Parasitol Res (2004) 94: 201–206 DOI 10.1007/s00436-004-1178-5
O R I GI N A L P A P E R
Viviane de Oliveira Vasconcelos Æ John Furlong Glaucia Marques de Freitas Æ Claudia Dolinski Marineide Mendonc¸a Aguillera Regina Celia Devitte Rodrigues Æ Ma´rcia Prata
Steinernema glaseri Santa Rosa strain (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora CCA Strain (Rhabditida: Heterorhabditidae) as biological control agents of Boophilus microplus (Acari: Ixodidae) Received: 26 November 2003 / Accepted: 22 June 2004 / Published online: 1 September 2004 Springer-Verlag 2004
Abstract The present study was carried out to evaluate the action of Steinernema glaseri Santa Rosa strain and Heterorhabditis bacteriophora CCA strain as biological control agents of Boophilus microplus. Engorged females ticks were distributed on Petri dishes containing different concentrations of infective juvenile (IJ) nematodes (0, 375, 500, 750, 1,500, 2,500, 5,000 and 25,000). The data showed a reduction of approximately 90% in the eggs laid at a concentration of 5,000 S. glaseri IJs and approximately 80% at a concentration of 1,500 H. bacteriophora IJs. The female mortality increased linearly with the increase in S. glaseri concentrations. However, in the tests with H. bacteriophora this linearity was not observed. The effectiveness of the treatment with both species of entomopathogenic nematodes was compatible with other control methods. The results show the potential of S. glaseri and H. bacteriophora as biological control agents for the control of B. microplus under laboratory conditions.
V. de Oliveira Vasconcelos Æ G. Marques de Freitas Universidade Federal de Juiz de Fora, Campus Universita´rio, Martelos, 36033-330 Juiz de Fora, Minas Gerais, Brazil J. Furlong (&) Æ M. Prata Embrapa Gado de Leite, Rua Eugeˆnio do Nascimento, 610, Bairro Dom Bosco, 36038-330 Juiz de Fora, Minas Gerais, Brazil E-mail:
[email protected] Tel.: +55-32-32494886 Fax: +55-32-32494821 C. Dolinski Universidade Estadual do Norte Fluminense, CCTA/LPP, 28015-620, Campos dos Goytacazes, Rio de Janeiro, Brazil M. Mendonc¸a Aguillera Æ R. C. Devitte Rodrigues Universidade Federal de Sa˜o Carlos, CCA, 13600–970, Araras, Sa˜o Paulo, Brazil
Introduction Entomopathogenic nematodes (Rhabditida: Nematoda) are promising biological control agents due to their efficiency in controlling different insect pests (Doucet et al. 1999). During their life cycle, there is a free-living stage in the soil (J3) commonly called the infective juvenile (IJ) stage. An IJ can easily be distinguished by its additional cuticle, which originates from its previous stage (J2) and increases resistance to environmental variation, including low humidity and high temperature. The IJs invade hosts through natural openings (mouth, spiracles or anus) or directly through the cuticle. When reaching the haemocoel, they release symbiotic bacteria that rapidly multiply, killing the host within 24–48 h. When food becomes scarce in the host due to a high population of nematodes, multiplication stops and the second-stage juveniles molt but retain their cuticle, becoming the third stage with an additional cuticle. The third-stage juveniles, now IJs, retain symbiotic bacteria in the anterior part of their intestine . Entomopathogenic nematodes of the genus Heterorhabditis possess Photorhabdus as symbiotic bacteria, while Steinernema carry Xenorhabdus. Biological control has become a promising alternative for controlling ticks, since it minimizes the problems caused by chemical control and, moreover, solves the problem of acaricide resistance. Exposure of engorged Boophilus annulatus Say, 1821 females to an inoculum of 100 Steinernema carpocapsae IJs/tick or 150 Heterorhabditis bacteriophora IJs/tick resulted in 100% mortality after 4 days of infection. The LD50 values obtained against engorged females of B. annulatus are similar to nematode preparations against insects (Samish and Glazer 1991a, 1991b). Zhioua et al. (1995) showed that engorged Ixodes scapularis females Say, 1821 were susceptible to Steiner-
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nema glaseri and S. carpocapsae. Kocan et al. (1998b) tested the ability of S. feltiae and S. riobrave to enter and kill engorged females of different tick species, including Dermacentor variabilis Say, 1821, Rhipicephalus sanguineus Latreille, 1806, Amblyomma maculatum Koch, 1844, A. americanum Linneus, 1758, and A. cajennense Fabricius, 1787. These species were considered susceptible, since the nematodes caused 80% mortality. Based on Zhioua et al. (1995), S. glaseri and S. carpocapsae not only cause high mortality in engorged I. scapularis females but also affect the quality and quantity of eggs laid by the treated females. Kocan et al. (1998a) also made the same observation on the significant reduction of the egg mass weight of ticks submitted to different entomopathogenic nematode species. The level of susceptibility not only differs among tick species, but also varies between the developmental stages of the ticks, as the engorged females are more susceptible to nematode infection than other stages (Hill 1998; Kocan et al. 1998a; Samish et al. 1999, 2000; Kaaya et al. 2000; Glazer et al. 2001). This study was carried out with the objective of controlling the population of Boophilus microplus Canestrini, 1887 using the entomopathogenic nematodes S. glaseri strain Santa Rosa and H. bacteriophora strain CCA as biological control agents against engorged females.
Materials and methods The experiments were carried out at Laborato´rio de Acarologia, Embrapa Gado de Leite, Juiz de Fora —MG, from August 2001 to July 2002. For the experiments, engorged B. microplus females cepa Porto Alegre acaricide susceptible were used. The ticks came from a colony maintained in Coronel Pacheco Experimental Station, EMBRAPA Gado de Leite, MG. ‘‘In vivo’’ production of entomopathogenic nematodes IJs of S. glaseri strain Santa Rosa and H. bacteriophora strain CCA were reared ‘‘in vivo’’ using the last instar of Galleria mellonella L., 1758 larvae (Pyralidae: Lepidoptera).
females were added to the Petri dishes. The Petri dishes were sealed with parafilm and kept in germination chambers (26±1C and RH>80%). After 72 h in the germination chamber, the engorged females were taken from the sand dishes, washed with distilled water and transferred to Petri dishes without sand or nematodes. When oviposition was detected, each female was weighed and the value termed initial weight (IW). The eggs were removed daily, weighed and transferred to disposable plastic syringes previously prepared and covered with cotton. The plastic syringes were also maintained in germination chambers at 26±1C and RH>80%. After the oviposition period, the engorged females were weighed again and the residual weight (RW) was obtained. Other biological parameters were observed and calculated: pre-oviposition period (POP), egg mass weights (EW), oviposition period (OP), and larval hatching percentage (%H). 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 larval hatching percentage was verified daily from the first day of hatching to the last day through visual estimates at 5% intervals. The number of dead ticks was observed and recorded daily after 10 days of infection and the percentage mortality was calculated. The estimated reproduction (ER) and the percentage of control (%C) were calculated following Drummond et al. (1973): Estimated reproduction ¼
egg masses weightsðgÞ Initial engorged female weightsðgÞ hatching rate (%) 20000
Percentage of controlð% CÞ P P ER control group ER treated group P 100 ¼ ER control group The reproductive efficiency index (REI) and nutritional efficiency index (NEI) were calculated following Bennet (1974):
Laboratory bioassays of B. microplus exposed to entomopathogenic nematodes
REI ¼
EWðgÞ 100 IWðgÞ
The concentrations of 375, 500, 750, 1,500, 2,500, 5,000 and 25,000 IJs of each nematode species were suspended in 4 ml distilled water and added to 5-cm-diameter Petri dishes with 15 g autoclaved sand. Five engorged females were added to each Petri dish, with four replicates, giving a total of 20 engorged females per treatment. The untreated group (control group) was submitted to the same climatic conditions as the treated groups, although only 4 ml distilled water without nematodes and 20 engorged
NEI ¼
EWðgÞ 100 IWðgÞ RWðgÞ
Statistical analysis The data were analysed using the non-parametric Kruskall-Wallis test and Dunn’s test with P £ 0.05 being considered significant.
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The v2 test was also used to test the linear response related to the increase of IJ concentration treatments and mortality. The values obtained in percentage were transformed into 2arcsin x, for further analysis. The program PROBIT was used to calculate the lethal concentration for 50% and 90% of engorged females (LC50 and LC90, respectively).
Results and discussion Pre-oviposition and oviposition periods There were no significant differences between the results for the control group and the treated group with respect to the pre-oviposition period of engorged B. microplus females when exposed to various concentrations of S. glaseri or H. bacteriophora IJs (H=1.564, P=0.9551 and H=1.884, P=0.9660, respectively; Tables 1, 2). This suggests that the entomopathogenic nematode infections did not interfere with the metabolic conversion process or production of the necessary nutrients for manufacturing eggs during the pre-oviposition period. A similar result was observed for the oviposition period of engorged females exposed to S. glaseri and H. bacteriophora IJs (H=4.699, P=0.5829 and H=3.489, P=0.384, respectively; Tables 1, 2). The values which we obtained are similar to those cited by Gloria et al. (1993) and Santos and Furlong (2002).
Egg mass weight
Samish and Glazer (1992) showed that infection by IJs of the S. carpocapsae DT strain did not interfere with the average weight of egg masses for engorged B. annulatus females. They suggested that the short exposure period of the ticks to the IJs (i.e. 24 h) could have been a factor in their result. Kocan et al. (1998b) demonstrated that infection with S. glaseri IJs did not interfere with the fecundity of A. americanum engorged females but that different concentrations of S. riobrave and S. feltiae IJs reduced their egg mass weights. Thus, different strains or species of IJs may have different influences on the fecundity of engorged females of different tick species. Larval hatching percentage IJs of either nematode species do not appear to interfere with the larval hatching of B. microplus. The larval hatching percentage showed no significant differences for various IJ concentrations of either species and ranged from 85% to 90% when the ticks were treated with S. glaseri IJs (H=2.044, P=0.9156), and from 82% to 91.3% when treated with H. bacteriophora IJs (H=09.332, P=0.2297). Similar results were found in studies of B. microplus by Snowball (1957) and Glo´ria et al. (1993). Reproductive efficiency index and nutritional efficiency index
The infection by S. glaseri IJs interfered with the oviposition of treated engorged B. microplus females with respect to the weight of the egg mass. This was lower for engorged females in the treated group (1,500 and 2,500 IJs) than in the untreated group (H=19.449, P=0.0035; Table 1). In contrast, for females infected by H. bacteriophora IJs, there were no significant differences between the treated and untreated groups for any concentration of IJs tested (H=3.506, P=0.8346).
The REI for the conversion of blood to egg mass for engorged females exposed to different concentrations of S. glaseri IJs ranged from 35.2%±15.93 to 45.4%±3.43, whereas 48.1%±6.20 was converted to egg mass in the control group, a difference which was not significant. For the NEI, 50.9%±12.28 to 63.4%±17.01 of the ingested food was consumed in physiological activities such as breathing and moving in the treated groups, whereas 65.26±9.03 was consumed in the untreated group, a difference which was also not significantly different (Tables 1, 2).
Table 1 Kruskall-Wallis test between the average values for the biological parameters of Boophilus microplus engorged females that survived at different concentrations of Steinernema glaseri Santa Rosa strain IJs (POP pre-ovoposition period, OP ovoposition period, EW egg mass weight (mg), %H hatching percentage, REI
reproductive efficiency index, NEI nutritional efficiency index). Values with same letters in the same column do not differ statistically. Mean values±SD are given. The numbers in the brackets are the numbers of surviving engorged females for which the biological parameters could be determined
Number of IJs/ Petri dish (n) Control (20) 375 (14) 500 (13) 750 (10) 1,500 (13) 2,500 (6) 5,000 (2) 25,000 (0)
Biological parameters POP (days)
OP (days)
EW
H%
REI
NEI
3.20a±0.41 3.07a±0.46 3.20a±0.41 3.16a±0.32 3.29a±0.47 3.25a±0.46 3.00a±0.00 –
12.50a±1.05 11.60a±2.20 11.40a±2.72 11.60a±2.12 10.82a±2.72 10.75a±2.91 10.50a±3.54 –
112a±016 100ab±032 100ab±024 080ab±037 084bc±025 078bc±020 086ac±004 –
86.50a±07.45 85.00a±09.44 86.00a±08.49 88.50a±12.48 88.53a±09.80 86.86a±11.00 90.00a±14.14 –
48.09a±06.20 41.55a±12.97 44.43a±10.56 35.21a±15.93 37.58a±11.22 35.44a±10.31 45.43a±03.43 –
65.26a±09.03 56.52a±16.17 63.41a±17.01 55.02a±19.68 56.30a±11.88 50.92a±12.28 62.32a±03.08 –
204 Table 2 Kruskall-Wallis test between the average values for the biological parameters of B. microplus engorged females that survived to different concentrations of Heterorhabditis bacteriophora CCA strain IJs (POP pre-ovoposition period, OP ovopostion period, EW egg mass weight (mg), H% hatching percentage, REI Number of IJs/ Petri dish (n) Control (20) 375 (13) 500 (14) 750 (13) 1,500 (4) 2,500 (9) 5,000 (11) 25,000 (10)
reproductive efficiency index, NEI nutritional efficiency index). Values with same letters in the same column do not differ statistically. Mean±SD are given. Numbers in the brackets are the numbers of surviving engorged females for which the biological parameters could be observed
Biological parameters POP (days)
OP (days)
EW
H%
REI
NEI
3.20a±0.41 3.13a±0.35 3.06a±0.44 3.15a±0.37 3.00a±0.00 3.11a±0.33 3.18a±0.40 3.20a±0.42
13.30a±1.78 11.80a±3.36 11.88a±3.62 13.46a±1.64 13.67a±0.58 13.44a±1.51 13.00a±2.19 13.00a±1.83
136a±039 121a±059 138a±049 134a±044 146a±036 124a±036 131a±029 142a±028
82.25a±10.19 91.33a±06.40 86.18a±11.25 87.69a±08.32 87.50a±15.00 88.88a±09.29 90.00a±12.04 82.00a±14.94
50.22a±14.01 47.10a±20.84 51.63a±18.46 49.12a±16.06 56.26a±13.25 49.00a±14.21 47.50a±10.72 51.80a±09.78
68.01a±14.44 63.00a±25.46 64.34a±21.40 68.04a±17.33 75.66a±13.60 63.37a±18.19 70.81a±10.78 66.94a±15.95
The REI and NEI results for infections with H. bacteriophora IJs were similar to those for S. glaseri IJs. In this case, 47.1%±20.84 to 56.26%±13.25 of the ingested blood of females from the treated group was converted into egg masses (REI), whereas 50.22%±14 was converted in the untreated group, a difference which was not significantly different (Tables 1, 2). For NEI, the percentage of the blood used in metabolic activities was 63%±25.46 to 75.66%±13.6 in the treated group and 68.01±14.44 in the control group, a difference which was not significantly different (Tables 1 and 2). These results show that the conversion process of the ingested blood in egg masses was similarly efficient for engorged females infected by either S. glaseri or H. bacteriophora IJs. The values found for REI and NEI in the present study are similar but lower than those reported by Bennett (1974), who found 58% for REI and 77.1% for NEI. These differences may be related to the strain of ticks used in each experiment, since our results are similar to the values presented by Santos and Furlong (2002), who used the same tick strains. Overall, the potential of the nematode-bacteria complex as a biological acaricide is indicated by the reduction of egg mass weight for female B. microplus. In treatments with high concentrations of S. glaseri IJs (e.g. 2,500 IJs), the percentage reduction in the total weight of egg masses reached 70% due to the high mortality found at these concentrations (Fig. 1). In contrast, a correlation between the concentrations of H. bacteriophora IJs and the percentage reduction in the weight of egg masses was found, but there was a reduction of 50–80% for some concentrations (Fig. 1). Ultimately, if similar results can be obtained under field conditions then nematode applications could have an important impact on tick populations by reducing egg viability.
S. glaseri IJs, mortality increased with IJ concentration (v2=5.819, df=6, P=0.40,) and 90% of B. microplus engorged females were killed within 3 days post-infection. In infections with H. bacteriophora IJs, mortality reached 80% within 2–3 days post-infection with concentrations of 1,500 IJs, but a linear increase in mortality with an increasing concentration of IJs was not observed (v2=18.560, df=6, P=0.005) (Table 3). Applications of entomopathogenic nematodes could be effective for controlling ticks in pastures because engorged females are quickly killed before beginning oviposition. Maule´on et al. (1993) observed that B. microplus and A. variegatum engorged females were resistant to 17 different strains of entomopathogenic nematodes from the genera Steinernema and Heterorhabditis. Overall, there can be considerable variation in infectivity when different strains of nematodes are used against different tick species (Samish et al. 1999). The LC50 and LC90 values of 874 and 1,930, respectively, for H. bacteriophora IJs per Petri dish were low compared to the values for S. glaseri IJs (i.e. 1,250 and 4,160 IJs/Petri dish, respectively). A high mortality index using low nematode concentrations is indicative of the high susceptibility of a specific tick species to a nematode species (Samish et al. 1999). The high susceptibility
Percentage mortality of B. microplus engorged females In this study, engorged female B. microplus were susceptible to both the S. glaseri Santa Rosa strain and the H. bacteriophora CCA strain. In treatments with
Fig. 1 Percentage reduction in Boophilus microplus egg mass total weight when females were treated with different concentrations of Steinernema glaseri Santa Rosa strain and Heterorhabditis bacteriophora CCA strain infective juveniles
205 Table 3 Mortality of B. microplus engorged females infected with different IJ concentrations of S. glaseri and H. bacteriophora calculated 10 days post-inoculation. Twenty engorged females were used for each concentration.T indicates the number of days between infection and the death of engorged females
Infective juveniles/ S. glaseri H. bacteriophora Petri dish Number of dead ticks T (mean±SD) Number of dead ticks T (mean±SD) and percentage and percentage Control 375 500 750 1,500 2,500 5,000 25,000
0 06 07 10 07 14 18 20
(30) (35) (50) (35) (65) (90) (100)
– 3.83±2.56 4.43±3.21 2.78±0.44 6.71±3.50 2.69±1.38 3.00±1.76 2.65±0.50
0 07 06 07 16 11 09 10
(35) (30) (35) (80) (55) (45) (50)
– 4.00±2.77 4.71±2.81 2.57±0.53 2.31±0.47 2.64±0.50 2.33±0.50 2.40±0.52
of B. microplus engorged females to H. bacteriophora IJs might be explained by the presence of a tooth on the anterior end of the IJs which facilitates penetration and infection. Dead engorged female B. microplus were easily differentiated from ticks in the control group by the change in their natural colour to an intense red. This change in colour is typical of heterorhabditid infections and is caused by the presence and multiplication of symbiotic bacteria (Samish and Glazer 1992; Glazer and Samish 1993; Zhioua et al. 1995). Some ticks inoculated with H. bacteriophora at a concentration of 25,000 IJs per Petri dish showed cuticle rupture and haemolymph leakage, which could have been caused by the high population of juveniles in the host. Hill (1998) observed similar ruptures in approximately 60% of H. bacteriophora infected ticks and attributed this to the perforation of membranes during host penetration.
respectively). These results show that the efficiency of entomopathogenic nematodes is comparable to other biological control agents. For example, the entomopathogenic fungi Bauveria bassiana is highly efficient in controlling B. microplus engorged females (above 80%) and an increase in mortality with increased concentrations is observed (Bittencourt et al. 1997). In chemical control, the acaricide group of organophosphates had an efficiency above 90% in controlling B. microplus but the development of resistance to these acaricides is becoming very common (Leite et al. 1995). In this study, it was shown that the entomopathogenic nematodes S. glaseri Santa Rosa strain and H. bacteriophora CCA strain can be efficient in controlling B. microplus engorged females, especially in relation to the high mortality of engorged females and the decrease of egg masses on pastures. More studies are necessary to show the potential of these nematodes under field conditions.
Treatment efficiency using different concentrations of S. glaseri and H. bacteriophora infective juveniles
Acknowledgements The authors thank Embrapa Gado de Leite (Empresa Brasileira de Pesquisa e Abastecimento) and Universidade Federal de Juiz de Fora, Minas Gerais for technical assistance and Dr. Richard Ian Samuels and Dr. Robin Stuart for reviewing the manuscript. This work was supported by CAPES.
In this study, concentrations of 2,500, 5,000 and 25,000 IJs of S. glaseri per Petri dish induced mortality above 70% (Fig. 2). Mortality of 80% was achieved with concentrations of 1,500 H. bacteriophora IJs per Petri dish (Fig. 2). A linear increase in mortality with increased IJ concentration (i.e. a dose response) was not detected in the treatments with either S. glaseri or H. bacteriophora IJs (v2=49.92, P=0.0001 and v2=70.24, P=0.0001,
Fig. 2 Percentage efficiency of different concentrations of S. glaseri Santa Rosa strain and H. bacteriophora CCA strain infective juveniles against B. microplus engorged females
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