Efficacy of collars with allomones on dogs to control Rhipicephalus sanguineus sensu lato infestations under field conditions.

Benzaldehyde and 2-hexanone are allomones produced by beagle dogs that reduce infestation by Rhipicephalus sanguineus sensu lato on these animals and on susceptible dogs which artificially release these repellents. These observations were obtained in previous laboratory tests or artificial infestations of susceptible dogs under controlled conditions. Here we evaluated the efficacy of collars delivering these repellents for suppressing the loads of R. sanguineus s.l. on naturally infested mixed-breed dogs under field conditions. Thirty dogs naturally infested with R. sanguineus s.l. were separated into two groups with 15 dogs each. The dogs from the treatment group received the collars with slow-release formulations of the allomones, and the dogs from the control group received collars without such compounds. Collar effectiveness tests were carried out over 30 days. All ticks found were removed from each dog, identified, and counted every collection day. The density of the different life stages of R. sanguineus s.l. varied greatly between groups and across evaluation times. Adult ticks were the stage most abundant and prevalent on infested dogs. The counts for larvae, nymphs and adults were similar between the control and the treatment groups within each tick collection day. Conversely, when we considered the total number of ticks over 30 days and gathered all life stages collected in the same animal, the dogs from the control group had significantly higher loads (35.3 ± 4.27) of ticks than the dogs from the treatment group (21.8 ± 2.96) (P < 0.01). In addition, the efficacy of treatment with repellent collars was high for adult ticks (30.8%) but was extremely low for reducing larval or nymphal infestation (0 or 2.6%, respectively). The present study demonstrates, for the first time, the efficacy of these volatile compounds on naturally infested dogs under field conditions. The development of a technologically enhanced device for slow release of the allomones tested here can be an advantageous alternative for reducing R. sanguineus s.l. infestation on dogs in residential environments.

Conidia and blastospores of Metarhizium spp. and Beauveria bassiana s.l.: Their development during the infection process and virulence against the tick Rhipicephalus microplus.

The current study compared the virulence of conidia and blastospores of Metarhizium robertsii (IP 146), M. anisopliae sensu lato (s.l.) (IP 363) and Beauveria bassiana s.l. (IP 361 and CG 307) against unfed larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae). In addition, the development of fungal propagules on tick cuticle was investigated. Tick larvae were treated with fungal suspensions at 106, 107 or 108 propagules mL-1, and percent mortality was assessed every two days. Engorged females were immersed in fungal suspensions (1.0 × 107 propagules mL-1) for 1 min, and their biological parameters monitored daily. The virulence of conidia and blastospores against larvae varied considerably among the isolates tested. Only females treated with blastospores of IP 146 or IP 361 presented lower nutrient and egg production indices than the control group; the higher percent control was reached when females were treated with blastospores of IP 146 (98%), IP 363 (79%), or IP 361 (93%) in comparison to the groups treated with conidia, 71%, 59%, or 63%, respectively. Engorged females treated with conidia or blastospores of IP 146 or IP 361 were also examined by scanning electron microscopy (SEM). Germination of blastospores of IP 146 and IP 361 was observed on tick cuticle after 4 h incubation at 27 ±â€¯1 °C and RH > 90%, whereas germ tubes from conidia of both isolates were observed at 48 h. Appressoria in developing blastospores of B. bassiana IP 361 were observed after 4 h incubation, whereas no appressoria were seen in developing blastospores of M. robertsii IP 146. Blastospore penetrations by both fungal isolates through natural openings was also evidenced by SEM; fine sections of R. microplus engorged females treated with blastospores of IP 146 or IP 361 confirmed that these isolates penetrated through their cuticle and natural openings. Blastospores might be promising for use in biocontrol of ticks, since they are virulent against R. microplus and present rapid development on their cuticle.

Heat-stressed Metarhizium anisopliae: viability (in vitro) and virulence (in vivo) assessments against the tick Rhipicephalus sanguineus.

The current study investigated the thermotolerance of Metarhizium anisopliae s.l. conidia from the commercial products Metarril® SP Organic and Metarril® WP. The efficacy of these M. anisopliae formulations against the tick Rhipicephalus sanguineus s.l. was studied in laboratory under optimum or heat-stress conditions. The products were prepared in water [Tween® 80, 0.01 % (v/v)] or pure mineral oil. Conidia from Metarril® SP Organic suspended in water presented markedly delayed germination after heating to constant 40 °C (for 2, 4, or 6 h) compared to conidia suspended in mineral oil. Metarril® SP Organic suspended in oil and exposed to daily cycles of heat-stress (40 °C for 4 h and 25 °C for 19 h for 5 consecutive days) presented relative germination of conidia ranging from 92.8 to 87.2 % from day 1 to day 5, respectively. Conversely, germination of conidia prepared in water ranged from 79.3 to 39.1 % from day 1 to day 5, respectively. Culturability of Metarril® WP decreased from 96 % when conidia were cultured for 30 min prior to heat exposure (40 °C for 4 h) to 9 % when conidia were cultured for 8 h. Tick percent control was distinctly higher when engorged females were treated with oil suspensions rather than water suspensions, even when treated ticks were exposed to heat-stress regimen. Oil-based applications protected fungal conidia against heat-stress. Although Metarril® is not registered for tick control, it may be useful for controlling R. sanguineus, especially if it is prepared in mineral oil.

Effect of heat stress and oil formulation on conidial germination of Metarhizium anisopliae s.s. on tick cuticle and artificial medium.

The effect of heat stress (45°C) versus non-heat stress (27°C) on germination of Metarhizium anisopliae sensu stricto (s.s.) isolate IP 119 was examined with conidia formulated (suspended) in pure mineral oil or in water (Tween 80, 0.01%), and then applied onto the cuticle of Rhipicephalus sanguineus sensu lato (s.l.) engorged females or onto culture medium (PDAY). In addition, bioassays were performed to investigate the effect of conidia heated while formulated in oil, then applied to blood-engorged adult R. sanguineus females. Conidia suspended in water then exposed to 45°C, in comparison to conidia formulated in mineral oil and exposed to the same temperature, germinated less and more slowly when incubated on either PDAY medium or tick cuticle. Also, conidial germination on tick cuticle was delayed in comparison to germination on artificial culture medium; for example, germination was 13% on tick cuticle 72h after inoculation, in contrast to 61.5% on PDAY medium. Unheated (27°C) conidia suspended in either water or oil and applied to tick cuticle developed appressoria 36h after treatment; whereas only heat-stressed conidia formulated in oil developed appressoria on tick cuticle. In comparison to conidia heated in mineral oil, there was a strong negative effect of heat on germination of conidia heated in water before being applied to arthropod cuticle. Nevertheless, bioassays [based primarily on egg production (quantity) and egg hatchability] exhibited high percentages of tick control regardless of the type of conidial suspension; i.e., water- or oil-formulated conidia, and whether or not conidia were previously exposed to heat. In comparison to aqueous conidial preparations, however, conidia formulated in oil reduced egg hatchability irrespective of heat or no-heat exposure. In conclusion, mineral-oil formulation protected conidia against heat-induced delay of both germination and appressorium production when applied to the cuticle of R. sanguineus.