Daniele Pereira Castro

Background: Leishmaniasis presents a major public health problem for a large number of countries requiring effective integrated management of the vector, sand flies, for sustained control. Such strategies need to be economically and environmentally sustainable and adaptable to the behaviour of local vectors. One such tool is Attractive Targeted Sugar Baits (ATSB) that exploit the necessity of sand flies to acquire sugars between bloodmeals. Here we explored the kinetics and cues for sugar and blood feeding to improve the efficacy of ATSBs against sand flies. Methods: A fluorescent assay was developed to quantify sugarmeals to assess the feeding efficiency of colony-reared female Lutzomyia longipalpis sand flies. Results: Sand flies showed a range of preferences for different sugars presented on cotton wool and could be manipulated to deposit them into the crop and/or midgut. We found that the combination of 10% sucrose and 10% fructose allowed flies to obtain the largest sugarmeals taken to the crop. Sugarmeals were taken to both the crop and midgut when it contained 200 mM bovine serum albumin (BSA) as a source of protein and 1 mM adenosine triphosphate (ATP) as a phagostimulant. Using this combination, the efficacy of the ingested insecticide fipronil was significantly increased; reducing the 50% lethal concentration from 584 µM to 1.65 µM in a sugarmeal that promoted the simultaneous uptake of the insecticide into the midgut as well as the crop. Conclusions: In this study we highlight the potential of understanding the cues used by vectors to sugar feed and blood feed. By incorporating blood feeding phagostimulants, such as BSA and ATP, in ATSB we vastly improve their killing efficiency against sand flies. This demonstrates a new approach to target these disease vectors. Author Summary: Many insect vectors of disease use sugars, such as nectar, as fuel to enable them to obtain blood, that in turn sustains parasite transmission. In most blood feeding Diptera sugars are diverted to the crop where it is stored and released into the midgut for energy whereas blood is taken directly to the midgut where it is digested to provide nutrients for egg development. For sand flies the cues required to switch between sugar and blood feeding programmes are unknown but if they were they could be exploited to improve Attractive Targeted Sugar Baits (ATSB) as a means of poisoning sand flies when they sugar feed. In this study we find that the sand fly, Lutzomyia longipalpis , uses a combination of physical cues (biting/piercing vs sucking) and meal composition to allow them to efficiently feed on nectar and blood. We show that they sense protein and adenosine triphosphate (ATP) to direct blood to the midgut and we demonstrate that this can be turned to our advantage to improve the lethality of ATSB. We show that inclusion of bovine serum albumin as a source of protein and the phagostimulant ATP can trick sand flies to blood feed on sugar and direct the insecticide fipronil to the midgut where it was 355-fold more potent than in sugar alone. These results show the potential of phagostimulants to improve the efficacy and selectivity of ATSB towards blood feeding arthropods, including sand flies, and highlights the value of understanding the feeding programmes of disease vectors in general.

The application of microorganisms as bio-control agents against arthropod populations is a need in many countries, especially in tropical, subtropical, and neotropical endemic areas. Several arthropod species became agricultural pests of paramount economic significance, and many methods have been developed for field and urban applications to prevent their, the most common being the application of chemical insecticides. However, the indiscriminate treatment based upon those substances acted as a selective pressure for upcoming resistant phenotype populations. As alternative tools, microorganisms have been prospected as complementary tools for pest and vectorial control, once they act in a more specific pattern against target organisms than chemicals. They are considered environmentally friendly since they have considerably less off-target effects. Entomopathogenic fungi are organisms capable of exerting pathogenesis in many vector species, thus becoming potential tools for biological management. The entomopathogenic fungi Metarhizium sp. have been investigated as a microbiological agent for the control of populations of insects in tropical regions. However, the development of entomopathogenic fungi as control tools depends on physiological studies regarding aspects such as mechanisms of pathogenicity, secreted enzymes, viability, and host-pathogen aspects. The following review briefly narrates current aspects of entomopathogenic fungi, such as physiology, cellular characteristics, host-pathogen interactions, and its previous applications against different insect orders with medical and economic importance. Approaches integrating new isolation, prospection, characterization, delivery strategies, formulations, and molecular and genetic tools will be decisive to elucidate the molecular mechanisms of EPFs and to develop more sustainable alternative pesticides.

Currently, approximately 70% of new cases of Chagas disease (CD) in Brazil are attributed to oral transmission, particularly through foods such as açaí, bacaba, and sugarcane juice, primarily in the northern and northeastern regions of the country. This underscores the imperative need to control the spread of the disease. The methods utilized to conduct quality control for food associated with outbreaks and to assess the potential for the oral transmission of CD through consuming açaí primarily rely on isolating the parasite or inoculating food into experimental animals, restricting the analyses to major research centers. While there are existing studies in the literature on the detection and quantification of T. cruzi DNA in açaí, the evaluation of parasites’ viability using molecular methods in this type of sample and differentiating between live and dead parasites in açaí pulp remain challenging. Consequently, we developed a molecular methodology based on RT-qPCR for detecting and quantifying viable T. cruzi in açaí pulp samples. This protocol enables the stabilization and preservation of nucleic acids in açaí, along with incorporating an exogenous internal amplification control. The standardization of the RNA extraction method involved a simple and reproducible approach, coupled with a one-step RT-qPCR assay. The assay underwent validation with various T. cruzi DTUs and demonstrated sensitivity in detecting up to 0.1 viable parasite equivalents/mL in açaí samples. Furthermore, we investigated the effectiveness of a bleaching method in eliminating viable parasites in açaí samples contaminated with T. cruzi by comparing the detection of DNA versus RNA. Finally, we validated this methodology using açaí pulp samples positive for T. cruzi DNA, which were collected in a municipality with a history of oral CD outbreaks (Coari-AM). This validation involved comparing the detection and quantification of total versus viable T. cruzi. Collectively, our findings demonstrate the feasibility of this methodology in detecting viable forms of T. cruzi in açaí pulp samples, emerging as a crucial tool for monitoring oral outbreaks of Chagas disease resulting from açaí consumption.

Chagas disease is a human infectious disease caused by Trypanosoma cruzi and can be transmitted by triatomine vectors, such as Rhodnius prolixus. One limiting factor for T. cruzi development is the composition of the bacterial gut microbiota in the triatomine. Herein, we analyzed the humoral immune responses of R. prolixus nymphs treated with antibiotics and subsequently recolonized with either Serratia marcescens or Rhodococcus rhodnii. The treatment with antibiotics reduced the bacterial load in the digestive tract, and the recolonization with each bacterium was successfully detected seven days after treatment. The antibiotic-treated insects, recolonized with S. marcescens, presented reduced antibacterial activity against Staphylococcus aureus and phenoloxidase activity in hemolymph, and lower nitric oxide synthase (NOS) and higher defensin C gene (DefC) gene expression in the fat body. These insects also presented a higher expression of DefC, lower prolixicin (Prol), and lower NOS levels in the anterior midgut. However, the antibiotic-treated insects recolonized with R. rhodnii had increased antibacterial activity against Escherichia coli and lower activity against S. aureus, higher phenoloxidase activity in hemolymph, and lower NOS expression in the fat body. In the anterior midgut, these insects presented higher NOS, defensin A (DefA) and DefC expression, and lower Prol expression. The R. prolixus immune modulation by these two bacteria was observed not only in the midgut, but also systemically in the fat body, and may be crucial for the development and transmission of the parasites Trypanosoma cruzi and Trypanosoma rangeli.

Beta-1,3-glucanases are enzymes that hydrolyze beta-1,3-glucans, and they are essential for the metabolism of seaweed, plants and fungi. These enzymes also participate in the digestion of herbivore and fungivore animals. Because of the importance of these enzymes in insects, beta-1,3-glucanase inhibitors may be used for the development of new control strategies against agricultural pests and disease vectors. Beta-1,3-glucanase inhibitors have been described in the brown seaweed Laminaria cichorioides, but were never recorded in Brazilian seaweed species. We evaluated the presence of beta-1,3-glucanase inhibitors in samples of Padina gymnospora, Dictyota sp., Colpomenia sinuosa, and Lobophora sp., collected in Arraial d'Ajuda (Bahia). Ethanolic or buffer extracts were used in inhibition tests against the beta-1,3-glucanase of Trichoderma sp. Extracts in buffer showed no inhibition, but ethanolic extracts from all species showed different extents of inhibition. Samples from Dictyota sp. and P. gymnospora showed inhibitions above 75% (absolute ethanol) or 50% (ethanol 50%). In summary, extraction with absolute ethanol resulted in better inhibitions, and P. gymnospora showed the higher inhibitions. Brazilian seaweed may be good sources of beta-1,3-glucanase inhibitors for biochemical and physiological studies of these enzymes. Besides that, these molecules show potential for the development of new biotechnological tools for insect control.

Serratia marcescens is a bacterium with the ability to colonize several niches, including some eukaryotic hosts. S. marcescens have been recently found in the gut of hematophagous insects that act as parasite vectors, such as Anopheles, Rhodnius, and Triatoma. While some S. marcescens strains have been reported as symbiotic or pathogenic to other insects, the role of S. marcescens populations from the gut microbiota of Rhodnius prolixus, a vector of Chagas’ disease, remains unknown. Bacterial colonies from R. prolixus gut were isolated on BHI agar. After BOX-PCR fingerprinting, the genomic sequences of two isolates RPA1 and RPH1 were compared to others S. marcescens from the NCBI database in other to estimate their evolutionary divergence. The in vitro trypanolytic activity of these two bacterial isolates against Trypanosoma cruzi (DM28c clone and Y strain) was assessed by microscopy. In addition, the gene expression of type VI secretion system (T6SS) was detected in vivo by RT-PCR. Comparative genomics of RPA1 and RPH1 revealed, besides plasmid presence and genomic islands, genes related to motility, attachment, and quorum sensing in both genomes while genes for urea hydrolysis and type II secretion system (T2SS) were found only in the RPA1 genome. The in vitro trypanolytic activity of both S. marcescens strains was stronger in their stationary phases of growth than in their exponential ones, with 65–70 and 85–90% of epimastigotes (Dm28c clone and Y strain, respectively) being lysed after incubation with RPA1 or RPH1 in stationary phase. Although T6SS transcripts were detected in guts up to 40 days after feeding (DAF), R. prolixus morbidity or mortality did not appear to be affected. In this report, we made available two trypanolytic S. marcescens strains from R. prolixus gut to the scientific community together with their genomic sequences. Here, we describe their genomic features with the purpose of bringing new insights into the S. marcescens adaptations for colonization of the specific niche of triatomine guts. This study provides the basis for a better understanding of the role of S. marcescens in the microbiota of R. prolixus gut as a potential antagonist of T. cruzi in this complex system.

Rhodnius prolixus is an insect vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. Nuclear factor-κB (NF-κB) transcription factors (TF) are conserved components of the innate immune system in several multicellular organisms including insects. The drug IMD-0354 [N-(3,5-bis-trifluoromethyl-phenyl)-5-chloro-2-hydroxy-benzamide] is a selective inhibitor of IκB kinases. It blocks IκBα phosphorylation thus preventing nuclear translocation of the NF-κb TF. In humans, NF-κB is involved in several biological processes such as inflammation, cell proliferation and immunity. In insects, the activation of the immune system upon microbial challenge can be controlled by signaling pathways such as the immune deficiency (IMD) and Toll, to combat infection. These activated pathways signal to downstream NF-κB TF to stimulate specific immune genes, triggering the synthesis of several molecules such as the antimicrobial peptides. In Drosophila melanogaster, the activation and regulation of NF-κB TF have been elucidated, while in triatomines these mechanisms are not fully understood Therefore, the present study investigated the effects of oral administration of the drug IMD-0354 on the R. prolixus immune response to challenge with bacteria and T. cruzi, as well as the impact on the gut bacterial microbiota. R. prolixus were fed with rabbit blood containing IMD-0354 and Escherichia coli, Staphylococcus aureus, or T. cruzi. The effects of IMD-0354 on insect mortality and antimicrobial activity in insect midgut samples, as well as the relative expression of R. prolixus immune genes were recorded. The bacterial microbiota was analyzed, and viable parasites were counted in insect midgut samples. The IMD-0354 treatment modulated antibacterial activity and the gene expression patterns of defensin A, defensin B, defensin C, and prolixicin, and the genes involved in the IMD and Toll pathways. Additionally, there was an increase of bacterial microbiota in treated insects. Insects treated with IMD-0354 and concomitantly infected with bacteria or T. cruzi through the blood meal had increased mortality, while the T. cruzi population in R. prolixus midgut was reduced. The inhibitory effect of IMD-0354 indicates the importance of NF-κB TF in the innate immune responses involved in the control of bacteria and parasite infections in the R. prolixus midgut.