Group Title: host-parasite relationship of Graphognathus spp. larvae and Neoaplectana dutkyi /
Title: The Host-parasite relationship of Graphognathus spp. larvae and Neoaplectana dutkyi
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Title: The Host-parasite relationship of Graphognathus spp. larvae and Neoaplectana dutkyi
Physical Description: 75 leaves : ill. ; 28 cm.
Language: English
Creator: Harlan, Donald Paris, 1941-
Publication Date: 1973
Copyright Date: 1973
 Subjects
Subject: White-fringed beetles   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1973.
Bibliography: Includes bibliographical references (leaves 67-74).
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Donald Paris Harlan.
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Bibliographic ID: UF00097578
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000427703
oclc - 37704132
notis - ACH6820

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THE HOST-PARASITE RELATIONSHIP OF
GRAPHOGNATHUS SPP. LARVAE AND NEOAPLECTALNA DUTKY!








by



Donald Paris Harlan


A DISSERTATION PRESENTED TO THE GRADUATE
COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL
FULFIL MENT OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPiiY






UNIVERSITY OF FLOPIDA
1973














ACKNOWLEDGEMENTS


I wish to express my appreciation to Dr. W. H. Whitcomb, Dr. V. G.

Perry, and Dr. W. G. Eden for their kindness, understanding and guidance.

Appreciation is also express to Drs. S. H. Kerr, G. E. Allen, and

G. C. Smart, Jr. of the Department of Entomology and Nematology for their

assistance; to Dr. H. N. Miller and Dr. F. W. Zettler of the Department

of Plant Pathology for their guidance; to Drs. S. R. Dutky, R. E. Lowe,

D. E. Weidhaas, B. J. Smittle, R. H. Roberts, D. F. Martin, and R. G.

Dahms of the United States Department of Agriculture for their courtesy

and assistance; to Dr. H. R. Gross, Jr., Mr. F. J. Bartlett, Mr. G. R.

Padgett, Mr. J. A. Mitchell, Mr. Z. A. Shaw, and Mrs. Billie Alley who

assisted in this investigation at the Whitefringed Beetle Investigations

Laboratory; to the employees of the Animal and Plant Health Inspection

Service in Louisiana, Alabama, and Mississippi for their assistance;

and finally, to my wife, Hazel, and daughters, Cathy and Donna, for

their sacrifices, indulgence, motivation and affection that made this

struggle possible and meaningful.














TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS.................................................. ii

LIST OF TABLES .................................................... vi

LIST OF FIGURES.................................................... viii

ABSTRACT........................................................... ix

INTRODUCTION........................................................ 1

LITERATURE REVIEW

HOST

Taxonomy....................................................... 2

Biology....................................................... 3

Control....................................................... 6

PARASITE

Taxonomy....................................................... 8

Biology........................................................ 9

Laboratory and field tests .................................... 11

MATERIALS AND METHODS

Calco dye tests............................................... 14

DD-136 test on adult whitefringed beetles...................... 15

DD-136 + Aqua-Gro wetting agent on whitefringed beetle larvae
in balled and burlapped, and potted nursery plants......... 16

DD-136 from Nutrilite Products, Inc., on whitefringed beetle
larvae in balled and burlapped plants...................... 17

Whitefringed beetle larvae in pots of rye treated with DD-136
nematodes from Nutrilite Products, Inc...................... 18








Page


DD-136 treatment of whitefringed beetle larvae in potted
nursery plants held at different temperatures.............. 19

Rate of reproduction of the DD-136 nematode in whitefringed
beetle larvae .............................................. 20

DD-136 nematodes against whitefringed beetle larvae in small
field plots, 1966.......................................... 21

DD-136 nematodes applied in fall or spring to control white-
fringed beetle larvae .................................... 23

DD-136 nematodes against a natural population of whitefringed
beetle larvae .............................................. 24

DD-136 nematodes against whitefringed beetle larvae in small
field plots, 1968.......................................... 26

Effects of environmental factors on the host and the parasite. 27

RESULTS
Calco dye tests............................................... 30

DD-136 test on adult whitefringed beetles..................... 30

DD-136 + Aqua-Gro wetting agent on whitefringed beetle larvae
in balled and burlapped, and potted nursery plants......... 30

DD-136 from Nutrilite Products, Inc. on whitefringed beetle
larvae in balled and burlapped plants...................... 33

Whitefringed beetle larvae in pots of rye treated with DD-136
nematodes from Nutrilite Products, Inc..................... 33

DD-136 treatment of whitefringed beetle larvae in potted
nursery plants held at different temperatures.............. 33

Rate of reproduction of the DD-136 nematode in whitefringed
beetle larvae .............................................. 38

DD-136 nematodes against whitefringed beetle larvae in small
field plots................................................ 38

DD-136 nematodes applied in fall or spring to control white-
fringed beetle larvae ..................................... 40

DD-136 nematodes against a natural population of whitefringed
beetle larvae .............................................. 44

DD-136 nematodes against whitefringed beetle larvae in small
field plots, 1968........................................ 44









Page

Effects of environmental factors on the host and the parasite. 46

SUMMARY............................................................ 60

DISCUSSION......................................................... 62

REFERENCES CITED.................................................... 67













LIST OF TABLES


Table Page

1. Mortality of adult whitefringed beetles 12 days after feed-
ing on peanut foliage treated with DD-136 nematodes...... 31

2. Whitefringed beetle stages recovered 60 days after treatment
with 200,000 DD-136 nematodes per balled and burlapped
or potted plant.......................................... 32

3. Insecticides found by GLC of soil from plants used in test
of DD-136 nematodes from Nutrilite Products, Inc. on
whitefringed beetle larvae............................... 34

4. Whitefringed beetle larvae recovered from balled and bur-
lapped plants 4 weeks after treatment with 1 million
DD-136 nematodes from Nutrilite Products, Inc............ 35

5. Whitefringed beetle larvae recovered from pots of rye 30
days after treatment with 1 million DD-136 nematodes from
Nutrilite Products, Inc.................................. 36

6. Whitefringed beetle larvae recovered from Ilex crenata var.
hetzi plants 28 days after treatment with 50,000 active
infective stage DD-136 nematodes per plant............... 37

7. Average numbers of whitefringed beetle larvae/ft2 in small
plots treated with DD-136 nematodes Dec. 13, 1966........ 39

8. Soil cores taken from plot no. 17 which was used to monitor
the nematode and larval whitefringed beetle population.
1966...................................................... 41

9. Mortality of whitefringed beetle larvae exposed to nematodes
extracted from field plot soil samples May 13, 1967...... 42

10. Whitefringed beetle larvae recovered from plots treated with
DD-136 nematodes. 1967-68............................... 43

11. Effect of DD-136 nematodes upon a natural population of
whitefringed beetle larvae 11 months after nematodes were
introduced by surface spraying........................... 45








Table Page

12. Rainfall recorded for small field plots treated with
DD-136 nematodes. 1968................................... 47

13. Effect of DD-136 nematodes upon the whitefringed beetle
larval population 3 months after the nematodes were
introduced onto the surface of small plots............... 49

14. Whitefringed beetle larvae recovered from plots treated with
DD-136 nematodes Apr. 12, 1972............................ 55

15. Weight of whitefringed beetle larvae before introduction
into and after recovery from field plots. 1972........... 57

16. Head capsule width of whitefringed beetle larvae recovered
from plots. 1972....................................... 58













LIST OF FIGURES


Figure Page

1. Avg percentage moisture in the top 10 in. of soil
in small field plots, 1968............................... 48

2. Avg percentage soil moisture for watered vs. nonwatered
plots, 1972.............................................. 50

3. Soil temperatures at 2 in. depth in watered vs. nonwatered
plots, 1972.............................................. 51

4. Soil temperatures at 4 in. depth in watered vs. nonwatered
plots, 1972.............................................. 52

5. Soil temperatures at 6 in. depth in watered vs. nonwatered
plots, 1972.............................................. 53


viii













Abstract of Dissertation Presented to the
Graduate Council of the University of Florida in Partial
Fulfillment of the Requirements for the Degree of Doctor of Philosophy



THE HOST-PARASITE RELATIONSHIP OF
GRAPHOGNATHUS SPP. LARVAE AND NEOAPLECTANA DUTKYI



By

Donald Paris Harlan

August, 1973



Chairman: Dr. W. H. Whitcomb
Major Department: Entomology and Nematology



A larval population of the whitefringed beetle, Graphognathus spp.,

in a Louisiana grassland field was reduced 38% by Neoaplectana dutkvi

Jackson applied at 40,000 nematodes/ft2. In Mississippi an introduced

larval population was reduced 50% by the nematode applied at 50,000/ft2.

Tests were conducted to study some of the environmental factors

in the field and to determine how they affected the host and the para-

site. There were no significant differences in host survival between

the treated and check plots or between watered and nonwatered plots.

There were significant differences in the survival of larvae at 3 soil

depths, with the survival being greater at the 4 in. than the 2 or 6 in.

depth. There was a significant difference in weight after 42 days be-

tween larvae from nonwatered and watered plots. There were no signifi-

cant differences in larval head capsule widths for watered vs. nonwatered

ix







or the 3 soil depths.

Ilex crenata var. hetzi Thunberg potted plants containing feeder

and nonfeeder larvae were held for 28 days at 160 or 270C after treat-

ment with 50,000 nematodes per plant. There were significant differ-

ences in larval survival between treatments, temperatures and stages.

The higher temperature yielded better control of both feeder and non-

feeder stage larvae by the nematode. The survival of both stages in

the checks was lower at the higher temperature. The number of treated

feeder stage larvae was significantly lower than the check larvae

recovered at 160C. This was not true for the nonfeeder larvae at this

temperature.

Ten whitefringed beetle larvae were injected with nematodes to

determine the rate of reproduction of the parasite in this host. The

average nematode yield was 6000 per larva, or 162/mg of body weight.













INTRODUCTION


The various species of the genus Graphognathus are individually

and collectively called whitefringed beetles. Tissot (1938) classified

the whitefringed beetle as: Order Coleoptera; Family Curculionidae;

Subfamily Otiorhynchinae; Genus and species Naupactus leucoloma Boheman.

The whitefringed beetle, native to South America, was first found

in the continental United States in 1936 devastating crops in northwest

Florida and southern Alabama (Watson 1937a, b). Populations of white-

fringed beetles now extend from Florida north to Virginia and Missouri

and west to eastern Texas (Gross et al. 1972b). In these areas, the use

of chlorinated hydrocarbon insecticides has until recently held damage

by this insect to a minimum. However, with the restricted usage of

these persistent pesticides and the development of resistance to them

(Harlan et al. 1972), damage by whitefringed beetle larvae has begun

to increase.

Larvae of the whitefringed beetle remain in the soil from 6 months

to 3 years (Young et al. 1950). The moisture and temperature conditions

vary greatly during this period; thus the potential for use of a non-

chemical control such as a biological agent appears promising. Studies

were conducted at the Whitefringed Beetle Investigations Laboratory,

Gulfport, Miss., from 1966 to 1972 to determine if the nematode Neo-

aplectana dutkyi Jackson could control larval whitefringed beetles.













LITERATURE REVIEW


HOST


Taxonomy. The whitefringed beetle was first described by Boheman

in 1840 as Nauoactus leucoloma. Buchanan (1939) stated that the white-

fringed beetle belonged to the Pantomorus Naupactus complex, which

included 240 or more named species, all indigenous to the New World.

Of these about 193 were natives of South America and about 45 of Mexico

or Central America. In 1939 the majority of the South American species

were catalogued in Naupactus, most of the others were in Pantomorus.

Buchanan's 1939 publication dealt with the taxonomy of 14 species and

varieties then known from the United States; all these were assigned

to Pantomorus. Graphcgnathus was assigned (Buchanan 1939) to sub-

generic rank under the genus Pantomorus. Buchanan (1939) identified

2 species of the whitefringed beetle that were discovered in the United

States. His identifications included Fantomorus (Graphognathus) leu-

coloma Blheman and a new species, Fantomorus (Graphognathus) p~iginus

Buchanan.

Buchanan (1942) listed 4 new species of whitefringed beetles frcm

the southeastern United States. These were: Paricmorus (Grahonatus

minor Buchanan, Pantomorus (Grap cL gna.thus) pixosus Buchanan, _Fantonoorus

(Graphognathus) stratus Buchanan. and Pantomorus (Graphogpnathus) a'-bus

Buchanan.

After further study, Buchaxnan (1947) found that the crue leucoloma







described by Boheman did not exist in the United States and also raised

Graphognathus to generic rank. The 2 species peregrinus and minor were

distinguishable enough to remain as species, but the other species

(pilosus and dubious) described previously along with 2 additional ones,

fecundus and imitator, became races in the leucoloma complex.

Anderson (1938) used a brief key to separate larvae of the white-

fringed beetle found in the United States from the larvae of closely

related species.

V. H. Owens (personal communication 1973) has prepared a manuscript

which I believe will simplify the taxonomy of the whitefringed beetle

in the United States, since this paper will redescribe and recognize

only 4 species of Graphognathus from the United States, based on

characters different from those used by Buchanan.

Biology. Until the whitefringed beetle was first found in the

United States in 1936, very little was known about its biology. The

whitefringed beetle Graphognathus spp. is considered to be native to

South America, and has been reported from Peru, Chile, Brazil, Argentina

and Uruguay (Berry 1939, 1947, Bosq 1934, Buchanan 1939, Bullock 1940,

Cortes 1941). Only in recent years has crop damage by whitefringed

beetles been reported from South America, where alfalfa, potatoes,

lentils, beans, and peppers have been the crops most severely affected,

and only in local areas are they considered major pests (Young et al.

1950).

The whitefringed beetle was recorded in Australia in 1933 (Wallace

1940) and in New Zealand on North Island in 1960 (Cumber 1960, Todd

1964) and on South Island in 1965 (Perrott 1966). The whitefringed

beetle was first found in the United States severely damaging crops in









Walton and Okaloosa Counties, Florida, in 1935 and 1936 (Watson 1937a).

The form originally found in the United States was Graphognathus leu-

coloma fecundus (Young et al. 1950).

The whitefringed beetle is now distributed from Florida, north to

Virginia and Missouri and west tc eastern Texas (Gross et al. 1972b).

Watson (1937b) gave one of che first reports on the biology of

the whitefringed beetle in the United States. Other descriptions of

its biology in the United States were made by Creighton (1939), Roberts

(1952), Young and App (1939), and Young et al. (1938).

The larvae and adults of the whitefringed beetle are general feeders

upon a wide range of plant species. The combined list of host plants

on which larvae or adults have been observed to feed in the field in-

cludes 385 species, distributed in 287 genera, 99 families, and Ll orders

(Senn 1969). The adults are known to feed on more than 170 species of

plants, including most field and garden crops, as well as ornamentals

and many weed species (Young et al. 1950). Most of the damage is caused

by the larvae which often destroy the root system of planes. Larvae

are known to feed on 240 species of plants.

The adult whitefringed beetle is incapable of flight, having the

elytra fused together (Young and App 1939). So far as is known, there

are no male whitefringed beetle, and all reproduction is parthenogenetic

(Tissot 1938). However, the female reproductive system contains a

spermatheca and a copulatory pouch.

After emerging from the soil the adults usually crawl onto plants

or other objects nearby and remain quiet for a few hours until their

body coverings harden. They then begin to search for food. Senn (1969)

reported a definite preference by the adults for night fcdding. Egg








deposition begins 5 to 25 days after adult emergence. Gross and Bartlett

(1972) found that the beetles produced more eggs in the laboratory when

held in constant darkness than when they were held in combinations of

light and darkness or constant light. The egg-laying period ranges

from 30 to 60 days. Adults may live as long as 150 days after emergence,

but most of them die within 90 days.

Eggs are deposited in masses of up to 60 or more, but the usual

number per mass is from 11 to 14. Adults fed on peanut foliage at

Florala, Ala., deposited an average of 1590 eggs, those fed on grasses

deposited an average of 3.6 eggs (Young et al. 1950). The freshly laid

egg is milky white, but after 4 or 5 days the color changes to a dull

light yellow. The individual eggs and masses are covered with a sticky

gelatinous substance which hardens after drying and causes the eggs to

adhere to one another and to objects such as plant stems, roots, and

soil. Eggs deposited in June, July, and August hatch in 11 to 30 days,

the average being about 17 days. Those laid in the fall and winter

require much longer periods for incubation, more than 100 days being

required for those deposited in December. Although larvae complete

their embryonic development within the eggs under dry conditions, they

require moisture to emerge (Young et al. 1950).

The entire larval stage is spent in the soil. Although the number

of larval instars has not been determined, there appears to be either

4 or 5 instars (Rarlan unpublished). Most larvae are found in the upper

9 in. of soil, but some have been taken to a depth of 24 in., and a few

at even greater depths.

Most larvae pupate from 2 to 6 in. below the soil surface. During

the summer months the length of the pupal period is ca. 13 days; in








cooler weather it is longer.

All species of whitefringed beetles discovered in the United States

have only 1 generation per year. They usually pass the winter in the

larval stage, and some individuals have remained in the egg stage

throughout the winter. Most of the larvae that hatch during 1 summer,

emerge as adults the following summer, but a few remain in the larval

stage as long as 2 years. A few larvae of G. peregrinus required 3 years

to reach maturity in the Gulf coastal area of Mississippi (Young et al.

1950).

Other reports on the biology of the whitefringed beetle including

laboratory rearing, range of host plants, crop damage and adult attrac-

tants have been published by Barnes and Bass (1972), Bartlett et al.

(1967), Bass and Barnes (1969a, b), Cherry (1966), Gross and Bartlett

(1972), Gross et al. (1972a, b), Harlan et al. (1970, 1971), Jarvis

(1968), Senn (1969), and Stone et al. (1971).

Control. The research on the biological control of this insect

includes that of Glaser et al. (1940) who reported on the nematode

Neoaplectana glaseri Steiner as a parasite in laboratory tests. Swain

(1943) described tests with whitefringed beetle larvae and N. glaseri

and also an undescribed species of Neoaplectana which was found in ca.

2% of field collected larvae. Swain (1945) also described the associa-

tion of nematodes of the genus Diplogaster with whitefringed beetles,

but concluded that these nematodes were not important in natural control

of this insect. Harlan et al. (1971) reported field tests with N. dutkyi

Jackson and an average reduction in whitefringed beetle larval popula-

tions of 50% compared with untreated check populations of ca. 34/ft2.

Young et al. (1950) stated that unfavorable weather and soil








conditions, parasites, predators, and diseases are important factors

in keeping whitefringed beetles in check. They also reported that a

fungus, Metarrhizium anisopliae (Metchnikoff), was known to attack the

soil-inhabiting stages of the whitefringed beetle in the Gulf coastal

area, but killed few beetles. Berry (1939) explored throughout the

whitefringed beetle infested countries of South America for natural

control agents. He found that 2 species of birds feed on the larvae,

but probably had little effect in reducing the populations, as they had

access to the larvae only during cultivation.

English and Grahaa (1933) tested several types of treatments on

whitefringed beetle larvae in balled and burlapped plants including

hot water and various chemical treatments. They concluded that potas-

sium cyanide in aqueous solution showed toxicity to whitefringed beetle

larvae, but killed the plants. Livingstone et al. (1940), Livingstone

and Swank (1942), McClurkin (1953), Swank (1949), and Swank and Latta

(1950) reported the use of methyl bromide fumigation on soil as satis-

factory for destruction of whitefringed beetle larvae. Harlan et al.

(1972) stated that D-D mixture used at 100 gal/acre killed all white-

fringed beetle larvae in the soil. Young (1944) and Keiser and Hender-

scn (1951) stated that the use of DDT formulations provided good control

of adult whitefringed beetles and Henderson et al. (1952) reported the

use of this insecticide for control of the larvae. Bennett (1967) used

dieldrin to protect direct-seeded longleaf pines from larval white-

fringed beetles. Bartlett et al. (1963) reported the use of disulfoton

as a systemic insecticide provided excellent control of adult white-

fringed beetles on peanuts in pots of soil. Press et al. (1970) and

Vardell et al. (1973) stated that the use of dichlorvos treatments








prevented the spread of whitefringed beetle adults in harvested wheat.

Other workers (Anonymous 1962, 1969, Denmark 1957, and Young et al.

1950) recommended using DDT, chlordane or dieldrin for controlling

whitefringed beetles in cropland and residential areas. However, Harlan

et al. (1972) reported resistance to dieldrin in larval whitefringed

beetles. Woodham and Bartlett (1973) compared the use of GLC to bio-

logical tests to determine resistance to dieldrin in larval whitefringed

beetles. Bowman et al. (1965) listed minimal concentrations of aldrin,

dieldrin, and heptachlor for the control of whitefringed beetle larvae

as determined by parallel gas chromatographic and biological assays.


PARASITE


Taxonomy. The nematode is classified as: Phylum Nematoda;

Class Secernentea; Order Rhabdita; Superfamily Rhabditoidea; Family

Neoaplectanidae and Genus and species, Neoaplectana dutkvi Jackson

(Turco et al. 1971).

The nematode N. dutkyi was first found in 1954 in larvae of the

codling moth, Carpocapsa pomonella (L.) (Dutky and Hough 1955). This

nematode was examined by Dr. G. Steiner who considered it to be a

member of the Steinernematidae very close to N. chresima Steiner

(Anonymous 1955). However, the nematode wan not described and has

been known by the code number DD-136 which was applied to it by Dutky.

Jackson (1965) made serological and scant morphological comparisons of

N. glaseri Steiner, N. carpocapsae Weiser, and DD-136. In this paper

Jackson referred to DD-136 as N. dutkii Welch, but a description was

never published by Welch. In 1953, Weiser (1955) found and later

described N. carpocapsae from diseased codling moth larvae in Czechoslo-









vakia. Poinar (1967) proposed that DD-136 be considered the DD-136

strain of N. carpocapsae and that the original N. carpocapsae be called

the Czechoslovakian strain. He described DD-136 and showed that the

mating of N. carpocapsae with DD-136 resulted in infective juvenile

progeny. He also found no consistent qualitative morphological char-

acters which could be used to separate the 2 nematodes. These facts

could be explained if the specimens that Poinar received from Weiser

in Czechoslovakia as N. carpocapsae were in reality part of the original

DD-136 culture which Dutky had previously sent to Weiser (Dutky personal

communication 1969). Since the original specimens of N. carpocapsae

were destroyed, it is impossible to substantiate this occurrence (Turco

et al. 1971). However, both of the cultures tested by Poinar were

probably DD-136. Turco et al. (1971) described the DD-136 nematode

and redescribed the other species of Neoaplectana. He contended that

N. dutkyi Jackson was a valid species name.

Biology. Dutky and Hough (1955) reported the DD-136 nematode in

larvae of the codling moth. They also reported that a bacterium was

associated with the nematode. Poinar and Thomas (1965) described a

new bacterium, Achromobacter nematophilus discovered in the intestinal

lumen of the DD-136 nematode. Poinar (1966) reported that studies showed

that most infective juveniles of DD-136 contained cells of A. nema-

tophilus Poinar and Thomas in the ventricular portion of their intestinal

lumen. hrnen the infective stage penetrated into the body cavity of a

suitable host, the bacteria were released through the anus and multi-

plied rapidly in the host's body, resulting in a fatal septicemia.

Poinar and Thomas (1966) found that the infective-stage juveniles of

the DD-136 nematode were able to penetrate and kill the insect host








without the presence of A. nematophilus or any other bacterium. However,

without accompanying bacteria, the nematode was unable to reproduce.

Dutky (1956) described the life cycle of the DD-136 nematode. The

infective stage of the nematode is the ensheathed second-stage larva.

The sheath is actually the cuticle of the second-stage larva which is

retained and the new cuticle for the third-stage larva is formed under

this outer cuticle. The nematode seeks out the host insect or is eaten

with the food, enters by way of the mouth parts, exsheaths, penetrates

the intestinal wall, after which the associated bacterium is released

into the body cavity of the host. This induces the septicemia that kills

the host. The entire process from exposure to death at 300C takes less

than 24 hours.

After the death of the host, the invading nematodes maturate and

become adults. If both males and females are present (the sex ratio is

1:1), they mate and give rise to young.

The young are born matricidally, since fertile ova produce embryos

within the ovaries of the gravid female. The eggs hatch, and the young

feed on the tissues of the adult female. They escape after her death

as second-stage larvae. Some of these (about 80%) are ensheathed and

do not develop further. Others maturate, producing adults that mate and

again produce young. Several generations may be completed until the

host cadaver is filled with ensheathed larvae. These ensheathed

(infective-stage) larvae then emerge from the cadaver in search of a

new host. At 25-30C the nematode life cycle is completed in 8 days.

Poinar and Leutenegger (1968) studied the fine structure of the

infective and normal third-stage juveniles of DD-136 and found pro-

nounced differences in the structure of the sensory organs, digestive









tract, hypodermal chords, excretory system, and somatic musculature.

They found that the digestive tract of the normal juvenile was functional

while that of the infective stage was not, and that the amphids and

somatic muscles were more highly developed in the infective stage.

The infective stage of DD-136 can survive for long periods under

proper conditions of temperature and moisture without loss of infec-

tivity. The infective stage nematode is also resistant to many chemicals

including most of the insecticides and fungicides in common use (Dutky

1969).

The nematode is not resistant to drying and is quickly killed by

desiccation. It also requires a moist surface in order to migrate in

search of a host.

The DD-136 nematode can be propagated in enormous numbers in the

laboratory. Dutky et al. (1964) described a technique for mass propa-

gation using larvae of the greater wax moth, Galleria mellonella (L.)

as the host. House et al. (1965) described the use of a dog biscuit

medium for successfully propagating the DD-136 nematode.

Dutky et al. (1967a, b) determined some of the sterol requirements

of the DD-136 nematode.

Laboratory and field tests. Niklas (1969) listed more than 120

species of insects as hosts for the DD-136 nematode. The DD-136 nema-

tode has been field and laboratory tested as a biological control

agent against several soil insects with varying degrees of success,

This nematode infected over 30 species of Canadian insects in laboratory

tests (Anonymous 1960). Harlan et al. (1971) reported a reduction in

the whitefringed beetle larval population of 50% compared with the un-

treated check of ca. 34 larvae/ft2. Creighton et al. (1968) reported








variable control in field tests on coleopterous larvae. Poinar and

Himsworth (1967) described the parasitism of the greater wax moth by

the DD-136 nematode and Poinar and Thomas (1967) reported the nature

of the associated bacterium as an insect pathogen. Reed and Carne (1967)

stated that DD-136 readily infected the larvae of the pruinose scarab,

Sericesthis geminata Boisduval, and the dark soil scarab, Othnonius

batesi Olliff, in the laboratory, but low mortality of S. geminata was

obtained in the field. Moore (1962, 1965, 1970) reported successful

field tests with the DD-136 nematode on some garden insects and the

forest insect Dendroctonus frontalis Zimmerman. Drooz (1960) reported

its use on the larch sawfly, Pristiphora erichsonii (Hartig), and

Webster and Bronskill (1968) used an evaporation retardant material in

testing the nematode on this insect. Nash and Fox (1969) also used

evaporation retardants and other solutions with the nematode on the

Nantucket pine tip moth, Rhyacionia frustrana (Comstock). Schmiege

(1963) also discussed the feasibility of using the DD-136 nematode for

control of various forest insects. Chamberlain and Dutky (1958) re-

ported an 85% reduction in larval population in field tests on the

tobacco budworm, Heliothis virescens (F.) on tobacco. Cheng and Bucher

(1972) indicated that the nematode controlled Hyleya spp. on tobacco

as well as did the standard insecticide, diazinon. Jaques (1967)

reported mortality of 5 apple insects induced by the DD-136 nematode.

Welch and Briand (1961a, b) had some success in controlling the

Colorado potato beetle, Leptinotarsa decemlineata (Say), and the cabbage

maggot, Hylemya brassicae (Bouche). Lam and Webster (1972) used DD-136

and a preparation of Bacillus thuringiensis var. thuringiensis Berliner

for controlling Tipula paludosa Meigen larvae. Welch and Bronskill





13


(1962) reported parasitism of mosquito larvae by the DD-136 nematode,

but also found that some of the nematodes were encapsulated by the

larvae after penetrating the gut wall. Reviews on the use of the DD-136

nematode in insect control have been written by Dutky (1967), Nickle

(1972), Steinhaus (1964), and Welch (1962, 1963, 1965).













MATERIALS AND METHODS


Infective stage DD-136 nematodes used in tests described herein

were produced by Dr. S. R. Dutky, USDA, Agr. Res. Serv., Insect

Pathology Pioneering Research Laboratory, Beltsville, Md.; Nutrilite

Products, Incorporated, Buena Park, Calif.; or USDA, Agr. Res. Serv.,

Whitefringed Beetle Investigations Laboratory, Gulfport, Miss. Those

produced by Dutky or the Gulfport Laboratory were propagated in larvae

of the greater wax moth according to the method of Dutky et al. (1964).

Those produced by Nutrilite were reared on dog food biscuits by the

method of House et al. (1965).

The whitefringed beetle larvae used in these tests were from

several locations where different species of this insect are found.

The larvae cannot be identified to species. Therefore, in this section

the whitefringed beetles are referred to as Graphognathus spp..

Calco dye tests. Most infection by DD-136 is through entry of

the host's mouth with the food. Therefore, a test was set up to deter-

mine if whitefringed beetle larvae ingest soil and might consume the

nematode also. Calco N-1700 powdered dye was mixed with benzene to

make a 1% dye solution. This solution was then mixed with sandy loam

soil at a rate of 1 ml/3 g soil. The soil was then air-dried to re-

move the benzene, and used to fill 40 plastic pots 1.5 inches in diam.

Two field collected whitefringed beetle larvae were then placed in

each pot and held at ca. 240C. The larvae were examined for a 10-day

period to determine if there was dye in the alimentary canal. The








first group of larvae were examined after 3 days and daily thereafter.

After concluding the first dye test, a second was set up using the

same procedures as the first. Fifty larvae were placed 2/pot in 25 pots

of dyed soil. The larvae were examined during the next 7 days beginning

1 day after they were placed in the soil.

DD-136 test on adult whitefringed beetles. To determine the

susceptibility of whitefringed beetle adults to DD-136 nematodes, a

feeding test was conducted on peanut foliage. A spray treatment of

the nematodes (furnished by Dutky) was applied to peanut leaves with

a one-quart hand sprayer. A water suspension of the nematodes was

sprayed onto the leaves of 5 freshly cut peanut plant branches. The

number of active nematodes applied per branch was calculated to be

165,000.

A treatment was also applied by dipping peanut branches having

ca. equal numbers of leaves into a water suspension of the nematodes.

This method was calculated to give 2000 active nematodes on each of 5

peanut branches. Three peanut branches were dipped in tap water and

used as checks.

After treatment each peanut branch was placed in a cage similar to

that described by Gross and Bartlett (1972). These consisted of cylin-

drical scree cages 12 in. high, over 6 in. earthenware flower pots

filled to within 1 in. of the top with clay loam soil. A 2 oz bottle

of water was imbedded in the center of the pot and supported the peanut

foliage. Twenty field collected whitefringed beetle adults were placed

in each cage and held in a screened insectary. The branches of peanut

foliage were removed after 5 days and replaced with fresh branches.

The test was concluded after 20 days.









DD-136 + Aqua-Gro wetting agent on whitefringed beetle larvae in

balled and burlapped, and potted nursery plants. This test was con-

ducted to determine if more parasitism of whitefringed beetle larvae by

the DD-136 nematodes could be attained when a wetting agent was used on

the soil. The theory was that one reason the nematode was not infecting

a greater number of the whitefringed beetle larvae in the soil was be-

cause the nematode was unable to move freely through the soil due to

the high surface tension of the water on the soil particles. The nema-

tode requires a free film of moisture on which to move when seeking out

a host (Dutky 1956). If a wetting agent was used to lower the surface

tension of the water on the soil particles more water would be avail-

able for nematode movement and therefore possibly greater infectivity of

the host.

Aqua-Gro, a wetting agent which is used on golf courses to make

more water available to the grass roots by lowering the surface tension

of the water, was tested with the nematode on whitefringed beetle larvae

in nursery plants.

Two year old azalea nursery plants for the test were bought from

a wholesale nursery near Mobile, Ala. The plants consisted of 21 balled

and burlapped, and 21 potted in 1 gal containers. One ounce of Aqua-

Gro granular formulation per gal of water was used to make the solution

for dipping 8 of the balled and burlapped plants. The plant ball was

soaked in this solution for 30 sec. Eight potted plants were treated

by placing 0.5 tsp of Aqua-Gro in each pot and watering it into the

soil.

One week later 20 field collected wnitefringed beetle larvae were

placed in each container. The nematode treatment was made 2 days after








introducing the larvae. Five hundred milliliters of nematode solution

was used to treat each of 16 balled and burlapped plants, 8 of which had

been treated with Aqua-Gro. Five balled and burlapped and 5 potted

plants were treated with 500 ml of distilled water and used as checks.

The nematodes were in a solution of 0.1% formalin and each 500 ml

contained ca. 200,000 active infective stage DD-136 nematodes. These

nematodes were also furnished by Dutky.

After treatment the plants were maintained outside for 60 days

at which time the soil was washed and the number of larvae remaining

was recorded. During this holding period the plants were kept watered.

DD-136 from Nutrilite Products, Inc.., on whitefringed beetle larvae

in balled and burlapped plants. In order to test DD-136 nematodes

produced by Nutrilite Products, Inc., Lakeview, Calif., the nematodes

were used to treat whitefringed beetle larvae in balled and burlapped

azaleas. Fifteen 2 year old azalea plants were bought from a nursery

near Mobile, Ala. and taken to Gulfport, Miss. where the tests were

conducted. Twenty-five field collected whitefringed beetle larvae were

placed in each soil ball. The soil moisture content of the plant balls

was determined by the gravimetric method (Gardner 1965) on 100 ml of

soil from each plant. A soil sample was also taken to determine if

insecticides were in the soil by GLC (Woodham and Bartlett 1973).

One-hundred milliliters of nematode solution and 50 ml of water (used

to rinse the container) was poured on each of 10 plant balls. This

produced about 1 million active infective stage nematodes per plant.

Five check plants were treated with 50 ml of water. The nematodes were

used in the test the same day they were received from the supplier. The

nematodes were shipped via air freight in an insulated jug containing








a solution of 0.09% sodium chloride, 1% Gelgard and l:4x105 Thimerosol.

After treatment each soil ball was placed in a plastic bag to keep the

soil moisture at a high level. The bag was taped at the top with the

above-soil part of the plant out of the bag. The plants were then

placed in a room at 270C, 90% RH, and 8 hr of light per day. The

soil moisture of 4 of the plants was determined 2 wk after treatment

and that of 4 of the others was determined 4 wk after treatment.

Surviving whitefringed beetle larvae were retrieved 4 wk after treat-

ment by washing the soil from the plant balls through 8, 16, and 24

mesh/in, screen and floating the larvae from the screens into a water-

filled pan (Harlan et al. 1971).

Whitefringed beetle larvae in pots of rye treated with DD-136

nematodes from Nutrilite Products, Inc. In an effort to determine if

a high concentration of insecticides found in the soil had adversely

affected the results in the recovery of larvae in the previous test,

another test was conducted with insecticide-free sandy loam soil, which

had been tested and found to be free from detectable amounts of in-

secticide. The soil was used to fill twenty 6 in. diam clay pots, and

then were watered and planted to 'Elbon' rye, Secale cereal L.. Four-

teen days later, 20 field collected whitefringed beetle larvae were

placed in the soil in each pot. Then after 8 days 12 of the pots were

treated with 1 million active infective stage DD-136 nematodes contained

in 40 ml of solution. These nematodes were also from Nutrilite Products,

Inc., and the carrying solution was the same as described for the previous

test. Eight of the pots were held as untreated checks. The pots were

maintained outside on a wooden bench where they were watered as needed.

Thirty days after treatment the soil from the pots was washed and the








larvae retrieved.

DD-136 treatment of whitefringed beetle larvae in potted nursery

plants held at different temperatures. A test was conducted to deter-

mine the effects of temperature and host developmental stage on control

of whitefringed beetle larvae by DD-136 nematodes. Twenty-four 2 year

old Ilex crenata var, hetzi Thunberg plants in 1 gal plastic pots were

purchased from a nursery near Mobile, Ala. Whitefringed beetle larvae

were collected near Dothan, Ala. and taken to Gulfport, Miss. The

larvae were divided into 2 groups according to feeder and nonfeeder

stage. The feeder stage was determined by the gut content which was

easily observed through the integument and appeared very dark in color.

The nonfeeder or prepupal stage was determined by the white appearance

of the gut. Six days after the larvae were collected they were placed

10/pot in the soil of the 24 Ilex hetzi plants. Twelve pots were in-

fested with feeder stage and 12 with nonfeeder stage larvae. Then 6

pots containing feeder and 6 containing nonfeeder larvae were placed

in a chamber with a temp of 160C, 80% RH, and 10 hr of light per day.

The procedure was then duplicated with the remaining 12 pots at 270C

and the same relative humidity and light conditions. Three days later

3 of the pots from each temp and larval stage were treated with 100 ml

of distilled water containing 50,000 active infective stage DD-136

nematodes. Duplicate pots were treated with 100 ml of distilled water

without the nematodes and held as checks. After treatment, the pots

were returned to their respective environments. The nematodes for this

test were produced in the laboratory at Gulfport, Miss. They were used

in the test the same day they were harvested from the culture pans. The

pots were watered as needed to keep the soil moist during the following









28 days after which the soil was washed as previously described and

the larvae were recovered.

Rate of reproduction of the DD-136 nematode in whitefringed beetle

larvae. Before conducting field tests with the DD-136 nematode on

whitefringed beetle larvae, it was necessary to know the potential rate

of increase of the nematode in this host. Ten field collected larvae

were each weighed and placed individually in petri dishes containing a

disc of no. 2 moistened filter paper 9 cm in diam. Each larva was

then injected with 3 ul of nematode suspension containing 5 nematodes.

The injection was performed with an ISCO Model M micro-applicator with

a calibrated 0.25 ml syringe and a 30 gauge needle. The larvae were

returned to the petri dish after injection and held for 24 hr at 290C.

One day after treatment the dead larvae were removed from the petri

dishes and placed on a nematode-trapping container (White 1927). The

trap was made by using a 4 cm x 4 cm x 2 cm high watch glass which was

wrapped with no. 42 Watman filter paper and placed inverted in a 9 cm

diam x 5 cm high dish. A 1:1000 formalin to water solution was added

to the dish to a depth ca. 1 cm. The whitefringed beetle larva was

placed on the filter paper on the inverted bottom of the watch glass,

so when the infective stage nematodes began emerging from the insect

larva they could move down the moistened filter paper, and into the

formalin solution where they could be harvested and counted. The

larvae were held in these dishes at 270C for ca. 24 days or until the

nematodes ceased emerging. Beginning 14 days after injection, the nema-

todes were collected and counted by determining the numbers in ten

0.1 ml samples from each collection. These numbers were then used to

estimate the number of nematodes produced per mg of host tissue.









DD-136 nematodes against whitefringed beetle larvae in small field

plots, 1966. Tests were conducted near Gulfport, Miss., in 1966-67

to study the effects of various treatments of DD-136 nematodes on white-

fringed beetle larvae, and the ability of the nematode to become estab-

lished in the soil. This test was conducted with an introduced popula-

tion of whitefringed beetle larvae in a field that was planted to 'Elbon'

rye on November 17, 1966. The field, Ruston sandy loam soil type,

was divided into sixteen 10x10 ft plots, with 10 ft borders between

plots. A Latin square design with 4 replicates of each of 4 treat-

ments was used to determine differences in treatments. A 17th plot

was also set up for sampling purposes. Newly hatched whitefringed

beetle larvae, from eggs collected in the laboratory, were introduced

into each plot at the rate of 80/ft2 during Nov. 11-21. Just prior

to introducing the larvae the plots were watered with a garden hose to

runoff to insure adequate moisture while the larvae were becoming estab-

lished. On Dec. 13, the plots were treated with 0; 10,000; 40,000; or

100,000 active infective stage DD-136 nematodes/ft2, in 2 gal of water

per plot with a 2 gal watering can. One-half of the 17th plot was also

treated with the highest rate while the other one-half was left untreated.

At a depth of 3 in., the soil was 13C, and the air temp was 5.50C at

the time of treatment. The nematodes for the test were supplied by

Dr. S. R. Dutky. The nematodes were personally delivered to Gulfport

by Dr. Dutky, who assisted in applying them to the test plots. After

treatment, the whirefringed beetle larval populations were determined

at monthly intervals (except for Apr.) through May by taking six 4 in.

diam soil cores per plot to a depth of 8 in. The cores were placed in

plastic bags and taken to the laboratory where they were washed to re-









cover and count the larvae as previously described.

At ca. weekly intervals throughout the test period, the treated

and untreated halves of the 17th plot were sampled to study the larval

whitefringed beetle and nematode populations. On Dec. 5, four random

4 in. diam soil cores 4 in. deep were taken from this plot since it

had not been treated at this time. However, on the dates following

treatment, the samples were taken by collecting two 4 in. diam soil

cores 8 in. deep from the treated and untreated halves of the plot.

The cores were divided into a top one-half (0-4 in.) and a bottom one-

half (5-8 in.). The 2 halves from the corresponding depths and treat-

ments were mixed together and enough soil removed to fill one 4 in.

diam petri dish, to which was added 5 large feeder-stage whitefringed

beetle larvae. The dishes were then held at 270C for a 2 wk period

after which the larval mortality was determined and the dead larvae

were examined for DD-136 nematodes. The remainder of the soil not used

for the petri dishes was washed through screens and the larvae floated

out in a pan of water and their numbers recorded.

On May 12, after most of the test reported here was concluded, the

nematode population from 2 replications of each of the 4 treatments was

also checked by bioassay with whitefringed beetle larvae. Two 4 in.

diam soil cores 6 in. deep were taken from each of 8 plots. These soil

cores were washed through a 60 and a 325 mesh/in, sieve. Material from

the 325 mesh sieve was washed into 4 centrifuge tubes and centrifuged

at 300 g for 4 min. After centrifuging, the water was decanted and a

sucrose solution was added to the mud and nematodes in the bottom.

This sucrose solution was made by dissolving 1 lb of sugar with enough

water to total 1 qt. The tubes were shaken well to mix the mud with









the sugar solution and then were centrifuged for 3 min. The supernatant

sugar solution was then poured through a 325 mesh sieve. The nematodes

caught on the sieve were gently washed with water to remove all sugar

and then washed into a beaker. The material in the beaker was allowed

to settle. The supernatant was decanted leaving 3 ml volume which was

poured onto a no. 3 Watman filter paper disc in the bottom of a 4 in.

petri dish. Five feeder-stage whitefringed beetle larvae were placed

in each dish which was then held at 270C for a 2 wk period after which

the larval mortality and per cent nematode infectivity was determined.

DD-136 nematodes applied in fall or spring to control whitefringed

beetle larvae. The test was begun in Sept. 1967 to determine if a

nematode treatment was more effective when applied in the fall or spring

on an introduced field population of whitefringed beetle larvae. The

test was conducted in the same general field area described in the pre-

vious test. A different portion of the field was used. The soil in

this area was fumigated with methyl bromide at 1 lb/100 ft2 on Aug. 14.

A soil sample was taken Sept. 1 for chemical analysis to determine if

there was any bromine residue. Thirty-two 10x10 ft plots were set up

with 10 ft borders between plots. They were arranged as 4 plots across

and 8 plots deep, and divided into 2 groups of 16 plots each, with I

group to be used for the fall treatment and the other for the spring

treatment. Each set of treatments was arranged in a Latin square design

with 4 replicates of each of 4 treatments including a check. The plots

were sown with 'Elbon' rye Sept. 1. Newly hatched whitefringed beetle

larvae, from eggs collected in the laboratory, were introduced at the

rate of 80/ft2 into each plot to be treated in the fall during Sept, 1 -

Oct. 11. Larvae were introduced into the plots to be treated in the








spring, during Oct. 13-20. On Sept. 12, the fall treatments were made

with 0; 10,000; 40,000; or 100,000 active infective stage DD-136 nema-

todes/ft2 in 2 gal of water per plot with a 2 gal watering can. The

air temp at the time of treatment was 270C. The nematodes for this test

were also produced by Dr. S. R. Dutky and were shipped from Beltsville,

Md. to Gulfport via air express the day before they were distributed

onto the plots. Sampling of the larval population in these plots was

begun Nov. 13 by taking six 4 in. diam soil cores 6 in. deep from each

plot. Another sample was taken in Dec. However, only 3 soil cores were

taken per plot from Jan. through June. The soil cores were taken to the

laboratory and washed to recover larvae as previously described. A

portion of the samples from each plot was placed in a petri dish to which

4 last-instar wax moth larvae were added to determine nematode infec-

tivity. Wax moth larvae were used because they have been reported to be

one of the preferred host of the nematode (Dutky et al. 1964). The

dishes were held at 270C for a 2 wk period after which time larval mor-

tality and nematode infectivity was determined.

The spring treatment plots were treated Mar. 15, 1968 at the same

rates used on the fall treated plots. These plots were sampled in Apr.,

May and June by taking three 4 in. diam by 6 in. deep soil cores from

each plot and washing the cores to recover the larvae. The cores were

also used to check for nematodes by exposing 4 wax moth larvae to soil

in a petri dish.

DD-136 nematodes against a natural population of whitefringed

beetle larvae. In order to test the nematodes in natural field con-

ditions, a test was conducted against a natural population of white-

fringed beetle larvae in a 12 acre grassland field on a Stough fine








sandy loam soil type located near Franklinton, La. Twelve 100x100 ft

plots separated by 20 ft borders were used for the test. Larval popu-

lations were checked before the treatment by counting the number present

in 25 samples (Ix1xl ft) from each plot (5 rows 20 ft apart).

Four treatments of 0; 10,000; 20,000; or 40,000 active infective-

stage DD-136 nematodes/ft2 were applied to the 12 plots (3 replicates

in a row) on Apr. 24, 1968, with a piston-type pump sprayer mounted

on a jeep. The sprayer had 11 cone-type nozzles spaced 1 ft apart

which covered a swath 10 ft wide. The nematodes used were supplied by

Dr. S. R. Dutky. Nematodes were stored in insulated jugs at 50,000/ml,

and 7.10C, and transported at a concentration of 100,000/ml. They were

brought to Gulfport by airplane by Dr. S. R. Dutky (who also assisted

in applying them) and transported by automobile to La. The insulated

containers were always protected from heat. The nematodes were applied

at the rate of 12 gal of water per plot at an operating pressure of

12 14 psi. The soil temp was 190C at the time of application, and

1 in. of rain had fallen 4 hr before the treatments were applied.

The plots were sampled monthly for nematodes by taking twenty-five

2 in. diam soil cores per plot to a depth of 6 in. (taken in 5 rows,

20 ft apart, cores within a row 20 ft apart), avoiding resampling the

exact location of previous months. Aliquots of a composite sample

of moist soil, composed of the cores from each plot, were portioned

out in 3 petri dishes, 5 last-instar wax moth larvae were placed in each

dish. The dishes were then held for 2 wk at 290C and any dead wax moth

larvae were examined each wk for DD-136 nematodes. One month post-

treatment the plots were sampled for whitefringed beetle larvae by the

same method described for the pretreatment samples. In addition, on








March 26, 1969, 11 months after the nematodes were applied, the larval

population was again checked by collecting seventy-five 2 in. diam soil

cores 6 in. deep from each plot (5 rows of 15 cores each), placing them

in plastic bags, and taking them to Gulfport where they were washed and

the larvae counted.

DD-136 nematodes against whitefringed beetle larvae in small field

plots, 1968. A test was conducted with an introduced population of

whitefringed beetle larvae in a field near Gulfport that had been

planted to rye on Sept. 15, 1968. The area which was in the same field

described in 2 of the previous tests was arranged as thirty-two 10x10 ft

plots set up in a randomized complete block design with 8 replicates of

each of 4 treatments. Newly hatched whitefringed beetle larvae, from

eggs collected in the laboratory, were introduced into each plot at the

rate of 100/ft2 during Oct. 2 6. Just prior to introducing the larvae,

the plots were watered with a garden hose to runoff to insure adequate

moisture while the larvae were becoming established. On November 15,

the plots were treated with 0; 5000; 20,000; or 40,000 active infective-

stage DD-136 nematodes/ft2, in 2 gal of water per plot with a 2 gal

watering can. The area had received a 2 in. rain 4 days before appli-

cation of the nematodes and the moisture level was high. The 2 in. soil

temp was 180C, and the air temp was 220C at the time of treatment. The

soil pH was 5.2. After treatment, the average moisture for the top 10 in.

of soil was determined weekly by taking two 2 in. diam cores 10 in. deep

from 2 plots and using the gravimetric method to determine percentage of

moisture. Rainfall was also recorded from a rain gauge. Two 2 in. cores

were taken each month to a depth of 10 in. in each plot to determine

nematode infectivity. This soil was put into petri dishes, and 5 wax








moth larvae were held in each dish at 290C for 1 wk after which the

dead larvae were removed and examined for nematodes. On Feb. 25, 1969,

about 3 months posttreatment, populations of whitefringed beetle larvae

were sampled by taking six 2 in. diam soil cores 10 in. deep in each

plot (2 rows of 3 cores each) placing the cores in plastic bags, and

taking them to the laboratory where they were washed and the larvae

collected.

Effects of environmental factors on the host and the parasite. -

In the spring of 1972 effects of environmental factors in the field on

whitefringed beetle larvae and the DD-136 nematode were determined.

Initially an attempt was made to establish an introduced population with

newly hatched whitefringed beetle larvae as described for previous tests.

However, the newly hatched larvae which were introduced in Dec. 1971 were

present in the plots in very low numbers in Feb. 1972. This was apparent-

ly the result of poor condition of the larvae due to adverse storage con-

ditions of the eggs, since larvae used in other tests in the laboratory

and field at this time also died. To overcome this problem, field

collected larvae were used in the test.

Larvae were collected Mar. 15, 1972 from a field near Dothan, Ala.

and taken to Gulfport. One wk later the larvae were divided into groups

of 21 and placed in petri dishes. Each group of larvae was weighed and

the average weight per larva calculated. After weighing, the larvae

were used to infest 6 plots 5x5 ft with 5 ft borders between the plots.

These plots were located near Gulfport in the same area described for

some of the previous tests. Each plot was divided into 7 rows spaced

8 in. apart. The 21 larvae in each petri dish were used to infest a

row. The larvae were placed 2 in. apart alternately at 2, 4 and 6 in.








deep in each row. A total of 147 larvae was placed in each plot. On

Apr. 12, the plots were treated with 0; 50,000; and 100,000 active

infective-stage DD-136 nematodes/ft2 of soil surface with 2 repli-

cations per treatment. The nematodes were reared in the Gulfport

laboratory. They were applied in the field the same day they were har-

vested. The plots were watered to runoff and the nematodes were applied

in 1 gal of water with a 2 gal watering can. One gal of water was also

applied to the check plots. After watering, the plots avg 22% soil

moisture. The air temp was 300C. Small rye plants were growing on the

plots.

One of the plots receiving each treatment was watered daily on

wk days throughout the test. The higher soil moisture level was main-

tained to determine if this would increase the infectivity of the nema-

tode on the beetle larvae. Beginning the day the nematodes were applied,

the soil moisture, soil temp and rainfall were recorded from the plots

until the test was terminated. The temp was recorded from one watered

plot throughout the test at the levels of 2, 4 and 6 in. deep in the

soil. The last 2 wk of the test, these same levels were also recorded

from a nonwatered plot. A YSI Model 47 automatic scanning telethermom-

eter with Type 401 probes was used in conjunction with a YSI Model 80

strip chart recorder. The temp was monitored at each depth at hourly

intervals. The Trapezoidal Rule (Fisher and Ziebur 1965) was used on

this data to determine the area under the curve for the temp data for

each day. The formula for this rule is: AT=h[C(Yo+Yn)+YI+Y2+...+Yn-l].

The soil moisture was determined from each plot in the pm Monday through

Friday of each wk. The moisture was measured with a Troxler Model 217

surface moisture gauge. This gauge employs a neutron source and was









used with a Troxler Model 1651 scaler-ratemeter. The gauge yields

a soil moisture determination based on the top 8 12 in. of soil,

but biased toward the more moist zone. After determining the moisture

content for each plot, 3 of the plots were watered to runoff. A small

U. S. Forest Service size rain gauge was used to collect rainfall data.

Two wk after the plots were treated with nematodes, 1 row in each

plot was dug with a shovel and the whitefringed beetle larvae remaining

in that row were recovered. Later, at 2 wk intervals, the larvae were

recovered from 2 rows in each plot. The depth of the larva in the

soil was noted and the larvae were returned to the laboratory where on

some dates they were weighed and/or the head capsule width was measured.

On May 5 and June 1, two 2 in. diam soil cores were taken from each

plot at depths of: 0-2 in.; 2-4 in and 4-6 in. The 2 cores from each

plot at each depth were used to fill 3 petri dishes into which were

placed 5 wax moth larvae each. These were held at 270C for 2 wk before

they were examined for dead wax moth larvae containing DD-136 nematodes.













RESULTS


Calco dye tests. The results of this test showed that white-

fringed beetle larvae definitely ingest soil. Only 1 day or less was

required for the dye to be found in the alimentary canal. This indi-

cates that the whitefringed beetle larvae probably ingest enough soil

in 1 or 2 days to consume challenging concentrations of a pathogenic

organism which might be in the soil. ,

DD-136 test on adult whitefringed beetles. The results of this

test are shown in Table 1. The nematode treatment did not appear to

have any effect on the whitefringed beetle mortality when compared to

the untreated checks. Dead adults examined from the treated cages

showed no evidence of nematode activity. These results differ from

those of Turco et al. (1970), who reported parasitism of adults of

the rice water weevil, Lissorhoptrus oryzophilus Kuschel, and the

spotted cucumber beetle, Diabrotica undecimpunctata howardi Barber,

by Neoaplectana glaseri.

DD-136 + Aqua-Gro wetting agent on whitefringed beetle larvae in

balled and burlapped, and potted nursery plants. Thirty days after

treatment with 200,000 active infective stage DD-136 nematodes, the soil

from 1 balled and burlapped and 1 potted plant was washed to recover

the whitefringed beetle larvae; the remaining plants were washed after

60 days. The recovery of live larvae was very low from the balled and

burlapped, and potted plants (Table 2). There were no significant

differences between treatments and checks. There was, however, a much














Table 1. Mortality of adult whitefringed beetles 12 days after
feeding on peanut foliage treated with DD-136 nematodes.




Spray Dip Check
No. No. No. No. No. No.
Replicate alive exposed alive exposed alive exposed


1 13 20 17 20 5 20

2 13 20 4 20 15 20

3 13 20 0 20 4 20

4 14 20 9 20 -

5 7 20 13 20 -

Total 60 100 43 100 24 60

% alive 60 43 40

















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lower recovery from the potted plants than from the balled and burlapped

plants. This recovery was different at the 0.01 level of significance

by analysis of variance.

DD-136 from Nutrilite Products, Inc. on whitefringed beetle larvae

in balled and burlapped plants. Soil moisture of the test plants avg

33.5% before treatment and 37 and 26.6% at 2 and 4 wk, respectively,

after treatment. A soil analysis by GLC yielded the results shown in

Table 3. Surviving whitefringed beetle larvae were recovered from the

soil balls 4 wk after treatment. The results (Table 4) indicate an

avg of 7.2 and 24.8% recovery of larvae from the treated and check

plants respectively.

Whitefringed beetle larvae in pots of rye treated with DD-136 nema-

todes from Nutrilite Products, Inc. Thirty days after treatment with

1 million active infective stage nematodes per 6 in. pot, the soil was

washed and the remaining whitefringed beetle larvae were retrieved.

The results (Table 5) indicate a 27 and 38% live recovery rate from the

treated and check pots respectively.

DD-136 treatment of whitefringed beetle larvae in potted nursery

plants held at different temperatures. The Ilex hetzi plants containing

feeder or nonfeeder larvae were held 28 days at 16 or 270C after treat-

ment with 50,000 DD-136 nematodes/plant. They were then washed and the

larvae were recovered. As shown in Table 6, there were significant

differences at the 0.01 level by analysis of variance between treat-

ments, temp and stages. Only 1 feeder stage larva was recovered alive

of the 30 treated with nematodes and held at 270C. However, 9 feeder

stage larvae were recovered alive in the checks for this temp. Seven

treated feeder stage larvae were recovered alive at 160C compared with














Table 3. Insecticides found by GLC of soil from plants used in test
of DD-136 nematodes from Nutrilite Products, Inc. on whitefringed beetle
larvae.


Insecticide


PPM in soil


p,p' DDT

o,p' DDT

p,p' DDE

p,p' TDE

Chlordane


Heptachlor epoxide


3.84

1.24

2.62

0.92

17.49

0.85














Table 4. Whitefringed beetle larvae recovered from balled and
burlapped plants 4 weeks after treatment with 1 million DD-136 nema-
todes from Nutrilite Products, Inc.




No. larvae recovered
Treatment Alive Dead


Treated 18 92

% of 250 7.2

Check 31 8

% of 125 24.8














Table 5. Whitefringed beetle larvae recovered from pots of rye
30 days after treatment with 1 million DD-136 nematodes from Nutrilite
Products, Inc.




No. larvae recovered
Treatment Alive Dead


Treated 65 39

% of 240 27

Check 61 16

% of 160 38














Table 6. Whitefringed beetle larvae recovered from Ilex crenata
var. hetzi plants 28 days after treatment with 50,000 active infective
stage DD-136 nematodes per plant




No. larvae recovered
Stage
Feeder Nonfeeder
Treatment Temp Alive Dead Alive Dead


Treated 160C 7b 23 20b 10

Check 160C 17 13 19 11

Treated 270C 1 29 7 23

Check 270C 9 21 17 13


a There were significant differences at the 0.01 level by analysis
of variance between treatments, temperatures, and stages.


b Total for 3 pots of 10 larvae each.








17 in the checks. From the nonfeeder stage larvae 7 and 17 were found

alive from the treated and checks respectively at 270C. From the 160C

nonfeeder stage larvae, 20 and 19 were recovered alive in the treated

and checks respectively.

These results indicate that the higher temperature is better for

control of both stages of the whitefringed beetle larvae by the DD-136

nematode. However, the survival of both stages in the checks was lower

at the higher temperature. The optimum temperature for both stages of

the host appears to be below 270C. The number of treated feeder stage

larvae was significantly lower than the check larvae recovered at 160C.

However, this was not true for the nonfeeder larvae at this temperature.

This is probably due to the nonfeeder larvae being very inactive at the

lower temperature and,therefore, no nematodes were ingested to kill the

larvae.

Rate of reproduction of DD-136 nematodes in whitefringed beetle

larvae. Ten field collected larvae were injected with nematodes to

insure an infection in the host. As the infective stage nematodes

emerged from the host they were collected and counted. The average

nematode yield was 6000/larva, or 162/mg of body weight. This compares

with an average yield of 1370 DD-136 nematodes/mg of wax moth larva

reported by Dutky et al. (1964).

DD-136 nematodes against whitefringed beetle larvae in small field

plots. The whitefringed beetle larval populations were determined

after treatment at monthly intervals (except for Apr.) through May by

taking six 4 in. diam soil cores per plot to a depth of 8 in. The re-

sults of these samples are shown in Table 7. A statistical analysis of

the data collected May 10 showed no significant differences. The highest














Table 7. Average numbers of whitefringed beetle larvae/ft2 in
small plots treated with DD-136 nematodes Dec. 13, 1966.




Avg no. larvae/ft2a
Treatment Date sampled (1967)
nematodes/ft2 Jan. 18 Feb. 15 Mar. 22 May 10


10,000 11.0 7.7 29.6 16.2

40,000 42.5 20.1 25.3 8.2

100,000 7.7 13.9 12.9 6.7

Check 15.7 19.6 26.8 18.7


a Avg of 4 plots determined
from each plot.


from 6 4 in. diam 8 in. deep soil cores


b Statistical analysis performed on data collected May 10.









treatment of 100,000 active infective stage DD-136 nematodes/ft2

evidently had a reducing effect on the whitefringed beetle larval

population when compared to the untreated check plots. The lower treat-

ment of 40,000 nematodes/ft2 also appeared to be effective.

Plot 17 was used to monitor the larval whitefringed beetle and

DD-136 nematode population throughout the test period. This was also

accomplished by removing soil cores. These results are presented in

Table 8. The whitefringed beetle larvae were seldom found below the

4 in. soil depth in the treated or untreated halves of this plot. The

results from these samples also indicated a lower average larval popu-

lation in the treated portion of the plot than in the untreated portion.

The larger number of samples from which no larvae were recovered can

also be seen. This method also showed that the nematode was present

and infective on whitefringed beetle larvae throughout the test period

in plot 17.

On May 12, after most of the test was concluded, the nematode

population from 2 replicates of each of the 4 treatments was checked

by the centrifuge method. These results are presented in Table 9.

It is evident from these results that the DD-136 nematode was still

present in the plots and infective on whitefringed beetle larvae.

DD-136 nematodes applied in fall or spring to control whitefringed

beetle larvae. Tests were begun in Sept. 1967 to compare a fall and

a spring treatment of the nematodes on an introduced population of

whitefringed beetle larvae. The results of this test are presented in

Table 10. Neither the fall nor spring nematode treatments caused a

significant reduction in the whitefringed beetle larval population in

the plots. The nematode was not recovered from the fall treatments by














Table 8. Soil cores taken from plot no. 17 which was used to
monitor the nematode and larval whitefringed beetle population. 1966.




Date
cores Depth of No. larvae/ft2 Nematode
taken cores taken (in.) Check Treated infectivity


12-5

12-22

12-30


1-12


2-2

2-9


2-27


3-8


3-28

4-3

4-11

4-17

4-24


a Two 4 in. diam cores were taken from
the plot except on 12-5 when 4 cores
the other was not sampled.


the treated and the check of
were taken from the check and














Table 9. Mortality of whitefringed beetle larvae exposed to nema-
todes extracted from field plot soil samples May 12, 1967.




Plot No. larvae No. larvae
treatment dead infected with DD-136


10,000 4 4

10,000 4 2

40,000 3 3

40,000 3 3

100,000 5 5

100,000 3 1

Check 1 -

Check 0


a Infective DD-136 nematodes/ft2

b Counts and numbers infected determined on 5-24-67.
















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the wax moth larva bioassay at any time after the treatment was applied.

However, this same technique showed that the nematode was present in

the spring treatment plots for at least 90 days after the treatments

were applied. One of the factors which may have contributed to our

inability to find the nematodes after the fall treatment was the presence

of 20 ppm Br in the soil on Sept. 1. This fact was not known at the

time the nematodes were applied, since the analysis was not completed

until October. Even though the nematode is not effected by many pesti-

cides (Dutky 1969) low dosages of Br are lethal (Dutky personal communi-

cation).

DD-136 nematodes against a natural population of whitefringed beetle

larvae. A test was conducted against a natural population of white-

fringed beetle larvae in a 12 acre field near Franklinton, La. The pre-

treatment populations avg 9.3, 7.3, 8.8 and 7.8 whitefringed beetle

larvae/ft2 in the plots that were treated with the 40,000; 20,000; 10,000

nematodes/ft2 and for the check, respectively. One month posttreatment,

there were no apparent differences in the populations of beetle larvae

among plots. However, soil moisture had been very low, which may have

reduced the infectivity of the nematodes. When the larval populations

were checked again at 11 months posttreatment, the results were those

shown in Table 11. Plots treated with the greatest number of nematodes

had 38% fewer larvae than the check plots. However, the differences

between treatments and checks were not statistically significant. The

monthly sampling for nematodes showed that infective nematodes were present

in the treated plots the last month of the test.

DD-136 nematodes against whitefringed beetle larvae in small field

plots, 1968. After introducing newly hatched whitefringed beetle














Table 11. Effect of DD-136 nematodes upon a natural population of
whitefringed beetle larvae 11 months after nematodes were introduced by
surface spraying.




No. larvae/ft2
Treatmenta
Replicates 0 (Check) 10,000 20,000 40,000


1 25.2 28.2 16.0 19.6

2 35.6 36.2 21.5 18.4

3 29.4 9.8 20.9 17.8

Total 90.2 74.2 58.4 55.8

% of check 100 82 65 62


a Number of DD-136 nematodes introduced/ft2









larvae into the plots, rainfall records were maintained as shown in

Table 12. A total of 20.58 in. of rain was recorded during the test

period. The effect of this rainfall on the soil moisture in the top

10 in. of soil is shown in Fig. 1. The avg moisture ranged from 14.7

to 22% throughout the test period. About 3 months after the nematodes

were applied, the populations of whitefringed beetle larvae were deter-

mined by taking six 2 in. diam soil cores 10 in. deep in each plot and

washing the cores to remove the larvae. The results of these samples

are presented in Table 13. The beetle larval populations were 50% lower

in the most heavily treated plots than in the check plots. The differ-

ences were not significant at the 0.05 level by analysis of variance.

Infective nematodes were present in the treated plots at the end of the

test.

Effects of environmental factors on the host and the parasite. -

After the nematodes were distributed on the plots, the soil moisture,

soil temperature and rainfall were recorded. The soil moisture for the

watered and nonwatered plots is shown in Fig. 2. The soil moisture avg

16.29 and 13.05% for the watered and nonwatered plots, respectively.

The average for the watered plots is biased toward the minimum since

the plots were watered each time after the moisture was determined. A

total of 8.52 in. of rainfall during the test period contributed to the

average moisture for the unwatered plots.

The soil temperature was recorded hourly for the 2, 4 and 6 in.

depths throughout the test in one of the watered plots. However, the

same was recorded for a nonwatered plot only during the last 2 wk of

the test. A comparison of the soil temp at these 3 depths for the

watered and nonwatered plots is shown in Fig. 3, 4 and 5. The Trape-














Table 12. Rainfall recorded for small field plots treated with
DD-136 nematodes 1968.




In. of rain
Date Amount Cumulative


11-4-68 0.3 0.3

11-12-68 2.0 2.3

11-25-68 1.16 3.46

12-2-68 2.65 6.11

12-9-68 0.84 6.95

12-16-68 0.90 7.85

12-23-68 0.68 8.53

1-6-69 4.09 12.62

1-13-69 0.03 12.65

1-20-69 2.51 15.16

1-27-69 0.15 15.31

2-3-69 2.68 17.99

2-10-69 0.27 18.27

2-17-69 1.45 19.71

2-24-69 0.87 20.58



































22



20



18



5 16



n 14



12



10




10 31 11 20 12 10 12 30 1 19 2 8 2 28
Dote





Fig. 1. Avg percentage moisture in the top 10 in. of soil in small
field plots, 1968.














Table 13. Effect of DD-136 nematodes upon the whitefringed beetle
larval population 3 months after the nematodes were introduced onto the
surface of small plots.




No. larvae/ft2
Treatment
Replicate 0 (Check) 5000 20,000 50,000


1 15 31 15 23

2 46 8 8 31

3 31 8 31 0

4 15 115 54 8

5 15 15 15 31

6 108 92 31 0

7 46 31 31 15

8 0 15 54 31

Total 276 315 239 139

% of check 100 114 87 50


a Number of DD-136 nematodes introduced/ft2.






































............ non-watered
-------watered
rainfall


Fig. 2. -
plots, 1972.


Avg percentage soil moisture for watered vs. nonwatered


30








S20


0





~10


LM LM


In NCO
Ln LMLM


I






51






































........ ................ ... .* .................. ........... ... ........ ....... ..


----------- -------------














............ non-watered

------------watered


I I


25 26 27 28 29 30 31 1 2
May


3 4
June


5 6 7


Fig. 3. Soil temperatures at 2 in. depth in watered vs. nonwatered
plots, 1972.


, n I I


*






52





































---------------------- -- --












............. non-wate red

------------ watered
I I I I I I I I I I I I


25 26 27 28 29 30 31 1 2 3 4
May June


5 6 7


Fig. 4. Soil temperatures at 4 in. depth in watered vs. nonwatered
plots, 1972.










































* ... .. ..*.. .. .. .. .....""".""".."...... .. ...... ... ............. ........... ... **..... .. ....... .......... o










K



...""......* non-watered
------- watered


25 26 27 28
May


29 30 31 1 2


3 5 6 7


3 4
June


Fig. 5. Soil temperatures at 6 in. depth in watered vs. nonwatered
plots, 1972.


35


30


25


20
"C

15


10


5


0


^p


5 6 7








zoidal Rule was used on this data to determine the area under the curve.

There was a significant difference (0.01) between the nonwatered and

watered plots with mean areas being 619.5 and 544.2, respectively.

Therefore the nonwatered plot was significantly warmer than the watered.

There were no significant differences in temperature for the 3 depths

measured.

The larvae were recovered from the plots at 2 wk intervals. The

numbers of larvae recovered are shown in Table 14. There were no

significant differences between the treated and nontreated (check)

plots. There also were no significant differences in larval survival

between the watered and nonwatered plots. However, there were signifi-

cant differences (0.05) in the survival of larvae at the 3 soil depths,

with the survival being greater at the 4 in. than the 2 or 6 in. depth.

The larvae were weighed before they were introduced into the plots, and

after they were recovered 42 days later. The weights are given in

Table 15. There was a significant (0.01) difference in weight after

42 days between the larvae from the nonwatered and watered plots with

the mean weights being 43.5 and 22.9 mg, respectively. The higher

soil moisture seems to have had a selecting effect towards the smaller

larvae. There were no significant differences in weight for the 3 soil

depths. The width of the larval head capsule was also measured in an

attempt to determine differences in growth. These measurements are

given in Table 16. There were no significant differences in head

capsule width for the watered vs. nonwatered or the 3 soil depths. The

lack of differences in head capsule width is in contrast to the highly

significant differences in larval weights.

The use of the wax moth larvae as a bioassay for the DD-136
























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-4


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H


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*w
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zd (V




C* 0lc


N *O r I r *: I r-
I c c Io e c c e c c
CM -i- l *Hl i- rl -il H l CM~ N *i-< *~I- *-l

hl M l \O m i CM~ Nl ^O U- M a-
























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'-4















,-4 -


r-
a-i
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U
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en 0 -
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4-


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000
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6=1 1=















Table 15. Weight of whitefringed beetle larvae before introduction
into and after recovery from field plots. 1972.


Before
introduction


Mean wt (mg)
After
introduction


Nonwatered

Watered

Treated

Check

2 in. depth

4 in. depth

6 in. depth


26.1

27.9

24.8


22.0

32.0

34.1

23.5

35.5

39.2


a Weighed 147 larvae/plot.

b Weighed no. of larvae'shown in Table 16, May 24. Difference signifi-
cant (0.01) between nonwatered and watered plots.


----














Table 16. Head capsule width of whitefringed beetle larvae recovered
from plots. 1972.




Date Mean head capsule width (mm)
Depth Check LT" HTb Watered Nonwatered


4-26C 1.47 1.61 1.30 1.52 1.40

5-16

2 in. 1.39 1.55 1.58 1.52 1.49

4 in. 1.54 1.48 1.54 1.57 1.47

6 in. 1.51 1.50 1.55 1.58 1.45

5-24

2 in. 1.37 1.37 1.41 1.42 1.34

4 in. 1.51 1.55 1.52 1.51 1.54

6 in. 1.62 1.46 1.59 1.54 1.57

6-8

2 in. 1.55 1.57 1.54 1.44 1.62

4 in. 1.55 1.46 1.56 1.54 1.50

6 in. 1.48 1.38 1.27 1.44 1.37


a 50,000 nematodes/ft2.

b 100,000 nematodes/ft2.

c 4-26 larvae from different depths measured together.





59


nematodes in the treated plots showed some nematodes present. However,

this method did not show any differences in numbers or infectivity

between the watered and nonwatered plots.














SUMMARY


The purpose of this research was to study the whitefringed beetle

and the DD-136 nematode in the laboratory and field. Tests were con-

ducted to try to better understand the host-parasite relationship be-

tween these 2 organisms. Laboratory and field tests were conducted over

a 7 year period under various temperature, moisture, plant, soil, and

population conditions.

In laboratory and field tests, the whitefringed beetle larva does

not appear to be as attractive a host for the DD-136 nematode as some

of the insects of the order Lepidoptera, e.g. the greater wax moth

larva. A nematode treatment applied to peanut leaves did not appear to

have any effect on adult whitefringed beetles.

A wetting agent was added to the nematode solution used to treat

balled and burlapped, and potted plants containing whitefringed beetle

larvae. There were no significant differences between the treatments

and checks. However, there was a significantly higher number of larvae

recovered from the balled and burlapped than the potted plants.

Under controlled environmental conditions the feeder-stage white-

fringed beetle larvae were shown to be more susceptible to attack by

the nematode than were the nonfeeder stage larvae. In the checks, both

stages survived better at 160C than at 27C. Better control of both

stages of the larvae was achieved at the higher temperature.

DD-136 nematodes produced by Nutrilite Products Corp., Dr. S. R.

Dutky, and our laboratory at Gulfport, were used in field tests with








varying degrees of success. Overall, the nematodes produced by Dutky

yielded the best results.

With the different conditions used in the field tests over the

7 year period, the reduction in larval population of 50% compared to

the check population of ca. 34 larvae/ft2 and achieved with a neraatode

treatment of 40,000/ft2, was the best control documented.

Much more information was obtained from these tests on the effects

of the environment on the insect than could be substantiated for the

nematode.

The nematode was shown to be present in some of the field plots

for over a year after it was applied. However, the insects were not

economically controlled in any tests. This may be partly due to the

nematode being less attached to this insect than to others. It is

also hard to determine the number of active infective stage nematodes

which must be present in the soil at any one time for the insect to

become infected.













DISCUSSION


This study, conducted over a 7 year period, is one of the most

extensive investigations yet reported on testing of DD-136 nematodes

for possible control of one host insect group. The results of this

study indicate that the whitefringed beetle larval population can often

be reduced by applying DD-136 nematodes. This reduction in host popu-

lation might be sufficient for practical control of pests of certain

row crops such as potatoes, peanuts, and cotton. Creighton et al. (1968)

found that applications of this nematode reduced larval injury to

Centennial sweet potatoes by Diabrotica and Systena spp. and to Nugget

sweet potatoes by Conoderus falli Lane and Chaetocnema confinis Crotch;

however, the protection provided was not adequate when the potatoes

were graded for insect injury. The nematode was tested against white-

fringed beetle larvae in nursery plant containers to determine if it

could be used on plants which are under whitefringed beetle quarantine

regulations. The results obtained indicate that this nematode cannot

be considered for this use, since complete control must be certain.

Better control of both feeder and nonfeeder-stage beetle larvae

was achieved at 270C than 160C. The higher temperature may have caused

the larvae to feed more actively and consequently caused them to ingest

more nematodes than at the lower temperature. Also, the nematodes

were probably more active at the higher temperature. Reed and Carne

(1967) found that at 300C the infective juveniles were very active,

but at temperatures near 160C the generation time was doubled, adult








size and fecundity were reduced, and dispersal activity of the nema-

tode could be virtually eliminated. The feeder-stage whitefringed

beetle larvae were controlled better than the ncnfeeder-stage larvae at

both 160C and 270C. This data was presumably influenced by the feeder-

stage ingesting more food and nematodes than the nonfeeder-stage which

would be expected to feed very little. Most nematodes of this type are

accidentally ingested by the host in the course of feeding (Reed and

Came 1967).

The DD-136 nematode was found in field plots for more than a year

after it was introduced. However, the beetle larval populations which

were treated were not reduced below ca. 50% of the nontreated. This

occurrence may be due to the low reproductive rate of the nematode in

this host. The average nematode yield was 6000/larva or 162/mg of body

weight. This compares with an average yield of 1370 DD-136 nematodes/mg

of body weight in wax moth larva as reported by Dutky et al. (1964).

The low reproductive rate may hinder the parasite from being present in

a population dense enough for individuals of a sparse beetle population

to ingest the nematode. This may be true only if one assumes that no

other host insects are present in the soil of the treated field. Since

Niklas (1969) listed more than 120 species of insects as hosts of DD-136,

there could possibly be other hosts present. The low reproductive rate

of the nematode in the whitefringed beetle larva may be due to a lack

of adequate quantities of certain sterols in this host which are neces-

sary for nematode reproduction and growth. Dutky (1967) and Dutky et al.

(1967a, b) reported that sterols derived from insect tissue are essen-

tial for growth, development, and reproduction of the nematode. The

associated bacterium, Achromobacter nematophilus, must also be present








for nematode reproduction (Poinar and Thomas 1966). If environmental

conditions such as temperature are not optimum for replication of the

bacterium, some of the infective stage nematodes might not carry the

bacterium. Soil moisture is also important for nematode movement and

survival. Dutky (personal communication) found that a soil moisture

of ca. 80% of the ball point of the soil (this was determined by adding

water to dry soil until the soil would form a ball) was optimum for

nematode movement. I found that with most of the sandy loam soils used

in the nematode tests, the 80% of ball point level was ca. equal to

16-20% moisture. This is the moisture level that was sought in the

soil moisture studies. The tests did not demonstrate significant

differences in nematode infectivity caused by moisture alone.

The infectivity of the nematode varies greatly with different

propagation hosts, temperatures, and storage conditions. Some of the

problems encountered in storing the nematodes may be avoided if the

propagation host is distributed in the field. W. W. Neel and P. P.

Sikorowski (personal communication) are distributing bollworm larvae

containing DD-136 nematodes onto test plots for controlling pecan weevil

larvae. This method also furnishes protection for the nematodes under

dry or hot soil conditions until they can become established in the soil.

When the nematodes are in a storage solution, the percent activity (no.

moving) is usually determined before they are distributed in the field.

Even though the activity is low, if adequate numbers of nematodes are

used the resulting insect mortality can be significant. W. L. Tedders

and E. J. Wehunt (personal communication) obtained 67% mortality of

pecan weevil larvae in small field tests when they used nematodes with

an average activity of only ca. 19%. However, they applied ca. 1 1.5








million active nematodes/ft2 of soil surface.

In the past the nematode like many other biological control agents

(e.g. bacteria, fungi and viruses) has been applied by techniques

developed for testing insecticides. The nematodes were sprayed onto

the plants or soil and insect mortality was later determined. In-

vestigators are now realizing that environmental factors such as soil

moisture, temperature and other factors must be studied along with

nematode application rates.

I have attempted to study the effects of soil moisture, tempera-

ture, nematode application rates, and host developmental stage on the

host-parasite relationship of the DD-136 nematode and the whitefringed

beetle larva. Under optimum temperatures (apparently ca. 270C) good

control of the host was achieved. However, control in the laboratory

and the field was not consistent. I do not know whether these erratic

results were caused by the condition of the nematodes when they were

introduced or by conditions present after they were introduced. However,

both presumably contributed to the results.

Proper environmental conditions are also important for survival

of whitefringed beetle larvae. In 1 experiment, the results indicated

a significantly lower survival of larvae in potted than balled and

burlapped nursery plants. The potted plants contained a potting mix-

ture which consisted of pine bark, peat moss, sand and soil. The balled

and burlapped plants contained only sandy loam soil. Harlan et al.

(1971) found that larvae survived better in potting material consisting

of sandy loam soil alone than in mixtures with other materials.

The beetle larval survival in a field test was significantly higher

at the 4 in. than the 2 or 6 in, soil depth. The higher survival rate








may have been influenced by a combination of differences in moisture and

temperature, even though these conditions individually did not cause

significant differences in survival. Gross et al. (1972b) stated that

declines in whitefringed beetle larval populations during April and May

are an annual occurrence, the causes of which are not understood. This

decline in larval populations in the Gulf coastal states is usually

preceded by an increase in soil temperature and an increase in soil

moisture caused by precipitation of ca. 1 in. or more. During this

period many of the larvae which die are infected by fungi e.g. Metarr-

hizium anisopliae and Beauveria bassiana (Balsamo), unidentified

bacteria, or nematodes. Young et al. (1950) stated that unfavorable

weather and soil conditions, parasites, predators, and diseases are

important factors in keeping whitefringed beetles in check. They also

reported that a fungus M. anisopliae is known to attack the soil

inhabiting stages of the beetle in the Gulf coastal area, but killed

few beetles. Swain (1943) was the first to report a parasite of the

whitefringed beetle. He stated that a nematode (which appeared to be

a species of Neoaplectana) was found in more than 2% of the soil stages

of whitefringed beetles collected from Harrison County, Miss.

Nematodes and other naturally occurring parasites and pathogens

of whitefringed beetle larvae have been found. I believe the DD-136

nematode would fit into a pest management program for this insect and

many other soil insects. The major need now is for a commercial source

of large numbers of the nematode which are propagated and stored properly

so that they maintain their activity and infectivity.














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BIOGRAPHY


Donald Paris Harlan was born near Kosciusko, Mississippi on

February 9, 1941. He graduated from West Memphis High School, West

Memphis, Arkansas, in 1959. He received his B. S. degree from Missis-

sippi State University in 1963, with a major in General Agriculture.

After graduation he accepted the position of agricultural field repre-

sentative with Swift and Company. He held this position for one year

while living in Columbia, Missouri. He then entered graduate school

at the University of Missouri where he received his M. S. degree in

Entomology in January, 1966. In February, 1966, he accepted the position

of research entomologist at the United States Department of Agriculture,

Agricultural Research Service, Whitefringed Beetle Investigations Labora-

tory, Gulfport, Mississippi. He held this position until June, 1972,

when he transferred to the Bioenvironmental Insect Control Laboratory,

Stoneville, Mississippi, where he is now conducting research on the

biology and control of livestock insects. He attended the University

of Florida from September, 1967, through August, 1968, as part of the

USDA educational program.

He was married to the former Miss Hazel Jean Little in December,

1960. They have two daughters, Cathy, aged 11, and Donna, aged 10.

They live at 105 Peninsula Drive, Leland, Mississippi.














I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Willard H. Whi tconb,' Chairman
Professor of Entomology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.




Vernon G. Pero
Professor o Nematology


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



7-. tV- ^ -r Y c -
Stratton H. Kerr
Professor of Entomology



I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy. ,


4-1- 1-

Francis W. Zettler
Associate Professor of Plant Pathology














This dissertation was submitted to the Dean of the College of
Agriculture and to the Graduate Council, and was accepted as partial
fulfillment of the requirements for the degree of Doctor of Philosophy.


August, 1973


Dean, College


of Agriculture


Dean, Graduate School




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