Presence of Native Prey Does Not Divert Predation on Exotic Pests by Harmonia axyridis in Its Indigenous Range

In China, two invasive pests, Bemisia tabaci MEAM1 (Gennadius) and Frankliniella occidentalis (Pergande), often co-occur with the native pest, Aphis gossypii (Glover), on plants of Malvaceae and Cucurbitaceae. All three are preyed on by the native ladybird, Harmonia axyridis (Pallas); however, the native predator might be expected to prefer native prey to the exotic ones due to a shared evolutionary past. In order to clarify whether the presence of native prey affected the consumption of these two invasive species by the native predator, field-cage experiments were conducted. A duplex qPCR was used to simultaneously detect both non-native pests within the gut of the predator. H. axyridis readily accepted both invasive prey species, but preferred B. tabaci. With all three prey species available, H. axyridis consumption of B. tabaci was 39.3±2.2% greater than consumption of F. occidentalis. The presence of A. gossypii reduced (by 59.9% on B. tabaci, and by 60.6% on F. occidentalis), but did not stop predation on the two exotic prey when all three were present. The consumption of B. tabaci was similar whether it was alone or together with A. gossypii. However, the presence of aphids reduced predation on the invasive thrips. Thus, some invasive prey may be incorporated into the prey range of a native generalist predator even in the presence of preferred native prey.


Introduction
Problems associated with biological invasions, both environmental and economic, have grown exponentially over the past decades [1]. Invasive species threaten natural habitats and can affect native organisms in various ways [2] and at different levels of biological organization [3,4] typically because predation pressure on the invader from natural enemies is relaxed [5], as well as due to the potential disruption of trophic interactions between local prey and local enemies [6]. Native natural enemies are one factor in the 'biotic resistance' concept of invasion biology [7], but it is unknown how many invasion episodes fail due to such activity. The activity of native natural enemies, in the form of biological control, is usually considered a powerful

Biological materials
Insect colonies of pest species were reared at the Department of Biological Invasions, Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS). A colony of Bemisia tabaci MEAM1 (henceforth Bt) was started from individuals collected on field-grown cabbage in the outdoor plots of the institute 5 years ago, and maintained on tomato plants (Lycopersicon esculentum L., cv. Zhongshu 5), under greenhouse conditions [47]. A colony of F. occidentalis (henceforth Fo) started with individuals collected on sweet pepper grown at the same location 4 years ago, and maintained on fresh kidney bean (Phaseolus vulgaris L., cv. Gongjizhe) pods in an insect rearing room [48]. A colony of A. gossypii (henceforth Ag) was started from individuals collected from outdoor cotton plants during the 2009 field season, and maintained on cucumber plants (Cucumis sativus L., cv. Zhongnong 18), under greenhouse conditions at 20-26°C and 60±10% RH with a 16 h:8 h light:dark photoperiod. Cucumber plants were cultivated in a mixed potting medium (soil, vermiculite and plant ash, 1:1:1) in plastic pots (130×175 mm in diameter), and were kept in a clean seedling greenhouse. The plants were checked daily and watered as needed. When the cucumber plants had 4-5 fully extended true leaves, they were put in whitefly-and thrips-proof screen cages (60×60×60 cm high, tent-like) (NingBoSafe Experimental Instrument Co., Ltd., Ningbo, China) for rearing the cotton aphid. Each cage contained eight plants. Every

Field cage trials
In order to test if there is a difference in prey consumption depending on the nature and diversity of prey present, cages with one (Bt or Fo alone), two (Bt and Ag, Fo and Ag, Bt and Fo, respectively), three (Bt, Fo and Ag) prey species were used in field cage trial. Since our method (i.e. duplex TaqMan quantitative PCR [qPCR]) did not allow us to quantify consumption of A. gossypii, there was no cage with only Ag. The field cage trials were conducted at the Experimental Station of CAAS, Langfang, Hebei, China (39°30'36.96"N, 116°36'31.87"E) during the 2010 growing season. Cucumber plants were grown from seeds in plots protected from insect infestation by screen cages (2×2×2 m) and without application of pesticides. Each cage contained 20 cucumber seedlings, planted at 39-41 cm × 34-36 cm plant distances in 4 rows by 5 columns, facing the south. Distances between seedlings and cage edges were~40 cm. Plants were checked daily and watered as needed. When the cucumber plants had 4-5 fully expanded true leaves, we established the following combinations, each in triplicate. Moreover, because of possible mortality after transfer, the densities of the exotic and native prey species had to be adjusted one week later. Since about 10-20% Fo adults rest in crevices, count of about 80-90% adults indicated that all adults were present (GF Zhang personal experience).
Bt alone. About 100 Bt adults / plant (sex ratio = 1:1; 3-5 d old; sexed under a stereomicroscope, 180× magnification) were released in each cage. Seven days later, the density of Bt was adjusted to 100 adults / plant by adding or removing whiteflies as needed. There were 50-70 eggs / plant at this time.
Fo alone. About 100 Fo adults (3-5 d old) / plant were released in each cage. The sex ratio was adjusted to 2:1 (female:male) as in nature; individuals were sexed under a stereomicroscope (180× magnification). Seven days later, the density of Fo was adjusted to 90-100 adults / plant by adding or removing adults as needed. At this stage, there were also about 30 eggs in tender tissues and about 30 1st-2nd nymphs / plant.
Bt and Ag. About 50 Bt adults / plant (sex ratio = 1:1) and 30 Ag adults / plant were released in each cage. Seven days later, the densities were checked and adjusted. Bt density was adjusted to 50 adults / plant by adding or removing whitefly adults, plus 20-30 eggs / plant. The density of Ag was adjusted to 50-60 adults / plant.
Fo and Ag. About 50 Fo adults / plant (sex ratio = 2:1) and 30 Ag adults / plant were released in each cage, and allowed to reproduce for 7 days, whereupon Fo density was adjusted to 40-50 adults / plant. In addition, there were 10-20 eggs in tender tissues and 10-20 nymphs (1st-2nd instar) per plant. The density of Ag was adjusted to 50-60 adults / plant.
Bt and Fo. About 50 adults / plant each of Bt (sex ratio = 1:1) and Fo (sex ratio = 2:1) were released in each cage. Seven days later, the density of Bt was adjusted to 50 adults / plant plus 20-30 eggs / plant and that of Fo to 40-50 adults / plant plus 10-20 eggs in tender tissues and 10-20 1st-2nd nymphs.
Eighty newly emerged H. axyridis adults were released into each cage. All the treatments and replications were conducted simultaneously. At given times (1h, 2 h, 4 h, 8 h, 12 h, 24 h and 36 h after predator release), 10 H. axyridis adults were collected using a mouth aspirator, individually put into single 1.5 mL microcentrifuge tubes filled with 95% ethanol, and kept at -40°C until analysis. We were concentrating on prey consumption and not on biological control effect, so the exact numbers of each prey species were not checked, only that there were still prey available at the end of the experiment. There were about 20 prey individuals left on each plant when the last sample was taken (36 h after predator release), so we consider that prey availability was ad libitum. Five randomly selected beetles were individually homogenized and tested for the presence of Bt and/or Fo in their guts for each time period from each field cage. The DNA of prey species in the predator gut was detected and quantified by using qPCR and external calibration (standard curve).

DNA extraction
Bt or Fo adults were individually homogenized in 1.5 mL mortar-and-pestle microcentrifuge tubes (Haimen Sanhe Huasheng Glasses Materials Ltd., Jiangsu, China) in 200 μL of DNA extraction buffer supplemented with SDS to 1% and proteinase K to 20 mg mL -1 [46]. The homogenate was vortexed briefly and incubated in a water bath at 60°C for 1 h, with a remixing after 30 min. After incubation, the tubes were boiled for 5 min to inactivate the proteinase K, after which the lysate was extracted with 200 μL of chloroform-isoamyl alcohol mix (24:1, v/v), kept on ice for 30 min and centrifuged at 12 000 g for 20 min. This process was repeated twice, and the aqueous phase was mixed with 400 μL of ice-cold 100% ethanol. DNA was pelleted by centrifugation, dried and re-suspended in 20 μL ultra-pure water and stored at -40°C. The same DNA extraction procedure was used for non-target whitefly and thrips species (see section Specificity of the detection system), as well as for the predators collected from the field cage treatments. Before DNA extraction, individual predators were washed twice with ice-cold, pure water and blotted thoroughly with a clean filter paper, and the wings and legs were cut off.

Primers and probes used for duplex TaqMan qPCR
Based on an established single tube, one-step duplex PCR [46], primers and probes for the Bt specific (Bt1 isolate) [9] and Fo specific (Fo1 isolate) (present study) DNA quantification were designed by using the PRIMER EXPRESS software (Applied Biosystems). The primers and probes (Table 1) were synthesized by Shanghai GeneCore BioTechnologies.
Two gene sequences from genomic DNA were simultaneously targeted in this duplex qPCR procedure. The first targeted sequence was a 93 bp long segment present only in Bt [9], and the second was a 138 bp segment specific to Fo (present study). The system and conditions used for the duplex TaqMan

Specificity of the detection system
To confirm the efficacy and specificity of the duplex qPCR, we used the following prey species, these are the most likely other species possibly present in the field especially those closely related which are more likely to present false positive for the specific primers, as negative controls [46]: • two non-target, cryptic species of B. tabaci (Asia II 3 and Asia II 1), • four non-target whitefly species (Trialeurodes vaporariorum [Westwood], Dialeurodes citri • the aphid A. gossypii, and • starved H. axyridis adults.
In all instances, primers and probes against Bt and Fo showed positive fluorescent signals; but we obtained no positive fluorescent signal from any of the above species, indicating target specificity. Quantification of prey number consumed by H. axyridis The duplex qPCR standard curves for Bt and Fo were generated using DNA from the purified plasmid Bt1 and Fo1 isolates originated from our laboratory. The DNA concentrations were determined by a NanoPhotometer P-Class (Implen, Munich, Germany) and the numbers of target DNA sequence copies were calculated using the expression: number of target DNA copies ¼ ðDNAmass ½mg=DNAmolarmassÞ Â 6:023 Â 10 23 : A volume of 2 μL was used as template in each duplex TaqMan qPCR assay. The qPCR standard curves were established using 10-fold serial dilutions of the number of target DNA copies of Bt (Bt1 isolate) and Fo (Fo1 isolate) from 9.958×10 6 to 9.958×10 2 and 1.217×10 7 to 1.217×10 3 , respectively. Three repetitions of the assay were undertaken.
Data acquisition and analysis were performed with the ABI PRISM 7500 software. The values of threshold cycle (C t ) correspondence to the five DNA copy numbers were 19 Numbers of Bt-and Fo-specific DNA copies from field-cage collected samples were estimated by extrapolation from the DNA standard curves amplified in the same PCR run. All samples generating fluorescence signals that exceeded a defined background threshold (i.e. C t 35 in this study) were classified as positive [49].

Data analysis
Percentage data, i.e. percentage of predator individuals that consumed Bt or Fo as detected by the molecular method, were arcsine transformed and the numbers of Bt and Fo adults were log-transformed before analysis of variance (ANOVA). The field-cage experiment and duplex qPCR analysis produced mean numbers of Bt and Fo adults and data on the percentage of predation by H. axyridis on Bt or Fo. Such data were subjected to two-way (treatment and time after predator release) and one-way (same time after predator release among different treatments, or same treatment among different time after predator release) ANOVA, and means were tested using Fishers LSD test [50]. The differences in same focal prey species among different treatments were compared by paired samples t-test. The numbers of Bt and Fo adults and percentage of predation by H. axyridis on Bt or Fo were reported as means and standard error (SEM). Since there were no significant differences in numbers of Bt and Fo adults consumed by H. axyridis among cages, the data of the three field cages were pooled and analyzed together.

Predation on single prey species
Adult ladybirds readily attacked both Bt and Fo in a no-choice situation. From 1 to 8 h after release, 90% of predators contained prey DNA; between 12 h and 36 h, a non-significant decrease to >80% was registered (Bt: P = 0.531, Fo: P = 0.557; Fig 1A 1 and 1B 1 ). The maximum of Bt prey was about 0.78 adults 24 h after predator release, while for Fo, a peak of 0.55 adults / predator was reached 2 h after predator release before a later decrease. Time after predator release (Bt: P = 0.008, Fo: P < 0.001) had significant effects on prey consumption although the effects varied (Fig 2A 1 and 2B 1 ).

Predation of prey species combinations
Invasive plus native prey: Bt or Fo plus Ag. Adult ladybirds readily attacked both invasive species in the presence of Ag: up to 80% of predator guts contained Bt and 60% contained Fo, without a significant temporal trend (Bt: P = 0.393, Fo: P = 0.729; Fig 1A 2 and 1B 2 ). In the presence of Ag, the percentage of predation on Bt over 36 h was 24.5% higher than on Fo (Paired t-test, t = 5.605, df = 6, P = 0.001; Fig 1A 2 and 1B 2 ). The maximum amount of invasive prey consumed over 36 h followed similar dynamics without a significant temporal trend (Bt: P = 0.559, Fo: P = 0.948; Fig 2A 2 and 2B 2 ), but the predator consumed 4.1-46.0% more Bt than Fo over this period (Paired t-test, t = 3.30, df = 3, P = 0.046; Fig 2A 2 and 2B 2 ).
Invasive prey species paired: Bt plus Fo. Adult ladybirds readily attacked both invasive species when they were encountered simultaneously: 90% of predators contained Bt DNA and 80% contained Fo DNA, with some decrease in Bt detected 8 h after release but this reduction was not significant (Bt: P = 0.463, Fo: P = 0.117; Fig 1A 3 and 1B 3 ). The dynamics over 36 h was similar, but predation was 6.1% higher on Bt than on Fo (Paired t-test, t = 3.286, df = 6, P = 0.017; Fig 1A 3 and 1B 3 ). The maximum amount of prey adults found over 36 h differed, but followed similar dynamics. For Bt, a maximum of about 1.19 adults was detected 4 h after release, compared to 0.59 Fo adults (Fig 2A 3 and 2B 3 ). Time after predator release had no significant effect on Bt consumption (P = 0.283) but a marginally significant one on Fo consumption (P = 0.052; Fig 2A 3 and 2B 3 ). Moreover, 9.2-58.5% more Bt was consumed over 36 h compared to Fo (Paired t-test, t = 3.080, df = 6, P = 0.022; Fig 2A 3

and 2B 3 ).
Three prey species together: Bt plus Fo plus Ag. Adult ladybirds readily attacked both invasive species even in this treatment. Up to 80% of predators contained invasive prey DNA, with some decrease 36 h after release but this reduction was not significant (Bt: P = 0.551, Fo: P = 0.318; Fig 1A 4 and 1B 4 ). The dynamics of percentage of predation on both invasive prey species over 36 h was similar (Fig 1A 4 and 1B 4 ). The maximum amount of prey consumed over 36 h differed between species but followed similar dynamics. For Bt, the maximum amount of prey was about 0.28 adults after 36 h, while that of Fo was 0.16, significantly less (Paired t-test, t = 9.257, df = 6, P < 0.001; Fig 2A 4 and 2B 4 ). Time after H. axyridis release had no significant effect on the consumption of either invasive prey (Bt: P = 0.324, Fo: P = 0.388).
Summary of prey consumption by H. axyridis. Percentage of predation: Neither prey species combination (P = 0.238) nor time (P = 0.253) had any significant effect on the percentage of H. axyridis preying on Bt, but the dynamics of predation were affected ( Table 2; Fig 1). However, presence of the native Ag did not influence predation on Bt in any combination (P = 0.111-0.457; Fig 1).
Prey consumption: There were significant effects of prey species combination (P < 0.001) and time after predator release (P = 0.018) on Bt consumption by H. axyridis (Table 2 and S1 Table; Fig 2A 1 -2A 4 ). The highest Bt consumption occurred when Bt was together with Fo (0.33-1.19 adults), followed by the Bt-Ag combination (0.23-0.44 adults). Bt consumption was the lowest (0.16-0.28 adults) when all three species were present. Thus, the presence of Fo triggered an increase in Bt consumption by H. axyridis (Table 2; Fig 2A 1 -2A 4 ), whereas no such effect was caused by Ag (P = 0.455; Fig 2A 1 and 2A 2 ).

Discussion
The data in our present study showed unequivocally that the presence of native prey does not disrupt predation by a native predator on exotic prey. H. axyridis still attacked both invasive species in the presence of (superfluous numbers of) native aphids. This was indicated by the results of both the paired and three-prey experiments in which ample prey remained at the end of all experiments, ruling out the possibility of preferred prey exhaustion. Although Berkvens et al. warn about possible bias when laboratory-reared predators are used in preference experiments [51], this potential source of bias can also be ruled out because we used field-collected H. aryridis adults. Because other species of whiteflies (e.g. T. vaporariorum) [52] and thrips (e.g. Th. tabaci, F. intonsa) [53] are native to China, it seems likely the predator may have pre-adapted to these prey types. Prey acceptance and nutritional value are often uncorrelated in coccinellids [54]. However, H. axyridis is highly polyphagous, and able to reproduce on many different kinds of prey species [51,[54][55][56], including aphids [51] and whiteflies [7,9]. We observed no mortality of H. aryridis adults in any of the experimental cages, so we assume all three preys were nutritionally adequate [56]. However, this is not to infer that predator development and reproduction on these prey species will be equivalent [54,56].  The duplex qPCR assay developed here should be useful in tracking the dynamics of control efficacy of native predators on two of the most economically important invasive pest species, B. tabaci MEAM1 and F. occidentalis. In order to get reliable quantification, the primer half-life must be determined. Whereas the half-life of primer and probe sets for B. tabaci MEAM1 in Propylea japonica (Thunberg) is known [9], DNA amplification in the H. axyridis gut is not, so the efficiency of the primers used here could have influenced our results. However, because both frequency of predation and quantification point to the same trends, we believe that the data are mutually supportive, even though strict quantification cannot be claimed, and thus the conclusion that H. axyridis prefers B. tabaci MEAM1 to F. occidentalis is valid.
Although there is no experimental evidence of differences in prey quality of these two species, the relative profitability of the two exotic prey species could differ because of their differences in size and escape abilities. Thrips are smaller, and they also have a higher surface: volume ratio, hence increasing the relative amount of indigestible chitin. Therefore, they are probably less profitable to the coccinellid predators than whiteflies. Additionally, there is a difference in prey vulnerability. B. tabaci MEAM1 eggs are laid on the abaxial surface of leaves and thus available to predators, while the eggs of F. occidentalis are inside the tender plant tissues, and are more protected. F. occidentalis nymphs can also jump, which makes them more difficult to catch than those of the immobile whitefly nymphs.
Why do H. axyridis adults continue to prey on inferior, novel prey in the presence of more preferred, native prey? One possible reason is nutritional complementation via dietary mixing [57], as observed in other invertebrate predators, including spiders [58] and carabids [59]. Predators under field conditions are usually hungry [60,61], thus a lack of discrimination, i.e. attacking whatever potential prey they find, may be effective due to simple parsimony. Coccinellids can display both a lack of discrimination (e.g. even consuming toxic prey) [62] and an ability to discriminate using kairomones [63]. Additional behavioral studies could clarify interactions with various prey types.
Indirect interactions between herbivores, mediated by natural enemies may vary in nature and time [64][65][66][67][68][69]. The effects of relative prey densities linked to landscape complexity and/or predator behavior, such as switching [66,70,71], could be developed in regards to the differences in predation between the two exotic species. Usually, these indirect interactions are positive for the prey at time scales shorter than the predator generation time because of shared predation pressure on multiple pests, but become negative at longer time scales because of the predator numerical response [65,72]. The lepidopteran invader, Tuta absoluta Meyrick had a similar effect on the local pest B. tabaci MED controlled by the mirid predator Macrolophus pygmaeus Rambur on tomato crops [73]. In our present study, time after predator release had no significant effect on the percentage of H. axyridis individuals preying on B. tabaci MEAM1 or F. occidentalis, although our time scale was short (36 h). In spite of this, time after predator release had significant effects on number of prey consumption with a reduced predation rate after eight hours in multiple treatments, which could be related to the feeding behavior of the ladybird beetle, e.g. ladybird beetles usually search actively during the day.
The results of this study indicate that certain invasive prey species can be incorporated into the prey range of this native predator even when preferred native prey are available. This is an example of biotic resistance to invasion (that is standard terminology in invasion ecology). Such resistance can be an important factor in the process of both plant [74] and vertebrate [10] invasions. Evidence of biotic resistance during invertebrate invasions has been found for other invasive pests, with signs of preadaptation by native predators [75][76][77][78]. Due to the increase of such invasions world-wide [79], quantification of such biotic resistance would greatly help our understanding, and managing invasions. H. axyridis is used as an effective control agent worldwide and may at the same time causing ecological damage [55]. Further work should be conducted so as to assess which conditions favor it as being an effective control agent (versus a potential pest). Moreover, larger scale studies to assess the potential of the native generalist predator to control biological invasions should be included.
Supporting Information S1 Table. One-way ANOVA (same time after predator release among different treatments) evaluating effects on percentage of predation and number of prey adults consumed by Harmonia axyridis on Bemisia tabaci MEAM1 (Bt) or Frankliniella occidentalis (Fo). (DOC)