wMel, wAlbB, and wild-type Ae. aegypti mosquitoes were derived from previously generated lines (7 (link)), sharing the same genetic background. Colonies were maintained at 27°C and 70% relative humidity with a 12-h light/dark cycle. Larvae were fed with tropical fish pellets (Tetramin, Tetra, Melle, Germany) and adults maintained with 5% sucrose solution ad libitum. Blood meals were provided using an artificial blood-feeding system (Hemotek, UK) using human blood (Scottish National Blood Transfusion Service, UK). Eggs were collected on a wet filter-paper (Grade 1 filter paper, Whatman plc, GE health care, UK). Eggs were desiccated for 5 days and later hatched in deionized water containing 1g/L bovine liver powder (MP Biomedicals, Santa Ana, CA, USA).
Artificial blood feeding system
Manufactured by Hemotek
Sourced in United Kingdom
The Artificial blood-feeding system is a lab equipment designed to provide a controlled environment for the study of blood-feeding insects. It simulates the conditions necessary for insects to feed on blood, allowing researchers to observe and analyze their behavior and physiology under controlled settings.
Automatically generated - may contain errors
Lab products found in correlation
Bovine liver powder, by MP Biomedicals (5 mentions)
Human blood, by Hemotek (3 mentions)
I 36vl, by Percival (2 mentions)
Drb030, by Avantor (2 mentions)
Trizol, by Merck Group (2 mentions)
Grade 1 filter paper, by Cytiva (2 mentions)
Grade 1 filter paper, by GE Healthcare (2 mentions)
Adenosine triphosphate (atp), by Avantor (1 mentions)
9 protocols using artificial blood feeding system
1
Maintaining Aedes aegypti Mosquito Colonies
2
Rearing Aedes aegypti Mosquitoes
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The A. aegypti wild-type line used was colonized from Selangor State, Malaysia, in the 1960s. Colonies were maintained under standard rearing conditions, at 27°C and 70% relative humidity with a 12-h light/dark cycle. Larvae were fed on tropical fish pellets (Tetramin; Tetra, Melle, Germany), and adults were maintained with 5% sucrose solution ad libitum. Blood meals to maintain the colony were provided using an artificial blood-feeding system (Hemotek, UK) using human blood (Scottish National Blood Transfusion Service, UK). Eggs were collected on a wet filter paper (grade 1 filter paper; Whatman plc, GE Healthcare, UK), desiccated for 5 days, and later hatched in deionized water containing 1 g/L bovine liver powder (MP Biomedicals, Santa Ana, CA, USA).
3
Mosquito Blood Feeding and Egg Laying
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CFAV- or mock-infected mosquitoes were allowed to access 37oC-warmed rabbit blood for 15 min using an artificial blood-feeding system (Hemotek, UK). The blood-fed mosquitoes were counted and placed individually into fly vials containing cotton with water-wet paper on top for oviposition and with access to a 10% sucrose solution. At day 7 after blood-feeding, the eggs laid on the paper were counted and transferred to a 12-well plate containing 2 ml water. The number of hatched larvae was counted 5 days after the egg transfer.
4
Mosquito Infection with Sindbis Virus
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Aedes aegypti mosquitoes either -infected and -uninfected with Wolbachia (wAlbB strain) (generously provided by Dr. Zhiyong Xi, Michigan State University, USA), were reared in an insect incubator (Percival Model I-36VL, Perry, IA, USA) at 28°C and 75% humidity with 12 h light/dark cycle. Four to six-day old mated female mosquitoes were allowed to feed for 1h on approximately 108 PFUs of SINV (TE12-untagged) containing citrated rabbit blood (Fisher Scientific DRB030) supplemented with 1mM ATP (VWR) and 10% sucrose using a Hemotek artificial blood feeding system (Hemotek, UK) maintained under constant temperature of 37°C. Engorged mosquitoes were then isolated and reared at 28°C in the presence of male mosquitoes. Infected mosquitoes were harvested 5–7 days post blood meal before being snap frozen in liquid nitrogen and stored at −80°C before further processing. Samples for qPCR and qRT-PCR were homogenized in TRiZOL (Sigma Aldrich) reagent and further processed for nucleic acid extractions using manufacturer’s protocols.
5
SINV Infection in Aedes aegypti Mosquitoes
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Aedes aegypti mosquitoes either -infected and -uninfected with Wolbachia strain wAlbB (generously provided by Dr. Zhiyong Xi, Michigan State University, USA), were reared in an insect incubator (Percival Model I-36VL, Perry, IA, USA) at 28 °C and 75% humidity with 12 h light/dark cycle. Four to six-day old mated female mosquitoes were allowed to feed for 1h on approximately 10 8 PFUs of SINV (TE12-untagged) containing citrated rabbit blood (Fisher Scientific DRB030) supplemented with 1mM ATP (VWR) and 10% sucrose using a Hemotek artificial blood feeding system (Hemotek, UK) maintained under constant temperature of 37 °C. Engorged mosquitoes were then isolated and reared at 28 °C in the presence of male mosquitoes. Infected mosquitoes were either harvested whole or dissected at 5-7 days post blood meal using the following method. At specified time points, mosquitoes were anesthetized following a short 5 min exposure to cold, before they were transferred to a CO2 pad for dissections. Dissected salivary glands were collected in sterile 1XPBS (Phosphate Buffered Saline) before being snap frozen in liquid nitrogen and storage at -80 °C for further processing. Samples for qRT-PCR were homogenized in TRiZOL (Sigma Aldrich) reagent and further processed for RNA extractions.
6
Maintenance of Aedes Mosquito Colonies
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The Ae. albopictus wild-type strain (JF) collected in the Jalan Fletcher area of Kuala Lumpur, Malaysia, and maintained for >20 generations, was provided by W.A. Nazni, Institute for Medical Research, Kuala Lumpur. A wAu-infected Ae. aegypti line [17 (link)] was used as the Wolbachia source. The wMel single-infected Ae. albopictus line was generated as previously described [31 (link)]. Mosquito colonies were maintained at standard 27°C and 70% relative humidity with a 12-hour light/dark cycle. Larvae were fed on tropical fish pellets (Tetramin, Tetra, Melle, Germany) and adults were offered 5% sucrose solution ad libitum. Blood meals were provided using a Hemotek artificial blood-feeding system (Hemotek, UK) using defribrinated sheep blood (TCS Biosciences, UK). Eggs were collected on wet filter paper (Grade 1 filter paper, Whatman plc, GE healthcare, UK) and desiccated for 5–10 days before hatching in de-ionized water containing 1g/L bovine liver powder (MP Biomedicals, Santa Ana, California, USA).
7
Rearing Ae. aegypti Mosquitoes with wAlbB
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All mosquito colonies were maintained at 27 °C and 70% humidity with 12-h light/dark cycles. Larvae were fed tropical fish pellets (Tetra) whilst adults had access to 5% sucrose solution ad libitum. Blood meals were provided using a Hemotek artificial blood-feeding system (Hemotek Ltd) and human blood (Scottish National Blood Bank). Damp Grade 1 filter-paper (Whatman plc) was provided as an oviposition source for egg collection. Eggs were desiccated for 5–10 days before hatching in water containing 1 g/l bovine liver powder (MP Biomedicals). The wAlbB-carrying Ae. aegypti line used in this study was generated previously through embryo microinjection40 (link).
8
Mosquito Colony Maintenance and Bloodmeal Provision
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The C. quinquefasciatus wild‐type was the Pel line originally colonized in Sri Lanka. The Wolbachia‐free PelU line was created by antibiotic treatment (Pinto et al., 2013 ). The source of wAlbA and wAlbB Wolbachia for cytoplasmic transfers was from transinfected Ae. aegypti colonies (Ant et al., 2018 ). All mosquito colonies were maintained at 27 °C and 70% relative humidity with a 12‐h light/dark cycle. Larvae were fed tropical fish pellets (Tetramin, Tetra, Melle, Germany) and adults were given access to a sucrose meal ad libitum. Bloodmeals were provided using a Hemotek artificial blood‐feeding system (Hemotek, Blackburn, UK) using defribrinated sheep blood (TCS Biosciences, Botolph Claydon, UK). Eggs were collected by providing a bowl of water for oviposition 3–4 days post blood‐feeding.
9
Ae. aegypti Mosquito Rearing Protocol
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The Ae. aegypti wild-type line used was colonized from Selangor State, Malaysia in the 1960s. All mosquito colonies were maintained at 27°C and 70% relative humidity with a 12-hour light/dark cycle. Larvae were fed tropical fish pellets (Tetramin, Tetra, Melle, Germany) and adults were given access to a sucrose meal ad libitum. Blood meals were provided using a Hemotek artificial blood-feeding system (Hemotek, UK) using defribrinated sheep blood (TCS Biosciences, UK). Eggs were collected by providing damp filter-paper (Grade 1 filter paper, Whatman plc, GE healthcare, UK) for oviposition. Eggs were desiccated for 5–10 days prior to hatching in water containing 1g/L bovine liver powder (MP Biomedicals, Santa Ana, California, USA).
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