SARS-CoV-2 Spatiotemporal Genomic and Molecular Analysis of the First Wave of the COVID-19 Pandemic in Macaé, the Brazilian Capital of Oil.

The SARS-CoV-2 virus infection led to millions of deaths during the COVID-19 pandemic. Hundreds of workers from several other Brazilian cities, as well as from other countries, arrive daily in Macaé to work in the oil supply chain, making this city a putative hotspot for the introduction of new viral lineages. In this study, we performed a genomic survey of SARS-CoV-2 samples from Macaé during the first outbreak of COVID-19, combined with clinical data and a molecular integrative analysis. First, phylogenomic analyses showed a high occurrence of viral introduction events and the establishment of local transmissions in Macaé, including the ingression and spread of the B.1.1.28 lineage in the municipality from June to August 2020. Second, SARS-CoV-2 mutations were identified in patients with distinct levels of COVID-19 severity. Third, molecular interactions of the mutated spike protein from three B.1.1.33 local samples and human ACE2 showed higher interactions than that of the wild-type spike protein from the ancestral virus. Altogether, these results elucidate the SARS-CoV-2 genomic profile in a strategic Brazilian city and further explore the functional aspects of SARS-CoV-2 with a characterization of emerging viral mutations associated with clinical data and the potential targets for drug development against SARS-CoV-2.

Microsatellite markers for the endangered franciscana dolphin (Pontoporia blainvillei).

The franciscana (Pontoporia blainvillei) is the most threatened small cetacean in the South Atlantic. In this study we report the development of 13 microsatellite markers for franciscanas through next-generation sequencing, and the characterization of those loci in 38 samples from the species' northernmost population (Espírito Santo, Brazil). Besides providing diversity indices for the new, specific loci, we also report on the transferability of heterologous loci which had not been screened in franciscanas before, and review all loci used in previous studies. Expected heterozygosity in the new loci ranged between 0.107 and 0.595, and all but one were in Hardy Weinberg Equilibrium. These are the first microsatellite loci isolated from franciscanas, and they are an important addition to heterologous markers that were available previously.

Transcriptome-based molecular systematics: Rhodnius montenegrensis (Triatominae) and its position within the Rhodnius prolixus-Rhodnius robustus cryptic-species complex.

BACKGROUND: Rhodnius montenegrensis (Triatominae), a potential vector of Chagas disease, was described after R. robustus-like bugs from southwestern Amazonia. Mitochondrial cytb sequence near-identity with sympatric R. robustus (genotype II) raised doubts about the taxonomic status of R. montenegrensis, but comparative studies have reported fairly clear morphological and genetic differences between R. montenegrensis and laboratory stocks identified as R. robustus. Here, we use a transcriptome-based approach to investigate this apparent paradox. RESULTS: We retrieved publicly-available transcriptome sequence-reads from R. montenegrensis and from the R. robustus stocks used as the taxonomic benchmark in comparative studies. We (i) aligned transcriptome sequence-reads to mitochondrial (cytb) and nuclear (ITS2, D2-28S and AmpG) query sequences (47 overall) from members of the R. prolixus-R. robustus cryptic-species complex and related taxa; (ii) computed breadth- and depth-coverage for the 259 consensus sequences generated by these alignments; and, for each locus, (iii) appraised query sequences and full-breadth-coverage consensus sequences in terms of nucleotide-sequence polymorphism and phylogenetic relations. We found evidence confirming that R. montenegrensis and R. robustus genotype II are genetically indistinguishable and, hence, implying that they are, in all likelihood, the same species. Furthermore, we found compelling genetic evidence that the benchmark 'R. robustus' stocks used in R. montenegrensis description and in later transcriptome-based comparisons are in fact R. prolixus, although likely mixed to some degree with R. robustus (probably genotype II, a.k.a. R. montenegrensis). CONCLUSIONS: We illustrate how public-domain genetic/transcriptomic data can help address challenging issues in disease-vector systematics. In our case-study, taxonomic confusion apparently stemmed from the misinterpretation of sequence-data analyses and misidentification of taxonomic-benchmark stocks. More generally, and together with previous reports of mixed and/or misidentified Rhodnius spp. laboratory colonies, our results call into question the conclusions of many studies (on morphology, genetics, physiology, behavior, bionomics or interactions with microorganisms including trypanosomes) based on non-genotyped 'R. prolixus' or 'R. robustus' stocks. Correct species identification is a prerequisite for investigating the factors that underlie the physiological, behavioral or ecological differences between primary domestic vectors of Chagas disease, such as R. prolixus, and their sylvatic, medically less-relevant relatives such as R. robustus (s.l.) including R. montenegrensis.

Evolution, Systematics, and Biogeography of the Triatominae, Vectors of Chagas Disease.

In this chapter, we review and update current knowledge about the evolution, systematics, and biogeography of the Triatominae (Hemiptera: Reduviidae)-true bugs that feed primarily on vertebrate blood. In the Americas, triatomines are the vectors of Trypanosoma cruzi, the etiological agent of Chagas disease. Despite declining incidence and prevalence, Chagas disease is still a major public health concern in Latin America. Triatomines occur also in the Old World, where vector-borne T. cruzi transmission has not been recorded. Triatomines evolved from predatory reduviid bugs, most likely in the New World, and diversified extensively across the Americas (including the Caribbean) and in parts of Asia and Oceania. Here, we first discuss our current understanding of how, how many times, and when the blood-feeding habit might have evolved among the Reduviidae. Then we present a summary of recent advances in the systematics of this diverse group of insects, with an emphasis on the contribution of molecular tools to the clarification of taxonomic controversies. Finally, and in the light of both up-to-date phylogenetic hypotheses and a thorough review of distribution records, we propose a global synthesis of the biogeography of the Triatominae. Over 130 triatomine species contribute to maintaining T. cruzi transmission among mammals (sometimes including humans) in almost every terrestrial ecoregion of the Americas. This means that Chagas disease will never be eradicated and underscores the fact that effective disease prevention will perforce require stronger, long-term vector control-surveillance systems.

Weaving through a cryptic species: Comparing the Neotropical ants Camponotus senex and Camponotus textor (Hymenoptera: Formicidae).

Camponotus senex (Fr. Smith 1858) and Camponotus textor Forel, 1899 are commonly confused species in the New World tropics. We provide morphological characteristics based on the larvae and adults, behavioural differences, together with evidence from molecular markers (cuticular hydrocarbon profiles, venom differences, nuclear ribosomal ITS-1, and mtDNA COI sequence comparisons) to separate the two species, demonstrating they are not immediately closely related. In conclusion we suggest new reliable morphological characters which can benefit from deeper phenetic analysis, and support the contextual usefulness of non-morphological tools in resolving sibling ant species.

The complete mitochondrial genome of Crassostrea gasar (Bivalvia: Ostreidae).

The complete mitochondrial genome of Crassostrea gasar was sequenced using the Ion Proton technology in combination with 454 Roche GS-FLX plataform data. We assembled a 17,686 bp complete circular mitochondrial genome, containing 13 protein-coding genes, a major non-coding region (MNR), two ribosomal RNA genes and 24 transfer RNA genes. Phylogenetic analysis of concatenated amino acid sequences from mitochondria showed monophyletic clades formed with high bootstrap values. This is the first complete mitochondrial sequence of an oyster from South America. Mitogenome sequence was deposited in GenBank under the accession number KR856227.

Molecular and Morphological Differentiation of Common Dolphins (Delphinus sp.) in the Southwestern Atlantic: Testing the Two Species Hypothesis in Sympatry.

The taxonomy of common dolphins (Delphinus sp.) has always been controversial, with over twenty described species since the original description of the type species of the genus (Delphinus delphis Linnaeus, 1758). Two species and four subspecies are currently accepted, but recent molecular data have challenged this view. In this study we investigated the molecular taxonomy of common dolphins through analyses of cytochrome b sequences of 297 individuals from most of their distribution. We included 37 novel sequences from the Southwestern Atlantic Ocean, a region where the short- and long-beaked morphotypes occur in sympatry, but which had not been well sampled before. Skulls of individuals from the Southwestern Atlantic were measured to test the validity of the rostral index as a diagnostic character and confirmed the presence of the two morphotypes in our genetic sample. Our genetic results show that all common dolphins in the Atlantic Ocean belong to a single species, Delphinus delphis. According to genetic data, the species Delphinus capensis is invalid. Long-beaked common dolphins from the Northeastern Pacific Ocean may constitute a different species. Our conclusions prompt the need for revision of currently accepted common dolphin species and subspecies and of Delphinus delphis distribution.

An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.

Polymorphic microsatellite loci for two Atlantic oyster species: Crassostrea rhizophorae and C. gasar.

Using a CA/CAA enriched library screening procedure, we isolated and characterised a total of seventeen polymorphic microsatellite loci for two species of Crassostrea with recognised economic importance. Eleven microsatellite loci were developed for C. rhizophorae, a Western Atlantic species for which no microsatellites were previously known. Another six loci were developed for C. gasar, a species that occurs on both sides of the South Atlantic, adding to the ten loci previously described for the species. The levels of polymorphism were estimated using 24 C. rhizophorae from Southeast Brazil (São Paulo) and 23 C. gasar individuals from North Brazil (Maranhão). The number of alleles per polymorphic locus varied from 3 to 27, and the observed and expected heterozygosities ranged between 0.174 and 0.958 and between 0.237 and 0.972 in C. rhizophorae and C. gasar, respectively. No linkage disequilibrium was found between any locus pair, and four of them exhibited deviations from Hardy-Weinberg expectations. Of the 17 loci developed, 8 cross-amplified in C. gigas and 13 in C. virginica. These markers are useful for evolution and population genetics studies of Crassostrea species and may provide fundamental data for the future cultivation of native oysters in Western Atlantic.

Phylogeographic pattern and extensive mitochondrial DNA divergence disclose a species complex within the Chagas disease vector Triatoma dimidiata.

BACKGROUND: Triatoma dimidiata is among the main vectors of Chagas disease in Latin America. However, and despite important advances, there is no consensus about the taxonomic status of phenotypically divergent T. dimidiata populations, which in most recent papers are regarded as subspecies. METHODOLOGY AND FINDINGS: A total of 126 cyt b sequences (621 bp long) were produced for specimens from across the species range. Forty-seven selected specimens representing the main cyt b clades observed (after a preliminary phylogenetic analysis) were also sequenced for an ND4 fragment (554 bp long) and concatenated with their respective cyt b sequences to produce a combined data set totalling 1175 bp/individual. Bayesian and Maximum-Likelihood phylogenetic analyses of both data sets (cyt b, and cyt b+ND4) disclosed four strongly divergent (all pairwise Kimura 2-parameter distances >0.08), monophyletic groups: Group I occurs from Southern Mexico through Central America into Colombia, with Ecuadorian specimens resembling Nicaraguan material; Group II includes samples from Western-Southwestern Mexico; Group III comprises specimens from the Yucatán peninsula; and Group IV consists of sylvatic samples from Belize. The closely-related, yet formally recognized species T. hegneri from the island of Cozumel falls within the divergence range of the T. dimidiata populations studied. CONCLUSIONS: We propose that Groups I-IV, as well as T. hegneri, should be regarded as separate species. In the Petén of Guatemala, representatives of Groups I, II, and III occur in sympatry; the absence of haplotypes with intermediate genetic distances, as shown by multimodal mismatch distribution plots, clearly indicates that reproductive barriers actively promote within-group cohesion. Some sylvatic specimens from Belize belong to a different species - likely the basal lineage of the T. dimidiata complex, originated ~8.25 Mya. The evidence presented here strongly supports the proposition that T. dimidiata is a complex of five cryptic species (Groups I-IV plus T. hegneri) that play different roles as vectors of Chagas disease in the region.

A nuclear single-nucleotide polymorphism (SNP) potentially useful for the separation of Rhodnius prolixus from members of the Rhodnius robustus cryptic species complex (Hemiptera: Reduviidae).

The design and application of rational strategies that rely on accurate species identification are pivotal for effective vector control. When morphological identification of the target vector species is impractical, the use of molecular markers is required. Here we describe a non-coding, single-copy nuclear DNA fragment that contains a single-nucleotide polymorphism (SNP) with the potential to distinguish the important domestic Chagas disease vector, Rhodnius prolixus, from members of the four sylvatic Rhodnius robustus cryptic species complex. A total of 96 primer pairs obtained from whole genome shotgun sequencing of the R. prolixus genome (12,626 random reads) were tested on 43 R. prolixus and R. robustus s.l. samples. One of the seven amplicons selected (AmpG) presented a SNP, potentially diagnostic for R. prolixus, on the 280th site. The diagnostic nature of this SNP was then confirmed based on the analysis of 154 R. prolixus and R. robustus s.l. samples representing the widest possible geographic coverage. The results of a 60% majority-rule Bayesian consensus tree and a median-joining network constructed based on the genetic variability observed reveal the paraphyletic nature of the R. robustus species complex, with respect to R. prolixus. The AmpG region is located in the fourth intron of the Transmembrane protein 165 gene, which seems to be in the R. prolixus X chromosome. Other possible chromosomal locations of the AmpG region in the R. prolixus genome are also presented and discussed.

Very low levels of genetic variation in natural peridomestic populations of the Chagas disease vector Triatoma sordida (Hemiptera: Reduviidae) in southeastern Brazil.

Levels of genetic variation and population structure were determined for 181 Triatoma sordida insects from four populations of southeastern Brazil, through the analysis of 28 allozyme loci. None of these loci presented fixed differences between any pair of populations, and only two revealed polymorphism, accounting for low levels of heterozygosity (H(e) = 0.027), and low genetic distances (D < 0.03) among populations. F(ST) and Contingency Table results indicated the existence of genetic structure among populations (F(ST) = 0.214), which were incompatible with the isolation by distance model (Mantel test: r = 0.774; P = 0.249).

Genetic variation and population structure of two species of neo-tropical mud-mussels (Mytella spp).

Mytella guyanensis Lamarck (1819) and Mytella charruana d'Orbigny (1846) are widespread euryhaline bivalves that have become commercially important in Brazil. Despite their importance, however, no genetic information that would be useful to orient governmental policies is available for these species. We analyzed, through allozyme electrophoresis, populations of M. guyanensis and M. charruana along 3,500 km of Brazilian coast. Pairwise comparisons among gene frequencies in M. guyanensis resulted in high levels of pairwise gene identity (I = 0.976 to 0.998). Conversely, significant levels of population structure were found in both M. guyanensis (FST = 0.089) and M. charruana (FST = 0.102). Heterozygosity levels for both species were high (H(e) = 0.090 to 0.134 in M. guyanensis and H(e) = 0.191 to 0.228 in M. charruana). The larger population size of M. charruana could explain, at least partially, the higher levels of genetic variability for this species. These levels of genetic variability yield an effective population size estimate of about 300,000 for M. guyanensis, and 540,000 for M. charruana, based on neutralist expectations. Remarkably, these numbers are much smaller than the estimated actual population sizes. This distortion might be explained by unstable population sizes and it suggests that long-term genetic variability studies are crucial to prevent artifactual viability analysis data for these commercially exploited species.

Population genetic structure of Brazilian shrimp species (Farfantepenaeus sp., F. brasiliensis, F. paulensis and Litopenaeus schmitti: Decapoda: Penaeidae)

Penaeid shrimps are important resources for worldwide fisheries and aquaculture. In the Southwest Atlantic, Farfantepenaeus brasiliensis, F. paulensis, F. subtilis, Farfantepenaeus sp. and Litopenaeus schmitti are among the most important commercially exploited species. Despite their high commercial value, there is little information available on the different aspects of their biology or genetics and almost no data on their stock structure. We used allozymes to estimate variability levels and population genetic structure of F. brasiliensis, F. paulensis, L. schmitti and the recently detected species Farfantepenaeus sp. along as much as 4,000 km of Brazilian coastline. No population heterogeneity was detected in F. brasiliensis or L. schmitti along the studied area. In contrast, F ST values found for Farfantepenaeus sp. and F. paulensis indicate that the populations of those two species are genetically structured, comprising different fishery stocks. The largest genetic differences in F. paulensis were found between Lagoa dos Patos (South) and the two populations from Southeast Brazil. In Farfantepenaeus sp., significant differences were detected between the population from Recife and those from Fortaleza and Ilhéus.

DOES COSMOPOLITANISM RESULT FROM OVERCONSERVATIVE SYSTEMATICS? A CASE STUDY USING THE MARINE SPONGE CHONDRILLA NUCULA.

The sponge species Chondrilla nucula has a simple morphology and a very wide geographical distribution. To verify whether the latter might be an artifact of the former, samples of this species were collected across 10,000 km of its range, in the Mediterranean, the Caribbean, and the southwestern atlantic. The classical (spicule morphology) and molecular (allozymes) systematic approaches were compared, to try to define the geographic limits between populations and detect possible cryptic species. We found five distinct genetic forms within C. nucula that sometimes showed morphological homogeneity and other times plasticity. The difference in size of spicules could not be related to the clear-cut genetic differences, suggesting that the use of spicule sizes for sponge systematics should be reappraised. The population of one of the genetic forms along 3000 km of the Brazilian coast was highly structured (FST = 0.21; Ne m = 0.96). Our results reject the null hypothesis of cosmopolitanism of C. nucula and indicate that the putative worldwide distribution of some marine sponges, and possibly many other benthic invertebrates, may be the result of overly conservative systematics. Cryptic species appear to be particularly prevalent when genera are well defined but species are characterized by only a few morphological characters.