A rearing technique for Pediobius foveolatus (Crawford) (Hymenoptera Eulophidae),

Material Information

A rearing technique for Pediobius foveolatus (Crawford) (Hymenoptera Eulophidae), a parasite of the Mexican bean beetle
Series Title:
Bulletin Agricultural Experiment Stations, University of Florida
Nong, Limhuot, 1939-
Sailer, R. I ( Reece Ivan ), 1915-
Place of Publication:
Gainesville [Fla]
Agricultural Experiment Stations, Institute of Food and Agricultural Sciences, University of Florida
Publication Date:
Physical Description:
iii, 21 p. : ill. ; 23 cm.


Subjects / Keywords:
Mexican bean beetle -- Biological control ( lcsh )
Mexican bean beetle -- Parasites ( lcsh )
Pediobius ( lcsh )
Alachua County ( local )
City of Gainesville ( local )
Larvae ( jstor )
Beetles ( jstor )
Parasite hosts ( jstor )
bibliography ( marcgt )
non-fiction ( marcgt )


Bibliography: p. 20-21.
General Note:
"May 1985."
Bulletin (University of Florida. Agricultural Experiment Station) ;
Statement of Responsibility:
Limhuot Nong and Reece I. Sailer.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
022594056 ( ALEPH )
14268961 ( OCLC )
ADA2673 ( NOTIS )


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Full Text

1 May 1985

A Rearing Technique
for Pediobius Foveolatus (Crawford)
(Hymenoptera: Eulophidae),
a Parasite of the Mexican Bean Beetle

Limhuot Nong and Reece I. Sailer

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Agricultural Experiment Stations
Institute of Food and Agricultural Sciences
University of Florida, Gainesville

Bulletin 854 (technical)


A Rearing Technique
for Pediobius Foveolatus (Crawford)
(Hymenoptera: Eulophidae),
a Parasite of the Mexican Bean Beetle

Limhuot Nong and Reece I. Sailer

Limhuot Nong is a Biological Scientist II and Reece I. Sailer is a Graduate
Research Professor, Department of Entomology and Nematology, University of
Florida, Gainesville, Florida 32611.


INTRODUCTION ................... .......................... 1
GROWING HOST PLANTS .............. .................. 6
Direct Seeding
Use of Laboratory-Germinated Seeds
Precautions and Care
Equipment and Procedures The Adults
Equipment and Procedures The Larvae
Precautions and Care
Equipment and Procedures
Precautions and Care
PARASITE PRODUCTION .............. .................. 17
REFERENCES CITED......................................... 20

The Mexican bean beetle (MBB), Epilachna varivestis Mulsant
(Coleoptera: Coccinellidae: Epilachninae) (Fig. 1), first described by
Mulsant in 1850, was also known under the name of Epilachna
corrupt Mulsant (Chapin 1936). The original home of the beetle is
southern North America; it occurred in many parts of Mexico and
Guatemala (Howard and English 1924). Present distribution, as
shown by the Commonwealth Institute of Entomology (1954), in-
cludes southern Canada, the USA, Mexico, and Guatemala. Gordon
(1975) added El Salvador, Honduras, Nicaragua, and Costa Rica to
the list.
The life history of the beetle was studied by Thomas (1924), who
reported that the developmental period from egg deposition to adult
emergence was on the average 30 days (25 days in summer, 56 days in
early spring). Studies at the Biological Control Laboratory of the
University of Florida (Florida Department of Agriculture and Con-
sumer Services; Division of Plant Industry; Gainesville, Florida)
confirmed a developmental period of 30 days at temperatures of


Fig. 1. Mexican bean beetle, Epilachna varivestis Mulsant. Extreme left vertical
row: 4 adult beetles; upper middle: six 2nd-instar host mummies parasitizedd host
larvae); upper right: six 3rd-instar host mummies; bottom horizontal row: four
4th-instar host mummies.

22-24C. Both larval and adult stages are destructive to host plants.
The Entomology Research Division of the United States Department
of Agriculture (1958) listed beans, such as snap (green or string),
kidney, pinto, navy, and lima beans, as primary food plants, while
also mentioning that the beetle can reproduce successfully on cow-
peas and soybeans. The beetle's second choice of food was identified as
beggarweed, Desmodium tortuosum (SW) D.C. (Fig. 2), which grows
wild throughout the southeastern states. Sherman and Todd (1939)
reported snapbeans (bush and pole), lima beans, soybeans, velvet
beans, crotalaria, alfalfa, peanut, beggarweed, and kudzu as food
plants in decreasing order of preference.
Additional host species mentioned by Howard and English (1924)
are tepary bean (Phaseolus acutifolius A. Gray), hyacinth bean
(Dolichos lablab L.), adzuki bean (Vigna angularis (Willd.) Ohwi &
Ohashi), sweet clover (Melilotus alba Desr.) and two species of Des-
modium (D. canescens (L.) DC. and D. viridiflorum (L.) DC). They
noted that adult beetles and larvae usually died when confined to
diets of either alfalfa or sweet clover. Kogan (1972) found a wide
spectrum of responses when MBB larvae were fed on different plants
reported to be hosts and showed adult preferences that closely fol-
lowed the nutritional suitability of the diets for larvae. Of the 22 diets
tested by Kogan, 17 were cultivars of soybean (Glycine max (L.)
Merr.). Preferred host plants were snapbeans (P. vulgaris L.) and
Fordhook lima beans (P. lunatus L.), closely followed by seven of the
soybean cultivars. Beggarweed was not included in the diets. When
confined to sweet clover, the larvae consumed more than when fed on
snap or lima beans, but they did not survive to the adult stage. Rye
(Secale cereale L.) is the only reported non-legume host (Turner
1932). The report was based on damage caused by adults that had
recently emerged from pupae in a nearby field of frost-killed beans.
Prior to 1972, control of the beetle in the USA was exclusively
dependent on the use of chemicals. Howard and Landis (1936) re-
ported that, despite a considerable list of known natural enemies, the
beetle had been practically unimpeded by parasites and predators.
This situation remained unchanged until introduction of the
eulophid Pediobiusfoveolatus (Crawford) (Fig. 3) from India in 1966
by the Insect Identification and Parasite Introduction Research
Branch of the USDA at Moorestown, New Jersey (Angalet et al.
1968). P. foveolatus is an arrhenotokous species characterized by an
inbreeding mating system (Nong 1982). The host from which the
original stock derived was the epilachnine beetle, Henosepilachna
sparsa (Herbst.), a pest of eggplant in India (Angalet et al. 1968).
Successful parasitization has been observed on 2nd-, 3rd-, and 4th-
instar MBB larvae. The life cycle of P. foveolatus (oviposition to adult

Fig. 2' Beggr-ee p D o ('.

Alt r

emergnce, t 22,); was f by Steen e al' ( to rn from

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D.C., at fruiting stage.

emergence, at 22C) was found by Stevens et al. (1975b) to range from
10 to 23 days. Under University of Florida laboratory conditions
(22-24C), it was observed to be in the range of 16 to 21 days, the
higher the temperature the shorter the egg to egg cycle.
The first account of successful use of P. foveolatus to control the
MBB in the USA was reported by Stevens et al. (1975a). The parasite
has also been successfully used to control the same pest in Florida
(Sailer, Nong, and Olesky, unpublished data). In Maryland, annual
inoculative releases of comparatively small numbers of parasites as
soon as host larvae are present in the fields has provided effective
control (Stevens et al. 1975a).
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In Florida, control resulted from inoculative releases of P. foveola-
tus in 1975 and 1976. For some years prior to 1975, all garden and
commercially grown green beans and lima beans grown in northcen-
tral and north Florida required chemical treatment. Otherwise all
foliage would be destroyed before first crop harvest. Following re-
lease of 4000 P. foveolatus distributed among 27 sites in Alachua
County and border areas of adjacent counties in late April and early
May of 1975, no unparasitized 4th-instar MBB could be found in
Alachua County after mid-July. By early November parasitized
MBB larvae were found as far north as Commerce, Georgia, 400
miles from the nearest release site (Sailer and Olesky, unpublished
data). At the end of 1975, residual MBB populations were known to
be present in Marion County to the south and in the counties north of
Alachua. Inoculative releases again totaling 4000 P. foveolatus were
made in 1976, beginning in Marion County in the first week of April
and in the northern counties of Union, Columbia, Hamilton, and
Madison during the first half of May. By September no unparasitized
3rd- and 4th-instar MBB could be found in Florida or in southeastern
Alabama and the southern third of Georgia (Sailer and Nong, unpub-
lished data). During 1977 and 1978, no infestations of economic
significance were observed or reported in Florida. In 1979, localized
infestations were present in home gardens in the northernmost coun-
Alachua County remained free of MBB until 1984, when a moder-
ate population was found in September, on Desmodium along the
northern border.
Two factors appear to be responsible for the magnitude of the
effects observed after the 1975 and 1976 inoculative releases. The
first is time. In both years there was sufficient time for 9 or 10
generations of the parasite between dates of release and the end of
the season in late fall. The second, and more important factor, was
the ubiquitousness of beggarweed, Desmodium. This excellent host
for MBB occurs throughout much of the southeastern United States,
growing abundantly in fence rows, roadsides, cornfields, melon fields,
and recently abandoned crop land. As an annual plant, it is available
to MBB from late June until late October. Prior to 1976 few beggar-
weed plants that failed to show evidence of MBB feeding could be
found in north and west Florida after July. Because of the high and
relatively uniform distribution of MBB larvae on this plant, P.
foveolatus was able to increase exponentially.
The MBB populations have increased from the very low number
present in 1978. Beginning in 1981 more and more gardeners in
north and west Florida have found it necessary to use insecticide to

Fig. 3. Pediobius foveolatus (Crawford), parasite of Epilachna spp. (approx-
imately 60X).

protect their bean plantings. However, these high populations were
local and until the fall of 1984 were rarely found on Desmodium
(Sailer and Nong, unpublished data). Because of the patchy and
widely dispersed character of these populations, achieving control
equal to that provided by the inoculative releases of 1975 and 1976
would have required the release of much larger numbers of P.
foveolatus, and at considerably more locations.
Although its remarkable ability to find a host and the reproductive
potential ofP. foveolatus enable the parasite to overtake and suppress
an MBB population in the course of a season, it has not successfully
survived the winter at any of the many places it has been released in
the United States (Schaefer et al., in press). Presumably, failure to
establish is due to prolonged absence of host larvae during the winter
months and lack of a diapause mechanism that might enable the
parasite to survive the period. Thus, if advantage is to be taken of this
parasite, cultures must be continuously available for periodic re-
leases at such times as MBB populations rebuild to economic levels.
Because of the shorter growing season in Maryland and the large
acreage of soybeans annually subject to damaging infestation of
MBB, it has been necessary to produce P. foveolatus on a compara-
tively large scale there (Stevens et al. 1975a). Large-scale production
of the parasite for nationwide distribution has also been undertaken

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by the Animal and Plant Health Service, USDA, Biological Control
Satellite Facility at Niles, Michigan (unpublished reports). The De-
partments of Agriculture of Maryland and New Jersey are also en-
gaged in large-scale rearing of P. foveolatus for release in those
states. However, the situation in Florida is different from that pre-
vailing over much of the rest of the United States. In Florida, MBB is
primarily a pest of home garden and commercially grown green
beans and lima beans, and prior to 1976 most of the overwintering
population of the beetle was produced on beggarweed during late
summer and fall. For most effective use of P. foveolatus, compara-
tively small numbers should be released in selected home gardens
during April and May in localities where MBB populations are ex-
pected to be high on beggarweed in late summer. In more northern
locations where protection of soybeans is the principal concern, much
larger numbers are needed for release in June and July. The rela-
tively unimportant status of MBB as a pest of soybeans in Florida is
probably a consequence of the beetle's preference for beggarweed.
Because of its general distribution and abundance, this weed func-
tions as a trap crop during the period of soybean production.
These considerations suggest that future control of the MBB in
Florida will require continuous maintenance of a culture of P.
foveolatus capable of producing 10,000 to 100,000 adults during April
and May. Apart from the advantages of being better able to schedule
production to suit conditions peculiar to Florida, there is a question of
the availability of stock from other sources. Present production cen-
ters are situated at more northern locations and during April and
May these centers need to increase their breeding stock to meet the
demands for large-scale releases in June and July. Therefore, one
objective of this bulletin is to describe a technique for production ofP.
foveolatus suited to the special needs of Florida. It should also be
useful to anyone wishing to produce the parasite for sale to home
gardeners or commercial producers of green beans and lima beans.

Successful production of host plants is a prerequisite to culture
maintenance and large-scale production of E. varivestis and P.
foveolatus. Kogan (1971) reported development of an artificial diet
(sol-casein) on which MBB taken past the second molt on beans could
then be reared to adults; however, it was necessary for the adults to
feed on bean leaves in order to obtain sustained egg production. No
investigation has yet been made in regard to production of P. foveo-
latus through MBB larvae reared on this artificial medium. 'Jackson

Wonder' and 'Henderson' lima beans have proven to be more suitable
than other host plants in terms of disease resistance and length of
growth period prior to senescence. These qualities outweigh the bee-
tle's preference for green beans (Kogan 1972) and the advantage of
longer duration of turgidity of green bean leaves after excision.

Beans can be grown either by direct seeding or by planting labora-
tory-germinated seeds into soil-filled benches (15 cm deep and 1 m
wide, see Fig. 4). Benches should be about 0.8 m above ground level
for convenience. Beans should be planted in potting soil or a fumi-
gated soil and peat moss mixture (approximately 3/4 low-organic
matter soil and 14 peat moss).

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Fig. 4. Young greenhouse-grown lima bean plants (var. Jackson Wonder).

Direct Seeding
Dry soil should be moistened and stirred daily for 4 to 5 days prior
to seeding. Fertilizer in compound form may be incorporated in the
soil immediately before the third daily stirring and watering. For a
very dry soil, i.e., soil left without plants over a long period of time,
more than one daily watering is recommended. The number and the
size of the plots used for plant production vary according to the

anticipated level of host insect-parasite production. Prior to seeding,
the soil is stirred for the last time and leveled. V-shaped furrows
approximately 12 cm apart and 3 cm deep can be made with a garden
trowel. Bean seeds are placed in the furrows at intervals of 3 to 4 cm.
The sides of the furrows are then pressed together to cover the seeds
with soil. Care should be taken to insure that the soil is firmly closed
around the beans and that they are planted at a uniform depth. After
planting, water is added as needed to maintain soil moisture. This
should be applied between the rows by means of a narrow-spout
watering can. Direct application of water on the rows should be
avoided to prevent washing off seed treatment chemicals and/or
exposure of the seeds. If additional fertilizer is needed, this should be
placed between furrows after the seeds are planted. This will prevent
direct contact of the fertilizer with the seeds. Covering the beds with
a single sheet of newspaper after seeding helps conserve soil moisture
and enhances rapid and uniform emergence of the seedlings. The
cover should be removed when the young plants reach a height of
about 5 cm.

Use of Laboratory-Germinated Seeds
Bed preparation is the same as in the case of direct seeding, except
that the furrows are made deeper to accommodate the larger size of
the germinating seeds. Although planting of laboratory-germinated
seeds requires more time than direct seeding, a uniform stand of
plants of equal size can be assured by selecting only germinating
seeds with vigorous sprouts. The result is maximum production of
foliage per unit of area. Time is saved through elimination of replant-
ing, and uniformity of plants allows harvesting leaves of uniform
quality and size. This reduces possible variation in the rate of de-
velopment and quality of the beetle larvae.
Germination of seeds under laboratory conditions consists of plac-
ing a single layer of seeds on moistened toweling paper in a flat-
bottom container. The bottom sheet should be sufficiently firm to
prevent root penetration. To avoid excessive loss of moisture and
assure a desirable level of humidity, the seeds are covered with
another sheet of toweling paper, and the container is closed with a
suitable cover or lid. Excess or deficient moisture in the germination
container should be avoided. Germinating seeds are ready to be
transferred to the bed in the greenhouse when their hypocotyls are
about 1 to 2 cm long. This normally requires 3 to 4 days at 24C. The
germinating seeds are then removed and placed individually in the
furrows 12 cm apart and 3 cm deep as for direct seeding. The furrows

are carefully closed, using a small spatula, taking care to avoid
damage ofhypocotyls. Water is applied directly over the rows to wash
surrounding soil into good contact with the germinating seeds. If
additional fertilizer is needed, it should be applied in furrows be-
tween the rows. Use of newspaper as described for direct seeding will
enhance rapid and uniform emergence of the plant through conserva-
tion of soil moisture. The newspapers should be left in place until
emerging plants are about 5 cm tall. Plants are ready for harvest
when four or five trifoliate leaves appear. Only older leaves (lower
leaves) are then picked for beetle feeding. Well-cared-for plants will
produce suitable leaves for MBB forage for up to three months.

Precautions and Care
Repeated plantings of beans on the same beds for a long period of
time increases susceptibility to soil-born diseases, such as damping-
off and root-rot. Bean plants show much better health, vigor, and
uniform growth on fresh soil. Fall-planted beans benefit most from
the positive effect of fresh fertile soil, as this tends to compensate for
the negative effects of shortening days and cooler temperatures.
Continuous operation of a greenhouse air-conditioning system dur-
ing alternately hot and cold weather may cause wilting of the plants.
When plants wilt in hot and dry summer weather conditions, turgid-
ity can be quickly restored by copious watering. Plants wilted be-
cause of cold should be treated with a water mist. After the foliage is
dampened, single sheets of newspaper should be laid over the plants
until weather conditions become more favorable. Plants that wilt
frequently do not yield good quality leaves.
While soil should be moist at all times, excessive watering will
result in poor plant growth. As need for water will vary from day to
day because of changing ambient humidity and light conditions,
frequency of watering, as well as the quantity used, must be deter-
mined on the basis of day-to-day judgment. While automatic water-
ing systems are available, installation of such systems is expensive
and once installed the hazard of malfunction requires daily surveil-
Rotation of the plots in a manner that allows one or more to be free
of vegetation and unwatered for 3 to 6 weeks enhances growth of
subsequent plants. The length of time allocated to the resting of soil
will depend on the number of plots needed for plant production.
During the warmer period of the year when beans can be grown
outdoors, most or all plots should remain free of vegetation and
allowed to dry out completely.

Fertilizer should be applied as needed. Experienced personnel who
tend the plants daily will recognize symptoms that indicate need for
fertilizer and act accordingly. With inexperienced personnel, fertil-
izer should be applied at intervals in accordance with recommen-
dations that normally accompany fertilizer packaged for use on small
gardens or house plants. In the latter case, soil fertility should also be
monitored at regular intervals by appropriate tests. In our experi-
ence, fertilizing as needed with a 10-10-10 formulation containing
minor elements will permit production for three months of leaves
suitable for feeding both the larvae and adult MBB.
Aphids, whiteflies, thrips, leaf-miners, mites, sowbugs (= pillbugs,
Isopoda), and root-knot nematodes may occasionally cause problems
in the production of host plants. Generally, these problems arise
when host plants are allowed to remain on the beds for a prolonged
period of time. The insects and mites can be controlled by short-lived
insecticides and/or acaricides. Pesticides should be applied only to
infested plots and scheduled so that residues do not adversely affect
host insect production. Nematode infestation can be prevented by use
of fumigated soil and by avoiding contamination from infested soils
or plants. Sowbugs generally feed on germinating seeds that are still
under paper cover. The use of laboratory-germinated seeds helps
reduce damage, since the young plants remain under paper cover for
a shorter time than those grown through direct seeding.
Ideally, these pests should be controlled biologically. This is cur-
rently possible in the case of tetranychid mites and the greenhouse
whitefly, Trialeurodes vaporariorum (Westwood). The predacious
mite Phytoseiulus persimilis Athias-Henriot can provide adequate
control of the mites (Hussey and Bravenboer 1971), and the parasitic
wasp Encarsia formosa Gahan can control the greenhouse whitefly.
Both are available commercially and can be used successfully by
experienced personnel (Tauber and Helgesen 1978). However, the
prospect that greenhouse cultures will become infested by aphids,
leafminers or other pests for which no biological controls are avail-
able renders exclusive reliance on biological control hazardous.

Equipment and Procedures-The Adults
Clear plastic containers of 31 cm x 22.9 cm x 10.9 cm (Fig. 5) are
used to obtain eggs of the Mexican bean beetle, E. varivestis. Ventila-
tion to avoid water condensation, particularly when external relative
humidity is high, is assured by circular holes (4.4 cm diameter), two

Fig. 5. Containers and tools for Mexican bean beetle rearing. Left: clear plastic
container for rearing of adult beetles; middle: plastic container and petri dishes
for rearing of beetle larvae; lower right: camel hair brush and locally made
bamboo tweezers.

at either end and two to four in the cover of each container, all
screened with 80-mesh brass strainer or suitable nylon fabric. Paper
toweling is placed at the bottom of the container underneath food
material to help reduce soilage. Ten to 15 pairs of adult beetles are
placed in each container. Food should be renewed daily in order to
ensure continued vigor of the adult beetles and obtain best egg
production. Containers should be changed at 3- or 4-day intervals,
depending on the degree of soilage. Female beetles normally begin
laying eggs about seven days after emergence from pupae. Egg
masses may be collected daily at the time of each food renewal. Adult
beetles are discarded when egg production is observed to decline or
about 4 to 5 weeks after emergence from pupae.

Equipment and Procedures-The Larvae

Portions of plant leaves on which eggs are deposited are cut into
small squares. These squares are then incubated in petri dishes until
the eggs hatch. Under dry atmospheric conditions, a portion of fresh
bean pod or a few drops of water may be provided to each dish to
improve egg hatch. The bottom of each dish should be covered by a
disc of paper toweling. Eggs hatch on about the sixth day after they

are laid. The newly closed larvae, still in aggregation on the empty
egg shells of the mass, are transferred to petri dishes (15 cm diam,
Fig. 5) containing food leaves. Larvae from two or three egg masses
(approximately 100 larvae) are reared in these dishes through 2nd-
instar. To obtain uniformity in size, larvae should be reared from egg
masses that hatch at the same time. The transfer of the newly
hatched larvae to the rearing dishes while they are still in aggrega-
tion on the empty egg shells helps avoid injury that might otherwise
result from individual handling. Clear plastic containers (18.4 cm x
13.2 cm x 8.9 cm and 31 cm x 22.9 cm x 10.8 cm, Fig. 5) can be used
to rear 100 3rd- and 4th-instar larvae. These smaller containers do
not need vented lids but have two screened circular vents (3.5 cm
diam) at either end. Bean or beggarweed leaves are made available
daily to the beetle larvae. The leaves are placed directly on the
toweling paper covering the bottom of the rearing container. The
frequency of change for the rearing units varies with the degree of

Precautions and Care
Under conditions of low relative humidity, excised leaves lose
water rapidly and soon become unsuitable as food for either adults or
larvae of the MBB. High humidity and water condensation inside the
rearing units also create conditions that are unfavorable for the
insects. Under dry conditions, plant leaves can be kept fresh for up to
two days by inserting their petioles or portions of the plant through
holes in the plastic top of a suitable container partially filled with
water. Another alternative, more suitable for rearing of both beetle
adults and larvae, is to place plant leaves directly on the toweling
paper at the bottom of the rearing containers and adjust vent hole
closures in accordance with the external humidity.
Where egg production should be delayed, adult beetles may be kept
for a few days at 4-5C. Whether newly emerged or reproductively
active, adult beetles should be fed well for 3 to 4 days prior to placing
them in the cool chamber. Few eggs will be produced during the first
two days following removal from the cold chamber.
When it is necessary to accumulate a quantity of hosts for produc-
tion of a desired number of parasites, 4th-instar larvae can be kept for
a few days in a refrigerator at 4.4C. Fourth instar larvae that have
developed to full maturity or prepupal stage should not be stockpiled,
since they are likely to pupate during storage. It should be noted that
mortality due to the cold treatment is progressively higher with each
younger instar.

Daily attention to food requirements of adult beetles needed for
production of eggs and of the larvae is essential in obtaining a high
rate of successful parasitization and production of vigorous, long-
lived adult parasites.
Overloading rearing units is counterproductive, as it results in
crowding stress to the developing larvae. These larvae are less vigor-
ous when harvested, and after parasitization they are likely to die
before the parasite larvae can complete their development.


Equipment and Procedures
Clear plastic 12 cm x 9.3 cm x 7 cm containers (Fig. 6) are used for
emergence of P. foveolatus from host mummies parasitizedd host
larvae) and to house the adult stock. To assure air circulation, each

Fig. 6. Containers and tools for rearing and handling of parasite adults. Upper
left: clear plastic container for culture maintenance of parasite adults; upper
middle (in glass petri dish cover): small glass tubes for isolation of parasite pupae
until emergence and sexing of the virgin adults; upper right: clear plastic con-
tainer topped with acetate sheet, used as mating chamber for study on sex ratios
resulting from sequentially mated males (Nong 1982, Chapter 6) and study on
multiple matings of females (Nong 1982, Chapter 10); lower left: aspirator for
transfer and isolation of mated females from container at upper right; lower
middle: plastic tubes for isolation and confinement of single females or female-
male separate pairs; lower right: standard aspirator for collection of adults.

rearing unit is provided with three screened circular vents (3.5 cm
diam), one on either lateral side and one on the cover. An additional
circular hole (2.8 cm diam) in one end of the unit facilitates exposure
of the host larvae to the parasites and removal of empty host mum-
mies left after the emergence of the parasite adults; at other times,
the hole is plugged with a cork or rubber stopper. To prevent the
parasite adults from being caught between the wall and the cover of
the rearing unit, the inner rim of the container is lined with adhesive
tape. Honey is used as food for the adult parasites. It is applied in
streaks on the bottom of the rearing unit and covered with a sheet of
paper toweling of appropriate size, which is then moistened with
several drops of water to assure even spread. Water for the adult
parasites is supplied in a small glass test tube (1 cm diam and 10 cm
long) loosely plugged with cotton and placed inside the rearing unit
in a slightly inclined position. A shallow plastic cap (about 4 cm
diam) placed in the unit near the plugged hole may be used to hold the
host mummies, thereby preventing them from sticking to the honey-
impregnated paper toweling.
Although 2nd-, 3rd-, and 4th-instar host larvae are all susceptible
to parasitization, for economy of effort and the highest production of
vigorous parasite adults only the 4th instars should be used both for
maintenance of laboratory cultures and for production of parasites
for field release. Fourth-instar larvae not only produce the highest
number of parasite adults per host mummy (average of 17/host
mummy compared to about eight and four for 3rd- and 2nd-instar
larvae respectively), but they are also least subject to premature
death following parasite oviposition. Given free choice, the 4th-instar
is also the stage most preferred by the parasite for oviposition (Nong
Host larvae are exposed to the parasite in a back-lighted insect
sorting hood (Fig. 7). Host larvae, previously collected in a 15 cm
diam petri dish (Fig. 5), are quickly transferred one by one, using a
pair of tweezers through the open hole into the adult parasite unit
(Fig. 5). The number of host larvae to be introduced each time into the
parasite unit for parasitization depends on the eagerness of the
female parasites to oviposit. This varies with different times of the
day and, to some degree, with the age of the females. On-the-spot
judgement, based on prior experience, is required. An insufficient
number of host larvae exposed to eagerly ovipositing females may
result in excessive superparasitization, causing premature death of a
high percentage of the parasitized hosts and production of undersized
parasite adults from those that survive to maturity.
A female parasite in the process of oviposition can be recognized by

Fig. 7. Hood for handling of parasite adults
(designed by R.I. Sailer).

her more or less vertical stance relative to the body of the host larva
and the upward position of her wings while her ovipositor is inserted
through the skin of the host larva. Stung host larvae are removed
from the rearing unit and placed in a separate, 15 cm diam petri dish
until the desired number have been obtained. They are then trans-
ferred to suitable rearing units containing food, i.e., large petri dishes
(15 cm diam) or roomier containers (18.4 cm x 13.2 cm x 8.9 cm or 31
cm x 22.9 cm x 10.8 cm), depending on the numbers of stung host
larvae to be reared. Upon termination of oviposition, the parasite
females are collected with an aspirator and returned to the original
parasite stock unit. Exposed host larvae are reared until cessation of
feeding. Parasitized host larvae, while retaining the normal larval
shape, begin to turn brown about the sixth or seventh day after the
parasite eggs are deposited. Host mummies may be transferred to the
parasite emergence and holding units about two or three days prior to
the parasite emergence (under 22-24C at the University of Florida
Biological Control Laboratory, 13 to 14 days after parasite ovi-
Mummies can be transferred to the adult parasite units with the
fingers if they are attached to a soft substratum, such as toweling
paper or plant leaves. If attached to the plastic walls of the container,
the mummies must be carefully loosened with a finely pointed blade,

as survival and adult behavior are adversely affected when the host
mummies are punctured or otherwise damaged. To facilitate transfer
of host mummies to the adult parasite rearing containers, a long pair
of flexible tweezers is used (Fig. 5). Twenty to 25 4th-instar host
mummies may be placed in each rearing container (Fig. 6). Empty
host mummies should not be removed from the rearing units before
the third day after emergence of the first parasite adults.
Although some parasite females in units initially containing 300 to
400 individuals have been observed to survive as long as three
months, with a few still capable of producing progeny of both sexes,
stock cultures should be renewed at intervals by introduction of
females and associated males that are no more than three weeks old.

Precautions and Care
Healthy, well-nourished host larvae reduce the percentage of pre-
maturely dead parasitized hosts for any of the susceptible larval
instars; however, the percentage of prematurely dead host larvae
increases with decreasing order of larval instars. Fourth-instar lar-
vae, in addition to being preferred by the parasite for oviposition and
most easily handled, also have the advantage of producing the high-
est percentage of successful parasitization (low host mortality due to
parasitization) and yield the largest number of adult parasites per
host mummy. However, a high percentage of larvae may escape
parasitization if 4-day old or prepupal larvae are used. The use of
3-day old 4th-instar larvae (17 to 20 days after egg deposition, under
22-24C), seems optimal.
At laboratory room temperature (22-24C), emergence of the para-
site adults generally occurs on the 16th or 17th day after oviposition
by the parasite females. However, cycles (from egg deposition to
emergence of adults) of up to 21 days have sometimes been observed,
primarily because of lower room temperature. Working in Maryland,
Stevens et al. (1975b) reported that the life cycle of P. foveolatus
varied from 10 to 23 days. Although the parasite life cycle may vary
among colonies reared at different times of the year, primarily be-
cause of temperature change, emergence of parasite adults from
mummies resulting from hosts subjected to simultaneous parasitiza-
tion consistently occurred within no more than a 2-day period. Reten-
tion of the mummies in the rearing unit until the third day following
first adult emergence insures complete emergence and mating of all
females. Field release of less than 2-day-old females is undesirable,
as they might remain unmated and thus produce only male progeny.
Since oviposition capability diminishes with age, use of females

that are less than 15 days old is recommended for production of
parasites for field release. However, for renewal of the laboratory
stock cultures, the female age may be extended up to four weeks, as
only a limited number of host larvae need be parasitized by the
approximately 300 females still available in a culture unit.
It should be noted that females gradually lose mating receptivity
after the age of about 15 days. Male mating capability, on the other
hand, appears to be less affected by age (Nong 1982). Thus, any
studies requiring segregation of sexes before or at emergence should
take account of this age factor if the females are to be mated later.

The following plan does not envision production on a scale of
millions. Rather it concerns a method of maintaining a stock culture
of approximately 500 high-quality female Pediobius (with associated
males). Depending on requirements for inoculative field release and
on available resources, this basic stock unit may be used in in-
cremental increases of production to levels required for a single
location and release date or for multiple release sites to be scheduled
at specified dates over a period of time.
In Florida, production needs may be expected to peak during the
period from mid-April to the end of May, when beetle populations
build up on beans in home gardens and fields of commercially grown
snap and lima beans. Production of adults for release during this
period must be based on greenhouse-grown bean foliage. During the
period of July through September, the beetle population may build up
on beggarweed. If so, inoculative releases may be required primarily
to reduce the population that would otherwise overwinter and infest
home garden beans the following year. During this period, produc-
tion of Pediobius can be based on garden-grown beans or on leaves of
The schematic production plan shown in Fig. 8 is designed to reflect
the requirements of three levels of production. It shows the number of
rearing units and number of adult beetles needed to produce the
4th-instar larvae required to produce a projected quantity of parasite
females on or near a specified date. All figures given are intended to
be conservative. A similar plan for host plants is not included, as
seasonal and weather factors will influence their production. The
reason for this omission rests on the frequency of plantings, under
both greenhouse and field conditions. At the University of Florida
(Gainesville), greenhouse production of host plants would be re-
quired only during a 6-month period, i.e., from October to March.

During this time, a total of four 2 m x 1 m plots of beans planted at 4-
to 7-day intervals in October will supply sufficient leaves to feed the
host insects for production of the number of parasites specified in
Level I of the production plan for a 3-month period. Decline of produc-
tivity of the standing plants, particularly of plots planted early, is
indicative of the need to plant new beans in order to maintain the
required level of food supply for the beetle at this production level.
If parasites at Level II of the diagram are to be produced for field
releases as early as April, then additional beans should be planted in
January to meet the requirements of food supply for the host. This
will require eight additional 2 m x 1 m plots of beans planted at
about 3- to 5-day intervals. From the total of 12 plots, it is expected
that the plants would provide sufficient food for parent beetles as well
as the number of beetle larvae required to produce 5000 female
parasites at about 4-day intervals over a 9-week period. A 10 m x 5 m
greenhouse provided with three 10 m x 1 m benches would be
appropriate for this purpose. For more frequent production of para-
sites, a larger planting area would be needed. Garden-grown beans
should be available for continued production after April 1.
Although production of parasites at the number projected in Level
II of the plan can be handled by one trained technician, temporary
assistance may be required for production at Level III. Production of
parasites at Level III proceeds through Level II, just as production at
Level II must progress through Level I since each preceding level of
production provides insect stocks necessary to the next higher level.
Under Gainesville (Florida) climatic conditions, beans can be planted
as early as the end of February, and production at Level II can be
maintained almost entirely on field-grown bean plants. Host and
parasite stocks for preceding Levels I and II, as well as for scaling-up
host larva production for initiation of parasite production at Level III,
rely entirely on greenhouse-grown beans. Once female parasites
from one production level have been used to parasitize the number of
host larvae needed for production at the next level, they become
available for field release.
Despite all precautions and safeguards, occasional shortages of
3-day-old 4th instars should be anticipated. When such a shortage
occurs, two options are available. (1) Two-day-old 4th-instar hosts
can be stockpiled by accumulating them over a period of several days
and holding the larvae in a refrigerator at 4.4C. They can then be
removed, fed for about 1 day, and exposed to parasitization. (2) Host
larvae may be parasitized as soon as they are 3-day-old 4th-instars.
The parasitized larvae should then be held at normal rearing temper-
ature. As soon as they mummify (between 6 to 14 days), the mummies


Hos ad praitestck

Host and parasite stocks
maintained in off-season

(Level I)

Increased production of
hosts and parasites
(Level II)

Increased production of
hosts and parasites
(Level III)

Number of

of host

Number of
host larvae

Number of
adult beetle
units (each
15Y +156)**

500 < 40 50 1
(reared in 2
standard units used

(reared in 20 ---

standard units) torst

50000 4000 5000 30
(reared in larger

*Based on (a) female to male sex ratio of 5:1 and (b) 15 individual parasites (2 and d) per host mummy.
**Based on daily egg production of 4-5 egg masses per unit and 40 eggs/mass; any shortage of host larvae can be compensated for by adjustment of
larval development in refrigerator set at 4.4C.

Fig. 8. Schematic diagram showing requirements for scaling up production of Pediobius foveolatus from culture maintenance to
50,000 adults for use in inoculative releases against Mexican bean beetle.

are placed in a refrigerator at 4.4C. None should be held at this
temperature longer than seven days before removal for adult para-
site emergence. This will allow accumulation of parasitized 4th-
instars over a 7-day period and delay emergence from the oldest
mummies 8 to 10 days. Of these two methods, the first has the
disadvantage of a somewhat higher pre-parasite emergence mortal-
ity rate, while the second tends to prolong the period of adult emer-
Ultimately, successful use of P. foveolatus for control of Mexican
bean beetle is contingent on availability of high quality adult stock
that can be released in a timely manner at selected locations. This
bulletin provides information and instruction about a technique for
maintaining a culture of the parasite and scaling up production as
required for inoculative releases at either the garden or regional
In any effort to maintain plant or animal cultures, success depends
on knowledge of the organism's requirements. In the case of P.
foveolatus, the problem is compounded by need to maintain three
such cultures-the bean plant, the Mexican bean beetle, and the
parasitoid. With knowledge of requirements and techniques needed
to satisfy them, success is still contingent on the availability of
personnel sufficiently experienced to be sensitive to the organism's
needs. Finally, personnel responsible for such cultures must be able
to schedule their time as required to accommodate the needs of the
organisms they have in culture.

Angalet, G.W., L.S. Coles, and J.A. Stewart. 1968. Two potential parasites of
the Mexican bean beetle from India. J. Econ. Entomol. 61:1073-1075.
Chapin, E.A. 1936. Correct name for Mexican bean beetle. J. Econ. Entomol.
Commonwealth Institute of Entomology. 1954. Distribution maps of pests.
Series A, Map no. 46: Epilachna varivestis Mulsant.
Entomology Research Division, Agricultural Research Service, USDA. 1958.
The Mexican bean beetle in the east and its control. U.S. Dep. Agric.
Farmers Bull. 1624. 17 pp.
Gordon, R.D. 1975. A revision of the Epilachninae of the Western Hemi-
sphere (Coleoptera: Coccinellidae). U.S. Dep. Agric. Tech. Bull. 221.
Howard, N.F., and L.L. English. 1924. Studies of the Mexican bean beetle in
the southeast. U.S. Dep. Agric. Bull. 1243.
Howard, N.F., and B.J. Landis. 1936. Parasites and predators of the Mexican
bean beetle in the United States. U.S. Dep. Agric. Circ. 418. 12 pp.

Hussey, N.W., and L. Bravenboer. 1971. Control of pests in glasshouse
culture by introduction of natural enemies. In C.B. Huffaker, ed.,
Biological Control. Plenum Press, New York. 788 pp.
Kogan, M. 1971. Feeding and nutrition of insects associated with soybeans:
I Growth and development of the Mexican bean beetle, Epilachna
varivestis Mulsant (Coleoptera: Coccinellidae) on artificial media. Ann.
Entomol. Soc. Am. 64:1044-1050.
Kogan, M. 1972. Intake and utilization of diets by the Mexican bean beetle,
Epilachna varivestis a multivariate analysis. In J.G. Rodriguez, ed.,
Insect and mite nutrition significance and implications in ecology and
pest management. North-Holland Publ. Co., Amsterdam. 702 pp.
Nong, L. 1982. Aspects of the reproductive biology of Pediobius foveolatus
(Crawford) (Eulophidae: Hymenoptera), parasite of Epilachna spp. (Coc-
cinellidae: Coleoptera). Ph.D. Diss., Univ. of Florida, Gainesville. 193 pp.
Schaefer, P.W., R.J. Dysart, R.V. Flanders, T.L. Burger, and K. Ikebe. Mex-
ican bean beetle (Coleoptera: Coccinellidae) larval parasite Pediobius
foveolatus (Hymenoptera: Eulophidae) from Japan: Field release in the
United States. Environ. Entomol. (in press).
Sherman, F., and J.N. Todd. 1939. The Mexican bean beetle in South Caro-
lina. S.C. Agric. Exp. Stn. Bull. 322. 24 pp.
Stevens, L.M., A.L. Steinhauer, and J.R. Coulson. 1975a. Suppression of
Mexican bean beetle on soybeans with annual inoculative releases of
Pediobius foveolatus. Environ. Entomol. 4:947-952.
Stevens, L.M., A.L. Steinhauer, and T.C.Elden. 1975b. Laboratory rearing of
the Mexican bean beetle and the parasite Pediobius foveolatus, with em-
phasis on parasite longevity and host-parasite ratios. Environ. Entomol.
Tauber, M.J., and R.G. Helgesen. 1978. Implementing biological control
systems in commercial greenhouse crops. Bull. Entomol. Soc. Am. 24:424-
Thomas, F.L. 1924. Life history and control of the Mexican bean beetle. Ala.
Agric. Exp. Stn. Bull. 221.
Turner, N. 1932. Mexican bean beetle injuring rye. J. Econ. Entomol.

This public document was promulgated at an annual cost of
$1,072.70, or 71 cents per copy, to provide gardeners and com-
mercial growers with information on rearing Pediobius
Foveolatus for control of Mexican bean beetle.

All programs and related activities sponsored or assisted by the Florida
Agricultural Experiment Stations are open to all persons regardless of race,
color, national origin, age, sex, or handicap.

ISSN 0734-8452