Cold-acclimation increases the predatory efficiency of the aphidophagous coccinellid Adalia bipunctata

Biological Control 65 (2013) 87–94

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Cold-acclimation increases the predatory efficiency of the aphidophagous coccinellid Adalia bipunctata Christian Hougaard Sørensen a, Søren Toft a,⇑, Torsten Nygaard Kristensen a,b a

Department of Bioscience, Aarhus University, Ny Munkegade 116, DK-8000 Aarhus C, Denmark Department of Molecular Biology and Genetics, Aarhus University, Blichers Allé 20, DK-8830 Tjele, Denmark

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" Cold acclimation may increase the

Rel. no. of aphids consumed (%)

b

biocontrol efficiency of a ladybird. " Acclimated beetles had the highest biocontrol efficiency at all temperatures. " Cold acclimation increased bodysize but reduced pupal survival and heat resistance. " Acclimation should be considered in augmentative release programs.

Rearing temp. 100

15 oC

95 90 85

20 oC

80 75

25 oC

70 15

20

25

Test temperature (oC)

a r t i c l e

i n f o

Article history: Received 21 May 2012 Accepted 24 September 2012 Available online 16 October 2012 Keywords: Acclimation Biological control Coccinellidae Heat resistance Predation Sitobion avenae

a b s t r a c t Ladybirds are used in integrated pest management and augmentative biological control programs all over the world. Typically, commercial rearing of the commonly used ladybird, Adalia bipunctata, takes place at a constant temperature (25 °C) which maximizes reproductive output and survival in the laboratory. However, insects are known to acclimate via physiological adjustments to their thermal environment and performance is often higher at temperatures to which they are acclimated. Thus rearing A. bipunctata at 25 °C may not be optimal if they are to effectively manage aphid pests under different thermal regimes. Here, we report on the effects of rearing temperature (15, 20 and 25 °C) of A. bipunctata on aphid predation at similar test temperatures and under cold semi-natural conditions. Furthermore we assessed the upper thermal critical limit of ladybirds from the three rearing temperatures using a heat knock down assay as well as the effects of rearing temperature on pupal survival and adult mass. We demonstrate that ladybirds acclimated to a certain temperature consume more aphids at that temperature than ladybirds acclimated to other temperatures. Acclimating ladybirds to cold temperatures also increased their bodysize but reduced pupal survival and heat resistance, suggesting costs associated with acclimation. Our findings have implications for the application of ladybirds as bio-control agents in different thermal environments. The results can be used to improve the efficiency of pest management in biological control programs. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Predaceous ladybirds (family Coccinellidae) have received attention from ecologists, because of their use in biological control

⇑ Corresponding author. Fax: +45 87154326. E-mail address: [email protected] (S. Toft). 1049-9644/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.biocontrol.2012.09.016

as predators of agricultural pests e.g. aphids, diaspids, coccids, aleyrodids and mites (Obrycki and Kring, 1998; Omkar and Pervez, 2005). They have been used as a component of integrated pest management and in augmentative control programs since the early 20th century (Hodek, 1970). Today, ladybirds are commercially produced and sold as bio-control agents, in particular against aphids which damage for billions of dollars of crops annually worldwide (Oerke, 1994). Adalia bipunctata (L.) is one of the best

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studied ladybirds due to its potential use against aphid pests and because it is one of the most common aphidophagous predators in arboreal habitats of Europe and Central Asia (Hodek and Hoñek, 1996). Because A. bipunctata is used in augmentation biocontrol programs, it is of interest to optimize its efficiency against aphid pests. Population growth parameters reported for A. bipunctata indicate that the species may be useful both in greenhouse systems (e.g. against Myzus persicae Sulzer) and in a number of outdoor crops (e.g. against M. persicae and Acyrthosiphon pisum Harris) (Jalali et al., 2009). Therefore, individuals released for bio-control measures are exposed to a wide range of temperatures. Temperature is an important factor influencing developmental rate, survival, adult size and feeding activity of many insects (Wratten, 1973; Schüder et al., 2004; Jalali et al., 2010). The predation rates of larval and adult stages of A. bipunctata on aphids increase with temperature in the range of approximately 10–30 °C (Gotoh et al., 2004). Typically, commercial rearing of A. bipunctata use only a single constant temperature (25 °C), which is optimal for reproduction, survival and development (BioBest, Belgium, Carl De Coninck – personal communication; Schüder et al., 2004). The thermal rearing regime may affect their ability to effectively manage aphid pests when employed at temperatures well below (or above) temperatures that are optimal in the laboratory. One method that may help to improve the efficiency of predators in biological control programs is thermal acclimation (e.g. Chidawanyika and Terblanche, 2011; Hart, 2002). Acclimation is often defined as a phenotypic alteration in physiology that occurs in response to the environmental conditions experienced by an animal and is often thought to enhance performance, thereby improving fitness (Angilletta, 2009). This view has been termed the beneficial acclimation hypothesis which predicts that ‘‘. . . acclimation to a particular environment gives an organism a performance advantage in that environment over another organism that has not had the opportunity to acclimate to that particular environment’’ (Leroi et al., 1994). Although the beneficial acclimation hypothesis is controversial and has been rejected as a general rule (Gibbs et al., 1998; Krebs and Loeschcke, 1994; Woods and Harrison, 2001), many studies on acclimation responses, especially those dealing with temperature, have supported the hypothesis (Chidawanyika and Terblanche, 2011; Nunney and Cheung, 1997; Thomson et al., 2001). For instance Chidawanyika and Terblanche (2011) showed that thermal acclimation can give mass-reared codling moths, Cydia pomonella, a significant performance advantage when released at temperatures similar to their developmental temperature. However, it may be costly to acclimate to a certain environment if it leads to trade-offs in performance under different thermal conditions (Chevin et al., 2010; Kristensen et al., 2008; Loeschcke and Hoffmann, 2007). Evidence for trade-offs were found in a study by Kristensen et al. (2008), testing for effects of larval and adult cold-acclimation on field released Drosophila melanogaster. At low release temperatures, flies acclimated at 15 °C were recaptured at baits almost 100 times more often than flies acclimated at 25 °C, indicating strong benefits of cold-acclimation to cope with cold conditions in the field. However, this advantage came at a huge cost at higher temperatures, where flies acclimated at 25 °C were up to 36 times more likely to find food than flies acclimated at 15 °C. Thus, costs and benefits of acclimation are contingent on the conditions the animal encounters subsequently. The aim of this study was to test the effects of acclimation to three different developmental temperatures in a common aphidophagous predator, A. bipunctata. To fulfill this objective, we examined the ability of ladybirds, previously acclimated to developmental temperatures of 15, 20 or 25 °C, to consume aphids at four different temperature regimes (constant 15, 20 or 25 °C and one at fluctuating temperatures with a mean of 8.5 °C). We

concentrate on responses to low acclimation temperatures since this is most representative for outdoor thermal conditions that are prevailing in Central and Northern Europe. The ability of A. bipunctata to control aphids was tested using a microcosm design which partly simulates the complex habitat structure of a cereal field. Furthermore, we tested acclimation effects on heat resistance in order to determine whether or not acclimation to low temperatures entails costs in terms of reduced ability to withstand high temperatures. Developmental effects of the temperature treatments were also investigated by scoring pupal survival and the mass of the adult ladybirds. We hypothesized that (1) developing at a particular temperature enhances the predatory performance of ladybirds in terms of an increased feeding rate on aphids at that temperature (according to the beneficial acclimation hypothesis); (2) that acclimation to low temperatures imposes a cost to the ladybirds in the form of reduced heat resistance; (3) that there is an association between developmental temperature and body-size with beetles maturing to larger sizes at lower developmental temperatures, as found in many other studies (e.g. Alpatov, 1930; Azevedo et al., 2002) and (4) that pupal survival is highest at 25 °C since this temperature is considered optimal for the species (Schüder et al., 2004). We confirmed all four hypotheses and discuss the implications of our results for the application of ladybirds as bio-control agents in different thermal environments and for the efficiency of biological control systems in general. 2. Materials and methods 2.1. Predator culture A. bipunctata larvae were purchased from Biobest NV (www.biobest.be). The colony in our laboratory was infused with new individuals from the same commercial source three times during establishment in our laboratory. In our experiments we only used the melanic form, quadrimaculata (black with four red spots) which comprised approximately 80% of the emerging adults. At Biobest, all lifestages were continuously reared at 25 °C and fed pea aphids, A. pisum. In our laboratory, the ladybirds were reared for two generations on an ad libitum supply of grain aphids, Sitobion avenae (Fabricius), prior to experiments. During that time the stock colony of the predator was maintained in a growth chamber at 25 ± 0.5 °C, and a 17:7 L:D photoperiod. Individual larvae were isolated in plastic tubes (height 8.1 cm; diameter 3.4 cm) containing a piece of dry filter paper. The larvae were fed aphids offered ad libitum on wheat leaves three times a week. 2.2. Prey culture For all experiments, S. avenae was obtained from a laboratory culture, reared at room temperature (ca. 22 °C) and a 16:8 L:D photoperiod on wheat seedlings of mixed cultivars. The culture was purchased at EWH BioProduction ApS (www.bioproduction.dk) where they also were reared on wheat seedlings. The grain aphid is known to be a suitable prey species for A. bipunctata that supplies the predator with all essential nutrients (Schüder et al., 2004). 2.3. Microcosm study To measure predator performance two separate microcosm experiments were run; one at three constant temperatures in the laboratory (15, 20 and 25 °C), and one at natural fluctuating temperatures in an outdoor environment. The two microcosm experiments were run at different dates and varied in some details of their experimental design. The microcosm set-up was roughly

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similar to that described by Madsen et al. (2004). Plastic flowerpots (diameter 11.5 cm, height 10 cm) were filled with a substrate of peat, leca and clay granules and with transparent cylinders (diameter 9.5 cm, height 21 cm) on top. To confine aphids and ladybirds, two pieces of tulle covered the top of the cylinder, secured by a rubber band. Water was supplied by placing the pots in water filled trays (25  35 cm). Wheat seedlings were grown in small aluminum trays filled with VermiculiteÒ growth medium. Three days after the seedlings appeared, they were picked one by one from the trays and transplanted to the flowerpots, ten to each pot. The seedlings were given 2 days to settle in the microcosm before aphids were added. The aphids were chosen at random, so a similar distribution of adults and nymphs in each microcosm can be assumed. Aphids were allowed approximately 4 h to settle on the plants before one ladybird was added to each microcosm. The ladybirds used for both experiments were adults from the third generation after introduction to our laboratory. Adults from the second generation were sexed according to guidelines in Baungaard (1980). One male and one female were transferred to each of 63 plastic vials containing a piece of filter paper. Here they were fed aphids ad libitum and given 3 days to mate before being separated. The tubes were inspected every other day for eggs. From each pair and from every egg cluster, eggs were assigned equally to the three different developmental temperatures (15, 20 and 25 °C), all with a 17:7 L:D photoperiod. One week after hatching, larvae from the three development temperatures were separated from each other in order to prevent cannibalism and reared singly to pupation at the respective temperatures. For each temperature regime, survival during the pupal stage was recorded (N = 189, 166 and 168, respectively). Pupae that had not moulted into an adult 7 days after normal development time (approximately 14, 9 and 5 days when reared at 15, 20 and 25 °C, respectively (Schüder et al., 2004)) were scored as dead. For the constant temperature experiment, emerging adult ladybirds (<48 h old) were sexed and fed an ad libitum supply of aphids for 7 days before being weighed on a Mettler A30 precision scale (MettlerÒ Instruments AG, Greifensee, Switzerland). They were then transferred to separate microcosms (see Table 1 for experimental design) to which 200 aphids had previously been added. Each treatment group (i.e. combination of rearing and test temperature) had 30 microcosm replicates (half of them with females and half with males). These experiments were run for exactly 4 days each at various times from November 2011 to January 2012. Replicates of all combinations of rearing and test temperature groups were mixed and spread over the experimental period as the ladybirds became available, approximating a randomly mixed design.

The fluctuating temperature microcosm experiment followed the same procedure but with a few minor changes. All replicates were run for exactly 3 days from 17 October 2011, 120 aphids were added to each microcosm, and 20 microcosm replicates were used per treatment group. In order to be able to run the treatments concurrently with similar-aged ladybirds, rearing of the ladybirds was timed so that the difference in age between animals from the temperature treatments was <36 h (they were used 3–5 days after emergence from the pupae). Temperatures in the microcosms were measured by a data logger (Tinytalk II, Orion Components, Chichester, UK). During the three experimental days the mean temperature was 8.5 °C (Tmin 4.9 °C; Tmax 11.9 °C). At the end of each microcosm experiment the wheat plants were cut at soil level and remaining aphids were counted together with any aphids off the plants. The consumption of aphids was calculated as the difference between the number of aphids added and the number remaining. Although this neglects aphid reproduction during the experimental period, the majority of the aphids added were juveniles that would not have had time to reproduce within the experimental period. Also, since this period only spanned 3 or 4 days reproduction by adult aphids can be assumed to be neglectable, at least at the lower temperatures. Still, as aphid reproduction increases with temperature, our estimate of aphid consumption will be less accurate at the higher test temperatures. This bias does not affect our main comparison, which is the predatory performance of ladybirds from different acclimation temperatures at the same test temperature.

2.4. Heat knockdown assay For estimating heat resistance, a knockdown test was used (see e.g. Kellett et al., 2005). Thirty adult ladybirds (15 males and 15 females) from each acclimation temperature were taken directly from the predatory performance experiment and tested. Only individuals that had experienced the same temperature during rearing and predation test were used (Table 1). The ladybirds were placed individually in 5 mL glass vials and exposed acutely to 43 °C by immersion in a preheated water bath. Initially the high temperature exposure caused the ladybirds to become very active, but soon they became increasingly lethargic. Heat knockdown time was scored as the time it took for individual ladybirds to lose muscular function. Movement of the mouthparts, in particular the palps, mandibles and labium, was used to determine muscular function as these were the last body parts to cease moving. In order to distinguish lethargy from inactivity a flashlight and a steel

Table 1 Experimental design and distribution of replicates on rearing temperature, test temperature, weight data and heat knockdown assay. The first number in the third column represents the temperature at which the ladybirds were tested while numbers in () indicates number of replicates. Ninety individuals from each rearing temperature were weighed before the performance experiment started and thirty individuals were tested at each rearing/test temperature combination. Heat knockdown assay were performed on thirty individuals from each rearing temperature. Rearing temperature (°C)

Weighing (number of replicates)

Microcosm test temperature (°C) (number of replicates)

Heat knockdown assay (number of replicates)

15 15 15 15 20 20 20 20 25 25 25 25

(30) (30) (30)

15 20 25 Fluctuating 15 20 25 Fluctuating 15 20 25 Fluctuating

(30)

(30) (30) (30) (30) (30) (30)

(30) (30) (30) (20) (30) (30) (30) (20) (30) (30) (30) (20)

(30)

(30)

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tapping-pole were used to stimulate movement during inspections. Each ladybird was inspected at least every minute.

25 °C had a 64.9% better chance of surviving the pupal stage than ladybirds reared at 15 °C and a 9.1% better chance than ladybirds reared at 20 °C.

2.5. Statistical analyses 3.3. Adult mass For statistical analysis JMP (8.0 by SAS Institute) was used. The untransformed data from the predatory performance tests were in all cases normally distributed (tested by Shapiro–Wilk W-tests) and showed homogeneity of variances (confirmed with Bartlett’s tests). Data from the constant temperature experiments were analyzed with full-factorial three-way ANOVAs to test for the effect of rearing temperature, test temperature and sex as fixed factors on number of aphids consumed. Acclimation would be indicated by a significant interaction between rearing and test temperature. Aphid consumption data from the fluctuating temperature experiment as well as the data on body-size and heat knockdown time were analyzed with two-way ANOVAs with rearing temperature and sex as fixed factors. Post-hoc comparisons were made by one-way ANOVAs for each combination of rearing and test temperature. For the graphical presentations the consumption data have been transformed to percentages (Fig. 1). Chi-square tests were used to test the effect of rearing temperature on pupal survival. 3. Results 3.1. Predatory performance experiments In the constant temperature laboratory experiment, microcosm rearing temperature and test temperature significantly affected the number of aphids consumed, while sex and its interactions did not (Table 2). The interaction between rearing temperature and test temperature was highly significant (Table 2), indicating that thermal acclimation had an effect on predation rate. There was a positive relationship between test temperature and number of aphids consumed for all rearing temperatures (Fig. 1a–c). Note that this pattern would have been even more prominent if we had been able to adjust for the presumably enhanced aphid reproduction at higher temperatures. Ladybirds tested in the thermal environment to which they had been acclimated, consumed more aphids compared to individuals acclimated to a different environment (Fig. 1d–f). Post-hoc pair-wise comparisons of data from each test temperature showed that ladybirds reared at 15 °C consumed significantly more aphids at 15 °C than ladybirds reared at 20 and 25 °C (22 and 23% more, respectively); ladybirds from the latter rearing temperatures did not differ in the amount of aphids consumed at this test temperature (Fig. 1d). When tested at 20 °C, ladybirds reared at 20 °C consumed significantly more aphids than ladybirds reared at 15 °C, whereas no statistical difference between ladybirds reared at 20 and 25 °C was detected (Fig. 1e). Tested at 25 °C all acclimation groups were significantly different from each other with ladybirds reared at 25 °C consuming 14.6% more aphids than ladybirds reared at 20 °C which consumed 18.8% more aphids than ladybirds reared at 15 °C (Fig. 1f). The results from the fluctuating temperature outdoor microcosm experiment showed a clear trend for cold-acclimated ladybirds to have a higher feeding rate at low fluctuating temperatures than ladybirds acclimated at higher temperatures although it was not statistically significant (Table 3, Fig. 2). Ladybirds reared at 15 °C consumed 7.3% and 24.7% more aphids compared to those reared at 20 and 25 °C, respectively. 3.2. Pupal survival The effect of rearing temperature on pupal survival was highly significant (v2 = 102.2, d.f. = 2, P < 0.0001) showing a positive effect of increasing temperature on survival (Fig. 3). Ladybirds reared at

Effects of rearing temperature and sex on body mass were both significant, whereas the interaction term was not (Table 2). For both sexes, body mass increased at lower rearing temperatures (Fig. 4). Ladybirds reared at 15 °C were on average 5.0 and 9.2% heavier than ladybirds reared at 20 and 25 °C, respectively. By gender, the difference between body mass at the two extreme rearing temperatures was 11.1% for females and 7.2% for males. Across rearing temperatures, females were on average 2.7% heavier than males. 3.4. Heat knockdown resistance The effect of rearing temperature on heat resistance was highly significant (F2,84 = 26.50, P < 0.001) while the effect of sex was not (F1,84 = 0.01, P = 0.91). The interaction between rearing temperature and sex was not significant (F2,84 = 0.33, P = 0.71). Increasing rearing temperature increased adult heat resistance, although a significant difference could only be observed between ladybirds reared at 15 °C and those from the other two temperature regimes (Fig. 5). Ladybirds reared at 15 °C had 39.8% and 33.6% lower heat resistance compared to ladybirds reared at 25 and 20 °C, respectively. 4. Discussion 4.1. Effects of rearing temperature We found that a laboratory bred population of A. bipunctata responded plastically to developmental temperature and that this response strongly enhanced its ability to consume aphids at that particular temperature (Figs. 1 and 2 and Graphical Abstract). The response to developmental temperature also affected other fitness components, and costs of cold acclimation were observed in several traits. Acclimation to low temperatures resulted in a rather drastic decrease in upper thermal resistance of the ladybirds and a substantial increase in pupal mortality. Our data also show that both rearing temperature and sex have an effect on body-size with lower temperatures leading to larger size and with females being larger than males. The higher mass of ladybirds reared at the lower temperatures could explain the higher predatory performance observed at 15 °C (Fig. 1d). However, the ladybirds reared at 15 °C would then also be expected to consume more aphids at higher temperatures due to their larger size, but they did not (Fig. 1e and f). This means that the predatory performance of the beetles raised at 15 °C would be lower at these temperatures, if controlled for size; thus the patterns observed in Fig. 1e and f would be conservative. The lack of sex effects on aphid consumption in the predatory performance experiments (Tables 2 and 3) also speaks against such a body mass effect. 4.2. Benefits of cold acclimation Laboratory studies on thermal acclimation often keep external conditions constant while only temperature is being altered (Angilletta, 2009 and references therein). Although this approach has many benefits for elucidating and isolating underlying physiological mechanisms of acclimation, it simplifies the often much more complex situation that ectothermic animals encounter in nature. In the present study, we combined laboratory studies on

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(a) Rearing temp. 15 oC 60

(b) Rearing temp. 20 oC

(c) Rearing temp. 25 oC C

60

60

B

50

50

50 B

B B AB

40

40

A

40

A

No. aphids consumed (%)

30

A

30

20 15

20

25

20

30

15

20

Rearing temperature

25

20

(e) Test temp. 20 oC

(d) Test temp. 15 oC

15

20

25

(oC)

(f) Test temp. 25 oC

60

60

60

50

50

50

C

B

B AB A

40

40

A

B

40

B

30

20

A

30

15

20

25

20

30

15

20

Rearing temperature

25

20

15

20

25

(oC)

Fig. 1. Mean (+SE) number (%) of aphids consumed by ladybirds in laboratory predatory performance experiments. Ladybirds reared at three different temperatures (15, 20 and 25 °C) were tested at each of these same temperatures. The number of aphids consumed is calculated as percentage of aphids that disappeared from the microcosms in relation to the number of aphids initially added. The two panels show the same data but arranged according to either test or rearing temperature for clarity. Different letters indicate significant difference between treatments.

acclimation with studies performed under semi-natural conditions at fluctuating temperatures. Our results from the laboratory showed that acclimation to a particular thermal environment enhances predation rate on aphids by the ladybirds at the acclimated temperature compared to other test temperatures. Although not significant, results from the predatory performance study performed at natural fluctuating temperatures revealed the same trend; ladybirds acclimated at lower temperatures consumed more aphids than ladybirds acclimated at higher temperatures. The trend was in the predicted direction since the ambient temperature was lower than any of the rearing temperatures. Lower statistical power due to a lower number of replicates in the outdoor experiment compared to the laboratory microcosm experiments might explain the lack of significance in the experiments performed outdoor. Overall results from the microcosm experiments suggest that acclimation can be utilized in biological control systems. Our results emphasize that plastic physiological responses

to developmental temperature can enhance predator performance against its prey in environments similar to the thermal conditions at which they have been acclimated. Temperature is known to affect feeding activity in various arthropod species (Giroux et al., 1995; Hill, 1980; Klinger et al., 1986), but this is the first study to show that rearing temperature can effectively be used as a tool for optimizing coccinellid feeding activity at various temperatures. Generally body-size has been found to have effects on several physiological and ecological parameters of a wide range of organisms (Van Voorhies, 1996). Several studies have found a positive effect of mass on fecundity (e.g. Hoñek, 1993; Reeve et al., 2000; Savalli and Fox, 1998). Hoñek (1993) found a positive intraspecific relationship between body mass and fecundity in 53 of 57 insect species. Large body-sizes also have a positive effect on longevity in D. melanogaster (Reeve et al., 2000) and on immune function in males of the ant Formica exsecta (Vainio et al., 2004). Considering these results, low rearing temperatures may have

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Table 2 Results of full-factorial ANOVA of the performance experiments at constant temperatures (data presented in Fig. 1 and 4). The upper part of the table shows the effect of the fixed factors (rearing temperature, test temperature, sex) on the number of aphids consumed while the bottom part shows rearing temperature and sex in relation to body-mass.

a b

d.f.a

MSb

F ratio

P

Dependent variable: number of aphids consumed Rearing temp. Test temp. Sex Rearing temp.  test temp. Rearing temp.  sex Test temp.  sex Rearing temp.  test temp.  sex Error

2 2 1 4 2 2 4 252

1770 3  104 911 4017 212 454 110 427

4.19 69.98 2.13 9.50 0.50 1.06 0.26

0.016 <0.0001 0.15 <0.0001 0.61 0.35 0.91

Dependent variable: weight (mg) Rearing temp. Sex Rearing temp.  sex Error

2 1 2 264

17.99 4.74 1.61

<0.0001 0.030 0.20

1.46  10 1.92  10 1.30  10 8.10  10

4 6 6 7

Degrees of freedom. MS, mean square.

Table 3 Results of full-factorial ANOVA of the performance experiments at fluctuating temperatures (data presented in Fig. 2). The table shows the effect of the fixed factors (rearing temperature and sex) on the number of aphids consumed.

Dependent variable: number of aphids consumed Rearing temp. Sex Rearing temp.  sex Error a b

d.f.a

MSb

F ratio

P

2 1 2 48

294 10 33 250

1.17 0.04 0.13

0.32 0.84 0.88

Degrees of freedom. MS, mean square.

36 100

90 B

80

28

Survival (%)

No. aphids consumed (%)

C

32

24

70

60

A

50

20 15

20

25

Rearing temperature (oC)

N:

189

166

168

40 15

20

25

Fig. 2. Mean (+SE) number of aphids consumed in an outdoor predatory performance experiment, calculated as percentage of aphids that disappeared from the microcosms in relation to the number of aphids initially added. The ladybirds were reared at three different temperatures and tested over 3 days at fluctuating temperatures under semi-natural conditions (mean = 8.5 °C, Tmin = 4.9 °C; Tmax = 11.8 °C).

Fig. 3. Mean pupal survival (%) of ladybirds at three rearing temperatures. N = samples sizes. Different letters indicate significant difference between treatments as determined by pairwise chi-square tests.

positive effects on certain life-history traits and generate more robust individuals which are better capable of coping with some environmental stresses (but see our data on heat knock-down

resistance). Thus we suggest that the larger size of ladybirds developed at lower temperatures is an advantage in relation to their use in biological control at low temperatures.

Rearing temperature (oC)

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10.0

A Females Males

a

9.5

B

Mass (mg)

b C

9.0

c

8.5

93

The difference in pupal survival at low and higher developmental temperatures could be explained by the inability of cold acclimated larvae to gather enough energy to complete the pupation process. In a study by Schüder et al. (2004) exploitation efficiency, defined as the proportion of offered food eaten before next feeding event, was examined in relation to rearing temperature and starvation in A. bipunctata. Here, larvae tested at 15 °C had an overall exploitation rate of only 75% compared with 100% in larvae tested at 20 and 25 °C. This suggests that predators at low temperatures have difficulty exploiting the complete food supply, thus having a lower energy uptake compared to predators at higher temperatures. This, in combination with lower feeding activity at low temperatures, could explain the high mortality rates during pupation found in this study. 4.4. Practical implications

8.0 15

20

25 o

Rearing temperature ( C) Fig. 4. Mean live mass + SE of adult ladybirds reared at different temperatures. The animals were derived from the constant temperature experiment. Different letters indicate a significant difference between treatments. Capital letters are used for females and lower case letters are used for males.

B

Time to heat knockdown (min)

80

B

70

60

50

A

40

30 15

20

25 o

Rearing temperature ( C) Fig. 5. Mean time + SE (minutes) to heat knockdown for ladybirds reared at different temperatures. Heat knock-down temperature: 43 °C; N = 30 per group. Different letters indicate significant difference between treatments.

4.3. Costs of cold-acclimation We detected several costs associated with cold acclimation. The relative predatory performance of cold-acclimated ladybirds was reduced at higher test temperatures (Figs. 1 and 2, Graphical Abstract), and developing at low temperatures decreased pupal survival (Fig. 3) and reduced tolerance to high temperatures (Fig. 5). Costs of cold acclimation are commonly observed both in laboratory and field studies on ectotherms (Basson et al., 2012; Chidawanyika and Terblanche, 2011; Kristensen et al., 2008; Thomson et al., 2001). Benefits of cold acclimation in some traits sometimes entail a trade-off by costs in other traits, and studies of cost and benefits of acclimation have provided insight into the evolution of plasticity and may partly explain why selection does not always favour higher plasticity (Chevin et al., 2010).

In our study we examined the effects of acclimation on the adult life-stage of A. bipunctata. For biological control measures second to fourth instar larvae are normally used (Schüder et al., 2004). Our suggestions on the practical implications for biological control systems are based on the assumption that responses to acclimation are similar at all life stages. If this assumption is true our results have wide applicability to pest management systems using A. bipunctata and other insect predators. In this experiment, predators from a single genetically variable population were acclimated at different thermal conditions according to the conditions in which they are to be used. Our results suggest a 23% increase in numbers of aphids consumed by 15 °C reared ladybirds when tested in a 15 °C environment compared to ladybirds reared at 25 °C. With 1.000 released ladybirds into a cold habitat (15 °C) this would result in an estimated increase of 3.242 aphids consumed per day compared to a situation where 1.000 ladybirds acclimated to 25 °C were released. This means that fewer ladybirds are necessary to generate the same impact on aphid pest populations. However in judging the net benefit of this approach there are a number of issues that should be considered. As evident from our data there are costs associated with exposure to low temperatures. These fitness costs have economic and ecological consequences. The economic consequences for the producers of ladybirds may be due to the lower viability observed at lower developmental temperatures and ecological consequences could be due to the lower heat resistance observed in the cold acclimated ladybirds. This will especially be a problem in variable thermal environments where temperatures may suddenly increase dramatically. Furthermore, a longer developmental time at lower temperatures and the need to have ladybirds developing at lower temperatures may cause practical (and economic) problems for the producers. Due to these concerns it would be important to test whether a short term exposure to a cold temperature (cold hardening) elicits a similar response to the one observed here. Strong effects of cold hardening in the form of increased cold resistance later in life of the Mediterranean fruit fly Ceratitis capitata (Basson et al., 2012), the leaf-beetle Ophraella communa (Zhou et al., 2011), and several Drosophila species (Nyamukondiwa et al., 2011) suggest that this approach may be successful. Thus, assuming that a short term exposure to cold temperatures elicits the same response as observed in our study, hardening treatments (or some treatment of intermediate duration) may be more attractive for producers of ladybirds both from practical and economic points of view. If our results have general applicability to other species, the implications for predators used in biological control could be substantial. Although acclimation often leads to trade-offs in critical thermal limits this may not be relevant for augmentative control programs as they often focus on short term pest suppression (Jalali

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et al., 2010). Augmentative bio-control programs often span only a few weeks which makes chances of encountering lethal upper or lower temperatures small in Northern Europe. Our results showed that ladybirds acclimated to the temperature at which they were tested, performed significantly better, in terms of consuming aphids, compared to ladybirds acclimated to a different thermal environment. Hence, producers of bio-control agents should pay more attention to their rearing conditions so that e.g. rearing temperatures to a larger extent mimic those that the bio-control agent is expected to perform under. This study demonstrated the potential value and practical feasibility of thermal acclimation for counteracting reduced predation efficiency at field temperatures below those that are optimal in the laboratory. The results are therefore of critical importance to ladybird–aphid bio-control systems, and have broad applicability to other pest management programs which employ natural enemy release methods to suppress pests. Acknowledgments We thank Doth Andersen, Vickie Gordon Christensen and Peter H. Sørensen for helpful assistance in the laboratory and Ingmar Birkeland, Volker Loeschcke, Jesper G. Sørensen, John Terblanche and Thure P. Hauser for valuable discussions and comments on early drafts of the manuscript. We are also indebted to two anonymous reviewers for their useful comments. S.T and T.N.K were supported by a grant from the Carlsberg Foundation and the Danish Research Council (STENO stipend), respectively. References Alpatov, W.W., 1930. Phenotypical variation in body and cell size of Drosophila melanogaster. Biological Bulletin 58, 85–103. Angilletta, M.J., 2009. Thermal Adaptation. A Theoretical and Empirical Synthesis. Oxford University press, Oxford. Azevedo, R.B.R., French, V., Partridge, L., 2002. Temperature modulates epidermal cell size in Drosophila melanogaster. Journal of Insect Physiology 48, 231–237. Basson, C.H., Nyamukondiwa, C., Terblanche, J.S., 2012. Fitness costs of rapid cold hardening in Ceratitis capitata. Evolution 66, 296–304. Baungaard, J., 1980. A simple method of sexing Coccinella septempunctata L. (Coleoptera Coccinellidae). Entomologiske Meddelelser 48, 26–28. Chevin, L.M., Lande, R., Mace, G.M., 2010. Adaptation, plasticity, and extinction in a changing environment: towards a predictive theory. PLoS Biology 8, e1000357. Chidawanyika, F., Terblanche, J.S., 2011. Costs and benefits of thermal acclimation of codling moth, Cydia pomonella (Lepidoptera: Tortricidae): implications for pest control and the sterile insect release programme. Evolutionary Applications 4, 534–544. Gibbs, A.G., Louie, A.K., Ayala, J.A., 1998. Effects of temperature on cuticular lipids and water balance in a desert Drosophila: is thermal acclimation beneficial? Journal of Experimental Biology 201, 71–80. Giroux, S., Duchesne, R.M., Coderre, D., 1995. Predation of Leptinotarsa decemlineata (Coleoptera, Chrysomelidae) by Coleomegilla maculata (Coleoptera, Coccinellidae) – comparative effectiveness of predator developmental stages and effect of temperature. Environmental Entomology 24, 748–754. Gotoh, T., Nozawa, M., Yamauchi, K., 2004. Prey consumption and functional response of three acarophagous species to eggs of the two-spotted spider mite in the laboratory. Applied Entomology and Zoology 39, 97–105. Hart, A.J., 2002. Effects of temperature on the establishment potential in the UK of the non-native glasshouse biocontrol agent Macrolophus caliginous. Physiological Entomology 27, 112–123. Hill, B.J., 1980. Effects of temperature on feeding and activity in the crab Scylla serrate. Marine Biology 59, 189–192. Hodek, I., 1970. Coccinellids and the modern pest management. BioScience 20, 543– 552.

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