Ornithodoros cerradoensis n. sp. (Acari: Argasidae), a member of the Ornithodoros talaje (Guérin-Méneville, 1849) group, parasite of rodents in the Brazilian Savannah

Ornithodoros cerradoensis n. sp. is described from field-collected and laboratory reared nymphs, males, females, and larvae parasitizing the rodents Cavia aperea and Thrichomys sp. in the Brazilian Savannah. This new species is morphologically and genetically related with the Ornithodoros talaje group and can be separated from other Neotropical species using the following combination of characters: larva with 18 pairs of setae on dorsum (seven anterolateral, four central and seven posterolateral), hypostome with median dentition 2/2; adults provided with large mammillae; dorsal disks surrounded by bulked marginal ridges delimiting barely pebbled areas; three disks in the anterolateral file, and median disk not merging with the posteromedian file. Feeding assays in the laboratory demonstrated that (1) larvae of O. cerradoensis are slow-feeders (∼6 days), (2) first nymphal instar (N1) molts to second instar (N2) without feeding, and (3) N2 and third nymphal instar (N3) engorge rapidly (minutes). With the exception of Ornithodoros hasei nymphs that depict flattened bodies, O. cerradoensis N1, N2, and N3 highly resemble homologous instars of other species in O. talaje sensu lato, therefore are not suitable for morphological comparisons within the group. In addition to morphological signature of larvae and adults that separate this new species; results of cross-mating attempts between O. cerradoensis and Ornithodoros guaporensis a morphologically and phylogenetically closely related species that also parasitizes rodents in the Brazilian Savannah; a Principal Component Analysis using larval characters; and a phylogenetic analysis using mitochondrial markers, support O. cerradoensis as an independent lineage within the Ornithodorinae.

Repellent Effects of Encapsulated Carvacrol on the Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Rhipicephalus (Boophilus) microplus (Canestrini) is a problem for livestock production systems, and its control has become challenging due to the selection of tick populations resistant to synthetic chemical acaricides. The use of repellent compounds prevents contact between the arthropod parasite and the host and can, thus, contribute to increases in the efficacy of these acaricides. Carvacrol monoterpenic phenol is a possible alternative method for controlling R. (B.) microplus; however, this compound is highly volatile, and its volatilization can be decreased through microencapsulation, which results in the timed release of the compound. The cell wall of Saccharomyces cerevisiae can be utilized for the protection of volatile molecules. The aim of this study was to evaluate the in vitro repellent effect of yeast cell wall-encapsulated carvacrol on susceptible R. (B.) microplus larvae. Specifically, the vertical filter paper bioassay was employed to analyze the repellent activity of encapsulated carvacrol, nonencapsulated carvacrol, and N,N-diethyl-meta-toluamide at concentrations ranging from 0.75 to 0.001 mg/cm2, and the repellent activities were evaluated. Both carvacrol and encapsulated carvacrol exhibited repellent effects on R. (B.) microplus larvae, and the encapsulated compound showed the highest repellent activities at the lowest concentrations. Carvacrol encapsulated exhibited a low repellent concentration in all times (≤0.05 mg/cm2), whereas the carvacrol nonencapsulated ranged CR50 from 0.13 to 0.27 mg/cm2 at 1- to 6-h posttreatment. The present paper provides the first description of the use of a microencapsulation technique for achieving the highest repellent effect of carvacrol and indicates that this technique might be used to obtain new delivery systems for volatile and hydrophobic compounds.

The first assessment of the stress inducible defense of Leucaena leucocephala with acaricidal potential effect against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Plants respond to wounding caused by mechanical stress or herbivory by synthesizing defense proteins. There are no studies reporting the action of induced plant proteins against ticks. The aim of this study was to investigate the effect of mechanically wounded Leucaena leucocephala leaves against Rhipicephalus (Boophilus) microplus. Initially, we carried out time course experiments to evaluate the impact of mechanical wounding on the protein content and the peroxidase, catalase and protease inhibitor activities in L. leucocephala. We then evaluated the acaricidal activity on R. (B.) microplus from protein extract collected from L. leucocephala after mechanical wounding. L. leucocephala leaves were artificially wounded, and after 6, 12, 24 and 48h, the leaves were collected for protein extraction. Quantitative and qualitative analyses of the proteins were performed. The protein content and peroxidase and protease activities increased 12h after wounding, and the acaricidal activity of this protein extract was evaluated using engorged R. (B.) microplus females. The protein extract obtained after wounding reduced egg production (8.5%) compared to those without wounding. Furthermore, the extract reduced egg hatching by 47.7% and showed an overall efficacy of 56.3% at 0.1 mgP/mL of the protein. We demonstrated that L. leucocephala defensive proteins could be effective against R. (B.) microplus.

Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks.

Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

Gut transcriptome analysis on females of Ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks / Transcriptoma do intestino de fêmeas de Ornithodoros mimon (Acari: Argasidae) e inferência filogenética de carrapatos

Abstract Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

Resumo Ornithodoros mimon é um carrapato argasídeo parasita de morcegos, aves e marsupiais, além de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de moléculas vitais no processo de digestão e na relação parasito-hospedeiro, além de fornecer também informações sobre a evolução dos artrópodes hematófagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais moléculas expressas no intestino de uma espécie de carrapato argasídeo após o repasto sanguíneo, através de uma análise transcritômica descritiva do intestino de fêmeas ingurgitadas de O. mimon, utilizando um sequenciamento de RNA de nova geração da plataforma 454. Além de inferir a relação filogenética de carrapatos através de um conjunto de dados transcritômicos. O transcriptoma do intestino revelou diversos transcritos associados com a digestão da hemoglobina, como proteinases das classes serino, cisteína, aspártica e metalo. Registramos a presença de um transcrito de uma cisteína peptidase do tipo catepsina O em carrapatos. A inferência filogenética baseada em conjunto de dados transcritos homólogos tem uma resolução topológica similar a de outros conjuntos de dados moleculares. Foram depositados no banco de dados gênico público 2213 transcritos de O. mimon. Os achados obtidos no presente estudo podem contribuir para compreensão dos importantes processos, como digestão, nutrição e imunidade dos carrapatos da família Argasidae, além de fornecer informações sobre a filogenia da ordem Ixodida.

The first assessment of the stress inducible defense of Leucaena leucocephala with acaricidal potential effect against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) / Primeira avaliação de extratos proteicos de Leucaena leucocephala induzidos por injúria mecânica com atividade carrapaticida sobre Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

Abstract Plants respond to wounding caused by mechanical stress or herbivory by synthesizing defense proteins. There are no studies reporting the action of induced plant proteins against ticks. The aim of this study was to investigate the effect of mechanically wounded Leucaena leucocephala leaves against Rhipicephalus (Boophilus) microplus. Initially, we carried out time course experiments to evaluate the impact of mechanical wounding on the protein content and the peroxidase, catalase and protease inhibitor activities in L. leucocephala. We then evaluated the acaricidal activity on R. (B.) microplus from protein extract collected from L. leucocephala after mechanical wounding. L. leucocephala leaves were artificially wounded, and after 6, 12, 24 and 48h, the leaves were collected for protein extraction. Quantitative and qualitative analyses of the proteins were performed. The protein content and peroxidase and protease activities increased 12h after wounding, and the acaricidal activity of this protein extract was evaluated using engorged R. (B.) microplus females. The protein extract obtained after wounding reduced egg production (8.5%) compared to those without wounding. Furthermore, the extract reduced egg hatching by 47.7% and showed an overall efficacy of 56.3% at 0.1 mgP/mL of the protein. We demonstrated that L. leucocephala defensive proteins could be effective against R. (B.) microplus.

Resumo As plantas respondem a injúria causada por estresse mecânico ou por ataque de herbívoros através da síntese de proteínas de defesa. Não há estudos de proteínas induzidas de plantas contra carrapatos. O objetivo deste estudo foi verificar a atividade acaricida de extratos protéicos de folhas Leucaena leucocephala após injúria mecânica, sobre Rhipicephalus (Boophilus) microplus. Inicialmente foram conduzidos experimentos em diferentes intervalos de tempo para avaliar o impacto da injúria mecânica no conteúdo de proteína, atividade de peroxidase, catalase e inibidor de protease de L. leucocephala. Em seguida foi avaliada a atividade acaricida sobre R. (B.) microplus de um extrato protéico após injúria mecânica. Folhas de L. leucocephala foram artificialmente feridas e após 6, 12, 24 e 48h, as folhas foram coletadas para extração de proteínas. Análises quantitativas e qualitativas das proteínas foram realizadas. A quantidade de proteína e atividades de peroxidase e protease aumentaram 12h após a injúria. O extrato proteico obtido após injúria (12h) reduziu a produção de ovos (8,5%) em comparação com extratos de plantas sem injúria. O extrato reduziu 47,7% a eclosão de ovos e apresentou eficácia geral de 56,3% a 0,1 miligrama de proteína por mL (mgP/mL). Apresentamos que proteínas de defesa de L. leucocephala podem ter atividade sobre R. (B.) microplus.

Gut transcriptome analysis on females of ornithodoros mimon (Acari: Argasidae) and phylogenetic inference of ticks / Transcriptoma do intestino de fêmeas de Ornithodoros mimon (Acari: Argasidae) e inferência filogenética de carrapatos

Ornithodoros mimon is an argasid tick that parasitizes bats, birds and opossums and is also harmful to humans. Knowledge of the transcripts present in the tick gut helps in understanding the role of vital molecules in the digestion process and parasite-host relationship, while also providing information about the evolution of arthropod hematophagy. Thus, the present study aimed to know and ascertain the main molecules expressed in the gut of argasid after their blood meal, through analysis on the gut transcriptome of engorged females of O. mimon using 454-based RNA sequencing. The gut transcriptome analysis reveals several transcripts associated with hemoglobin digestion, such as serine, cysteine, aspartic proteases and metalloenzymes. The phylogenetic analysis on the peptidases confirmed that most of them are clustered with other tick genes. We recorded the presence a cathepsin O peptidase-coding transcript in ticks. The topology of the phylogenetic inferences, based on transcripts of inferred families of homologues, was similar to that of previous reports based on mitochondrial genome and nuclear rRNA sequences. We deposited 2,213 sequence of O. mimon to the public databases. Our findings may help towards better understanding of important argasid metabolic processes, such as digestion, nutrition and immunity.

Ornithodoros mimon é um carrapato argasídeo parasita de morcegos, aves e marsupiais, além de ser bastante agressivo aos humanos. O conhecimento dos transcritos presentes no intestino dos carrapatos auxilia no entendimento do papel de moléculas vitais no processo de digestão e na relação parasito-hospedeiro, além de fornecer também informações sobre a evolução dos artrópodes hematófagos. Desta maneira, o presente estudo teve como objetivo conhecer e identificar as principais moléculas expressas no intestino de uma espécie de carrapato argasídeo após o repasto sanguíneo, através de uma análise transcritômica descritiva do intestino de fêmeas ingurgitadas de O. mimon, utilizando um sequenciamento de RNA de nova geração da plataforma 454. Além de inferir a relação filogenética de carrapatos através de um conjunto de dados transcritômicos. O transcriptoma do intestino revelou diversos transcritos associados com a digestão da hemoglobina, como proteinases das classes serino, cisteína, aspártica e metalo. Registramos a presença de um transcrito de uma cisteína peptidase do tipo catepsina O em carrapatos. A inferência filogenética baseada em conjunto de dados transcritos homólogos tem uma resolução topológica similar a de outros conjuntos de dados moleculares. Foram depositados no banco de dados gênico público 2213 transcritos de O. mimon. Os achados obtidos no presente estudo podem contribuir para compreensão dos importantes processos, como digestão, nutrição e imunidade dos carrapatos da família Argasidae, além de fornecer informações sobre a filogenia da ordem Ixodida

New records for Amblyomma sculptum (Ixodidae) on non-passerine birds in Brazil.

The aim of this paper was to provide new records of Amblyomma sculptum on two species of terricolous birds in two areas of the Cerrado (savannah- like) bioma: two specimens of Cariama cristata were captured in the state of Goiás and one specimen ofCrax fasciolata was captured in the state of Minas Gerais. One of the C. cristata was parasitized by 15 larvae, six nymphs, one male and two females whereas the C. fasciolata was parasitized by seven larvae and eight nymphs. This paper presents a new locality for occurrence of parasitism A. sculptum in C. cristata and a new host for C fasciolata.

Laboratory life cycle of Ornithodoros brasiliensis (Acari: Argasidae): An endemic tick from southern Brazil.

ORNITHODOROS BRASILIENSIS: Aragão is an endemic tick restricted to the highlands of the state of Rio Grande do Sul, Brazil. This species is very aggressive toward humans, causing fever, great pain and intense inflammatory response at the bite site. It is also very aggressive toward dogs, and tick toxicosis syndrome has been reported in this host. In order to elucidate the biology of this tick, the present study describes its life cycle under laboratory conditions, using guinea pigs as hosts for two generations. In the nonparasitic phase, the ticks were maintained in sand, in an incubator under controlled conditions. The larvae molted to the nymphal stage without feeding, and five nymphal stages (N1, N2, N3, N4 and N5) were observed in both generations. In both generations emergence of adults started from N3 when sex ratio was 0.85:1 (23 males and 27 females) in F1 and 0.63:1 (34 males and 54 females) in F2. For both generations, N4 generated more females, while N5 only produced females. The pre-ecdysis period of the nymphs ranged from 31.1 to 38.6 days. Two gonotrophic cycles were observed, and the first one presented a higher average number of eggs deposited (N=139) than the second (N=73.8). The mean duration of the life cycle (egg to egg) of O. brasiliensis was 215.4 days for the first generation and 195 days for the second.

New records for Amblyomma sculptum (Ixodidae) on non-passerine birds in Brazil / Novos registros de Amblyomma sculptum (Ixodidae) em aves não-passeriformes no Brasil

Abstract The aim of this paper was to provide new records of Amblyomma sculptum on two species of terricolous birds in two areas of the Cerrado (savannah- like) bioma: two specimens of Cariama cristata were captured in the state of Goiás and one specimen ofCrax fasciolata was captured in the state of Minas Gerais. One of the C. cristata was parasitized by 15 larvae, six nymphs, one male and two females whereas the C. fasciolata was parasitized by seven larvae and eight nymphs. This paper presents a new locality for occurrence of parasitism A. sculptum in C. cristata and a new host for C fasciolata.

Resumo O objetivo deste trabalho foi apresentar novos registros de Amblyomma sculptum em duas espécies de aves terrícolas em duas áreas do bioma Cerrado: dois espécimes de Cariama cristata foram capturados no Estado de Goiás e um exemplar de Crax fasciolata foi capturado no Estado de Minas Gerais. Um dos exemplares de C. fasciolataestava parasitado por 15 larvas, seis ninfas, um macho e duas fêmeas, enquantoC. fasciolata estava parasitada por sete larvas e oito ninfas. Neste registro são apresentados nova localidade para ocorrência do parasitismo de A. sculptum em C. cristata e novo hospedeiro para C. fasciolata.

Laboratory life cycle of Ornithodoros brasiliensis (Acari: Argasidae): An endemic tick from southern Brazil

Ornithodoros brasiliensis: Aragao is an endemic tick restricted to the highlands of the state of Rio Grande do Sul, Brazil. This species is very aggressive toward humans, causing fever, great pain and intense inflammatory response at the bite site. It is also very aggressive toward dogs, and tick toxicosis syndrome has been reported in this host. In order to elucidate the biology of this tick, the present study describes its life cycle under laboratory conditions, using guinea pigs as hosts for two generations. In the nonparasitic phase, the ticks were maintained in sand, in an incubator under controlled conditions. The larvae molted to the nymphal stage without feeding, and five nymphal stages (N1, N2, N3, N4 and N5) were observed in both generations. In both generations emergence of adults started from N3 when sex ratio was 0.85:1 (23 males and 27 females) in F1 and 0.63:1 (34 males and 54 females) in F2. For both generations, N4 generated more females, while N5 only produced females. The pre-ecdysis period of the nymphs ranged from 31.1 to 38.6 days. Two gonotrophic cycles were observed, and the first one presented a higher average number of eggs deposited (N=139) than the second (N=73.8). The mean duration of the life cycle (egg to egg) of O. brasiliensis was 215.4 days for the first generation and 195 days for the second

Ornithodoros faccinii n. sp. (Acari: Ixodida: Argasidae) parasitizing the frog Thoropa miliaris (Amphibia: Anura: Cycloramphidae) in Brazil.

BACKGROUND: Most argasid ticks from the Neotropical region are parasites of mammals and birds, with a few records from reptiles. Many species of the genus Ornithodoros are known only through larval descriptions, and their chaetotaxy and morphological characteristics have been used to separate the taxa. In the present study, we describe the larva and the nymph of first instar of a new species of the genus Ornithodoros that was collected from frogs of the species Thoropa miliaris. METHODS: Larvae of Ornithodoros were collected from frogs of the species T. miliaris at waterfalls in the state of Rio de Janeiro, southeastern Brazil. The larval and nymphal description was based on optical and scanning electron microscopy. Molecular analysis using the argasid 16S rRNA sequences available in GenBank was also conducted. RESULTS: Ornithodoros faccinii sp. n. is closely related to Ornithodoros clarki Jones & Clifford, Ornithodoros marinkellei Kohls, Clifford & Jones, Ornithodoros capensis Neumann and Ornithodoros sawaii Kitaoka & Susuki. However, the larval morphology of the new species is unique. The mitochondrial 16S rDNA partial sequence of O. faccinii generated in the present study was deposited in GenBank under the number KP861242. CONCLUSIONS: The larvae collected from Thoropa miliaris are a new species, Ornithodoros faccinii n. sp. This is the first report of argasid ticks on frogs in Brazil, the second on frogs and the third on Amphibia in the Neotropical region.

Immature argasid ticks: diagnosis and keys for neotropical region / Carrapatos argasídeos imaturos: diagnoses e chaves para a região neotropical

Many argasid tick species are known only through their larval descriptions, in which the chaetotaxy, together with other external morphological characteristics, has been used to separate genera and species. However, the illustrations of these features are based on optical microscopy alone and many of these features are not clearly defined. Because of the difficulties in determining the larval and nymph stages of some genera, we have prepared illustrated keys for the immature stages of argasids, including an up-to-date list of the known species of the Neotropical region. We have also included an illustrated key for larvae of the Ornithodoros species from Brazil, based on scanning electron microscopy.

Muitos carrapatos argasídeos são conhecidos somente por descrições larvais, nas quais a quetotaxia associada a outros caracteres morfológicos tem sido usada para separar gêneros e espécies. No entanto, as ilustrações sobre esses caracteres são baseadas somente em microscopia óptica e muitos deles não estão claramente definidos. Devido às dificuldades em determinar estágios larvais e ninfais de alguns gêneros, elaboramos chaves ilustradas para os estágios imaturos de argasídeos, incluindo uma lista atualizada de espécies conhecidas da região Neotropical. Incluímos também uma chave ilustrada para larvas das espécies de Ornithodoros do Brasil baseada em microscopia eletrônica de varredura.

New host records of Ixodes luciae (Acari: Ixodidae) in the State of Para, Brazil.

The aim of this paper is to record new hosts for Ixodes luciae Sénevet in the State of Para, Brazil, and present a case of malformation (teratogeny) in a nymph of this species. The new host records are Marmosa murina (parasitized by females) and Philander opossum (parasitized by nymphs). One of these nymphs showed malformation in the posterior margin of the opisthosoma resulting in a heart shaped posterior end.

New host records of Ixodes luciae (Acari: Ixodidae) in the State of Para, Brazil / Registros de novos hospedeiros para Ixodes luciae (Acari: Ixodidae) no estado do Pará, Brasil

The aim of this paper is to record new hosts for Ixodes luciae Sénevet in the State of Para, Brazil, and present a case of malformation (teratogeny) in a nymph of this species. The new host records are Marmosa murina (parasitized by females) and Philander opossum (parasitized by nymphs). One of these nymphs showed malformation in the posterior margin of the opisthosoma resulting in a heart shaped posterior end.

O objetivo deste trabalho foi registrar novos hospedeiros para Ixodes luciae Sénevet no estado do Pará, Brasil, e um caso de malformação em uma ninfa dessa espécie de carrapato. Os novos hospedeiros são Marmosa murina (parasitado por fêmeas) e Philander opossum (parasitado por ninfas). Uma dessas ninfas apresentou uma malformação na parte posterior do opistossoma em forma de coração.

Immature argasid ticks: diagnosis and keys for Neotropical region.

Many argasid tick species are known only through their larval descriptions, in which the chaetotaxy, together with other external morphological characteristics, has been used to separate genera and species. However, the illustrations of these features are based on optical microscopy alone and many of these features are not clearly defined. Because of the difficulties in determining the larval and nymph stages of some genera, we have prepared illustrated keys for the immature stages of argasids, including an up-to-date list of the known species of the Neotropical region. We have also included an illustrated key for larvae of the Ornithodoros species from Brazil, based on scanning electron microscopy.

Description of nymphal instars of Ornithodoros mimon Kohls, Clifford & Jones, 1969 (Acari: Argasidae).

Ornithodoros mimon is an argasid tick common on Chiroptera in the Neotropical region, where it also bites humans aggressively. Here we describe for the first time all nymphal instars (N1, N2 and N3) of O. mimon based on optical and scanning electron microscopy. Although the nymphal instars of O. mimon resemble each other closely, there are characters that differentiate them: the N3 are taller that N1 and N2; the genital primordium occurs in some N2 and all N3; the spiracular plate in N1 and N2 is cone-like, but in N3 it is semicircular; and the submarginal dorsal groove is less distinct in N1 but more evident in N3. Nymphs of 0. mimon closely resemble the bat-associated species of the genus Ornithodoros included in the Alectorobius group. We review prior descriptions of nymphs of the Alectorobius group and make comparisons with nymphs of O. minon, highlighting characters with diagnostic information, such as the idiosomal shape, presence of discs and hood and absence subapical protuberance of tarsus I. The description of nymphal instars of O. mimon herein presented, improves the taxonomy of the family Argasidae, performing a work more detailed about the immature stage of this species.