Yara Maria Traub-Csekö

The 11th International Symposium on Phlebotomine Sand flies (ISOPS XI) took place in Portorož, Slovenia, in September 2024 and brought together experts from around the world to discuss recent advances in the biology, ecology and control of phlebotomine sand flies and the pathogens they transmit. This report summarises the key findings of the symposium and is organised thematically by session. Key topics included the development of refined experimental models of Leishmania transmission, new insights into the interactions between vector, parasite and microbiota, and the detection of Leishmania donovani in new geographic regions. Advances in molecular diagnostics and surveillance technologies were emphasised, as were emerging concerns about insecticide resistance. The potential of paratransgenesis and symbiont-based vector control approaches was also emphasised. In a separate session, the CLIMOS project was presented, which integrates climate monitoring, ecological modelling and public health tools to develop an early warning system (EWS) for sand fly-borne diseases. Overall, the contributions to the symposium reflect the dynamic development of sand fly research in response to global environmental change and emphasise the importance of international collaboration in combating emerging vector-borne diseases.

There is increasing evidence on the occurrence of Crithidia spp. in patients presenting either cutaneous or visceral leishmaniasis, solely or associated with Leishmania. We analyzed growth, morphology, and temperature tolerance of two C. fasciculata strains, the reference strain COLPROT048 and patient isolate COLPROT606. We also evaluated their co-cultivation with L. braziliensis, macrophage infectivity, and infections in hamsters, BALB/c mice, and sandflies. In culture, both Crithidia strains survived at 32 °C for 96 h, showing major morphological alterations and decreased mitochondrial membrane potential, with ΔΨm reducing to 52% in COLPROT606. At 34 °C, the patient isolate showed an 80% reduction in cell number. Mixed cultivation of Crithidia-Leishmania led to recovery of only Crithidia. In macrophages, C. fasciculata alone was virtually eliminated, and in co-infection only Leishmania was detected. No Crithidia lesion or RNA were found in infected mice or hamsters, while L. braziliensis reached 1145–1625 parasites/mg of tissue. In sandflies, C. fasciculata successfully established infection for up to 7 days, both alone and in coinfections. Amphotericin B IC₅₀ values at 72 h were 4- to 5-fold higher in C. fasciculata strains compared to L. braziliensis. Our results indicate that both C. fasciculata strains are unable to reproduce the pathogenic effect in vitro and in vivo models.

Sand flies, which transmit diseases like leishmaniases, bartonellosis, and certain viruses, pose a significant public health threat. Our research focuses on the immune responses of Lutzomyia longipalpis, the primary vector for visceral leishmaniasis in the Americas. We use L. longipalpis LL5 cells as a model to study how sand flies respond to pathogens. These cells exhibit robust immune reactions, producing molecules mainly regulated by the Toll, IMD, Jak-STAT, and RNAi pathways. In previous studies, we detected a non-specific antiviral response in LL5 cells following double-stranded RNAs (dsRNAs) transfection. A previous complete secretome of these cells showed molecules resembling an interferon-like antiviral response when transfected with polyinosinic–polycytidylic acid (poly I:C), a synthetic dsRNA analog. In the current study, we analyzed soluble proteins secreted by LL5 cells after poly I:C transfection. Using comparative mass spectrometry, we examined protein composition of conditioned media depleted of exosomes at 24 h and 48 h. Most proteins uniquely expressed in the transfected groups had low abundance compared to the overall expressed proteins. Interactome prediction analysis revealed that at 24 h, the proteins uniquely found in the secretome of the transfected group were involved in RNA degradation and purine metabolism, while at 48 h they were linked to ribosomal proteins and signaling pathways such as Hedgehog, Transforming Growth Factor-beta (TGF-β), and Wingless/integrated (Wnt). We highlight increased abundance of the TGF-β-induced protein ig-h3 (24 h and 48 h), a Toll-like receptor 3 (48 h), and a hemocytin (48 h) in the secretion of transfected groups compared to the controls. We also performed an interaction analysis of proteins more secreted by the treated group at 24 h and 48 h. Unlike the interactome of uniquely identified proteins, few interactions were observed at 24 h, with a predominance of extracellular matrix and cell adhesion proteins. The set of proteins more secreted at 48 h presented more interactions than at 24 h, with emphasis on catabolic processes, including RNA degradation. These findings indicate that poly I:C transfection in LL5 cells induces the secretion of proteins involved in cellular defense and repair, revealing molecules involved in the LL5 non-specific antiviral response.

Background: Sand flies of the species Nyssomyia umbratilis (Diptera: Psychodidae: Phlebotominae) are vectors of the parasite Leishmania (Viannia) guyanensis, one of the etiological agents of cutaneous leishmaniasis in the Amazon region. In addition, Ny. umbratilis is a cryptic species, with populations showing differences in their ability to transmit the parasite. For instance, populations of Ny. umbratilis from the Manacapuru municipality (MAN), located on the south bank of the Negro river, in the Amazonas state of Brazil, shows refractoriness to Leishmania infection, while populations from Rio Preto da Eva municipality (RPE), located on the north bank of the Negro river, are susceptible to infection. This lack of vectorial capacity may be caused by several factors, including the intestinal bacterial microbiota of sand flies. Methods: In this work, we carried out a metagenomic study of the intestinal microbiota of Ny. umbratilis populations from MAN and RPE. Ny. umbratilis females were collected in forested areas, sand fly midguts were dissected, DNA was extracted, and the 16 S rRNA gene sequenced to identify the bacterial composition of the microbiota. Results: In total, 16 phyla, 33 classes, 49 orders, 93 families, and 112 genera of bacteria were identified. The phylum Proteobacteria was the most frequent (85.9%) in both localities, followed by the phyla Bacteroidetes, Actinobacteria, and Firmicutes with, 9.9%, 4.9%, and 4.4%, respectively. In MAN, 84 genera were identified and 79 in RPE, with MAN having a greater richness compared with RPE. Among these, the genera Rickettsia, Prevotella, Porphyromonas, Peptostreptococcus, and Caulobacter were the most prevalent in MAN, and the genera Rickettsia, Prevotella, Cryocola, Porphyromonas, and Caulobacter were the most prevalent in RPE. Conclusions: Bacterial microbiota from MAN insects presents a greater diversity in relation to the RPE insects. Some of the identified bacteria have the potential to be used in alternative transmission control approaches as the development of transgenic vectors, and also, bacteria found exclusively in MAN sand flies may be candidates for a future transmission control approach to combat leishmaniasis in the Amazon region. Graphical

There is increasing evidence on the occurrence of Crithidia spp. in patients presenting either cutaneous or visceral leishmaniasis, solely or associated with Leishmania. We analyzed the influence of temperature in the growth rate and morphology of two Crithidia fasciculata strains (a reference strain and one isolated from a patient), and the effect of the co-cultivation of Leishmania and Crithidia in parasite isolation, in the infection of macrophages in vitro, and also in infections of hamsters, BALB/c mice and sandflies. In culture, both Crithidia strains could undergo 32oC for 96 h, although major morphological alterations and a decrease in mitochondrial membrane potential were observed. At 34°C, there was an 80% reduction on the number of cells from the patient strain. Mixed cultivation of Crithidia-Leishmania led to the recovery of only Crithidia. In macrophages, C. fasciculata alone was virtually eliminated, and in the co-infection only Leishmania was recovered. The same was observed in vivo. Curiously, C. fasciculata is more resistant to Amphotericin B. Our results indicate that both C. fasciculata strains are unable to reproduce the pathogenic effect in vitro and in vivo models.

The northern region of Brazil is endemic for American Tegumentary Leishmaniasis (ATL) primarily caused by Leishmania guyanensis and transmitted by the sand fly Nyssomyia umbratilis. The disease occurs at different rates in the municipalities of Manacapuru (MAN) and Rio Preto da Eva (RPE), located in the state of Amazonas. Despite their geographic proximity and separation by the Rio Negro, MAN has a low incidence, whereas RPE reports a significantly higher number of cases. Since the vector is present in both locations, potential biological differences in N. umbratilis may influence transmission. Previous studies suggested genotypic and phenotypic differences in N. umbratilis from both localities. To investigate the molecular factors underlying their potentially differential vectorial capacities, we performed a comparative proteomic analysis of dissected insect intestines from both localities. Our results revealed that sand flies from MAN showed a higher abundance of proteins related to gene transcription, protein translation, amino acid and proton transport, innate immune response and intestinal motility. Since the importance of microbiota has previously been shown in parasite–vector interactions, we also identified bacteria from both vector populations. We detected bacteria specific to each population and, exclusively in MAN, some species described in the literature as having parasiticidal properties. These findings highlight molecular and microbial peculiarities that could contribute to the observed difference in ATL prevalence in the two areas.

Sandflies are known vectors of leishmaniasis. In the Old World, sandflies are also vectors of viruses while little is known about the capacity of New World insects to transmit viruses to humans. Here, we relate the identification of RNA sequences with homology to rhabdovirus nucleocapsids (NcPs) genes, initially in the Lutzomyia longipalpis LL5 cell lineage, named NcP1.1 and NcP2. The Rhabdoviridae family never retrotranscribes its RNA genome to DNA. The sequences here described were identified in cDNA and DNA from LL-5 cells and in adult insects indicating that they are transcribed endogenous viral elements (EVEs). The presence of NcP1.1 and NcP2 in the L. longipalpis genome was confirmed in silico. In addition to showing the genomic location of NcP1.1 and NcP2, we identified another rhabdoviral insertion named NcP1.2. Analysis of small RNA molecules derived from these sequences showed that NcP1.1 and NcP1.2 present a profile consistent with elements targeted by primary piRNAs, while NcP2 was restricted to the degradation profile. The presence of NcP1.1 and NcP2 was investigated in sandfly populations from South America and the Old World. These EVEs are shared by different sandfly populations in South America while none of the Old World species studied presented the insertions.

Introduction: Sand flies (Diptera: Phlebotominae) belonging to the Lutzomyia genus transmit Leishmania infantum parasites. To understand the complex interaction between the vector and the parasite, we have been investigating the sand fly immune responses during the Leishmania infection. Our previous studies showed that genes involved in the IMD, Toll, and Jak-STAT immunity pathways are regulated upon Leishmania and bacterial challenges. Nevertheless, the parasite can thrive in the vectors’ gut, indicating the existence of mechanisms capable of modulating the vector defenses, as was already seen in mammalian Leishmania infections.Methods, results, and discussion: In this study, we investigated the expression of Lutzomyia longipalpis genes involved in regulating the Toll pathway under parasitic infection. Leishmania infantum infection upregulated the expression of two L. longipalpis genes coding for the putative repressors cactus and protein tyrosine phosphatase SHP. These findings suggest that the parasite can modulate the vectors’ immune response. In mammalian infections, the Leishmania surface glycoprotein GP63 is one of the inducers of host immune depression, and one of the known effectors is SHP. In L. longipalpis we found a similar effect: a genetically modified strain of Leishmania amazonensis over-expressing the metalloprotease GP63 induced a higher expression of the sand fly SHP indicating that the L. longipalpis SHP and parasite GP63 increased expressions are connected. Immuno-stained microscopy of L. longipalpis LL5 embryonic cells cultured with Leishmania strains or parasite conditioned medium showed cells internalization of parasite GP63. A similar internalization of GP63 was observed in the sand fly gut tissue after feeding on parasites, parasite exosomes, or parasite conditioned medium, indicating that GP63 can travel through cells in vitro or in vivo. When the sand fly SHP gene was silenced by RNAi and females infected by L. infantum, parasite loads decreased in the early phase of infection as expected, although no significant differences were seen in late infections of the stomodeal valve.Conclusions: Our findings show the possible role of a pathway repressor involved in regulating the L. longipalpis immune response during Leishmania infections inside the insect. In addition, they point out a conserved immunosuppressive effect of GP63 between mammals and sand flies in the early stage of parasite infection.

We previously showed that L. (Leishmania) amazonensis promastigotes and amastigotes of the PH8 strain generated larger lesions in mice than LV79, and that lesion-derived amastigotes from the two strains differ in their proteomes. We recently reported that PH8 promastigotes are more phagocytized by macrophages. Promastigotes’ membrane-enriched proteomes showed several differences, and samples of each strain clustered based on proteomes. In this paper, we show phenotypic differences between PH8 and LV79 promastigotes that may explain the higher virulence of PH8. We compared in vitro macrophage infections by day 4 (early) and day 6 (late stationary phase) cultures, resistance to complement, and LPG characteristics. PH8 promastigotes showed a higher infectivity and were more resistant to murine complement. LPG was different between the strains, which may influence the interaction with macrophages and survival to complement. We compared the infection of the permissive vector Lutzomyia longipalpis. PH8 was more abundant in the vector’s gut 72 h after feeding, which is a moment where blood digestion is finished and the parasites are exposed to the gut environment. Our results indicate that PH8 promastigotes are more infective, more resistant to complement, and infect the permissive vector more efficiently. These data suggest that PH8 is probably better adapted to the sand fly and more prone to survive in the vertebrate host.