Triazophos induced lethal, sub-lethal and transgenerational effects on biological parameters and demographic traits of Pectinophora gossypiella using two sex life table

2.1 Field insect collection; colony preparation; and insecticide

Pectinophora gossypiella adults and larvae were collected from cotton fields located at Adda Peer Murad, District Vehari fields for colony preparation. Insects after collection from the fields were kept in cages with cotton bolls were provided to them for feeding and plant terminals held in water for egg laying in the colonies. Cotton plants in green houses were also grown for continuous food supply of insects. For this purpose, plastic pots of 6x10x20 cm were used in green house. Bolls from plants were collected and were brought to the laboratory. Insecticide free cotton bolls were kept for colony preparation. Temperature and relative humidity were 27 ± 1 °C and 70 % respectively. Adults were fed with 10 % honey solution. While adults for their egg laying were provided with voiles having plant terminals held in water as substrate for egg laying. Insects were reared for 2 generations in the laboratory. For bioassay purpose, triazophos (Jaffer agro services) 40 % EC having active ingredients of 10 % per liter purchased from local market in package of 1000 ml.

2.2 Bioassay for lethal concentration of insecticide

This experiment was conducted to investigate the direct toxicity of triazophos on insect biology. Bioassay of newly hatched larvae (hatching within 12 h) was conducted by feeding them on insecticide treated bolls. Bolls was cut diagonal sections to make it convenient for larvae to feed on it. Pre-liminary range finding dilutions (0.050–10 mg/L) was already conducted to get a series of concentrations which provided mortality of 0–80 %. Dilutions of 0,1,2,3,4,5,6 mg/L were prepared and already diagonally cut bolls were dipped in insecticides and made it dry for 20 min on towels at room temperature. Same procedure as Gao et al. (2018) leaf-dip bioassay with modifications was conducted. Bioassay was conducted for each instar of larvae. One larva per vile was tested and 80 larvae per instar for each replicate was tested. For each treatment total 240 larvae in 3 replications were used at each dose. Each treatment was repeated 3 times and mortality was observed after 24 h till 7 days. Individuals not showing movement by touching the brush were considered as dead.

For F₁ generation, bioassay was conducted with newly hatched larvae evolved from F₀. These insects were separate from two sex table studies larvae. Same procedure of F₀ was followed with each treatment repeated 3 times.

2.3 Evaluation of LC₂₅ and sublethal LC₁₀ sublethal effects of triazophos on life history traits of P. gossypiella at F₀ and F₁ generations

This experiment was aimed to know the direct impact of low or sublethal concentration on pink bollworm larvae on F₀ generation and further impacts on F₀ evolved generation F₁ (without triazophos exposure). Different concentrations which were determined from baseline toxicity experiments were used to determine the LC₂₅ and sublethal LC₁₀sublethal effects of triazophos on life cycle parameters of P. gossypiella. 1500 adults were transferred to clean cage with tissue papers and cotton bolls inside to lay eggs on them. After 48 h they were removed from the cage and egg laying was transferred to other cage until eggs were hatched (within 12 h) and their first instar was used for F₀ generation. 60 larvae were transferred to other cage for LC₂₅ and LC₁₀sublethal treatments in glass vials. Cotton bolls as diet was changed after one day interval till 2nd instar, while changed cotton bolls each day on becoming 3rd instar. For pairing the insects, newly hatched pupae were chosen and observed for male or female genital appearance (CABI 2010) and were kept in vile, they were paired and mated (30–32 per treatment). Fecundity and longevity were observed until mortality of pair. Oviposition period was observed for each female insect (Chi et al., 2018). Data was recorded after 12 h till death of individual insect. Each experiment was repeated 3 times. Honey-soaked cotton was given to adult moths.

2.4 Transgenerational effects of triazophos on F₁ generations of P. gossypiella

Two eggs from each pair of each group of F₀ (LC₁₀; LC₂₅; LC_50; Control) were selected and kept in voiles for starting F₁ generation. Hatched offspring from these eggs were used as F₁ generation observations. Diagonally cut cotton bolls were placed in voiles for food. The transgenerational effects were studied at all stages of insect. At pupal stage, insects were paired and kept in voiles for egg laying. Data was recorded after 12 h till death of individual insect. Characteristics such as developmental time; adult longevity; and fecundity were observed. Temperature was kept at 27 ± 1 °C with relative humidity 75 %.

2.5 Statistical analysis

Lethal and sublethal concentrations were calculated using POLO-Plus and Probit analysis (LeOra Software 2006). ANOVA was used to examine mortality. TWO-SEX MS CHART assessed life table characteristics utilizing age-stage two-sex table (Chi 1988). (Chi 2015). Means and SEs were determined using bootstrap (n = 100,000) (Huang and Chi, 2012). Various life table metrics, including age-stage specific survival rate (sxj), life expectancy (exj), which tells us if a person will survive to an age appropriate for that stage, and age-stage specific net reproductive value, are available (vxj). As of the calculation, other population factors such as the intrinsic rate of rise (r), finite rate of increase (λ), net reproductive rate (R₀), and mean generation time (T) (Chi and Liu 1985).

The Euler-Lotka equation was used to compute the intrinsic rate of rise (r) for the Age-stage, two sex life table, with age indexed as (Goodman 1982). $$\sum _{x=0}^{\infty }{e}^{-r\left(x+1\right)}lxmx=1$$

R₀ (Net Reproductive Rate) which is total number of offsprings that an individual can produce during lifetime will be calculated as $$\mathit{Ro}=\sum _{x=0}^{\infty }lxmx$$

Net Reproductive Rate (R₀) and mean female fecundity (F) relationship was calculated as $$R0=F\frac{\mathit{Nf}}{N}$$

N = total number of individuals, F = number of females adults in this study (Chi 1988).

The finite rate (λ) λ = e^r,

Mean generation time (T) = time span that population needs to increase R₀ folds of its size. Value of T was calculated as. $$T=\frac{\ln R0}{r}$$ $$\mathit{GRR}=\sum \mathit{mx}$$

Life expectancy (e_xj) was calculated as $$\mathit{exj}=\sum _{i=x}^{\infty }\sum _{y=j}^{k}siy$$

fe probability (s_xj) was calculated as $$\mathit{siy}=1$$

e-stage reproductive value (v_xj) was calculated as $$\mathit{Vxj}=\frac{e^{-r\left(x+1\right)}}{\mathit{Sxj}}\sum _{i=x}^{\infty }{e}^{-r\left(x+1\right)}\sum _{y=j}^{k}siyfiy$$

3.1 Lethal effects of triazophos against F₀ and F₁ generations

The LC₅₀ values for P. gossypiella at 0, 1, 2, and 4 instars were 2.72, 2.75, 3.34, and 3.27 mg/L of triazophos (Table 1). The LC₁₀ for triazophos was 1.984, 1.859, 2.380, and 2.297 mg/L for the first, second, third, and fourth instars of the F₀ generation, respectively (Table 1). Triazophos's LC₂₅ for the F₀ generation was 2.307 mg/L for the first instar, 2.241 mg/L for the second instar, 2.798 mg/L for the third instar, and 2.781 mg/L for the fourth instar (Table 1). The LC₉₀ values of triazophos for first, second, third, and fourth instar P. gossypiella larvae of the F₀ generation were 3.75, 4.09, 4.70, and 4.67 mg/L, respectively (Table 1).

For larvae, the LC₅₀ values for the first, second, third, and fourth instars of the F₁ generation (a different set of insects other than the two-sex table group) were 1.852, 2.008, 2.627, and 2.637 mg/L, respectively (Table 1). Mortality concentration response lines were used to calculate the LC₁₀ and LC₂₅. For the F₁ generation, the LC₁₀ values were 0.987, 1.042, 1.555, and 1.776 mg/L for the first, second, third, and fourth instar larvae, respectively (Table 1). The LC₂₅ values in the first, second, third, and fourth instars of P. gossypiella larvae were 1.330, 1.422, 1.993, and 2.142 mg/L, respectively, for the F₁ generation (Table 1). First generation P. gossypiella larvae had an LC₉₀ value of 3.4763 mg/L for the first instar, 3.8673 mg/L for the second instar, 4.4382 mg/L for the third instar, and 3.915 % mg/L for the fourth instar when exposed to triazophos (Table 1).

3.2 LC₂₅, LC₁₀ and transgenerational effects of triazophos on biological and reproductive parameters of F₀, F₁ generations

Triazophos application to determine the LC₂₅; sublethal LC₁₀sublethal and transgenerational effects on biological and reproductive parameters on all instars and pupae of F₀ and F₁ (Tables 2 and 3) showed that significant differences occurred when compared to control. There was no significant difference in hatching duration of F₀ (F = 4.20; df = 57; P = 0.013); F₁ (F = 2.12; df = 57; P = 0.156) generations (Table 2). There was significant difference in 1st instar larval duration of F₀ (F = 5.17; df = 57; P = 0.0001) at LC₁₀ and LC₂₅ showing prolonged duration as compared to control; F₁ (F = 2.64; df = 57; P = 0.0002) generation (Table 2). For 2nd instars there was no significant difference in larval duration of F₀ at LC₁₀ and LC₂₅ (F = 3.27; df = 57; P = 0.217); F₁ (F = 1.94; df = 57; P = 0.143) generation (Table 2). For 3rd instars there were no significant difference in instar larval duration of F₀ (F = 6.32; df = 57; P = 0.301); F₁ (F = 2.29; df = 57; P = 0.244) generation (Table 2). For 4th instars there were no significant difference in instar larval duration of F₀ (F = 3.31; df = 57; P = 0.0433); F₁ (F = 1.87; df = 57; P = 0.0037) generation (Table 2). For pupa there were significant difference in duration of F₀ (F = 2.24; df = 57; P = 0.0172); F₁ (F = 3.76; df = 57; P = 0.0564) generations (Table 2).

Reproductive parameters for F₀ and F₁ when used LC₁₀ and LC₂₅ for P. gossypiella are shown in (Table 3). Pre-oviposition of F₀ at LC₁₀ and LC₂₅ showed significant differences as compared to control (F = 0.82; P = 0.0013); significant difference occurred for F₁ (F = 1.24; P = 0.0029). Fecundity for F₀ at LC₁₀ and LC₂₅ showed significant differences as compared to control (F = 0.93; P = 0.002); F₁ (F = 0.88; P = 0.034). Female adult longevity for F₀ at LC₁₀ and LC₂₅ showed significant differences as compared to control (F = 0.84; P = 0.002); F₁ (F = 1.13; P = 0.0001); (Table 3). Oviposition period for F₀ at LC₁₀ and LC₂₅ showed significant differences with longer or shorter oviposition period as compared to control (F = 0.68; P = 0.021); F₁ (F = 1.27; P = 0.002); (Table 3).

3.3 Effect of triazophos on demographic traits of F₀ and F₁ generations

Table 4 displays the results two-sex-stage-specific life table regarding the effects of triazophos on population characteristics (demographic attributes). The intrinsic rate of increase (r) for the F₀ generation was negatively correlated with concentration and did not vary significantly from the control group (Table 4). There was no statistically significant difference between the mean finite rate of growth of insects treated with LC₁₀ or LC₂₅ and those in the control group (Table 4). Control had a greater net reproduction rate (R0), but LC₁₀ and LC₂₅ drastically reduced it. As shown in Table 4, the longer mean generation time (T) for insects treated with LC₁₀ and LC₂₅ compared to the control group would lead to fewer generations of insects in cotton fields. Comparing the control group to the LC₁₀ and LC₂₅ treated groups, the gross reproductive rate (GRR) of the latter was found to be considerably lower (Table 4).

When comparing LC₁₀ and LC₂₅ to the control group, the F₁ generation showed an intrinsic rate of rise (r) that was inversely proportional to concentration (Table 4). Both the LC₁₀ and LC₂₅ groups showed no statistically significant differences from the control group in terms of the mean finite rate of increase (Table 4). Insects treated with LC₁₀ and LC₂₅ had a lower net reproduction rate (R₀) than controls, and this decline accelerated with increasing concentration (Table 4). Insects treated with LC₁₀ and LC₂₅ had a longer mean generation time (T) compared to controls (Table 4). Insects treated with LC₁₀ and LC₂₅ have dramatically reduced GRR compared to controls (Table 4).

Using bootstrap analysis, we were able to determine the age-stage specific survival rate (sxj). The sxj demonstrated the likelihood that a freshly placed egg would survive from age × to stage j when applied to the F₀ generation. The LC₁₀ and LC₂₅ groups had an increase in total life span when compared to the control group (Fig. 1). The fact that the age-stage specific life expectancy exj was greater in LC₁₀ and LC₂₅ populations as compared to control (Fig. 2) demonstrated the likelihood that LC₁₀ and LC₂₅ treated insects would have a longer lifespan than control. Age-stage specific reproductive rate (vxj) indicates that the overall reproduction rate decreased in LC₂₅ populations; further, LC₁₀ populations also shown less reproductive rate as compared to control (Fig. 2), which demonstrated that although a population can survive longer, it will reproduce fewer offspring in the generations to come.

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Fig. 1

Age Stage Specific Survival Rate (Sxj) and Age Stage Life Expectancy (Exj) of F₀ generation P. gossypiella.

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Fig. 2

Age Stage Reproductive Value (Vxj) of F₀ generation and Age Stage Specific Survival Rate (Sxj) of F₁ generation pink bollworm P. gossypiella.

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To determine the survival rate (sxj) for each age group, a bootstrap sample was generated. For the F₁ generation of a species, the survival probability from age x to age j was denoted by the symbol sxj. The LC₁₀ and LC₂₅ had significantly longer median ages than control (Fig. 3). Life expectancy at different ages exj was greater in the LC₁₀ and LC₂₅ populations than in the control group (Fig. 3), suggesting that treated insects lived longer than their untreated counterparts. Compared to controls, LC₂₅ populations have a lower age-specific reproductive rate (vxj) of F₀ (Fig. 3), indicating that longer-lived populations do not generate more offspring than those who perish earlier in their lives.

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Fig. 3

Age Stage Life Expectancy (Exj) and Age Stage Reproductive Value (Vxj) of F₁ generation P. gossypiella.

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