COMPARISON OF LIFE CYCLE TRAITS OF A heloPeltis theiVoRa WATERHOUSE ( HETEROPTERA : MIRIDAE ) POPULATION INFESTING ORGANIC AND CONVENTIONAL TEA PLANTATIONS , WITH EMPHASIS ON DELTAMETHRIN RESISTANCE

The tea mosquito bug, Helopeltis theivora (Waterhouse), is an important economic pest of tea in India. The development of resistance in H. theivora populations obtained from a conventional plantation as compared with a strain from an organic plantation was studied in the laboratory for five generations, and associated changes in life cycle traits were assessed. Selection using sublethal concentrations of deltamethrin resulted in a 5.19-fold increase in insecticide resistance ratio from the F1 to the F5 generation in the H. theivora population from the conventional plantation. By the F5 generation, nymphal duration and total developmental duration and adult longevity were higher and fecundity was lower in the deltamethrin-selected strain than in the non-selected strain from a conventional plantation and the susceptible strain from an organic plantation. These findings have practical implications for insecticide resistance management of this important sucking pest of tea.


INTRODUCTION
The tea mosquito bug, Helopeltis theivora (Waterhouse) (Heteroptera: Miridae), is one of the most important sucking pests of tea responsible for increasing economic losses in tea production (Gurusubramanian et al., 2008;Roy et al., 2009aRoy et al., , 2010aRoy et al., ,b, 2011)).In recent years, destructive outbreaks of H. theivora have been increasing in northeast India due to change in climate and a deteriorating ecological environment (Grusubramainan et al., 2008;Mukhopadhyay and Roy, 2009).Insecticide resistance was an important factor contributing to the outbreaks (Gurusubramanian and Bora, 2008;Gurusubramanian et al., 2009;Roy et al. 2009a).
For over two decades, synthetic pyrethroid insecticides have been widely used by planters because of their efficacy in controlling a wide range of tea pests at low doses and at extremely low cost (Gurusubramanian et al., 2008).The use pattern of insecticides in tea was 5.21-7.49l/ha, of which synthetic pyrethroids represent 36.6% (Sannigrahi and Talukdar, 2003;Roy et al., 2008), deltamethrin and cypermethrin being the most popular.In the regions surveyed, an average of 7-12 times of insecticide applications to tea are common (Roy et al., 2008).Of the synthetic pyrethroids, deltamethrin is the most widely used insecticide.Hence, deltamethrin was chosen as representative molecule to monitor resistance to synthetic pyrethroid insecticides in tea against H. theivora.An area-wide survey evidenced the presence of resistant genes among field-sampled populations in 2007, while first treatment failures occurred in some tea fields in Dooars during 2005 (Roy et al., 2009a).Variations in the relative toxicity of commonly used insecticides have been observed in H. theivora populations from Jorhat, Assam (Gurusubramanian and Bora, 2008), Darjeeling (Bora et al., 2007), and the sub-Himalayan Dooars region of India (Roy et al., 2009a(Roy et al., , 2011)).
In addition, qualitative and quantitative changes could be recorded in the enzyme patterns of insecticide-exposed H. theivora, due to formation of greater amounts of esterases (Sarker and Mukhopadhyay, 2003), glutathione S-transferase and acetylcholinesterase (Sarker and Mukhopadhyay, 2006a, b), indicating an adaptation for higher insecticide tolerance.Though the distribution of this pest is ubiquitous in all tea growing regions of India, no work has been undertaken to assess the cumulative resistance acquired by the pest through generations against the most commonly used pesticide, deltamethrin.Resistance to insecticides has resulted in changes in the biological characteristics of the strains of different species (Bhatia andPradhan, 1968 and1971;Saxena and Bhatia, 1980;Senapati and Satpathi, 1981;Campanhola et al., 1991;Yamada et al., 1993).The assessment of changes in the biological characteristics of populations that are resistant to insecticides is very important in resistance management programs (Kumar and Kumar, 1997).Most resistance management tactics involve the reduction of fitness of resistant genotypes relative to susceptible genotypes by either preserving susceptible homozygotes or eliminating heterozygotes and resistance homozygotes (Leeper et al., 1986).A need was therefore felt to quantify the potential of development of resistance to the synthetic organic pesticide, deltamethrin (the most commonly used insecticide against the pest in tea), and to compare the fitness costs in this pest under three situations, i.e. (i) deltamethrin selected strains (D), (ii) a strain derived from conventional tea plantation but non-selected (C), and (iii) a susceptible strain derived from population of organic tea plantation (S) in their 5 th generation.

Development of resistance to deltamethrin
The laboratory culture of H. theivora was initiated with about 500 nymphs collected during the first week of September 2010 from tea plantations of the Dooars, where the tea bushes were managed by conventional insecticide treatment.The nymphs were kept in the laboratory at 26 ± 2°C in a BOD incubator and were provided with tealeaves of a TV1 (Tocklai variant) clone.The leaves were changed every 24 h until the insects attained the adult stage.After one generation, the stock was divided into two groups and named D and C for laboratory testing.While group C was maintained without any deltamethrin treatment for five generations, group D was subjected to sublethal treatment (laboratory screening with deltamethrin) through 5 generations (the selection procedure being described later on).
The biological parameter of the specimen (strains) obtained in the 5 th generation from the groups D and C were compared with each other as well as with the specimen obtained in the 5 th generation of the stock brought from the organic plantation (Makibari tea estate) that had never been treated with synthetic pesticide (Roy et al., 2010a ).The later stock was taken to be a susceptible strain -S (Fig. 1).

Bioassay and laboratory selection
Serial dilutions of the insecticide deltamethrin (Decis 2.8 EC; Bayer Crop Science Ltd., Mumbai, India) were prepared in distilled water from 1% stock solution.Toxicity assays were conducted as per the standard 'Leaf Dipped Method' recommended by FAO Method No. 10a (FAO 1980).Healthy shoots of TV 1 clones (three leaves and a bud) were collected from the experimental garden and brought to the laboratory.The leaves were washed thoroughly with distilled water and air-dried.Fifteen tea shoots for each treatment were dipped for five seconds in the pesticide solutions to ensure complete wetting and then kept in glass vials (2 cm long X 2 cm diameter) containing water with their bases wrapped in cotton.The treated tea shoots were kept under ceiling fans for 15 min for the emulsion to evaporate.The glass vials along with the treated shoots were placed in Petri dishes (10 cm diameter) toweled with blotting paper.The whole arrangement was covered with a hurricane lamp glass chimney (12.5 cm wide mouth, base opening of 9.0 cm, and 18.0 cm height) with its mouth covered with muslin cloth.This whole set-up was kept in a BOD incubator (26 ± 2°C; 12: 12 L/D period and 80% humidity) in a culture room.
Dose selection is a very important element in each insecticide bioassay test.More than five preliminary tests were done to find the proper doses.The process of dose selection was based on Robertson et al. (1984) and Robertson and Preisler (1991).
Based on the results of preliminary tests, 30 insects (15males and 15 females) were exposed to each concentration of deltamethrin.Six concentrations of deltamethrin were tested to obtain a concentrationprobit mortality curve.A set of controls (with water only) was also maintained with each exposure to work out the corrected mortalities.The survivors obtained at higher concentrations (>LC 80 ) were shifted to clean rearing glass vials and provided with fresh TV1 tealeaves.The progeny of the first surviving lot was designated the F1 generation.The deltamethrin treatments and selections were continued to the 5 th generation.

Biological studies
Thirty newly emerged 5 th generation adults of the three strains, D, C and S, were kept separately in three groups inside hurricane lamp glass chimneys with the mouth covered with a nylon mesh for aeration.Two to three healthy TV1 tea shoots (with three leaves and a bud) were kept with their bases immersed in water-filled glass vials (2cm long x 2 cm diameter) through a cotton plug.The vials were placed in a Petri dish (12 cm diameter) with paper toweling.The whole set-up was covered by the previously mentioned glass chimney and maintained in the laboratory at 25±2 o C and 70-80% RH and 16: 10 L/D photoperiod.Three male and three female individuals of each strain were introduced into the glass chimney.Five such replications were studied in every strain.Mated females were allowed to oviposit in the shoots and the egg-laden shoots were removed.By using a strong magnifying glass, micropylar processes (in the form of hairs) of eggs could be seen sticking out from the plant surface where they had been inserted.The bases of the egg-laden twigs were kept immersed in glass tubes to avoid desiccation.Fresh tender shoots were maintained in the tube until the emergence of the nymphs.Water mixed with 0.1% carbendazim was added to prevent fungal growth on the shoots.Observations on percent hatching were recorded cumulatively up to 28 days (Gope and Handique, 1991;Roy et al., 2009b).Those eggs that did not hatch after this period were regarded as nonviable.Newly emerging first instar nymphs were transferred individually to a Petri dish (10 cm diameter) and daily provided with tender host shoots moistened with wet cotton around the petiole.Third instar nymphs (n=10) were reared in each glass chimney as mentioned.The shoots and the glass chimney were replaced every day with new ones.Nymphs were carefully removed from old shoots using a camel hairbrush and transferred to new shoots.This process continued until the emergence of adults.The life history traits, such as pre-oviposition period, oviposition period, fecundity, incubation period, hatchability, total nymphal duration, longevity of male and female and sex ratio were recorded from such a culture.

Statistical analysis of the data
The degree of development of resistance through different generations was determined by generating the LC 50 values in each generation using the mortality data.These data were converted to percent mortality and subjected to probit analysis (Finney, 1971), then computed for the resistance ratio.The resistance ratio for In none of the cases was the data found to be significantly heterogeneous at P =0.05.df, degrees of freedom; y, probit kill; x, log (concentration x 10 3 ); LC50, concentration required for 50% mortality.any generation was assessed by dividing LC 50 for that generation with the LC 50 value of the parental generation.Significant difference among the biological traits of the three strains was determined using ANOVA test.Means were compared by Tukey's multiple range test (p ≤0.05) (Snedecor and Cochran, 1989).

Deltamethrin selection
Helopeltis theivora were selected for five generations against various discriminating concentrations of deltamethrin to obtain maximum mortality at the highest concentration in each generation.In the F 1 generation, minimum survival of H. theivora (17%) was observed at the highest deltamethrin concentration used (0.35%) (Table 1).The tolerance of the bug to increasing concentrations of deltamethrin increased from the F 1 to the F 5 generation (Table 1).The LC 50 value for the F 1 generation of strain D of H. theivora, obtained from a conventional tea plantation in the Dooars, was 0.1921%.This increased progressively with generation to 0.9965% in the F5 generation.The insect's resistance to deltamethrin thus increased 5.19-fold within five generations (Table 2).Dosage-mortality response data for H. theivora in the successive generations, when subjected to a chi-square test, indicated a good fit of probit responses in all the bioassays, showing that there was no heterogeneity between observed and expected responses (Table 3).

Comparative biology
Fecundity, total nymphal duration and total developmental duration, were adversely affected in the deltamethrin-selected H. theivora strain (D).The susceptible form (S) showed a shorter oviposition period and a high rate of fecundity (157.34 eggs/ female), the fecundity in H. theivora strain derived from the conventional plantation but not selected (C) was 135.47 eggs/female, which was at par with the deltamethrin-selected strain (D), i.e. 122.96 eggs/ female/day (Table 3).
The D and C strains had a longer nymphal development period compared to the susceptible strain (S) (Table 3).Therefore, the prolongation of the overall developmental period of the resistant strains was mainly due to longer nymphal duration.Strains D and C had a significantly increased adult longevity (male -5.56-7.13days and female -11.84-13.97days) than S strain (Table 3).

Deltamethrin selection
The data explains the propensity of H. theivora to develop resistance with repeated exposure to deltamethrin when experimentally reared in laboratory on tea.Further, it was observed that the Dooars population of H. theivora was quick to develop resistance to deltamethrin within a few generations.Thus, an increase in the use of this group of insecticide for the control of the pest in question may lead to control failures.
Though several studies (Gurusubramanian and Bora, 2008;Bora et al., 2007, Gurusubramanian et al., 2009;Roy et al., 2009aRoy et al., , 2011) ) have indicated the development of various levels of resistance against chlorinated hydrocarbons in H. theivora, the present study upholds for the first time a generation-wise accrual and increase in resistance in this pest against deltamethrin.

Comparative biology
It is evident from the data that the selection pressure of deltamethrin resulted in considerable reduction of the egg-laying capacity of H. theivora (D strain) compared to the susceptible strain (S).The reduction in the rate of egg laying in the deltamethrin-challenged population might have resulted from a decreased amount of energy available for reproduction.The major share of the metabolic energy possible was allocated and utilized in developing biochemical and physiological defense related to detoxification of the insecticide (Price, 1974;Ribeiro et al., 2001).
It seems that the resistant forms of H. theivora, despite passing through a bottleneck during screening, developed certain lifecycle traits such as a longer nymphal developmental period, prolonged oviposition period and lower fecundity, in order to adapt to the low available metabolic energy level and withstand the stressed condition resulting from repeated pesticide exposures.Similar changes in lifecycle traits were reported by Yaqoob and Arora (2005) and Yaqoob et al. (2006) for endosulfan and carbaryl selected populations of Helicoverpa armigera, and Roy et al. (2010a) for endosulfan selected populations of H. theivora in Dooars tea plantation.It was established that the reduced fecundity in Epilachna sparsa resulted from metabolic resistance to insecticides (Senapati and Satpathi, 1981).Furthermore, it was advocated by various researchers that the females of H. armigera (Forrester et al., 1993;Glenn et al., 1994;Xia et al., 2001) and Plutella xylostella (Yamada et al., 1993), with changes in fecundity and oviposition periods, had a relatively higher tendency for developing resistance to insecticides.It was evident that deltamethrin use at a very high concentration can induce rapid change in the lifecycle traits, possibly leading to the development of biotype.In view of this, it is possible that the field recommended dose used for several decades for controlling H. theivora of the Dooars tea plantations has become sublethal, hence less effective.Therefore it could be suggested that the use of deltamethrin be either discontinued or the field recommended dose reviewed.The present findings have practical implications in the insecticide resistance management (IRM) of the major sucking pest of tea, H. theivora.

Fig. 1 .
Fig. 1.Schematic presentation of the strains of Helopeltis theivora used in the study of comparative biological traits.
± SE of ten individuals.Figures in parentheses represent range.Means followed by the same letter in a column are not significantly different (P < 0.0001) in Tukey's multiple comparison Test, NS, not significant.

Table 1 .
Screening of population of H. theivora through generations by using deltamethrin insecticide

Table 2 .
Toxicity of deltamethrin in different generations of a strain of Helopeltis theivora from a conventional tea plantation

Table 3 .
Biological attributes of F5 generation of deltamethrin-selected strain (D), conventional plantation strain (C) and susceptible organic plantation strain (S) of Helopeltis theivora