Spiracles as attraction source and entrance for entomopathogenic

Spiracles as attraction source and entrance for
entomopathogenic nematode species in Rhipicephalus
microplus (Acari: Ixodidae) engorged females
Inês Ribeiro Machadoa, Liliana Parente Ribeiroa and Claudia Dolinskia*
Nematology Research Group, Laboratório de Entomologia e Fitopatologia (LEF), Centro de Ciências e Tecnologias Agropecuárias
(CCTA), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes (RJ) Brazil
*claudia.dolinski@censanet.com.br
a
HIGHLIGHTS
• Entomopathogenic nematodes (EPNs) were attracted to spiracles, but not to the mouth or anus.
• The attraction was highest in the nematodes with cruiser foraging behavior.
• The highest mortality of engorged females happened one week after infection by heterorhabdtids.
ABSTRACT: Although entomopathogenic nematodes (EPNs) have been proved an efficient biological control agent
against ticks, as yet there has been no report about how the infection occurs. The infection process takes place
after infective juveniles (IJs) recognize the tick as a host and make entry. The objective of this study was to find out
the main entrance for IJs in engorged females of Ripicephalus microplus. We hypothesized that infection success
may be better for some foraging strategies than others. We used Heterorhabditis baujardi LPP7 and H. indica LPP4,
both natives of Brazil, Steinernema feltiae Sn and S. carpocapsae All, both exotics. Besides the attractiveness, we
also evaluated female mortality one week post infection. The IJs were only attracted to spiracles, and the EPN most
attracted was H. baujardi LPP7, followed by H. indica LPP4, S. feltiae Sn and finally S. carpocapsae All. This attraction
is related to foraging strategies since heterorhabditids have a cruiser strategy. The entrance was directly related to
mortality, since the highest mortalities were also caused by heterorhabditids. These findings may have an impact
on the control of the cattle tick since it was clear what cruiser EPNs were better capable to find and enter the host,
causing greater mortality.
Keywords: infection process, behavior, ecology, biological control, Steinernematidae, Heterorhabditidae, LPP4, LPP7.
Cite as
Machado IR, Ribeiro LP, Dolinski C. Spiracles as attraction source and entrance for entomopathogenic nematode species in
Rhipicephalus microplus (Acari: Ixodidae) engorged females. Nematoda. 2015;2:e052015. http://dx.doi.org/10.4322/nematoda.05015
Received: Apr. 30, 2015 Accepted: July 1, 2015
INTRODUCTION
Due to the extensive economic damage that the cattle tick, Rhipicephalus microplus (Canestrini),
causes to milk and meat production systems around the world, researchers have been searching
for alternatives to control it[1, 2]. Laboratory and field studies have shown that entomopathogenic
nematodes (EPNs) from the families Steinernematidae Chitwood & Chitwood and Heterorhabditidae
Poinar represent a promising alternative for the control of R. microplus[3, 4, 5, 6, 7, 8, 9]. The biological
method using EPNs focuses on control of the non–parasitic phase of ticks, since engorged females at
the time of oviposition seek in the soil moisture and protection from solar radiation environments, a
habitat that also favors EPNs[10].
Entomopathogenic nematodes in the genera Heterorhabditis and Steinernema (Rhabditida) are
obligate parasites of insects. These nematodes have a symbiotic relationship with bacteria in the genera
Nematoda. ISSN 2358-436X. Copyright © 2014. This is an Open Access article distributed under the terms of the Creative Commons Attribution
Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.
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Spiracles as attraction source and entrance for EPNs in Rhipicephalus microplus engorged females
Photorhabdus and Xenorhabdus, respectively. Infective juveniles (IJs), the only stage of the nematodes
found in the soil, enter hosts through natural openings such as the mouth, anus or spiracles, but
heterorhabditis IJs can also enter through the cuticle. After penetrating into the host’s hemocoel, the
nematodes release their symbiotic bacteria, which usually kill the host within 24 to 48 h. The bacteria are
also responsible for antibiotic production and for providing nutrition for the nematodes. The nematodes
feed, develop, mate, and often complete 2-3 generations within the host cadaver. When resources
within the cadaver are depleted, a new generation of IJs is produced and leaves the cadaver to search
for new hosts[11].
Entomopathogenic nematodes’ behavior and ecology have been widely studied in an attempt to
improve their use as biological control agents[12]. The infective juvenile (IJ) stage is the only non‑feeding,
free-living stage in this nematode’s life cycle. Their foraging strategies can be predicted from the
behaviors they exhibit during host search. They are a continuum from sit-and-wait (ambusher) to
cruising (cruiser)[13]. Infective juveniles that ambush potential hosts forage near the soil surface and
are generally best adapted to attacking mobile insects. Ambusher IJs raise most of their bodies off
the substrate, a movement called “tail standing”, and jump towards the host-associated volatile cues
as a host passes nearby[14, 15]. Cruisers, when comparing to ambushers, are typically found deeper
in the soil profile, and are more effective at attacking sedentary hosts because they move towards
host‑associated cues[16, 17].
Indeed, their possible utility as biological control agents has spurred most of the research so far
conducted on entomopathogenic nematodes. One important question is how the infection process takes
place. Four progressive stages of the infection process were outlined by Doutt[18]: host habitat‑finding
(finding the habitat that favors the presence of the host), host finding, host acceptance, and host
suitability. Each host selection step serves to narrow host range, involving interactions between host
and EPNs.
Entomopathogenic nematodes can reach the host’s hemocoel through the cuticle, through the gut wall
when entrance is via the anus/mouth, or through the tracheal cuticle when entrance is via spiracles.
Thus, putative penetration stimuli may be located at these sites. Penetration into a host can potentially
be fatal to a nematode, due either to defense against infection (a lethal composition of gut fluids or a
strong immune response against the nematode) or other conditions that can disrupt host infection [19].
Some studies have pointed to the spiracles as the main or only IJ entrance, for example in human
head lice, Pediculus humanus capitis De Geer (Anoplura: Pediculidae)[20]. In others the anus is the only
entrance, as observed in adult insects of hazelnut weevil, Curculio nucum (Coleptera: Curculionidae)
[21]
, and in still others entrance is only through the mouth, but with size limitation as in Culex pipiens
L. larvae (Diptera: Culicidae)[22]. In any case, it seems that nematode species and insect stage both play
an important role in juvenile penetration, consequently infection[21].
The goal of this study was to observe the infection process of Heterorhabditis baujardi Phan, Subbotin,
Nguyen & Moens LPP7, H. indica Poinar, Karunakara, & David LPP4, Steinernema feltiae (Filipjev) Sn
and S. carpocapsae (Weiser) All in susceptible engorged R. microplus. We aimed to find out how the IJs
would behave in the presence of engorged females. We hypothesized that because foraging behavior
varies, we would expect to see higher activity for cruisers than ambushers, with intermediate foraging
nematodes showing mixed reaction.
MATERIAL AND METHODS
Three hundred and sixty engorged females of a susceptible R. microplus strain Porto Alegre were
used in this assay. These females were from a line of females that had never had any contact with
acaricides.
The nematodes H. baujardi LPP7, H. indica LPP4 were from the Amazon Forest (Monte Negro,
Rondônia) and were identified based on morphology and molecular biology[23]. In turn, S. feltiae Sn
was from France and S. carpocapsae All from Georgia, USA. All nematodes were multiplied in the last
instar of Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. There were no native steinernematids
available for this experiment. Groups of 5 larvae with an average weight of 250 mg were placed in
9-cm diameter Petri dishes lined with two sheets of filter paper (Whatman No. 1), and 100 IJs diluted
in 1 mL of distilled water, sealed and placed in a climate-controlled chamber at 27 ± 1 °C and RH> 80
± 10% for 48 hours. Dead larvae were transferred to modified White traps[24] and placed in a climatecontrolled chamber under the same conditions described above. After 11 to 12 days, when the first
IJs began to emerge from G. mellonella cadavers, they were collected on alternate days using Pasteur
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pipettes. Infective juveniles were kept in cell culture bottles (40 mL) and stored in a climate-controlled
chamber at 16 ± 1 °C and RH> 80 ± 10% for up to one week before being used in the test.
To observe the IJs’ attraction and how they would enter engorged females, we used a concentration of
960 IJs / ♀ in 0.5 mL of distilled water. Previous to this experiment, we tested different IJ concentrations,
but it was only with 960 IJs that we could truly observe the IJs moving on the cuticle of engorged
females. To obtain this specific concentration, a Peters slide was used under a light microscope to count
the IJs, using 20 aliquots of 10 μℓ each, swirling the solution before taking each aliquot. The engorged
females had fallen off the host (adult dairy cows) on the day before the experiments, and they were
weighed and separated into groups with about same weigh (260-270 mg), excluding the smaller and
bigger ones for both assays.
For each species, we used three 24-well plates (1.5 cm of diameter × 2 cm high), each well containing
4 g of sifted and autoclaved sand, 960 IJs in 0.5 mL of distilled water and one engorged female. For
control, we used three 24-well plates, each well containing 4 g of sifted and autoclaved sand, 0.5 mL
of distilled water free of nematodes and one engorged female. The nematodes were added to the sand
before the ticks. The plates were covered and placed in a climate-controlled chamber at 25 ± 1 °C and
RH> 80 ± 10% for 72 h. Each treatment (four species and control) had 72 replicates (24 wells in 3 plates).
Monteiro et al.[25] showed that the highest mortality was reached after 72 h of contact between
H. bacteriophora HP88 and the R. microplus engorged female. Since we wanted to follow the infection
process, we decided to observe the ticks every 12 h until reaching 72 h of observation. Therefore, every
12 h for 5 min, three people at the same time observed the engorged females, focusing on their mouth,
anus and pair of spiracles; counting IJs during this time when they clustered and/or stopped moving.
The entire infection process for each species used in this study was noted and randomly photographed
with a Kodak EasyShare® C743 digital camera with 3X optical zoom and 7.1 Megapixels, coupled to
the objective lens of the Tecnival stereomicroscope.
Data were tested for normality (Lilliefors test) and homogeneity of variance (Cochran and Bartlett
tests). Percent mortality were square root transformed (x1 = √ (X + 0.5) and submitted to ANOVA with
Sistema de Análises Estatísticas and Genéticas[26]. Differences in treatment means were compared using
Tukey’s honestly significant difference procedure at P<0.05. The percentages shown in Figure 1 are
transformed data. A fitted polynomial regression model was used to identify the relationship between
the number of IJs in and around spiracles (dependent variable) and time (independent variable).
The program used was Excel version 2007 with alpha 0.05. The entire experiment was repeated after
the first (Assay 1 and 2). The results are shown separately for better visualization.
RESULTS AND DISCUSSION
Infective juveniles from all species tested were found around ticks’ spiracles only. They crawled over
the entire tick, but only stopped and clustered around spiracles. For heterorhabditids, the number of
IJs clustering around spiracles grew over time. Tick mortality only occurred one week after infection,
with the lowest mortality caused by S. carpocapsae All. This is the first reported observation of EPN
Figure 1. Percent mortality (mean + standard error) of adult females of Ripicephalus microplus after exposure to
various entomopathogenic nematodes at 72 hours. Different letters above bars indicate significant differences (P < 0.05).
H. i.= Heterorhabditis indica LPP4; H. b.= H. baujardi LPP7; S. f.=Steinernema feltiae Sn and S. c.= S. carpocapsae All.
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penetration via spiracles in R. microplus, and indeed the first ever report of H. indica LPP4 and
H. baujardi LPP7 penetration in any host (Figures 2a-d).
Monteiro et al.[25] showed that the highest mortality was reached after 72 hours of contact between
H. bacteriophora HP88 and the R. microplus engorged female. Since we wanted to follow the infection
process, we decided to observe the ticks every 12 hours until reaching 72 hours of observation. So,
when the ticks were observed, care was taken not to touch or misplace them, so the IJs would not
be disturbed. The number of IJs around all spiracles was counted every time the engorged females
were observed, and this number for H. baujardi LPP7 and H. indica LPP4 reached the maximum of
6 and 4, respectively. Both S. feltiae Sn and S. carpocapsae All were observed with up to 2 IJs around
the spiracle at each time (Figures 2a-d). The numbers shown in Figure 3 are the sum of IJs observed
from 72 females. The IJs that had already entered the females were not dealt with, since the females’
dark color made it impossible to count them (Figures 4a, b).
Infective juveniles of the genus Heterorhabditis are known to penetrate the hemocoel of insect-hosts
through the cuticle, via anus/mouth or through spiracles. Depending on the host and nematode species,
different penetration routes can be taken[21]. In this study, we demonstrated that penetration in female
cattle ticks occurs only through spiracles by all species tested. However, the frequency of attraction or
penetration may vary depending on EPN species (Figures 3a, b and 5a, b). The ticks’ tegument is very
hard, and if the IJs tried to perforate they did not succeed. Otherwise, there would be an external blood
leakage. This leakage or hemorrhage only happened one week after the infection, due to rupture of
tissues from inside out (Figure 4b).
The number of IJs found around the spiracles was higher for H. baujardi LPP7 and H. indica LPP4,
probably due to their cruiser behavior and greater sensitivity to host-produced volatiles such as carbon
dioxide (CO2). Carbon dioxide, coming from the spiracles and other sources, has been confirmed as
a cue for IJs to locate a host, since CO2 indicates biological activity[27, 28]. Hinton[29] suggested that gas
Figure 2. (a) Photo showing the exact moment that infective juveniles of Heterorhabditis indica LPP4 penetrated
through the spiracle of a Rhipicephalus microplus engorged female susceptible to acaricides at 72 hours. (b) Group
of H. baujardi LPP7 infective juveniles penetrating through female tick spiracle at 72 hours. (c) Penetration of
Steinernema feltiae Sn infective juvenile through female tick spiracle at 72 hours. (d) Penetration of S. carpocapsae
All infective juvenile through female tick spiracle at 72 hours. Bar = 1 mm.
Figure 3. Sum of the number of infective juveniles (IJs) in and around spiracles of 72 engorged females observed
during 5 minutes at different times (hours). Heterorhabditis baujardi LPP7 (H. b.), H. indica LPP4 (H. i.), Steinernema
feltiae Sn (S. f.) and S. carpocapsae All (S. c.) (a) Assay 1. (b) Assay 2.
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exchange between the tick and the environment occurs via airfoils through the spiracles. The demand
for oxygen and release of carbon dioxide must be balanced by the need to protect the animal against
excessive water loss[30]. Therefore, it is possible to infer that the carbon dioxide released by the female
cattle ticks was the compound that attracted the nematodes to the spiracles.
Polynomial regression analysis predicted the number of IJs in and around spiracles through time
for all species tested in both assays. The coefficient of determination was higher for heterorhabditids,
which are cruiser forager strains (Figures 5a, b). There was a clear tendency for the infection to
increase over time in these species. Ambush foraging nematodes also respond to volatile cues, but
their behavioral responses can be different from those of cruise foragers and are context-specific. On
smooth substrates, ambush foragers like S. carpocapsae exhibit wide-ranging search behavior, but
they are not attracted to host volatile cues like carbon dioxide without prior contact with a host[16, 31].
This response does not appear to result from limitation of their sensory systems, because artificial
selection can produce populations of nematodes that are attracted to host insects[32]. Ambush foragers
also do not switch to area-concentrated search in response to contact with host cues such as cuticle
and feces, as does S. glaseri (Steiner)[27].
Considering the total number of IJs found around the spiracles, we can infer that S. feltiae SN IJs
were more attracted by the spiracles than S. carpocapsae All. S. feltiae is an intermediate forager, and
more attracted to carbon dioxide than S. carpocapsae All. Moreover, many S. carpocapsae All IJs were
found bouncing on the female ticks, meaning that they failed to recognize the tick as a host. One could
infer that size may be negatively influencing the entrance, but we observed at least one specimen from
Figure 4. Photos of engorged females of Ripicephalus microplus after infection with Heterorhabditis baujardi LPP7.
(a) Dead female ticks at 72 hours. (b) Tick presenting hemorrhage, after 7 days of infection.
Figure 5. Polynomial curves relating number of infective juveniles of Heterorhabditis baujardi LPP7 (H. b.), H. indica
LPP4 (H. i.), Steinernema feltiae Sn (S. f.) and S. carpocapsae All (S. c.) found in and around spiracles at observation
time (hours). (a) Assay 1. (b) Assay 2.
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each species entering the spiracle. Different observation was done by de Doucet et al.[20] in which IJs
were seen entering through spiracles which were never greater than 30 µg of diameter.
During the assay, mortality was low (data not shown). However, female mortality reached its
maximum after 72 hours, when the mean mortality was compared (Figures 1a, b). Significant differences
in mortality were detected between different species tested and control, in both assays (ANOVA, Assay 1:
F = 18.305; df = 4; P=0.00043 and Assay 2: F = 31.701; df = 4; P=0.00006). H. baujardi LPP7, H. indica
LPP4, S. feltiae Sn and S. carpocapsae All caused 83 and 97, 66, and 76, 53 and 69, 29 and 18% mortality
in Assay 1 and 2, respectively. This high percentage of mortality is probably related to the number
of IJs entering the female tick and releasing their symbiotic bacteria. There was no mortality in the
control treatment (Figure 3a, b).
These findings may have an impact on the control of the cattle tick since it was clear what cruiser
EPNs were better capable to find and enter the host, causing greater mortality.
CONCLUSIONS
Penetration via spiracles seems to be the only route taken by IJs to infect engorged females of
R. microplus. Thus, we conclude that cruiser foraging nematodes may find female ticks more easily
than steinernematids, recognize them as hosts and make entrance through spiracles. After 72 hours,
mortality reaches its maximum, and hours after that, females undergo external bleeding (hemorrhage).
ACKNOWLEDGEMENTS
We thank CAPES (Higher Education Personnel Training Coordination) and CNPq (National Council
of Technological and Scientific Development) for scholarships.
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