Effect of Rearing Galleria mellonella and Plodia interpunctella Naturally and Artificially on Their Biological Aspects and The Morpho-Biological Features of Trichogramma turkestanica

The present study was initiated to validate whether the artificial or natural diets are the best for Galleria mellonella and Plodia interpunctella hosts for rearing Trichogramma turkestanica Meyer and to assess the effect of reared host eggs on the morpho-biological features of Trichogramma turkesta, nica and their suitability for parasitism. Results for the life tables parameters indicate that the shortest generation time and the lowest doubling time (DT) were recorded Plodia interpunctella which reared artificially ( Plodia A), while the highest net reproductive rate (R 0 ) was recorded for Galleria mellonella which reared artificially ( Galleria A). The artificial diets are better than those for the natural diet for rearing G . mellonella and P . interpunctella and their generation periods in terms of the measured parameters. Results concerning the morphological features of Trichogramma females that were reared on the eggs of Galleria fed on artificial and natural diets were hosts better than Plodia A., Sitotroga N., and Plodia N under laboratory conditions. Also, the obtained data prove that the maximum mean percentage of successful parasitism and the maximum number of emerged female parasitoids were for Galleria A. In comparison, the rate of emerged adults from parasitized eggs was for Plodia A. It is of interest to point herein that the role of the host diet leads to improving the morpho-biological parameters of Trichogramma which can enhance the success of biological control programs as an essential part of Integrated Pest Management (IPM). This contributes to achieving the Sustainable Development Goals (SDGs).


INTRODUCTION
In the current global agricultural scenario, several management tactics are employed, either alone or in association with other procedures to combat insect pests that produce economic damage to different crops.Among the control tactics is biological control.It can significantly reduce the impact of chemical insecticides on the environment and other side effects.The use of parasitoids as biological control agents can be highly effective, particularly, due to their high host specificity (Sani et al., 2016;Cherif et al., 2021).
Recently, Trichogramma species have become the most widely used insect as natural enemy in the world because they are easy to mass-produce and to use against many important crop insect pests.Trichogramma wasps parasitize the eggs of butterflies and moths (Lepidoptera), eggs of flies (Diptera), beetles (Coleoptera), other wasps (Hymenoptera), true bugs (Heteroptera), and lacewings (Neuroptera) (Sarwar and Salman, 2015;Shah et al., 2015).Trichogramma helps in the early control of insect pests before economic damage occurs.Relatively economical mass-rearing and short life cycles make them available for use in several crop protection programs.Also, Trichogramma wasps are used by artificial releases for pest control (Salas Gervassio et al., 2019).It is a valuable alternative for the sustainable control of many insect pests on various crops, such as vegetable and tree crops, as well as stored products (Parsaeyan et al., 2020).
Natural hosts are widely used to rear large quantities of parasitoids providing enough hosts to parasitoids and the time plays a critical role in sustaining the mass-rearing system (King et al., 1985;Wang et al., 2021).Consequently, improving the rearing of the host can be considered as one of the fundamental steps in mass production.
Artificial diets are one of the best ways to obtain standardized insect sources in insect mass-rearing (Vanderzant, 1974;Singh, 1983;Cohen, 2001;and Wang et al., 2021).Evaluation of insect fitness on either natural or artificial diets has been achieved by several recent studies (Chi, 2020;Wang et al., 2021).The importance of a natural and artificial host diets as a key factor influencing the nutritional quality of host eggs and consequently the fitness of reared Trichogramma returns to laboratory studies which found that the body length of Trichogramma adult is dependent on the size of the host egg, which it developed in (Cherif et al., 2021).
So, the objectives of this study are to determine which diets natural or artificial are most effective for rearing Trichogramma turkestanica Meyer in Galleria mellonella and Plodia interpunctella hosts egg.It also aims to evaluate the impact of reared hosts eggs on the morpho-biological characteristics of Trichogramma turkestanica and their suitability for parasitism.

MATERIALS AND METHODS
Two hosts of Trichogramma, i.e., the greater wax moth Galleria mellonella L., (Lepidoptera: Pyralidae), and the Indian meal moth Plodia interpunctella Hb., (Lepidoptera: Pyralidae) were reared naturally and artificially at the Biological Control Lab., Plant Protection Dept., Assiut University, Assiut, Northern Upper Egypt (375 km South of Cairo).

1-Rearing Techniques:
1.1-The Greater Wax Moth, Galleria mellonella (L.): The initial stock of the greater wax moth was collected from different apiaries and transferred to the laboratory.It was reared for at least two generations before experimental studies.Steps of rearing on a natural medium were carried out as explained by Hosamani et al. (2017), while steps of rearing on an artificial diet were done according to Kulkarni et al. (2012).

1.2-The Indian Meal Moth, Plodia interpunctella (Hb.):
The Indian meal moth, Plodia interpunctella, larvae collected from infested dates.The larval stage was reared in plastic containers (1kg) containing 100 g of wheat flour and corn flour.The plastic containers were covered with muslin cloth and fastened with a rubber band.The culture was maintained at 23±2°C, 75±5% RH, and was reared for at least two generations before the experiment.Steps of rearing on a natural diet were carried out as explained by Bouayad et al. (2008) and Predojević et al. (2017), while steps of rearing on an artificial diet were done according to Kulkarni et al. (2012).

2-Effect of Rearing Media on The Immature Stages of G. mellonella and P. interpunctella 2.1-Egg stage:
Eggs (<24 hours old) were kept in separate containers and checked every 24 hours, and the incubation period (in days) and hatchability (%) were calculated.

2.2-Larval Stage:
Newly hatched larvae were transferred into a separate glass tube per larva (7.5×2.5 cm), covered with plastic wrap, and containing (5 g) of the two-host diet daily.Replicas were checked to calculate larval duration and survival percentage.

2.3-Pre-Pupal Stage:
Pre-pupal stages were transferred into a separate glass tube per one (7.5×2.5 cm), covered with plastic wrap.Replicas were checked to calculate pre-pupal duration and survival percentage.

2.4-Pupal Stage:
Newly formed pupae were collected on the same day of pupation, placed in a labeled glass tube (2.0×7.5 cm) (One pupa/tube), and plugged tightly with a piece of cotton, observed daily until adult emergence.Pupal duration and survival percentage were calculated.

2.5-Adult Stage:
Ten newly emerged moths were transferred on the same day of emergence to plastic containers, ten replicates, each with two adults (1♂+1♀).Daily observations were made to record adult longevity and fecundity.

3-Certain Life Table Parameters of G. mellonella and P. interpunctella:
Life table parameters, including gross reproduction rate or net reproduction rate, generation time, doubling time, and rate of increase were calculated according to Carey (1993).

3.1-Net Reproductive Rate (R0) = ∑ lx. Mx:
The ratio of individuals in a population at the start of one generation to the number at the beginning of the previous generation is measured in units of ♀/♀/generation.3.2.Generation Time (T) =∑lx.mx.x/∑lx.Mx: The mean time from the birth of the parent to the birth of offspring measured in days.The time required for a given population to double its numbers is measured in days.

3.5-Finite Rate of Increase (λ) = e rm :
The number of times the population will multiply itself per unit of time, measured in units of female/female/day.x = age of individuals in days lx = number of individuals alive at age (x) as a proportion of one mx = the number of female offspring per female in age interval (x) 4-Collecting and Rearing Trichogramma Parasitoid: The egg parasitoid Trichogramma was collected from Qus province, Qena Governorate (26° 09' 51.05" N, 32° 43' 36.16" E).Trichogramma wasps were collected in glass tubes.It was identified by sequencing the ITS2 region, which matches Trichogramma turkestanica Meyer in the GenBank under the accession number of MW459187.1 (Abdel-Galil et al., 2023).It was raised using the hosts eggs of G. mellonella and P. interpunctella which were artificially and naturally reared for two generations under laboratory conditions (23±2°C, 75±5% RH, L 16:D 8) in the Biological Control Lab., Plant Protection Dept., Assiut University, Assiut, Egypt.

5-Effect of Rearing Trichogramma turkestanica on Different Insect Host's Eggs:
Mounting adults of egg parasitoid T. turkestanica were processed to be permanent specimens for abdominal female measurements using the methods applied by Abdel-Galil et al. (2018) and Abdel-Galil et al. (2023).According to Greenberg and Leppla (2017), fecundity and body length in the mature female of T. turkestanica are positively associated.The body length of T. turkestanica adult females is measured from the front to the tip of the abdomen.However, in the present study, the length of the abdomen was only measured from the front of the abdomen to the tip and the abdominal area measurement to differentiate between T. turkestanica reared on different host's eggs.Also, the measuring unit was changed from (mm) to (μm).

6-Evolution of the Quality of Host's Eggs on T. turkestanica under Laboratory Conditions:
Mated females of T. turkestanica parasitoid were singly transferred to Eppendorf Safe-Lock Tubes (2.0 mL).Fresh eggs from hosts that were reared on both natural and artificial diets, G. mellonella and P. interpunctella, were exposed to the females of T. turkestanica parasitoid (20 eggs /Trichogramma female) (n= 10).Experiments were conducted under incubation conditions of 23±2°C and 70±5 RH % for 24 hours.Female parasitoids were removed from the tubes, and the parasitized eggs were incubated.After five days, eggs were examined for initial parasitism (black eggs).Then, daily examinations were conducted until adult parasitoid emergence.

7-Analysis of Data:
Data were expressed as mean ± SE and were statistically subjected to a one-way analysis of variance by Statistix 8.1 software (Analytical software, 2003).

1-Effect of Natural And Artificial Diets on The Immature Stages:
The design of Integrated Pest Management (IPM) programs depends on characterizing and measuring biological factors, nutritional needs, and insect behavior.These studies are made possible for the insects for mass rearing in laboratories.The diet was able to support development that is comparable to that of natural foods as reported by Marzban et al. (2001), Razazzian et al. (2015), and Kandel et al (2020).
Data in Table 1, show the effect of different diets on the duration of immature stages and longevity of G. mellonella and P. interpunctella.The results indicate that immature stage periods differed in both diets and hosts except for the egg incubation period.The egg incubation period for G. mellonella reared on both nutritional diets was not different from each other (9.68±0.054and 9.86±0.056days for artificial and natural diets, respectively).Also, this period was not different for P. interpunctella (8.85± 0.054 and 8.84±0.054days for artificial and natural diets, respectively).
However, comparing the two hosts, the egg incubation periods were different.The egg duration period is a very crucial criterion for the effectiveness of the parasitoid against the host.At the same time, it's important for parasitoid augmentation in the field, as the incubation period elongates giving the parasitoids more chances of parasitism than a short egg incubation period.These results are very interesting because the egg incubation period for the standard host (Sitotroga cereallela Oliv.) in lab rearing was 5 days, while these hosts recorded nine and ten-day egg incubation periods.The host eggs' age had a bigger effect on parasitism.These results agreed with Paraiso et al., (2012), who found that T. fuentesi parasitized on two-day-old host eggs, which was the most suitable host egg age for parasitism.Concurrently, T. fuentesi females continued the parasitism process until the 13day-old host egg age of Cactoblastis cactorum.In our results, the host with the longest egg incubation period is recommended for Trichogramma.
The longest larval stage period was 32.21±0.20 days for Galleria A, followed by 20.33±0.21 and 17.43±0.19days for Plodia N and Galleria A, respectively.Also, the shortest period was 11.67±0.21days for Plodia A. However, the longest pre-pupal stage period was 5.56±0.107days for Galleria N, followed by 4.33±0.105and 2.16±0.10days for Plodia N, and Plodia A respectively.Also, the shortest period was 1.56±0.097days for Galleria A. In the pupal stage periods, the longest period recorded 16.51±0.16days for Galleria N, followed by 9.09±0.14and 7.9±0.16days for Galleria A and Plodia N, respectively.However, the shortest period was 7.04±0.17days for Plodia A. The longest total immature stage periods for both nutritional diets and hosts were 64.47±0.47daysfor Galleria N, and the shortest period was 29.70±0.36days for Plodia A. These results indicate that Plodia A has the shortest immature stages which means many generations in a short period.Consequently, Trichogramma has many generations in a short period.
The longevity period for G. mellonella reared on both nutritional diets was not different (5.96 and 5.97 days for artificial (A) and natural (N) diets, respectively).Also, this period was not different for P. interpunctella (5.33 and 5.36 days for the artificial (A) and natural (N) diets, respectively).However, when comparing the two hosts, the period was significantly different.
According to the above-mentioned results, the total developmental period of the immature stages of the two hosts reared on artificial diets was shorter than that of the natural diet.Perhaps the components of the artificial diet have a positive effect on the growth and developmental stages of both hosts.Similar results were reported by Kandel et al., (2020) who studied the effect of natural and artificial diets on the life duration of G. mellonella stages.The laboratory incubation period was 8.0±0.1, the larval stage was 37.10±1.4,the pupal stage was 12.41±1.7,and longevity was 25.33±2.8.In this respect, Marzban et al. (2001) recorded that the egg, larval, pupal, and adult developmental periods of P. interpunctella at 27±1 °C, 50-60% relative humidity, on pistachio with an average of 2.5, 31.08, 8 and 7 days, respectively.
Data presented in Table 2, indicate that the highest percentage of hatchability was recorded for Galleria A, Plodia A, and Plodia N (80%), whereas the lowest was recorded for Galleria N (76%).The embryogenesis data show that Galleria A, Plodia A, and Plodia N were the most suitable hosts for the completion of the embryogenesis.The population parameters, such as generation time (T), doubling time (DT), net reproductive rate (R0), the intrinsic rate of increase (rm), and the finite rate of increase (λ) are shown in Table 3.
The longest generation time (T) was recorded for Galleria N (65.91days)> Plodia N (41.85 days) > Galleria A (38.92 days), while the shortest generation time was recorded for Plodia A (30.98 days).The doubling time (DT) was affected by different diets.The descending order of the doubling time was Galleria N (8.77days) > Plodia N (6.41 days) > Galleria A (4.62 days), while the lowest value was recorded for Plodia A (4.08 days).The highest net reproductive rate (R0) was recorded for Galleria A (379.05 days) followed by Galleria N (186.07days), and Plodia A (188.45 days) while the lowest was recorded for Plodia N (94.89 days).The highest Intrinsic rate of increase (rm) was recorded for Plodia A (0.169 days) followed by Galleria A (0.15 days) > N (0.079 days) > and Plodia N (0.108 days).The highest Finite rate of increase (λ) was recorded for Plodia A (1.18 days), followed by Galleria A (1.16 days) > Plodia N (1.11 days) > while the lowest was recorded for Galleria N (1.08 days).
In this respect, Kandel et al (2020) studied the effect of natural and artificial food on the life table parameters of G. mellonella.The net reproductive rate (R0) was recorded 49.4 and 31.5 days.However, mean generation time (T) was 57.4 and 68.4 days.The intrinsic rate of increase (rm) recorded 0.07 and 0.05 days.The finite rate of increase (λ) recorded 1.07-and 1.05-days.Time of population doubling (DT) was recorded 10.5 and 14.7 days.Also, Razazzian et al. (2015) studied the life table parameters of Plodia interpunctella which reared on four commercial pistachio cultivars.He found that the intrinsic rate of increase (rm) ranged from 0.0961 to 0.1382 (female/female/day) on Kalehghouchi and Akbari, respectively.The mean generation time (T) was found to be 35.67 and 31.83days on Kalehghouchi and Akbari, respectively.

Table 3: Certain life table parameters of hosts reared on different diets.
By discussing the above-mentioned results obtained from life table parameters, the artificial diet is better than the natural diet for rearing G. mellonella and P. interpunctella in the laboratory, and the short life cycle in generation periods thus makes them available for using in the biological control programs.4, indicate that the length of the Trichogramma turkestanica female abdomen produced from Galleria eggs (N) measured from the front to the tip reached 322.141±11.92μm.Also, the average abdominal area reached 65291.4±2858.43μm 2 (Fig. 1a), while in Plodia eggs (N), the length of Trichogramma turkestanica female abdomen was 217.64±12.58μmand the average abdominal area reached 37543.72±4961.96μm 2 (Fig. 1b).

The Measurements of Trichogramma turkestanica Female Abdomen from Galleria mellonella and Poldia interponctella Eggs Reared Artificially (A):
Data in Table 4, indicate that the length of the Trichogramma turkestanica female abdomen produced from Galleria eggs (A) was 390.263±7.86μm.Also, the average abdominal area reached 79936.208±1530.53μm 2 (Fig. 1c), meanwhile, in Plodia eggs (A), the length of Trichogramma turkestanica female abdomen was 271.72±5.79μmand the average abdominal area reached 56854.51±416.70μm 2 (Fig. 1d).The above-mentioned results of morphological features of Trichogramma turkestanica female evaluate the efficacy of rearing Trichogramma turkestanica on Galleria mellonella and Plodia interpunctella eggs naturally and artificially under laboratory conditions to select the best host for rearing Trichogramma turkestanica for biological control.
According to Greenberg and Leppla (2017) fecundity and body length in mature female Trichogramma are positively associated.Trichogramma adult females are classified into four quality groups based on the length of their bodies measured from the tip of their abdomen to the front as follows: Class 1 >0.421 mm, Class 2 from0.290 to 0.420 mm, Class 3 from 0.188 to 0.289 and Class 4 <0.187mm (Non-standard).
In the present study, the length of the abdomen was only measured from the front of the abdomen to the tip to differentiate between T. turkestanica reared on different host's eggs.Also, the length of female abdomen measurement is classified into four quality groups as follows: Class 1 > 401μm, Class 2 from 311 to 400 μm, Class 3 from 200 to 310 μm and Class 4 <199 μm (Non-standard).Concerning to the area of female abdomen measurement is classified into five quality groups as follows: Class 1 > 82644μm 2 , Class 2 from 69813 to 82643 μm 2 , Class 3 from 48221 to 69812 μm 2 , Class 4 from 30953 to 48220μm² and Class5 <30952 μm 2 (Non-standard).
Concerning to the area of female abdomen measurement values of five hosts eggs naturally and artificially Galleria A, Galleria N, Plodia A, Sitotroga N, and Plodia N were 79936.2, 65291.4, 56854.51, 43274.3, and 37543.72 μm².So, the Galleria A (79936.2μm²) is considered in class 2 (69813 -82643 μm 2 ).While the Galleria N and Plodia A (65291.4,56854.51μm²,respectively) are considered class 3 (48221 -69812 μm 2 ).However, the Sitotroga N, and Plodia N (43274.3, 37543.72 μm², respectively) are considered class 4 (30953 -48220μm²) By discussing the above-mentioned results concerning morphological features of T. turkestanica females produced from eggs of Galleria A and Galleria N (Artificially and Naturally) is the best host to reared Trichogramma turkestanica under laboratory conditions followed by Plodia A, Sitotroga N, and Plodia N. It is important to point out herein that the size of the host female is an important quality parameter for selecting the best host for mass-rearing Trichogramma in the Biological Control Lab.

3-Host Egg Suitability for T. turkestanica:
The current results show the suitability of the two-host eggs for Trichogramma turkestanica under laboratory conditions (25±2°C, 70±5% RH), as the hosts were reared on different diets, natural and artificial.The hosts were G. mellonella reared on natural wax combs and reared on an artificial diet and P. interpunctella which was reared on whole grain corn flour and the same artificial diet as G. mellonella.
Biological criteria include percentages of parasitism (=successful parasitized eggs), Hatched parasitized eggs, emerged adult parasitoids, and emerged female parasitoids.Twenty eggs per Trichogramma female were replicated ten times.Galleria N, Galleria A, Plodia N, and Plodia A (egg age < 24 hour-old).Data in Table 6, show the effect of the host egg on the percentage of successful parasitized eggs, emerged adult parasitoids from parasitized eggs, and emerged female parasitoids for the tested hosts, Galleria N, Galleria A, Plodia N, and Plodia A. The minimum percentage of successful parasitized eggs for Plodia A was 78.5±13.75%,while the maximum mean percentage of successful parasitized eggs was 94±6.43% for Galleria A. However, the successfully parasitized eggs of Galleria N and Plodia N were 84±12.20%.The percentage of emerged adults from parasitized eggs was affected significantly (F =3.30*) with a maximum mean of 95.39±6.49% for Galleria A. The percentages of emerged adult parasitoids from parasitized eggs for Galleria N and Plodia N were the same (87.35±10.31%),while the minimum for Plodia A was 85.28±12.41%.
Emerged female parasitoids were not affected (F =1.47 ns ).The maximum mean was 69.97±8.85%for Galleria A, followed by Plodia N with a mean of 63.28±4.19%,then Galleria N with a mean of 62.78±3.78%and the minimum was for Plodia A with a mean of 61.79±3.97%.From these results, it can be concluded that the parameter % varied between the tested hosts.So, Galleria A appeared as the best host with successful parasitized eggs, emerged adult parasitoids from parasitized eggs, and emerged female parasitoids.It can compare with the above-mentioned results with those obtained by many authors who use G. mellonella and P. interpunctella for mass rearing of Trichogramma but do not study host-parasite relationships, such as Haque et al. (2021), they study the cold storage-mediated rearing of T. evanescens on eggs of P. interpunctella and G. mellonella.
In conclusion, it is of interest to point out herein that the role of the host diet in the development and on the other morphological parameters of Trichogramma turkestanica can improve the success of biological control programs as an essential part of Integrated Pest Management (IPM).Of which will contribute to achieving the Sustainable Development Goals (SDGs).

Fig. 1 :
Fig. 1: The abdomen of Trichogramma turkestanica female reared on different hosts: a, b Galleria and Plodia eggs nurtured naturally and c, d Galleria and Plodia eggs nurtured artificially, respectively.

Fig. 2
Fig. 2: a) Female abdominal lengths and b) Female abdominal area for the Trichogramma turkestanica hosts (N, hosts reared on a natural diet, A, hosts reared on an artificial diet)

Table 1 :
Duration of immature stages (mean ± SE) and longevity of Galleria mellonella and Plodia interpunctella reared on different diets.
Means in a column followed by the same letter are not significantly different at 0.05 probability level.*: A, hosts reared on artificial diet, N, hosts reared on natural diet.

Table 2 :
Survival of the immature stages of Trichogramma turkestanica hosts reared on different diets.

Effect of Rearing Trichogramma turkestanica on Different Insect Host's Eggs: 2.1. Morphological Features: 2.1.1. The Measurements of Trichogramma turkestanica Female Abdomen from Galleria mellonella and Poldia interponctella Eggs Reared Naturally
(N): Data in Table

Table 5 :
Trichogramma turkestanica female abdominal measurements reared on different insect host's eggs.

Table 6 :
Biological criteria of host's egg suitability for Trichogramma turkestanicaMeans having the same letter in a column are not significant at 5% level of probability according to Duncan's multiple range test.#: A, hosts reared on artificial diet, N, hosts reared on natural diet Ethical Approval is not applicable.Authors Contributions: Dr. Farouk A. Abdel-Galil and Dr. Mervat A. B. Mahmoud made a great effort in the technique of the experiment.Dr. Sara E. Mousa and Ms. Aya A. M. Ahmed made great efforts to breed different the stages of insect hosts and egg parasitoid Trichogramma turkestanica used in this experiment.Dr. Farouk A. Abdel-Galil, Ms. Aya A.