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.

Genomic surveillance and serological profile of SARS-CoV-2 variants circulating in Macaé and nearby cities, southeastern Brazil.

Introduction: A characteristic of the COVID-19 pandemic has been the sequential emergence and global dissemination of SARS-CoV-2 variants, noted for their enhanced transmission efficiency. These variants with mutations in the Spike glycoprotein (S-glycoprotein), which interacts with ACE2 receptors in human cells is critical for infection, affects the transmissibility of the virus, which is a matter of great concern for public health. Objective: This research analyses the effects these variants on a cohort of vaccinated and naturally infected individuals from the cities of Macaé-RJ, Rio das Ostras-RJ, and Campos dos Goytacazes-RJ, Brazil, from March 2021 to March 2023. Methods: This investigation encompasses the Alpha (B.1.1.7), Gamma (P.1), Delta (B.1.617.2, B.1.671.3), and Omicron (BQ.1, BQ.1.1 sublines, and BF.7) variants, focusing on their genomic surveillance and implications for the disease's epidemiology. The experimental analysis included a control group (vaccinated and uninfected subjects), and an infected group (post-vaccinated subjects). Samples from nasopharyngeal swabs underwent viral detection via RT-qPCR for diagnosis confirmation. RNase H-dependent RT-qPCR (rhAmp-PCR) and third-generation sequencing were used to detect SARS-CoV-2 variants. Anti-S-glycoprotein immunoglobulins were also evaluated for vaccinated infected and noninfected volunteers. Symptoms from infected individuals were compiled in order to reveal patterns of clinical signs associated with viral infection. Results: The study included 289 participants, with infections identified by Gamma (n = 44), Delta (n = 189), and Omicron (n = 56) variants. The prevalent symptoms among the naturally infected participants were cough, fever, sore throat, headache, and runny nose. For Omicron, cognitive symptoms such as memory loss and concentration issues were reported. Interestingly, the infected vaccinated group had higher anti-S-glycoprotein IgM production (n = 28, 0.2833 ± 0.09768 OD) compared to the uninfected vaccinated group (n = 14, 0.1035 ± 0.03625 OD). Conversely, anti-S-glycoprotein IgG production was higher in the control group (n = 12, 1.770 ± 0.1393 OD) than in the infected vaccinated group (n = 26, 1.391 ± 0.1563 OD). Conclusion: This comprehensive study enables monitoring of predominant variants and their correlation with clinical cases, providing valuable insights for public health. Our research group continues to survey circulating variants, contributing to the global understanding of the pandemic.

The long-term dynamics of serum antibodies against SARS-CoV-2.

Objective: To analyze the long-term dynamics of antibodies against SARS-CoV-2 and understand the impact of age, gender, and viral load on patients' immunological response. Methods: Serum samples were obtained from 231 COVID-19 positive patients from Macaé, in Rio de Janeiro state, in Brazil, from June 2020 until January 2021. The production of IgA, IgM, IgG, and IgE against S glycoprotein was analyzed using the S-UFRJ assay, taking into account the age, gender, and viral load. Results: Analysis of antibody production over 7 months revealed that IgA positivity gradually decreased after the first month. Additionally, the highest percentage of IgM positivity occurred in the first month (97% of patients), and declined after this period, while IgG positivity remained homogeneous for all 7 months. The same analysis for IgE revealed that almost all samples were negative. The comparison of antibody production between genders showed no significant difference. Regarding the age factor and antibody production, patients aged ≥60 years produced almost twice more IgA than younger ones (17-39 years old). Finally, a relationship between viral load and antibody production was observed only for older patients. Conclusions: Our work provides an overview of long-term production of antibodies against SARS-CoV-2, suggesting prolonged production of IgA and IgM antibodies for 3 months and continued IgG production for over 7 months. In addition, it identified a correlation between viral load and IgM titers in the older group and, finally, different IgA production between the age groups.

Molecular testing and analysis of disease spreading during the emergence of COVID-19 in Macaé, the Brazilian National Capital of Oil.

The Brazilian strategy to overcome the spread of COVID-19 has been particularly criticized due to the lack of a national coordinating effort and an appropriate testing program. Here, a successful approach to control the spread of COVID-19 transmission is described by the engagement of public (university and governance) and private sectors (hospitals and oil companies) in Macaé, state of Rio de Janeiro, Brazil, a city known as the National Oil Capital. In 2020 between the 17th and 38th epidemiological week, over two percent of the 206,728 citizens were subjected to symptom analysis and RT-qPCR testing by the Federal University of Rio de Janeiro, with positive individuals being notified up to 48 h after swab collection. Geocodification and spatial cluster analysis were used to limit COVID-19 spreading in Macaé. Within the first semester after the outbreak of COVID-19 in Brazil, Macaé recorded 1.8% of fatalities associated with COVID-19 up to the 38th epidemiological week, which was at least five times lower than the state capital (10.6%). Overall, considering the successful experience of this joint effort of private and public engagement in Macaé, our data suggest that the development of a similar strategy countrywise could have contributed to a better control of the COVID-19 spread in Brazil. Quarantine decree by the local administration, comprehensive molecular testing coupled to scientific analysis of COVID-19 spreading, prevented the catastrophic consequences of the pandemic as seen in other populous cities within the state of Rio de Janeiro and elsewhere in Brazil.

Larvicidal Potential of the Halogenated Sesquiterpene (+)-Obtusol, Isolated from the Alga Laurencia dendroidea J. Agardh (Ceramiales: Rhodomelaceae), against the Dengue Vector Mosquito Aedes aegypti (Linnaeus) (Diptera: Culicidae).

Dengue is considered a serious public health problem in many tropical regions of the world including Brazil. At the moment, there is no viable alternative to reduce dengue infections other than controlling the insect vector, Aedes aegypti Linnaeus. In the continuing search for new sources of chemicals targeted at vector control, natural products are a promising alternative to synthetic pesticides. In our work, we investigated the toxicity of a bioactive compound extracted from the red alga Laurencia dendroidea J. Agardh. The initial results demonstrated that crude extracts, at a concentration of 5 ppm, caused pronounced mortality of second instar A. aegypti larvae. Two molecules, identified as (-)-elatol and (+)-obtusol were subsequently isolated from crude extract and further evaluated. Assays with (-)-elatol showed moderate larvicidal activity, whereas (+)-obtusol presented higher toxic activity than (-)-elatol, with a LC50 value of 3.5 ppm. Histological analysis of the larvae exposed to (+)-obtusol revealed damage to the intestinal epithelium. Moreover, (+)-obtusol-treated larvae incubated with 2 µM CM-H2DCFDA showed the presence of reactive oxygen species, leading us to suggest that epithelial damage might be related to redox imbalance. These results demonstrate the potential of (+)-obtusol as a larvicide for use against A. aegypti and the possible mode of action of this compound.

small ORFs: A new class of essential genes for development.

Genes that contain small open reading frames (smORFs) constitute a new group of eukaryotic genes and are expected to represent 5% of the Drosophila melanogaster transcribed genes. In this review we provide a historical perspective of their recent discovery, describe their general mechanism and discuss the importance of smORFs for future genomic and transcriptomic studies. Finally, we discuss the biological role of the most studied smORF so far, the Mlpt/Pri/Tal gene in arthropods. The pleiotropic action of Mlpt/Pri/Tal in D. melanogaster suggests a complex evolutionary scenario that can be used to understand the origins, evolution and integration of smORFs into complex gene regulatory networks.

Toll signals regulate dorsal-ventral patterning and anterior-posterior placement of the embryo in the hemipteran Rhodnius prolixus.

BACKGROUND: Insect embryonic dorso-ventral patterning depends greatly on two pathways: the Toll pathway and the Bone Morphogenetic Protein pathway. While the relative contribution of each pathway has been investigated in holometabolous insects, their role has not been explored in insects with a hemimetabolous type of development. The hemimetabolous insect Rhodnius prolixus, an important vector of Chagas disease in the Americas, develops from an intermediate germ band and displays complex movements during katatrepsis that are not observed in other orders. However, little is known about the molecular events that regulate its embryogenesis. Here we investigate the expression and function of genes potentially involved in the initial patterning events that establish the embryonic dorso-ventral axis in this hemipteran. RESULTS: We establish a staging system for early embryogenesis that allows us to correlate embryo morphology with gene expression profiles. Using this system, we investigate the role of Toll pathway genes during embryogenesis. Detailed analyses of gene expression throughout development, coupled with functional analyses using parental RNA interference, revealed that maternal Toll is required to establish germ layers along the dorso-ventral axis and for embryo placement along the anterior-posterior axis. Interestingly, knockdown of the Toll pathway effector Rp-dorsal appears to regulate the expression of the Bone Morphogenetic Protein antagonist Rp-short-gastrulation. CONCLUSIONS: Our results indicate that Toll signals are the initiating event in dorso-ventral patterning during Rhodnius embryogenesis, and this is the first report of a conserved role for Toll in a hemipteran. Furthermore, as Rp-dorsal RNA interference generates anteriorly misplaced embryos, our results indicate a novel role for Toll signals in establishment of the anterior-posterior axis in Rhodnius.

Alpha-glucosidase promotes hemozoin formation in a blood-sucking bug: an evolutionary history.

BACKGROUND: Hematophagous insects digest large amounts of host hemoglobin and release heme inside their guts. In Rhodnius prolixus, hemoglobin-derived heme is detoxified by biomineralization, forming hemozoin (Hz). Recently, the involvement of the R. prolixus perimicrovillar membranes in Hz formation was demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Hz formation activity of an alpha-glucosidase was investigated. Hz formation was inhibited by specific alpha-glucosidase inhibitors. Moreover, Hz formation was sensitive to inhibition by Diethypyrocarbonate, suggesting a critical role of histidine residues in enzyme activity. Additionally, a polyclonal antibody raised against a phytophagous insect alpha-glucosidase was able to inhibit Hz formation. The alpha-glucosidase inhibitors have had no effects when used 10 h after the start of reaction, suggesting that alpha-glucosidase should act in the nucleation step of Hz formation. Hz formation was seen to be dependent on the substrate-binding site of enzyme, in a way that maltose, an enzyme substrate, blocks such activity. dsRNA, constructed using the sequence of alpha-glucosidase gene, was injected into R. prolixus females' hemocoel. Gene silencing was accomplished by reduction of both alpha-glucosidase and Hz formation activities. Insects were fed on plasma or hemin-enriched plasma and gene expression and activity of alpha-glucosidase were higher in the plasma plus hemin-fed insects. The deduced amino acid sequence of alpha-glucosidase shows a high similarity to the insect alpha-glucosidases, with critical histidine and aspartic residues conserved among the enzymes. CONCLUSIONS/SIGNIFICANCE: Herein the Hz formation is shown to be associated to an alpha-glucosidase, the biochemical marker from Hemipteran perimicrovillar membranes. Usually, these enzymes catalyze the hydrolysis of glycosidic bond. The results strongly suggest that alpha-glucosidase is responsible for Hz nucleation in the R. prolixus midgut, indicating that the plasticity of this enzyme may play an important role in conferring fitness to hemipteran hematophagy, for instance.

Perimicrovillar membranes promote hemozoin formation into Rhodnius prolixus midgut.

Rhodnius prolixus is a hematophagous insect that ingests large quantities of blood in each blood-feeding session. This ingested blood provides important nutrients to sustain the insect's oogenesis and metabolic pathways. During the digestive process, however, huge amounts of heme are generated as a consequence of the hemoglobin breakdown. Heme is an extremely dangerous molecule, since it can generate reactive oxygen species in the presence of oxygen that impair the normal metabolism of the insect. Part of the hemoglobin-derived heme can associate with the perimicrovillar membranes (PMM) in the gut lumen of R. prolixus; in this study we demonstrate the participation of the PMM in a heme detoxification process. These membranes were able to successfully induce heme aggregation into hemozoin (Hz). Heme aggregation was not dependent on the erythrocyte membranes, since the contribution of these membranes to the process was negligible, demonstrating that the ability to induce heme aggregation is a feature of the PMM, possibly representing a pre-adaptation of the hemipterans to feeding on blood.