Protocol for assembling and implementing a partially automated system for rearing and handling Anopheles stephensi mosquitoes

Abstract

Live mosquitoes are required to comprehensively study vector-borne diseases, including transmission. Traditional mosquito-rearing protocols are laborious and time consuming. Here, we present a protocol for assembling and implementing a partially automated system for rearing and handling Anopheles stephensi mosquitoes. We describe steps for assembling a pupation station, self-emptying bucket, pupal funnel and dish vacuum, automatic aspirator, and sugar tubes. We also detail the application of these systems, along with specific limitations.

Keywords: Immunology; Microbiology; Model Organisms.

Graphical abstract

Figure 1

Schematic of the pupation station which facilitates the semi-automated rearing of Anopheles mosquitoes from day 3 larvae to pupae (A) Completed pupation station. (B) Pupation station frame. For a detailed description of each beam and assembly instruction view Figure S1. (C) Side and bottom views of one of six pans that make up the Pupation Station. Assembly instructions can be found in Figure S2. (D) Valve and splash eliminator used for controlling the flow of water between pans.

Figure 2

The self-emptying bucket (SEB) serves to increase efficiency of transferring liquid waste to the sink in the insectary facility (A) Photograph of the side view of SEB. (B) Photograph of the top-down view of SEB, showing the sump pump.

Figure 3

The pupal funnel facilitates the separation of pupae from larvae (A) Schematic of completed pupal funnel. (B) Side and top-down view of engineering diagram for 3D printed adapter. (C) Side view of engineering diagram for 3D printed pupal funnel holder.

Figure 4

Schematic of the pupal dish vacuum which facilitates the rapid transfer of water out of pupal dishes The pupal dish vacuum comprises a vacuum pump, vacuum flask, tubing, and a pipette.

Figure 5

The automatic aspirator facilitates the transfer of adult mosquitoes between cages (A) Schematic of automatic aspirator set-up. (B) Side and top-down view engineering diagrams of the transfer cage lid. (C) Photograph of transfer cage. (D) Photograph of the transfer tube.

Figure 6

Schematic of sugar tube set-up which facilitates the feeding of adult mosquitoes for extended periods The set-up depicts a 5 mL culture tubes filled with sugar water with a cotton ball at the end.

Figure 7

Pupation station produces similar mosquito yields and does not reduce mosquito infectivity with Plasmodium compared to traditional rearing methods (A) Photograph of pans used to rear 10⁵–10⁶ mosquitoes using traditional methods. (B) Mosquitoes produced per source colony cage using traditional rearing methods and the pupation station. Each dot represents a generation of mosquitoes produced from a batch of eggs from a single colony cage. Data were analyzed by paired t-test. p > 0.05 was considered not significant (ns). (C) Number of P. yoelii sporozoites per mosquito day 14 post-infection in mosquitoes produced by traditional rearing methods and the pupation station. Each point represents the average number of parasites isolated from the salivary glands of an independent batch of infected mosquitoes. An average of 19 mosquitoes were dissected per batch. Data were analyzed by paired t-test. p > 0.05 was considered not significant (ns).

Figure 8

Automatic aspirator does not reduce mosquito lifespan compared to manual mouth aspiration (A) Survival percentage of mosquitoes immediately post manual or automatic aspiration. Each dot represents an independent aspiration event. Data were analyzed by paired t-test. p > 0.05 was considered not significant (ns). (B) Representative survival curve showing survival percentage for mosquitoes aspirated into a cage by manual mouth aspiration or using the automatic aspirator. Three independent experiments were performed using roughly 100 mosquitoes per group. Data were analyzed using the Log-rank (Mantel-Cox) test. p > 0.05 was considered not significant (ns).