Microsclerotial pellets of Metarhizium spp.: thermotolerance and bioefficacy against the cattle tick.

The cattle tick, Rhipicephalus microplus (Acari: Ixodidae), is a multi-billion dollar ectoparasite of global importance affecting beef and milk production. Submerged cultures of cosmopolitan entomopathogenic fungal species of the genus Metarhizium typically produce microsclerotia that provide both long-term survival and environmental resistance. Microsclerotia hold great potential as an unconventional active propagule to control this tick under laboratory and semi-field conditions. However, heat stress caused especially by elevated temperatures poses a critical environmental constraint for the successful development and efficacy of microsclerotia under tropical conditions. First, we screened six strains of Metarhizium anisopliae, Metarhizium robertsii and Metarhizium humberi for their ability to produce microsclerotia by submerged liquid cultivation. In addition, we assessed the biological fitness and bioefficacy of dried microsclerotial pellets under amenable (27 °C) and heat-stressed (32 °C) incubation against engorged adult females of R. microplus. Microsclerotia in pelletized formulation prepared with carriers based on diatomaceous earth and microcrystalline cellulose exhibited conidial production at different extents according to the fungal strain and the incubation temperature, but most strains displayed reduced sporogenesis when exposed to 32 °C. Engorged tick females exposed to sporulated microsclerotia from pelletized M. anisopliae CG47 or IP 119 had fewer number of hatching larvae in comparison to the control group, irrespective of the incubation temperature tested. The minimum dosage of microsclerotial pellets that effectively reduced hatchability of tick larvae was estimated to be 2 mg per plate (equivalent to 6.0 kg per hectare). Metarhizium microsclerotial pellets exhibited significant tolerance to 32 °C and pronounced acaricidal activity against this economically important ectoparasite of cattle, even under simulated environmental heat stress. KEY POINTS: • Heat stress affects conidial production by microsclerotia of most pelletized Metarhizium strains • Heat stress does not impair the acaricidal performance of pelletized microsclerotia • Pellet formulation of Metarhizium microsclerotia is a promising mycoacaricide.

Lethal and sub-lethal activity of Brevibacillus laterosporus on the mosquito Aedes albopictus and side effects on non-target water-dwelling invertebrates.

The biocidal potential of Brevibacillus laterosporus against mosquitoes of major medical importance has been widely documented, but its effects on non-target invertebrates are still poorly known. In this study, we determined the lethal and sub-lethal effects of B. laterosporus strain UNISS 18, an entomopathogenic bacterium known for its effectiveness against synanthropic Diptera, on the larvae of the Asian tiger mosquito Aedes albopictus, a vector of several pathogens to humans. Moreover, we compared the larvicidal activity with the lethal action on the invasive snail Physella acuta and on two non-target water-dwelling species: the mayfly Cloeon dipterum, and the harlequin fly Chironomus riparius. B. laterosporus exhibited significant lethal effects on all the tested species with a concentration-dependent activity. However, the susceptibility varied among species, with a higher susceptibility of Ae. albopictus (LC50 = 0.16 × 107 spores mL-1) than the other species (LC50 = 0.31, 0.33, and 0.30 × 107 spores mL-1 for C. dipterum, C. riparius, and P. acuta, respectively). While 1st instar mosquito larvae were very susceptible to the bacterial infection, no effects on preimaginal development stages and adult emergence were observed at sub-lethal spores' concentrations. Even if the efficacy of B. laterosporus against Ae. albopictus and the invasive freshwater snail P. acuta is promising for their control, the susceptibility of non-target beneficial aquatic insects, highlights the need of accurate evaluations before applying B. laterosporus for pest management in water environments.

Thymol and eugenol microemulsion for Rhiphicephalus sanguineus sensu lato control: Formulation development, field efficacy, and safety on dogs.

The present study aimed to develop a microemulsion formulation containing thymol and eugenol for field control of Rhipicephalus sanguineus sensu lato on dogs, as well to evaluate its safety and the physical characteristics of the formulation. The microemulsion using thymol and eugenol (5.0 + 5.0 mg/mL) had as vehicles water, propylene glycol, polysorbate 80 and canola oil. On the next day the preparation (formulation freshly prepared) and after 24 months, the size of the microemulsion droplets, polydispersion index (PdI), organoleptic properties (color, viscosity), and presence of precipitate in the microemulsion were evaluated. For the field assay, on day -1, 10 English Cocker Spaniel dogs were experimentally infested with 200 larvae, 100 nymphs and 30 adults of R. sanguineus s.l. On day 0, after tick counts, the animals were divided into two groups: treated with the freshly prepared microemulsion (10 mL/kg), and control, which received the vehicle (10 mL/kg). Tick counts on dogs were performed daily for three more days. Engorged females were recovered from the dogs and their biological and reproductive parameters were monitored. The dogs' clinical parameters (temperature, mucosa color, and general physical condition) were evaluated daily. In addition, blood samples were collected before infestation to verify hematological (packed cell volume) and biochemical parameters (total serum protein, albumin, globulins, creatinine, urea, alanine transaminase, aspartate aminotransferase, and alkaline phosphatase). Freshly prepared and 24-month aged microemulsions had droplets with mean sizes of 30.94 nm and 27.93 nm, and PdI values of 0.214 and 0.161, respectively. In addition, no difference in the organoleptic properties and no precipitation formation were observed, indicating physical stability. Treatment with the microemulsion resulted in reduction of larvae (p < 0.05) parasitizing the dogs on day 1 while the number of nymphs and adults was not reduced (P> 0.05). In the evaluation of the reproductive biology of engorged females, the larval hatchability (%) was compromised (p < 0.05), and the microemulsion had control rate of 85.5 %. The microemulsion and its vehicles did not change the clinical, hematological and biochemical parameters of the dogs. We concluded that the microemulsion was efficient against R. sanguineus s.l. by reducing the number of larvae and affecting the reproductive parameters of engorged females, safe for dogs, and stable (physical stability) during a two-year interval.

Inorganic pellets containing microsclerotia of Metarhizium anisopliae: a new technological platform for the biological control of the cattle tick Rhipicephalus microplus.

This study was sought to devise pellets containing inorganic materials and microsclerotia of Metarhizium anisopliae strain IP 119 for biological control of Rhipicephalus microplus, the most economically important tick in Brazilian cattle industry. In addition, we evaluated the storage stability of the pellets, their tolerance to ultraviolet radiation (UV-B), and efficacy against ticks under laboratory conditions. Fungal microsclerotia were produced by liquid culture fermentation and mixed with pre-selected inorganic matrices: vermiculite powder, diatomaceous earth, and colloidal silicon dioxide (78:20:2, w/w/w). The microsclerotial pellets were then prepared by a two-stage process involving extrusion and spheronization. Pellet size averaged 525.53 ± 7.74 µm, with a sphericity index of 0.72 ± 0.01, while biomass constituents did not affect the wet mass properties. Conidial production from microsclerotial pellets upon rehydration ranged from 1.85 × 109 to 1.97 × 109 conidia g-1 with conidial viability ≥ 93%. Conidial production from pellets stored at 4 °C was invariable for up to 21 days. Unformulated microsclerotia and microsclerotial pellets were extremely tolerant to UV-B compared with aerial conidia. Engorged tick females exposed to conidia from sporulated pellets applied to soil samples and upon optimal rehydration exhibited shorter oviposition time length, shorter life span, and reduced number of hatched larvae. In summary, microsclerotial pellets of M. anisopliae IP 119 effectively suppressed R. microplus and showed outstanding UV-B tolerance in laboratory tests. Prospectively, this formulation prototype is promising for targeting the non-parasitic stage of this tick on outdoor pasture fields and may offer a novel mycoacaricide for its sustainable management. KEY POINTS: • Pellets with microsclerotia and inorganic materials are innovative for tick control. • Metarhizium microsclerotia show superior UV-B tolerance in relation to conidia. • Pellets of Metarhizium microsclerotia produce infective conidia against ticks.

Insecticidal potential of Brevibacillus laterosporus against dipteran pest species in a wide ecological range.

In order to increase our understanding of the insecticidal potential of the entomopathogenic bacterium Brevibacillus laterosporus strain UNISS 18 against insect pests, investigations were conducted on a selection of dipteran species including fruit flies, house flies, blow flies, and mosquitoes, characterized by adaptations to very diverse habitats. According to lethal concentration (LC50) values, the common house mosquito Culex pipiens (LC50 = 0.10 × 106 spores/mL) and the yellow fever mosquito Aedes aegypti (LC50 = 0.18 × 106 spores/mL) were significantly more susceptible than the flies. The blow flies were the second taxon in term of susceptibility to B. laterosporus spores, with a higher mortality in Calliphora vomitoria (LC50 = 78.84 × 106 spores/mL) than Lucilia caesar (LC50 = 148.30 × 106 spores/mL). The effectiveness of B. laterosporus spores was reduced by half in the house fly Musca domestica (LC50 = 82.41 × 106 spores/mL). The lowest susceptibility was observed in the fruit flies, among which the spotted wing drosophila (SWD), Drosophila suzukii, was the most susceptible (LC50 = 217.51 × 106 spores/mL) in comparison with the medfly Ceratitis capitata and the olive fly Bactrocera oleae (LC50 = 2567.32 and 2567.36 × 106 spores/mL, respectively). The present study demonstrated that significantly different degrees of susceptibility are associated with diverse dipteran species including plant and animal parasites, and we suggest that B. laterosporus established different relationships with dipteran species in different ecosystems.

Persistence and efficacy of a new formulation based on dog allomonal repellents against Rhipicephalus sanguineus sensu lato tick / Persistência e eficácia de uma nova formulação baseada em repelentes alomônios de cães contra o carrapato Rhipicephalus sanguineus sensu lato

Abstract Beagles are less susceptible to Rhipicephalus sanguineus sensu lato tick due to the production of the allomones benzaldehyde and 2-hexanone. Our previous published work showed that these compounds can reduce tick burden on susceptible dogs. Here we tested the hypothesis that an increase in repellent dose and release rate could increase repellent efficacy and persistence. Slow-release formulations of these compounds, with higher doses and release rates, were tested on artificially-infested dogs. Ten dogs were randomly assigned to two groups with five dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted once a day for 40 days. No significant increase in repellent efficacy was observed with the higher doses and release rates, whereas a greater persistence in repellent activity was observed. Treatment with the formulations resulted in a two-to-three-fold reduction in the number of immature stage ticks for up to three weeks. However, the number of adults was similar in both groups. Loss of repellent activity after the third week of testing coincided with a marked change in the relative release rates for the two compounds. It is hypothesized that relative amounts, rather than absolute amounts, of repellent release from slow-release formulations are important for repellent activity. We also hypothesize that the avoidance of less-preferred hosts by ticks relies on olfactory-mediated perception of specific blends of volatile cues from less preferred hosts.

Resumo Beagles são menos suscetíveis ao carrapato Rhipicephalus sanguineus sensu lato devido à produção de benzaldeído e 2-hexanona. Nosso trabalho anterior já publicado mostrou que esses compostos podem reduzir a carga de carrapatos em cães suscetíveis. Aqui testamos a hipótese de que um aumento na dose destes repelentes e na taxa de liberação poderia aumentar a eficácia e a persistência do efeito repelente. As formulações de liberação lenta destes compostos, com doses e taxas de liberação mais elevadas foram testadas em cães infestados artificialmente. Dez cães foram distribuídos aleatoriamente em dois grupos com cinco cães cada. O grupo tratado recebeu colares contendo formulações de liberação lenta dos compostos, enquanto o grupo controle recebeu colares com formulações limpas. Cinco infestações ambientais foram realizadas, com o número de carrapatos (em todas as fases) nos cães sendo contados, uma vez ao dia, por 40 dias. Não se observou aumento significativo na eficácia do repelente com doses e taxas de liberação mais elevadas e, enquanto observou-se maior persistência na atividade repelente. O tratamento com as formulações resultou em uma redução de duas a três vezes no número de carrapatos dos estágios imaturos, por até três semanas. No entanto, o número de adultos foi semelhante em ambos os grupos. A perda de atividade repelente após a terceira semana de teste coincidiu com uma mudança nas taxas de liberação relativa para os dois compostos. A hipótese é que as quantidades relativas, ao invés das quantidades absolutas de liberação lenta, são importantes para a atividade repelente. Então, a hipótese é de que a repelência apresentada por hospedeiros menos susceptíveis aos carrapatos depende da percepção pelos carrapatos de misturas específicas de voláteis liberados por estes hospedeiros.

Persistence and efficacy of a new formulation based on dog allomonal repellents against Rhipicephalus sanguineus sensu lato tick.

Beagles are less susceptible to Rhipicephalus sanguineus sensu lato tick due to the production of the allomones benzaldehyde and 2-hexanone. Our previous published work showed that these compounds can reduce tick burden on susceptible dogs. Here we tested the hypothesis that an increase in repellent dose and release rate could increase repellent efficacy and persistence. Slow-release formulations of these compounds, with higher doses and release rates, were tested on artificially-infested dogs. Ten dogs were randomly assigned to two groups with five dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted once a day for 40 days. No significant increase in repellent efficacy was observed with the higher doses and release rates, whereas a greater persistence in repellent activity was observed. Treatment with the formulations resulted in a two-to-three-fold reduction in the number of immature stage ticks for up to three weeks. However, the number of adults was similar in both groups. Loss of repellent activity after the third week of testing coincided with a marked change in the relative release rates for the two compounds. It is hypothesized that relative amounts, rather than absolute amounts, of repellent release from slow-release formulations are important for repellent activity. We also hypothesize that the avoidance of less-preferred hosts by ticks relies on olfactory-mediated perception of specific blends of volatile cues from less preferred hosts.