Influence of natural and non-natural diets on the fitness and rearing of Pectinophora gossypiella Saunders

In order to develop integrated management approaches for Pectinophora gossypiella, basic studies are crucial. The two-sex life table is the most important tool for describing the fitness and population parameters of both sexes (male and female) of an insect, while the traditional life table only explains the female sex of an insect. However, no study has reported on the biology of P. gossypiella using two-sex life table tools. Therefore, this study explains the rearing dynamics of P. gossypiella on a cotton seed-based artificial diet and a natural diet (mature cotton bolls). According to the results, the oviposition period of P. gossypiella was recorded to be longer on the artificial diet (9.07 ± 0.24) compared to the natural diet (7.40 ± 0.11). The total fecundity of P. gossypiella was greater on the artificial diet (125.94 ± 3.06) in comparison to the natural diet (60.37 ± 1.10). The population parameters, including intrinsic rate of increase, finite rate of increase, gross reproductive rate, and net reproductive rate of P. gossypiella were highest on the artificial diet in comparison to the natural diet. This study concluded that the cotton seed-based artificial diet was most suitable for the rearing of P. gossypiella. In the future, P. gossypiella may be studied in depth in light of the findings in this study.

www.nature.com/scientificreports/creates a selection pressure for resistant caterpillars.Increasing the number of Bt cotton acres increases its ability to resist the toxin, resulting in a population that is more resistant than before.Because larvae of P. gossypiella are oligophagous or mostly monophagous 9 , its initial stages are more vulnerable to any detrimental effect.e.g., second instar of bollworm for insecticide bioassay.first instar cannot be used due to small size and susceptibility 10,11 .While in case of P. gossypiella, the 1st instar enters inside the cotton bolls from tip within 30-45 min just after hatching 9 .
The P. gossypiella is the most economically harmful pest and feeds on reproductive parts of cotton crops in an inconspicuous manner [12][13][14] .Nearly 4.5 million bales of cotton yield were lost in Pakistan due to their attacks during 2015 and 2016 15 .Furthermore, because of the oligophagous or mostly monophagous nature of feeding on cotton fruiting parts, the P. gossypiella has developed resistance against Bt-toxin [16][17][18] .Practice of growing non Bt cotton as refugia is not prevailing in Pakistan 19,20 .This is the main reason for resistance development in monophagous or oligophagous insects so it can develop resistance easily because genes cannot be diluted with susceptible population compared to those feed on alternative host 14,21 .
The study of basic information of insect pests is necessary for their integrated pest management (IPM) 22,23 .However, the biology and rearing of P. gossypiella have been reported on cotton bolls 24 , okra 25 , artificial diets including cotton seed, chickpea flours 25,26 , Bt and non-Bt cotton bolls 27 , and wheat germ 28 via traditional life table tools.To study the basic information on artificial diet is more convenient.Meanwhile, it is challenging to employ a natural diet, e.g., cotton bolls, throughout the year in laboratory rearing of P. gossypiella.And artificial diet must be have sufficient quality and quantity of protein and nutrients that are essentials for the fitness and reproduction of insects 29 , in other words artificial diet must be alternate to natural host that are easily available to insect in fields.Two-sex life traits are crucial when aiming at gaining a better understanding of a pest, e.g., when the population of pest or predator is vulnerable and resistant [30][31][32] .
Therefore, this study explains the fitness parameters using two-sex life table tools.Due to COVID-19 issues, ingredients of the artificial diet for P. gossypiella have become expensive to import or purchase from developed countries, e.g., the USA and China.In our study, the cotton seed-based artificial diet was prepared for the rearing of P. gossypiella in the laboratory.The ingredients of this diet (Table 1) are easily available and cheaper in local markets in Pakistan.Additionally, this study would be more beneficial for laboratory and field research as well as understanding how to utilize pesticides and biological control agents when the population is at its lowest, most vulnerable, and least resistant.The biology and fitness of P. gossypiella on a cotton seed-based artificial diet compared with a natural diet (cotton bolls) by using two-sex life table tools are, however, lacking.

Biological parameters
There was a significant difference in egg developmental time or incubation period (time between laying and hatching) of P. gossypiella; it was shorter on the artificial diet (2.64 ± 0.09 d) compared to on the natural diet (4.35 ± 0.09 d), as shown in Table 2.However, there was no major difference in the first, second, third, and fourth instars' duration on the artificial and the natural diet.Pupal duration of P. gossypiella was significantly (p < 0.0001) longer on the artificial diet (9.25 ± 0.12 d) than on the natural diet (6.91 ± 0.13 d).Female adult duration was longer than male adult duration on both the artificial and the natural diet.Furthermore, male adult longevity was greater on the artificial diet (15.83 ± 0.4 d) than on the natural diet (14.95 ± 0.43 d).Similarly, female adult

Population Parameters
There was a small difference in the intrinsic rate of increase (r) and the finite rate of increase (ʎ) between the two diets.However, values of r and λ were a bit higher on the artificial diet (r = 0.10, λ = 1.11) in comparison to the natural diet (r = 0.09, λ = 1.09) (Table 3).There was a remarkable difference in gross reproductive rate (GRR ) of P. gossypiella reared on the two different diets; the GRR was significantly greater on the artificial diet (67.54 offspring) than on the natural diet (33.49offspring).There was also a significant difference in the net reproductive rate R 0 (offspring/individual) of P. gossypiella between the artificial and natural diets; R 0 was noticeably higher on the artificial diet (54.16 offspring/individual) compared to the natural diet (27.17 offspring/individual) (Table 3).
There was no significant difference in the mean generation time between the artificial and natural diets (38.31 and 37.86 d, respectively) (Table 3).There was a significant difference in doubling time (DT); it was shorter (6.65 d) on the artificial diet than on the natural diet (7.96 d).There was no significant difference in survival rate (S), birth rate (B), and death rate (R) between the artificial and natural diets (Table 3).

Survival rate
The comprehensive age-stage survival rate (s xj ) of P. gossypiella on different diets was determined (Fig. 1).Findings showed the possibility of a freshly hatched larva surviving to age x and stage j.Because developing rates varied across individuals on different diets, substantial differences in intersecting plotted graphs for different developmental phases were also noted (Fig. 1).The projected curves exhibited completely different layouts at The survival rate of male adults was higher on the artificial diet (0.42 s xj ) than on the natural diet (0.40 s xj ) (Fig. 1).

Age-specific fecundity
The peak noted value of age-stage specific fecundity (f xj ) was higher on the artificial diet compared to the natural diet (Fig. 2).There was a direct relation in age-specific maternity (l x *m x ) of the P. gossypiella (Fig. 2).As survival rate increased, fecundity also increased in both cases (artificial and natural) (Fig. 2).The constant peak points of age-specific maternity (l x *m x ) of the P. gossypiella were 54.15 eggs/d from 50 to 69 d, which was higher than the natural diet (27.16 eggs/d from 46 to 59 d) (Fig. 2).The m x curves indicated that reproduction started comparatively earlier on the natural diet than the artificial diet.The highest value of age-specific fecundity (m x ) was higher on the artificial diet than on the natural diet (Fig. 2).

Life expectancy
The effects of different diets on the population's life expectancy (e xj ) at each stage of P. gossypiella were determined (Fig. 3).The longevity of the newly hatched P. gossypiella eggs was longer on the artificial diet compared to the natural diet (Fig. 3).The e xj was 45.62 d, which was higher than that on the natural diet (42.92 d) (Fig. 3).The peak value of e xj in female adults of P. gossypiella was maximum on artificial diet in comparison to natural diet.Overall, higher e xj of all stages of the P. gossypiella were observed on the artificial diet than on the natural diet (Fig. 3).The age distribution mortality of each stage of P. gossypiella was higher on the natural diet than on the artificial diet as shown in Fig. 5.

Discussions
The biology/fitness of P. gossypiella has been covered on various natural host plants and cotton, corn and wheat based artificial diets using old/simple table analysis in a number of studies 28,[33][34][35] .When study the biology of an insect on artificial diet as compared to growing on natural food, feeding promote fitness reduces the amount of work, time, space, and money needed to cultivate the host plants.Additionally, the availability of artificial food enables easier agreement of insect growth 36 .In the case of P. gossypiella, providing natural hosts all year around is difficult.As an alternative to natural diet, observing the fitness of P. gossypiella can be more convenient with alternative diet.Study of fitness parameters via two-sex life table is the most effective for describing the age-stage dynamic of both male and female insects 30,31,37 : as compared to old life table tools that only give the data on female adults only and cannot explain the age-stage parameter of male and female insects, while the Two-sex life table parameters of P. gossypiella had not been used on cotton-seed based artificial diet.Therefore, the two-sex life table features of P. gossypiella were described in our work for the first time.Food plays the most important role in the growth and developmental rate of the Bactrocera dorsalis (Diptera: Tephritidae) 38,39 .Proteins and other nutrients help insects' development, especially enhancement of insect fecundity, which is why they are important nutritional elements in diets used to mass-raise insect pests in laboratories 40,41 .Female adults of bollworms that were fed on any of the artificial diets, regardless of the protein, are unable to produce viable and sufficient eggs 42 .In our study, cotton seed-based artificial diets enhanced the fecundity and fitness of P. gossypiella.Because cotton seeds are rich source high-quality protein and nutrients 29 .
The incubation period on the artificial diet was shorter than on the natural diet (Table 1).This finding was in contrast to the results of Jothi et al. ( 2016), who reported a longer incubation period of P. gossypiella because of diet ingredients, e.g., cotton seed flour and chickpea flour (processed) 43 .Diet constituents are more important in enhancement of incubation period as well as eggs.The survival of bollworms is more negatively impacted by protein less diets than as compared to carbohydrate deficits.Similarly, another study revealed that the incubation period of P. gossypiella was shorter i.e., 4.9 days on artificial food 35 using the same ingredients and procedure as described by the authors of 43 .Another study showed that the incubation period of P. gossypiella was 4 days on a natural diet (cotton bolls) and 3.89 days when fed on an okra diet 44 .So, these studies reported shorter incubation period of P. gossypiella.These results are in contrast to present findings because we used a different controlled conditions (27 ± 2 °C and 65 ± 5% RH) and cotton-seed based diet that is enrich in protein 42 .
The larval duration of P. gossypiella was in the range of 18-21 days on a chickpea flour-and wheat-grambased diet 28 .However, in another study it was reported that the average larval duration of P. gossypiella was 25 days on an artificial diet 43 .These findings were different to the present study due to different rearing techniques and, mainly, difference of artificial diet ingredients.While on a natural diet, the larval period of P. gossypiella was reported to be about 22 days 24 , which is close to our results of larval duration when fed on mature cotton bolls.
Pupal duration of P. gossypiella was in the range of 8-9 days on an artificial diet 26,28,45 , while another study revealed that the pupal period of P. gossypiella was 16 days 46 .Despite the natural diet, the pupal developmental period of P. gossypiella has been reported as 9-10 days on a natural diet 25,27 , Pupal development and fecundity are important parameter for insect development and fitness 47,48 .while in our study the pupal duration was longer on the artificial diet in comparison to the natural diet.In this study, the fecundity of female P. gossypiella adults was higher on artificial diets.Female adult insects often live longer than male adults, according to the previous literature, which highlights the difference in longevity between the sexes 24,28,44 .Similarly, another study reported that females of Earias vittella (Lepidoptera: Noctuidae) lived longer than males when fed on cotton bolls and okra 49 .Similar results were observed on both foods in this study.
Oviposition period in insects is a significant component in understanding species' presence and population levels 50 .In the current study, we found that the oviposition period of P. gossypiella was highest on the artificial diet in comparison to the natural diet.A different study reported that the oviposition period of P. gossypiella was almost similar to the current study 35 , while another study revealed that the oviposition period of P. gossypiella was shorter than that in the current study because of differences in diet components 25 .Insects' ability to produce healthy larvae or nymphs depends on when they ovulate and their eggs hatch 51 .Different species have different fecundity rates 52 .Egg laying in insects has a significant effect on the dynamics of the insect population 48 .Fecundity depends on the insect food 53 .In our results, we found higher fecundity and longer oviposition period of P. gossypiella when fed on the cotton based artificial diet.Two-sex life table studies help us understand the ecology and fitness of an organism since they account for both sexes while building the right population curve for future populations which will help researcher in designing pest management strategies against any pest 31 .When defining how food affects an insect pest's fitness, reproductive traits r, GRR λ, and R 0 values are crucial 54 .In order to represent how an insect has adapted to a food supply, r may be the most appropriate population index if it is greater than 0. In our study, P. gossypiella's artificial diet with a cotton base resulted in lower mortality and increased rates of reproduction and birth.The immature life span of P. gossypiella did not appear to differ for either diet.Results concluded that the fecundity of P. gossypiella was highest on the cotton seed-based artificial diet as compared to natural diet.Because cotton bolls were not sufficient diet for larvae of P. gossypiella in laboratory environment.Future work on the P. gossypiella in laboratory and fields will be benefit from our study.

Methodology Mass production
The P. gossypiella population (larvae and pupae) was collected from several cotton fields of Khanewal and Kacha Khu (Location: 30° 26′ 11″ N and 72° 13′ 55″ E, respectively).Larvae collected from infested rosette flowers and infested cotton bolls were kept in plastic jars (0.09 × 0.09 × 0.12 m) with cotton seed based artificial diet for pupation covered with muslin cloth.Pupae were placed into a Petri dish of 0.09 m diameter and placed into a cage (0.25 m × 0.25 m × 0.12 m).Adults food containing cotton swabs soaked in aqueous solution of honey and vitamins in equal ratio were given into the cage.Adult diet source were changed daily.After 12 h, cotton shoots about 0.07 m long were fitted in a test tube (0.37 × 1 m) containing water and placed inside the cage for egg laying.The adults of P. gossypiella laid fertilized eggs on shoots having flowers and leaves spread in the cage.Eggs of pink bollworm are whitish green color.Two stock cultures of P. gossypiella were maintained in the laboratory conditions (27 ± 1 °C, 65 ± 5% R.H.) separately on dissected locules of mature cotton boll and artificial diet.Both of cultures were raised at least for two generations to acclimatize on food and in laboratory conditions.

Life traits on natural diet
One hundred eggs of P. gossypiella laid on small cotton shoots were collected and allowed to hatch.After egg hatching (2-4 days), a jar was placed upside down on a table.A piece of white paper was placed on the table to observe the 1st instar larvae.Newly hatched larvae were fed singly on a locule of non-Bt cotton (CIM-717) bolls.All bolls were washed with simple water followed by drying with tissue paper for 5 min.After that, cotton bolls were placed in a jar containing 0.1% formalin solution (for 2 min), following the methodology of Fand et al. 24 .All bolls were washed again with simple water.A small hole measuring 1 mm in diameter was made in each cotton boll with the help of needle.Holes should be about 1-2 cm long so larvae can easily live in the burrow-like holes and also feed with ease.First instar larvae were put through the holes into the cotton bolls.When larvae hatch from eggs, both of male and female' first instar larvae are white in color having brown head.Third instar larvae have visible pink marks with creamy to ivory body.Larvae went through four growth stages (instars), starting to turn pink in the fourth instar.While, the male larvae (in 3rd instar) have two light spots on dorsal side of the abdomen, while in 4th instar these two spots becomes darker.One hundred larvae were observed till adult formation.Mature cotton bolls were replaced after 2 days.The bolls were first gently washed with tap water to remove any inherent dirt, then disinfected by dipping in a 0.1% formalin solution for two to three minutes to prevent any secondary infections, further rinsed with tap water to remove any remaining formalin, and finally shade dried in an open space.Each jar contained one cotton boll and was covered with muslin cloth.Larval instars were checked after every two days by opening the cotton boll with sharp knife.Cutting is done in the line of locule that was totally safe for immature of P. gossypiella.Pair of male and female pupae of P. gossypiella were separated and placed into the jars (0.18 × 0.18 × 0.16 m) 55 .The cotton shoots about 0.07 m long (for egg laying) were fitted in a test tube (0.37 × 1 m) containing water and placed inside the cage; replaced daily after counting eggs under the stereo microscope.In total, 10 pairs were used to observe the reproductive parameters, i.e., mating, pre-oviposition, oviposition, and fecundity.Data were recorded on a daily basis until the death of the male and female parents.

Life traits on the artificial diet
The cotton seed-based artificial diet was prepared following the methodology of Ihsan et al. 28 , with some modifications as follows: fraction (A): cotton seed flour, chickpea flour, and sucrose were mixed in 200 mL water, fraction (B): agar-agar was boiled in water with continuous stirring, and fraction (C): basin salt, streptomycin sulphate, dried yeast powder, methyl 4-hydroxy benzoate, multivitamin, casein, cystiene, wesson salt, ascorbic acid, and sorbic acid were mixed.After preparation of these three fractions, they were mixed together in a plastic tray and put in a refrigerator.The exact quantity of diet components was measured using an electronic weight balance as mentioned in Table 1.
One hundred eggs from acclimatized population of P. gossypiella that were reared on artificial diet were collected.Newly hatched larvae were singly released on artificial diet in petri-dishes of 0.5 m diameter each covered with lids having aeration holes.The lids were sealed with the help of parafilm warped around.Diet was replaced after every 48 h.Data of life table traits regarding larval instars and pupal duration were recorded daily.The pupae were placed in glass jars and moths emerged were allowed to mate (Supplementary material).Ten pairs of P. gossypiella were isolated and kept in glass jars with liquid food.Some important life traits like durations of mating, pre-oviposition and oviposition along with fecundity were recorded.

Statistical analysis
Means of each biological parameter for P. gossypiella were analyzed in the SPSS Statistics 15.0.The TWOSEX program was used to assess the P. gossypiella, age-stage-specific survival rate (S xj ), age-stage life expectancy (e xj ), age-specific fecundity (m x ), age-specific survival rate (l x ), and age-stage reproductive value (v xj ), as well as criteria for the life table (finite rate of increase r, net reproductive rate R 0 , mean generation time T, and number of stages k) 56 .
Equations of l x , m x , and R 0 are as follows, (1) where k is no. of stages.In this study, the Euler-Lotka formula's iterative bisection approach was used to estimate the r, with the age index starting at 0 as in Eq. ( 2) 57 .
Finally, the bootstrap method was used to determine two-sex life table traits 58 .Using the TWOSEX-MS-Chart application, 200,000 new samples of the bootstrap method were run 37 .

Table 1 .
Ingredients used for artificial diet of P. gossypiella.

Table 2 .
Stimulus of artificial and natural diets on the biological parameters of P. gossypiella.d: days, n: no. of individuals, APOP: adult pre-oviposition period of a female adult, TPOP: total pre-oviposition period of a female counted from emergence.The standard errors of the mean values of each biological parameter were estimated using two-sex life table software.The independent t-test was used to estimate the difference between the two treatments (artificial and natural diets) showed by different lower-case letters.And significantly higher mean values are represented in bold.

Table 3 .
Impact of artificial and natural diets on the reproductive and population parameters of P. gossypiella.r: intrinsic rate of increase, λ: finite rate of increase, GRR : gross reproductive rate (offspring), R 0 : net reproductive rate, SASD: stable age-stage distribution; d: day.Higher mean values are represented in bold.Vol:.(1234567890)Scientific Reports | (2023) 13:13666 | https://doi.org/10.1038/s41598-023-40712-6 each developmental stage.Individual survival rates rapidly dropped as age increased, and it showed an inverse relationship between survival and age (Fig. 1).The developmental time of female adults of P. gossypiella was longer on the artificial diet (36-49 d) in comparison to the natural diet (36-45 d), while the survival rate of female adults was shorter (0.43 s xj ) on the artificial diet in comparison to the natural diet (0.45 s xj ).In the case of male adults, the developmental time was shorter (31-38 d) on the artificial diet in comparison to the natural diet (32-40 d).
Age-stage reproductive value (v xj ) explains an individual's contribution to the future population (i.e., the population forecasting scale) at age x and stage j.Results showed that reproductive value v xj significantly increased (79.92 v xj on 33 d) on the artificial diet and also increased (40.37 v xj on 33 d) on the natural diet.However, the results revealed that the age-stage reproductive value (v xj ) of the P. gossypiella is higher on the artificial diet than on the natural diet.The artificial diet had more positive effects on the reproductive value (v xj ) of P. gossypiella (Fig.4).Stimulus of artificial and natural diets on the age-stage-specific life expectancy (e xj ) of the P. gossypiella.L1: 1st instar larvae, L2: 2nd instar larvae, L3: 3rd instar larvae, L4: 4th instar larvae.