African Journal of Agricultural Research Vol. 7(23), pp. 3473-3484, 19 June, 2012 Available online at http://www.academicjournals.org/AJAR DOI: 10.5897/AJAR11.2073 ISSN 1991-637X ©2012 Academic Journals

Full Length Research Paper

Detection of predators within Brassica crops: A search for predators of diamondback (Plutella xylostella) and other important pests

Reza Hosseini1*, Otto Schmidt2 and Michael A. Keller2

1Department of Plant Protection, College of Agriculture, University of Guilan, P. O. Box 41635-1314, Rasht, Iran. 2Discipline of Plant and Food Science, School of Agriculture, Food and Wine, Waite Campus, University of Adelaide, SA 5005, Australia.

Accepted 28 March, 2012

Techniques based on the Polymerase Chain Reaction (PCR) have been shown to be powerful tools for ecological studies of predator-prey interactions. By using developed species-specific primers from the cytochrome oxidase subunit I (COI) gene for six pests of Brassica crops (Plutella xylostella, Pieris rapae, hydralis, Helicoverpa punctigera, Brevicoryne brassicae, and Myzus persicae) trophic relationships of selected predators and their prey in Brassica fields demonstrated the potential of DNA-based techniques to screen predator communities and to identify their prey. In this investigation, all examined predators including kinbergii (Heimptera: ), Oechalia schellenbergii (: ), Micromus tasmaniae (Neuroptera: Hemerobiidae), Hippodamia variegata, Coccinella transversalis (Coleoptera: Coccinellidae) and four wolf spider species including Trochosa expolita, Venatrix pseudospeciosa, Venator spenceri and Hogna kuyani (Araneae: Lycosidae) showed polyphagy to some extent. All tested positive for P. xylostella, hence can be considered as predators of this pest. Although, the records of DNA from the guts of predators probably represent instances of real predation, but interpretation of predation data because of some errors is very difficult. There are limitations for this type of interpretation which has been comprehensively discussed in this paper.

Key words: Molecular markers, predator, gut contents, Brassica crop, biological control.

INTRODUCTION

The value of predators in the biological control of insect of predators on pest populations and especially critical in pests in integrated pest management programs has been evaluating the effectiveness of a predator as a biological highlighted by many investigators (Hagler and Durand, control agent (Hayes and Lockley, 1990). There are 1994; Symondson et al., 2002). Identification of trophic inherent difficulties associated with the study of the diet relationships between predators and particular pests is breath of predators in nature because of the relatively one of the first important steps in determining the impact small size of both predator and prey, and their often elusive and nocturnal activity (Greenstone, 1996; Hoogendoorn and Heimpel, 2001). Consequently, there is a lack of knowledge about predators’ feeding behaviour *Corresponding author. E-mail: [email protected], in the natural environment; because of this difficulty, little [email protected]. Fax: +98-131-6690281. is known about predator-prey trophic interactions and the

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effects of predators on pests of Brassica crops. However, Studies showed that Brassica crops have a rich fauna of to elucidate the role of predators in the control of these predators. Schmaedick and Shelton (2000) have pests, some experiments have been done under documented a list of predators associated with P. rapae laboratory conditions or in field cages (Schmaedick and in cabbage fields of New York State. Hosseini et al. Shelton, 1999), but these studies do not necessarily (2006b) reported a range of predators associated with accurately simulate field conditions. So far, the most Brassica pests in South Australia. The impact of effective and least disruptive method for studying predators on pests of Brassica crops has not been predation has been the development of biochemical thoroughly studied and therefore their potential in diagnostic technologies, monoclonal and polyclonal suppression of major pests of Brassica has not been antibodies (Symondson et al., 2002) and enzyme- elucidated. Hooks et al. (2003) found broccoli plants electrophoresis (Traugott, 2003) can be used to protected by birds and spiders as predators sustained determine which prey has been consumed by a predator. less damage from caterpillars and the plants had greater However, amplification of specific prey DNA using the productivity compared to control plants. Cage exclusion polymerase chain reaction has proven to be more experiments have shown that predation varies practical and cost-effective in detecting prey remains. considerably in space and time. Once it is developed, this assay can be used to Wang et al. (2004) estimated between 7 and 81% of efficiently and sensitively test large numbers of field- immature stages of P. xylostella were lost due to collected predators for evidence of feeding on a particular predation on farms in Queensland, Australia. prey species, such as key agricultural pest (Symondon, Subsequently, more extensive research (Furlong et al., 2002). PCR-based techniques are rapidly replacing other 2004) confirmed estimated losses of P. xylostella to molecular techniques because molecular biology facilities predation of the same magnitude, between 2 and 85%. A are widely available and prey-specific primers can be better understanding of the identities of key predators used in different contexts such as ecological, taxono- and factors that affect their feeding activities could enable mical, behavioral study of (Hoy, 2003) once they farmers and pest managers to conserve key predators. have been designed. This technique has been This would ensure that they more consistently kill 80% or successfully used for detection of a variety of prey more of immature diamondback . Hence, for this remains in predators’ gut contents (Zaidi et al., 1999; one insect, predators are known to cause considerable Chen et al., 2000; Hoogendoorn and Heimpel, 2001; mortality at times. It is essential to develop a reliable Agustí et al., 2003a, 2003b; Harper et al., 2005; Juen and technique to evaluate the diets of key predatory species Traugott, 2005; Read et al., 2006; Harwood et al., 2007; in order to understand their role in suppressing pests like Zhang et al., 2007; Kuusk and Agusti, 2008; Schmidt et P. xylostella. Ma et al. (2005) developed a species- al., 2009; Monzó et al., 2010). Brassica vegetables and specific marker for P. xylostella based on the internal oilseeds are economically important crops; approximately transcribed spacer (ITS-1) of the ribosomal gene. This 3.1 and 26.1 million ha respectively were grown specific primer pair was used to detect prey in the gut worldwide in 2004 (Food and Agriculture Organization of contents of two polyphagous predators, Nabis kinbergii the United Nations, 2009). These crops are associated and Trochosa expolita (reported as Lycosa sp.). In a pilot with several destructive and widespread insect pests. study of predation of diamondback moth on cauliflower Total damage caused by these pests is substantial; for and broccoli farms near Virginia, South Australia, the example, management costs for Plutella xylostella alone remains of P. xylostella were detected in the gut contents were estimated at US$ 1 billion annually in 1997 (Shelton of both species of field-collected predators. In the current et al., 1997). In Australia, the pests of Brassica crops study, cytochrome oxidase subunit I (COI) was selected include the lepidopterans P. xylostella, Pieris rapae, as a potentially diagnostic gene. As a mitochondrial gene, Hellula hydralis and Helicoverpa punctigera, and two it occurs as multiple copies in each cell (Hoy, 2003), aphids, Brevicoryne brassicae and Myzus persicae which increases the likelihood of successful amplification (Hosseini et al., 2006a). Integrated pest management of prey residues in the predators’ gut contents. This gene (IPM) systems and the use of biological control methods is a protein-coding gene that has a high level of are preferred approaches to controlling these pests over interspecific variability (Zhang and Hewitt, 1996), which insecticides due in part to the prevalence of insecticide allows closely related species to be separated. resistance in diamondback moth (Shelton et al., 1997). In the present study, a PCR-based technique was used Generalist predators can play a major role in the control for the first time to study predator-prey interactions of agricultural pests (Symondson et al., 2002); but this is extensively in Brassica crops. The aim of this research considered controversial (Rosenheim et al., 1993). reported in this paper was to determine the trophic

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relationships among predators and prey, mainly focusing primer pairs and 4 μl of DNA extract. The thermocycling program on the identification of predators of Brassica crops key consisted of an initial step of 2 min at 95°C, followed by 35 cycles of pest “diamondback moth” and other common pests. 30 s at 94°C, 30 s at the specific annealing temperature for each prey specific primer (Table 1), 1 min at 72°C and a final elongation step of 5 min at 72°C. PCR products were visualised following electrophoresis on 1.8% agarose gel in TAE containing 0.5 μl/ml MATERIALS AND METHODS ethidium bromide for DNA staining and then photographed. Each PCR assay had a positive (DNA of relevant species) and a negative Sampling predators (water) control. Percentage of positive and frequency of prey species detected were calculated for each predator species. Most predator samples (n = 437) were collected from a broccoli field on a commercial vegetable farm at Currency Creek, South Australia (35° 41´ S, 138° 75´ E). Predators were collected from unsprayed crop residues and regrowth. Presence and relative RESULTS abundance of lepidopteran phytophagus insects and two aphids were checked by direct observation on these plants. Another sample of wolf spiders (n = 82) only was collected in mixed field of Direct observations of plants on collected sites indicated broccoli and cabbage at Cudlee Creek, South Australia (34°84´ S, that all six Brassica pests were relatively abundant. 138° 85´ E). Wolf spiders were collected at night (7 to 9 PM) by Abundant predatory insect species that were collected by searching with a headlamp among cultivated rows, under leaves insect net and vacuum sampler included N. kinbergii and litter, and on the soil surface around the bases of broccoli and (Heimptera: Nabidae), Oechalia schellenbergii cabbage plants. Collected specimens were put individually into a 5 ml vial. Other predators were collected from vegetation by an insect (Hemiptera: Pentatomidae), Micromus tasmaniae net or a vacuum sampler (Makita model RBL 250, Makita (Neuroptera: Hemerobiidae), Hippodamia variegata and Corporation, Japan), as the sampler walked randomly across the Coccinella transversalis (Coleoptera: Coccinellidae). field. All collected samples, including collection bags containing Among ground-dwelling predators, wolf spider species predators were kept chilled on ice until they were transferred to the were relatively abundant including Trochosa expolita, laboratory. Venatrix pseudospeciosa, Venator spenceri and Hogna Mostly, collected specimens were released in an insect cage (70 × 50 × 50 cm) and after separation and identification of selected kuyani (Araneae: Lycosidae). Result of molecular assays predators; each predator individually was kept in a 1.5 ml for each predator species is as follows: microcentrifuge tube at -80°C for subsequent molecular assay.

DNA extraction Nabis kinbergii

In order to avoid possible external contamination with prey N. kinbergii proved to be a polyphagus predator with a contents, before DNA extraction, each predator specimen individually was washed with washing buffer1 by the following wide range of prey. At least four prey species were found procedure; 1 ml of washing buffer was added to 1.5 ml in the gut contents of 54.6% of the tested specimens microcentrifuge containing predator sample and gently vortexed for (Figure 1). Among examined specimens, a high 5 min. Then, the tube was centrifuged for 2 min at 10000 rpm. After percentage had tested positive for the presence of the centrifugation, the supernatant was removed and washing was DNA of three lepidopteran pests (Figure 1). Overall, the repeated once more. Finally, from all collected predators, their results clearly demonstrated that N. kinbergii feeds on abdomens individually were treated using a DNA extraction method involving silica (Hosseini 2007). All extracted DNA were kept at - diamondback moth at high amount and five other pests 20°C for subsequent molecular tests. present in Brassica crops.

Molecular assay by PCR and analysis of predation Oechalia schellenbergii Extracted DNA from each predator was examined separately in PCR with already designed and tested specific primer pairs for DNA from all six pests was found in the gut contents of detection of six pests of Brassica crops (Table 1) (Hosseini et al., 2011). PCR conditions were as follows: amplification was O. schellenbergii, which indicates that it is a polyphagous performed in a 25 μl reaction volume containing 150 μM dNTPs predator (Figure 2). Molecular analysis of the gut (Fisher Scientific Inc., USA), 2 mM MgCl2 (Fisher Scientific Inc., contents of O. schellenbergii showed high frequency of USA), 2.5 μl of 10× reaction buffer, 1 U of Taq DNA polymerase positive tests for three lepidopteran species (Figure 2). (Biotech international Ltd., Australia), 0.4 μM each of the respective Moreover, like N. kinbergii, aphids are another source of food for O. schellenbergii, where 39% of tested

1 specimens were positive to B. brassicae. 969 μl H2O; 20 μl 5 M NaCl; 10 μl 1 M Tris-HCl pH 8.0; 1 μl 1 M MgCl2

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Table 1. Species-specific primer sequences designed from the COI mtDNA of six common pests of Brassica crops, optimal PCR annealing temperatures, amplification fragment sizes and GeneBank accession numbers for the COI gene fragments (Hosseini et al., 2011).

Species Primer name Sequence Annealing temperature (°C) Fragment size DBM-F-2 5-TGTTTATCCTCCTTTATCTTCA-3 58 293 P. xylostella DBM-R1-1 5-CTCCTGCAGGATCAAAGAAG-3

PR-F-1 5-AGTGTACCCCCCACTTTCTT-3 60 222 P. rapae PR-R-1 5-ACTGGTAATGATAATAGTAAAAGT-3

HP-F 5-CTCATGGAGGAAGATCTGTA-3 64 270 H. punctigera HP-R 5-CTCCTCCTCCAGCAGGAT-3

BB-F-1 5-TTGATTACTCCCTCCATCAC-3 60 307 B. brassicae BB-R 5-TCCAGCTAATACTGGGAGA-3

MP-F 5-TGATTATTACCACCCTCAT-3 58 247 M. persicae MP-R 5-TGGAAATAAAGGGATTTGG-3

HH-F-1 5-TGGTGGAAGATCAGTTGATC-3 58 200 H. hydralis HH-R-1 5-CTCCAGCTAATACTGGTAGT-3

Hippodamia variegata be an aphidophagus predator, although, almost was so low, the results were combined. Results 20.8% of all examined specimens (n = 24) were interestingly showed that more specimens were Although, aphids are often regarded as found positive to P. xylostella compared to 8.3% found positive to P. xylostella (44.9%) than aphids coccinellids preferred prey, this ladybird also for B. brassicae (Figure 4). Also, this species (16.3%) (Figure 5). The results reported here proved to be a generalist predator. Among all seems to be a voracious predator to P. xylostella, clearly show that M. tasmaniae feeds on both specimens examined (n = 58), 13.8 and 12% however, it is not clear which developmental and aphids and possibly other tested positive to P. xylostella and H. hydralis, stages of prey are preferred by these predators. . Nearly 2/5 of examined specimens respectively, while only 1.7% detected positive for show no positive signals for prey DNA (Figure 6), B. brassicae (Figure 3). which suggests possible feeding on non-prey food Micromus tasmaniae or other species. Compared to other predators, M.

tasmaniae showed a limited diet as 51% of Coccinella transversalis In this investigation, apart from 6 specimens that examined specimens were found feeding on one were larvae, the rest of examined specimens were prey only. As this species is commonly collected Similar to H. variegata, C. transverslais seems to adults. Because the number of collected larval

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Figure 1. Percentage detection of different prey species in N. kinbergii. Numbers beside bars indicate percentages.

Figure 2. Percentage detection of different prey species in O. schellenbergii. Numbers beside bars indicate percentages.

in Brassica crops, further study aimed at elucidating the Table 2. diet of this species is warranted.

DISCUSSION Lycosidae (4 species) A total of 519 predators belonging to 5 different insect Results for four species of Lycosids are summarized on species and 4 wolf spider species were screened for the

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Figure 3. Percentage detection of different prey species in H. variegata. Numbers beside bars indicate percentages.

Figure 4. Percentage detection of different prey species in C. transversalis. Numbers beside bars indicate percentages.

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Figure 5. Percentage detection of different prey species in M. tasmaniae. Numbers beside bars indicate percentages.

Figure 6. Frequency of prey species detected in different predators. Numbers beside bars indicate percentages.

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Table 2. Frequency of prey species detection rate in different wolf spider species collected from two fields (numbers in table indicate percentage).

Trochosa expolita Venator spenceri Venatrix pseudospeciosa Hogna kuyani Prey species Field 1 (n = 44) Field 2 (n = 2) Field 1 (n = 37) Field 2 (n = 8) Field 1 (n = 13) Field 2 (n = 71) Field 1 (n = N/A) Field 2 (n = 1) P. xylostella 63.6 100 59.4 87.5 38.4 35.2 - 0 H. hydralis 31.8 0 16.2 0 23 14 - 100 P. rapae 56.8 100 64.8 100 69.2 59.1 - 100 H.punctigera 38.6 0 10.8 0 7.7 1.4 - 0 B.brassicae 20.4 0 29.7 25 30.7 18.3 - 0 M. persicae 0 0 0 0 0 0 - 0

Field 1 = Currency Creek; Field 2 = Cudlee Creek.

presence of prey DNA in their guts (Table 3). 1982). They feed voraciously on insect crop pests schellenbergii is a polyphagus species. This Results showed that it was possible to identify the such as pea aphids Acyrthosiphon pisum (Harris), species has previously been reported to feed on a residues of all potential prey species in the gut diamondback moth P. xylostella L., Australian wide range of immature stages of moths, beetles, contents of the various predators crop mirid Sidnia kinbergi (Stal) (Siddique and sawflies and weevils (Mensah, 1999). In Australia collected from the field. The specificity of each Chapman, 1987), native bud worm Helicoverpa and New Zealand, this species is a predator of primer pair was shown previously (Hosseini et al., punctigera (Wallengren) (Awan, 1990) and P. several lepidopteran (Awan, 1990) and 2011), therefore, the DNA bands of the size rapae (Kapuge et al., 1987). Ma et al. (2005) used coleopteran pests (Edwards and Suckling, 1980). allocated for each candidate species was a PCR-based method for detection of specific assumed to be diagnostic for the correct species. DNA fragments to show that N. kinbergii collected Detection of prey was successfully done in from the field had the remains of P. xylostella in H. variegata various predators with different types of their gut contents. In their investigations, 67.6% of mouthparts. Predators include: examined specimens tested positive for the In this study we showed this species’ polyphagy presence of DNA from P. xylostella, while this behavior. H. variegata has been reported not only amount in this study was 93.8%. Results revealed as predator of Aphis gossypi Glover on cotton N. kinbergii that N. kinbergii could be an important predator of (Fan and Zhao, 1988), but also as a predator of aphids, where 71.5% of specimens tested positive noctuid larvae (Araya et al., 1997) and other This species like other members of this family of for B. brassicae (Figure 1). This clearly shows N. insects. Results revealed that about three insects, are thought to feed mostly on small kinbergii will vary in its capacity to capture and quarters of the total number of H. variegata invertebrates especially the eggs and larvae of consume different prey despite it being a specimens tested in this study were not positive to Lepidoptera (Cordingly, 1981). Our studies polyphagous predator. any of the six potential prey species. A possible showed that this potentially important predator is explanation for this result can be the consumption abundant in Brassica crops like other field and O. schellenbergii of non-prey food, such as pollen. Two horticultural crops, such as cotton (Mensah, observations support this hypothesis. Firstly, field 1999), soybean (Evans, 1985) and lucerne (Awan, Our results clearly demonstrated that O. observation showed that H. variegata was mostly

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Table 3. Summary of prey species detection in the gut contents of field-collected predators. (+ detected and - undetected).

Predator No. tested Prey P. xylostella H. hydralis P. rapae H. punctigera B. brassicae M. persicae N. kinbergii 130 O. schellenbergii 82 H. variegata 58 - - C. transversalis 24 - - M. tasmaniae 49 - - Lycosidae (4 species) 176 -

active on flowers and possibly feeding on pollen. For this However, it is also reported as an important predator of reason, almost all adults of H. variegata were collected eggs of Helicoverpa sp. in cotton (Mensah, 1999) and P. from older plants in flower. Secondly, entomophagous rapae (Kapuge et al., 1987). The results reported here insects, especially coccinellids are known to feed on non- were confirmed that M. tasmaniae feeds on both prey food including nectar, fungal spores, and prey lepidoptera and aphids and possibly other arthropods. products such as honeydew or pollen (Lundgren et al., 2005). This strategy may be adopted by some predators when prey is scarce or during specific life stages for their Lycosidae (4 species) growth and development. In the current study, it is possible that abundant non- Wolf spiders (Lycosids) were commonly found and prey food such as pollen may actually have reduced the collected from harvested broccoli crops and within the predation rate and consequently this species’ impact on crops from early to late in the growing season. This pests (Pfannenstiel and Yeargan, 2002). suggests they are active throughout the year. The result clearly demonstrated that wolf spiders are polyphagous predators. This has been shown in other studies which C. transversalis indicate that they have this ability to feed on a variety of pests (Nyffeler et al., 1994). Pearce et al. (2004) showed Transverse ladybird is a predaceous insect occurring in wolf spiders feed on H. armigera (Hubner). Bishop (1978) every region of Australia and is commonly found primarily observed lycosids feeding on cotton looper larvae and feeding on aphids in agricultural and horticultural fields rough bollworm larvae in cotton fields, and Kapuge et al. (Omkar Bind, 1993). The data showed positive signals for (1987) found 33.3% of tested field-collected lycosids three lepidopteran pests. In earlier investigations, it was were positive to P. rapae by immunological assays. also reported as one of the major predators feeding on Despite wolf spiders being ground active predators, eggs and young larvae of Helicoverpa punctigera surprisingly, a noticeable number of specimens tested Wallengern and H. armigera (Hubner) (Lepidoptera, positive to aphid residues (Table 2). In a study of Noctuidae) in Australian cotton fields (Evans, 2000) and arthropod predation on aphids in lucerne crop, 25% of P. rapae L. (Kapuge et al., 1987). It is not surprising that Lycosa sp. tested gave positive precipitin reactions using C. transversalis, like H. variegata, feeds on non-prey serological methods (Leathwick and Winterbourn, 1984). food, such as cotton nectar (Adjei-Maafo and Wilson, Another investigation also showed 20 to 24% of the diet 1983). This may account for the observation that more of lycosids consisted of aphids (Sunderland, 1988). than half of examined specimens (66.7%) were not Results reported here and other investigations clearly positive to any of the six prey species. demonstrated that lycosid spiders could be considered as predators of aphids. Results in this study showed that 23.4% of lycosids collected from Currency Creek were M. tasmaniae found positive to H. punctigera compared with only 1.21% in the examined lycosids collected from Cudlee Creek. Investigations by Leathwick and Winterbourn (1984) This difference in detection may have been due to showed 73% of M. tasmaniae were positive to aphid differences in the population density of H. punctigera in specific proteins using serological techniques, which the two different locations. support the view that this species is aphidophagus. As long as we do not know the density or even

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occurrence of all pest species in the field, the ranking of predators maybe able to eat or kill more pests compared predators for frequency of feeding on particular pests is to ground dwelling predators because more prey impossible. Laboratory observations showed that wolf populations are available for them, whilst ground dwelling spiders are highly aggressive predators and easily feed predators only rely on prey that have fallen or walked on other predators and even show cannibalism (Hosseini away from the crop (Losey and Denno, 1998). Therefore, et al., 2008). Therefore, some of the positive signals it is impossible to compare the rate of impact of these two could be due to cannibalism (wolf spiders consuming groups of predators with each other. Some species of target prey, which in turn were consumed by a bigger Coccinellidae and even wolf spiders have been reported wolf spider) or intraguild predation (non-wolf spider to scavenge on dead insects. Also, secondary predation predators consumed target prey, which in turn were was demonstrated to be a potential source of error consumed by a wolf spider). (Hosseini et al., 2008). Thus, a predator testing positive in a DNA-based assay does not necessarily indicate the role of it in pest population control. For comparison of Potential of PCR techniques in predator-prey ecology predators, the half-life of detection (the time of which half studies of the predators tested positive) may be used in an initial ranking of predators (Hosseini et al., 2008). For example, The current study demonstrated the usefulness of PCR- if more positive signals were observed for P. xylostella in based techniques towards development of a better wolf spiders compared to H. variegata (Figures 1 and 3), understanding of predator-prey ecology in Brassica crop it could be attributed to the longer retention time after ecosystems. In this investigation, for the first time, large feeding the target prey, because the half-life was longer numbers of field-collected predators were examined for for wolf spiders compared to H. variegata (Hosseini et al., residues of six pests of Brassica crops and the results of 2008). Therefore, if this value was available for all the study provided information on the predators’ diet predators, then comparison of the relative importance of ranges, especially P. xylostella. Study of predator-prey confirmed predators may become easier. trophic interactions in Brassica crops indicated that N. Molecular approaches used in post mortem gut analysis kinbergii, O. schellenbergii and Lycosids are abundant usually estimate the consumption rate of prey by and most of the selected prey species were found in their predators rather than the predation rate. It should be diets. However, ranking of predators in their ability to considered that consumption rate and predation rate are suppress particular pests is incomplete, because two different concepts. Study of consumption rate is differences among predator species in detection or being relevant for the energetic relationships of trophic positive to different prey species such as P. xylostella interactions among predators and prey, which has been could be influenced by many factors (Hosseini et al., investigated in this study, but predation rate is essential 2008), such as: 1) temperature, 2) time since feeding, 3) and applicable to pest control by predators. Currently, prey species, 4) prey size and developmental stage, 5) with the available technologies, it is impossible to prey availability or prey population density, 6) predator’s distinguish between large meals eaten a long time ago by species, behaviour, level of hunger and digestion rate, 7) a predator and small meals eaten more recently, because partial prey consumption or killing without consumption, both of these situations can provide the same signal. 8) scavenging, 9) secondary predation, and 10) factors Therefore, PCR-based techniques are basically like season, crop varieties, location, weather and many qualitative. As currently used methods are not able to others. The frequency of consumption of prey is likely to quantify the number or amount of prey consumed by depend on the abundance of each species in the field. predators, the development and evaluation of a method Since no data were collected for the densities of pests, based on qRT-PCR (Nejstgaard et al., 2007) to quantify differences in the frequency of consumption of different prey consumption might open a new window on the prey species could be due either to variable pest investigation of predation. Results obtained from this abundance or to preferences of the predators. study were essential in the determination of trophic Nocturnal or diurnal activity of predators is another interactions of predators and prey, but alone not sufficient important factor which should be considered for each to allow interpretation of predation data from the field. predator (Fewkes, 1961). Therefore, samples taken Overall, we are still in a long way from having reliable during the day are unlikely to be fully representative, and techniques for studying predator-prey food webs and to comparisons of data obtained at different times of the day get a complete view of their complex relationships in may be of limited value (Leatwick and Winterbourn, agroecosystems. Each available technique has its own 1984). Among predators, climbing or vegetation dwelling advantages and disadvantages. Therefore, it is advisable

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to combine results obtained by using more than one Edwards RB, Suckling DM (1980). Cermatulus nasalis and Oechalia method of study. In investigations of predator-prey schellenbergii (Hemiptera: Pentatomidae) as predators of eucalyptus tortoise beetle larvae, Paropsis charybdis (Coleoptera: interactions, at least one method should be used that has Chrysomelidae), in New Zealand. New Zealand Entomol. 7: 158-164. the least disruption of predators’ behaviour, which could Evans ML (1985). Arthropod species in soybeans in southeast be the identification of prey in the guts of predators. In Queensland (Australia). J. Aust. Entomol. Soc., 24: 169-177. conclusion, this study clearly has revealed the Evans EW (2000). Egg production in response to combined alternative foods by the predator Coccinella transversalis. Entomol. Exp. App., effectiveness of the DNA-based method for 94: 141-147. establishment of trophic interactions of predators and Fan GH, Zhao JF (1988). Functional response of Adonia variegata their prey in Brassica ecosystems. The results will enable (Goeze) (Coleoptera, Coccinellidae) to cotton aphids. Nat. Enem. Ins. future researchers to confidently identify predators that 10, 187-190. Fewkes DW (1961). Diel vertical movements in some grassland attack six pests of Brassica crops. This will open the way Nabidae (). Entomol. Month. Mag. 97: 128-130. for future studies on the assessment of predators as Food Agriculture Organization of the United Nations (2009). FAOSTAT: biological control agents. Core production data. faostat.fao.org [accessed 6 June 2009] Furlong MJ, Zuhua S, Shijan G, Yinquan L, Sheng LS, Zalucki M (2004). Quantitative evaluation of the biotic mortality factors affecting diamondback moth in south-east Queensland, Australia, pp. 185-193. 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