Tauber et al. stated that an effective storage technique must fulfill the following fitness requirements after storage: high survival, synchronous and predictable initiation of reproduction, and sustained high fecundity and fertility35. In this study, the indoor-reared H. axyridis adults were stored at 6°C with the longest period of 120 days, which was closed to that used for filed-collected pre-overwintering individuals29,30. After storage, the adults exhibited appreciable post-storage fitness, such as relatively high survival and a great power in reproduction with stable pre-oviposition period. However, there were also some noticeable negative effects that had rarely been reported before, which would encourage more studies to reveal the mechanisms and develop more effective storage techniques to evade these negative effects.
More than 90 percent individuals survived after 30 days of storage, but the survivals gradually decreased with the increasing of storage duration. Although those at 90 or 120 DIS were significantly lower, there were still more than 30 percent. Chilling injury during storage was an important factor causing mortality36. In addition, other indirect effects including accumulation of toxic metabolites and elimination of energy reserves have also been suggested as probable reasons for death1,3. Here, with prolonged storage, the weight loss gradually increased and, especially, the glycogen and trehalose contents sharply decreased even at 30 DIS. These results indicated that energy reserves, especially those directly related to cold tolerance, were rapidly consumed during storage. Specifically, at 60 DIS, the survivals of Ha-CK were about 70% which were similar to those reported in our previous studies that using the same pre-storage diet1. However, Ha-CK still had a survival rate of 31.3% (female) and 35.3% (male) at 120 DIS, which was superior to that reported before (all adults died at 90 DIS under similar conditions)1. These results confirmed our speculation that feeding H. axyridis with artificial diet for prolonged periods (10 versus 2 days) can improve the cold tolerance of indoor-reared H. axyridis adults. Moreover, compared to control diet treatment (Ha-CK), we found that feeding H. axyridis with β-carotene-amended artificial diet before storage (Ha-Car) can further improve the post-storage survivals when storage duration was prolonged to 120 days (> 50%). However, the weight losses of Ha-Car and Ha-CK were not significantly different at 120 DIS. As we know, carotenoids are powerful non-enzymatic antioxidants and supplementation of β-carotene can thus reduce the costs of innate immune response37. In addition, other physiological functions of carotenoids might also be benefit for survivals38,39.
In addition, after 90 and 120 days of storage, the females began to lay eggs following 6–7 days’ development and maintained a quite stable and high daily fecundity (around 30 eggs) within 15 days. In previous studies, persistence of high post-storage fecundity has been widely reported in field collected pre-wintering H. axyridis adults. For example, after 6 months of storage at 6°C, the adults had a daily oviposition rate of 21 eggs during the first month30; storage at 3°C for 120–150 days had no adverse effect on reproductive capacity29,32. For indoor-reared individuals, our previous research also found a high fecundity following 60 days of storage (39.9 eggs per day)1. Moreover, in this study, no significant difference was detected among the alive individuals of Ha-CK-90, Ha-CK-120, Ha-Car-90, and Ha-Car-120 feeding on M. persicae. Studies showed that aphid feeding during post-storage periods can cue ovarian development within several days which might reduce the differences originally existed between treatments40. These results also indicated that the fecundity of H. axyridis seems to be less sensitive to low temperature experiences. Actually, for many biocontrol agents, ability to survive after long-term cold storage with retaining reproductive capacity is their important property41.
Even so, obvious sublethal effects of long-term cold storage on fertility were detected in this study. In the four treatments, the daily egg hatchability dramatically decreased from day 6 and thereafter, and totally lost at day 14 and 15, which resulted in a relatively low average hatch rate (26–35%). Actually, our previous study had detected a relative lower egg hatch rate following 60 days of storage (28.1% vs 46.5% in non-stored control)1. Surprisingly, for field collected pre-overwintering adults, a very high hatch rate (85%) can be detected even after twofold durations of storage (up to eight months) at 6°C30. These results showed that the fertility of indoor-reared H. axyridis was much more sensitive to long-term cold storage, which means much more indirect chilling injuries were accumulated during storage3. Even so, the egg viability can be quickly recovered (day 3 post re-mating) and gradually increased by re-mating with a normal non-stored partner. To our knowledge, this study might be the first to report such mysterious negative effects of cold storage in coccinellids, while the possible reasons were still hard to deduce. Some studies reported that heat can cause negative effects on fertility through compromised spermatogenesis, dropped viability (or less competitive of sperm), or compromised motility of sperm (Reviewed by McAfee et al.42). Studies showed that the number of micropyles as well as their structures had crucial roles in the fertilization of eggs34, and eggs equipped with multiple micropyles offer several benefits43. In H. axyridis, the number of micropyles has been showed to be plastic but relatively stable (range from 18 to 21)34,37. Here, significantly reduced number of micropyles was detected on eggs in treatment Ovi-14 (19.0, egg hatch rate was 0) or ReM-8 (18.5, egg hatch rate was recovered to almost 50%) compared to that in Ovi-5 (21.0, egg hatch rate was almost 57%). These results further confirmed that the number of micropyles was plastic following long-term cold storage, but not directly related to the change of fertility.
More importantly, we found that the negative effects of cold storage on fertility can be transferred to the next generation. Eggs produced by the F1 offspring of Ha-Car still had significantly lower hatch rate compared to control treatment (19.6% versus 57.8%), but similar oviposition number was detected in these two treatments. However, Zhao et al. reported that cold acclimation (5°C) of H. axyridis parents for 5 days can cause negative effects on the fecundity of F1 offspring44. In addition, the number of micropyles in F1-11 (18.1) was still significantly lower than that in Ovi-5. The mechanism for how passing maternal effects of chilling damage to the F1 generation has not been fully revealed, and DNA methylation was supposed to be an important approach45. In future, the capability of recovering normal egg viability in F1 and following generations and how to realize still need to be revealed.
In summary, the indoor-reared H. axyridis (Ha-Car) can be stored for about 120 days at 6°C with the survivals of above 50%, and the alive adults had very high egg production capability on M. persicae. However, long-term cold storage had intra- and trans-generational negative effects on fertility, but it can be recovered by mating with a new non-stored male. Therefore, the stored H. axyridis showed to be applicable for releasing to control filed pests due to the fact that the egg hatchability can be recovered when their parents were re-mated with a wild partner.