Cas9-mediated gene-editing in the malaria mosquito Anopheles stephensi by ReMOT Control

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by Anopheles mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially Anopheles) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (Receptor-mediated Ovary Transduction of Cargo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector Anopheles stephensi without injecting embryos. In Anopheles, ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to Anopheles opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species

Geometric Morphometrics of Nine Field Isolates of <i>Aedes aegypti</i> with Different Resistance Levels to Lambda-Cyhalothrin and Relative Fitness of One Artificially Selected for Resistance

<div><p><i>Aedes aegypti</i>, a mosquito closely associated with humans, is the principal vector of dengue virus which currently infects about 400 million people worldwide. Because there is no way to prevent infection, public health policies focus on vector control; but insecticide-resistance threatens them. However, most insecticide-resistant mosquito populations exhibit fitness costs in absence of insecticides, although these costs vary. Research on components of fitness that vary with insecticide-resistance can help to develop policies for effective integrated management and control. We investigated the relationships in wing size, wing shape, and natural resistance levels to lambda-cyhalothrin of nine field isolates. Also we chose one of these isolates to select in lab for resistance to the insecticide. The main life-traits parameters were assessed to investigate the possible fitness cost and its association with wing size and shape. We found that wing shape, more than wing size, was strongly correlated with resistance levels to lambda-cyhalothrin in field isolates, but founder effects of culture in the laboratory seem to change wing shape (and also wing size) more easily than artificial selection for resistance to that insecticide. Moreover, significant fitness costs were observed in response to insecticide-resistance as proved by the diminished fecundity and survival of females in the selected line and the reversion to susceptibility in 20 generations of the non-selected line. As a practical consequence, we think, mosquito control programs could benefit from this knowledge in implementing efficient strategies to prevent the evolution of resistance. In particular, the knowledge of reversion to susceptibility is important because it can help in planning better strategies of insecticide use to keep useful the few insecticide-molecules currently available.</p></div

Wing size variation of both lambda-cyhalothrin selected and non-selected lines.

<p>Violin plots enclose box plots. Each box is divided by the median (black circles), which top and bottom correspond to 25th and 75th quartiles, respectively. CS: centroid-size; P: parental Comuneros isolate; F9-NS and F20-NS: ninth and twenty generations non-selected for lambda-cyhalothrin resistance; F9-S and F20-S: ninth and twenty generations selected for lambda-cyhalothrin resistance. Sample-sizes were 51 parental females, 42 F9-NS, 49 F9-S, 50 F20-NS, and 56 F20-S.</p

Relative fitness and parameters used to calculate it. Fitness was calculated relative to the ROCK strain.

<p><i>l_x</i>: age specific survival, <i>m_x</i>: fecundity. ROCK: the Rockefeller strain, F9-S and F10-S: the selected line at ninth and tenth generations, and F9-NS and F10-NS: the non-selected line at ninth and tenth generations.</p

Dendrogram showing the relationships in wing shape of isolates from Cúcuta and Quibdó.

<p>Cophenetic correlation coefficient: 0.8935737. Isolates from Cúcuta are colored in red, and those from Quibdó in green. Between parentheses is the RR₅₀ to lambda-cyhalothrin. ROCK: the Rockefeller strain.</p

Origin and resistance status to lambda-cyhalothrin of <i>Ae. aegypti</i> isolates.

<p>MUNIC.: Municipality, NEIGHB.: Neighborhood, COORD.: Geographical coordinates, RR₅₀-LAMB: Resistance Ratios 50 to lambda-cyhalothrin, DATE OF COLLEC.: date of collection. N: sample size; CODE: isolate code; Ref.: Reference of susceptibility.</p

Dendrogram showing the relationships in wing size of isolates from Cúcuta and Quibdó.

<p>Between parentheses is the RR₅₀ to lambda-cyhalothrin. Cophenetic correlation coefficient: 0.892522. isolates from Cúcuta are colored in red, and those from Quibdó in green ROCK: the Rockefeller strain.</p

Arrangement of landmarks digitized on right and left wings of 758 <i>Aedes aegypti</i> females.

<p>The landmarks were digitized in a clockwise sequence from the number one.</p