Mosquito pornoscopy: Observation and interruption of <i>Aedes aegypti</i> copulation to determine female polyandric event and mixed progeny

<div><p>Ades aegypti is the most important arbovirus vector in the world, and new strategies are under evaluation. Biological studies mentioning the occurrence of a second mate in <i>Aedes aegypti</i> can interfere with vector control program planning, which involves male mosquito release technique. This study presents different experiments to show the occurrence of mixed progeny. Mixed male crosses (using a combination of different type of males in confinement with virgin females) showed no polyandric female. Individual crosses with male substitution in every gonotrophic cycle also did not show any polyandric female. Individual crosses with a 20 minutes interval, with subsequent male change, showed that only a few females presented mixed offspring. The copulation breach in three different moments, group A with full coitus length, group B the coitus was interrupted in 5–7 seconds after the start; and group C, which the copulation was interrupted 3 seconds after started. In summary, group A showed a majority of unique progeny from the first male; group B showed the higher frequency of mixed offspring and group C with the majority of the crosses belonging to the second male. To conclude, the occurrence of a viable second mate and mixed offspring is only possible when the copulation is interrupted; otherwise, the first mate is responsible for mixed progeny.</p></div

Average number of eggs, mean hatch rate and number of crosses with mixed/unique type of progeny in 20 minutes interval for first male cross.

<p>Average number of eggs, mean hatch rate and number of crosses with mixed/unique type of progeny in 20 minutes interval for first male cross.</p

Model probability distribution of crosses among the progeny type and the initial male.

<p>Model probability distribution of crosses among the progeny type and the initial male.</p

Number of crosses of each interrupted mating group according to the initial male.

<p>Number of crosses of each interrupted mating group according to the initial male.</p

Average number of eggs and hatch rate through a consecutive male change throughout different gonotrophic cycles and its progeny status.

<p>Average number of eggs and hatch rate through a consecutive male change throughout different gonotrophic cycles and its progeny status.</p

Total number of crosses among the progeny type and the initial male.

<p>Total number of crosses among the progeny type and the initial male.</p

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

Method illustration of the male exchange experiment.

<p>Method illustration of the male exchange experiment.</p

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

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