The role of the peritrophic matrix and red blood cell concentration in Plasmodium vivax infection of Anopheles aquasalis

Abstract

Background: Plasmodium vivax is predominant in the Amazon region, and enhanced knowledge of its development inside a natural vector, Anopheles aquasalis, is critical for future strategies aimed at blocking parasite development. The peritrophic matrix (PM), a chitinous layer produced by the mosquito midgut in response to blood ingestion, is a protective barrier against pathogens. Plasmodium can only complete its life-cycle, and consequently be transmitted to a new host, after successfully passing this barrier. Interestingly, fully engorged mosquitoes that had a complete blood meal form a thicker, well-developed PM than ones that feed in small amounts. The amount of red blood cells (RBC) in the blood meal directly influences the production of digestive enzymes and can protect parasites from being killed during the meal digestion. A specific study interrupting the development of the PM associated with the proteolytic activity inhibition, and distinct RBC concentrations, during the P. vivax infection of the New World malaria vector An. aquasalis is expected to clarify whether these factors affect the parasite development.

Results: Absence of PM in the vector caused a significant reduction in P. vivax infection. However, the association of chitinase with trypsin inhibitor restored infection rates to those of mosquitoes with a structured PM. Also, only the ingestion of trypsin inhibitor by non-chitinase treated mosquitoes increased the infection intensity. Moreover, the RBC concentration in the infected P. vivax blood meal directly influenced the infection rate and its intensity. A straight correlation was observed between RBC concentrations and infection intensity.

Conclusions: This study established that there is a balance between the PM role, RBC concentration and digestive enzyme activity influencing the establishment and development of P. vivax infection inside An. aquasalis. Our results indicate that the absence of PM in the midgut facilitates digestive enzyme dispersion throughout the blood meal, causing direct damage to P. vivax. On the other hand, high RBC concentrations support a better and thick, well-developed PM and protect P. vivax from being killed. Further studies of this complex system may provide insights into other details of the malaria vector response to P. vivax infection.

Keywords: Chitinase; Hematocrit; Malaria; Peritrophic matrix; Plasmodium vivax; Trypsin.

Fig. 1

Morphology of An. aquasalis midguts after normal and chitinase-containing blood meals. Histology (a) and SEM (c) of An. aquasalis midguts 24 h after a normal blood meal. The thick PM is visible isolating the midgut epithelium from the partially digested blood meals. Similar histology (b) and SEM (d) of An. aquasalis midguts 24 h after a chitinase-containing blood meal. The PM is absent inside the midgut, and the blood meal is in direct contact with the epithelium. Abbreviations: PM, peritrophic matrix; Ep, epithelium; Blood, blood meal. Scale-bars: a-d, 50 μm

Fig. 2

Effect of exogenous chitinase and trypsin enzyme inhibitor on the infection of Anopheles aquasalis with Plasmodium vivax. The intensity of infection of each experimental group is presented in the top graph as the oocyst number per midgut (dots), the black lines represent the mean (Kruskal-Wallis H-test: χ² = 41.333, df = 3, P < 0.0001). The infection rate is represented in the bottom graph as the percentage of P. vivax infected mosquitoes (light blue pie section) (ANOVA: F_{(3, 8)} = 10.63, P = 0.0036)

Fig. 3

Effect of blood meal hematocrit (RBC concentration) on the infection of An. aquasalis with P. vivax. The intensity of infection of each experimental group (red 15% hematocrit, green 30% hematocrit, blue 40% hematocrit) is presented in the top graph as the oocyst number per midgut (dots), the black lines represent the mean (Kruskal-Wallis H-test: χ² = 19.090, df = 2, P < 0.0001). The infection rate is represented in the bottom graph as the percentage of P. vivax infected mosquitos (light blue pie section) (Kruskal-Wallis H-test: χ² = 8.578, df = 2, P = 0.0093)