Parasitol Res (2009) 104:12031206 DOI 10.

1007/s00436-008-1313-9

ORIGINAL PAPER

Evaluation of the efficacy of strains of Steinernema carpocapsae Santa Rosa and ALL (Steinernematidae: Rhabditida) to control engorged female Anocentor nitens (Acari: Ixodidae)
G. M. Freitas-Ribeiro & V. O. Vasconcelos & J. Furlong & C. Dolinski

Received: 26 March 2008 / Accepted: 6 December 2008 / Published online: 3 January 2009 # Springer-Verlag 2008

Abstract In view of the need to combat the generalized spread of resistance in ticks to commercial acaricides, the objective of this study was to evaluate the action of entomopathogenic nematodes (Steinernema carpocapsae, strains Santa Rosa and ALL) on engorged female Anocentor nitens. Five ticks per Petri dish were exposed to concentrations of 500, 5,000, or 25,000 infective juveniles of S. carpocapsae for 72 h. After transferring the ticks to clean plates, biological parameters were analyzed. Related to strains Santa Rosa, the period of pre-oviposition (p = 0.0001), oviposition (p =0.041), and the mass weight of eggs (p =0.005) showed significant differences between the

control group and treated group. When the strain ALL was tested, the control and treated groups differed between the periods of pre-oviposition (p =0.001), oviposition (p =0.001), and egg mass weight (p =0.01). The egg mass conversion was less significant in the groups when exposed to strains Santa Rosa (p =0.002) and ALL (p =0.001) relative to the control. The efficacy of both entomopathogenic nematode strains used in this study was comparable to other biological control agents, showing their potential against A. nitens in the laboratory.

Introduction
G. M. Freitas-Ribeiro Departamento de Biologia Geral, Ps-graduao em Biologia Celular e Estrutural, Universidade Federal de Viosa (UFV), Campus Universitrio, CEP: 36570-000 Viosa, MG, Brazil V. O. Vasconcelos (*) Departamento de Parasitologia, Instituto de Cincias Biolgicas (ICB), Universidade Federal de Minas Gerais (UFMG), Av. Presidente Antnio Carlos, 6627, Campus Pampulha, CEP: 31270-910 Belo Horizonte, MG, Brazil e-mail: vivianeleo@hotmail.com J. Furlong Embrapa Gado de Leite, Rua Eugnio do Nascimento, 610, Bairro Dom Bosco, CEP: 36038-330 Juiz de Fora, MG, Brazil C. Dolinski Universidade Estadual do Norte Fluminense, Av. Alberto Lamego, 2000, Parque Califrnia, CEP: 28013-620 Campos dos Goytacazes, RJ, Brazil

The tick Anocentor nitens (Neumann) Schulze 1937 is an ectoparasite found principally on the auditory meatus of equids (Arago and Fonseca 1961). Ticks suck blood and irritate the animals, causing dermatitis and creating portals for secondary infections and myiases. They are also vectors of Babesia caballi, the etiological agent of equine pyroplasmosis, a disease that limits the performance of racehorses and restricts international trade in these animals, which has increased considerably in recent years (Pfeifer 1995). The principal control method against this ectoparasite has been the use of acaricides, but the generalized spread of resistance in tick populations, together with toxic reactions in animals and man due to the presence of chemical residues in the environment, has led to the search for safer, nonchemical alternatives. There are many studies on the biological control of ticks using their natural predators such as ants, spiders, birds, and so forth (Sutherst et al. 2000). In Brazil, the Empresa Brasileira de Pesquisa Agropecuria (EMBRAPA), with other research institutions, has been high-

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lighted as a leading company in biological control (Pacheco and Corra-Ferreira 2000; vila and Melhorana 1999; Castro et al. 2001). Brum and Teixeira (1992) had highly efficient results in the control of Boophilus microplus engorged females using the bacteria Cedecea lapagei and Escherichia coli and the entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae (Bittencourt et al. 1997) under laboratory conditions. Entomopathogenic nematodes have become increasingly important in biological control programs as microbial agents, supplementing or replacing the use of chemical insecticides against agricultural pests (Acevedo and Lpez 2002). The juvenile infective (IJ) stages of nematodes of the genus Steinernema harbor symbiotic bacteria of the genus Xenorhabdus (Burnell and Stock 2000; Forst and Clarke 2002). When these juveniles penetrate the natural orifices of the host (mouth, spiracles, and anus) or directly cross the cuticle and reach the hemocoel, bacteria are liberated, multiply rapidly, and kill the host by septicemia within 24 48 h (Adams and Nguyen 2002). The effectiveness of entomopathogenic nematodes as biological control agents has already been demonstrated against several tick species, including B. microplus where a 90% reduction was observed in oviposition when exposed to IJ of S. glaseri strain Santa Rosa and 80% when exposed to IJ of Heterorhabditis bacteriophora strain ALL (Vasconcelos et al. 2004; Freitas-Ribeiro et al. 2005). A 100% death rate was observed in engorged females of B. annulatus when exposed to concentrations of 1,000 and 5,000 IJ of S. carpocapsae (Samish and Glazer 1991a, b). Highly efficient results were obtained with other species of ticks such as Ixodes scapulari, Dermacentor variabilis, Rhipicephalus sanguineus, Amblyomma maculatum, A. americanum, and A. cajennense, showing that the nematodes are not species-specific (Maulon et al. 1993; Zhioua et al. 1995; Kocan et al. 1998). The objective of the present study was to evaluate the action of the entomopathogenic nematodes Steinernema carpocapsae strains Santa Rosa and ALL on engorged females of the tick species, Anocentor nitens.

sand (Zhioua et al. 1995). The IJ doses applied in the tests were similar to those used in previous studies (Vasconcelos et al. 2004; Freitas-Ribeiro et al. 2005), thus giving continuity to the line of research conducted at EMBRAPA. Entomopathogenic nematodes are found in soil and thus sand was used as a substrate in order to simulate the environmental conditions favorable to the survival of these parasites. The use of sand substrate can also facilitate movement during foraging because it increases the mechanical contact between nematodes and sand. Five engorged female A. nitens were added to each of six Petri dishes, giving a total of 30 engorged females per treatment. Control group ticks were submitted to the same conditions as those of infected groups, but received only 4 mL distilled water. The size of the sample was limited by the availability of ticks (maximum quantity of similar weight females collected in the same day). For the experiments, engorged acaricide-susceptible A. nitens females were used. The ticks were obtained from Coronel Pacheco Experimental Station, EMBRAPA Gado de Leite, Mina Gerais, Brazil and removed directly from more than one host. Petri dishes containing ticks were stored in incubation chambers (261C and RH>80%). After 72 h, engorged females were removed and transferred to empty plates (without nematodes and sand) and replaced in the incubator. Before the initiation of oviposition, female ticks were weighed to obtain their initial weights (IW). The egg mass of each female was weighed (EW) and transferred to a plastic syringe previously prepared and stoppered with cotton wool. The syringe was stored in an incubation chamber at 261C and RH>80%. The biological parameters evaluated were pre-oviposition period (POP), oviposition period (OP), and egg mass weight (EW). The pre-oviposition period embraced the days between collecting the engorged females from the Petri dishes with sand until the beginning of oviposition. The oviposition period consisted of the number of days between the beginning of oviposition and the day when the last egg was laid. The reproductive efficiency index (REI) was calculated following Bennett (1974). Statistical analysis

Materials and methods Experiments were performed in the parasitology laboratory of EMBRAPA in Juiz de Fora, Minas Gerais, Brazil. Nematodes of S. carpocapsae strains Santa Rosa and ALL were cultured in the laboratory by successive passages in larvae of Galleria mellonella (Lindegren et al. 1993). Concentrations of 500, 5,000, and 25,000 (IJ) were suspended in 4 mL distilled water and added to 5 cm diameter Petri dishes containing 15 g of autoclaved, moist Analysis of variance (ANOVA) and the TukeyKramer test at p 0.05 were used for each parameter to confirm the existence of significant differences between experimental groups exposed to different concentrations of entomopathogenic nematodes. Parameters in which the difference between standard deviations was highly significant based on Bartletts test (indicating non-normal sample distribution) were analyzed using the KruskalWallis and Dunn nonparametric tests at

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p <0.05, rather than ANOVA and the TukeyKramer test, respectively.

Results and discussion Related to the Santa Rosa strain, the pre-oviposition period (p =0.0001, F =21.169, df =3), oviposition (p =0.041, F = 2.981, df =3), and mass weight of eggs (p =0.005, F =4.665, df =3) showed significant differences between the groups. When the ALL strain was tested, the control and treatment groups also differed between the pre-oviposition period (p =0.001, F =24.673, df =2), oviposition period (p =0.001, F =20.118, df =2), and egg mass weight (p =0.01, F =6.746, df =3) (Table 1). In this study, a longer pre-oviposition period was observed in A. nintes exposed to high concentration of S. carpocapsae Santa Rosa and ALL. Freitas-Ribeiro et al. (2005) also found a longer pre-oviposition period in B. microplus exposed to S. carpocapsae Santa Rosa (5.40 days) compared to controls (3.03 days). However, these results differ from the findings of Samish and Glazer (1992) in which female B. annulatus that survived exposure to the S. carpocapsae strain DT began laying eggs about the same time as those in the control group. The infection by the S. carpocapsae strain ALL interfered with the oviposition of treatment of engorged A. nitens females with respect to the egg mass weight. The egg

mass weight was lower for engorged females in the treatment group (5,000 and 25,000 IJ) than in the control group. Zhioua et al. (1995) found that egg mass weight of engorged females of I. scapularis was lower than the control group when exposed to S. carpocapsae ALL but no difference was observed between treatment groups (different concentrations of nematodes). Vasconcelos et al. (2004) observed that when B. microplus was exposed to S. glaseri strain Santa Rosa, a reduction occurred in the egg mass weight compared to the control groups of ticks. However, this reduction was not observed when engorged females of B. microplus were exposed to IJ of H. bacteriophora strain CCA. The authors suggested that different strains or species of entomopathogenic nematodes can interfere in the fecundity of engorged females of different species of ticks. Reduction in egg mass as a result of nematode penetration is relevant because it affects the number of ticks in the next generation (Kaaya et al. 2000). With respect to the REI, 26.5525.67% and 19.37 24.06% of ingested blood was converted into eggs by ticks exposed to concentrations of 500 and 25,000 IJ of S. carpocapsae Santa Rosa, respectively, significantly lower than the values obtained for unexposed ticks (56.90 7.043%; p =0.002, F =7.839, df =3). Engorged females, exposed to 5,000 and 25,000 IJ of S. carpocapsae ALL, converted into eggs 18.1426.88% and 6.3319.40%, respectively, of the blood they ingested (significantly lower than the control group values p =0.001, F =12.598, df =3).

Table 1 Mean values of biological parameters in engorged female A. nitens exposed to different concentrations of S. carpocapsae strains Santa Rosa and ALL and maintained in the laboratory under controlled conditions (261C and RH>80%) Treatment Concentration of Initial weight of POP (days) infective juveniles/plate engorged female (IW) 0 500 5,000 25,000 S. carpocapsae ALL 500 5,000 25,000 222.6075.96a 15 242.1366.97a 15 244.9374.77a 15 236.4771.71a 15 221.3364.70a 15 217.3354.06a 15 221.0755.38a 15 4.930.26b 15 6.300.67ab 10 6.200.42ac 10 7.001.13ac 12 5.460.66bc 13 7.000.63a 6 6.000.00* 2 OP (days) EW (mg) REI (%)

Control S. carpocapsae Santa Rosa

14.600.74a 15 12.903.57a 10 14.000.00a 10 11.254.98ab 12 13.460.52a 13 8.334.93b 6 2.500.71* 2

127.9353.90a 15 56.2059.79abc 15 91.3377.59ab 15 53.9356.09abc 15 90.8763.90ab 15 43.1371.18bc 15 1.876.70c 15

56.907.43a 15 26.5525.67bcd 15 35.4926.29abd 15 19.3724.95bcd 15 41.0724.06ab 15 18.1426.88bcd 15 6.3319.40d 15

The numbers below the biological parameters refer to the meanstandard deviation and sample size (n). Different letters in the same column represent a significant difference (p <0.05) POP pre-oviposition period, OP oviposition period, EW egg mass weight, REI reproductive efficiency index *Statistical analysis not carried out due to reduced sample size

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Parasitol Res (2009) 104:12031206 Bittencourt VRCP (1997) Avaliao da eficcia in vitro de dois isolados do fungo entomopatognico Beauveria bassiana (Bals.) em fmeas ingurgitadas de Boophilus microplus (Canestrini, 1887). Rev Bras Parasitol Vet 6:4252 Brum JGW, Teixeira MO (1992) Acaricidal activity of Cedecea lapagei on engorged females of Boophilus microplus exposed to environment. Arq Bras Med Vet Zootec 44:543544 Burnell AM, Stock P (2000) Heterorhabditis, Steinernema and their bacterial symbiontslethal pathogens of insects. Nematology 2:3142 Castro MEB, Ribeiro ZMA, Souza ML (2001) Estudo de Especificidade do Vrus da Lagarta da Soja (AgMNPV) em Cultura de Clulas. Ruralnet-Trabalhos Cientficos 20 nov Forst S, Clarke D (2002) Bacterianematode symbiosis. In: Gaugler R (ed) Entomopathogenic nematology. CABI, New York, pp 5777 Freitas-Ribeiro GM, Furlong J, Vasconcelos VO, Dolinski C, LouresRibeiro A (2005) Analysis of biological parameters of Boophilus microplus Canestrini, 1887 exposed to entomopathogenic nematodes Steinernema carpocapsae Santa Rosa and All Cepas (Steinernema: Rhabditida). Braz Arch Biol Technol 48:911919 Kaaya GP, Samish M, Glazer I (2000) Laboratory evaluation of pathogenicity of entomogenous nematodes to African ticks species. Ann N Y Acad Sci 916:303308 Kocan KM, Pidherney MS, Blouin EF, Claypool PL, Samish M, Glazer I (1998) Entomopathogenic nematodes as a potential biological control method of ticks. Ann N Y Acad Sci 9:355364 Lindegren JE, Valero KA, Mackey BE (1993) Simple in vivo production and storage methods for Steinernema carpocapsae infective juvenile. J Nematol 25:193197 Maulon H, Barr N, Panoma S (1993) Pathogenicity of 17 isolates of entomophagous nematodes (Steinernematidae and Heterorhabditidae) for the ticks Amblyomma variegatum (Fabricius), Boophilus microplus (Canestrini) and Boophilus annulatus (Say). Exp Appl Acarol 17:831838 Pacheco DJP, Corra-Ferreira BS (2000) Parasitismo de Telenomus podisi Ashmead (Hymenoptera: Scelionidae) em populaes de percevejos pragas da soja. An Soc Entomol Bras 29:295302 Pfeifer BI (1995) Epidemiological aspects of equine babesioses in a herd of horses in Brazil. Vet Parasitol 58:18 Samish M, Glazer I (1991a) Pathogenicity of parasitic nematodes to ticks. Modern acarology, vol. 2. Academia, Prague, pp 629632 Samish M, Glaze I (1991b) Killing ticks with parasitic nematodes of insects. J Invertebr Pathol 58:281282 Samish M, Glazer I (1992) Infectivity of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) to female ticks of Boophilus annulatus (Acari: Ixodidae). Entomol Soc Am 29:614618 Samish M, Glazer I (2001) Entomopathogenic nematodes for the biocontrol of ticks. Trends Parasitol 17:368371 Schulze P (1937) Anocentos columbianus. Zool Anzeiger 120(1-2): 2427 Sutherst RW, Wilson LJ, Cook IM (2000) Predation of the cattle tick, Boophilus microplus (Canestrini) (Acarina: Ixodidae), in three pastures Australian. Aust J Entomol 39:7077 Vasconcelos VO, Furlong J, Freitas GM, Dolinski C, Mendona AM, Devitte RCR, Prata M (2004) Steinernema glaseri Santa Rosa cepa (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora CCA Cepa (Rhabditida: Heterorhabditldae) as biological control agents of Boophilus microplus (Acari: Ixodidae). Parasitol Res 94:201206 Zhioua E, Brun RA, Ginsberg HS, Aeschlimann A (1995) Pathogenicity of Steinernema carpocapsae and Steinernema glaseri (Nematoda: Steinernematidae) to Ixodes scapularis (Acari: Ixodidae). J Med Entomol 32:900905

These results indicate that conversion of ingested blood by ticks into eggs was affected by the presence of nematodes of both strains used in the experiments, in contrast to the findings of Vasconcelos et al. (2004) in the same conditions of temperature and humidity. In the present study, contact time between nematodes and ticks was 3 days, producing a reduction in the oviposition period, egg mass weight, and reproductive rate. However, a high mortality rate was observed when exposed to 5,000 IJ of S. carpocapsae strain ALL only with 7 days postinfection. Freitas-Ribeiro et al. (2005) used the same nematode strains and exposure times, noting alterations in all biological parameters observed for engorged females of B. microplus. Samish and Glazer (1992) observed that engorged females of B. annulatus, when exposed to 1,000 IJ of S. carpocapsae strain DT, mortality reached 100% within 2 days postinfection. Engorged females of B. microplus were susceptible to both the S. glaseri Santa Rosa and the H. bacteriophora CCA, as mortality reached 90% and 80% with 3 days postinfection, respectively (Vasconcelos et al. 2004). Applications of entomopathogenic nematodes could be effective for controlling ticks in pastures because engorged females are quickly killed before oviposition begins. Our findings demonstrate the efficacy of entomopathogenic nematodes as biocontrol agents, based on the reduced egg mass weights and egg production rates of ticks exposed to different IJ concentrations. As with other biocontrol agents such as bacteria, fungi, viruses, and insects, nematodes are demonstrated to be as efficient as chemical products (Brum and Teixeira 1992; Samish and Glazer 2001).

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