Introduction
Ambient temperature is an important factor influencing many aspects of insect ecology and development. It is clear that the change in global climate includes not only an increase in average temperatures, but also a high frequency of extreme climatic events such as heat waves (Easterling et al., Reference Easterling, Evans, Groisman, Karl, Kunkel and Ambenje2000; Hansen et al., Reference Hansen, Sato and Ruedy2012). Extreme temperatures are highly stressful and are known to have detrimental effects on insects (Rinehart et al., Reference Rinehart, Yocum and Denlinger2000; Malmendal et al., Reference Malmendal, Overgaard, Bundy, Sørensen, Nielsen, Loeschcke and Holmstrup2006; Hance et al., Reference Hance, van Baaren, Vernon and Boivin2007; Terblanche et al., Reference Terblanche, Hoffmann, Mitchell, Rako, le Roux and Chown2011; Hoffmann et al., Reference Hoffmann, Chown and Clusella-Trullas2013). High temperatures threaten the survival of insects on a daily basis (Rinehart et al., Reference Rinehart, Yocum and Denlinger2000). Mortality may occur immediately for several insects of economic importance (Armstrong, Reference Armstrong1992; Hansen, Reference Hansen1992), or may result in a failure to complete development (Denlinger et al., Reference Denlinger, Joplin, Chen, Lee, Lee and Denlinger1991). In addition, insects that survive heat stress may pay a price manifested in their life history traits (Chihrane et al., Reference Chihrane, Laugé and Hawlitzky1993; Krebs & Loeschcke, Reference Krebs and Loeschcke1994; Chihrane & Laugé, Reference Chihrane and Laugé1996; Rinehart et al., Reference Rinehart, Yocum and Denlinger2000; Ma et al., Reference Ma, Hau and Poehling2003; Mahroof et al., Reference Mahroof, Subramanyam and Flinn2005a; Jørgensen et al., Reference Jørgensen, Sørensen and Bundgaard2006; Sisodia & Singh, Reference Sisodia and Singh2006; Cui et al., Reference Cui, Wan, Xie and Liu2008; Xie et al., Reference Xie, Hou and Zhang2008). Furthermore, heat stress in adults can also influence their offspring, reducing the rate of egg hatching (Silbermann & Tatar, Reference Silbermann and Tatar2000), or inducing changes in morphology (Andersen et al., Reference Andersen, Pertoldi, Scali and Loeschcke2005).
Parasitoids are likely extremely susceptible to changes in environmental conditions, as they rely on a series of adaptations to the ecology and physiology of their hosts for survival (Hance et al., Reference Hance, van Baaren, Vernon and Boivin2007). Generally, the success of using parasitoids as biological control agents relies not only on the time and the numbers of parasitoids released, but also on their quality (longevity and fecundity) (Dutton et al., Reference Dutton, Cerutti and Bigler1996). Therefore, the ability of a parasitoid to tolerate the physical environment is a critical factor in evaluating their biological control potential (Tillman & Powell, Reference Tillman and Powell1991; Miller & Gerth, Reference Miller and Gerth1994). Trichogrammatoidea bactrae Nagaraja (Hymenoptera: Trichogrammatidae) is an important natural enemy of many species of lepidopterous pests (Lin, Reference Lin1994). This parasitoid is mostly found in many tropical and subtropical countries, such as India, Malaysia, Thailand, Pakistan, Australia, and can also be encountered in some provinces of China (i.e., Guangdong, Hainan, Fujian and Taiwan) (Lin, Reference Lin1994; Huang et al., Reference Huang, Chen and Shen2002; Liu et al., Reference Liu, Cooper, Llewellyn, Elson-Harris, Duff, Furlong and Zalucki2004; Tian & Lin, Reference Tian and Lin2009). Many studies showed that T. bactrae has great parasitization potential on eggs of the diamondback moth, Plutella xylostella (Hübner) (Wuhrer & Hassan, Reference Wuhrer and Hassan1993; Vasquez et al., Reference Vasquez, Shelton, Hoffmann and Roush1997; Guo et al., Reference Guo, Zhu and Li1999); thus, this parasitoid is considered as a suitable candidate to control the diamondback moth. It is necessary and important to estimate the efficacy of this parasitoid under field conditions, where temperature conditions can be subjected to daily variations.
In a recent study, T. bactrae were exposed to an increasing range of constant temperatures during their development and their fitness parameters were assessed. Chen et al. (Reference Chen, He, Lu, Lu and Huang2005) reported that the minimum and maximum temperatures for T. bactrae development were 11.9 and 38.95 °C, respectively. Nadeem & Hamed (Reference Nadeem and Hamed2008) described that only 36.1% adult T. bactrae emerged successfully from host eggs at 35 °C. However, the effects of heat stress on this parasitoid have not been reported yet.
The goal of this study was to measure the effects of heat stress on biological parameters (emergence percentage and deformity rate of adults, longevity and fecundity of surviving females, and life-table parameters) of T. bactrae, under conditions simulating high temperatures of field microclimate, in order to make some realistic recommendations for field applications of this parasitoid.
Materials and methods
Insect origin and rearing
T. bactrae were reared from parasitized diamondback moth egg masses (about 100 eggs) collected in May 2011 from Chinese Kale (Brassica alboglabra Bailey) fields located near Guangzhou, China. The stock population (about 20,000 parasitoids) was maintained and reared on eggs of Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) in glass tubes (2.3 cm internal diameter, 7.4 cm internal height) at 25±1 °C, 80±5% R.H. and a 14L:10D photoperiod in the laboratory. These rearing conditions of the parasitoids were as the ‘control conditions’ throughout our study. The eggs of C. cephalonica (approximately 2000 eggs) were stuck to 3×2 cm cardboard strip and exposed to UV light (30 W) for 30 min before experiments and rearing of T. bactrae. C. cephalonica was reared following the procedure described in Wang et al. (Reference Wang, He, Guo and Luo2012). To feed the adults of T. bactrae, a droplet of 25% honey solution was placed in the glass tube prior to or upon their emergence.
To obtain standardized parasitoids for experiments, approximately 150 fed, newly emerged (<6 h after emergence) and mated females of T. bactrae were supplied with fresh host eggs (approximately 2000 eggs) on cardboard strip (3×2 cm). After 8 h, parasitoids were removed with a brush from the egg card. Then the card was cut into small egg sheets (1×0.5 cm, about 160 eggs of C. cephalonica per sheet), inserted into small glass vials (1 cm internal diameter, 5.5 cm internal height) and maintained under control conditions. The entrances of the glass vials were closed with cotton balls to allow air circulation and to prevent the escaping of wasps.
Experimental design
The experiment was designed to test three factors: heat stress temperature, developmental stage of parasitoids exposed to high temperature, and duration of exposure.
Heat stress was applied to the four preimaginal developmental stages: embryo-first instar larvae (24 h after egg laying), second instar larvae (48 h after egg laying), prepupae (96 h after egg laying), and pupae (144 h after egg laying) (Chen et al., Reference Chen, Liu, Fang and Huang2004).
To test the resistance of T. bactrae to heat stress, we used climatic temperature controlled cabinets (Ningbo Jiangnan RXZ-380A, Ningbo, Zhejiang Province, China). The temperatures (33, 36, and 39 °C) were chosen based on the field microclimate temperatures (recorded by a hygrothermograph (Smartsensor AR847, HongKong, China) in preliminary experiments) in Southern China during summer months. The exposure times (2, 4, 6, and 8 h) were selected, in order to simulate the theoretical duration of extreme temperatures. Parasitoids maintained continuously at 25 °C, 80±5% R.H. and a 14L:10D photoperiod served as controls.
Measured traits
Development aspects
In order to test the effects of heat stress on survival and development of T. bactrae, we calculated the emergence percentage and deformity rate of adults. After each heat exposure, egg sheets were placed at 25 °C as described above. Emergence percentage and deformity rates of adults were measured after the parasitoids completely emerged from host eggs. Emergence was defined as empty eggs with eclosion hole on them. We considered adults as deformed when their wings were folded or incomplete. Ten replicates were carried out for each treatment.
The general linear model (GLM) procedure (SPSS 13.0, SPSS Inc., Chicago, IL) with interaction terms was used to analyze the effects of heat stress temperature (33, 36, and 39 °C), developmental stage of parasitoids exposed to high temperature (embryo-first instar larvae, second instar larvae, prepupae, or pupae), duration of exposure (2, 4, 6, or 8 h) and all their interactions on the emergence percentage and deformity rate of T. bactrae adults subjected to heat stress. Under the condition of the same developmental stage and same temperature, the variability of the emergence percentage and deformity rate as functions of the heat stress duration were analyzed assuming a GLM. When statistical differences existed between data sets (P<0.05), Tukey's HSD test was used to determine statistically significant differences between means.
Reproduction aspects
To determine the effects of heat stress on reproduction of T. bactrae, the life-table method of the surviving parasitoids (Chen et al., Reference Chen, He, Lu, Lu and Huang2005; Wang et al., Reference Wang, He, Guo and Luo2012) was applied. After heat exposure, egg sheets of parasitoids were maintained in the small glass vials under control conditions. Five replicates (egg sheets) were carried out for each treatment.
After the parasitoids emerged, they were fed a 25% honey solution and were placed under control conditions for 2 h to mate. The surviving and mated female parasitoids were introduced individually into a clean glass tube, and supplied with a 25% honey solution on the internal wall as food. As previously described, tubes were closed with cotton balls. For each treatment, 20 surviving females were randomly selected from the five replicates to further investigate the longevity and fecundity of surviving female adults, as well as the daily daughter output. To determine the fecundity of each female parasitoid an egg sheet (1×0.5 cm) with about 160 C. cephalonica eggs stuck to cardboard strips was inserted into each tube, and was replaced with a new egg sheet after 24 h and substituted daily until all individuals had died. Survival of females was recorded daily, and honey solution was added when necessary. The egg sheets were labeled and placed into small glass vials and incubated for 13 days under control conditions, long enough for complete emergence of offspring from the initial parasitism. Finally, the vials were placed into −10 °C for 1 h to kill offspring, and the number of parasitized eggs (black), and daughters were recorded for each treatment under a dissecting microscope (Zeiss Stemi 2000-CS, Shanghai, China).
Daily schedules of survival rate and fecundity were integrated into a life-table format (Dastjerdi et al., Reference Dastjerdi, Hejazi, Ganbalani and Saber2009; Bayram et al., Reference Bayram, Salerno, Onofri and Conti2010) and used to estimate life-table parameters: probability of an individual surviving to age x (l x), the number of daughters produced by a female at age x (m x), net reproduction rate (R 0=Σl xmx), mean generation time (T=Σxl xmx/Σl xmx), intrinsic rate of natural increase (r m=ln R 0/T), finite rate of increase (λ=erm), and doubling time (DT=ln 2/r m).
The effects of heat stress temperature, developmental stage of parasitoids exposed to high temperature, duration of exposure and their interactions with each response variable (longevity and fecundity of female adults and life-table parameters of T. bactrae) were analyzed using GLM as the model (SPSS 13.0, SPSS Inc., Chicago, IL). For the same developmental stage and temperature, GLM was used to analyze the effect of different duration of exposure on each response variable. Means were compared using Tukey's HSD test with α=0.05.
Results
Effects of heat stress on the development of T. bactrae
Emergence rate of T. bactrae adults was remarkably affected by the developmental stage at the time of heat stress, heat stress temperature, duration of exposure, and all the possible interactions of these three factors (table 1). After heat stress, the emergence rate of T. bactrae adults decreased with the increase in exposure time (fig. 1). Only 19.90% adults emerged after they had been exposed to 39 °C for 8 h during pupal stage (fig. 1D). Compared with other developmental stages, significantly fewer adults emerged if they had been exposed to heat stress at the embryo-first instar larval stage (P<0.05, Tukey's HSD test).
Fig. 1. Emergence rate (%) of T. bactrae adults from heat treated stage (A: embryo-first instar larvae; B: second instar larvae; C: prepupae; or D: pupae). Under similar temperature and developmental stage conditions, only if statistically significant differences between data sets of different exposure times were detected (P>0.05), subsequent multiple comparisons were performed. Different letters in different durations of exposure denote statistically significant differences (P<0.05, Tukey's HSD test), the same below.
Table 1. Three-factor ANOVA statistics for analyzing emergence and deformity rate of T. bactrae adults, the mean longevity and fecundity of females treated by heat stress.
a: developmental stage of parasitoids exposed to high temperature; b: heat stress temperature; c: duration of exposure.
All three factors, developmental stage during heat stress, heat stress temperature and exposure time, and their interactions had significant effects on the deformity rate of T. bactrae adults (table 1). The deformity rate was significantly higher when the wasps were heat stressed during pupal stage compared to any other stage. And more than 14% adults emerged with abnormal wings when they were exposed as pupae to 39 °C for 6 or 8 h (fig. 2D).
Fig. 2. Deformity rate (%) of T. bactrae adults that emerged from treated stage (A: embryo-first instar larvae; B: second instar larvae; C: prepupae; or D: pupae).
Effects of heat stress on the reproduction of T. bactrae
The mean longevity of T. bactrae females was negatively influenced by developmental stage during heat stress, heat stress temperature, duration of exposure, and all the possible interactions between these three factors, except for the triple interaction of developmental stage, heat stress temperature, and duration of exposure (table 1). After heat stress during each preimaginal developmental stage, the longevity of females was reduced when compared to control condition. Furthermore, compared to other heat stress temperatures, exposure to 39 °C significantly reduced longevity of T. bactrae females (fig. 3). In comparison with their longevity at other treatment stages, females exposed as prepupae to heat stress lived significantly shorter (P<0.05, Tukey's HSD test). In particular, females exposed as prepupae to 39 °C for 8 h lived for only 1.45 days (fig. 3C).
Fig. 3. Mean longevity (days) of T. bactrae females that emerged from treated stage (A: embryo-first instar larvae; B: second instar larvae; C: prepupae; or D: pupae).
The mean fecundity of T. bactrae females was negatively affected by the developmental stage of heat stress, heat stress temperature, duration of exposure, and the temperature in interaction with the duration of heat stress (table 1). When exposed to 39 °C during each preimaginal developmental stage, emerged females parasitized fewer eggs than untreated ones (fig. 4). Moreover, females exposed as prepupae to 39 °C for 8 h only parasitized on average 23.5 eggs in their lifetime (fig. 4C). In addition, the parasitic ability of females was also significantly influenced by exposure to 33 °C during embryo-first instar larval or second instar larval or prepupal stage, 36 °C during prepupal stage (fig. 4A–C). Fewer eggs were parasitized when females were heat stressed during prepupal stage compared to all other stages (P<0.05, Tukey's HSD test).
Fig. 4. Fecundity (no. eggs parasitized) of T. bactrae females that emerged from treated stage (A: embryo-first instar larvae; B: second instar larvae; C: prepupae; or D: pupae).
Effects of heat stress on the life-table parameters of T. bactrae
Both heat stress temperature and duration of exposure had significant effects on R 0 (net reproduction rate) and r m (intrinsic rate of natural increase) of T. bactrae, as did the possible double interactions of developmental stage, heat stress temperature, and duration of exposure. Developmental stage had a statistically significant effect on r m but not on R 0. All three factors, developmental stage during heat stress, heat stress temperature and duration of exposure had significant influences on λ (finite rate of increase) of T. bactrae, as did the possible interactions among them. The T (mean generation time) and DT (doubling time) of T. bactrae were significant affected by developmental stage of heat stress, heat stress temperature, duration of exposure, and all the possible double interactions among them, except for the interaction of developmental stage and duration of exposure (table 2).
Table 2. Three-factor ANOVA statistics for analyzing life-table parameters of T. bactrae treated by heat stress.
a: developmental stage of parasitoids exposed to high temperature; b: heat stress temperature; c: duration of exposure.
After heat stress, R 0, r m and λ were lower than untreated parasitoids, and this tendency was especially pronounced for exposure to 39 °C during each preimaginal developmental stage. In addition, heat stress treatment can shorten the mean generation time and increase the doubling time of T. bactrae (table 3).
Table 3. Life-table parameters of T. bactrae after exposure to heat stress treatments.
1 Parasitoids without exposure to heat stress treatment (control).
2 Means followed by the same lower case letter in different durations of exposure at same temperature are not significantly different (P<0.05, Tukey's HSD test).
3 No multiple comparison, since no statistically significant differences between data sets of different durations of exposure at same temperature (P>0.05).
Discussion
Our results show that all the preimaginal developmental stages of T. bactrae are susceptible to heat stress. This susceptibility manifests itself in a reduction in emergence rate, longevity, and fecundity, as well as an increase of adult malformation incidences. In addition, heat sensitivity was enhanced as heat stress temperatures increased.
Generally, high temperature can affect emergence of parasitoids (Chihrane et al., Reference Chihrane, Laugé and Hawlitzky1993; Liu & Tsai, Reference Liu and Tsai2002). After heat stress, the emergence rate of T. bactrae adults decreased with the increase in exposure time, especially when parasitoids were exposed to 39 °C. After exposure to 39 °C for 8 h during pupal stage, the emergence rate was only 19.90%, and more than 14% adults were deformed. Insects are especially vulnerable to desiccation during the pupal stage since they are close to adult molt (Chihrane et al., Reference Chihrane, Laugé and Hawlitzky1993). Female parasitoids with abnormal wings will not fly normally, in which case they would have little chance to encounter food or host eggs. Thus, heat stress is bound to result in reduced survival and parasitic (or reproductive) ability of female parasitoids.
Our study shows that preimaginal heat stress is harmful to the survival of T. bactrae females. The mean life span of T. bactrae females that survive heat stress was shortened; in particular females exposed as prepupae to 39 °C for 8 h survived only for 1.45 days (fig. 1). Similar results have previously been reported for Trichogramma brassicae females where heat stress treatment during pupal stage decreased their mean survival rates (Chihrane et al., Reference Chihrane, Laugé and Hawlitzky1993; Chihrane & Laugé, Reference Chihrane and Laugé1996). However, when adult parasitoid of Aphidius ervi was exposed to high temperatures, the mean life span of surviving wasps was also reduced (Ismaeil et al., Reference Ismaeil, Doury, Desouhant, Dubois, Prevost and Couty2013). Insect metabolic rate depends on temperature which influences their longevity so that insects live for a shorter time under the condition of higher temperatures (Denlinger & Yocum, Reference Denlinger, Yocum, Hallman and Denlinger1998; Speight et al., Reference Speight, Hunter and Watt1999). Thus, the negative effect of heat stress on the longevity of T. bactrae females is likely to be due to immediate and direct effects of short-term high temperature on their physiology.
Heat stress not only influenced the survival of female parasitoids but also decreased their fecundity. Many studies reported that high temperatures can interrupt the normal functioning of the reproductive system in both sexes of insects (Arbogast, Reference Arbogast1981; Saxena et al., Reference Saxena, Sharma, Thappa and Tikku1992; Mahroof et al., Reference Mahroof, Zhu and Subramanyam2005b). In our study, T. bactrae females that emerged from each treatment parasitized fewer eggs than untreated ones. Females exposed as prepupae to 39 °C for 8 h only parasitized 23.5 eggs per female in their lifetime compared to 87.9 in the control treatment (fig. 4). Similar results were reported by Roux et al. (Reference Roux, Le Lann, van Alphen and van Baaren2010): after exposure to 36 °C for 1 h, the surviving females of A. avenae laid fewer eggs. The reduction in fecundity may be due to the direct cost of heat stress as reported for Drosophila melanogaster and for the parasitoid Trichogramma carverae (Krebs & Loeschcke, Reference Krebs and Loeschcke1994; Scott et al., Reference Scott, Berrigan and Hoffmann1997), alternatively reproductive structures might get directly damaged. Mironidis & Savopoulou-Soultani (Reference Mironidis and Savopoulou-Soultani2010) reported that heat shock can cause injury to oocytes and ovarian development; heat treated female T. brassicae had abnormally formed ovaries, or ovaries arrested in their juvenile state (Chihrane & Laugé, Reference Chihrane and Laugé1997).
Life-table parameters of T. bactrae were also affected by heat stress in our study. When exposed to 39 °C during each preimaginal developmental stage, R 0, r m, λ, and T were significantly decreased compared to control values. Doubling time (DT) is an important parameter that provides an indication of population growth. When exposed to 39 °C during each preimaginal developmental stage of T. bactrae, DT was significantly increased compared to control values.
Trichogramma are more often used in biological control programs than any other natural enemy (Stinner, Reference Stinner1977). Adult Tricogramma have short life spans in the field and their parasitism success depends on their ability to cope under severe environmental conditions including heat stress (Naranjo, Reference Naranjo1993; Ramesh & Baskaran, Reference Ramesh and Baskaran1996; Maisonhaute et al., Reference Maisonhaute, Chihrane and Laugé1999). Our data indicate that even small variations in temperature can potentially influence quality and efficacy of T. bactrae as IPM agents. Therefore, this parasitoid may be more effectively controlling lepidopterous pests during cooler weather conditions. Furthermore, in order to maintain high parasitoid activity under hot conditions, it is necessary to increase release numbers. However, the cost of heat stress has only been assessed for a limited number of traits and under laboratory conditions. In our study, only developmental aspects (emergence and deformity rate of parasitoids) and reproduction aspects (longevity and fecundity of females, life-table parameters of parasitoids) were examined. Unfortunately, other fitness components such as host localization and mating success were neglected. These important aspects might also be influenced by heat stress. In Trichogramma field fitness will rely on the ability of parasitoids to mate and find host eggs as well as on parasitism rate and longevity. These components still need to be tested under field condition.
Acknowledgements
We thank Li Hua Lu (Guangdong Academy of Agricultural Sciences, China) for helpful suggestion on data analysis of the manuscript. We are grateful to Irmgard Seidl-Adams (Pennsylvania State University, USA) for helpful suggestion and revision on the manuscript. This research was supported by National Basic Research Program of China (973 project, 2013CB127604) and the Special Fund for Agro-scientific Research in the Public Interest of China (201103021).
