(Proboscis had been observed at this site just prior to initiation of time-lapse video.) Bar?=?100 m for all frames. relatively long exposure times of 1 1 sec per frame were required. Bar?=?100 m for all videos.(2.26 MB MOV) ppat.1000399.s001.mov (2.1M) GUID:?0E607522-CEBA-49AA-8BF0-0297F2F9E0E7 Video S2: Intravital fluorescence video showing mosquito proboscis introducing saliva into mouse ear pinna after passive immunization with fluorescent antibodies (heterologous antigen and antibodies). Mosquitoes had salivary gland infection of sporozoites. Mouse had received passive IV transfer of anti-sporozoite monoclonal antibodies conjugated with Tedizolid (TR-701) FITC. Saliva contained sporozoites in addition to secreted, soluble CS protein. Probing proboscis is visualized as orange due to autofluorescence. No proboscis-associated green densities were ever seen during our observations.(1.30 MB MOV) ppat.1000399.s002.mov (1.2M) GUID:?CDF2176F-9F41-4732-8BE8-E24B5EC6733E Video S3: Intravital fluorescence video showing mosquito proboscis introducing saliva into mouse ear pinna after passive immunization with antibodies (negative control). Mosquitoes had no salivary gland infections with sporozoites. Mouse had received passive IV transfer of anti-sporozoite monoclonal antibodies conjugated with FITC. Saliva contained neither sporozoites nor sporozoite antigen. Probing proboscis is visualized as orange due to autofluorescence. No proboscis-associated green densities were ever seen during our observations, although secreted droplets of saliva (arrows) can be seen associated with two of the three probes within this sequence.(2.31 MB MOV) ppat.1000399.s003.mov (2.2M) GUID:?454C6C63-0EF7-4275-A6CD-E8A0135464BD Video S4: Secretion of saliva and sporozoites from proboscis of immobilized mosquito into medium on microscope slide: ADFP Medium contained FITC-conjugated BSA. Sporozoites were freely released Tedizolid (TR-701) into medium. Secretion of saliva can be seen Tedizolid (TR-701) as a dark globule at the tip of proboscis. Green streak running along middle of distal end of proboscis indicates that some medium was being sucked back by mosquito. (Real-time duration of video was 5 min, 32 sec; each frame was captured for 1 sec.)(5.52 MB AVI) ppat.1000399.s004.avi (5.2M) GUID:?D37EEB4F-C99C-4BA4-88BA-5DFAD947888E Video S5: Secretion of saliva and sporozoites from proboscis of immobilized mosquito into medium on microscope slide: Medium contained FITC-conjugated 3D11. Relatively few sporozoites were released into medium. Some of these have become coated with antibody and fluoresce green. Green plug at end of proboscis is associated with stasis of unreleased, red-fluorescing sporozoites within proboscis, suggesting formation of an immune complex that inhibits Tedizolid (TR-701) sporozoite release. (Real-time duration of Video S5 was 2 min, 22 sec; each frame was captured for 1 sec.)(4.31 MB AVI) ppat.1000399.s005.avi (4.1M) GUID:?404DCE59-08B2-4316-993B-1CDDB76E5883 Video S6: Secretion of saliva and sporozoites from proboscis of immobilized mosquito into Tedizolid (TR-701) medium on microscope slide: Medium contained FITC-conjugated 3D11. Relatively few sporozoites were released into medium. Some of these have become coated with antibody and fluoresce green. Green plug at end of proboscis is associated with stasis of unreleased, red-fluorescing sporozoites within proboscis, suggesting formation of an immune complex that inhibits sporozoite release. (Real-time duration of Video S6 was 3 min, 28 sec; each frame was captured for 1 sec.)(3.44 MB AVI) ppat.1000399.s006.avi (3.2M) GUID:?3B1AE716-436F-4BAD-9D2E-D07BBAF651C1 Abstract Malaria is initiated when the mosquito introduces sporozoites into the skin of a mammalian host. To successfully continue the infection, sporozoites must invade blood vessels in the dermis and be transported to the liver. A significant number of sporozoites, however, may enter lymphatic vessels in the skin or remain in the skin long after the mosquito bite. We have used fluorescence microscopy of sporozoites expressing a fluorescent protein to evaluate the kinetics of sporozoite disappearance from the skin. Sporozoites injected into immunized mice were rapidly immobilized, did not appear to invade dermal blood vessels and became morphologically degraded within several hours. Strikingly, mosquitoes introduced significantly fewer sporozoites into immunized than into non-immunized mice, presumably by formation of an immune complex between soluble sporozoite antigens in the mosquito saliva and homologous host antibodies at the proboscis tip. These results indicate that protective antibodies directed against sporozoites may function.