Rapid Plant Identification Using Species- and Group-Specific Primers Targeting Chloroplast DNA

Plant identification is challenging when no morphologically assignable parts are available. There is a lack of broadly applicable methods for identifying plants in this situation, for example when roots grow in mixture and for decayed or semi-digested plant material. These difficulties have also impeded the progress made in ecological disciplines such as soil- and trophic ecology. Here, a PCR-based approach is presented which allows identifying a variety of plant taxa commonly occurring in Central European agricultural land. Based on the trnT-F cpDNA region, PCR assays were developed to identify two plant families (Poaceae and Apiaceae), the genera Trifolium and Plantago, and nine plant species: Achillea millefolium, Fagopyrum esculentum, Lolium perenne, Lupinus angustifolius, Phaseolus coccineus, Sinapis alba, Taraxacum officinale, Triticum aestivum, and Zea mays. These assays allowed identification of plants based on size-specific amplicons ranging from 116 bp to 381 bp. Their specificity and sensitivity was consistently high, enabling the detection of small amounts of plant DNA, for example, in decaying plant material and in the intestine or faeces of herbivores. To increase the efficacy of identifying plant species from large number of samples, specific primers were combined in multiplex PCRs, allowing screening for multiple species within a single reaction. The molecular assays outlined here will be applicable manifold, such as for root- and leaf litter identification, botanical trace evidence, and the analysis of herbivory

Amplification facilitators and multiplex PCR: Tools to overcome PCR-inhibition in DNA-gut-content analysis of soil-living invertebrates

Polymerase chain reaction (PCR) can be used to detect prey within the gut contents of predators and allows specific trophic interactions to be studied among soil-dwelling invertebrates which cannot be examined by other approaches. PCR-inhibitory substances, however, are commonly found in DNA prepared from soil organisms or from biological material contaminated with soil. This can lead to false-negative results and the risk of not detecting trophic connections or of underestimating predation rates in field studies. In the present study, we developed mitochondrial DNA markers to detect Amphimallon solstitiale (Coleoptera: Scarabaeidae) in the gut contents of invertebrate predators. Larvae of A. solstitiale can cause serious damage in grasslands, field crops, and forests by feeding on roots. Adequate methodologies to study predation on these pests are lacking, and their invertebrate predator guild is, therefore, barely known. To test the new molecular markers for prey detection, larvae and eggs of A. solstitiale were fed to Poecilus versicolor larvae (Coleoptera: Carabidae), which are abundant below-ground predators in grassland ecosystems. Unfortunately, even when specific DNA extraction and purification methods were used, DNA extracts from predators were of poor quality and not amplifiable by PCR; this yielded false-negative results and a dramatically lower prey-detection rate. We overcame PCR-inhibition by applying 1.28 μg μl−1 bovine serum albumin to the PCR reaction mix. This enabled us to detect A. solstitiale DNA within fed carabid larvae up to 48 and 40 h post-feeding for 127 and 463 bp sized DNA fragments, respectively. When single A. solstitiale eggs were consumed by the carabid larvae, predation could be verified in 100% of the predators within the first 8 h of digestion; some carabid larvae even tested positive 32 h after feeding. Moreover, by multiplexing primers targeting both prey and predator, we were able to simultaneously screen for prey consumption and check for co-purified PCR inhibitors. Sensitivity in prey detection was not reduced compared to singleplex PCR. We recommend the multiplex approach because it considerably reduces time and costs compared to singleplex assays. We also show that multiplex PCR not only detects specific prey, but also can identify the predator itself. This allows the identification of taxa which are difficult or not identifiable based on morphological characters, such as soil-dwelling predatory beetle larvae.Anita Juen and Michael Traugotthttp://www.elsevier.com/wps/find/journaldescription.cws_home/332/description#descriptio

Spatial distribution of epigaeic predators in a small field in relation to season and surrounding crops

The arthropod predator community of an organically cultivated 0.3 ha field was sampled by pitfall trapping during 1 year and analysed for small-scale spatial distribution patterns and their seasonal variation. Due to the small size of the study site, most of the investigated species were able to colonise the whole field. Ordination analyses revealed significant differences in community composition (i) between field centre and margin in summer and (ii) between field parts adjacent to meadows or conventionally cultivated arable land in all seasons. Results changed considerably if only adult carabids or spiders were used instead of the whole predator community. During summer the following significant differences in community parameters were found: (i) total catches in the centre were nearly twice as high as near the margin, (ii) predator richness did not differ between centre and margin while significantly more species were found close to meadows and (iii) evenness and heterogeneity were significantly higher at the margin versus the centre. The arthropod predator community even of a small field site showed distinct spatial distribution patterns with seasonal changes. Selecting appropriate taxonomic groups is crucial for the outcome of such studies and should be tailored to the aim of the investigation.Anita Juen and Michael Traugotthttp://www.elsevier.com/wps/find/journaldescription.cws_home/503298/description#descriptio

Detecting predation and scavenging by DNA gut-content analysis: a case study using a soil insect predator-prey system

The original publication can be found at www.springerlink.comWhite grubs (larvae of Coleoptera: Scarabaeidae) are abundant in below-ground systems and can cause considerable damage to a wide variety of crops by feeding on roots. White grub populations may be controlled by natural enemies, but the predator guild of the European species is barely known. Trophic interactions within soil food webs are difficult to study with conventional methods. Therefore, a polymerase chain reaction (PCR)-based approach was developed to investigate, for the first time, a soil insect predator-prey system. Can, however, highly sensitive detection methods identify carrion prey in predators, as has been shown for fresh prey? Fresh Melolontha melolontha (L.) larvae and 1- to 9-day-old carcasses were presented to Poecilus versicolor Sturm larvae. Mitochondrial cytochrome oxidase subunit I fragments of the prey, 175, 327 and 387 bp long, were detectable in 50% of the predators 32 h after feeding. Detectability decreased to 18% when a 585 bp sequence was amplified. Meal size and digestion capacity of individual predators had no influence on prey detection. Although prey consumption was negatively correlated with cadaver age, carrion prey could be detected by PCR as efficiently as fresh prey irrespective of carrion age. This is the first proof that PCR-based techniques are highly efficient and sensitive, both in fresh and carrion prey detection. Thus, if active predation has to be distinguished from scavenging, then additional approaches are needed to interpret the picture of prey choice derived by highly sensitive detection methods

Earthworm primers for DNA-based gut content analysis and their cross-reactivity in a multi-species system

Specific primers can be used in polymerase chain reactions (PCR) to amplify prey DNA from the gut content of generalist predators at high specificity and sensitivity. A prerequisite for applying this approach to field studies, however, is to confirm that primers are actually targeting specific prey species or prey groups and do not produce false positive results by amplifying DNA either from predator species or from the wide range of potential alternative prey found under natural conditions. Here, we report on a new group-specific primer pair for earthworms designed from cytochrome oxidase c subunit 1 (COI) sequences of 11 earthworm species found in Central Europe that can be used to detect consumption of earthworms by invertebrate predators. Besides inter-specific also considerable intra-specific variation was found for COI sequences among most of the earthworm species. We, therefore, combined a universal forward primer with an earthworm-specific reverse primer which amplified a 523 bp product from all 11 species tested. Earthworm DNA amplification was also successful in the presence of excess DNA of a predator species. The primer pair was tested against 82 non-target invertebrate species commonly found in the same habitats, including potential prey for generalist predators and predators themselves. The earthworm primer was highly specific: only one of the non-target species showed a product of similar length as the earthworms, whereas PCR with 12 non-target species produced amplicons whose length differed from that of earthworms. We conclude that the new primer will be a useful tool to investigate the role earthworms play as a food resource in soil food-webs. Moreover, we suggest that future studies utilizing DNA-based approaches for prey detection should select non-target species for cross-reactivity tests according to their abundance and importance rather than choosing representatives of taxonomic units; this will help validate the results achieved using species- or group-specific primers and guarantee their meaningful ecological interpretation.Belayneh Admassu, Anita Juen and Michael Traugotthttp://www.elsevier.com/wps/find/journaldescription.cws_home/332/description#descriptio

Results of a 4-year plant survey and pitfall trapping in Bt maize and conventional maize fields regarding the occurrence of selected arthropod taxa

In the Oderbruch region, Germany, an outbreak area of the European corn borer (Ostrinia nubilalis), we investigated the density and community composition of arthropods in Bacillus thuringiensis (Bt) and conventional (CV) maize fields from 2000 to 2003. Aphids, thrips, bugs and aphid predators on the plants were counted to obtain density estimates. Community structure of arthropod taxa on plants taken from field as well as ground-dwelling carabids caught in pitfall traps was investigated by ordination analysis. The density of most taxa varied considerably between the years. We detected a few significant differences between Bt and CV maize, but no specific tendencies. However, the ordination analyses revealed a small but significant influence of maize variety on the arthropod community. The percentage of total variance explained by maize variety was only 2.7% in ground dwellers and 2.1% in maize dwellers. The present data show that the influence of Bt maize on nontarget arthropods is small compared to field characteristics and yearly changing environmental conditions

Impact of wildflower strips on biological control of cabbage lepidopterans

In a 2-year experiment we investigated whether wildflower strips can be used to enhance the control of cabbage moth, Mamestra brassicae L., and cabbage white butterfly, Pieris rapae L. At two sites, including six organically cultivated fields, M. brassicae egg parasitism and predation rates were determined along with an assessment of larval parasitism rates in M. brassicae and P. rapae using a DNA-based approach. Within each field, plots with and without wildflower strips were sampled and a grid design of 3 m x 3 m was used to analyse the spatial pattern of parasitism. The provision of wildflower strips provided an idiosyncratic effect on the control of lepidopterans: parasitism rates in M. brassicae eggs and larvae were not affected, whereas parasitism rates of larval P. rapae were significantly enhanced by the wildflower strips at one of the two sites. Moreover, at one site predation rates on M. brassicae eggs were significantly enhanced in the wildflower strip plots. Geostatistical analysis showed no distinct spatial patterns in parasitism rates. These results demonstrate that the provision of wildflower strips does not necessarily enhance biological control of lepidopteran cabbage pests and suggest that site-specific environmental factors strongly affect the impact of wildflower strips. (c) 2008 Elsevier B.V