Functional response and predation of Nabis kinbergii （Hemiptera. Nabidae） to Plutella xylostella （Lepidoptera. Plutellidae）

The functional response of adult Nabis kinbergii （Hemiptera： Nabidae） to density of diamondback moth Plutella xylostella （Lepidoptera： Plutellidae） was investigated under laboratory conditions. Holling＇ s （1959） type Ⅱ model was found to be a good fit for the observed functional response of this predator. The numbers of P. xylostella consumed increased with temperature from 15℃ to 35℃. The maximum number of prey killed was observed at 35℃, with average of 10.3 and 8.3 forth instar larvae consumed by adult females and males of N. kinbergii, respectively. The predation of N. kinbergii on P. xylostella increased with successive immature stages. The number of prey consumed by predators decreased as the body size of prey increased. An average of 131 eggs or 95 larvae of P. xylostella were killed by a single of female adult in 24 hours at 24＂C. The pupae of P. xylostella were observed to be eaten by fifth instar nymphs and adults N. kinbergiiin numbers of less than an average of 0.7 pupae per predator in 24 hours at 24＂C. Predation preference by N. kinbergii was also investigated. The number of P. xylostella and Myzus persicae killed by female N. kinbergii was not significantly different, but males killed significantly more P. xylostella than M. persicae. Both eggs and larvae of P. xylosteUa were killed in significantly greater number than those of Pieris rapae in the same feeding arena.     

A comparative study on the efficacy of a pest-specific and prey-marking enzyme-linked immunosorbent assay for detection of predation

The efficacy of two different antigen–antibody combinations to detect predation on eggs of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) was compared. The first method was an indirect enzyme‐linked immunosorbent assay (ELISA) using monoclonal antibody‐based gut content analysis that detects H. armigera egg protein. The second method was a sandwich ELISA that detects an exotic protein [rabbit immunoglobulin G (IgG)] applied as an external marker to H. armigera eggs. The target predators were the predatory beetles Dicranolaius bellulus (Guerin‐Meneville) (Coleoptera: Melyridae) and Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae). Beetles were fed with H. armigera eggs that had been marked with rabbit IgG and then held at various intervals after prey consumption. Each individual beetle was then assayed by both ELISA techniques to identify the prey remains in their guts. The two ELISA methods were further tested on field‐collected predators. Specifically, protein‐marked egg masses were strategically placed in a cotton field. Then, predators from surrounding cotton plants were collected at various time intervals after the marked eggs were exposed and assayed by both ELISAs to detect the frequency of predation on the marked eggs. The rabbit IgG‐specific sandwich ELISA had a higher detection rate than the H. armigera‐specific indirect ELISA under controlled and field conditions for both predator species. Moreover, a greater proportion of field‐collected D. bellulus tested positive for predation than H. variegata. The advantages and disadvantages of using prey‐marking ELISAs instead of pest‐specific ELISA assays are discussed.     

Detection and decay rates of prey and prey symbionts in the gut of a predator through metagenomics

DNA methods are useful to identify ingested prey items from the gut of predators, but reliable detection is hampered by low amounts of degraded DNA. PCR‐based methods can retrieve minute amounts of starting material but suffer from amplification biases and cross‐reactions with the predator and related species genomes. Here, we use PCR‐free direct shotgun sequencing of total DNA isolated from the gut of the harlequin ladybird Harmonia axyridis at five time points after feeding on a single pea aphid Acyrthosiphon pisum. Sequence reads were matched to three reference databases: Insecta mitogenomes of 587 species, including H. axyridis sequenced here; A. pisum nuclear genome scaffolds; and scaffolds and complete genomes of 13 potential bacterial symbionts. Immediately after feeding, multicopy mtDNA of A. pisum was detected in tens of reads, while hundreds of matches to nuclear scaffolds were detected. Aphid nuclear DNA and mtDNA decayed at similar rates (0.281 and 0.11 h^?1 respectively), and the detectability periods were 32.7 and 23.1 h. Metagenomic sequencing also revealed thousands of reads of the obligate Buchnera aphidicola and facultative Regiella insecticola aphid symbionts, which showed exponential decay rates significantly faster than aphid DNA (0.694 and 0.80 h^?1, respectively). However, the facultative aphid symbionts Hamiltonella defensa, Arsenophonus spp. and Serratia symbiotica showed an unexpected temporary increase in population size by 1–2 orders of magnitude in the predator guts before declining. Metagenomics is a powerful tool that can reveal complex relationships and the dynamics of interactions among predators, prey and their symbionts.     

Ranking common predators of Bemisia tabaci in Georgia (USA) agricultural landscapes with diagnostic PCR: implications of primer specific post-feeding detection time

Developing a successful biological control program relies on understanding predator–prey interactions in agroecosystem field settings. Among several methods used, molecular gut content analysis (MGCA) has become a popular method to measure predator contributions to pest control services. Once MGCA is applied to diagnose predator–prey interactions, the DNA detectability half-life is often applied to adjust for differences in prey digestion time among predators. Although MGCA best practices are well established, with many primers available, further work is needed to rank among published primers for MGCA. Using a combination of laboratory feeding trials and application of diagnostic MGCA to field collected predators, we investigated Bemisia tabaci post-feeding detection times in three dominant predator functional groups (chewing, piercing/sucking, and spiders). This was based on three published B. tabaci-specific primers. These data reveal that primer choice generated significantly different B. tabaci DNA half-lives in predator gut content. The primers with longer half-life resulted in higher field predation frequency estimation. Our field data using the primer with the longest half-life suggest several abundant predators, including Hippodamia convergens, Geocoris punctipes, Orius spp., Thomisidae spider, and fire ants (Solenopsis invicta), are actively feeding on B. tabaci in cotton fields. Orius spp. and fire ants were the most abundant predator species in our study area and contributed the most to B. tabaci control. Our results suggest that primers can be classified based on their specific DNA half-lives and can be used to address different ecological questions such as how to study time-specific predation detection (nocturnal or diurnal).

     

Foraging responses of Coccinella septempunctata,Hippodamia variegata and Chrysoperla carnea to changing in density of two aphid species

The successful use of predators in classical biocontrol programmes needs several background laboratory investigations, one of which is the evaluation of predator behavioural responses to changes in the density of their prey. The impact effect of the density of two prey species [Myzus persicae Sulzer and Aphis craccivora Koch (Hemiptera: Aphididae)] on the predation rates of third-instar Chrysoperla carnea Stephens (Chrysopidae: Neuroptera) and fourth-instar Coccinella septempunctata L. and Hippodamia variegata Goeze (Coccinellidae: Coleoptera) larvae was studied. Although prey species, predator species, prey density, and their interactions all had significant effects on the numbers of aphids consumed, the type of functional response did not vary, remaining a type II response in all treatments. However, the type II parameters differed among predator species on the same prey species, and for each predator species on the two prey species. Chrysoperla. carnea on M. persicae and H. variegata on A. craccivora were more voracious than other predators. In the context of functional response and biological control, the release of these predators, that show inverse density-dependent mortality, has to be started in early season to build up their population on low aphid densities and attack later high aphid populations.     

Feeding mode and prey detectability half-lives in molecular gut-content analysis: an example with two predators of the Colorado potato beetle

The time during which prey remains are detectable in the gut of a predator is an important consideration in the interpretation of molecular gut-content data, because predators with longer detectability times may appear on the basis of unweighted data to be disproportionately important agents of prey population suppression. The rate of decay in detectability, typically expressed as the half-life, depends on many variables; one that has not been explicitly examined is the manner in which the predator processes prey items. The influence of differences in feeding mode and digestive physiology on the half-life of DNA for a single prey species, the Colorado potato beetle Leptinotarsa decemlineata (Say), is examined in two predators that differ dramatically in these attributes: the pink ladybeetle, Coleomegilla maculata (DeGeer), which feeds by chewing and then ingesting the macerated material into the gut for digestion; and the spined soldier bug, Podisus maculiventris (Say), which physically and enzymatically processes the prey extra-orally before ingestion and further digestion in the gut. In order to standardize the amount of DNA consumed per predator, a single L. decemlineata egg was used as the prey item; all predators were third instars. The PCR assay yields estimated prey DNA half-lives, for animals maintained under field temperatures, of 7.0 h in C. maculata and 50.9 h in P. maculiventris. The difference in the prey DNA half-lives from these two predators underscores the need to determine detectabilities from assemblages of predators differing in feeding mode and digestive physiology, in order to weight positives properly, and hence determine the predators' relative impacts on prey population suppression.     

Molecular gut-content analysis of a predator assemblage reveals the effect of habitat manipulation on biological control in the field

Habitat manipulation in agroecosystems can influence predator–prey interactions. In this study, we collected foliar predators from field potato plots with different mulch treatments and assayed them for DNA of the target prey, Leptinotarsa decemlineata (Say), using species-specific primers. Concurrently, L. decemlineata larval abundance and plant damage were recorded from the same plots. Predator species abundance and diversity were not influenced by habitat manipulation, while prey density was highest in plots without mulch. Gut-content analysis revealed that the highest incidence of predators positive for L. decemlineata DNA was in plots without mulch, where target prey abundance was highest. Therefore, the lower prey abundance in mulched plots was not due to predation. The most abundant species in the predator assemblage was Coleomegilla maculata, which had the lowest proportion of L. decemlineata DNA in the gut. Podisus maculiventris, Perillus bioculatus, and Lebia grandis were less abundant but had a higher incidence of target prey DNA in the gut. DNA detectability half-lives were used to adjust for inter-specific variation in DNA digestive rates of the four predator species. Using this information to adjust actual number of positives for prey DNA, we compared proportions positive for L. decemlineata and found that P. maculiventris is the most effective predator species in the complex.     

A new method for immunologically marking prey and its use in predation studies

We introduce a new method for immunologically examining predator gut contents. It differs from previously described gut content analyses because it does not require the development of prey-specific antibody probes. Instead, insect prey were marked with a readily available antigen, rabbit immunoglobulin G (IgG). We then assayed predators that had fed on IgG labeled prey with an enzyme-linked immunosorbent assay (ELISA) using goat anti-rabbit IgG. Of the predator species that fed on the IgG labeled prey, 98.8% of those with chewing mouthparts scored positive for IgG 1 h after feeding. Our prey-labeling ELISA was less efficient for detecting IgG prey remains in predators with piercing/sucking mouthparts. Only 29.5% of these individuals scored positive for rabbit IgG in their guts 1 h after feeding. An additional study was conducted to measure the retention time of IgG-labeled prey in the guts of two species of predators with chewing mouthparts. Results from this experiment showed that the retention time varied depending on the predator and prey species examined. Results from these studies indicate that this marking technique could have widespread use for analyzing the gut contents of predators with chewing mouthparts, but it has limited application for those predators with piercing/sucking mouthparts. This article presents the results of research only. Mention of a proprietary product does not constitute an endorsement or recommendation for its use by the USDA.     

Linking habitat complexity with predation of pests through molecular gut-content analyses

Improving the diversity of farm systems or landscapes can lead to more effective biological control by providing refuge and alternative resources for colonising natural enemies. Within an experimental cabbage agroecosystem, we examined the effects of habitat management (i.e. herbicide use and cover crops) on pest populations and predator community structure, and report one of the first studies on the trophic links in this system using molecular gut-content analysis. In response to herbicide and cover crop management treatments designed to create different levels of habitat diversity, we quantified the abundance of two pests, Plutella xylostella Linnaeus (Lepidoptera: Plutellidae) and Pieris rapae Linnaeus (Lepidoptera: Pieridae), and predators. We designed species-specific primers to detect prey DNA in predators' guts. Pieris rapae were significantly more abundant in plots where cover crops were killed early in the season, and habitat management generated unique predator communities in response to weed management treatments. Thirty-five per cent of predators tested positive for prey DNA, and habitat management had interactive effects on predation of P. xylostella. Combined we found that habitat management has variable effects on natural enemy–pest interactions.     

Detection of Bemisia tabaci remains in predator guts using a sequence-characterized amplified region marker

Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) B biotype is an invasive species (biotype) in China. In order to understand the role that native natural enemies might play in its control, techniques were developed for detecting B. tabaci DNA within the gut of predators. A species-specific DNA fragment, ca. 350 bp, was identified by random amplified polymorphic DNA analysis. This fragment was absent in other closely related or co-occurring prey species, cotton, and other select predator species. After cloning and sequencing the fragment, one pair of sequence-characterized amplified region (SCAR) primers was developed, which amplified a single band of 240 bp. Specificity tests performed with the primers showed the presence of the 240-bp band for B. tabaci in all developmental stages and both sexes, in adult Propylaea japonica (Thunberg) (Coleoptera: Coccinellidae) fed on B. tabaci nymphs in the laboratory, and in predators collected in cotton fields. Following consumption of a single red-eyed B. tabaci nymph, prey DNA was detectable in 100% of P. japonica at t = 0, decreasing to 20% after 12 h of digestion, and no B. tabaci DNA detected at t = 24 h. In total, we analyzed the gut contents of 185 field-collected predators, representing four different orders. All nine field-collected predator species (namely, P. japonica, Harmonia axyridis, Scymnus hoffmanni, Coccinella septempunctata, Orius sauteri, Chrysopa pallens, Chrysopa formosa, Erigonnidium graminicolum, and Neoscona doenitzi) contained DNA from B. tabaci and are assumed predators of this pest insect. Overall, the B. tabaci was eaten by more than 50% of field-collected predator individuals, including larvae of the coccinellids (P. japonica and H. axyridis) and lacewings (C. pallens and C. formosa) and adults of O. sauteri and the spiders (E. graminicolum and N. doenitzi). There was a trend of a higher percentage of larval than adult coccinellids and lacewings that preyed on B. tabaci in the field. This study provides a framework for the future use of molecular gut content analysis in arthropod conservation ecology and food web research, with considerable potential for quantifying threats to invasive or endemic pest species in China and elsewhere.     

Relationship between predation by Podisus nigrispinus and developmental phase and density of its prey,Plutella xylostella

This study evaluated the predation by Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) at various densities of larvae and pupae of the pest Plutella xylostella (L.) (Lepidoptera: Plutellidae). We tested predator behavior of female P. nigrispinus at six experimental densities (1, 5, 10, 15, 20, or 25 prey items in a 1‐l transparent plastic container, replicated 15 times for each density) of both the fourth instar and pupae of P. xylostella. The number of prey consumed was monitored every 15 min for 12 h and was subsequently monitored at 24 h. Podisus nigrispinus females were weighed before and after the experiments to determine the effect of different densities of prey on their weight gain. Female predators had a Type‐II functional response, with attack rate estimated at 1.387 and 0.260 and a handling time of 0.091 and 0.183 h^?1 for larvae and pupae, respectively. Podisus nigrispinus consumed on average 10.9 larvae or 5.5 pupae in 24 h. Despite the similarity of the response type, P. nigrispinus preferred to feed on larvae, rather than on pupae.     

The influence of time and temperature on molecular gut content analysis： Adalia bipunctata fed with Rhopalosiphum padi

Gut content analysis is a useful tool when studying arthropod predator-prey interactions. We used polymerase chain reaction （PCR） technique to examine how detection of prey DNA in the gut content of predators was influenced by digestion time and temperature. Such knowledge is critical before applying PCR-based gut content analysis to field collected predators. Larvae of the two-spotted ladybeetle （Adalia bipunctata L.） were fed with the bird cherry-oat aphid （Rhopalosiphum padi L.） at either 21℃ or 14℃. After consuming one aphid, the predators were allowed to digest the prey for a range of time periods up to 24 hours. The influence of temperature on A. bipunctata feeding behavior was also recorded. From the fed larvae, total DNA was extracted and PCR reactions with R. padi specific primers were run. The number ofA. bipunctata that tested positive for R. padi DNA was negatively related to the length of digestion time. Temperature influenced larval feeding behavior but did not have a significant effect on R. padi DNA detection. After pooling the data from both temperature treatments we estimated the time point when R. padi DNA could be amplified from 50% of the fed A. bipunctata by PCR to be 4.87 hours. With such a rapid decrease in prey DNA detection success, positive PCR reactions will most likely be the result of predation events occurring shortly before capture. If a defined digestion temperature range has proven not to influence prey detection, PCR data obtained from predators collected within that particular range can be interpreted in the same way.     

The significance of facultative scavenging in generalist predator nutrition: detecting decayed prey in the guts of predators using PCR

Gut-content analyses using molecular techniques are an effective approach to quantifying predator-prey interactions. Predation is often assumed but scavenging is an equally likely route by which animal DNA enters the gut of a predator/scavenger. We used PCR (polymerase chain reaction) to detect scavenged material in predator gut homogenates. The rates at which DNA in decaying slugs (Mollusca: Pulmonata) and aphids (Homoptera: Aphididae) became undetectable were estimated. The detectability of DNA from both carrion types in the guts of the generalist predator Pterostichus melanarius (Coleoptera: Carabidae) was then determined. The effects of carrion age and weight, as well as beetle sex, on detection periods, were quantified. Laboratory trials measured prey preference of beetles between live and decaying prey. Further experiments measured, for the first time, feeding by P. melanarius on dead slugs and aphids directly in the field. In both field and laboratory, P. melanarius preferentially fed on dead prey if available, but preference changed as the prey became increasingly decayed. Disappearance rates for slug carrion in wheat fields and grasslands were estimated and P. melanarius was identified as the main scavenger. Comparison of the retention time for dead slugs in the field, with the detection period for decaying slug material in the guts of the predators, showed that PCR-based techniques are not able to distinguish between predated and scavenged food items. This could potentially lead to overestimation of the impact of predation on slugs (and other prey) by carabids. Possible implications of facultative scavenging by invertebrate predators for biocontrol and food-web research are discussed.     

Influence of intraguild predation between Episyrphus balteatus and Hippodamia variegata on their prey

Intraguild predation (IGP) is an interaction that frequently occurs in natural enemy communities, especially aphidophagous predators. This research investigated IGP intensity between Episyrphus balteatus De Geer (Diptera: Syrphidae), with Hippodamia variegata Goeze (Coleoptera: Coccinellidae). Five predator combinations including second and third larvae of H. variegata and third instar larvae of E. balteatus plus control treatment (totally six treatments) were tested. The effect of IGP on cotton aphid, Aphis gossypii Glover (Hemiptera: Aphididae) population density was investigated on sweet pepper seedlings under laboratory microcosms. In most combinations, the third instar larvae of E. balteatus alone reduced an A. gossypii population more efficiently than ladybird larvae and their combinations. Furthermore, IGP between third instar of E. balteatus and second larvae of H. variegata was asymmetrical; second instar H. variegata larvae were always the intraguild prey for third instar E. balteatus. The obtained result showed that outcome of IGP interaction on cotton aphid density was non-additive.     

Comparing three types of dietary samples for prey DNA decay in an insect generalist predator

The rapidly growing field of molecular diet analysis is becoming increasingly popular among ecologists, especially when investigating methodologically challenging groups, such as invertebrate generalist predators. Prey DNA detection success is known to be affected by multiple factors; however, the type of dietary sample has rarely been considered. Here, we address this knowledge gap by comparing prey DNA detection success from three types of dietary samples. In a controlled feeding experiment, using the carabid beetle Pterostichus melanarius as a model predator, we collected regurgitates, faeces and whole consumers (including their gut contents) at different time points postfeeding. All dietary samples were analysed using multiplex PCR, targeting three different length DNA fragments (128, 332 and 612 bp). Our results show that both the type of dietary sample and the size of the DNA fragment contribute to a significant part of the variation found in the detectability of prey DNA. Specifically, we observed that in both regurgitates and whole consumers, prey DNA was detectable significantly longer for all fragment sizes than for faeces. Based on these observations, we conclude that prey DNA detected from regurgitates and whole consumers DNA extracts are comparable, whereas prey DNA detected from faeces, though still sufficiently reliable for ecological studies, will not be directly comparable to the former. Therefore, regurgitates and faeces constitute a useful, nonlethal source for dietary information that could be applied to field studies in situations when invertebrate predators should not be killed.     

Consumption of cereal leaf beetle,Oulema melanopus,by generalist predators in wheat fields detected by molecular analysis

The cereal leaf beetle (CLB), Oulema melanopus L. (Coleoptera: Chrysomelidae), is a major pest of cereal crops that has recently been reported in western Canada. We developed a set of primers to detect CLB DNA in the gut of six common predator taxa in wheat fields: lady beetles (20 positives of 143 individuals), nabid bugs (73 positives of 206 individuals), and wolf spiders (2 positives of 25 individuals). Nabis americoferus Carayon (Hemiptera: Nabidae) and Coccinella septempunctata L. (Coleoptera: Coccinellidae) were the most abundant predators in cereal fields, with 0.35 and 0.05 proportion of samples positive for CLB DNA, respectively. The prey DNA half-lives were used to adjust the estimates for N. americoferus to 0.22, due to its longer DNA detectability relative to C. septempunctata. Overall, Hippodamia parenthesis (Say) (Coleoptera: Coccinellidae) had the highest proportion of positives at 0.43. There was a positive association between CLB abundance and proportion of N. americoferus and C. septempunctata positives for CLB DNA. This study highlights the contribution of generalist predators to CLB mortality and their important role in integrated management for CLB. Furthermore, we provide a molecular tool that can be used to identify predators of CLB and predation frequency in agricultural fields .     

Gut content examination of the citrus predator assemblage for the presence of Homalodisca vitripennis remains

A two-year study was conducted in a citrus orchard, Citrus sinensis L., to determine frequency of predation on glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar). A total of 1,578 arthropod predators, representing 18 taxa, were collected and assayed for the presence of GWSS egg protein by an enzyme-linked immunosorbent assay using a Homalodisca-species and egg-specific monoclonal antibody and then by polymerase chain reaction using a H. vitripennis-specific DNA marker. The gut content analyses revealed the presence of GWSS remains in the gut of 2.28 % of the total arthropod predator population, with 3.09 % of the spiders and 0.59 % of the insect predators testing positive. Moreover, a comparison of the two assays indicated that they were not equally effective at detecting GWSS remains in predator guts. Low frequencies of GWSS detection in the gut of predators indicated that GWSS are not a primary prey and that predators may contribute little to suppression of this pest in citrus.     

Disentangling mite predator‐prey relationships by multiplex PCR

Gut content analysis using molecular techniques can help elucidate predator‐prey relationships in situations in which other methodologies are not feasible, such as in the case of trophic interactions between minute species such as mites. We designed species‐specific primers for a mite community occurring in Spanish citrus orchards comprising two herbivores, the Tetranychidae Tetranychus urticae and Panonychus citri, and six predatory mites belonging to the Phytoseiidae family; these predatory mites are considered to be these herbivores’ main biological control agents. These primers were successfully multiplexed in a single PCR to test the range of predators feeding on each of the two prey species. We estimated prey DNA detectability success over time (DS₅₀), which depended on the predator‐prey combination and ranged from 0.2 to 18 h. These values were further used to weight prey detection in field samples to disentangle the predatory role played by the most abundant predators (i.e. Euseius stipulatus and Phytoseiulus persimilis). The corrected predation value for E. stipulatus was significantly higher than for P. persimilis. However, because this 1.5‐fold difference was less than that observed regarding their sevenfold difference in abundance, we conclude that P. persimilis is the most effective predator in the system; it preyed on tetranychids almost five times more frequently than E. stipulatus did. The present results demonstrate that molecular tools are appropriate to unravel predator‐prey interactions in tiny species such as mites, which include important agricultural pests and their predators.     

Detecting frogs as prey in the diets of introduced mammals: a comparison between morphological and DNA‐based diet analyses

Amphibians are currently the most threatened group of vertebrates worldwide, and introduced fauna play a major role in their decline. The control of introduced predators to protect endangered species is often based on predation rates derived from diet studies of predators, but prey detection probabilities using different techniques are variable. We measured the detectability of frogs as prey, using morphological and DNA‐based diet analyses, in the stomachs and faeces of four mammal species that have been introduced to many areas of the world. Frogs (Litoria raniformis) were fed to rats (Rattus norvegicus and R. rattus), mice (Mus musculus) and hedgehogs (Erinaceus europaeus). DNA‐based analysis outperformed morphological analysis, increasing the prey detection rate from 2% to 70% in stomachs and from 0% to 53% in faeces. In most cases, utilizing either stomachs or faeces did not affect the success of prey DNA detection; however, using faeces extended the detectability half‐life from 7 to 21 h. This study is the first to measure prey DNA detection periods in mammalian stomachs, and the first to compare prey DNA detection periods in the stomachs and faeces of vertebrates. The results indicate that DNA‐based diet analysis provides a more reliable approach for detecting amphibians as prey and has the potential to be used to estimate the rate of predation by introduced mammals on endangered amphibians.     

Unnecessary roughness? Testing the hypothesis that predators destined for molecular gut‐content analysis must be hand‐collected to avoid cross‐contamination

Molecular gut‐content analysis enables detection of arthropod predation with minimal disruption of ecosystem processes. Mass‐collection methods, such as sweep‐netting, vacuum sampling and foliage beating, could lead to regurgitation or rupturing of predators along with uneaten prey, thereby contaminating specimens and compromising resultant gut‐content data. Proponents of this ‘cross‐contamination hypothesis’ advocate hand‐collection as the best way to avoid cross‐contamination. However, hand‐collection is inefficient when large samples are needed, as with most ecological research. We tested the cross‐contamination hypothesis by setting out onto potato plants immature Coleomegilla maculata and Podisus maculiventris that had been fed larvae of either Leptinotarsa decemlineata or Leptinotarsa juncta, or unfed individuals of these predator species along with L. decemlineata larvae. The animals were then immediately re‐collected, either by knocking them vigorously off the plants onto a beat cloth and capturing them en masse with an aspirator (‘rough’ treatment) or by hand‐searching and collection with a brush (‘best practice’). Collected predators were transferred in the field to individual vials of chilled ethanol and subsequently assayed by PCR for fragments of cytochrome oxidase I of L. decemlineata and L. juncta. Ten to 39 per cent of re‐collected fed predators tested positive by PCR for DNA of both Leptinotarsa species, and 14–38% of re‐collected unfed predators contained L. decemlineata DNA. Overall levels of cross‐contamination in the rough (31%) and best‐practice (11%) samples were statistically different and supported the cross‐contamination hypothesis. A pilot study on eliminating external DNA contamination with bleach prior to DNA extraction and amplification gave promising results.