Toxicity and Sublethal Effects of Lambda-Cyhalothrin Insecticide on Parent and Filial Generations of Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae)

1. Introduction

The 28-spotted ladybug Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae) is a crop pest that occurs widely throughout China, and elsewhere in countries such as Korea, Japan, Russia, and Australia. This ladybug is a phytophagous pest that feeds mainly on crops such as Solanaceae, Leguminosae, Cruciferae, and Cucurbitaceae, with adults and larvae eating the tender leaves and stems [1,2]. When leaves are eaten, a semi-transparent parallel-shaped depression is formed, and in severe cases, only leaf veins and the epidermis remain, causing the leaves to wilt, yellow, and die. China is an important producer of potato—a globally important food crop—and H. vigintioctomaculata seriously affects potato agriculture [2,3,4]. Changes in microclimates and promotion of intercropping of corn, vegetables, and potatoes have also led to H. vigintioctomaculata becoming both an increasingly serious pest in potato plantations in Yunnan, Guizhou, and Sichuan regions, and a main spreader of potato brown spot disease, which also poses a threat to local potato production. In severe cases, yield can be reduced by 50% [5,6,7].

The insecticide lambda-cyhalothrin is highly efficient at controlling leaf-eating pests [8]. After spraying, insecticide residue in general gradually decreases over time, and with crop growth [9]. The physiology and behavior of some pests exposed to insecticides at sublethal doses may change [10,11]. Most sublethal insecticide doses have inhibitory or delayed effects on pest populations, but some can also stimulate growth and development, and promote pest proliferation [12]. These sublethal effects on pests can lead to physiological and biochemical changes and insecticide resistance. Therefore, understanding the sublethal effects of insecticides on pests is important to evaluate their efficacy and assess risks associated with their use [13]. Pests can reduce the effects of insecticides by regulating detoxification enzyme activities [14]. Among enzymes, glutathione S-transferase (GST), carboxylesterase (CarE), and multifunctional oxidase (MFO) are the most important. Low concentrations of insecticides or plant secondary metabolites can induce or inhibit the activity of various enzymes, thereby affecting pest metabolic processes, promoting resistance and providing continuous selection pressure, reducing the effectiveness of insecticides for pest control [15,16].

While the median lethal concentration (LC₅₀) or median lethal dose (LD₅₀) have been commonly used to evaluate the effects of insecticides on pests, they only report the response to insecticides at certain developmental stages [17]. Life tables can be used to more comprehensively analyze the effects of pesticides on pests at the population level. Compared with traditional life tables, an age–age bisexual life table increases statistics for males, accurately describes age differentiation of insects, differentiates between pre- and total-oviposition periods of adults, and can comprehensively describe changes in the entire population [18,19,20]. Beta-cypermethrin, phoxim, and abamectin pesticides are mostly used to control potato ladybird adults [4].While the sublethal effects of insecticides on target species are being increasingly explored, the effects of lambda-cyhalothrin lethal concentration (LC) on growth, development, and fecundity of H. vigintioctomaculata are imperfectly known. We first determine the sublethal concentration (SLC) of lambda-cyhalothrin on the F₀ generation, and its effect on the F₁ generation at SLCs. We construct an age–instar bisexual life table for the F₁ population and compare changes in parameters such as developmental duration, survival rate, fecundity, and longevity at different lambda-cyhalothrin concentrations. We also report detoxifying enzyme activities after SLC treatment of F₀ adults. Results provide a theoretical basis for a comprehensive assessment of the potential of lambda-cyhalothrin to control H. vigintioctomaculata, and for more informed application of this insecticide.

2. Materials and Methods

2.1. Insect Rearing and Insecticide

Henosepilachna vigintioctomaculata were collected from Luliang County, Qujing City potato fields, Yunnan Province (103°48ˊ52.02˝E, 25°17ˊ20.74˝N, altitude 1878 m) in September 2023. Insects were placed in an insect-feeding cage (50 × 50 × 50 cm) at room temperature. Potted potato (Yunshu 108) grown at (25 ± 1) °C, relative humidity 70% ± 5%, and a photoperiod 16:8 h L/D were cultivated as a host; fresh leaves were cut to feed ladybugs when plants had grown to 15–20 cm. Before experimentation, adult ladybugs were reared for at least three generations with no exposure to insecticide. Three-day-old adults were used as the initial insect source. Insect feeding conditions were consistent with potato cultivation conditions.

2.2. Insecticide

The stock insecticide solution was 2.5% lambda-cyhalothrin WG (Henan Yongguan Qiaodi Agricultural Science and Technology Co., Ltd.). Multifunctional oxidase (MFO) activity detection kits (A162-1-1), Carboxylesterase (CarE) activity detection kits (A133-1-1) and Glutathione S-transferase (GSH-ST) activity detection kits (A004-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute Co., Ltd.

2.3. Determining Biological Activity

The sensitivity of H. vigintioctomaculata to lambda-cyhalothrin was determined using a leaf film method [21]. Five lambda-cyhalothrin treatments (1.25, 1.00, 0.75, 0.50, and 0.25 mg L⁻¹) were initially established; water was used as a control. Each treatment contained 30 × 3-d-old similarly sized adult ladybugs, and was replicated three times. Before experimentation, adult ladybugs were starved for 48 h. Potato leaves were immersed in each treatment concentration for 20 s, then removed, placed in an insect box (15 × 12 × 5 cm), dried, and their petiole wrapped with water-soaked cotton wool. Ladybugs were then added. Ladybug mortality was recorded after 48 h of treatment. Feeding conditions were the same as in section 2.1. Ladybugs were pronounced dead if touched by a brush and no reaction occurred. A virulence regression curve, 95% confidence intervals, chi-square values (χ²), and degrees of freedom (df) were calculated for LC₁₀, LC₂₀, and LC₄₀ concentrations.

2.4. Effects of Lambda-cyhalothrin Sublethal Treatment on F₀ and F₁ H. vigintioctomaculata

Potato leaves were immersed in LC₁₀, LC₂₀, and LC₄₀ concentrations for 20 s, then removed and dried; water was used as a control. Sixty healthy 3-d-old similarly sized male and female adults (1:1, morphological identification) were fed potato leaves and reared in an artificial climate box. After 48 h of exposure to leaves immersed in different concentrations of insecticide (without changing leaves), surviving H. vigintioctomaculata were removed, paired male and female, placed in a Petri dish, and fed fresh, untreated potato leaves. The initial stage of the F₀ generation experiment involved approximately 30 individuals, with three biological replicates. At least 15 pairs of ladybug from each insecticide treatment were paired; fresh potato leaves were replaced daily in the insect box. The oviposition date, oviposition amount, and adult longevity of single females were observed regularly (8:00 and 18:00 h). If a male died before a female, it was replaced. We refer to adult contemporary H. vigintioctomaculata treated with SLCs of lambda-cyhalothrin as parent generation (F₀), and offspring produced by natural mating of these parents filial generation (F₁) [2].

Eggs (90, oviposition < 5 h) laid on the same day by an F₀ female in each lambda-cyhalothrin treatment and the control group were used as the F₁ generation. These eggs were placed into circular plastic, numbered Petri dishes (diameter 60 mm) with moist filter paper, and then moved to an artificial climate box for hatching, where hatching rate was determined. Feeding conditions were the same as in section 2.1. Individual newly hatched F₁ generation larvae treated with different insecticide concentrations were inoculated separately into Petri dishes for feeding. Fresh potato leaves were placed into Petri dishes, and ladybug development was monitored every 24 h. After 24 h of pupation, pupae were weighed, then transferred into individual centrifuge tubes sealed with absorbent cotton, and returned to the climate chamber. After adult eclosion, pupal duration was determined. For each insecticide concentration treatment, female and male adults that emerged on the same day were randomly selected and paired 1:1 and placed into new culture dishes. A wet cotton ball was placed in the dish to provide moisture, and fresh leaves were replaced the next day. Test insects ate and deposited eggs. The number of eggs laid by a single female and the longevity of adult females and males were observed daily (8:00 and 18:00 h) until all adults had died. The number of eggs hatched / the total number of eggs laid by females represents the egg-hatching rate. The time from newly hatched larvae to pupation represents the larval development period, and the time from pupation to adult eclosion represents the pupal development period. The percentage of the number of pupae that could break the shell and become adults divided by the total number of pupae × 100 represents the eclosion rate.

2.5. Determining Detoxifying Enzyme Activities

Detoxifying enzyme activities were determined using the method of Jiang et al [22]. F₀ generation adult ladybugs were fed fresh leaves treated with LC₁₀, LC₂₀, and LC₄₀ concentrations of lambda-cyhalothrin, with four replicates per treatment. After 48 h, surviving adults were collected and placed into centrifuge tubes. Each replicate contained 10 individuals, frozen in liquid nitrogen and stored at −80 °C for enzyme activity determination. Ten similarly sized adults were selected from each concentration treatment. PBS (pH 7.4) was added to an ice bath homogenate at a weight (g): volume (mL) ratio of 1:9; the homogenate was centrifuged at 4 °C and 12,000 r/min for 30 min. The supernatant was recovered to determine GST, CarE, and MFO enzyme activities, and protein concentration, following kit instructions.

2.6. Data Analysis

Probability unit analysis was used to analyze the toxicity regression equation of lambda-cyhalothrin using SPSS 26.0 (IBM Co., Ltd., Armonk, NY, USA); SLCs were determined. One way-ANOVA was used on data related to detoxification enzymes. A Tukey’s multiple range test was used for multiple comparisons, and Student’s t tests were used to identify significant differences between treatments in pairwise comparisons. Original growth and development, survival rate, and fecundity data for the F₁ generation were collated and imported into Twosex-MSChart software (v 5/7/2024) to calculate developmental duration, adult longevity, adult preoviposition period (APOP), total preoviposition period (TPOP), fecundity, net reproductive rate (R₀), finite rate of increase (λ), intrinsic rate of increase (r), and average generation cycle (T) for each stage [23,24,25,26]. All variances and standard errors were obtained by using 100,000 random sampling bootstraps, and differences for each parameter in insecticide treatments were evaluated by paired bootstrap tests [27,28]. Plots were generated using SigmaPlot 14.0. Specific calculation formulas are:

$$l_x={\displaystyle \sum _{j=1}^{\beta }{s}_{xj}}$$

(1)

$$M_x={\displaystyle \frac{{{\displaystyle \sum }}_{j=1}^{\beta }{S}_{xj}{f}_{xj}}{{{\displaystyle \sum }}_{j=1}^{\beta }{S}_{xj}}}$$

(2)

$$R_0={\displaystyle \sum _{x=0}^{\infty }{l}_x{m}_x}$$

(3)

$${\displaystyle \sum _{x=0}^{\infty }{e}^{-r(x+1)}{l}_x{m}_x}=1$$

(4)

λ = e^r

(5)

$$T={\displaystyle \frac{\ln (R_0)}{r}}$$

(6)

3. Results

3.1. Toxicity of Lambda-cyhalothrin to Henosepilachna vigintioctomaculata Adults

After 48 h treatment, LC₁₀, LC₂₀, and LC₄₀ values for lambda-cyhalothrin exposure to H. vigintioctomaculata adults were 0.193, 0.251 and 0.355 mg L⁻¹, respectively. Three concentration values overlapped with other 95% CL values for subsequent experiments (Table 1).

3.2. Effect of Sublethal Concentration of Lambda-cyhalothrin on Longevity and Fecundity of F₀ H. vigintioctomaculata

LC₁₀, LC₂₀, and LC₄₀ concentrations of lambda-cyhalothrin significantly affected the longevity and average fecundity of F₀ adults (Table 2). Compared with the control group, the higher the concentration of lambda-cyhalothrin, the shorter the adult life span (female and male), with the life span of females being slightly longer. After exposure to lambda-cyhalothrin concentrations (LC₁₀, LC₂₀, and LC₄₀), individual egg production and F₁ egg hatching rates also significantly decreased compared with the control group.

3.3. Effect of Sublethal Concentration of Lambda-cyhalothrin on Growth, Development, Fecundity, and Pupal Weight of F₁ H. vigintioctomaculata

LC₁₀, LC₂₀, and LC₄₀ concentrations of lambda-cyhalothrin affected each F₁ generation developmental stage (Table 3). Compared with the control, the duration of egg, larval, and pupal stages in the LC₁₀ and LC₂₀ treatment groups differed significantly (P < 0.05); the lifespan of male and female adults in the LC₄₀ concentration treatment group was 16.57% and 19.76% significantly shorter than control group adults. APOP and TPOP were prolonged with increase lambda-cyhalothrin concentration (control group APOP and TPOP durations were the shortest (6.20 d and 23.53 d), and those in the LC₄₀ treatment were the longest (7.10 d and 30.45 d, respectively). Compared with other treatments, the numbers of eggs laid by females exposed to the LC₄₀ concentration was lowest (13.40 eggs/female); the number of eggs laid by females in the control group (55.59 eggs/female) was 1.13×, 1.70×, and 4.15× that of LC₁₀, LC₂₀, and LC₄₀ treatments, respectively. Additionally, compared with controls, the pupal weight and adult emergence rate of the F₁ generation were also significantly lower in the LC₁₀, LC₂₀, and LC₄₀ treatments (P < 0.05), with the effect stronger with increased insecticide concentration (Fig. 1, Table 3).

3.4. Population Parameters

Compared with the control, LC₁₀, LC₂₀, and LC₄₀ treatments significantly affected F₁ generation life table parameters. Values for r, λ, and R₀ of the F₁ population decreased with increased lambda-cyhalothrin concentration, and T increased (Table 4).

3.5. Age–Stage Specific Maternity

At each concentration the l_x curves follow a similar trend: there is a period of stability, then gradual, then steep decline (Fig. 2), indicating that death of individuals in the H. vigintioctomaculata F₁ population mainly occurred in later stages. The f_x curves are more variable, indicating greater variation in the emergence and oviposition of female adults, resulting in the curve appearing high and low. For LC₁₀, LC₂₀ and LC₄₀ lambda-cyhalothrin concentrations, peak adult female f_x values were 13.77 (37 d), 14.11 (55 d), and 8.46 (58 d) (Fig. 2). The peak number of eggs of f_x in the control group was greatest (15.62 eggs d⁻¹). Over time, the age-specific survival rate l_x of the control group trended down. The m_x curve indicated that the control group began reproducing at 33 d, whereas in other concentrations reproduction began 2–11 d later. Multiple peaks appear in the mx curve, indicating changes in individual spawning periods. The age-specific oviposition rate l_xm_x in LC₄₀-treated individuals decreased sharply, from 5.00 individuals d⁻¹ in the control group to 1.70 individuals d⁻¹ (Fig. 2).

3.6. Effect of Lambda-cyhalothrin Exposure on Detoxifying Enzyme Activity

Detoxifying enzyme activities in adult H. vigintioctomaculata treated with SLCs of lambda-cyhalothrin trended upwards (Fig. 3). After 48 h exposure at LC₂₀ and LC₄₀ concentrations, female GST activities decreased significantly by 28.88% and 44.88% compared with control values, and male activities decreased significantly by 39.39% and 44.95%, respectively (P < 0.05); at LC₁₀ there was an increase in GST activity. CarE activity first increased, then decreased with increased lambda-cyhalothrin concentration; enzyme activities in the LC₄₀ treatment were significantly lower than those in either other treatment (P < 0.05). With increased treatment concentration, MFO activity was inhibited. Additionally, the GST activity of adults in the LC₂₀ treatment and CarE activity in the LC₁₀ and LC₄₀ treatments differed significantly (P < 0.05).

4. Discussion

Insecticides are used extensively in agriculture, and they contribute to the development of pest resistance [17]. Integrated pest-management programs underscore the importance and necessity of moderate insecticide use [29]. Lambda-cyhalothrin is an efficient, broad-spectrum and quick-acting pyrethroid insecticide developed by ICI, UK. It has contact and stomach poisoning effects, and has no internal absorption effect. It is mainly used to control pests with chewing or piercing and sucking mouthparts [8,9,14]. Using a leaf-dip method we evaluate the bioactivity of lambda-cyhalothrin against adult H. vigintioctomaculata [4]. Our LC₄₀ value of 0.355 mg L⁻¹ indicates that this insecticide exhibits potent toxicity toward H. vigintioctomaculata. Chemical control is the main strategy to manage H. vigintioctomaculata. However, post-application in the field, insecticide toxicity generally decreases because of various abiotic environmental factors, producing SLCs [30]. This reduced toxicity can induce sublethal effects in certain pest individuals that may be passed on to subsequent generations. Sublethal effects may influence the growth, development, behavior, and reproductive capacity of target pests and their offspring, and change population dynamics [30,31,32].

Many studies have reported sublethal insecticide concentrations to exert significant inhibitory effects. For instance, treatment with chlorantraniliprole at LC₁₀ concentrations significantly reduces the survival rate and longevity of F₁ generations of Sogatella furcifera [33]. Studies on development in Cydia pomonella under lambda-cyhalothrin stress have yielded similar results [34]. We report SLCs of lambda-cyhalothrin (LC₁₀, LC₂₀, and LC₄₀) to significantly reduce the lifespan and average oviposition of the F₀ generation H. vigintioctomaculata compared with control group ladybugs (Table 2). Furthermore, exposure to LC₄₀ concentrations significantly reduced the F₁ population size, with delays observed in egg, larval, and pupal developmental duration, further indicative of sublethal effects (Table 3). These findings are consistent with those for exposure of Paracoccus marginatus to SLCs of spirotetramat, with extended nymphal periods from F₀ to F₂ generations and pre-adult delays [35]. We also report pupal weight—an important indicator of insect stress resistance and environmental adaptability—of F₁ generation H. vigintioctomaculata to decrease with increased SLC of lambda-cyhalothrin [36]. Leaves treated with LC₄₀ concentrations also showed negligible feeding damage, likely attributable to the antifeedant properties of lambda-cyhalothrin [37]. Reduced feeding reduced growth and development of H. vigintioctomaculata, and reduced body weight.

The impact of insecticides on pest fecundity manifests their sublethal effects [31]. The toxic excitatory response is influenced by various factors, with exposure duration (or generational exposure) being a key determinant [38]. Low doses of imidacloprid stimulate peach aphids, leading to increased methylation levels in the F₂ generation compared with the F₁ generation. This phenomenon may be linked to genetic adaptability induced by low-dose insecticide stress [39]. We report SLCs of lambda-cyhalothrin to also induce transgenerational effects in H. vigintioctomaculata. Specifically, the average oviposition, r, λ, and R₀ values of both F₀ and F₁ generations decreased with increased lambda-cyhalothrin concentration, limiting F₁ population growth (Table 2 and Table 3). A similar phenomenon is reported for pests such as Paracoccus marginatus, Aphis gossypii, and Rhizoglyphus robini [9,14,40]. These findings suggest that insecticide treatments can effectively slow transgenerational population growth of a variety of species. This inhibitory effect may be attributed to downregulation of gene expression associated with vitellogenin (Vg) and its receptor synthesis in females exposed to sublethal doses of insecticide. For example, the reduced expression of Vg and vitellogenin receptor (VgR) genes leads to decreases in respective contents [40,41,42]. Furthermore, age–stage specific maternity curves (Figure 2) indicate that lambda-cyhalothrin stress significantly suppresses the F₁ generation reproductive capacity, with the effect increasing at higher concentrations. Interestingly, many insecticides (e.g., triazophos, thiamethoxam) regulate pest fecundity by modulating expression of Vg and VgR through the TOR protein kinase and juvenile hormone signaling pathways [43,44]. Whether lambda-cyhalothrin similarly regulates the reproductive capacity of H. vigintioctomaculata adults via these pathways is unknown.

The reduced sensitivity of insects to insecticides typically involves multiple mechanisms, with the most prominent being the regulation of insecticide metabolism through detoxifying enzymes [45]. These enzymes play important roles in the metabolic processing of chemicals in insects. Insecticides can induce or inhibit their activity, and by exerting continuous selective pressure, drive the evolution of insecticide resistance [46]. Insecticides can affect various population parameters in insects such as developmental periods, reproductive capacity, and adult longevity. Additionally, they can influence the activity of detoxifying enzymes [47]. Different insecticides or plant secondary metabolites have distinct inhibitory or inducing effects on detoxifying enzyme activities in the same pest species [31]. For example, after 24 h of spirotetramat exposure, low concentrations induced a significant increase in GST and CarE activity in Bradysia odoriphaga [48]. Similarly, treatment with lambda-cyhalothrin significantly increased GST activity in Cydia pomonella, but suppressed CarE activity [49]. We report that after 48 h of exposure to lambda-cyhalothrin, LC₁₀ concentrations induced an increase in both GST and CarE activities in adult H. vigintioctomaculata. At LC₂₀, the CarE activity in adult males and females was significantly higher than in the control group, but lower than in the LC₁₀ treatment group, possibly because of the lower insecticide dose (where CarE was activated to participate in insecticide metabolism). Additionally, the GST activity of adults in the LC₂₀ treatment and CarE activity in the LC₁₀ and LC₄₀ treatments differed significantly (P < 0.05). Similar GST and CarE activity results were reported by Kinareikina [50]. At increased dosage, enzyme activity was progressively suppressed. The degree of MFO activity correlated positively with insecticide dosage. At low lambda-cyhalothrin concentrations the activities of CarE and GST in H. vigintioctomaculata were induced, but MFO activity was significantly reduced. Further molecular-level studies on higher resistance strains are required to elucidate the underlying mechanisms of resistance to lambda-cyhalothrin.

After exposure of adult H. vigintioctomaculata to SLCs of lambda-cyhalothrin in the F₀ generation, F₁ population growth was significantly reduced, and the degree of reduction increased with increased insecticide concentration. This suggests that field application of lambda-cyhalothrin exerts transgenerational effects on H. vigintioctomaculata. In agricultural practice, the rational use of lambda-cyhalothrin to mitigate damage caused by H. vigintioctomaculata to crops is viable. However, the dosage and frequency of application must be managed to avoid enhancing pest fitness or promoting resistance through prolonged use of a single class of insecticide. Furthermore, the implementation of insecticide rotation—using two or more insecticides with different modes of action—could delay development of resistance and support sustainable pest management strategies.

5. Conclusions

As a new insecticide with high efficiency, low toxicity, and low residue, lambda-cyhalothrin has high insecticidal activity against Coleoptera pests. We report the effects of SLCs of this insecticide on the growth and development, longevity and fecundity of H. vigintioctomaculata using an age–age bisexual life table. Compared with the control, male and female longevity and fecundity all decreased, and other life table parameters deceased with increased SLC. At low concentrations this insecticide also increases the activity of detoxifying metabolic enzymes. Field application of lambda-cyhalothrin has a cross-generational effect on H. vigintioctomaculata that may affect F₁ generations. This provides a reference for how to improve evaluation of the effects of insecticides.