Lethal and Sublethal Effects of Long-term Cold Storage on Indoor-reared Harmonia Axyridis Adults

Indoor-reared natural enemy with high quality after long-term cold storage is crucial for sustainable pest management. However, besides survivals, the sublethal effects were not widely been reported. In this study, Harmonia axyridis (Pallas), an important biological control agent in Asia, was reared with β-carotene-amended articial diet (Ha-Car) before storing at 6°C. After 30, 60, 90 and 120 days of storage, a series of biological parameters were measured to evaluate the tness of H. axyridis. We found that: (1) Survivals signicantly decreased with prolonged storage, and more Ha-Car individuals survived at day 120 compared to Ha-CK (control); (2) The contents of glycogen and trehalose dramatically decreased following storage, and the weight losses gradually increased; (3) The average egg production and hatch rates within 15 days were not signicantly different among treatments Ha-Car and Ha-CK following long-term storage (90 and 120 days), while the daily hatch rates gradually decreased from relatively high to zero at day 14 and 15; (4) The number of micropyles deposited on eggs also gradually decreased along with oviposition period. After re-mating with a new non-stored partner, the egg viability gradually increased again, while low egg viability was still detected in F1 generation. Moreover, reduced number of micropyles were detected on their eggs. In summary, Ha-Car can be cold stored for about 120 days with relatively high survivals and fecundity, but long-term storage produced remarkable intra- and trans-generational negative effects on fertility. Even though, the cold-stored H. axyridis had great potential being used in biological control program with inevitable promiscuity with eld individuals.


Introduction
Long term cold storage of laboratory-reared natural enemy is very important for developing feasible biological control programs 1 , especially for those relying on large number releasing of natural enemies 2 .
However, somatic damages causing by direct and/or indirect chilling injuries may accumulate during storage, e.g. accumulation of toxic metabolites and elimination of energy reserves 3 . In practice, some quanti ed parameters can be measured to evaluate the lethal and sublethal effects induced by cold storage. For most studies, signi cantly lowered survivals have been detected with prolonged storage (reviewed by Rathee and Ram 2 ), while the sublethal effects were not widely been reported 4 . Even so, the sublethal effects still needed to be closely monitored when make a comprehensive evaluation of a storage technique.
Speci cally, the sublethal effects generally include delayed oviposition, lower fecundity and fertility and lower voracity 4 or parasitism 5 . Until now, these effects were mostly conducted on parasitoids and rarely quanti ed in predators 4 . For example, the parasitism rate and fecundity of Habrobracon hebetor (Say) (Hymenoptera: Braconidae) signi cantly decreased after cold storage at 3°C or 5°C 6 ; fecundity of Trichogramma brassicae Bezdenko (Hymenoptera, Trichogrammatidae) also decreased at a lower rate after storage at 10°C 7 . For the mymarid wasp, Gonatocerus ashmeadi Girault (Hymenoptera: Mymaridae), cold storage of immature wasps for 40 days even caused 44% female sterility, and tremendous parasitism and fecundity reduction of 70% and 73%, respectively 8 . Moreover, studies showed that some sublethal effects can even be passed to their offsprings, called "transgenerational effects" 9 . For G. ashmeadi, when cold storage of the adult parents was more than 20 d, delayed development, decreased fecundity, reduced longevity, and increased male production were observed in F1 generation 10 . For predators, signi cantly decreased fecundity was found in Hippodamia variegate (Goeze) (Coleoptera: Coccinellidae) when the adults were stored at 6°C (> 35 days) or at 12°C/0°C (> 45 days) 4 ; similar reduced fecundity has also been detected in Cryptolaemus montrouzieri Mulsant (Coleoptera, Coccinellidae) when the adults were stored at 15°C for 20 days 11 and in Rhyzobius lophantae Blaisdell (Coleoptera Coccinellidae) when the adults were stored at 12°C (10-40 days) 12 .
The multicolored Asian ladybird beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae), native to Asia, has established populations worldwide and has also been considered as an important invasive insect species [13][14][15] . At present, this predatory ladybird beetle has been commercially reared and widely used as a biocontrol agent in many agricultural elds 16,17 due to its excellent voracity, dispersal capacity and multivoltine life-cycle 18,19 . However, H. axyridis is a "chill intolerant" predator 20 , and the logistics of low-temperature storage has been shown to be one of the most important limitations in using it in biological control programs 21,22 . Abundant studies have been done to improve the cold storage techniques, but most of them only focused on the eld collected pre-overwintering individuals 1 . For example, the quality of pre-storage diet has been con rmed to have notable effect on the post-storage tness of H. axyridis, and injured wine grape berries or sugar showed to be more optimal than water in decreasing mortality of overwintering beetles 23 . The indoor-reared individuals have been reported to be much more chilling intolerant 24,25 . For these populations, we recently found that the meridic arti cial diet is fruitful pre-storage nutrition for long-term cold storage (almost 60 days) 1 . As is well known, carotenoids are ubiquitous in organisms and have many important biological and physiological roles, e.g. light detection, photo-protection, coloration, hormone precursors and antioxidants 26,27 . In our previous study, we have con rmed that supplementation of β-carotene in arti cial diet can greatly increase the larval survival of H. axyridis 28 . Here, we speculated that feeding H. axyridis adults with carotenoid-amended arti cial diet before storage might contribute to improving their cold tolerance.
As for the post-storage tness of H. axyridis adults, previous studies reported that cold storage generally duration dependent, had obvious lethal effects on survival rate 1,29−31 . For sublethal effects, the persistence of high egg hatch rate (75-85% during two months) 30 and shortened pre-oviposition duration 29 has been reported by a few studies. Besides, no adverse effects on fecundity has also been reported before 1,29,30,32 .
In this study, we still focused on the effect of cold storage on indoor-reared H. axyridis adults, and the adopted storage techniques were similar to those used in our previous studies with some modi cations 1 .
Brie y, β-carotene-amended arti cial diet was supplemented as pre-storage nutrition, and the rearing period was prolonged to 10 days due to the deduction that rearing on arti cial diet for longer periods might result in higher post-storage survivals 1,33 . After that, the adults were stored at 6°C for different periods (30, 60, 90, or 120 days), and their post-storage tness as well as offspring's reproductive capabilities were measured to evaluate the lethal and sublethal effects. In addition, the contents of trehalose and glycogen that play important roles in cold hardiness of insects 22 were measured to evaluate their dynamic changes during storage. Besides, the number of micropyles located on eggshell, which play important roles in egg fertilization 34 , was determined to reveal the potential mechanisms of cold storage on fertility.

Determination of glycogen and trehalose content
Among the four storage durations, the content of glycogen was not signi cantly different, while the content of trehalose was signi cantly lower in Ha-Car-120 than in Ha-Car-30. However, compared to nonstored control (Ha-Car-0), both the glycogen and trehalose contents were signi cantly lower in individuals that had cold storage experiences (trehalose: Kruskal-Wallis χ 2 = 28.355, p < 0.001; glycogen: Kruskal-Wallis χ 2 = 19.277, p < 0.001) (Fig. 3).
Post-storage reproductive capabilities F0 generation. After removing from refrigerator, the adults began to lay eggs at day 6-7, which were similar among the four treatments (Kruskal-Wallis χ 2 = 2.914, p = 0.4051) (Fig. 4A). Daily egg production was quite stable within 15 days and showed to be uctuated around 30 (Fig. 4B). However, the daily egg hatch rates were relatively high only at the rst 5 days (above 50% in Ha-Car-120, Ha-CK-90, and Ha-CK-120), and dramatically decreased since then. Especially at day 12 and 13, the hatch rates were close to 0 (In fact, at day 11, 12 and 13, only small proportion of hatchable eggs can be detected from one pair of adults), and all eggs absolutely lost viability at day 14 and 15 ( At day 16, each Ha-Car-120 female or male was paired with a refreshed new partner (neither mated nor being exposed to cold storage before). After pairing, the daily egg productions in treatments ReM-were generally higher than those in ReM-within 7 days, but the average number per day was not signi cantly different between these two treatments (F 1, 9 =0.556, p = 0.4750) (Fig. 5A, B). The average egg hatch rate per day was also similar in treatments ReM-and ReM-(F 1, 9 =0.111, p = 0.7460) (Fig. 5C), while the daily egg hatch rates in treatments ReM-were also generally higher than those in ReM-within 7 days, and they all gradually increased with prolonged oviposition period and nally reached to about 50% (Fig. 5D).

Discussion
Tauber et al. stated that an effective storage technique must ful ll the following tness requirements after storage: high survival, synchronous and predictable initiation of reproduction, and sustained high fecundity and fertility 35 . 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 led-collected pre-overwintering individuals 29,30 . After storage, the adults exhibited appreciable post-storage tness, 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 signi cantly lower, there were still more than 30 percent. Chilling injury during storage was an important factor causing mortality 36 . In addition, other indirect effects including accumulation of toxic metabolites and elimination of energy reserves have also been suggested as probable reasons for death 1,3 . 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. Speci cally, 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 diet 1 . 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 con rmed our speculation that feeding H. axyridis with arti cial 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 arti cial 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 signi cantly different at 120 DIS. As we know, carotenoids are powerful non-enzymatic antioxidants and supplementation of βcarotene can thus reduce the costs of innate immune response 37 . In addition, other physiological functions of carotenoids might also be bene t for survivals 38,39 .
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 eld 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 rst month 30 ; storage at 3°C for 120-150 days had no adverse effect on reproductive capacity 29,32 . 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 signi cant 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 treatments 40 . 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 property 41 .
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 eld 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°C 30 . 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 storage 3 . 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 rst 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 eggs 34 , and eggs equipped with multiple micropyles offer several bene ts 43 . In H. axyridis, the number of micropyles has been showed to be plastic but relatively stable (range from 18 to 21) 34,37 . Here, signi cantly 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 con rmed 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 signi cantly 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 offspring 44 . In addition, the number of micropyles in F1-11 (18.1) was still signi cantly 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 approach 45 . 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 led pests due to the fact that the egg hatchability can be recovered when their parents were re-mated with a wild partner.

Insects
The ladybird beetles H. axyridis were from continuous laboratory rearing colony and they were reared with the mixture of Aphis craccivora Koch (Hemiptera: Aphididae) and Acyrthosiphon pisum Harris (Hemiptera: Aphididae) that co-infesting on broad bean seedlings. Large number of H. axyridis larvae were reared with these two aphids and used for producing adults for cold storage. In addition, the colony of Myzus persicae (Sulzer) (Hemiptera: Aphididae), a serious pest of many crops 46 , was established on pepper seedlings to evaluate the post-storage tness of H. axyridis. All these insects as well as their host plants were maintained in an insectary (24 ± 1°C, 60% RH, and 16: 8 L: D).

Preparation of pre-storage diets
The fresh pork liver-based arti cial diet was prepared following the methods described in Sun et al. 1 , but with the additional additive of carotenoids. Brie y, the standard (> 98.00%) (Yuanye Biotechnology Company, Shanghai, China) was weighted and fully dissolved in the lin-seed oil + olive oil (1 + 1.4 by volume) using an ultrasonic cleaner to get a solution of 475 µg/ml. After that, 1.9 ml β-carotene oily solution was added into the basic components. All ingredients were fully stirred in a beaker (100 ml), and the prepared diet was divided into groups (kept in 20 ml plastic vials) and stored in a − 20°C refrigerator until use. The diet without any carotenoid additives was used as control.

Cold storage treatments
The cold storage procedures were also similar to those reported by Sun et al 1 .
Step 1, newly emerged H. axyridis adults were reared in plastic Petri dishes (9 cm in diameter) with the density of 6-8 in each. These adults were respectively fed with su cient β-carotene-amended arti cial diet or control diet for 10 days (respectively named Ha-Car and Ha-CK). During this period, the diet was refreshed every other day.
Step 2, the adults were transferred to a climate chamber (15 ± 1°C) for cold acclimation for 2 days (continue feeding with diet).
Step 3, at day 13, adults were individually weighed with an AE224C electronic balance (SDPTOP, China) and then transferred to a small plastic Petri dish (3 cm in diameter).

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Five or six plastic Petri dishes were placed one on top of another and fastened together with a para lm.
Step 4, the Petri dishes were packed in a carton box (30×10×10 cm) and stored at 6°C under full darkness in a refrigerator (6°C). The storage durations were set as 30, 60, 90, or 120 days, and the storage treatments as well as number of individuals used in each treatment were shown in Table 1. Step 5, following different days in storage (DIS), the adults were transferred back to 25 ± 1°C, and their survivals were calculated following the equation: The alive individuals were weighed again to calculate the weight loss during storage (pre-storage weight -post-storage weight), and 10-13 individuals of both sexes were stored in a − 80°C refrigerator for measuring nutrition. Specially, during storage, the ladybird beetles were transferred back to 15 ± 1°C for one-hour intermittent recovery every 30 days.

Determination of glycogen and trehalose content
The glycogen and trehalose content in survived adults were determined using a glycogen and trehalose assay kit (Comin Biotechnology Co., Ltd. Suzhou, China) 47 . To obtain enough samples, the Ha-Car females and males at same DIS were incorporated and 3-4 individuals (elytra discarded, almost 0.05 g) were used as one replicate. The extractions were conducted following the instructions, and the absorption at 620 nm was measured four times for each replicate. In total, 6 and 8 replicates were respectively conducted for measuring glycogen and trehalose content.
Evaluation of post-storage reproductive capabilities F0 generation. In order to determine the effects of long-term cold storage on reproductive capabilities of H. axyridis, the alive-adults of Ha-Car and Ha-CK following 90 and 120 days of storage were paired and separately reared in plastic Petri dishes (9 cm in diameter, 1.5 cm high). Su cient M. persicae infesting on pepper leaves was supplied as food and refreshed every day. Their oviposition was monitored, and, once egg production initiated, the eggs in each Petri dish was carefully counted and incubated in another plastic Petri dish with an immersed cotton ball for keeping moisture. Specially, at day 5, 8, 11, and 14, 6-8 eggs were randomly selected from the egg cluster of each pair (respectively named Ovi-5, Ovi-8, Ovi-11, and Ovi-14) and used for determination of the number of micropyles. The hatching of eggs was monitored once a day, and newly hatched larvae were immediately picked out to avoid cannibalizing on unhatched eggs. Egg hatch rates were calculated following the equation: In total, 6-7 pairs were used for each treatment and their egg production within 15 days was recorded.
In this study, we found that egg hatch rates gradually decreased with prolonged oviposition period and nally decreased to zero at day 14 and 15. Here, we specially designed a re-mating experiment to test whether the egg viability could be recovered. That was, at day 16, six females or ve males (one male dead) each was re-mated with a normal unmated male or female (15-20 days old) (these two treatments were respectively named ReM-and ReM-), and their egg production as well as hatch rates were recorded for another 7 days. The average egg production and hatch rates were calculated as described above. At day 8, 10 eggs from each pair were also randomly selected for determination of micropyles (named ReM-8).

F1 generation.
In order to test whether long-term cold storage had transgenerational effects on reproductive capabilities of F1 offsprings, the neonate larvae of Ha-Car-120 (from eggs produced at day 8-9) were reared with M. persicae until pupation. The newly emerged adults were paired and reared with M. persicae in a plastic Petri dish. The pre-oviposition period, egg production and hatch rates within 10 days were recorded, and those larvae from pairs that had not been cold stored were used as control. In total, 10 pairs were used in each treatment. Specially, after recording of 10 days, at day 11, the eggs from F1 offsprings were collected for determining the number of micropyles (named F1-11).
Determination of the number of micropyles Number of micropyles located on the egg shell was examined with Hitachi S-3400N scanning electron microscope (SEM) (Hitachi Science Systems, Ltd., Japan). For SEM photographing, the sample preparations were following the methods described by Sun et al 48 . Brie y, the collected egg samples were immediately transferred to a 1.5 ml centrifuge tube containing 10% ethanol. Then, they were dehydrated in a graded series of 30%, 50%, 70%, 80%, 90% ethanol for 20 min each and in 100% ethanol, twice for 30 min. Specimens were transferred to a mixed solution of ethanol and tert-butanol (3:1, 1:1, and 1:3, by volume) for 15 min each, and nally to 100% tert-butanol for 30 min. After that, the specimens were dried with a freeze-drier (VFD-21S, SHINKKU VD, Japan) for 2 hr. The dried specimens were mounted on aluminum stubs and coated with gold/palladium (40/60) in a high-resolution sputter coater (MSP-1S, SHINKKU VD, Japan). In total, 80 available images were selected for each treatment to determine the number of micropyle located on the top area of each egg.

Data analysis
All data analyses were conducted with R software (version 4.0.2). Post-storage survivals were analyzed using a generalized log-linear model (function glm. model) with pre-storage diet treatments (Ha-Car and Ha-CK) and cold storage durations (DIS) as independent xed factors. Weight loss during cold storage was analyzed using two-way analysis of variance (ANOVA). If the interactions of pre-storage diets and storage durations were signi cant, one-way ANOVA were then used to examine the effects of one factor within each level of the other factor. For the parameters number of micropyles (after log10 transformation), daily average fecundity and fertility in F0 and F1 generation, and pre-oviposition period in F1 generation, one-way ANOVA was used to analyze the differences among different treatments; while the non-parametric Kruskal-Wallis test was used to analyze the differences of glycogen and trehalose content and the pre-oviposition period in F0 generation.

Data Availability
The datasets generated and analyzed during the current study are available in the fgshare repository, https://doi.org/10.6084/m9. gshare.13663613.   Weight losses of H. axyridis female (A) and male (B) adults after different periods of cold storage at 6 °C.

Male
In each graph, different lowercase and uppercase letters represents signi cant differences among four storage durations in Ha-Car and Ha-CK, respectively (Tukey HSD test, p < 0.05). Asterisk represents a signi cant difference between Ha-Car and Ha-CK at each storage period, ns means no signi cant difference (Tukey HSD test, p < 0.05).