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EXPERIMENTAL TECHNIQUES TO
EVALUATE NATURAL ENEMIES
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Introduction In order to improve success rates in biological control, an
understanding of events in past successful introduction programs is essential
Luck et al. (1999). Successful cases can be used to test hypotheses about
predator/prey interactions, and develop criteria for identifying effective
natural enemies. Bellows & Van Driesche (1999) review the analytical
bases developed in the late 1980's to estimate total losses from parasitism.
Thay state 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). 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)." Bellows
& Van Driesche (1999) continue, "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." 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) deat rate analysis, and (4) experimental manipulations in
the field. The primary goal is to determine whether regulation of the host
population exists and to identify the agents responsible for regulation. 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. 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). There
is probably no single method which can provide conclusive evidence that
natural enemies are regulating a population. 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). 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). 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). The
development of an appropriate sampling routine is essential for the evaluation of natural
enemies. 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. 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. 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). 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). When
evaluating indigenous natural enemy populations, it is necessary to determine
whether biological control of the hosts exists. 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. Time is required to
reestablish interactions between natural enemy and prey/host populations.
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. 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. 1990a, 1990b; 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). 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). 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. 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). 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). 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. 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.
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). 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). 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). 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). 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). 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). Removal by
Insecticide Treatment.--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). 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. 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%. 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. 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). Removal of
Natural Enemies by Hand.--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. 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). Prey Enhancement.--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). 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. 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). Methods
For Detecting Predation/Parasitism Serology.--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). 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). 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). 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. 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). 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). 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. 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). 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. Electrophoresis &
Isoelectric Focusing.--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. 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. 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. 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). 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. 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. Marking Prey.--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. 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. 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. ( 1990a, 1990b ) 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. Visual Counts.--There are
several advantages of using visual counts over many of the exclusion
techniques. There is no manipulation of the environment required. Prey can be
added or predators removed to determine the response of the predator to
changes in prey density. A visual record reveals the predator's diet in the
field. Perhaps serology and electrophoresis share these three advantages, but
the latter require considerable technology. visual counts require a
substantial commitment of time to observe the predation and to determine the feeding
rates for different combinations of predator/prey stages. Vision cannot be
used if the predator is cryptic, easily disturbed or escapes from the
observer. Also, the time a predators spends consuming a prey may vary
depending on the range of prey stages attacked, the hunger level of the
predator, interference or stimulation by other predators or prey that are
active in the area, and differences among individual predators due to genera,
reproductive stage or molting (Luck et al. 1999). These in turn can determine
the probability that a predator will be observed in the field with a prey.
Laboratory data on the time spent by four predators consuming prey was highly
variable, leaving the investigators pessimistic about the visual method's
utility for estimating predation rates (Kiritani et al. 1972). But the
approach may still be valid for some predators, and has been used to
determine the fraction of diurnal predation for each predator species in a
complex (Elvin et al. 1983). Statistical Sampling.--Obtaining
field samples during parasitoid/predator liberation periods can provide
useful information about the ability of a species to effect its host/prey
density. Parasitoid impact was thus measured on the pink bollworm, Pectinophora gossypiella (Saunders). A significant
positive relationship was found between the total number of parasitoids
released and the host density, which was most pronounced during a mid autumn
period (Legner & Medved 1979). Releases of
egg-larval and larval-parasitoids produced small measurable reductions in P. gossypiella moth emergence from mature cotton bolls, but
did not significantly reduce % boll infestation. By releasing parasitoids at
three densities, it was possible to show significant differences between
controls and a low and medium release rate, but excessive parasitoid
dispersal out of the release areas into the cotton field at large explained a
leveled slope after the medium release rate (Legner & Medved 1979). The
potential of Goniozus spp.
and Pentalitomastix plethorica Caltagirone to
regulate navel orangeworm, Amyelois
transitella (Walker) was
judged from seasonal positive functional responses to host density and with
k-value analyses (Varley et al. 1974, Legner & Silveira-Guido 1983 ). Application
of this technique requires that the host show minimal overlapping of
generations, however. Goniozus
emigratus (Rohwer) and Goniozus legneri Gordh demonstrated a significant capacity to
recognize and respond in a regulative fashion in mid summer by increasing
attack rates on higher host densities. However, no such tendency was
indicated during cooler periods of late autumn. Indigenous
parasitism of Rhagoletis completa Cresson in its native
range of western Texas and southeastern New Mexico was also assessed with
k-value analysis,which showed a significant impact of combined natural
mortality on host reduction (Legner & Goeden 1987). Biosteres
sublaevis Wharton
demonstrated the greatest measurable activity as a cause of natural
mortality. Legner
& Brydon (1966 ) were able to
show an increased parasitism and house fly host mortality closer to
liberation sites of parasitoids. Legner et al. (1990a, 1990b ) also charted
increases and spread of muscoid fly parasitism from release sites. The importance
of proper field sampling, measurement of host destruction and unpredicted
upsets to organisms in different guilds in these and similar studies was
emphasized (Legner & Bay 1964, Legner 1979, Legner 1983a, 1986 ). Long
term sampling of the width of aquatic weed masses following the introduction
of phytophagous cichlid fish established a regulatory capacity for Tilapia zillii Gervais in California irrigation canals (Legner
& Fisher 1980 , Legner &
Murray 1981). The data clearly showed seasons of high,
medium and low regulatory capacity. MOLECULAR TECHNIQUES Unruh
(1999) discussed various molecular techniques and procedures as they may be
applied to classical biological control work. Diehl & Bush (1984) pointed
out that emphasis is often to seeking new races or biotypes of beneficial
species which had demonstrated partial control of a pest. Usually this effort
is directed at finding races which are better suited for climates in specific
parts of the pest's range. Collections of morphologically identical
populations of a natural enemy from geographically separate and ecologically
distinct habitats often result. In such cases scientists are left with the
problems of evaluating the subsequent collections for their inherent
variability for a variety of biological attributes, such as climatic,
reproductive, sexual or host preference. Populations of the various races are
usually released in a random way followed by biological studies of those
which become established. Such studies have revealed cryptic species or races
with distinct biological attributes, as exemplified by host specific races of
Comperiella bifasciata (Howard) which
attack the Chinese race of the California red scale, and the Japanese race of
the yellow scale (Diehl & Bush 1984). Nature screens the adaptive
diversity of natural enemies, which is believed more accurate and cost
effective than any regimen of prerelease screening (van Lenteren 1980). This
procedure nevertheless precludes the development of new procedures and theory
for evaluating and using races that are morphologically indistinguishable
(Unruh 1999). Three
frequent errors of omission in many biological control programs are (1) the
low priority allotted to pre- and post-release evaluation of natural enemy
effectiveness. Traits such as developmental rates, diapause, host range, sex
ratio and resistance to encapsulation are of special importance; (2) the
tools for identifying and studying infraspecific categories of natural
enemies both prior and subsequent to their release are not used; and (3)
descriptions of the ecological and genetic relations of natural enemies in
their native distribution are rare. In
spite of increasing sophistication in the theories of natural enemy/pest
interactions, it is still not possible to choose the best natural enemies
even when give sound ecological studies in a laboratory or in the native
range (van Lenteren 1980). In order to improve the understanding of
biological control introductions, particularly those employing biotypes, it
is necessary to have better methods for discriminating populations. Traits to
identify populations must reside in the organism itself. although short term
field or laboratory studies may often employ dyes, heavy metals or
radioactive tracers. Where morphology cannot distinguish, the next level of
identification is biological or molecular markers. In some instances where
biological traits under study change through time, molecular differences must
be sought among species, races, strains or even individuals. Molecular traits
reflect a natural hierarchical organization that begins with the DNA sequence
and ends with the protein composition of the organism (as well as other
phenotypic expressions). DNA sequences are the most difficult and expensive
to obtain and estimates of the electrophoretic mobility of particular
proteins, such as enzymes, are the least expensive and most straightforward. Hutchinson
(1965) compared evolution to a play and the ecological environment to a
theater. Unlike most researchers of natural variation in organisms,
biological control workers remove populations from their home town
theaters and attempt to run
many plays simultaneously at
an out of town stage (Unruh 1999). The
problems encountered may partially explain the seeming preoccupation with the
concepts of biotype, race, geographic, sibling, semi-, and cryptic species
(DeBAch 1969, Rosen & DeBach 1973, Rosen 1978, Gonzalez et al. 1979). In
biological control it must be considered how previous evolutionary history affects
adaptation to a novel ecological milieu. The ecological and genetic dynamics
which arise when two or more species are released into the same area against
the same host need to be understood. When questioning the likelihood of pre-
and postzygotic isolating mechanisms of geographic species, the query is more
than academic. The idea that biological control programs represent
evolutionary experiments on a grand scale (Myers 1978) is important, but very
little effort has been expended on postrelease study of natural enemy
adaptations to their new home. Only concerted effort in these aspects of a
biological control program can transform an empirical practice into an
evolutionary experiment. Molecular
methods are important for biological control research because they represent
a set of tools to employ in dissecting the biology of natural enemies (Unruh
1999). Sometimes these tools may be irreplaceable but in many cases the
questions posed may be addressed by a different route. Using molecular
methods to the exclusion of more traditional methods, or at the expense of
developing navel methods, is not logical. Avoiding molecular methods because
of a perception that they are too complicated is also foolish. With natural
enemies systematic investments in molecular approaches should yield rapid
returns. However, studies of the biological control practice of importing few
specimens, rearing them in the laboratory and releasing their progeny into a
hostile environment may uncover radical effects associated with colonization
(Unruh 1999). Hunter
& Markert (1957) and Unruh (1999) discussed gel electrophoresis and
histochemical localization of enzymes within the supporting gel matrix, which
is a powerful method with which to observe genetic variation within and
between animal populations (Hubby & Lewontin 1966, Lewontin 1974).
Differences in mobility among allelic variants of specific enzyme gene
products is the most accessible and practical form of biochemical trait
variation available. Protein
electrophoresis does not reveal all amino acid sequence differences that may
exist in a protein nor all nucleic acid differences in the genes which code
for proteins. About 29% of the nucleotide substitutions which occur are
synonymous, arising from redundancy in the genetic code. Of the remaining 71%
which cause amino acid substitutions, only about 25-30% change the net charge
of a protein (Nei & Chakraborty 1973). A few substitutions that do not
change charge may affect conformation (tertiary structure) and be detectable
by electrophoresis (Lewontin 1974). Even variants differing in charge may not
be detectable with a single buffer system of those commonly employed. This
hidden genetic variation (Johnson 1977) may be exposed by use of sequential
electrophoresis (multiple electrophoretic separations using a sequence of
buffer pHs to detect heterogeneity in a given mobility class) with starch and
acrylamide gel supports or by isoelectric focusing (Coyne 1982, Aquadro &
Avise 1982), or with cellulose acetate supports (Easteal & Boussy 1987).
No single set of conditions has proven able to detect all differences,
therefore the careful worker usually tries several buffer systems and
potentially two or more supporting matrices. Isoelectric focusing is not
significantly superior to zone electrophoresis in detecting hidden variation
(Aquadro & Avise 1982, Coyne 1982). But isoelectric focusing is much
superior in its ability to concentrate proteins while it separates them (Righetti
1983) and is potentially superior for studies with minute insects where
enzyme concentrations in homogenates are very dilute. Isozyme
data are typically of the form of allele (mobility class) frequencies at one
to several enzyme loci. Phenotypes of individuals as seen on the stained gels
should be interpretable under a genetic model that corresponds to the
insect's ploidy and mode of inheritance of the gene in question. This may
require genetic analysis in order to eliminate nongenetic variation (Richardson
et al. 1986). Enzyme
electrophoresis is in such wide use as a tool in systematics and genetic
studies that a comprehensive enumeration for the insects alone would be
impractical. But, numerous reviews in relation to systematics of insects and
other animals exist (Avise 1974, 1983; Berlocher 1984, Bush & Kitto 1978,
Buth 1984, Thorpe 1983), as well as many useful references on technique
(Harris & Hopkinson 1976, Eastel & Boussy 1987, Richardsson et al.
1986, Selander et al. 1971, Shaw & Prassad 1970). As of 1991 few studies
have been conducted on natural enemies of insects and there have been limited
in scope (Unruh et al 1986). These
data can be used in two basically distinct ways in systematics: (1) presence
or absence of alleles can be analyzed directly as character states; (2)
allele frequency data may be summarized into a matrix of distances among taxa
which is then analyzed. The distance, phenetic, or so called quantitative
approach (Avise 1983) has been most popular, but persuasive arguments for the
superiority of a character state approach exist. This topic has been
variously reviewed by Avise (1983), Berlocher (1984), Buth (1984),
Felsenstein (1982) and Hillis (1987). This dichotomy in analytical approaches
exists for all discrete types of biochemical characteristics including
restriction fragment polymorphisms and sequence data, but for analyses
involving DNA the character states approach is preferred because restriction
fragment or sequence differences can be unambiguously polarized (Hillis 1987).
The discrete nature of isozyme, restriction fragment, and sequence data
contrast with immunological distance and DNA-DNA hybridization which yield
only distance data (Unruh 1999). DNA
Restriction Fragment Analyses A
critical step in the growth of molecular genetics came with the discovery and
utilization of restriction enzymes in the 1970's (Roberts 1982). Their use in
characterizing polymorphisms in natural populations followed within 10 years
(Avise et al. 1979). Restriction enzymes are a group of endonucleases that
cleave DNA at characteristic 4-6 base sequences. For example the restriction
enzyme known as EcoRI cleaves 5'-GAATTC-3' between G and A. Differences
between individuals in the presence or absence of these specific recognition
sequences throughout a DNA sequence are detectable as different length pieces
of cleaved DNA upon separation in an electrophoretic assay. The variants are
called restriction fragment length polymorphisms (RFLP) or more briefly
restriction polymorphisms. Some 350 different restriction enzymes are known
(Roberts 1982) and >50 are available commercially. Typically, purified DNA
is digested and the fragments, which are separated by electrophoresis based
on size, are visualized by a direct DNA stain. Alternatively, in a method which
is ca. 100X more sensitive, the DNA is transferred from the gel to a
nitrocellulose membrane and visualized there by autoradiography, or by
hybridization with a preselected radioactive or biotinilated (Dykes et al.
1986) DNA probe (Lansman et al. 1981). probes are pieces of DNA (or RNA)
which, by virtue of their complementary sequence, bind to the DNA (or RNA) of
interest. The combined procedures of transferring nucleic acid to a membrane
and detecting complementary sequences bound there is called blotting (see
Maniatis et al. 1982 and Unruh 1999). Studies
of natural populations have emphasized the mitochondrial genome because of
several attributes of this extrachromosomal DNA (Brown 1983, Moritz et al.
1987): (1) mtDNA is a relatively small molecule of 15-20 thousand base pairs
which are arranged in a covalently closed circle; its small size allows the
molecule, or a few fragments from it, to be analyzed with a single
electrophoretic procedure. (2) mtDNA is easily isolated because mitochondria,
themselves, are discretely sized organelles, easily segregated by
centrifugation, and numerous in almost all cells. Also, mtDNA often has a
buoyant density significantly different from nuclear DNA further facilitating
its isolation. (3) The molecule is quite simple compared to nuclear DNA (ca.
4 order of magnitude shorter; has the same 37 genes in all taxa; each gene is
represented by only a single copy of the mtDNA molecule). (4) It is haploid
precluding the high possibility of heterozygotic individuals for RFLP as
might be seen in a region of genomic DNA. This fact allows for
straightforward analysis of the resulting fragments on the gels. (5) mtDNA is
maternally inherited simplifying genetic analysis. (6) Usually all
mitochondria within an individual have identical DNA sequences (=
homoplasmy). (see Moritz et al. 1987, and Unruh 1999). Restriction
fragment length polymorphism and mapping in mtDNA is most useful at or below
the species level (Wilson et al. 1985, Avise et al. 1987). For higher
taxonomic categories its utility is limited by the rapid accumulation of
transitional nucleotide substitutions (versus base transversions).
Transitional substitutions apparently become saturated within about 10
million years in mammals, after which substitution rates fall 5-10 fold to ,
or below, that characteristic of mammalian nuclear DNA (Brown 1983). The
situation in invertebrates is less clear (Powell et al. 1986). Restriction
polymorphism studies of nuclear DNA are technologically more difficult than
that for mtDNA because of the great size of the nuclear genome. In mtDNA
there are 37 genes in about 18,000 base pairs; in eukaryotic nuclear genomes
there are 40,000 or more genes in 200 million base pairs (Spradling &
Rubin 1981, Lewin 1985). In most mtDNA (except yeast mtDNA) there is almost
no spacer DNA, no intervening sequences, no satellite DNA and each gene is
unique (in some cases sequences overlap). By contrast, eukaryotic nuclear
genes (cistrons) are often repeated, interrupted and surrounded by non-coding
sequences (introns and exons) which are often composed of characteristic
repeating sequences. In addition there are large blocks of DNA called
satellite DNA (the heterochromatin visible in cytological studies) that
consist solely of highly repetitive sequences. About half of eukaryotic
genomic DNA consists of moderately to highly repetitive DNA (Unruh 1999). To
study RFLP in nuclear DNA, the sequence of interest must first be isolated.
This requires cloning the sequence. For example, if restriction fragment
variation in alcohol dehydrogenase (ADH) is to be examined, the major tasks
would be (1) to develop a probe specific for the ADH sequence and (2) to
clone the region of DNA containing the gene (for which the probe is
complementary). The second step may be necessary for each individual or
population to be assayed. Since the ADH sequence represents only about 1 X 10
-5% of the total genomic sequence, developing a probe is a major
effort demanding the facilities and expertise of a fully operational molecular
biochemistry laboratory. Although
quite a few genomic and mitochondrial DNA regions have been sequenced from a
variety of animals, no complex eukaryotic genome has been completely sequenced.
In 1983 the complete mtDNA sequences was available for only three species:
the mouse, humans and the cow (Avise & Lansman 1983). There are numerous
articles treating of the merits of a unified effort to completely sequence
the human genome. Comparative studies of sequences of genomic DNA have been
conducted only where a history of protein study precedes the work (ADH in Drosophila species) (Dreitman
1983, Coyne & Kreitman 1986), or, more often, in humans and diseases of
humans and animals (MacIntyre 1985). Generally, the same difficulties
encountered in restriction fragment analysis is also encountered in DNA
sequencing. This is because sequencing must be preceded by restriction
analysis and cloning of each of the restriction fragments. Selected cloned
fragments are finally sequenced. About 1,000 bases may be sequenced in a day,
and with automated gel scanning sequencers can attain a rate of 600 bases per
hour (Prober et al. 1987). However, the techniques to isolate a specific
sequence of interest from the billion base4 pairs comprising the genome
remains a formidable obstacle. This topic is discussed in Beckendorf &
Hoy (1985), Legin (1985), and the mathematics of analyzing sequence data has
been reviewed by Nei (1987). In
biological control, the cost of DNA sequencing is excessive. The technical
difficulty and expense of DNA sequencing
presently exceeds that of protein sequencing (see Kimura 1983). There
is only one exception toe the technological constraints posed by the large
size of eukaryotic genomes. Ribosomal RNA (rRNA) is an important functional
component of the ribosomes, occurring in multiple, in tandem repeating copies
which are often localized in a part of the genome (one or a few chromosomes).
In insects as much as 1% of the total genomic DNA is rDNA (ribosomal DNA).
Probably because of the structural role rRNA plays in the ribosome, its
sequence is highly conserved among all life (excepting viruses). Across the
eukaryotes rRNA sequence homology is above 70%. Sequence divergence of rRNA
among bacteria represents the most compelling evidence supporting the recent
recognition of a third kingdom of organic life, the archaebacteria (Woese
1981). In addition to the highly conserved rRNA, the genes for rRNA spacer
regions show considerably less sequence conservation than mature rRNA (the
RNA remaining after all excision of spacers). Creating genomic libraries for
rDNA is facilitated by its high copy number and probes developed for
completely unrelated taxa retaining sufficient homology to be useful for
restriction studies as well as sequencing. Phylogenetic
studies based on restriction site variation of rDNA have begun to appear in
the literature (e.g., for Rana
spp. by Hillis & Davis 1986). The organization of rDNA in insects was reviewed
by Beckingham (1982). With restriction analysis, we may concentrate on space
DNA (both transcribed and nontranscribed) where sequence homology can be
quite low and genetic difference among taxa are well known for insects
(Beckingham 1982). Heterogeneity within the spacer regions of Drosophila populations has also
been observed by restriction analysis (Coen et al. 1982). Probably
more significant for systematic application is a rapid sequencing technique
developed by Lane et al. (1985). It was observed that the highly conserved
nature of rRNA allowed side stepping the development of a probe; instead it
is possible to synthesize or purchase a probe based on published sequences
for other taxa in the same phylum or kingdom. Then, this short oligonucleotide
(ca. 20 bases) "probe" is used as a primer in more routine
procedures to sequence from a highly conserved domain into one that is
variable and taxonomically informative. Because rRNA exists in enormous
proportions in the cell (10% by dry weight in E. coli;
Lewin 1985), the rRNA from a single, or few individuals, may be sufficient to
sequence >200 without a need for cloning or probe development. The highly
conserved nature of rRNA, although permitting this method, may also restrict
its value to the systematics of higher categories, such as families and
genera. Direct
Estimation of Genetic Distance Two
methods are used to measure genetic distance directly, as compared to
averaging of many qualitative differences: Immunological distance and DNA-DNA hybridization. The application of both methods is
limited to phylogenetic questions because they provide only distances. Immunological
methods to measure genetic distance among related organisms was reviewed in
Bush & Kitto (1978) and Beverly & Wilson (1985). The basis of the
technique is to isolate a protein, produce antibodies to it and use the
purified antibody in microcomplement reactions within and between taxa. The
resulting distance measurements are phenotypic, and subject to the peculiarities
of antigen-antibody interactions. Problems
with the method include significant stochasticity in the rate at which amino
acids are substituted in antigenic proteins and immunological distance is not
always proportional to the number of amino acid substitutions (Nei 1987).
Both sources of error combine to make the molecular clock from immunological
distance slightly more inaccurate than that from amino acid sequence data
(Wilson et al. 1977). However, the correlation between immunological distance
and nucleotide substitution usually is high (r = 0.9). This association holds
for proteins differing by as many as 30% of their amino acids, or, on another
scale, comparisons remain possible where divergence times approach 150
million years (Wilson et al. 1977). Thus, the technique has an important role
in estimating phylogeny of higher taxonomic ranks and remains useful down to
specific differences. Microcompliment
fixation is highly sensitive and repeatable and the amount of antibody
harvested from each immunized rabbit is adequate for thousands of
comparisons. The methods greatest fault is the technical difficulty and
expense of isolating sufficient quantities of pure protein for immunization
from each test population. The protein selected as antigen must be homologous
and relatively abundant. Larval hemolymph protein was used by Beverly &
Wilson (1985) to examine drosophilids. It is not certain what an ideal
protein would be for parasitic Hymenoptera. The difficulty in obtaining pure,
homologous protein from the taxa of interest has limited the use of this
method. DNA-DNA
hybridization provides a direct physical estimate of sequence homology over
the whole coding genome making its use for quantifying genetic distance among
taxa theoretically attractive. it is based on the proportionality of the
thermal stability of a DNA duplex and sequence homology between reannealing
strands. The procedures consist of purifying the DNA, shearing or digesting
it into standardized size pieces, disassociating the duplexes, and estimating
the amount of reassociation of mixtures between and within taxa at various
temperatures. The
occurrence of repetitive sequences throughout the genome represents a
nuisance in DNA hybridization studies. About 20-40% of the DNA in insects is
repetitive (Spradling & Rubin 1981). When a sequence exists as a single
copy in a mixture of segments of DNA from the organism, its probability of
encountering (= rate of hybridizing with) its complementary sequence is the
length of the sequence divided by the total genome size. When a sequence
exists in multiple copies, the probability of one copy encountering a
complement is this ratio multiplied by the copy number of the sequence. Thus,
repetitive sequence hybridize much more rapidly than unique sequences. The genome
has been classified into fast, intermediate, and slow components which
corresponds to their rate of hybridization and copy number (Lewin 1985). In
actuality, the nonrepetitive, or slow, DNA component is isolated from the
fast and intermediate components by thermal chromatography and then used for
hybridization (Britten et al. 1974). A small amount (0.1%) of a tracer
composed of radio-labelled single copy DNA from one test organism is mixed
with a large mount of unlabelled single copy DNA (driver DNA) from another
organism and reassociation of the strands is estimated from the measured
radioactivity of different aliquots from a second thermal chromatographic
separation. Based
on DNA "melting" experiments with thousands of bird species and
primates, Sibley & Ahlquist (1983, 1984), estimated that a one degree C.
difference in melting temperature between homo- and heteroduplex comparisons
(T5OH) corresponds to about 4-5 million years since divergence of genomes. In
contrast, for cage crickets (Coccone & Powell 1987), Drosophila (Powell et al. 1986) and sea urchins (Britten
1986), one degree C. corresponds to ca. 1 million years since divergence.
Therefore, invertebrate DNA seems to be evolving at 4-5 times the rate of
mammalian DNA. But
DNA-DNA hybridization requires several grams of tissue in order to extract
sufficient DNA to do all the reciprocal hybridization reactions required to
compare a modest number of populations. As the number of populations
increase, the amount of material required increases geometrically. Therefore,
completely orthogonal comparisons among a large number of populations or
species is impossible. The use of single copy DNA hybridization for insects
dates to 1975 (Sohn et al. 1975). The large part of the genome examined and
the physico-chemical precision of hybridization of single copy DNA represents
a strong argument for its use as an evolutionary clock in phylogenetic
analyses (Gould 1985). Therefore,
immunological distance offers a highly repeatable estimate of genetic
(phenotypic) distance of a single homologous gene product, which may be
compared across many taxa because of the large amount of antibody produced,
and can be used to compare clearly differentiated species and up to genera,
families and probably superfamilies depending on the rate of divergence of
the protein selected. DNA hybridization offers an estimate of total (single
copy) genome divergence for a smaller group of populations, and in insects is
probably restricted to comparisons ranging from among well differentiated
populations (or cryptic species) to comparisons among genera. Uses
of Molecular Techniques in Biological Control There
have been no insect bio-control agents studied with molecular involving
nucleic acids, and only a few studies have employed the isozyme method (Unruh
et al. 1986). However, there has been considerable use of molecular methods
with insect viruses (Crook et al. 1985), entomogenous bacteria (Oeda et al.
1987) and with viruses found in calyx fluid of some parasitic wasps (Theilman
& Summers 1986). The problem in analyzing the genome of these forms of
life (viruses and bacteria) is much less than for comparable studies of
higher eukaryotes. Systematics.--Unruh (1999) pointed out that
systematists often reiterate the maxim that from phylogenetically sound
classifications emerges the ability to predict characteristics of yet unseen
species. For example, all known species in the aphelinid genus Marietta are hyperparasitoids
of diaspidid scale insects (Clausen 1972). Therefore, it would be safely predicted
that any newly encountered Marietta
also had this habit. It is obvious that when higher taxonomy, especially at
the generic and subgeneric levels, corresponds to such biological attributes
it is very useful to the biological control researcher. In many important
parasitoid genera, molecular techniques would be valuable in both organizing
species within species groups and these within genera and by helping
taxonomists find natural gaps on which to split genera into more workable
entities. Assumptions
in the use of isozymes for phylogenetic analysis are that amino acid
substitutions accrue at an about equitable rate among lineages within a taxon
or, at least, that novel amino acid substitutions are much more likely than
reversions to a previous sequence. Procedures and theory for phylogeny
estimation using isozymes (Berlocher 1984b) and other molecular data
(Felsenstein 1982, Nei 1987) and for consensus between molecular and
morphological data (Hillis 1987) are very extensive topics. It is possible to
compare the association between genetic distance from isozyme surveys to
proposed taxonomic rank of taxa. Judging taxonomic rank by genetic distance
measures is both overtly phenetic and typological, but genetic distances can
alert us to problems when groups are observed which display considerable
morphological variation without associated genetic differences, or
conversely, high genetic differentiation without associated morphological
differences. Genetic distances observed at various taxonomic ranks for several
allozyme studies of insects are summarized in Unruh (1999). The mean values
of Nei's standard genetic distance for studies employing 15 or more genetic
loci are shown. Nei's distance is defined as D = -1n(I) where I = Jxy/(JxJy)1/2
and J represent estimates of sums of squares for the proportion of shared
alleles. Nei's distance, as opposed to many other distance measures commonly
employed in isozyme studies, has well characterized theoretical properties in
relation to rate of amino acid substitutions and evolutionary time (Nei
1987). However, the measure cannot be used for some methods of phylogeny
estimation because it is not metric (Farris 1972, Hillis 1987). Isozyme
studies sometimes prompt authors to relabel infraspecific ranks or to suggest
revision of current taxonomic rank. The
association of genetic distance to presumed taxonomic rank is very
inconsistent, particularly at levels below morphologically distinct species.
Comparisons among species groups or genera result in D values of 1 or >1.
Interspecific comparisons are less consistent, ranging between ca. 0.1-1.4
(see Unruh 1999). Most interesting are those cases where morphology, behavior
or cytology has indicated specific rank while isozyme data reveals few
differences. An example is the relationship between 10 species of Speyeria which includes one
cryptic species pair: S. mormonia and S. egleis. These morphologically identical species are among
the most genetically distinct of the 10 species (D = 0.224). In contrast, S. callipe, another species nearly indistinguishable
morphologically from S. egleis but differing from it in
karyotype, is very similar in isozymes (D = 0.015). Generally, the Speyeria species studied
(Brittanacher et al. 1978) are a very homogenous group which does not include
some of the more distinctive species in the genus (Brussard et al. 1985). Another
example is found in the study of Gryllus
by Harrison (1979). A cryptic species pair, G. veletis
and G. pennsylvanicus, previously thought to be products of
allochronic speciation, and which display broadly overlapping distributions,
were found to be quite distinct (D = 0.165). In contrast, three
morphologically distinct and either macro- or microgeographically allopatric
species differed very little (G.
pennsylvanicus, G. firmus, and G.
ovisopis; D<0.03). The
parasitoid genus Muscidifurax
consists of five distinct species and various biotypes, based on both
morphological and ethological (mating) behavior data (Kogan & Legner 1970, Legner 1987, van den Assem & Povel 1973). Isozyme
analyses have borne out very closely the morphological and behavioral data,
and strain differences are clearly shown (Kawooya 1983). Unique extranuclear
phases to inheritance in one species, M.
raptorellus were mentioned
previously (Legner 1989, 1991). Examples
of very low differentiation of species include Magicicada spp. and Drosophila
silvestris and D. heteroneura. Although each may have speciated recently,
they still represent cases where biological or morphological differentiation
has outstripped genetic differentiation detected by isozyme variation. Each
displays aspects of their biology, and probably history, which are consistent
with rapid development of reproductive isolation. In Magicicada reproductive isolation is imposed by 13 and 17
year life cycles; isolation may have been reinforced by a history of
glaciations which fragmented the species' ranges (Simon 1979). In the
Hawaiian drosophilids, D. silvestris and D. heteroneura, highly ritualized courtship behavior (Speith
1981) may have mediated rapid isolation after colonization-founder events
triggered strong genetic shifts (Templeton 1980). More examples of lower than
expected genetic distances exist in many of the other studies but are hidden
in averaging (e.g., Berlocher & Bush 1982, Bentz & Stock 1986,
Pashley et al. 1985, Unruh 1999) or by taxonomic confusion (e.g., Stock &
Castrovillo 1981). Low
genetic distances among some species contrasts markedly with high distances
among geographic populations of single species. This is true of cave crickets
where some species dwell in forests and populations are relatively free to
interbreed compared to species which inhabit caves and populations are
strongly subdivided. In such comparisons D ranged from 0.02 for forest
inhabiting species versus values exceeding 0.2 for cave inhabiting
"cave" crickets (Caccone & Sbordoni 1987). Genetic differences
exceeding 0.2 are probably associated with postreproductive isolating
barriers in cave inhabiting invertebrates (see Caccone & Sbordoni 1987).
High D is also seen for intraspecific populations of butterflies in the genus
Euphydryas. The
genetic distance among entities variously defined as biotypes, subspecies or
semispecies are highly variable (Unruh 1999). These must be interpreted in
light of the dispersal abilities of the organisms (level of gene exchange
among populations), rate of amino acid substitutions (the molecular clock),
the effective size of populations (probability of fixation of genes through
drift) and any behavioral or physiological attributes of the insects life
system which may reinforce isolation among subpopulations (host mediated mate
finding, allochrony) or which are associated with divergent selection.
Historical phenomena, such as founder events and hybridization, may also
produce unexpected patterns in genetic distances. Genetic
distances calculated from isozymes, particularly those that are higher than
expected may prove useful in judging the rank of taxa. Low genetic distances
among taxa known to be distinct based on other characteristics is a common
but often inexplicable observation. Such studies can reveal new species and
provide evidence for the existence of species complexes. Bush
& Kitto (1978) suggested that DNA-Hybridization was valuable for
comparisons at taxonomic levels of species to class. Recent studies of
parthenogenetic populations of Drosophila
mercatorum (Caccone et al.
1987) and cave crickets (Caccone & Powell 1987) show that DNA melting provides
resolution below the species level. Phenograms for cave crickets from DNA
hybridization (Caccone & Powell 1987) are similar but not identical to
those derived from isozymes (Caccone & Sbordoni 1987) and were also
consistent with those based on morphology save a few exceptions. Caccone
& Powell (1987) argued that the DNA hybridization data are more
compelling evidence of genetic divergence than other techniques because so
much more of the genome is analyzed and because amino acid substitutions
(measured with isozyme, protein sequencing and immunological distance
methods) may be subject to greater selective constraint than total single
copy DNA. This resolution derives partly from the rate of differentiation of
invertebrate DNA (high compared to mammalian DNA) and in part from the highly
isolated (and genetically differentiated) nature of cavernicolous camel
crickets and parthenogenetic lineages. The high rate of evolution of
invertebrate DNA (Caccone & Powell 1987, Powell et al. 1986, Britten 1986)
also suggests that DNA melting will prove unreliable much beyond the generic
level in these taxa. Mitochondrial
DNA restriction patterns may prove to be even more sensitive in these kinds
of studies. At the species level, mtDNA RFLP patterns for the homosequential
(uniform inversion patterns on chromosomes) Hawaiian Drosophila have been used to clarify phylogenies already
addressed by morphology, behavior, allozymes and DNA hybridization (DeSalle
& Giddings 1986). Harrison (Harrison et al. 1987) used the method to
analyze the structure of a hybrid zone between two Gryllus species and reviewed other similar studies. Therefore,
Unruh (1999) concluded that at specific and generic levels, isozymes, single
copy DNA-DNA hybridization and rDNA restriction analysis are all potentially
valuable. mtDNA analysis offers the greatest resolution for infraspecific
groupings (Avise et al. 1987), but there are still too few data from insect
groups to form a clear picture of its upper range of resolving power (moritz
et al. 1987). At the range of species and genus, DNA-DNA hybridization and
ribosomal DNA restriction analysis are most powerful. Only the latter method
can be subjected to cladistic analyses. At higher taxonomic ranks, genera to
family, rRNA sequencing appears to hold most promise. Immunological distance
may also be considered for interspecific through family comparisons. Biotypes.--Populations that fall into the region between species and
clearly undifferentiated populations have been classified many ways. Diehl
& Bush (1984) classified insect biotypes on a combination of genetic and
geographic relationships. They began with the premise that some biological
trait is variable and suggested that there are five categories into which
biotypic variation falls: nongenetic polyphenisms, genetic polymorphisms,
geographic variation, host races and species. In
contrast, Gonzalez et al. (1979) provided a nomenclatoral classification of all
infraspecific categories. They suggested biotypes are reproductively
compatible populations which display differences in some biological
attributes. This definition of biotype is more narrow and perhaps more
functional, than popular usage. Neither classification excludes the other,
nor collectively do they completely treat the biological patterns seen in
infraspecific categories. Molecular
methods have disclosed that sympatric biotypes associated with different
hosts (or habitat, etc.) are probably species if each is also characterized
by striking isozyme differences (fixed allelic differences). These same
isozyme markers would only allow a definition of biotypes as "geographic
races," semispecies or subspecies if populations were allopatric. Most
morphologically indistinguishable entities dealt with in biological control
were once allopatric. Whether more genetic variation in adaptive traits
exists between such allopatric populations compared to within sympatric
populations is questionable (Gould 1983, Diehl & Bush 1984, Fox &
Morrow 1981) When
allozymes differ only in frequency among geographic populations they can no
longer provide unambiguous markers of races. Here mtDNA restriction mapping
could be extremely valuable. mtDNA restriction differences can be ordered
into a series from which infraspecific phylogenies (clonal lineages of the
mtDNA molecule itself) can be constructed (Avise et al. 1987). Differences in
the presence or absence of restriction sites among populations can be ordered
into networks connecting haplotypes by the minimum number of steps. The
ability to overlay these clonal phylogenies onto the geographic distribution
of sexually breeding populations may allow us to separate historical and
adaptive processes responsible for producing racial or biological differences
in animal populations. If two biotypes are sympatric but show an mtDNA
phylogeny that segregates them, species status is indicated. One
potential of mtDNA lies with the ability to find additional variation in
restriction studies beyond that found with allozymes, a case made in several
vertebrate taxa (Avise et al. 1987), but less well documented for arthropod
species. More important is that carefully mapped restriction fragment
differences can be ordered into a series (or an unrooted phylogeny) whereas
frequency variation in allozyme loci cannot (Felsenstein 1982). Establishment, Introgression and
Spread of Biotypes--An important use of molecular methods for
biological control is to document the establishment of new natural enemy
races or species and monitoring their spread subsequent to colonization.
Studies of postcolonization adaptation and introgression of races of natural
enemies is substantially dependent on these molecular classifications. There
are a few examples of this application of the allozyme method. Aphidius ervi Halliday is one of several
species that have been released into North America to control the pea and
blue alfalfa aphids (Gonzalez et al. 1978). After several releases,
comprising thousands of specimens from populations of A. ervi
complex, A. eadyi, A. smithi,
Praon barbatum and Ephydrus
sp. at various sites in southern California, A. ervi
has become the dominant parasitoid of these aphids (Unruh 1999). Isozyme
analysis of established populations of A.
ervi from southern
California dna of early generation samples of populations from throughout its
wide Palearctic distribution have clarified their genetic relationship.
Genetic differentiation among four A.
ervi populations from
western Europe and north Africa was low (D = 0.016-0.029). This contrasts
with the relatively high differentiation found in populations from Pakistan
and Japan (Unruh 1999). At
one liberation site in Riverside, California, established A. ervi populations were identical to the African type (Unruh
et al. 1986). A recent analysis of A.
ervi from a second site in
Escondido showed that these were of the Japan type. Two diagnostic allelic
differences exist between the African and Japan types and not intermixing has
yet been found in the field. Sexual compatibility between the Japan and
African races is only slightly lower than between intrapopulation comparisons
and there is no evidence of postreproductive barriers (Unruh 1999). Isozymes
were also used to clarify the status of a natural enemy of weeds. Rhinocyllus conicus, a weevil specializing
on the flower heads of thistles in the Palearctic has been introduced into
North America to control milk, musk and Italian thistles. Several studies
suggested that at least two distinct races of R. conicus
exist, one adapted to milk thistle (Silybum
marianum) and another adapted
to Italian thistle (Carduus nutans), and probably a third
adapted to musk thistle (Carduus
nutans). Studies of weevil
and thistle species associations throughout Europe showed that R. conicus feeds on a small subset of potential hosts in a
given region (some plants are hosts in one region but not in another, and
vice versa) (Zwölfer & Preiss 1983). Early attempts to establish this
weevil in California on milk thistle failed, probably because the weevils
belonged to a biotype adapted to musk thistle. Eventually, all these races
were established, each on its own thistle, presumably because they were
matched corresponding to their original host in Europe (Goeden & Kok
1986). Subsequent to establishment of these biotypes, an electrophoretic
analysis showed that the milk thistle biotype was genetically distinct from
the other two biotypes at one enzyme locus and was different in allele
frequencies at two others (Goeden et al. 1985). In
1985 about a decade after the races attacking milk and Italian thistle were
established on their respective hosts in southern California, R. conicus was discovered feeding and reproducing on two
species of native north American thistles, Cirsium californicum
and C. proteanum. Electrophoretic analysis of these populations
showed that only the Italian thistle race had shifted to the native, nonpest,
thistles despite an apparently equal opportunity for each race to do so
(Unruh & Goeden 1987). The results were not surprising, however, since
the Carduus attacking
biotypes are known to accept European Cirsium
spp. in their range (Zwölfer & Preiss 1983). These observations suggested
that isozyme analysis, and perhaps mtDNA restriction studies, could
effectively supplement biological screening (host range studies) of
candidates for biological control of weeds (Unruh 1999). Testing of Cloned or Selected Lines.--Allozymes have been
useful in verifying the establishment of two synthetic pyrethroid resistant
mite predators in apple orchards (Whalon et al. 1982). Unique to this study
was that the isozyme loci assayed also identify the biological
distinctiveness of the strains. That is, susceptible and resistant mite
populations were characterized by differences in general esterases. Resistant
populations displayed distinctive mobility differences in their major
esterase bands and these stained much more intensely. This intensity of
staining is probably reflective of higher enzyme titer (as opposed to greater
activity per molecule) in the resistant forms. The esterases themselves are
important in detoxifying both organophosphates and pyrethroids (Chang &
Whalon 1986). Esterase of green peach aphid has been used to follow the
seasonal flux of resistant and susceptible pest populations in the field
(Baker 1979). Immunological procedures which detect the titer of a specific
esterase have proven even more sensitive and specific than esterase assays
(Devonshire et al. 1986). The future use of allozyme markers to follow the
progress of genetically engineered strains is likely (Unruh 1999). Only
discrete characters can be effectively used as markers in studies of
population phenomena. Among the methods that yield discrete characters only
isozymes and to a lesser extent mtDNA are practical for large surveys of many
individuals. Unruh (1999) discussed several potential applications of these
methods in biological control. Effects of Colonization.--Baker &
Moeed (1987) emphasized that populations of animals or plants introduced by
humans to ecologically and climatically disparate regions of the world are of
great interest to evolutionary biologists because they provide developing
case histories of differentiation under the constraint of reduced population
size in the founding population. The effects of these genetic bottlenecks are
of special interest in light of theories suggesting that rapid genetic shifts
may occur in founders and may mediate speciation (Templeton 1980). The
genetic systems of populations may respond to drift and a novel selective
environment by a rapid shift to a new adaptive peak, which Templeton (1980)
called genetic transilience. These genetic events are more apt to occur when
the originating population is large and panmictic and when there are few
founders. Such a shift has been postulated as an important force in the
speciation of Drosophila silvestris - D. heteroneura (Templeton 1980). There are several changes
likely to be evident in the genetic makeup of a newly colonized population
following a bottleneck. First, most alleles which were rare in the endemic source
population will be lost (Nei et al. 1975). Second, inbreeding is likely to
increase as is gametic phase and linkage disequilibrium (Templeton 1980).
Third, when several isolated populations are founded they are likely to
display more interpopulation variation in gene frequencies (greater Fst) than
would population from the native range of the introduced species. Each
of these phenomena can be estimated using polymorphic enzyme loci. In
contrast, only the loss of alleles is measurable with restriction analysis of
mtDNA. But, since there is only one mtDNA haplotype inherited from a mating,
opposed to four of genomic DNA, the effective population size of mtDNA is
one-fourth that of chromosomal genes and should be much more sensitive
indicators of population bottlenecks (DeSalle & Giddings 1986). With
mtDNA this would be measured as the number of restriction polymorphisms in
the source population versus that in the colonized population. Molecular
studies of colonized populations are rare but they are consistent with the
predicted effects. Berlocher (1984) noted higher Fst and linkage
disequilibrium for eight colonized populations of the walnut husk fly. Baker
& Moeed (1987) saw significantly fewer rare alleles in colonized Myna birds and St. Louis &
Barlow (1988) saw the same in the Eurasian tree sparrow. The adaptive nature
of these changes were not discussed, however. Latorre
et al. (1986) used mtDNA restriction maps to show that Drosophila subobscura, populations which recently colonized the
Nearctic were derived from European (as opposed to African) progenitors.
Although not especially sensitive in the last case, mtDNA studies may be very
sensitive in discovering the source of pest populations and thereby
facilitate a search for adapted natural enemies. Genetic
bottlenecks occurring during and after colonization may not result in
reproductive isolation and speciation but may still induce genetic shifts in
natural enemies that may improve or reduce their efficacy. Also, other
genetic changes, especially in highly heritable quantitative traits, may be
driven by selection alone, without invoking reduced population size. However,
as noted previously, the nature of polygenic inheritance is still not clearly
understood in many species, and certainly the parasitic Hymenoptera are known
to show some bizarre inheritance patterns (Legner 1987, 1989, 1991).
Phenomena associated with colonization of biological control agents have not
been examined using molecular methods, but some work with quantitative
genetic changes has been reported. Myers & Sabath (1980) detected changes
in polygenic traits in colonized populations of the cinnabar moth, a
phytophage of tansy ragwort. Stearns (1983) observed changes in life history
traits of Gambusia fish
introduced to water reservoirs in Hawaii that corresponded with the stability
of water levels in the reservoirs through time. Genetic bottlenecks were not
implicated in either of these cases. In most biological control projects,
bottlenecks are common because of the few founder individuals introduced. Population Structure and Mating Systems.--The distribution of genetic
variation among individuals within a population may be a consequence of
habitat structure or phenomena intrinsic to the ecology and behavior of a species. Studies
at this level can illuminate other, more far reaching aspects of natural
enemy behavior and evolution. For example, Templeton's theory of genetic
resilience may be particularly inapplicable to highly inbred parasitic
Hymenoptera. Unfortunately, the degree of inbreeding in most parasitoids is
unknown with the exception of those whose biology allows direct enumeration
of the number of founders to a patch of hosts and the interactions of their
progeny which issue from it. The
level of inbreeding characteristic of a particular parasitic life style has
implications beyond the tendency to form species. For example, inbreeding may
influence the optimal sex ratio of a population (Hamilton 1967, Charnov 1982,
Waage 1986). The levels of inbreeding in a population can be estimated using
isozyme variation if the discrete patches utilized by the progeny of founding
female parasitoids can be adequately defined. This estimate can be used to
the predicted sex ratio based on local mate competition (LMC) theory
(Hamilton 1967) and compared to the observed sex ratio. Deviations from LMC
predictions may arise from fitness differences between females and males
developing in different size hosts and from various deviations from the
assumptions of LMC (Charnov 1982, Waage 1986). Methods of estimating
relatedness of individuals occurring in groups such as nests of eusocial
Hymenoptera (but directly applicable to discrete patches of parasitized
hosts) using isozyme or related characters has been reviewed by Pamilo (1984)
and Weir & Cockerham (1984). Because mtDNA is clonally inherited it would
not be useful unless sequence variation was extremely high in local
populations. Population
structure at a more crude level can provide other valuable insights but
caution is needed in interpreting the data. Allozyme frequency differences
among populations may reflect three forces: selective differences, isolation
and effective population size. Also, historical phenomena which may include
the accumulated effects of one or more of these is very important but
difficult, if not impossible, to separate from ongoing, dynamic (equilibrium)
processes. These phenomena can seldom be isolated with certainty, and often
they are hopelessly confounded. For example, allozyme based methods to
estimate gene flow (Slatkin 1987), would be questionable when the assumptions
of the estimation methods are violated by recent colonization events or other
perturbations. Significant genetic differentiation among geographic
populations could mean that populations have experienced one or several
bottlenecks and gene exchange has been or is too low to erase the evidence of
genetic drift, or, that there are disruptive selective forces which have
shaped and maintain these differences. Although selection may be suspected if
populations displayed different host use patterns or some other
characteristic biological attribute, in general allozyme variation is treated
as if selectively neutral (Lewontin 1974). In fact, populations may appear
undifferentiated electrophoretically but still display distinctive biological
attributes, such as host races (Unruh 1999). Other Uses for
Molecular Methods Molecular
methods may be used to detect internal parasitoids; both immunological
methods and isozymes have been valuable (Luck et al. 1987). Isozymes have
been used to detect parasitoids of Lepidoptera (Menken 1982), aphids (Wool et
al. 1978, castanera et al. 1983) and of whiteflies (Wool et al. 1984). New
technology, such as cDNA probes (complementary to parasitoid DNA) in
"dot-blot" assays, could also be used to detect internal
parasitoids. These should prove both more sensitive and more selective than
immunological techniques. Probes of this sort are having wide application in
plant virus detection (Jayasena et al. 1984). Comparative
studies of the level of a polymorphism in animal populations have raised the
question of why do some taxa, such as Hymenoptera or aphids, display
significantly genetic variation than other insect groups? The subject has
been addressed for Hymenoptera (Sheppard & Heydon 1986, Unruh et al.
1986) and for aphids (Wohrmann et al. 1986, Suomalainen et al. 1980). Isozymes have been
valuable in quality control assessment of insect laboratory populations (Bush
& Neck 1976, Berlocher & Friedman 1981, Pashley & Proverbs 1981,
Stock & Robertson 1982, Unruh et al. 1983). as should other molecular
markers if they become less expensive. However, changes in allele frequencies
and decline in isozyme variation do not necessarily mean reduced performance
of laboratory stocks. In mass cultures of screw worm an allele frequency
changes was shown to be responsible for a loss in competitiveness (fitness)
(Bush & Neck 1976), but in other studies such an association has not been
made. If it is accepted that most allozymes detected by electrophoresis are
selectively neutral, then loss of variation in these genes under laboratory
culture provides an estimate of inbreeding and drift but not of selective
changes. For some species, highly inbred lines may be as fit as their outbred
natural counterparts (Unruh 1999). Exercise 73.1: What do biologists typically measure when they sample arthropod
populations? Exercise 73.2: How may it be determined that natural enemies are responsible for regulating a population? Exercise 73.3: What must be considered in the evaluation of a biological control
agent? Exercise 73.4: List and explain the five principal techniques used for evaluating
biological control organisms. Exercise 73.5: What are five common methods used for detection of predation or
parasitism? GENERAL REFERENCES: <bc-73.ref.htm> [Additional references may be
found at MELVYL
Library ] |