Introduction

The multicolored Asian ladybird beetle, Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae) is native to China, Japan, Korea, Mongolia and Siberia (Brown et al. 2008). Currently, the ladybird has gained much attention because it has become a threat to native biodiversity, a potential pest in certain fruit crops and a human nuisance in its invasive range in North America and Europe (Roy and Wajnberg 2008). However, the generalist ladybird has successfully been used for the biological control of some pests in greenhouses and outdoor crops. Several aphid species, such as Aphis gossypii Glover, Macrosiphum rosae (L.), Toxoptera piricola Matsumura, and Brachycorynella asparagi Mordvilko, were successfully suppressed by releasing H. axyridis in China (Li et al. 1995; Sun et al. 1996; Ma et al. 2005). In North America and Europe, the ladybird has been used in the biocontrol of A. gossypii (Wells et al. 2001; Gil et al. 2004), A. glycines (Landis et al. 2004; Mignault et al. 2006), M. rosae (Ferran et al. 1996), Acyrthosiphon pisum (Harris) (Cardinale et al. 2003), and Phorodon humuli (Schrank) (Trouve et al. 1997). Furthermore, this species has also exhibited effective biocontrol on scale insects, such as Matsucoccus massonianae Yang and Hu in China (Wang 1982), M. matsumurae (Kuwana) in Japan (McClure1986), and Pseudaulacaspis pentagona (Targioni) in Korea (Choi et al. 1995). The decline in the populations of the pseudococcid, Nesticoccus sinensis Tang and the eriococcid, Rhizococcus transversus (Green), both important pests of bamboo in Jiangsu, China (Xu and Wu 1989), and of the curculionid, Diaprepes abbreviatus L., a major pest of citrus in Florida (Stuart et al. 2002), were largely due to this predator.

There are abundant natural resources of H. axyridis in northeastern China. This ladybird is the most common aphidophagous predator in the local forest ecosystems and in orchards and agricultural fields (Yuan et al. 1994). Rising numbers of H. axyridis have been found in recent years in Jilin Province, which may be related to the occurrence of a large number of corn leaf aphids, Rhopalosiphum maidis (Fitch), with the expansion of maize planting areas (personal observation). In mid-October in northeastern China, massive swarms of ladybirds flew out of the cornfields toward the sunlit south-southwestern sides of buildings or rocks, searching for overwintering sites. The migrations were usually aggravated once the noontime temperature rose above 18°C following a cool period (Huelsman et al. 2002).

Overwintering adults of H. axyridis are very resistant to winter conditions and may survive for short periods, even at −30°C (Pervez and Omkar 2006). They overwinter in cracks and crevices of rocks and buildings or caves, which offer them a degree of protection from low temperatures (Sakurai et al. 1992). In addition, they acquire cold hardiness by myo-inositol accumulation (Watanabe 2002) and protect themselves against low temperatures by supercooling (Koch et al. 2004; Berkvens et al. 2010). The adults of a Japanese strain may survive up to 200 days at −5°C (Watanabe 2002).

Cold storage is a valuable tool for insects used in biological control programs. It not only provides a steady supply of insects for research but also yields flexibility and efficiency in mass production, allows for the synchronization of a desired developmental stage for release, and lengthens their availability to consumers (Leopold 1998). Long-term cold storage of some parasitoids has been successfully achieved by inducing diapause or quiescence without decreasing their fitness (López and Botto 2005; Chen et al. 2008). Recently, Riddick and Wu (2010) tested long-term storage of the predatory mite Phytoseiulus persimilis Athias-Henriot with cryoprotectant and carbohydrate chemicals. The possibility of cold storage has been studied for many coccinellids including Coccinella septempunctata L., Adalia bipunctata L. (Hamalainen 1977), C. undecimpunctata L. (Abdel-Salam and Abdel-Baky 2000) and Coleomegilla maculata lengi Timberlake (Gagné and Coderre 2001). Several studies have investigated the cold hardiness of H. axyridis (Ma et al. 1997; Watanabe 2002; Koch et al. 2004; Berkvens et al. 2010), but the effect of cold storage on fitness of the ladybird has rarely been documented. Ma et al. (1997) examined the effect of temperature and relative humidity (RH) on the survival of the pre-wintering adults of H. axyridis from northeastern China and found that 0–4°C and 70–80% RH were the most favorable conditions. However, the reproductive parameters of H. axyridis after long-term cold storage have not been evaluated until now.

With the swarming and gathering behavior in pre-wintering adults of H. axyridis in autumn, massive natural supplies are easily collected manually or mechanically from the walls of buildings. Developing effective methods to store biocontrol agents without compromising their fitness is crucial to releasing them in augmentative biological control programs (Leopold 1998). We hypothesized that an optimal duration of cold storage would not influence the fitness of pre-wintering H. axyridis in terms of survival, predation capacity and fecundity. Storing pre-wintering adults of H. axyridis may be useful for the biological control of insect pests in the following season.

Materials and methods

Insects

Pre-wintering adults of Harmonia axyridis were collected at Jilin Agricultural University (43°48′N, 125°23′E) on October 10, 2009 while they swarmed on and around the walls of buildings. The adults were separated into two groups of females and males according to labrum pigmentation (McCornack et al. 2007). The two groups were fed a 20% honey solution for two days at 11°C. Then approximately 200 females or males were put into plastic Ziploc bags (length 16 cm, width 13 cm) with pinholes on each side for ventilation and filled with folded filter papers. These adults were immediately used for the following long-term cold storage experiments or as unstored controls.

Survival of adult H. axyridis during storage

The female or male ladybirds in plastic Ziploc bags as described above were transferred to four different incubators set at different temperatures (−3, 0, 3, 6 ± 1°C) for five different durations (30, 60, 90, 120, 150 days) under full darkness and 60–70% RH. A total of fifteen plastic Ziploc bags containing about 200 females (or males) were prepared for each storage temperature. Considering the adverse impacts of sudden temperature changes, the adults that were taken out of the incubators could not be returned to continue cold storage once survival was determined. Three plastic Ziploc bags containing females (or males) at various storage durations were randomly removed from the incubator and initially placed at 10 ± 1°C for 24 h, then transferred to standard conditions (25 ± 1°C, 60 ± 5% RH and 16:8 L:D). The adults were considered alive if they were still mobile after 24 h at standard conditions.

Post-storage fitness of adult H. axyridis

After the survival investigation mentioned above, the fecundity of adult H. axyridis was immediately determined after 60, 90, 120 or 150 days of storage, as was the fecundity of the unstored adults. In this experiment, pairs of female and male adults that had experienced various storage durations at each storage temperature were introduced in individual Petri dishes (diameter 9 cm, height 2 cm) with about 400–500 cowpea aphids Aphis craccivora Koch on the detached leaves of horsebean, Vicia faba L. The cowpea aphids were cultured for more than ten generations on horsebean plants in the laboratory. Every 24 h, the total number of eggs laid was recorded, and the live females and males were transferred to a new Petri dish containing a similar number of aphids. During the experiment, only a single male was used to mate with each female. This process lasted for 20 days after the ladybird couples were introduced. Twenty-five pairs of adults for each treatment were initially used, and the data from 18 to 23 replicates were analyzed because several adults escaped during manipulation or were parasitized by a Perilitus species.

At the end of long-term cold storage, we investigated the predation capacity on aphids by the adults. In this experiment, pairs of female and male adults that had experienced a 150-day storage at 3°C were introduced in individual Petri dishes with 400 older-stage aphids (third-fourth instar nymphs and wingless adults) on the detached leaves of horsebean. To prevent aphids from escaping, the bottom of the dish was covered with a moist filter disk and inverted. Every 24 h, the total number of aphids consumed by the adult couple was recorded, and the live female and male were transferred to a new Petri dish containing a similar number of aphids. This process lasted for 15 days. Pairs of unstored adults were used as control in this study. A total of twenty replicates were used for both treatments, but only the data from 15 replicates of each treatment were analyzed because several adults escaped or were parasitized. All experiments were conducted under standard conditions (25 ± 1°C, 60 ± 5% R.H. and 16:8 L:D).

Statistical analysis

For the comparison of the survival, pre-oviposition period and fecundity of adult H. axyridis under different cold storage conditions, a two-factor analysis of variance (ANOVA) was performed. The two factors were storage temperature (four levels) and storage duration (survival: five levels; fecundity: four levels). Before analysis, data on the percentage of survival were arcsine square-root transformed. When an overall ANOVA indicated significant effects of the factors at P < 0.05, the means were compared using Tukey’s honestly significantly difference (HSD) test. A paired t-test was used to compare the number of aphids consumed by adult pairs that experienced 150-day storage at 3°C and those that were unstored. All statistical analyses were done using the statistical software package SAS (SAS Institute 2006).

Results

Survival of adult H. axyridis during storage

The survival of adult H. axyridis after storage at −3, 0, 3 and 6°C is shown in Fig. 1. Adult survival varied significantly among storage temperatures (Female: F 3, 40 = 13.68, P < 0.0001; Male: F 3, 40 = 12.66, P < 0.0001) and storage duration (Female: F 4, 40 = 6.78, P = 0.0003; Male: F 4, 40 = 7.21, P = 0.0002), and a significant storage temperature × duration interaction was detected (Female: F 12, 40 = 63.38, P < 0.0001; Male: F 12, 40 = 98.75, P < 0.0001). A similar dynamic tendency existed for both females and males at various storage conditions (Fig. 1 a, b). The survival of females and males decreased clearly at −3°C with 38.3–44.7% of survival after 90-day storage. All adults had died after a 150-day storage. More than 87% of female and male adults survived after 90-day storage at 0°C. After that, the ladybird showed a relatively rapid decrease in survival. At 150 days, survival had dropped to near 50%. Female and male adults showed more than 82% survival after 150-day storage at both 3°C and 6°C. Specifically, female adult survival was the highest (near 90%) at the end of the 3°C storage.

Fig. 1
figure 1

Survival (means ± SE) of adult H. axyridis stored for different durations at −3, 0, 3 and 6°C. a Female b Male

Full size image

Post-storage fitness of adult H. axyridis

Fecundity Both storage temperature and storage duration had no significant effect on the fecundity of H. axyridis (Storage temperature: F 3,304 = 1.78, P = 0.1506; Storage duration: F 3, 304 = 0.88, P = 0.4517), but a significant storage temperature × duration interaction was detected (F 9,304 = 85.77, P < 0.0001). Similarly, neither storage temperature nor storage duration had a significant effect on the pre-oviposition duration of H. axyridis (Storage temperature: F 3,304 = 0.76, P = 0.5173; Storage duration: F 3,304 = 1.66, P = 0.1757), but a significant storage temperature × duration interaction was detected (F 9,304 = 165.98, P < 0.0001).

Harmonia axyridis that were stored for 90 days at 0°C laid more eggs than those of the unstored control but laid similar egg numbers than those stored for 90–150 days at 3°C and 120–150 days at 6°C. The beetles that were stored for 90–150 days at 3°C laid more eggs than those stored for 60 days at the same temperature. The number of eggs laid was increased with prolonged storage duration at 6°C until the storage duration reached 120 days. However, the number of eggs laid was decreased with a prolonged storage duration at −3°C until the storage duration reached 150 days and all individuals were dead (Fig. 2a).

Fig. 2
figure 2

Number of eggs laid a and pre-oviposition duration b (means + SE) of H. axyridis females over 20 days at different storage treatments. Different letters above the bars indicate significant differences (Tukey’s HSD test, P < 0.05)

Full size image

The pre-oviposition duration of H. axyridis that were stored for 120 days was longer than for those stored for 60–90 days at −3°C. In contrast, the pre-oviposition duration tended to shorten with a prolonged storage duration at 0–6°C, with the exception of the 0°C 120-day treatment (Fig. 2b).

Predation There was a tendency that the individuals that were stored for 150 days at 3°C consumed more aphids per day than the unstored controls from the second day onward (t = 1.2601–9.1698, df = 28, P-value between 0.0001 and 0.2180) with the exception of 12th day (t = 3.2246, df = 28, P = 0.0032) (Fig. 3). Over a period of 15 days, cold-stored H. axyridis couples consumed significantly more aphids than the unstored controls (3534.6 ± 26.7 vs. 3184.6 ± 33.3; t = 8.1898, df = 28, P < 0.0001).

Fig. 3
figure 3

Number of aphids killed (means ± SE) by adult pairs of H. axyridis stored for 150 days at 3°C or without cold storage for 15 days

Full size image

Discussion

The results of this study indicate that long-term storage at or below 0°C had negative effects on the survival of adult H. axyridis. More than 50% of adults died when they were stored for 90 days at −3°C, and no individuals survived 150-day storage at −3°C. In contrast, the ladybirds showed more than 80% survival when they were stored for 150 days at both moderately low temperatures (3°C and 6°C). Harmonia axyridis, a ‘chill intolerant’ insect, can survive at freezing temperatures by moving to physically protected areas or by acquiring cold hardiness by cold acclimation and supercooling (Pervez and Omkar 2006). A temperature of −3°C would result in more cold damage, especially in swarming adults (collected in early October), which have not acquired adequate cold tolerance yet. In addition, the cold tolerance of adult H. axyridis changes seasonally. The supercooling point of adults collected in Japan between December and February reached −18°C, far lower than the supercooling point of those collected in October (−7°C) (Watanabe 2002). All adults kept for 150 days at −3°C died in the current study. Whereas in the study of Labrie et al. (2008), about 45% of adults collected from the field in Canada in November were still alive when they were stored for at least 150 days at −5°C. These differences were probably related to different quantities of accumulated myo-inositol between the two collections (Watanabe 2002). Here, female and male adults exhibited a similar dynamic tendency in survival at various storage conditions, which is in accordance with previous studies of Koch et al. (2004) and Berkvens et al. (2010).

The effects of cold storage on the fitness of numerous mass-reared parasitoids (López and Botto 2005; Pandey and Johnson 2005; Chen et al. 2008) and predators (Abdel-Salam and Abdel-Baky 2000; Gagné and Coderre 2001; Riddick and Wu 2010) have been assessed. However, the effect of cold storage on the fitness of pre-wintering H. axyridis have rarely been documented, except for some studies focusing on survival (Koch et al. 2004; Berkvens et al. 2010). Our results indicated that long-term cold storage had different effects on the fecundity of H. axyridis at different temperatures. Prolonged cold storage at both 3°C and 6°C shortened pre-oviposition duration and had no adverse effect on reproductive capacity. Compared with unstored individuals, there was a significantly shorter pre-oviposition duration at shorter storage durations for those kept at −3°C. Subsequently, prolonged cold storage exhibited deleterious effects that occurred in the form of extended pre-oviposition duration and reduced fecundity. Interestingly, the individuals that experienced 90-day storage at 0°C had the shortest pre-oviposition duration and the largest reproductive capacity. Nevertheless, 3–6°C appear to be suitable storage temperatures for the swarming adult H. axyridis that were collected in October. These temperatures resulted in higher survival and fecundity. The ladybirds that were stored for 150 days at 3°C consumed more aphids than the unstored controls, which further demonstrated that there was no adverse effect of long-term cold storage on their fitness. The higher predation capacity of adults post-storage could be explained in part by the fact that H. axyridis is a temperate insect with a facultative winter diapause (Koch 2003). Although these individuals survive much longer at a moderately low temperature (3°C), they must consume a certain amount of energy reserves to maintain their metabolism and/or produce cryoprotectant compounds, such as myo-inositol (Watanabe 2002). The ladybirds emerging from hibernation are in a status of starvation. Thus, they have the potential to consume more prey to replenish energy and promote oogenesis.

Many factors might have affected the survival of the predatory natural enemies in the cold storage experiments, including the food given prior to cold storage (Coudron et al. 2009) or during storage (Tauber et al. 1997; Thorpe and Aldrich 2004), cryoprotectants and carbohydrates sprayed prior to cold storage (Riddick and Wu 2010), and populations tested in storage (Berkvens et al. 2010). For the assessment of the cold tolerance of H. axyridis, it would be more practical to use individuals from laboratory populations than those collected from the field (Bazzocchi et al. 2004; van Lenteren et al. 2008). However, the cold tolerance of individuals collected from the field was much higher than that of laboratory-reared individuals (Berkvens et al. 2010). Therefore, it appears that freshly collected field populations are more suitable for long-term cold storage. A honey solution was offered to adult ladybirds for two days prior to storage in the current study. Further research is necessary to investigate the effects of other foods prior to storage, including natural and unnatural foods (Dong et al. 2001) on the survival, reproduction and predation capacity of H. axyridis.

In summary, we conclude that swarming H. axyridis adults collected in October can be safely stored with a high survival rate at 3°C for 150 days without any reduction in reproductive and predatory capacity. The cold storage methods show the potential of using field-collected pre-wintering ladybirds for pest suppression in the following season. As adult ladybirds tend to fly away from the crop soon after release, their effectiveness is constrained (Adachi-Hagimori et al. 2011). Nevertheless, these stored adults could be used to mass-produce eggs or larvae in the laboratory prior to inundative release. Furthermore, these results can be used to maintain laboratory and mass-reared colonies of H. axyridis. Although there was no adverse effect of long-term cold storage on the fecundity and predation of adult H. axyridis in the laboratory, further field studies are needed to investigate the success of stored ladybirds under natural conditions, particularly in dispersal, predation capacity and prey location.