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Acclimation increases the efficiency of the aphidophagous coccinellid predator

Christian Hougaard Sørensen

Integrative Ecology and

Supervisors:

Søren Toft

Torsten Nygaard Kristensen

Acclimation increases the efficiency of the aphidophagous coccinellid predator Adalia bipunctata on aphid pests

Master Thesis 2012

by

Christian Hougaard Sørensen

Integrative Ecology and Evolution, Department of Bioscience Aarhus University

Denmark

Acclimation increases the efficiency of the aphidophagous on aphid pests

Evolution, Department of Bioscience

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Acclimation increases the efficiency of the aphidophagous coccinellid predator Adalia bipunctata on aphid pests

M. Sc. Thesis by Christian Hougaard Sørensen

20052622

Department of Bioscience, Integrative Ecology and Evolution Aarhus University, Denmark.

Supervisors

Søren Toft

Integrative Ecology and Evolution, Department of Bioscience, Aarhus University, Denmark.

Torsten Nygaard Kristensen

Department of Molecular Biology and genetics, Aarhus University, Denmark.

Defence 08.03.2012

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Preface

The aim of this Master’s Thesis was to tests the effects of acclimation to three different developmental temperatures in a common aphidophagous predator, A. bipunctata. In the manuscript, the effects of thermal acclimation on numbers of aphids consumed at different test temperatures were investigated using a microcosm design which partly simulates the complex habitat structure of a cereal field. Effects of thermal acclimation on heat resistance, body-size and pupae-to-adult survival were also investigated. This manuscript demonstrate the potential value and practical feasibility of thermal acclimations for counteracting negative efficiencies in predation activities below thermal conditions that seem optimal in the laboratory. The implications for this do not only concern biological control programs using A.

bipunctata, but have wide applicability to other pest management systems using insect predators.

Acknowledgements

Many people deserve credit for supporting me and for their contribution to this thesis:

− I would like to thank my supervisors Torsten N. Kristensen and Søren Toft for their indispensable guidance and instructions throughout my Master’s Thesis.

− Volker Loeschcke (Aarhus University) and Jesper G. Sørensen (Aarhus University) for valuable discussions and comments on experimental design.

− Vickie G. A. Christensen and Peter H. Sørensen for moral support and for help in the laboratory.

− Ingmar Birkeland for comments on early drafts of the manuscript.

− Anne Marie V. Henten for the beautiful cover illustration.

− Lest but not least I would like to thank my family and my girlfriend for their everlasting support and confidence to me and my work.

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Acclimation increases the efficiency of the aphidophagous coccinellid predator Adalia bipunctata on aphid pests

Abstract

A variety of ladybird species are sold and used in integrated pest management and in 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 fitness is often higher at temperatures that they are acclimated to. 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 acclimation of A. bipunctata to three different developmental temperatures (15, 20 and 25 °C) on their predation rate, weight, pupae-to-adult survival and upper thermal critical limits. We demonstrate that thermal acclimation has positive effects on ladybird feeding rates of aphids when tested in thermal environments similar to their acclimation temperature. Ladybirds reared at 15 °C was found to consume 22 and 23 % more aphids than ladybirds reared at 20 and 25 °C, respectively, when tested in an 15 °C environment. Acclimating ladybirds to cold temperatures also reduced pupae-to-adult survival, heat resistance and increased body-size significantly. We discuss the wide-ranging implications of our results for the application of ladybirds as bio-control agents in different thermal environments and how our findings might help improve the efficiency of pest management in biological control programs.

Key words: Adalia bipunctata, Sitobion avenae, biological control, thermal acclimation, predation activity, heat resistance, body-size, pupae-to-adult survival

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Introduction

Predaceous ladybirds (family: Coccinellidae) have received attention from ecologists all over the world, because of their use in biological control as a predator on e.g. aphids, diaspids, coccids, aleyrodids and mites (Omkar and Pervez, 2005). Ladybirds have been used as a component of biological control in integrated pest management and in augmentative programs since the early 20th century (Hodek, 1970). Today, ladybirds are commercially produced and sold as bio-control agents in particular of aphids which annually are responsible for billions of dollars of crop damage worldwide (Oerke, 1994). Aphidophagous ladybirds are used in many different environments such as greenhouses, fields, gardens, orchards and flowerbeds. In biological control perspectives Adalia bipunctata is one of the most well studied ladybirds due to its potential as a biological control agent against aphid pests and because it is one of the most common aphidophagous predators occurring in arboreal habitats of Europe, Central Asia and North America (Hodek and Honek, 1996).

Because ladybirds such as A. bipunctata have potential as bio-control agents in augmentative pest programs, it is of interest to optimize their efficiency against aphid pests. Population growth parameters reported for A. bipunctata indicate that the species has sufficient environmental plasticity to be used as a biological control agent both in greenhouse systems and in a number of outdoor crops, (Jalali et al. 2009) Therefore, individuals released for bio- control measures are exposed to a wide range of temperatures and other environmental variables. Several studies have shown that temperature is an important factor influencing developmental rate, mortality and feeding activity of ladybirds and insects in general (Wratten, 1973; Schüer et al. 2004; Jalali et al. 2010). The predation rates of larval and adult stages of A. bipunctata on aphids have been reported to increase with temperature in the range of approximately 10-30 °C (Gotoh et al. 2004). Typically, commercial rearing of A.

bipunctata use only constant temperatures (25 °C), which is optimal for reproduction survival and development time (BioBest, Belgium, Carl De Coninck - personal communication). This may have negative impacts on their ability to effectively manage aphid pests when employed in environments where temperature regimes may be well below (or above) temperatures that are optimal in the laboratory. Such thermal discrepancies could result in suboptimal pest management and affect the success of ladybirds as bio-control agents.

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One method that may help to improve the efficiency of predators in biological control programs is thermal acclimation. 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 being adaptive (Angilletta, 2009). This view has been termed the beneficial acclimation theory 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 theory is controversial and has been rejected as a general rule (Woods and Harrison, 2001; Gibbs et al. 1998; Krebs and Loeschcke, 1994), many studies on acclimation responses, especially those dealing with temperature, have found results supporting the theory. Chidawanyika and Terblance (2011) measured costs and benefits of thermal acclimation for laboratory and field responses of codling moths, Cydia pomonella. The study showed that thermal acclimation can give mass- reared codling moth a significant performance advantage enabling them to cope better with varying field temperatures when released in temperatures similar to their developmental temperature. However while the beneficial acclimation theory implicitly assumes that acclimation imposes no cost, this is often not the case. Indeed it may be costly to acclimate to a certain environment if it leads to trade-offs in performance under different thermal conditions. 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, cold-acclimated flies were recaptured at baits almost 100 times more often than control flies acclimated at 25 °C, indicating strong benefits of cold acclimation to cope with conditions in the field. However, this advantage came at a huge cost at higher temperatures, where control flies were up to 36 times more likely to find food than cold-acclimated flies. This clearly indicates that both costs and benefits should be taken into account when evaluating the physiological responses to acclimation.

The aim of this study was to test the effects of acclimation to three different developmental temperatures in a common aphidophagous predator, A. bipunctata. We did this by examining the ability of ladybirds, acclimated to the three developmental temperatures (15, 20 and 25

°C), to consume aphids at four different test temperatures (three constant and one fluctuating).

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This was done using a microcosm design which partly simulates the complex habitat structure of a cereal field. Furthermore we tested for acclimation effects on heat resistance in order to determine whether or not thermal acclimation alters the ability of the ladybirds to withstand extreme high temperatures. Responses to acclimation were also investigated by scoring pupae-to-adult survival and the weight of ladybirds from each of the three developmental temperatures. To our knowledge this is the first time a ladybird species has been subjected to such assays.

We hypothesized; 1. that acclimation to a particular thermal environment enhances performance in terms of an increased feeding rate of aphids by ladybirds at temperatures similar to acclimation temperatures; 2. that an association between developmental temperature and body-size is observed with larger sizes at lower developmental temperatures; 3. that acclimation to low temperatures imposes some cost to the ladybirds in the form of reduced heat resistance; and 4. that acclimation at lower temperatures reduces pupae-to-adult survival.

Finally, we discuss the wide-ranging implications of our results for the application of ladybirds as bio-control agents in different thermal environments and how our findings might help improve the efficiency of biological control systems in general.

Materials and methods

Predator culture

A. bipunctata larvae were purchased from Biobest NV (www.biobest.be). The colony in our laboratory was repeatedly infused with new individuals from the same commercial source. At Biobest, the larvae are continuously reared at 25 °C and are being fed with live pea aphids, Acyrthosiphon pisum. After receiving the ladybirds they were reared on live grain aphids, Sitobion avenae. Upon introduction to our laboratory, the ladybirds were reared for two generations on an ad libitum supply of adult and different nymphal stages of the grain aphid prior to experimental start. During that time the stock colony of the predator was maintained in a growth chamber at 25 ± 1 °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 filter paper.

The tubes were closed with a rubber-foam stopper. No water was added to the tubes. Three times a week aphids were offered to the larvae ad libitum.

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In our experiments we only used the melanic form, quadrimaculata (black with four red spots), of A. bipunctata. Studies have shown a lower cuticular reflectance for melanic ladybirds resulting in higher body temperatures and activities under certain conditions (Brakefield and Wilmer 1985). This is because a dark ectothermic insect will heat up faster thereby reaching a higher equilibrium temperature. It would have been relevant to test both forms in our study, but quadrimaculata was chosen since approximately 80 % of the emerging adults were of this type.

Prey culture

For all experiments S. avenae was obtained from a laboratory culture, reared on wheat seedlings of mixed cultivars. The grain aphid 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 common prey species for A. bipunctata, as this prey supplies the predator with all essential nutrients (Schüder et al. 2004).

Microcosm study

We used a microcosm set-up roughly similar to the arrangement described by Madsen et al.

(2003). The microcosm consisted of a plastic flowerpot (ɸdiameter 11.5 cm, height 10 cm) filled with a substrate of peat, leca and clay granules and with a transparent cylinder (ɸdiameter 9.5 cm, height 21 cm) on top. To confine the aphids and ladybirds, two pieces of tulle covered the top of the transparent cylinder, secured by a rubber band. Water was supplied by placing the pots in water filled trays (25 cm x 35 cm). Wheat seedlings were grown in small aluminum trays, filled with Vermiculite® growth medium. Three days from the day the seedlings appeared, the seedlings were picked one by one from the trays and transplanted to the flowerpots. Ten seedlings were transplanted to each flowerpot. The seedlings were given two days to settle in the microcosm before aphids were added. In total four series of microcosm experiments were run; three at constant temperatures in the laboratory and one at fluctuating temperature in an outdoor environment.

Emerging adult ladybirds from the second generation after introduction to our laboratory were sexed according to Baungaard (1980). One male and one female were transferred to each of

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63 plastic vials containing a piece of filter paper. Here they were fed with aphids ad libitum and given three days to mate before being separated. The tubes were inspected every other day for eggs. From each pair eggs were assigned equally to three different acclimation temperatures (15, 20 and 25 °C), all with a 17:7 LD photoperiod. One week after hatching the larvae were separated from each other in order to prevent cannibalism and reared singly to pupation. The emerging adults from the three acclimation temperatures (<48h old) were sexed and fed an ad libitum supply of aphids for seven days before being weighed and transferred to separate microcosms. The ladybirds were weighed in plastic tubes with a rubber-foam stopper using a Mettler A30 precision scale (Mettler® Instruments AG, Greifensee, Switzerland). The microcosm assays at the fluctuating temperature consisted of 20 replicates from each acclimation temperature while the assays at the constant temperatures each consisted of 30 replicates (See Fig. 1 for an explanation of the experimental design). Half of the replicates were with males and half with females. To each microcosm was added 120 and 200 aphids in the fluctuating and constant temperature treatments, respectively. Aphids added to the microcosms were chosen at random, so a similar distribution of adult and nymph sizes in each microcosm can be assumed. Aphids were allowed approximately four hours to settle before one ladybird was added to each microcosm.

For each acclimation temperature pupae-to-adult survival was recorded. Pupae that had not moulted into an adult seven days after normal development time (approximately 13.7, 8.5 and 5.1 days when reared at 15, 20 and 25 °C, respectively (Schüder et al. 2004)) were scored as dead.

The fluctuating temperature microcosm experiment was run for exactly three days from October 17th. During this period the mean temperature was 8.5 °C (Tmin 4.9 °C; Tmax 11.9

°C, see Fig. 2). Temperatures were measured by a data logger (Tinytalk II, Orion Components, Chichester, U.K.). The constant temperature experiments in the laboratory were run for exactly four days each at various times from November 2011 to January 2012. At the end of each microcosm experiment the wheat plants were cut at soil level and remaining aphids were counted together with the aphids away from the plants in each microcosm.

Heat knockdown assay

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For estimating heat resistance a knockdown test was used (see Kellett et al. 2005). Thirty adults (15 males and 15 females) from each acclimation temperature were tested. Prior to testing, teneral ladybirds were fed aphids ad libitum for 12 days and kept in growth chambers at their respective acclimation temperatures with a 17:7 L:D photoperiod. The ladybirds were placed individually in 5 ml glass vials and exposed acutely to 43 °C by immersion in a preheated water bath. Heat knockdown time was scored as the time it took for individual ladybirds to get knocked down and loose muscular function. A flashlight and a tapping-pole were used to inspect consciousness.

Statistical analyses

For statistical analysis JMP (8.0 by SAS Institute) was used. The untransformed microcosm data were in all cases normally distributed (tested by Shapiro-Wilk W-tests, results not shown), and showed homogeneity of variances (confirmed with Bartlett’s tests, results not shown). Full-factorial two-way ANOVAs were used to test for the effect of rearing temperature, test temperature (excluded in microcosm experiment with fluctuating temperature) and sex (fixed factors) on number of aphids consumed (dependent variable).

To test for the effects of rearing temperature at each test temperature on number of aphids consumed in the microcosm experiments performed at constant temperatures in the laboratory, one-way ANOVAs were performed with rearing temperature as a fixed factor and number of aphids consumed as the dependent variable. In the same way the effects of test temperature at each rearing temperature on number of aphids consumed were tested using one-way ANOVAs with test temperature as a fixed factor and number of aphids consumed as the dependent variable.

To test for effects of rearing temperature (15, 20 and 25 °C) and sex on body-size, full- factorial two-way ANOVAs were used. Normality and homogeneity of variances were generally confirmed by Shapiro-Wilk W-tests (results not shown) and Bartlett’s tests (results not shown). However, in a single case the assumptions for performing ANOVAs were violated. ANOVAs are quite robust to deviations from normality and homogeneity of variances as long as sample sizes are high and equal, and therefore parametric tests were justified. Two-way ANOVAs were also used to test the effects of rearing temperature and sex

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on heat knockdown time while one-way ANOVAs were used to test the effect of rearing temperature on pupae-to-adult survival.

Results

Microcosm experiments

Rearing temperature and test temperature significantly affected number of aphids consumed while sex and all interactions including sex did not (Table 1). Across test temperatures, ladybirds reared at 25 °C consumed 11.6 and 3.7 % more aphids than ladybirds reared at 15 and 20 °C, respectively (data not shown). Across rearing temperatures, the ladybirds consumed 59.5 and 18.3 % more aphids at 25 °C compared to ladybirds tested at 15 and 20

°C, respectively (data not shown). The interaction term between rearing temperature and test temperature was highly significant, indicating that thermal acclimation has an effect on predation rate (Table 1). Even though there was a positive relationship between test temperature and number of aphids consumed for all rearing temperatures (Fig. 3 (a), (b) and (c)), data showed that ladybirds tested in the thermal environment to which they had been acclimated, consumed more aphids compared to individuals acclimated to a different environment (Fig. 3 (d), (e) and (f)). Post-hoc pairwise comparisons of individual rearing temperatures at each test temperature on number of aphids consumed, showed that 15 °C reared ladybirds tested at 15 °C consumed significantly more aphids than both 20 and 25 °C reared individuals (22 and 23 %, respectively) between which no significant difference was observed (Fig. 3 (d)). When tested at 20 °C , 20 °C reared ladybirds consumed significantly more aphids than individuals reared at 15 °C, while no statistical difference between individuals reared at 20 and 25 °C was detected (Fig. 3 (e)). Tested at 25 °C all acclimation lines were significantly different from each other with individuals reared at 25 °C consuming more aphids (14.6 %) than individuals reared at 20 °C which consumed more aphids (18.8 %) than ladybirds reared at 15 °C (Fig. 3 (f)).

Although there is no statistical evidence for concluding that cold-acclimated ladybirds have an increased feeding rate at cold fluctuating temperatures under semi-natural conditions compared with ladybirds reared at 20 and 25 °C (Table 2, Fig. 4), a clear tendency can be observed. Ladybirds reared at 15 °C consumed 7.3 and 24.7 % more aphids compared to those

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reared at 20 and 25 °C, respectively. This supports the results from the constant temperature microcosm experiments performed in the lab.

Weight data

In the microcosm experiment performed at constant temperatures, effects of rearing temperature and sex on body-size were all significant, whereas the interaction term was not (Table 1). For both sexes body-size increased with lower rearing temperatures (Fig. 5).

Ladybirds reared at 15 °C were on average 5.0 and 9.2 % larger than ladybirds reared at 20 and 25 °C, respectively. By gender, the difference between body-sizes 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.

Pupae-to-adult survival

The effect of rearing temperature on pupae-to-adult survival was highly significant (F2,701 = 59.49, P<0.001) showing a positive effect of increasing temperature on survival (Fig. 6). At the three rearing temperatures (15, 20 and 25 °C) the pupae-to-adult survival was 56.9, 86.0 and 93.9 %, respectively, showing that ladybirds reared at 25 °C has a 64.9 % better chance of surviving the pupae stage than ladybirds reared at 15 °C and a 9.1 % better chance than ladybirds reared at 20 °C.

Heat knockdown

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). There was a positive effect of increasing rearing temperature on adult heat resistance, although a significant difference could only be observed between ladybirds reared at 15 °C and ladybirds from the other two temperature regimes (Fig. 7). Ladybirds reared at 15 °C had 39.8 and 33.6 % lower heat resistance compared to ladybirds reared at 25 and 20 °C, respectively.

Discussion

Our results show that rearing temperature can have significant effects on performance, scored as number of aphids consumed, under various test temperatures. We found that a laboratory

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bred population of A. bipunctata respond plastically to development temperature and that this response enhanced its ability to consume aphids at that particular temperature (Fig. 8). The plastic response to developmental temperature however, was found to have some consequences for different fitness components. In our study, acclimation to low temperatures resulted in a rather drastic decrease in upper thermal resistance of the ladybirds. For example, acclimation to 15 °C which is just 10 °C below optimal temperature reduces heat resistance by 39.8 %. Also, acclimating ladybirds to 15 °C instead of the optimal temperature in the laboratory reduces pupae-to-adult survival from 93.9 to 56.9 %. Our results also show that both rearing temperatures and sex has an effect on body-size with rearing temperature regimes having the most significant effect.

Our results clearly demonstrate that acclimation to low temperatures induces a physiological response that has strong effects on multiple fitness components which can be utilized to improve the efficiency of A. bipunctata and other predators in biological control systems.

Responses to acclimation

Laboratory studies on thermal acclimation often maintain external conditions constant while only temperature is being altered. 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 acclimation with studies performed at semi-natural conditions at fluctuating temperatures. Our results from the laboratory showed that acclimation to a particular thermal environment enhances predation rate on aphids of the ladybirds at the acclimated temperature compared to other test temperatures. Although not significant, results from the microcosm study performed at fluctuating temperatures revealed the same trend. This suggests 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 are known to effect feeding activity in various arthropod species (Hill, 1980; Klinger et al. 1986; Giroux et al. 1995), but this is the first study to show that rearing temperature can effectively be used as a tool for optimizing feeding activity at various temperatures.

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Rearing temperature was also found to have a significant effect on body-size. Ladybirds reared at low temperatures showed an increase in body-size compared to ladybirds reared at higher temperatures. It has long been known that ectothermic organisms develop more slowly but show increased body-size, when reared under colder temperatures (e.g. Alpatov, 1930).

Ray (1960) examined body-sizes in 17 species of ectotherms grown at high and low temperatures and showed that nearly 80 % of the species grew larger at lower temperatures.

No species showed the opposite trend. Azevedo et al (2002) found that lower rearing temperatures increase epidermal cell size, with no significant change in cell number in D.

melanogaster. It is likely that these findings also apply to other cell types and other ectotherms, such as ladybirds.

Generally body-size has been found to have effects on several physiological and ecological parameters of a wide range of organism (Van Voorhies, 1996). Several studies have found a positive effect of body-size on fecundity (e.g. Savalli and Fox, 1998; Reeve et al 2000;

Honek, 1993). In the study by Honek (1993) an intraspecific relationship between body-size and fecundity in 57 insect species were investigated. In 53 of the 57 species there was a positive effect of body-size on fecundity in insect females. Large body-sizes have also been shown to have a positive effect on longevity in D. melanogaster (Reeve et al. 2000) and on immune function in males of the ant Formica exsects (Vainio et al. 2004). Considering these results, low rearing temperatures may have positive effects on certain life-history traits and generate more robust individuals which are more capable of coping with different environmental stresses. Thus we suggest that the larger sizes of ladybirds developed at lower temperatures is an advantage in relation to their use in biological control at low temperatures.

Costs of cold-acclimation

Although thermal acclimation to low temperatures resulted in an enhanced predation rate of aphids at low temperatures, it was also associated with significant costs. In our study pupae- to-adult survival was significantly influenced by acclimation temperature with pupae mortalities being much higher for ladybirds reared at low temperatures compared to ladybirds reared at higher temperatures. This difference could be explained by the inability of cold acclimated larvae to gather enough energy to complete the pupation process. In a study by

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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 % which was much lower than ladybirds tested at 20 and 25 °C which had an exploitation rate of 100

%. This suggests that predators tested at low temperatures have difficulty exploiting the complete food supply at 15 °C thus having a lower energy uptake compared to predators tested 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.

Our results also demonstrated that being acclimated to low temperatures imposed huge costs with regard to upper critical thermal limits. This result is in accordance with other studies, examining the effects of cold-acclimation on upper critical thermal limits (e.g. Kristensen et al. 2008; Chidawanyika and Terblance, 2011). Although not examined here, many studies of different insects have shown that low temperature acclimation reduces the lower critical thermal limit (increasing cold resistance) (Terblanche et al. 2005; Mitchell et al. 1993). This may also be valid for ladybirds suggesting that pre-conditioning to low temperatures not only can enhance performance, with regard to number of aphids consumed, but also induce higher resistance to low fluctuating temperatures increasing fitness in cold harsh environments.

Practical implications

In the following our results will be discussed in biological control perspectives, for our test organism and in general. In our study we examined the effects of acclimation on the adult life-stage of A. bipunctata, but for biological control measures second-fourth instar larvae are normally used (Schüder et al. 2004). Although our discussion on practical implications for biological control systems are based on the assumption that responses to acclimation are similar at all life stages, our results still have wide applicability to pest management systems using A. bipunctata and other insect predators.

Generally there are two methods to improve the efficiency of predators in bio-control programs. It can be through genetic approaches such as selection or through ecological approaches such as acclimation. With selection you select individuals that have a phenotype (and a genotype) that is beneficial under the conditions that they are expected to be exposed

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to. It could be increased cold or heat resistance, longevity, reproductive output or superior ability to consume prey. Although selection can be very effective, it requires that artificial selection for the desired trait is continuously performed, that genetic variation is available in the population and that tailor made populations are ‘created’ for different environments. This is not an easy neither a cheap task. An alternative method - the method used in this experiment - is acclimation. Here, the predator is acclimated at different thermal conditions and the consequences of acclimation temperature are investigated at different test temperatures. Our results suggests an 23 % increase in numbers of aphids consumed by 15 °C reared ladybirds when tested in an 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 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.

If our results have general applicability to other species, the implications for predators used in biological control could be substantial. Although acclimation often lead to trade-offs in thermal critical limits this may not be relevant for augmentative control programs as they often focuses on short term pest suppression (Jalali et al. 2010). Augmentative bio-control programs often span few weeks which makes chances of encountering lethal upper or lower temperatures minimal. For predators used in classical bio-control programs, investigations of the effects of acclimation on reproductive output at low temperatures may be important.

Several studies have shown that reproductive output significantly affects the numerical response of a predator on prey density, thereby being an important trait of an efficient bio- control agent (Britto et al. 2009; Caron et al. 2011).

Acclimating organisms to temperatures similar to those under which they are to perform in biological control systems may not only enhance predation of that organism in that thermal environment, but may also make it more resistant to other environmental stresses. In a study by Bubliy et al. (2011), evidence for cross-resistance was found using D. melanogaster. Here it was shown that acclimation to one stress in D. melanogaster can confer plastic responses, which leads to increased resistance to other stresses. They found that acclimating flies to desiccation increased resistance to heat compared to control flies. Starvation acclimated flies

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were also found to be more resistant to desiccation than control individuals. Acclimation to low temperatures may induce similar cross-resistance, but this needs further investigation.

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 consider having more flexibility in their rearing conditions so that e.g. rearing temperatures to a larger extent mimic those that the bio-control agent is going to perform under. This study demonstrated the potential value and practical feasibility of thermal acclimations for counteracting negative efficiencies in predation activities below thermal conditions that seem optimal in the laboratory. These results are of critical importance to ladybird–aphid bio- control systems, and have broad applicability to other pest management programs which employ natural enemies release methods to suppress pests.

Acknowledgements

We thank Doth Andersen, Vickie Gordon Christensen and Peter Hougaard Sørensen for helpful assistance in the laboratory and Ingmar Birkeland for valuable comments on early drafts of the manuscript. We also thank Volker Loeschcke and Jesper Givskov Sørensen for helpful comments on experimental design and Anne Marie Vestergaard Henten for the beautiful cover illustration.

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Baungaard, J. 1980. A simple method of sexing Coccinella septempunctata L. (Coleoptera Coccinellidae). Entomologiske Meddelelser 48:26-28.

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22 Table 1. Results of full-factorial ANOVA of the microcosm experiments at constant

temperatures (data presented in figure 1 and figure 7). 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-size.

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23 Table2. Results of full-factorial ANOVA of the microcosm experiments at fluctuating

temperatures (data presented in figure 3 and figure 4). The table shows the fixed factors (rearing temperature and sex) tested against number of aphids consumed.

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Figure legends

Figure 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 microcosm 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.

Figure 2. Temperature during the microcosm experiment under semi-natural conditions, with registrations every 10. minute for 72 h after start of the experiment. Temperature mean was 8.5 °C (Tmin 4.9 °C; Tmax 11.8 °C).

Figure 3. Mean number of aphids consumed (%) ± SE at three different test temperatures by ladybirds reared at 15 °C (a), 20 °C (b) and 25 °C (c). In (d), (e) and (f) ladybirds are reared at three different temperatures and tested at 15 °C, 20 °C and 25 °C, respectively. Note that number of aphids consumed is calculated as percentage of aphids eaten in relation to aphids initially added. Different letters indicate significant difference between regimes. At all rearing temperatures (a), (b) and (c), most aphids are consumed at 25 °C. (d), (e) and (f) show that ladybirds acclimated to a certain environment consume most aphids at that particular environment.

Figure 4. Mean number of aphids consumed (%) + SE calculated as percentage of aphids eaten in relation to aphids initially added. Coccinellid predators were reared at three different temperatures and tested at fluctuating temperatures under semi-natural conditions (mean 8.5

°C, Tmin 4.9 °C; Tmax 11.8 °C).

Figure 5. Mean weight ± SE at different rearing temperature for the two sexes. Body-size decreases with increasing developmental temperature in both males and females. Different letters indicate a significant difference between regimes.

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Figure 6. Mean pupae-to-adult survival ± SE at the three rearing temperatures. Pupae-to-adult survival increases with increasing rearing temperatures in the range of 15-25 °C. Different letters indicate significant difference between regimes.

Figure 7. Mean time to heat knockdown ± SE. For each rearing temperature 30 replicates were tested at 43 °C. Heat knockdown time was scored as the time for individual ladybirds to be knocked down and immobilized. Different letters indicate significant difference between regimes.

Figure 8. Relative percentage of aphids consumed at three different test temperatures ± SE.

Note that the percentage of aphids consumed at each test temperature is calculated as the number of aphids consumed relative to the maximal from that particular test temperature which is then set to 100 %. The figure shows that ladybirds acclimated to a certain environment consume most aphids at that particular environment at all test temperatures. Note that line between data points are extrapolations. Different letters indicate significant difference between test temperatures.

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Fig. 1

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Fig. 3

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Fig. 4

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Fig. 6

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Fig. 8

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