Infectivity of Entomopathogenic Nematodes (Steinemematidae and Heterorhabditidae) to Female Ticks of Boophilus annulatus (Arachnida: Ixodidae) MICHAEL SAMISHi AND ITAMAR GLAZER2

J. Med. Entomol. 29(4): 614-618 (1992)

KEY WORDS Arachnida, Boophilus annulatus, tick, entomopathogenic nematodes

HARD TICKS (Acarina: Ixodidae) are economically important pests in practically all parts of the world, mainly as vectors of different animal and human diseases. Fully engorged females of the one-host tick Boophilus annulatus (Say) drop off the host in search of a hiding place and lay their eggs in the upper layer of the soil. Emerging larvae search for a new host, where they remain until they become fully engorged adults. This tick species is established in Mexico and Central America as well as in large sectors of Africa, the Near East, and Russia (Graham & Price 1966). The ticks cause major damage to the cattle industry, mostly as vectors of different diseases, mainly babesiosis. Tick damage is currently reduced mainly by spraying or dipping of animals with chemical acaricides. With increased reports on growing resistance of ticks to acaricides (Solomon 1983) and the intensifying interest in substitutes for acaricides, the potential use of biological control agents is being studied, but as yet without marked success. Several nonspecific predators, parasitic wasps, and some pathogens of ticks have been described (Cole 1965, Wilkinson 1970, Lipa 1971, Arthur 1973, Doube & Heath 1975, Rechav 1981, Bowman et al. 1986, Geevarghese 1988, Mwangi 1989, Barre et al. 1990). However, cur1

Kimron Veterinary Institute, Bet Dagan 50250, Israel. Department of Nematology, A.R.O., The Volcani Center, Bet Dagan 50250, Israel. 2

rently they are not used commercially against ticks. Among measures for the biological control of insect pests, the facultatively parasitic rhabditoids from the families Steinemematidae and Heterorhabditidae were especially promising as bioinsecticides (Georgis 1990a,b). The only freeliving stage of these entomopathogenic nematodes is the third-stage infective juvenile. Under natural conditions, this stage persists primarily in the upper layer of the soil, where it can survive for months until it finds a new host (Kaya 1985, Akhurst 1986). The infective juvenile finds its insect host by a chemotactic response (Gaugler et al. 1980). Once a suitable host has been found, it is invaded by the infective juvenile, which releases symbiotic bacteria into the hemolymph. The bacteria proliferate and kill the host within 24-48 h (Poinar 1986). Steinernematids and heterorhabditids generally are considered to be specific to insects. Numerous insect species, including many with economic importance, are susceptible to these nematodes (Poinar 1979). The nematodes are most effective as biological control agents against insects that inhabit the soil, which is the natural environment of the nematodes (Kaya 1990). In contrast, little is known about the susceptibility of other invertebrates to nematodes. Some representatives of the classes Gastropoda, Symphyla, Collembola, Curutacea, Diplopoda, and even Arachnida can be killed under laboratory

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ABSTRACT Exposing Boophilus annulatus (Say) to different concentrations of Steinernema carpocapsae (Filipjev) infective juveniles in petri dishes (50-10,000 nematodes per dish) resulted in high mortality (>90%) at nematode concentrations as low as 500 nematodes per dish within 8 d. At a concentration of 10,000 nematodes per dish, 100% of the ticks died within 2 d after infestation. After exposure to 500 nematodes per dish, complete mortality was achieved with the Heterorhabditis bacteriophora strain 'HP88' within 4 d. During the same period, only 15 and 40% mortality were recorded with the 'Mexican' and 'All' strains of S. carpocapsae, respectively. In a lethal dose analysis, S. carpocapsae strain 'DT" was the most infective strain with the lowest LD 50 and LDgo values (15 and 165 infective juveniles per tick, respectively). The 'All' strain of S. carpocapsae was the least infective of the four strains tested, with L D ^ and L D ^ values of 372 and 9,251 infective juveniles/tick, respectively. Optimal temperature for tick control by the nematodes was between 22 and 26°C. Mortality rate was reduced at 18 and 30°C. The susceptibility of fully engorged ticks was not influenced by the weight of the replete females. Nematode infection did not have an adverse effect on egg laying by surviving ticks.

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Materials and Methods Three strains of Steinernema and one strain of Heterorhabditis were used in the current study: Steinernema carpocapsae (Filipjev) (=Neoaplectana carpocapsae Weiser) strains 'Mexican', 'All', and 'DT' (the 'DT' strain is a subpopulation of the 'All' strain that was selected for enhancement of desiccation tolerance [I.G., unpublished data]); and the H. bacteriophora strain 'HP88'. The nematodes were reared on artificial media according to the method of Bedding (1981). An isolate of B. annulatus (Say, 1821) from Ramat HaGolan has been maintained in the laboratory during the past 6 yr. The colony was fed every 2 mo on healthy Friesian calves (1^3 mo old). To determine the effect of different nematode concentrations on the mortality rate of B. annulatus females, infective juveniles from the various strains listed above were suspended at various concentrations in deionized water. A 0.5-ml volume of each nematode suspension was applied to four 5-cm-diameter petri dishes padded with filter paper (Whatman No. 1). The petri dishes contained various concentrations of infective juveniles (i.e., 50, 200, 500, 1,000, or 10,000 per dish). In the control, only water was applied. Five fully engorged B. annulatus-replete females that had dropped off during the last 24 h were placed in each dish. Each treatment was replicated with four dishes. The dishes were incubated in the dark at 26°C. Tick mortality was determined daily. The experiment was repeated twice. The infectivity of the nematode strains was evaluated by comparing the LD 5 0 and LD 9 0 values, which were derived from the mortality data after exposure of the ticks for 4 d. The data were subjected to probit analysis using the SAS statistical package (SAS Institute 1982). The LD 5 0 and LD 9 0 values were not considered to be significant whenever the ranges overlapped. The effect of temperature on nematode activity against ticks was determined by incubating fully engorged females of B. annulatus together with 2,000 infective juveniles of S. carpocapsae strain 'DT' in each dish at 14, 18, 22, 26, or 30°C for 18

2 4 6 Days post infestation Fig. 1. Effect of different numbers of S. carpocapsae strain 'DT' infective juveniles per petri dish on the mortality rate of B. annulatus females, during 8 d of exposure at 26°C.

d. Tick mortality was recorded 2, 4, 7, 14, and 18 d after inoculation. Control treatment consisted of ticks placed in nematode-free dishes that contained filter paper and 0.5 ml of water. Each treatment consisted of four replicates (five ticks per dish per temperature). To observe the effect of nematodes on egg laying of single females, the ticks were placed on trays (8.5 by 17 cm) padded with filter paper. The trays were preinoculated with the DT strain of S. carpocapsae at concentrations of 4,16, 64, or 256 infective juveniles per cm2. In the control treatment, only water was applied to each tray. Two trays were used for each treatment. A cardboard divider was placed to divide the tray surface into 40 squares (one for each tick). Each female was weighed on an analytical balance with a 0.1-mg accuracy before it was placed in its square. After 1 d of incubation, the ticks were rinsed thoroughly with tap water and transferred individually into a 1.5-ml well in a multiwell perspex plate (24 wells per plate). Tick mortality was recorded daily, as was the date of onset of egg laying for each female tick. Egg masses were removed and weighed 20 d after female repletion. Results The mortality rate of B. annulatus-replete females was proportional to the amount of nematodes to which they were exposed (Fig. 1). At a concentration of 10,000 infective juveniles per dish, 100% of tick mortality was recorded within 2 d after infestation. Ninety-five percent mortality was reached also with concentrations as low as 500 infective juveniles/dish, but in this case all ticks died after only 8 d. At concentrations of

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conditions by steinernematid and heterorhabditid nematodes (Poinar & Thomas 1985; Poinar 1988, 1989). However, ticks were found to be resistant to some entomopathogenic rhabditids (Barre et al. 1990). Recently, Samish & Glazer (1991) found that the hard tick B. annulatus was readily killed under laboratory conditions by steinernematid nematodes. The aim of the current study was to elucidate the host—parasite interactions between various steinernematid and heterorhabditid nematodes and their non-insect host B. annulatus-replete females.

B. annulatus

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Stroins o SC 'Mexican A SC 'All' A HB'HP88'

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Fig. 3. Effect of temperature (°C) on the mortality rate of B. annulatus females infested with S. carpocapsae strain 'DT' (2,000 infective juveniles per dish).

incubation together with the nematodes. At 30°C the rate of tick mortality was slower than at 26°C 200 or 50 infective juveniles per dish, 100% mor- and complete mortality was recorded only 10 d tality was not observed even after 8 d (Fig. 1). after infestation. Female ticks kept at 14, 18, 22, The rate of tick mortality was affected by the 26, and 30°C started to lay eggs >22,18, 7,5, and nematode strain (Fig. 1 and 2). After exposure to 2 d after repletion, respectively. 500 infective juveniles, complete mortality was The susceptibility of B. annulatus-replete feachieved with H. bacteriophora strain 'HP88' males according to their weight and effect of (Fig. 2) within 6 d. High mortality (90%) was re- nematode infestation on egg laying of surviving corded also after 4 d of exposure among tick fe- females is presented in Table 2. A 24-h exposure males infested with S. carpocapsae strain 'DT' to the infective juveniles resulted in partial mor(Fig. 1). During the same period, only 15 and 40% tality that was proportional to the increase in mortality were recorded with the 'Mexican' and nematode concentrations. The average weight of 'All' strains of S. carpocapsae, respectively (Fig. fully engorged ticks that died later (232.3 ± 51.5 2). mg [x ± SD]) did not differ significantly (P > Differences in nematode infectivity also are 0.05) from the weight of ticks that survived the seen in the results obtained from lethal dose (LD) nematode infestation (229.6 ± 45.9 mg). Ticks analysis (Table 1). S. carpocapsae strain 'DT' was that survived the exposure to nematodes started the most infective strain, with the lowest LD 5 0 to lay eggs on the average 3.4 d after repletion, and LD 9 0 values, which differed significantly whereas nonexposed ticks in the control started from all other nematode strains. The LD 5 0 and to lay eggs 3.3 d after repletion (Table 2). The LD 9 0 values of the H. bacteriophora strain 'HP88' egg mass from females that survived exposure to were significantly lower than those of the 'All' nematodes weighed (on average) 111.7 mg; and strain of S. carpocapsae. The latter strain was the from nonexposed females, 125.7 mg. (Of 40 feleast infective of the four strains tested. males used for each replicate, one to three laid The effect of various temperatures on nema- black and dry eggs. These females and eggs were tode activity against tick females is shown in Fig. not included in the data analysis.) 3. The most rapid mortality occurred at 26 and 22°C (100% mortality within 4 and 6 d, respecDiscussion tively). Complete mortality at 18°C was achieved after 18 d, which is 4.5 times slower than at 26°C. Poinar & Thomas (1985) showed in laboratory At 14°C, only 85% of the ticks died after 18 d of trails that S. carpocapsae and H. heliothidis (=H. Table 1. Infectivity of different entomopathogenic nematode strains to engorged females of the tick B. annulatus (number of nematodes per tick) Strain

LD 50

Range

LDgo

Range

Slope

S. carpocapsae (DT) S. carpocapsae (All) S. carpocapsae (Mexican) H. bacteriophora (HP88)

15a 372c 124bc 81b

7- 29 188-573 92-285 53-146

165a 9,251c 5,486bc 394b

125- 257 6,352-12,511 3,919-10,649 191- 623

1.35 0.93 1.10 1.25

Data were calculated following exposure of ticks to six concentrations of nematodes (0, 10, 40, 100, 200, and 2,000 nematodes per tick) for 4 d. Within columns, values followed by the same letter did not differ significantly (t test, P = 0.05, df = 28).

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8 3 4 5 6 7 Days post infestation Fig. 2. Effect of different entomopathogenic nematode strains on the mortality rate of B. annulatus females, after exposure to 500 infective juveniles per dish for 8 d at 26°C.

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Table 2. Effect of the nematode 5. carpocapsae strain 'DT' on surviving females of B. annulatus Nematode concn, infective juveniles per cm2 0 4 16 64 256 Average

Beginning of egg laying (d post-repletion) 3.3 ± 0.7 3.1 ±0.5 3.8 ± 0.7 3.7 ± 0.7 3.5 ± 1.4 3.5 ± 0.8

Avg. wt of engorged female, mg Survived 2.5 7.5 60.0 72.5 85.0

234.0 ± 48.7 218.0 ± 51.7 244.0 ± 67.3 242.0 ± 68.7 222.0 ± 22.1 232.0 ±51.5

Died 216.2 dt28.4 231.0 dt42.4 240.0 it57.4 232.0 db51.2 229.0 dt45.1 229.6 it44.9

Avg. eggs (mg per female) 125.7 ± 29.8 103.8 ± 33.1 123.4 ± 45.3 113.3 ± 36.2 114.7 ± 10.3 115.6 ± 30.9

bacteriophora) are infective against aerial and ground spiders (Arachnida). Whereas thousands of steinernematid and heterorhabditid infective juveniles were needed to kill these spiders, the data show that as few as 100 infective juveniles per female in a petri dish produced >90% mortality of B. annulatus ticks. The results demonstrate that engorged B. annulatus females are highly susceptible to infection by steinernematid and heterorhabditid nematodes compared with other noninsect hosts from the same class and similar to results obtained with susceptible insects (Poinar 1986). The 'DT strain of S. carpocapsae was the most pathogenic, with the lowest LD50, of the four strains tested, whereas the 'All' strain of the same species was the least infective. Because the 'DT' population was derived from the 'All' population after repeated selection cycles at low relative humidity (I.G., unpublished results), the significant differences in infectivity between the 'DT' and 'All' strains to B. annulatus could be attributed to some trait that was selected unintentionally. The nature of this trait is still obscure. Among the other strains tested (Fig. 2), the most rapid mortality of B. annulatus was obtained with infective juveniles of H. bacteriophora strain 'HP88'. Thisfindingmight be attributed to the ability of heterorhabditid infective juveniles to penetrate through soft cuticle and thin membranes with the help of a cuticular tooth in their head region (Poinar & Georgis 1990). The optimal temperature for insecticidal activity of steinernematid and heterorhabditid nematodes is between 18 and 28°C (Georgis 1990a). Therefore, the current findings on the lower rate of tick mortality at 14°C and the inhibition of tick mortality at 30°C (Fig. 3) can be attributed to the exposure of nematodes to extreme temperature conditions. However, B. annulatus mainly inhabits zones with warm and temperate climates in nature. Furthermore, the optimal temperature for rearing this tick in the laboratory is