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Short Communication - Susceptibility of Adult Engorged Ticks, Rhipicephalus microplus (Acari: Ixodidae) to a Native Heterorhabditid Isolate (Nematoda: Heterorhabditidae) in Colima, Mexico.

Byline: Jaime Molina-Ochoa, Edelmira Galindo-Velasco, Ana Maria Rosales-Gutierrez, Martin Gonzalez-Ramirez, Roberto Lezama-Gutierrez, Wilberth Chan-Cupul, Steven R. Skoda, Muhammad Irfan Ullah, Luis Jorge Garcia-Marquez and John E. Foster


A survey of entomopathogenic nematodes (EPNs) belonging to the families Steinernematidae and Heterorhabditidae was conducted in the municipality of Tecoman, Colima, Mexico in an effort to obtain alternative controls for cattle ticks. Soil cores from grasslands were baited with last instars of the great wax moth (GWM), Galleria mellonella L., and an EPN isolate was obtained, it belonged to the genus Heterorhabditis and identified as JMO71. The nematodes were cultivated in GWM larvae, and the emerging infective juveniles (IJs) were collected from White traps, and pooled in canted neck vented plastic flasks. The susceptibility of an important livestock arthropod, the cattle tick, Rhipicephalus microplus Canestrini to JMO71 was determined.

Ticks were manually collected from cross-bred beef cattle, and then the heterorhabditid nematodes assayed against these ticks using the Petri dish assay. Concentrations of 0, 500, 1000, 2000, 4000 and 8000 IJs were applied in 1 mL of sterile distilled water dispensed on the surface of a couple of moistened filter papers. For each treatment concentration, 10 adult engorged ticks were placed in the Petri dishes, and each treatment was replicated four times; the experiment was replicated two times. Tick mortality was recorded daily for 9 days. Our EPNs were able to parasitize engorged ticks, and tick mortality ranged between 70-100% after 9-d post-exposure. The faster and higher tick mortality was obtained with the concentration of 2000 IJs causing 100% mortality after 3 days post-exposure. This is the first report of a native Mexican heterorhabditid nematode causing cattle tick mortality.

Key words

Heterorhabditis, Mexico, Grasslands, Cattle ticks, Rhipicephalus (Boophilus).

Entomopathogenic nematodes (EPNs) belong to the families Steinernematidae and Heterorhabditidae, they are microbial obligate pathogens that infect a wide range of insects and other arthropods in the laboratory. In field conditions, they infect mainly the soil-dwelling forms of insects such as caterpillars, cutworms, crown borers, grubs, corn root worms, cranefly, thrips, fungus gnat, and beetles (Miles et al., 2012), and few other soil arthropods. EPNs in the genera Steinernema and Heterorhabditis are considered lethal pathogens of insects due their association with the symbiont bacteria, Xenorhabdus, and Photorhabdus, respectively (Burnell and Stock, 2000), and the third-stage infective juvenile nematodes (IJs) are proven to be the most effective as biological control organisms (Kaya and Gaugler, 1993).

In Mexico, the Universidad de Colima has conducted studies to determine the potential of exotic, and native EPNs to control insect pests, evaluating them in laboratory and field conditions against Mexican fruit flies, Anastrepha spp., fall armyworm, Spodoptera frugiperda (J. E. Smith), and agave weevil, Scyphophorus acupunctatus Gyllenhal. However, few years ago, a native steinernematid strain, Steinernema diaprepesi, was isolated in a survey from grasslands in Colima, Mexico, and evaluated against engorged cattle ticks, Rhipicephalus aBoophilus microplus Canestrini, and was found to cause significant adult tick mortality (Molina-Ochoa et al., 2009).

Cattle tick is one of the main ectoparasitic pests of bovine in tropical and subtropical regions around the world. Ticks are controlled commonly with chemical acaricides; however, ticks have developed resistance to most of these products (George et al., 2004). The EPNs, their infectivity to and susceptibility of female ticks have been previously reported in Israel (Samish and Glazer, 1992), Brazil (de Oliveira-Vasconcelos et al., 2004) and Mexico, recently (Molina-Ochoa et al., 2009; Rosales-Gutierrez, 2015). In an effort to obtain alternative controls for cattle ticks, we surveyed grasslands in Tecoman, Colima, Mexico, and a heterorhabditd isolate was recovered, and it was bioassayed against adult engorged ticks under laboratory conditions.

Materials and methods

Soil cores were collected from grasslands cultivated with a native Panicum sp. called "Zacaton", in the municipality of Tecoman, Colima, Mexico. Each soil sample, weighing approximately 1 Kg, was a composite of five random subsamples collected at least 100 m apart each site at a depth of 10-20 cm in an area of 20 m2. Soil samples were placed in polyethylene bags to avoid the loss of moisture and kept in coolers containing refrigerant gel packs during the transit to the laboratory and stored at 15AdegC in the Entomopathogenic Nematology Laboratory of the Universidad de Colima at Tecoman, Colima, Mexico.

Stored soil samples were processed within a 1 week of collection. To bait the soils for the recovery of EPNs each sample of 1 Kg was thoroughly mixed, and a ca. 240 cc was placed into a 250 CC plastic container. Then, five last instar larvae of the great wax moth (GWM), Galleria mellonella L. were placed in the soil sample, and the container was covered with a lid and inverted (Kaya and Stock, 1997). The container was held at room temperature (20+-3AdegC) for a period of 7-8 days.

GWM larvae were infected and killed by EPNs during the 15-day baiting period. Cadavers of GWM were recovered from the baited traps, disinfected with a solution of Sodium hypochlorite 1% during 3 min, rinsed with distilled water for 3 times. They were collected at 3-day intervals over 15 days after set-up and transferred to White traps to collect the emerging IJs (Kaya and Stock, 1997). IJs that emerged were pooled from each sample and were used to infect fresh GWM last instars; IJs were collected in 200 mL canted neck tissue culture flasks, and were stored in an aqueous suspension at low temperature (7-10AdegC) and used within 1 month of collection from the White traps to verify their pathogenicity and allow for progeny production for identification at the genera level, considering the characteristic color and other signs of the GWM cadavers infected with heterorhabditids (Woodring and Kaya, 1988; Kaya and Stock, 1997), as well as to carry out the bioassays.

Soil samples that were negative for EPN during the first round were baited again with GWM last instars; then, all the soil samples were maintained at room temperature. An isolate of heterorhabditid nematode was recovered from a sandy loam soil and designated as Heterorhabditis sp. JMO71.

Adult engorged ticks were manually collected from naturally infested cross bred cattle in the municipality of Tecoman, Colima. Ticks were deposited in plastic containers of 500 ml and then covered with a perforated lid. They were transported to the laboratory, disinfected with 0.1% sodium hypochlorite for 10 seconds and then rinsed three times with distilled water to remove the hypochlorite. Ticks were then placed in Petri plates 90 x 100 mm containing a double layer of filter paper (Whatman No. 1; Whatman International Ltd., England) moistened with distilled water and incubated at 25 +- 1AdegC and a 12 h light-12 h dark photoperiod, and 90% RH. Adult engorged ticks were not sexed.

We used the Petri dish bioassay procedure (Freitas-Rivero et al., 2005; Reis-Menini et al., 2008) to evaluate the susceptibility of adult engorged ticks to concentrations of 0, 500, 1000, 2000, 4000, a 8000 IJs of Heterorhabditis sp. JMO71. EPN concentrations were applied in 1 mL of sterile distilled water dispensed with a pipet on the surface of a couple of moistened filter papers (Whatman No. 1) in Petri dishes, only sterile distilled water (1 mL) was added to the controls. For each treatment concentration, 10 adult engorged ticks were placed in the Petri dishes (60 x 15 mm) and each treatment was replicated 4 times; and the experiment was replicated two times. The Petri dishes of each replication were subsequently placed in double plastic bags and incubated in the dark at room temperature (23 +- 2Adeg C) to avoid the desiccation. Tick mortality was recorded every 24 h for 9 d (de Oliveira-Vasconcelos et al., 2004).

Ticks were considered as dead by the absence of movement after stimulation, showing gummy body and reddish cuticle (Woodring and Kaya, 1988). Cadavers were incubated and later examined to verify the presence of EPN.

Analysis of variance was used for determining differences between treatments on tick mortality, and among different nematode concentrations and times post-exposure. Angular transformation was performed on the percent of mortality by square-root transformation before the analysis. A bifactorial arrangement (6 X 9; 54 treatments) of a completely random design was used; factor A was the nematode concentrations and factor B the exposure times. Means were separated by the Tukey test (P=0.05). Mean lethal concentration values (LC50), and mean survival time (ST50) were calculated by Probit analysis (Finney, 1971) using a software SAS version 7.12 (SAS, 1998).

Results and discussion

Current study effort was focused on isolating EPNs that may ubiquitous to grasslands in the municipality of Tecoman, Colima, Mexico. Ten soil samples were collected from grasslands. A heterorhabditid nematode isolate was recovered from 1 of the 10 soil samples with a prevalence of 10%. Heterorhabditis sp. JMO71 was highly pathogenic to engorged ticks, R. microplus in laboratory. Mortality rates increased with nematode concentration (r =0.478, P< 0.001) and time post-exposure (r = 0.501, P < 0.001), similar results were reported by Freitas-Rivero et al. (2005) with Steinernema carpocapsae, Molina-Ochoa et al. (2009) with S. diaprepesi, Monteiro et al. (2010) and (2012) with H. bacteriophora HP88 and Rodrigues et al. (2012) with H. indica against B. microplus. According to the authors mentioned above, longer exposure times allow greater number of nematodes to locate and penetrate the host, which apparently increase the effects on mortality of engorged ticks, B. microplus measured in this study.

Highest mortalities (80.6 % +- 34, 79.9% +- 28, and 75.4% +- 34) were obtained with the concentrations 2000, 8000 and 4000 IJ, respectively, after 9-d post-exposure (Supplementary Table SI), but concentration of 2000 IJ caused 100% mortality after 5 day post-exposure; the LC50 for the 5th day had 801.8, and 1364.4 IJs as lower and upper 95% confidence intervals (CI) (P < 0.001, Y = -0.55 + 0.0004 X) (Supplementary Table SII). The survival time for percent mortality of engorged ticks by the application of different concentrations of EPNs showed also that 2000 IJs had the lowest mean survival time (2.3 +- 0.768; 2.1, 2.4 lower, and upper CI; X2 = 404.2; P < 0.001; Y = 2.94 - 1.30 X) (Supplementary Table SIII), and needed less than three days for causing 100% mortality (Supplementary Table SI).

Molina-Ochoa et al. (2009) reported that cattle ticks are not specific hosts of S. diaprepesi, speculating that this is one reason for their low cumulative mortalities (40%) obtained; they mentioned also that the nematodes body length and width (1002 um, and 34 um, respectively) could reduce the opportunities to penetrate the natural openings of the engorged ticks. Comparing the Molina-Ochoa et al. (2009) results for S. diaprepesi (Colimense strain) with ours reported here, Heterorhabditis sp. isolate JMO71 caused almost 200% higher mortality with similar nematode concentrations.

We speculate that Heterorhabditis sp. isolate JMO71 caused higher mortalities after 9-d because: 1) Heterorhabditis species have smaller range of body lengths and body widths in the IJ (<800 um, and about 29-20 um, respectively) (Nguyen 2010); 2) when in contact with an insect host, the IJs exsheath the previously retained J2 cuticle and enter the hemocoel through the insect mouth, anus or spiracles or by penetrating the exoskeleton using a buccal 'tooth'-like structure (Bedding and Molyneux, 1981), Monteiro et al. (2010) reported the rupture of the tick cuticle due to the penetration of the IJs using this keratinous tooth; even when Samish et al. (2008) reported that penetration of ENs in ticks occurs primarily through the anal or genital pore, and 3) the cruiser foraging behavior could be another reason (Lewis et al., 1995).

The present study demonstrates that under laboratory conditions, B. microplus engorged ticks had high susceptibility to Heterorhabditis sp. JMO71, with mortality rates above those found with the use of other nematodes, such as H. bacteriophora CCA, Steinernema glaseri Santa Rosa, and S. carpocapsae Santa Rosa and All strains (Vasconcelos et al., 2004; Freitas-Rivero et al., 2005), but similar to those from Monteiro et al. (2010) and (2012) with H. bacteriophora HP88, and Rodrigues et al. (2012) with H. indica with 100% of control.

We speculate that the EPNs could be applied as a strategy of the Integrated Pest Management (IPM) in controlling adult cattle engorged ticks in the host animal interfering with the biological parameters of the non-parasite phase of engorged females as discussed by Vasconcelos et al. (2004), Freitas-Rivero et al. (2005) and Reis-Menini et al. (2008).

Morphometrical and molecular characterizations are the next steps for identifying this Heterorhabditis sp. (isolate JM071) to the level of species. We will also investigate the potential of Heterorhabditis sp. isolate JMO71 for control of other pests of veterinary importance such as the dog ticks, Rhipicephalus sanguineus (Acari: Ixodidae).


A native heterorhabditid nematode was recovered from a survey conducted in grasslands in Tecoman, Colima, Mexico. Adult engorged tick mortality rates were associated with nematode concentration and time exposure, and 100% mortality rate of engorged ticks was obtained with the concentration of 2000 IJs of Heterorhabditis sp. JMO71 after 5 day post-exposure.


Mention of a proprietary product does not constitute endorsement or recommendation for its use by the USDA. USDA is an equal opportunity provider and employer. The authors thank M. A. Jose Eduardo Hernandez Nava, Rector of the Universidad de Colima for supporting the research and senior author.

Supplementary material

There is supplementary material associated with this article. Access the material online at:

Statement of conflict of interest

Authors have declared no conflict of interest.


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Author:Molina-Ochoa, Jaime; Galindo-Velasco, Edelmira; Rosales-Gutierrez, Ana Maria; Gonzalez-Ramirez, Mart
Publication:Pakistan Journal of Zoology
Article Type:Report
Date:Apr 30, 2018
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