Gene Editing in the Chagas Disease Vector Rhodnius prolixus by Cas9-Mediated ReMOT Control.

Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. However, transgenesis and gene editing protocols to advance the field are still lacking. Here, we tested protocols for the maternal delivery of CRISPR-Cas9 (clustered regularly spaced palindromic repeats/Cas-9 associated) elements to developing R. prolixus oocytes and strategies for the identification of insertions and deletions (indels) in target loci of resulting gene-edited generation zero (G0) nymphs. We demonstrate successful gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) with the ovary-targeting BtKV ligand. These results provide proof of concepts for generating somatic mutations in R. prolixus and potentially for generating germ line-edited lines in triatomines, laying the foundation for gene editing protocols that could lead to the development of novel control strategies for vectors of Chagas disease.

Caracterização epidemiológica e distribuição geográfica de potenciais vetores da doença de Chagas na região do Maciço de Baturité, Ceará, Brasil / Epidemiological characterization and geographical distribution of potential vectors of Chagas Disease in Maciço Baturité region, Ceara, Brazil

Objetivo: identificar potenciais vetores da doença de Chagas na região do Maciço de Baturité, Ceará. Métodos: no período de2014 a 2018, foi realizado estudo parasitológico em fezes de triatomíneos para Trypanosoma cruzi, em oito municípios da região do maciço de Baturité. Os triatomíneos foram capturados em ambiente intradomiciliar e peridomiciliar. Resultados: foram examinados 594 vetores, a diagnose revelou infecção natural por Trypanosoma cruzi em 79 (13,3%) espécimes, sendo, 53 machos (8,9%), 25 fêmeas (4,2%) e 1 ninfa (0,2%). Conclusão: os resultados evidenciam a região como endêmica para doença de Chagas com sete casos humanos confirmados em três municípios, existindo vários focos silenciosos em que pode haver a transmissão do agente etiológico.

Objective: to identify potential vectors of Chagas disease in the Baturité Massif region, Ceará Methods: during the period from 2014 to 2018, a parasitological study was performed on triatomine feces for Trypanosoma cruzi, in eight municipalities in Maciço Baturité region. Triatomines were captured indoors and peridomiciliary by endemic agents and population as well. Results: a total of594 vectors were examined, diagnosis revealed natural infection by Trypanosoma cruzi in79 (13,3%) specimens, being, 53 males (8,9%), 25 females (4,2%) and 1 nymph (0,2%). Conclusion: results evidenced the region as endemic to Chagas Disease with seven confirmed human cases in three municipalities with several silent foci where the causative agent might be transmitted.

Quantitative 1 H NMR spectroscopy (qNMR) in the early process development of a new quorum sensing inhibitor.

2-methyl-5,6,7,8-tetrahydro-2H-chromen-4(3H)-one (called 6-oxo) is presented as a new AI-1 quorum sensing inhibitor for Vibrio harveyi. The development of a chemical process to afford traceable materials for new biological assays demands the development of analytical methods to ensure their purity and quality. This work describes the use of quantitative 1 H nuclear magnetic resonance (NMR) spectroscopy (qNMR) to assess the purity of a sample of 6-oxo (99.88%) and a sample of its major process impurity (E)-1-(2-hydroxycyclohex-2-en-1-yl)but-2-en-1-one (called HCB; 98.28%). To explore the scope of the use of qNMR to quantify the amount of low-content components in samples related to the chemical process for 6-oxo synthesis, this work also determined the amount of 6-oxo in two HCB samples: (a) the high-purity HCB sample described above and (b) a crude HCB sample collected during the chemical process. Despite the complexity of the crude sample, the amount of 6-oxo was readily assessed and could help to estimate the extent to which 6-oxo was already formed during the HCB synthesis. This information can help the understanding of how the process parameters can be modified to improve the performance of the whole process, by controlling the reaction mechanisms working at each step of this chemical process. In this context, our results reinforce qNMR as a complementary analytical tool for the quantification of the main component found in a sample, contributing to the standardization of reference materials and thus allowing the development of analytical methods for process control and traceability of the samples used for biological assays.

Rickettsia sp. Strain NOD Infecting Ticks ( Amblyomma nodosum) in an Endemic Area of Spotted Fever in Brazil.

Amblyomma nodosum ticks were collected from one collared anteater ( Tamandua tetradactyla) in the Caatinga biome, Brazil. From one sample, we isolated a Rickettsia sp. that was phylogenetically close to Rickettsia sp. strain NOD, with 99.9, 100.0, and 99.8% identity for gltA, htrA, and ompA genes, respectively.

Rickettsia rickettsii infecting Rhipicephalus sanguineus sensu lato (Latreille 1806), in high altitude atlantic forest fragments, Ceara State, Brazil.

In Brazil, Spotted Fever (SF) is caused by Rickettsia rickettsii and Rickettsia parkeri strain Atlantic Forest. In recent years, several human cases of a milder SF have been reported from the Maciço de Baturité region of Ceará State. Previous studies in this region found R. parkeri strain Atlantic Forest to be present in Rhipicephalus sanguineus sensu lato and Amblyomma ovale ticks. The present study isolated and identified the Rickettsia spp. present in this new endemic area in Brazil. In March 2015, R. sanguineus s.l. and A. ovale were collected in rural areas of the Maciço de Baturité region, and subjected to the isolation technique. A bacterium was isolated from one R. sanguineus s.l., which phylogenetic analysis clustered to the R. rickettsii group. In conclusion, R. rickettsii bacteria is circulating in the studied area and may in future have an impact on the clinical diagnoses and consequently cause changes in the profile of the disease in the region. In addition, we suggest the increase of epidemiological and environmental surveillance in the area, in order to prevent Brazilian Spotted Fever cases.

Rickettsia (Rickettsiales: Rickettsiaceae) Vector Biodiversity in High Altitude Atlantic Forest Fragments Within a Semiarid Climate: A New Endemic Area of Spotted-Fever in Brazil.

Rickettsioses are re-emerging vector-borne zoonoses with a global distribution. Recently, Rickettsia sp. strain Atlantic rainforest has been associated with new human spotted-fever (SF) cases in Brazil, featuring particular clinical signs: eschar formation and lymphadenopathy. These cases have been associated with the tick species, Amblyomma ovale From 2010 until 2015, the Brazilian Health Department confirmed 11 human SF cases in the Maciço de Baturité region, Ceará, Brazil. The present study reports the circulation of Rickettsia spp. in vectors from this entirely new endemic area for SF. A total of 1,727 ectoparasites were collected in this area from the environment, humans, and wild and domestic animals. Samples (n = 887) were screened by polymerase chain reaction (PCR), targeting the gltA and ompA rickettsial genes. Sequencing and phylogenetic analyses of gltA gene amplicons were carried out for 13 samples positive for both screening PCRs. Fragments of gltA and ompA from three samples were cloned, sequenced, and analyzed further. A. ovale and Rhipicephalus sanguineus specimens, collected from dogs, were found to be infected with Rickettsia sp. str. Atlantic rainforest, suggesting the importance of dogs in the epidemic cycle. Candidatus Rickettsia andeanae, Rickettsia felis, and Rickettsia bellii were also found infecting ticks and fleas in five municipalities, demonstrating the broad diversity of rickettsiae in circulation in the studied area. This study reports, for the first time, evidence of infection with Rickettsia sp. strain Atlantic rainforest in A. ovale and R. sanguineus in Ceará, and Ca. R. andeanae in an Atlantic rainforest environment of Brazil.