Fossil Mermithid from Amber: Poinar 375
mm long when the adult mosquito emerged
(2). Obviously, the development ofH. dominicus was considerably slower than that of
E. riouxi in the postlarval stages of the host.
Heydenius dominicus is the first fossil mermithid described from a probable mosquito host and the first one from a Neotropical
area.
A list of described fossil mermithids is
presented in Table 1. Based on probable
host insect and general morphology, H.
dominicus does not appear similar to any
previously described mermithid.
Dominican amber is thought to have
been formed sometime between the Miocene and Oligocene eras some 25 million
years ago (9).
LITERATURE CITED
1. Dollfus, R. Ph. 1950. Liste des N~mathelminthes connus ~ l'6tat fossile. Compte Rendu Soci~t6
G~ologique de France 5:82-84.
2. Doucet, M. 1980. Contribution a 1'etude d'Empidomermis riouxi n. sp. (Nematoda: Mermithidae).
Ph.D. thesis, University of Montpellier.
3. Von Heyden, C. 1860. Mermisantigua, ein fossiler Eigeweidewurm. Entomolisches Zeitschrift, Stettin 21:38.
4. Larsson, S. G. 1978. Baltic a m b e r - - A paleobiological study. Entomonograph 1, Klampenborg,
Denmark.
5. Menge, A. 1863. Ueber ein Rhipidopteron und
einige Andere im Bernstein eingeschlossene Tiere.
Schriften der Naturforschenden Gesellschaft in Danzig 1 : 1-8.
6. Menge, A. 1872. Ueber eine im Bernstein eingeschlossens Mermis. Schriften der Naturforschenden
Gesellschaft in Danzig 3:1-2.
7. Poinar, Jr., G. O. 1975. Entomogenous nematodes. Leiden: E. J. Brill.
8. Rennie,J. 1925. AmermithidparasiteofTipula
paludosa Meigen. Proceedings of the Physiological Society of Edinburgh 21:1-3.
9. Sanderson, M. R., and T. H. Farr. 1960. Amber
with insect and plant inclusions from the Dominican
Republic. Science 131 : 1313.
10. Schlee, D., and W. Gl~chner. 1978. Bernstein.
Stuttgarter Beitritge zur Naturkunde: Serie C, Heft
8:72 pp.
11. Taylor, A. L. 1935. A review of the fossil
nematodes. Proceedings of the Helminthological Society of Washington 2:47-49.
Journal of Nematology 16(4):375-379. 1984.
© The Society of Nematologists 1984.
Reproductive Isolation and Taxonomic Differentiation of
Romanomermis culicivorax Ross and Smith, 1976 and
R. communensis Galloway and Brust, 1979
J. CURRAN AND j. M. WEBSTERL
Abstract: The infertility of hybrid progeny ofRomanomermis communensisand R. culicivoraxsupports
their retention as distinct species. Their taxonomic separation on the basis of morphometric data
and possession of a cone-shaped spicule guide is rejected. However, differences in the enzyme
patterns of peptidase and phosphoglucomutase and the restriction fragment length differences in
repetitive genomic DNA provide sensitive diagnostic characters that confirm the differentiation
into two species.
Key words: reproductive isolation, Romanomermis communensis, R. culicivorax, taxonomy.
Romanomermis communensis Galloway and
Brust, 1979 and R. culicivorax Ross and
Smith, 1976, both parasites of larval mosquitoes in North America, were recognized as distinct species on the basis of morphological differences (10). However, the
Received for publication 27 February 1984.
1Department of Biological Sciences, Simon Fraser University, Burnaby, Vancouver, British Columbia, Canada V5A
1S6.
validity of separating them into two species
has been questioned recently 1) on morphological grounds, following analyses of
the nature and extent of intraspecific morphological variation in R. culicivorax (2,3),
and 2) by cross-mating experiments which
produced viable hybrids (8). In this latter
study preparasitic juveniles were obtained
from the reciprocal crosses, but they developed into males only. Lack of females
prevented testing of hybrid fertility.
376 Journal of Nematology, Volume 16, No. 4, October 1984
TABLE 1. Survival o f males and females and gravid o r postoviposition females f r o m reciprocal and selfcrosses of Romanomermis communensis (com) and R. culicivorax (cul) after 50, 75, and 100 days.*
Surviving d
Cross
50
75
Surviving
gravid or postoviposition
Surviving 9
100
50
75
100
50
(a) N e m a t o d e s r e a r e d at 20 C t h r o u g h o u t e x p e r i m e n t
com ~ x com ¢
89
t't"
t
100
t
t
98
corn ~ x cul ~
92
t
t
84
t
t
88
cul ~ x corn ~
86
88
88
90
100
88
1
cul 9 x cul ~
70
100
100
100
78
84
6
cul 9
100
94
91
0
com ~
90
93
68
0
(b) N e m a t o d e s r e a r e d at 20 C for 50 days, followed by 27 C for an additional 50 days
cul ~ × corn ~
86
33
26
90
95
88
1
cul ~ x cul d
70
80
t
100
100
100
t
75
100
t
t
2.3
7.7
0
0
t
t
0
4.8
0
0
7.3
96
2.6
t
* Expressed as percentage surviving 0-50 days, 50-75 days, 75-100 days for respective columns.
~"t = experiment terminated at previous sampling data.
T o help clarify the taxonomic status of
these two nominal species, we report on
further cross-mating trials to test hybrid
fertility and on isoenzyme and genomic
DNA restriction endonuclease analysis to
partially characterize both genotypes.
M A T E R I A L S AND M E T H O D S
Postparasitic juveniles of R. communensis
(reared from Aedes communis larvae collected from the nematode type locality at
Goose Creek, Churchill, Manitoba) and R.
culicivorax (reared from Aedes aegypti larvae, the nematode culture originating from
the laboratory culture of Dr. J. J. Petersen,
Lake Charles, Louisiana) were sexed and
the separate sexes allowed to molt at 20 C
(R. communensis) and 27 C (R. culicivorax)
over a period of 2-3 weeks. T h e resulting
adults were placed in a glass Petri dish containing acid-washed, coarse-grained (1-2mm grain size) silica sand (1-2 cm deep)
overlain with glass-distilled water (1 cm
deep) in the following groupings:
R. communensis 100 ~ x R. culicivorax
100
R. communensis 100 ~ x R. communensis
100
R. culicivorax 100 ~ x R. culicivorax
100
R. culicivorax 100 ~ x R. communensis
100
R. communensis 100
R. culicivorax 100
After 50 days at 20 C, the number of
survivors and of surviving gravid or postoviposition females was recorded (8). In
view of the results obtained (see below) it
was speculated that R. culicivorax females
did not mate well at 20 C. To test this
hypothesis, the surviving nematodes from
each cross were subdivided into two groups
and maintained at either 20 C or 27 C for
an additional 50 days. T h e number of survivors and of surviving gravid or postoviposition females was recorded after 25 and
50 days (i.e., 75 and 100 days after the start
of the experiment). In addition, to ascertain if sperm transfer had taken place at
20 C in the R. culicivorax selfcross, five females were stained in Hoechst 33258 and
the spermatheca examined for the presence of spermatozoa (5).
T h e viability and fertility of R. communensis × R. culicivorax hybrid progeny was
determined as follows: hybrid eggs were
maintained at 20 C and the percentage of
fully embryonated eggs recorded after 100
days (eggs were flushed from sand with distilled water). First instar of A. aegypti were
infected with newly emerged hybrid preparasitic juveniles (approximately 5:1 ratio
(2), and the number and sex of postparasitic juveniles produced was recorded. To
test hybrid fertility, these hybrid postparasitic juveniles were placed in moist sand
in the wells of tissue culture plates (2) at a
ratio of approximately 3 ~ to 1 ~ per well.
N u m b e r s o f surviving n e m a t o d e s and
numbers of gravid or postoviposition females was recorded after approximately 50
Romanomermis, Reproductive Isolation: Curran, Webster 377
days at 20 C and again after the temperature was elevated to 27 C for 25 days.
Isoenzyme analysis of virgin adult females of each species reared at 20 C utilized standard starch gel techniques (1,9)
with the inclusion of 15 mg O-dianisidine.
HC1 in the peptidase stain. A known Drosophila sample was included in each gel as
a positive control for enzyme activity. Restriction endonuclease analysis of the genomic DNA was performed on each species
as described by Curran et al. (6).
RESULTS
Percentage survival of males and females
and percentage of surviving females that
were gravid or had laid eggs were recorded
after 50, 75, and 100 days (Table 1). After
50 days at 20 C, both the R. communensis
2 x R. communensis ~ and R. communensis
2 x R. culicivorax ~ crosses produced a high
percentage of gravid or postoviposition females while crosses involving R. culicivorax
females had a low percentage of gravid or
postoviposition females. This pattern continued throughout the experiment at 20
C. However, in the concurrent experiment, following elevation of the rearing
temperature after 50 days to 27 C, the majority of females from the R. culicivorax
selfcross were gravid or postoviposition 25
days later whereas the R. culicivorax ~ x R.
communensis ~ cross produced few gravid or
postoviposition females. No evidence for
parthenogenesis was found in the two
species.
The low percentage of gravid or postoviposition females obtained at 20 C from
the R. culicivorax setfcross was surprising,
since males and females were frequently
observed in copula. In light of this low mating success, it was hypothesized that sperm
transfer may not occur at 20 C. None of
the five R. culicivorax females examined
contained sperm within the spermatheca.
Eggs resulting from R. communensis ~ x
R. communensis ~ and R. communensis ~ x R.
culicivorax ~ (crosses maintained at 20 C)
were examined 75 and 100 days postoviposition. At the respective sampling date,
7 / 2 0 0 and 5 / 2 0 0 of the R. communensis
x R. culicivorax ~ hybrid eggs were embryonated, compared with 1 9 4 / 2 0 0 and
1 9 6 / 2 0 0 of the R. communensis 2 x R. communensis ~ eggs. T o o few R. culicivorax 2 ×
R. communensis ~ embryonated hybrid eggs
(a)
Phosphoglucomutase
+re
mmm
/
l
I
l
origin
/
1
cut com cuL c~'ncuLcomdro
(b)
Peptidase
R
m
R
+re
I
m
m
D
origin
corn c u t c o m c u l c o m dro
FIG. 1. Comparison of iosenzyme patterns of individual Romanomermis culicivorax and R. communensis
virgin females. (a) Phosphoglucomutase. (b) Peptidase. cul = R. culicivorax; corn = R. communensis;
dro = Drosophila sp., used as positive control for enzyme activity.
were obtained to permit further study of
their development.
T h e R. communensis 2 x R. culicivorax
parasitic juveniles reared in A. aegypti
emerged over a 5-day period; 52 postparasiticjuvenile males (6 dead on emergence)
and 15 postparasitic juvenile females (2
dead on emergence) were collected and
used in mating trials to test hybrid fertility.
After 50 days at 20 C ten adult males and
three females were recovered. Numerous
dead postparasiticjuveniles were observed,
many of which had failed to complete ecdysis. The surviving males and females were
placed at 27 C for an additional 25 days,
but no females became gravid. R. communensis males and females were reared under
identical conditions and after 50 days at 20
C all females were gravid.
Differences were detected between the
isoenzyme patterns of virgin females of R.
culicivorax and those ofR. communensis (Fig.
la, b) for phosphoglucomutase and peptidase. However, the malate enzyme-malate
dehydrogenase patterns (not shown) for
both species were identical. Differences in
the restriction fragment lengths of repetitive DNA of R. communensis and R. culicivorax were detected (Fig. 2a, b) as can be
seen by comparing the relative positions of
repetitive DNA fragments (arrowed in figures).
5
378 Journal of Nematology, Volume 16, No. 4, October 1984
[a)
[bl
eul
corn
sis and R. culicivorax should be maintained
eul
corn
E
FIG. 2. Restriction fragment length differences of
repetitive DNA between Romanarnermis eulicivorax (cul)
and R. communensis (corn). (a) Photograph of ethidium
bromide stained UV illuminated (260 nm) 0.7% agarose gel of EcoR1 cut genomic DNA (1 #g per lane).
Arrows indicate selected diagnostic repetitive DNA
bands. (b) Tracing, from the negative, indicating all
repetitive DNA bands, and EcoR1 cut ~ DNA (Lane
E) as size marker, bands at 21.7, 7.5, 5.9, 5.5, 4.9, 3.4
kilobases top to bottom.
DISCUSSION
T h e s e e x p e r i m e n t s confirm previous
findings that cross-mating occurs between
R. communensis and R. culicivorax (8). Furthermore, as with previous findings, more
gravid females were obtained in our study
from the R. communensis 2 x R. culicivorax
cross than the R. culicivorax 2 x R. communensis ~ cross. However, the present
study has demonstrated that, despite the
readiness with which mating occurred, most
hybrid eggs failed to embryonate; of those
that did, many of the hybrid postparasitic
juveniles failed to complete ecdysis, and of
the few adult males and females produced,
none were fertile. This hybrid infertility
supports the conclusion that R. communen-
as distinct species (8).
Originally, R. communensis was taxonomically separated from other species in the
genus on the basis of a cone-shaped spicule
guide as well as several morphometric differences (7). Later studies on intraspecific
variation with R. culicivorax demonstrated
that morphometric data was highly variable and influenced by environmental factors, and, consequently, that its use in
species diagnosis within the genus was
questionable (2). Furthermore, examination of adult males of R. communensis (provided by T. D. Galloway) revealed that the
structure previously described as a spicule
guide is, in fact, the lining of the cloaca.
T h e so-called spicule guide is likely a fixation ar'tifact because it is absent in live
specimens. A similar structure can be observed in lactophenol processed R. culicivorax (4). Hence, the morphological characters used to differentiate R. communensis
and R. culicivorax are of little diagnostic
value.
The virgin females of these two species
can be clearly differentiated from each other by their phosphoglucomutase and peptidase isoenzyme patterns. These biochemical characters may be subject to phenotypic
variability, but by direct characterization
of the genotype, using comparative restriction fragment length differences of repetitive DNA, the distinctiveness of the two
species is reliably confirmed.
In conclusion, the biological, biochemical, and genetic data presented here confirms the maintenance of R. communensis
and R. culicivorax as two distinct species.
LITERATURE CITED
1. Ayala, R. J., J. R. Powell, M. L. Tracey, C. A.
Mourao, and S. Perez-Salas. 1972. Enzyme variability in Drosophila willistoni group. IV. Genic variation
in natural populations o f Drosophila willistoni. Genetics
70:113-139.
2. Curran, J. 1981. Morphometrics of Romanomermis culicivorax Ross and Smith, 1976 (Nematoda:
Mermithidae). Canadian Journal of Zoology 59:23652374.
3. Curran,J. 1982. Morphological variation in Romanomermis culicivorax Ross and Smith, 1976 (Nematoda: Mermithidae). Canadian Journal of Zoology
60:1007-1011.
4. Curran, J., and W. M. Hominick. 1980. Effect
of mounting methods on taxonomic characters of adult
male mermithids (Nematoda: Mermithidae). Nematologica 26:455-466.
5. Curran, J . , a n d J . M. Webster, 1983. Post-em-
Romanomermis, Reproductive Isolation: Curran, Webster 379
bryonic growth of Romanomermis culicivorax Ross and
Smith, 1976: An example of accretionary growth in
the Nematoda. Canadian Journal of Zoology 61:17931796.
6. Curran,J., D. L. Baillie, andJ. M. Webster. 1984.
Use of genomic DNA restriction fragment length differences to identify nematode species. Parasitology
(submitted).
7. Galloway, T. D., and R. A. Brust. 1979. Review
of the genus Romanomermis (Nematoda: Memithidae)
with a description of R. communensis sp. n. from Canada. Canadian Journal of Zoology 57:281-289.
8. Galloway, T. D., and R. A. Brust. 1982. Crossmating of Romanomermis culicivorax and R. communensis (Nematoda: Mermithidae). Journal of Nematology
14:274-276.
9. Prakash, S. 1973. Patterns of gene variation in
central and marginal populations of Drosophila robusta. Genetics 75:347-369.
10. Ross, J. F., and S. M. Smith. 1976. A review
of the mermithid parasites (Nematoda: Mermithidae)
described from North American mosquitoes (Diptera: Culicidae) with descriptions of three new species.
Canadian Journal of Zoology 54:1084-1102.
Journal of Nematology 16(4):379-386. 1984.
© The Society of Nematologists 1984.
Nematode Population and Community Dynamics in
Soybean-Wheat Cropping and Tillage Regimes 1
SONYA MOBBS BAIRD AND ERNEST C. BERNARD 2
Abstract: The nematode community structures of various soybean-wheat regimes and of a singlecropped, conventionally tilled soybean regime were studied at two sites in Tennessee. Each of the
100 nematode species identified in the study was placed in one of five trophic groups, the most
diverse being plant parasites (31 species), followed by Dorylaimida (26 species), bacterivores (23
species), fungivores (15 species), and predators (5 species). No significant differences in overall
diversity and dominance among treatments and trophic groups were found. Densities o f Heterodera
glycines Ichinohe infective juveniles were significantly higher in single-cropped, conventionally tilled
soybeans in July. When data were subjected to ordination analysis, it was shown that plant-parasitic
nematode communities produced an aggregation of conventionally tilled, single-cropped soybean
plots when compared to all double-cropped treatments. Ordination of overall nematode communities
yielded similar results.
Key words: community ordination techniques, ecology, Filenchus species, Glycine max, Heterodera
glycines, soybean cyst nematode, no-till, Triticum aestivum, trophic groups.
Mounting concern with soil erosion,
water pollution, and land and pest management has increased utilization of minimum or no-tillage planting techniques in
the United States (15) and in other countries (4). Conservation tillage practices reduce soil erosion (15) and water pollution
(19) and may increase crop yields (8).
Received for publication 20 April 1984.
Portion of a thesis submitted by the senior author in partial fulfillment of the requirements for the M.S. degree in
Entomology and Plant Pathology, University of Tennessee,
Knoxville.
2 Former Graduate Research Assistant and Associate Professor, respectively, Department of Entomology and Plant
Pathology, University of Tennessee, Knoxville, TN 37901.
Current address of senior author: Department of Entomology
and Nematology, Nematology Lab., Bldg. 78, IFAS, University of Florida, Gainesville, FL 32611.
We thank Albert Y. Chambers and Charles Graves for
allowing us to take soil samples from their experimental fields.
We also thank Tim E. Rickard for his extensive work in
adapting the computer program WORM for use in this study.
Previous studies of nematode populations in various conservation tillage systems have yielded widely varied results
(4,17,18). Populations of plant-parasitic
nematodes were greater in conventionally
tilled field plots than in no-till plots of grain
sorghum followed by winter rye in Georgia
(17). In Nigeria, numbers of Helicotylenchus
pseudorobustus (Steiner) Golden and Meloidogyne incognita (Kofoid and White) Chitwood juveniles were greater in no-till
plantings t h a n in conventionally tilled
plantings of maize in rotation with several
crops (4). In Indiana, Pratylenchus scribneri
Steiner was more evenly distributed in notill plots than in conventional tillage plots
(1).
T h e present study was undertaken to
c h a r a c t e r i z e the n e m a t o d e c o m m u n i t y
structure of no-till soybean-wheat fields
and to assess the effects o f various cropping