REARING OF APHIDIUS AVENAE AND APHIDIUS GIFUENSIS

Colonies of A. avenae and A. gifuensis were initiated from individual mummies collected from wheat plants at the experimental station of Shandong Agricultural University, China (36.15°N, 117.15°E), and reared at a 16:8 h L:D photoperiod and 22 °C on wheat ‘Lumai 2' seedlings infested with S. avenae. Parasitoids were allowed to complete at least 1 generation under these conditions before use in the experiments. To obtain adult parasitoids, synchronized 1-d-old mummies were collected. All mummies were placed in glass vials (10 per vial), closed with plastic lids that were modified to allow for the movement of air through a small hole (1 cm diameter) covered by 75 µm mesh muslin. In each vial, a cotton wool wick saturated in 10% honey and water solution served as food and moisture sources for emerging parasitoids. Each day, newly emerged wasps were collected into clean glass tubes (2.5 × 20 cm) with access to both water and honey solution. Parasitoids were used in experiments less than 24 h after emergence. Experimental individuals of each species were measured to make sure they were of the same size for each species.

SUPERCOOLING POINT AND FREEZING POINT

The SCP and FP were determined by attaching a copper-constantan thermocouple (0.125 mm diameter) with a small amount of Vaseline grease to the upper surface of each adult within a polypropylene tube. The tube was sealed with cotton wool and inserted in a larger polypropylene tube placed in the chamber of a controlled rate freezer (DCW23506, Ningbo, China). Changes in temperature were continuously monitored with an electronic recorder. Prior to immersion in a refrigerated alcohol bath, the tubes were enclosed in an aluminum cylinder that acted as a heat sink and provided temperature stabilization during cooling. The temperature of the bath was lowered from 10 °C to −30 °C at a rate of approximately 1 °C/min. The lowest temperature reached before an exothermic event occurred due to release of latent heat was taken as the SCP of the individual. The burst of heat during the formation of ice crystals would raise the temperature of the individual to its FP.

MINIMUM TEMPERATURE FOR WALKING AND CHILL COMA

Temperature was decreased from 22 °C (the rearing or acclimation temperature) to −15 °C at a rate of 0.2 °C/min, representative of rates experienced in nature, to avoid other responses being induced in the small subject specimens, such as rapid cold hardening (Kelty & Lee 1999). The minimum temperatures were selected on the basis of preliminary experiments, in which they resulted in 100% entry into chill coma. The temperatures at which each individual lost the ability to walk (minimum temperature for walking, WTmin) and entered chill coma were recorded. In the experiments, chill coma was defined as the last movement of an appendage (i.e., leg or antenna) as described by Hazell et al. (2008).

CHILL COMA RECOVERY AND ACTIVITY RECOVERY

Chill coma recovery and activity recovery were observed in a fresh sample of parasitoids. Temperature was decreased from 22 to −15 °C at a rate of 0.2 °C/min, thereby reaching temperatures that ensured 100% of the parasitoids were in chill coma. Specimens were held at these temperatures for 5 min before being warmed to their rearing or acclimation temperature at a rate of 0.2 °C/min. The temperatures at which each individual regained the ability to move their appendages (chill coma recovery) and then walk (activity recovery) were recorded.

MAXIMUM TEMPERATURE FOR WALKING AND HEAT COMA

Temperature was increased at a rate of 0.2 °C/min from 22 to 50 °C. This maximum temperature was selected on the basis of preliminary experiments, in which it induced heat coma in all individuals. The temperatures at which an individual was not able to walk (maximum temperature for walking, WTmax) and entered heat coma (last movement of an appendage) were recorded.

It was not possible to record the activity recovery temperature after WTmax and heat coma exposure because these 2 temperatures were typically lethal.

STATISTICAL ANALYSES

Differences in SCPs and FPs of male or female adults of A. avenae and A. gifuensis were analyzed using 1-way analysis of variance (ANOVA), and means were separated using Tukey's honestly significant difference (HSD) test. For thermal activity thresholds, individual comparisons were made using the Bonferroni 95% confidence intervals. SPSS 16.0 for Windows was used for calculations.

SUPERCOOLING POINT AND FREEZING POINT

There were no differences in both SCP and FP between the male and female wasps of A. gifuensis (SCP: P = 0.328, FP: P = 0.545) or A. avenae (SCP: P = 0.732, FP: P = 1.000) (Table 1). The mean SCP of A. gifuensis females was lower than that of A. avenae males (P = 0.031). A difference was observed in FP between female wasps of A. gifuensis (−19.8 ± 0.4 °C) and females of A. avenae (−18.1 ± 0.3 °C) (P = 0.007) or males of A. avenae (−18.0 ± 0.4 °C) (P = 0.01).

MINIMUM TEMPERATURES FOR WALKING, CHILL COMA, CHILL COMA RECOVERY, AND ACTIVITY RECOVERY

With decreasing temperature, the wasps lost the ability to move their appendages and then were unable to walk. The temperatures at which these thermal thresholds, i.e., WTmin, chill coma, chill coma recovery, and activity recovery, occurred are shown in Table 2.

Differences in WTmin between the females or the males of the 2 parasitoid species, and between the females and males of A. gifuensis were significant (F = 9.9; df = 3; P < 0.001). Females of A. gifuensis had the lowest WTmin (−5.1 ± 0.3 °C). No difference was observed in the temperature at which the parasitoids entered chill coma (F = 1.3; df = 3; P = 0.284).

A difference was observed in the temperature at which the parasitoids began to recover after chill coma (F = 6.3; df = 3; P = 0.001). The males of A. avenae had the highest temperature (5.9 ± 0.3 °C) at which they began to recover after chill coma, followed by the females of A. avenae (5.1 ± 0.2 °C), males of A. gifuensis (4.6 ± 0.1 °C), and the females of A. gifuensis (4.1 ± 0.4 °C). The females of A. gifuensis had a lower temperature (5.9 ± 0.3 °C) for activity recovery after chill coma compared with the females of A. avenae (7.8 ± 0.9 °C) (P = 0.034).

MAXIMUM TEMPERATURE FOR WALKING AND HEAT COMA

There was no difference (F = 1.3; df = 3; P = 0.292) in WTmax between the males and females or between the 2 species (Table 3). A difference was observed for the temperature at which the parasitoids entered heat coma between females of A. avenae (43.0 ± 0.3 °C) and males of A. gifuensis (41.3 ± 0.7 °C) (P = 0.024).

THERMAL TOLERANCES MATCH SEASONAL OCCURRENCE OF THE TWO PARASITOID SPECIES

Although there was no significant difference in SCP between the male or female wasps of the 2 species, the mean FP of A. gifuensis females was significantly lower than that of A. avenae females (Table 1). Moreover, WTmin and the temperature at which the females of A. gifuensis regained the ability to walk after chill coma were significantly lower than those of A. avenae females (Table 2). Females of A. gifuensis had lower thermal activity thresholds than females of A. avenae, which might account for their smaller mean body size compared with the females of A. avenae, because WTmin was previously shown to correlate with surface-area-to-volume ratio (SVR) in other parasitoids, where species with higher SVR can maintain activity at lower temperatures than those with lower SVR (Renault et al. 2003; Le Lann et al. 2011). This trait could lead to the occurrence of A. gifuensis in large quantity in the early spring season, when Dong (2012) found its populations reaching peak numbers about 10 d earlier than populations of A. avenae in wheat fields.

Once the parasitoids survive the winter, a gradually increasing temperature in spring would then allow significant movement of individuals from overwintering sites, both as adult parasitoids and as eggs or larvae within aphids (Starý 1988). Both A. gifuensis and A. avenae have been occurring from late Mar to early Jun (early spring to early summer) in the main cultivating area of wheat in China. The females of A. avenae had significantly higher heat coma temperatures than the males of A. gifuensis (Table 3). The variation in content and composition of heat shock protein in these 2 parasitoid species may be the key factor that caused the different high-temperature tolerance capacity. In addition, a previous study found that Wolbachia-infected parasitoids were more tolerant to high-temperature stress than uninfected individuals (Wu et al. 2015). As temperatures rise, the enhanced tolerance of A. avenae to high-temperature stress would allow this species to produce more eggs to develop into adults locating the increased number of host aphids, and become the major parasitoid in the late grain filling and milk ripe stages (Dong 2012). The sympatric species A. gifuensis and A. avenae from the same ecological guild have different thermal tolerances, which may allow them to occur in the same wheat field habitat, and protect them from severe competition within the same niche of S. avenae, especially when the aphid number is low in early spring.

DEVELOPMENT OF RELEASE TECHNIQUES

In Taian, Shandong Province, the mean minimum temperature in late Mar was 2 °C, and the extreme low temperature was −8 °C in the past 10 yr. The parasitoid A. gifuensis was the most dominant species from wheat jointing to early grain filling stages (Dong 2012). Therefore, large numbers of A. gifuensis could be reared and released in the early spring season to control aphids in the fields when the temperature is above 5.9 °C. This recommendation may guide the farmers to develop an efficient aphid control practice based on conservation and augmentation biological control techniques in the fields. Furthermore, our results indicate that A. avenae was more suitable than A. gifuensis for the biological control of aphids in high-temperature areas or greenhouse environments. To develop and release the 2 parasitoid species in fields or greenhouses under optimum conditions, we suggested that the temperature fluctuation could be from 5.9 to 40.2 °C for A. gifuensis and from 7.8 to 40.6 °C for A. avenae.

Table 2.

Temperatures (°C) of minimum temperature for walking, chill coma, chill coma recovery, and activity recovery in Aphidius avenae and Aphidius gifuensis.

Table 3.

Temperatures (°C) of maximum temperature for walking and heat coma of Aphidius avenae and Aphidius gifuensis

In combination with promising field practices such as intercropping, semiochemical release, and intercropping combined with semiochemical release, the increasing numbers of parasitoids A. avenae and A. gifuensis were important in suppressing wheat aphid populations in the field (Liu et al. 2009; Wang et al. 2009; Wang et al. 2011; Dong et al. 2012). To efficiently develop parasitoid conservation strategies and to formulate more efficient biological control programs, further studies should be conducted. More detailed work is needed on whether other factors such as fecundity, long-distance migration, and hyperparasitism could influence the occurrence and density of the parasitoids.