Collagen-binding protein, Aegyptin, regulates probing time and blood feeding success in the dengue vector mosquito, Aedes aegypti

Abstract

Mosquito salivary glands have important roles in blood feeding and pathogen transmission. However, the biological relevance of many salivary components has yet to be determined. Aegyptin, a secreted salivary protein from Aedes aegypti, binds collagen and inhibits platelet aggregation and adhesion. We used a transgenic approach to study the relevance of Aegyptin in mosquito blood feeding. Aedes aegypti manipulated genetically to express gene-specific inverted-repeat RNA sequences exhibited significant reductions in Aegyptin mRNA accumulation (85-87%) and protein levels (>80-fold) in female mosquito salivary glands. Transgenic mosquitoes had longer probing times (78-300 s, P < 0.0001) when feeding on mice compared with controls (15-56 s), feeding success was reduced, and those feeding took smaller blood meals. However, no differences in feeding success or blood meal size were found in membrane feeding experiments using defibrinated human blood. Salivary gland extracts from transgenic mosquitoes failed to inhibit collagen-induced platelet aggregation in vitro. Reductions of Aegyptin did not affect salivary ADP-induced platelet aggregation inhibition or disturb anticlotting activities. Our results demonstrate the relevance of Aegyptin for A. aegypti blood feeding, providing further support for the hypothesis that platelet aggregation inhibition is a vital salivary function in blood feeding arthropods. It has been suggested that the multiple mosquito salivary components mediating platelet aggregation (i.e., Aegyptin, apyrase, D7) represent functional redundancy. Our findings do not support this hypothesis; instead, they indicate that multiple salivary components work synergistically and are necessary to achieve maximum blood feeding efficiency.

Keywords: RNAi; evolution; hematophagy; saliva; transgenesis.

Fig. 1.

Schematic representations of the dsAegyptin and 30KExGM transgenes. (A) The plasmid containing the transgene dsAegyptin is engineered to express an anti-Aegyptin dsRNA under the control of the 30K bidirectional promoter. 30K a drives the expression of the reporter gene, eGPF, and 30K b, the Aegyptin promoter, drives the Aegyptin-specific dsRNA. The transformation marker gene DsRED is under control of the 3XP3 eye-specific promoter (11). (B) The transgene inserted in the control transgenic line 30KExGM has a similar organization and was engineered to express Mnp, an antidengue dsRNA. Detailed descriptions of pMos_dsAegyptin and pMos_30KExGM are in Fig. S1 and Mathur et al. (11), respectively. Dashed arrows indicate direction of transcription.

Fig. 2.

Quantification of Aegyptin gene expression in transgenic mosquitoes. (A) Accumulation of Aegyptin mRNA in salivary glands was quantified by using qRT-PCR. Expression of dsRNAi in dsAegyptin mosquitoes reduced significantly the Aegyptin mRNA level relative to the 30KExGM control (P < 0.0001). Biological replicates (n = 3) containing 20 salivary gland pairs each were assayed in duplicate. (B) dsRNA-mediated silencing of Aegyptin transcription products reduces significantly corresponding protein levels in transgenic dsAegyptin mosquitoes. ****P < 0.0001. n.s, not significant.

Fig. 3.

Suppressing Aegyptin transcription products results in the reduction of collagen binding activity of salivary gland extracts and saliva. SPR experiments were carried out to evaluate collagen binding activity of salivary glands and saliva of transgenic dsAegyptin mosquitoes. (A) Saliva of dsAegyptin mosquitoes showed lower collagen-binding capacity compared with control mosquitoes (Higgs and 30KExGM). (B) Single mosquito salivary gland pairs were homogenized in Hepes saline-buffered solution supplemented with 0.05% (vol/vol) surfactant P20 and flowed over immobilized collagen (HBS-V). Salivary gland homogenates from dsAegyptin have a significantly lower collagen-binding activity than Higgs and 30KExGM control mosquitoes. **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s, not significant.

Fig. 4.

Effect of reduced Aegyptin on platelet aggregation and blood clotting. (A) Salivary gland extracts of dsAegyptin mosquitoes failed to inhibit collagen-induced platelet aggregation. Platelet-rich plasma was preincubated with two pairs of salivary glands for 60 s before adding 2 µg/mL of fibrillar collagen. Platelet aggregation was measured by light transmittance. (B) Suppressing Aegyptin did not impair the ability of salivary gland homogenates to inhibit ADP-induced platelet aggregation. (C) Prothrombin time was measured in a coagulometer by using normal reference plasma from healthy donors. Homogenates of two pairs of salivary gland were incubated with human plasma for 60 s before triggering clotting with thromboplastin reagent. No significant differences were found in the anticoagulant activity of salivary gland homogenates from dsAegyptin, Higgs and 30KExGM mosquitoes. All samples were assayed in triplicate. Arrows indicate addition of either collagen (A) or ADP (B). ***P < 0.001.

Fig. 5.

Probing time on mice is prolonged by reduced Aegyptin in transgenic mosquitoes. Probing time was significantly longer in dsAegyptin mosquitoes then in controls (n = 24, P < 0.0001). Transgenic mosquitoes expressing dsRNAi had the first sign of blood in their midgut at least 78 s from insertion of their mouth part in the host’s skin, whereas control mosquitoes were able to start ingesting blood at 15–21 s. dsAegyptin mosquitoes with a probing time of more than 300 s were not included in the statistical analysis. Horizontal bars represent the median value. ****P < 0.0001.

Fig. 6.

Feeding success of transgenic mosquitoes on mice and artificial membranes. (A) Reduced levels of Aegyptin significantly lowered the feeding success of mosquitoes on anesthetized mice. Percentages of successful feeding were analyzed by using a χ² test. (B) Transgenic mosquitoes expressing dsRNAi against Aegyptin mRNA had significantly smaller blood content in their abdomens. Blood meal size (expressed as OD_540nm units) ingested by mosquitoes that fed on mice was quantified by measuring the total hemoglobin content using Drabkin’s reagent as described in Materials and Methods. (C) dsAegyptin mosquitoes did not differ in their feeding success compared with controls when fed on an artificial membrane and a bloodmeal lacking collagen and collagen-induced platelet activation and aggregation. (D) Measurement of blood meal size revealed that dsAegyptin and 30KExGM had ingested similar amounts of blood when fed on artificial membrane system. Results were analyzed by using a χ² test with 95% confidence interval. ***P < 0.001 and ****P < 0.0001. n.s, not significant.