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

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.Aedes aegypti, the main vector of dengue, chikungunya, and yellow fever viruses, is an anautogenous mosquito that requires a blood meal for egg development. Female mosquitoes probe the skin of a suitable vertebrate host to acquire a blood meal. Hematophagy (blood feeding) to repletion usually takes from seconds to a few minutes (1). The stylets used to pierce the vascular beds of the vertebrate hosts cause local damage that triggers host hemostatic responses.Primary hemostasis is characterized by vascular contraction, platelet adhesion, and formation of a soft aggregate plug that begins immediately after endothelial disruption. Injury causes temporary local contraction of vascular smooth muscle. Vasoconstriction slows blood flow, enhancing platelet adhesion and activation. The soft platelet plug is stabilized to form a clot during secondary hemostasis (2, 3). Hematophagic arthropods have evolved a complex mixture of salivary components that counteract platelet aggregation, blood coagulation, vasoconstriction, and inflammation to overcome this efficient and redundant vertebrate hemostatic system (4–6).Aedes aegypti, Anopheles stephensi, and Simulim nigrimanum express salivary collagen-binding proteins that prevent collagen-induced platelet aggregation and adhesion by blocking its interaction with the platelet receptors glycoprotein VI and Integrin α₂β₁, and von Willebrand factor (vWF) (7–10). Aegyptin is an A. aegypti salivary protein that binds directly through its C-terminal domain to the vWF binding site in collagen (7, 8). Moreover, Aegyptin prevents carotid thrombus formation in vivo without causing excessive bleeding in mice.We used a transgenic approach to reduce the products of the Aegyptin gene (also known as the 30K b gene; ref. 11) to study its relevance in blood feeding. Our results show that reduced levels of Aegyptin mRNAs and protein result in a phenotype in which the probing times of transgenic mosquitoes are increased significantly, and their salivary gland extracts fail to inhibit collagen-induced platelet aggregation in vitro. Reductions in Aegyptin did not affect either the salivary gland anticoagulant or ADP-induced platelet aggregation activities. We interpret these data to indicate that the altered feeding capabilities of transgenic insects results from their inability to inhibit collagen-induced platelet aggregation.Although mosquito salivary antihemostatic activity has been demonstrated, it remains to be proven whether each particular salivary component is relevant or necessary to acquire a successful blood meal. It was proposed that redundancy of salivary function reinforces the efficiency of blood feeders (12, 13). However, our results do not support this general hypothesis. It appears that the diversity and complexity of antihemostatic proteins found in salivary secretions is not entirely redundant because reduction of Aegyptin alone impairs significantly the ability of the mosquito to acquire a blood meal.     

The birth of Dolly and xenotransplantation 25 years on

A number of reviews have been written recently celebrating the 25th anniversary of the birth of Dolly the cloned sheep and the effect this breakthrough has had on various fields of research. However, arguably the biggest impact Dolly has had is on the field of xenotransplantation, described here based on our own experience and that of others.     

Clonal segregation of oligodendrocytes and astrocytes during in vitro differentiation of glial progenitor cells.

To study the clonal lineage of the glial progenitor population, isolated from newborn rat brain (Lubetzki et al. J Neurochem 56:671, 1991), we combined somatic transgenesis using a retroviral vector encoding a modified bacterial beta-galactosidase with nuclear localization, and triple immunofluorescence labeling with A2B5, anti-galactosylceramide, and anti-glial acidic fibrillary protein antibodies. This allowed clonal analysis of the postnatal glial lineage with precise phenotypic identification of each cell within the lacZ-positive clones. When infected cells were cultivated under constant conditions, in the presence of either 1% or 10% fetal calf serum (FCS)-containing medium, all the 250 lacZ-positive clusters examined were homogeneous, i.e., either oligodendroglial or astroglial. Mixed astrocyte-oligodendroglial clones were observed when cells cultivated in the presence of 1% FCS were switched to a 10% FCS-containing medium, confirming the bipotentiality of glial progenitor cells (Temple and Raff Nature 313:223, 1985). However, even under the switch culture conditions, segregation into homogeneous clones of either oligodendrocytes or astrocytes still predominated, and the percentage of mixed clones dropped from 25 to 8 or to 3, when the switch took place at 8, 16, or 22 days in vitro, respectively. Two additional observations lead us to suggest that microenvironmental factors are responsible for the clonal segregation of glial progenitor cells: 1) the uneven distribution of oligodendrocyte and astrocyte clusters, the latter being seen mostly on the edge of the coverslips; and 2) the presence, in the vicinity of an homogeneous lacZ-positive clone, of some lacZ-negative cells expressing the same phenotype.     

Establishing genetic transformation for comparative developmental studies in the crustacean Parhyale hawaiensis

The amphipod crustacean Parhyale hawaiensis has been put forward as an attractive organism for evolutionary developmental comparisons, and considerable effort is being invested in isolating developmental genes and studying their expression patterns in this species. The scope of these studies could be significantly expanded by establishing means for genetic manipulation that would enable direct studies of gene functions to be carried out in this species. Here, we report the use of the Minos transposable element for the genetic transformation of P. hawaiensis. Transformed amphipods can be obtained from approximately 30% of surviving individuals injected with both a Minos element carrying the 3xP3-DsRed fluorescent marker and with mRNA encoding the Minos transposase. Integral copies of the transposon are inserted into the host genome and are stably inherited through successive generations. We have used reporter constructs to identify a muscle-specific regulatory element from Parhyale, demonstrating that this transformation vector can be used to test the activity of cis-regulatory elements in this species. The relatively high efficiency of this transgenic methodology opens new opportunities for the direct study of cis-regulatory elements and gene functions in Parhyale, allowing functional studies to be carried out beyond previously established model systems in insects.