Naturally Occurring Incompatibilities between Different <em>Culex pipiens pallens</em> Populations as the Basis of Potential Mosquito Control Measures

<div><h3>Background</h3><p>Vector-borne diseases remain a threat to public health, especially in tropical countries. The incompatible insect technique has been explored as a potential control strategy for several important insect vectors. However, this strategy has not been tested in <em>Culex pipiens pallens</em>, the most prevalent mosquito species in China. Previous works used introgression to generate new strains that matched the genetic backgrounds of target populations while harboring a new <em>Wolbachia</em> endosymbiont, resulting in mating competitiveness and cytoplasmic incompatibility. The generation of these incompatible insects is often time-consuming, and the long-term stability of the newly created insect-<em>Wolbachia</em> symbiosis is uncertain. Considering the wide distribution of <em>Cx. pipiens pallens</em> and hence possible isolation of different populations, we sought to test for incompatibilities between natural populations and the possibility of exploiting these incompatibilities as a control strategy.</p> <h3>Methodology/Principal Findings</h3><p>Three field populations were collected from three geographic locations in eastern China. Reciprocal cross results showed that bi-directional patterns of incompatibility existed between some populations. Mating competition experiments indicated that incompatible males could compete with cognate males in mating with females, leading to reduced overall fecundity. F1 offspring from incompatible crosses maintained their maternal crossing types. All three populations tested positive for <em>Wolbachia</em>. Removal of <em>Wolbachia</em> by tetracycline rendered matings between these populations fully compatible.</p> <h3>Conclusions/Significance</h3><p>Our findings indicate that naturally occurring patterns of cytoplasmic incompatibility between <em>Cx. pipiens pallens</em> populations can be the basis of a control strategy for this important vector species. The observed incompatibilities are caused by <em>Wolbachia</em>. More tests including field trials are warranted to evaluate the feasibility of this strategy as a supplement to other control measures.</p> </div

Reproductive compatibilities between three <i>Cx. pipiens pallens</i> field populations.

<p>Three <i>Cx. pipiens pallens</i> field populations NJ, WX and TK were used in reciprocal crosses to test their reproductive compatibilities. For each cross, an equal number of virgin females and males were kept in a cage for 48 hours before blood feeding. After oviposition, egg rafts were collected and allowed to hatch. The average hatching rates of these crosses were compared. (A) Mating combinations of NJ and WX populations, (B) Mating combinations of TK and WX populations, (C) Mating combinations of NJ and TK populations. Error bars represent standard errors; ns, non-significant <i>P</i>-value; *** <i>P</i><0.0001 by <i>t</i>-test.</p

Genes used for <i>Wolbachia</i> typing and their PCR amplification primers.

<p>Genes used for <i>Wolbachia</i> typing and their PCR amplification primers.</p

Collecting sites of <i>Cx. pipiens pallens</i> field populations.

<p>(A) The mosquito populations were collected from eastern China; (B) Three collecting sites Tangkou, Nanjing and Wuxi are shown in detail. The mosquito populations collected are designated TK, NJ and WX, respectively.</p

Effect of subsequent encounter with cognate males on the fecundity of the females retrieved from incompatible crosses.

<p>Females were retrieved from the two incompatible crosses as shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002030#pntd-0002030-g002" target="_blank">Figure 2B</a>. TK or WX females were equally divided into two subgroups, with one mixed with cognate males and the other kept alone (no male). After a second blood feeding and oviposition, egg rafts were collected and allowed to hatch. The average hatching rates of these subgroups were compared. Error bars represent standard errors; ns, non-significant <i>P</i>-value.</p

Correlation between group average hatching rate and the percentage of incompatible males.

<p>The average hatching rate of a group was plotted against the percentage of incompatible males which was calculated by dividing the number of incompatible males by the total number of males (incompatible and cognate). (A) TK females with TK and WX males; (B) WX females with WX and TK males.</p

Reproductive compatibilities of F1 offspring from incompatible crosses.

<p>F1 offspring from TK and WX crosses were mixed with TK, WX or the F1. The hatching rates were compared. (A) F1 from TK♀×WX♂ cross; (B) F1 from WX♀×TK♂ cross. Error bars represent standard errors; ns, non-significant <i>P</i>-value; *** <i>P</i><0.0001 by <i>t</i>-test.</p

Elimination of <i>Wolbachia</i> rendered TK and WX populations fully compatible.

<p>WX females were crossed with an equal number of tetracycline-treated TK males (TK_tet♂), untreated TK males (TK♂) or WX males (WX♂). TK females were crossed with an equal number of tetracycline-treated WX males (WX_tet♂), untreated WX males (WX♂) or TK males (TK♂). Hatching rates were calculated for these crosses. Error bars represent standard errors; ns, non-significant <i>P</i>-value; *** <i>P</i><0.0001 by <i>t</i>-test.</p

Differential expression of <i>Anopheles. sinensis</i> detoxification genes between deltamethrin-susceptible and -resistant strains.

<p>Differential expression of <i>Anopheles. sinensis</i> detoxification genes between deltamethrin-susceptible and -resistant strains.</p

The phylogenetic analysis of cytochrome P450s, glutathione-S-transferases and choline/carboxylesterases.

<p>(A) Unrooted distance neighbor-joining tree showing the phylogeny of cytochrome P450s from the genomes of <i>Anopheles sinensis</i> (red circle), <i>Aedes aegypti</i> (green square) and <i>Culex pipiens quinquefasciatusin</i> (aqua triangle). (B) Unrooted distance neighbor-joining tree showing the phylogeny of glutathione-S-transferases from the genomes of <i>Anopheles sinensis</i> (red circle), <i>Anopheles gambiae</i> (blue triangle), <i>Aedes aegypti</i> (green square), <i>Culex pipiens quinquefasciatusin</i> (aqua triangle) and <i>Drosophila melanogaster</i> (pink rhombus). (C) Unrooted distance neighbor-joining tree showing the phylogeny of choline/carboxylesterases from the genome of <i>Anopheles sinensis</i> (red circle), <i>Aedes aegypti</i> (green square) and <i>Culex pipiens quinquefasciatusin</i> (aqua triangle). The percentage of bootstrap confidence values greater than 70% (1000 replicates) is indicated at the nodes.</p