through an increase in primary production and rice crop vegetation growth (Simpson et al., 1994a), which would provide more food, substrate for egg-laying, and vegetative cover. This pattern fits the model of initial toxicity followed by higher suitability of nitrogen treated plots. At later crop stages, once nitrogen has been assimilated into
Fig. 5. Mean abundance of Pomacea canaliculata per nitrogen fertiliser treatment (A) and the mean proportion of snails within each size category (mm; shell height) in plots that were applied with 0 kg/ha (B), 60 kg/ha (C) and 150 kg/ha (D) of nitrogen fertiliser over the duration of a dry season rice crop at the International Rice Research Institute Experimental Station.
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the plant, snails are no longer considered a crop production constraint, but may in fact be beneficial as herbivores of weeds and algae (Joshi et al., 2006) and thereby release nutrients into the rice ecosystem (Horgan et al., 2014). At this time, the snails are likely to benefit from the nitrogen assimilated into weeds and algae, as has also been demonstrated for other aquatic algae feeders that inhabit rice fields (Simpson et al., 1994a,b; Sunish and Reuben, 2002; Mutero et al., 2004). Interestingly, it was also observed by Muturi et al. (2007) that despite the toxic effects of inorganic nitrogen fertiliser on aquatic mosquito larvae in laboratory studies, mosquito larval populations increase following inorganic nitrogen fertiliser applications to rice fields. It has been suggested that this increase in mosquito larval populations may be due to an increasing availability of microscopic prey items (e.g., algae) following nitrogen assimilation, or because of a reduction in water turbidity making the fields more attractive for oviposition (Simpson et al., 1994b; Sunish and Reuben, 2002; Mutero et al., 2004). Apple snails continue to be a problem for rice production and it is apparent that no single management method will be sufficient to reduce snail damage to rice (Litsinger and Estano, 1993; Teo, 2003; Wada, 2004). Therefore, farmers are recommended to adopt several cultural control methods, such as planting older plants that are raised in low-density seedbeds to increase the overall resistance of rice plants (Yanes Figueroa et al., 2014), or by water management strategies to reduce the mobility of snails (Litsinger and Estano, 1993). Few cultural control options are aimed at reducing snail densities (most aim at reducing damage levels). We believe that careful management of fertilisers can increase snail mortality and improve protection of the crop at its most vulnerable stage. For example, use of complete fertiliser or urea is expected to increase snail mortality if applied to saturated or recently drained soil followed immediately by shallow flooding to limit snail movement. To avoid inefficient use of fertilisers that may incur financial losses and negative effects on the environment, site-specific nutrient recommendations should be followed (Buresh et al., 2007). However, in snail-infested areas, the first nitrogen fertiliser application could be increased from 30 to 50% of the total nitrogen input for a single rice crop when transplanting older seedlings (>24 days old) and shortduration varieties (Buresh et al., 2007). This is expected to maximise snail mortality during the vulnerable stages of the rice crop, as well as minimise snail mortality during later crop stages, when they are considered to be a beneficial herbivore of weeds (Joshi et al., 2006; Hidaka et al., 2007; Horgan et al., 2014). Because fertilisers, and the method of application are likely to also affect other benthic organisms in the rice fields, optimal fertiliser application regimes should also focus on minimizing the negative effects on beneficial species. We found fertilisers to have no effect on the native snail, Melanoides tuberculata (Müller) in similar experiments to those reported here (Stuart et al., unpublished data); however, more exhaustive testing, that includes a range of benthic organisms, would be welcome. Further long-term studies are also needed to examine the role nitrogen plays on apple snail population dynamics and plant susceptibility within tropical rice-based ecosystems over several rice-cropping seasons. This will help to better understand how crop management practices affect snails and to develop improved recommendations to management apple snails in rice. Acknowledgements We thank Alberto Naredo, Vincent Vertudes, Reyuel Quintana, Felisa Navor and Marol Recide for their assistance in sampling and measuring snails from the field and for monitoring the field experiment at IRRI farm. We thank two anonymous reviewers for helpful comments that improved the manuscript. This study was funded by the Global Rice Science Partnership (GRiSP). ANP was
funded through a training and mobility grant from the Spanish Government and administered through Becas Faro (Faro Global). References Borlongan, I.G., Coloso, R.M., Mosura, E.F., Sagisi, F.D., Mosura, A.T., 1998. Molluscicidal activity of tobacco dust against brackishwater pond snails (Cerithidea cingulata Gmelin). Crop Prot. 17, 401e404. Buresh, R.J., Witt, C., Pasuquin, J.M.C., 2007. Fertiliser best management practices in Southeast Asia. In: Proceedings of the IFA International Workshop on Fertiliser Best Management Practices, 7e9 March 2007, Brussels, Belgium. International Fertiliser Industry Association, Paris, France, pp. 221e229. Cooke, G.M., King, A.G., Miller, L., Johnson, R.N., 2012. A rapid molecular method to detect the invasive golden apple snail Pomacea canaliculata (Lamarck, 1822). Conserv. Genet. Resour. 4, 591e593. De La Cruz, M.S., Joshi, R.C., Martin, A.R., 2001. Basal application of fertiliser reduces golden apple snail population. Int. Rice Res. Notes 26, 20e21. FAO, 2008. Current World Fertiliser Trends and Outlook to 2011/12. Food and Agriculture Organization of the United Nations, Rome. FAO, 2012. Current World Fertiliser Trends and Outlook to 2016. Food and Agriculture Organization of the United Nations, Rome. Galloway, J.N., Aber, J.D., Erisman, J.W., Seitzinger, S.P., Howarth, R.H., Cowling, E.B., Cosby, B.J., 2003. The nitrogen cascade. BioScience 53, 341e356. Hayes, K.A., Joshi, R.C., Thiengo, S.C., Cowie, R.H., 2008. Out of South America: multiple origins of non-native apple snails in Asia. Divers. Distrib. 14, 701e702. Hidaka, K., Mineta, T., Tokuoka, M., 2007. Impact evaluation of apple snail, Pomacea canaliculata (Lamarck) invasion on vascular plant flora in rice fields: some notes in a case of rice fields using conventional herbicides in Matsuyama City. J. Rural Plan. Assoc. 2, 46e55 (in Japanese). Horgan, F.G., Stuart, A.M., Kudavidanage, E.P., 2014. Impact of invasive apple snails on the functioning and services of natural and managed wetlands. Acta Oecol. 54, 90e100. Joshi, R.C., Martin, E.C., Wada, T., Sebastian, L.S., 2006. Role of golden apple snail in organic rice cultivation and weed management. In: Joshi, R.C., Sebastian, L.S. (Eds.), Global Advances in Ecology and Management of Golden Apple Snails. Philippine Rice Research Institute, Nueva Ecija, pp. 483e488. Litsinger, J.A., Estano, D.B., 1993. Management of the golden apple snail Pomacea canaliculata (Lamarck) in rice. Crop Prot. 2, 363e370. Mikkelsen, D.S., De Datta, S.K., Obcemea, W., 1978. Ammonia volatilization losses from flooded rice soils. Soil. Sci. Soc. Am. J. 42, 725e730. Mohammed, A., 2013. Why are early life stages of aquatic organisms more sensitive to toxicants than adults? In: Gowder, S. (Ed.), New Insights into Toxicity and Drug Testing. InTech, Rijeka, Croatia, pp. 49e62. Mutero, C.M., Ng'ang'a, P.N., Wekoyela, P., Githure, J., Konradsen, F., 2004. Ammonium sulphate fertiliser increases larval populations of Anopheles arabiensis and culicine mosquitoes in rice fields. Acta Trop. 89, 187e192. Muturi, E.J., Jacob, B.G., Shililu, J., Novak, R., 2007. Laboratory studies on the effect of inorganic fertilisers on survival and development of immature Culex quinquefasciatus (Diptera: Culicidae). J. Vector Borne Dis. 44, 259e265. Norman, R.J., Wilson, C.E., Slaton, N.A., Griggs, B.R., Bushong, J.T., Gbur, E.E., 2009. Nitrogen fertiliser sources and timing before flooding dry-seeded, delayedflood rice. Soil. Sci. Soc. Am. J. 73, 2184. Overrein, L.N., Moe, P.G., 1967. Factors affecting urea hydrolysis and ammonia volatilization in soil. Soil. Sci. Soc. Am. J. 31, 57e61. Palis, F.G., Buresh, R.G., Singleton, G.R., Flor, R.J., 2007. Adoption of fertiliser best management practices: the need for a sociological approach. In: Proceedings of the IFA International Workshop on Fertiliser Best Management Practices, 7e9 March 2007, Brussels, Belgium. International Fertiliser Industry Association, Paris, France, pp. 99e110. Santos, C.A.Z., Penteado, C.H.S., Mendes, E.G., 1987. The respiratory responses of an amphibious snail Pomacea lineata (Spix, 1827), to temperature and oxygen tension variations. Comp. Biochem. Physiol. 86A, 409e415. Simpson, I.C., Roger, P.A., Oficial, R., Grant, I.F., 1994a. Effects of nitrogen fertiliser and pesticide management on floodwater ecology in a wetland ricefield. III. Dynamics of benthic molluscs. Biol. Fertil. Soils 18, 219e227. Simpson, I.C., Roger, P.A., Oficial, R., Grant, I.F., 1994b. Effects of nitrogen fertiliser and pesticide management on floodwater ecology in a wetland ricefield: I. Experimental design and dynamics of the photosynthetic aquatic biomass. Biol. Fertil. Soils 17, 129e137. Simpson, I.C., Roger, P.A., Oficial, R., Grant, I.F., 1994c. Effects of nitrogen fertiliser and pesticide management on floodwater ecology in a wetland ricefield. II. Dynamics of microcrustaceans and dipteran larvae. Biol. Fertil. Soils 17, 138e146. Smith, C.W., Diday, R.H., 2003. Rice: Origin, History, Technology and Production. John Wiley & Sons, New Jersey. Stewart, W.M., Dibb, D.W., Johnston, A.E., Smyth, T.J., 2005. The contribution of commercial fertiliser nutrients to food production. Agron. J. 97, 1e6. Sunish, I.P., Reuben, R., 2002. Factors influencing the abundance of Japanese encephalitis vectors in ricefields in India e II. Biotic Med. Veter. Entomol. 16, 1e9. Tanaka, K., Watanabe, T., Higuchi, H., Miyamoto, K., Yusa, Y., Kiyonaga, T., Kiyota, H., Suzuki, Y., Wada, T., 1999. Density-dependent growth and reproduction of the apple snail, Pomacea canaliculata: a density manipulation experiment in a paddy field. Res. Popul. Ecol. 41, 253e262.
A.M. Stuart et al. / Crop Protection 64 (2014) 78e87 Tchounwou, P.B., Englande Jr., A.J., Malek, E.A., 1991. Toxicity evaluation of ammonium sulphate and urea to three developmental stages of freshwater snails. Arch. Environ. Contam. Toxicol. 21, 359e364. Teo, S.S., 2003. Damage potential of the golden apple snail Pomacea canaliculata (Lamarck) in irrigated rice and its control by cultural approaches. Int. J. Pest Manag. 49, 49e55. Tilman, D., Lehman, C., 2001. Human-caused environmental change: impacts on plant diversity and evolution. Proc. Natl. Acad. Sci. U.S.A. 98, 5433e5440. Vitousek, P.M., 1994. Beyond global warming: ecology and global change. Ecology 75, 1861e1876. Wada, T., 2004. Strategies for controlling the apple snail Pomacea canaliculata (Lamarck) (Gastropoda: Ampullariidae ) in Japanese direct-sown paddy fields. Jpn. Agric. Res. Q. 38, 75e80. Yanes Figueroa, J., Almazan, M.L.P., Horgan, F.G., 2014. Reducing seed-densities in rice seedbeds improves the cultural control of apple snail damage. Crop Prot. 62, 23e31.
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Yoshida, K., Hoshikawa, K., Wada, T., Yusa, Y., 2009. Life cycle of the apple snail Pomacea canaliculata (Caenogastropoda: Ampullariidae) inhabiting Japanese paddy fields. Appl. Entomol. Zool. 44, 465e474. Youens, A.K., Burks, R.L., 2007. Comparing apple snails with oranges: the need to standardize measuring techniques when studying Pomacea. Aquat. Ecol. 42, 679e684. Yusa, Y., Wada, T., Takahashi, S., 2006. Effects of dormant duration, body size, selfburial and water condition on the long-term survival of the apple snail, Pomacea canaliculata (Gastropoda: Ampullariidae). Appl. Entomol. Zool. 41, 627e632. Zhu, Z.L., Chen, D.L., 2002. Nitrogen fertiliser use in China e contributions to food production, impacts on the environment and best management strategies. Nutr. Cycl. Agroecosyst. 63, 117e127.
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Effects of fertiliser applications on survival and recruitment of the apple snail, Pomacea canaliculata (Lamarck) Alexander M. Stuart, Alvaro Nogues Palenzuela, Carmencita C. Bernal, Angelee Fame Ramal, Finbarr G. Horgan* Crop and Environmental Sciences Division, International Rice Research Institute, DAPO Box 7777, Metro Manila, Philippines
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 January 2014 Received in revised form 28 May 2014 Accepted 30 May 2014 Available online
Since its introduction to Asia in the 1980s, the golden apple snail, Pomacea canaliculata (Lamarck), has represented a major constraint to the profitability of rice (Oryza sativa L.) farming by damaging rice seedlings during crop establishment. This study describes a series of experiments designed to determine the effects of nitrogenous fertilisers on snail fitness. We examined the possibility of a two-phase model of snail response to nitrogen, whereby fertilisers initially increase snail mortality through toxicity, but once assimilated into the rice ecosystem, eventually favour snail reproduction and survival. In experimental arenas, fertiliser had lethal effects: Complete fertiliser (14:14:14), urea, ammonium sulphate and organic fertilisers were associated with snail mortality, generally affecting adult snails more than juvenile snails, and with greater effects when applied to saturated soil that was subsequently flooded (as opposed to direct application to flooded soil). Snail mortality was found to decline considerably when snails were added to arenas one day after fertiliser application e this occurred in arenas with soil and water, but not in arenas with water only, suggesting that soil can reduce the toxic effects of fertilisers. In a field experiment, snail numbers declined in both fertilised and non-fertilised plots at the time of crop establishment. Numbers increased in all plots after rice tillering, with significantly more recruitment in plots with high nitrogen. Although the responses were generally weak in the field experiment, they did support the two-phase model. The consequences of fertiliser applications for snail management and ecosystem health are discussed. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Ammonium sulphate Benthic organisms Crop establishment Invasive species Nitrogen Urea
1. Introduction Pressures to increase food production to meet a growing world population and global food demands have led to an increasing use of chemical and organic inputs in agriculture; particularly nitrogenbased chemical fertilisers (FAO, 2008). The global demand for fertilisers is forecast to grow annually by 1.9%, from around 180 million tonnes in 2012 to around 194 million tonnes by 2016 (FAO, 2012). The use of nitrogen in agriculture has been linked to sustaining agricultural productivity (Stewart et al., 2005) and due to limitations in land availability, increasing fertiliser use on existing cropland is considered the most likely path farmers will take to increase production (FAO, 2008). However, high fertiliser use has a number of negative effects on ecosystems, such as altering plant community composition (Vitousek, 1994; Tilman and Lehman, 2001);
* Corresponding author. Tel.: þ63 2 580 5600x2708. E-mail address: [email protected] (F.G. Horgan). http://dx.doi.org/10.1016/j.cropro.2014.05.020 0261-2194/© 2014 Elsevier Ltd. All rights reserved.
increasing greenhouse gas (nitrous oxide) emissions; and contaminating groundwater, rivers, lakes and coastal waters, causing eutrophication (Zhu and Chen, 2002; Galloway et al., 2003). Rice (Oryza sativa L.) is the only major cereal crop that is grown in standing water, thus the use of nitrogen fertilisers during rice production is likely to have major consequences for the aquatic flora and fauna that inhabit rice ecosystems. Many aquatic organisms are susceptible to water-borne chemicals, including fertilisers, and to changes in floodwater chemistry as a result of chemical inputs (Simpson et al., 1994a). However, few studies have examined the effects of nitrogen fertilisers on aquatic organisms in rice ecosystems. Aquatic biomass, in particular green algae, followed by populations of ostracods and chironomid as well as mosquito larvae have been shown to increase following inorganic nitrogen fertiliser applications in rice fields (Simpson et al., 1994a,b; Sunish and Reuben, 2002; Mutero et al., 2004). Simpson et al. (1994a) indicated that benthic molluscan communities change over the course of rice crop development and that this may be associated with changing water chemistry resulting from fertiliser use.
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Furthermore, De La Cruz et al. (2001) demonstrated that inorganic nitrogen fertilisers cause mortality of golden apple snails (Pomacea canaliculata [Lamarck]) within a few days of application during land preparation for rice crops. Applications of nitrogen fertilisers from land preparation to within 14 days of crop establishment, followed by one to two further applications during the rice cropping season, are common practices in rice fields in South-East Asia (Buresh et al., 2007; Palis et al., 2007). The addition of nitrogen to the rice crop initially increases photosynthesis levels and pH and reduces concentrations of dissolved oxygen in the floodwater (Mikkelsen et al., 1978; Simpson et al., 1994a). However, assimilation of nitrogen also increases plant vigor, which benefits the crop, and increases primary productivity in the aquatic environment. This includes new growth of macrophytes and algae. Increasing plant and algal productivity can also benefit aquatic herbivores by increasing food quality and availability (Horgan et al., 2014). For aquatic herbivores, nitrogen fertilisers can therefore have both negative effects through toxicity and by altering the physio-chemical properties of the water, and positive effects by increasing the production (biomass) and quality of aquatic vegetation. Because of the dynamic nature of nitrogen in aquatic agro-ecosystems like rice, including sudden peaks during application and gradual declines post-application, interactions between nitrogen fertilisers and aquatic herbivores will be complex. The present study examines the effects of fertilisers on the survival of P. canaliculata (Lamarck) in rice systems. P. canaliculata was introduced to much of Asia, including the Philippines, during the 1980s and is considered a major pest of rice during early crop stages, but a beneficial herbivore of weeds during later crop stages (Joshi et al., 2006; Hidaka et al., 2007; Horgan et al., 2014). Previous field experiments by De La Cruz et al. (2001) indicated a reduction in rice damage from snails (missing hills) as a result of basal applications of inorganic nitrogen fertilisers, but the effects of compound fertilisers on snail mortality were largely inconclusive. Furthermore, as plants are affected by fertiliser treatments, i.e., increased growth rates, increased tolerance, and an increased capacity to compensate for damage, field experiments that monitor missing hills or other forms of damage to the crop do not clearly differentiate between the direct effects of snails on the plants and indirect effects mediated through snail mortality. Therefore, we conducted a series of controlled experiments to examine the direct effects of fertilisers on snail mortality. Furthermore, we addressed a two-phase model of snail responses to fertilisers, whereby mortality is increased soon after nitrogen application, but where survival and fitness is eventually enhanced once the excess nitrogen has been assimilated into plant tissues, creating a more favorable environment for herbivore growth and development.
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et al., 1999; Yusa et al., 2006). Shell height (also known as shell length; Youens and Burks, 2007) was measured from the tip of the apex to the lower margin of the aperture (Tanaka et al., 1999; Yoshida et al., 2009). 2.2. Experiment 1 e fertiliser (type) induced mortality The effects on snail mortality caused by four different fertilisers were examined. The fertilisers tested were ammonium sulphate [21-0-0 NPK], complete fertiliser [14-14-14 NPK], urea [46-0-0], and organic fertiliser (chicken manure) [2.6-3.0-3.0]. All treatments were replicated six times and a replicate set of control pots without fertiliser was maintained. In addition, we examined two flooding treatments (flooded, delayed flooding), and two sizes of snails (juvenile, adult) were tested. The fertiliser applications and flooding regimes were designed to simulate two contrasting farmer practices. One hundred and twenty pots (20.5 cm diameter) were filled with 10 cm of dry garden soil followed by the addition of water to a depth of 2 cm above the soil surface. In half the pots (60), the water was allowed to settle with 2 cm of standing water (flooded treatment). In the remaining pots, the water was drained over a 48 h period so that the soil was completely saturated but lacked standing water (delayed flooding treatment). Each fertiliser was added, based on the weight of soil in the pots, to contribute the equivalent of 80 kg/ha of nitrogen. Fertiliser was added directly to the pots by sprinkling over the water in the flooded pots and directly onto the saturated soil in the delayed flooding pots. Snails of two sizes (10e15 mm; 20e25 mm) were added immediately to the pots. Ten snails were added to each pot, maintaining snail size as a treatment. Thus, each pot either received ten juvenile snails or ten adult snails. After 2 h, water was added to the delayed flooded pots to a level of 2 cm. After a further 22 h, water was added to all pots to a level of 5 cm to prevent the snails from aestivating. Pots were interspersed on a greenhouse bench as a completely randomized design. The bench was under shade netting to prevent direct sunlight reaching the pots and to prevent unusually high temperatures. A fan was also used during the hottest daytime periods. Snails were provided with an ample supply of fresh lettuce throughout the experiment. Each pot was covered with a fine mesh lid to prevent snails from escaping. The greenhouse temperature during the experiment ranged from 24.4 to 36.3 C. Daylight hours were from about 0600 h to 1800 h. Daily pH readings, taken from treated pots without snails, ranged from 6.96 to 8.59 with no apparent effects of any of the treatment regimes. Snail mortality was monitored daily for the first five days, with an extra observation after seven days. Where snails appeared inactive, they were gently prodded to evaluate condition (dead or alive). Dead snails were removed from the pots daily.
2. Materials and methods 2.3. Experiment 2 e fertiliser (level) induced mortality 2.1. Study species The P. canaliculata in our experiments were collected from lowland irrigated rice fields at the International Rice Research ~ os, Institute Experimental Station (henceforth IRRI), Los Ban Philippines (14110 N, 121150 E). Although several Pomacea species have been introduced into Asia (and the Philippines), recent molecular analysis has indicated P. canaliculata as the most common and perhaps only apple snail species present in the Philippines (Hayes et al., 2008). Using published primers (Cooke et al., 2012), we verified P. canaliculata as the species occurring at our field sites. We separated adult and juvenile snails based on shell height, roughly classifying those with a shell height of 10e15 mm as juveniles and with a shell height of 20e25 mm as adults (Tanaka
The effects of three different application rates of nitrogen fertiliser on snail mortality were examined. In addition, we tested two snail introduction times in relation to fertiliser application times (snails added at the same time as fertiliser application, snails added 24 h after fertiliser application), two flooding treatments (drained, non-drained) and two sizes of snail (juvenile, adult). All treatment combinations were replicated six times. The water regimes were designed to simulate contrasting farmer practices and the delayed introduction of snails was designed to simulate snails moving into areas treated with fertiliser. One hundred and forty-four pots (20.5 cm diameter) were filled with 10 cm of homogenized paddy soil (with all gastropods removed by hand), water was added and allowed to settle in all the
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pots to a depth of 2 cm of standing water. Pots received three nitrogen treatments (ammonium sulphate equivalent to 0, 40 and 80 kg/ha nitrogen). The fertiliser was added directly to the pots, as a single application, by sprinkling over the standing water. Ten snails of each size category (10e15 mm or 20e25 mm) were added to the pots, maintaining snail size as a treatment, with either ten juvenile snails or ten adult snails added per pot. In half of the pots (72), fertiliser was applied 24 h before snails were added. In the remaining pots, fertiliser was applied immediately before snails were added. In half of each group of pots (36 2), the water level was increased to 5 cm 24 h after adding fertiliser (non-drained treatment) to prevent snails from aestivating. In the other half of each group, pots were gradually drained of standing water (by percolation through the soil) 24 h after fertiliser applications and maintained without water for five days, after which time water was added again to a depth of 5 cm (drained treatment). All pots were interspersed on a greenhouse bench under shade netting as a completely randomized design. The greenhouse temperature during the experiment ranged from 25.4 to 36.1 C. Daylight hours were from about 0600 h to 1800 h. Snails were provided with an ample supply of fresh lettuce throughout the experiment. Each pot was covered with a fine mesh lid to prevent snails escaping. Snail mortality was monitored over a 14-day period; one day after their introduction into pots and every other day thereafter. In pots that were drained, snails were monitored one day after their introduction into the pots, one day after the drained pots were re-flooded and every other day thereafter. Final observations were made after 14 days where snails had been added immediately and after 13 days where the snails had been added one day after nitrogen application. 2.4. Experiment 3 e substrate influence on fertiliser induced mortality The effects of soil substrate on nitrogen fertiliser induced snail mortality were examined. Following the protocol described for Experiment 2, 48 pots were prepared, 24 with soil and 24 without soil. Water was added to a depth of 2 cm and all pots received nitrogen treatments (as above) to an equivalent of 80 kg/ha. Ten juvenile snails (10e15 mm) each were added to half the pots and ten adult snails (20e25 mm) each to the remaining pots; these were each divided into two groups of pots with snails added immediately after fertiliser application to half the pots and snails added after 24 h to the remaining pots. 2.5. Experiment 4 e field study During the 2013 dry season at IRRI, snails were sampled from a group of 18 rice plots (400 m2 plot size). Each plot had been transplanted with two rice varieties, IR62 and IR64, as two subplots. Variety had no apparent effect on snail life-histories within plots (Horgan, unpublished). Plots were treated with three nitrogen (ammonium sulphate) regimes (six plots each at 0, 60, and 150 kg/ ha), each with independent irrigation and drainage systems to avoid fertiliser spread and mixture. Each plot received 0, 20 or 50 kg nitrogen/ha per application on three dates over the rice cropping season. These included a basal application to recently rotavated plots prior to final soil preparation and rice transplanting, a second application during the rice tillering stage, and a third application at the rice flowering stage. During basal application, 47 kg of solophos and 34 kg of muriate of potash were also added to the nitrogen treated plots. To sample snails, five 1 1 m quadrats were randomly placed in each plot and visible snails were collected manually. In addition, five circular quadrats, using an open-bottomed bucket
(27 cm diameter; 0.053 m2), were randomly placed in each plot and the soil (to a depth of 5 cm below the soil surface) and water within the bucket were collected and passed through a sieve (17 cm diameter, 2 mm mesh) to extract all snails. Snails were sampled two days before transplanting (one day prior to the basal nitrogen application and final soil preparation), eight days after transplanting (DAT) when fields were first flooded after transplanting, and during subsequent flooding events at the tillering stage of the rice crop (20 and 27 DAT). Further samples were taken every two to three weeks thereafter until the fields were drained for harvest. Sampled snails were measured before being returned to their original plots. 2.6. Statistical analyses 2.6.1. Experiment 1 e fertiliser (type) induced mortality To investigate the effects of fertiliser type (including the control), snail size, and time of flooding relative to fertiliser application on cumulative snail mortality at the end of the experiment (after seven days), univariate GLM was performed using ranktransformed data. Post-hoc multiple comparisons were conducted using Tukey tests. To investigate the effects of fertiliser type (excluding the control) on daily snail mortality rates over time (Days 1, 2, 3, 4, 5, 7), a repeated measures GLM with a compound symmetry variance-covariance structure was performed. The main factors entered into the model were fertiliser type, snail size and time of flooding. Data was rank-transformed. The control was excluded from this analysis of mortality rate over time as no snail mortality was observed in the control treatments and at the end of the experiment accumulated mortality was found to be significantly higher among all pesticides treatment when compared to the control. The repeated measures analysis therefore examined patterns in mortality among the fertilizer treatments only. 2.6.2. Experiment 2 e fertiliser (level) induced mortality To analyse the effects of nitrogen level, time of snail introduction relative to nitrogen application, drainage and snail size on daily mortality rates over time (on days 1, 6e7, and 13e14 after snail addition to pots), a repeated measures GLM with a compound symmetry variance-covariance structure was performed using rank-transformed data. Post-hoc multiple comparisons were conducted using Tukey tests. 2.6.3. Experiment 3 e substrate influence on fertiliser induced mortality To investigate the effects of soil (versus without soil) on daily mortality, a repeated measures GLM with a compound symmetry variance-covariance structure was performed using ranktransformed data for 1, 3 and 5 days after fertiliser treatment. The main factors entered into the model were substrate (soil, no soil), time of snail introduction and snail size. 2.6.4. Experiment 4 e field study To analyze differences in snail abundance in the field between nitrogen levels and different crop stages, a repeated measures GLM with a compound symmetry variance-covariance structure was performed using log-transformed data. Post-hoc multiple comparisons were conducted using Tukey tests. Univariate GLM was used to analyse differences in mean shell height between nitrogen levels and crop stages from snails collected from the circular quadrats. All analyses were conducted using SPSS (version 18).
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3. Results 3.1. Experiment 1 e fertiliser (type) induced mortality Snail mortality at the end of the experiment was affected by fertiliser type/treatment and snail size (Table 1; Fig. 1), with the highest mortality occurring among adults. Post-hoc analysis revealed a significant difference (P < 0.05) in snail mortality between the control and all four fertiliser types, but no significant difference between the four fertilisers tested (Table 1; Fig. 1). There was higher adult mortality in pots that received either complete fertiliser or urea, but similar levels of mortality between adults and juveniles in the other treatments resulting in a significant fertiliser*snail size interaction (Table 1). There was also a significant fertiliser type*flooding treatment interaction (Table 1) because mortalities were similar between flooded and delayed flooding treatments with organic fertilizer and ammonium sulphate, but delaying flooding increased mortality due to complete and urea fertilisers (Fig. 1). The control pots were removed from the repeated measure analysis because no snails had died in the control pots. Among the remaining pots, adult snails had higher fertiliser-induced mortality than juveniles and mortality was higher where flooding had been delayed (Table 2; Fig. 2). As with the cumulative data (above), there was a significant fertilizer*snail size interaction (Table 2). Mortality increased over the course of the experiment, with mortality levels generally diverging as the experiment progressed: this resulted in significant time*fertiliser type and time*snail size interactions (Table 2; Fig. 2): After application of ammonium sulphate and complete fertiliser, snails (particularly adults) rapidly died e the highest mortality generally occurring within the first two days (Table 2, Fig. 2). In comparison, the lethal response to urea and organic fertiliser was slightly delayed, with higher levels of mortality observed at least three days after fertiliser application. The mortality of adults and juveniles in pots treated with organic manure were similar throughout the experiments (Fig. 2).
time*drainage*fertiliser timing and drainage*snail size*fertiliser timing interactions (Table 3; Fig. 3).
3.2. Experiment 2 e fertiliser (level) induced mortality Snail mortality increased over the course of the experiment and was positively related to nitrogen levels resulting in significant nitrogen level and time*nitrogen level effects (Table 3, Fig. 3). Tukey tests indicated significant increases in mortality with each increase in nitrogen level (P 0.05). Mortality was also higher when snails were added to the pots on the same day as nitrogen fertiliser. Mortality of adults was higher than mortality of juveniles when snails were added one day after fertiliser, resulting in significant 3and 4-way interaction terms (Table 3, Fig. 3). In general, the lowest mortality occurred in pots that had been drained and where snails were added one day after the fertiliser. This produced significant
Table 1 Results of univariate GLM of the effects of fertiliser type, snail size and flooding treatment on cumulative snail mortality following a 14-day observation period (see also Fig. 1).
Fertiliser type/treatment Snail size Flooding treatment Fertiliser*snail size Fertiliser*flooding treatment Snail size*flooding treatment Fertiliser type*snail size*flooding treatment Error
Fig. 1. The mean proportion of adult and juvenile Pomacea canaliculata that had died during a 7-day observation period following application of ammonium sulphate, complete fertiliser, organic manure and urea in delayed flooded (A) and flooded (B) arenas.
df
F
P
4 1 1 4 4 1 4 100
8.933 44.392 0.967 6.516 3.427 0.131 0.034
<0.001 <0.001 0.328 <0.001 0.011 0.718 0.998
3.3. Experiment 3 e substrate influence on fertiliser induced mortality Snail mortality gradually increased over time (Table 4). Snail mortality was lower in pots that contained soil compared to pots without soil and with water only (Table 4, Fig. 4). Adding snails to the pots one day after the fertiliser was added also reduced mortality (Table 4, Fig. 4). Mortality levels were similar on day 1 of the experiment, but diverged as the experiment progressed increasing to higher levels when snails, particularly adults, were added on the same day as the fertiliser and producing a significant time*snail size*fertiliser timing interaction (Table 4, Fig. 4). 3.4. Experiment 4 e field study Across all treatments, snail abundance fluctuated throughout the course of crop development (Table 5, Fig. 5A); abundance declined following the basal application of fertiliser and final soil preparation until the tillering stage of the rice crop and then tended to increase until the end of the rice cropping season. Tukey tests indicated a higher abundance of snails only during landpreparation with no significant differences among later crop stages (P 0.05). Although, both the decline in abundance during the early crop stages and the increase in abundance during the later crop stages were greater in the high nitrogen plots, the effect of
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Table 2 Results from repeated measures GLM of the effects of fertiliser type, snail size and flooding treatment on snail mortality (see also Fig. 2). df Between-subjects effects Fertiliser type Snail size Flooding treatment Fertiliser*snail size Fertiliser*flooding treatment Snail size*flooding treatment Fertiliser type*snail size*flooding treatment Error Within-subjects effects Time Time*fertiliser type Time*snail size Time*flooding treatment Time*fertiliser type*snail size Time*flooding treatment*snail size Time*fertiliser type*flooding treatment Time*fertiliser type*snail size*flooding treatment Error
F
P
3 1 1 3 3 1 3 80
1.809 30.342 4.305 3.092 1.373 2.358 0.522
0.152 <0.001 0.041 0.032 0.257 0.129 0.668
5 15 5 5 15 5 15 15 400
5.855 6.311 6.234 0.933 6.136 0.866 1.233 0.824
<0.001 <0.001 <0.001 0.460 <0.001 0.504 0.244 0.651
nitrogen level and the crop stage*nitrogen interaction were not statistically significant (Table 5). Over the rice cropping season, snail shell height ranged from 4.1 mm to 36.1 mm (Fig. 5B). Mean snail shell height fluctuated over the course of crop development (F6,255 ¼ 5.505, P < 0.001). There
was no significant effect of nitrogen level (F2,255 ¼ 0.582, P ¼ 0.560). However, there was a significant crop stage*nitrogen interaction (F12,255 ¼ 1.817, P ¼ 0.046) due to a large increase in juvenile snails (10 mm in shell height) in nitrogen-treated plots towards the end of the season (Fig. 5B). 4. Discussion Our study has shown that nitrogen fertilisers have lethal effects on apple snails. This confirms findings by De La Cruz et al. (2001) that nitrogen pulses in irrigated rice ecosystems initially cause a decrease in P. canaliculata survival. In our laboratory studies we found 10e60% mortality of adult snails when confined in the presence of fertiliser and in the field we found larger declines in snail numbers (18%) in fertilised plots compared to control plots. Complete fertiliser and urea appeared to be more toxic to snails compared to ammonium sulphate and organic fertiliser. However, the lethal response of adults to fertiliser was faster following application of ammonium sulphate and complete fertiliser than with urea and organic fertiliser. In one of three experiments conducted by De La Cruz et al. (2001), complete fertiliser was shown to cause a significant reduction in snail damage to rice when compared to urea. That experiment was conducted during only three days. However, our results indicate that the toxic affect of urea may be greater after 3e5 days. Our findings also demonstrate that the flooding conditions before and after fertiliser application play an important role in the
Fig. 2. The mean proportion of adult (A,C,E,G) and juvenile (B,D,F,H) Pomacea canaliculata that died over time following application of ammonium sulphate (A,B), complete fertiliser (C,D), organic manure (E,F) and urea (G,H) in flooded (open circles) and delayed flooded (closed circles) arenas, respectively.
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Table 3 Results of repeated measures GLM of the effects of nitrogen fertiliser level, drainage, snail size and timing of fertiliser application relative to snail introduction (fertiliser timing) on snail mortality (see also Fig. 3). df Between-subjects effects N2 level Drainage Snail size Fertiliser timing N2 level*drainage N2 level*snail size N2 level*fertiliser timing Snail size*fertiliser timing Drainage*snail size Drainage*fertiliser timing N2 level*drainage*snail size N2 level*drainage*fertiliser timing N2 level*snail size*fertiliser timing Drainage*snail size*fertiliser timing Error
2 1 1 1 2 2 2 1 1 1 2 2 2 1 122
F 13.739 0.104 0.261 36.715 0.017 0.192 1.256 1.226 0.260 0.401 1.886 1.803 0.273 4.995
P <0.001 0.748 0.610 <0.001 0.983 0.826 0.288 0.270 0.611 0.528 0.156 0.169 0.761 0.027
lethal impact of nitrogen fertiliser on P. canaliculata. Snails are more likely to survive if the water is drained or lost through percolation 24 h after fertiliser application, as often occurs after basal application during land preparation. Snails also survived better when the soil was flooded at the time of fertiliser application. In our experiments, snail mortality was greater when flooding was delayed, i.e. nitrogen applied to saturated soil which was subsequently
Within-subjects effects Time Time*N2 level Time*drainage Time*snail size Time*fertiliser timing Time*N2 level*drainage Time*N2 level*snail size Time*drainage*snail size Time*N2 level*fertiliser timing Time*drainage*fertiliser timing Time*snail size*fertiliser timing Time*N2 level*drainage*snail size Time*N2 level*drainage*fertiliser timing Time*N2 level*size*fertiliser timing Time*drainage*snail size*fertiliser timing Error
df
F
P
2 4 2 2 2 4 4 2 4 2 2 4 4 4 2 244
0.083 4.206 2.584 0.411 2.149 1.044 1.689 0.286 4.294 0.199 3.276 1.750 0.191 3.341 0.637
0.920 0.003 0.078 0.663 0.119 0.385 0.153 0.752 0.002 0.820 0.039 0.140 0.943 0.011 0.530
flooded and maintained in flooded conditions for several days, a practice recommended in rice cultivation to reduce losses from ammonia volatilization and/or nitrificationedenitrification (Smith and Diday, 2003; Norman et al., 2009). For many species of aquatic organisms, juveniles are usually more sensitive to toxicants than adults (Tchounwou et al., 1991; Mohammed, 2013). However, contrary to what might be
Fig. 3. The mean proportion of adult and juvenile Pomacea canaliculata that died over time following application of three levels of ammonium sulphate either immediately before snails were added (A, C) or 24 h before snails were added (B, D) to arenas that were continuously flooded (A, B, respectively) or were drained for a 5-day period (C, D, respectively).
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Table 4 Results of repeated measures GLM of the effects of substrate, snail size and timing of fertiliser application relative to snail introduction (fertiliser timing) on snail mortality (see also Fig. 4). df
F
P
Between-subjects effects Substrate Snail size Fertiliser timing Substrate*snail size Substrate*fertiliser timing Snail size*fertiliser timing Substrate*snail size*fertiliser timing Error
1 1 1 1 1 1 1 33
50.34 0.6 7.925 0.932 1.022 0.047 0.069
<0.001 0.808 0.008 0.341 0.319 0.829 0.795
Within-subjects effects Time Time*substrate Time*snail size Time*fertiliser timing Time*substrate*snail size Time*substrate*fertiliser timing Time*snail size*fertiliser timing Time*substrate*snail size*fertiliser timing Error
2 2 2 2 2 2 2 2 66
6.289 0.99 0.837 1.056 1.32 1.664 3.616 2.69
0.003 0.377 0.438 0.354 0.274 0.197 0.032 0.075
expected, our findings showed adult P. canaliculata to be more sensitive to nitrogen fertiliser than juvenile snails. These findings are similar to a study by Borlongan et al. (1998) in which tobacco dust caused higher mortality in larger and older brackish water pond snails (Cerithidea cingulata [Gmelin]) compared to smaller
Fig. 4. The mean proportion of adult and juvenile Pomacea canaliculata that died over time in arenas with soil and without soil following application of ammonium sulphate either immediately before snails were added (A) or 24 h before snails were added (B).
individuals. We found the lethal effects to also depend on the concentration of fertiliser: With ammonium sulphate, higher concentrations led to a higher mortality in snails. The same effects have been found in a previous laboratory study investigating the toxicity of ammonium sulphate and urea on Helisoma trivolvis (Say) and Biomphalaria havanensis, (Pfeiffer) two freshwater snails that inhabit rice fields in Cameroon (Tchounwou et al., 1991). Snail mortality may also have been underestimated in the study of Tchounwou et al. (1991) since their experiments were restricted to the first 48 h after fertiliser application. Snail mortality was reduced in our study by draining water for a period after the application of fertiliser or by adding snails to experimental arenas one day after the fertilizer was added. This indicates that the toxicity effects are diluted or diminished over time, through elutriation, volatilization, and/or neutralization. In an experiment where we removed soil from the arenas, we found the mortality of adults to decline slightly when snails were added one day later, indicating a possible dilution effect. However, in arenas with soil, mortality of both adults and juveniles declined considerably when added one day later, suggesting that soil plays a large role in neutralizing the toxic effects of fertilisers. This has consequences for the management of apple snails, as draining the field during the early stages of the rice crop is recommended to reduce snail movement and therefore reduce damage to seedlings (Litsinger and Estano, 1993). Our results indicate that a better management option might be to maintain fields flooded with a low water depth (i.e., 2 cm) following fertiliser application. This would limit the movement of larger snails, which are more likely to damage rice than smaller snails, but still expose the snails to the toxic effects of the fertilisers. Snails that move into treated rice fields after fertiliser application are less likely to be affected; however, the movement of large snails between rice fields is apparently rare (Stuart, unpublished data). We have described the gross responses of snails to fertiliser types and concentrations, but little is known of the mechanisms involved. Simpson et al. (1994c) have suggested a number of explanations for declining gastropod numbers in rice fields in the Philippines treated with fertilisers. These included indirect responses such as interspecific competition due to an increase in competitive invertebrates, changes in floodwater chemistry, such as lower dissolved oxygen caused by an increase in photosynthetic aquatic biomass, or because of a shift in balance from nitrogenfixing algae to non-nitrogen-fixing algae. However, the rapid lethal effects of some fertiliser types and the lower mortality of snails that were introduced one day after fertiliser treatments in our experiments suggest that P. canaliculata mortality is more likely the result of a direct response to nitrogen fertilizers and not due to increasing oxygen demands in the floodwater. Furthermore, under low oxygen conditions, Pomacea spp. use their siphons to breath air (Santos et al., 1987). Hydrolysis or ammonia volatilization following fertiliser application (Overrein and Moe, 1967; Mikklesen et al., 1978) could possibly have toxic effects on snails. However, there was no major change in the pH levels of water following fertiliser application in our experiments. Direct consumption of fertiliser by snails is another possible cause of snail mortality. Higher mortality of adults compared to juveniles in arenas applied with urea and complete fertiliser suggests that mortality is likely determined by the total amount of fertiliser consumed. Observations by De La Cruz et al. (2001) of intensified mucus secretion by snails after fertiliser applications are similar to observations of toxicity from molluscicides (Horgan, pers. obvs.), further suggesting that the lethal effects of fertilisers are likely due to poisoning after ingestion. Furthermore, if the toxic effects worked through the skin, we would expect higher mortality in juveniles due to higher surface area to body ratios in small snails, but we found smaller snails to better survive
A.M. Stuart et al. / Crop Protection 64 (2014) 78e87 Table 5 Results of repeated measures GLM of the effects of nitrogen level on snail abundance over different crop stages of a dry season rice crop (see also Fig. 5A). df
F
Between-subjects effects N2 level Error
P
2 15
0.559
0.583
Within-subjects effects Crop stage Crop stage*N2 level Error
6 12 90
5.209 1.017
<0.001 0.440
fertiliser applications. Information on the mechanisms of fertiliserinduced mortality of apple snails is still lacking, but would represent a convenient model for assessing the effects of fertilisers on benthic organisms in general.
85
In the field experiment we found reduced (but not significantly so) snail numbers at the start of the crop in plots with higher nitrogen. These effects were likely masked by the overall decrease in abundance across all treatments as a result of harrowing activities during final soil preparation, a management practice known to cause mortality in snails (Wada, 2004), and possibly as a result of intermittent flooding and draining during the early crop stages to reduce snail damage. Numbers increased after tillering, mainly due to recruitment, and this led to a significant change in snail size in the plots, suggesting the plots with higher nitrogen were more favourable for snail reproduction. This is likely to be through an increase in primary production and rice crop vegetation growth (Simpson et al., 1994a), which would provide more food, substrate for egg-laying, and vegetative cover. This pattern fits the model of initial toxicity followed by higher suitability of nitrogen treated plots. At later crop stages, once nitrogen has been assimilated into
Fig. 5. Mean abundance of Pomacea canaliculata per nitrogen fertiliser treatment (A) and the mean proportion of snails within each size category (mm; shell height) in plots that were applied with 0 kg/ha (B), 60 kg/ha (C) and 150 kg/ha (D) of nitrogen fertiliser over the duration of a dry season rice crop at the International Rice Research Institute Experimental Station.
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the plant, snails are no longer considered a crop production constraint, but may in fact be beneficial as herbivores of weeds and algae (Joshi et al., 2006) and thereby release nutrients into the rice ecosystem (Horgan et al., 2014). At this time, the snails are likely to benefit from the nitrogen assimilated into weeds and algae, as has also been demonstrated for other aquatic algae feeders that inhabit rice fields (Simpson et al., 1994a,b; Sunish and Reuben, 2002; Mutero et al., 2004). Interestingly, it was also observed by Muturi et al. (2007) that despite the toxic effects of inorganic nitrogen fertiliser on aquatic mosquito larvae in laboratory studies, mosquito larval populations increase following inorganic nitrogen fertiliser applications to rice fields. It has been suggested that this increase in mosquito larval populations may be due to an increasing availability of microscopic prey items (e.g., algae) following nitrogen assimilation, or because of a reduction in water turbidity making the fields more attractive for oviposition (Simpson et al., 1994b; Sunish and Reuben, 2002; Mutero et al., 2004). Apple snails continue to be a problem for rice production and it is apparent that no single management method will be sufficient to reduce snail damage to rice (Litsinger and Estano, 1993; Teo, 2003; Wada, 2004). Therefore, farmers are recommended to adopt several cultural control methods, such as planting older plants that are raised in low-density seedbeds to increase the overall resistance of rice plants (Yanes Figueroa et al., 2014), or by water management strategies to reduce the mobility of snails (Litsinger and Estano, 1993). Few cultural control options are aimed at reducing snail densities (most aim at reducing damage levels). We believe that careful management of fertilisers can increase snail mortality and improve protection of the crop at its most vulnerable stage. For example, use of complete fertiliser or urea is expected to increase snail mortality if applied to saturated or recently drained soil followed immediately by shallow flooding to limit snail movement. To avoid inefficient use of fertilisers that may incur financial losses and negative effects on the environment, site-specific nutrient recommendations should be followed (Buresh et al., 2007). However, in snail-infested areas, the first nitrogen fertiliser application could be increased from 30 to 50% of the total nitrogen input for a single rice crop when transplanting older seedlings (>24 days old) and shortduration varieties (Buresh et al., 2007). This is expected to maximise snail mortality during the vulnerable stages of the rice crop, as well as minimise snail mortality during later crop stages, when they are considered to be a beneficial herbivore of weeds (Joshi et al., 2006; Hidaka et al., 2007; Horgan et al., 2014). Because fertilisers, and the method of application are likely to also affect other benthic organisms in the rice fields, optimal fertiliser application regimes should also focus on minimizing the negative effects on beneficial species. We found fertilisers to have no effect on the native snail, Melanoides tuberculata (Müller) in similar experiments to those reported here (Stuart et al., unpublished data); however, more exhaustive testing, that includes a range of benthic organisms, would be welcome. Further long-term studies are also needed to examine the role nitrogen plays on apple snail population dynamics and plant susceptibility within tropical rice-based ecosystems over several rice-cropping seasons. This will help to better understand how crop management practices affect snails and to develop improved recommendations to management apple snails in rice. Acknowledgements We thank Alberto Naredo, Vincent Vertudes, Reyuel Quintana, Felisa Navor and Marol Recide for their assistance in sampling and measuring snails from the field and for monitoring the field experiment at IRRI farm. We thank two anonymous reviewers for helpful comments that improved the manuscript. This study was funded by the Global Rice Science Partnership (GRiSP). ANP was
funded through a training and mobility grant from the Spanish Government and administered through Becas Faro (Faro Global). References Borlongan, I.G., Coloso, R.M., Mosura, E.F., Sagisi, F.D., Mosura, A.T., 1998. Molluscicidal activity of tobacco dust against brackishwater pond snails (Cerithidea cingulata Gmelin). Crop Prot. 17, 401e404. Buresh, R.J., Witt, C., Pasuquin, J.M.C., 2007. Fertiliser best management practices in Southeast Asia. In: Proceedings of the IFA International Workshop on Fertiliser Best Management Practices, 7e9 March 2007, Brussels, Belgium. International Fertiliser Industry Association, Paris, France, pp. 221e229. Cooke, G.M., King, A.G., Miller, L., Johnson, R.N., 2012. A rapid molecular method to detect the invasive golden apple snail Pomacea canaliculata (Lamarck, 1822). Conserv. Genet. Resour. 4, 591e593. De La Cruz, M.S., Joshi, R.C., Martin, A.R., 2001. Basal application of fertiliser reduces golden apple snail population. Int. Rice Res. Notes 26, 20e21. FAO, 2008. Current World Fertiliser Trends and Outlook to 2011/12. Food and Agriculture Organization of the United Nations, Rome. FAO, 2012. Current World Fertiliser Trends and Outlook to 2016. Food and Agriculture Organization of the United Nations, Rome. Galloway, J.N., Aber, J.D., Erisman, J.W., Seitzinger, S.P., Howarth, R.H., Cowling, E.B., Cosby, B.J., 2003. The nitrogen cascade. BioScience 53, 341e356. Hayes, K.A., Joshi, R.C., Thiengo, S.C., Cowie, R.H., 2008. Out of South America: multiple origins of non-native apple snails in Asia. Divers. Distrib. 14, 701e702. Hidaka, K., Mineta, T., Tokuoka, M., 2007. Impact evaluation of apple snail, Pomacea canaliculata (Lamarck) invasion on vascular plant flora in rice fields: some notes in a case of rice fields using conventional herbicides in Matsuyama City. J. Rural Plan. Assoc. 2, 46e55 (in Japanese). Horgan, F.G., Stuart, A.M., Kudavidanage, E.P., 2014. Impact of invasive apple snails on the functioning and services of natural and managed wetlands. Acta Oecol. 54, 90e100. Joshi, R.C., Martin, E.C., Wada, T., Sebastian, L.S., 2006. Role of golden apple snail in organic rice cultivation and weed management. In: Joshi, R.C., Sebastian, L.S. (Eds.), Global Advances in Ecology and Management of Golden Apple Snails. Philippine Rice Research Institute, Nueva Ecija, pp. 483e488. Litsinger, J.A., Estano, D.B., 1993. Management of the golden apple snail Pomacea canaliculata (Lamarck) in rice. Crop Prot. 2, 363e370. Mikkelsen, D.S., De Datta, S.K., Obcemea, W., 1978. Ammonia volatilization losses from flooded rice soils. Soil. Sci. Soc. Am. J. 42, 725e730. Mohammed, A., 2013. Why are early life stages of aquatic organisms more sensitive to toxicants than adults? In: Gowder, S. (Ed.), New Insights into Toxicity and Drug Testing. InTech, Rijeka, Croatia, pp. 49e62. Mutero, C.M., Ng'ang'a, P.N., Wekoyela, P., Githure, J., Konradsen, F., 2004. Ammonium sulphate fertiliser increases larval populations of Anopheles arabiensis and culicine mosquitoes in rice fields. Acta Trop. 89, 187e192. Muturi, E.J., Jacob, B.G., Shililu, J., Novak, R., 2007. Laboratory studies on the effect of inorganic fertilisers on survival and development of immature Culex quinquefasciatus (Diptera: Culicidae). J. Vector Borne Dis. 44, 259e265. Norman, R.J., Wilson, C.E., Slaton, N.A., Griggs, B.R., Bushong, J.T., Gbur, E.E., 2009. Nitrogen fertiliser sources and timing before flooding dry-seeded, delayedflood rice. Soil. Sci. Soc. Am. J. 73, 2184. Overrein, L.N., Moe, P.G., 1967. Factors affecting urea hydrolysis and ammonia volatilization in soil. Soil. Sci. Soc. Am. J. 31, 57e61. Palis, F.G., Buresh, R.G., Singleton, G.R., Flor, R.J., 2007. Adoption of fertiliser best management practices: the need for a sociological approach. In: Proceedings of the IFA International Workshop on Fertiliser Best Management Practices, 7e9 March 2007, Brussels, Belgium. International Fertiliser Industry Association, Paris, France, pp. 99e110. Santos, C.A.Z., Penteado, C.H.S., Mendes, E.G., 1987. The respiratory responses of an amphibious snail Pomacea lineata (Spix, 1827), to temperature and oxygen tension variations. Comp. Biochem. Physiol. 86A, 409e415. Simpson, I.C., Roger, P.A., Oficial, R., Grant, I.F., 1994a. Effects of nitrogen fertiliser and pesticide management on floodwater ecology in a wetland ricefield. III. Dynamics of benthic molluscs. Biol. Fertil. Soils 18, 219e227. Simpson, I.C., Roger, P.A., Oficial, R., Grant, I.F., 1994b. Effects of nitrogen fertiliser and pesticide management on floodwater ecology in a wetland ricefield: I. Experimental design and dynamics of the photosynthetic aquatic biomass. Biol. Fertil. Soils 17, 129e137. Simpson, I.C., Roger, P.A., Oficial, R., Grant, I.F., 1994c. Effects of nitrogen fertiliser and pesticide management on floodwater ecology in a wetland ricefield. II. Dynamics of microcrustaceans and dipteran larvae. Biol. Fertil. Soils 17, 138e146. Smith, C.W., Diday, R.H., 2003. Rice: Origin, History, Technology and Production. John Wiley & Sons, New Jersey. Stewart, W.M., Dibb, D.W., Johnston, A.E., Smyth, T.J., 2005. The contribution of commercial fertiliser nutrients to food production. Agron. J. 97, 1e6. Sunish, I.P., Reuben, R., 2002. Factors influencing the abundance of Japanese encephalitis vectors in ricefields in India e II. Biotic Med. Veter. Entomol. 16, 1e9. Tanaka, K., Watanabe, T., Higuchi, H., Miyamoto, K., Yusa, Y., Kiyonaga, T., Kiyota, H., Suzuki, Y., Wada, T., 1999. Density-dependent growth and reproduction of the apple snail, Pomacea canaliculata: a density manipulation experiment in a paddy field. Res. Popul. Ecol. 41, 253e262.
A.M. Stuart et al. / Crop Protection 64 (2014) 78e87 Tchounwou, P.B., Englande Jr., A.J., Malek, E.A., 1991. Toxicity evaluation of ammonium sulphate and urea to three developmental stages of freshwater snails. Arch. Environ. Contam. Toxicol. 21, 359e364. Teo, S.S., 2003. Damage potential of the golden apple snail Pomacea canaliculata (Lamarck) in irrigated rice and its control by cultural approaches. Int. J. Pest Manag. 49, 49e55. Tilman, D., Lehman, C., 2001. Human-caused environmental change: impacts on plant diversity and evolution. Proc. Natl. Acad. Sci. U.S.A. 98, 5433e5440. Vitousek, P.M., 1994. Beyond global warming: ecology and global change. Ecology 75, 1861e1876. Wada, T., 2004. Strategies for controlling the apple snail Pomacea canaliculata (Lamarck) (Gastropoda: Ampullariidae ) in Japanese direct-sown paddy fields. Jpn. Agric. Res. Q. 38, 75e80. Yanes Figueroa, J., Almazan, M.L.P., Horgan, F.G., 2014. Reducing seed-densities in rice seedbeds improves the cultural control of apple snail damage. Crop Prot. 62, 23e31.
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Yoshida, K., Hoshikawa, K., Wada, T., Yusa, Y., 2009. Life cycle of the apple snail Pomacea canaliculata (Caenogastropoda: Ampullariidae) inhabiting Japanese paddy fields. Appl. Entomol. Zool. 44, 465e474. Youens, A.K., Burks, R.L., 2007. Comparing apple snails with oranges: the need to standardize measuring techniques when studying Pomacea. Aquat. Ecol. 42, 679e684. Yusa, Y., Wada, T., Takahashi, S., 2006. Effects of dormant duration, body size, selfburial and water condition on the long-term survival of the apple snail, Pomacea canaliculata (Gastropoda: Ampullariidae). Appl. Entomol. Zool. 41, 627e632. Zhu, Z.L., Chen, D.L., 2002. Nitrogen fertiliser use in China e contributions to food production, impacts on the environment and best management strategies. Nutr. Cycl. Agroecosyst. 63, 117e127.