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INFECTIVITY AND REPRODUCTION OF THREE HETERORHABDITID
NEMATODES (RHABDITIDA: HETERORHABDITIDAE) IN TWO INSECT HOSTS
Richard K. Jansson1
Merck Research Laboratories
1Previous address
Abstract
The infectivity, incubation time, and reproduction
of three heterorhabditid nematodes, Heterorhabditis sp. Bacardis
and FL2122 strains and H. bacteriophora Poinar HP88 strain were
studied in two insect hosts, an apionid weevil, Cylas formicarius
(F.), and a pyralid moth, Galleria mellonella (L.). Two of the
nematodes, Heterorhabditis sp. Bacardis and FL2122 strains, were
of tropical or subtropical origin, whereas the third nematode,
H. bacteriophora HP88 strain, was of temperate origin. Infectivity
did not differ among nematodes within each host; however, it did
differ between hosts for the Bacardis strain. Cylas formicarius
was more susceptible to this nematode than G. mellonella. Incubation
times also did not differ among nematodes within hosts; however,
incubation times were 3.2-4.3 d shorter in C. formicarius than
in G. mellonella. Progeny production differed (although not significantly
consistent) among nematodes and was highest for Heterorhabditis
sp. Bacardis followed by the Heterorhabditis sp. FL2122 and H.
bacteriophora HP88 in both hosts. Percentages of infected cadavers
that produced progeny were consistently higher for the tropical
and subtropical nematodes, Heterorhabditis sp. Bacardis and FL2122,
than for the temperate nematode, H. bacteriophora HP88, in both
hosts. Patterns of emergence from cadavers were consistent in
G. mellonella; most progeny emerged by 23 d after inoculation
and emergence lasted for up to 48 d after inoculation. Conversely,
emergence patterns varied markedly in C. formicarius. Emergence
lasted for up to 29 d after inoculation and peak emergence varied
between 12 and 28 d after inoculation. Progeny production in C.
formicarius was not related to the biomass of the host cadaver.
Key Words: Heterorhabditis spp., Cylas formicarius,
Galleria mellonella, infectivity
Resumen
Fueron estudiadas la infectividad, el tiempo de incubación
y la reproducción de tres nemátodos heterorhabdítidos,
las cepas Bacardis y FL2122 de Heterorhabditis sp., y la cepa
HP88 de H. bacteriophora Poinar, en dos insectos hospedantes,
un gorgojo apiónido, Cylas formicarius (F.), y una polilla
pirálida, Galleria mellonella (L.). Dos de los nemátodos,
las cepas Bacardis y FL2122 de Heterorhabditis sp., fueron de
origen tropical o subtropical, mientras que el tercer nemátodo,
la cepa HP88 de H. bacteriophora Poinar, fue de origen templado.
La infectividad no difirió entre los nemátodos dentro
de cada hospedante; sin embargo, difirió entre los hospedantes
para la cepa Bacardis. Cylas formicarius fue más susceptible
a este nemátodo que G. mellonella. Los períodos
de incubación tampoco difirieron entre los nemátodos
dentro de los hospedantes; sin embargo, los tiempos de incubación
fueron 3.2-4.3 días más cortos en C. formicarius
que en G. mellonella. La producción de progenie difirió
(aunque no con significación consistente) entre los nemátodos
y fue más alta para Heterorhabditis sp. Bacardis, seguida
por Heterorhabditis sp. FL2122 y por H. bacteriophora HP88 en
ambos hospedantes. Los porcentajes de cadáveres infectados
que produjeron progenie fueron consistentemente más altos
para los nemátodos tropicales y subtropicales, Heterorhabditis
sp. Bacardis y Heterorhabditis sp. FL2122, que para el templado,
H. bactriophora HP88, en ambos hospedantes. Los patrones de emergencia
de los cadáveres fueron consistentes en G. mellonella;
la mayoría de la progenie emergió antes de los 23
días posteriores a la inoculación y la emergencia
duró al menos 48 días después de la inoculación.
Por el contrario, los patrones de emergencia variaron marcadamente
entre los 12 y los 28 días después de la inoculación.
La producción de progenie en C. formicarius no estuvo relacionada
con la biomasa del cadáver del hospedante.
Two population parameters of entomopathogenic nematodes
that affect their suitability as a biological control agent against
specific target insects are their level of infectivity and reproductive
capacity. Infectivity refers to the ability of nematodes to cause
infection in a target insect (Tanada & Fuxa 1989) and has
been shown to vary among nematodes within specific target hosts
(Bedding et al. 1983, Molyneux et al. 1983, Morris et al. 1990,
Mannion 1992) and among hosts for a given nematode species or
strain (Bedding et al. 1983, Morris et al. 1990). The reproductive
capacity of nematodes was also shown to differ among nematodes
within target insects (Morris et al. 1990, Mannion & Jansson
1992), and among hosts within specific nematode species or strains
(Morris et al. 1990). Nematodes with higher levels of infectivity
and reproduction within a specific target host may be more effective
at controlling a particular insect under field conditions. These
two population parameters are also central to long-term persistence.
Morris et al. (1990) noted that a high infection rate of soil
insects followed by a high rate of reproduction is critical to
ensure reinfestation of the habitat by nematode progeny.
Recent studies conducted in the laboratory that used
a variety of different bioassay systems showed that heterorhabditid
nematodes, especially an undescribed nematode isolated in Florida,
Heterorhabditis sp. FL2122 strain, were most suitable as biological
control agents of the sweetpotato weevil, Cylas formicarius (F.)
(Mannion 1992). She found that heterorhabditid nematodes had some
of the lowest LC50 and LC90 values, produced more progeny per
cadaver, had higher levels of infectivity in sand, soil, and Petri
plates, killed more hosts within sweet potato storage roots, and
had a greater ability to exit infected weevil cadavers within
storage roots and infect new hosts in the soil than steinernematid
nematodes. Her data concur with previous reports (Jansson 1991,
Jansson et al. 1990, 1992, 1993) that found heterorhabditid nematodes
to be more efficacious against C. formicarius. Jansson et al.
(1992, 1993) also found that heterorhabditid nematodes persisted
longer than steinernematids in the field.
A recent study that determined the potential for
applying nematode-infected wax moth, Galleria mellonella (L.),
cadavers for controlling C. formicarius in the field found that
an undescribed nematode isolated from Puerto Rico, Heterorhabditis
sp. Bacardis strain, produced more progeny per G. mellonella cadaver
than H. bacteriophora Poinar HP88 strain (Jansson et al. 1993).
The present studies were conducted to more fully compare the suitability
of certain nematodes as biological control agents of C. formicarius
and determined if fitness parameters of three heterorhabditid
nematodes differed between two insect hosts, C. formicarius and
G. mellonella.
Materials And Methods
Two insect hosts were used in these experiments:
late instar wax moth, G. mellonella, and third instar sweetpotato
weevil, C. formicarius. Galleria mellonella larvae were obtained
from a commercial supplier (JA-DA Bait, Antigo, Wisconsin). Cylas
formicarius were reared in the laboratory using methods described
previously (Mannion & Jansson 1992).
Three heterorhabditid nematodes were tested: H. bacteriophora
HP88 strain, and two undescribed nematodes, Heterorhabditis sp.
FL2122 and Bacardis. The two latter nematodes were of tropical
or subtropical origin. The FL2122 isolate was found in central
Florida and the Bacardis nematode was isolated near the San Juan
harbor in Puerto Rico (R. K. J. et al., unpublished). These two
undescribed isolates are presumably local variants of a previously
unrecorded species of Heterorhabditis (J. Curran, personal communication)
reported earlier as Heterorhabditis sp. B in Poinar (1990). The
HP88 strain was of temperate origin (Poinar 1990). Nematodes were
reared in vivo (Dutky et al. 1964) in G. mellonella larvae as
previously described (Mannion & Jansson 1992). Infective juveniles
used in these tests were less than two weeks old at the time of
the experiment.
Infectivity
The level of infectivity was determined in each host
using a single nematode/single host bioassay system (Miller 1989).
Single infective juveniles were removed from an aqueous suspension
taken from laboratory cultures and pipetted (0.3 ml) onto a double
layer of filter paper in individual wells (1.5 cm diam) of a Multiwell
TM 24 well, flat bottom tissue culture plate (Falcon R, model
3047, Becton Dickinson and Co., Lincoln Park, New Jersey). One
host larva was then placed on the filter paper in each well. Filter
paper treated with deionized water served as the control. Microsoap
(International Products Corp., Burlington, New Jersey) was added
(1.25 ml/liter) to nematode suspensions to improve efficiency
of nematode transfer. Tissue culture plates were covered, sealed
with parafilm and stored in the dark at 25±2°C. Larvae
were checked daily for nematode-induced mortality for four consecutive
days. Two trials were conducted against G. mellonella larvae and
five trials were conducted against C. formicarius larvae. A total
of four (G. mellonella) and twelve (C. formicarius) tissue culture
plates (96 and 288 larvae, respectively) per nematode treatment
were used to determine infectivity of these nematodes.
Efficiency of nematode transfer was determined by
removing an aliquot (0.3 ml) with a single nematode from suspensions,
dispensing each aliquot onto a Petri plate, and then counting
the number of infective juveniles in each aliquot. A total of
80 aliquots were removed from each suspension on the day of the
experiment. The efficiency of transferring single nematodes were
93.7±3.7, 97.5±1.4, and 95.0±2.9 for H. bacteriophora
HP88, Heterorhabditis sp. FL2122, and Heterorhabditis sp. Bacardis,
respectively. These efficiencies were then used to adjust infectivity
data to estimate real infectivity of each nematode.
Incubation Time and Reproduction
All nematode-infected G. mellonella larvae and only
those C. formicarius larvae infected in the first three trials
were removed from the tissue culture plates and placed in individual,
modified White traps (White 1924) and incubated in the dark at
25±2°C. Cadavers were inspected daily for nematode emergence.
The time required for infective juveniles to emerge from each
cadaver was recorded. The percentage of cadavers that produced
infective juveniles was recorded in both trials with G. mellonella,
but in only the first and third trials with C. formicarius. Once
emergence began, infective juveniles were removed from the outer
well of each White trap once per week and counted. The time that
emergence ceased was also recorded at which time cadavers were
dissected and the numbers of infective juveniles within each cadaver
were recorded. In the first two trials with C. formicarius, the
biomass of each larva was recorded before each trial to determine
if progeny production was related to host biomass.
Data Analysis
Most data were analyzed by least squares analysis
of variance or regression techniques, accordingly (Zar 1984).
Percentage infectivity was compared among nematodes within hosts
by chi-square analysis (Conover 1980). Mean incubation periods
and numbers of progeny produced were compared among nematodes
by the Waller-Duncan K-ratio t test (Waller & Duncan 1984).
Numbers of progeny produced per C. formicarius cadaver in the
first two trials were pooled and regressed on biomass to determine
if reproduction was dependent upon host biomass.
Results
Infectivity
Infectivity to G. mellonella larvae did not differ
(X2 = 1.71, df = 2, P > 0.05) among nematodes. Adjusted percentages
of infective juveniles that invaded larvae were 15.4, 16.0, and
26.7% for Heterorhabditis sp. Bacardis, Heterorhabditis sp. FL2122,
and H. bacteriophora HP88, respectively.
Infectivity to C. formicarius also did not differ
(X2 = 1.9, df = 2, P > 0.05) among nematodes. Adjusted percentages
of infective juveniles that invaded C. formicarius larvae were
30.3, 25.9, and 20.4% for Heterorhabditis sp. Bacardis, Heterorhabditis
sp. FL2122, and H. bacteriophora HP88, respectively.
Infectivity of the Heterorhabditis sp. Bacardis nematode
was affected by the insect host (X2 = 4.9, df = 1, P < 0.05).
A higher percentage of infective juveniles invaded C. formicarius
larvae than G. mellonella larvae. Infectivity of Heterorhabditis
sp. FL2122 and H. bacteriophora HP88 did not differ between hosts
(X2< or =2.3, df = 1, P > 0.05).
Incubation Time
The incubation times of nematodes within the two
hosts also did not differ (F< or =1.1, df = 2,4, P > 0.05)
among nematodes. In G. mellonella larvae, Heterorhabditis sp.
Bacardis, Heterorhabditis sp. FL2122, and H. bacteriophora HP88
required 11.9±0.1, 12.1±0.1, and 12.0±0.1 d, respectively,
to emerge from cadavers. In C. formicarius larvae, these nematodes
required only 8.7±0.2, 7.8±0.2, and 8.1±0.2 d,
respectively, to emerge from hosts. Incubation periods for each
nematode were significantly shorter in C. formicarius than in
G. mellonella, which is probably related to differences in host
size.
Reproduction
In the first trial, percentages of G. mellonella
cadavers that produced progeny differed among nematodes (X2 =
10.7, df = 2, P < 0.01). Higher percentages of G. mellonella
infected with Heterorhabditis sp. Bacardis (66.7%; n = 6) and
FL2122 (71.4%; n = 7) produced progeny than those infected with
H. bacteriophora HP88 (38.5%; n = 13). In the second trial, percentages
of cadavers that produced progeny did not differ among nematodes
(X2 = 3.6, df = 2, P > 0.05). All nematode-infected cadavers
produced progeny in high percentages and were 75% (n = 8), 100%
(n = 8), and 91% (n = 11) for Bacardis, FL2122, and HP88, respectively.
Patterns of progeny production were consistent for
each nematode in G. mellonella larvae (Fig. 1). Most progeny emerged
from cadavers infected with Heterorhabditis sp. Bacardis (67.7-79.3%),
Heterorhabditis sp. FL2122 (69.0-81.3%) and H. bacteriophora HP88
(85.6-96.2%) within 23 d after inoculation. Emergence of progeny
declined 23 d after inoculation for all nematodes and lasted for
up to 48 d after inoculation.
Total progeny production in G. mellonella cadavers
did not differ (F = 2.3, df = 2,12, P > 0.05) among nematodes
in the first trial, but did differ (F = 4.4, df = 2,12, P <
0.05) among nematodes in the second trial (Table 1). Trends in
the data were consistent between the two trials. More progeny
were consistently produced by Heterorhabditis sp. Bacardis followed
in decreasing order by Heterorhabditis sp. FL2122 and H. bacteriophora
HP88. Considerably more progeny were produced in the second trial
than in the first trial, and the reasons for this are unclear.
Percentages of C. formicarius cadavers that produced
progeny differed among nematodes in the two trials (X2> or =15.3, df = 2, P < 0.001). In the first trial, the highest percentages
of C. formicarius cadavers produced progeny when infected with
Heterorhabditis sp. Bacardis (90%; n = 20) followed in decreasing
order by those infected with Heterorhabditis sp. FL2122 (41.7%;
n = 12) and H. bacteriophora HP88 (35.3%; n = 17). In the third
trial, cadavers infected with Heterorhabditis sp. FL2122 had the
highest percentage of nematode production (90%, n = 20) followed
by Heterorhabditis sp. Bacardis (58.8%; n = 17) and H. bacteriophora
HP88 (46.7%; n = 15).
No consistent patterns of emergence of infective
juveniles from C. formicarius cadavers were found in the three
trials (Fig. 2). In the first trial, emergence of Heterorhabditis
sp. Bacardis peaked 19 d after inoculation, whereas those of H.
bacteriophora HP88 and Heterorhabditis sp. FL2122 peaked 28 d
after inoculation. In trial 2, emergence of H. bacteriophora HP88
peaked 12 d after inoculation, whereas those of Heterorhabditis
sp. FL2122 and Bacardis peaked 19 d after inoculation. Few progeny
emerged after 19 d. In the third trial, emergence of all three
nematodes peaked 16 d after inoculation and few progeny emerged
after 16 d.
Total progeny production in C. formicarius cadavers
did not differ (F< or =1.3, df = 2,24, P > 0.05) among nematodes
in the first two trials, but did differ (F = 11.2, df = 2,32,
P < 0.001) in the third trial (Table 1). As found in G. mellonella,
more progeny were consistently produced (although not consistently
significant) by Heterorhabditis sp. Bacardis followed in decreasing
order by Heterorhabditis sp. FL2122 and H. bacteriophora HP88.
Table 1. Progeny production of three heterorhabditid
nematodes in two insect hosts, Cylas formicarius and Galleria
melonella.
Host × Nematode
1Data are means ± sem. Means within a column
followed by the same letter do not differ by the Waller-Duncan
K-ratio t test (Waller & Duncan,1969).
The nematode host influenced progeny production.
Approximately 28.1-, 30.5-, and 43.4-fold more progeny were produced
in G. mellonella than in C. formicarius for Heterorhabditis sp.
Bacardis, Heterorhabditis sp. FL2122, and H. bacteriophora HP88,
respectively.
Progeny production in C. formicarius was not related
to host biomass for any of the three nematodes (Bacardis: Y =
1054 + 646713X, F = 1.3, df = 1,31, P > 0.05, r2 = 0.04; FL2122:
Y = 91 + 687518X, F = 1.2, df = 1,13, P > 0.05, r2 = 0.09;
HP88: Y = -1901 + 510346X, F = 2.4, df = 1,6, P > 0.05, r2
= 0.29).
Discussion
Few differences in infectivity were found among nematodes
within insect hosts; however, infectivity differed between hosts
for Heterorhabditis sp. Bacardis strain. This nematode was more
infective against the weevil, C. formicarius, than against the
lepidopteran, G. mellonella, despite the fact that these nematodes
were all reared in vivo in G. mellonella before experiments. Bedding
et al. (1983) showed that H. bacteriophora was least infective
against the host from which it was isolated, Heliothis punctigera
(Wallengren). They also found that Steinernema feltiae (Filipjev)
(= Neoaplectana bibionis) isolated from Lucilia cuprina (Wiedemann)
and Otiorhynchus sulcatus (F.) was least infective to these insects.
Kaya (1987) suggested that the host from which a nematode is isolated
in soil probably has little, if any, affect on the suitability
of the nematode/host encounter. Entomopathogenic nematodes attack
a broad spectrum of insects; thus, isolation from soil is, in
part, due to a chance encounter between the isolation host and
the nematode. Kaya (1987) also suggested that, in certain cases,
continued association with the same insect species may reduce
virulence rather than enhance it.
Incubation times also did not differ among nematodes
within each species; however, incubation times were considerably
shorter for all nematodes in the weevil, C. formicarius, than
in the lepidopteran, G. mellonella. It is well known that emergence
of infective juveniles is related to depletion of food reserves
and crowding within the host cadaver (Kaya 1985, 1987).
Patterns in total reproduction of nematodes differed
among the three nematodes in both hosts. Heterorhabditis sp. Bacardis
consistently produced more (although not consistently significant)
progeny than the other two nematodes in both hosts. These data
concur with previous studies (Jansson & Lecrone 1994, Jansson
et al. 1993) which showed that Heterorhabditis sp. Bacardis produced
more progeny per cadaver than H. bacteriophora HP88 in G. mellonella
larvae. Progeny production of Bacardis was higher in the previous
study (range, 272,576-396,598 per cadaver) than in the present
study (range, 137,229-238,692 per cadaver). However, a dose of
20 infective juveniles per larva was used in the previous study
compared with only 1 per larva in the present study. Progeny production
of H. bacteriophora HP88 was comparable between the two studies
despite large differences in dose (range of previous study, 76,260-219,181
per cadaver; range of present study, 74,992-194,222 per cadaver).
Patterns of emergence from cadavers were consistent
in G. mellonella, but not in C. formicarius. In G. mellonella,
most infective juveniles emerged within 23 d after inoculation.
In C. formicarius, emergence patterns varied among trials. As
noted earlier, emergence of infective juveniles is related to
depletion of food reserves and crowding (Kaya 1985, 1987). These
factors may have been less apparent to emerging infective juveniles
from C. formicarius due to the smaller size of this host compared
with G. mellonella.
Total progeny production of heterorhabditid nematodes
in C. formicarius concurred with a previous report (Mannion &
Jansson 1992), although progeny production was higher for FL2122
and lower for HP88 in the present study. The dose used might have
affected these data. Mannion & Jansson (1992) used a dose
of 25 infective juveniles per larva compared with 1 per larva
in the present study.
It is recognized that a laboratory bioassay that
predicts performance of entomopathogenic nematodes in the field
is needed to facilitate selection of nematodes in biological control
programs (Hominick 1990, Mannion 1992). Mannion (1992) conducted
Petri dish, sand, soil, and simulated field bioassays to select
suitable entomopathogenic nematodes for the biological control
of C. formicarius and consistently found that heterorhabditids
were superior to steinernematids in all bioassay systems tested.
The present bioassay system may also have potential for selecting
suitable entomopathogenic nematodes, especially heterorhabditid
nematodes, for C. formicarius.
Morris et al. (1990) noted that both infectivity
and reproduction within hosts were important attributes of nematodes
capable of reinfesting new hosts in the field. The present study
demonstrated that Heterorhabditis sp. Bacardis was most infective
and reproductive against C. formicarius, and, for this reason,
it should be a suitable nematode for use in biological control
of this weevil. Recent results from field studies confirm this
belief. Heterorhabditis sp. Bacardis was shown to be very efficacious
at controlling weevil damage to storage roots and persisted at
levels higher than those of all other nematodes tested, including
H. bacteriophora HP88, S. carpocapsae (Weiser) All and S20, and
S. feltiae N27 (Jansson et al. 1993). Collectively, these data
suggest that the use of this single nematode/single host bioassay
may be an important tool for identifying potential candidate heterorhabditid
nematodes in biological control programs of target insect pests.
More work is needed, however, to confirm this belief.
These data also suggest that certain Neotropical
and subtropical nematodes, Heterorhabditis sp. Bacardis and FL2122,
may be more suitable as biological control agents against the
pantropical sweetpotato weevil, C. formicarius, than the temperate
nematode, H. bacteriophora HP88 strain. Recent studies by Lawrence
(1994) also suggested that certain Neotropical nematodes may be
more suitable as biological control agents of this weevil; however,
not all tropical and subtropical isolates were superior to all
temperate isolates.
Acknowledgments
I thank K. Ericsson for assistance with data collection
and R. Miller (Biosys Inc., Columbia, MD), R. Gaugler (Rutgers
University, New Brunswick, NJ), and C. M. Mannion and R. J. Zimmerman
(University of Florida, I.F.A.S., T.R.E.C., Homestead) for critically
reviewing the manuscript. Thanks are also extended to R. Miller
for technical assistance. I thank G. C. Smart, Jr. (University
of Florida, Gainesville) for providing Heterorhabditis sp. FL2122.
Heterorhabditis sp. Bacardis was collected near San Juan, Puerto
Rico by W. Figueroa (University of Puerto Rico, Rio Piedras) as
part of a study [supported by the U.S. Department of Agriculture
under CSRS Special Grant No. 91-34135-6134 (to R.K.J.)] to survey
the Caribbean for entomopathogenic nematodes. These nematodes
are currently being held at the University of Florida, I.F.A.S.,
Entomology and Nematology Department, Gainesville, FL and at the
Department of Entomology, Rutgers University. The present research
was supported by the U.S. Department of Agriculture under CSRS
Special Grant No. 91-34135-6134 (to R.K.J.) managed by the Caribbean
Basin Administrative Group (CBAG). Research was conducted at the
University of Florida, I.F.A.S., T.R.E.C., Homestead. The manuscript
was prepared, in part, at Merck & Co., Inc.
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Fig. 2. Emergence patterns of infective juveniles
from C. formicarius cadavers infected with three different heterorhabditid
nematodes: Heterorhabditis sp. Bacardis strain, Heterorhabditis
sp. FL2122 strain, and H. bacteriophora HP88 strain. Numbers counted
on the last sample date of each trial are the sum of those that
emerged plus any remaining infective juveniles within cadavers.
Fig. 1. Emergence patterns of infective juveniles
from G. mellonella cadavers infected with three different heterorhabditid
nematodes: Heterorhabditis sp. Bacardis strain, Heterorhabditis
sp. FL2122 strain, and H. bacteriophora HP88 strain. Numbers counted
on the last sample date of each trial are the sum of those that
emerged plus any remaining infective juveniles within cadavers.
P.O. Box 450
Hillsborough Road
Three Bridges, NJ 08887
University of Florida
Institute of Food and Agricultural Sciences
Tropical Research and Education Center
Homestead, FL 33031
Trial
1
2
3
C. formicarius
Heterorhabditis sp. Bacardis
6,522.7(1,844.0)a1
7,919.5(1,061.0)a
8,625.6(1,162.6)a
Heterorhabditis sp. FL2122
5,610.6(1,086.5)a
6,896.8(1,167.1)a
4,834.6(676.7)b
H. bacteriophora HP88
1,880.8(469.0)a
3,387.0(259.0)a
2,071.6(388.7)c
G. mellonella
Heterorhabditis sp. Bacardis
194,222.5(54,228.0)a
238,692.2(45,442.2)a
Heterorhabditis sp. FL2122
122,580.2(34,579.4)a
229,903.4(30,825.3)a
H. bacteriophora HP88
74,992.5(30,940.0)a
137,228.8(13,072.1)b