Claudia Dolinski, Eleodoro E. Del Valle, Rogério S. Burla & Inês Ribeiro Machado ARTIGO
Biological Traits of Two Native Brazilian Entomopathogenic Nematodes
(Rhabditida: Heterorhabditidae)
Claudia Dolinski, Eleodoro E. Del Valle, Rogério S. Burla & Inês Ribeiro Machado
Universidade Estadual do Norte Fluminense, Laboratório de Entomologia e Fitopatologia CCTA, Av. Alberto Lamego
2000, 28015-620, Campos dos Goytacazes (RJ), Brasil.
Corresponding author: claudia.dolinski@censanet.com.br
Received for publication on February 06, 2007. Acept on July 20, 2007
Resumo – Dolinski, C., E.E. Del Valle, R.S. Burla & I.R. Machado. 2007. Características biológicas de dois
nematóides entomopatogênicos nativos do Brasil (Rhabditida: Heterorhabditidae).
Nematóides entomopatogênicos (NEPs) causam doença e morte a vários insetos em diferentes ordens.
Esses nematóides estão sendo usados como agentes de controle biológico com grande sucesso em várias
culturas. Em pomares onde os insetos-praga estão concentrados sob a copa, os NEPs estão sendo aplicados na
forma de insetos-cadáver. Para aumentar o controle da praga, é muito importante saber a quantidade de juvenis
infectantes (JIs) produzidos e o tempo para emergência desses do inseto-cadáver de cada linhagem aplicada ao
campo. Essas duas características foram avaliadas para duas linhagens nativas brasileiras: Heterorhabditis baujardi
LPP7 e LPP1, nas temperaturas de 25 e 28 oC. LPP7 e LPP1 produziram em média 147.680 ± 24.126 e 165.813
± 20.830 JIs a 25 oC e 122.350 ± 8.905 e 131.980 ± 8.774 JIs a 28 oC, respectivamente. O tempo para os JIs
emergirem dos cadáveres foi de 14,7 ± 2,2; 14,4 ± 0,8; 11,4 ± 1,1 e 10,7 ± 1,7 dias, para LPP7 e LPP1 às
temperaturas de 25 e 28 oC, respectivamente. A temperatura afetou significativamente ambas as variáveis. As
linhagens avaliadas diferiram significativamente para o parâmetro produção de JIs a 25 oC, sendo que H. baujardi
LPP1 apresentou a produção mais alta.
Palavras-chaves: produção de juvenis infectantes, influência da temperatura, tempo para emergência, insetocadáver, Nemata.
Summary - Dolinski C., E.E. Del Valle, R.S. Burla & I.R. Machado. 2007. Biological traits of two native
Brazilian entomopathogenic nematodes (Rhabditida: Heterorhabditidae).
Entomopathogenic nematodes (EPNs) cause disease and death to a number of insects in different orders.
Those nematodes are being used as biological control agents with great success in various crops. In orchards
where the insect pests are concentrated under the canopy, the EPNs are being applied as insect-cadavers. In
order to increase the pest control, it is very important to know from each strain applied in the field the number
of infective juveniles (IJs) produced and their emergence time from the insect-cadaver. Those characteristics
were evaluated for two Brazilian native strains: Heterorhabditis baujardi LPP7 and LPP1, under the temperatures
of 25 and 28 oC. LPP7 and LPP1 strains produced average of 147,680 ± 24,126 and 165,813 ± 20,830 IJ at 25
o
C and 122,350 ± 8,905 and 131,980 ± 8,774 IJ at 28 oC, respectively. The IJ emergence time were 14.7 ± 2.2,
14.4 ± 0.8, 11.4 ± 1.1 and 10.7 ± 1.7 days, for LPP7 and LPP1 at 25 and 28 oC, respectively. The temperature
significantly affected both IJ production and emergence time. The strains tested differed significantly for the
variable IJ production at 25 oC, with higher production for H. baujardi LPP1.
Key words: infective nematode yield, temperature influence, emergence time, insect- cadaver, Nemata.
180 Vol. 31(3) - 2007
Biological Traits of Two Native Brazilian Entomopathogenic Nematodes (Rhabditida: Heterorhabditidae)
Introduction
Steiner matids and heterorhabditids are
entomopathogenic nematodes (Nemata: Rhabditida),
which are capable to kill many insect species in soil
and cryptic habitats. Those nematodes are obligated
pathogens that kill insects with the aid of two
symbiotic bacterium carried in the intestine,
Xenorhabdus spp. and Photorhabdus spp., associated
respectively with the genera Steinernema spp. and
Heterorhabditis spp. (Poinar, 1990; Forst & Clarke,
2002).
The infective juvenile (IJ) is soil-dwelling, nonfeeding, third-stage juvenile protected by the secondstage cuticle. Once in contact with the insect, IJ
penetrate the insect via natural opening or directly
through the cuticle. They release the bacteria into the
insect’s hemocoel for its development. The bacteria
then multiply, produce endotoxins that kill the insect
in 24-48 hours by septicemia (Dowds & Peters, 2002).
The bacteria provide nourishment for the nematodes
that develop, make ecdises and become adults. In the
case of steinermatids, males and females compose the
first generation; they mate and produce two or three
generations in the same insect-cadaver. The life cycle
of heterorhabditis is similar except that the IJ develop
into hermaphrodite adults capable to self-fertilization,
and that their progeny are formed by males and
females. In both families, after two or three generations
within the host, IJ emerge from the cadaver to seek
new hosts (Poinar, 1990).
Entomopathogenic nematodes can be produced
in vivo or in vitro depending on the scale production
needed. Small-scale production, as for small farmer
and laboratories, uses in vivo cultivation, while larger
farmers will need in vitro in liquid or solid culture
(Ehlers, 2001). Larvae of Galleria mellonella (L.)
(Pyralidae: Lepidoptera) have been used for in vivo
rearing of these nematodes due to its high
susceptibility to EPNs to a wider range of nematodes
species (Gaugler & Han, 2002). In average 100,000150,000 IJs may emerge from each G. mellonella larvaecadaver, depending on the nematode species and/or
strain (Woodring & Kaya, 1988).
Two features that may affect the EPNs suitability
as a biological control agent against specific insect-
pests are their level of infectivity and reproductive
capacity. Infectivity refers to the ability of nematodes
to cause infection in different insects, and may vary
with the insect target and the nematode species or
strains, e.g. the effective control of Diaprepes abbreviatus
(L.) by Steinernema riobrave (Cabanillas, Poinar &
Raulston) 1994 355 (Stuart et al. 2004), Conotrachelus
nenuphar (Herbst) by S. car pocapsae (Weiser) All
(Shapiro-Ilan et al. 2002), and Conotrachelus psidii
Marshal by H. baujardi Phan, Subbotin, Nguyen &
Moens LPP7 (Dolinski et al., 2006).
The reproductive capacity and emergence time can
be affected by abiotic and biotic factors. Temperature
is the most important factor limiting the reproductive
success of EPNs. Each strain has its own temperature
range to which it is best-adapted, and an understanding
of the effects of soil temperature on nematode
reproduction and development helps to improve the
accuracy of field applications (Kaya, 1990; Zervos et
al., 1991; Choo et al. 2002). As for biotic factors, density
of invading IJs (Selvan et al., 1993; Koppenhöfer &
Kaya, 1995; Boff et al., 2000), as well as the insect
host (Jansson, 1996; Molina et al., 2004) and nematode
strain (Jansson, 1996; Zervos et al., 1991) may affect
the nematode development.
In this study, we tested the influence of
temperature and strain type on JI production and
emergence time. Also, the method for counting
nematode progenies was detailed.
Material and Methods
Nematodes, Galleria mellonella larvae, and
experimental design. The nematode strains used in
this study, H. baujardi LPP7 and H. baujardi LPP1, were
reared in G. mellonella larvae at 25 oC, according to
procedures in Woodring & Kaya (1988). Harvested IJ
were kept at 16 oC for less than one week before the
tests. G. mellonella larvae were reared under laboratory
conditions in plastic pots (30 x 15 x 10 cm), on a diet
based on cereals, sugar cane and honey. These strains
were obtained from the Tropical Forest of
Montenegro (RO), Brazil, and being described
elsewhere.
One thousand IJ of H. baujardi LPP7 were
suspended in 0.5 ml of distilled water and distributed
Nematologia Brasileira 181
Piracicaba (SP) Brasil
Claudia Dolinski, Eleodoro E. Del Valle, Rogério S. Burla & Inês Ribeiro Machado
Average Ijs Production
(103)
evenly onto a 9 cm-diameter plastic Petri dish with
paper filter (Whatman no 1) on the bottom. Five lastinstar G. mellonella larvae (250-280 mg) were placed in
each dish. Since at that time best IJ concentration was
not known, the amount of about 200 IJs / larva was
used in order to avoid overcrowding and / or subinfections. A total of five Petri dishes with five larvae
in each were placed in plastic bags and incubated in
the dark at 25 ± 2 oC and 28 ± 2 oC (60 % humidity).
The same procedure was repeated for H. baujardi
LPP1. The Petri dishes were placed in plastic bags to
avoid temperature oscillation during the infection
period. Larval mortality was recorded after 4 days and,
when dead insects showed typical symptoms
(homogeneous brownish color), they were transferred
individually to modified White traps (White, 1927),
arranged side by side on the germination chamber
shelves, and inspected daily for nematode emergence.
The time required for IJs to emerge from each cadaver
was also recorded. The IJs were collected from each
White trap to 50 ml-erlenmeyers during one week.
After that, the volume of each erlenmeyer was adjusted
to 40 ml with distilled water and kept in a germination
chamber at 16 ºC.
Counting. A total of 20 erlenmeyer flasks of each
strain in each temperature were used for counting
nematodes, each considered a replicate. The
erlenmeyer flasks containing the nematode
suspensions were shaken and homogenized for 10
200
175
B
seconds by hand every time an aliquot was taken. The
number of IJs in each harvested flask was estimated
from the average number of IsJ present in five aliquots
of 20 µl, counted on a counting slide under a
stereomicroscope.
Statistical analysis. Average IJ production and
emergence time were subjected to analysis of variance
(ANOVA) using Sistema de Análises Estatísticas
(SAEG, 1990). Significant differences among different
temperatures and strains were determined using
Tukey’s Honestly Significant Difference Test at P =
0.05. Data are presented as means ± standard error.
Results and Discussion
This study showed the influence of the
temperature on the IJ production and emergence time
of H. baujardi LPP1 and H. baujardi LPP7, two
entomopathogenic nematodes strains.
Temperature significantly affected IJ production
of both strains tested in G. mellonella larvae, H. baujardi
LPP7 and H. baujardi LPP1. The data shows the
average juvenile yield significantly different between
temperatures for H. baujardi LPP7 (df = 1, 38; F =
13.63; P = 0.05), with yields at 25 oC greater than at
28oC (147,680 ± 24,126 vs. 122,350 ± 8,905 IJ). For H.
baujardi LPP1, the same was observed (F = 74.53),
with average IJ production higher at 25 oC as well
(165,813 ± 20,830 vs. 131,980 ± 8,774 IJ at 25 oC and
28 oC, respectively) (Figure 1).
A
C
150
C
Hb LPP7
125
100
75
Hb LPP1
50
25
0
25
25
28
28
Temperature (ºC)
Figure 1 - Temperature effect on infective juveniles production (± SD) of Heterorhabditis baujardi LPP7 and H. baujardi LPP1 in Galleria
mellonella larvae. Bars with the same letter are not significantly different (P = 0.05).
182 Vol. 31(3) - 2007
Biological Traits of Two Native Brazilian Entomopathogenic Nematodes (Rhabditida: Heterorhabditidae)
Hussaini et al. (2005) showed that temperature
affects the progeny production of different nematode
strains in different hosts. Specifically in G. mellonella,
the IJ production was significantly higher at 25 oC in
all strains tested, including H. indica EN, a very close
related strain of H. baujardi. In this present study, the
temperature of 25 oC was also the most indicated
temperature in both strains tested. That reassures the
necessity of irrigating the soil in order to lower soil
temperature before applying the nematodes in the field
(Georgis and Gaugler, 1991).
H. baujardi LPP1 produced significantly more IJ
than H. baujardi LPP7 at 25 oC (Figure 1). In general,
it can be concluded that optimal IJ production varies
within nematode strain, but also depends on the
number of inoculated IJ and temperature, in this case
25 oC was the optimum temperature for both strains.
Temperature also affected significantly the
emergence time of H. baujardi LPP7, (14.7 ± 2.2 vs.
11.4 ± 1.1 days at 25 and 28 oC, respectively; F = 34.77),
and H. baujardi LPP1 (14.4 ± 0.8, and 10.7 ± 1.7 days
at 25 and 28 oC, respectively; F = 73.85). The time for
IJ to emerge from the cadavers was not different
between the two strains at 25 oC (F = 0.31) nor at 28
o
C (F = 2.10) (Figure 2).
The emergence day was significantly lower at 28
o
C for both strains tested. This lower number of days
has positive and negative views. As positive, the sooner
the juveniles leave the cadaver and start searching for
Emergence Time
(Days)
20
new hosts in the soil, the better. This characteristic
also influences the nematode production, as it speeds
up the process, making the cycle shorter (Ehlers, 2001).
On the other hand, Schirocki and Hague (1997)
mentioned that if the nematode development in the
host is longer, for example under lower temperatures,
that increases the nutrient absorption by the
nematodes and it could be expressed as longer IJs. As
a negative view, at 28oC IJs are produced faster, but
they may have less nutrient reserves in their bodies,
what could affect their ability to search and invade a
host. This hypothesis could be tested by analyzing the
IJ lipid contents of both strains in both temperatures.
Bedding et al. (1983) suggested applying EPNs at
a concentration of 100 IJs / insect as a preliminary
assessment of host susceptibility. It has been shown
for a number of EPNs species, that when the number
of penetrating IJs exceeds an optimal level,
intraspecific competition occurs among the developing
nematodes in the insect cadaver. This competition is
mainly for nutrients and space, affecting nematodes’
fecundity, producing smaller progeny (Koppenhöfer
and Kaya, 1996; Zervos et al., 1991; Boff et al., 2000).
Selvan et al. (1993) showed an optimal survival and
nematode reproduction at a density of approximately
100 IJs per host, agreeing with Bedding et al. (1993).
Within the genera Heterorhabditis, this optimal
number of entering IJs also varies; as for H. heliothidis
(a synonym of H. bacteriophora) NC1, the inoculum
A
A
15
B
B
Hb LPP7
10
Hb LPP1
5
0
25
25
28
28
Temperature (ºC)
Figure 2 - Temperature effect on emergence time in days (± SD) of Heterorhabditis baujardi LPP7 and H. baujardi LPP1 produced in
Galleria mellonella larvae. Bars with the same letter are not significantly different (P = 0.05).
Nematologia Brasileira 183
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Claudia Dolinski, Eleodoro E. Del Valle, Rogério S. Burla & Inês Ribeiro Machado
level that yields the highest number of IJs is 25 IJs
per G. mellonella larvae (Zervos et al. 1991). For H.
megidis NLH-E 87.3, the best concentration is 300 IJs
(Boff et al. 2000). In this study, a tentative
concentration of 200 IJs / larvae was used aiming to
avoid intraspecific competition. Tests on best yields
based on entering IJs of H. baujardi LPP7 and H.
baujardi LPP1 are in progress (results not shown).
Several unknown factors affect nematode
production in vivo, and these factors interact in
unpredictable ways. This reflects on a high variability
in the number of juveniles produced per host, which
is indicated by high standard error values. Specifically
in this study, we tried to correct that using more
replicates (20 progenies for each treatment), but in a
few treatments the standard error was still high.
These results herein presented are of interest
mainly to visualize and understand what takes place
in the soil when G. mellonella larvae infected with EPNs
are applied in the field. We expect that native
nematodes such as H. baujardi LPP7 and H. baujardi
LPP1, that are well adapted to local climate and soil
conditions, could be very effective as biological control
agents against local pests.
Acknowledgments
The authors thank Ricardo M. Souza for critically
reading an earlier version of this manuscript. We also
thank Grace Kelly dos Santos (TECNORTE
fellowship) for rearing the G. mellonella and the
technicians of ICB5 / USP for helping on the
nematode sampling. Rogério S. Burla and Inês R.
Machado received scholarships from Universidade
Estadual do Norte Fluminense Darcy Ribeiro
(UENF). Eleodoro E. Del Valle received scholarship
from Fundação de Amparo à Pesquisa do Governo
do Estado do Rio de Janeiro (FAPERJ).
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