Journal of Invertebrate Pathology 127 (2015) 81–86
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Fitness costs of Cry1F resistance in two populations of fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from Puerto Rico and Florida q Vikash Dangal, Fangneng Huang ⇑ Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA, 70803, USA
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Article history: Received 23 December 2014 Revised 3 March 2015 Accepted 9 March 2015 Available online 16 March 2015 Keywords: Spodoptera frugiperda Bacillus thuringiensis Cry1F Zea mays Resistance Fitness costs
a b s t r a c t The development of resistance in target pest populations is a threat to the sustainability of transgenic crops expressing Bacillus thuringiensis (Bt) proteins. Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major target pest of Bt maize in North and South America. This insect is the first target pest that has developed field resistance to Bt maize at multiple locations in these regions. The objective of this study was to assess the fitness costs associated with the Cry1F resistance in two populations of S. frugiperda collected from Puerto Rico (RR-PR) and Florida (RR-FL). In the study, fitness costs were evaluated by comparing survival, growth, and developmental time of seven populations of S. frugiperda on (1) non-Bt meridic diet and (2) non-Bt maize leaf tissue and non-Bt diet. The seven populations were RR-PR, RR-FL, a Bt-susceptible strain (Bt-SS), and four F1 populations developed from reciprocal crosses between Bt-SS and the two resistant populations. Biological parameters measured were neonate-to-adult survivorship, neonate-to-adult developmental time, 10 day larval weight on non-Bt maize leaf tissue, pupal weight, and sex ratios. Results of the study show that the Cry1F resistance in both RR-PR and RR-FL was associated with considerable fitness costs, especially for the Florida population. Compared to the Bt-susceptible population, RR-PR showed an average of 61.1% reduction in larval weight, 20.4% less in neonate-to-adult survivorship, and 3.7 days delay in neonate-to-adult developmental time. These fitness costs for RR-FL were 66.9%, 31.7% and 4.4 days, respectively. The fitness costs of RR-PR and RR-FL appeared to be nonrecessive. The results indicate that a diversified genetic basis may exist for the Cry1F resistance in S. frugiperda. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Field maize, Zea mays L., is one of the major crops that have been genetically modified to express Bacillus thuringiensis (Bt) proteins targeting herbivorous insect pests. Maize is also a major crop in the U.S. with a total area of 37.1 million hectares planted in 2014, out of which 80% was Bt maize (NASS, 2014). Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a common pest targeted by Bt maize and Bt cotton in North and South America (Storer et al., 2010; Farias et al., 2014; Huang et al., 2014). It is also a major pest of many other crops in the tropical and subtropical regions. Because of resistance development, most traditional chemical
q This paper reports research results only. Mention of a proprietary product name does not constitute an endorsement for its use by Louisiana State University Agricultural Center. ⇑ Corresponding author. Tel.: +1 0 225 5780111; fax: +1 0 225 5781643. E-mail address: [email protected] (F. Huang).
http://dx.doi.org/10.1016/j.jip.2015.03.004 0022-2011/Ó 2015 Elsevier Inc. All rights reserved.
control tactics are unable to produce satisfactory control results against S. frugiperda (Siebert et al., 2008). Transgenic maize containing the event TC1507 expressing the Cry1F protein was registered in the U.S. in 2001 for controlling various pests including S. frugiperda (Siebert et al., 2008). In 2003, Cry1F maize was first commercially planted in Puerto Rico for silage and dairy farms (Storer et al., 2010). However, soon after the commercialization, unprecedented damage of Cry1F maize plants was reported in Puerto Rico (Storer et al., 2010). The unexpected field survival and damage of S. frugiperda were then confirmed to be due to resistance development to the Cry1F protein in the plants. Recently, field resistance that resulted in reduced efficacy or control failure of Cry1F maize in S. frugiperda has also been documented in Brazil (Farias et al., 2014) and the southeast coastal region of the U.S. (Huang et al., 2014). Until now, S. frugiperda is the only target pest that has developed field resistance to commercial Bt crops at multiple locations across different countries and continents. Because Cry1F is a common Bt protein
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expressed in many currently used Bt maize and Bt cotton products, the widespread of Cry1F resistance in S. frugiperda could represent a great challenge for the sustainable use of the Bt crop technology (Huang et al., 2014). Fitness costs of Bt resistance occur when insect genotypes conferring at least one allele of resistance has lower fitness than those individuals without any resistance allele in the absence of selection pressure (Gassmann et al., 2009). Fitness costs are regarded as one of the major factors influencing the evolution of resistance (Tabashnik et al., 2008; Carrière and Tabashnik, 2001; Carrière et al., 2010; Gassmann et al., 2009; Huang et al., 2011). In many cases where resistance to Bt has been detected in the laboratory, there has been rapid decline in resistance level after the selection pressure is removed (Gassmann et al., 2009). Mathematical modeling also suggests that fitness costs could play a key role in delaying resistance by selecting against resistant genotypes in the refuges where the Bt protein is not present (Gassmann et al., 2009). Fitness costs of Cry1F resistance in S. frugiperda have recently been investigated in two Puerto Rico populations. The results showed lack of fitness costs in both populations (Vélez et al., 2013; Jakka et al., 2014). The main objective of this study was to determine if fitness costs were associated with Cry1F resistance in two other resistant populations of S. frugiperda that were collected from Florida of the U.S. mainland and the southern Puerto Rico. In addition, comparisons of results from this study are made with those from the previous two studies by Vélez et al. (2013) and Jakka et al. (2014) to determine if the lack of fitness costs of the Cry1F resistance is consistent among different populations of S. frugiperda. Data generated from this study should be useful in understanding the mechanism of resistance and developing effective strategies for managing the Cry1F resistance in S. frugiperda.
2. Materials and methods 2.1. Sources of insects Three insect populations, a Bt-susceptible population (Bt-SS) of S. frugiperda collected from maize fields near Weslaco, Texas and two Cry1F-resistant populations obtained from Puerto Rico (RRPR) and Florida (RR-FL) were used as the original insect sources in this study. The Bt-SS strain was collected in 2013 and had never been exposed to Bt proteins or any other insecticides in the laboratory. Laboratory bioassays and greenhouse whole plant tests have shown that Bt-SS was susceptible to both Cry1F plants and purified Cry1F protein in diet (Huang et al., 2014). RR-PR was originated from >300 larvae collected from maize fields in southern Puerto Rico during 2011 (Niu et al., 2013). The field-collected population had been selected on Cry1F maize (Pioneer 31D59) leaf tissue for at least four generations before it was used in the current study. RR-PR is highly resistant to purified Cry1F protein (>769fold) and Cry1F maize plants (Niu et al., 2013, 2014). RR-FL was developed from an F2 screen with two-parent families derived from a field population collected from maize plants in south Florida in 2011 (Huang et al., 2014). It was confirmed that RR-FL possessed a major resistance allele to allow the insect to survive and complete normal development on commercial Cry1F maize plants. RR-FL is highly resistant to purified Cry1F protein (>270fold) in diet-incorporated bioassays (Huang et al., 2014). To ensure that the three populations have a similar genetic background, both RR-PR and RR-FL were backcrossed to Bt-SS to generate the F1 generations and then F1 populations were backcrossed with SS one more time to produce F2 populations. The F2 populations were sib-mated to produce F3 populations. F3 progeny were selected for Cry1F resistance, by rearing the populations on Cry1F maize leaf tissue for three generations before they were used
in the study. The methods used in the reselection of Cry1F resistance were the same as described in Niu et al. (2013). In the selection process, 2–4 pieces of Cry1F maize leaf tissue were placed in each well of the 32-well C-D International trays (Bio-Ba-32, C-D International, Pitman, NJ). Approximately 5–10 newly hatched larvae were released in each well. For each crossed population, a total of 1000–1500 neonates were selected on Cry1F maize leaf tissue. After 7 days, approximately 120–150 survivors of each population were transferred into 30-ml plastic cups (Fill-Rite, Newark, NJ) containing a meridic diet (Ward’s Stonefly Heliothis diet, Rochester, NY). In the selection, only the survivors with a relatively big body size (P3rd instars) were transferred and used to develop the next generation (Niu et al., 2013). The larval-rearing cups were held in 30-well trays (Bio-Serv, Frenchtown, NJ) and placed under the room conditions until pupation. In addition, four F1 hybrid populations were derived from the reciprocal crosses between Bt-SS and the two reselected resistant populations (RR-PR and RR-FL). The two F1 populations generated from the reciprocal cross between Bt-SS and RR-PR were denoted as PRmSSf (cross between males of RR-PR and females of Bt-SS) and PRfSSm (cross between females of RR-PR and males of Bt-SS). The two F1 populations developed by crossing Bt-SS and RR-FL were denoted as FLmSSf and FLfSSm, respectively. Fitness costs associated with the seven insect populations was evaluated using two assays: (1) a non-Bt diet and (2) a combined rearing of non-Bt maize leaf tissue and non-Bt diet. 2.2. Maize plants A non-Bt hybrid maize, Pioneer 31P40 (Pioneer Hi-Bred, Johnston, Iowa), was planted in 5 gallon plastic pots filled with approximately 5 kg of a standard potting mixture (Perfect Mix, Expert Gardener products, St. Louis, MO) as described in Wu et al. (2007). The pots were held in a greenhouse at the Louisiana State University Agricultural Center in Baton Rouge, Louisiana. Two plants per pot were maintained with regular irrigation and fertilization. 2.3. Growth and development of S. frugiperda on non-Bt diet To determine if fitness costs were associated with the Cry1F resistance in S. frugiperda, growth and development of the seven populations of S. frugiperda were first examined on a non-Bt meridic diet. In this assay, approximately 1 g of non-Bt diet (WARD’S Stonefly Heliothis diet, Rochester, NY) was placed into each cell of the 128-cell bioassay trays (Bio-Ba-128, C-D International Inc. Pitman, NJ) as described in Zhang et al. (2014). One neonate ( 0.05) pupal weight as their resistant parents, while it for PRfSSm was similar (P > 0.05) to that of Bt-SS. For the crosses between PP-FL and Bt-SS, the papal weight of the two F1 populations was not significantly different (P > 0.05) for both males and females and the values were between those of Bt-SS and RR-FL. 3.2. Neonate-to-adult survivorship of S. frugiperda reared on non-Bt diet The effect of insect population on neonate-to-adult survivorship of insects reared on non-Bt diet was significant (F = 12.04; df = 6, 18; P < 0.0001). The survivorship rate was similar (P > 0.05) between RR-PR and RR-FL with an average of 67.6%, which was significantly (P < 0.05) less than that (91.4%) of Bt-SS (Table 1). Survivorship was similar (P > 0.05) among the four F1 populations, which was also no significantly (P > 0.05) different compared to the survivorship of Bt-SS. However, the survivorship rates of the F1 populations were, in general, significantly greater (P < 0.05) than that of the resistant populations. 3.3. Neonate-to-adult emergence time of S. frugiperda reared on nonBt diet The effect of insect population on neonate-to-adult emergence time of S. frugiperda reared on non-Bt diet was significant for both males and females (F = 9.79; df = 6, 18; P < 0.0001 for male and F = 11.85; df = 6, 18; P < 0.0001 for female) (Table 1). The averaged neonate-to-adult emergence time for Bt-SS males was 21.8 days and females was 20.4 days (Table 1). The developmental time for males and females of RR-PR and RR-FL was longer (P < 0.05) than Bt-SS. Resistant males needed an average of 24.9 days and females needed 24.1 days to develop to adults. There were no significant (P > 0.05) differences in the development time among the four F1 populations. The development time of the two F1 populations of the crosses between Bt-SS and RR-PR was similar (P > 0.05) to that of Bt-SS for both males and females, but it was significantly (P < 0.05) shorter than that of RR-PR. For the two F1 populations derived from the crosses of Bt-SS and RR-FL, the difference in the development time was significant (P < 0.05) between FLfSSm and
Table 1 Pupal weight (mean ± SEM), neonate-to-adult survivorship (% mean ± SEM), neonate-to-adult-emergence time, and sex ratio (mean ± SEM) of seven populations of Spodoptera frugiperda on non-treated diet. Insect populationa
Bt-SS RR-PR PRmSSf PRfSSm RR-FL FLmSSf FLfSSm Analysis of Variance
Pupal weight (mg/pupa)b,c Male
Female
231.0 ± 2.2 ab 223.5 ± 4.4 bc 214.4 ± 1.0 c 237.0 ± 2.1 a 199.2 ± 2.0 d 220.9 ± 3.7 bc 215.2 ± 3.4 c F6, 18 = 26.83 P < 0.0001
237.2 ± 5.8 a 215.6 ± 3.3 bc 215.9 ± 2.8 bc 235.0 ± 2.8 a 203.6 ± 4.0 c 224.0 ± 3.1 ab 213.2 ± 5.0 bc F6, 18 = 14.92 P < 0.0001
Neonate-to-adult survivorship (%)b,c
Neonate-to-adult emergence time (days)b,c Male
Female
91.4 ± 1.49 a 73.4 ± 6.05 bc 88.2 ± 1.4 ab 95.3 ± 2.01 a 61.7 ± 4.3 c 85.9 ± 2.7 ab 89.1 ± 3.2 ab F6, 18 = 12.04 P < 0.0001
21.8 ± 0.2 cd 25.1 ± 0.3 a 23.0 ± 0.9 bcd 21.7 ± 0.09 d 24.7 ± 0.7 ab 23.7 ± 0.4 abc 22.8 ± 0.7 bcd F6, 18 = 9.79 P < 0.0001
20.4 ± 0.2 d 24.0 ± 0.4 a 21.2 ± 0.5 cd 20.6 ± 0.1 d 24.2 ± 0.7 ab 23.0 ± 0.7 abc 21.8 ± 0.4 bcd F6, 18 = 11.85 P < 0.0001
Sex ratiob,c
0.98 ± 0.09 a 1.45 ± 0.39 a 0.80 ± 0.11 a 0.85 ± 0.09 a 1.05 ± 0.10 a 1.02 ± 0.13 a 1.09 ± 0.18 a F6, 18 = 1.21 P = 0.3393
a Populations of S. frugiperda: Bt-SS = Cry1F-susceptible population obtained from Texas; RR-PR = Cry1F resistant population obtained from Puerto Rico; RR-FL = Cry1Fresistant population obtained from Florida; PRmSSf = F1 hybrid of RR-PR males and Bt-SS females; PRfSSm = F1 hybrid of RR-PR females and Bt-SS males; FLmSSf = F1 hybrid of RR-FL males and Bt-SS females; FLfSSm = F1 hybrid of RR-FL females and Bt-SS males. b Sample size for each insect population was 128 larvae for measuring neonate-to-adult survivorship and 79–117 for assaying neonate-to-adult development time, pupal weight, and sex ratio. c Mean values within a column followed by a same letter are not significantly different (P > 0.05; by Tukey’s honest significance difference test).
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RR-FL, but it was not significant (P > 0.05) between FLmSSf and its parent populations.
3.4. Sex ratio of S. frugiperda reared on non-Bt diet The effect of the populations on sex ratios was not significant (F = 1.21; df = 6, 18; P = 0.3393). The overall sex ratio of the seven populations was 1.03:1 (male:female) (Table 1).
3.5. Larval weight of S. frugiperda at the 10th day reared on non-Bt maize leaf tissue The effect of population on larval weight after 10 days on nonBt maize leaf tissue was significant (F = 9.34; df = 6, 18; P < 0.0001). Bt-SS larvae at 10th day feeding on non-Bt maize leaf tissue weighed 235.6 mg, which was significantly (P < 0.05) greater than that of the other six populations (Table 2). Larval weight of the resistant populations after 10 days feeding on non-Bt maize leaf tissue was 91.7 mg/larva for RR-PR and 78.1 mg/larva for RR-FL. There was a reduction of 61.1 for RR-PR and 66.9% for RR-FL in the weight. In general, larval weight of the four F1 populations was similar (P > 0.05), but it was significantly (P < 0.05) greater than that of their resistant parental populations except for PRmSSf.
3.6. Pupal weight of S. frugiperda in the combined rearing of non-Bt maize leaf tissue and non-Bt diet The effect of population on pupal weight of S. frugiperda in the combined rearing was significant for both sexes (F = 3.59; df = 6, 18; P = 0.0161 for male and F = 4.16; df = 6, 18; P = 0.0066 for female) (Table 2). For males and females, pupal weight of both resistant populations was numerically less than that of Bt-SS, but the differences were not significant (P > 0.05). Pupal weight of the F1 populations did not vary significantly (P > 0.05) from the pupae weight of their parental populations except for PRmSSf. Pupal weight of PRmSSf was greater (P < 0.05) than the weight of RR-PR pupae (Table 2).
3.7. Neonate-to-adult survivorship of S. frugiperda in the combined rearing of non-Bt maize leaf tissue and non-Bt diet The effect of population on neonate-to-adult survivorship rate was significant (F = 4.93; df = 6, 18; P = 0.0027). An average of neonate-to-adult survivorship rate of Bt-SS was 88.2%, while it was 69.5% for RR-PR and 60.9% for RR-FL (Table 2). The difference between Bt-SS and RR-FL was significant (P < 0.05), but it was not significant (P > 0.05) between Bt-SS and RR-PR. The survivorship of the F1 populations derived from the crosses between BtSS and RR-PR was similar and it did not differ (P > 0.05) from the parental populations. The survivorship (84.3%) of FLmSSf was not significantly different compared to Bt-SS and FLfSSm (78.1%), but it was significantly greater (P < 0.05) than the survivorship of RRFL. There were no significant (P > 0.05) differences in the survivorship rates between the two resistant populations or among the four F1 populations.
3.8. Neonate-to-adult development time of S. frugiperda in combined rearing of non-Bt maize leaf tissue and non-Bt diet The effect of insect population on the neonate-to-adult developmental time in the combined rearing was significant for both males and females (F = 17.77; df = 6, 18; P < 0.0001 for male and F = 16.69; df = 6, 18; P < 0.0001 for female) (Table 2). S. frugiperda females developed into adults approximately one day earlier than the males (Table 2). For Bt-SS neonates, it took the males an average of 21.2 days and females an average of 20.7 days to develop into adults. Compared to Bt-SS, development of the two resistant populations was significantly (P < 0.05) delayed. For RRPR, it took the males 25.0 days and female 24.7 days to develop into adults. For RR-FL, it took the males 27.4 days and females 25.3 days to complete the same development. The differences between the two resistant populations were not significant (P > 0.05) for both sexes. There were also no significant (P > 0.05) differences in the development time among the four F1 populations. In general, the development time of F1 hybrid populations was similar (P > 0.05) to that of their susceptible parental population, but it was significantly (P < 0.05) shorter than the time of the resistant parental populations.
Table 2 Insect body weight (mean ± SEM), neonate-to-adult survivorship (% mean ± SEM), neonate-to-adult emergence time (mean ± SEM), and sex ratio (mean ± SEM) of seven populations of Spodoptera frugiperda on non-Bt maize leaf tissues transferred to non-treated diet at 10th day. Insect populationa
Insect body weight (mg/individual)b,c Larval weight at 10th day on leaf
Bt-SS RR-PR PRmSSf PRfSSm RR-FL FLmSSf FLfSSm Analysis of Variance
235.6 ± 7.8 a 91.7 ± 8.1 de 120.6 ± 9.8 cd 145.9 ± 7.3 b 78.1 ± 5.3 e 174.9 ± 10.8 b 150.0 ± 8.8 bc F6, 18 = 9.34 P < 0.0001
Pupal weight
Neonate-to-adult Survivorshipb,c (%)
Male
Female
195.2 ± 8.8 ab 192.9 ± 6.5 ab 222.7 ± 3.5 a 192.9 ± 6.5 ab 188.9 ± 10.2 b 213.3 ± 1.9 ab 213.1 ± 3.8 ab F6, 18 = 3.59 P = 0.0161
201.7 ± 7.7 ab 189.8 ± 7.1 b 217.8 ± 2.8 a 201.2 ± 2.8 ab 201.6 ± 4.6 ab 213.7 ± 1.3 a 212.6 ± 1.1 a F6, 18 = 4.16 P = 0.0066
88.2 ± 2.6 a 69.5 ± 5.1 ab 70.3 ± 7.0 ab 92.9 ± 2.3 a 60.9 ± 7.4 b 84.3 ± 4.5 a 78.1 ± 3.8 ab F6, 18 = 4.93 P = 0.0027
Neonate-to-adult emergence time (days)b,c Male
Female
21.2 ± 0.3 c 25.0 ± 0.6 ab 22.5 ± 0.2 c 23.3 ± 0.5 bc 27.4 ± 1.4 a 22.2 ± 0.4 c 22.1 ± 0.3 c F6, 18 = 17.77 P < 0.0001
20.7 ± 0.1 b 24.7 ± 0.6 a 21.1 ± 0.2 b 22.5 ± 0.4 b 25.3 ± 0.9 a 20.9 ± 0.3 b 21.4 ± 0.3 b F6, 18 = 16.69 P < 0.0001
Sex ratiob,c
0.89 ± 0.15 a 1.3 ± 0.17 a 1.02 ± 0.16 a 1.0 ± 0.13 a 1.5 ± 0.51 a 0.89 ± 0.1 a 0.94 ± 0.1 a F6, 18 = 1.12 P = 0.3900
a Populations of S. frugiperda: Bt-SS = Cry1F-susceptible population obtained from Texas; RR-PR = Cry1F resistant population obtained from Puerto Rico; RR-FL = Cry1Fresistant population obtained from Florida; PRmSSf = F1 hybrid of RR-PR males and Bt-SS females; PRfSSm = F1 hybrid of RR-PR females and Bt-SS males; FLmSSf = F1 hybrid of RR-FL males and Bt-SS females; FLfSSm = F1 hybrid of RR-FL females and Bt-SS males. b Sample size for each insect population was 120 larvae for measuring neonate-to-adult survivorship, 78–128 for assaying 10 day larval weight, pupal weight, sex ratio and neonate-to-adult development time. c Mean values within a column followed by a same letter are not significantly different (P > 0.05; by Tukey’s honest significance difference test).
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3.9. Sex ratio of S. frugiperda in combined rearing of non-Bt maize leaf tissue and non-Bt diet As observed in the diet bioassay, the effect of insect population on sex ratio in the combined rearing was not significant (F = 1.12; df = 6, 18; P = 0.3900). The overall ratio of the seven populations was 1.07:1(male:female) (Table 2). 4. Discussion Results from comparisons of fitness parameters in this study show that there are fitness costs associated with the Cry1F resistance in both RR-PR and RR-FL. The fitness costs in the resistant populations of S. frugiperda resulted in reduced growth, increased mortality, and delayed development. In the assay with non-Bt diet, both resistant populations exhibited fitness costs in three of the four biological parameters measured. Compared to Bt-SS, RR-PR showed an average of 6.1% reduction in pupal weight, 19.7% reduction in neonate-to-adult survivorship, and 3.5 days delay in neonate-to-adult developmental time. The fitness costs for RR-FL were even more significant. In the diet assay, RR-FL had an average of 14.0% reduction in pupal weight, 32.5% reduction in the insect survivorship, and 3.4 days delay in the developmental time. Results of the combined rearing were generally consistent with those observed in the diet assay with few variations. Compared to Bt-SS, RR-PR had an average of 61.1% reduction in larval weight after 10 days feeding on non-Bt maize leaf tissue, 21.2% reduction in neonate-to-adult survivorship and 3.9 days delay in neonate-toadult developmental time. These fitness costs in the combined rearing for RR-FL were 66.9%, 31.0%, and 5.4 days, respectively. In recent studies, fitness costs of Cry1F resistance in S. frugiperda were evaluated by Vélez et al. (2013) and Jakka et al. (2014). Both studies found no evidence of fitness costs in the Cry1F resistant S. frugiperda populations from Puerto Rico. However, in the current study, there were fitness costs with RRPR and RR-FL populations. The variations in the results of the current and previous studies may be due to a diverse genetic variation in the resistant populations. However, factors like different assay methods and test substances could affect the results. Various studies have shown that the intensity of fitness costs associated with Bt resistance can vary depending on the test environmental conditions such as host plants, insect pathogens, intraspecific competition, and other factors (Gassmann et al., 2009; Janmaat and Myers, 2011; Kruger et al., 2012). There is the possibility that the Cry1F resistance observed in the southeastern coastal US mainland region may be due to migrations of resistant populations from Puerto Rico through Caribbean islands (Huang et al., 2014). However, the results from the current study suggest that the genetics of Cry1F resistance in RR-FL and RR-PR populations could be different from the populations used in the previous studies by Vélez et al. (2013) and Jakka et al. (2014). As discussed, the diverse genetics of Cry1F resistance in S. frugiperda populations, as well as, the interactions of genotypeenvironment may be responsible for the variations in the results reported. More studies are needed to determine and understand whether field resistance in US mainland was due to migrations or local selections. Disadvantages in life-history traits of homozygous resistant strains (RR) might be sometimes due to the reasons that are independent to resistance (Amin et al., 1984; Roush and Croft, 1986). If fitness costs associated to Bt resistance cause the RR individuals to be less fit than the homozygous susceptible individuals (SS), it is important to know if differences exist between SS and heterozygous individuals (RS). This is because, during the early stages of resistant evolution in the field, RS individuals are much more abundant than RR insects. Therefore,
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non-recessive fitness costs of resistance could be an important factor in delaying resistance development in field pest populations (Gassmann et al., 2009). Published data showed that Bt resistance is often associated with fitness costs, but most of the costs are recessively inherited (Anilkumar et al., 2008; Gassmann et al., 2009). Results from the current study suggest that fitness cost associated with the Cry1F resistance in RR-PR, and RR-FL varied from intermediate to recessive depending on the feeding methods, biological parameters measured, and populations. For example, on non-Bt diet, fitness costs in the neonate-to-adult survivorship were inherited recessively, but the fitness costs in the 10 day larval weight were intermediate. The non-recessive fitness costs of the Cry1F resistance in S. frugiperda identified in the current study could be useful in developing resistance management strategies. Acknowledgments This article is published with the approval of the Director of the Louisiana Agricultural Experiment Station as manuscript No. 2014234-19927. This project represents work supported by the Louisiana Soybean and Feed Grain Promotion Board and Hatch funds from the USDA National Institute of Food and Agriculture. References Amin, A.M., White, G.B., 1984. Relative fitness of organophosphate resistant– and susceptible strains of Culex quefasciatus Say (Diptera: Culicidae). Bul. Entomol. Res. 74, 591–598. Anilkumar, K.J., Pusztai-carey, M., Moar, W.J., 2008. Fitness costs associated with Cry1Ac-resistant Helicoverpa zea (Lepidoptera: Noctuidae): A factor countering selection for resistance to Bt cotton? J. Econ. Entomol. 101, 1421–1431. Carrière, Y., Tabashnik, B.E., 2001. Reversing insect adaptation to transgenic insecticidal plants. Proc. R. Soc. Lond. Sci. Ser. B 268, 1475–1480. Carrière, Y., Crowder, D.W., Tabashnik, B.E., 2010. Evolutionary ecology of insect adaptation to Bt crops. Evol. Appl. 3, 561–573. Farias, J.R., Andow, D.A., Horikoshi, R.J., Sorgatto, R.J., Fresia, P., Santos, A.C., Omoto, C., 2014. Field-evolved resistance to Cry1F maize by Spodoptera frugiperda (Lepidoptera: Noctuidae) in Brazil. Crop Protect. 64, 150–158. Gassmann, A.J., Carrière, Y., Tabashnik, B.E., 2009. Fitness costs of insect resistance to Bacillus thuringiensis. Ann. Rev. Entomol. 54, 147–163. Huang, F., Andow, D.A., Buschman, L.L., 2011. Success of the high dose/refuge resistance management strategy after 15 years of Bt crop use in North America. Entom. Exp. App. 140, 1–16. Huang, F., Qureshi, J.A., Meagher Jr., R.L., Reisig, D.D., Head, G.P., Andow, D.A., Ni, X., Kerns, D., Buntin, G.D., Niu, Y., Yang, F., Dangal, V., 2014. Cry1F resistance in fall armyworm Spodoptera frugiperda: single gene versus pyramided Bt maize. PLoS ONE 9 (11), e112958. http://dx.doi.org/10.1371/journal.pone.0112958. Jakka, S.R.K., Knight, V.R., Jurat-Fuentes, J.L., 2014. Fitness costs associated with field-evolved resistance to Bt maize in Spodoptera frugiperda (Lepidoptera: Noctuidae). J. Econ. Entomol. 107, 342–351. Janmaat, A.F., Mayers, J.H., 2011. Genetic variation in fitness parameters associated with resistance to Bacillus thuringiensis in male and female Trichopulsia ni. J. Invertibrt. Pathol. 107, 27–32. Kruger, M., Van Rensburg, J.B.J., Van den Berg, J., 2012. Transgenic Bt maize: farmers perceptions, refuge compliance and reports of stem borer resistance in South Africa. J. Appl. Entomol. 136, 38–50. NASS (National Agricultural Statistics Service); 2014. Acreage. (12.18.2014). Niu, Y., Meagher Jr., R.L., Yang, F., Huang, F., 2013. Susceptibility of field populations of fall armyworm (Lepidoptera: Noctuidae) from Florida and Puerto Rico to purified Cry1F protein and maize leaf tissue containing single and pyramided Bt genes. Florida Entomol. 96, 701–713. Niu, Y., Yang, F., Dangal, V., Huang, F., 2014. Larval survival and plant injury of Cry1F-susceptible, –resistant, and –heterozygous fall armyworm (Lepidoptera: Noctuidae) on non-Bt and Bt corn containing single or pyramided genes. Crop Protect 59, 22–28. Roush, R.T., Croft, B.A., 1986. Experimental population genetics and ecological studies of pesticide resistance in insects and mites. In: Pesticide Resistance. Strategies and Tactics for Management. Natl. Acad. Press, Washington, DC, pp. 257–270. SAS Institute Inc., 2010. SAS/STAT: 9.3 User’s third ed., SAS Institute Inc., Cary, NC. Siebert, M.W., Tindall, K.V., Leonard, B.R., Van Duan, J.W., Babcock, J.M., 2008. Evaluation of corn hybrids expressing Cry1F (HerculexÒ insect protection) against fall armyworm (Lepidoptera: Noctuidae) in the Southern Unites States. J. Entomol. Sci. 43, 1–51. Storer, N.P., Babcock, J.M., Sclenz, M., Meade, T., Thompson, G.D., Bing, J.W., Huckaba, R., 2010. Discovery and characterization of field resistance to Bt
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maize: spodoptera frugiperda (Lepidoptera: Noctuidea) in Puerto Rico. J. Econ. Entomol. 103, 1031–1038. Tabashnik, B.E., Gassmann, A.J., Crowder, D.W., Carrière, Y., 2008. Insect resistance to Bt crops: evidence versus theory. Nat. Biotechnol. 26, 199–202. Vélez, A.M., Spencer, T.A., Alves, A.P., Crespo, A.L.B., Siegfried, B.D., 2013. Fitness costs of Cry1F resistance in fall armyworm, Spodoptera frugiperda. J. Appl. Entomol. 138, 315–325.
Wu, X., Huang, F., Leonard, B.R., Moore, S.H., 2007. Evaluation of transgenic Bacillus thuringiensis maize hybrids against Cry1Ab –susceptible and –resistant sugarcane borer (Lepidoptera: Crambidae). J. Econ. Entomol. 100, 1880–1886. Zhang, L., Leonard, B.R., Chen, M., Clark, T., Konasale, A., Huang, F., 2014. Fitness and stability of Cry1Ab resistance in sugarcane borer, Diatraea saccharalis (F.). J. Invertebr. Pathol. 117, 26–32.
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Fitness costs of Cry1F resistance in two populations of fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from Puerto Rico and Florida q Vikash Dangal, Fangneng Huang ⇑ Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA, 70803, USA
a r t i c l e
i n f o
Article history: Received 23 December 2014 Revised 3 March 2015 Accepted 9 March 2015 Available online 16 March 2015 Keywords: Spodoptera frugiperda Bacillus thuringiensis Cry1F Zea mays Resistance Fitness costs
a b s t r a c t The development of resistance in target pest populations is a threat to the sustainability of transgenic crops expressing Bacillus thuringiensis (Bt) proteins. Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a major target pest of Bt maize in North and South America. This insect is the first target pest that has developed field resistance to Bt maize at multiple locations in these regions. The objective of this study was to assess the fitness costs associated with the Cry1F resistance in two populations of S. frugiperda collected from Puerto Rico (RR-PR) and Florida (RR-FL). In the study, fitness costs were evaluated by comparing survival, growth, and developmental time of seven populations of S. frugiperda on (1) non-Bt meridic diet and (2) non-Bt maize leaf tissue and non-Bt diet. The seven populations were RR-PR, RR-FL, a Bt-susceptible strain (Bt-SS), and four F1 populations developed from reciprocal crosses between Bt-SS and the two resistant populations. Biological parameters measured were neonate-to-adult survivorship, neonate-to-adult developmental time, 10 day larval weight on non-Bt maize leaf tissue, pupal weight, and sex ratios. Results of the study show that the Cry1F resistance in both RR-PR and RR-FL was associated with considerable fitness costs, especially for the Florida population. Compared to the Bt-susceptible population, RR-PR showed an average of 61.1% reduction in larval weight, 20.4% less in neonate-to-adult survivorship, and 3.7 days delay in neonate-to-adult developmental time. These fitness costs for RR-FL were 66.9%, 31.7% and 4.4 days, respectively. The fitness costs of RR-PR and RR-FL appeared to be nonrecessive. The results indicate that a diversified genetic basis may exist for the Cry1F resistance in S. frugiperda. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Field maize, Zea mays L., is one of the major crops that have been genetically modified to express Bacillus thuringiensis (Bt) proteins targeting herbivorous insect pests. Maize is also a major crop in the U.S. with a total area of 37.1 million hectares planted in 2014, out of which 80% was Bt maize (NASS, 2014). Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a common pest targeted by Bt maize and Bt cotton in North and South America (Storer et al., 2010; Farias et al., 2014; Huang et al., 2014). It is also a major pest of many other crops in the tropical and subtropical regions. Because of resistance development, most traditional chemical
q This paper reports research results only. Mention of a proprietary product name does not constitute an endorsement for its use by Louisiana State University Agricultural Center. ⇑ Corresponding author. Tel.: +1 0 225 5780111; fax: +1 0 225 5781643. E-mail address: [email protected] (F. Huang).
http://dx.doi.org/10.1016/j.jip.2015.03.004 0022-2011/Ó 2015 Elsevier Inc. All rights reserved.
control tactics are unable to produce satisfactory control results against S. frugiperda (Siebert et al., 2008). Transgenic maize containing the event TC1507 expressing the Cry1F protein was registered in the U.S. in 2001 for controlling various pests including S. frugiperda (Siebert et al., 2008). In 2003, Cry1F maize was first commercially planted in Puerto Rico for silage and dairy farms (Storer et al., 2010). However, soon after the commercialization, unprecedented damage of Cry1F maize plants was reported in Puerto Rico (Storer et al., 2010). The unexpected field survival and damage of S. frugiperda were then confirmed to be due to resistance development to the Cry1F protein in the plants. Recently, field resistance that resulted in reduced efficacy or control failure of Cry1F maize in S. frugiperda has also been documented in Brazil (Farias et al., 2014) and the southeast coastal region of the U.S. (Huang et al., 2014). Until now, S. frugiperda is the only target pest that has developed field resistance to commercial Bt crops at multiple locations across different countries and continents. Because Cry1F is a common Bt protein
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expressed in many currently used Bt maize and Bt cotton products, the widespread of Cry1F resistance in S. frugiperda could represent a great challenge for the sustainable use of the Bt crop technology (Huang et al., 2014). Fitness costs of Bt resistance occur when insect genotypes conferring at least one allele of resistance has lower fitness than those individuals without any resistance allele in the absence of selection pressure (Gassmann et al., 2009). Fitness costs are regarded as one of the major factors influencing the evolution of resistance (Tabashnik et al., 2008; Carrière and Tabashnik, 2001; Carrière et al., 2010; Gassmann et al., 2009; Huang et al., 2011). In many cases where resistance to Bt has been detected in the laboratory, there has been rapid decline in resistance level after the selection pressure is removed (Gassmann et al., 2009). Mathematical modeling also suggests that fitness costs could play a key role in delaying resistance by selecting against resistant genotypes in the refuges where the Bt protein is not present (Gassmann et al., 2009). Fitness costs of Cry1F resistance in S. frugiperda have recently been investigated in two Puerto Rico populations. The results showed lack of fitness costs in both populations (Vélez et al., 2013; Jakka et al., 2014). The main objective of this study was to determine if fitness costs were associated with Cry1F resistance in two other resistant populations of S. frugiperda that were collected from Florida of the U.S. mainland and the southern Puerto Rico. In addition, comparisons of results from this study are made with those from the previous two studies by Vélez et al. (2013) and Jakka et al. (2014) to determine if the lack of fitness costs of the Cry1F resistance is consistent among different populations of S. frugiperda. Data generated from this study should be useful in understanding the mechanism of resistance and developing effective strategies for managing the Cry1F resistance in S. frugiperda.
2. Materials and methods 2.1. Sources of insects Three insect populations, a Bt-susceptible population (Bt-SS) of S. frugiperda collected from maize fields near Weslaco, Texas and two Cry1F-resistant populations obtained from Puerto Rico (RRPR) and Florida (RR-FL) were used as the original insect sources in this study. The Bt-SS strain was collected in 2013 and had never been exposed to Bt proteins or any other insecticides in the laboratory. Laboratory bioassays and greenhouse whole plant tests have shown that Bt-SS was susceptible to both Cry1F plants and purified Cry1F protein in diet (Huang et al., 2014). RR-PR was originated from >300 larvae collected from maize fields in southern Puerto Rico during 2011 (Niu et al., 2013). The field-collected population had been selected on Cry1F maize (Pioneer 31D59) leaf tissue for at least four generations before it was used in the current study. RR-PR is highly resistant to purified Cry1F protein (>769fold) and Cry1F maize plants (Niu et al., 2013, 2014). RR-FL was developed from an F2 screen with two-parent families derived from a field population collected from maize plants in south Florida in 2011 (Huang et al., 2014). It was confirmed that RR-FL possessed a major resistance allele to allow the insect to survive and complete normal development on commercial Cry1F maize plants. RR-FL is highly resistant to purified Cry1F protein (>270fold) in diet-incorporated bioassays (Huang et al., 2014). To ensure that the three populations have a similar genetic background, both RR-PR and RR-FL were backcrossed to Bt-SS to generate the F1 generations and then F1 populations were backcrossed with SS one more time to produce F2 populations. The F2 populations were sib-mated to produce F3 populations. F3 progeny were selected for Cry1F resistance, by rearing the populations on Cry1F maize leaf tissue for three generations before they were used
in the study. The methods used in the reselection of Cry1F resistance were the same as described in Niu et al. (2013). In the selection process, 2–4 pieces of Cry1F maize leaf tissue were placed in each well of the 32-well C-D International trays (Bio-Ba-32, C-D International, Pitman, NJ). Approximately 5–10 newly hatched larvae were released in each well. For each crossed population, a total of 1000–1500 neonates were selected on Cry1F maize leaf tissue. After 7 days, approximately 120–150 survivors of each population were transferred into 30-ml plastic cups (Fill-Rite, Newark, NJ) containing a meridic diet (Ward’s Stonefly Heliothis diet, Rochester, NY). In the selection, only the survivors with a relatively big body size (P3rd instars) were transferred and used to develop the next generation (Niu et al., 2013). The larval-rearing cups were held in 30-well trays (Bio-Serv, Frenchtown, NJ) and placed under the room conditions until pupation. In addition, four F1 hybrid populations were derived from the reciprocal crosses between Bt-SS and the two reselected resistant populations (RR-PR and RR-FL). The two F1 populations generated from the reciprocal cross between Bt-SS and RR-PR were denoted as PRmSSf (cross between males of RR-PR and females of Bt-SS) and PRfSSm (cross between females of RR-PR and males of Bt-SS). The two F1 populations developed by crossing Bt-SS and RR-FL were denoted as FLmSSf and FLfSSm, respectively. Fitness costs associated with the seven insect populations was evaluated using two assays: (1) a non-Bt diet and (2) a combined rearing of non-Bt maize leaf tissue and non-Bt diet. 2.2. Maize plants A non-Bt hybrid maize, Pioneer 31P40 (Pioneer Hi-Bred, Johnston, Iowa), was planted in 5 gallon plastic pots filled with approximately 5 kg of a standard potting mixture (Perfect Mix, Expert Gardener products, St. Louis, MO) as described in Wu et al. (2007). The pots were held in a greenhouse at the Louisiana State University Agricultural Center in Baton Rouge, Louisiana. Two plants per pot were maintained with regular irrigation and fertilization. 2.3. Growth and development of S. frugiperda on non-Bt diet To determine if fitness costs were associated with the Cry1F resistance in S. frugiperda, growth and development of the seven populations of S. frugiperda were first examined on a non-Bt meridic diet. In this assay, approximately 1 g of non-Bt diet (WARD’S Stonefly Heliothis diet, Rochester, NY) was placed into each cell of the 128-cell bioassay trays (Bio-Ba-128, C-D International Inc. Pitman, NJ) as described in Zhang et al. (2014). One neonate ( 0.05) pupal weight as their resistant parents, while it for PRfSSm was similar (P > 0.05) to that of Bt-SS. For the crosses between PP-FL and Bt-SS, the papal weight of the two F1 populations was not significantly different (P > 0.05) for both males and females and the values were between those of Bt-SS and RR-FL. 3.2. Neonate-to-adult survivorship of S. frugiperda reared on non-Bt diet The effect of insect population on neonate-to-adult survivorship of insects reared on non-Bt diet was significant (F = 12.04; df = 6, 18; P < 0.0001). The survivorship rate was similar (P > 0.05) between RR-PR and RR-FL with an average of 67.6%, which was significantly (P < 0.05) less than that (91.4%) of Bt-SS (Table 1). Survivorship was similar (P > 0.05) among the four F1 populations, which was also no significantly (P > 0.05) different compared to the survivorship of Bt-SS. However, the survivorship rates of the F1 populations were, in general, significantly greater (P < 0.05) than that of the resistant populations. 3.3. Neonate-to-adult emergence time of S. frugiperda reared on nonBt diet The effect of insect population on neonate-to-adult emergence time of S. frugiperda reared on non-Bt diet was significant for both males and females (F = 9.79; df = 6, 18; P < 0.0001 for male and F = 11.85; df = 6, 18; P < 0.0001 for female) (Table 1). The averaged neonate-to-adult emergence time for Bt-SS males was 21.8 days and females was 20.4 days (Table 1). The developmental time for males and females of RR-PR and RR-FL was longer (P < 0.05) than Bt-SS. Resistant males needed an average of 24.9 days and females needed 24.1 days to develop to adults. There were no significant (P > 0.05) differences in the development time among the four F1 populations. The development time of the two F1 populations of the crosses between Bt-SS and RR-PR was similar (P > 0.05) to that of Bt-SS for both males and females, but it was significantly (P < 0.05) shorter than that of RR-PR. For the two F1 populations derived from the crosses of Bt-SS and RR-FL, the difference in the development time was significant (P < 0.05) between FLfSSm and
Table 1 Pupal weight (mean ± SEM), neonate-to-adult survivorship (% mean ± SEM), neonate-to-adult-emergence time, and sex ratio (mean ± SEM) of seven populations of Spodoptera frugiperda on non-treated diet. Insect populationa
Bt-SS RR-PR PRmSSf PRfSSm RR-FL FLmSSf FLfSSm Analysis of Variance
Pupal weight (mg/pupa)b,c Male
Female
231.0 ± 2.2 ab 223.5 ± 4.4 bc 214.4 ± 1.0 c 237.0 ± 2.1 a 199.2 ± 2.0 d 220.9 ± 3.7 bc 215.2 ± 3.4 c F6, 18 = 26.83 P < 0.0001
237.2 ± 5.8 a 215.6 ± 3.3 bc 215.9 ± 2.8 bc 235.0 ± 2.8 a 203.6 ± 4.0 c 224.0 ± 3.1 ab 213.2 ± 5.0 bc F6, 18 = 14.92 P < 0.0001
Neonate-to-adult survivorship (%)b,c
Neonate-to-adult emergence time (days)b,c Male
Female
91.4 ± 1.49 a 73.4 ± 6.05 bc 88.2 ± 1.4 ab 95.3 ± 2.01 a 61.7 ± 4.3 c 85.9 ± 2.7 ab 89.1 ± 3.2 ab F6, 18 = 12.04 P < 0.0001
21.8 ± 0.2 cd 25.1 ± 0.3 a 23.0 ± 0.9 bcd 21.7 ± 0.09 d 24.7 ± 0.7 ab 23.7 ± 0.4 abc 22.8 ± 0.7 bcd F6, 18 = 9.79 P < 0.0001
20.4 ± 0.2 d 24.0 ± 0.4 a 21.2 ± 0.5 cd 20.6 ± 0.1 d 24.2 ± 0.7 ab 23.0 ± 0.7 abc 21.8 ± 0.4 bcd F6, 18 = 11.85 P < 0.0001
Sex ratiob,c
0.98 ± 0.09 a 1.45 ± 0.39 a 0.80 ± 0.11 a 0.85 ± 0.09 a 1.05 ± 0.10 a 1.02 ± 0.13 a 1.09 ± 0.18 a F6, 18 = 1.21 P = 0.3393
a Populations of S. frugiperda: Bt-SS = Cry1F-susceptible population obtained from Texas; RR-PR = Cry1F resistant population obtained from Puerto Rico; RR-FL = Cry1Fresistant population obtained from Florida; PRmSSf = F1 hybrid of RR-PR males and Bt-SS females; PRfSSm = F1 hybrid of RR-PR females and Bt-SS males; FLmSSf = F1 hybrid of RR-FL males and Bt-SS females; FLfSSm = F1 hybrid of RR-FL females and Bt-SS males. b Sample size for each insect population was 128 larvae for measuring neonate-to-adult survivorship and 79–117 for assaying neonate-to-adult development time, pupal weight, and sex ratio. c Mean values within a column followed by a same letter are not significantly different (P > 0.05; by Tukey’s honest significance difference test).
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RR-FL, but it was not significant (P > 0.05) between FLmSSf and its parent populations.
3.4. Sex ratio of S. frugiperda reared on non-Bt diet The effect of the populations on sex ratios was not significant (F = 1.21; df = 6, 18; P = 0.3393). The overall sex ratio of the seven populations was 1.03:1 (male:female) (Table 1).
3.5. Larval weight of S. frugiperda at the 10th day reared on non-Bt maize leaf tissue The effect of population on larval weight after 10 days on nonBt maize leaf tissue was significant (F = 9.34; df = 6, 18; P < 0.0001). Bt-SS larvae at 10th day feeding on non-Bt maize leaf tissue weighed 235.6 mg, which was significantly (P < 0.05) greater than that of the other six populations (Table 2). Larval weight of the resistant populations after 10 days feeding on non-Bt maize leaf tissue was 91.7 mg/larva for RR-PR and 78.1 mg/larva for RR-FL. There was a reduction of 61.1 for RR-PR and 66.9% for RR-FL in the weight. In general, larval weight of the four F1 populations was similar (P > 0.05), but it was significantly (P < 0.05) greater than that of their resistant parental populations except for PRmSSf.
3.6. Pupal weight of S. frugiperda in the combined rearing of non-Bt maize leaf tissue and non-Bt diet The effect of population on pupal weight of S. frugiperda in the combined rearing was significant for both sexes (F = 3.59; df = 6, 18; P = 0.0161 for male and F = 4.16; df = 6, 18; P = 0.0066 for female) (Table 2). For males and females, pupal weight of both resistant populations was numerically less than that of Bt-SS, but the differences were not significant (P > 0.05). Pupal weight of the F1 populations did not vary significantly (P > 0.05) from the pupae weight of their parental populations except for PRmSSf. Pupal weight of PRmSSf was greater (P < 0.05) than the weight of RR-PR pupae (Table 2).
3.7. Neonate-to-adult survivorship of S. frugiperda in the combined rearing of non-Bt maize leaf tissue and non-Bt diet The effect of population on neonate-to-adult survivorship rate was significant (F = 4.93; df = 6, 18; P = 0.0027). An average of neonate-to-adult survivorship rate of Bt-SS was 88.2%, while it was 69.5% for RR-PR and 60.9% for RR-FL (Table 2). The difference between Bt-SS and RR-FL was significant (P < 0.05), but it was not significant (P > 0.05) between Bt-SS and RR-PR. The survivorship of the F1 populations derived from the crosses between BtSS and RR-PR was similar and it did not differ (P > 0.05) from the parental populations. The survivorship (84.3%) of FLmSSf was not significantly different compared to Bt-SS and FLfSSm (78.1%), but it was significantly greater (P < 0.05) than the survivorship of RRFL. There were no significant (P > 0.05) differences in the survivorship rates between the two resistant populations or among the four F1 populations.
3.8. Neonate-to-adult development time of S. frugiperda in combined rearing of non-Bt maize leaf tissue and non-Bt diet The effect of insect population on the neonate-to-adult developmental time in the combined rearing was significant for both males and females (F = 17.77; df = 6, 18; P < 0.0001 for male and F = 16.69; df = 6, 18; P < 0.0001 for female) (Table 2). S. frugiperda females developed into adults approximately one day earlier than the males (Table 2). For Bt-SS neonates, it took the males an average of 21.2 days and females an average of 20.7 days to develop into adults. Compared to Bt-SS, development of the two resistant populations was significantly (P < 0.05) delayed. For RRPR, it took the males 25.0 days and female 24.7 days to develop into adults. For RR-FL, it took the males 27.4 days and females 25.3 days to complete the same development. The differences between the two resistant populations were not significant (P > 0.05) for both sexes. There were also no significant (P > 0.05) differences in the development time among the four F1 populations. In general, the development time of F1 hybrid populations was similar (P > 0.05) to that of their susceptible parental population, but it was significantly (P < 0.05) shorter than the time of the resistant parental populations.
Table 2 Insect body weight (mean ± SEM), neonate-to-adult survivorship (% mean ± SEM), neonate-to-adult emergence time (mean ± SEM), and sex ratio (mean ± SEM) of seven populations of Spodoptera frugiperda on non-Bt maize leaf tissues transferred to non-treated diet at 10th day. Insect populationa
Insect body weight (mg/individual)b,c Larval weight at 10th day on leaf
Bt-SS RR-PR PRmSSf PRfSSm RR-FL FLmSSf FLfSSm Analysis of Variance
235.6 ± 7.8 a 91.7 ± 8.1 de 120.6 ± 9.8 cd 145.9 ± 7.3 b 78.1 ± 5.3 e 174.9 ± 10.8 b 150.0 ± 8.8 bc F6, 18 = 9.34 P < 0.0001
Pupal weight
Neonate-to-adult Survivorshipb,c (%)
Male
Female
195.2 ± 8.8 ab 192.9 ± 6.5 ab 222.7 ± 3.5 a 192.9 ± 6.5 ab 188.9 ± 10.2 b 213.3 ± 1.9 ab 213.1 ± 3.8 ab F6, 18 = 3.59 P = 0.0161
201.7 ± 7.7 ab 189.8 ± 7.1 b 217.8 ± 2.8 a 201.2 ± 2.8 ab 201.6 ± 4.6 ab 213.7 ± 1.3 a 212.6 ± 1.1 a F6, 18 = 4.16 P = 0.0066
88.2 ± 2.6 a 69.5 ± 5.1 ab 70.3 ± 7.0 ab 92.9 ± 2.3 a 60.9 ± 7.4 b 84.3 ± 4.5 a 78.1 ± 3.8 ab F6, 18 = 4.93 P = 0.0027
Neonate-to-adult emergence time (days)b,c Male
Female
21.2 ± 0.3 c 25.0 ± 0.6 ab 22.5 ± 0.2 c 23.3 ± 0.5 bc 27.4 ± 1.4 a 22.2 ± 0.4 c 22.1 ± 0.3 c F6, 18 = 17.77 P < 0.0001
20.7 ± 0.1 b 24.7 ± 0.6 a 21.1 ± 0.2 b 22.5 ± 0.4 b 25.3 ± 0.9 a 20.9 ± 0.3 b 21.4 ± 0.3 b F6, 18 = 16.69 P < 0.0001
Sex ratiob,c
0.89 ± 0.15 a 1.3 ± 0.17 a 1.02 ± 0.16 a 1.0 ± 0.13 a 1.5 ± 0.51 a 0.89 ± 0.1 a 0.94 ± 0.1 a F6, 18 = 1.12 P = 0.3900
a Populations of S. frugiperda: Bt-SS = Cry1F-susceptible population obtained from Texas; RR-PR = Cry1F resistant population obtained from Puerto Rico; RR-FL = Cry1Fresistant population obtained from Florida; PRmSSf = F1 hybrid of RR-PR males and Bt-SS females; PRfSSm = F1 hybrid of RR-PR females and Bt-SS males; FLmSSf = F1 hybrid of RR-FL males and Bt-SS females; FLfSSm = F1 hybrid of RR-FL females and Bt-SS males. b Sample size for each insect population was 120 larvae for measuring neonate-to-adult survivorship, 78–128 for assaying 10 day larval weight, pupal weight, sex ratio and neonate-to-adult development time. c Mean values within a column followed by a same letter are not significantly different (P > 0.05; by Tukey’s honest significance difference test).
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3.9. Sex ratio of S. frugiperda in combined rearing of non-Bt maize leaf tissue and non-Bt diet As observed in the diet bioassay, the effect of insect population on sex ratio in the combined rearing was not significant (F = 1.12; df = 6, 18; P = 0.3900). The overall ratio of the seven populations was 1.07:1(male:female) (Table 2). 4. Discussion Results from comparisons of fitness parameters in this study show that there are fitness costs associated with the Cry1F resistance in both RR-PR and RR-FL. The fitness costs in the resistant populations of S. frugiperda resulted in reduced growth, increased mortality, and delayed development. In the assay with non-Bt diet, both resistant populations exhibited fitness costs in three of the four biological parameters measured. Compared to Bt-SS, RR-PR showed an average of 6.1% reduction in pupal weight, 19.7% reduction in neonate-to-adult survivorship, and 3.5 days delay in neonate-to-adult developmental time. The fitness costs for RR-FL were even more significant. In the diet assay, RR-FL had an average of 14.0% reduction in pupal weight, 32.5% reduction in the insect survivorship, and 3.4 days delay in the developmental time. Results of the combined rearing were generally consistent with those observed in the diet assay with few variations. Compared to Bt-SS, RR-PR had an average of 61.1% reduction in larval weight after 10 days feeding on non-Bt maize leaf tissue, 21.2% reduction in neonate-to-adult survivorship and 3.9 days delay in neonate-toadult developmental time. These fitness costs in the combined rearing for RR-FL were 66.9%, 31.0%, and 5.4 days, respectively. In recent studies, fitness costs of Cry1F resistance in S. frugiperda were evaluated by Vélez et al. (2013) and Jakka et al. (2014). Both studies found no evidence of fitness costs in the Cry1F resistant S. frugiperda populations from Puerto Rico. However, in the current study, there were fitness costs with RRPR and RR-FL populations. The variations in the results of the current and previous studies may be due to a diverse genetic variation in the resistant populations. However, factors like different assay methods and test substances could affect the results. Various studies have shown that the intensity of fitness costs associated with Bt resistance can vary depending on the test environmental conditions such as host plants, insect pathogens, intraspecific competition, and other factors (Gassmann et al., 2009; Janmaat and Myers, 2011; Kruger et al., 2012). There is the possibility that the Cry1F resistance observed in the southeastern coastal US mainland region may be due to migrations of resistant populations from Puerto Rico through Caribbean islands (Huang et al., 2014). However, the results from the current study suggest that the genetics of Cry1F resistance in RR-FL and RR-PR populations could be different from the populations used in the previous studies by Vélez et al. (2013) and Jakka et al. (2014). As discussed, the diverse genetics of Cry1F resistance in S. frugiperda populations, as well as, the interactions of genotypeenvironment may be responsible for the variations in the results reported. More studies are needed to determine and understand whether field resistance in US mainland was due to migrations or local selections. Disadvantages in life-history traits of homozygous resistant strains (RR) might be sometimes due to the reasons that are independent to resistance (Amin et al., 1984; Roush and Croft, 1986). If fitness costs associated to Bt resistance cause the RR individuals to be less fit than the homozygous susceptible individuals (SS), it is important to know if differences exist between SS and heterozygous individuals (RS). This is because, during the early stages of resistant evolution in the field, RS individuals are much more abundant than RR insects. Therefore,
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non-recessive fitness costs of resistance could be an important factor in delaying resistance development in field pest populations (Gassmann et al., 2009). Published data showed that Bt resistance is often associated with fitness costs, but most of the costs are recessively inherited (Anilkumar et al., 2008; Gassmann et al., 2009). Results from the current study suggest that fitness cost associated with the Cry1F resistance in RR-PR, and RR-FL varied from intermediate to recessive depending on the feeding methods, biological parameters measured, and populations. For example, on non-Bt diet, fitness costs in the neonate-to-adult survivorship were inherited recessively, but the fitness costs in the 10 day larval weight were intermediate. The non-recessive fitness costs of the Cry1F resistance in S. frugiperda identified in the current study could be useful in developing resistance management strategies. Acknowledgments This article is published with the approval of the Director of the Louisiana Agricultural Experiment Station as manuscript No. 2014234-19927. This project represents work supported by the Louisiana Soybean and Feed Grain Promotion Board and Hatch funds from the USDA National Institute of Food and Agriculture. References Amin, A.M., White, G.B., 1984. Relative fitness of organophosphate resistant– and susceptible strains of Culex quefasciatus Say (Diptera: Culicidae). Bul. Entomol. Res. 74, 591–598. Anilkumar, K.J., Pusztai-carey, M., Moar, W.J., 2008. Fitness costs associated with Cry1Ac-resistant Helicoverpa zea (Lepidoptera: Noctuidae): A factor countering selection for resistance to Bt cotton? J. Econ. Entomol. 101, 1421–1431. Carrière, Y., Tabashnik, B.E., 2001. Reversing insect adaptation to transgenic insecticidal plants. Proc. R. Soc. Lond. Sci. Ser. B 268, 1475–1480. Carrière, Y., Crowder, D.W., Tabashnik, B.E., 2010. Evolutionary ecology of insect adaptation to Bt crops. Evol. Appl. 3, 561–573. Farias, J.R., Andow, D.A., Horikoshi, R.J., Sorgatto, R.J., Fresia, P., Santos, A.C., Omoto, C., 2014. Field-evolved resistance to Cry1F maize by Spodoptera frugiperda (Lepidoptera: Noctuidae) in Brazil. Crop Protect. 64, 150–158. Gassmann, A.J., Carrière, Y., Tabashnik, B.E., 2009. Fitness costs of insect resistance to Bacillus thuringiensis. Ann. Rev. Entomol. 54, 147–163. Huang, F., Andow, D.A., Buschman, L.L., 2011. Success of the high dose/refuge resistance management strategy after 15 years of Bt crop use in North America. Entom. Exp. App. 140, 1–16. Huang, F., Qureshi, J.A., Meagher Jr., R.L., Reisig, D.D., Head, G.P., Andow, D.A., Ni, X., Kerns, D., Buntin, G.D., Niu, Y., Yang, F., Dangal, V., 2014. Cry1F resistance in fall armyworm Spodoptera frugiperda: single gene versus pyramided Bt maize. PLoS ONE 9 (11), e112958. http://dx.doi.org/10.1371/journal.pone.0112958. Jakka, S.R.K., Knight, V.R., Jurat-Fuentes, J.L., 2014. Fitness costs associated with field-evolved resistance to Bt maize in Spodoptera frugiperda (Lepidoptera: Noctuidae). J. Econ. Entomol. 107, 342–351. Janmaat, A.F., Mayers, J.H., 2011. Genetic variation in fitness parameters associated with resistance to Bacillus thuringiensis in male and female Trichopulsia ni. J. Invertibrt. Pathol. 107, 27–32. Kruger, M., Van Rensburg, J.B.J., Van den Berg, J., 2012. Transgenic Bt maize: farmers perceptions, refuge compliance and reports of stem borer resistance in South Africa. J. Appl. Entomol. 136, 38–50. NASS (National Agricultural Statistics Service); 2014. Acreage. (12.18.2014). Niu, Y., Meagher Jr., R.L., Yang, F., Huang, F., 2013. Susceptibility of field populations of fall armyworm (Lepidoptera: Noctuidae) from Florida and Puerto Rico to purified Cry1F protein and maize leaf tissue containing single and pyramided Bt genes. Florida Entomol. 96, 701–713. Niu, Y., Yang, F., Dangal, V., Huang, F., 2014. Larval survival and plant injury of Cry1F-susceptible, –resistant, and –heterozygous fall armyworm (Lepidoptera: Noctuidae) on non-Bt and Bt corn containing single or pyramided genes. Crop Protect 59, 22–28. Roush, R.T., Croft, B.A., 1986. Experimental population genetics and ecological studies of pesticide resistance in insects and mites. In: Pesticide Resistance. Strategies and Tactics for Management. Natl. Acad. Press, Washington, DC, pp. 257–270. SAS Institute Inc., 2010. SAS/STAT: 9.3 User’s third ed., SAS Institute Inc., Cary, NC. Siebert, M.W., Tindall, K.V., Leonard, B.R., Van Duan, J.W., Babcock, J.M., 2008. Evaluation of corn hybrids expressing Cry1F (HerculexÒ insect protection) against fall armyworm (Lepidoptera: Noctuidae) in the Southern Unites States. J. Entomol. Sci. 43, 1–51. Storer, N.P., Babcock, J.M., Sclenz, M., Meade, T., Thompson, G.D., Bing, J.W., Huckaba, R., 2010. Discovery and characterization of field resistance to Bt
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