The tree that hides the forest: Cryptic diversity and phylogenetic relationships in the Palaearctic vector Obsoletus/Scoticus Complex (Diptera: Ceratopogonidae) at the European level

Background: Culicoides obsoletus is an abundant and widely distributed Holarctic biting midge species, involved in the transmission of bluetongue virus (BTV) and Schmallenberg virus (SBV) to wild and domestic ruminants. Females of this vector species are often reported jointly with two morphologically very close species, C. scoticus and C. montanus, forming the Obsoletus/Scoticus Complex. Recently, cryptic diversity within C. obsoletus was reported in geographically distant sites. Clear delineation of species and characterization of genetic variability is mandatory to revise their taxonomic status and assess the vector role of each taxonomic entity. Our objectives were to characterize and map the cryptic diversity within the Obsoletus/Scoticus Complex. Methods: Portion of the cox1 mitochondrial gene of 3763 individuals belonging to the Obsoletus/Scoticus Complex was sequenced. Populations from 20 countries along a Palaearctic Mediterranean transect covering Scandinavia to Canary islands (North to South) and Canary islands to Turkey (West to East) were included. Genetic diversity based on cox1 barcoding was supported by 16S rDNA mitochondrial gene sequences and a gene coding for ribosomal 28S rDNA. Species delimitation using a multi-marker methodology was used to revise the current taxonomic scheme of the Obsoletus/Scoticus Complex. Results: Our analysis showed the existence of three phylogenetic clades (C. obsoletus clade O2, C. obsoletus clade dark and one not yet named and identified) within C. obsoletus. These analyses also revealed two intra-specific clades within C. scoticus and raised questions about the taxonomic status of C. montanus. Conclusions: To our knowledge, our study provides the first genetic characterization of the Obsoletus/Scoticus Complex on a large geographical scale and allows a revision of the current taxonomic classification for an important group of vector species of livestock viruses in the Palaearctic region.[Figure not available: See fulltext.

Colonização e lesão em fêmeas ingurgitadas do carrapato Rhipicephalus sanguineus causadas pelo fungo Metarhizium anisopliae Colonization and lesions on engorged female Rhipicephalus sanguineus, caused by Metarhizium anisopliae

O presente trabalho teve como objetivo verificar a forma de penetração do fungo Metarhizium anisopliae [METSCH. (SOROKIN, 1883)] em carrapatos da espécie Rhipicephalus sanguineus (LATREILLE, 1806), assim como as lesões infringidas nos tecidos internos do ácaro. A forma de aderência e penetração do fungo foi estudada através da microscopia eletrônica de varredura e a ação do fungo nos tecidos internos avaliada em secções histológicas convencionais. Para observação destes eventos, realizaram-se infecções experimentais em 11 grupos de fêmeas ingurgitadas do carrapato R. sanguineus contendo 12 fêmeas ingurgitadas cada. Para tal, as fêmeas ingurgitadas foram banhadas durante 3 minutos, sob agitação manual, em suspensão com concentração 108 conídios/mL. No caso dos grupos controle o banho foi realizado apenas no veículo da suspensão. Os carrapatos foram processados para histopatologia e microscopia eletrônica em diversos tempos após a infecção, a saber: 1 e 18h, e um, dois, três, quatro, cinco, seis, sete, nove e onze dias. Observou-se que a maior parte dos conídios germinou em até 18h após a inoculação e que o fungo penetrou no ácaro através do tegumento 48h após a infecção. Após a penetração, o fungo invadiu o corpo do hospedeiro promovendo uma colonização difusa, sem preferência aparente por tecidos específicos. Dentre as lesões nos tecidos internos do ácaro, ressalta-se o rompimento da parede intestinal e vazamento do conteúdo para a hemocele. A morte do hospedeiro ocorreu entre 96 e 120h pós-infecção, e a esporulação do patógeno sobre o cadáver do ácaro iniciou-se em torno de 120 a 144h pós-infecção. Espera-se, com este trabalho, contribuir para o desenvolvimento e viabilização de técnicas de controle biológico dos carrapatos por fungos como alternativa ao uso de acaricidas.<br>The objective of this work was to verify the penetration of the fungus Metarhizium anisopliae [METSCH. (SOROKIN, 1883)], in to the Rhipicephalus sanguineus ticks (LATREILLE, 1806), as well as the lesions caused in the tissues of the acarus. The form of adherence and penetration of the fungus in the deep tissue was studied through scanning microscopy and the fungus action on the internal tissues was evaluated in conventional hystological sections. In order to evaluate these events, experimental infestations were conducted in 11 engorged females groups of the tick R. sanguineus, containing 12 engorged females in each. Aiming this goal the engorged females were submited to a bath in a conidial suspension in a concentration of 108 conidia/mL during three minutes under manual agitation. In case of control groups the bath was done only in the suspension vehicle. Ticks were processed for histological and scanning microscopy surveys at different intervals after the infection: 1 and 18h and one, two, three, four, five, six, seven, eight, nine and eleven days. It has been observed that the major part of the conidians have germinated within 18h after the inoculation and that the fungus the penetrated the acarus body, trough it&acute;s tegument 48h after the infection. After the penetration the fungus colonized the tick’s body in a diffuse manner, without any preference for a specific tissue. Among the lesions caused by the fungus, rupture of the tick’s intestinal wall and the dispersion of the fluid into the hemocel were the most outstanding features. Death of the host occurred 96h pos-infection and the sporulation of the pathogen over the dead acarus began 120 to 144h pos-infection. This work aims to contribute to the establishement of techniques for biological control of the ticks by fungus as an ecological alternative for acaricides