Considerations for Rearing and Maintaining<i>Anopheles</i>in the Laboratory

Leite, Laura N; Bascuñán, Priscila; Dotson, Ellen M.; Benedict, Mark Q.

doi:10.1101/pdb.top107802

Cited by 9 publications

(4 citation statements)

References 18 publications

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“…Laid eggs were placed in plastic trays containing 500 mL of demineralized water for hatching. 48 hours later, L1 larvae were transferred to clean trays containing 500ml of demineralized water at a density of 250 larvae per tray and provided with fish food solution daily until pupation, according to standard protocols 30 .…”

Section: Methodsmentioning

confidence: 99%

Targeting mosquito X-chromosomes reveals complex transmission dynamics of sex ratio distorting gene drives

Haber,

Arien,

Lamdan

et al. 2023

Preprint

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Engineered sex ratio distorters (SRDs) have been proposed as a powerful component of both self-limiting and self-sustaining genetic control strategies designed to suppress harmful insect pest or disease vector populations such as malaria mosquitoes. Two different CRISPR-based SRD mechanisms, X-shredding which eliminates X-bearing sperm in males and X-poisoning which causes postzygotic mortality of daughters inheriting disrupted X-linked genes, have been proposed. The nature of the sex distortion mechanism is predicted to affect the population dynamics of released Y-chromosome SRDs: A Y-linked X-shredder is invasive constituting a classical meiotic Y-drive, whereas Y-linked X-poisoning is self-limiting, unable to invade but also insulated from selection and disappearance. Here, we established the first X-poisoning SRD system in the malaria vectorAnopheles gambiaetargeting three X-linked ribosomal protein (RP) genes during spermatogenesis. Surprisingly, when we characterized transgenic strains showing a significant male bias, we found that sex distortion was primarily driven by a loss of X-bearing sperm with limited evidence for postzygotic lethality of daughters. By leveraging theDrosophilamodel, we show unambiguously that engineered SRD traits can operate differently in these two insects. Unlike X-shredding, X-poisoning could theoretically operate at early stages of spermatogenesis. We therefore explored premeiotic Cas9 expression to target the mosquito X-chromosome. We find that, by pre-empting the onset of meiotic sex chromosome inactivation, this approach may enable the development of Y-linked SRDs, once trade-offs between sex chromosome transmission distortion and male fertility are functionally balanced.

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Section: Methodsmentioning

confidence: 99%

Targeting mosquito X-chromosomes reveals complex transmission dynamics of sex ratio distorting gene drives

Haber,

Arien,

Lamdan

et al. 2023

Preprint

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“…See Introduction: Considerations for Rearing and Maintaining Anopheles in the Laboratory (Leite et al 2023a) for more information Scissors Sealed container (see Step 14) Sponge cloth (optional; see Step 17) Squat, wide-opening plastic cups ( 250-mL) Stainless-steel forceps Suction device to collect adult mosquitoes (e.g., Fulton RHM 200 aspirator) Vacuum filtration system A Nalgene 500-mL vacuum filtration system can be adapted to this method by breaking off the upper chamber and removing the membrane. Connect it to a vacuum system with a trap, which is composed of an Erlenmeyer filtration flask capped with a two-holed stopper.…”

Section: Cotton Ballsmentioning

confidence: 99%

AnophelesEgg Collection, Disinfection, and Hatching

Leite

Bascuñán

Dotson

et al. 2023

Cold Spring Harb Protoc

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Gravid (i.e., with fully developed eggs), matedAnophelesfemales typically lay their eggs directly on water ∼48–72 h after a blood meal. Unlike some other mosquito species,Anopheleseggs cannot be desiccated and stored for long durations, and, hence, colonies must be reared continuously. In this protocol, we discuss methods for egg collection, including individual anden masseoviposition; egg disinfection to avoid the transmission of infectious agents to the next generation; and egg hatching for colony maintenance or experimentation. We also include optional methods for estimating life history traits such as fecundity, fertility, and larval mortality rates from egg counts.

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“…Mosquito heterogeneity and its impact on bioassays was rst described in 1971 [21], and has been shown to impact insecticide susceptibility [20]. It is reduced, but not eliminated by careful mosquito rearing [22].…”

Section: Introductionmentioning

confidence: 99%

Complete series method (CSM): a convenient method to reduce daily heterogeneity when evaluating the regeneration time (RT) of insecticide-treated nets (ITNs)

Lugenge,

Odufuwa,

Mseti

et al. 2024

Preprint

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Background: "Regeneration time" (RT), denotes the time required to obtain a stable mortality rate for mosquitoes exposed to insecticide-treated nets (ITNs) after three consecutive washes of a net in a day. The RT informs the wash interval of ITNs used to artificially age ITNs to simulate their lifetime performance under user conditions (20 washes). RT was estimated following World Health Organization (WHO) longitudinal method (LM) bioassay procedures. However, longitudinal evaluation may introduce daily heterogeneity due to mosquito batch variability, complicating RT determination. To overcome this, nets at each stage of regeneration (i.e., 1, 2, 3, 5 &7 days post wash) were prepared in advance and refrigerated, then a complete regeneration series was tested with a single mosquito batch on one testing day over 4 days (N=1 per timepoint /day). This study validated the complete series method (CSM) against the LM. Methods: The RT of two incorporated alpha-cypermethrin and piperonyl butoxide (PBO) and one incorporated permethrin with PBO ITNs were determined using laboratory-reared resistant Anopheles arabiensis under standard laboratory conditions. LM methods and CSM were compared in two experiments 1) refrigerated nets acclimatized for two hours (Test 1), and 2) refrigerated nets acclimatized for three hours (Test 2). Four replicates per day of regeneration were tested per ITN product with 50 mosquitoes exposed per replicate to give an equivalent sample size to that of LM. The RTs and overall heterogeneity from these methods were compared descriptively. Results: The RT measured by the CSM showed 100% agreement with that measured by LM. The intra-method variability for unwashed pieces was minimal, with variance of 1.26 for CSM and 1.18 for LM. For unwashed nets LM had substantially greater variance, ratio of LM:CSM was 2.66 in test one and 2.49 in test two. The magnitude of mortality measured in bioassays depended on sample acclimation after refrigeration. Conclusion The CSM is a convenient method for determining the regeneration times of ITNs that are prepared in advance, and a complete series of samples is removed from the fridge, defrosted and evaluated on a single day allowing easy facility scheduling and reduced daily heterogeneity. The CSM gave comparable estimates of regeneration time as the LM.

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Considerations for Rearing and MaintainingAnophelesin the Laboratory

Cited by 9 publications

References 18 publications

Targeting mosquito X-chromosomes reveals complex transmission dynamics of sex ratio distorting gene drives

Targeting mosquito X-chromosomes reveals complex transmission dynamics of sex ratio distorting gene drives

AnophelesEgg Collection, Disinfection, and Hatching

Complete series method (CSM): a convenient method to reduce daily heterogeneity when evaluating the regeneration time (RT) of insecticide-treated nets (ITNs)

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