Baseline Susceptibility of the Field Populations of Ostrinia furnacalis in Indonesia to the Proteins Cry1A.105 and Cry2Ab2 of Bacillus thuringiensis

Genetically modified MON 89034 corn (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins, viz. Cry1A.105 and Cry2Ab2, is a biotechnological option being considered for the management of the major corn pest in Indonesia, the Asian corn borer (Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae)). As a part of a proactive resistance-management program for MON 89034 corn in Indonesia, we assessed the baseline susceptibility of field-collected populations of O. furnacalis to Cry1A.105 and Cry2Ab2 proteins. Dose–response bioassays using the diet-dipping method indicated that the lethal concentration (LC50) values of Cry1A.105 and Cry2Ab2 in 24 different field populations of O. furnacalis ranged from 0.006 to 0.401 µg/mL and from 0.044 to 4.490 µg/mL, respectively, while the LC95 values ranged from 0.069 to 15.233 µg/mL for Cry1A.105 and from 3.320 to 277.584 µg/mL for Cry2Ab2. The relative resistance ratios comparing the most tolerant field populations and an unselected laboratory population were 6.0 for Cry1A.105 and 2.0 for Cry2Ab2 based on their LC50 values. Some field populations were more susceptible to both proteins than the unselected laboratory population. The LC99 and its 95% fiducial limits across the field populations were calculated and proposed as candidate diagnostic concentrations. These data provide a basis for resistance monitoring in Bt Corn and further support building resistance-management strategies in Indonesia.


Introduction
The Asian corn borer, Ostrinia furnacalis (Guenée), is one of the major pests of corn (Zea mays Linnaeus) in Southeast Asia, including the Indonesian archipelago, Vietnam, the Philippines, and China [1].Areekul [2] and Camarao [3] reported two generations of O. furnacalis infestation in each growing season in tropical areas with 24-30 total development days for each generation.Damage from O. furnacalis occurs not only in the stem of corn plants but also in the whorl, tassel, and ears [4,5].In vegetative stage plants, the newly hatched larvae feed on young leaf whorls, resulting in holes in the leaves that can widen as the larvae grow and feed more vigorously.In the reproductive stage, the larvae feed on tassel, then bore into the stem, making tunnels, and might continue to feed on the ears [2].
Da-Lopez et al. [6] reported that the presence of O. furnacalis egg masses was as high as nine egg masses per plant in fields in Sleman, Yogyakarta.A recent study (2018)(2019) in Lampung and Central Java showed that O. furnacalis infestations were present in as many as 95% of corn plants with an average of four holes per stalk and gallery lengths of 4-6 cm [7].This level of damage was above the economic threshold for this insect, estimated as one larva (hole) per stalk [8,9].One O. furnacalis larva boring per plant during V10, R1, or R2 resulted in grain yield losses of 4.94%, 4.56%, or 3.76%, respectively [9].
The economic damage on corn indicates the need for effective and ecologically sound management practices for O. furnacalis in Indonesia and other countries in Southeast Asia [10,11], where growers have similar challenges due to this species attacking corn plants [5,12,13].Chemical control using insecticides is the most commonly practiced control measure in Indonesia for the management of O. furnacalis [7].Genetically modified corn plants expressing Bacillus thuringiensis (Bt) insecticidal proteins have been commercialized and are widely grown in corn-growing countries, such as the United States, Brazil, Argentina, Canada, South Africa, the Philippines, and Vietnam for the successful management of corn stalk borers, corn ear feeders, and fall armyworm (Spodoptera frugiperda (J.E.Smith)) [14][15][16].Several Bt corn technologies are currently in the process of registration in Indonesia [17], including the MON 89034 corn.MON 89034 is a pyramided transgenic corn event expressing two Bt proteins, Cry1A.105 and Cry2Ab2, that are highly effective against key lepidopteran corn pests [18][19][20].MON 89034 has been approved for commercialization in the Philippines since 2010 and in Vietnam since 2015 as a viable alternative for the sustainable control of O. furnacalis and other corn lepidopteran pests [15,21,22].In September 2021, MON 89034 was approved as a registered product to control S. frugiperda in the Philippines [23].
The development of resistance in populations of the target pests poses the risk to the sustainability of Bt crops [24][25][26].In Indonesia, the risk of resistance development could be higher due to the year-round cultivation of corn which may result in continuous pressure of selection once MON 89304 is commercialized.The planting of refuges and the adoption of technologies expressing multiple Bt proteins with an independent mode of action significantly reduces the risk of resistance development in populations of the target pests [27,28].Because MON 89034 corn expresses two Bt proteins of different mechanisms of action targeting O. furnacalis, its inherent resistance risk is likely lower than that for single-gene Bt plants [29].Nevertheless, it is important to monitor the susceptibility of O. furnacalis populations to Cry1A.105 and Cry2Ab2 proteins after the introduction of MON 89034 in Indonesia.Establishing the baseline susceptibility of O. furnacalis to Cry1A.105 and Cry2Ab2 is important for resistance-monitoring programs in Indonesia.The assessment of the baseline susceptibility would not only allow the assessment of the natural variation among field populations but can also be used to document shifts in susceptibility likely resulting from selection for resistance [30,31].We hypothesize that there will be differences in the sensitivity of O. furnacalis populations.The goals of this study were to establish the baseline susceptibility of O. furnacalis to Cry1A.105 and Cry2Ab2 proteins in Indonesia and to estimate Cry1A.105 and Cry2Ab2 concentrations to be used as candidate diagnostic concentrations to monitor the development of resistance in O. furnacalis in Indonesia.

Susceptibility of O. furnacalis to Cry1A.105
Populations of O. furnacalis collected from six provinces in 2013-2015 showed variation in susceptibility to Cry1A.105 (Table 1).The LC 50 values for the field-collected populations ranged from 0.006 to 0.401 µg/mL, and for the laboratory population, these were 0.067 µg/mL.The LC 95 of the laboratory population was 0.890 µg/mL, while those of the field-collected populations varied from 0.069 to 15.233 µg/mL.The resistance ratios (RRs) of the 24 field populations relative to the laboratory population were 0.1-6.0 based on the LC 50 values (Table 1).Some field populations were more susceptible (RR < 1) to the protein than the laboratory population, while other field populations were more resistant (RR > 1) than the laboratory population.The difference between LC 50 values of any two populations from Cry1A.105 assays was considered significant if their 95% fiducial limits (FLs) did not overlap [32,33].

Susceptibility of O. furnacalis to Cry2Ab2
Populations of O. furnacalis collected from six provinces in 2013-2015 also showed variation in susceptibility to Cry2Ab2, as evidenced by the 0.1-2.0RR values based on the LC 50 (Table 2).The LC 50 values of Cry2Ab2 against 24 field-collected populations ranged from 0.044 µg/mL to 4.490 µg/mL, whereas that of the laboratory population was 2.276 µg/mL.The LC 95 value of the laboratory population was 22.984 µg/mL, and for the 24 field populations, it ranged from 3.320 to 277.584 µg/mL.Similarly to responses observed in the Cry1A.105assays, some field populations were more susceptible to Cry2Ab2 than the laboratory population.The difference between LC 50 values of any two populations from Cry2Ab2 assays was considered significant if their 95% fiducial limits (FLs) did not overlap [32,33].

Candidate Diagnostic Concentrations
The O. furnacalis LC 99 value for Cry1A.105 was significantly higher than its LC 95 value as indicated by non-overlapping lower and upper limits of the 95% FL (Table 3).However, there was no significant difference observed between LC 99 and LC 95 values for Cry2Ab2.We propose the LC 99 and its upper and lower fiducial limits (95% FLs) as the candidate diagnostic concentrations: 13.240 (6.716-33.831)µg/mL for Cry1A.105 and 127.320 (46.616-676.792)µg/mL for Cry2Ab2.These concentrations need to be tested with several field populations for further validation, followed by the selection of one diagnostic concentration for each protein to be used in monitoring programs.

Discussion
There is a need to develop a robust insect resistance-management (IRM) strategy for MON 89034 in Indonesia to prolong its durability in the field and, thus, delay the development of practical resistance once the event is approved for cultivation [27,34,35].Baseline susceptibility data are essential for resistance-monitoring purposes, particularly to provide information on the level of susceptibility of O. furnacalis to Cry1A.105 and Cry2Ab2 before the introduction of MON 89034 corn and to provide benchmark data for future comparison to detect susceptibility shifts.In this study, the baseline susceptibility of O. furnacalis to Cry1A.105 and Cry2Ab2 was established based on the populations collected from major corn-producing provinces in Indonesia.
Differences in susceptibility to Bt proteins before the onset of resistance have been reported among geographically distinct populations in many different species of insects attacking corn [20,[36][37][38].For O. furnacalis, several studies have been conducted using five proteins, i.e., Cry1Ab, Cry1Ac, Cry1F, Cry1A.105, and Cry2Ab2.Field-collected populations of O. furnacalis in Vietnam differed in susceptibility to Cry1Ab by up to three-fold, which was reflective of natural variability among the 11 populations used in the study [39].In China, the differences in susceptibility for this species were up to eight-fold for Cry1Ab [40], and in different study, up to two-fold for Cry1Ab, Cry1Ac, and Cry1F [41].Contrastingly, in yet another study that assayed the bioactivity of Cry1Ab with 25 field populations of O. furnacalis in China [33], 80-fold and 309-fold variations at LC 50 and LC 95 , respectively, were demonstrated.Furthermore, Alcantara et al. [20] reported that the differences in the Philippines were six-and seven-fold to Cry1A.105 and Cry2Ab2, respectively.
We reported higher levels of variation in the susceptibility of O. furnacalis field populations to Cry1A.105 and Cry2Ab2 than were reported in the Philippines.However, direct comparison between these two studies (diet dipping vs. diet overlay) was not possible since different bioassay procedures were employed.Differences in the susceptibility of O. furnacalis to these two proteins may represent natural variation among populations because transgenic corn has not yet been planted commercially in Indonesia, and commercial Bt formulations for controlling O. furnacalis are not commonly utilized by Indonesian farmers [4].A high difference in the susceptibility of field-collected populations was previously reported with O. furnacalis to Cry1Ab [33] and in Helicoverpa armigera to Cry1Ac (67-fold) in India [42].In addition, the innate heterogeneity within insect populations tested in routine bioassays using the same methodology may account for three-to six-fold, or even twelve-fold, variation in laboratory-reared population comparisons [43,44].Therefore, the heterogeneity may be even greater across field-derived populations, and the method chosen for bioassay needs careful consideration in the context of resistance monitoring.
Cry1A.105 was more toxic than Cry2Ab2 against O. furnacalis in Indonesia (Tables 1 and 2) and in the Philippines [20].Similar results were reported for one species of corn stem borer, Chilo partellus, in India [38].In contrast, Cry2Ab2 was more toxic than Cry1A.105against the other species of corn borer in India, Sesamia inferens [38].The same researchers also reported that Cry1A.105 was more toxic than Cry2Ab2 against H. armigera.Hernandez-Rodriguez et al. [45] also reported that Cry1A.105 was more toxic than Cry2Ab2 to Ostrinia nubilalis and S. frugiperda.These studies provide evidence that different species of corn borers may have different levels of susceptibility to different proteins and that their susceptibility may differ among geographically distinct populations.
A laboratory concentration of Cry1A.105 and Cry2Ab2 that reliably causes 99% mortality provides a candidate for use as a discriminating concentration [46].The proposed diagnostic concentrations of 6.7, 13.2, and 33.8 µg/mL for Cry1A.105 and of 46.6, 127.3, and 676.8 µg/mL for Cry2Ab2 need to be validated against field-collected populations.Based on these tests, the determination of diagnostic concentrations for each protein is planned and these should be available before the commercialization of MON 89304.
The expression of Cry1A.105 and Cry2Ab2 in MON 89034 corn varies depending on the tissues with the highest expression occurred in the young leaves of V2-V4 with the average of 520 and 180 µg/g dry weight tissue [47].This information in combination with baseline data may be used in developing IRM strategies in Indonesia.

Conclusions
This study reports the baseline susceptibility of distinct geographical O. furnacalis populations of Indonesia to Cry1A.105 and Cry2Ab2 proteins.Our findings demonstrate that O. furnacalis populations are highly sensitive to Cry1A.105 and Cry2Ab2 proteins, although there are differences in sensitivity among populations, which is a natural variation.The baseline susceptibility data are used to establish candidate diagnostic concentrations for further validations.The data on baseline susceptibility and the diagnostic concentrations provide valuable tools for future resistance-monitoring programs to detect early shifts in sensitivity among the field populations of O. furnacalis once transgenic corn expressing these two proteins is commercialized in Indonesia.

Field-Collected Populations
Egg masses, larvae, and pupae of O. furnacalis were collected from six provinces in Indonesia: three on Java Island (East Java, Central Java, and Yogyakarta Special Region), two on Sumatra Island (North Sumatra and Lampung), and the province of Gorontalo on Sulawesi (Figure 1).With the exception of Yogyakarta, these provinces are the major corn production sites in Indonesia, and insect pressure in these provinces were prominent based on the previous field observations.Field collections were made from non-Bt corn plants between 55 and 70 days after sowing with the intention of collecting insects from the second generation of O. furnacalis.Twenty-four populations were collected from 14 districts distributed in the six selected provinces between November 2013 and May 2015.The number of collected O. furnacalis from each location varied, and 83% of the collected populations were between 65 and 113 larvae and pupae.Only three of the collections were egg masses, each obtained from three different sites.The lowest number of O. furnacalis collected from one population was 12 larvae + 12 pupae + 1 egg mass.Each population was collected from at least two corn farms in one village to capture the genetic variability among individuals within a population and also because of the low infestations of O. furnacalis during the period of collection.

Insect-Rearing Procedure
The field-collected and laboratory O. furnacalis populations were reared using a similar artificial diet and standardized laboratory procedures [48].Larvae were fed on a redbean-based artificial diet (2-3 larvae per ~5 g of diet) in clear plastic cups (3.3 cm in diameter and 4.3 cm height) until pupation.A maximum of fifty cups were placed in a plastic tray.The trays were labelled with the locations of the populations.Pupae were collected daily and approximately 200 pupae were placed in a Petri dish (9 cm in diameter).The Petri dish containing the pupae were placed in the middle of a mating cage made of wire (21 cm in diameter and 21 cm height) covered with white paper on the top for oviposition.Emerging adults were fed with a 10% honey solution, and wet cotton was placed in the cage for maintaining high relative humidity during the day.The paper containing egg masses was removed every other day or daily as needed to collect larvae of similar ages.Egg masses were incubated in glass jars (6.5 cm in diameter and 15.5 cm height) containing moistened filter paper until hatching.A portion of the newly hatched larvae (ca.300 larvae) was transferred individually into plastic cups as described above for the next generations, and the other portion was used for bioassays.All insect life stages were incubated at room temperature (24-28 °C) with relative humidity ranges of 60-85%.All equipment for making the artificial diet as well as for rearing was semi-sterilized by dipping in 10% sodium hypochlorite (SC Johnson, Indonesia).If mortality occurred during mass rearing, dead larvae were removed immediately from the colony to prevent contamination during mass rearing.

Susceptibility of O. furnacalis to Cry1A.105 and Cry2Ab2
The proteins (Cry1A.105and Cry2Ab2) were provided by Bayer CropScience, St. Louis, USA.Bioassays were carried out using mostly F2 generation O. furnacalis neonates O. furnacalis at different life stages collected from the fields were handled using different methods.O. furnacalis egg masses were transferred into jars (18 cm tall, 7 cm diameter) layered with wetted filter paper to maintain the freshness of the eggs.Newly hatched larvae and bigger larvae were transferred individually into clear plastic containers with a screw cap (4.3 cm tall, 3.3 cm diameter) containing an artificial diet (ca. 5 g) [48] to minimize field-derived diseases.Collected pupae were put together in a container cup (the same size as for the larvae layered with filter paper) with a maximum of 10 pupae in each container.All collected larvae were healthy and pupated.Pupae produced from collected larvae, as well as those collected directly from the field, were placed into a Petri dish (9 cm diameter) layered with filter paper, and the Petri dish with the pupae was placed in a wire mesh cage (20 cm in diameter and 20 cm in height) upon arrival in the laboratory.Moisture was maintained by adding water to the filter paper in the plastic cups containing egg masses.Ostrinia furnacalis populations collected from different locations were reared in separate cups, trays, and cages to avoid contamination between populations.Additional details are provided below in "Insect-Rearing Procedure".

Insect-Rearing Procedure
The field-collected and laboratory O. furnacalis populations were reared using a similar artificial diet and standardized laboratory procedures [48].Larvae were fed on a red-beanbased artificial diet (2-3 larvae per ~5 g of diet) in clear plastic cups (3.3 cm in diameter and 4.3 cm height) until pupation.A maximum of fifty cups were placed in a plastic tray.The trays were labelled with the locations of the populations.Pupae were collected daily and approximately 200 pupae were placed in a Petri dish (9 cm in diameter).The Petri dish containing the pupae were placed in the middle of a mating cage made of wire (21 cm in diameter and 21 cm height) covered with white paper on the top for oviposition.Emerging adults were fed with a 10% honey solution, and wet cotton was placed in the cage for maintaining high relative humidity during the day.The paper containing egg masses was removed every other day or daily as needed to collect larvae of similar ages.Egg masses were incubated in glass jars (6.5 cm in diameter and 15.5 cm height) containing moistened filter paper until hatching.A portion of the newly hatched larvae (ca.300 larvae) was transferred individually into plastic cups as described above for the next generations, and the other portion was used for bioassays.All insect life stages were incubated at room temperature (24-28 • C) with relative humidity ranges of 60-85%.All equipment for making the artificial diet as well as for rearing was semi-sterilized by dipping in 10% sodium hypochlorite (SC Johnson, Indonesia).If mortality occurred during mass rearing, dead larvae were removed immediately from the colony to prevent contamination during mass rearing.

Susceptibility of O. furnacalis to Cry1A.105 and Cry2Ab2
The proteins (Cry1A.105and Cry2Ab2) were provided by Bayer CropScience, St. Louis, MO, USA.Bioassays were carried out using mostly F 2 generation O. furnacalis neonates and a few using the F 1 or F 3 generation.The F 3 generation was used when neonates of F 2 was not sufficient to do the whole bioassays.The larvae of F 2 and F 3 were used if the F 1 neonates in uniform age were inadequate for the conduction of the bioassay.Bioassays were carried out by following the diet-dipping procedure described by Trisyono et al. [49] by dipping the diet in protein solutions or distilled water.A cube of diet (1 cm × 1 cm × 1 cm) was dipped for 10 s in a treated or control solution and then air dried for 20 min.After drying, the diet was transferred into a plastic cup of similar size to the ones used for rearing.For each replication, ten newly hatched O. furnacalis larvae were transferred individually into two plastic cups containing the treated or control diet (5 larvae per cup).Each treatment was replicated 3-5 times.In the Cry1A.105bioassays, 10 concentrations from 0.002 to 48 µg/mL Cry1A.105(three-fold dilutions) were tested to determine LC 50 and LC 95 of 24 field-collected O. furnacalis populations and the laboratory population.For Cry2Ab2, 10 concentrations ranging from 0.0007 to 15.5 µg/mL (three-fold dilutions) and the control were used for all bioassays to determine LC 50 and LC 95 against the same F 1 -F 3 of fieldcollected and laboratory populations.Based on the results from the preliminary bioassays, the concentrations used for Cry1A.105 and Cry2Ab2 were different due to varying response of O. furnacalis to each protein.The selected concentrations based on the preliminary test were expected to result in larval mortality ranging from 2% to 98%.Prior to the actual assays, preliminary tests of both proteins were carried out using 10 newly hatched larvae per concentration in three replicates.Larvae were exposed to the treated or control diet continuously, and observed mortality was recorded 7 days after they were placed in the plastic cups containing the artificial diet.

Data Analysis
Probit analysis [50] was carried out using the PoloJR program within PoloSuite, Version 2.1 (LeOra Software 2016) to determine LC 50 and LC 95 values and their 95% fiducial limits (FLs) for each protein and population.The relative resistance ratios (RR) were calculated by dividing the values of LC 50 of field populations by that of the laboratory population [51].The LC 95 and LC 99 and their lower and upper limits for the Cry1A.105and Cry2Ab2 proteins were determined from pooled baseline concentration-mortality data (24 fieldcollected populations) using the program PoloJr, and the estimated LC 99 and its lower and upper limits of 95% were proposed as candidate diagnostic concentrations for each protein, as they killed 99% of the susceptible population [52,53].

Figure 1 .
Figure 1.The six provinces in Indonesia selected for collecting field populations of Ostrinia furnacalis in 2013-2015 during the peak corn season in each province.With the exception of the Yogyakarta Special Region, these provinces were the top corn-producing areas in the country.

Figure 1 .
Figure 1.The six provinces in Indonesia selected for collecting field populations of Ostrinia furnacalis in 2013-2015 during the peak corn season in each province.With the exception of the Yogyakarta Special Region, these provinces were the top corn-producing areas in the country.

Table 1 .
Susceptibility to Cry1A.105 of field-collected populations of Ostrinia furnacalis from major corn-growing areas in Indonesia.
N = number of larvae.SE = standard error.95% FL = 95% fiducial limits.a Resistance ratios (RRs) were calculated by dividing the values of LC 50 of field populations by that of the laboratory population.b Collected from Yogyakarta in 2009 and maintained in the laboratory without Bt or insecticide selection.* The program did not give the 95% FL values.c Double asterisk (**) indicates significant differences based on the p-value (α = 0.05).

Table 2 .
Susceptibility to Cry2Ab2 of field-collected populations of Ostrinia furnacalis from major corn-growing areas in Indonesia.

Table 2 .
Cont. = number of larvae.SE = standard error.95% FL = 95% fiducial limits.a Resistance ratios (RRs) were calculated by dividing the values of LC 50 of field populations by that of the laboratory population.b Collected from Yogyakarta in 2009 and maintained in the laboratory without Bt or insecticide selection.c No significant differences based on the p-value (α = 0.05). N

Table 3 .
Candidate diagnostic concentrations of Cry1A.105 and Cry2Ab2 estimated using baseline susceptibility data of 24 field-collected populations of Ostrinia furnacalis.= standard error.95% FL = 95% fiducial limits.For each protein, LC 95 and LC 99 values followed by different letters were significantly different based on non-overlapping 95% fiducial limits. SE This research was funded by Universitas Gadjah Mada and PT Branita Sandhini Indonesia (Bayer Indonesia) grant number [032/RA Dept/PTBS/2012] or [2137/PN/TU/VII/2012] and The APC was funded by Universitas Gadjah Mada and Bayer CropScience Indonesia.Institutional Review Board Statement: Not applicable. Funding: