Complete assembly, annotation of virulence genes and CRISPR editing of the genome of Leishmania amazonensis PH8 strain.

We report the sequencing and assembly of the PH8 strain of Leishmania amazonensis one of the etiological agents of leishmaniasis. After combining data from long Pacbio reads, short Illumina reads and synteny with the Leishmania mexicana genome, the sequence of 34 chromosomes with 8317 annotated genes was generated. Multigene families encoding three virulence factors, A2, amastins and the GP63 metalloproteases, were identified and compared to their annotation in other Leishmania species. As they have been recently recognized as virulence factors essential for disease establishment and progression of the infection, we also identified 14 genes encoding proteins involved in parasite iron and heme metabolism and compared to genes from other Trypanosomatids. To follow these studies with a genetic approach to address the role of virulence factors, we tested two CRISPR-Cas9 protocols to generate L. amazonensis knockout cell lines, using the Miltefosine transporter gene as a proof of concept.

Identification of Candidate Chemosensory Gene Families by Head Transcriptomes Analysis in the Mexican Fruit Fly, Anastrepha ludens Loew (Diptera: Tephritidae).

Insect chemosensory systems, such as smell and taste, are mediated by chemosensory receptor and non-receptor protein families. In the last decade, many studies have focused on discovering these families in Tephritidae species of agricultural importance. However, to date, there is no information on the Mexican fruit fly Anastrepha ludens Loew, a priority pest of quarantine importance in Mexico and other countries. This work represents the first effort to identify, classify and characterize the six chemosensory gene families by analyzing two head transcriptomes of sexually immature and mature adults of A. ludens from laboratory-reared and wild populations, respectively. We identified 120 chemosensory genes encoding 31 Odorant-Binding Proteins (OBPs), 5 Chemosensory Proteins (CSPs), 2 Sensory Neuron Membrane Proteins (SNMPs), 42 Odorant Receptors (ORs), 17 Ionotropic Receptors (IRs), and 23 Gustatory Receptors (GRs). The 120 described chemosensory proteins of the Mexican fruit fly significantly contribute to the genetic databases of insects, particularly dipterans. Except for some OBPs, this work reports for the first time the repertoire of olfactory proteins for one species of the genus Anastrepha, which provides a further basis for studying the olfactory system in the family Tephritidae, one of the most important for its economic and social impact worldwide.

Trypanosoma cruzi genetic diversity: impact on transmission cycles and Chagas disease

Trypanosoma cruzi, the agent of Chagas disease (ChD), exhibits remarkable biological and genetic diversity, along with eco-epidemiological complexity. In order to facilitate communication among researchers aiming at the characterisation of biological and epidemiological aspects of T. cruzi, parasite isolates and strains were partitioned into seven discrete typing units (DTUs), TcI-TcVI and TcBat, identifiable by reproducible genotyping protocols. Here we present the potential origin of the genetic diversity of T. cruzi and summarise knowledge about eco-epidemiological associations of DTUs with mammalian reservoirs and vectors. Circumstantial evidence of a connection between T. cruzi genotype and ChD manifestations is also discussed emphasising the role of the host's immune response in clinical ChD progression. We describe genomic aspects of DTUs focusing on polymorphisms in multigene families encoding surface antigens that play essential functions for parasite survival both in the insect vector and the mammalian host. Such antigens most probably contributed to the parasite success in establishing infections in different hosts and exploring several niches. Gaps in the current knowledge and challenges for future research are pointed out.

The thermal proteome stability profile of Trypanosoma cruzi in epimastigote and trypomastigote life stages.

Trypanosoma cruzi is a flagellate protozoa being the etiological agent of Chagas disease, a neglected tropical disease, which still poses a public health problem worldwide. The intricate molecular changes during T. cruzi-host interaction have been explored using different largescale omics techniques. However, protein stability is largely unknown. Thermal proteome profiling (TPP) methodology has the potential to characterize proteome-wide stability highlighting key proteins during T. cruzi infection and life stage transition from the invertebrate to the mammalian host. In the present work, T. cruzi epimastigotes and trypomastigotes cell lysates were subjected to TPP workflow and analyzed by quantitative large-scale mass spectrometry-based proteomics to fit a melting profile for each protein. A total of 2884 proteins were identified and associated to 1741 melting curves being 1370 in trypomastigotes (TmAVG 53.53 °C) and 1279 in epimastigotes (TmAVG 50.89 °C). A total of 453 proteins were identified with statistically different melting profiles between the two life stages. Proteins associated to pathogenesis and intracellular transport had regulated melting temperatures. Membrane and glycosylated proteins had a higher average Tm in trypomastigotes compared to epimastigotes. This study represents the first large-scale comparison of parasite protein stability between life stages. SIGNIFICANCE: Trypanosoma cruzi, a unicellular flagellate parasite, is the etiological agent of Chagas disease, endemic in South America and affecting more that 7 million people worldwide. There is an intense research to identify novel chemotherapeutic and diagnostic targets of Chagas disease. Proteomic approaches have helped in elucidating the quantitative proteome and PTMs changes of T. cruzi during life cycle transition and upon different biotic and abiotic stimuli. However, a comprehensive knowledge of the protein-protein interaction and protein conformation is still missing. In order to fill this gap, this manuscript elucidates the T. cruzi Y strain proteome-wide thermal stability map in the epimastigote and trypomastigote life stages. Comparison between life stages showed a higher average melting temperature stability for trypomastigotes than epimastigotes indicating a host temperature adaptation. Both presented a selective thermal stability shift for cellular compartments, molecular functions and biological processes based on the T. cruzi life stage. Membrane and glycosylated proteins presented a higher thermal stability in trypomastigotes when compared to the epimastigotes.

Chromosomal organization of four classes of repetitive DNA sequences in killifish Orestias ascotanensis Parenti, 1984 (Cyprinodontiformes, Cyprinodontidae).

Orestias Valenciennes, 1839 is a genus of freshwater fish endemic to the South American Altiplano. Cytogenetic studies of these species have focused on conventional karyotyping. The aim of this study was to use classical and molecular cytogenetic methods to identify the constitutive heterochromatin distribution and chromosome organization of four classes of repetitive DNA sequences (histone H3 DNA, U2 snRNA, 18S rDNA and 5S rDNA) in the chromosomes of O. ascotanensis Parenti, 1984, an endemic species restricted to the Salar de Ascotán in the Chilean Altiplano. All individuals analyzed had a diploid number of 48 chromosomes. C-banding identified constitutive heterochromatin mainly in the pericentromeric region of most chromosomes, especially a GC-rich heterochromatic block of the short arm of pair 3. FISH assay with an 18S probe confirmed the location of the NOR in pair 3 and revealed that the minor rDNA cluster occurs interstitially on the long arm of pair 2. Dual FISH identified a single block of U2 snDNA sequences in the pericentromeric regions of a subtelocentric chromosome pair, while histone H3 sites were observed as small signals scattered in throughout the all chromosomes. This work represents the first effort to document the physical organization of the repetitive fraction of the Orestias genome. These data will improve our understanding of the chromosomal evolution of a genus facing serious conservation problems.

Evolutionary Dynamics of rDNAs and U2 Small Nuclear DNAs in Triportheus (Characiformes, Triportheidae): High Variability and Particular Syntenic Organization.

Multigene families correspond to a group of genes tandemly repeated, showing enormous diversity in both number of units and genomic organization. In fishes, unlike rDNAs that have been well explored in cytogenetic studies, U2 small nuclear RNA (snRNA) genes are poorly investigated concerning their chromosomal localization. All Triportheus species (Characiformes, Triportheidae) studied so far carry a ZZ/ZW sex chromosomes system, where the W chromosome contains a huge 18S rDNA cistron. In some species the syntenic organization of rDNAs on autosomes was also verified. To explore this particular organization, we performed three-color-fluorescence in situ hybridization using 5S, 18S rDNA, and U2 snRNA genes as probes in eight Triportheus species. This work represents the first one analyzing the chromosomal distribution of U2 snRNA genes in genomes of Triportheidae. The variability in number of rDNA clusters, and the divergent syntenies for these three multigene families, put in evidence their evolutionary dynamism, revealing a much more complex organization of these genes than previously supposed for closely related species. Our study also provides additional data on the accumulation of repetitive sequences in the sex-specific chromosome. Besides, the chromosomal organization of U2 snDNAs among fish species is also reviewed.

High similarity of U2 snDNA sequence between A and B chromosomes in the grasshopper Abracris flavolineata.

B chromosomes are frequently enriched for a wide variety of repetitive DNAs. Among grasshoppers in the species Abracris flavolineata (Ommatolampidinae) the B chromosomes are submetacentric, C-negative and harbor repetitive DNAs such as, U2 snDNA, C 0 t-1 DNA, two Mariner-like elements and some microsatellites. Here, we provide evidence showing the intragenome similarity between the B chromosome and the A complement in A. flavolineata, combining analysis of microdissection and chromosome painting and B chromosome-specific amplification through polymerase chain reaction (PCR) of U2 snDNA. Chromosome painting revealed signals spread through the C-negative regions, including the A and B chromosomes. Moreover, significant clustered signals forming bands were observed in some A chromosomes, and for the B chromosome, significant signals were located on both arms, which could be caused by accumulation of repetitive DNA sequences. The C-positive regions did not reveal any signals. Sequence comparison of U2 snDNA between that obtained from a genome without the B chromosome and that from µB-DNA revealed high similarity with the occurrence of four shared haplotypes, one of them (i.e., Hap1) being highly prevalent and putatively ancestral. The highest divergence from Hap1 was observed for Hap3, which was caused by only six mutational steps. These data support an intraspecific origin of the B chromosome in A. flavolineata that is highly similar with the A complement, and the low U2 snDNA sequence diversity observed in the B chromosome could be related to its recent origin, besides intrachromosomal concerted evolution for U2 snDNA repeats in the B chromosome.