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                                                  EXPERIMENTAL DESIGN & SAMPLING

 

I.  Luck et al. (1999) emphasized that in order to improve success rates in biological control, an understanding of events in past

    successful introduction programs is essential. 

 

A.  Successful cases can be used to test hypotheses about predator/prey interactions, and develop criteria for identifying effective natural enemies. 

 

B.  Van Driesche et al. (1991) reviewd the analytical bases developed in the late 1980's to estimate total losses from parasitism.  Thay stated that, "Because the population of an insect stage typically begins to lose members through death or development to the next stage in the life cycle before the entire recruitment to the stage is completed, at no time are all members of the generation present to be counted.  This idea is analogous to a sink partly filled with water (i.e., the population), into which water is flowing (recruitment) and from which water is draining (death or advancement to the next stage)..."  "To construct a life table, we need to know the total numbers that enter a stage (in this analogy, the total amount of water entering the sink). 

 

C.  What biologists typically measure, however, is the number of animals present per sample unit at points in time (which is analogous to the amount of water in the sink at any given time).  Although it is true that the volume of water present at any time is determined by the moment-to-moment balance of cumulative influx minus cumulative outflow, if these latter quantities are not known, it is not possible to determine total inflow from even the most detailed set of observations on the quantity of water in the tank at fixed moments in time.  What is needed is a continuous record of recruitment for the whole period over which animals enter the stage of interest for the generation.  This can be achieved by measuring recruitment for a series of contiguous intervals spanning the whole period when recruitment occurs (e.g., Van Driesche & Bellows 1988)."

 

D.  Van Driesche et al. (1991) continued, "When the goal is to assess not only how many insects enter a given life stage over the course of a generation, but also to determine how many of that number subsequently become parasitized, the problem is compounded because the basic problem discussed above now applies to two quantities that must be measured; i.e., the total number of hosts recruited and the number that subsequently become parasitized.  The linkages between these value are both dynamic and complex..."  "Although there are some systems in which biology and life history characteristics are such as to produce nondynamic systems not subject to these problems (for example, cases where the sampled stage is in a diapause stage and accumulates without loss as, for example, is approximately the case for gypsy moth eggs, because dead or parasitized eggs remain countable) or systems such as some leafminers in which lost insects continue to be traceable in samples through their remains, the majority of insects do have overlapping recruitment and losses.  For these cases, densities and percentage parasitism values seen in samples do not measure adequately the level of parasitoid effect."

 

                II.  Approaches in the evaluation process include  1.  life table analysis, which is a descriptive method;  2.  stage frequency

                      analysis;  3.  direct measurement of recruitment; 4.  death rate analysis; 5.  experimental manipulations in the field. 

 

                                A.  The primary goal is to determine whether regulation of the host population exists and to identify the agents

                                      responsible for  regulation. 

 

B.  Luck et al. (1999) defined regulation as the biological processes involving natural enemies that suppress prey or host densities below levels that prevail in the absence of natural enemies.  It must be determined whether the populations are regulated, measure the level of regulation and identify the forces involved in regulation.  If the populations are not regulated, if the regulation is intermittent, or if the level of suppression is inadequate, then other options to consider are (1) introduction of additional natural enemies, (2) inoculative or inundative releases, (3) development of plant resistance, (4) change the cultural practices, etc. 

 

C.  There are other key references pertaining to measurement of natural enemy impact (Thompson 1955, Richards et al. 1960, Hafez 1961, Kirtitani & Nakasuji 1967, Manly 1974, 1976, 1977, 1989; Ruesink 1975, Russell 1987, Kolodny-Hirsch 1988, Schneider et al. 1988, Van Driesche 1988, Bellows et al.  1989, Gould et al. 1989, Keating 1989, McGuire & Henry 1989, Van Driesche et al. 1989, 1991a,b, Buonaccorsi, J. P. & J. S. Elkinton 1990, Gould, J. R. 1990a,b, Hazzard et al. 1991).

 

III.  There is probably no single method which can provide conclusive evidence that natural enemies are regulating a population. 

 

A.  Natural enemies are not the only factor involved in many interactions, and the plant can significantly affect the natural enemies' ability to regulate (Flanders 1942, Starks et al. 1972, Price ta al. 1980). 

 

B.  Luck et al. (1999) conclude that no research method if free of technical problems, and management decisions are made with insufficient knowledge.  Therefore research aimed at developing an integrated pest management program is a continuous process in which hypotheses are continually being refined and tested (Way 1973).  Classical biological control and augmentive biological control are important IPM tactics, but they must be pursued and expanded to include situations for which they have not ben emphasized (DeBach 1964, 1974, Ridgway & Vinson 1976, Carl 1982).  Indigenous biological control forms the foundation for pest management and therefore must be utilized if IPM is to become more effective.  Its presence in an agroecosystem can be demonstrated by disrupting it with insecticides (Folsom & Brondy 1930, Woglum et al. 1974, Brown 1951, Pickett & Patterson 1953, Ripper 1956, Bartlett 1968, Smith & van den Bosch 1967, Wood 1971, Ehler et al. 1973, Eveleens et al. 1973, Croft & Brown 1975, Luck & Dahlsten 1975, Luck et al. 1977, Reissig et al. 1982, Kenmore et al. 1984), or by comparing unsprayed, abandoned orchards with treated orchards.  Insecticidal disruption provides one of the best experimental techniques for evaluating natural enemies.  It can reveal the amount of control provided by indigenous entomophages (Stern et al. 1959, Smith & van den Bosch 1967, Falcon et al. 1968, MacPhee & MacLellan 1971, Wood 1971, Flint & van den Bosch 1981, Jones 1982, Metcalf & Luckmann 1982, Kenmore et al. 1984).

 

                               C.  In the experimental evaluation of biological control, testing whether regulation exists and which natural enemies are

                 responsible for the regulation, life tables and their analyses provide a quantitative framework in which to explore the consequences

                of a predator/prey interaction and to generate hypotheses.  However, life tables cannot demonstrate the efficacy of natural enemies

                in suppressing a host or prey population in the field; only experimental methods can do this (Luck et al. 1999).  Some populations

                cannot be manipulated with available technology because they are based on untested assumptions.  Evidence is that natural

                enemies suppress host/prey populations and experimental results suggest that a host plant's nutritional quality, its physical

                structure and its chemical defenses play a role in pest suppression (Denno & McClure 1983, Futuyma & Peterson 1985, Whitham et

               al. 1984, Mattson 1980). 

 

               IV.  The development of an appropriate sampling routine is essential for the evaluation of natural enemies.

 

A.  The design is determined by the objectives of the experiment, the biology of the organisms involved and the cost of acquiring the information to meet the objectives.  The sampling procedure used to acquire data and the statistical techniques used to analyze data must be decided before field evaluation begins. 

 

B.  Appropriate experimental designs require preliminary studies to identify variation sources.  Preliminary samples can save time and resources (Green 1979).  For example Legner (1979, 1983, 1986) and Van Driesche (1983) described some of the problems associated with estimating and interpreting percent parasitism from field samples, while Van Driesche & Bellows (1988) discussed analytical procedures for dealing with some of the problems. 

 

C.  Statistical randomness is important in population sampling and in the assignment of treatments.  Randomness includes locating field plots and selecting sample plants and sample units.  Each sample unit must have an equal chance of being selected.  Nonrandom sampling makes analysis of the data questionable because of the uncertainty associated with the estimation of the values.  Texts and articles on sampling and experimental design should be consulted before an evaluation of natural enemies or of biological control is begun (Morris 1955, 1960, Cochran 1963, Stuart 1976, Elliot 1977, Jessen 1978, Southwood 1978, Green 1979).

 

               V.  Evaluation in biological control must consider the following:  Do natural enemies affect pest population densities; what natural

                     enemies kill a pest; how quickly will an natural enemy kill a pest; how many pests will a natural enemy kill; how does an natural

                     enemy respond to changes in pest densities in the field; and how do environmental changes affect the predator-prey/parasitoid-

                     host interaction (Luck et al. 1999).

 

A.  When evaluating indigenous natural enemy populations, it is necessary to determine whether biological control of the hosts exists. 

 

B.  An effective means compares pest densities in an area not treated with pesticides to pest densities in an area subjected to traditional pesticide practices.  Ceasing the use of pesticides in parts of a field does not constitute a previously unsprayed area, as prolonged pesticide use reduces natural enemies and alternate prey or hosts upon which the natural enemies depend. 

 

C.  Time is required to reestablish interactions between natural enemy and prey/host populations. 

 

D.  Also, the untreated area must be large enough to buffer the plots from pesticide drift and to insulate arthropod populations within from the dynamics and interactions of those in the adjacent areas.  Estimating the degree of regulation exerted by the natural enemies residing in plots subjected to disruptive effects almost always underestimates the amount of potential biological control (Luck et al. 1999).  Pesticide trials in which a small untreated block within a sprayed area is used to estimate the amount of control from factors other than the pesticide treatments are not adequate in that populations in the unsprayed area are overwhelmed by the dynamics of those in the surrounding treated blocks.

 

                VI.  Techniques For Evaluation

 

                               A.  Introduction / Augmentation of Natural Enemies.--In classical and indigenous biological control, a prey population is expected to be self sustaining.  Control derived from augmentive releases is only temporary, lasting one season or less.  The evaluation of each method poses different problems.  In Classical biological control a natural enemy's impact can be demonstrated by comparing the change in a pest's density in the initial release sites with a control site of similar characteristics but lacking the natural enemy (Huffaker et al. 1962, Legner & Silveira-Guido 1983).  A drop in the pest's abundance in the release site compared with the control site suggests that the natural enemy is responsible for the pest's decline.  This conclusion is further supported if the pest's density in the control site also declines following the subsequent introduction or immigration of the natural enemy to that site.  Replication of release and control sites adds confidence to the evaluation if the pattern of decrease is consistent across the experimental plot.  A similar design can evaluate augmentive releases, but the results may be confounded if closely related or morphologically similar indigenous and released natural enemies attack the same pest (see Legner & Brydon 1966).  However, Oatman & Platner (1971, 1978) showed that release and control plots are never identical ecologically.  Exclusion, inclusion or interference methods are required to assess the difference between resident and released natural enemies.  Introducing genetically marked individuals that differ from the resident population only in the genetic marker can also distinguish between resident and introduced populations (Legner et al. 1990, 1991; Luck et al. 1999). 

 

 

The translocation of natural enemies to areas invaded by pest species and subsequent classical biological control gives additional proof that indigenous natural enemies can have a significant role in regulation of native populations (Wilson 1960, Dowden 1962, McGugan & Coppel 1962, McLeod 1962, DeBach 1964, CIBC 1971, Greathead 1971, Laing & Hamai 1971, Rao et al. 1971, Clausen 1978, Luck 1981, Kelleher & Hulme 1984, Cock 1985).  Further proof is given when the introductions are repeated at several locations with similar results (DeBach 1964, Laing & Hamai 1976).

 

 

B.  Exclusion / Inclusion of Natural Enemies.--Cages and other barriers have been used in exclusion and inclusion procedures to evaluate natural enemies (Smith & DeBach 1942, DeBach et al. 1949, DeBach 1955, Sparks et al. 1966, Lingren et al. 1968, Way & Banks 1968, van den Bosch et al. 1969, DeBach & Huffaker 1971, Ashby 1974, Campbell 1978, Richman et al. 1980. Aveling 1981, Faeth & Simberloff 1981, Frazer et al. 1981b, Jones 1982, Elvin et al. 1983, Chambers et al. 1983, Linit & Stephen 1983, Barry et al. 1984, Kring et al. 1985).  Cages to exclude natural enemies were first deployed by Smith & DeBach (1942), using paired sleeve cages to test whether the introduced parasitoid Metaphycus helvolus (Compere) regulated the black scale, Saissetia oleae (Bern.).  Comparison of the black scale in the open and closed cages showed that less black scale survived in the open cages.  This technique was modified by using insecticide impregnated netting to kill natural enemies that emerged in the closed cages when the methods was used to evaluate other classical biological control projects (DeBach et al. 1949, DeBach 1955, DeBach & Huffaker 1971). 

 

  1.  Cages with different sizes of mesh have been used to exclude natural enemies based on their size (Campbell 1978, Kring et al. 1985).  Three types employed were (1) a complete exclusion cage with small mesh netting and sealed at both ends, (2) a control cage with similar netting and open at both ends and (3) a partial exclusion cage with large mesh netting and closed at both ends.  The latter excluded large predators but allowed access of small predators and parasitoids. 

 

 

                  2.  Sleeve and field cages with more complex designs, such as those which enclosed whole plants, accompanied by samples of the prey and natural enemy populations, showed that the spring increase of predators eliminated black bean aphid, Aphis fabae Scop., colonies on its overwinter host, Euonymus europaenus L., after June (Way & Banks 1968).  If spring aphid populations had been dense on the tree, the predators that remained after the aphids emigrated to their summer hosts prevented recolonization of spindle tree by late fundatrices during the summer, even though the spindle tree was capable of supporting an increasing aphid population.  Closed field cages covered with dieldrin treated netting coupled with hand removal excluded natural enemies from some spindle trees whereas open field cages constructed with slatted walls allowed access of the natural enemies to the aphids on the uncaged trees but provided the same degree of shading as the closed cage (Way & Banks 1958, 1968).  Such experiments and making census of populations on the sample twigs document the importance of predators in excluding aphids from the overwintering host plant during the summertime (Luck et al. 1999).

 

 

                 3.  The evaluation of indigenous natural enemies of cereal aphids was done in large field cages and accompanying population samples.  The experimental design combined field cages erected at several intervals after the aphids immigrated into a winter wheat field.  The growth rates and peak densities of the aphid populations within the cages was compared with those in several open plots of similar size (Chambers et al. 1983).  Samples showed that the abundance of Coccinella 7-punctata L. was negatively correlated with aphid abundance in the open plots but the incidence of parasitism and disease was not negatively correlated with aphid abundance.  These latter two factors were more common in the caged plots.  If the difference between the aphid densities in the cage and open plots was converted to per capita aphid consumption, based on the sampled coccinellid densities, the calculated values were within the range of known values.  Coccinellids appeared to be the key agents limiting the growth rate and peak abundance of cereal aphids during mid season but they were unable to do so early in the season (Rabbinge et al. 1979, Carter et al. 1980).

 

                  4.  Field cages with open field controls were used to determine whether the predator complex aggregated at dense patches of the pea aphid, Acrythosiphon pisum (Harris) (Frazer et al. 1981b).  The cages excluded the predators and allowed the aphid population to increase to about 5X that of the open control plots.  When the cages were removed the aphid populations declined to the densities that prevailed in the control plots and the decline was correlated with increased predator numbers aggregating at the denser aphid patches.  Large field cages have also been used to evaluate the potential of predators in cotton to reduce egg and larval populations of the tobacco budworm, Heliothis virescens (F.) (Lingren et al. 1968).  Evening releases of budworm moths initiated the prey populations within the cages.  Fewer prey survived in the cages with predators than in cages excluding predators.  Similar studies were conducted in California cotton to evaluate predation on the survival of larval populations of the cotton bollworm, Heliothis zea (Boddie) (van den Bosch et al. 1969).  The cotton plants within the predator-free cages were treated with an insecticide to eliminate resident predators before bollworm larvae were introduced.  Significantly fewer prey survived in the untreated cages and significantly more predators were collected from the untreated cages.

 

  5.  In order to determine whether indigenous natural enemies or microclimatic changes within a cage explained the increased survival of caged European corn borer, Ostrinia nubialis (Hübner) larvae, caged and uncaged plots and plots of similar size but enclosed with a cage within a cage were used (Sparks et al. 1966).  The double cages was designed so that the screened panels on the inside cage were opposite that unscreened panels on the outside cage and vice versa.  This arrangement allowed predators access to the plants inside while maintaining the same level of shading and air flow in both the complete cage and cage within a cage plots.  Entomopathogenic fungi (Deuteromycotina) effects were also tested with cages for the black bug, Scotinophara coarctata F., in rice (Rombach et al. 1986a.).  Adult bugs were introduced into screened cages and applications of fungi Beauveria bassiana (Bals.) Vuill, Metarhizium anisopliae (Metsch.) and Paecilomyces lilacinus Thom. were made with a backpack sprayer.  The black bugs were significantly less abundant in all treatments when compared with untreated controls, with effects lasting to nine weeks.  Similarly caged brown planthoppers, Nilaparvata lugens Stal, were treated with entomopathogenic hyphomycetes (Fombach et al. 1986b).  Mortality from fungal infections ranged from 63-98% three weeks after application. 

 

  6.  Ground predators, principally carabids, were excluded with trenches that contained insecticide soaked straw, from the cabbage root fly, Erioischia brassicae (Bouché) (Wright et al. 1960, Coaker 1965).  Polythene barriers were used to exclude predators from two of three treatments in which the predator density was manipulated to determine its effect on the density of aphid populations (Winder 1990).  Sticky bands around selected branches of a spindle tree were used to exclude the walking predators of Aphis fabae (Way & Banks 1968) and around the plant base to exclude walking predators of Trichoplusia ni (Hübner) (Jones  1982).  Sticky circles around Trichoplusia ni eggs were used to exclude predators and parasitoids from attacking the eggs (Jones 1982).

 

  7.  Studies relating cage densities to the densities of resident field populations of predators outside the cages have been used for aphids and Lepidoptera (Frazer & Gilbert 1976, Campbell 1978, Aveling 1981, Frazer et al. 1981b, Chambers et al. 1983), providing useful hypotheses (Way & Banks 1968, van den Bosch et al. 1969, Campbell 1978, Carter et al. 1980, Aveling 1981, Faeth & Simberloff 1981, Frazer et al. 1981b, Chambers et al. 1983).  Cages can provide quantitative information on predation rates (Elvin et al. 1983) but not without limitations.  Small sleeve cages inhibit predator or prey movement and are good for experiments with sessile species or species with low vagility (smith & DeBach 1942).  The abundance of citrus red mite, Panonychus (= Metatetranychus) citri (McG.), within sleeve cages was sometimes 12X greater than outside sleeve cages (Fleschner 1958) even though the mite population outside the cages was kept predator-free by continuous hand removal of predators.  It was thought that the cage prevented the reproductive females from emigrating, that the microclimate within the cages favored rapid growth of the mite population, or both factors influenced population growth (Fleschner et al. 1955, Fleschner 1958).

 

  8.  It is not possible to identify which members of a predator/parasitoid complex are regulating a host population with exclusion cages unless the complex consists of one or a few species (Jones 1962).  Partial exclusion cages may show whether small predators, pathogens or parasitoids regulate in the absence of large predators, but they cannot show whether large predators regulate prey in the absence of parasitoids or small predators (Luck et al. 1999).  Cages may also inhibit predator or prey movement or interfere with natural enemy oviposition.  Two leaf mining species on oak failed to reproduce within whole tree cages and a third species failed to reproduce in one cage (Faeth & Simberloff 1981).  Aphid alates cannot emigrate from a cage, thus caged versus uncaged aphid populations may show differences in density because alate immigration reduces the uncaged aphid population.  Some predator species aggregate at patches of high prey density in a numerical response (Readshaw 1973, Frazer et al. 1981a. Kareiva 1985).  Such behavior may be inhibited by cage size because the spatial pattern in nature to which the predator species responds is larger than that present within the cage.  Also, confining predators to a cage may causae them to search areas more frequently and thereby increases the likelihood that they will encounter prey.  Under these conditions the predator may reduce prey densities to levels below normal, and in this way inclusion studies resemble laboratory experiments in which predators are confined with prey (van Lenteren & Bakker 1976, Luck et al. 1979). 

 

  9.  Erroneous interpretations can result when prey are placed into a cage without consideration of their preferences for oviposition sites, their density and distribution patterns or their preferred feeding sites under field conditions.  Some predators and parasitoids use kairomones to find their prey and hosts (Hassell 1980, Nordlund et al. 1981).  Some kairomones are associated with feeding activity.  Placing prey or hosts in new sites influences their risk of detection.  Food quality may affect a phytophage's feeding time and increase its risk to predation because of the kairomones released while feeding (Nordlund et al. 1981).  Detailed studies of a predator's searching behavior and capture rates and a prey's oviposition and feeding behavior are important (Fleschner 1950, Dixon 1959, Frazer & Gilbert 1976. Gilbert et al. 1976, Rabbinge et al. 1979, Carter et al. 1980. Baumbaertner et al. 1981, Frazer & Gill 1981, Sabelis 1981).

 

  10.  Whenever predator free controls are employed, it is difficult to exclude all predators, even when they have been treated with insecticides (van den Bosch et al. 1969, Irwin et al 1974, Elvin et al. 1983).  Some predators may pass through excluding screens when in small developmental stages (Sailer 1966, Way & Banks 1968), or they are difficult to exclude because they become buried in the soil (Frazer et al. 1981a, Elvin et al. 1983).  Cages also alter the microclimate through shading and inhibiting air flow.  Exclusion and partial exclusion cages using terylene netting reduced the light intensity inside cages by 24-37% (Campbell 1978) and saran screen reduced solar radiation by 19% (Hand & Keaster 1967).  Such shading required the use of a more shade tolerant cotton cultivar than was normally planted in the region (van den Bosch et al. 1969).  Shading also affects plant physiology and thus may affect the plant's quality as a substrate for the host or prey population (Scriber & Slansky 1981).  Temperatures within cages used in a corn borer study were 8-10°F lower than the temperature outside.  The humidity fluctuated more moderately within and was 5-10% higher than that outside the exclusion cages (Sparks et al. 1966). 

 

 

  11.  Solar radiation changes cause differences in leaf temperature by as much as 13°C (Hand & Keaster 1967).  Leaf temperatures and moisture availability influence photosynthetic rates and evapotranspiration (Gates 1980).  Leaf temperatures probably affect the behavior and feeding rates of phytophagous hosts and prey.  Temperature related interactions between the growth rates of aphids and the searching rates of their predators are important (Frazer & Gilbert 1976, Frazer et al. 1981a).  Screening also reduced wind speed within a cage by as much as 48% (Hand & Keaster 1967) which, depending on RH and wind velocity outside and inside a cage, influences the leaf's boundary layer within the cage (Gates 1980, Ferro & Southwick 1984).  Instrumentation allows the monitoring of many of these effects but their influence on predator/prey interactions must be assessed (Luck et al. 1999).

 

C.  Removal by Insecticide Treatment.

 

  1.  Natural enemy complex impact may be assessed through the application of insecticides.  The method was first used to kill natural enemies of the long-tailed mealybug, Pseudococcus longispinus (Targ.), without affecting the mealybugs (DeBach 1946).  Insecticides have been used to determine whether indigenous predator populations in cotton suppress populations of the beet armyworm, Spodoptera exigua (Hübner), and cabbage looper, Trichoplusia ni (Ehler et al. 1973, Eveleens et al. 1973).  Early season insecticides applied to cotton were thought to interfere with natural controls (Ehler et al. 1973, Eveleens et al. 1973).  Large blocks (3-4 square miles) were treated with an insecticide scheduled during early season, early and midseason and early, mid- and late season.  A fourth plot served as an unsprayed control.  Samples and observations showed that the absence of predators in the treated plots was correlated with the increased survival of beet armyworm eggs and first generation small larvae of the cabbage looper.  The hemipteran predators, Geocorus pallens Stal, Orius tristicolor (White) and Nabis americoferus Carayon were implicated as the most important predators since they were the most affected by the treatments whereas Chrysoperla carnea Stephen was not so strongly affected.  Insecticide treatment showed that the suppression of cabbage looper densities in celery arising from egg parasitism by Trichogramma spp. and predation of eggs and young larvae by Hypodamia convergens Guer. and O. tristicolor was sufficient to prevent economic damage before the production of the first marketable petiole in celery (Jones 1982).

 

  2.  Insecticides were also used to test whether the coccinellid, Stethorus sp. regulated the density of the two spotted spider mite, Tetranychus urticae (Koch), in a previously untreated apple orchard in Australia (Readshaw 1973).  Two applications of malathion increased the density of the mite populations.  Tetranychus urticae, unlike the predator fauna associated with it, was resistant to malathion.  Stethorus regulated the mite population by numerically responding both aggregatively and reproductively to the denser mite patches.  Even with insecticide disruption and stimulation of the mite reproduction (Chaboussou 1965, Bartlett 1968, van de Vrie et al. 1972, Dittrich et al. 1974), Stethorus was able to prevent the mite population from attaining an economic density of 100 mites/leaf on most trees.

 

 

  3.  The action of two parasitoids of the olive scale, Parlatoria oleae (Colvee), was evaluated using insecticides (Huffaker & Kennett 1966).  This scale is bivoltine on olive in the San Joaquin Valley of Calviornia.  One generation occurs during the autumn and spring and the second generation during summer.  Aphytis paramaculicornus DeBach & Rosen and Coccophagoides utilis Doutt was introduced for biological control (Rosen & DeBach 1978).  Aphytis dominated during the autumn and spring scale generation whereas Coccophagoides dominated during summer.  Three DDT treatments were used to exclude the parasitoids: (1) a spring treatment to exclude Aphytis, (2) as summer treatment to exclude Coccophagoides and (3) a spring and summer treatment to exclude both parasitoids.  Untreated trees were left as controls.  It was thought that DDT residues on the foliage and twigs inhibited the parasitoids but did not affect the scale's reproduction and survival.  Treatments which excluded Coccophagoides had higher scale densities than the untreated controls but lower densities than the treatments which excluded Aphytis.  Treatments that excluded only one of the parasitoids had lower scale densities than treatments that excluded both parasitoids.  Treatments also indicated that together the parasitoids provided better biological control than either did alone even though the mortality contributed by Coccophagoides was only about 5%. 

 

  4.  Inoculation of fumigated (12 hrs with methyl bromide) and unfumigated poultry manure with  Musca domestica L. eggs demonstrated 53.4 to 99.4% mortality in the presence of predatory and scavenger arthropods (Legner 1971).  Significant negative correlations of parasitization with increasing host densities were explained by parasitoid behavior.  Inherently, single female parasitoids without interference from other individuals of the same or different species respond positively with increases in host density; parasitization rates increase, which appears to be correlated with increases in the production of progeny (Legner 1967).  However, when groups of parasitoids concentrate their search among several host pupae, as is common in nature, their efficiency per female is decreased through mutual interference, that apparently involves combinations of physical interruption and chemical effects.  There was some evidence that female parasitoids were strongly attracted to denser concentrations of their hosts in their habitat (e.g., Legner 1969), which evidence further tends toward increases in the interference factor at natural high host densities.  Furthermore, any interference that would deter some female parasitoids from oviposition during the first few days of adult life would lower fecundity and longevity (Legner & Gerling 1967).  Operating collectively, these several forces would tend to produce the observed apparent negative correlation between parasitization and host density. 

 

  5.  Several problems are associated with interpreting results from an insecticide treatment, however.  The pesticide may stimulate reproduction of the prey population.  There may be a pesticide induced sex ratio bias, and pesticide induced physiological effects on the plant may arise.  Mites that are exposed to sublethal doses of some pesticides are stimulated reproductively and occasionally even increase female biased sex ratios (Charboussou 1965, Bartlett 1968, van de Vrie et al. 1972, Dittrich et al. 1974, Maggi & Leigh 1983, Jones & Parrella 1984).  Such effects may also extend to aphids (Bartlett 1968, Mueke et al. 1978), and delphacids (Chelliah et al. 1980, Reissig et al. 1982).  Differential mortality resulting from pesticide treatments has also been reported.  Male black pineleaf scale, Nucalaspis californica (Coleman) (Edmunds & Alstad 1985), and California red scale, Aonidiella aurantii (Maskell) (Shaw et al. 1973) are more susceptible to pesticides than females.  Plant physiology is also affected by insecticide applications (Kinzer et al. 1977, Jones et al. 1983).  Row crops treated with certain insecticides become attractive oviposition sites for Lepidoptera (Kinzer et al. 1977).  Interactions between aphid reproduction, insecticides and cultivars have been reported on alfalfa (Mueke et al. 1978).  Knowledge of the biology and interactions is required to properly time an insecticide application to disrupt the natural enemy populations while minimizing their effects on prey or host.  Because insecticides potentially stimulate arthropod reproduction and effect plant physiology, estimates of predation rates with this exclusion method should be done cautiously.  Although insecticide treatments stimulated the brown planthopper, Nilaparvata lugens Stal, reproduction, the amount of stimulation could not account for the high levels of resurgence.  Only the reduction of natural enemies could.  Insecticides can be used to determine the relative importance of natural enemies when the complex is composed of a few species showing temporal separation of their effects, in seasonal occurrence or in the generations they attack (Luck et al. 1999).

 

 

D.  Removal of Natural Enemies by Hand.

 

  1.  Although laborious, hand removal has been used to evaluate the predators of tetranychid mites on citrus and avocado and to compare results obtained with other exclusion methods (Fleschner et al. 1955, Fleschner 1958).  It has also been used to evaluate the mirid, Crytorhinus fulvus Knight, introduced to control the taro leafhopper, Tarophagus proserpina (Kirkaldy) (Matsumoto & Nishida 1966).  Predation of Aphis fabae was also assessed in part by removing adult predators by hand when they flew onto predator free branches (Way & Banks 1968).  A sticky band at the base excluded walking predators from feeding on A. fabae individuals placed on the branch.

 

  2.  Luck et al. (1999) believe that the hand removal method deserves more attention, especially as a method of checking for bias in other exclusion methods.  However, it seems to be limited to studies of predator/prey interactions with species of low vagility, those that occur at reasonable densities and are diurnally active or are undisturbed by night lights (Luck et al. 1999).

 

E.  Prey Enhancement.

 

 

  1.  Prey may be placed directly on plants in the field to stimulate predator attraction.  This procedure involves tethering prey to a substrate (Weseloh 1974, 1982) or placing them on leaves or other plant parts where they would normally occur (Ryan & Medley 1970, Elvin et al. 1973, van Sickle & Weseloh 1974, Weseloh 1974, 1978, 1982; Torgensen & Ryan 1981).  Some studies marked the prey with dyes before placing them in the field (Hawkes 1972, Elvin et al. 1973).  The prey were visited frequently to measure predation, and if predation was observed, the predator's identity was noted.  Predators such as spiders can be observed in the field with their prey *Kiritani et al. 1972), and web spinning spiders leave cadavers in or beneath their webs (Turnbull 1964).

 

  2.  It is sometimes more practical to use greenhouse grown plants of the same age, size and variety as plants used in field studies.  Plants can be caged in the greenhouse or field for pest oviposition.  Then the infested plants are transferred to the field and monitored for parasitism and predation.  Van der Berg et al. (1988) used eggs of several foliage-feeding rice pests to determine predation.  The egg chorion showed that eggs were attacked by predators with chewing or sucking mouthparts.

 

  3.  Predation and parasitism was thought to alternate as principal mortality factors during the year in studies that followed the seasonal incidence of predation and parasitism of eggs of the yellow stemborer of rice, Scirpophaga incertulas (Walker) (Shepard & Arida 1986).  The technique of prey enhancement may be used to advantage with cages and or insecticides.  However, a major limitation is that prey must be limited to sessile forms such as eggs, pupae or some scale insects, although there are possibilities with tethered hosts (Weseloh 1974).  Kairomones and other chemical cues may be important to establishing the appropriate interaction (Nordlund et al. 1981). 

 

 

VII.  Methods For Detecting Predation/Parasitism

 

A.  Serology.

 

                 1.  Predators have been associated with their prey with serological methods (Dempster et al. 1959, Dempster 1960, 1964, 1967; Rothshild 1966, 1970, 1971; Frank 1967, Ashby 1974, Vickermann & Sunderland 1975, Boreham & Ohiagu 1978, Sunderland & Sutton 1980, Gardner et al. 1981, Greenstone 1983).  Predations rates have also been estimated with serology (Dempster et al. 1959, Dempster 1960, 1964, 1967).  A precipitin assay has been also used (Boreham & Ohiagu 1978, Ohiagu & Boreham 1978, Southwood 1978).  Other methods are the enzyme-linked immunosorbent assay (ELISA) (Vickermann & Sunderland 1975, Fichter & Stephen 1979, 1981, 1984; Ragsdale et al. 1981, Crook & Sunderland 1984, Sunderland et al. 1987, Sopp & Sunderland 1989), and an assay based on passive hemagglutination inhibition (PHI) (Greenstone 1977, 1979).  Agglutination assay employs polystyrene latex particles coated with antibody (Boreham & Ohiagu 1978, Ohiagu & Boreham 1978).  Such methods detect prey particles in the gut of predators by its reaction with antibodies obtained from a vertebrate, such as a rabbit, that has been sensitized to the prey.  The reaction is a visible precipitate.  (Also see Boreham & Ohiagu 1978, Miller 1978 and Sunderland 1988).

 

  2.  Detection of prey in a predator's gut is influenced by the size of prey, size of meal, time since the meal was taken, the rate of digestion, whether the natural enemy is a sucking or chewing predator, the abundance of taxonomically closely related prey and the sensitivity of the test.  Sensitivity of the assay can be increased if the antibody is linked to an enzyme (ELISA).  When the antibody reacts with prey, the enzyme carried with the antibody allows amplification of the reaction because one enzyme molecule can convert many molecules of substrate.  This assay may detect hemolymph dilutions of more than 260,000 (Fichter & Stephen 1981) and is often sufficient to differentiate among prey stages (Ragsdale et al. 1981).

 

  3.  Both precipitation and ELISA techniques are useful for identifying the prey in a predator's diet and estimating predation rates (Sunderland 1988).  ELISA is more sensitive to the presence of small amounts of antigen (prey protein or carbohydrate), is suitable for large scale testing and can be used with a minimum of equipment.  Material necessary for the tests may be prepared and stored under refrigeration for six months (Sunderland 1988).

 

  4.  The passive haemagglutination assay (PHA) is a method for increasing sensitivity of the precipitin test.  Sheep red blood cells (rbc) are chemically coated with the antigen of the suspected prey.  Antigen coated rbc's are added to a solution of specific antibody and combine with the antigen molecules on the rbc to form a mat (agglutination).  Small amounts of antibody cause agglutination.  In antibody-free controls the rbc's do not agglutinate and this inhibition forms the basis of the assay.  The amount of antibody required to cause agglutination is determined and added to an extract of a predator's gut contents.  If prey protein or carbohydrate (antigen) is present it binds with the antibody.  When antigen coated rbc's are added, they will not agglutinate because the antigen from the predator's gut has been bound by the antibodies (Luck et al. 1999).  A small amount of antigen produces inhibition which explains the assay's greater sensitivity than that of a comparable precipitin assay (Greenstone 1979).  Freshly sensitized erythrocytes have to be prepared each time an assay is conducted (Boreham & Ohiagu 1978), and this requires skilled operators.

 

                                  5.  The precipitin test was originally used to document arthropod predation of mosquito larvae (Bull & King 1923, Hall et al. 1953, Downe & West 1954) and latter was applied to terrestrial predator/prey interactions (Downe & West 1954).  The first prey for which estimates were attempted from field samples was a chrysomelid beetle Gonioctena (= Phytodecta) olivacea (Forster) feeding on broom (Dempster 1960).  Tests revealed six mirids, two anthocorids, a nabid, a dermaptern and red mites feeding on the beetle in the field.  Laboratory tests showed that only the older mirid and anthocorid stages fed exclusively on younger stages of G. olivacea.  A single laboratory feeding by the mirids and anthocorids could be detected 24 hrs after they had ingested a meal, and feeding by a dermapteran could be detected 60 hrs after it had fed (see Luck et al. 1999).

 

                                  6.  The degree of overlap between older stages of the predator and younger stages of the beetle influenced the number of beetles preyed upon.  Densities of prey and predators were estimated from field samples.  The fraction of positive responses in predator samples estimated the fraction of the predator population that had fed on G. olivacea.  Because G. olivacea were scarce in the field while alternative prey were abundant, encounters between G. olivacea and the predators were infrequent.  Therefore, if a predator tested positive to G. olivacea antibody, it was interpreted as a single predation event.  Then the number of beetles preyed upon by each predator could be estimated suing the equation:

 

                            Pa = (NpiFpiTpi) / Rpi

 

 

where Pa is the number of prey killed; Npi the density of the predator (or stage of predator) i; Fpi the fraction of positive tests of the ith predator in a sample; Tpi the duration in days that the appropriate prey and predator stages are coincident in the field; and Rpi the retention time of a single prey feeding by ith predator (or stage of predator).  Estimates from the precipitin test of egg and larval mortality due to predator for two beetle generations were found to agree closely with the independent estimates of "unknown" losses of eggs and young larvae during the same two beetle generations (Richards and Waloff 1961). 

 

                7.  The precipitin test also was used to identify the predator species and to determine the fraction of Pieris rapae (L.) eggs and young larvae that died due to predation (Dempster 1967).  Because of the relative scarcity of P. rapae a positive precipitin test was interpreted as one predation event.  Studies of the delphacid Conomelus anceps (Germar) employed precipitin tests to identify ten of 91 potential predators (Rothchild 1966).  The precipitin test could not be used to estimate predation rates because multiple predation events were possible. 

 

                 8.  For estimating predation rates with the precipitin test it is necessary to have information about predator and prey densities, densities of alternate prey, the period during which a meal can be detected in each predator and prey and predator stages involved.  Precipitin tests estimate predation rates of prey which form a small fraction of the available prey or infrequent predation events.  A slight bias may arise in such estimates if predators have fed on other predators that have fed on the prey, if a suspected predator is phytophagous but ingests sessile prey stages while feeding on the plant or if a suspected predator feeds on prey carrion (Boreham & Ohiagu 1978).  The precipitin test may also yield biased estimates of predation rates from cross reactions between the antibodies of closely related species.  Therefore, a knowledge of the local fauna which might serve as prey and the predator's propensity for local movement is essential to the successful application of this test (Luck et al. 1999).  Also the serum developed from one prey stage may not react with the antigen of another (Boreham & Ohiagu 1978).  Sufficient resources must be committed in order to use this technique:  prey must be collected in sufficient numbers to elicit an immunological response when injected into the vertebrate.  As such the procedure is not ideal when applied to small prey such as mites (Murray & Solomon 1978).

 

                                9.  When used in conjunction with other population studies, precipitin assays may be very helpful.  Few other methods can provide quantitative estimates of predation rates under natural field conditions.  Although they cannot be used to estimate predation rates under all situations, they are valuable for identifying predator species or stages that feed on a prey.  This method deserves more attention especially as more sensitive tests such as ELISA are available (Vickermann & Sunderland 1975, Fichter & Stephen 1981, 1984; Ragsdale et al. 1981, Crook & Sunderland 1984, Sunderland et al. 1987, Soop & Sunderland 1989).  A great advantage is that predation is allowed to occur naturally.

 

                  B.  Electrophoresis & Isoelectric Focusing.

 

                                1.  Predators may be associated with the prey with electrophoretic techniques.   Electrophoresis separates proteins based on charge and size differences in an electrical field.  Differences in charge and size commonly occur among isoenzymes (proteins catalyzing the same reaction) from different taxa.  If the prey and predator have isoenzymes with different electrophoretic mobilities, the analysis of homogenates prepared from predators fed on prey should exhibit protein bands corresponding to the predator and the prey.  Also if there are several potential prey of a predator, and if the prey have electrophoretically distinct isoenzymes, analysis of predator homogenates can reveal the prey species inside the predator.

 

                                 2.  Electrophoresis can be successful if the prey isoenzymes are detectable after predator feeding, and electrophoretic variation occurs among the prey and predator isoenzymes.  Isoenzyme detection depends on prey size, in vitro activity of the isoenzyme, presence and volume of the predator foregut, and the type of electrophoresis employed (Murray & Solomon 1978, Giller 1984, Lister et al. 1987, Soop & Sunderland 1989).  Electrophoretic variation depends on the suite of isoenzymes available for comparison and the type of electrophoresis.  Standard electrophoretic procedures (starch gel and polyacrylamide gel electrophoresis) can detect prey isoenzyme activity for several isoenzyme types involving relatively large prey (>2-3 mm body length).  Under this size, the number of detectable prey isoenzymes is diminished and hence the chance of distinguishing closely related prey is decreased.

 

                                 3.  Enhanced sensitivity of electrophoretic methods include conventional electrophoresis in cellulose acetate membranes (Easteal & Boussy 1987, Höller & Braune 1988) and isoelectric focusing (IEF) (see Luck et al. 1999).  IEF has advantages over other techniques involving small and large prey.  In IEF, proteins are "focused" into narrow bands along relatively broad pH gradients.  Focusing enhances the detection of enzymes compared to other techniques which gradually spread the proteins into diffuse bands.  In addition, because relatively broad pH gradients are used in IEF, enzymes with different charges, such as may occur between unrelated prey taxa, will remain sharply focused on the gel.  The fine resolution of IEF does not affect the ability to distinguish enzymes with very similar charges.  With standard techniques, these contrasting problems are difficult to solve simultaneously as one set of conditions (buffer type and pH, gel type) may be optimal for one prey type but not others.

 

                                4.  The prey of several arthropod predators have identified with electrophoresis.  Polyacrylamide gradient gel electrophoresis was used to detect prey protein (esterases) in the gut of predators after they had fed on known prey (Murray & Solomon 1978).  The technique detected esterases of Panonychus ulmi (Koch) in the predaceous mite Typhlodromus pyri (Scheuten), and in two anthocorids, Anthocoris nemoralis (F.) and Orius minutus (L.) that had fed on the mite in the laboratory.  Dicke & DeJong (1986) used methods to determine whether T. pyri and Amblyseius finlandicus (Oudemans) also fed on the apple rust mite, Aculus schlechtendali (Nalepa) as an alternate host in the field.  Electrophoresis was also used to identify the prey species exploited by A. nemoralis on alders in the field (an aphid Pterocallis alni [DeGeer]) (Murray & Solomon 1978).  Electrophoresis with polyacrylamide disc gels detected esterases of several prey species in the gut of the waterboatman Notonecta glauca L. (Giller 1982, 1984, 1986).  A meal was detectable from 17-48 hrs depending on temperature and meal size, and was strongly correlated with the length of time the meal spent in the foregut (Giller 1984).  Giller (1986) used electrophoresis to identify the prey of N. glauca and N. viridis Delcourt in the field.  Lister et al. (1987) used polyacrylamide gel electrophoresis and electrophoresis and esterase allozymes to determine the diet of some microarthropods and the predation rate by the acarine predator Gamasellus racovitzai (Trousessart).

 

  5.  Predation rate estimates with serological methods and electrophoresis requires substantial resources.  The techniques call for the development of antibodies or methods for identifying the isozymes of the prey species or stage, the development of methods to estimate the predator and prey densities, including those needed to estimate the densities of alternate prey, and the identification of the predator and prey stages involved.  Initially the use of these techniques to estimate predation rates appeared limited to prey populations which form a small fraction of the available prey or in which predation events by a predator are frequent.  Frequent predation confounds interpretation of a positive test because a single large meal cannot be distinguished from several small meals.

 

                  6.  Immature parasitoids within aphids have been detected with electrophoresis (Wool et al. 1978, Castanera et al. 1983), and in whiteflies (Wool et al. 1984).  The parasitoid Aphidius matricariae Hal. was detected in the green peach aphid Myzus persicae (Sulz) and parasitism of the white fly, Bermesia tabaci (Gennadius) by the endoparasitoids Encarsia lutea (Masi) and Eretmocerus mundus (Mercet), was detected with electrophoresis and histochemical staining for esterases.  But the whitefly parasitoids could not be identified to species (Wool et al. 1984).  Electrophoresis allows the processing of large numbers of hosts to estimate the fraction that are parasitized and sometimes the parasitoid species involved.  This contrasts with the traditional methods in which field samples are dissected while fresh or reared.  Electrophoresis can detect within a host immature parasitoids without dissection and parasitoid enzyme activity within a prey cannot be confused with host's enzyme activity.

 

C.  Marking Prey.

 

                   1.  Predator species and/or predation rates have been identified with marking techniques.  Markers have included radioactive isotopes -151europium (Ito et al. 1972), 32phosphorus (Jenkins & Hassett 1950, Pendleton & Grundmann 1954, Jenking 1963, McDaniel & Sterling 1979, McCarty et al. 1980, Elvin et al. 1983) and 137cesium (Moulder & Reichle 1972), 14carbon (Frank 1967), rare elements (Stimmann 1974, Shepard & Waddill 1976), and dyes (Hawks 1972, Elvin et al. 1983).  Prey are fed (Elvin et al. 1983, Frank 1967, Room 1987) or injected (McDaniel & Sterling 1979, McCarty et al. 1980) with the radioactive isotope and the radioactivity is detected in a predator with scintillation, a Geiger counter, or autoradiography.  For autoradiography suspected predators are collected after exposure to labelled prey and are glued to paper, which is placed against X-ray film (McDaniel & Sterling 1979).  The film is developed, and dark spots on the film produced by the rays from 32phosphorus indicate labelled predators.  Methods involving isotopes require training and necessary equipment to perform the assays.  Safety regulations and environmental considerations may limit the use of the method in some situations.  Other disadvantages, as with electrophoresis and serological techniques, include the inability to detect whether a predator had fed on other predators that had consumed labelled prey or whether a prey was scavenged (Luck et al. 1999).  Experiments using isotopes, especially those using autoradiography, are simpler to conduct than serological and related techniques.  Methods using labelled elements require several manipulations, but they provide more information per unit effort than other kinds of marker tests.

 

                   2.  Such rare elements as rubidium and strontium also have application as labels.  They can be sprayed on foliage or placed in the diet of the prey, incorporated into the prey's tissues and then transferred to the predators or parasitoids who feed on labelled hosts (Stimmann 1974, Shepard & Waddill 1976).  The mark should be retained for life, and self-marking is possible via a labelled plant.  However, the technique requires an atomic absorption spectrophotometer, which is expensive, and placement of the labelled prey on plants may expose them to abnormal predation rates.  Phytophages seldom choose feeding or oviposition sites on their plant hosts at random (Ives 1978, Wolfson 1980, Denno & McClure 1983, Guerin & Stadler 1984, Whitham et al. 1984, Myers 1985, Papaj & Rausher 1987).  Parasitoids and predators do not search their habitats uniformly (Weseloh 1974, 1982; Fleschner 1950).  Therefore, without the proper behavior studies, the degree of bias in determining the natural enemy complex or in estimating predation rates is unknown.

 

 

                                   3.  Genetic markers have been used to track parasitoids and assess their impact against hosts, such as common muscoid flies.  Legner & Brydon (1966) liberated a thelytokous race of parasitoid on poultry farms which they were able to tract and derive host mortality data from.   Legner et al. (1990, 1991) derived similar information by releasing gregarious strains of Muscidifurax raptorellus Kogan & Legner, and a temporary interference of several weeks with resident parasitism during the establishment phase was detected.  However, this was later overcome when the released strain had a chance to multiply naturally at the site.

 

                 D.  Visual Counts.