Description of three new species of frog–biting midges (Diptera: Corethrellidae) from the Central Brazilian Amazon

Three species of Corethrella Coquillett, 1902 from the state of Amazonas, Brazil are described as new to science based on female adult specimens. Corethrella cabocla Feijó, Belchior, Marialva & Pessoa sp. nov. possesses four large setae on the frons between the ventromedial area of ommatidia, a wide clypeus with 1–4 setae, a wing with the apex of R2 basal to the apex of M2 and with a midlength band, and with the abdomen entirely dark brown. Corethrella ielemdei Feijó, Ramires, Lima & Pessoa sp. nov. possesses an elongated coronal suture, four large setae on the frons between the ventromedial area of ommatidia, a clypeus squarish with 42–43 setae, a wing with the apex of R2 basal to the apex of M1 and with a midlength band and dark scales on the basal and subbasal areas of the anterior margin, legs with dark scales, and with the abdomen entirely dark brown. Corethrella menini Feijó, Picelli, Ríos-Velásquez & Pessoa sp. nov. possesses wings with the apex of R2 basal to the apex of M2 and a midlength band, with darker basal scales along all veins, basal band dark scales on C, Sc, R, M, and Cu and the abdomen entirely dark brown. With the addition of the new species, the numbers of frog-biting midges described in the Amazon basin, Brazil and in Neotropical region are now 31, 49 and 80 species, respectively

The Role of Reactive Oxygen Species in <em>Anopheles aquasalis</em> Response to <em>Plasmodium vivax</em> Infection

<div><p>Malaria affects millions of people worldwide and hundreds of thousands of people each year in Brazil. The mosquito <i>Anopheles aquasalis</i> is an important vector of <i>Plasmodium vivax</i>, the main human malaria parasite in the Americas. Reactive oxygen species (ROS) have been shown to have a role in insect innate immune responses as a potent pathogen-killing agent. We investigated the mechanisms of free radicals modulation after <i>A. aquasalis</i> infection with <i>P. vivax</i>. ROS metabolism was evaluated in the vector by studying expression and activity of three key detoxification enzymes, one catalase and two superoxide dismutases (SOD3A and SOD3B). Also, the involvement of free radicals in the mosquito immunity was measured by silencing the catalase gene followed by infection of <i>A. aquasalis</i> with <i>P. vivax</i>. Catalase, SOD3A and SOD3B expression in whole <i>A. aquasalis</i> were at the same levels of controls at 24 h and upregulated 36 h after ingestion of blood containing <i>P. vivax</i>. However, in the insect isolated midgut, the mRNA for these enzymes was not regulated by <i>P. vivax</i> infection, while catalase activity was reduced 24 h after the infectious meal. RNAi-mediated silencing of catalase reduced enzyme activity in the midgut, resulted in increased <i>P. vivax</i> infection and prevalence, and decreased bacterial load in the mosquito midgut. Our findings suggest that the interactions between <i>A. aquasalis</i> and <i>P. vivax</i> do not follow the model of ROS-induced parasite killing. It appears that <i>P. vivax</i> manipulates the mosquito detoxification system in order to allow its own development. This can be an indirect effect of fewer competitive bacteria present in the mosquito midgut caused by the increase of ROS after catalase silencing. These findings provide novel information on unique aspects of the main malaria parasite in the Americas interaction with one of its natural vectors.</p> </div

Characterization of Catalase cDNA.

<p>A: Schematic representation of <i>A. aquasalis</i> catalase (AqCAT) deduced protein. Red - clade 3 of the heme-binding catalase domain. B: Phylogenetic tree for catalase constructed based on the neighbor-joining method. C: Multiple aminoacid sequence alignment of insect catalase related proteins. Accession numbers of catalase sequences from: <i>A. aquasalis</i> (Aq) (HQ659100), <i>A. gambiae</i> (Ag) (XP_314995.4), <i>A. aegypti</i> (Aa) (XP_001663600.1), <i>Culex quinquefasciatus</i> (Cq) (XP_001848573.1) and <i>D. melanogaster</i> (Dm) (NP_536731.1).</p

Molecular analysis of catalase silencing.

<p>A and B – Effect of dsCatalase (dsCat) injections on catalase mRNA expression (A) and activity (B) in the mosquito midgut. C – H₂O₂ production in the midgut of insects silenced for catalase and β-gal (control) genes. D – Prevalence of <i>P. vivax</i> infection in insects after β-gal and catalase dsRNA injections. E and F – Oocysts numbers in the midguts of dsβ-gal and dsCat injected mosquitoes 3–5 days after <i>Plasmodium</i> infection. The arrows show <i>P. vivax</i> oocysts in the <i>A. aquasalis</i> midgut. G: Bacterial load based on 16 s gene in the gut of mosquitoes silenced with dsCat and dsβ-gal. In figure A, data were analyzed by the ANOVA test with multiple comparisons of Tukey or Games-Howell or Kruskal-Wallis test with multiple comparisons of Dunn's; in figures B and C by unpaired t-test; in the figure E by the Mann-Whitney test.</p

Characterization of SOD3A and SOD3B cDNA.

<p>A: Schematic representation of SOD3A (A) and 3B (B) protein from <i>A. aquasalis</i> (AqSOD3A and SOD3B). Green: iron/manganese superoxide dismutases alpha-hairpin domain; blue: iron/manganese superoxide dismutases C-terminal domain; red: Cu-Zn_superoxide_dismutase domain. B: Phylogenetic tree for SOD constructed based on the neighbor-joining method. C: Multiple aminoacid sequence alignment of mosquito SOD related proteins. Accession numbers of SOD sequences from: <i>A. aquasalis</i> (Aq) (SOD3A - HQ659101 and SOD3B HQ659102), <i>A. gambiae</i> (Ag) (SOD1 - XP_314490.3, SOD2 - XP_314137.4, SOD3A - XP_311594.2 and SOD3B - XP_001230820.1).</p

Expression levels and activity of SOD3A and SOD3B in <i>A. aquasalis</i> according to gender and feeding regimens.

<p>A and D: mRNA relative expression of SOD3A (A) and SOD3B (E) in whole body of sugar-fed males and females, B and D: mRNA relative expression of SOD3A (B) and SOD3B (E) in whole body of sugar-fed females (dotted line), and females after blood feeding and after <i>P. vivax</i> infection. C and F: mRNA relative expression of SOD3A (C) and SOD3B (F) in midgut of sugar-fed females (dotted line), and females after blood feeding and after <i>P. vivax</i> infection. G: SOD activity in sugar-fed females, 24 h after blood feeding (BFC) and after <i>P. vivax</i> infection (BFI) reported as units per minute per micrograms of protein (U/mg ptn). Data are reported as the mean ± SEM. * p<0.05, ** 0.03>p>0.01, *** p<0.01. In figures A and D the data was analyzed by t-student or the Wilcoxon tests, in figures B,C, E and F by ANOVA test with multiple comparisons of Tukey or Games-Howell or Kruskal-Wallis test with multiple comparisons of Dunn's, and in figure G by ANOVA test with Dunnett's Multiple Comparison Test.</p

Additional file 4: of Infection of Anopheles aquasalis from symptomatic and asymptomatic Plasmodium vivax infections in Manaus, western Brazilian Amazon

Table S4. Plasmids dilutions containing the sequence of the respective PCR product were used both as assay standards and to determine the limit of detection (LoD) of each assay. Generation of the plasmids is described in [35, 38]. (DOC 29 kb

Additional file 2: of Infection of Anopheles aquasalis from symptomatic and asymptomatic Plasmodium vivax infections in Manaus, western Brazilian Amazon

Table S2. PCR cycling conditions used to detect parasites and gametocytes of Plasmodium vivax parasites. (DOC 32 kb

Additional file 1: of Infection of Anopheles aquasalis from symptomatic and asymptomatic Plasmodium vivax infections in Manaus, western Brazilian Amazon

Table S1. PCR setup of Plasmodium-specific qPCR (QMAL) and P. vivax specific qPCR based on detection of 18S rRNA genes and RT-qPCR detecting pvs25 transcripts. For sequences of primers and probes see [35, 38]. (DOC 31 kb

Additional file 3: of Infection of Anopheles aquasalis from symptomatic and asymptomatic Plasmodium vivax infections in Manaus, western Brazilian Amazon

Table S3. Data from each membrane feeding assay of Anopheles aquasalis performed with samples from symptomatic and asymptomatic individuals of Plasmodium vivax. (DOC 151 kb