Relative Fitness and Feeding Capacity of Imidacloprid Resistant Nilaparvata Lugens

Imidacloprid is a neonicotinoid insecticide that is recommended for controlling Nilaparvata lugens. In Asian countries, such as, China, Vietnam, India, and Thailand, imidacloprid has caused resistance to N. lugens. Imidacloprid has also caused resistance to N. lugens based on some previous studies in Indonesia. The aim of this study was to determine the fitness and feeding capacity of imidacloprid-resistant N. lugens. The population of N. lugens used in this study had a resistance level of 50.64 times compared to the susceptible population. When the resistant and susceptible population of N. lugens did not receive any exposure to imidacloprid, the susceptible population had better fitness than the resistance one. However, the fitness of the resistant population increased when this population was resistance which sublethal cencentration (LC50 & LC20) of imidacloprid. The increase fitness of this resistant population most likely related to the increase in feeding capacity of the resistant population when they were treated which sublethal imidacloprid. These findings suggest that the field population of N. lugens that have developed resistance would increase the probability of outbreak if they were sprayed with imidacloprid. INTISARI Imidakloprid adalah insektisida neonicotinoid yang direkomendasikan untuk mengendalikan Nilaparvata lugens. Di negara Asia, seperti, China, Vietnam, India, dan Thailand, imidakloprid telah menyebabkan resistensi terhadap N. lugens. Di Indonesia, berdasarkan beberapa penelitian sebelumnya dilaporkan imidakloprid juga menyebabkan resistensi terhadap N. lugens. Tujuan penelitian ini untuk mengetahui kebugaran relatif dan kemampuan makan N. lugens resisten terhadap imidakloprid. Populasi N. lugens yang digunakan dalam penelitian ini mempunyai tingkat resistensi 50,64 kali dibandingkan dengan populasi peka. N. lugens populasi resisten dan peka apabila tidak dipapar dengan imidakloprid, populasi peka mempunyai kebugaran lebih baik dari pada resisten, namun kebugaran dari N. lugens populasi resisten meningkat ketika populasi tersebut dipapar dengan imidakloprid konsentrasi subletaal (LC50 & LC20). Peningkatan kebugaran populasi N. lugens resisten diduga terkait dengan peningkatan kemampuan makan akibat dipapar lagi dengan imidakloprid subletal. Hasil penelitian ini menunjukkan bahwa ledakan N. lugens akan terjadi, apabila populasi N. lugens yang telah resisten terhadap imidakloprid dipapar lagi dengan imidakloprid.


INTRODUCTION
The brown planthopper, Nilaparvata lugens Stal (Hemiptera: Delphacidae), is a major pest of rice, which infests rice planted in many countries like Australia, Bangladesh, Bhutan, Myanmar, Cambodia, China, Fiji, India, Indonesia, Japan, North and South Korea, Laos, Malaysia, Nepal, Pakistan, Papua New Guinea, the Philippines, SriLanka, Taiwan, Thailand, and Vietnam (Chen et al., 2012;Liu et al., 2012) (Botrell &Schoenly, 2012). The insect is a serious threat to worlds' rice sustainability by affecting to the instability of rice production, monetary loss, and harvest failure (Catindig et al., 2009).
Insect pest resistance to insecticides is a major issue in many countries including Indonesia since it is directly related to the success in insect pest control. N. lugens resistance has been reported occuring in many rice producing countries like China, Thailand, India, and Vietnam (Catindig et al., 2009;Liu et al., 2012). In China, the resistance to buprofezin reached 28.8 times. In Thailand, the resistance to fenobucarb ranged between 3-5.6 times. In India the resistant level to thiamethoxam and clothianidin were 10.7 and 4.9 times, respectively (Catindig et al., 2009). N. lugens resistance cases were reported in many regions in Java especially to diazinon, carbaryl, carbofuran, carbamate, and fentoat (Sutrisno, 1989). Melhanah et al. (2002) found that N. lugens developed resistance to fipronil with resistance level of 59.5 times compared to the susceptible population after four generations of laboratory selections. These studies show the capability of N. lugens to develop resistance to many different insecticides.
Imidacloprid belongs to neonicotinoid class which was released in 1991. It is sold US $ 1.560.000.000 annually or almost 17% of the global marketed insecticides (Jeschke & Nauen, 2005;Elbert et al., 2008). Neonicotinoid is consisted of at least seven main chemicals with market share of 25% of the global insecticides (Bass et al., 2015). The effectivity of this insecticide intensified its use which eventually created the resistance in N. lugens. During 2005During −2006 in China, the resistance of N. lugens to imidacloprid raised to 79.1-81.1 times compared to susceptible N. lugens Catindig et al., 2009). Resistance to imidacloprid was found in N. lugens collected from some locations in Central and East Java (Y.A. Trisyono, 2012, personal communication).
The exposure of insects to sublethal dose or concentration of insecticide is one factor causing resistance. Sublethal dose affects pest biology  such as the length of insects' stadium. The length of larval and adult stages could be longer, however the developmental and consumption rates decreased (Herbert & Harper, 1987). The reduction of eggs viability, oviposition period and prolonged larval stadium were related to the development of resistance in Leptinotarsa decemlineata selected with Bacillus thuringiensis CryIIIA delta-endotoxin (Trisyono & Whalon, 1997). Liu et al. (2012) found that unselected population developed faster to the next generation, while selected population endured higher fitness cost covering the significant reduction of larval longevity, adult eclosion, the rate of copulation, fecundity and hatching rate, and relative fitness of resistant N. lugens on the next generation.
Insects' fitness components like fecundity is related to insects' feeding capacity and and insects' food use, which are the main component in the life of an organism (Leather, 1995). Ratna (2011) reported that resistant N. lugens showed different feeding capacity compared to the susceptible one. Chelliah and Heinrichs (1984) found that reproduction rate of N. lugens increased when they were fed with rice sprayed with deltamethrin, methyl parathion, cypermethrin and fenvalerat. They suspect this condition was the consequence of insecticide residue or its metabolites, which induced chemical changes within the sprayed host plants. The rise of reproduction rates in N. lugens may be caused by the increase of consumption. This study was aimed to determine the fitness and feeding capacity of imidacloprid resistant N. lugens. The results of this study can be used as the bases to develop management tactics to delay or combat resistance to imidacloprid in N. lugens.

Mass rearing of Nilaparvata lugens
N. lugens population used in this study was collected in 2010 from the District Klaten. The Laboratory susceptible population was used as a comparison. The laboratory population has been reared in Pesticide Toxicology Laboratory (PLT) of the Faculty of Agriculture, Universitas Gadjah Mada (FA-UGM) since 1986 without any exposure to insecticides and no additional field population. These population were reared using an establish laboratory rearing procedures. One hundred g of sorted Cisadane seeds were carefully cleaned and soaked for 24 h. The seeds were germinated in a plastic jar (diameter = 30 cm, height = 32 cm), and covered with gauze. Seven d-old seedlings were used as the oviposition media and the diet of N. lugens.
Resistant adults of N. lugens (75−100 pairs) were moved to rice seedlings for oviposition. After 10 d or after eggs hatching, N. lugens nymphs were moved to a new jar previously filled with fresh rice seedlings. Used seedlings were placed above these fresh ones in an upside down position, held by wires attached to the side of the jar to allow the newly hatched nymphs to move to the fresh seedlings. The replacement of the seedlings was conducted every 6−7 d until N. lugens reached adult stage. Newly formed adults were directly transferred into a new jar containing with fresh 7 d old seedlings to develop the next generation.

Insecticide
The insecticide used was imidacloprid (Confidor 5 WP; PT Bayer Indonesia). Imidacloprid is a wide spectrum neonicotinoid, systemic, worked by contact and stomach poisoning and this chemical is recommended to control plant pests including N. lugens (Elbert et al., 2008).

The Resistance of Nilaparvata lugens
The resistance of N. lugens to imidacloprid after the five generations of selection was 50.64 times compared to the susceptible population (Londingkene et al., 2016, in press). LC 50 and LC 20 of imidacloprid to resistant N. lugens was 33.93 and 1.18 ppm, respectively. These sublethal concentrations were used in the following studies.

Study 1. Relative Fitness (preoviposition, oviposition, fecundity, egg hatching percentage, adult longevity, and life cycle) of Nilaparvata lugens
The fitness of N. lugens was studied using CRD with four treatments and 20 replications. The treatments were resistant N. lugens without exposure to imidacloprid (unexposed), resistant N. lugens exposed to 33.93 ppm (LC 50 ) and 1.18 ppm (LC 20 ) of imidacloprid, and susceptible N. lugens without exposure to imidacloprid. There were three applications administered to three consecutive generations. The insects were fed with the 6 d old seedlings previously dipped thoroughly in insecticide solution or water.
Preoviposition and oviposition experiment was conducted by placing a pair of newly formed adults into a test tube filled with 5-d old rice seedling. The seedling was replaced and examined daily to ensure the oviposition. The examination of preoviposition period was stopped when the first egg was laid, and right after the observation of the oviposition period was started.
Fecundity was examined by placing a pair of newly molted adults (1-d old) into a plastic cup (d = 10 cm, h = 11 cm) filled with 10 rice seedlings of 5-d old. The seedlings were replaced every four days. The number of eggs laid was examined by dissecting the leaves sheath toward the stem to count eggs mass under the dissecting microscope. The egg hatching percentage or eggs' viability was counted (Heinrichs et al., 1981).
The longevity of female adults was determined by placing a newly formed female adult into a test tube filled with a 5 d old seedling. The seedling was replaced every three days. The observation was ended when the insect died. Nymphal stages were determined by placing a newly hatched nymph into plastic cup having rice seedlings. Subsequently, the second instar that emerged from the first instar was placed into a new plastic cup. Procedure for observing the following stages were similar until the nymphs reached adults. If N. lugens dead during the test, they were substituted with N. lugens of the same age.

Study 2. Feeding capacity of Nilaparvata lugens
Feeding capacity of N. lugens was determined with ninhydrin method (Heinrichs et al., 1981) with modifications. The study was aimed to compare the feeding capacity of the imidacloprid exposed resistant N. lugens to the unexposed resistant ones. Three times exposure were made at 28, 53, and 78 days after planting (DAP), and each was replicated 10 times. Trays with the size of 50×50 cm were used for planting the seeds. The 14 DAP seedlings were removed from the trays and planted in the pots (d = 12 cm, h = 12 cm) and allowed to grow until 28, 53, and 78 DAP. The rice hill of 28 DAP were cut to leave only one stem in the pot. Every pot was enclosed with a plastic cup (d = 12 cm, h = 25 cm) equipped with a hole and cotton on its top to free the upper part of the stem, while keeping N. lugens inside the cup. The base of the cup was layered with plastic plate to allow the placement of filter paper and to prevent the moisture around the soil to enter the cup. A square opening of 4×4 cm was placed on the side of the cup and enclosed with gauze to allow air circulation. The surface of the stem was brushed with imidacloprid (33.93 and 1.18 ppm) or with water for the control (the unexposed resistant N. lugens and susceptible one). A Whatman filter paper (No 42, d = 12 cm) was placed around the lower stem. On every treatment five brachypteran female N. lugens adults (≤ 1 d old) were placed. These insects were previously starved for ± 3 h. After 24 h, the filter paper was retrieved, sorted according to the treatments and sprayed with 0.1% ninhydrin in aceton. The papers were then picked up with a pair of forceps and dried in oven for 5 min, at 100 0 C. Feeding capacity of N. lugens was observed by measuring the spots of amino acid (mm 2 ) left by the brachypteran females. The spots were the honey dew excretion of N. lugens containing amino acid which became purple after reacted with ninhydrin. The spots were measured with Leaf Area Meter (Heinrichs et al., 1981).

Calculation
The preoviposition period was calculated from the formation of female adult until its first oviposition. The oviposition period was determined from the first egg was laid until no more eggs were deposited. Fecundity was counted as the number of deposited eggs and unhatched ones by a female. Eggs viability represented the ratio between the number of new nymphs and deposited eggs. The life cycle of N. lugens counted since eggs were placed to the adults. The longevity of female adults was observed since the emergence of female adult to its death.

Data Analysis
LSD on 5% level was used to analyse every variable of fitness and feeding capacity. The significance test was performed when anova showed significant difference among treatments. The statistical analysis was conducted using Statistix 8.0.

The Fitness of Nilaparvata lugens
The application frequency of imidacloprid did not affect the fitness (preoviposition, oviposition, fecundity, female age, eggs viability, and life cycle) of N. lugens. However, there was a significant difference between resistant N. lugens (control), exposed resistant N. lugens, and susceptible N. lugens (Table 1.).
The application of imidacloprid affected all fitness variables including preoviposition, oviposition, female age, fecundity, and eggs viability of N. lugens. Fecundity of resistant N. lugens exposed to imidacloprid was significantly higher compared to the unexposed resistant one for all applications (Table 1). This suggest that imidacloprid increase the fecundity of resistant females. Chelliah and Heinrichs (1984) explained that reproduction rate of N. lugens raised when they were fed with rice plants sprayed with deltamethrin, methylparathion, cypermethrin, and fenvalerate. Furthermore, the increase of N. lugens reproduction was also enhanced by the increase of nutrition consumption capacity due to the sublethal treatment of insecticide. Basit et al. (2012) said that insect resistant to insecticide influences other fitness component such as longevity, development, fecundity, and fertility. According to Moriarty (1969), the sublethal dose of insecticide affected insect population by altering their capacity to live, reproduce, and the genetic materials of the survival generation. The application of triazophos and fenvalerate on sublethal concentration increased the reproduction of female N. lugens (Bao et al., 2009). The increase of reproductive capacity happens through the stimulation of fecundity (Yin et al., 2008). Cutler et al. (2009) reported that the sublethal concentration of imidacloprid and azadirachtin stimulated the reproduction of Myzuz persicae. Wang et al. (2005) informed that imidacloprid applied to first instar larvae of Tryporyza incertulas increased the fecundity of its adult, due to the increase of juvenil III hormone on female adult. Both studies showed a lesser fitness than the susceptible population of N. lugens.
In general, the application of imidacloprid to the resistant N. lugens prolonged the period of egg and nymph stages. Both resistant N. lugens (exposed and non exposed to imidacloprid) had longer egg and nymphl period compared to the susceptible N. lugens (Table 2). This might be due to the lesser fitness level of resistant N. lugens compared to susceptible N. lugens which was better in fitness. Liu and Han (2006) found that the fitness of imidacloprid resistant N. lugens can drop to the initial state if the use of imidacloprid is stopped. Abbas et al. (2012) reported that the fitness of imidacloprid resistant Spodoptera litura was far below the susceptible one, in term of the length of larvae, pupae and adult stadia. Kliot and Ghanim (2012) mentioned that the population of unselected insects could develop very fast, while the fitness of selected population was very low in term of larval longevity, the number of molted adults, and the rate of copulation, fecundity, and hatching rate. All of these were significantly lower which caused a bigger fitness cost.

Feeding capacity of Nilaparvata lugens
The frequency of 1 st , 2 nd , and 3 rd application of imidacloprid application did not affect feeding capacity of N. lugens. However, its feeding capacity was affected by the kind of treatments used (Table 3). Feeding capacity of imidacloprid exposed N. lugens (LC 50 dan LC 20 ) was higher than to the unexposed resistant N. lugens, but the feeding capacity of these two populations was less than the susceptible population. Elsa et al. (2003) suggested that feeding capacity was an important factor that influenced N. lugens fecundity which determined whether pest resurgence will happen or not. Kumar and Champan (1984) reported that application of LC 50 like permethrin and fenvalerate decreased the habit and feeding activity. Increasing the feeding capacity of the resistant population when they were treated with imidacloprid might have contributed to the increase of their fecundity as indicated in the previous linkings/findings: Together which the fact the field population of N. lugens has developed resistance and continuations use of imidacloprid would result increase probability of outbreak.  Table 1. The effect of imidacloprid to the fitness of Nilaparvata lugens Note: A pair of brachypteric Nilaparvata lugens was released two days after imidacloprid application. Means in each colum for each application followed by similar letter is not significantly defferent (LSD; α = 0.05). d = day

CONCLUSION
Although the fitness and feeding capacity of imidacloprid-resistant N. lugens were less than the susceptible population, application of imidacloprid at sublethal cocentration to the resistant population increased their fitness and feeding capacity. These effects could contribute to the outbreak of N. lugens. In other words, the probability of outbreak is higher when imidacloprid is applied to field population of N. lugens.

ACKNOWLEDGEMENT
We are thank to the Nuffic project-PT MDF. Pacific-Indonesia for providing the scholarship for pursuing doctoral program. Thanks also to Mr. Sriyanto and Mr. Tugimin for technical assistance.