D. M. Glen

Rapid screening of invertebrate predators for multiple prey DNA targets.

DNA‐based techniques are providing valuable new approaches to tracking predator–prey interactions. The gut contents of invertebrate predators can be analysed using species‐specific primers to amplify prey DNA to confirm trophic links. The problem is that each predator needs to be analysed with primers for the tens of potential prey available at a field site, even though the mean number of species detected in each gut may be as few as one or two. Conducting all these PCRs (polymerase chain reactions) is a lengthy process, and effectively precludes the analysis of the hundreds of predators that might be required for a meaningful ecological study. We report a rapid, more sensitive and practical approach. Multiplex PCRs, incorporating fluorescent markers, were found to be effective at amplifying degraded DNA from predators’ guts and could amplify mitochondrial DNA fragments from 10+ species simultaneously without ‘drop outs’. The combined PCR products were then separated by size on polyacrylamide gels on an ABI377 sequencer. New primers to detect the remains of aphids, earthworms, weevils and molluscs in the guts of carabid predators were developed and characterized. The multiplex‐sequencer approach was then applied to field‐caught beetles, some of which contained DNA from as many as four different prey at once. The main prey detected in the beetles proved to be earthworms and molluscs, although aphids and weevils were also consumed. The potential of this system for use in food‐web research is discussed.

The rhabditid nematode Phasmarhabditis hermaphrodita as a potential biological control agent for slugs

A nematode, Phasmarhabditis hermaphrodita, known to be associated with slugs but not previously thought to be parasitic, was shown to be a parasite capable of killing the pest slug Deroceras reticulatum. The parasite infects slugs in the area beneath the mantle surrounding the shell, causing a disease with characteristic symptoms, particularly swelling of the mantle. Infection leads to death of the slug, usually between seven and 21 days afterwards. The nematode then spreads and multiplies in the cadaver. In an experiment where individual D. reticulatum were exposed to different numbers of P. hermaphrodita, a significant positive relationship was found between nematode dose and slug mortality. In two experiments on host range, the nematode was found to infect and kill all pest slug species tested: Deroceras caruanae, Arion distinctus, Arion silvaticus, Arion intermedius, Arion ater, Tandonia sowerbyi and T. budapestensis, in addition to D. reticulatum.

Do earthworms help to sustain the slug predator Pterostichus melanarius (Coleoptera: Carabidae) within crops? Investigations using monoclonal antibodies

Earthworms provide a major potential source of alternative food for polyphagous predators, such as carabid beetles, that are natural enemies of slugs, aphids and other agricultural pests. Non‐pest prey may foster larger numbers of natural enemies, which then help to control pests, or alternatively may help to divert the predators away from pest control. An earthworm‐specific monoclonal antibody was developed to study carabid–earthworm interactions in the field and assess the role of earthworms as alternative prey. The antibody could identify as little at 7 ng of earthworm protein in an ELISA, and could detect earthworm remains in the foregut of the carabid beetle Pterostichus melanarius for 64 h after consumption. Thirty‐six per cent of field‐collected beetles contained earthworm remains. Quantities of earthworm proteins in the beetle foreguts were negatively related to total foregut biomass, suggesting that earthworm consumption increased as total prey availability declined. There was also a negative relationship between foregut biomass and beetle numbers, but both quantities and concentrations of earthworm proteins in beetle foreguts were positively related to beetle numbers. This suggests that as beetle activity–density increased, total prey availability declined, or, as prey availability declined, beetles spent more time searching. In these circumstances, beetles fed to a greater extent on earthworms, an acceptable but nonpreferred food item. Earthworms may, therefore, provide an ideal alternative prey for P. melanarius, helping to sustain it when pest numbers are low but allowing it to perform a ‘lying‐in‐wait’ strategy, ready to switch back to feeding on pests when they become available.

DYNAMICS OF THE RELATIONSHIP BETWEEN A GENERALIST PREDATOR AND SLUGS OVER FIVE YEARS

Although communities of generalist invertebrate predators are known to be capable of suppressing pests, little is known about the long-term population dynamics of individual species of generalist predators and single classes of prey in the field. We present evidence of a dynamic interaction between a generalist insect predator, the carabid beetle Pterostichus melanarius, and its slug prey. We analyzed the numbers of slugs and beetles in an arable field over a 5-yr period, during the main activity period of the beetles from June to September. The field contained 25 plots comprising five replicates of five cultural treatments. An index of the nutritional status of the 8497 beetles collected was obtained by weighing the crop of each beetle (a measure of total prey availability per predator). There was a strong relationship between the crop mass of the beetles and slug numbers in the soil, indicating that slugs were a major part of the diet of the beetles. The change in the beetle population from year to year was strongly related to both slug numbers in the soil and the crop mass of the beetles. This indicated that the slugs influenced the nutritional status, and hence the reproductive success, of the beetles. The predators had a significant effect on slug population growth between years but not between months within years. The temporal effect of these processes was a between-year coupling of beetle and slug population dynamics, buffered by feeding on other prey. The relationship appears to be similar to that between mammalian predators and limited numbers of prey species in the subarctic, where one prey species forms a substantial proportion of the total available food resources. Such relationships may be common in agroecosystems, where species diversity is low, and may be a significant factor driving periodic fluctuations in the abundance of both predators and pests.

Biodiversity vs. biocontrol: positive and negative effects of alternative prey on control of slugs by carabid beetles

Environment-friendly farming techniques seek to increase invertebrate biodiversity in part with the intention of encouraging greater numbers of predators that will help to control crop pests. However, in theory, this effect may be negated if the availability of a greater abundance and diversity of alternative prey diverts predators away from feeding on pests. The hypothesis that access to alternative prey can lead to reduced pest suppression under semi-field conditions was tested. Alternative prey type and diversity were manipulated in 70 mesocosms over 7+ weeks in the presence of the carabid Pterostichus melanarius (Illiger), a known predator of slugs, and reproducing populations of the slug Deroceras reticulatum (Müller). Significantly fewer slugs survived where no alternative prey were provided. Maximum slug numbers and biomass were found in treatments containing either carabids plus a high diversity of alternative prey (many species of earthworm and three of Diptera larvae) or a single additional prey (blowfly larvae, Calliphora vomitoria Linnaeus). In these treatments slug numbers and biomass were as high as in plots lacking predators. The effects of alternative prey were taxon-specific. Alternative prey strongly affected carabid fitness in terms of biomass and egg load. The fittest predators (those with access to high alternative prey diversity or C. vomitoria larvae) reduced slug numbers the least. The mean individual slug weights were greater in treatments with alternative prey than where no alternative prey were provided to the carabids. These results suggest that pests may survive and reproduce more rapidly in patches where predators have access to alternative prey.

Predation and prey size choice by the carabid beetle Pterostichus melanarius (Coleoptera: Carabidae): the dangers of extrapolating from laboratory to field

The impact of predation by the generalist carabid beetle Pterostichus melanarius (Illiger) on populations of the field slug Deroceras reticulatum (Müller), and the effects of prey size on the predator-prey interaction, were measured under semi-field conditions. It was hypothesized that environmental heterogeneity would lead to very different patterns of comparative mortality than might be deduced from size choice experiments conducted in the laboratory. Results from outdoor mini-plots, emulating conditions in a field of wheat, demonstrated that P. melanarius significantly reduced numbers of slugs from all size classes, with no apparent preferences. This was in marked contrast to results from earlier laboratory studies, where this beetle fed preferentially on the smallest slugs. The slugs in the mini-plots ranged in size from 2-100 mg and the numbers in the mini-plot reflected the size frequency distribution in the field. Beetles in mini-plots containing high densities of slugs increased significantly in weight, in contrast to beetles in mini-plots with low slug density or no added slugs, which did not. Enzyme-linked immunosorbent assays (ELISA), using anti-slug monoclonal antibodies, showed that where there was a higher density of slugs there was more slug protein in the guts of the beetles. It was concluded that environmental heterogeneity probably provided a greater number and diversity of refugia for smaller than for larger slugs, counteracting laboratory-measured size preferences measured in arenas without refugia. These results have implications for a range of ecological studies involving inter- and intra-specific prey size choice, and emphasize the dangers of extrapolating from the laboratory to the field.

Mass cultivation and storage of the rhabditid nematode Phasmarhabditis hermaphrodita, a biocontrol agent for slugs

The rhabditid nematode Phasmarhabditis hermaphrodita (a parasite capable of killing pest slugs) was grown in vitro, in association with a mixed bacterial flora on foam chips impregnated with a kidney‐based nutrient medium in aerated bags, to provide sufficient numbers for laboratory and field experiments. The feasibility of producing nematodes in liquid culture was investigated using 250 ml flasks. Baffled flasks containing 25 or 50 ml or liquid were found to be better than baffled flasks containing 100 ml or unbaffled flasks. Inoculum densities ranging from 50 to 330 ml‐1 did not affect final yield. Dauer larvae in aerated water died rapidly at temperatures of 26–35°C. Survival was progressively better at 22°C and 15°C, and best at 5°C or 10°C.

Transgenic 'Arabidopsis' leaf tissue expressing a modified oryzacystatin shows resistance to the field slug 'Deroceras reticulatum' (Muller).

Transgenic Arabidopsis thaliana has been developed which expresses the oryzacystatin mutant OC‐IΔ86, which is an inhibitor of the major proteinase present in the digestive gland of the slug, Deroceras reticulatum. When fed on leaf tissue from plants expressing this inhibitor the growth of juvenile slugs was significantly reduced by 31% compared with those feeding on control leaf tissue. Furthermore, while surviving slugs did not individually consume less when feeding on leaf tissue expressing OC‐IΔ86, the total amount of leaf tissue eaten was 50% less, due to reduced survival of slugs. The synthetic cysteine proteinase inhibitors E‐64 and leupeptin also significantly reduced slug weight gain (by at least 40%) and digestive gland cysteine proteinase activity when administered in an artificial diet, indicating that their antimetabolic effects are due to direct inhibition of gut proteolytic activity. These results suggest that transgenic crop plants expressing phytocystatins could be used to suppress the growth rates of slug populations in the field.

Biocontrol of slugs in protected lettuce using the rhabditid nematode Phasmarhabditis hermaphrodita.

In two experiments, the rhabditid nematode Phasmarhabditis hermaphrodita, a parasite of slugs, was cultured in vitro and applied as a drench to soil at four dose rates (3 108, 1 109, 3 109 and 1 1010 ha-1) 1 or 4 days before planting lettuce seedlings in a polythene tunnel. The effects of the four nematode doses on slug damage during the first 3 weeks after planting and on the numbers of slugs found within and below lettuce plants at harvest were measured. Results were then compared with untreated plots and with plots where methiocarb pellets were applied at the recommended field rate. In the first experiment, methiocarb pellets significantly reduced the percentage of plants damaged by slugs, but the nematode did not. In the second experiment, methiocarb pellets and the second highest dose of nematodes significantly reduced the percentage of plants damaged by slugs. The different effect of the nematode in the two experiments may have resulted from differences in the timing of nematode application and/or d...

Laboratory tests of the potential of entomopathogenic nematodes for the control of field slugs (Deroceras reticulatum)

The entomopathogenic nematodes, Steinernema feltiae (UK 76) and Heterorhabditis sp. (North West European Group, UK 211), and the slug-parasitic nematode Phasmarhabditis hermaphrodita (UK 1) were tested for their ability to kill the slug Deroceras reticulatum using petri dish and soil bioassays. The entomopathogenic nematodes did not kill slugs in either bioassay at 14 and 23°C, although they were shown to kill tenebrionid insect larvae (Tenebrio molitor and Zophobas morio) at 14°C. The symbiotic bacteria of S. feltiae and Heterorhabditis sp. (Xenorhabdus bouienii and Photorhabdus luminescens, respectively) were injected in large numbers into the hemocoel of D. reticulatum. X. bovienii was found to be of low pathogenicity and P. luminescens was not pathogenic, whereas slugs were rapidly killed by injection of a strain of Pseudomonas fluorescens isolated from a cadaver of D. reticulatum. P. hermaphrodita killed D. reticulatum at 14°C but not at 23°C and did not kill T. molitor or Z. morio at 14°C. It is concluded that S. feltiae and Heterohabditis sp. have no potential as biocontrol agents against slugs, whereas P. hermaphrodita is effective at the relatively low temperatures at which slugs are active.