Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

Schmid, Michael; Kauffman, Elizabeth B.; Payne, Anne F.; Harris, Eva; Kramer, Laura D.

doi:10.21769/bioprotoc.2407

Cited by 16 publications

(14 citation statements)

References 15 publications

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“…As a result, severe undersampling of mosquito and pathogen populations remains a major concern around the globe 11 . In addition, molecular analysis of mosquitoes in the lab or field is usually performed using either (i) whole body sampling of mosquitoes by homogenizing them 12 , (ii) analysis of mosquito saliva after extraction of salivary glands 13 , or (iii) forced mosquito salivation 14 to determine the prevalence of pathogens in mosquito populations. In addition to requiring sample preparation of mosquitoes, these methods do not represent actual biting events thus providing limited information about the transmission dynamics such as the infectious viral load of a bite.…”

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confidence: 99%

A microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

et al. 2021

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Mosquito bites transmit a number of pathogens via salivary droplets deposited during blood-feeding, resulting in potentially fatal diseases. Little is known about the genomic content of these nanodroplets, including the transmission dynamics of live pathogens. Here we introduce Vectorchip, a low-cost, scalable microfluidic platform enabling high-throughput molecular interrogation of individual mosquito bites. We introduce an ultra-thin PDMS membrane which acts as a biting interface to arrays of micro-wells. Freely-behaving mosquitoes deposit saliva droplets by biting into these micro-wells. By modulating membrane thickness, we observe species-dependent differences in mosquito biting capacity, utilizable for selective sample collection. We demonstrate RT-PCR and focus-forming assays on-chip to detect mosquito DNA, Zika virus RNA, as well as quantify infectious Mayaro virus particles transmitted from single mosquito bites. The Vectorchip presents a promising approach for single-bite-resolution laboratory and field characterization of vector-pathogen communities, and could serve as a powerful early warning sentinel for mosquito-borne diseases.

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confidence: 99%

A microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

et al. 2021

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“…Proteins from a total of 152 A. aegypti females were processed, of which 39 were coinfected with Wolbachia and ZIKV (WZ); 36 non-infected (A); 35 infected with Wolbachia (W) only and 42 infected with ZIKV (Z). On 14 days post-infection, each mosquito head plus the salivary gland was separated from the body using needles and a scalpel blade which were sterilized after every single use (Schmid et al, 2017). Proteins were extracted by lysis with a buffer containing 7 M urea, 2 M thiourea, 50 mM HEPES pH 8, 75 mM NaCl, 1 mM EDTA, 1 mM PMSF, and protease/phosphatase inhibitor cocktails (Roche).…”

Section: Protein Extraction For Proteomicsmentioning

confidence: 99%

Comprehensive Quantitative Proteome Analysis of Aedes aegypti Identifies Proteins and Pathways Involved in Wolbachia pipientis and Zika Virus Interference Phenomenon

et al. 2021

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Zika virus (ZIKV) is a global public health emergency due to its association with microcephaly, Guillain-Barré syndrome, neuropathy, and myelitis in children and adults. A total of 87 countries have had evidence of autochthonous mosquito-borne transmission of ZIKV, distributed across four continents, and no antivirus therapy or vaccines are available. Therefore, several strategies have been developed to target the main mosquito vector, Aedes aegypti, to reduce the burden of different arboviruses. Among such strategies, the use of the maternally-inherited endosymbiont Wolbachia pipientis has been applied successfully to reduce virus susceptibility and decrease transmission. However, the mechanisms by which Wolbachia orchestrate resistance to ZIKV infection remain to be elucidated. In this study, we apply isobaric labeling quantitative mass spectrometry (MS)-based proteomics to quantify proteins and identify pathways altered during ZIKV infection; Wolbachia infection; co-infection with Wolbachia/ZIKV in the A. aegypti heads and salivary glands. We show that Wolbachia regulates proteins involved in reactive oxygen species production, regulates humoral immune response, and antioxidant production. The reduction of ZIKV polyprotein in the presence of Wolbachia in mosquitoes was determined by MS and corroborates the idea that Wolbachia helps to block ZIKV infections in A. aegypti. The present study offers a rich resource of data that may help to elucidate mechanisms by which Wolbachia orchestrate resistance to ZIKV infection in A. aegypti, and represents a step further on the development of new targeted methods to detect and quantify ZIKV and Wolbachia directly in complex tissues.

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“…aegypti dilakukan dengan teknik mikrodiseksi yaitu metode pembedahan tubuh nyamuk yang dilakukan dibawah mikroskop untuk memisahkan kelenjar saliva Ae. aegypti dari bagian tubuh nyamuk lainnya (Schmid et al 2017). Pada proses mikrodiseksi, tubuh nyamuk diletakkan di atas tetesan larutan 0,5% NaCl.…”

Section: Isolasi Dan Analisisunclassified

PURIFIKASI PROTEIN IMUNOGENIK 31 DAN 56 kDa DARI KELENJAR SALIVA <em>Aedes aegypti</em>

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et al. 2020

J Bioteknol Biosains Indones

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Purification of 31 and 56 kDa Immunogenic ProteinsÂ from the Salivary Glands of Aedes aegyptiThe salivary gland of arthropod vector contains various bioactive compounds and plays a role in the transmission of pathogens to the host. The host develops anti-salivary antibodies against vector saliva exposure. Our previous research has identified two immunogenic proteins with molecular weights of 31 and 56 kDa from the Aedes aegypti salivary gland protein extract. However, the role of the 31 and 56 kDa immunogenic proteins from saliva Ae. aegypti is not fully known, so it is necessary to purify two immunogenic protein fractions to better specify the target of developing a dengue vaccine. This study aimed to purify the 31 and 56 kDa immunogenic protein fractions by electroelution and dialysis methods. The purification of the two protein fractions has been successful which were confirmed by the SDS-PAGE by the existence of single-band parallel to the positive control. These results were further supported by the dot blot analysis which showed a positive reaction in the form of dark spots in the two protein fractions which were reacted with dengue patients' serum, endemic healthy people, and neonates. These results indicated that the purified 31 and 56 kDa immunogenic protein fraction can be identified by specific antibodies.Keywords: dialysis, electroelution, immunogenic, purification, saliva Â ABSTRAKKelenjar saliva vektor arthropoda mengandung berbagai senyawa bioaktif dan berperan dalam transmisi patogen ke tubuh inang. Tubuh inang mengembangkan antibodi anti-saliva terhadap paparan saliva vektor. Penelitian kami sebelumnya telah mengidentifikasi dua protein imunogenik dengan berat molekul 31 dan 56 kDa dari ekstrak protein kelenjar saliva Aedes aegypti. Namun demikian, peranan protein imunogenik 31 dan 56 kDa dari saliva Ae. aegypti belum diketahui sepenuhnya sehingga perlu dilakukan purifikasi dua fraksi protein imunogenik untuk lebih menspesifikkan target pengembangan vaksin dengue. Tujuan penelitian ini untuk melakukan purifikasi fraksi protein imunogenik 31 dan 56 kDa melalui metode elektroelusi dan dialisis. Keberhasilan purifikasi dua fraksi protein 31 dan 56 kDa terbukti dari hasil konfirmasi SDS-PAGE dengan terbentuknya pita tunggal sejajar dengan kontrol positif. Hasil tersebut diperkuat dengan analisis dot blot yang menunjukkan reaksi positif dengan munculnya noktah gelap pada dua fraksi protein tersebut ketika direaksikan dengan serum pasien DBD, penduduk sehat endemik dan neonatus. Hasil ini mengindikasikan bahwa fraksi protein imunogenik 31 dan 56 kDa hasil purifikasi dapat dikenali oleh antibodi spesifik.

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Preparation of Mosquito Salivary Gland Extract and Intradermal Inoculation of Mice

Cited by 16 publications

References 15 publications

A microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

A microfluidic platform for highly parallel bite by bite profiling of mosquito-borne pathogen transmission

Comprehensive Quantitative Proteome Analysis of Aedes aegypti Identifies Proteins and Pathways Involved in Wolbachia pipientis and Zika Virus Interference Phenomenon

PURIFIKASI PROTEIN IMUNOGENIK 31 DAN 56 kDa DARI KELENJAR SALIVA <em>Aedes aegypti</em>

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