Temperature dependent functional response of Diaeretiella rapae ( Hymenoptera : Aphidiidae ) to the cabbage aphid , Brevicoryne brassicae ( Hemiptera : Aphididae )

Diaeretiella rapae MacIntosh (Hymenoptera: Aphidiidae) is one of the most common and successful parasitoids of the cabbage aphid. The functional response of D. rapae towards cabbage aphids was examined in laboratory studies at three constant temperatures, 17°C, 25°C and 30°C. D. rapae exhibited a type II functional response at all three temperatures. The search rates were uninfluenced by temperature whereas handling times differed significantly between 17°C and 25°C, and between 17°C and 30°C, but not between 25°C and 30°C. This study is a first-step in the evaluation of the effectiveness of D. rapae as a biocontrol agent of Brevicoryne brassicae at different temperatures.

The functional response of D. rapae to the cabbage aphid has been studied (e.g.Fathipour et al., 2006) but no one has considered the effect of temperature.Consequently, the aim of this study was to determine the influence of temperature on the searching efficiency and handling time of D. rapae attacking cabbage aphid nymphs.

Insect rearing
Cabbage aphids and D. rapae were originally collected from infested ornamental cabbage, Brassica oleracea L. (var. acephala), on the Zanjan University campus in October 2010 and subsequently kept in separate cultures on B. oleracea in net covered cages (40 × 40 × 40 cm) at 24 ± 2°C, 45 ± 5% R.H. and a photo-period of 16L : 8D in the Insect Ecology and Biocontrol Laboratory, the Research Institute of Physiology and Biotechnology, Zanjan University.

Functional response experiments
Mummified cabbage aphids were collected from the colony and placed individually in small vials (approx.4 ml).After 24 h, newly emerged parasitoids were removed and placed in plexiglas cylindrical cages (15 × 35 cm) and fed a 10% honey solution.Twenty four hours later mated females were recovered and kept individually in similar small vials for 2-3 h before using them in the experiments.
The experimental arena consisted of a cabbage leaf placed in a Petri dish (9 cm diameter) with two droplets of honey and a piece of wet cotton wool added as food and a source of water.Two,4,6,8,16,24,40 or 50 second instar nymphs of B. brassicae were placed randomly on the leaf.The number of replicates was 15 except for the density 8 nymphs per leaf at 17°C and 50 at 30°C, for which there were 14 replicates.One female D. rapae was added to each arena, which was then placed in a growth chamber at 17, 25 or 30°C, 65 ± 5% R.H. and a photoperiod of 16L : 8D.After 24 h the parasitoids were removed and the arenas were kept in the same conditions for a further 2 weeks after which the numbers of mummified aphids were recorded.The present study thus only examines the influence of temperature on the functional response in terms of the parasitization activity of the adult parasitoids.All replicates at each temperature were conducted simultaneously over a 3-week period.

Statistical analysis
The overall effect of temperature on the rate of parasitization of B. brassicae by D. rapae was analysed by comparing the number of mummified aphids recorded at the different temperatures (one-way ANOVA or Kruskal-Wallis test, SAS Institute Inc. 2004).
Subsequently, the type of functional response and the parameters characterizing it were estimated for each temperature (Juliano, 2001).Logistic regressions of the proportions of aphids parasitized at different host densities were used to determine the type of functional response (Trexler et al., 1988;Trexler & Travis, 1993) using the quadratic model: (1) where Ne is the number of parasitized aphids, N0 is the host density and P0, P1 and P2 are the parameters to be estimated.Significant negative values of P1 indicate a type II functional response, whereas positive significant values indicate a type III functional response (Juliano, 2001).Non-linear least-square regression (NLIN procedure, SAS Institute Inc., 2004) of the number of mummified aphids recorded at different host densities was used for estimating searching rates and handling times (Juliano & Williams, 1987;Juliano, 2001) based on either the Random Parasitoid Equation (Rogers, 1972) (Equation 2), or if that failed to provide an adequate fit or meaningful parameter estimates, Holling's Disc Equation (Holling, 1961) (Equation 3).
(2) Roger's model N e  N 0 1  exp  aT Where a is the search rate, T the total available time (here 24 h) and Th the handling time (Juliano & Williams, 1987;Juliano, 2001).The individual parameters for each pair of temperatures were compared using t-tests (SAS/STAT, NLMIXED, SAS Institute Inc., 2004).
Temperature did not affect the type of functional response recorded for D. rapae as the linear term of the logistic regression in all cases was significantly negative, i.e. the number of cabbage aphids parasitized increased with a decreasing slope, which indicates a type II functional response (Table 2, Fig. 1).
Roger's Random Parasitoid Equation (Rogers, 1972) did not fit the data and the functional response parameters were consequently estimated using Holling's Disc Equation (Holling, 1961) (Table 3).The search rates were not influenced by temperature (t-test, 17°C vs. 25°C, t = -0.152,P = 0.879; 17°C vs. 30°C: t = -0.243,P = 0.809; 25°C vs. 30°C: t = -0.098,P = 0.922) whereas handling times recorded at 17°C and 25°C and at 17°C and 30°C differed significantly, but not at 25°C and 30°C (17°C vs. 25°C: t = 3.876, P < 0.001; 17°C vs. 30°C: t = 2.762, P = 0.006; 25°C vs. 30°C: t = -1.138,P = 0.256).The maximum attack rates (T/Th) were 14. 54, 24.74  second instar aphid nymphs per day at 17°C, 25°C and 30°C, respectively.DISCUSSION D. rapae showed a type II functional response to increasing densities of cabbage aphid nymphs at all the temperatures tested.Type II functional responses are commonly recorded for parasitoids, especially under controlled conditions in the laboratory (Fernandez-Arhex & Corley, 2003).In fact, more than three quarters of such studies carried out on parasitoids between 1959-2001 report type II functional responses (Fernandez-Arhex & Corley, 2003).This may be due to the generally artificial experimental design in which parasitoids have a fixed time to search for hosts and there is a lack of any spatial complexity within patches (Fernandez-Arhex & Corley, 2003).
A type II functional response of D. rapae is reported for the cabbage aphid (Fathipour et al., 2006), Russian wheat aphid (Bernal et al., 1994;Tazerouni et al., 2011Tazerouni et al., , 2012) ) and green peach aphid (Yu et al., 1993).The rate at which this parasitoid attacks cabbage and green peach aphids is estimated to be 0.033 h -¹ (Fathipour et al., 2006) and 0.031 h -¹ (Yu et al., 1993), respectively, which is in accordance with our results, whereas the rate at which it attacks the Russian wheat aphid is higher (0.056 h -¹ (Bernal et al., 1994) and 0.072 h -¹ (Tazerouni et al., 2011), respectively).These higher rates of attack might be due to the differences in the sizes of the hosts, as the Russian wheat aphid is smaller than the cabbage aphid (Antolin et al., 2006), and handling time in aphid parasitoids is thought to be inversely related to host size (Hofsvang & Hagvar, 1986).
Temperature had a significant effect on the number of cabbage aphids parasitized by D. rapae only at the higher cabbage aphid densities, which is reflected in the significantly higher handling time recorded at the lowest temperature.No influence of temperature on attack rate was found.The longer handling time recorded at 17°C is most likely a result of an increase in non-searching activities (e.g.resting).A similar temperature-dependent influence on functional response parameters with a decrease in handling time with increase in temperature without significant effects on search rates is recorded for predators, e.g. the predatory mite Neoseiulus californicus (McGregor) attacking Tetranychus urticae Koch (Ahn et al., 2009).Considering the effect of global warming and climate change, the effect temperature has on the handling time of D. rapae indicates that this parasitoid performs best at temperatures around 25°C and it is likely to be less effective at higher temperatures.
Compared with the parasitization of other aphid species by D. rapae, such as D. noxia, M. persicae and Lipaphis erysimi (Kaltenbach), the parasitization of cabbage aphids in the present study was slightly higher than that recorded for D. noxia (Tazerouni et al., 2012) and lower than for the other two species (Blande et al., 2004).The highest number of aphids parasitized by D. rapae in the present study was 15.8 ± 1.17 at the density of 50 at 25°C (Table 1), which is slightly lower than recorded by Fathipour et al. (2006) (17.3 ± 0.87) for the same host-parasitoid system at a similar temperature and host density.This discrepancy may be due to differences in the parasitoid biotypes or size of experimental arena used.
The results of the present research indicate that D. rapae can be used as a biocontrol agent of B. brassicae and probably other aphid species, such as the green peach aphid, at temperatures ranging between 17 and 30°C.However, for a comprehensive evaluation of the effectiveness of D. rapae in controlling cabbage aphids infesting other host plants more natural and field-based studies are needed (Bernal et al., 1994;Montoya et al., 2000).The present results additionally provide a basis for using D. rapae for augmentative or conservational biocontrol using the banker plant method (Huang et al., 2011) by developing a system consisting of crop or noncrop plants infested with a specialist crucifer-feeding aphid species as a non-pest alternative prey.

Fig. 1 .
Fig. 1.Functional response of Diaeretiella rapae to Brevicoryne brassicae nymphs at 17°C (solid line, black circles), 25°C (dotted line, white circles) and 30°C (dashed line, black triangles).The symbols represent average values (with standard errors) and the lines the fitted model.

TABLE 2 .
Logistic regression analysis of the proportion ofBrevicoryne brassicae nymphs parasitized by Diaeretiella rapae at different host densities at different temperatures.