Introduction
Transgenic crops expressing Bacillus thuringiensis (Bt) endotoxins have become one of the most important tools for managing maize and cotton insect pests in the US and other countries (James, Reference James2004; NASS, 2005). The widespread adoption of transgenic Bt crops could place a high selection pressure on the target insect populations and accelerate development of resistance, raising concerns about the long-term durability of Bt plants (Ostlie et al., Reference Ostlie, Hutchison and Hellmich1997; Gould, Reference Gould1998). One of the key factors for a successful resistance management plan to prolong the durability of Bt plants is to have a cost-effective resistance monitoring system. The monitoring system should provide information about the initial Bt resistance allele frequencies at low levels. Early shifts in the resistance allele frequencies of field insect populations should be detected so that proactive measures for managing resistance can be deployed before a field control failure occurs.
Several methods have been used, or suggested, to detect and monitor insecticide resistance in field insect populations (Andow & Alstad, Reference Andow and Alstad1998; Huang, Reference Huang2006). Among these, the dose-response technique has often been suggested because it is a relatively simple procedure (Bolin et al., Reference Bolin, Hutchison, Andow and Ostlie1998; Marçon et al., Reference Marçon, Young, Steffey and Siegfried1999; Reed & Halliday, Reference Reed and Halliday2001; Trisyono & Chippendale, Reference Trisyono and Chippendale2002; Wu et al., Reference Wu, Guo, Lv, Greenplate and Deaton2002; Farinós et al., Reference Farinós, Poza, Hernández-Crespo, Ortego and Castañera2004). However, the dose-response method is not suitable for monitoring resistance for Bt crops because it is inefficient (Roush & Miller, Reference Roush and Miller1986) and not sufficiently sensitive to detect resistance alleles before field resistance occurs (Andow & Alstad, Reference Andow and Alstad1998).
A more common method used to detect and monitor resistance alleles is the diagnostic dose technique (Marçon et al., Reference Marçon, Siegfried, Spencer and Hutchison2000; Tabashnik et al., Reference Tabashnik, Patin, Dennehy, Liu, Carrière, Sims and Antilla2000). The diagnostic dose method is a more powerful technique than the dose-response method (Roush & Miller, Reference Roush and Miller1986) because all sample insects are phenotypically characterized individually at a single diagnostic dose rather than as a population for dose-response bioassays. The diagnostic dose method is very effective for detecting dominant resistance alleles (Plapp et al., Reference Plapp, Campanhola, Bagwell, McCutcheon, Roush and Tabashnik1990) or when recessive resistance allele frequencies in pest populations are at high levels. The diagnostic dose method was modified to screen F1 iso-female lines for non-recessive Bt resistance alleles in bollworm, Helicoverpa zea (Boddie), in the US (Burd et al., Reference Burd, Gould, Bradley, Vanduyn and Moar2003) and in cotton bollworm, H. armigera, in China (Li et al., Reference Li, Wu, Gould, Feng, He and Guo2004). However, the diagnostic dose method cannot detect heterozygous individuals if resistance is recessive. Most insect resistance to Bt-maize is genetically or functionally recessive (Tabashnik et al., Reference Tabashnik, Carriere, Dennehy, Morin, Sisterson, Roush, Shelton and Zhao2003). Screening a large number (e.g. >1,000,000) of field-collected individuals at the diagnostic dose is necessary to estimate the frequency of a rare recessive allele (e.g. <0.001) in a field population (Andow & Alstad, Reference Andow and Alstad1998). Therefore, the diagnostic dose method by itself is usually not a feasible technique for detecting rare recessive Bt resistance.
Andow and Alstad (Reference Andow and Alstad1998) introduced an F2 screening technique. As originally proposed, this technique involves inbreeding iso-female families and screening their F2 progeny on Bt plants or exposure to a diagnostic dose. Theoretically, this technique can detect heterozygous individuals even if the resistance is recessive. The F2 screen, therefore, should be more powerful than the diagnostic dose method alone, provided the resistance gene is recessive. Since 1998, more than 2000 iso-line families of the European corn borer, Ostrinia nubilalis (Hübner), have been screened for Bt resistance by using the F2 screening method, but no major resistance alleles that allowed larval development on high dose commercial Bt plants were detected (Andow et al., Reference Andow, Alstad, Pang, Bolin and Hutchison1998, Reference Andow, Olson, Hellmich, Alstad and Hutchison2000; Bourguet et al., Reference Bourguet, Chaufaux, Séguin, Buisson, Hinton, Stodola, Porter, Cronholm, Buschman and Andow2003; Stodola et al., Reference Stodola, Andow, Hyden, Hinton, Roark, Buschman, Porter and Cronholm2006).
Based on the principles of the F2 screen proposed by Andow and Alstad (Reference Andow and Alstad1998), a cost-effective modification of the F2 screening method was developed to detect Bt resistance in sugarcane borer, Diatraea saccharalis (F.). Diatraea saccharalis is a corn pest and a primary target of Bt-maize in Louisiana (Castro et al., Reference Castro, Riley, Leonard and Baldwin2004). This paper details the use of the F2 screening technique for detecting a major Bt resistance in a population of D. saccharalis collected from northeast Louisiana.
Materials and methods
The F2 screening method for detecting Bt resistance in D. saccharalis involved modifications in (i) collecting larvae from maize field; (ii) establishing two-parent families from the field-collected insects; (iii) screening F2 neonates of each two-parent family for survival on Bt-maize leaf tissues; and (iv) confirming Bt resistance on commercial Bt-maize plants in a greenhouse and on Bt-maize leaf tissues in the laboratory (fig. 1).
Illustration of F2 screening procedures for detecting Bt resistance alleles in Diatraea saccharalis.
Insect collection
The F2 screen originally proposed by Andow and Alstad (Reference Andow and Alstad1998) was initiated with mated feral adult females. In the present study, feral larvae of D. saccharalis were collected from maize fields near Winnsboro, Louisiana during the first and second field generations. Field-collected larvae were reared individually until the pupal stage in 30-ml plastic cups (Fill-Rite, Newark, NJ) each containing approximately 10 ml of a meridic diet (Bio-Serv, Frenchtown, NJ). The larval-rearing cups were held on 30-well trays (Bio-Serv, Frenchtown, NJ) and placed in environmental growth chambers (Percival Scientific, Perry, IA) maintained at 27°C, 50% RH and a 16:8 h (L:D) photoperiod.
Establishment of two-parent families
Pupae collected from the larval-rearing cups were sexed using characters described by Butt and Cantu (Reference Butt and Cantu1962). To establish two-parent families, a pair of newly-eclosed virgin male and female adults was introduced into a 450-ml paper container (Huhtamaki Foodservice, De Soto, Kansas). A 30-ml plastic cup containing cotton wadding saturated with a 10% sugar water solution was placed in the centre of the adult container. For insects collected in the second generation, approximately 10 g of vermiculite (Sun Gro, Pine Bluff, AR) were also added to each of the containers. The inside wall of each container was covered with wax paper. The open end of the container was covered with cotton gauze. The wax paper served as an adult oviposition substrate. Progeny from each pair comprised an iso-line family. F1 neonates hatched from eggs of each two-parent family were reared on a meridic diet as previously described. The F1 adults were sib-mated within each family in a 3.8-l cardboard carton (Neptune Paper Products, Newark, NJ) containing approximately 100 g of vermiculite (Huang et al., Reference Huang, Leonard and Gable2006). The cardboard cartons were placed in environmental chambers maintained at the same conditions as mentioned above.
Screening on maize leaf tissues – negative controls
To establish the validity of using maize leaf tissue for screening F2 neonates, we evaluated larval survival on negative controls of non-Bt-maize leaf tissue. Five seeds of DK697 (Monsanto, St Louis, MO), a commercial non-Bt-maize cultivar, were planted in 18.9-l pots containing approximately 5000 g of a standard potting soil mixture (Perfect Mix™, Expert Gardener Products, St. Louis, MO) in a greenhouse on the campus of the Louisiana State University AgCenter in Baton Rouge, Louisiana. Plants were thinned to three plants per pot after two weeks. When plants reached the V6–V8 stages (Ritchie et al., Reference Ritchie, Hanway, Benson and Herman1993), fully-expanded maize leaves were removed from the plants and cut into approximately 7-cm lengths across the entire leaf width. Six pieces of the leaf tissues were placed in each well of an 8-well C-D international tray (Bio-Smart-8, C-D International, Pitman, NJ). Each well contained approximately 10 ml of 3.5% solidified agar solution to maintain high moisture (Huang et al., Reference Huang, Leonard and Gable2006). Thirty neonates (<24 h old) of a susceptible D. saccharalis colony (LA-NE-A) derived from a collection near Winnsboro, Louisiana in 2004 were released on the leaf tissues in each well. The trays were placed in an environmental chamber and maintained at the same conditions as described above. Preliminary tests showed that most larvae of D. saccharalis were late 2nd instars or 3rd instars after 10 days on non-Bt-maize leaf tissues. Large larvae (≥3rd instar) of D. saccharalis usually bore into plant stalks and do not feed on fully-expanded leaves in the field. Thus, bioassays with non-Bt-maize leaf tissues were terminated 10 days after larval release. Larval survival was evaluated at 5 days and 10 days after inoculation. Leaf tissues were replaced with fresh leaf tissues every 3 days. For each test duration, there were five replications (N=150).
Screening F2 neonates on Bt-maize leaf tissues
Seeds of the DKC69-70 hybrid (Monsanto, St Louis, MO) were planted in a greenhouse as described above. DKC69-70 is a Bt-maize hybrid expressing the Cry1Ab protein. This cultivar is commonly planted in Louisiana to control a corn borer complex of D. saccharalis and the southwestern corn borer, D. grandiosella Dyar (Castro et al., Reference Castro, Riley, Leonard and Baldwin2004). It is genetically similar to the DK697 non-Bt-maize hybrid. F2 neonates from each two-parent family were screened in 8-well trays (C-D International) containing Bt-maize leaf tissues dissected from fully-expanded leaves of V6–V8 greenhouse Bt plants. The F2 screening trays were maintained at the same environmental conditions as used for assessing larval survivorship on non-Bt-maize leaf tissues, except the non-Bt leaf tissues were replaced with Bt-maize leaf tissues. In each family, approximately 60 F2 neonates were screened in each of five wells (300 larvae per family). Previous laboratory bioassays showed that susceptible larvae of D. saccharalis survived on Bt-maize leaf tissues for more than 4 days (Huang et al., Reference Huang, Leonard and Gable2006). However, larval growth and development of D. saccharalis on Bt-maize leaf tissues were delayed considerably compared to on non-Bt-maize leaf tissues. Therefore, to increase the ability for detecting Bt resistance in D. saccharalis, F2 neonates of each family were screened on Bt-maize leaf tissues for 15 days, 5 days longer than the time that larvae were exposed to non-Bt-maize leaf tissues. The number of surviving larvae was recorded at 5 days, 10 days and 15 days after inoculation. Leaf tissues were replaced every 3 days. Surplus eggs (if any) for each family were held in an environmental chamber maintained at 21°C, 50% RH and a 16:8 h (L:D) photoperiod. These eggs (or larvae hatched from the eggs) were discarded if no F2 larvae survived in the F2 screen. If any larvae survived, the surplus larvae of the family (if available) were continuously reared on a meridic diet and used as a backup strain for that family.
Resistance confirmation on Bt plants
Data from the F2 screen indicated at least one family (family 52) might possess a major Bt resistance allele. F3 larvae (LA-NE-B) of this family were selected on Bt-maize plants in a greenhouse. DKC69-70 was planted and thinned to one plant per pot two weeks later. At the V12–V13 stages, ten F3 neonates (<24 h old) of family 52 (LA-NE-B strain) were manually placed into the whorl of each of 27 plants. After 27 days, all live F3 larvae recovered from Bt plants were transferred into 30-ml plastic cups containing 10 ml of a meridic diet, and survivors were reared to the pupal stage. Pupae were removed from the larval rearing cups and sexed. Newly-emerged virgin moths from the F3 survivors were individually transferred into 450-ml paper containers (Huhtamaki Foodservice, De Soto, KS), one moth per container. The design of the moth containers was similar to that used in the moth pairing for establishing two-parent families. To facilitate colony establishment, each of the four earliest emerged moths derived from the F3 survivors was immediately provided with three newly emerged virgin males from the susceptible LA-NE-A strain if it was a female or three newly emerged virgin females of the LA-NE-A strain if it was a male (backcross, BC1). The pooled backcross neonates (BC1F3) produced from the four backcrosses were reared on a meridic diet. The BC1F3 adults were mated in 3.8-l cardboard cartons (Neptune Paper Products, Newark, NJ) under the same conditions as described above. BC1F4 eggs of the backcross were harvested from wax papers attached against the inside wall of the cartons.
To confirm resistance, survival of BC1F4 neonates (LA-NE-C) was evaluated on Bt plants in the greenhouse. Five Bt-maize seeds were planted in each pot (18.9 l) within the greenhouse. The plants were thinned to three plants per pot after two weeks. At V12–V13 stages, approximately 80 neonates (<12 h) of LA-NE-C strain were placed into the whorl of each Bt plant using the same methods as described above. A total of approximately 6000 larvae were exposed to 75 Bt plants in 25 pots. In addition, 21 Bt plants (seven pots) were infested with neonates of the susceptible LA-NE-A strain (control). All Bt plants were tested for the Cry1A expression with Cry1Ab/Cry1Ac Lateral Flow QuickStix™ Strip Kit (Envirologix, Portland, ME) before insect infestations. To determine the termination time of these tests, larval development on non-Bt-maize plants was monitored by infestation of 10 LA-NE-C neonates on each of 12 non-Bt plants in four pots in the same greenhouse room. To reduce the risk of larvae moving from non-Bt plants to Bt plants, the four pots with non-Bt plants were placed in one corner of the greenhouse and separated from Bt plants by >3 m. During the initial screen of family 52 (LA-NE-B strain), larval development on Bt-maize plants was significantly delayed compared to the larvae on non-Bt plants. Therefore, during the resistance confirmation tests, larval development was monitored on Bt plants. After the first pupa was observed on non-Bt-maize plants, all remaining non-Bt-maize plants were removed from the greenhouse. Larval development on Bt plants was then monitored twice a week (Monday and Friday) by recording the number of surviving larvae on three Bt-maize plants in three pots, one plant from each pot. The greenhouse tests were terminated 57 days after inoculation, when pupae were first observed on Bt plants. At the point of test termination, 55 Bt plants were still available for data collection. These Bt plants were dissected to record number and locations of surviving insects.
Resistance confirmation on Bt leaf tissues
Pupae and pre-pupae (mature larvae) of BC1F4 recovered from Bt plants after 57 days were transferred into cardboard cartons (Neptune Paper Products, Newark, NJ) for adult emergence, mating and oviposition. A Bt ‘resistant’ insect strain (LA-NE-D) was established from the eggs produced from these survivors. Larval mortality of the susceptible LA-NE-A and the resistant LA-NE-D were assayed on leaf tissues of non-Bt and Bt-maize plants in the 8-well trays using a similar method as described above. In the bioassays, 20 neonates were released on the maize leaf tissues in each well of the 8-well trays. Larval mortality was recorded every day for 6 days. There were four replications for each combination of insect strain, maize cultivar and test duration, and each replicate included 80 neonates in four wells (N=320). Each replication was treated separately.
Larval mortality of D. saccharalis on Bt-maize leaf tissues was corrected using the method of Abbott (Reference Abbott1925). The corrected mortality data were transformed using arcsine (x 0.5) (Zar, Reference Zar1984). Differences in larval mortality between LA-NE-A and LA-NE-D strains at a specific observation time were analyzed using t-tests (SAS Institute, 1999). Treatment means were separated using the conservative Cochran and Cox test at the α=0.05 level (Cochran & Cox, Reference Cochran and Cox1950). Untransformed data are presented in fig. 2.
Larval mortality (%) of Bt-susceptible (LA-NE-A) and Bt-resistant (LA-NE-D) strains of Diatraea saccharalis on DKC69-70 Bt-maize leaf tissues. Mean values within a specific test period followed by a different letter are significantly different (P<0.05; Cochran and Cox's t-test).
Results
Establishment of two-parent families
A total of 228 pairs of D. saccharalis adults were established from a collection of 629 larvae during the first field generation. However, only 45 families (19.7%) produced enough F2 progeny (>300 neonates) for the F2 screen. An average of 44±3 (mean±SEM) F1 pupae for each family were used to produce the F2 progeny for the insects collected in the first generation. The success rate was higher for insects collected during the second generation. A total of 168 iso-line families from 197 adult pairs (85.3%) produced enough F2 neonates for the F2 screen. An average of 41±2 F1 pupae per family were used to produce F2 larvae for the insects collected in the second generation.
Larval survivorship of D. saccharalis on non-Bt-maize leaf tissues
D. saccharalis neonates feeding on non-Bt-maize leaf tissues survived well in the laboratory. The larval survival rate (mean±SEM) was 94±1% after 5 days and 91±1% after 10 days.
Screening F2 neonates on Bt-maize leaf tissues
All F2 larvae of 163 families out of the 213 iso-line families were killed on Bt-maize leaf tissues after 5 days (table 1). Only 40 F2 larvae from five families survived after 5 days, for the insects collected during the first field generation, and all of these larvae were dead after 10 days. A total of 608 larvae in 45 families, established from insects collected during the second field generation, survived the Bt-maize leaf tissues for ≥5 days. Among these, 37 larvae in four families and 26 larvae in three families survived after 10 days and 15 days, respectively. Twenty of the 26 survivors after 15 days were from one family (family 52). The other six survivors were from families 146 and 176. Three larvae of family 147 also were alive after 10 days, but died after 15 days. No success in establishing colonies from these survivors was achieved because of the limited number of larvae and differences in larval development and moth emergence time.
Summary of an F2 screen searching for Bt resistance alleles in a Louisiana population of Diatraea saccharalis.
1 Days after exposure.
Resistance confirmation on Bt-maize plants
All Bt-maize plants in the greenhouse tested positively for Cry1A endotoxin expression. No surviving larvae were found on the Bt plants 57 days after inoculation with neonates of the susceptible LA-NE-A strain. A total of 14 F3 larvae of family 52 (LA-NE-B) were recovered from nine Bt plants after 27 days. A large number (>10,000) of BC1F4 progeny (LA-NE-C) were produced from the backcross. On the 57th day after larval inoculations, a total of 351 larvae and pupae of the LA-NE-C strain were recovered from 51 out of the 55 Bt plants tested. These survivors included 20 pupae, 48 large larvae (pre-pupae or 5th instars) and 283 small larvae (2nd–4th instars). The pupae and larvae were viable and were located within the stalks (182 insects), on the ears (79 insects) and in the ear shanks (90 insects). All moths derived from the pupae and pre-pupae emerged normally as observed for those reared on the meridic diet. The emerged moths laid viable eggs (see below).
Resistance confirmation on Bt-maize leaf tissues
Larval mortality on non-Bt-maize leaf tissues during the 6 day test period ranged from 4±1% (mean±SEM) to 13±1% for the LA-NE-A susceptible strain and from 3±1% to 9±3% for the LA-NE-D resistant strain. The difference in larval mortality on non-Bt leaf tissues between the two D. saccharalis strains was not significant (t⩽1.16, df=3, P≥0.3316).
Neonates of the LA-NE-D resistant strain were more tolerant to Bt-maize leaf tissues than were those of the LA-NE-A susceptible strain (fig. 2). No mortality occurred after 1 day for either strain on Bt-maize leaf tissues. However, significant mortality, 21±6% (mean±SEM), of the LA-NE-A strain was observed after 2 days. Mortality of the susceptible strain on Bt leaf tissues was 98±1% after 4 days and 100% after 5 days. Larval mortality of the resistant LA-NE-D on Bt leaf tissues was significantly lower (t<3.12, df=3, P<0.05) than that of the LA-NE-A strain across the test periods except at 1 day. Significant mortality of LA-NE-D did not occur on Bt leaf tissues until 5 days (63±3%) and 6 days (72±3%).
Discussion
Since the F2 screen was proposed as a method for identifying Bt resistance alleles in O. nubilalis by Andow and Alstad (Reference Andow and Alstad1998), several modifications of the screening protocol have been evaluated to detect Bt resistance in O. nubilalis (Andow et al., Reference Andow, Alstad, Pang, Bolin and Hutchison1998, Reference Andow, Olson, Hellmich, Alstad and Hutchison2000; Bourguet et al., Reference Bourguet, Chaufaux, Séguin, Buisson, Hinton, Stodola, Porter, Cronholm, Buschman and Andow2003; Stodola & Andow, Reference Stodola and Andow2004); D. grandiosella (Huang et al., Reference Huang, Leonard, Cook, Lee, Andow, Baldwin, Tindall and Wu2007); a rice stem borer, Scirpophage incertulas (Walker) (Bentur et al., Reference Bentur, Andow, Cohen, Romena and Gould2000); a coleopterous poplar leaf beetle, Chrysomela tremulae F. (Génissel et al., Reference Génissel, Augustin, Courtin, Pilate, Lorme and Bourguet2003); and diamondback moth, Plutella xylostella (L.) (Zhao et al., Reference Zhao, Li, Collins and Shelton2002). Indeed, Stodola and Andow (Reference Stodola and Andow2004) indicated that many potential modifications could be made to the F2 screen to adapt it to other species and to improve its efficiency.
The present study used a new method to develop two-parent families for the F2 screen. The families of D. saccharalis were established from field-collected larvae rather than field-collected adult males or mated females. Starting with field-collected larvae of D. saccharalis was cost-effective for several reasons. Larvae of D. saccharalis can be easily collected from maize plants, particularly from late maize plantings. For example, in 2004, >700 1st generation larvae of D. saccharalis and >600 2nd generation larvae were collected from late-planted maize fields near Winnsboro, Louisiana, in only a few hours. In addition, there is no sex pheromone available for trapping male D. saccharalis, and adult moths of D. saccharalis are calm during the daytime so that they are easy to pair mate.
Although the success rate (<20%) of screening two-parent families was low for samples from the first field generation, success (>85%) was substantially higher for pairs from the second field generation. The reasons for the considerable improvement in the success rate are unknown. The addition of a small amount (10 g) of vermiculite in each adult container for the insects derived from the 2nd generation collection might have improved the environmental conditions for adult mating and oviposition.
Another notable modification of the F2 screen developed in the present study was the use of Bt-maize leaf tissues for screening F2 neonates rather than directly screening F2 larvae on Bt plants (Andow et al., Reference Andow, Alstad, Pang, Bolin and Hutchison1998, Reference Andow, Olson, Hellmich, Alstad and Hutchison2000; Bourguet et al., Reference Bourguet, Chaufaux, Séguin, Buisson, Hinton, Stodola, Porter, Cronholm, Buschman and Andow2003) or on a diet containing Bt protoxins (Zhao et al., Reference Zhao, Li, Collins and Shelton2002; Bourguet et al., Reference Bourguet, Chaufaux, Séguin, Buisson, Hinton, Stodola, Porter, Cronholm, Buschman and Andow2003). The use of Bt-maize leaf tissues may have several advantages above screening on whole Bt plants. First, when screening a large number of iso-line families, using Bt-maize leaf tissues saves considerable space and can be conducted in growth chambers in the laboratory. If whole Bt plants are used, it must be done in the field or in large greenhouses. This also reduces the cost and labour involved in an F2 screen. Secondly, field collections of corn borers (e.g. O. nubilalis or D. grandiosella) are often done during the second field generation, which typically occurs in June to October, depending on geographical location. Because the insects are reared in a laboratory for two generations, screens would most likely be conducted during the winter season. In the northern US, whole plant assays could only be done in greenhouses at considerable expense compared to leaf tissue assays, which require significantly fewer plants. Thirdly, it is much easier to recover survivors from maize leaf tissues in small bioassay trays than from whole Bt plants. The high larval survival and recovery rates of D. saccharalis on non-Bt-maize leaf tissues observed in the present study also suggest that the use of maize leaf tissues is an effective method to assay larval mortality of D. saccharalis. Finally, it minimizes the effect of any potential Bt concentration variations that might occur within plants. Although this has not been demonstrated, research with Bt-broccoli whole plant assays has suggested that a whole plant assay may produce more false negatives than is desirable (Zhao et al., Reference Zhao, Li, Collins and Shelton2002).
In addition, the method has some advantages over diet assays. Firstly, it does not require synthesis, purification and quantification of Cry toxin. Secondly, it does not require determination and validation of a diagnostic dose for detecting resistance alleles. Thirdly, it is unnecessary to use a toxin model when it is difficult to purify the toxin as expressed in the plant. For example, use of more readily obtained Bt protoxin, trypsinized Bt toxin or microbial formulations, such as Dipel, may not accurately reflect the status of insect resistance to Bt-maize because the Bt proteins expressed in Bt plants can be different from these model systems (National Research Council, 2002).
Selection of properly aged leaves is important in the F2 screen with Bt-maize leaf tissue. In preliminary tests, leaves removed from older maize plants (V12–V13) could be maintained longer in the laboratory than younger leaves (data not shown). However, these data also showed that old leave tissues excised from non-Bt-maize plants could cause higher larval mortality to D. saccharalis than younger leaves. In our study, mortality of D. saccharalis larvae feeding on non-Bt-maize leaf tissues excised from fully expanded leaves of V6–V8 plants was low, <9% after 10 days. These tissues can be maintained in the screening trays for at least 3 days, suggesting that fully expanded leaves of V6–V8 Bt plants are suitable for assays for Bt resistance alleles. D. saccharalis has three overlapping generations a year on maize in the mid-southern region of the US (Castro et al., Reference Castro, Riley, Leonard and Baldwin2004). Neonates of first generation typically occur during V8–V12 maize plant stages, while the second and third generations occur during reproductive plant stages. Therefore, any potential resistance identified in the laboratory bioassays with leaf tissues needs to be further evaluated in intact whole Bt plants to confirm the resistance, because Bt concentration variations might occur in different plant stages and Bt expression in leaf tissues might be different compared to whole plants.
In this study, we were unable to establish colonies directly from the F2 survivors after screening on Bt-maize leaf tissues to confirm resistance because of the limited number of survivors and differences in developmental times. Instead, the insect population was increased by rearing the surplus F2 larvae of family 52 to F3 generation. F3 progeny of family 52 were screened on greenhouse Bt-maize plants. The F3 survivors recovered from Bt plants were then backcrossed with a susceptible family. Finally, the F2 offspring of this backcross were selected again on Bt-maize plants in the greenhouse to verify and isolate resistance in a single resistant line (LA-NE-D). However, if the F2 screen survivors were immediately backcrossed to a susceptible strain and the F2 progeny of the backcross were screened and selected on Bt plants, resistance could be verified and isolated at a lower cost compared to the present method.
Bt resistance confirmed in family 52 using intact Bt-maize plants in the greenhouse indicates that the 15 day screening procedure was effective and feasible for detecting Bt resistance alleles in D. saccharalis. In contrast, the 5 day screen apparently was not sufficiently severe for detecting Bt resistance in D. saccharalis because many families (23%) survived on Bt-maize leaf tissues for ≥5 days, but most of these survivors died on Bt-maize leaf tissues after 10 days. Data from the present study are not sufficient to conclude that the 10 day screening procedure is a valid approach for detecting Bt resistance in D. saccharalis. Three of the four families (including family 52) that survived the Bt-maize leaf tissues in the 10 day screen procedure also survived after 15 days. However, except family 52, survival rates of the other three families were very low, ⩽1.7% after 10 days and ⩽1.3% after 15 days. The results suggest that the 10 day screening procedure might be used to detect both major and partial resistance alleles in D. saccharalis. However, additional laboratory bioassays are needed to validate such abilities for the 10 day screening procedure. In summary, we recommend, at this point, the 15 day procedure for screening Bt resistance alleles in D. saccharalis.
One disadvantage associated with the use of Bt-maize leaf tissues for the F2 screen, compared to the use of whole Bt plants, is that the concentration or biological activity of the Bt toxin (Cry1Ab) in leaf tissues may change after removal from the whole plants. Preliminary tests showed that insecticidal activity of Bt-maize leaf tissues did not significantly change for at least 4 days after removal from whole plants unless the leaf tissues were chlorotic yellow (FH, BY & BRL., unpublished data). Laboratory bioassays have shown that leaves removed from transgenic Bt poplar trees were lethal to larvae of C. tremulae, regardless of leaf age, and maintained the insecticidal activity for at least 3 days in the laboratory conditions (Génissel et al., Reference Génissel, Augustin, Courtin, Pilate, Lorme and Bourguet2003). In addition, dissociated leaf tissues may produce additional defense mechanisms that may have affected the survival of Bt-resistant larvae. However, bioassays with neonates of D. saccharalis and two other Bt-susceptible maize borer species, O. nubilalis and D. grandiosella, did not show significant mortality on dissociated non-Bt-maize leaf tissues (Huang et al., Reference Huang, Leonard and Gable2006), suggesting such effect, if any, should be limited and not be practically important in the F2 screen.
The F2 screen technique developed in this study could be easily modified for detecting rare Bt resistance alleles in other similar corn stalk borers, such as O. nubilalis and D. grandiosella (Huang et al., Reference Huang, Leonard, Cook, Lee, Andow, Baldwin, Tindall and Wu2007). In fact, the protocol may be even more suitable for screening these two corn borer species than for D. saccharalis. Larvae of O. nubilalis and D. grandiosella are more susceptible to Bt-maize leaf tissues than D. saccharalis (Huang et al., Reference Huang, Leonard and Gable2006). For example, under the same environmental conditions as used in the present study, 100% of larvae of O. nubilalis and D. grandiosella were killed on DKC69-70 Bt-maize leaf tissues within 4 days (Huang et al., Reference Huang, Leonard and Gable2006), suggesting that a 4 day screening period could be used for these two species instead of the 5 day screening used for D. saccharalis. As discussed above, a 4 day screening period implies that changes of leaf tissues in the screen trays may not be necessary. Thus, cost of screening O. nubilalis and D. grandiosella would be further reduced.
Acknowledgements
We thank Drs Seth Johnson, Michael Stout, Boris Castro and Kelly Tindall for reviewing an earlier draft of the manuscript. We also thank Joshua Temple, Karla Emfinger, Xiaoyi Wu, Xiaoyun Kan and several other students for insect collection and rearing. This article is approved as publication 05-26-0723 from the Louisiana Agricultural Experiment Station and represents work sponsored by Louisiana Soybean and Feed Grain Promotion Board, National Science Foundation Center for IPM, NC-205 and the hatch fund from Department of Entomology, Louisiana State University AgCenter.
