Fitness costs in clothianidin-resistant population of the melon aphid, Aphis gossypii

Clothianidin is a second-generation neonicotinoid insecticide, widely used against sap-sucking insect pest including melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae). This pest causes severe economic damage to Cucurbitaceae plants worldwide. In this study, we investigated clothianidin resistance development under continuous selection pressure. Moreover, the age-stage, two-sex life table approach was used to evaluate the impact of clothianidin resistance on the fitness of A. gossypii. A clothianidin resistant strain (CT-R) with a 23.17-fold resistance level was developed from a susceptible strain (CT-S) after continuous selection for 24 generations. Life table results showed a significant reduction in the relative fitness (0.847) of CT-R strain compared to the CT-S strain of A. gossypii. The developmental duration, oviposition days, total pre-oviposition period (TPOP), longevity, and fecundity of CT-R strain were found to be significantly lower when compared to CT-S strain. The demographic parameters, including the intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R0), and mean generation time (T) were also significantly decreased in CT-R strain compared to the CT-S strain. Both the reproductive and survival rates were affected by clothianidin resistance in CT-R strain compared with the CT-S strain of A. gossypii. Overall, our results demonstrate that in-depth knowledge about the trade-off at play between resistance degree and fitness cost might be useful to design resistance management strategies against A. gossypii.

Fitness cost affects insecticide resistance evolution and the rate of resistance increase in insects [41,42]. Resistance costs energy and that may influence fitness in the absence of the insecticide stressor [42]. Prior studies showed evidence about the fitness costs associated with insecticide resistance in several insects including Bradysia odoriphaga Yang and Zhang  [15,45]. Several studies have reported the development of insecticide resistance accompanied by fitness costs in different insect pests [15,[46][47][48][49].
To our knowledge, no study to date has examined selection-induced clothianidin resistance development accompanied with fitness costs in A. gossypii. The overall goal of this work was to analyze the risk of clothianidin resistance in A. gossypii under continuous selection pressure. To examine the impact of clothianidin resistance on fitness, we used the age-stage two-sex life table approach to accurately quantify the life history traits of resistant (CT-R) and susceptible (CT-S) strains of A. gossypii. This gives an in-depth knowledge about the optimal application of clothianidin insecticides against A. gossypii.

Insects and insecticide
Aphis gossypii were initially collected from melon plants at Weifang City, Shandong Province, China. The population was maintained under standard laboratory conditions (25 ± 1˚C; 75% RH; 16:8 L: D) at China Agricultural University using insecticide-free cucumber plants. Technical grade clothianidin (95% of the active ingredient) was purchased from Bayer CropScience Co. Ltd (Monheim, Germany). Triton X-100 was obtained from Sigma-Aldrich Co. Saint Louis, USA.

Toxicity of clothianidin against A. gossypii
The bioassays of clothianidin were conducted under laboratory conditions using the leaf-dipping procedure. The stock solution of clothianidin was prepared in acetone, and further dilution was set up in distilled water containing 0.05% (v/v) Triton X-100. Cucumber leaf discs were dipped in the required diluted concentrations of clothianidin or in 0.05% (v/v) Triton X-100 water as a control for 15 s. The treated discs were allowed to dry on the disposable transparent plastic gloves and then placed the adaxial side of leaf discs on 2% (w/v) agar bed (2 ml) in a 12-well cell culture plate. Twenty adult melon aphids were placed on each leaf disc, and Chinese art paper (Xuan rice paper) was used to cover the plate to prevent the aphid's escape. There were three leaf disks for each concentration, and the entire experiment was repeated three times for a total of 180 aphids tested at each concentration. The mortality was recorded at 72 h after treatment. The LC 50 values of clothianidin were calculated by probit analysis using POLO Plus 2.0 statistical software.

Establishing the resistant colony
The resistant strain of clothianidin (CT-R) was established from an originally collected susceptible population of A. gossypii through continuous selection pressure for 24 generations. The acute toxicity of clothianidin was recorded for each generation. Based on the results of the bioassays of the parent aphids, the clothianidin concentrations were gradually increased throughout the selection experiment. The mortality rate was maintained at 60-80%. The resistance ratio (RR) was determined at each generation by dividing the LC 50 of resistant strain to the LC 50 of the susceptible strain of A. gossypii. The susceptible strain (CT-S) was maintained on cucumber plants without any pre-or post-exposure of clothianidin. Both strains were kept under standard laboratory conditions (25 ± 1˚C; 75% RH; 16:8 L: D) in the Department of Entomology, China Agricultural University, Beijing, China.

Fitness comparisons
Fitness of the susceptible and resistant strains of A. gossypii was compared using age-stage, two-sex life table approach. About 500 apterous adults were inoculated to insecticide-free cucumber seedlings. After 24 h, ninety newly born nymphs of A. gossypii were collected from both susceptible and resistant populations. Both strains were transferred to insecticide-free cucumber seedlings and were maintained separately under laboratory conditions. Each individual aphid growing on one insecticide-free cucumber seedling was considered as a single replicate [8,19]. Nymphs from both populations were observed individually and we recorded development duration, mortality, longevity, and fecundity. The life table data of susceptible (CT-S) and resistant (CT-R) strains of A. gossypii were subjected to the TWOSEX-MSChart computer program [50] to analyze the population parameters including age-stage specific survival rates (s xj ), age-specific survival rate (l x ), age-specific fecundity (m x ), age-specific maternity (l x m x ), age-stage specific life expectancy (e xj ) and age-stage reproductive value (v xj ) following age-stage two-sex life table procedure [51]. The s xj shows the probability that a newly born nymph will survive to age x and stage j. The l x represents a simplified form of the survival history and the probability that a newly-born nymph will survive to age x. The m x indicates the age-specific fecundity, while the l x m x shows age-specific maturity. The v xj depicts the devotion to future offspring for A. gossypii individuals of at age x and stage j. The e xj describes the expected duration of time an individual of age x at stage j that will survive after the age x. The newly born nymphs produced by females during the reproductive period were counted and removed daily. Fresh cucumber seedlings were replaced after 5 days without any insecticide exposure throughout the experiment. The aphids were individually transferred to new seedlings using a soft brush.

Statistical analysis
The age-stage two-sex life table procedure was applied to analyze the developmental duration, adult longevity, and fecundity for all individual aphids using the TWOSEX-MSChart computer program [50][51][52][53]. The population parameters including the intrinsic rate of increase (r), finite rate of increase (λ), net reproductive rate (R 0 ), mean generation time (T), gross reproduction rate (GRR), adult pre-oviposition period (APOP), total pre-oviposition period (TPOP), oviposition days (Od), age-stage specific survival rates (s xj ), age-specific survival rate (l x ), age-specific fecundity (m x ), age-specific maternity (l x m x ), age-stage specific life expectancy (e xj ) and age-stage reproductive value (v xj ) were investigated following Chi and Liu [52] and Chi [53] using TWOSEX-MSChart computer program [50]. The means and standard errors of the population parameters between CT-S and CT-R strains were analyzed using paired bootstrap tests via 100,000 bootstrap replicates [51, 54,55]. All figures were constructed using Sig-maPlot 12.0 (Systat Software Inc., San Jose, CA, USA).

Clothianidin resistance development
The clothianidin resistant strain (CT-R) was established from the susceptible strain (CT-S) through continuous exposure with clothianidin for 24 generations under controlled conditions ( Table 1).
The LC 50 value of the CT-S was 0.38 mg L −1 . In the first 10 generations (F 2 -F 10 ) of the CT-R population, the LC 50 values were slowly increased from 0.52 mg L −1 to 2.23 mg L −1 . However, these values were steeply increased in the following generations (F 12 -F 24 ) with LC 50 values 2.79, 3.52, 4.47, 5.56, 6.78, 7.72, and 8.76 mg L −1 , respectively. After the selection

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Fitness costs in clothianidin-resistant population of the melon aphid, Aphis gossypii for 24 generations, the resistance ratio was increased (23.17-fold) compared to the CT-S strain ( Table 1).

Impact of clothianidin resistance on the life history traits of A. gossypii
The life-history traits, including developmental time, longevity, fecundity, and oviposition days between clothianidin resistant (CT-R) and susceptible strains (CT-S) of A. gossypii are presented in Table 2. The mean developmental durations of 1 st instar, 3 rd instar, and 4 th instar nymph of CT-R strain was significantly shorter than that of the CT-S aphids (P <0.001). The pre-adult period, adult duration, and total pre-oviposition period (TPOP) were significantly shorter in CT-R strain compared to the CT-S strain ( Table 2). No significant differences were observed for the adult pre-oviposition period (APOP) between both strains of A. gossypii. The oviposition days, total longevity and fecundity were significantly lower in CT-R strain of A. gossypii (P <0.05).
The demographic traits (r, λ, R 0 , T, and GRR) of CT-R and CT-S strains of A. gossypii were evaluated by a paired bootstrap technique based on the life table (Table 3). When compared to CT-S strain, the r and λ of CT-R strain were significantly increased (P <0.001). While the R 0 , T, and GRR in CT-R strain were markedly decreased as compared to the CT-S strain of A. gossypii (Table 3). In the absence of insecticide exposure, the overall fitness of clothianidin-resistant strain (CT-R) of A. gossypii was 0.847 as compared to the susceptible strain (CT-S).
The s xj shows the probability of neonate nymph that will survive to age x and stage j. The overlaps among different stages occurred due to the stage differentiations between CT-R and CT-S individuals (Fig 1). The s xj curves show apparent differences, with a lower survival rate of nymphal and adult female stages in CT-R strain compared to the CT-S strain. The adult female survival rate of CT-R strain started to decline on 10 th day, while decline occurred on 12th day in CT-S strain (Fig 1).
The l x , m x and l x m x differences among CT-R and CT-S strains are presented in Fig 2. The l x curves show a lower survival rate of CT-R strain at the age of 12-32 days compared to the CT-S strain of A. gossypii (Fig 2). The maximal survival duration of CT-R strain was 32 days,

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Fitness costs in clothianidin-resistant population of the melon aphid, Aphis gossypii which was lower than that of the CT-S strain (34 days). The m x and l x m x values in CT-R strain was decreased after 18 th day, which shows lower fecundity as compared to the CT-S strain (Fig 2). In the first 12 days, values of m x and l x m x for CT-R strain were higher as compared to CT-S strain; however, after that, this trend has been reversed, and CT-R strain displayed lower fecundity as compared to the CT-S strain (Fig 2). The e xj curves in CT-R strain show the shorter survival expectancy of the developmental as well as adult stage compared to the CT-S strain of A. gossypii (Fig 3). The v xj shows the devotion of individuals of age x and stage j towards future offspring (Fig 4). The pattern of v xj was recorded lower for the CT-R strain as compared to the CT-S of A. gossypii (Fig 4).

Discussion
Chemical applications are still crucial for the control of A. gossypii in China [6, [56][57][58]. Clothianidin belongs to second-generation neonicotinoid insecticide that acts as an agonist of the nicotinic acetylcholine receptors (nAChRs) [27]. Due to excellent insecticidal activity, clothianidin has been broadly used against many insect pests including hemipterans and many species of beetles [8,  [15,45]. Beet armyworm, Spodoptera exigua Hübner (Lepidoptera: Noctuidae) showed 69.76-and 113.29-fold resistance against deltamethrin and gossypol, respectively following 10 generations selections [63]. There have been numerous studies to investigate selection-induced resistance in many Table 3.

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Fitness costs in clothianidin-resistant population of the melon aphid, Aphis gossypii insect and insecticide combinations [64][65][66][67][68]. The resistance ratio was lower in CT-R strain of A. gossypii, as compared to other insects and insecticides. The difference might be due to differences in the field collected material, their prior exposure to insecticides, and the length of time the colony was maintained insecticide-free in the laboratory. The resistance ratio might be higher if the population exposed to clothianidin for more generations, which is a future prospect. Also, different insect species show different responses to insecticides. Fitness costs associated with resistance have been broadly studied in several insect species including B. odoriphaga, T. hawaiiensis, P. xylostella, N. lugens and M. domestica [15,41,45,[69][70][71]. In the current study, two strains with similar genetic backgrounds have been used to accurately assess the resistance-linked fitness costs [45]. The results have shown the shorter developmental duration of 1 st instar, 3 rd instar, and 4 th instar nymphs of CT-R strain as compared to the CT-S strain of A. gossypii. The pre-adult period of CT-R was also shorter than that  [44,72]. The developmental duration was also decreased in deltamethrin and gossypol resistant strains of S. exigua compared to susceptible strain [63]. In contrast, others have shown increased developmental durations in the resistant insect pests compared to the susceptible strain [71,73]. For example, the developmental period of B. odoriphaga significantly enhanced in clothianidin resistant strain [15].
In current study, the adult longevity, TPOP, oviposition period, total longevity, and fecundity were decreased significantly in CT-R strain. From the results, it is clear that the clothianidin resistance developed at the cost of the reduced fecundity. Overall these findings are in accordance with findings reported by Ullah et al. and Gul et al. that development of insecticide resistance affects the life-history traits, including fecundity and longevity of the resistant strain
The demographic traits can explain the growth potential of insect pest populations [79,80]. The r and λ were significantly increased in CT-R compared to the CT-S. However, the R 0 and T were decreased in clothianidin resistant strain of A. gossypii. The findings are directly in line with previous findings of Ma et al. showing that the r and λ were increased, while R 0 and T were decreased in sulfoxaflor-resistant A. gossypii [43]. The R 0 was lowered in the laboratory selected resistant strains of M. persicae and B. odoriphaga [15,44,45]. The demographic traits,

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Fitness costs in clothianidin-resistant population of the melon aphid, Aphis gossypii including r, λ, R 0 , and T were also affected by clothianidin and gossypol resistance in S. exigua [63].
The s xj , l x , m x , l x m x , e xj and v xj were significantly decreased in clothianidin resistant strain of A. gossypii. Our findings are consistent with Ullah et al. and Gul et al. showing similar results in clothianidin and chlorfenapyr resistant strain of B. odoriphaga [15,45]. Many prior reports have showed similar effects on several insect and insecticide combinations including sulfoxaflor-resistant A. gossypii [43], imidacloprid-resistant S. litura [75], deltamethrin and indoxacarb-resistant Heliothis virescens Fabricius (Lepidoptera:Noctuidae) [77] and spinosadresistant P. xylostella [81]. From the results, it is clear that A. gossypii has the potential to develop resistance against widely used clothianidin insecticide. Our study also provides a comprehensive understanding of the fitness costs in CT-R as compared to CT-S.