Pesticide-induced changes in fecundity and rice stripe virus transmission ability in Laodelphax striatellus (Homoptera: Delphacidae)

Journal of Asia-Pacific Entomology 20 (2017) 830–834

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Pesticide-induced changes in fecundity and rice stripe virus transmission ability in Laodelphax striatellus (Homoptera: Delphacidae)

MARK

Guo-Qing Yang, Xin Gao, Nan-Nan Zhang, Dan-Yu Chen, Fang Liu⁎, Jian-Xiang Xu⁎, Jin-Cai Wu School of Horticulture and Plant Protection & Institute of Insect Ecology, Yangzhou University, Yangzhou 225009, China Joint International Research Laboratory of Agriculture & Agri-Product Safety, the Ministry of Education, Yangzhou University, Yangzhou 225009, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Small brown planthopper (SBPH) Rice stripe virus (RSV) Pesticides Fecundity

The widespread use of pesticides in rice has caused the resurgence of the small brown planthopper (SBPH), Laodelphax striatellus. In addition to inciting damage by feeding on phloem cells, SBPH also functions as a vector for rice stripe virus (RSV), which can cause serious yield losses. In general, studies focused on pesticide-induced stimulation of SBPH populations have focused on the insects; little information is available on the impact of pesticides on RSV transmission by SBPH. The present study investigated the effects of two pesticides (validamycin and triazophos) on the fecundity and RSV transmission ability of SBPH. Our results demonstrated that the fecundity of non-viruliferous (naïve) or viruliferous SBPH was enhanced by exposure to triazophos at the LC20 and validamycin at 200 mg L− 1. Meanwhile, the increased number of eggs produced by viruliferous females treated with pesticides was larger than those from naïve females. Exposure to validamycin caused an increase in horizontal transmission of RSV; furthermore, vertical viral transmission rates of SBPH were significantly increased by exposure to triazophos. The present study provides valuable information for understanding the mechanisms underlying the resurgence of SBPH and subsequent outbreaks of RSV.

Introduction The small brown planthopper (SBPH), Laodelphax striatellus, is one of the most important insect pests of rice and wheat in Asia. SBPH damages rice plants by phloem-feeding and by functioning as a vector that transmits rice stripe virus (RSV), which can cause serious crop losses (Deng et al., 2013). Economic losses due to SBPH have escalated in eastern China and western Japan due to the population density of SBPH and the increased frequency of viruliferous insects carrying RSV (Wang et al., 2008a; Wei et al., 2009; Sanada-Morimura et al., 2011). Several factors are associated with the population dynamics of SBPH; these include changes in the agro-ecosystem, crop rotation, climate change, and pesticide application (Yamamura et al., 2006; Zhang et al., 2008; Jing et al., 2015; Sanada-Morimura et al., 2011). Pesticides are generally used to control SBPH in rice; however, widespread application has resulted in insecticide resistance in SBPH (SanadaMorimura et al., 2011) and reduced populations of natural enemies (Chelliah and Heinrichs, 1980). A resurgence in the SBPH population often occurs when insecticides are applied at a sublethal doses. In the field, insecticide degradation occurs via photolysis and hydrolysis, thereby exposing insect pests to sublethal concentrations of insecticides for long periods of time (Desneux et al., 2005). Recent investigations

⁎

have shown that the application of the fungicide carbendazim and the insecticide chlorpyrifos increased the population of SBPH in rice or wheat fields (Shen et al., 2007; Xu et al., 2008). To our knowledge, studies have not been undertaken to evaluate pesticide effects on the fecundity of viruliferous and naïve SBPH populations, which commonly occur in rice fields. Furthermore, it is unclear whether pesticide applications can influence RSV transmission by SBPH. The lack of comprehensive and integrated considerations of fecundity and RSV transmission after pesticide use has limited our understanding of the underlying mechanisms in the resurgence of SBPH and dissemination of RSV. The objective of the present study was to examine the effects of the fungicide validamycin and the insecticide triazophos on SBPH fecundity and RSV transmission. Materials and methods Rice varieties, insects, and pesticides Wuyujing No. 3, a rice variety susceptible to RSV (Jing et al., 2015), was used in all experiments. Rice seedlings were sown in plastic cups (5 cm diameter, 7 cm depth), and plants at the third-leaf stage were used in the experiments.

Corresponding author. E-mail addresses: [email protected] (F. Liu), [email protected] (J.-X. Xu).

http://dx.doi.org/10.1016/j.aspen.2017.05.005 Received 10 March 2017; Received in revised form 15 May 2017; Accepted 16 May 2017 Available online 17 May 2017 1226-8615/ © 2017 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.

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and the total represented the number of eggs laid per female. Each treatment was replicated five times.

SBPH populations were previously collected from an experimental farm located at Yangzhou University and were maintained in the laboratory for nearly six years. These laboratory populations were utilized in the present study because the occurrence of highly viruliferous SBPH was < 10% in rice fields near Yangzhou (Jing et al., 2015). Planthoppers were reared on Wuyujing No. 3 at 25 ± 1 °C, 60–80% RH with a 14:10 h (L:D) photoperiod. Plants were grown in plastic boxes (22 × 15 × 16 cm) inside sponges; these were enclosed in nylon mesh after the insects were introduced. Individual planthoppers were transferred to new rice seedlings (2–3 cm tall) every 10–14 days to assure sufficient nutrition. Individual viruliferous (RSV-infected) and naïve (non-infected) planthoppers were screened for RSV as described by Liu et al. (2007). Approximately 24 h after emergence, SBPH mating pairs were transferred to rice seedlings in plastic cups with transparent cylindrical plastic cages and incubated for 2 days to ensure that the female was fertilized. The mated females were subsequently labeled and raised individually in plastic cups containing rice seedlings (2–3 cm tall) for oviposition. Females were collected after they died and analyzed for RSV using the dot immunobinding assay (DIBA) described by Zhou et al. (2004). If a female was naïve, her progeny was also considered naïve, and these progeny were used as naïve insects in subsequent experiments. Validamycin and triazophos, which are commonly used pesticides in Jiangsu Province, were evaluated in this study. The fungicide validamycin (Jiangsu Shenhua Biochemical Co. Ltd., Jiangsu, China) is applied for control of sheath blight in rice and wheat, which is caused by Rhizoctonia solani. Validamycin was applied to rice during the booting and heading stages at 112.5–255 g a.i. ha− 1. Triazophos (Changqin Pesticide Co. Ltd., Jiangsu, China) is typically used for controlling rice borers and leaffolders at 180–300 g a.i. ha− 1.

Effect of pesticides on RSV transmission RSV transmission was evaluated by monitoring the acquisition of RSV by naïve planthoppers feeding on infected rice leaves and by measuring RSV transmission via viruliferous SBPH to healthy rice plants. Virus acquisition by naïve SBPH feeding on RSV-infected, pesticidetreated rice. RSV-infected plants were prepared by incubating healthy rice plants with viruliferous planthoppers. Plants were maintained under ambient conditions in the laboratory until rice stripe symptoms were observed. Twenty naïve SBPH nymphs (3rd or 4th instars) that had been starved for 5–6 h were then transferred into cups containing RSV-infected rice plants, which were previously treated with triazophos at the LC20 or validamycin at 200 mg L− 1. After a 48-h acquisition period, SBPH nymphs were removed and a 7-day feeding period ensued to facilitate circulative transmission. The nymphs were then individually tested for RSV by the DIBA method (Zhou et al., 2004). The RSV acquisition rate was defined as the ratio of viruliferous SBPH to the total number of tested SBPH. A replication consisted of twenty naïve SBPH nymphs in a single cup, and each treatment consisted of eight replications. RSV transmission rates by viruliferous SBPH nymphs treated with pesticides. A newly-emerged female from a viruliferous parent and a newly-emerged male from a non-viruliferous parent were caged in cups containing rice seedlings treated with triazophos or validamycin at the concentrations described above. After a 48-h mating period, the male was removed, and the mated female was maintained on the rice seedlings for oviposition until it died. Fifty mating pairs of SBPH were prepared for each treatment. The next-generation nymph of a putative, viruliferous planthopper was then placed in the cup that contained healthy plants to facilitate RSV transmission. After 48 h, next-generation nymphs were individually tested for RSV by the DIBA method (Zhou et al., 2004). Only viruliferous nymphs were considered valid individuals, and naïve nymphs were discarded. Test plants were maintained for approximately 15 days under ambient laboratory conditions to facilitate the reproduction of RSV. Rice plants were then analyzed for RSV by RT-PCR as described by Ji et al. (2005). The RSV transmission rate was defined as the ratio of infected rice plants to the number of viruliferous SBPH. Each treatment contained 30 replications.

Effects of pesticides on SBPH fecundity Prior to investigating the effect of triazophos on fecundity, the susceptibility of naïve and viruliferous planthoppers to triazophos was evaluated using the rice seedling immersion method (Wang et al., 2008b). Triazophos solutions were prepared in dimethyl sulfoxide (DMSO) and five serial dilutions (400 mg L− 1, 200 mg L− 1, 100 mg L− 1, 50 mg L− 1, and 25 mg L− 1) were made. Tap water containing 0.5% DMSO was used as a control. Rice seedlings were immersed in the triazophos solutions for 30 s, air-dried, and five seedlings were placed into individual plastic cups (5 cm diameter, 7 cm depth). Fifteen 3rd instar nymphs of SBPH were then transferred introduced into the cups, which were maintained at 25 ± 1 °C, 60–80% RH, with a 14:10 h (L:D) photoperiod. The viability of planthoppers was checked 72 h after treatment, the number of dead insects was recorded, and sublethal concentrations of triazophos were calculated with DPS statistical software developed by Tang and Feng (2002). Each treatment was replicated five times. To evaluate the number of eggs laid per female after pesticide treatment, ten rice seedlings (third-leaf stage) were planted in plastic cups (5 × 7 cm) and exposed to the two pesticides using the immersion method described above. Triazophos concentrations were as follows: LC10, LC20, and LC30 for triazophos; 100 mg L− 1, 200 mg L− 1, and 400 mg L− 1 for validamycin according to the normal concentration (200 mg L− 1) used in the rice field. Tap water containing 0.5% DMSO was used as a control. Thirty newly-emerged SBPH nymphs of viruliferous and naïve offspring were reared on the rice plants in plastic cups. All cups were covered with transparent, cylindrical cages and covered with nylon mesh as described above. Planthoppers were transferred to new rice seedlings every 10 days to ensure adequate nutrition. Mating pairs of SBPH were maintained on seedlings as described above, and fertilized females were separated and raised in cups containing three rice seedlings. When 3rd instar nymphs appeared, all nymphs (1st, 2nd, and 3rd instars) and unhatched eggs were counted

Pesticide effects on vertical RSV transmission rates by SBPH A newly-emerged female from a viruliferous parent and an emergent male from a non-viruliferous parent were caged in plastic cups containing rice seedlings treated with triazophos (LC20) or validamycin (200 mg L− 1). After 48 h, the male was removed, and the mated female was maintained on the rice seedlings for oviposition until it died. Twenty SBPH mating pairs were prepared for each treatment. Dead females were analyzed for RSV by the DIBA method (Zhou et al., 2004). Infected females were considered valid individuals, and naïve females were abandoned. Within 24 h after hatching, nymphs were individually placed in plastic cups containing fresh rice seedlings until they matured to the 3rd instar stage. Nymphs were then analyzed for RSV using the DIBA method (Zhou et al., 2004). The vertical transmission rate was defined as the ratio of viruliferous individuals produced by a single mother to the number of offspring produced by the same mother. A viruliferous, mated female (e.g. a mother) comprised one replication. Each population contained 15 replications. Statistical analysis Statistical tests were conducted with DPS statistical software developed by Tang and Feng (2002). Normal distributions and 831

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Table 1 Toxicity of sublethal triazophos concentrations to 3rd instars nymphs of SBPH. Nymphs of SBPH

Regression equation

Correlation coefficient

Lethal concentration (95% confidence limit) (mg L− 1)

Naïve

y = 1.03x + 3.38

r = 0.94

y = 1.32x + 2.67

r = 0.98

LC10 = 2.14 (0–13.47) LC20 = 5.73 (0–23.83) LC30 = 11.65 (0–36.60) LC10 = 6.24 (0.07–19.71) LC20 = 13.41 (0.48–32.77) LC30 = 23.30 (1.92–48.21)

100

Naïve

A

Viruliferous

a 80

Validamycin b c

c

c

40

Nnumber of eggs laid per female

Viruliferous

bc

60

homogeneity of variances (determined using the Bartlett test) were verified before analysis of variance (ANOVA). A one-way ANOVA was performed for vertical RSV transmission rates by SBPH exposed to pesticides. A two-way ANOVA was performed for the number of eggs produced per female treated with pesticides. Multiple comparisons of means were conducted based on Fisher's protected least significant difference (LSD) test. The data for virus acquisition and transmission efficiency by pesticide-treated SBPH were analyzed by a chi-squared (χ2) test. All data expressed in percentages were calculated by inverse sine transformation.

d d

20

0

CK

100 mg L-1

200 mg L-1

400 mg L-1

160

Naïve

B

a

Viruliferous

140

Triazophos

b

120

cd

cde

100

c de

e

cde

80

Results

60

Effects of pesticides on eggs laid by naïve and viruliferous SBPH

40

The toxicity of sublethal levels of triazophos to 3rd instar SBPH nymphs is shown in Table 1. The LC10, LC20, and LC30 values for viruliferous SBPH were greater than those for naïve SBPH by 191.6%, 134.0%, and 100.0%. This suggests that naïve SBPH were more susceptible to triazophos than viruliferous SBPH. The application of validamycin significantly altered the number of eggs laid by naïve and viruliferous SBPH females (F1, 89 = 65.02; P < 0.001). Changes in fecundity were also observed for different validamycin concentrations (F3, 89 = 50.95; P < 0.001) where the two variables significantly interacted (F3, 89 = 5.62; P = 0.002) (Fig. 1A). Triazophos had a significant impact on fecundity with respect to RSV (viruliferous vs. naïve; F1, 96 = 4.92; P = 0.03) and pesticide concentration (F3, 96 = 24.53; P < 0.001) (Fig. 1B). In the control, the number of eggs laid by naïve planthoppers was higher than viruliferous insects. In general, the two pesticides increased the number of eggs laid both in naïve and viruliferous insects, with the exception of naïve insects treated with validamycin at 100 and 400 mg L− 1. In viruliferous insects, multiple comparisons showed that validamycin at 200 mg L− 1 and triazophos at the LC20 increased the fecundity of SBPH more profoundly than the control and the other two pesticide concentrations. Furthermore, the increased number of eggs produced by viruliferous females treated with pesticides was larger than naïve females. For example, viruliferous females exposed to triazophos at the LC20 produced 45% more eggs than the control. Similarly, viruliferous females exposed to validamycin at 200 mg L− 1 produced 60.2% more eggs than the control, and this increase was substantially higher than the 28.7% increase observed for naïve insects (Fig. 1A). Consequently, validamycin at 200 mg L− 1 and triazophos at the LC20 were used to further investigate pesticide effects on RSV transmission by SBPH.

20 0

LC10

CK

LC20

LC30

Fig. 1. Number of eggs laid by SBPH females exposed to pesticides. Vertical columns represent mean ± SE, and columns labeled with different letters indicate significant differences between treatments (P < 0.05, Fisher's protected LSD test). Table 2 RSV transmission to healthy rice by next-generation nymphs of viruliferous SBPH treated with pesticides. Pesticide

Concentration (mg L− 1)

No. of valid insects

No. of rice plants acquiring RSV

Virus transmission rates (%)

χ2 test

Validamycin

0 200

40 27

5 8

12.50 29.63

Triazophos

0 13.41

25 31

3 5

12.00 16.13

χ2 = 2.03 df = 1 P = 0.15 χ2 = 0.44 df = 1 P = 0.51

Table 3 RSV acquisition rates by naïve, pesticide-treated SBPH feeding on virus-infected rice.

Effects of two pesticides on horizontal RSV transmission by naïve and viruliferous SBPH RSV transmission rates to healthy rice plants by nymphs of viruliferous, pesticide-treated planthoppers are shown in Table 2. The χ2 832

Pesticides

Concentration (mg L− 1)

No. of valid insects

No. of nymphs of acquiring RSV

Virus transmission rates (%)

χ2 test

Validamycin

0 200

39 59

12 44

30.77 74.58

Triazophos

0 13.41

26 62

15 32

57.69 51.61

χ2 = 16.65 df = 1 P < 0.001 χ2 = 0.08 df = 1 P = 0.77

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Vertical transmission rates (%)

100

80

infected plants (Table 3), indicating that exposure to validamycin enhanced horizontal transmission of RSV. Jing et al. (2015) reported that the virus acquisition rate of naïve SBPH feeding on infected resistant rice variety was markedly lower than that on comparative susceptible rice variety. We deduce that the altered virus transmission rate with validamycin treatment might be the changes in rice plants (more resistant or susceptible) or insects. The antibiotic validamycin (MW 497) is a fungicide used for control of R. solani in rice and has little effect on insect pests. The active ingredient in validamycin is a large water-soluble glucoside compound, which is unlikely to penetrate the wax layer of the insect cuticle. Zhu et al. (2014) showed that application of validamycin as a foliar spray stimulated the reproduction of N. lugens; however, topical treatments with the fungicide did not enhance fecundity. It is tempting to speculate that foliar sprays with validamycin induced changes beneficial for virus transmission in rice plants infested with naïve SBPH. Validamycin changes the physiology and biochemistry of rice plants by decreasing resistant substances to herbivory in rice plant (Luo et al., 2002; Wu et al., 2003), which favors SBPH feeding. Our ongoing work using electrical penetration graphs (EPG) to monitor the feeding behavior of SBPH will help elucidate the mechanisms underlying the changes in RSV transmission. It remains possible that validamycin might induce changes that facilitate virus replication, thus increasing RSV acquisition rates by naïve SBPH, but this remains unproven. Persistent, propagative viruses can replicate in different organs of insect vectors, and some of these viruses can be transmitted vertically from female parent to offspring in a transovarial manner. For example, RSV can be transmitted transovarially to the progeny of planthoppers for 40 generations (Hibino, 1996). We found that vertical RSV transmission by triazophos-treated SBPH increased significantly as compared to the untreated control plants (Fig. 2); this suggests that a higher percentage of viruliferous progeny was produced from mothers treated with triazophos. Deng et al. (2013) reported that the highest RSV accumulation was in the SBPH ovary, followed by the midgut lumen and epithelial cells. Recently, vitellogenin (Vg) was shown to play a critical role in enabling RSV to enter SBPH nurse cells in the germarium via Vgmediated endocytosis (Huo et al., 2014). Thus, a plausible explanation for our results is that triazophos exposure positively impacted Vg in SBPH, which then enhanced fecundity and RSV propagation. Interestingly, our previous studies showed that triazophos exposure induced more Nlvg expression in N. lugens (Ge et al., 2010), which was confirmed by Bao et al. (2010); however, it unclear whether triazophos increased Vg expression in SBPH. It is also important to note that the fecundity of adult females and RSV transmission is regulated by many factors. For example, after Vg is incorporated into oocytes, it is stored in a crystalline form of vitellin (Vn) and functions as a food source for the future embryo. In conclusion, the molecular mechanisms underlying pesticide-induced effects on SBPH fecundity and RSV replication warrant further investigation.

a b

b

CK

Validamycin

60

40

20

0

Triazophos

Fig. 2. Vertical transmission of RSV by SBPH treated with pesticides. Vertical columns represent mean ± SE. Columns labeled with different letters indicates significant differences among treatments at P < 0.05 (Fisher's protected LSD test).

test indicated no significant differences in RSV transmission rates between the pesticide treatments and controls. However, RSV acquisition rates in naïve, pesticide-exposed SBPH were altered obviously in comparison with controls not treated with pesticide (Table 3). The RSV acquisition rate of naïve SBPH treated with validamycin was approximately two-fold higher than the control, which indicates that validamycin can enhance the horizontal transmission of RSV by SBPH. There were no obvious changes in the RSV acquisition rates in naïve SBPH exposed to triazophos. Effects of two pesticides on vertical RSV transmission rates of SBPH Vertical transmission rates of RSV by SBPH changed after treatment with triazophos but not validamycin (F2, 41 = 10.48, P < 0.001) (Fig. 2). The ratio of RSV-positive individuals in the progeny of viruliferous mothers treated with triazophos was significant higher than the control and validamycin treatments by 18.3 and 17.2%, respectively. These results indicate that triazophos at the LC20 fosters vertical transmission of RSV by infected planthoppers. Discussion Considerable quantities of pesticides have been applied in rice fields to control SBPH and other insects. However, pesticide application has caused a resurgence in planthopper populations due to a stimulation in fecundity (Chelliah and Heinrichs, 1980; Wu et al., 2001; Wang et al., 2010), and this phenomenon includes SBPH in rice fields (Shen et al., 2007; Xu et al., 2008). Triazophos was shown to stimulate the reproduction of SBPH and two other planthoppers (Nilaparvata lugens and Sogatella furcifera); whereas validamycin enhanced fecundity in N. lugens but not S. furcifera (Zhang et al., 2014). Our results show that both triazophos and validamycin impacted the fecundity of viruliferous and non-viruliferous SBPH. To our knowledge, this is the first report describing differences in the fecundity of viruliferous and non-viruliferous SBPH due to pesticides. Interestingly, we found that viruliferous SBPH was more tolerant to triazophos than naïve insects (Table 1), and the pesticides-stimulated fecundity effects of viruliferous insects were more profound than those from naïve SBPH (Fig. 1). There is evidence that the relationship between RSV and SBPH might be mutualistic (Wan et al., 2015). RSV infection accelerated nymphal development, increased weight, enhanced the abundance of yeast-like symbionts, and modified the feeding behavior of SBPH (Wan et al., 2015). These virusmediated changes in vector physiology, endosymbionts and behavior support a mutualistic relationship between SBPH and RSV. RSV acquisition rates increased when naïve SBPH fed on virus-

Acknowledgments This research was financially supported by National Natural Science Foundation of China (No 31171842), the National Key Research and Development Program of China (No 2016YFD0300706), the Fund of Science and Technology of Jiangsu Province, People's Republic of China (No. BE2015342), the College Students Practice Innovation Training Program of Jiangsu Province University (20151117065Y), and the Jiangsu Agricultural Scientific Self-Innovation Fund (No cx [14]2030). We thank the two anonymous referees and the editor for helpful comments and suggestions on an earlier version of the article. References Bao, Y.Y., Li, L., Liu, Z.B., Xue, J., Zhu, Z.R., Cheng, J.A., Zhang, C.X., 2010. Triazophos up-regulated gene expression in the female brown planthopper, Nilaparvata lugens. J.

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