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The persistence of Phasmarhabditis hermaphrodita (Rhabditida: Rhabditidae) in different substrates

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The effect of different substrates on persistence of the facultative slug parasitic nematode Phasmarhabditis hermaphrodita was studied. Four different substrates (sand, leaf compost, organic horticultural substrate and garden soil) were inoculated with P. hermaphrodita strain B1, isolated in central Bohemia, and the persistence of this nematode was observed for 8 months. A new artificial trap was used to isolate nematodes from the substrates. We found that P. hermaphrodita is able to persist in slightly wet sand at least for 5 months. The density of dauer juveniles decreased markedly in sand. The persistence in compost was very low, probably because of large amount of antagonistic organisms. Organic horticultural substrate and garden soil provide the best condition for persistence of P. hermaphrodita. Nematodes are able to persist in these substrates in very high density, for more than 8 months. We demonstrated that P. hermaphrodita preferably persists in organic soil or habitats with a high content of organic matter and readily reacted to the presence of our artificial trap.
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Russian Journal of Nematology, 2012, 20 (1), 61 - 64
61
The persistence of Phasmarhabditis hermaphrodita
(Rhabditida: Rhabditidae) in different substrates
Jiří Nermuť 1 2
1 Institute of Entomology, Biology Centre, ASCR, v. v. i., Branišovská 31, 370 05 České Budějovice, Czech Republic
2 Faculty of Agriculture, University of South Bohemia, Studentská 13, 370 05 České Budějovice, Czech Republic
e-mail: Jirka.Nermut@seznam.cz
Accepted for publication 20 January 2012
Summary. The effect of different substrates on persistence of the facultative slug parasitic nematode
Phasmarhabditis hermaphrodita was studied. Four different substrates (sand, leaf compost, organic horticultural
substrate and garden soil) were inoculated with P. hemaphrodita strain B1, isolated in central Bohemia, and the
persistence of this nematode was observed for 8 months. A new artificial trap was used to isolate nematodes from the
substrates. We found that P. hermaphrodita is able to persist in slightly wet sand at least for 5 months. The density of
dauer juveniles decreased markedly in sand. The persistence in compost was very low, probably because of large
amount of antagonistic organisms. Organic horticultural substrate and garden soil provide the best condition for
persistence of P. hermaphrodita. Nematodes are able to persist in these substrates in very high density, for more than
8 months. We demonstrated that P. hermaphrodita preferably persists in organic soil or habitats with a high content
of organic matter and readily reacted to the presence of our artificial trap.
Key words:, nematode isolation, slug parasitic nematodes, survival,. trap.
The slug parasitic nematode Phasmarhabditis
hermaphrodita is a bacteriophagous nematode;
nevertheless it does not live in a close association
with only one species of bacteria as do
entomopathogenic nematodes (EPN), but is able
to feed on many bacterial species that are
common in a habitat (Wilson et al., 1995). This
nematode is capable of killing many slug and
snail species, such as Deroceras reticulatum. The
dauer larvae infect slugs in the area beneath the
mantle surrounding the shell, causing a disease
with characteristic symptoms, particularly
swelling of the mantle. Infection leads to the
death of the slug, usually between 7 and 21 days
afterwards. New dauer juveniles emerge from the
slug cadaver, spread into the soil and look for new
hosts (Wilson et al., 1993).
Survival of entomopathogenic nematodes in soil
is strongly influenced by many factors such as
predation, desiccation or starvation. Indirect
evidence for lethal effects of nematode antagonists
is seen in the fact that infective juveniles survived
longer in sterilized than in unsterilized media
(Ishibashi & Kondo, 1986, 1987; Timper et al.,
1991). Starvation of nematodes is correlated with
lipid reserves, that decline during storage (Patel et
al., 1997) and it is probably one of the main causes
of nematodes mortality (Fitters, 1999; Qui &
Bedding, 2000). The level of lipid reserves is also
dependent on the movement activity of
entomopathogenic nematodes and moistness of the
soil. Nematodes are more active in the moist soil
than in the dry soil and more active nematodes
utilise lipid reserves more quickly, which is the
reason for the better survival of EPNs in soil with
lower water content (Hass et al., 2002). On the other
hand, desiccation (extremely low water content) is
very important factor that negatively influences
survival of entomopathogenic nematodes (Grant &
Villani, 2003; Preisser et al., 2005).
Phasmarhabditis hermaphrodita is a facultative
parasite that is able to live and reproduce in many
rich organic substrates (Rae et al., 2009), e.g., leaf
litter (MacMillan et al., 2009) or compost (Nermuť,
unpublished). So far no study has assessed the
persistence of P. hermaphrodita in different
substrates. No data about the persistence are
available for P. hermaphrodita even though this
information can improve understanding to the
biology of this species. Our data shows the ability of
P. hermaphrodita to exploit different substrates
(sand, soil, horticultural substrate and leaf compost)
and helps to understand where and how this
nematode can persist. This study is the first that
presents the influence of different substrates on
persistence of P. hermaphrodita.
J. Nermuť
62
MATERIALS AND METHODS
The B1 strain of P. hermaphrodita was used for
this experiment. This strain was isolated in central
Bohemia. B1 strain is kept in laboratory culture and
reared on freeze-killed slugs Deroceras reticulatum.
Sand and horticultural substrate were bought in a
hobby market, compost and garden soil were
obtained from the author’s private garden.
Substrates were not sterilised.
Persistence of P. hermaphrodita was assessed in
10 l plastic pots with a lid. These pots were filled
with four different substrates: sand (grain diam. 1.3
mm), leaf compost, organic horticultural substrate
and garden soil and each pot were inoculated with
14 000 dauer juveniles. Each experiment consisted
of four replicates and control without added
nematodes. Pots with substrates were kept at 15°C
in dark and substrates were maintained slightly wet.
A new artificial trap was made from plastic test
tube 1.5 ml (Eppendorf). The bottom of this tube
was cut and substituted with a sieve with large loops
(diam. 1 mm). Twelve holes (diam. 1 mm) were
made in the sides of the trap. Sand (size of particles
1.3 mm) was placed inside the trap. Then 100 µl of
attractant was pipetted onto sand in the trap. The
attractant was made from homogenised and
sterilised (autoclaved, 121°C, 15 min) bodies of
grey garden slugs Deroceras reticulatum (1 g of
sterilised slug body + 2 ml of tap water), that were
collected in the garden of Institute of Entomology in
České Budějovice in the Czech Republic.
The presence of P. hermaphrodita was
checked monthly using the artificial traps. The
last (6th) isolation was performed 3 months after
the 5th isolation (8 months from the start of the
experiment). Traps were placed into the substrate
in each pot. Traps were kept in the pots for 48 h.
After this time, traps were removed and placed
into Petri dishes with moist filter paper on the
bottom and incubated at 15°C in dark for next 72
h. After this, the sand was rinsed with a small
amount of tap water. Water with sand was shaken
and the supernatant was poured on the counting
dish. Phasmarhabditis females, that are relatively
easily distinguished (Hooper et al., 1999), were
collected and counted under the stereomicroscope.
Phasmarhabditis females were identified based on
morphology (Hooper et al., 1999; Andrássy,
1983).
Statistical analyses were performed using two
way factorial ANOVA and Tukey HSD test
(Statistica program, version 7, StatSoft Inc.).
Logarithmic transformation was necessary to meet
the homogeneity of variance.
RESULTS
Results of analyses of variance confirm that
both time (F = 2.74, df = 5, P = 0.025) and
substrate (F = 44.90, df = 3, P  0.001) have a
significant influence on the persistence of P.
hermaphrodita in different substrates.
According to the data gained from the
experiment it seems that sand and also leaf
compost are not suitable substrates for the
persistence of P. hermaphrodita. The number of
isolated nematodes in sand decreased with time
and at the last isolation (after 8 months) there
were no Phasmarhabditis females. Leaf
compost also provides poor conditions for the
persistence of P. hermaprodita. After 5 months
there were no Phasmarhabditis females, but
after 8 months, in the last isolation, we isolated
one Phasmarhabditis female. The other
substrates, horticultural substrate and garden
soil, provide the best condition for persistence
of P. hermaphrodita. The numbers of isolated
Phasmarhabditis females was very high, in
comparison with numbers from leaf compost
and sand (Fig. 1). There was no significant
difference between garden soil and horticultural
substrate (P = 0.999) and between sand and leaf
compost (P = 0.440), but both garden soil and
horticultural substrate had significantly higher
amount of isolated Phasmarhabditis females
than sand (both P < 0.001) and leaf compost
(both P < 0.001). No P. hermaphrodita was
isolated in the control pots. Other nematodes
were found in the traps, identified as
Diplogasterids and Rhabditids.
DISCUSSION
Survival of nematodes, including EPNs, is
dependent on many factors, e.g., starvation,
predation and moistness of soil (Ishibashi &
Kondo, 1986; Fitters, 1999, Haas et al., 2002).
The advantage for P. hermaphrodita is that it is
a facultative parasite (Rae et al., 2007, 2009),
that lives on many organic material (MacMillan
et al., 2009) and does not need a live host as
required by EPNs and other obligatorily
parasites. We found that P. hermaphrodita is
not able to survive more than 8 months in
slightly wet sand. The probable reason is that
this substrate does not provide any host or other
organic substrate for continued existence.
Nematodes are active in the moist sand, starve,
utilise their lipid reserves and die (Hass et al.,
2002).
The persistence of Phasmarhabditis hermaphrodita
63
Fig. 1: Average number of Phasmarhabditis hermaphrodita females isolated from different substrates during 8
months (isolation periods 1–6).
The opposite situation should occur in leaf
compost; P. hermaphrodita is able to grow and
reproduce in this substrate (Nermuť, unpublished).
Similar results were observed by MacMillan et al.
(2009) who found that P. hermaphrodita
reproduces on leaf litter. Therefore, the expectation
was that P. hermaphrodita will survive and persist
in leaf compost; however, our results showed the
opposite. The number of isolated nematodes was
very low for the whole period of our experiment, in
comparison with other substrates. Only one
Phasmarhabditis female was isolated from four
pots, at the end of the experiment. We suppose that
P. hermaphrodita was overcome by predators and
other antagonistic organisms such as mites,
collembolans or fungus, that are present in this leaf
compost in large amount and that strongly
influence survival of nematodes (Strong, 2002;
Wilson & Gaugler, 2004). Low number of isolated
Phasmarhabditis females in the leaf compost could
also be caused by aeration of substrate.
Horticultural substrate and garden soil are very
well aerated, whereas the leaf compost used was
more compact matter with lower aeration and this
can cause higher mortality of nematodes.
According to our results, garden soil and
horticultural substrate seem to be very good
habitat for P. hermaphrodita. Nematodes were
able to persist in these substrates for the whole
period of our experiment, at least for 8 months,
in high densities. The horticultural substrate
used was similar to the leaf compost or other
organic soils but the absence of predators and
other antagonistic organisms was beneficial for
P. hermaphrodita. The study confirmed the
ability of P. hermaphrodita to reproduce on
substrates with the high content of organic
matter as it was shown with the leaf litter by
MacMillan et al., 2009.
Thus, the persistence of P. hermaphrodita is
better in substrates with the higher content of
organic matter but lacking increased numbers of
antagonists. The new method for P.
hermaphrodita isolation is proposed..
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J. Nermuť. Устойчивость рабдитид Phasmarhabditis hermaphrodita к различным субстратам.
Резюме. Исследовано воздействие различных субстратов на выживание нематод Phasmarhabditis
hermaphroditaфакультативных паразитов наземных моллюсков. Нематод P. hemaphrodita
штамма B1, изолированных в центральной Богемии, вносили в 4 различных субстрата: (песок,
листовой компост, органический субстрат для цветоводства и садовую почву). Наблюдения
проводили в течение 8 месяцев. Было показано, что P. hermaphrodita способны выживать в слегка
увлажненном песке в течение как минимум 5 месяцев. При этом численность дауэр-лиичнок в
песке значительно сокращалась. Выживаемость к компосте была низкой, предположительно из-за
обилия организмов-антагонистов. Органический субстрат для цветоводства и садовая почва
представляли наилучшие условия для выживания P. hermaphrodita. Нематоды сохранялись в этих
субстратах при высокой численности в течение более 8 месяцев. Было показано, что P.
hermaphrodita выживает наилучшим образом в органических субстратах, активно реагируя на
предлагаемые им искусственные приманки.
The persistence of Phasmarhabditis hermaphrodita
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A nematode, Phasmarhabditis hermaphrodita, known to be associated with slugs but not previously thought to be parasitic, was shown to be a parasite capable of killing the pest slug Deroceras reticulatum. The parasite infects slugs in the area beneath the mantle surrounding the shell, causing a disease with characteristic symptoms, particularly swelling of the mantle. Infection leads to death of the slug, usually between seven and 21 days afterwards. The nematode then spreads and multiplies in the cadaver. In an experiment where individual D. reticulatum were exposed to different numbers of P. hermaphrodita, a significant positive relationship was found between nematode dose and slug mortality. In two experiments on host range, the nematode was found to infect and kill all pest slug species tested: Deroceras caruanae, Arion distinctus, Arion silvaticus, Arion intermedius, Arion ater, Tandonia sowerbyi and T. budapestensis, in addition to D. reticulatum.
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Phasmarhabditis hermaphrodita Schneider (Nematoda: Rhabditidae) is a nematode that parasitises a wide range of slug and snail species. It has been formulated into a biological control agent (Nemaslug) and was commercialised in 1994. It is now available in fourteen European countries. A review is given of all research on P. hermaphrodita, including basic biology, mass cultivation, formulation, host range, application strategies, field efficacy and effects on non-target organisms. The many critical gaps in present knowledge are highlighted, and future research is proposed that will lead to greater understanding of this unusual parasite and may enable its more widespread use in the management of mollusc pests.
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The slug parasitic nematodes Phasmarhabditis hermaphrodita and P. neopapillosa are described and figured including observations with the scanning electron microscope. P. hermaphrodita is a protandrous hermaphrodite and males were not found in most of the cultures derived from slugs. Populations in which males were common were identified as P. neopapillosa. Apart from their mode of reproduction, these two species are morphologically identical. Protein electrophoresis using the Pharmacia PhastSystem or a cellulose acetate system stained for phosphoglucose isomerase resulted in very different patterns for the two species, indicating that their different specific identities are warranted. Quelques observations sur la morphologie et les profils proteiniques des nematodes parasites de limaces Phasmarhabditis hermaphrodita et P. neopapillosa (Nematoda: Rhabditida) - Les nematodes parasites de limaces Phasmarhabditis hermaphrodita et P. neopapillosa sont decrits et illustres notamment en microscopie electronique a balayage. L'electrophorese des proteines a l'aide du Pharmacia PhastSystem ou d'une procedure utilisant un systeme a l'acetate de cellulose colorant la phosphoglucose isomerase definit des profils tres differents chez les deux especes, confirmant ainsi a chacune leur statut d'espece valide.
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Entomopathogenic nematodes are commonly applied to soil and crops for biocontrol of insects. Typically, when nematodes are applied, recoverable numbers decline quickly. Because ultra-violet light is known to kill entomopathogenic nematodes in the laboratory, many researchers blame poor field persistence on the action of ultra-violet light. We conducted a field experiment to test this hypothesis, using Heterorhabditis bacteriophora applied to turf as a model system. We compared persistence of surface-applied nematodes with subsurface-applied nematodes and found no difference in persistence. Numbers of potential nematode antagonists (mites and collembola) were also monitored. Poor persistence was positively correlated with numbers of mites and collembola in plots where nematodes were surface-applied, but not in plots where they were subsurface-applied.
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Survival of the entomogenous nematodes Steinernema carpocapsae, S. glaseri, and Heterorhabditis bacteriophora was lower in nonsterilized soil than in sterilized soil. The nematode-parasitic fungus Hirsutella rhossiliensis was the dominant antagonist in the soil. In the nonsterilized soil, more S. glaseni than S. carpocapsae or H. bacteriophora were parasitized by H. rhossiliensis; consequently, survival of S. glaseri was lower than that of the other two nematode species. Differential survival of the three nematode species in the presence of H. rhossiliensis may be due to differences in their retention of the second-stage (J2) cuticle, susceptibility to fungal adhesion, and motility. When H. bacteriophora with and without its protective J2 cuticle was placed in H. rhossiliensis-infested soil, only 22% of the nematodes without the cuticle survived after 4 days compared with 100% survival of those possessing the cuticle. The nematode species were not equally susceptible to adhesion of H. rhossiliensis conidia. When individual nematodes contacted five conidia, fewer conidia adhered to the J3 cuticle of S. carpocapsae than to the J3 cuticle of S. glaseri or H. bacteriophora. Highly motile nematodes will encounter more H. rhossiliensis conidia than will less motile nematodes. The relative motility of the nematode species in soil was S. glaseri > H. bacteriophora > S. carpocapsae. Entomogenous nematodes that retain their J2 cuticle, exhibit low motility, and are refractory to fungal adhesion may be suitable for controlling insects in habitats with abundant parasitic fungi such as H. rhossiliensis.
Article
The commercially available parasitic nematode Phasmarhabditis hermaphrodita is an effective biocontrol agent for slugs and particularly Deroceras reticulatum, a widespread pest species. Use of the nematode is currently limited by cost and it may be that by developing a fuller understanding of the ecology and behaviour of this nematode, more cost effective application strategies can be developed. We investigated the ability of two strains of P. hermaphrodita (one newly isolated and one that had been maintained in vitro for >15 years) to move through mineral soils and organic media. Active dispersal of both strains was found to be greatest in organic media (bark chips and leaf litter, and to a lesser extent peat) and the nematode was capable of growth and reproduction in leaf litter. Conversely, active dispersal was poor in mineral soils. Nematodes moved further in a clay loam compared with a sandy loam, and moved more at a bulk density of 1.0 vs. 1.2 Mg m(-3). However. P. hermaphrodita was capable of moving greater distances in mineral soils by using the earthworm Lumbricus terrestris as a phoretic host. Our data suggest that P. hermaphrodita is a facultative parasite that is adapted to living in leaf litter and organic material where slugs frequently rest. The implications of these findings for using the nematode as a biological control agent for slugs are discussed.
Article
The effect of soil moisture on entomopathogenic nematode virulence was examined in the laboratory. Objectives were to determine the virulence of several species and isolates of entomopathogenic nematodes at various soil moisture contents and temperatures, and after fluctuations in soil moisture. Studies included up to five isolates of entomopathogenic nematodes: Heterorhabditis bacteriophora Poinar (Oswego and Tuscarora strains), Steinernema glaseri (Steiner) (NC1 strain), S. feltiae (Filipjev) (Biosys 369 strain), and S. carpocapsae (Weiser) (NY001 strain). Nematodes were applied to sandy loam soils ranging in soil moisture content from below the permanent wilting point of plants to near saturation. In all experiments, a rainfall or irrigation event was simulated by adding water to rehydrate soils to high moisture levels (near saturation). Nematode virulence was evaluated periodically by measuring insect mortality in Galleria mellonella (L.) larval bioassays, before and after rehydration. Nematode virulence increased with soil moisture content for all species and isolates tested. Our studies demonstrated that the virulence of entomopathogenic nematodes in low moisture conditions could be restored by rehydrating the soil. Insect mortality was generally low in low-moisture, nematode-infested soils before rehydration, but increased to high levels posthydration. Moisture effects were evident from the onset of each experiment, whereas the effect of soil temperature on nematode-induced insect mortality was delayed and nonsignificant until 14 wk after the initiation of the third experiment.