Toxicological effects and resistance to pyrethroids in Boophilus microplus from Goiás, Brazil

Efeitos toxicológicos e resistência a piretróides em Boophilus microplus de Goiás

Abstracts

Estudaram-se os efeitos de cipermetrina, deltametrina e permetrina sobre larvas de uma cepa de campo de Goiânia, com o objetivo de monitorar a susceptibilidade de Boophilus microplus para esses acaricidas e fomentar medidas de controle. Larvas em jejum com 14 a 21 dias, imersas em soluções desses piretróides, foram mantidas a 27±1°C e UR% > ou = 80% e observadas por 24h ao estereoscópio, contidas em dispositivo desenvolvido originalmente para estudos da fase não parasitária do ciclo evolutivo. O material utilizado em sua confecção, placa de petri descartável, tecido organza e parafina, não foi tóxico para as larvas. Os seguintes efeitos toxicológicos foram observados: excitabilidade, movimentação repetitiva, diminuição da capacidade locomotora, desprendimento, paralisia, knock-down e proliferação cuticular de líquidos e gases. A mortalidade média na 24ªh foi de 76,3%, 87,5%, 77,6%, 91,2%, 86,20%, e 100,0%, respectivamente, para deltametrina 25ppm e 50ppm, cipermetrina 150ppm e 300ppm e permetrina 1250ppm e 2500ppm. O ixodídeo foi resistente às concentrações comerciais de deltametrina e cipermetrina. Apenas permetrina 2500ppm provocou índice de mortalidade superior ao valor recomendado pelo Ministério da Agricultura para acaricidas.

Ixodidae; Boophilus microplus; controle de carrapatos; piretróide; resistência a acaricida

Toxicological effects and resistance to pyrethroids in Boophilus microplus from Goiás, Brazil

[Efeitos toxicológicos e resistência a piretróides em Boophilus microplus de Goiás]

F.F. Fernandes

Instituto de Patologia Tropical e Saúde Pública da Universidade Federal de Goiás

Caixa Postal 131

74605-050 - Goiânia, GO

Recebido para publicação em 5 de setembro de 2000.

Recebido para publicação, após modificações, em 28 de março de 2001

E-mail: [e-mail]

ABSTRACT

In order to monitor the susceptibility of Boophilus microplus to acaricides and improve control measures, the effects of cypermethrin, deltamethrin and permethrin on larvae obtained in the city of Goiânia in the Brazilian state of Goiás were studied. Although these pyrethroids are already sold as acaricides, their cost-benefit efficiency has been questioned. Fasting 14-21 day-old larvae were immersed in solutions of the acaricides under test, maintained at 27±1° C, and relative humidity over 80%, and observed under the stereoscope within an apparatus originally designed for studying the non-parasitic phase of the tick life cycle. The observed toxicological effects were: excitability, repetitive motion, decreased motor ability, detachment, paralysis, knock-down and cuticular proliferation of liquids and gases. The materials used in the manufacture of this apparatus consisted of disposable Petri dishes, "organza" cloth and paraffin, none of which are toxic to tick larvae. Mean death rates after 24h were 76.3%, 87.5%, 77.6%, 91.2%, 86.2% and 100% for 25 and 50ppm deltamethrin, 150 and 300ppm cypermethrin and 1250 and 2500ppm permethrin, respectively. The ticks were resistant to commercial concentrations of deltamethrin and cypermethrin. Only 2500ppm permethrin produced the mortality recommended by the Brazilian Ministry of Agriculture.

Keywords: Ixodidae, Boophilus microplus, tick control, pyrethroid, acaricide resistance.

RESUMO

Estudaram-se os efeitos de cipermetrina, deltametrina e permetrina sobre larvas de uma cepa de campo de Goiânia, com o objetivo de monitorar a susceptibilidade de Boophilus microplus para esses acaricidas e fomentar medidas de controle. Larvas em jejum com 14 a 21 dias, imersas em soluções desses piretróides, foram mantidas a 27±1°C e UR% ³80% e observadas por 24h ao estereoscópio, contidas em dispositivo desenvolvido originalmente para estudos da fase não parasitária do ciclo evolutivo. O material utilizado em sua confecção, placa de petri descartável, tecido organza e parafina, não foi tóxico para as larvas. Os seguintes efeitos toxicológicos foram observados: excitabilidade, movimentação repetitiva, diminuição da capacidade locomotora, desprendimento, paralisia, knock-down e proliferação cuticular de líquidos e gases. A mortalidade média na 24^ah foi de 76,3%, 87,5%, 77,6%, 91,2%, 86,20%, e 100,0%, respectivamente, para deltametrina 25ppm e 50ppm, cipermetrina 150ppm e 300ppm e permetrina 1250ppm e 2500ppm. O ixodídeo foi resistente às concentrações comerciais de deltametrina e cipermetrina. Apenas permetrina 2500ppm provocou índice de mortalidade superior ao valor recomendado pelo Ministério da Agricultura para acaricidas.

Palavras-chave: Ixodidae, Boophilus microplus, controle de carrapatos, piretróide, resistência a acaricida

INTRODUCTION

Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae) is the most economically important tick species between the latitudes 30° N and 30^oS, including Brazil where it is one of the principal causes of low productivity in cattle. Originally from Asia, it colonized most tropical and sub-tropical countries via infestations of imported cattle (Warton, 1974). Blood diseases, sting stress, allergic reactions to tick saliva, hide damage, toxicosis, metabolic alterations, infection with Babesia, Anaplasma and Wad Medani virus, anorexia and death are all consequences of B. microplus parasitism (Brossard & Wikel, 1997). In addition to the losses caused by these ailments, an estimated one billion dollars is spent on B. microplus control in Brazil each year, including the cost of labor and chemical acaricides (Brasil, 1985). These have been the principal control methods worldwide since this species was introduced in Australia by the end of the last century (Martins et al., 1995). B. microplus quickly developed resistance to the acaricides and in some countries, this has involved several chemical groups, including arsenicals, organochlorines, organophosphates, mixed base products, amidines and pyrethroids (Leite, 1988; Leite et al., 1995). Monitoring of susceptibility and/or resistance of B. microplus to acaricides is essential to improve control measures, avoid the use of inefficient products, decrease costs and minimize the environmental impact.

The aim of this study was to determine in vitro larvicidal activity and toxicological effects of different concentrations of the pyrethroids deltamethrin and cypermethrin on B. microplus from Goiânia in Brazil.

MATERIALS AND METHODS

Sampling was carried out on cattle farms in the municipality of Goiânia in Goiás state, Brazil. Ticks were collected from naturally infected cattle of undefined breed that had not been treated with acaricides for at least 45 days prior to the study. They were placed in glass flasks and transported to the laboratory, where they were washed in distilled water and dried with paper towels. The largest individuals, which moved more quickly and did not present mutilations or malformations, were selected under the stereoscope and transferred into individual polyethylene egg-laying tubes containing moistened cotton wool attached to the inner surface of the screw cap. All eggs laid by the ticks on a particular day were removed with a stainless-steel spatula and pooled in a single egg-hatching tube, which was identified, sealed and stored in an incubator.

The acaricides were diluted in distilled water and tested at the following concentrations: 25 and 50ppm deltamethrin, 150 and 300ppm cypermethrin and 1,250 and 2,500ppm permethrin. The solutions were homogenized by shaking and poured into a white-based Petri dish that permitted larvae to be seen clearly at the moment of immersion. The hatching tube with the highest eclosion rate (90 - 100%) was selected and placed in a 1000ml beaker, sitting on a dish of aluminium foil that was half-filled with water to act as a moat and prevent ticks form escaping. The hatching tube cap was quickly perforated using a flamed pointer and the larvae stimulated to initiate questing behavior by heat or the CO₂ (Stange & Stowe, 1999) of the researcher’s breath. They were then collected immediately using a white n° 4 paint brush and immersed for 2 min in the acaricide solutions (Fernandes, 2000).

A holding apparatus was used that permitted observation of the larvae throughout the experiment. Pyrethroids were selected for testing because of their low mammalian toxicity and rapid degradation in the environment, their proven effectiveness in the control of arthropods of medical and veterinary importance (Escuret & Scheid, 1993; Fernandes & Silva, 1999) and their extensive use by cattle ranchers in the study area, which may have been responsible for reports of reduced efficacy.

Larvae were held in an experimental apparatus developed by Fernandes (1997) in order to maintain ixodid tick colonies in studies of the free-living phase of their life cycle. After immersion, the larvae were placed close to each other on a 12cm² square of white synthetic "organza" (» organzine cloth) of mesh size 2590/cm², placed on top of a sheet of paper attached to the bench. The larvae were then covered with the base of a disposable Petri dish (9cm diameter x 1.4cm height) that was fixed in place by slightly pressing it onto the substrate. Fused paraffin was brushed along the outer edge of the Petri dish base and once solidified it fixed the "organza" to the paper and sealed the apparatus. The surplus material was then cut away, leaving a border of 0.6cm around the base. Paraffin was fused by heating in a stainless steel container, kept on a hot plate. The apparatus border was reinforced by brushing more paraffin along the perimeter.

A total of 1735 fasting larvae were used in this study. Ten units of the apparatus were made for each concentration with a minimum of 20 larvae in each. Larvae of the control group were immersed into water. The test group was composed of 1470 larvae and the control group of 265 larvae, with means of 24.5 to 26.5 larvae per unit, respectively. After treatment all larvae were maintained in a BOD chamber at 27 ± 1° C, with RH ³80%. Acaricide action and tick mortality was monitored by observing the units under the stereoscope at 6, 12 and 24h after treatment (Fernandes, 2000). All larvae used in the bioassays were 14-21-day old. Mortality was calculated by assuming all immobile larvae to be dead. Mean percentage mortality was calculated and adjusted by Abbot's formula. Replicates in which control mortality exceeded 5% were not considered (FAO, 1995). Larval mortality rates at different concentrations were compared using the c² test at the 95% significance level and the efficiency of the treatments were interpreted according to WHO guidelines (1970), values ³80% indicating susceptibility and <80% resistance to an acaricide; or those of the Brazilian Ministry of Agriculture, which stipulates 95% as the minimum mortality obtained for an acaricide concentration to be recommended for use against ixodids (Brasil, 1990).

RESULTS

Results are shown on Table 1. The mean percentage mortalities of the test group at 24h according to FAO (1995) standards, were 76.3%, 87.5%, 77.6%, 91.2%, 86.2% and 100.0%, for 25 and 50ppm deltamethrin, 150 and 300ppm cypermethrin and 1250 and 2500ppm permethrin, respectively. Statistically significant differences (P=0.05) were found between the mean mortality values of the two concentrations of deltamethrin (c² = 10.60 > c²_.05(1) = 3.84), cypermethrin (c² = 16.22 > c²_{.05 (1)} = 3.84) and permethrin (c² = 32.93 > c²_{.05 (1)} = 3.84) after 24h. Mortality values at different times were also significantly different (c² = 47.67 > c²_{.05 (1)} = 3.84).

The apparatus used (Fernandes, 1997) held the larvae safely during the observation period and was non-toxic them (no mortality in control group). The paraffin used to seal the apparatus did not trap the larvae, while the "organza" mesh maintained the conditions of humidity, oxygen levels and temperature at similar levels to those of outside. The sequence of toxicological effects, observed under the stereomicroscope, did not differ among the pyrethroids and consisted of excitation, repetitive movements, progressive decrease of locomotory capacity, tremblings, prostration, stretching of the pods resulting in detachment, locomotion capacity recovered, although sometimes only of the palps, paralysis, leading to death (killing effect) or recovery (knock-down effect) (Hervé, 1983). The knock-down effect was observed in this study at 12h after exposure in 1.8%, 1.2%, 0.9% and 1.0% of the larvae submitted to 25 and 50ppm deltamethrin, 150ppm cypermethrin and 1250ppm permethrin respectively. On the other hand, 2.4% of the surviving larvae showed proliferation of liquid and gases inside their bodies, producing dilation of small areas of cuticle areas on the proterosome, histerosome or pods which subsequently led to progressive compromising of the idiosome and death.

DISCUSSION

B. microplus was found to be resistant to both deltamethrin and cypermethrin at commercially used concentrations (WHO, 1970). The tick was however susceptible to permethrin at 2500ppm, the mortality rates being higher than the minimum levels officially recommended for acaricides by the Ministry of Agriculture (Brasil, 1990). Ticks were susceptible to double the commercial concentrations of deltamethrin and cypermethrin, as well as to 1250ppm permethrin, although the mortality rates produced were below the levels recommended for control. Toxicological assays should however be carried out on cattle prior to recommendation of these high concentrations.

Carneiro et al. (1985), working in the dairy farm region of Goiânia, observed that either deltamethrin or flumethrin produced the highest mortality of B. microplus teleogens in vitro. Later, decreased cypermethrin efficacy on ticks from the same region was reported by Sobrinho et al. (1997). Fernandes (2000) discovered Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) resistance to deltamethrin in the urban area of Goiânia. The present study demonstrated for the first time resistance of B. microplus to deltamethrin and cypermethrin at the commercial concentrations recommended for use on cattle in this area. These results corroborate local farmers observations of reduced cost-benefit efficiency of these products recommended for tick control. It is believed that this phenomenon will spread to other parts of Goiás state, requiring increased monitoring of B. microplus susceptibility levels.

Researchers working in other Brazilian states have noted the loss of activity of pyrethroid acaricides to B. microplus by means of in vitro larval and teleogen tests. In Bahia, Almeida et al. (1994) reported deltamethrin and cypermethrin to be ineffective at concentrations up to four times higher than those commercially recommended. In Pernambuco inefficacy at the commercially recommended dosage (Pena et al., 1994), was shown for deltamethrin and later also for cypermethrin (Faustino et al., 1996). In Minas Gerais, deltamethrin exposure resulted in low tick mortality (Leite et al., 1995) while in Rio Grande do Sul strains of B. microplus were found to be resistant to all pyrethroids used in the region (Martins et al., 1995).

Resistance of this tick to pyrethroids has been reported in other countries including USA (He et al., 1999), Mexico (Redondo et al., 1999), Colombia (Benavides et al., 1997), New Caledonia (Beugnet & Chardonnet, 1995), and Australia (Jonsson et al., 2000). In contrast to the verified in R. sanguineus (Fernandes, 2000) and B. microplus from Goiânia municipality, resistance to permethrin was detected in Dermanyssus gallinae (Degeer, 1778) (Acari: Dermanyssidae), the red poultry mite, in France (Beugnet et al., 1997).

The characteristic knock-down effect of pyrethroids on arthropods, which may be misinterpreted as death caused by reversible inhibition of movement (Hervé, 1983), was observed for B. microplus in the present study and also by Fernandes (2000) and Fernandes et al. (2001) in R. sanguineus larvae exposed to the same compounds. Since knock-down may persist until 24h after exposure mortality may sometimes have to be assessed a day or more after exposure. However in the present study the effect was not seen in ticks later than 12h after exposure. The sealed holding apparatus ensured that living larvae could be easily distinguished from dead ones that might be moved inadvertently by the breath of the observer. Receptacles used in previous studies, including filter paper envelopes (FAO, 1995) or even tea-bags (Gladney & Dawkins, 1976), did not allow the sequence of toxicological effects to be observed, since larvae could only be viewed at the end of the experiment, when the apparatus was opened. Glass test tubes (Sobrinho et al., 1997) and disposable syringes (Leite, 1988) allow restricted viewing but cannot be fitted easily under the stereoscope, and do not permit individual larvae to be distinguished. The apparatus used in the present study was transparent and fitted under the lens of the stereomicroscope, permitting the larvae to be observed clearly.

The increasing loss of effectiveness of acaricides on B. microplus shows the necessity of continual monitoring of susceptibility levels and rational application, together with the use of alternative control measures with lower environmental impact. These include: methods based on detailed knowledge of the habitat, life cycle and population dynamics of the tick, vaccines (Redondo et al., 1999), genetic improvement by crossing different breeds of cattle breeds to increase natural resistance (Furlong, 1993), phyto-sanitation (Tucci et al., 1998), and use of entomopathogenic microorganisms (Bittencourt et al., 1996; Kaaya et al., 1996).

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Publication Dates

• Publication in this collection
  18 June 2002
• Date of issue
  Oct 2001

History

• Reviewed
  28 Mar 2001
• Received
  05 Sept 2000