Fitness costs and biological adaptability of Cry toxin-resistant field strains of Pink bollworm, Pectinophora Gossypiella (Saunders) from India

Abstract

Pink bollworm strains (F₀) collected from 59 geographically different locations across India were advanced to F₁ and screened for Cry toxin resistance using 21-days diet incorporation bioassay. The survived population from five locations with the highest LC₅₀ values for Cry1Ac were grouped as highly resistant, while those from five locations with lower LC₅₀ values were grouped as relatively less resistant. Similarly, populations with high and relatively low resistance to Cry2Ab were also developed. Further, these populations advanced to next generation. One-day-old neonate larvae from all populations, along with a susceptible strain (F₁₉₄), were exposed to various selected doses to evaluate the precise impact of these concentrations on biological fitness. The results indicated that higher concentrations of Cry toxin prolonged the larval, pupal, and total developmental durations, while reducing adult longevity, fecundity, and egg hatchability. Egg incubation period, however, remained unaffected. Susceptible strains on toxin-free diet exhibited the shortest development times (larval: 18.48 ± 0.95 d; pupal: 7.22 ± 0.36 d; total: 39.41 ± 2.52 d), longest adult life (9.78 ± 0.35 d), highest fecundity (123.15 ± 4.95 eggs/female), and greatest survival rates (> 95%). Their growth (5.28) and survival indices (0.97) reflected robust development under non-stress conditions. Conversely, toxin exposure increased mortality and malformations, particularly in susceptible strains. Fitness costs due to Cry1Ac exposure were highest in susceptible strains (84.42% at 1.0 µg/ mL), followed by less resistant (79.46%) and highly resistant strains (60.07% at 10.0 µg/ mL). For Cry2Ab, the relatively less resistant strain showed the highest fitness cost (74.51%), followed by susceptible (58.82%) and highly resistant strains. Overall, fitness costs were closely linked to reduced fecundity, egg hatchability, larval survival, adult emergence, and prolonged development.

Introduction

In the vast canvas of cotton fields spanning 32.6 million hectares worldwide, with a yield reaching a staggering 116.10 million bales¹ India emerges as the star player, reigning supreme with 130.61 lakh hectares and a bale count of 343.47 lakh, each weighing 170 kg with the productivity of 445 kg lint per hectare². More than 1,326 insect species worldwide pose significant threats to cotton cultivation³. In India alone, around 130 different insect pests attack cotton crops at various stages of growth⁴. However, in the USA, cotton is primarily attacked by Heliothis virescens (Fabricius) and Helicoverpa zea (Boddie)⁵. In India, Helicoverpa armigera (Hübner) is the most significant bollworm, and in Australia, H. armigera, along with H. punctigera Wallengren, pose major threats⁶. In India, over 43 per cent of the cost of cotton production is dedicated to insect pest management, with 80 per cent of that targeting bollworm control, particularly H. armigera⁷. Heliothines present a serious threat to upland cotton production worldwide, including in India, due to their high resistance to classical insecticides such as organophosphates, carbamates, and pyrethroids since the late 1980s⁸. In India, cotton bollworms have shown significant levels of resistance to the organochlorine, organophosphate, carbamate and pyrethroid group of insecticide⁹. By the mid-1990s, cotton cultivation became largely uneconomical in major cotton-growing countries, especially in the Indian subcontinent and China, due to the high cost of insecticides with novel chemistry^7,10.

To mitigate the burdens associated with insecticide resistance, the development and deployment of genetically modified crops expressing Bacillus thuringiensis (Bt) toxins have emerged as a transformative technology¹¹. Consequently, Bt varieties/hybrids have been developed and released for commercial cultivation in several cotton growing countries. In USA, during 1996 Bt cotton expressing Cry genes was first released for commercial cultivation¹². Following this, Bt cotton was released for commercial cultivation in many countries; Australia (1996), China (1997), Argentina (1997), South Africa (1998), Mexico (1998), Indonesia (2001), Colombia (2002) and India (2002)¹³. In all these countries H. armigera, H. zea, H. virescense, Earias spp, and Pectinophora gossypiella (Saunders) were controlled successfully and reduced their damage^14,15. Despite the initial success of Bt cotton in managing the bollworms, there have been instances of pink bollworm populations developing resistance to these toxins in India. During 2010, pink bollworm recorded resistance to Bollgard event (Cry1Ac) in Gujarat, India¹⁶ and subsequently to Bollgard II (Cry1Ac + Cry2Ab) with 40–80 per cent of the bolls harboured surviving larvae as recorded from Amreli and Bhavnagar districts of Gujarat, India during 2014¹⁷. The pink bollworm is a notorious pest, practically monophagous that targets cotton flowers, bolls and feed on seeds, damaging the lint, undergone selection pressure on Bt cotton. As, it first feeding itself target the flowers which express the Cry1Ac concentration of 0.16–1.47 µg/ g^18,19,20 and Cry2Ab concentration of 2.01–18.94 µg/ g²⁰. As the generation advances, its starts feeding on boll rind and raw seed expressing the Cry1Ac toxin concentration of 0.10–0.95 µg/ g and 0.15–1.74 µg/ g, respectively^17,18,19. Similarly, Cry2Ab expression varied from 7.95 to 40.68 µg/ g and 2.01–18.94 µg/ g in boll rind and raw seed, respectively^17,18,19. Over the years, development of resistance to Bt toxins arises from a dynamic interaction of genetic, ecological, and evolutionary influences that collectively shape the adaptive landscape of pest populations^20,21. In resistant insects, fitness costs often stem from the detrimental pleiotropic effects associated with resistance-conferring genes²². These costs are evident when, in toxin-free environments, resistant individuals exhibit reduced fitness compared to their susceptible counterparts²³. Such resistance-associated fitness penalties can substantially affect vital life-history traits, including survival and growth^24,25,26 the regulation of diapause²⁵ and reproductive performance such as mating success²². Fitness costs can hinder the development of resistance to Bt toxins and may help in postponing or even preventing such resistance^27,28. Evaluating the fitness of resistant individuals in environments without Bt selection pressure such as in non-Bt cotton fields or refuge crops is vital for gaining insight into the mechanisms of resistance evolution²⁷. Consequently, a thorough understanding of the life history traits and ecological interactions of pink bollworm is fundamental to identifying the drivers of resistance to Cry toxins²⁹. This study examines the developmental biology and reproductive fitness of Cry toxin resistant pink bollworm strains from India. Key metrics include larval duration, pupation, adult longevity, fecundity, egg viability, and fitness indices. Survivability and malformation rates are analysed under toxin stress to assess adaptability^30,31. Findings aim to reveal potential fitness costs and trade-offs associated with resistance evolution. We hypothesized that Cry toxin resistance in pink bollworms imposes significant fitness costs, which vary across resistant and susceptible strains. To test this, we compared developmental parameters, survivability, and reproductive fitness among strains exposed to varying Cry1Ac and Cry2Ab concentrations.

Results

The biology of highly resistant, relatively less resistant and lab reared susceptible pink bollworm strains revealed that there were noteworthy distinctions observed in larval development, pupation period and adult longevity on artificial diet incorporated with varied concentration of Cry1Ac and Cry2Ab separately. Similarly, significant variance was noted on survivability, mortality and malformation in different stages of pink bollworm reared on the artificial diet containing Cry1Ac (0.1, 1.0 and 10.0 µg/ mL of artificial diet) and Cry2Ab (1.0, 5.0 and 10.0 µg/ mL of artificial diet). The different stages of pink bollworm represented under Fig. 1.

Fig. 1

Different stages of pink bollworm (a) Eggs (b) Matured eggs (c) Fourth instar larva (d) Pupa (e) Freshly emerged moth (f) Adult female moth.

Larval, pupal and adult longevity

The larval and pupal period of pink bollworm increased with increasing concentration of both the Cry toxins in highly resistant, relatively less resistant and lab reared susceptible strains. In contrast, adult longevity was reduced with increasing concentration of Cry toxin and egg incubation period was non-significant across all the Cry toxin concentration in various strains of pink bollworm.

The highest larval (27.88 ± 1.31 days) and pupal (9.14 ± 0.58 days) period was recorded in the highly resistant field strains reared on diet incorporated with 10 µg/ mL of Cry1Ac, which was on par with the larval (26.72 ± 1.48 days) and pupal (9.05 ± 0.54 days) period of relatively less resistant field strains fed on diet with 10 µg/ mL of Cry1Ac (Fig. 2). Similarly, on diet incorporated with 10 µg/ mL of Cry2Ab recorded highest larval (27.05 ± 1.25 and 25.89 ± 1.46 days, respectively) and pupal (8.89 ± 0.39 and 8.80 ± 0.51 days, respectively) period in both highly resistant and relatively less resistant field PBW strains (Fig. 3). Whereas, all the susceptible pink bollworm larvae died on diet incorporated with Cry1Ac at 10 µg/ mL, and Cry2Ab at 5 and 10 µg/ mL. In contrast, highest adult longevity was recorded in the susceptible strain reared on artificial diet without Cry toxins (9.78 ± 0.35 days) followed by highly resistant field strain fed on Cry2Ab at 1.0 µg/ mL of diet (8.98 ± 0.35 days) and Cry1Ac at 0.1 µg/ mL of diet (8.85 ± 0.40 days), which was on par with the adult longevity of relatively less resistant and susceptible strains obtained by rearing on Cry1Ac at 0.1 µg/ mL of diet (8.68 ± 0.35 and 8.62 ± 0.34 days, respectively) and Cry2Ab at 1.0 µg/ mL of diet (8.88 ± 0.55 and 8.82 ± 0.62 days, respectively).

Fig. 2

Effect of Cry1Ac on the biology of resistant and susceptible pink bollworm strains.

Fig. 3

Effect of Cry2Ab on the biology of resistant and susceptible pink bollworm strains.

The shortest life cycle (39.41 ± 2.52 days) was observed in the susceptible strain reared on a diet without Cry toxins. Whereas, the longest life cycle was observed in the highly resistant field PBW strain (48.61 ± 2.89 days), which was statistically comparable to the moderately resistant or relatively less resistant field strain (47.23 ± 2.95 days) reared on a diet containing Cry1Ac at 10.0 µg/ mL. Similarly, complete life cycle of highly resistant and relatively less resistant field PBW strains were observed 47.62 ± 2.66 and 46.26 ± 3.06 days respectively, when reared on a diet containing with Cry2Ab at 10.0 µg/ mL (Figs. 2 and 3).

Fecundity and egg hatchability

The fecundity and egg hatching percentage of highly, relatively less resistant and susceptible pink bollworm strains decreased as the concentration of Cry toxins in the diet increased. Significant differences were observed in fecundity and per cent egg hatching among the highly resistant, relatively less resistant and susceptible strains at each concentration of Cry toxin. The susceptible strain reared on the diet without Cry toxins recorded higher fecundity and egg hatching compared to the highly resistant, relatively less resistant and susceptible pink bollworm strains, when reared on diets with either Cry1Ac or Cry2Ab toxins. Among the two different Cry toxins, the PBW larvae reared on diet with Cry2Ab laid more eggs with higher hatchability than the strain reared on diet with Cry1Ac toxin.

The susceptible strain reared on the diet without Cry toxins laid a greater number of eggs (123.15 ± 4.95 number / female) and egg hatching (97.75 ± 4.12%).Whereas, highly resistant PBW larvae fed with Cry1Ac at 0.1 µg/ mL of diet documented an average of 104.35 ± 4.98 eggs per female with 88.52 ± 3.13 per cent egg hatching (Fig. 4). Similarly, highly resistant pink bollworm strain fed with Cry2Ab at 1.0 µg/ mL of diet recorded the fecundity of 107.53 ± 5.62 per female and egg hatching of 90.47 ± 4.35 per cent (Fig. 5). Whereas, relatively less resistant strain recorded lowest fecundity (73.38 ± 4.25 /female and 76.27 ± 3.28/ female on Cry1Ac at 10 µg/ mL and Cry2Ab µg/ mL, respectively) and per cent egg hatching (72.55 ± 3.65% and 73.64 ± 2.64% on Cry1Ac at 10 µg/ mL and Cry2Ab µg/ mL, respectively).

Fig. 4

Effect of Cry1Ac on the fecundity and egg hatching of resistant and susceptible pink bollworm strains.

Fig. 5

Effect of Cry2Ab on the fecundity and egg hatching of resistant and susceptible pink bollworm strains.

Survival and growth metrics

Significant differences were observed in larval survivability, pupation, adult emergence, and adult survivability among highly resistant, relatively less resistant, and susceptible pink bollworm strains at each Cry toxin concentration. Across strains, all biological parameters were consistently higher when reared on Cry2Ab than on Cry1Ac. The susceptible strain reared on a toxin-free diet showed the highest values across all stages, with larval survivability (98.33%), pupation (97.53%), adult emergence (96.73%) and adult survivability (95.93%). In contrast, the highly resistant strain reared on 0.1 µg/ mL Cry1Ac exhibited lower values of 80.96, 78.31, 76.11 and 75.71 per cent, respectively (Fig. 6). At 1.0 µg/ mL Cry1Ac, the susceptible strain showed drastic declines in all parameters, with adult survivability dropping to 7.20 per cent. A similar pattern was observed with Cry2Ab. At 1.0 µg/ mL, the highly resistant strain retained relatively high performance (e.g., 77.12% adult survivability), whereas the susceptible strain recorded notably lower values (17.20% adult survivability; Fig. 7).

Fig. 6

Effect of Cry1Ac on the biological parameters of resistant and susceptible pink bollworm strains.

Fig. 7

Effect of Cry2Ab on the biological parameters of resistant and susceptible pink bollworm strains.

The growth and survival index are a key indicator of strain viability under varying Cry toxin concentrations. Higher values reflect better survival and development, while lower values suggest stress or adverse effects. This index is instrumental in assessing adaptation and resistance development in insect populations. In pink bollworm strains, the index declined progressively with increasing concentrations of Cry1Ac and Cry2Ab. The susceptible strain on a toxin-free diet showed the highest growth (5.28) and survival index (0.97), whereas the lowest values (0.76 and 0.12) occurred at 1.0 µg/ mL Cry1Ac (Fig. 8). Similarly, Cry2Ab at the same concentration yielded indices of 0.94 and 0.19 (Fig. 9).

Fig. 8

Effect of Cry1Ac on the growth and survival index of resistant and susceptible pink bollworm strains.

Fig. 9

Effect of Cry2Ab on the growth and survival index of resistant and susceptible pink bollworm strains.

Mortality and malformation in different developmental stages of PBW

Mortality and malformation rates in pink bollworm strains increased with higher concentrations of Cry1Ac and Cry2Ab toxins. As shown in Fig. 10, the larval stage exhibited the highest susceptibility, with greater mortality and malformation compared to pupal and adult stages. Complete (100%) mortality was recorded in the susceptible strain exposed to Cry1Ac (10.0 µg/ mL) and Cry2Ab (5.0 and 10.0 µg/ mL). In contrast, the lowest mortality rates in larval (1.67%), pupal (0.0%), and adult (0.0%) stage were observed in the susceptible strain on a toxin-free diet, along with minimal malformation of 0.80% in larvae and none in pupae and adults. The highest malformation rates in larvae (10.00%), pupae (6.80%) and adult (3.80%) stage were noted in the relatively less resistant strain at 10.0 µg/ mL Cry1Ac, and similarly 9.51% of larvae, 6.40% of pupae and 3.40% of adult stage malformation was recorded at 10.0 µg/ mL Cry2Ab (Supplementary Figs. 1 and 2).

Fig. 10

Malformation in different stages of Cry toxin fed pink bollworm. a, b & c. Malformed larva d, e & f. Malformed pupa g & h. Malformed Adult.

Weight of the different developmental stages of PBW

The weight of different developmental stages in the susceptible pink bollworm strain significantly decreased with increasing concentrations of Cry toxins. In contrast, the weights of the resistant strains reared on various Cry toxin concentrations, as well as the susceptible strain reared on a toxin-free diet, showed no significant changes.

Across different strains, larval, pupal, and adult weights ranged from 21.45 to 29.96 mg, 18.26–21.91 mg, and 14.84–18.53 mg, respectively, under varying Cry1Ac concentrations. Similarly, for Cry2Ab, the respective weight ranges were 24.26–29.96 mg (larvae), 19.11–21.91 mg (pupae), and 15.13–18.53 mg (adults).

At 0.1 µg/ mL Cry1Ac, the highly resistant strain showed the highest larval, pupal, and adult weights (28.94 ± 0.83, 21.28 ± 0.77, and 18.26 ± 0.76 mg), followed closely by the relatively less resistant strain. The susceptible strain recorded lower weights (24.96 ± 2.12, 20.11 ± 1.90, and 16.13 ± 1.14 mg) (Fig. 11). Similarly, at 1.0 µg/ mL Cry2Ab, the highest weights were found in the highly resistant strain, comparable to the relatively less resistant strain, while the susceptible strain again showed reduced weights (Fig. 12).

Fig. 11

Effect of Cry1Ac on the weight in different stages of resistant and susceptible pink bollworm strains.

Fig. 12

Effect of Cry2Ab on the weight in different stages of resistant and susceptible pink bollworm strains.

Relative fitness and fitness cost of Cry toxin resistant field strains of PBW

The relative fitness of the susceptible pink bollworm strain was highest on a diet without Cry toxins, compared to diets containing Cry toxins. Among Cry1Ac-exposed groups, the highest relative fitness was recorded in the susceptible strain reared on a toxin-free diet, followed by the highly resistant (0.74) and relatively less resistant (0.67) strains on a diet with 0.1 µg/ mL Cry1Ac (Supplementary Fig. 3). Similarly, relative fitness values for the highly resistant and relatively less resistant strains on 1.0 µg/ mL Cry2Ab were 0.79 and 0.71, respectively (Supplementary Fig. 4).

Cry1Ac exposure imposed the highest fitness cost (84.42%) on the susceptible strain at 1.0 µg/ mL, followed by the relatively less resistant (79.46%) and highly resistant (60.07%) strains at 10.0 µg/ mL (Fig. 13). For Cry2Ab, the greatest fitness cost (74.51%) occurred in the relatively less resistant strain at 10.0 µg/ mL, followed by the susceptible (58.82%) and highly resistant (57.48%) strains at 1.0 and 10.0 µg/ mL, respectively (Fig. 14). These findings indicate that fitness cost is closely linked to fecundity, egg hatchability, larval survival, developmental time, and adult emergence.

Fig. 13

Fitness cost of field resistant pink bollworm against Cry1Ac.

Fig. 14

Fitness cost of field resistant pink bollworm against Cry2Ab.

Discussion

Developmental delays in resistant strains

The resistance developed against Cry toxins in pink bollworm have significant implications for their fitness, encompassing both costs and benefits. Understanding these impacts is crucial for designing effective pest management strategies. The study observed significant differences in the larval and pupal periods among highly resistant, relatively less resistant and lab-reared susceptible pink bollworm strains when reared on an artificial diet incorporated with varying concentrations of Cry1Ac and Cry2Ab. The extension of larval and pupal periods with increasing Cry toxin concentrations aligns with previous studies. The larval duration of each instar was longer on non-Bt cotton bolls compared to Bt cotton bolls³²and an increased larval period in resistant pink bollworm (20.8 ± 0.4 days) on Bt cotton compared to the susceptible strain on non-Bt cotton, with an average developmental time 12 days (41%) longer on Bt cotton (42.4 days) than on non-Bt cotton (29.8 days)²⁰. Similar findings recorded in diamond back moth (Plutella xylostella)²² and other lepidopterans^{33,34,35,36,37} where the development time increased in the populations exposed to Bt toxins. This suggests that extended larval duration at higher concentration of Cry toxins may be necessary to repair tissue damage and accumulate sufficient nutrients for pupation and adult development. Hosts may respond to Cry toxin exposure through various mechanisms, including detoxification and tissue repair³⁸.

The increased pupal duration and decreased adult longevity of pink bollworm^37,39,40,41 on an artificial diet with Cry toxins were consistent with our findings. However, the reduction in adult longevity of resistant H. armigera when exposed to higher concentrations of Bt toxins⁴² and the reduced longevity in resistant populations may be indicative of the physiological costs associated with resistance⁴³. Similarly, pink bollworm took more days to complete its life cycle on different Bt cotton events compared to non-Bt cotton^41,44ranging from 40 to 55 days (mean 46.82 ± 1.20 days)⁴⁵ and from 36.5 to 49 days (mean 42.8 ± 4.66 days)⁴⁶ in Bt cotton.

Reasons for extended life cycle

The increased life cycle of Cry toxin-resistant pink bollworm fed on Bt cotton may be due to several factors. These include viz., (1) Impact of cry proteins: Cry proteins produced by Cry toxin can adversely affect the growth and development of pink bollworm, contributing to delayed maturation (2) Toxicity effects: Sublethal exposure to Cry toxins can disrupt normal physiological processes in larvae. This often results in energy being redirected from growth to detoxification and tissue repair, ultimately slowing development (3) Physiological stress: Continued exposure to Cry toxins may induce stress responses in the insect, causing developmental delays during both larval and pupal stages (4) Altered feeding behaviour: Cry toxin exposed larvae often exhibit reduced feeding efficiency, which can lower nutrient intake and slow growth, leading to prolonged developmental periods (5) Adaptive response and hormonal regulation: Slower growth and extended development may serve as adaptive survival strategies in response to Bt toxins. Notably, increased levels of juvenile hormone (JH) have been reported in resistant insects such as Helicoverpa armigera, following Bt exposure^47,48. Similar effects were observed by Pérez-Hedo et al.⁴⁹and Muñoz et al.⁵⁰where Bt ingestion led to elevated JH levels, likely contributing to prolonged larval stages, altered lifespan, and reduced reproductive potential in resistant populations.

The Midgut receptor modifications critically impair the binding efficiency of Cry toxin, effectively neutralizing its toxic action and enabling larvae to survive on Bt cotton. As such, PgCad1 mutations serve as a molecular hallmark of high-level resistance in PBW. Understanding these midgut receptor alterations not only enhances our mechanistic insight into resistance but also provides potential molecular targets for next-generation Bt traits and diagnostic tools for early resistance detection in field populations. Additionally, the energy and resources diverted towards developing and maintaining resistance mechanisms often result in slower growth rates and extended developmental periods. Resistant insects may experience lower metabolic efficiency, leading to prolonged larval stages and delayed maturation. Physiological changes associated with resistance, such as altered enzyme activity and changes in gut microbiota, also contribute to extended developmental durations. These adaptations, necessary for detoxifying or tolerating the toxins, come at the cost of overall growth and reproductive speed⁴³.

Reproduction trade-offs

Fecundity and egg hatchability decreased with increasing concentrations of Cry toxins in both resistant and susceptible strains, though resistant strain exhibited higher fecundity and hatchability than susceptible strain, when reared on Cry toxin incorporated diet. Pink bollworm reared on an artificial diet without Cry proteins laid more eggs compared to those reared on a diet with Cry proteins. There was a significant difference in the fecundity of gravid pink bollworm adults reared on an artificial diet with Cry toxins compared to those without. These findings are consistent with Mushtaq et al.⁵¹. who recorded an average of 111.5 ± 13.96 eggs per female pink bollworm on Bt cotton. Similarly, higher fecundity and egg hatching recorded in PBW grown on non Bt cotton^37,41. Whereas, Liu et al.³⁷. found that fecundity and egg hatchability were higher in Bt-resistant strains of pink bollworm compared to susceptible ones.

Groeters et al.²² and Sayyed et al.⁵² found that Plutella xylostella females resistant to Bt toxins laid fewer eggs than those that were susceptible. Similarly, Campanhola⁵³ observed that Helicoverpa armigera females resistant to Bt produced around 1,200 eggs each, while the susceptible ones laid about 2,500 eggs. These results suggest that reduced egg-laying in resistant females may be a result of fitness costs linked to their resistance mechanisms. Zhang et al.⁵⁴ also reported significantly lower egg production in Bt-resistant H. armigera compared to non-resistant ones. One explanation is that resistant insects may use more energy for developing resistance like detoxifying the Bt toxins which leaves less energy available for reproduction. This indicates that resistance to Cry toxins may lower reproductive success, yet still allow resistance genes to persist and spread within the pest population.

Growth and survival indices

The growth and survival indices decreased with enhanced Cry toxin levels. These indices were notably higher in resistant strain as compared to susceptible strain grown on diet containing Cry toxin, similar to findings by Carrière et al.²⁷. in Bt-resistant pink bollworm, which showed better growth and survival under Bt toxin exposure. Whereas, susceptible strain growth and survival indices were higher on the diet without toxins as compared to resistant strain on diet with toxins, these results are in line with findings of Liu et al.³⁷. This highlights the adaptive advantage of resistant strains in environments with high Bt toxin concentrations.

Mortality and malformation rates were highest in all the different biological stages, particularly in susceptible strain, consistent with the findings of different authors^{32,33,41,44,55} as they recorded highest mortality percentage of all stages on Bt cotton. Similarly, continuous selection with Cry1Ac improved the adaptability of H. armigera with decline in mortality and other deformities with the progression of each generation⁵⁶. The 100 per cent mortality observed in susceptible strain at the highest Cry toxin concentrations underscore the potent efficacy of Cry toxins against non-resistant pink bollworm, emphasizing the importance of resistance management strategies.

Body weight in resistant and susceptible strains

The weight of the different stages of susceptible pink bollworm strain was significantly affected by varying concentrations of Cry toxins. In contrast, the weight of various stages of resistant and relatively less resistant strain was comparable to that of the susceptible strain reared on a toxin-free diet. Our findings are consistent with those reported by Rajput et al.. (2018)³²who documented larval weights of 13.84 mg and 20.24 mg on Bt and non-Bt cotton, respectively and pupa obtained from the larvae reared on Bt cotton has lower weight as compared to non Bt cotton. The weight of male and female pupae on non-Bt was 21.20 and 23.10 mg, respectively and on Bt was 13.10 and 17.70 mg, respectively³⁷. Similarly, larval and pupal weight of PBW was lower on different events of Bt cotton as compared to non Bt cotton⁴⁴. Adkisson et al.³⁹. recorded the average adult weight of 13.20, 14.90, 16.20 and 15.10 mg when reared in bolls, 5 per cent cotton seed meal, 1 per cent cotton seed meal and wheat germ medium, respectively and these results are in partial accordance with the current experiment results. Several factors may have contributed to the decreased weight of susceptible pink bollworms fed Cry toxins. First, Cry toxins exhibit insecticidal properties that target specific receptors in the insect’s gut. The consumption of these toxins can disrupt normal physiological processes, leading to reduced feeding efficiency, impaired digestion, and poor nutrient absorption, ultimately resulting in decreased weight gain. Additionally, exposure to Cry toxins induces metabolic stress in insects. The energy required to detoxify or tolerate the toxins may divert resources from growth and development, negatively impacting overall weight gain. Furthermore, the presence of Cry toxins may alter the feeding behaviour of pink bollworms. They may consume less food or exhibit aversion to the artificial diet containing the toxins, leading to reduced nutrient intake and subsequent weight loss. Cry toxins can also interfere with various developmental processes in pink bollworms, such as moulting, metamorphosis, and reproductive physiology. Disruption in these processes can result in slower growth rates and lower final body weight. Lastly, the specific mode of action of each Cry toxin, targeting different receptors and pathways in the digestive system, can lead to variable effects on the growth and development of pink bollworms at different life stages. Overall, the reduced weight observed in Cry toxin-fed pink bollworms is likely the result of a combination of physiological, behavioural, and developmental effects induced by the toxins in the artificial diet.

Fitness cost and relative fitness

The relative fitness of susceptible strain is higher on diet without Cry toxins as compared to highly resistant, relatively less resistant and susceptible PBW strains reared on diet with variable concentration Cry toxins. Whereas, resistance to Cry toxins imposes a significant fitness cost on different PBW strains, particularly at higher toxin concentrations as compared to susceptible PBW strain reared on Cry toxin free diet. The evolution of resistance to Bt cotton imposes significant fitness costs on the pink bollworm^57,58,59 especially reduced fecundity and fertility, which are key traits influencing resistance selection^60,61these are in line with our results. Whereas, development of Cry toxin (Bt) resistance may cause fitness costs for the resistant strain in H. armigera^56,62H. zea⁶³Spodoptera litura⁶⁴ and P. xylostella^22,52. The overall fitness cost of Cry toxin resistance in H. armigera^48,53,61Spinetoram resistance in Spodoptera frugiperda⁶⁵ and Cypermethrin resistant H. armigera⁶⁶ was closely related to several factors, including the egg hatching rate, fecundity, emergence rate, larval survival rate, and the developmental duration to adults.

Reduced egg-laying at higher toxin concentrations in insects may occur because more energy is directed toward detoxification and defense mechanisms, leaving less energy available for reproduction. Another possible factor is the effect of juvenile hormone (JH-III). Studies suggest that higher JH levels under Cry toxin stress might reduce egg production, although the exact mechanism is still unclear. JH plays a key role in the production and uptake of vitellogenin, a protein essential for egg development, and changes in hormone levels can affect fertility⁶⁷. Additionally, JH transferred from males to females may influence reproductive processes like egg development and mating behaviour^68,69. Zhang et al.⁵⁴ found that Cry toxin resistant Helicoverpa armigera had smaller ovaries and less follicle development. This suggests that the lower fecundity observed in PBW may also result from Cry toxin related effects on ovarian development.

Broader ecological and economic threats

The emergence of Cry toxin-resistant strains, particularly in pests like the pink bollworm, poses a complex and far-reaching threat that extends well beyond the immediate confines of Bt cotton fields. Ecologically, resistance disrupts established food web dynamics and natural pest control mechanisms, as predators and parasitoids that depend on susceptible pest populations may decline due to reduced prey availability. This can lead to a rise in secondary pests and greater ecological instability. Additionally, the persistence and potential spread of Cry toxins in the environment may endanger non-target organisms, including beneficial insects such as pollinators and predatory arthropods⁷⁰. Over time, farmers have to spray more pesticides to manage new pests like aphids, spider mites, and lygus bugs that appear after the main pests are controlled. Besides asking farmers about these new pests, we also looked at how much they know about Bt cotton and what they think about its advantages and disadvantages (e.g., effectiveness, productivity, price, and pesticide use) as compared to regular (non-GM) cotton⁷¹. Spillover of resistant pests into non-Bt crops such as chickpea and okra further complicates integrated pest management, necessitating broader and more frequent chemical interventions. Moreover, the survival of resistant PBW increases the risk of gene flow across regions and pest populations, accelerating the evolution of resistance and diminishing the effectiveness of pest control strategies across larger agricultural landscapes⁷².

Economically, these ecological shifts translate into significant financial burdens for farmers. As the efficacy of Bt cotton declines, growers are forced to rely more heavily on costly and often environmentally harmful insecticides to manage resistant pest populations. This not only reduces farm profitability but also erodes the initial technological advantages that Bt cotton once offered, particularly for smallholder farmers⁷³. Despite reports of reduced fecundity in resistant PBW strains, their capacity to inflict major crop losses remains a pressing concern⁷⁴. The declining reliability of genetically modified pest-resistant crops threatens both farmer livelihoods and the broader stability of cotton-based economies, particularly in developing regions.

Biological insights into Bt resistance and management of pink bollworm

The biology of pink bollworm strains under varying concentrations of Cry1Ac and Cry2Ab toxins provides crucial insights into resistance development. Resistant strains exhibit prolonged larval and pupal durations⁷⁴which increase the window of exposure to natural enemies like predators and parasitoids⁷⁵. This suggests a potential for enhanced biological control. Observed reductions in adult longevity, fecundity, and egg hatchability indicate fitness costs associated with resistance⁷⁴. These biological costs can be exploited in resistance management strategies. Complete mortality in susceptible strains at higher Cry toxin doses confirms the continued efficacy of Bt toxins and supports the early-season use of Bt cotton. Growth and survival indices are effective markers for resistance monitoring and can help trigger timely control measures. Additionally, increased mortality and malformations in less resistant or susceptible strains emphasize the importance of sublethal effects in shaping integrated pest management (IPM) strategies. The increasing survivability of resistant strains, particularly under Cry toxin exposure, signals a trend toward evolving resistance and highlights the need for deploying pyramided Bt hybrids and rotating toxins. Although non-Bt refuges are critical, their inadequate implementation in India may be contributing to persistent resistance, pointing to the need for improved compliance and farmer awareness. Fitness trade-offs in resistant PBW - such as lower fecundity and developmental delays -could be leveraged through strategies like Bt-free zones, crop rotation, refuge strategies, targeting weaknesses in resistant strains (encouraging biological control), pyramided Bt toxins, insecticide timing. Bt toxins alone are not sufficient for sustainable pest control, highlighting the need for their integration into broader IPM strategies⁷⁶ that include agronomic practices, biological control, and selective pesticide use. Biological indicators like malformation rates and relative fitness can guide region-specific, adaptive resistance management. Therefore, long-term success of Bt cotton relies on comprehensive, integrated approaches rather than sole dependence on Bt toxins.

Implications for pest management

Effective pest management for PBW in Indian cotton-growing regions requires integrating biological insights with field-level practices and policy enforcement. A global review found 19 cases where pests in specific countries became resistant to Bt crops, reducing their ability to control those pests. Pink bollworm, a major cotton pest, shows different outcomes in the top cotton-producing countries. In the U.S., resistance was delayed and the pest was eventually eliminated using a mix of strategies like planting non-Bt cotton and releasing sterile moths. In China, resistance declined because farmers unknowingly planted hybrid Bt and non-Bt cotton, creating natural refuges⁷⁷. In contrast, India saw widespread resistance due to limited use of non-Bt refuges and now relies on integrated pest management practices⁷⁸ viz., Timely sowing and narrowing the sowing window to disrupt PBW life cycle; Field sanitation including removal of old stalks and partially opened or unopened bolls, to reduce pest carryover; Use of verified Bt cotton seeds to ensure genetic purity and effectiveness; Crop and toxin rotation to delay resistance buildup; Monitoring PBW through pheromone trap catches (8–10 moths for 3 consecutive days) and scouting for 10% flower or green boll damage (Economic Threshold Level); Mass trapping with pheromone traps (> 20 traps/ha) starting at 45 DAS (days after sowing); Ovicidal sprays at 60 DAS to target early pest stages; Two releases of Trichogrammatoidea bactrae @ 60,000 per acre between 75 and 85 DAS; Use of mating disruption tools such as SPLAT or PB rope-L; Application of synthetic pyrethroids after 100 DAS if needed; Timely crop termination to prevent late-season PBW buildup. Policymakers must enforce structured refuge strategies, invest in farmer education, and support innovations such as RNA interference (RNAi) and next-generation Bt traits. A region-specific, data-driven, and collaborative approach is essential to sustaining the effectiveness of Bt cotton and managing resistance in PBW populations.

Research gaps and future directions

While this study provides valuable insights into the fitness costs associated with Cry toxin resistance in pink bollworm, several critical research gaps remain. Long-term evolutionary studies, molecular investigations into resistance mechanisms, and exploration of cross-resistance, as well as environmental factors, are essential for a more comprehensive understanding of how resistance evolves and persists. Additionally, examining cross-resistance and validating findings in field conditions will offer a clearer understanding of the practical implications of resistance in natural ecosystems. Finally, exploring broader fitness costs, beyond just reproductive and survival metrics, will help refine pest management strategies. Addressing these research gaps will improve our ability to manage resistance and develop sustainable, long-term pest control solutions.

Conclusions

This study aimed to assess the fitness costs associated with Cry1Ac and Cry2Ab resistance in pink bollworm (Pectinophora gossypiella) field strains from India. Our results demonstrate that resistant strains, especially those with high resistance to Cry1Ac and moderate resistance to Cry2Ab, exhibit significant biological trade-offs. These include extended larval and pupal development periods, reduced adult longevity, decreased fecundity, lower egg hatchability, and compromised survival metrics across developmental stages. Notably, resistance to Cry1Ac was associated with greater biological penalties than resistance to Cry2Ab. The relatively less resistant strain exhibited the most severe fitness costs in several parameters, suggesting a non-linear relationship between resistance level and biological adaptability. These findings highlight that while resistance provides survival advantages under toxin exposure, it also imposes ecological and reproductive disadvantages. Incorporating fitness cost assessments into resistance monitoring programs can enhance our understanding of the evolutionary dynamics of resistance and improve predictive models of its spread. Furthermore, given the observed strain-specific responses and variable biological impacts, region-specific integrated pest management (IPM) strategies should be prioritized. These may include Cry toxin rotation, reduced toxin exposure levels, or integration with biological control methods to delay resistance development and sustain the efficacy of Cry-based technologies.

Methods

The experiments related to the study “fitness costs and biological adaptability of cry toxin-resistant field strains of pink bollworm from India” were conducted at ICAR-Central Institute for Cotton Research (CICR), Nagpur, Maharashtra (21.0366344, 79.0554520) during 2022–2023. The experimental flow chart is shown in Fig. 15 and the procedures followed are detailed in this section.

Fig. 15

The experimental flow chart for the study of fitness costs and biological adaptability of Cry toxin-resistant field strains of pink bollworm.

Artificial diet preparation for rearing pink bollworm

The in vitro rearing protocol for pink bollworm, as developed by Naik et al. (2022)⁷⁹was followed using a semi-synthetic diet. All ingredients, except agar, yeast, and formaldehyde, were weighed and mixed in a 2000 cm³ container with 600 mL of distilled water, stirring continuously to ensure homogeneity. Separately, a yeast solution was prepared by dissolving dried yeast in 300 mL of distilled water and bringing it to a boil. At the same time, agar was dissolved in another 300 mL of distilled water and gently heated until boiling. The yeast and agar solutions were then combined and cooked together for five to six minutes. This mixture was added to the main container containing the other ingredients and thoroughly blended using a hand-held mixer. The resulting diet was poured into a tray lid, spread evenly, and left to solidify under laminar airflow. Ultraviolet light in the laminar flow chamber was turned on to sterilize the solidified diet. Once set, the diet was cut into small cubes of approximately 1 cm³ and placed into the individual compartments of a multi-cavity plastic tray, where newly hatched pink bollworm larvae were introduced for feeding.

Pink bollworm rearing procedure

The present study was carried out under controlled environmental conditions at the ICAR-Central Institute for Cotton Research (CICR), Nagpur, Maharashtra, during 2022-23. The experimental setup maintained a temperature of 27 ± 2 °C, relative humidity of 65 ± 5%, and a photoperiod of 14:10 h (light: dark).

Pink bollworm larvae were collected from Bt cotton fields using random cotton boll sampling across 59 locations in the major cotton-growing regions of Southern, Central, and Northern India. Field-collected PBW strains were reared separately on toxin-free artificial diet until pupation. Sexing of the pupae was performed using distinct pupal characteristics, specifically the position of the mid-ventral setae on the 9th and 10th abdominal segments, which vary between males and females. Additionally, the distance between the genital and anal pores served as a reliable marker, with females showing more than twice the distance compared to males, as previously reported⁸⁰. The sexed pupae were transferred to emergence cages (55 × 55 × 80 cm) to facilitate adult moth eclosion.

The emerged adults were introduced into mating chambers at a 1:1 female-to-male ratio (5:5), and the process was replicated five times to produce a sufficient number of neonates for the study (Fig. 16). A cotton swab soaked in 10% honey solution served as the adult food source and was suspended in the cage using a thread. For oviposition, females were provided with cotton twigs. These twigs were replaced daily, and the egg-laid twigs were incubated separately in transparent plastic containers (15 × 15 × 30 cm) at room temperature and later placed in a growth chamber under the same controlled environmental conditions (27 ± 2 °C, 65 ± 5% RH). Newly hatched neonates (1-day-old larvae) from each location were subjected to bioassay tests using various concentrations of Cry toxins.

Fig. 16

Experimental setup for rearing resistant and susceptible pink bollworm strains on Cry toxin incorporated artificial diet. (a) Field collected pink bollworm larvae (b) Pupae kept for eclosion (c) Mating chamber (d) Egg incubation (e) Artificial diet incorporated with Cry toxin (f) Transfer of neonates individually on to diet plates.

Preparation of Cry toxin for bioassay

The Cry1Ac protein obtained from the freeze-dried commercial formulation MVP-II® (using the Cell-Cap® encapsulation system by Mycogen, San Diego, California, USA). MVP-II® is a lyophilized version of a liquid formulation containing Cry1Ac encapsulated in Pseudomonas fluorescens. This formulation was found to contain 19.7% (w/w) Cry1Ac protein, as determined by the diet-incorporation method⁸¹. A primary stock solution of Cry1Ac was prepared by vortexing 12.69 mg of MVP-II powder in 10 mL of 0.2% agar solution. From this stock solution, aliquots were taken to prepare working concentrations of 0.01, 0.1, 1.0, 5.0 and 10.0 µg/ mL through serial dilution^82,83.

Similarly, the Cry2Ab protein, derived from transgenic maize leaves, was used as a stable resource in bioassays and global resistance monitoring studies. The Cry2Ab protein comes from the leaf powder of transgenic maize plants (event MON 84006), with 3 mg of Cry2Ab protein per 1.0 g of corn leaf powder. To create the primary stock solution for Cry2Ab, 5.2 mg of maize leaf powder was vortexed in 125 mL of sterile water. Serial dilutions were then performed to produce working concentrations of 0.01, 0.1, 1.0, 5.0, and 10.0 µg/ mL^17,83.

Bioassay on pink bollworm strains from India

Samples from all 59 locations were used to identify highly resistant and relatively less resistant pink bollworm strains through a 21-day diet incorporation bioassay using Cry toxins⁸⁴. Based on the mortality data for different concentrations, probit analysis was carried out using LeOra Software⁸⁵ POLO PLUS^® as given by Finney⁸⁶. The bioassay results revealed the highest LC₅₀ values for Cry1Ac and Cry2Ab from the field strains collected from Nagpur (7.682 and 12.574 µg/ mL, respectively), Adilabad (6.866 and 9.504 µg/ mL, respectively), Jind (6.845 and 7.350 µg/ mL, respectively), Chandrapur (6.839 and 9.181 µg/ mL, respectively) and Guntur (6.755 and 8.823 µg/ mL, respectively) districts. Whereas, relatively lower LC₅₀ values for Cry1Ac and Cry2Ab was recorded in the field strains collected from Banswara (2.449 and 4.228 µg/ mL, respectively), Jodhpur (2.619 and 3.778 µg/ mL, respectively), Sri muktsar sahib (2.804 and 5.007 µg/ mL, respectively), Sriganganagar (2.825 and 4.436 µg/ mL, respectively) and Uttara Kannada (3.088 and 4.852 µg/ mL, respectively) districts⁸⁷.

The surviving larvae from various concentrations of Cry1Ac and Cry2Ab from Nagpur, Adilabad, Jind, Chandrapur, and Guntur were pooled separately to establish highly resistant PBW strains for Cry1Ac and Cry2Ab. Similarly, Banswara, Jodhpur, Sri muktsar sahib, Sriganganagar and Uttara Kannada were pooled separately to establish relatively less resistant PBW strains. The susceptible culture (F₁₉₄ generation) of pink bollworm, sourced from the ICAR-CICR, Nagpur, Maharashtra, has been maintained without exposure to Cry toxin for the past fifteen years.

Cry toxin concentrations used in the biology and fitness cost study

The three concentrations of Cry1Ac (0.1, 1.0 and 10.0 µg/ g of artificial diet) and Cry2Ab (1.0, 5.0 and 10.0 µg/ g of artificial diet) were used to analyse the fitness cost of resistance in highly resistant and relatively less resistant pink bollworm strains. The selection of these Cry toxin concentrations was based on the findings of Badiger et al. ¹⁷ and Shrilaxmi¹⁹ who reported that the field-level expression of Cry1Ac toxin in raw seeds of commercially available Bt cotton hybrids ranges from 0.15 to 1.74 µg/ g^18,20, while Cry2Ab expression ranges from 2.01 to 18.94 µg/ g²⁰.

Biology of resistant pink bollworm

After hatching, 500 neonates each from the highly resistant (F₁ from field-collected larvae), relatively less resistant (F₁ from field-collected larvae), and susceptible (F₁₉₄ from laboratory-reared larvae) strains were carefully transferred using a moist camel hair brush onto artificial diet plates. These diets were incorporated with varying concentrations of Cry1Ac (0.1, 1.0, and 10.0 µg/ mL) and Cry2Ab (1.0, 5.0, and 10.0 µg/ mL) (Fig. 16). The larval rearing plates were then placed in a Biochemical Oxygen Demand (BOD) incubator with controlled conditions of 27 ± 2 °C temperature and 65 ± 5% relative humidity.

The study encompassed the meticulous recording of observations on biological parameters viz., larval duration, pupal period, adult longevity, fecundity, egg hatching (%), growth and survival index. In addition, larval survivability (%), pupation (%), adult emergence (%), adult survivability (%), mortality (%), malformation (%) and fitness cost^28,43 in different life stages were calculated using the following formulae.

$${\text{Larval}}\,{\text{survivability}}(\% ) = \frac{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{surviving}}\,{\text{larvae}}}}{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{larvae}}\,{\text{tested}}}} \times 100$$

$${\text{Pupation}}(\% ) = \frac{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{pupae}}\,{\text{formed}}}}{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{larvae}}\,{\text{tested}}}} \times 100$$

$${\text{Adult}}\,{\text{emrgence}}(\% ) = \frac{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{adult}}\,{\text{month}}\,{\text{emerged}}}}{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{larvae}}\,{\text{tested}}}} \times 100$$

$${\text{Adult}}\,{\text{survivability}}(\% ) = \frac{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{adult}}\,{\text{month}}\,{\text{survived}}}}{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{larvae}}\,{\text{tested}}}} \times 100$$

$${\text{Egg}}\,{\text{hatching}}(\% ) = \frac{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{egg}}\,{\text{hatching}}}}{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{eggs}}\,{\text{kept}}\,{\text{for}}\,{\text{incubation}}}} \times 100$$

$${\text{Mortality}}\,{\text{of}}\,{\text{larvae}}/{\text{pupae}}/{\text{adults}}(\% ) = \frac{{{\text{Total}\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{dead}}\,{\text{larvae}}/{\text{pupae}}/{\text{adults}}}}{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{larvae}}\,{\text{tested}}}} \times 100$$

$${\text{Malformation}}\,{\text{of}}\,{\text{larvae}}/{\text{pupae}}/{\text{adults}}(\% ) = \frac{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{malformation}}\,{\text{larvae}}/{\text{pupae}}/{\text{adults}}}}{{{\text{Total}}\,{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{larvae}}\,{\text{tested}}}} \times 100$$

$${\text{Survival}}\,{\text{index}} = \frac{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{months}}\,{\text{emerged}}}}{{{\text{Total}}\,{\text{number}}\,{\text{of}}\,{\text{neonates}}\,{\text{tested}}}} \times 100$$

$$\:\text{G}\text{r}\text{o}\text{w}\text{t}\text{h}\:\text{i}\text{n}\text{d}\text{e}\text{x}\:=\frac{\text{P}\text{e}\text{r}\:\text{c}\text{e}\text{n}\text{t}\:\text{p}\text{u}\text{p}\text{a}\text{t}\text{i}\text{o}\text{n}}{\text{L}\text{a}\text{r}\text{v}\text{a}\text{l}\:\text{d}\text{e}\text{v}\text{e}\text{l}\text{o}\text{p}\text{m}\text{e}\text{n}\text{t}\:\text{p}\text{e}\text{r}\text{i}\text{o}\text{d}\:\left(\text{d}\text{a}\text{y}\text{s}\right)}$$

If the pupae not eclose after 15 days were considered dead and adult moth died within three days after emergence is regarded as adult mortality. Whereas, the adult moths survived more than three days after eclosion are considered as biologically potential adults (adult survivability). Furthermore, the weight of different life stages was documented. The mean and standard deviation regarding biology and growth parameters of different pink bollworm strains on different concentration of Cry toxin were calculated and were subjected to ANOVA using the WASP - web agri statistical package⁸⁸.

Relative fitness and fitness cost in resistance pink bollworm

The relative fitness was calculated^42,65,89 by dividing the r_m values of the resistant strain by the r_m value of the susceptible strain. The intrinsic rate of population increase (r_m) is calculated based on the formula given by Birch⁹⁰ and fitness cost (C) calculated by using the formula.

$${\text{Fitness}}\,{\text{cost}}({\text{C}}) = \left( {1 - \frac{{{\text{r}}_{{\text{m}}} \,{\text{of}}\,{\text{resistant}}\,{\text{individual}}}}{{{\text{r}}_{{\text{m}}} \,{\text{of}}\,{\text{susceptible}}\,{\text{individual}}}}} \right)100$$