TAXONOMY AND BIOLOGY OF Verutus volvingentis
N. GEN. N. SP. (TYLENCHIDA-NEMATA)
ROBERT PAUL ESSER
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
The author is deeply grateful to the chairmen of his
supervisory committee, Dr. V. G. Perry and Dr. A. C. Tarjan,
who have given considerable time and immeasurable assistance
during the term of this study.
Gratitude is also expressed to Dr. R. A. Dunn and
Dr. D. F. Rothwell for invaluable assistance and encourage-
ment, while serving as members of my supervisory committee.
A special debt of thanks must also go to Dr. G. C.
Smart, Mr. A. L. Taylor, and Dr. K. R. Langdon for sugges-
tions and assistance pertinent to this study.
Thanks are also given to Mr. W. W. Smith who origi-
nally found the new nematode, and provided much data and
material essential to expediting the objectives of this
I am also deeply grateful to Agricultural Commissioner
Doyle Conner, and H. L. Jones, director of the Division of
Plant Industry, for encouragement and very generous vouch-
safement in this endeavor.
Finally, I am very appreciative for considerable en-
couragement and forebearance from my wife Hannelore in this
term of trial.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS .. . ii
TABLE OF CONTENTS . ... iii
LIST OF TABLES . .. ... v
LIST OF FIGURES . ... vi
ABSTRACT . . x
INTRODUCTION ... . ... 1
HISTORY . . .. 2
TAXONOMY AND SYSTEMATICS . 3
Taxonomic Position of the New Genus 3
Taxonomy of the New Genus . 17
Anatomy .. . 36
REPRODUCTIVE DEVELOPMENT . .. 45
Early Development . 45
Male Development .. . 47
Female Development . .. 50
HOST-PARASITE RELATIONSHIPS . .. 54
Methods . .. 54
Behavior Studies . .. 56
HOST PLANT INVESTIGATIONS .
Host Plant . .
Host Habitat . .
Host Testing . .
Host Symptoms . .
Pathogenicity . .
Distribution of Verutus volvingentis
in Florida . ...
BIOLOGICAL CONTROL INTERACTIONS .
LITERATURE CITED . .
BIBLIOGRAPHY . .
BIOGRAPHICAL SKETCH . .
LIST OF TABLES
1. Comparative distinguishing characteristics
of females in genera of Heteroderidae .
2. Comparative distinguishing characteristics
of males contained in the Heteroderidae .
3. Male development . .
4. Number of fully developed eggs detected
in 123 mature females . .
5. Survival stage and numbers of nematodes
recovered before and after a longivity test
6. Larval hatch from eggs kept in fallow
soil 3 years . .
7. Phytoparasitic nematodes found in soil
associated with roots of buttonweed .
8. Verutus volvingentis host testing results
9. Effect of Verutus volvingentis on foliage
and seed pod production of inoculated
plants . . .
10. Nematode population density in treated
and untreated soil . .
11. Areas in Florida sampled for Verutus
volvingentis . .
12. Plants examined for the new nematode
in the Florida survey . .
LIST OF FIGURES
1. Meloidogyne sp. showing spheroid shape, and
terminal vulva of a mature female .
2. Meloidodera floridensis . .
3. Verutus n. gen. female in root tissue with
a single deposited egg . .
4. Cryphodera eucalypti . .
5. Hypsoperine graminus . .
6. Heteroderidae male tail types .
7. A comparison of female appearances in three
subfamilies. A) Verutinae, B) Nacobbinae,
C) Rotylenchulinae . .
8. First-stage larva of Verutus volvingentis
n. gen., n. sp . .
9. A comparison of first and second-stage larva
esophageal glands in the Heteroderidae .
10. Water agar en face method . .
11. Verutus volvingentis n. gen. n. sp.
Mature female . .
12. Verutus volvingentis n. gen. n. sp.,
female body shapes . .
13. Vestigial larval tail tip on posterior
area of a mature female . .
14. En face presentation of a mature female
15. Mature male of Verutus volvingentis
n. gen. n. sp. . .
16. Verutus volvingentis male en face view .
17. Female tail area showing lateral field
irregularities . .
18. Telorhabdions of a mature female posterior
view . . .
19. Posterior region of mature females .
20. Anus (left) and rectum of a mature female
21. Excreta exuded from the anus of a
mature female . .
22. Vulva and vulva muscles of a mature female
23. Lateral views of an unprolapsed (left) and
prolapsed (right) vagina of mature females
24. Uterus of a mature female .
25. Male tail showing tubus . .
26. Male tail in a ventral view .
27. Larval tail shapes . .
28. Ova . . .
29. Anterior portion of third-stage larval male
30. Rectal musculature . .
31. Anterior nervous system in a first-stage
larva . . .
32. Ventral nerve cord in the area of the
genital primordia . .
33. Nerves in the tail of a first-stage larva
34. Crystalline layer . .
35. Gubernaculum . .
36. Larvae forced from egg by applying
cover slip pressure . .
37. Spicular primordia cells in the cloacal
area of an early second-stage male .
38. Male reproductive system development .
39. Early third-stage female gonad .
40. Vaginal development of a late third-
stage female . .
41. Virgin female with gonad development
complete . . .
42. Final female ecdysis . .
43. Mature female . .
44. Anatomy of the female reproductive system
from vulva to uteri . .
45. A root map . .
46. Mode of root entry by larvae .
47. Larval head in contact with middle lamella
48. A mature female in a shallow, hand-cut
epidermal section . .
49. Tissue discoloration . .
50. Large lesion occupied by 4 larvae .
51. Attack sites . .
52. Spheroid bodies attached to anterior
region of a mature female .
53. Appearance of a single spheroid body .
54. Exudates on the anterior end of
Meloidodera floridensis . .
55. Female containing 7 eggs . .
56. Diodia virginiana in flower .
57. A mat of buttonweed mixed with
herbaceous plants . .
58. Paynes Prairie, Gainesville, Florida .
59. Young female in root, showing large
dark lesion at feeding site .
60. Appearance of inoculated and uninoculated
plants at the conclusion of the
pathogenicity trial . .
61. A comparison of leaves from inoculated
and uninoculated plants . ... 82
62. A comparison of seeds from inoculated
and uninoculated plants . ... 82
63. Biological control interactions ... 88
64. Sporangium of Rhizophidium sp. attached
to an egg . .... 88
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
TAXONOMY AND BIOLOGY OF Verutus volvingentis
N. GEN. N. SP. (TYLENCHIDA:NEMATA)
ROBERT PAUL ESSER
Chairman: Armen C. Tarjan
Major Department: Entomology and Nematology
A new subfamily Verutinae is proposed. Females dif-
fer from all other subfamilies in the Heteroderidae in pos-
sessing a sausage shaped body with an uncommonly large sub-
The genus and species named Verutus volvingentis is
described. Larval ecdysis was not noted within the egg.
Eggs are not deposited in a gelatinous matrix. Anatomical
features include a phasmid that appears only on tail of
cast cuticles of the first stage larvae. A previously un-
described muscle, the "median dilator vulvae,"was named.
A description of the rectal musculature and nervous system
is given. A detailed account of male and female develop-
ment is presented. Male development is completed in 6-15
days; female development took 17 days. Larvae entered the
root by pushing through the middle lamella between 2
epidermal cells. Tissue discoloration occurred 3-4 days
after feeding. Nuclei of invaded cells enlarged and exu-
date production by the host was incited. Larvae and eggs
survived at least 3 years in the absence of food. Eggs are
the dominant survival stage.
The nematode is widely distributed in Florida in
moist habitats. Testing of selected economic crops as
hosts proved negative. Host plants inoculated with a
minimal number of nematodes died in 13- months. Control
plants were maintained in a healthy vigorous condition.
Catenaria anguillulae killed males but not larvae or
eggs in biological control tests.
The family Heteroderidae contains a large number of
highly pathogenic species included in 17 genera, 14 of
which have been erected since 1956. Pathogenicity has not
been proved for the following genera: Atalodera Wouts &
Sher, 1971; Meloidoderita Poghossian, 1966; Meloidodorella
Khan, 1972; Meloinema Choi & Geraert, 1973; Punctodera
Mulvey & Stone, 1976; Sarisodera Wouts & Sher, 1971;
Sherodera Wouts, 1973; and Thecavermiculatus Robbins, 1978.
The principal objectives of this research were to
establish the systematic position of the new genus of nema-
todes described in this work and to investigate the patho-
genic potential of the new taxon.
Secondary objectives included: host testing, life
cycle and developmental studies, longevity, host-parasite
relationships and anatomical studies.
In March, 1969, Mr. Wayne W. Smith, "Agricultural
Products Specialist," with the Florida Department of Agri-
culture submitted 14 samples from a field near Apopka,
Florida,for regulatory analysis. Four of the samples were
infested with larvae that resembled Heterodera sp. A
search of the sample material for Heterodera cysts revealed
females that did not fit the generic concept of any known
nematode phytoparasitic genus described at that time.
In May, 1969,the site from which the samples origi-
nated was surveyed in an attempt to isolate and identify
the host plant of the undescribed nematode. The host was
found to be buttonweed (Diodia virginiana L.) and was sub-
sequently infected with the nematode in greenhouse culture.
TAXONOMY AND SYSTEMATICS
Taxonomic Position of the New Genus
Verutinae n. subf.
Diagnosis: Heteroderidae (Filipjev, 1934) Skarbilovitch,
Female: Mature female saccate, sausage to reniform-shaped
(Fig. 12), vulva uncommonly large, subequatorial in posi-
tion, vulval lips strongly protuberant (Fig. 11), ovaries
reflexed, anus subterminal, cyst stage absent, body striae
present, phasmid obscure, strong sexual dimorphism present.
Male: (Fig. 15) Body vermiform, caudal alae absent, tail
rounded flatly to truncate, body untwisted, one testis
Type genus Verutus n. gen. (from the Latin "armed with a
Affinities with the Family Heteroderidae
Females. Table 1 shows comparative female distin-
guishing characteristics of genera contained in the sub-
families of Heteroderidae.
Verutus n. gen. differs from all other members of the
Heteroderinae in lacking a cyst stage and a terminal vulva.
It differs from all members of the Ataloderinae, and
Meloidogyninae in lacking a terminal or subterminal vulva,
and a spheroid body (Fig. 1). Verutus is most closely re-
lated to Meloidoderinae, one member of which, Meloidodera
floridensis Chitwood, Hannon, & Esser, 1956, possesses a
subequatorial vulva (Fig. 2) and a spheroid or subspheroid
body shape. Verutus eggs are deposited as they mature
(Fig. 3), and not retained in large numbers within the fe-
male body as in females of Meloidoderinae (Fig. 4). The
body is completely annulated in the subfamilies Meloidoderi-
nae, Meloidogyninae, and Verutinae n. subfam. Members of
Ataloderinae and Heteroderinae possess an irregular body
pattern, or lack body markings. In some members of these
2 subfamilies, annulation may be present on the cervical
area or about the vulva, but not on the body. The vulva
is widely separated from the anus in the Verutinae (Fig. 11)
and in the genus Meloidodera (Fig. 2) in the Meloidoderinae.
In all other subfamilies the vulva is located in the peri-
neal area or near the anus (Fig. 4, Table 1). In some mem-
bers of Ataloderinae, Heteroderinae, and Meloidoderinae the
vulva is situated on a papule (Fig. 5). Neither Meloido-
derinae or Verutinae are so equipped. A labial disc (Fig.
14) is described only in Ataloderinae, Meloidoderinae, and
Verutinae. An attempt was made to utilize the presence of
a gelatinous matrix, or a sub-crystalline layer (Fig. 34)
in the diagnosis. This was not possible due to lack of
data concerning these criteria in many species descriptions.
Genera of the Heteroderidae were also compared to the new
Figure 3. Verutus n. gen.
female in root tissue with
a single deposited egg.
Figure 1. Meloidogyne sp.,
showing spheroid shape and
terminal vulva of a mature
Figure 2. Meloidodera floridensis
showing spheroid shape and equatorial
genus on the basis of the measurement (length/greatest body
width) alpha. This was found to be infeasible due to
omitted data and differences in the measurement criteria
used by some authors. Some measure the total body length,
others exclude the neck and head from the measurement
(Mulvey & Stone, 1976).
A key to the subfamilies of Heteroderidae based on
mature females is as follows.
Key to subfamilies of the Heteroderidae
1. Female forms a cyst---------------------Heteroderinae
Female does not form a cyst-------------------------2
2. Annulation absent or limited only to
cervical or vulva area---------------Ataloderinae
Annulation present on body (may be sparse)----------3
3. Body sausage or reniform-shaped vulva
uncommonly large---------------------Verutinae n. gen.
Body ovoid or pear-shaped, vulva not
4. Eggs retained in large numbers in female
body, labial disc present------------Meloidoderinae
Eggs not retained in body in large
numbers (exception Meloidoderita),
labial disc present------------------Meloidogyninae
Males. Table 2 compares selected male characteris-
tics of genera included in the subfamilies of the
Heteroderidae. It can be seen that few definitive differ-
ences exist between males. Only the subfamilies Ataloder-
inae and Verutinae contain genera without a twist in the
male body (Fig. 6-C). Males of both subfamilies also pos-
sess a truncate tail terminus, similar spicules and guber-
naculum, and a tubus (Fig. 6-A,B). Stylet and body length
Comparative distinguishing characteristics of
females in genera of Heteroderidae.
Cyst Cuticle Anus
Genus Stage Markings Gap Papule Disc tion HPR Host Sac C L Position
1/70 no head & very yes yes yes ? ? ? ? terminal
1/89 no none .... "
1/121 head & no semi- ? no yes "
2/118 yes pattern close no no no "
2/13 no & yes yes
2/99 pattern no endo yes ?
2/102 semi- no "
2/71 lace- very ? ? ? terminal
like close recessed
3/63 no striae not yes semi- no no yes terminal
3/16 far mid-body
3/95 not ? ? ? ? terminal
4/41 very yes no endo yes yes yes "
4/56 "V ? ? yes semi- no "
4/17 no no & no no endo "
4/92 striae close no ? ? ? ? ? "
5/130 striae far yes no semi- rare no yes mid-body
*HPR = Host parasite relationship, Semi-endo=semi-endoparasite, Endo=endoparasite.
**CL = Crystalline layer.
Legend Subfamily & Genus
l=Ataloderinae; 70=Atalodera, 89=Sherodera, 121=Thecavermiculatus
2=Heteroderinae; 118=Globodera, 13=Heterodera, 99=Meloidodorella, 102=Punctodera
3=Meloidoderinae; 63=Cryphodera, 16=Meloidodera, 95=Zeylandodera
4=Meloidogyninae; 41=Hypsoperine, 56=Meloidoderita, 17=Meloidogyne, 92=Meloinema
Egg filled cyst develops. Golden, 1976; Andrews et al, 1977.
<3x a > 3
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measurements also overlap in both subfamilies. Except for
the striated gubernaculum of the genus Sherodera it would
be difficult to differentiate between males of Ataloderidae
and Verutinae. The only other genus with a truncate tail
terminus and tubus is Sarisodera in the subfamily Hetero-
derinae. The Sarisodera male can be separated from males
of Ataloderinae and Verutinae by its long stylet (38-46 pm).
Meloidoderita possesses a sharp conoid tail and is the only
male in the Heteroderidae with caudal alae. All other
genera, not included in the aforementioned, have rounded
or bluntly conoid tails (Fig. 6-C) with or without a twist.
The only small males have been described in Meloidoderita
(350-432 pm), and Meloidodera (457-505 pm). Males in the
genus Meloinema stand apart from all other males in posses-
sing a distinct subacutely conoid tail (Fig. 6-D).
Larvae. The larvae of the new genus (Fig. 8) close-
ly resemble larvae in the subfamilies Atalodorinae,
Meloidoderinae, and all of the larvae in the genera of
Heteroderinae (except Meloidodorella, which has a short
stylet [11-16 vm and reduced telorhabdions]). Larvae of the
Meloidogyninae differ from the new genus, for the most part,
in having a small stylet, small telorhabdions, and fine
body striae. The principal character peculiar to first-
stage larvae of the new genus is the absence of a detect-
Figure 4. Cryphodera eucalypti (after
Colbran), showing egg retention in
the mature female, and separation
of the vulva and anus.
,...;. .. '. *L i
',_ ', .-. ,
Figure 5. Hypsoperine graminus Sledge
and Golden, 1964. A mature female
showing a vulva situated on a papule.
A B C \D
Figure 6. Heteroderidae male tail types; A,B Truncate
with tubus. A Sherodera (redrawn from Wouts & Sher),
B Verutus, C Rounded twisted type. Meloidodera (after
Hopper), D Blunt conoid. Meloinema (redrawn from Choi
Comparisons were made of esophageal gland structures
in the 16 genera of the Heteroderidae to determine if the
glands could be used in generic or subfamily diagnosis.
Esophageal glands with two lobes, the anterior lobe over-
lapping the posterior lobe, were found in the Verutinae,
Meloidoderinae, and Heteroderinae (Fig. 9 A,D,F,H,J).
Considerable variation in esophageal gland structure was
noted in the 27 species descriptions examined in 4 genera
of the Heteroderinae (Fig. 9 F-K). Esophageal glands in
29 species of Meloidogynidae all consisted of a single
lobe. Meloinema differed from all other genera in having
an extremely long esophagus (300 pm). However, in the
description the esophagus length range was listed at 125-
130 pm, which contradicts the illustration. The genera
/,' s / im 5-----< "
I\ '(K \
Figure 7. A comparison of female appearances in three
subfamilies. A) Verutinae, B) Nacobbinae, C) Rotylenchulinae.
Figure 8. First stage larva of
Verutus volvingentis n. gen.,
Sherodera and Zelandodera, Wouts, 1973 are not included in
Figure 9 since neither genus is represented by an illustra-
tion of the larval esophageal glands in the literature.
The Verutinae also differ from the Ataloderinae and
Meloidogyninae in possessing two esophageal gland lobes.
Affinities with the Subfamily Rotylenchulinae, Husain &
The females of Verutus (Fig. 7-A) closely resemble
females in Rotylenchulinae (Fig. 7-C). Verutus females
differ in the absence of a well-defined tail tip, a dorsal
gland orifice that originates less than one stylet length
from the base of the telorhabdions, possession of a large
muscular uterus, and, principally, by the absence of a
vermiform, vulvate juvenile female stage.
Verutinae males differ markedly in general appearance
from Rotylenchulinae males which have a tapering conoid
tail, are usually less than 500 pm long, and assume a C-
shaped body position. Rotylenchulinae males possess an
elongate, truncate cephalic framework in contrast to the
slightly convex, shallow cephalic framework of Verutinae
Affinities with the Subfamily Nacobbinae, Chitwood &
Nacobbinae females differ from females of Verutinae
in body shape (Fig. 7-B), position of a posteriorly sit-
uated vulva, and in having a single gonad. Nacobbinae
E F G H I J K
^ @ @
Figure 9. A comparison of first and second-
stage larval esophageal glands in the
Heteroderidae. A) Verutinae; Verutus. B,C)
Ataloderinae; B) Atalodera, C) Thecavermiculatus.
D,E) Meloidoderinae; D) Meloidodera, E)
Cryphodera Colbran,1966. F,K) Heteroderinae;
F) Sarisodera, G) Meloidodorella, H) Punctodera,
I,J) Globodera or Heterodera. K) Heterodera.
L-O Meloidogynidae; L) Meloidoderita, M)
Meloidogyne, N) Hypsoperine, O) Meloinema.
(Esophageal glands are directly proportional
to the size of the metacorpus shown.)
females form prominent root galls while Verutinae females
Nacobbinae males possess conoid tails and caudal
alae, both of which are absent in Verutinae males.
Nacobbinae larvae differ from Verutinae larvae in
having a bluntly rounded tail tip.
Discussion. The new subfamily Verutinae does not fit
within the concepts of the subfamilies in the family
Heteroderinae, or the subfamilies, Nacobbinae or Rotylen-
chulinae. It is therefore proposed as a new subfamily.
Subfamilies proposed by Husain, 1976, but not included in
the analysis are Meloineminae and Meloidoderellinae.
The position of the Verutinae in the Animal Kingdom
is shown in the following scheme:
Phylum-Nemata (Rudolphi,1808), Cobb,1919
Class-Secernentia (von Linstow,1905)Chitwood,1958
In the above scheme categories above Phylum are based
on Storer and Usinger, 1957. Classifications below Section
are based on schemes proposed by Golden, 1971, Wouts, 1972,
Andrassy, 1976, Husain, 1976, and Stone, 1977.
Taxonomy of the New Genus
Methods; Measurement Preparation
Specimens to be measured were placed in water within
a "Zut" ring on a glass microscope slide (Esser, 1973-b), and
a cover slip placed on the zut. The nematodes ceased moving
in 3 to 5 min., and measurements were taken according to
the method proposed by Esser, 1971. While the nematodes
are in the quiescent state one has 20 to 30 min. to make
observations and measurements before deterioration, swelling
and/or shrinkage occur.
When specimens on slides are to be fixed permanently
the cover slip is removed and 2 or 3 drops of 2% formalin
are added to the exposed zut well. The specimens are then
transferred to a BPI watch glass for permanent fixation in
lactophenol (Esser, 1973-a).
En face Preparation
A new method was devised to study en face prepara-
tions. Live immobile females or males, or freshly killed
nematodes in 2% formalin were used as subjects.
Procedure. A 12 X 12 X 3 mm square of 1.7% water
agar is cut very evenly with a razor blade (Fig. 10-A) and
placed on a microscope slide. A 3- to 4-mm piece is pre-
cisely cut from the square (Fig. 10-A) and laid with the
outer face down (Fig. 10-B). Nematodes are placed on the
upper side of the cut piece with the longitudinal axis of
the head parallel with the outer edge of the cut piece
(Fig. 10-B). The cut piece is then placed back into the
same position it occupied in Fig. 10-A, then gently pushed
into its original position against the parent block (Fig.
10-C). A small (4-mm) drop of water is applied to a 15-mm
cover slip that is then placed waterside down over the cut
line (Fig. 10-C). A drop of immersion oil is applied to
the center of the cover slip at the junction of the cut
pieces. When the body of the nematode is properly aligned
the en face appears as in Fig. 10-D If the en face is
off-center or below the field of focus, the cover slip is
removed, the cut piece placed backside down, and the speci-
mens reoriented. Water must be added to the cover slip
each time it is placed on the agar block. It takes 10 to
15 min. to prepare an en face ready for viewing using this
technique. Locating the en face is rather easy since it
lies within the cut line.
Figure 10. Water agar en face method; A) 12 X 12 X 3 mm
square of water agar with a 3- to 4-mm piece cut off, B)
Specimens aligned on outer edge of the inner face of the
cut piece, C) Re-alignment of the separated agar pieces,
with cover slip in place, D) Closeup of en face in
junction line of re-aligned agar.
External Cuticle Preparation
Lateral lines and phasmids were not well-defined in
live or fixed specimens. Several stains were tested to
bring out the lateral lines including: methyl blue, acid
fuchsin, merthiolate, iodine, chlorazol black-E, and pro-
pionic carmine, none of which enhanced cuticular incisures
or phasmids. Lateral incisures were brought out clearly
by making squash mounts. Cut or uncut females, males, and
larvae were placed in a 4-mm drop of water, and a 18-mm
cover slip applied. A needle point was pressed against the
cover slip until the body contents gushed out. Examination
for the phasmid was made of each squash mount specimen.
Nervous System Preparation
Chlorazol black-E in lactophenol was used with the
4-min. fixation method (Esser, 1973-a).
Verutus n. gen.
Diagnosis: Verutinae, with characters of the subfamily.
Mature female (Fig. 11): Body swollen, reniform or sausage-
shaped (Fig. 12). Cephalic framework moderately sclerotized,
lips striated, set off, amphids obscure, oral disc hexagonal
(Fig. 14). Body striated, lateral lines irregular, some-
times indistinct. Crystalline layer present (Fig. 34).
Stylet tylenchoid, dorsal gland orifice near telorhabdion
base. Uncommonly large protuberant post-equatorial vulva.
n. gen. n. sp.
Figure 12. Verutus volvingentis n. gen. n. sp.,
female body shapes.
Figure 13. Vestigial larval
tail tip on posterior area
of a mature female.
Figure 14. En face view of
a mature female.
Gonads didelphic and amphidelphic. Ovaries reflexed. Anus
subterminal forming small depression (Fig. 13). Tail
vestigial (Fig. 13) or absent.
Female. (Table 1) Females differ from all other fe-
males in the Heteroderidae in possessing a reniform or
sausage-shaped body, with an uncommonly large vulva in a
post equatorial position with strongly protuberant lips.
Esophagus typically tylenchoid, procorpus moderately expan-
ded, metacorpus moderate in size. Isthmus narrower than
procorpus, esophageal gland a single lobe moderately over-
lapping the intestine. Deirids and phasmids not observed.
sausageshapedbody, wth an ncommo lylrevvaia
post~~~~~~~~~~~~ eqaoil oiinwihsrngypouern is
Esophagus tyial tlnhid rcrusmdrteyepn
Male. (Fig. 15) Body vermiform, monodelphic, lips
striated not set off, oral disc circular (Fig. 16), amphid-
ial openings elliptical on lateral lips. Body untwisted.
Spicules and gubernaculum tylenchoid. Caudal alae absent,
tail terminus angular truncate (Fig. 6-B). Phasmids or
deirids not detected.
Verutus volvingentis n. sp.
Female. (35 specimens) Total length = 662.7 (500-
930) pm; width = 141.4 (94-207) pm; tail = 10.2 (3.9-15.7)
pm; esophagus = 188.3 (150-290) pm; a = 4.7 (3.0-6.5); c =
69.4 (34-155); total stylet length = 26.1 (23.5-29.4) vm;
vulva % = 67.5 (50-75); excretory pore 139.8 (122-183) pm.
Female holotype. Total length = 540 pm; width =
118 pm; tail = 15 pm; esophagus = 148 pm; a = 4.6; b = 3.6;
c = 36; stylet = 27.2 pm; vulva % = 71.2; excretory pore =
114 pm from anterior end.
Female description. (Fig. 11) Body pearly white,
reniform or sausage-shaped (Fig. 12), anterior part of body
sometimes twisted upward lying in a different plane than
the posterior swollen portion. Head and neck occasionally
reflexed across the posterior body. Six equidistant lips
surround a hexagon-shaped oral disc (Fig. 14). Amphid
apertures or lip papillae not observed. Lips set off, com-
prised of 2 annules. Cuticle 9-10 pm thick, evenly stria-
ted, striae about 2.5 pm apart. The occurrence and
1volv = vulva, ingen = remarkable size.
n. gen. n. sp.
appearance of lateral lines are variable: lines may proceed
for a short distance beyond anus and fade out, or appear as
midline cuticular interruptions or irregularities extending
slightly past the vulva area. They appear as 1 or 2 lines
of irregular blocks in the cervical area. In the tail
area they appear as a mass of irregularities in the tail
tip area with sometimes a wide separation (5 pm) of the
striae (Fig. 17). In a few females lateral lines were not
observed. Stylet tylenchoid, telorhabdions (Fig. 11, 18),
4-5 wide by 1-2 ym long, directed posteriad. Prorhabdions
14 pm. Two stylets with tips protruding from the body
measured 25.5 and 25.6 pm, respectively. The dorsal gland
orifice appears 7-11 pm posterior to the telorhabdion base.
A moderately swollen procorpus narrows prior to the well-
developed metacorpus. A clearly defined metacorpal valve
is present. A short narrow isthmus leads from the meta-
corpus followed by a single distinct esophageal gland lay-
ing ventrally over the intestine. The intestine extends
from beneath the mid-area of the esophageal gland to the
rectal intestinal valve. The sclerotized portion of the
rectum is 12 pm long in a lateral view. The rectum dilates
anteriorly extending 30 pm beyond the sclerotized portion
(Fig. 19) as a finely sclerotized tube (15 pm long) which
joins the intestine. The oval anus (Fig. 20) lies in a
depression (Fig. 19). Tail is usually absent, occasionally
vestigial (Fig. 19). Differences in orientation of the body
do not permit an accurate tail annule count, or anal body
diameter measurement. One female was noted (Fig. 21) with
a granular mass over the anus, assumed to be excreta. The
excretory pore lies at the level of the esophageal gland
134.4 (113-183) pm from the oral opening. The nerve ring
appears as a mass of tissue surrounding the isthmus.
Gonad amphidelphic, anterior branch 136-147 pm long,
posterior branch 117-130 pm long. The vulva appears as a
transverse slit (Fig. 22) about 62 m wide. In some fe-
males the vulva lips protrude markedly. The vulva striae
do not form a distinctive pattern, but surround the vulva
rather uniformly (Fig. 22). In some females prolapse of
the vaginal walls causes the vulva to widen, and the vaginal
lining prolapses externally (Fig. 23). Wide muscle bands,
the dilator vulvae appear at either end of the vulva under-
lying the cuticle (Fig. 22). Vulva epitygma were not ob-
served. The vagina extends 42 to 70.5 pm into the body
where it joins the well-developed vagina uterina (58-70 pm;
Fig. 11). A constriction appears at the junction of the
vagina uterina and the uterus. The uterus is a large mus-
cular sac (70 X 35 pm) that joins directly with the sper-
matheca (Fig. 24). It comprises 4 to 5 rows of large cells
with a furrow in the center for expansion. The spermatheca
is a roughly circular thick walled chamber 35 to 40 pm in
diameter. The oviduct, a thickened area comprised of small
cells,lies between the spermatheca and the maturation zone
of the ovaries. The ovary is reflexed at the spermatheca,
Figure 16. Verutus
male en face view.
Figure 18. Telorhabdions of a
mature female posterior view.
Figure 17. Female tail area
showing lateral field
Figure 20. Anus (left) and
rectum of a mature female.
Figure 19. Posterior region of mature
females, showing vestigial larval tail
tips (T), and rectum (R).
I"' ":k' """^."" II
Figure 21. Excreta exuded Figure 22. Vulva and vulva
1/ \ \ \
/: / '/ ,'
Muscles, M-median dilator
vulvae muscles (striae cutaway
to show underlying muscles).
and or 2 times in the maturation zone area. The cap cell
and germinal zone cells are rarely delineated in live or
Males. (24 specimens, Fig. 15) Body length = 830.8
l -.( specimens, Fig."15)_.ody // lnt 330.8
(650-1020) pm; width = 28.9 (25.5-35.5) pm; tail = 9.5
(5-12.7) pm; esophagus = 153.5 (122-188) pm; a = 28.7
(24.3-32.8) 1jm; b = 5.4 (4.5-6.6) pm; c = 99.2 (59.1-178.6)
pm; total stylet length = 25.3 (21.5-27.4) pim; dorsal gland
orifice = 4.6 (2-6.8) pm behind the base of the telorhabdions;
orifice = 4.6 (2-6.8) Dm behind the base of the telorhabdions;
Figure 23. Lateral views of an unprolapsed (left)
and prolapsed (right) vagina of mature females
P=prolapsed vaginal tissue.
spicules = 40.4 (36.2-46.6) pm; gubernaculum = 16.2 (14.7-
Allotype. Total body length 790 pm; width = 25 pm;
tail = 6 im; esophagus = 140 pm; a = 31.6; b = 5.6; c =
131.7; total stylet length = 22 pm; dorsal gland orifice =
6 pm; spicules = 40 pm; gubernaculum = 15 pm.
Male description. Body vermiform, untwisted (Fig.
15), 6 equidistant lips (Fig. 16) surround a circular oral
disc that stands out clearly in profile. Crescent-shaped
amphids appear indistinctly on posterior margin of lateral
Figure 24. Uterus of a mature female:
1) spermatheca, 2) uterus, 3) egg.
Figure 25. Male tail showing tubus
lips. Labium moderately sclerotized, comprising 4 to 7
labial annules, counting from first reduced annule at onset
of cephalic sclerotization. Labium rounded, not set off,
papillae not observed. Body striae about 2 vm wide, some-
times ending irregularly at the terminus (Fig. 15). Four
unareolated lateral fields present, extending from region
of corpus to cloacal area. Phasmid not observed. Excre-
tory pore lying in posterior esophageal gland area, 103-
146 pm from oral disc (mean = 124 pm). Hemizonid 4 pm long,
located just posterior to excretory pore. Stylet typi-
cally tylenchoid. Telorhabdions sloping posteriorly.
Cheilorhabdions extending through lip annules 2 to 4.
Esophagus comprising a moderately swollen procorpus that
dilates just prior to oval, distinct metacorpus containing
a valve slightly smaller than that of female. A narrow
isthmus precedes a single ventral esophageal gland with
single nucleus. Cardia not observed. Nerve ring appear-
ing as an irregular band of tissue overlapping isthmus,
and extending past the esophageal gland about 1/3 of its
length (Fig. 15). Intestine overlapping about 1/2 of
esophageal gland and extending uniformly to cloaca. Tail
bluntly hemispherical to truncate; tail terminus annu-
lated. Anal lips in form of tubus (Fig. 25). Caudal
alae absent. Spicules equal and slightly arcuate when
seen in lateral view. Capitulum moderately swollen,
followed by slight constriction, and moderately swollen
calomus. Lamina wide at the center tapering at both
extremities. Sclerotized piece arising at junction of
lamina and calomus and projecting along ventral wall of
calomus. The gubernaculum with teeth on lateral sides of
cuneus seen in ventral.view when cuneus is situated between
spicules (Fig. 26). Male gonaduct originating from ventral
face of the cloaca. Narrow vas deferens (Fig. 15) about
90 pm long is followed by rather long seminal vesicle, usu-
ally filled with sperm. Germinal and growth zones sometimes
indistinguishable. Testes have been observed in which en-
tire tube was filled with sperm and an observable germinal
and growth zones were not present. Cephalids and deirids
Larval description. (49 first-stage larvae; Fig. 8)
Length = 492 (430-540) pm; width = 18.5 (16-20.2) pm; tail =
53.6 (46-64) m; esophagus = 163 (132-190) pm; a = 26.5 (22-
30); b = 3.2 (2.6-3.7); c = 9.1 (6.7-10.5); anal body diam-
eter = 4.5 (4.0-5.2) pm; stylet = 23.1 (21.5-24.5) pm; dorsal
gland orifice 6.4 (4-8.9) pm posterior to telorhabdions;
excretory pore 93.8 (79-103) pm from oral disc.
Body vermiform, labium rounded, cephalic framework
consisting of 16, C-shaped sclerotized pieces lying 4 pm
below oral disc. Head bearing 6 annules. Four lateral
incisures beginning as single line, 45 pm posterior to
oral disc forming 4 lines at median procorpus. The 4
lines resolve into a single line just posterior to anus
(30 pm from the tail tip). Width of lateral incisures at
mid-body is 5-7 pm. Excretory pore located in mid-isthmus
Figure 26. Male tail in a
ventral view, showing
toothed cuneus of
Figure 27. Larval tail
area. Hemizonid located 1 annule anterior to excretory
pore. Stylet well-developed, prorhabdion 10.8-12 pm long.
Rounded telorhabdions usually laying in an even plane, occa-
sionally sloping posteriorly. Procorpus (38 pm long by 6-7
pm wide), moderately swollen, narrowing just prior to the
well-developed metacorpus (15 pm long by 12 pm wide).
Metacorpus valve a wide oval shape. Isthmus narrow, 25 pm
long. Esophageal glands about 35 pm long, lying on ventral
side of body. Posterior lobe sometimes filled with coarse
granules (digestive fluid). Coarse granules also appearing
in anterior end of anterior lobe, in isthmus, and in a
large vesicle in posterior part of metacorpus (Fig. 8). A
single large nucleus present in the posterior esophageal
gland lobe. Anterior esophageal gland lobe not strongly
set-off. It is delineated by a weak line of demarcation on
posterior lobe, and has a very large nucleus surrounded by
a large clear area (Fig. 8). Esophageal glands overlapping
intestine by about 1/2 of their length, extending as a
straight tube to undilated rectum. Anus oval, 1 to 2 pm
wide. Nerve ring appearing either as group of nerve cells
(Fig. 31), or as fine band of tissue surrounding isthmus
(Fig. 8). Genital primordia appearing about 160 pm ante-
rior to tail tip (Fig. 8). Tail conoid (Fig. 27), with
26-29 annules. Tail tip usually awl-shaped. Hyaline area
of tail 25.4 (23.5-31.3) pm long. Deirids, phasmids and
cardia not observed.
Ova. (Fig. 28) Eggs broadly oval 50 X 100 pm. No
markings observed on shell. An en-utero egg was 52 X 103
Third-stage larvae. (10 specimens) Length = 572
(500-677) pm; width = 31.3 (27.4-34.3) pm; tail = 11.1
(9.8-13.7) pm; a = 19.5 (17.3-22.6); c = 54.5 (46.7-63.2);
dorsal gland orifice = 3.9 (2.9-6.8) pm; excretory pore =
115.9 (109-122) pm.
Body slightly swollen, tail rounded, head and esoph-
agus similar to that of first-stage larvae (Fig. 29).
Type specimens. Holotype collected May, 1969 by
Wayne W. Smith. Collection number B-5018; Allotype same
data as holotype. Type slides in Bureau of Nematology nema-
tode collection, Division of Plant Industry, Florida De-
partment of Agriculture.
Type habitat. Soil about roots, and roots of Diodia
virginiana growing near bodies of water.
Type locality. Irrigation ditch bank bordering Hwy
50, 4 miles west of Hwy 27 near Clermont, Florida. (Orig-
inal site now commercially developed.
These structures are very difficult to see in live or
fixed specimens even when various stains were used. It was
possible to see them, however, by squeezing out the body
contents and examining the lateral sides of the integument
'; .. -. ..' ^
*,,, _.r-. > ...
-: .. ;... *' .r -, ^ .-- _. .* ,
i,- -' 1.
a:. '-, s '-a.S .'
: .: -. .- .- .. .. .. .., .,.. .
; ^^ \
''I' ,- "- '. -'? :" l
'. .. .-.: .. -- -; ,.- .,,:; A W
;" ." "I^
-I ."". ..' -
...~ ~~U '..:.<.-. :.'
.. ... : .- .-- _* .- ,
C '. f.
A- ^ r4-?' '
i z-. I. ,.:": :..". : : '
*; -, ~:-. ","* -*, ,-"-"" ," "-a ,'* 3 -. -
Figure 23. Ova. Top to bottom:
2-cell, 3.cell, 4-c-], 5.cell,
tadpole stage, first-stage
,larva' "- ,
i.,- : ...-'*" '
Figure ~ '= 23 Ova.Top o botom
2-cll 3-el 4cl, 5ce
tadpol stage firsct-stag
Figure 29. Anterior
portion of third-
stage larval male.
using an oil immersion objective. This procedure was
not necessary for males and females.
Phasmid. Over a hundred each of males, females, and
first-stage larvae were examined for phasmids with negative
results in both ventral and lateral views. The phasmid and
its lining were only detected on the cast integument of
first-stage larvae early in the first molt. The phasmid
was located between the 14th and 18th annule from the tail
tip. When its location was known, fixed and living first-
stage larvae were examined to see if the phasmid was detect-
able; in no case was it observed.
Vulva. The dilator vulvae musculature are well devel-
oped in broad bands in mature females, extending from vagin-
al epithelium to a ventrolateral insertion in the hydodermis
(Fig. 22). A band of muscle also attaches to vagina on
either side of median part of vulva, herein called "median
dilator vulvae" (Fig. 22).
Rectal muscles. Rectal musculature was observed in a
third-stage female (Fig. 30). The H-shaped muscle surrounds
the rectum or rectal intestinal valve. The depressor ani
extends into the dorsal hypodermis, and the dilator ani is
inserted in ventral hypodermis. A sarcoplasm band nucleus
as described by Chitwood & Chitwood, 1937 was not observed.
Procorpus and Metacorpus
In third-stage larvae the procorpus is short and
stout while metacorpus is a well-developed, wide oval. The
esophagus of mature females is very similar to that of
third-stage larvae. Geraert, 1978, found that the metacor-
pus enlarges in saccate females (Heterodera carotae Jones,
1950), as was the case in V. volvingentis.
In males the esophagus is shorter, more slender and
the metacorpus is smaller and more elongate.
In males and females stained with chlorazol black-E
the circum-esophageal commissure appears as a flat band of
tissue that surrounds the posterior part of isthmus (Fig. 11,
15) and proceeds posteriorly a short distance past the ante-
rior part of the basal bulb as 2 ventral ganglion. Anterior
and posterior nerve cords were not seen. In first-stage
larvae stained with chlorazol black-E, the circum-esophageal
commissure appears looped around the isthmus, either as a
flat band of tissue (Fig. 8) or as an accumulation of nerve
cells (Fig. 31). The ventral ganglion proceeds posteriorly
a short distance, branching dorsally and ventrally. The
dorsal nerve arises from the dorsal portion of the ventral
ganglion, and becomes indistinguishable a short distance
posterior to the esophageal gland. The ventral nerve arises
from the ventral portion of the ventral gnaglion, and pro-
ceeds as a chain of ganglia (92 in 1 specimen) in the
Figure 30. Rectal musculature.
A) Rectal-intestinal valve
area: (1) sarcoplasm, (2)
depressor ani, (3) dilator ani, /'i
(4) rectal intestinal valve. .
B) Rectal area: (5) mid-rectum.
D) Dorsal side.
V) Ventral side.
Figure 31. Anterior nervous
system in a first-stage larva.
A) Anterior ventral nerve
cord; b) Circum-oral
commissure; C) Ventral nerve;
D) Dorsal nerve; E) Hemizonad.
hypodermis. The ganglial chain forms rectal commissures
that surround the rectum with 3 dorsal and 5 ventral ganglia
(Fig. 33). A dorsal rectal ganglion (Fig. 33) is present
where rectal commissures rejoin post-rectally in the dorsal
position. Three ganglia are present in the medial caudal
nerve (Fig. 33). Anteriorly, a large ganglion arises from
the dorsal portion of the nerve ring, and one from the ven-
tral side (Fig. 31). The 2 nerve cords extend around either
side of the metacorpus forming a small mass of nerve cells
just anterior to the metacorpus. Dorsal and ventral nerves
proceed from this ganglion to sclerotized area of the labium.
Cephalic nerves appear as elongate, spindle-shaped proc-
esses. Lateral and papillary nerves were not detected.
Brown et al, 1971, reported the subcrystalline layer
is a complex of long-chain fatty acids. It was hypothesized
that sugar exudates from the integument of Heterodera spp.
are converted to long chain fatty acids by soil fungi there-
by producing the crystalline layer. A crystalline layer was
observed on the integument of about 10% of the females of
the new genus. This layer assumes the form of the striae
and other designs and modifications present in the parent
integument (Fig. 34-B,C). The subcrystalline layer is usu-
ally fragmented and sloughs off the female body (Fig. 34-A,
Figure 32. Ventral nerve cord (arrow) in the area of the
Figure 33. Nerves in the tail of a first-stage larva. A)
ventral ganglion; b) rectal commissure; C) dorsal rectal
ganglion; D) dorsal view; E) medial caudal nerve; L)
"- ,. ..
gue 34 Crytaline ayer A)Seprto fth ae
from ih ancrren;BC aera ,-od;D ae
frgmnt~cjfrm ta~ area.i";
A gubernaculum was isolated from the surrounding tissue for
observation of the dorsal and ventral faces (Fig. 35). The
dorsal face is longer, measuring 16 pm, and shows serrated
margins on the cuneus (Fig. 35-D). The ventral face is
shorter (11 im), ventrally grooved, and serrations were not
observed in the focal plane (Fig. 35-V).
The female deposits a naked undivided egg in the
environment (gelatinous matrix absent).
Four-hundred eggs in lots of 50 were examined under
the oil immersion lens to determine if a molt occurred in
the egg as described for Heterodera rostochiensis Wollen-
weber by Hagemeyer, 1951, and in Meloidogyne sp. by
Christie and Cobb,1941. In no case was evidence of ecdysis
present. After examination, the larvae were expelled from
the eggs (Fig. 36) by exerting a gentle pressure with a
fine needle tip on the cover slip. None of the larvae ex-
pelled from the 400 eggs showed evidence of ecdysis. It
is concluded based on these data that a molt does not occur
in the egg.
First-stage larvae possess binucleate genital primor-
dia with posterior and anterior cap cells (Fig. 32, 38-A).
Shortly after the first molt, determination of sex is pos-
sible by examination of the rectal area. If spicular pri-
mordia cells are present (Fig. 37), a male is developing.
Absence of spicular primordia cells indicate a female is
Figure 35. Gubernaculum (top
is anterior). V) ventral face
(4 pm wide by 11 pm long);
D) dorsal face (5 pm wide by
16 m long); l=cuneus, 2=
Figure 36. Larvae forced
from egg by applying
cover slip pressure.
Figure 37. Spicular primordia
cells in the cloacal area of
an early second-stage male.
Phasmid is shown (arrow) on
Shortly after the first molt, and before the first-
stage integument is cast off, the genital primordium begins
to divide and proliferate posteriorly (Fig. 38 B,C). The
body widens, and the testes join the spicular primordia
shortly before, and after, the first molted integument is
lost (Fig. 38-D). Sperm cells are large and angular at
this stage. Following the second molt, little development
is evident in the testes and spicular primordia. The esoph-
agus is not clearly differentiated at this stage of devel-
opment. The gubernaculum is the anlage of sclerotization,
followed by the lamina of the spicules. The calomus and
capitulum are the last to become sclerotized. Development
proceeds to completion after the second exuviae is cast.
In 2 cases observed, the male left the third-stage exuviae
embedded in the root. Empty exuviae are not uncommon in in-
fected roots. Table 3 shows the length of time required for
development of males. Male development from penetration of
the first-stage larva until a fully developed male was ob-
served took place in a minimum of 6 days and a maximum of
15 days in roots growing in water agar. Three first-stage
larvae that entered a root about the same time all molted to
the second-stage in 48 hours. Twenty-four hours later, all
3 molted to the third-stage. Three days later the final
molt occurred for all 3 males within 92 hours. Total aver-
age time required was 10 days and 20 hours. Other periods
I1O,_ oo 0
;oo 0 00 .
Figure 38. Male reproduc-
tive system development:
A) genital primordium; B)
Genital primordium, 4-cell
stage; C) Genital primor-
dium, early third-stage
male; D) Reproductive
system in late third-
stage male. l=spicular
of male development observed were: a 15-day cycle; a 10-day,
4-hour cycle (Table 3); and a 6-day cycle.
Table 3. Male Development
1 I1 1
Third-stage larva emerged from
exuviae which remained in root.
Migration along root (length
700 im), stylet 24 im.
Migration to a new root, no
ecdysis (third & fourth)
= 0 days, 4 hours.
It is shown in Table 3 that the width of the feeding larva
increases with time until 176- hours have elapsed when a
maximum width of 51.7 pm is attained. After the second
ecdysis the width decreases until the male is fully devel-
oped with a width of 33.3 pm. Feeding has ceased at this
time and it is postulated that the decrease in width is due
to energy expended during ecdysis and migration.
Shortly after the first molt the body swells and the
genital primordium proliferates anteriorly, and posteriorly
(Fig. 38-A,B). The cells in the center bulge toward the
body wall forming the vaginal primordium, after which the
anterior and posterior branches elongate and develop (Fig.
39). The vagina first appears as a large opening with very
large vaginal primordia cells on either side (Fig. 40).
After the second molt the body swells and the gonad com-
pletes its development (Fig. 41). The reproductive system
is complete when the third-stage exuviae is cast (Fig. 42).
Mature females (Fig. 11,43) are usually swollen more than
virgin females, possess convoluted ovaries, and contain
sperm in the spermatheca (Fig. 43). The vagina uterina was
very narrow in a few females (Fig. 44-C). In older females
the vagina uterina is well-developed, with thick folds capa-
ble of containing several eggs. In several females a
severely prolapsed vagina was noted (Fig. 23-right).
Female Life History
Only 1 female developed to maturity in life history
tests. The onset of ecdysis was never observed. Vulva
development was seen 4 days after root penetration by the
first-stage larva. The ovaries were defined 7 days after
penetration, a fully developed female was evident 17 days
after penetration. Seventeen eggs were deposited on the
Figure 39. Early third-stage female gonad.
Figure 40. Vaginal
development of a
Figure 41. Virgin female with
gonad development complete.
Figure 44. Anatomy of the female reproductive
system from vulva to uteri: A=Egg in uterus;
B=uterus cells; C=Narrow vagina uterina; D=
Vagina; and E=Vaginal muscles.
^ A' ,. .* "f.".' '.
Figure 42. Final
Figure 43. Mature female.
same day development was considered complete. Males were
not observed near the female prior to oviposition.
Critical examination failed to reveal ecdysis in the
egg. Early development proceeded as known in most phyto-
parasitic nematodes. The distinct spicular primordia that
appeared early in male development was not noted in other
similar studies (Chitwood & Buhrer, 1946; Christie and Cobb,
1941; Hirschmann & Triantaphyllou, 1971; and Raski, 1950).
Another unique feature of male development occurred
when the male abandoned the third-stage larval integument,
leaving it embedded in the root following final ecdysis.
Male growth measured by body widths during develop-
ment has not been reported in other developmental reports
examined by the author. A loss in width of 18 im was shown
from a maximum of 52 pm. Time for male development varied
from 6 to 15 days.
The unique feature of female development was the
occurrence of huge vaginal primordia cells.
A variety of devices and ideas were tested to study
the host-parasite relationships and life history of the
new nematode species, all but one of which were unsuccess-
Wood chambers were patterned after rearing chambers
used by Dean, 1929,and by Minton, 1962. The box is 23-cm
square with a 2.3-cm chamber enclosed by glass and removable
wood sides. Buttonweed plants, well established in white
sand in the chamber, were inoculated with groups of 100
larvae at the site of healthy root flushes under the glass.
Development of the nematode either failed to occur or took
place in areas away from visible root sites. This method
was abandoned after a number of failures. In the next
trial plastic boxes,17.5 cm long by 9.3 cm wide by 3 cm
deep, were filled with either white, or with black volcanic
sand, planted with buttonweed and then inoculated with 100
larvae of the new species. In this system the activities of
the nematodes were obscured by the substrate. The macro-
observation boxes were used to observe nematodes on roots
growing in soil. Limitations of magnification and depth
of focus severely handicapped close observation by this
Trials were conducted utilizing small plastic boxes
of various sizes, and plastic petri dishes containing a
poured 4-mm layer of water agar. Success was assured using
the following procedure: A 4-mm layer of 1% sterile water
agar is poured into a 9.2-cm plastic petri dish lid. A
5-mm ring of water agar is removed from the outside peri-
meter of the agar ring after hardening. A 5-mm glass rod
is heated over a glass flame until slightly red,then used
to burn a hole into the side of the closed petri dish. The
burn area should be sanded so the dish can be easily sepa-
rated. A stem cutting of buttonweed with 1 or 2 small
leaves is inserted through the hole and into the agar with
the leaves external to the dish. When primary roots emerge
and grow into the agar, a 5-mm well is cut into the agar
1-cm lateral to a primary root. Twenty-five first-stage
larvae, and 5 mature males in a small drop of sterile water
were inoculated into the agar well. A root map was drawn
(Fig. 45) when larvae made contact with the root. Each
larvae that situated itself at a particular site on the root
was assigned an alphabetical letter which was placed at the
approximate site on the root map. For each observation, the
date, time, dish number, and larva letter was recorded. Ob-
servations and measurements were made using the oil
immersion lens by placing a small drop of water on a cover
slip, which was inverted and placed over the root site
where larvae were attached. Basic data taken when possible
included: time elapsed from the inoculation to the time
larvae penetrated the root, time elapsed between penetration
of the larva to the appearance of root discoloration, if and
when a larva left its feeding site, body width measurements,
and ecdysis observations.
Eighteen plates containing plants in agar were inocu-
lated. Life history activities were observed in 5 plates;
the remainder were abandoned due to plant death, visibility
problems, or severe bacterial contamination.
As soon as the water in the inoculation well dried
the males and larvae migrated into the agar.
Males migrated at random in the agar. A proclivity
to the root by males was not noted. Several males were
seen with lips in contact with the epidermis of a primary
root. In no case was stylet movement or metacorpus valve
pulsation noted in such contacts. One male lay quiescent
very close to a primary root the duration of the trial.
Most males migrated slowly through the agar after which
they became quiescent.
A total of 49 larvae of 450 inoculated was observed
penetrating roots. Data were taken until they departed,
ceased activity, or completed development.
Root penetration. Larvae migrated to the root follow-
ing pathways peculiar to most nematodes in agar (Wallace,
1964). One group of 4 larvae reached the root in 19, 23,
30, and 36 min., respectively,,following inoculation. Sty-
let movement was initiated about 4 min. after lip contact
with the root. Stylet thrusts were recorded at 92 per min.,
and 112 per min. by 2 larvae shortly after contact with the
root. Klinkenberg, 1963, recorded 69 thrusts per min. for
Pratylenchus crenatus Loof, 1960. Pressure was exerted on
the epidermal surface by thrusts of the nematode head and
stylet. The head slid over the cell surface as it thrust
until the stylet tip was over the middle lamella between 2
epidermal cells (Fig. 46-A). At this point the metacorpus
valve moved intermittently indicating digestive enzymes
were extruded into the attack site (Fig. 47). The lamella
between the 2 cells separated and the nematode slipped
laterally into the opening (Fig. 46-B). After penetration,
the larvae migrated obliquely 1-3 cells and 1-3 cells deep
(Fig. 46-C). One root with a 100 pm diameter was penetrated
61 pm laterally and 50 pm deep.
Feeding. Once the larvae were situated in the root,
feeding began immediately. In very small roots, feeding
Figure 45. A root map
charting the progress
of each nematode that
occupied a feeding
site in one of the
dishes. Each letter
represents a larva
at a feeding site.
Figure 46. Mode of root entry by
larvae. A=larva with head cen-
tered on middle lamella between
B=Penetration. C=Feeding site.
SJ' i' I r,*
: el i. -
Figure 47. Larval head in Figure 48. A mature female
contact with middle in a shallow, hand-cut
lamella. Note small area epidermal section.
of discoloration in front
of oral aperture.
occurred in the cortex or pericycle. One larva fed in a
cortical cell occupied by another larva feeding in the peri-
cycle. Feeding sites were rather shallow (1-4 epidermal
cells deep) in mature roots. A very shallow hand cut longi-
tudinal section (Fig. 48) underneath a feeding female rare-
ly cuts the female.
Tissue discoloration. Yellowing of the tissue (Fig.
49-A) appeared initially 3, 4, and 4- hours following pene-
tration. In some cases (Fig. 49-B) the discoloration was
confined to the cell wall. Larvae were also noted with the
stylet inserted in the cell wall (Fig. 49-B). Nuclei of
Figure 49. Tissue discoloration. A) Heavy stippled
area indicates yellow discoloration in cells 4
hours after larval entry. B) Stippled area indicates
yellowing in cell walls. Note difference in nuclei
size between healthy and attacked cell.
cells with discolored walls were consistently enlarged
(11-13 pm) in comparison to healthy cells containing nuclei
(5-7 pm, Fig. 49-B).
Feeding migration. A few larvae touched the root and
departed without entry. Eleven larvae entered the root,
fed, and after a few hours or several days departed. Some
of the departing nematodes took up a new feeding position
on the same root or entered a different root and resumed
feeding. Some larvae were never seen again after leaving a
feeding site. Entry of an epidermal site predisposes the
site for entry of searching larvae. Nine larvae were seen
feeding together in a single, large longitudinal lesion
(Fig. 50). Such lesions are usually abandoned by feeding
larvae. One assumes the excess of enzymes and metabolites
in such a large open lesion renders the site unfavorable
for the development of the nematode.
Attack sites. Larvae have been detected feeding at
root tips (Fig. 51-A), root scales (Fig. 51-B), along
feeder roots, and rhizomes, singly, or in groups (Fig. 51-
C,D). In mature rhizomes, females are commonly seen either
singly or in groups (Fig. 51-E). Females can almost always
be found at the junction of secondary roots emerging from
the rhizome (Fig. 51-F). Larvae and third-stage males
have also been observed on chlorophyll-bearing stem tissue
at the soil line. Larvae and mature females have been de-
tected in root leaf scales just below chlorophyll-bearing
aerial leaf scales (Fig. 51-B). Many females resembling
I I ill
li I yI
P Ij) )
Figure 50. Large lesion occupied
by 4 larvae.
,. < ;',. Hl
Figure 51. Attack sites. A) Larvae feeding at root tip;
B) Larvae feeding on root-leaf scale; C) Larvae feeding on
feeder root; D) Large group of larvae in rhizome; E) Group
of mature females in mature rhizome; F) Females at secon-
dary root juncture.
small white sausages lie appressed to large rhizome pieces
Host exudates. About 5% of the females examined pos-
sessed an accumulation of spheroid objects around the cervi-
cal region (Fig. 52). The exudates were closely associated
with the integument but did not adhere as do the cement
bodies adhering to the integument of cyst nematodes de-
scribed by Shepherd and Clark, 1978. The cement bodies are
depicted as brown hardened exudates originating from the
integument and grossly resemble the spheroid bodies of
V. volvingentis. Exudates of the new species differ in ap-
pearing to have a crystalline composition (Fig. 53).
The spheroid bodies appear to be exudates originating
from the host in response to feeding activities of the new
genus. Exudates have also been noted in Meloidodera flori-
densis (Fig. 54).
Oviposition and fecundity. Eggs are deposited naked
in the substrate. Five to 25 eggs usually lie inside the
ventral space formed by the body coil, or they are scattered
about the female body near the vulva. One large egg mass
contained 185 eggs in various states of development. Stan-
dard soil washing procedures usually wash the eggs from the
female so the number of eggs deposited is difficult to as-
certain. To determine the number of eggs contained in ma-
ture females, 123 female specimens were examined (Table 4).
Figure 52. Spheroid bodies
attached to anterior region
of a mature female.
-. .E *-
Figure 53. Appearance
of a single spheroid
Figure 55. Female
containing 7 eggs.
Figure 54. Exudates on the
anterior end of Meloidodera
Number of fully developed eggs detected
in 123 mature females.
Egg number Females
Most females contained 1 or 2 eggs with a maximum of
7 eggs in 1 female (Fig. 55). The largest egg measured was
128 X 70.5 pm, inside a female.
Longevity. All plants which had been inoculated with
V. volvingentis for pathogenicity trials had died by
September, 1973. Five 1.9 X 20.3 cm soil plugs were re-
moved from each of 7 pots and the nematode population
counted (Table 5). Most pots contained large numbers of
eggs. The pots were maintained in the greenhouse in a fal-
low condition until October, 1976,when a sample similar to
the soil sample in September, 1973,was taken and the nema-
tode population counted (Table 5).
No females survived the longevity test while male and
larval survival was minimal. It appears feasible that eggs
are the prime survival stage in this species.
Egg viability. A 15-cm clay pot in which buttonweed
was root-bound, but nematode free,was saturated with sterile
water. The pot was then placed in a beaker and an addition-
al 100 ml of sterile water added. The effluent was
Table 5. Survival stage and numbers of nematodes recovered
before and after a longevity test.
Pot Females Males Larvae Eggs
Sept. Oct. Sept. Oct. Sept. Oct. Sept. Oct.
1973 1976 1973 1976 1973 1976 1973 1976
1 2 0 0 0 18 6 552 257
2 0 0 18 0 8 0 1282 5
3 1 0 0 0 0 0 198 6
4 0 0 1 0 3 0 41 287
5 0 0 0 0 0 0 1 2
6 1 0 1 0 6 0 811 2
7 0 0 0 2 8 6 336 129
Total 4 0 20 2 43 12 3221 688
Mean .57 0 2.85 .28 6.1 1.7 460 98.2
collected, and added back to the pot. The collection and
addition procedure was repeated 9 times, after which the
final effluent was filtered and the leachate placed in an
Erlenmeyer flask in the refrigerator.
To test egg viability, 10 eggs from 3-year-old fal-
lowed soil were placed in a drop of sterile water in each
of 4 dishes. Ten drops of stock leachate was added to the
water containing eggs in each of the 4 dishes. Four simi-
lar dishes containing eggs in sterile water but without
leachate served as controls. Results of the test are
shown in Table 6.
Table 6. Larval hatch from eggs kept in fallow
soil 3 years.
Examination Sterile water Sterile water
date and leachate only
Dish no. Dish no.
10/26/76 0-0-0-0 0-0-0-0
10/27/76 0-0-0-0 0-0-0-0
10/28/76 1-0-2-0 0-0-0-0
10/29/76 0-0-1-0 0-0-0-0
11/ 1/76 1-0-0-0 0-0-0-0
11/ 4/76 0-0-1-0 0-0-0-0
11/ 8/76 1-3-4-2 0-0-0-0
Total 3-3-8-2 0-0-0-0
Results. Larvae emerged from eggs only to which
leachate was added. Eggs placed in sterile water failed to
hatch. The test demonstrated that eggs can survive 3 years
in fallow soil. Attempts to inoculate buttonweed with lar-
vae hatched from 3-year-old eggs from fallow soil were
unsuccessful. Failure is attributed to low inoculum levels
and reduced nematode viability.
Larvae entered roots by penetrating the middle lamel-
la between 2 epidermal cells. Tissue discoloration became
evident 3-4 hours after entry of the nematode into the root.
A number of larvae abandoned the site after actively feed-
ing. Nuclei in invaded cells were distinctly larger than
nuclei in cells not entered by the nematode.
Host exudates were noticeably extruded at attack
sites and these exudates adhered to the cervical area of
female feeding at the site.
In longevity tests, eggs and larvae survived 3 years
in the absence of a host. Results indicate that ova are
the survival stage of this nematode.
HOST PLANT INVESTIGATIONS
Diodia virginiana L. (buttonweed) in the family Rubi-
aceae is a perennial of no known economic importance.
Buttonweed (Fig. 56) is comprised of smooth, weedy stems
bearing lanceolate leaves. The plant creeps across the
soil as it grows forming a dense mat when abundant (Fig.
57). It usually is found in a mixture of herbaceous plants
peculiar to vegetation growing near bodies of water. The
vegetative form is found from Florida West to Texas, and
North to New England and Missouri (Small, 1933; Rickett,
Buttonweed is found growing on moist soil adjacent to
bodies of water such as lakes, ponds, water-bearing ditches,
swamps, and prairies (Fig. 58). Maximum growth appears from
1 to 40 meters from the water's edge. As soil becomes less
moist and elevation increases, buttonweed decreases until
none are found. When collecting buttonweed a body of water
will almost always be in sight.
Norton, 1978,lists 15 genera of phytoparasitic nema-
todes comprising 30 species in aquatic habitats. Eight
genera and 2 species listed by Norton were found associated
Figure 56. Diodia virginiana in flower. (A Susan
Anthony dollar is in the background for comparison.)
Figure 57. A mat of buttonweed mixed with
Figure 58. Paynes Prairie, Gainesville, Florida. A
prime habitat for buttonweed and Verutus volvingentis.
with buttonweed in an aquatic habitat. Ten genera and 23
species of phytoparasitic nematodes not included in Norton's
list were detected (Table 7).
A primary consideration, when a previously undescribed
phytoparasite is encountered, is the determination of its
potential as a parasite of economic crop plants. In the
original site where the new genus was collected corn and
soybean plantings were established adjacent to buttonweed
plants. The 2 aforementioned plants, in addition to 19
other plants, were placed in soil infested with the new
genus. Results of the host testing are shown in Table 8.
Table 7. Phytoparasitic nematodes found in soil
with roots of buttonweed.
Criconemoides curvatum Raski, 1952
mutabile Taylor, 1936
xenoplax Raski, 1952
Dolichodorus heterocephalus Cobb, 1914
Helicotylenchus crenicauda Sher, 1966
iI" dihystera (Cobb, 1893)
longicaudatus Sher, 1966
I" paxilli Yuen, 1964
Hemicriconemoides wessoni Chitwood &
Hemicycliophora zuckermani Brzeski, 1965
Meloidogyne arenaria (Neal, 1889)
Pratylenchus brachyurus (Gocfrey, 1929)
Trichodorus christ= e Allen, 1957
Trophonema arenariam (Raski, 1956) Raski,
Tylenchorhynchus irregularis Wu, 1969
Xiphinema americanvum Cobb, 1913
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Plants to be tested were seeded into or propagated
by cuttings in 20-cm clay pots filled with steamed soil.
When a healthy state of growth was evident, the pot was in-
oculated with 50 cc of soil and roots taken from a soil box
heavily infested with V. volvingentis. Plants were har-
vested in a minimum of 4 weeks. Roots were removed, washed,
and examined using a dissecting microscope to see if any
stage of the new nematode was in the root system. Five
1.9 X 20.3 cm soil plugs were removed from each inoculated
plant and processed for the nematode (Table 8). A few
plants were placed directly in a population maintainence
Females of the new taxon developed only in roots of
Ludwigia peruviana (Onagraceae), a weed of no economic im-
portance. Larvae were not detected in any of the other
host plants examined.
Results of this host test and because this nematode
has not been reported in numerous surveys of economic crop
plants indicate the nematode is doubtful as a threat to
economic crops, and very likely has a limited host range.
In the original site, the infested buttonweed was found
growing into plantings of both corn and soybeans, neither
of which proved to be hosts in field samples or host
tests. Since the nematode habitat includes irrigation
ditch banks serving as borders for numerous crops, the nema-
tode has ample opportunity to infest a variety of crops,
should they prove susceptible.
Aboveground symptoms of nematode injury were not
noted in any site examined. Symptoms were noted only in
inoculated population maintainence boxes, inoculated pots,
and in plants grown in water agar.
Generalized aboveground symptoms include stunting,
chlorosis, seed pod reduction, and death. Healthy green
stolons and leaves gradually turn chlorotic until the entire
plant is a pale yellow-green colo:. Plants begin to decline
and die 6 months after inoculation until no living plants
remain. In the final stages of decline, germinating seeds
produce small plants with 2-4 leaves that rarely assume a
healthy green color. Such plants rarely survive very long,
and their roots are almost always infected with females and
larvae of V. volvingentis.
Root symptoms are expressed as shallow epidermal
lesions of a pale yellow to brown or dark brown color.
Lesions comprise 3-15 cells and are present at feeding
sites. Lesions enlarge and darken with time (Fig. 59). In
a few cases, epidermal swelling in the form of a small
rounded protuberance appeared at the feeding site. In very
small roots lesions sometimes encircle the root resulting
in a necrotic constriction that causes blocking of
Figure 59. Young female in root, showing large
dark lesion at feeding site.
conductive elements in the root and subsequent detachment
of the root at the lesion site.
Feeder roots of buttonweed are very succulent and de-
tach quite easily. Thus, it is not practical to compare
root systems of infected and nematode-free plants under ex-
Twenty cuttings of buttonweed were taken from plants
grown in steamed soil and transplanted to steamed soil in
20-cm clay pots. Four months later all foliage extending
outside the pot perimeters were clipped off, and 25 larvae
of V. volvingentis were inoculated into each of 10 pots.
Ten pots were left as uninoculated checks. Pots were ar-
ranged in a randomized block design using "Tippets Random
Number Table" (Le Clerg, Leonard & Clark, 1966).
Pathogenicity was evaluated by clipping foliage ex-
tending beyond the pot perimeter, and calculating the dry
weights. Clippings were dried in a heat chamber for 3 weeks
at 570C. The total number of seed pods produced were coun-
ted on each plant just prior to taking perimeter clippings.
Eight 20 cm deep by 2 cm wide soil plugs were removed
from each pot to evaluate the nematode population. Soil
from the 8 plugs was thoroughly mixed and 150 cc subsamples
were removed from each of the mixtures. Samples were proc-
essed using the sugar-centrifuge technique.
Table 9 shows the severe effect of the nematodes on
the plant after 1 year. Thirteen days after final evalua-
tion data were taken, 4 chlorotic, severely stunted plants
still survived the initial inoculation (Fig. 60-A). Leaves
on the surviving plants were very chlorotic (Fig. 61) and
seed production was reduced considerably (Fig. 62). Inocu-
lated plants yielded 2.9 grams of seed pods, while check
plants yielded 15.7 grams. Seeds sprouting in infested
soil in pots inoculated with the nematode were attacked at
a very early stage of development and rarely produced more
than 1 or 2 yellowed leaves before dying.
Within 60 days of the final leaf evaluation, the 4
surviving inoculated plants were dead. All control plants
were in a healthystate of growth.
Table 10 shows the developmental stage and numbers of
individuals in each stage in the 150 cc of soil examined
from each replication. This table indicated that the egg
is the survival stage of the nematode.
The severe parasitism by the nematode resulted in
eventual death of all inoculated plants. This occurred
under greenhouse conditions when the plants were maintained
Figure 60. Appearance of inoculated (A) and
uninoculated (B) plants at the conclusion of
the pathogenicity trial.
Table 9. Effect of Verutus volvingentis on foliage and
seed pod production of inoculated plants.
Mean dry weight
of pot clippings
per plant in grams
Mean number of seed
pods per plant
untreated treated untreated treated
95 11.54 11.40 320.2 324.0
217 4.9 2.6* 0 0
398 24.0 7.2** 512.0 168.3
2 inoculated plants died.
2 inoculated plants died.
** 5 inoculated plants died.
Table 10. Nematode population density in treated and
Days after Mean numbers of eggs or nema- Uninoculated
inoculation todes from inoculated plants plants
eggs larvae males females
406 1144 26 7 3.0 0
504 728 9 2 0.4 0
Figure 62. A comparison of
seeds from inoculated (left)
and uninoculated (right).
Figure 61. Comparison of leaves
from inoculated (top), and
uninoculated plants (bottom).
several months past their normal annual cycle. Symptoms
produced in the greenhouse were never observed in the natu-
ral habitat. It is believed that the nematode population is
in ecological balance with the environment and is not large
enough to severely damage its host when buttonweed dies out
after about 6 months of growth, following the first frost.
During the next 6 months enough nematode eggs and seeds of
buttonweed survive to establish a well balanced association
between the host and its parasite. In the greenhouse there
is no intervening period to retard nematode population
growth. As a consequence the parasite increases unchecked,
severely damaging or killing its host. Factors in the nat-
ural habitat such as biological control agents and
population-limiting physical changes are also absent from
the controlled environment of the greenhouse.
Distribution of V. volvinqentis in Florida
A total of 266 soil samples, and an almost correspon-
ding number of root samples, were examined from collection
sites in 13 Florida counties (Table 11).
Ninety-eight samples were positive and 168 samples
were negative. Plants examined in the survey are shown in
Table 12. The nematode was detected in soil from 13 hosts
in the survey, but in roots of buttonweed only. It is
doubtful if the 12 plants surveyed are true hosts of the
new nematode since most of them were growing close to
The nematode was detected in Alachua, Lake, Orange,
and Sumter counties. The largest population of the nema-
tode occurred in Alachua county within Paynes prairie (Fig.
57), which is a relatively undisturbed, natural, ecological
unit in Florida. The prairie supports large plantings of
buttonweed. Samples taken at the waterline or from high
dry sites in the prairie are almost always free of the nem-
atode and buttonweed. About 90% of the negative sample
results originate from plants other than buttonweed in
Areas in Florida samples for Verutus volvingentis.
Buttonweed was not sampled at this site.
Plants examined for Verutus volvingentis in
the Florida Survey.
Andropoaon glomeratus (Wa]t)B.S.P,
Baccaris halinifolia L.
Bacopa caroliniana (Walt) G.L. Robinson
Bidens laevis (L.)B.S.P.
Brassica oleraceae L. (acephala group)
Cassia obtusifolia L.
Cephalanthus oc-identalis L.
Cuphea carthagensis (Jacq) Macbride
Cyperus odoratus L.
Diodia teres Walt
." virginiana L.
Geranium carolinianum L.
Clottidium vesicarium (Jacq) Mohr
Glvcines max (L.) Merr
Hyp~L cun r:iutilum L.
Juncus effusus L.
Lachnanthes caroliniana (Lam.) Dandy
Ludwigia arcuata Walt
leptocarpa (Nutt.) Hara
Mikania scandens (L.) Willd.
Nelumbo lutea (Willd.) Pers.
Paron.chia balwinii Fenzl
- ______ rlel- Steud.
Polygonum hydropiperoides Michx.
Ptilimnium capi1l1ceum (Michx.) Raf.
Rnexia mariana L.
Sabal palmeti o (Walt.) Todd ex. Schult
& Schult f.
Scoparla dulcis L.
Stenota hrum secundatum (Walt.)
Zea mays L.
dwarf St. Johns
mock bishop weed
St. Augustine grass
higher, less moist areas, or from areas in water or at the
V. volvingentis occurs in Florida in moist habitats.
Paynes Prairie contains large populations of the nematode
and its host. The host range appears to be very limited.
None of the economic host plants tested were susceptible to
the nematode. Three months following a low level inocula-
tion of the nematode on its host, little or no effect was
evident. Severe symptoms occurred 7 months after inocula-
tion. Fourteen months after inoculation all inoculated
plants were dead. All uninoculated plants in the test were
in a healthy vigorous condition. It is believed that death
of the inoculated plants was a result of growing an annual
plant in the greehouse, devoid of natural inimical ecologi-
cal factors present in its natural habitat, and past its
time of normal growth.
BIOLOGICAL CONTROL INTERACTIONS
Males and larvae were exposed to zoospores of Cate-
naria anguillulae Sorokin. Within 10 min., zoospores were
attached to the cephalic region of larvae (Fig. 63-A), and
males (Fig. 63-B), and to the male cloacal area (Fig. 63-C).
Development of the fungus was only completed in males.
Zoospores were released from an infected male 78 hours after
the initial infection.
Two-hundred eggs were exposed to a culture of zoo-
spores to test the susceptibility of ova to the fungus.
Zoospores became attached to 25% of the eggs in culture but
no thalli developed subsequently.
Eggs were also exposed to a culture of an aquatic
Phycomycete, Rhizophidium sp., a member of the Chytridiales,
known to attack eggs of invertebrates. Sporangia of the
fungus was observed attached to a number of eggs (Fig. 64);
however, further development of the fungus was not noted.
A natural population of the nematode was found infes-
ted with endospores of Pasteuria ramosa Metchnikoff (Fig.
63-D). A single larvae hatching from an egg in the popula-
tion was also noted with endospores attached.
:-I iit :rli ;
11 t rI %
I ~ C'
Figure 63. Biological control
anguillulae zoospores attached
to the cephalic region of a
larva (A), a male (B), male
cloacal region (C). Endospores
of Pasteuria ramosa on a
-- ". -
Figure 64. Sporangium of
attached to an egg.
Femaleswere not noted in biological control inter-
actions. Biological control agents attached to ova but
failed to penetrate the shell and complete development.