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1. Acclimation responses are normally assumed to increase the fitness of an organism, but supporting evidence is generally lacking, especially under field conditions. Even where an advantage arises from acclimation, this can be offset by other fitness costs. Heat hardening is a well-known form of acclimation in many invertebrates where exposure to high but sub-lethal temperatures protects against subsequent heat-induced death.
2. Previous laboratory work has shown that hardening occurs in wasp egg parasitoids of the genus Trichogramma because pretreatment with a mild temperature shock can increase survival at high temperatures. This system allowed fitness benefits and costs of hardening under the more stringent conditions in nature to be tested.
3. Heat hardening in Trichogramma carverae was considered at the pupal and adult stages and it was shown that hardening had a beneficial effect on heat resistance in the laboratory. Moreover, hardening enhanced adult fitness in the field under hot conditions. No costs of acclimation were detected under mild field and laboratory conditions. Conditions leading to hardening without costs were different from another Trichogramma species.
4. Hardening can therefore have fitness benefits without costs under field and laboratory conditions, and this process can be used to enhance parasitism rates in inundative commercial releases of Trichogramma against moth pests.
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The exposure of organisms to new environments often causes reversible changes in the expression of a variety of phenotypic traits that influence the fitness of organisms. The possibility that performance at a given temperature can be increased by prior acclimation to that temperature has been the subject of much interest in recent years. However, several recent laboratory studies contradict this beneficial acclimation hypothesis (Huey & Berrigan 1996); it seems that exposure to one temperature does not invariably enhance fitness at that temperature.
In Trichogramma egg parasitoids, however, it appears that heat acclimation is not invariably associated with costs. In particular, Hoffmann & Hewa-Kapuge (2000) showed that in T. nr. brassicae there was a pupal treatment involving exposure to 35 °C for 2 h day−1 that led to increased heat resistance in adults without detectable adverse effects on fitness traits in this species. This suggests that any adverse effects of acclimation are not associated with the acclimation process per se, but instead arise because of general damage associated with sub-lethal exposures.
In this paper we consider acclimation to heat in another Trichogramma species, T. carverae (Datmar & Pinto). Previous research has shown that this species can be acclimated to increase heat resistance after pupal and adult stages are exposed to high temperatures (Scott et al. 1997). However, there is also evidence that such treatments can lead to decreased parasitism rates. The work of Scott et al. (1997) is extended here and in contrast to their results, we report an acclimation treatment that is not associated with a cost. We have focused on T. carverae because field releases can be readily undertaken with this species to assess field fitness effects (Bennett & Hoffmann 1998). Thus this species is a good candidate for testing the beneficial and deleterious effects associated with acclimation under field conditions. Field work on fitness benefits of acclimation are particularly rare. There are some studies of the adaptiveness of other types of plasticity in the field such as wing melanization (Kingsolver & Huey 1998) and stress protein response (Bosch et al. 1988; Sanders et al. 1991; Roberts et al. 1997; Worthen & Haney 1999) but these do not directly test the benefits and costs of short-term plastic response such as that seen in laboratory acclimation.
Three main issues are addressed. First, are there laboratory conditions that lead to acclimation without detectable costs in this species and how do these conditions compare with those identified for T. nr. brassicae? Second, are beneficial effects evident under field conditions where wasps are exposed to hot temperatures? Finally, are there field costs associated with acclimation when temperatures are not extreme?
Materials and methods
Wasps were mass bred from parasitized Sitotroga cerealella (Olivier) eggs provided by a commercial insectary (Bioprotection, Warwick, Queensland) in February 1997 and reared on S. cerealella eggs in clear plastic vials (285 ml) at 25 °C, with a 16L : 8D photoperiod. Honey was provided as food. Under these conditions the life cycle of Trichogramma carverae is 11 days.
Control wasps were maintained at 25 °C throughout their 11-day life cycle. For pupal acclimation, pupae were acclimated on different days after the eggs had been parasitized (days 5 and 6, 7 and 8, 9 and 10, or only on day 10 just prior to emergence). Preliminary experiments indicated that the acclimation treatments effective with T. nr. brassicae (Hoffmann & Hewa-Kapuge 2000) did not cause acclimation in T. carverae, and previous acclimation treatments of T. carverae (Scott et al. 1997) demonstrated benefits but with costs. Different daily exposure periods were therefore used to generate acclimation. On each day, pupae were acclimated for 8 h day−1 by immersing parasitized eggs sealed in vials with Parafilm® in a water bath at 33 °C. For adult acclimation the vials were sealed with Parafilm® and immersed in a water bath for 30 min at 35 °C on the day of emergence. This resulted in six treatments: control wasps reared at the normal rearing temperature of 25 °C, wasps that had been acclimated as pupae for a total of 16 h (8 h on days 5 and 6, 7 and 8, 9 and 10) and 8 h (day 10 only) and adult wasps which were acclimated at 35 °C for 30 min 2 h before application of the heat shock. Wasps from the six treatments always emerged on the same day.
The effect of the acclimation treatments on survival of a heat shock was scored. For each replicate, 10 wasps from each treatment were placed in a vial, the vials sealed with Parafilm® and placed in a water bath at 40 °C for 90 min. Wasps 12–24 h postemergence were tested. After the heat shock, wasps recovered at 25 °C for 24 h before survival was scored. There were 10 wasps per treatment and the experiment was repeated four times.
To assess possible costs of acclimation, parasitism rates were compared for wasps acclimated at the different pupal stages, at the adult stage and without acclimation (controls). These experiments were undertaken without heat stress. For each treatment, 30 individual females were tested for parasitism rate. Each female was provided with ≈60−100 S. cerealella eggs presented on a small strip of paper from a Post-it® pad. All wasps were placed at 25 °C and given fresh S. cerealella eggs daily for 4 days. Parasitized S. cerealella egg cards were kept at 25 °C for 5 days until they turned black. The total number of eggs parasitized per female over the 4 days was calculated.
Two field trials were undertaken in a vineyard to assess the cost of acclimation in mild conditions and to test the benefit of acclimation under heat stress in nature. Three treatments (control, pupal and adult acclimation) were compared. The pupal acclimation treatment that yielded the most resistant wasps as measured by laboratory survival of heat shock after acclimation (8 h at 33 °C on day 10 – see below) was chosen as the developmental acclimation treatment for field tests. Field fitness was assessed by the ability of wasps to locate and parasitize eggs of Light Brown Apple Moth (Epiphyas postvittana[Walker]) laid on cards pinned to grapevine leaves. Parasitism rate on pinned eggs is similar to that on naturally laid egg masses (Glenn & Hoffmann 1997) and E. postvittana is the preferred natural host of T. carverae. The presence/absence of parasitism on egg cards was used rather than the number of eggs parasitized by each female as in the laboratory experiments. This was mainly because there was no control over the number of females potentially parasitizing each individual egg card unlike in the laboratory; in fact there were several instances where the entire egg mass was parasitized. Egg predation in the field was also encountered, which meant that reduced egg numbers were available for parasitism on some cards unlike in the laboratory where individual females could not exhaust their egg supply.
Twelve egg cards 30 cm apart were placed around a release point, and there were 10 release points per treatment. Release points were 70 m apart. Each egg card carried a single mass of 20–70 eggs. All releases were carried out at Rutherglen in Victoria (42°31′ S 171°10′ W). To avoid the problem of background parasitism, we carried out releases in a vineyard where T. carverae was not found. Previous experiments (Glenn & Hoffmann 1997; Bennett & Hoffmann 1998) have shown that parasitism from released Trichogramma is highest within 2 days of release. Within this time period, parasitism is largely restricted to a few metres from the release point along the release row of vines and in the adjacent row. Treatments were assigned randomly to release points in the vineyard. Because the area where the release was undertaken had only one vine variety at a uniform stage of development, we did not expect microclimatic differences among the release sites.
The first release, which compared the treatments under mild conditions (temperature range 20–30 °C), tested the cost of acclimation. The egg cards were collected 24 h after release of the wasps. A second release was done in summer, in hot weather. For this release, two sets of E. postvittana egg cards were used: the first set was collected after 3 h (temperature range 40–42 °C) and then replaced with a second set left overnight for a further 12 h when temperatures ranged from 25 to 35 °C. Collected egg cards were held at 25 °C and parasitism was scored as black (parasitized) eggs after 5 days. Egg cards were scored as parasitized or not parasitized and the percentage of parasitized egg cards around each release point calculated.
There was a consistent increase in survival of wasps exposed to 40 °C for 90 min after they had been pretreated as adults on day 11 at 35 °C for 30 min (Fig. 1a) (analysis of variance, F1,30 = 12·07, P = 0·003). An increase in survival was also found when T. carverae were exposed at the pupal stage to 33 °C for 8 h over 2 days or over the final day before eclosion (Fig. 1b) (analysis of variance, F4,130 = 12·99, P < 0·001). The increase was greatest when pupae were acclimated later in their development. Post hoc comparisons (Tukey B) indicated three homogeneous (P > 0·05) groups, the first consisting of the control and day 5–6 treatment, the second consisting of all remaining treatments except day 10, and the final one consisting of the day 10 treatment. This day 10 treatment was therefore used to examine acclimation effects in field releases.
There were no significant differences among the acclimation and control treatments (Kruskal–Wallis test χ2 = 4·26, df = 5, P = 0·512) when parasitism was measured at 25 °C (Fig. 2) in the laboratory. Thus there was no detectable cost of acclimation in the laboratory for this trait.
In the release undertaken in mild conditions there was no significant difference in the percentage of egg cards parasitized by the control, pupal acclimated or adult acclimated wasps (Kruskal–Wallis test χ2 = 0·25, df = 2, P = 0·881) (Fig. 3a). Thus there was no evidence for costs associated with acclimation under these conditions.
For the high-temperature release, in the initial 3-h period unacclimated wasps failed to parasitize any of the cards but there was a low level of parasitism in both acclimation treatments (Fig. 3b). Treatments differed significantly (Kruskal–Wallis test, χ2 = 8·00, df = 2, P = 0·018). Although wasps acclimated at the adult stage parasitized more egg cards than those acclimated at the pupal stage, this difference was not significant (Kruskal–Wallis test, χ2 = 0·01, df = 1, P = 0·902). For the second set of cards, some parasitism occurred overnight in the control treatment but parasitism rates remained substantially higher in the acclimated treatments and rates differed significantly among the treatments (Kruskal–Wallis test, χ2 = 16·22, df = 2, P = 0·0003) (Fig. 3c). In addition to significant differences between the control and acclimation treatments, wasps acclimated at the adult stage parasitized signi-ficantly more egg cards than those acclimated at the pupal stage (Kruskal–Wallis test, χ2 = 5·53, df = 1, P = 0·018).
These results indicate that both adult and pupal acclimation increased field fitness under hot conditions. The benefits of acclimation as measured by field parasitism were large. Overall, wasps from the pupal and adult acclimation treatments parasitized 4·7 and 8·7 times more egg masses, respectively.
Parasitism rates examined in field releases undertaken when ambient temperatures were mild (20–30 °C) indicated no reduction in performance as a result of either adult or pupal acclimation compared with controls under these conditions (χ2 = 1·56, df = 2, P = 0·457) (Fig. 3a). Three other releases under cool or mild conditions also failed to find an effect of acclimation on field parasitism (data not presented). Therefore neither pupal nor adult acclimation treatments had a detectable cost in the field.
We have shown that the acclimation effects found in the laboratory are also beneficial in the field under hot conditions. Our findings therefore support the beneficial acclimation hypothesis in contrast to other studies based on rearing organisms continuously under different conditions (Leroi et al. 1994; Hoffmann 1995; Padilla & Adolph 1996; Bennett & Lenski 1997). The results support earlier work on T. carverae (Scott et al. 1997), indicating that adult wasps can be heat hardened. However, by changing the acclimation conditions from those used by Scott et al. (1997) we were able to identify laboratory conditions that led to hardening without a reduction in parasitism.
One reason why experiments that involve acclimation via rearing may produce different results from those based on hardening is that rearing organisms in particular conditions throughout development has the potential to influence organisms in more ways. In contrast, the effects of short exposures to high temperatures may largely be mediated via heat shock proteins (Morimoto et al. 1990) which are also expressed in Trichogramma (Maisonhaute et al. 1999). While there is some evidence that expression of these proteins can have deleterious effects on fitness (Krebs & Loeschke 1994a; Krebs & Feder 1998), the data presented here and for T. nr. brassicae (Hoffmann & Hewa-Kapuge 2000) suggest that this is not always the case.
These findings have implications for the success of inundative and augmentative releases of parasitoids. Trichogramma are of particular interest because they are used in biological control programmes more than any other natural enemy (Stinner 1977). Adult Trichogramma have short life spans in the field and their parasitism success depends on coping with severe environmental conditions including heat stress (Naranjo 1993; Ramesh & Baskaran 1996; Maisonhaute et al. 1999). Our pupal acclimation results indicate that wasp performance can be improved in summer by exposing parasitized eggs at a late stage in their development to high temperatures prior to field release. It should therefore be possible for insectaries to produce wasps suitable for release under extreme conditions. For T. carverae, which is a parasitoid of eggs of Light Brown Apple Moth in vineyards and orchards (Glenn et al. 1997), this should increase the effectiveness of commercially available material.
This work was supported by grants from the Australian Research Council SPIRT Programme and Special Research Centre Programme, and from the Grape and Wine Research and Development Corporation. Thanks to Malcolm Campbell of Rutherglen for access to his vineyard.
Received 28 June 2000; revised 20 September 2000; accepted 22 September 2000