The large-scale implementation of T. podisi in soybean cropping systems is primarily constrained by the continuous availability of high-quality host eggs for mass rearing. In the present study, we demonstrate that long-term storage of E. heros eggs, particularly under ultra-low temperature (−80°C) and liquid nitrogen (−196°C) conditions, is feasible for periods of up to 12 months without substantially compromising parasitoid parasitism, emergence, or overall biological quality. These findings represent an important advance toward overcoming one of the major operational bottlenecks limiting the expansion of T. podisi releases in Brazil.

In recent years, considerable efforts have been devoted to defining optimal release rates, release intervals, and the soybean phenological stage for the application of T. podisi and these parameters are now relatively well established (Weber et al. 2022; Oliveira et al. 2024). However, such advances must be accompanied by strategies that ensure a reliable and continuos supply of host eggs for parasitoid production. The development of efficient mass-rearing systems for E. heros, including the automation of rearing procedures and the implementation of effective egg storage techniques, is therefore essential to meet the demands of large-scale Brazilian agriculture. In this context, low-temperature storage, particularly for long-term preservation, emerges as a key component for enabling continuous parasitoid production throughout the year and for buffering unexpected peaks in demand, which are common in a country with continental dimensions such as Brazil (van Lenteren and Tommasini 2003).

Although several studies have reported successful storage of pentatomid eggs at low temperatures (Favetti et al. 2014; Silva et al. 2019; Almeida et al. 2023; Ramos et al. 2025), to our knowledge, this is the first study to evaluate the long-term storage of E. heros eggs for periods of up to 12 months. When overall parasitism performance was considered, storage in an ultra-low temperature (ULT) freezer and in liquid nitrogen proved to be considered the most suitable preservation methods. Both treatments resulted in high mean parasitism rates (58.47% and 89.18%, respectively), which is similar those observed in the control treatment using freshly laid eggs (56.67%) In contrast, eggs stored in the conventional freezer exhibited substantially lower parasitism rates (37.39%), indicating that this method is the least suitable for the mass production of T. podisi. These findings are partially consistent with previous studies evaluating low-temperature eggs storage in pentatomids. Almeida et al. (2023), for example, assessed different storage methods and egg packaging strategies for E. heros and reported that eggs wrapped in aluminum foil and stored in a conventional freezer (−18°C) or in liquid nitrogen (−196°C) remained viable for parasitism for up to 60 days, although with significant variation among storage periods. However, the storage duration evaluated by those authors was limited to 60 days, whereas the present study extends this assessment to substantially longer periods, providing new evidence for the feasibility of long-term egg preservation. Similarly, Silva et al. (2019) reported lower parasitism rates when E. heros eggs were stored under ultra-low temperature conditions (−80°C and −196°C) for up to 70 days; in contrast the present results demonstrate that high parasitism levels can be maintained over much longer storage periods when appropriate protocols are applied.

The lower performance in parasitism observed for eggs stored in conventional freezer is likely related to cellular damage caused by ice crystal formation during freezing and thawing processes at moderately low temperatures (–15°C). At such temperatures, intracellular and extracellular ice crystallization may compromise egg tissue, thereby reducing host quality for parasitoid development (Canet 1989). Freezing speed is a critical factor for preservation cellular integrity, as rapid cooling, such as that achieved during vitrification in liquid nitrogen, minimizes ice crystal formation within cellular compartments (Milward-de-Azevedo et al. 2004). This mechanism likely explains the superior parasitism and emergence rates observed for eggs stored under ultra-low temperature and cryogenic conditions in the present study.

Eggs stored in an ultra-low temperature (ULT) freezer or in liquid nitrogen for up to 12 months exhibited emergence rates exceeding 85%, whereas eggs stored in a conventional freezer showed mean emergence values below 50%. These results are consistent with those reported by Silva et al. (2019), who documented significantly lower emergence from E. heros eggs stored at −15°C compared with those maintained under ultra-lower temperature and liquid nitrogen conditions, further emphasizing the limited suitability of conventional freezing for long-term egg preservation.

In contrast, the duration of parasitoid development was not affect by any of the storage methods evaluated. This finding is consistent with Oliveira et al. (2024), who reported no differences in the development time of T. podisi emerging from fresh eggs or eggs stored in liquid nitrogen under different rearing temperatures. However, Ramos et al. (2025) observed a slight delay of approximately one day in the development of T. podisi originating from cryopreserved eggs compared with those developing from fresh eggs. Suggesting that subtle effects on developmental dynamics may occur depending on storage duration and protocol.

Regarding transgenerational responses, only minor reductions were detected in specific biological parameters of the progeny originating from eggs stored in ULT freezer and liquid nitrogen. Nevertheless, considering the extended storage period evaluated (up to 12 months) and the overall biology of the parasitoid, these effects were limited and did not compromise the functional quality of the F1 generation. Similar conclusions were reached by Ramos et al. (2025) who reported no significant differences in parasitism, emergence, sex ratio, or longevity of T. podisi females developing from E. heros eggs stored in liquid nitrogen, although in that study eggs were preserved for only 30 days. Favetti et al. (2014) also reported high parasitism rates from E. heros eggs stored in liquid nitrogen for three and six months; however, the lack of detailed quantitative information regarding egg availability limits direct comparisons with the present results.

It is important to note that long-term egg storage outcomes may be further optimized by refining cryopreservation protocols, particularly thawing procedures prior to egg exposure to parasitoid females. In addition to freezing rate, thawing speed plays a crucial role in preserving biological quality, as rapid thawing in a controlled water bath (35–40°C) can prevent the fusion of microcrystals formed during freezing and reduce damage associated with devitrification (Santos 2000). Such refinements may further enhance egg viability and parasitoid performance following extended storage periods. From an applied perspective, the implications of long-term egg storage are substantial. The recommended release rate of T. podisi for the control of E. heros is approximately 6,500 parasitoids per hectare, applied in three consecutive releases at weekly intervals, resulting in a total requirement of nearly 19,500 parasitoids per hectare. As T. podisi is a solitary parasitoid, this release strategy requires at least one host egg per individual produced. Consequently, implementing biological control across the entire soybean-growing area in Brazil would require a minimum of approximately one billion E. heros eggs.

Considering that parasitism viability after 12 months of storage averages approximately 60% for eggs preserved in ULT freezers and liquid nitrogen, but only around 40% for eggs stored in conventional freezers, achieving one billion eggs viable for parasitism would require an increase in egg production of more than 150% when using conventional freezer storage, whereas ULT freezer and liquid nitrogen storage would require an increase of approximately 70%. Specifically, conventional freezing would require the production of approximately 2.5 billion eggs, compared with about 1.67 billion eggs required under ultra-low temperature or liquid nitrogen storage. These differences highlight the importance of incorporating cost–benefit analyses into decision-making processes regarding storage methodologies for large-scale biological control programs.

Overall, our results demonstrate that E. heros eggs can be stored in an ultra-low temperature freezer or in liquid nitrogen for up to 12 months without significantly affecting either the parental generation of T. podisi produced in the laboratory and released in the field or their progeny. Under favorable field conditions, such progeny may contribute to the persistence and continuity of parasitoid populations, thereby enhancing the effectiveness and sustainability of biological control strategies in soybean agroecosystems.