PCR-based species identification of Agriotes larvae

Click beetle larvae within the genus Agriotes (Coleoptera: Elateridae), commonly known as wireworms, are abundant ground-dwelling herbivores which can inflict considerable damage to field crops. In Central Europe up to 20 species, which differ in their distribution, ecology and pest status, occur in arable land. However, the identification of these larvae based on morphological characters is difficult or impossible. This hampers progress towards controlling these pests. Here, we present a polymerase chain reaction (PCR)-based approach to identify, for the first time, 17 Agriotes species typically found in Central Europe. Diagnostic sequence information was generated and submitted to GenBank, allowing the identification of these species via DNA barcoding. Moreover, multiplex PCR assays were developed to identify the nine most abundant species rapidly within a single-step reaction: Agriotes brevis, A. litigiosus, A. obscurus, A. rufipalpis, A. sordidus, A. sputator, A. ustulatus, A. lineatus and A. proximus. The latter two species remain molecularly indistinguishable, questioning their species status. The multiplex PCR assays proved to be highly specific against non-agrioted elaterid beetles and other non-target soil invertebrates. By testing the molecular identification system with over 900 field-collected larvae, our protocol proved to be a reliable, cheap and quick method to routinely identify Central European Agriotes species.     

Dispersal abilities of adult click beetles in arable land revealed by analysis of carbon stable isotopes

1 The dispersal abilities of agrioted beetles, serious pests on a variety of crops, are poorly known under natural conditions. This hampers their control. We used, for the first time, a stable isotope approach to assess dispersal of adult Agriotes obscurus in arable land.
2 After a diet switch from a C3- to a C4-plant, carbon isotope ratios of A. obscurus larvae significantly changed towards the isotopic signature of the new diet. Moreover, the larval δ¹³C signatures were transferred to the wing covers of the adult beetles with little distortion.
3 To assess the dispersal abilities under natural conditions, pheromone traps, lured for Agriotes sp., were installed at two study sites in Western Austria. Each site comprised a maize field (= C4-plant) and adjacent C3-grasslands with traps established along a transect of increasing distance to the maize.
4 δ¹³C signatures of wing covers revealed that adult male A. obscurus were able to migrate at least 80 m, which was the maximum distance that dispersal could be traced in the present study. The dispersal behaviour might have been influenced by site-specific factors.
5 The results obtained demonstrate a higher potential of adult male Agriotes to disperse than previously assumed. Moreover, the combination of pheromone trapping and stable isotope analysis proved to be an effective approach to study insect movement and dispersal in arable systems harbouring C3- and C4-crops.     

Predation of Thereva nobilitata (Fabricius) (Diptera: Therevidae) on Agriotes obscurus L. (Coleoptera: Elateridae)

Little is known about the natural enemies of wireworms (Coleoptera: Elateridae), but there are frequent anecdotal reports of (usually unnamed) stiletto fly larvae (Diptera: Therevidae) preying on various species. We observed larvae of Thereva nobilitata (Fabricius) feeding on larvae of the dusky wireworm, Agriotes obscurus L., during the summer of 2011, in Agassiz, British Columbia. This finding is of interest as: both the predator and the wireworm are introduced species to this area from Europe; T. nobilitata is uncommon in North America; and this predator has not been associated with any wireworm species previously. We observed that larvae of male and female T. nobilitata will feed on various sizes of A. obscurus larvae, most feeding being carried out by the smallest T. nobilitata larvae. These findings suggest future work should assess the potential for therevid larvae as top‐down regulators of Agriotes larvae under field conditions.     

Spinosad interacts synergistically with the insect pathogen Metarhizium anisopliae against the exotic wireworms Agriotes lineatus and Agriotes obscurus (Coleoptera: Elateridae)

We determined that spinosad interacts synergistically with the biocontrol agent Metarhizium anisopliae (Metch) Sorokin to increase the mortality of two wild-collected wireworm species, Agriotes lineatus (L.), and Agriotes obscurus (L.). Bioassays were performed using a M. anisopliae isolate originally acquired from a local wireworm cadaver. M. anisopliae was applied as a soil drench at 3.3 x 10(2) and 10(4) conidia per gram sand, respectively. Soil drenches also were prepared using a commercial formulation of the actinomycete toxins spinosyn-A and spinosyn-D (common name spinosad) at sublethal doses of 1.5, 3, and 6 ppm active ingredient per gram sand. Combined treatments of spinosad and M. anisopliae were synergistic in causing mortality for all spinosad concentrations. Wireworm feeding activity was reduced after exposure to both spinosad and M. anisopliae and was found to be concentration dependent. The high mortality and reduced rate of wireworm feeding suggest that spinosad and M. anisopliae treatment combinations should be tested in the field.     

Spatial relationships between two Agriotes click-beetle species and wireworms in agricultural fields

1 The production of new insect pheromones for pest monitoring proceeds at a greater rate than their evaluation, with the consequential possibility of premature introduction. Fundamental to their successful deployment is the determination of a consistent relationship between adult male pheromone trap catches and pest damage. In the present study, adult pheromone traps and larval bait traps were used to examine spatial relationships between two species of Agriotes beetle and wireworms at the field scale. 2 The spatial distributions of adult male Agriotes lineatus and Agriotes obscurus in two fields were determined and compared with the distribution of their larvae. Data were assembled as spatially referenced trap counts, and analysed for evidence of aggregation and clustering using Spatial Analysis by Distance IndicEs (SADIE) methodology. Spatial stabilities of adult populations between sampling dates were tested using association tests. Spatial and quantitative linkages between adult and larval trap catches were also tested. Moreover, a new way of adapting SADIE methodologies is presented for situations where two datasets within an area do not share the same sampling points. 3 There was no significant difference in variance : mean relationships for the two species but there were differences in their spatial distributions, and this is a definitive example of the general argument stating that it is important to consider spatial as well as count data in ecological studies. The spatial distribution of A. lineatus varied between sampling occasions at both sites whereas A. obscurus had consistently significant SADIE indices over time at one site, and adult catches could also be linked to larval distributions and counts. It is proposed that observed differences between the two species can be explained by interference between traps and dissimilar movement rates. There was some evidence of an edge effect at the field boundaries. 4 The distance between pheromone traps is related to the time that elapses before adjacent traps interfere with trap captures and this limits the detection of statistically significant spatial patterns. It is shown that the current practice of adding trap counts for different Agriotes species and treating them as numerically equivalent is insufficiently robust to be recommended at this stage. 5 The implications for the use of sex pheromone traps in wireworm pest management are considered. It is concluded that pheromone traps, as currently used, will not reliably indicate where wireworms occur in a field, and that the complexity of interpreting adult male trap counts limits quantitative predictions of population size.     

DNA barcoding identification and genetic diversity of bamboo shoot wireworms (Coleoptera: Elateridae) in South China

Wireworms are recognized as economically important oligophagous pests of Phyllostachys bamboo shoot, which have caused a huge economic loss in South China. Studies of bamboo shoot wireworm control and management were seriously hampered by the lack of reliable identification of larvae. DNA barcoding has been proved to be an important and useful tool in species identification of morphologically cryptic insects. Accurate knowledge of damage-causing species and the phylogenic structure of elaterids will provide insight into their sustainable and effective management. Here, we use interspecific variation in COI and EF1-a as a robust method of identification and consider the intra- and interspecific genetic variation of bamboo wireworms. This approach has revealed that three Melanotus larvae occurred in Phyllostachys forest. The dominance of one species in different sampling location suggests there maybe underlying difference in habit and host plant preference. These data provide a starting point for determining the distribution of wireworm species feeding on bamboo shoots in South China. The inclusions would further aid identification of wireworm species and relationships between certain wireworm species with specific environments.     

Differential responses of three Agriotes click beetle species to pheromone traps

1 Previous work had suggested that adult male click beetles (Agriotes spp.) show differential responses to species specific pheromone traps. This hypothesis was tested using mark–release–recapture methods to estimate the maximum sampling range and the effective sampling area of traps for three species. 2 Captured beetles of the species Agriotes lineatus, Agriotes obscurus and Agriotes sputator were marked to show the direction of release, the distance of the release point from the trap and the replicate. Analysis of variance showed that there were significant differences in recapture rates between species and release distances. There were no significant differences between release direction and replicates. 3 Calculated linear speeds suggested differences in movement rates in the order: A. lineatus > A. obscurus > A. sputator. There were also substantial differences between the species in the maximum sampling ranges and effective sampling areas of the traps. These placed the species in the same order. 4 The results are used to estimate the minimum cost of mass trapping programmes to prevent males from mating, giving values of €165/ha/year (A. lineatus), €247.5/ha/year (A. obscurus) and €2343/ha/year (A. sputator). 5 Implications for the use of pheromone traps in wireworm pest management are discussed. It is concluded that current U.K. recommendations based on the cumulative total catch of the three species over a sampling season can be improved by considering the spatial relationships between the adult trapping system and larval distribution. The current constraint to this is the general inability to separate wireworms into species.     

Carnivory and resource-based niche differentiation in anuran larvae: implications for food web and experimental ecology

1.  Food webs represent the paths of material and energy flow through organisms in an ecosystem. Anuran larvae are important components of pond food webs: they are abundant, consume large quantities of food and serve as prey for many organisms. However, there are very basic uncertainties about the feeding ecology of anuran larvae; for instance, as to which trophic level they belong and whether species differ in resource use. Because anuran larvae have been employed in model systems in experimental ecology for decades, these uncertainties could lead to misinterpretation of published experiments, or inadequate designs of experiments directed at general, conceptual issues in ecology.
2.  Using ¹³C and ¹⁵N stable isotope and gut content analyses of free-ranging and enclosed tadpoles of four ranid species ( Lithobates sylvaticus , L. pipiens , L. clamitans , L. catesbeianus ) in the food webs of six wetlands, we tested the following null hypotheses: (i) that anuran larvae are strict primary consumers; (ii) that they are non-selective feeders and therefore exhibit little feeding niche differentiation; (iii) that they are opportunistic consumers and (iv) that their diet remains unchanged through ontogeny.
3.  All four species consumed and assimilated substantial amounts of animal food; bullfrog larvae, in particular, appear to be predatory. Significant feeding niche differentiation among species occurred with respect to the sources of carbon, consumption of animal matter and nutritional quality of food ingested. We further documented opportunistic feeding habits and ontogenetic shifts in diet.
4.  Collectively, these studies revealed complex trophic relationships that might require a reconsideration of the role of anuran larvae in pond food webs, as well as a reinterpretation of results of previous studies employing anuran larvae in model experimental systems.     

Offspring performance in dynamic habitats: key factors for a riparian carabid beetle

Abstract.  1. Knowledge of the ecology of carabid species is largely restricted to adults, although larval mortality is assumed to be the key factor in overall mortality. As low-mobility larvae are unable to avoid unfavourable conditions, habitat selection of reproducing adults should be clearly affected by the habitat factors which determine offspring performance.
2. The present study examines the key habitat factors governing the distribution patterns of Bembidion velox larvae and adults on the river banks of the River Elbe by means of habitat suitability models. The validity of the determined habitat factors for offspring performance and survival was tested in laboratory experiments.
3. In the field, B. velox adults as well as larvae show a strong association with semi-terrestrial, sandy, open soil habitats.
4. In the laboratory, overall mortality of larvae reared in different substrates was lowest in sand of medium grain size mixed with fine and coarse sand. The first larval instars in particular reacted sensitively to variations in grain size. Furthermore, flood resistance of eggs was demonstrated, as 90% of larvae hatched under permanently flooded conditions.
5. Short development times were recorded, with 4–7 days for hatching of young larvae from eggs after oviposition and approximately 28 days for the development of adults from newly hatched larvae. This probably increases the survival probability for the pre-imaginal stages in dynamic habitats.
6. In conclusion, it can be said that reproductive success is strongly dependent on oviposition site selection by adults as this reflects the ecological demands of the immature stages.     

Antipredator Behavioral Traits of some <Emphasis Type="Italic">Agriotes</Emphasis> Wireworms (Coleoptera: Elateridae) and their Potential Implications for Species Identification

Many insects show a typical antipredator behavior. The two simplest tactics are immobilization and flight. The antipredator behavior of wireworms, the soil-dwelling polyphagous larvae of click beetles, has not yet been investigated. Birds are known to attack wireworms. Our experimental method (picking up and dropping wireworms) mimics the first attack of a predator, such as a bird. In Europe, some Agriotes species are of particular importance as crop pests. As these species differ considerably, in ecology, damage threshold, and pathogen resistance, knowing more about the species specific biology and behavior will support the establishment of successful control measures. In an extensive microcosm study, we observed the antipredator behavior of wireworms belonging to four different Agriotes species (A. lineatus, A. obscurus, A. sordidus, and A. ustulatus) before and during digging into the soil after a startle-stimulus. We recorded wireworm immobility frequency and duration, and locomotor activity at three temperatures. We also analyzed genetic and morphologic attributes of A. lineatus larvae from two different origins. Following behavior types were found reflecting different escape tactics: (a) distinct tonic immobility (A. lineatus), (b) brief/inconsistent tonic immobility (A. obscurus, A. sordidus), and (c) immediate fleeing/burying (A. ustulatus). Additionally, we found small but significant differences in morphology, behavior, and genetics (PCR results) between A. lineatus larvae originating from the Netherlands and those originating from Germany. The biological information we gathered about each species will further increase the understanding of these insects and improve interpretation of future experimental data. In addition, the described behavioral differences between Agriotes obscurus and A. lineatus wireworms may represent a useful additional criterion in morphological species identification.     

Peculiarities of the biotope distribution of click beetle larvae (Coleoptera, Elateridae) in the Irtysh River valley

The spatial distribution of Elateridae larvae in the Lower Irtysh valley has been analyzed. The larvae analyzed belonged to 19 Elateridae species inhabiting soils of the floodplain and the river terrace. It has been pointed out that the larvae of Paraphotistus nigricornis, Agriotes lineatus, Limonius parallelus, and Aplotarsus incanus are most tolerant to the regular spring flooding. It has been found that the species compositions of floodplains and terrace meadows are significantly different. As a rule, species with a wide tropical spectrum inhabit floodplain soils, including bush and forest habitats. At the same time, the terrace was dominated by species characteristic of open habitats, including the steppe zone. It has been revealed that the values of species diversity vary significantly in the floodplain and terrace plots, but the higher species richness (Menhinick index) was registered in the floodplain.     

Intrinsic competition between larval parasitoids with different degrees of host specificity

Abstract.  1. Interspecific competition among parasitoids may play a key role in the community dynamics of tritrophic plant–herbivore–parasitoid systems and has important implications for management of herbivorous insect pests.
2. A model system was used to explore the outcome of interspecific competition between parasitoids that differ in host specificity. The system included the lepidopteran pest Heliothis virescens , the generalist parasitoid Cotesia marginiventris , and two specialist parasitoids, Microplitis croceipes and Cardiochiles nigriceps .
3. The generalist, C. marginiventris , dominated intrinsic competition when given an 8-h developmental head start over C. nigriceps or when its oviposition was simultaneous with that of M. croceipes . Microplitis croceipes and especially C. nigriceps larvae prevailed when they were allowed to oviposit prior to C. marginiventris .
4. Rates of host mortality prior to parasitoid emergence varied with parasitoid species composition and with the order of oviposition.
5. Implications for integrated pest management and the adaptive significance of competition as related to host specialisation are discussed.     

Laboratory and field evaluation of entomopathogenic nematodes for control of Agriotes obscurus (L.) (Coleoptera: Elateridae)

The susceptibility of the dusky wireworm, Agriotes obscurus (L.) (Coleoptera: Elateridae), to different species and strains of entomopathogenic nematodes was tested in a virulence assay in the laboratory. Larvae were exposed to different nematode doses of 50 and 100 IJs/cm². At a dose of 50 IJs/cm², only a commercial strain Heterorhabditis bacteriophora Poinar and the native strain Steinernema carpocapsae (Weiser) B14 caused increased mortality compared with the control (11.1% and 13.3% mortality, respectively). At the higher dose tested, all strains (except Steinernema sp. D122) were virulent to A. obscurus larvae. Steinernema carpocapsae B14 caused higher mortality of wireworm (75.6%) and was used for the assay conducted in cages, with a dose of 100 IJs/cm², in field conditions. The results showed that S. carpocapsae B14 controlled 48.3% of A. obscurus larvae, demonstrating that some entomopathogenic nematodes have the potential to control larvae of A. obscurus. However, further work is needed to improve their efficacy.     

Note on the effect of wireworms of the genera Agriotes and Corymbites on crop yields

Experiments were made to illustrate the regular falling off of yield of crops due to increasing wireworm populations under controlled conditions. The genus Corymbites was shown to cause as much damage as Agriotes; Athous was less harmful. The damage caused to various cereals by Agriotes is contrasted.     

Parasitism by the mite Trombidium breei on four U.K. butterfly species

Abstract 1.  The incidence of parasitism by larvae of the mite species Trombidium breei was reported in one population of the lycaenid butterfly Polyommatus icarus , four populations of the satyrine butterfly Maniola jurtina , one population of the satyrine butterfly Aphantopus hyperanthus , and two populations of the satyrine butterfly Pyronia tithonus , as well as on one specimen of the dipteran Alophorus hemiptera . A considerable proportion of butterflies (11-50%) was infested in all study populations.
2. The pattern of infestation was examined in detail in M. jurtina . Males had a significantly higher incidence of infestation than females, and middle-aged butterflies had a higher incidence of infestation than old or young butterflies. The incidence of infestation peaked in the middle of the flight season, and this seasonal effect was independent of the effect of butterfly age.
3. Using a model based on capture-recapture data, it was estimated that a hypothetical ideal male M. jurtina that lives exactly the mean expected lifespan of 9-10 days has an approximately 75% chance of becoming infested with mites at least once during its lifetime, a mean time to first infestation of 3-4 days, and an average infestation persistence time of 2-3 days.
4. Capture-recapture data failed to show any effect of mite infestation on the lifespan or within-habitat movement rate of M. jurtina .
5. In experiments in which individual butterflies were taken out of their normal habitat and released, M. jurtina and P. tithonus that were infested with mite larvae did not differ from uninfested individuals in the efficiency with which they returned to suitable habitat. Thus, parasitism by T. breei larvae had no detectable effects on flight performance or orientation ability.
6. The results suggest that trombidiid mite larvae have limited potential in the biological control of insect pests.     

Transitional sublethal and lethal effects of insecticides after dermal exposures to five economic species of wireworms (Coleoptera: Elateridae)

During an insecticide toxicity study involving field-collected dusky wireworm, Agriotes obscurus (L.) (Coleoptera: Elateridae), wireworms exposed dermally to six classes of insecticides exhibited characteristic transitional symptoms of toxicity. These symptoms, collectively termed "morbidity," were categorized as "writhing," "leg and mouthpart movements," or "mouthpart-only body movements." These symptoms could persist for long periods, depending on insecticide and dose, with morbid wireworms ultimately recovering or dying. Additional LC50 and LD50 toxicity studies showed that these stages of morbidity also occurred in four other wireworm species, notably Agriotes sputator (L.), Limonius canus LeConte, Ctenicera pruinina (Horn), and Ctenicera destructor (Brown). In addition, all species exposed dermally to clothianidin moved in significant numbers to the surface of soil in posttreatment holding cups. This movement was not observed when these species were exposed to chlorpyrifos or the control solvent. These findings suggest that toxicity trials involving wireworms should include observations on morbidity, and the duration of trials should continue until symptoms of morbidity cease. The long-term morbidity and potential recovery or death of wireworms exposed to certain insecticides has implications for how laboratory and field studies can be better designed and interpreted in the future.     

Sex pheromone of Nearctic Agriotes mancus and its similarity to that of three Palearctic Agriotes invasive in North America

1. The wheat wireworm, Agriotes mancus (Coleoptera: Elateridae), is a predominant elaterid pest species in the Nearctic region, with a life history and morphology similar to those of Agriotes obscurus, Agriotes lineatus and Agriotes sputator, three Palearctic pest elaterids invasive in North America. Here, we report the identification and field testing of the sex pheromone of A. mancus.
2. We collected headspace volatiles from female beetles on Porapak Q, and analysed aliquots of Porapak extract by gas chromatography with electroantennographic detection (GC-EAD) and by GC-mass spectrometry. In GC-EAD recordings, two esters—geranyl butanoate and geranyl hexanoate—elicited antennal responses from A. mancus males. In field experiments, trap lures containing both geranyl butanoate and geranyl hexanoate afforded large captures of A. mancus males, which were—on average—approximately 30-fold higher than captures in traps baited with a single ester.
3. Traps baited with geranyl butanoate as a single-component lure captured a significant number of Palearctic A. sputator, indicating the establishment of A. sputator in its invaded Nearctic range.
4. With the A. mancus sex pheromone now known, it can be included in the development of pheromone-based programmes to monitor and manage native and invasive Agriotes pests in North America.

     

Effects of aggregation pheromone on individual behaviour and food web interactions: a field study on Drosophila

Abstract.  1. The effects of an aggregation pheromone on individual behaviour and food web interactions were investigated in two ecological communities, using Drosophila melanogaster and D. simulans as focal species.
2. Fruit substrates with aggregation pheromone were significantly more attractive to adult D. melanogaster and D. simulans than control fruit substrates, and the response was positively dose dependent. Competing species and natural enemies were also significantly attracted to substrates with the aggregation pheromone of D. melanogaster and D. simulans .
3. Significantly more eggs were deposited on pheromone-treated fruits than on control fruits, and the microdistribution of eggs within fruits was correlated to the microdistribution of the pheromone. The aggregation pheromone induced more females to share the breeding site.
4. The extremely high densities of fruit flies in the large aggregations appeared to reduce the oviposition rate of females. Physical interactions with conspecific and heterospecifics were frequently observed in the aggregations, and often led to patch leaving of the fruit flies.
5. Competition for food among larvae occurred at high densities and parasitism was density dependent. Aggregation pheromones can be directly responsible for these patterns through their effects on the con- and heterospecific behaviour.
6. The combined results show that aggregation pheromones affect a multitude of aspects in the ecology of interacting animals. The importance of incorporating the communication signals in ecological theory of aggregations is discussed.     

Dropping behaviour of larvae of aphidophagous ladybirds and its effects on incidence of intraguild predation: interactions between the intraguild prey, Adalia bipunctata (L.) and Coccinella septempunctata (L.), and the intraguild predator, Harmonia axyridis Pallas

Abstract.  1. Two experiments were performed in the laboratory to assess the behaviour of dropping from a host plant as a defence against intraguild predation in aphidophagous ladybird larvae.
2. In the first experiment, encounters were observed on bean plants between fourth instars of the intraguild predator species, Harmonia axyridis , and first instars of two other ladybird species, Adalia bipunctata (L.) and Coccinella septempunctata (L.). The percentages of first instars of the latter two species that dropped from the plant in response to attack differed dramatically, with 47.5% of C. septempunctata first instars dropping vs. 0% of A. bipunctata .
3. In the second experiment, first instars of A. bipunctata or C. septempunctata and a fourth instar of H. axyridis were allowed to forage together on bean plants for 3 h. During this time, 44.3% of C. septempunctata larvae dropped from the plant, but less than 2% of A. bipunctata larvae did so. In contrast, 95.0% of A. bipunctata larvae fell victim to intraguild predation by H. axyridis vs. only 54.5% of C. septempunctata larvae.
4. The significance of dropping behaviour of ladybird larvae as a defence against intraguild predation, and the relationship of dropping behaviour to species-specific habitat affinity of ladybirds, is discussed.     

Invertebrate pests of canola and their management in Australia: a review

Abstract  In Australia, canola is subject to attack by at least 30 species of invertebrate pests, although the composition of this pest complex can vary between regions. Mites (e.g. the redlegged earth mite Halotydeus destructor and the blue oat mites Penthaleus spp.), lucerne flea ( Sminthurus viridis ) and false wireworms (e.g. the grey false wireworm Isopteron punctatissimus and the bronzed field beetle Adelium brevicorne ) are the major pests threatening the seedling establishment, whereas aphids (the cabbage aphid Brevicoryne brassicae , the turnip aphid Lipaphis erysimi and the green peach aphid Myzus persicae ), the native budworm ( Helicoverpa punctigera ), the diamondback moth ( Plutella xylostella ) and the Rutherglen bug ( Nysius vinitor ) can cause irregular and unpredictable damage to the flowering and podding plants. Current tactics of pest management for canola rely largely on the use of synthetic pesticides, but this single-technology approach is likely to incur negative effects on natural enemies and the risk of pest resistance. Thus, the sustainable production of canola requires integrated pest management (IPM) strategies, in which cultural control, crop resistance and biological control are used as important components, with chemical inputs applied only when absolutely needed to restrict pests from reaching economically damaging densities. Such IPM strategies should be built around a fundamental understanding of pest ecology at both regional and local farm levels and the integration of renewable technologies. Therefore, future research efforts need to be focused on the canola-cropping system, with a particular emphasis on the impact of pest species, natural enemies of the pests, varietal resistance to pests and the spatial ecology of pest species.