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.

Establishment of suitable reference genes for studying relative gene expression during the transition from trophozoites to cyst-like stages and first evidences of stress-induced expression of meiotic genes in Trichomonas vaginalis.

Trichomonas vaginalis is a parasite of the human urogenital tract and the causative agent of trichomoniasis, a sexually transmitted disease of worldwide importance. This parasite is usually found as a motile flagellated trophozoite. However, when subjected to stressful microenvironmental conditions, T. vaginalis trophozoites can differentiate into peculiar cyst-like stages, which exhibit notable physiological resistance to unfavourable conditions. Although well documented in morphological and proteomic terms, patterns of gene expression changes involved in the cellular differentiation into cyst-like stages are mostly unknown. The real-time reverse transcription polymerase chain reaction (RT-qPCR) is recognized as a sensitive and accurate method for quantification of gene expression, providing fluorescence-based data that are proportional to the amount of a target RNA. However, the reliability of relative expression studies depends on the validation of suitable reference genes, which RNAs exhibit a minimum of variation between tested conditions. Here, we attempt to determine suitable reference genes to be used as controls of invariant expression during cold-induced in vitro differentiation of T. vaginalis trophozoites into cyst-like forms. Furthermore, we reveal that the mRNA from the meiotic recombinase Dmc1 is upregulated during this process, indicating that cryptic sexual events may take place in cyst-like stages of T. vaginalis.

Evolution and multiple roles of the Pancrustacea specific transcription factor zelda in insects.

Gene regulatory networks (GRNs) evolve as a result of the coevolutionary processes acting on transcription factors (TFs) and the cis-regulatory modules they bind. The zinc-finger TF zelda (zld) is essential for the maternal-to-zygotic transition (MZT) in Drosophila melanogaster, where it directly binds over thousand cis-regulatory modules to regulate chromatin accessibility. D. melanogaster displays a long germ type of embryonic development, where all segments are simultaneously generated along the whole egg. However, it remains unclear if zld is also involved in the MZT of short-germ insects (including those from basal lineages) or in other biological processes. Here we show that zld is an innovation of the Pancrustacea lineage, being absent in more distant arthropods (e.g. chelicerates) and other organisms. To better understand zld´s ancestral function, we thoroughly investigated its roles in a short-germ beetle, Tribolium castaneum, using molecular biology and computational approaches. Our results demonstrate roles for zld not only during the MZT, but also in posterior segmentation and patterning of imaginal disc derived structures. Further, we also demonstrate that zld is critical for posterior segmentation in the hemipteran Rhodnius prolixus, indicating this function predates the origin of holometabolous insects and was subsequently lost in long-germ insects. Our results unveil new roles of zld in different biological contexts and suggest that changes in expression of zld (and probably other major TFs) are critical in the evolution of insect GRNs.

Inorganic pyrophosphatase from the red flour beetle (Tribolium castaneum) modulates mitochondrial polyphosphate metabolism.

Polyphosphates (polyPs) have been found in all cell types examined to date and play diverse roles, depending on the cell type. In eukaryotic organisms, polyPs have been mainly investigated in mammalian cells, with few studies on insects. In this study, we investigated mitochondrial polyphosphate metabolism in the red flour beetle, Tribolium castaneum. Substrate specificity for different chain lengths demonstrated the presence of two exopolyphosphatase isoforms in mitochondria. T. castaneum mitochondrial polyP levels decreased after injection with soluble pyrophosphatase (Tc-sPPase) dsRNA, while the membrane exopolyphosphate activity increased. Mitochondrial respiration modulated exopolyphosphatase activity only in wild-type beetles. Tripolyphosphate was able to increase the F-ATPase activity in wild-type and Tc-sPPase RNAi beetles. We suggest that inorganic pyrophosphatase modulates polyphosphate metabolism in mitochondria and affects the link between mitochondrial activity and polyphosphate metabolism in T. castaneum.

A soluble pyrophosphatase is essential to oogenesis and is required for polyphosphate metabolism in the red flour beetle (Tribolium castaneum).

Polyphosphates have been found in all cell types examined to date and play diverse roles depending on the cell type. In eukaryotic organisms, polyphosphates have been mainly investigated in mammalian cells with few studies on insects. Some studies have demonstrated that a pyrophosphatase regulates polyphosphate metabolism, and most of them were performed on trypanosomatids. Here, we investigated the effects of sPPase gene knocked down in oogenesis and polyphosphate metabolism in the red flour beetle (Tribolium castaneum). A single sPPase gene was identified in insect genome and is maternally provided at the mRNA level and not restricted to any embryonic or extraembryonic region during embryogenesis. After injection of Tc-sPPase dsRNA, female survival was reduced to 15% of the control (dsNeo RNA), and egg laying was completely impaired. The morphological analysis by nuclear DAPI staining of the ovarioles in Tc-sPPase dsRNA-injected females showed that the ovariole number is diminished, degenerated oocytes can be observed, and germarium is reduced. The polyphosphate level was increased in cytoplasmic and nuclear fractions in Tc-sPPase RNAi; Concomitantly, the exopolyphosphatase activity decreased in both fractions. Altogether, these data suggest a role for sPPase in the regulation on polyphosphate metabolism in insects and provide evidence that Tc-sPPase is essential to oogenesis.

The mlpt smORF gene is essential for digestive physiology and molting during nymphal stages in the kissing bug Rhodnius prolixus.

Chagas disease affects around 8 million people globally, with Latin America bearing approximately 10,000 deaths each year. Combatting the disease relies heavily on vector control methods, necessitating the identification of new targets. Within insect genomes, genes harboring small open reading frames (smORFs - < 100 amino acids) present numerous potential candidates. In our investigation, we elucidate the pivotal role of the archetypal smORF-containing gene, mille-pattes/polished-rice/tarsalless (mlpt/pri/tal), in the post-embryonic development of the kissing bug Rhodnius prolixus. Injection of double-stranded RNA targeting mlpt (dsmlpt) during nymphal stages yields a spectrum of phenotypes hindering post-embryonic growth. Notably, fourth or fifth stage nymphs subjected to dsmlpt do not undergo molting. These dsmlpt nymphs display heightened mRNA levels of JHAMT-like and EPOX-like, enzymes putatively involved in the juvenile hormone (JH) pathway, alongside increased expression of the transcription factor Kr-h1, indicating changes in the hormonal control. Histological examination reveals structural alterations in the hindgut and external cuticle of dsmlpt nymphs compared to control (dsGFP) counterparts. Furthermore, significant changes in the vector's digestive physiology were observed, with elevated hemozoin and glucose levels in the posterior midgut of dsmlpt nymphs. Importantly, dsmlpt nymphs exhibit impaired metacyclogenesis of Trypanosoma cruzi, the causative agent of Chagas disease, underscoring the crucial role of proper gut organization in parasite differentiation. Thus, our findings constitute the first evidence of a smORF-containing gene's regulatory influence on vector physiology, parasitic cycle, and disease transmission.

The embryogenesis of the tick Rhipicephalus (Boophilus) microplus: the establishment of a new chelicerate model system.

Chelicerates, which include spiders, ticks, mites, scorpions, and horseshoe crabs, are members of the phylum Arthropoda. In recent years, several molecular experimental studies of chelicerates have examined the embryology of spiders; however, the embryology of other groups, such as ticks (Acari: Parasitiformes), has been largely neglected. Ticks and mites are believed to constitute a monophyletic group, the Acari. Due to their blood-sucking activities, ticks are also known to be vectors of several diseases. In this study, we analyzed the embryonic development of the cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). First, we developed an embryonic staging system consisting of 14 embryonic stages. Second, histological analysis and antibody staining unexpectedly revealed the presence of a population of tick cells with similar characteristics to the spider cumulus. Cumulus cell populations also exist in other chelicerates; these cells are responsible for the breaking of radial symmetry through bone morphogenetic protein signaling. Third, it was determined that the posterior (opisthosomal) embryonic region of R. microplus is segmented. Finally, we identified the presence of a transient ventral midline furrow and the formation and regression of a fourth leg pair; these features may be regarded as hallmarks of late tick embryogenesis. Importantly, most of the aforementioned features are absent from mite embryos, suggesting that mites and ticks do not constitute a monophyletic group or that mites have lost these features. Taken together, our findings provide fundamental common ground for improving knowledge regarding tick embryonic development, thereby facilitating the establishment of a new chelicerate model system.

Inhibition of Aedes aegypti DNA topoisomerase II by etoposide: Impact on survival and morphology of larvae and pupae.

DNA topoisomerase II enzymes maintain DNA stability during vital processes, such as genome replication, transcription and chromosomal segregation during mitosis and meiosis. In the present work, we analyzed functional aspects of the DNA topoisomerase II (AeTopII) enzyme of the mosquito Aedes aegypti. Here, we show that AeTopII mRNA is expressed at all stages of mosquito development. By in situ hybridization, we found that the AeTopII mRNA is concentrated along the ovarian follicular cells as well as in the region of the follicles. The observed expression profiles likely reflect increased topoisomerase II cellular requirements due to the intense ovarian growth and egg production following blood feeding in Ae. aegypti females. The drug etoposide, a classic inhibitor of topoisomerase II, was used for in vivo testing with 2nd stage larvae, in order to investigate the functional importance of this enzyme in Ae. aegypti survival and development. Inhibition of topoisomerase II activity with etoposide concentrations ranging from 10 to 200 µM did not leads to the immediate death of larvae. However, after 10 days of observation, etoposide treatments resulted in 30-40% decrease in survival, in a dose dependent manner, with persisting larvae and pupae presenting incomplete development, as well as morphological abnormalities. Also, approximately 50% of the treated larvae did not reach the pupal stage. Thus, we conclude that AeTopII is a vital enzyme in the development of Ae. aegypti and its sensitivity to inhibitors should be explored for potential chemical agents to be used in vector control.

Glycogen and glucose metabolism are essential for early embryonic development of the red flour beetle Tribolium castaneum.

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.