Biological attributes of Hadronotus pubescens (Motschoulsky) (Hymenoptera: Scelionidae) reared on refrigerated eggs of Riptortus pedestris Fabricius (Hemiptera: Alydidae)
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Abstract Hadronotus pubescens (Motschoulsky) (Hymenoptera, Scelionidae) is a type of wasp that lays its eggs in the eggs of Riptortus pedestris Fabricius (Hemiptera: Alydidae), which is a major pest for legume crops in Korea, Japan, and Bangladesh. This study assessed the quality of refrigerated R. pedestris eggs in relation to host-accepting behaviors and biological characteristics, including parasitism rate, development duration, emergence rate, sex ratio, and longevity. The hatchability of the refrigerated eggs was evaluated. Riptortus pedestris eggs that were refrigerated for over 30 days failed to hatch entirely. The refrigeration influences the average duration of each host acceptance behavior (i.e., drumming, drilling, oviposition, and marking) on host eggs. The number of parasitized host eggs per female, the male proportion, and the emergence rate of H. pubescens were unaffected by chilling for up to 30 days in comparison to fresh eggs. Moreover, the refrigerated host eggs did not influence the reproduction of the emerged adult parasitoids or the growth of their offspring. The longevity of H. pubescens was not influenced by the refrigeration of R. pedestris eggs. The refrigeration of host eggs for up to 30 days did not adversely affect any biological features of the second generation of H. pubescens. So, keeping H. pubescens eggs in the fridge can be a good way to produce a lot of the parasitoid, and the host eggs that are no longer alive after being stored in the cold can be used in the field to increase parasitism.
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Biological attributes of Hadronotus pubescens (Motschoulsky) (Hymenoptera: Scelionidae) reared on refrigerated eggs of Riptortus pedestris Fabricius (Hemiptera: Alydidae) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Biological attributes of Hadronotus pubescens (Motschoulsky) (Hymenoptera: Scelionidae) reared on refrigerated eggs of Riptortus pedestris Fabricius (Hemiptera: Alydidae) Md. Rasel Raju, Mst. Arifunnahar, Md. Alamgir Hossain, Md. Abdul Alim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6958307/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 15 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 12 You are reading this latest preprint version Abstract Hadronotus pubescens (Motschoulsky) (Hymenoptera, Scelionidae) is a type of wasp that lays its eggs in the eggs of Riptortus pedestris Fabricius (Hemiptera: Alydidae), which is a major pest for legume crops in Korea, Japan, and Bangladesh. This study assessed the quality of refrigerated R. pedestris eggs in relation to host-accepting behaviors and biological characteristics, including parasitism rate, development duration, emergence rate, sex ratio, and longevity. The hatchability of the refrigerated eggs was evaluated. Riptortus pedestris eggs that were refrigerated for over 30 days failed to hatch entirely. The refrigeration influences the average duration of each host acceptance behavior (i.e., drumming, drilling, oviposition, and marking) on host eggs. The number of parasitized host eggs per female, the male proportion, and the emergence rate of H. pubescens were unaffected by chilling for up to 30 days in comparison to fresh eggs. Moreover, the refrigerated host eggs did not influence the reproduction of the emerged adult parasitoids or the growth of their offspring. The longevity of H. pubescens was not influenced by the refrigeration of R. pedestris eggs. The refrigeration of host eggs for up to 30 days did not adversely affect any biological features of the second generation of H. pubescens . So, keeping H. pubescens eggs in the fridge can be a good way to produce a lot of the parasitoid, and the host eggs that are no longer alive after being stored in the cold can be used in the field to increase parasitism. Biological sciences/Biological techniques Biological sciences/Ecology Egg parasitoid Cold storage Stink bug Mass rearing Country bean Figures Figure 1 Figure 2 Figure 3 Introduction The bean bug, Riptortus pedestris (Fab.) (Hemiptera, Alydidae), is a major pest affecting soybean crops in East Asia 1 and has also been responsible for infestations in legume crops in Bangladesh 2,3 . It poses a serious threat to soybeans ( Glycine max (L.) 4,5 and fruit trees 6 . Riptortus pedestris attacks many cereal crops, such as sorghum ( Sorghum bicolor ), foxtail millet ( Setaria italica ), and barley ( Hordeum vulgare ) during their reproductive phases 6,7 , along with other field crops throughout Asia 8,9 . Adults and nymphs of this insect primarily target seeds, feeding through piercing and sucking, and their capacity to utilize various host plants increases the vulnerability of crops to damage 5 . The insects drain sap from seed pods, leading to incomplete pod maturation, browning, shrinking, and death. When pods are opened, seeds are often found to be shriveled, underdeveloped, or deformed. These damaged seeds frequently suffer from secondary fungal infections, such as yeast-spot disease caused by Eremothecium coryli 10 , which can devastate an entire crop 11 . Farmers typically apply synthetic chemical insecticides to manage R. pedestris infestations, but this approach is not always cost-effective for lower-value crops like mung beans ( Vigna radiata ) in Bangladesh. Chemical insecticides play a role in integrated pest management (IPM) due to their rapid action and ease of use 12 , but they can harm beneficial species, lead to pest resurgence, and have other negative environmental impacts 13 . As a result, the high cost of chemical pesticides may render them unfeasible, especially when combined with environmental concerns. While these chemicals provide rapid action, the mobility of R. pedestris makes its control more challenging 5,9 . To tackle this issue, cultural practices such as delayed planting and the use of resistant varieties have been suggested for managing stink bugs 14,9 . Additionally, biological control methods involving the introduction or enhancement of egg parasitoids have proven effective against stink bugs 15-17 and could provide a viable option for controlling R. pedestris in Bangladesh 2 . Cold storage is commonly employed to improve efficiency and provide flexibility in the mass rearing of insects for biological control programs 18 . The use of low temperatures to store host eggs is well-established in the rearing of parasitoids for stink bugs, including R. pedestris 16,19,20-22 . Studies have shown that refrigerated eggs of R. pedestris are suitable for parasitism by Gryon japonicum 22,23 . Hadronotus pubescens (Motschoulsky) (Hymenoptera: Scelionidae) is a solitary egg parasitoid that has been reported in Sri Lanka, India, particularly in Karnataka, and Bangladesh 24,2 . The biological parameters of H. pubescens on refrigerated R. pedestris eggs remain unknown, with no existing published data on the effects of host egg refrigeration on this parasitoid. Raju et al. 2 were the first to document the presence of H. pubescens in Bangladesh and studied its biological traits on R. pedestris eggs under laboratory conditions. In this study, we investigated how refrigerating R. pedestris eggs affects parasitism by H. pubescens in a lab environment. Specifically, we examined the number of parasitized eggs, parasitoid emergence, sex ratio, development time, longevity, and reproduction across the first and second generations of H. pubescens using refrigerated host eggs. Results Survival of refrigerated R. pedestris eggs. The hatching percentage of R. pedestris eggs was significantly affected by the duration of refrigeration ( χ 2 = 87.10, df = 6, P < 0.001). Survival decreased from 99% from the fresh (unrefrigerated) eggs to 7% from the eggs refrigerated for 25 days. In this study, no eggs hatched after 30 days of refrigeration due to loss of viability (Fig. 1 ). Effect of refrigeration host egg on host acceptance behavior of H. pubescens. All H. pubescens exhibited complete host acceptance behaviors toward the refrigerated host eggs, with the duration of these behaviors detailed in (Table 1 ). The duration of drumming increased with longer refrigeration times ( H C = 55.02, df = 5, P < 0.001). The eggs of R. pedestris refrigerated for 120 days showed the longest drumming duration by the parasitoid. Following the drumming, the female parasitoid begins drilling by inserting her ovipositor into the host eggs. The average time spent on oviposition increased for host eggs stored at various refrigeration periods compared to fresh eggs (unrefrigerated) ( H C = 56.57, df = 5, P < 0.001). Additionally, H. pubescens spent more time on marking behavior for eggs refrigerated for 30 days compared to fresh eggs (unrefrigerated) ( H C = 52.64, df = 5, P < 0.001). Table 1 Duration (Seconds ± SE) of host acceptance behaviors in different ages of refrigerated R. pedestris eggs Behaviors Age of host eggs (days) 0 (Control) 30 45 60 90 120 Drumming 38.32 ± 0.72 f 43.11 ± 0.51 e 50.44 ± 1.03 d 54.53 ± 0.73 c 60.22 ± 1.08 b 66.13 ± 1.00 a Drilling and Oviposition 415.14 ± 0.94 e 439.82 ± 1.49 d 494.48 ± 1.36 c 553.6 ± 1.49 b 591.85 ± 1.26 a 596.81 ± 0.82 a Marking 138.31 ± 1.01 e 173.21 ± 1.06 a 162.81 ± 1.86 b 154.65 ± 0.94 c 147.77 ± 1.19 d 143.19 ± 1.43 de Values in the same row with the same letter are not significantly different ( P > 0.05). See text for statistics. Effect of refrigerated host eggs on the biological parameters of H. pubescens. The number of host eggs parasitized by H. pubescens varied depending on the duration of refrigeration. For up to 30 days, H. pubescens can effectively parasitize host eggs without a notable decrease in parasitism. However, for the host egg of 120-day refrigeration time period, the parasitized host eggs decreased approximately 47.35% ( H C = 38.71, df = 5, P < 0.001) compared to the control (Table 2 ). The emergence rate ( χ 2 = 45.41, df = 5, P < 0.001) was significantly affected by host eggs refrigeration time, while the sex ratio ( χ 2 = 5.78, df = 5, P = 0.328) of H. pubescens was not significantly affected by host eggs refrigeration time (Table 2 ). The emerged parasitoids exhibited female-biased sex ratios, with no statistically significant differences observed across the different time periods. However, the overall emergence of parasitoids from the parasitized eggs significantly declined as the refrigeration duration increased (H C = 45.41, df = 5, P < 0.001). Mean development time (from eggs to adult) of H. pubescens male and female increased significantly as host eggs aged increased (male H C = 72.66, df = 5, P < 0.001; Female H C = 240.36, df = 5, P < 0.001) (Table 2 ). Also, less development time finds in control (0 day) whereas 120 days of refrigeration showed the highest mean time for development. The longevity of H. pubescens male and female showed significant decline following refrigeration of the host eggs for 120 days (male H C = 70.68, df = 5 P < 0.001; Female H C = 183.51, df = 5 P < 0.001) (Fig. 2 ). The highest longevity was found in 0 days of age compared to others. Table 2 Effect of refrigerated host eggs on the biological parameters of H. pubescens Age of host eggs (days) 0 30 45 60 90 120 Number parasitized host eggs / female ± SE 8.30 ± 0.37 a 8.00 ± 0.47 a 6.70 ± 0.21 b 6.10 ± 0.18 b 5.90 ± 0.23 b 4.38 ± 0.26 c Proportion of male 0.16 (13/83) a 0.20 (16/80) a 0.19 (13/67) a 0.24 (15/61) a 0.20 (12/59) a 0.34 (12/35) a Emergence rate 0.72(72/100) a 0.67(67/100) a 0.60(60/100) ab 0.50(50/100) b 0.51(51/100) b 0.30(30/100) c Development time, days ± SE Male 25.08 ± 0.18 e 25.94 ± 0.06 e 27.77 ± 0.17 d 33.13 ± 0.09 c 36.67 ± 0.33 b 43.33 ± 0.14 a Female 26.13 ± 0.11 e 26.17 ± 0.05 e 28.78 ± 0.12 d 33.72 ± 0.10 c 37.26 ± 0.07 b 43.91 ± 0.17 a Values in the same row with the same letter are not significantly different ( P < 0.05). See text for statistics. Effect of second-generation host eggs on the biological parameters of H. pubescens . The number of parasitized eggs by H. pubescens was not influenced by the progeny of different ages host eggs in second generation (Table 3 ). The progeny emerged from 120 days of refrigerated eggs, the parasitized host eggs decreased about 16.85% (H C = 14.03, df = 5, P < 0.015) compared to the control (Table 3 ). The emergence rate ( H C = 9.32, df = 5, P = 0.096) and the sex ratio ( χ 2 = 8.75, df = 5, P = 0.119) of H. pubescens were not influenced by the age of host eggs (Table 3 ). The emerged parasitoids exhibited a female-biased sex ratio across all refrigeration periods, although these differences were not statistically significant. The development time (from eggs to adult) of H. pubescens male and female increased after 60 days refrigerated eggs of progeny (male H C = 26.15, df = 5, P < 0.001; Female H C = 227.05, df = 5, P < 0.001) (Table 3 ). In this study, the highest mean development time was found as 120 days of age while the lowest in control (0 day). The longevity of H. pubescens males and females decreased after 45 days of refrigerated eggs progeny (male H C = 54.25, df = 5, P < 0.05; Female H C = 257.16, df = 5, P < 0.05) (Fig. 3 ). The highest longevity was found in 0 hour (control) of age compared to others. Therefore, refrigerating host eggs for up to 60 days can effectively facilitate the parasitization of R. pedestris by H. pubescens without negatively affecting the second generation of the parasitoid. Table 3 Effect of second-generation host eggs on the biological parameters of H. pubescens Age of the host eggs (days) 0 30 45 60 90 120 Number parasitized host eggs / female ± SE 8.90 ± 0.28 a 8.40 ± 0.50 ab 8.20 ± 0.36 ab 8.10 ± 0.41 ab 7.50 ± 0.34 ab 7.40 ± 0.31 b Proportion of male 0.12(11/89) a 0.08(7/84) a 0.19(16/82) a 0.12(10/81) a 0.08(6/75) a 0.06(5/74) a Emergence rate 0.87(87/100) a 0.80(80/100) a 0.79(79/100) a 0.77(77/100) a 0.73(73/100) a 0.71(71/100) a Development time, days ± SE Male 14.27 ± 0.24 c 14.57 ± 0.20 c 15.0 ± 0.13 bc 15.10 ± 0.09 b 15.67 ± 0.49 ab 15.80 ± 0.37 a Female 14.74 ± 0.07 c 14.68 ± 0.08 c 15.29 ± 0.10 b 15.34 ± 0.06 b 15.65 ± 0.06 a 15.75 ± 0.07 a Values in the same row with the same letter are not significantly different ( P < 0.05). See text for statistics. Discussion The length of refrigeration had a significant impact on the viability of R. pedestris eggs; after 30 days, the eggs completely failed to hatch because of their diminished viability. According to Alim and Lim 22 , R. pedestris eggs that are refrigerated for 30 days lose their viability. Similarly, Chen and Leopold 28 found that eggs of Homalodisca coagulata (Say) were killed after 5 days at 2°C chilling. Additionally, Mahmoud and Lim 21 discovered that Dolycoris baccarum L. egg hatching was not observed following 20 days of storage at 2.1°C. The refrigeration time of R. pedestris eggs significantly influences the behavioral responses of H. pubescens . The prolonged drumming duration was observed with increased refrigeration time. This may be attributed to alterations in the physical structure of the refrigerated host eggs as a result of desiccation 19 , 28 , leading the parasitoids to spend more time evaluating the host eggs. According to Mahmoud and Lim 21 , Trissolcus nigripedius Nakagawa displayed extended drumming behavior when interacting with refrigerated eggs of Dolycoris baccarum . Strand and Vinson 25 described that Telenomus heliothidis (Ashmead) drumming on Heliothis virescens (F.) eggs is an essential step in determining host acceptance. Drumming is characterized by the parasitoid moving its antennae up and down across the exposed surface of the eggs while remaining still or standing over them. Likewise, the longer oviposition period for refrigerated eggs demonstrates the parasitoids' capacity to adjust to host quality variations, possibly suggesting a compensation mechanism that overcomes the effects of refrigeration on host egg alterations. The slight rotation of the abdomen as the ovipositor enters the chorion is known as drilling 25 . During oviposition, the females remain still with their heads in a pumping motion, wings aligned parallel to their bodies, and hind legs firmly gripping the host egg 25 . After head pumping, the female stays motionless for 5–10 seconds until oviposition is complete. Notably, the extended marking behavior observed for eggs refrigerated for 30 days might reflect a preference or enhanced effort to secure those eggs as a resource. Since host marking behavior is influenced by the quality of host eggs 29 , the prolonged marking time would suggest that host eggs that have been chilled for 15 or 30 days are more suitable for parasitoids than fresh eggs. The female walks rearward after removing the ovipositor from the egg surface while swinging the posterior part of her abdomen laterally to brush along the eggs surface. This behavior, known as marking 30 , indicates the end of oviposition 31 , 15 , 32 . The parasitism rate of H. pubescens remained stable for host eggs refrigerated up to 30 days but showed a significant decrease for eggs refrigerated for 120 days. Refrigeration duration strongly impacted emergence rates, which decreased with extended refrigeration, though the sex ratio remained consistently female-biased. Development time for both male and female parasitoids increased with egg refrigeration duration, peaking at 120 days, while longevity was highest at 30 days but significantly reduced for 120-day-old eggs. Leopold 18 reported refrigeration might affect the physiological process of the host egg that is deleterious for the development of parasitoid. The significant variations in the quantity of parasitized host eggs, the total number of parasitoids, and the development time may be attributed to a reduction in the nutritional quality of eggs that have been refrigerated for extended durations 20 , 28 , 22 , 23 . The parasitization and emergence rates of H. pubescens were unaffected by the progeny of host eggs refrigerated up to 60 days, with a consistent female-biased sex ratio. However, the mean development time and longevity of the parasitoids significantly increased and decreased, respectively, after 60 days of refrigeration. Similar patterns were observed by, Alim and Lim 23 , Mahmoud and Lim 21 in a gregarious and scelionid parasitoid and by Chen and Leopold 28 in a mymarid parasitoid. The low nutritional quality attributed to older host eggs can slow down offspring development rate 29 . The parasitization rate and emergence of H. pubescens were not significantly influenced by the age of refrigerated host eggs in the second generation, with a consistent female-biased sex ratio. However, mean development time increased, and longevity decreased significantly after 45 days of refrigeration. Alim and Lim 23 also found that the longevity of Ooencyrtus nezarae Ishii females decreased after 60 days of refrigeration for the progeny of eggs. Our study indicates that H. pubescens can parasitize R. pedestris host eggs refrigerated at 2.0˚C for up to 30 days without a significant decline in parasitism rates. Additionally, refrigeration of host eggs did not impact the reproductive capabilities of adult parasitoids that developed from these refrigerated hosts (i.e., the second generation). Thus, refrigerating R. pedestris eggs could facilitate the mass rearing of H. pubescens . Additionally, these refrigerated eggs can be introduced into the field to enhance parasitoid populations, as they remain viable for oviposition for several days without resulting in the emergence of the pest, R. pedestris 33 . Methods Rearing of R. pedestris. Adults R. pedestris were collected from country beans fields ( Lablab purpureus L.) located in Hajee Mohammad Danesh Science and Technology University (HSTU) Agriculture Research field, Dinajpur, People’s Republic of Bangladesh (25° 41' 51'' N, 88° 39' 15'' E). R. pedestris were reared and maintained in the laboratory following the method of Raju et al. 2 . Both nymphs and adults were raised in acrylic cages (40L × 40W × 40H cm), featuring windows on three sides covered with mesh screens to allow for ventilation. The insects were raised under stable conditions of temperature (31.62°C ± 1) and relative humidity (79.25% ± 1.60), following the natural photoperiod. Breeding dishes containing dissolved ascorbic acid pellets along with soybean seeds were provided to the adult R. pedestris , while nymphs were sustained on a diet of soybean seeds and bean plants ( L. purpureus L.) with cotyledonous leaves. Oviposition substrates were created by positioning four pieces of gauze fabric in the upper and bottom corners inside the adult cages, with the fabric being replaced daily and the eggs collected for colony maintenance. The collected eggs were stored in zipper plastic bags at a temperature of 2.0 ± 0.7°C in a refrigerator for experiments. To record the temperature and relative humidity a data logger was used (U10-001; Onset Computer Corporation, Bourne, MA, USA). Rearing of H. pubescens. H. pubescens was obtained from naturally parasitized eggs of R. pedestris found in the same bean fields described earlier. These H. pubescens specimens were then raised on R. pedestris eggs in the same laboratory conditions described in the previous section. The identification of the parasitoid was conducted in accordance following the method outlined in Raju et al. 2 . Both male and female H. pubescens individuals were reared in 50 mL plastic centrifugal tube (SPL Life Sciences Co. Ltd., Korea), where they were provided with a surface layer of honey and a piece of moistened cotton at the bottom of the tube. The moistened cotton and honey were refreshed every three days. Survival of R. pedestris refrigerated eggs. Eggs of R. pedestris were kept in the zipper plastic bags and placed in a refrigerator on days 0, 10, 15, 20, 25, and 30 at 2.0 ± 0.7°C. After refrigeration, 50 host eggs from each refrigeration period were removed to a Petri dish (4 H × 10 D cm) and were kept in laboratory condition at 31.62°C ± 1, 79.25% ± 1 RH. Survival was quantified by calculating the proportion of hatched eggs to total eggs examined. Effects of host egg age refrigeration on host acceptance behavior by H. pubescens . After refrigeration for 0, 30, 45, 60, 90, and 120 days, 10 R. pedestris eggs from each time point were taken out and transferred to a (4 H × 10 D cm) Petri dish at room temperature. A single 4-day-old mated, and naive female H. pubescens was then introduced into each dish. Host acceptance behaviors were monitored under a stereomicroscope (Luxeo 6Z, LaboMed, USA) and classified into categories including drumming and drilling, oviposition and marking 25 , 26 . The duration of each behavior was recorded for eggs that elicited a full host acceptance response from the parasitoid. This procedure was repeated for 10 female parasitoids at each refrigeration interval. Effect of refrigerated host eggs on the biological parameters of H. pubescens . Host eggs refrigerated at 2.0 ± 0.7°C for the different time periods (0, 30, 45, 60, 90 and 120 days), 10 R. pedestris eggs on each period were removed from the refrigerator and placed individually in a petri dish (4 H × 10 D cm) for one hour in laboratory condition. A 4-day old, mated and inexperienced female H. pubescens was introduced and permitted to parasitize the eggs for 24 hours at 31.62°C ± 1, 79.25% ± 1 RH and then removed. The number of parasitoids that emerged from every egg was counted every day. The number of host eggs parasitized host eggs, sex ratio, emergence rate, longevity and development time of both male and female H. pubescens was recorded. Host eggs that contained parasitoids that did not develop or emerge were also classified as parasitized based on the dissection of each egg. These procedures were repeated ten times for each storage period. The parasitoid was collected and placed in a parasitoid rearing tube. Every three days, fresh honey and moistened cotton were given, and the parasitoids were reared by transferring them to new tubes as needed. Effect of second-generation host eggs on the biological parameters of H. pubescens . The impact of refrigerated host eggs on the parasitoid of second generation was evaluated. A 4-day old, inexperienced and previously mated, 10 female H. pubescens were randomly selected from the group of adult parasitoids that emerged from host eggs refrigerated for various durations (0, 30, 45, 60, 90 and 120 days). After that, each parasitoid was provided with 10 R. pedestris fresh eggs for 24 hours under the same laboratory condition and then removed after 24 hours. After 24 hours, the female H. pubescens was withdrawn, and the eggs were placed individually in 2 ml microtubes containing a drop of honey. The emergence of parasitoids was observed daily. The number of parasitized host eggs, sex ratio, adult emergence, development time and longevity of male and female of H. pubescens were observed. Host eggs that contained parasitoids which did not develop or emerge were also classified as parasitized based on the dissection of each egg. For every storage period age, these processes were repeated ten times. Statistical analysis. Emergence rate, adult sex ratio and survival percent were subjected to a chi-square test of a contingency table and a Tukey- type multiple comparison test for post hoc analysis 27 . The Kruskal-Wallis single-factor analysis of variance by ranks was used to examine the number of parasitized host eggs, the number of parasitoids that emerged, the duration of development, and the longevity of the adult. If significance was detected, multiple comparisons were conducted using Duncan test 27 . All statistical analyses were conducted using IBM SPSS Statistics software for Windows. Declarations Funding The People's Republic of Bangladesh's Ministry of Science and Technology provided funding for this study (Project no. 39.00.0000.009.14.011.21–745.ES 397). Author Contribution Md. Rasel Raju wrote the main manuscript text and prepared the tables and figures. Mst. Arifunnahar, Md. Alamgir Hossain, and Md. Abdul Alim reviewed the manuscript. Acknowledgement We sincerely thank Professor Dr. Kazunori Matsuo of Kyushu University, Japan, for assisting in the identification of the parasitoid genus. Special thanks to Matthew Moore and Natalie McGathey of the Florida Department of Agriculture and Consumer Services, Division of Plant Industry, for their respective contributions to DNA barcoding and imaging. We are deeply grateful to the Ministry of Science and Technology for their financial assistance (Project no. 39.00.0000.009.14.011.20-1332, The People's Republic of Bangladesh). Elijah Talamas received support from the Florida Department of Agriculture and Consumer Services, Division of Plant Industry. Data Availability All data generated or analysed during this study are included in this published article [and its supplementary information files]. References Fu, W. et al. Screening candidate effectors of the bean bug Riptortus pedestris by proteomic and transcriptomic analyses. Front. Ecol. Evol. 9 , 760368 (2021). Raju, M. 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Refrigerated eggs of Riptortus clavatus (Hemiptera: Alydidae) added to aggregation pheromone traps increase field parasitism in soybean. J. Econ. Entomol. 104 , 1833–1893 (2011). Leopold, R. A. Cold storage of insects for integrated pest management. In: (eds Hallman, G. J. & Denlinger, D. L.) Temperature Sensitivity in Insects and Application in Integrated Pest Management. Westview, Boulder, CO, 235–267 (1998). Corrêa-Ferreira, B. S. & Moscardi, F. Biological control of soybean stink bug by inoculative releases of Trissolcus basalis . Entomol. Exp. Appl. 79 , 1–7 (1996). Kivan, M. & Kilic, N. Effects of storage at low temperature of various heteropteran host eggs. J. Asia-Pac Entomol. 13 , 123–131 (2005). Mahmoud, A. M. A. & Lim, U. T. Evaluation of cold-stored eggs of Dolycoris baccarum (Hemiptera: Pentatomidae) for parasitization by Trissolcus nigripedius (Hymenoptera: Scelionidae). Biol. Control . 43 , 287–293 (2007). Alim, M. A. & Lim, U. T. Refrigeration of Riptortus clavatus (Hemiptera: Alydidae) eggs for the parasitization by Gryon japonicum (Hymenoptera: Scelionidae). Biocontrol Sci. Technol. 19 , 315–325 (2009). Alim, M. A. & Lim, U. T. Biological attributes of Ooencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) reared on refrigerated eggs of Riptortus pedestris (= clavatus ) Fabricius (Hemiptera: Alydidae). J. Asia-Pac Entomol. 13 , 139–143 (2010). Sharma, S. K. On some Scelionidae (Proctotrupoidea: Hymenoptera) from India. Rec Zool. Surv. India . 79 , 319–342 (1982). Strand, M. R. & Vinson, S. B. Host acceptance behavior of Telenomus heliothidis (Hymenoptera: Scelionidae) toward Heliothis virescens (Lepidoptera: Noctuidae). Ann. Entomol. Soc. Am. 76 , 781–785 (1983). Noda, T. Ovipositional strategy of Gryon japonicum (Hymenoptera: Scelionidae). Bull. Natl. Inst. Agro-Environ. Sci. 9 , 1–51 (1993). Zar, J. H. Biostatistical Analysis 5th edn (Prentice Hall, 2010). Chen, W. L. & Leopold, R. A. Progeny quality of Gonatocerus ashmeadi (Hymenoptera: Mymaridae) reared on stored eggs of Homalodisca coagulate (Hemiptera: Cicadellidae). J. Econ. Entomol. 100 , 685–694 (2007). Godfray, H. C. J. Parasitoid Behavioral and Evolutionary Ecology pp. 473 (Princeton University Press, 1994). Wiedemann, L. M., Canto-Silva, C. R., Romanowski, H. P. & Redaelli, L. R. Oviposition behavior of Gryon gallardoi (Hymenoptera: Scelionidae) on eggs of Spartocera dentiventris (Hemiptera: Coreidae). Braz J. Biol. 63 , 781–785 (2003). Rabb, R. L. & Bradley, J. R. Marking host eggs by Telenomus sphingis . Ann. Entomol. Soc. Am. 63 , 209–215 (1970). Higuchi, H. & Suzuki, Y. Host handling behavior of the egg parasitoid Telenomus triptus to the egg mass of the stink bug Piezodorus hybneri . Entomol. Exp. Appl. 80 , 475–479 (1996). Lim, U. T. & Mahmoud, A. M. A. Inoculation of refrigerated non-viable eggs of Riptortus clavatus (Heteroptera: Alydidae) to enhance parasitism by egg parasitoid in soybean fields. Appl. Entomol. Zool. 44 , 37–45 (2009). Additional Declarations No competing interests reported. Supplementary Files Supplimentaryfile.xlsx Cite Share Download PDF Status: Published Journal Publication published 15 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 20 Aug, 2025 Reviews received at journal 10 Aug, 2025 Reviews received at journal 08 Aug, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviewers agreed at journal 24 Jul, 2025 Reviews received at journal 15 Jul, 2025 Reviewers agreed at journal 15 Jul, 2025 Reviewers invited by journal 09 Jul, 2025 Editor assigned by journal 09 Jul, 2025 Editor invited by journal 09 Jul, 2025 Submission checks completed at journal 07 Jul, 2025 First submitted to journal 07 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6958307","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":485300200,"identity":"3e43efed-531d-4dc6-b178-c06647303116","order_by":0,"name":"Md. Rasel Raju","email":"","orcid":"","institution":"Gyeongkuk National University","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"Rasel","lastName":"Raju","suffix":""},{"id":485300201,"identity":"9dde7fc9-ddb1-42b6-a1c6-899ee80c9bad","order_by":1,"name":"Mst. Arifunnahar","email":"","orcid":"","institution":"Hajee Mohammad Danesh Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Mst.","middleName":"","lastName":"Arifunnahar","suffix":""},{"id":485300202,"identity":"25ea6dd1-c49f-4a6b-8d2f-2e9715333a25","order_by":2,"name":"Md. Alamgir Hossain","email":"","orcid":"","institution":"Hajee Mohammad Danesh Science and Technology University","correspondingAuthor":false,"prefix":"","firstName":"Md.","middleName":"Alamgir","lastName":"Hossain","suffix":""},{"id":485300203,"identity":"a03e6ba4-a835-46f9-8a00-6d89cdbe1c52","order_by":3,"name":"Md. Abdul Alim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYDACdsYGho8NUA5jAzMRWpgZGxhnNjBIkKIFiHhJ0sLfzNy62XZHXZ3BAeaHHxh3WBPWInGYse127pnDEgYH2IwlGM+kE+EwsJa2A0AtDGYMjG2HCeuQB2mxbKsDamH/RpwWA5AWxjZmoBYeIm0xBGq52dt2WHLmYZ5iiURi/CJ3vP3ZjZ9tdfx8x9s3fvhITIghAChGEkjRMApGwSgYBaMANwAAK8M5CnqWYCIAAAAASUVORK5CYII=","orcid":"","institution":"Hajee Mohammad Danesh Science and Technology University","correspondingAuthor":true,"prefix":"","firstName":"Md.","middleName":"Abdul","lastName":"Alim","suffix":""}],"badges":[],"createdAt":"2025-06-23 15:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6958307/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6958307/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-18638-y","type":"published","date":"2025-10-15T15:57:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86781675,"identity":"75386067-6403-4859-adf2-d38244222f52","added_by":"auto","created_at":"2025-07-15 13:34:12","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":121984,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival of refrigerated\u003cem\u003e R. pedestris \u003c/em\u003eeggs. Values labeled with different letters are significantly different according to multiple comparison procedures analogous to Tukey test (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05\u003cem\u003e).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6958307/v1/e32aba67acd857a0f4949d37.jpeg"},{"id":86781369,"identity":"15c5519d-3f7c-451f-8fc9-a881ba288110","added_by":"auto","created_at":"2025-07-15 13:26:12","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":156950,"visible":true,"origin":"","legend":"\u003cp\u003eLongevity of \u003cem\u003eH. pubescens\u003c/em\u003e (male \u0026amp; female) emerged from refrigerated egg of \u003cem\u003eR. pedestris\u003c/em\u003e. Values labeled with different letters are significantly different based on Kruskal-Wallis single factor analysis of variance by rank (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6958307/v1/b8fd41826b8942b8642fcd46.jpeg"},{"id":86781677,"identity":"8ce96ba1-df86-4b2e-b423-4257ed849693","added_by":"auto","created_at":"2025-07-15 13:34:12","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":164389,"visible":true,"origin":"","legend":"\u003cp\u003eLongevity of second-generation of \u003cem\u003eH. pubescens\u003c/em\u003e(male \u0026amp; female) emerged from refrigerated egg of \u003cem\u003eR. pedestris\u003c/em\u003e. Values labeled with different letters are significantly different based on Kruskal-Wallis single factor analysis of variance by rank (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6958307/v1/6060b3837858be5bde0ed41a.jpeg"},{"id":93956120,"identity":"eac771db-b59e-4b20-a73d-2ba6a5777c08","added_by":"auto","created_at":"2025-10-20 16:10:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1413123,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6958307/v1/b35a7b93-96d5-41f1-bfa9-406397b99cf3.pdf"},{"id":86781366,"identity":"939f33f4-5132-4261-98d0-2b772d25bee6","added_by":"auto","created_at":"2025-07-15 13:26:12","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":45468,"visible":true,"origin":"","legend":"","description":"","filename":"Supplimentaryfile.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6958307/v1/b35ed3473ef55c2f482b0677.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biological attributes of Hadronotus pubescens (Motschoulsky) (Hymenoptera: Scelionidae) reared on refrigerated eggs of Riptortus pedestris Fabricius (Hemiptera: Alydidae)","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe bean bug, \u003cem\u003eRiptortus pedestris\u003c/em\u003e (Fab.) (Hemiptera, Alydidae), is a major pest affecting soybean crops in East Asia\u003csup\u003e1\u003c/sup\u003e and has also been responsible for infestations in legume crops in Bangladesh\u003csup\u003e2,3\u003c/sup\u003e. It poses a serious threat to soybeans (\u003cem\u003eGlycine max\u003c/em\u003e (L.)\u003csup\u003e4,5\u003c/sup\u003e and fruit trees\u003csup\u003e6\u003c/sup\u003e. \u003cem\u003eRiptortus pedestris\u003c/em\u003e attacks many cereal crops, such as sorghum (\u003cem\u003eSorghum bicolor\u003c/em\u003e), foxtail millet (\u003cem\u003eSetaria italica\u003c/em\u003e), and barley (\u003cem\u003eHordeum vulgare\u003c/em\u003e) during their reproductive phases\u003csup\u003e6,7\u003c/sup\u003e, along with other field crops throughout Asia\u003csup\u003e8,9\u003c/sup\u003e. Adults and nymphs of this insect primarily target seeds, feeding through piercing and sucking, and their capacity to utilize various host plants increases the vulnerability of crops to damage\u003csup\u003e5\u003c/sup\u003e. The insects drain sap from seed pods, leading to incomplete pod maturation, browning, shrinking, and death. When pods are opened, seeds are often found to be shriveled, underdeveloped, or deformed. These damaged seeds frequently suffer from secondary fungal infections, such as yeast-spot disease caused by \u003cem\u003eEremothecium coryli\u003c/em\u003e\u003csup\u003e10\u003c/sup\u003e, which can devastate an entire crop\u003csup\u003e11\u003c/sup\u003e. Farmers typically apply synthetic chemical insecticides to manage \u003cem\u003eR. pedestris\u003c/em\u003e infestations, but this approach is not always cost-effective for lower-value crops like mung beans (\u003cem\u003eVigna radiata\u003c/em\u003e) in Bangladesh. Chemical insecticides play a role in integrated pest management (IPM) due to their rapid action and ease of use\u003csup\u003e12\u003c/sup\u003e, but they can harm beneficial species, lead to pest resurgence, and have other negative environmental impacts\u003csup\u003e13\u003c/sup\u003e. As a result, the high cost of chemical pesticides may render them unfeasible, especially when combined with environmental concerns. While these chemicals provide rapid action, the mobility of \u003cem\u003eR. pedestris\u003c/em\u003e makes its control more challenging\u003csup\u003e5,9\u003c/sup\u003e. To tackle this issue, cultural practices such as delayed planting and the use of resistant varieties have been suggested for managing stink bugs\u003csup\u003e14,9\u003c/sup\u003e. Additionally, biological control methods involving the introduction or enhancement of egg parasitoids have proven effective against stink bugs\u003csup\u003e15-17 \u003c/sup\u003eand could provide a viable option for controlling \u003cem\u003eR. pedestris\u003c/em\u003e in Bangladesh\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCold storage is commonly employed to improve efficiency and provide flexibility in the mass rearing of insects for biological control programs\u003csup\u003e18\u003c/sup\u003e. The use of low temperatures to store host eggs is well-established in the rearing of parasitoids for stink bugs, including \u003cem\u003eR. pedestris\u003c/em\u003e\u003csup\u003e16,19,20-22\u003c/sup\u003e. Studies have shown that refrigerated eggs of \u003cem\u003eR. pedestris\u003c/em\u003e are suitable for parasitism by \u003cem\u003eGryon japonicum\u003c/em\u003e\u003csup\u003e22,23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHadronotus pubescens\u003c/em\u003e (Motschoulsky) (Hymenoptera: Scelionidae) is a solitary egg parasitoid that has been reported in Sri Lanka, India, particularly in Karnataka, and Bangladesh\u003csup\u003e24,2\u003c/sup\u003e. The biological parameters of \u003cem\u003eH. pubescens\u003c/em\u003e on refrigerated \u003cem\u003eR. pedestris\u003c/em\u003e eggs remain unknown, with no existing published data on the effects of host egg refrigeration on this parasitoid. Raju et al.\u003csup\u003e2\u003c/sup\u003e were the first to document the presence of \u003cem\u003eH. pubescens\u003c/em\u003e in Bangladesh and studied its biological traits on \u003cem\u003eR. pedestris\u003c/em\u003e eggs under laboratory conditions. In this study, we investigated how refrigerating \u003cem\u003eR. pedestris\u003c/em\u003e eggs affects parasitism by \u003cem\u003eH. pubescens\u003c/em\u003e in a lab environment. Specifically, we examined the number of parasitized eggs, parasitoid emergence, sex ratio, development time, longevity, and reproduction across the first and second generations of \u003cem\u003eH. pubescens\u003c/em\u003e using refrigerated host eggs.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eSurvival of refrigerated\u003c/b\u003e \u003cb\u003eR. pedestris\u003c/b\u003e \u003cb\u003eeggs.\u003c/b\u003e The hatching percentage of \u003cem\u003eR. pedestris\u003c/em\u003e eggs was significantly affected by the duration of refrigeration (\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;87.10, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Survival decreased from 99% from the fresh (unrefrigerated) eggs to 7% from the eggs refrigerated for 25 days. In this study, no eggs hatched after 30 days of refrigeration due to loss of viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of refrigeration host egg on host acceptance behavior of\u003c/b\u003e \u003cb\u003eH. pubescens.\u003c/b\u003e All \u003cem\u003eH. pubescens\u003c/em\u003e exhibited complete host acceptance behaviors toward the refrigerated host eggs, with the duration of these behaviors detailed in (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The duration of drumming increased with longer refrigeration times (\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 55.02, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The eggs of \u003cem\u003eR. pedestris\u003c/em\u003e refrigerated for 120 days showed the longest drumming duration by the parasitoid. Following the drumming, the female parasitoid begins drilling by inserting her ovipositor into the host eggs. The average time spent on oviposition increased for host eggs stored at various refrigeration periods compared to fresh eggs (unrefrigerated) (\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 56.57, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Additionally, \u003cem\u003eH. pubescens\u003c/em\u003e spent more time on marking behavior for eggs refrigerated for 30 days compared to fresh eggs (unrefrigerated) (\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 52.64, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDuration (Seconds\u0026thinsp;\u0026plusmn;\u0026thinsp;SE) of host acceptance behaviors in different ages of refrigerated \u003cem\u003eR. pedestris\u003c/em\u003e eggs\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eBehaviors\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eAge of host eggs (days)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0 (Control)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e120\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrumming\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e38.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72 f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e50.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e54.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e60.22\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e66.13\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDrilling and Oviposition\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e415.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e439.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e494.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.36 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e553.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e591.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e596.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMarking\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e138.31\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e173.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e162.81\u0026thinsp;\u0026plusmn;\u0026thinsp;1.86 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e154.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.94 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e147.77\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e143.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.43 de\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eValues in the same row with the same letter are not significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eSee text for statistics.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of refrigerated host eggs on the biological parameters of\u003c/b\u003e \u003cb\u003eH. pubescens.\u003c/b\u003e The number of host eggs parasitized by \u003cem\u003eH. pubescens\u003c/em\u003e varied depending on the duration of refrigeration. For up to 30 days, \u003cem\u003eH. pubescens\u003c/em\u003e can effectively parasitize host eggs without a notable decrease in parasitism. However, for the host egg of 120-day refrigeration time period, the parasitized host eggs decreased approximately 47.35% (\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 38.71, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the control (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The emergence rate (\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;45.41, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was significantly affected by host eggs refrigeration time, while the sex ratio (\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;5.78, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.328) of \u003cem\u003eH. pubescens\u003c/em\u003e was not significantly affected by host eggs refrigeration time (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The emerged parasitoids exhibited female-biased sex ratios, with no statistically significant differences observed across the different time periods. However, the overall emergence of parasitoids from the parasitized eggs significantly declined as the refrigeration duration increased \u003cem\u003e(H\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 45.41, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Mean development time (from eggs to adult) of \u003cem\u003eH. pubescens\u003c/em\u003e male and female increased significantly as host eggs aged increased (male \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 72.66, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Female \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 240.36, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Also, less development time finds in control (0 day) whereas 120 days of refrigeration showed the highest mean time for development. The longevity of \u003cem\u003eH. pubescens\u003c/em\u003e male and female showed significant decline following refrigeration of the host eggs for 120 days (male \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 70.68, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Female \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 183.51, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest longevity was found in 0 days of age compared to others.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of refrigerated host eggs on the biological parameters of \u003cem\u003eH. pubescens\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eAge of host eggs (days)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e120\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber parasitized host eggs / female\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e5.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e4.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProportion of male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.16 (13/83) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.20 (16/80) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.19 (13/67) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.24 (15/61) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.20 (12/59) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.34 (12/35) a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEmergence rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.72(72/100) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.67(67/100) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.60(60/100) ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.50(50/100) b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.51(51/100) b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.30(30/100) c\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDevelopment time, days\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e25.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e25.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e33.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e36.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e43.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e26.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e28.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 d\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e33.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e37.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e43.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eValues in the same row with the same letter are not significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eSee text for statistics.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of second-generation host eggs on the biological parameters of\u003c/b\u003e \u003cb\u003eH. pubescens\u003c/b\u003e. The number of parasitized eggs by \u003cem\u003eH. pubescens\u003c/em\u003e was not influenced by the progeny of different ages host eggs in second generation (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The progeny emerged from 120 days of refrigerated eggs, the parasitized host eggs decreased about 16.85% (H\u003csub\u003eC\u003c/sub\u003e = 14.03, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.015) compared to the control (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The emergence rate (\u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 9.32, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.096) and the sex ratio (\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;8.75, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.119) of \u003cem\u003eH. pubescens\u003c/em\u003e were not influenced by the age of host eggs (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The emerged parasitoids exhibited a female-biased sex ratio across all refrigeration periods, although these differences were not statistically significant. The development time (from eggs to adult) of \u003cem\u003eH. pubescens\u003c/em\u003e male and female increased after 60 days refrigerated eggs of progeny (male \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 26.15, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Female \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 227.05, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In this study, the highest mean development time was found as 120 days of age while the lowest in control (0 day). The longevity of \u003cem\u003eH. pubescens\u003c/em\u003e males and females decreased after 45 days of refrigerated eggs progeny (male \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 54.25, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Female \u003cem\u003eH\u003c/em\u003e\u003csub\u003e\u003cem\u003eC\u003c/em\u003e\u003c/sub\u003e = 257.16, \u003cem\u003edf\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The highest longevity was found in 0 hour (control) of age compared to others. Therefore, refrigerating host eggs for up to 60 days can effectively facilitate the parasitization of \u003cem\u003eR. pedestris\u003c/em\u003e by \u003cem\u003eH. pubescens\u003c/em\u003e without negatively affecting the second generation of the parasitoid.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEffect of second-generation host eggs on the biological parameters of \u003cem\u003eH. pubescens\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eAge of the host eggs (days)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e60\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e90\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e120\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNumber parasitized host eggs / female\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e8.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e7.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProportion of male\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.12(11/89) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.08(7/84) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.19(16/82) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.12(10/81) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.08(6/75) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.06(5/74) a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEmergence rate\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.87(87/100) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.80(80/100) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.79(79/100) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.77(77/100) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.73(73/100) a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.71(71/100) a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDevelopment time, days\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 bc\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e15.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e15.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eValues in the same row with the same letter are not significantly different (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eSee text for statistics.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe length of refrigeration had a significant impact on the viability of \u003cem\u003eR. pedestris\u003c/em\u003e eggs; after 30 days, the eggs completely failed to hatch because of their diminished viability. According to Alim and Lim\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eR. pedestris\u003c/em\u003e eggs that are refrigerated for 30 days lose their viability. Similarly, Chen and Leopold\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e found that eggs of \u003cem\u003eHomalodisca coagulata\u003c/em\u003e (Say) were killed after 5 days at 2\u0026deg;C chilling. Additionally, Mahmoud and Lim\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e discovered that \u003cem\u003eDolycoris baccarum\u003c/em\u003e L. egg hatching was not observed following 20 days of storage at 2.1\u0026deg;C.\u003c/p\u003e\u003cp\u003eThe refrigeration time of \u003cem\u003eR. pedestris\u003c/em\u003e eggs significantly influences the behavioral responses of \u003cem\u003eH. pubescens\u003c/em\u003e. The prolonged drumming duration was observed with increased refrigeration time. This may be attributed to alterations in the physical structure of the refrigerated host eggs as a result of desiccation\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, leading the parasitoids to spend more time evaluating the host eggs. According to Mahmoud and Lim\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eTrissolcus nigripedius\u003c/em\u003e Nakagawa displayed extended drumming behavior when interacting with refrigerated eggs of \u003cem\u003eDolycoris baccarum\u003c/em\u003e. Strand and Vinson\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e described that \u003cem\u003eTelenomus heliothidis\u003c/em\u003e (Ashmead) drumming on \u003cem\u003eHeliothis virescens\u003c/em\u003e (F.) eggs is an essential step in determining host acceptance. Drumming is characterized by the parasitoid moving its antennae up and down across the exposed surface of the eggs while remaining still or standing over them. Likewise, the longer oviposition period for refrigerated eggs demonstrates the parasitoids' capacity to adjust to host quality variations, possibly suggesting a compensation mechanism that overcomes the effects of refrigeration on host egg alterations. The slight rotation of the abdomen as the ovipositor enters the chorion is known as drilling\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. During oviposition, the females remain still with their heads in a pumping motion, wings aligned parallel to their bodies, and hind legs firmly gripping the host egg\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. After head pumping, the female stays motionless for 5\u0026ndash;10 seconds until oviposition is complete. Notably, the extended marking behavior observed for eggs refrigerated for 30 days might reflect a preference or enhanced effort to secure those eggs as a resource. Since host marking behavior is influenced by the quality of host eggs\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, the prolonged marking time would suggest that host eggs that have been chilled for 15 or 30 days are more suitable for parasitoids than fresh eggs. The female walks rearward after removing the ovipositor from the egg surface while swinging the posterior part of her abdomen laterally to brush along the eggs surface. This behavior, known as marking\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, indicates the end of oviposition\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe parasitism rate of \u003cem\u003eH. pubescens\u003c/em\u003e remained stable for host eggs refrigerated up to 30 days but showed a significant decrease for eggs refrigerated for 120 days. Refrigeration duration strongly impacted emergence rates, which decreased with extended refrigeration, though the sex ratio remained consistently female-biased. Development time for both male and female parasitoids increased with egg refrigeration duration, peaking at 120 days, while longevity was highest at 30 days but significantly reduced for 120-day-old eggs. Leopold\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e reported refrigeration might affect the physiological process of the host egg that is deleterious for the development of parasitoid. The significant variations in the quantity of parasitized host eggs, the total number of parasitoids, and the development time may be attributed to a reduction in the nutritional quality of eggs that have been refrigerated for extended durations\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe parasitization and emergence rates of \u003cem\u003eH. pubescens\u003c/em\u003e were unaffected by the progeny of host eggs refrigerated up to 60 days, with a consistent female-biased sex ratio. However, the mean development time and longevity of the parasitoids significantly increased and decreased, respectively, after 60 days of refrigeration. Similar patterns were observed by, Alim and Lim\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, Mahmoud and Lim\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e in a gregarious and scelionid parasitoid and by Chen and Leopold\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e in a mymarid parasitoid. The low nutritional quality attributed to older host eggs can slow down offspring development rate\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. The parasitization rate and emergence of \u003cem\u003eH. pubescens\u003c/em\u003e were not significantly influenced by the age of refrigerated host eggs in the second generation, with a consistent female-biased sex ratio. However, mean development time increased, and longevity decreased significantly after 45 days of refrigeration. Alim and Lim\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e also found that the longevity of \u003cem\u003eOoencyrtus nezarae\u003c/em\u003e Ishii females decreased after 60 days of refrigeration for the progeny of eggs.\u003c/p\u003e\u003cp\u003eOur study indicates that \u003cem\u003eH. pubescens\u003c/em\u003e can parasitize \u003cem\u003eR. pedestris\u003c/em\u003e host eggs refrigerated at 2.0˚C for up to 30 days without a significant decline in parasitism rates. Additionally, refrigeration of host eggs did not impact the reproductive capabilities of adult parasitoids that developed from these refrigerated hosts (i.e., the second generation). Thus, refrigerating \u003cem\u003eR. pedestris\u003c/em\u003e eggs could facilitate the mass rearing of \u003cem\u003eH. pubescens\u003c/em\u003e. Additionally, these refrigerated eggs can be introduced into the field to enhance parasitoid populations, as they remain viable for oviposition for several days without resulting in the emergence of the pest, \u003cem\u003eR. pedestris\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eRearing of\u003c/b\u003e \u003cb\u003eR. pedestris.\u003c/b\u003e Adults \u003cem\u003eR. pedestris\u003c/em\u003e were collected from country beans fields (\u003cem\u003eLablab purpureus\u003c/em\u003e L.) located in Hajee Mohammad Danesh Science and Technology University (HSTU) Agriculture Research field, Dinajpur, People\u0026rsquo;s Republic of Bangladesh (25\u0026deg; 41' 51'' N, 88\u0026deg; 39' 15'' E). \u003cem\u003eR. pedestris\u003c/em\u003e were reared and maintained in the laboratory following the method of Raju et al.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Both nymphs and adults were raised in acrylic cages (40L \u0026times; 40W \u0026times; 40H cm), featuring windows on three sides covered with mesh screens to allow for ventilation. The insects were raised under stable conditions of temperature (31.62\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1) and relative humidity (79.25% \u0026plusmn; 1.60), following the natural photoperiod. Breeding dishes containing dissolved ascorbic acid pellets along with soybean seeds were provided to the adult \u003cem\u003eR. pedestris\u003c/em\u003e, while nymphs were sustained on a diet of soybean seeds and bean plants (\u003cem\u003eL. purpureus\u003c/em\u003e L.) with cotyledonous leaves. Oviposition substrates were created by positioning four pieces of gauze fabric in the upper and bottom corners inside the adult cages, with the fabric being replaced daily and the eggs collected for colony maintenance. The collected eggs were stored in zipper plastic bags at a temperature of 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u0026deg;C in a refrigerator for experiments. To record the temperature and relative humidity a data logger was used (U10-001; Onset Computer Corporation, Bourne, MA, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eRearing of\u003c/b\u003e \u003cb\u003eH. pubescens.\u003c/b\u003e \u003cem\u003eH. pubescens\u003c/em\u003e was obtained from naturally parasitized eggs of \u003cem\u003eR. pedestris\u003c/em\u003e found in the same bean fields described earlier. These \u003cem\u003eH. pubescens\u003c/em\u003e specimens were then raised on \u003cem\u003eR. pedestris\u003c/em\u003e eggs in the same laboratory conditions described in the previous section. The identification of the parasitoid was conducted in accordance following the method outlined in Raju et al.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Both male and female \u003cem\u003eH. pubescens\u003c/em\u003e individuals were reared in 50 mL plastic centrifugal tube (SPL Life Sciences Co. Ltd., Korea), where they were provided with a surface layer of honey and a piece of moistened cotton at the bottom of the tube. The moistened cotton and honey were refreshed every three days.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSurvival of\u003c/b\u003e \u003cb\u003eR. pedestris\u003c/b\u003e \u003cb\u003erefrigerated eggs.\u003c/b\u003e Eggs of \u003cem\u003eR. pedestris\u003c/em\u003e were kept in the zipper plastic bags and placed in a refrigerator on days 0, 10, 15, 20, 25, and 30 at 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u0026deg;C. After refrigeration, 50 host eggs from each refrigeration period were removed to a Petri dish (4 H \u0026times; 10 D cm) and were kept in laboratory condition at 31.62\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1, 79.25% \u0026plusmn; 1 RH. Survival was quantified by calculating the proportion of hatched eggs to total eggs examined.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffects of host egg age refrigeration on host acceptance behavior by\u003c/b\u003e \u003cb\u003eH. pubescens\u003c/b\u003e. After refrigeration for 0, 30, 45, 60, 90, and 120 days, 10 \u003cem\u003eR. pedestris\u003c/em\u003e eggs from each time point were taken out and transferred to a (4 H \u0026times; 10 D cm) Petri dish at room temperature. A single 4-day-old mated, and naive female \u003cem\u003eH. pubescens\u003c/em\u003e was then introduced into each dish. Host acceptance behaviors were monitored under a stereomicroscope (Luxeo 6Z, LaboMed, USA) and classified into categories including drumming and drilling, oviposition and marking\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. The duration of each behavior was recorded for eggs that elicited a full host acceptance response from the parasitoid. This procedure was repeated for 10 female parasitoids at each refrigeration interval.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of refrigerated host eggs on the biological parameters of\u003c/b\u003e \u003cb\u003eH. pubescens\u003c/b\u003e. Host eggs refrigerated at 2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u0026deg;C for the different time periods (0, 30, 45, 60, 90 and 120 days), 10 \u003cem\u003eR. pedestris\u003c/em\u003e eggs on each period were removed from the refrigerator and placed individually in a petri dish (4 H \u0026times; 10 D cm) for one hour in laboratory condition. A 4-day old, mated and inexperienced female \u003cem\u003eH. pubescens\u003c/em\u003e was introduced and permitted to parasitize the eggs for 24 hours at 31.62\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1, 79.25% \u0026plusmn; 1 RH and then removed. The number of parasitoids that emerged from every egg was counted every day. The number of host eggs parasitized host eggs, sex ratio, emergence rate, longevity and development time of both male and female \u003cem\u003eH. pubescens\u003c/em\u003e was recorded. Host eggs that contained parasitoids that did not develop or emerge were also classified as parasitized based on the dissection of each egg. These procedures were repeated ten times for each storage period. The parasitoid was collected and placed in a parasitoid rearing tube. Every three days, fresh honey and moistened cotton were given, and the parasitoids were reared by transferring them to new tubes as needed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of second-generation host eggs on the biological parameters of\u003c/b\u003e \u003cb\u003eH. pubescens\u003c/b\u003e. The impact of refrigerated host eggs on the parasitoid of second generation was evaluated. A 4-day old, inexperienced and previously mated, 10 female \u003cem\u003eH. pubescens\u003c/em\u003e were randomly selected from the group of adult parasitoids that emerged from host eggs refrigerated for various durations (0, 30, 45, 60, 90 and 120 days). After that, each parasitoid was provided with 10 \u003cem\u003eR. pedestris\u003c/em\u003e fresh eggs for 24 hours under the same laboratory condition and then removed after 24 hours. After 24 hours, the female \u003cem\u003eH. pubescens\u003c/em\u003e was withdrawn, and the eggs were placed individually in 2 ml microtubes containing a drop of honey. The emergence of parasitoids was observed daily. The number of parasitized host eggs, sex ratio, adult emergence, development time and longevity of male and female of \u003cem\u003eH. pubescens\u003c/em\u003e were observed. Host eggs that contained parasitoids which did not develop or emerge were also classified as parasitized based on the dissection of each egg. For every storage period age, these processes were repeated ten times.\u003c/p\u003e\u003cp\u003e\u003cb\u003eStatistical analysis.\u003c/b\u003e Emergence rate, adult sex ratio and survival percent were subjected to a chi-square test of a contingency table and a Tukey- type multiple comparison test for post hoc analysis\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The Kruskal-Wallis single-factor analysis of variance by ranks was used to examine the number of parasitized host eggs, the number of parasitoids that emerged, the duration of development, and the longevity of the adult. If significance was detected, multiple comparisons were conducted using Duncan test\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. All statistical analyses were conducted using IBM SPSS Statistics software for Windows.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe People's Republic of Bangladesh's Ministry of Science and Technology provided funding for this study (Project no. 39.00.0000.009.14.011.21\u0026ndash;745.ES 397).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMd. Rasel Raju wrote the main manuscript text and prepared the tables and figures. Mst. Arifunnahar, Md. Alamgir Hossain, and Md. Abdul Alim reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe sincerely thank Professor Dr. Kazunori Matsuo of Kyushu University, Japan, for assisting in the identification of the parasitoid genus. Special thanks to Matthew Moore and Natalie McGathey of the Florida Department of Agriculture and Consumer Services, Division of Plant Industry, for their respective contributions to DNA barcoding and imaging. We are deeply grateful to the Ministry of Science and Technology for their financial assistance (Project no. 39.00.0000.009.14.011.20-1332, The People's Republic of Bangladesh). Elijah Talamas received support from the Florida Department of Agriculture and Consumer Services, Division of Plant Industry.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eFu, W. et al. Screening candidate effectors of the bean bug \u003cem\u003eRiptortus pedestris\u003c/em\u003e by proteomic and transcriptomic analyses. \u003cem\u003eFront. Ecol. Evol.\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 760368 (2021).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRaju, M. R. et al. \u003cem\u003eHadronotus pubescens\u003c/em\u003e (Motschoulsky) (Hymenoptera: Scelionidae): redescription, biological attributes, and parasitism on eggs of \u003cem\u003eRiptortus pedestris\u003c/em\u003e (Fab.) (Hemiptera: Alydidae). \u003cem\u003eJ. Hymenoptera Res.\u003c/em\u003e \u003cb\u003e94\u003c/b\u003e, 139\u0026ndash;161 (2022).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eArifunnahar, M., Khatun, M. M., Hossain, M. A. \u0026amp; Alim, M. A. Toxicity evaluation of different chemical pesticides against \u003cem\u003eRiptortus pedestris\u003c/em\u003e (Hemiptera: Alydidae) under laboratory condition in Bangladesh. \u003cem\u003eJ. 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Host handling behavior of the egg parasitoid \u003cem\u003eTelenomus triptus\u003c/em\u003e to the egg mass of the stink bug \u003cem\u003ePiezodorus hybneri\u003c/em\u003e. \u003cem\u003eEntomol. Exp. Appl.\u003c/em\u003e \u003cb\u003e80\u003c/b\u003e, 475\u0026ndash;479 (1996).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLim, U. T. \u0026amp; Mahmoud, A. M. A. Inoculation of refrigerated non-viable eggs of \u003cem\u003eRiptortus clavatus\u003c/em\u003e (Heteroptera: Alydidae) to enhance parasitism by egg parasitoid in soybean fields. \u003cem\u003eAppl. Entomol. Zool.\u003c/em\u003e \u003cb\u003e44\u003c/b\u003e, 37\u0026ndash;45 (2009).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Egg parasitoid, Cold storage, Stink bug, Mass rearing, Country bean","lastPublishedDoi":"10.21203/rs.3.rs-6958307/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6958307/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eHadronotus pubescens\u003c/em\u003e (Motschoulsky) (Hymenoptera, Scelionidae) is a type of wasp that lays its eggs in the eggs of \u003cem\u003eRiptortus pedestris\u003c/em\u003e Fabricius (Hemiptera: Alydidae), which is a major pest for legume crops in Korea, Japan, and Bangladesh. This study assessed the quality of refrigerated \u003cem\u003eR. pedestris\u003c/em\u003e eggs in relation to host-accepting behaviors and biological characteristics, including parasitism rate, development duration, emergence rate, sex ratio, and longevity. The hatchability of the refrigerated eggs was evaluated. \u003cem\u003eRiptortus pedestris\u003c/em\u003e eggs that were refrigerated for over 30 days failed to hatch entirely. The refrigeration influences the average duration of each host acceptance behavior (i.e., drumming, drilling, oviposition, and marking) on host eggs. The number of parasitized host eggs per female, the male proportion, and the emergence rate of \u003cem\u003eH. pubescens\u003c/em\u003e were unaffected by chilling for up to 30 days in comparison to fresh eggs. Moreover, the refrigerated host eggs did not influence the reproduction of the emerged adult parasitoids or the growth of their offspring. The longevity of \u003cem\u003eH. pubescens\u003c/em\u003e was not influenced by the refrigeration of \u003cem\u003eR. pedestris\u003c/em\u003e eggs. The refrigeration of host eggs for up to 30 days did not adversely affect any biological features of the second generation of \u003cem\u003eH. pubescens\u003c/em\u003e. So, keeping \u003cem\u003eH. pubescens\u003c/em\u003e eggs in the fridge can be a good way to produce a lot of the parasitoid, and the host eggs that are no longer alive after being stored in the cold can be used in the field to increase parasitism.\u003c/p\u003e","manuscriptTitle":"Biological attributes of Hadronotus pubescens (Motschoulsky) (Hymenoptera: Scelionidae) reared on refrigerated eggs of Riptortus pedestris Fabricius (Hemiptera: Alydidae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-15 13:26:07","doi":"10.21203/rs.3.rs-6958307/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-20T21:55:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-11T01:36:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-08T17:22:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240349900906082759122052055654318978875","date":"2025-07-25T01:18:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84782310960007848074462674976012710672","date":"2025-07-24T14:37:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-15T17:56:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"72947528091433061418847462883524501388","date":"2025-07-15T16:07:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-09T15:04:53+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-09T14:43:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-09T09:43:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-07T11:55:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-07-07T11:51:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e9aa2533-1a20-4282-8d78-d00b9aab8275","owner":[],"postedDate":"July 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51518829,"name":"Biological sciences/Biological techniques"},{"id":51518830,"name":"Biological sciences/Ecology"}],"tags":[],"updatedAt":"2025-10-20T16:05:54+00:00","versionOfRecord":{"articleIdentity":"rs-6958307","link":"https://doi.org/10.1038/s41598-025-18638-y","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-15 15:57:29","publishedOnDateReadable":"October 15th, 2025"},"versionCreatedAt":"2025-07-15 13:26:07","video":"","vorDoi":"10.1038/s41598-025-18638-y","vorDoiUrl":"https://doi.org/10.1038/s41598-025-18638-y","workflowStages":[]},"version":"v1","identity":"rs-6958307","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6958307","identity":"rs-6958307","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}
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