Naturally Occurring Incompatibilities Between Different Culex Pipiens Pallens Populations As The Basis Of Potential Mosquito Control Measures

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 Culex pipiens pallens, 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 Wolbachia 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-Wolbachia symbiosis is uncertain. Considering the wide distribution of Cx. pipiens pallens hence possible isolation of different populations, we sought to test the incompatibilities between natural populations and the possibility of exploiting these incompatibilities as a control strategy.Three field populations were collected from three geographic locations in eastern China. Reciprocal cross results showed that incompatibilities 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. F1offspring from incompatible crosses maintained their maternal crossing types. All three populations were tested positive for Wolbachia. Removal of Wolbachia by tetracycline rendered these populations fully compatible. Our findings indicate that naturally occurring incompatibility between Cx. pipiens pallens populations can be the basis of a control strategy that is potentially sustainable. The observed incompatibilities are caused by Wolbachia. More tests including field tests are warranted to evaluate the feasibility of this strategy as a supplement to other control measures.1. Incompatibility exists between Cx. pipiens pattens populationsWe first tested the compatibilities between available Cx. pipiens pallens populations collected from three geographic locations in China. In the mating combinations of NJ and WX populations, no significant incompatibility was detected in either NJ♀×WX♂or WX♀×NJ♂group as compared to NJ♀×NJ♂and WX♀×WX♂control groups. In the mating combinations of TK and WX populations, bidirectional incompatibility was observed, with both TK♀×WX♂and WX♀×TK♂crosses having significantly reduced hatching rates as compared to the TK♀×TK♂and WX♀×WX♂control groups. Similarly, bidirectional incompatibility was detected in the mating combinations of NJ and TK populations.2. The incompatibility is not caused by mating failureTo make sure the observed reduction in hatching rate was not caused by mating failure, egg rafts were examined48hours after oviposition. The eggs from incompatible crosses showed clear embryonic development. Inside the eggs from TK♀×WX♂cross, there was segment formation along the axis. At the anterior end of the eggs, two red spots representing primitive eyes (stemmata) were visible. Consistent with previous reports, these embryos showed some abnormalities. One evident difference between TK♀×WX♂cross and compatible crosses was that the incompatible embryos had disorientated bristles. In the eggs from TK♀×NJ♂cross, similar development was observed.1-3(mostly2) pigmented stemmata were formed. Most stemmata were located in the head region, but some did not seem to have a specific localization. In some eggs, two stemmata were aligned anteroposteriorly instead of being side-by-side. In addition, there was segment formation along the axis in some eggs. Disoriented bristles were also observed in the eggs from TK♀×NJ♂cross. These observations further confirmed that the females in the incompatible crosses had mated.3. Mating with incompatible males prevents females from subsequent mating with cognate malesTK♀from TK♀×WX♂cross and WX♀from WX♀×TK♂cross were separated from WX♂and TK♂,respectively. Each group was equally divided into two subgroups, with one subgroup mixed with cognate males and the other kept alone. TK♀subgroup mixed with TK♂did not have higher hatching rate compared to TK♀subgroup kept alone without males. On the other hand, WX♀subgroup mixed with WX♂did not have higher hatching rate compared to WX♀subgroup kept alone without males. These results indicate that both TK♀and WX♀became refractory to subsequent mating after they mated with incompatible males.4. Incompatible males can competitively decrease females mating with compatible males and reduce overall fecundity in a dose-dependent mannerTwo sets of experiments were performed using two different females. In each set, equal number of females were placed in different cages together with no male (blank), equal number of compatible males (positive control), equal number of compatible males plus equal number of incompatible males, or equal number of males plus3x as many incompatible males. Subsequently, females were collected from each group and divided into two subgroups to be mixed with either no male or cognate males. The results show that in the positive control group (TK♀×TK♂) the average hatching rate was0.861±0.020. When an equal number of WX males were included in the cage [TK♀×(TK♂+WX♂)], the average hatching rate dropped to0.211±0.071. This value was further decreased to0.063±0.039in the group that included3x as many WX males [TK♀×(TK♂+3xWX♂)]. In comparison, average hatching rate of TK♀mixed with only WX♂was0.004±0.003and TK♀kept alone produced no larva. These data indicate TK? mated with TK♂or WX♂in the presence of mixed male population, i.e., even in the presence of TK♂, mating between TK? and WX♂still occurred, which resulted in reduced overall fecundity of the groups. The extent of fecundity reduction correlated with the ratio of WX♂to TK♂.Similarly, in the parallel experiment with WX♀(Table3, Fig.5B), the average hatching rate of WX♀was reduced from0.903±0.013in the positive control group (WX♀×WX♂) to0.607±0.066in the group with equal number of competing TK♂included [WX♀×(WX♂+TK♂)] and further to0.407±0.077in the group with3x competing TK♂included [WX♀×(WX♂+3xTK♂)]. The average hatching rate of WX♀mixed with only TK♂(WX♀×TK♂) was0.001±0.001. WX♀kept alone produced no larva. These data also indicate that the frequency of WX♀×TK♂mating in the presence of mixed male population can also be increased with increasing TK♂:WX♂ratio.The correlation between hatching rate and the proportion of incompatible males was plotted for both TK♀and WX♀. Based on the curves, the fecundity of TK♀could be diminished when inundated by excess WX♂.9×WX♂can achieve nearly complete suppression of TK♀fecundity. On the other hand, the maximal fecundity reduction of WX♀by TK♂is around90%in one generation. The r2value is0.92(P<0.05) for TK? curve, and0.9363(P<0.05) for WX♀curve.5. The incompatibility is preserved in the offspring from the crosses In the case of releasing WX♂to suppress TK population, both male and female F1offspring will be generated from TK♀×WX♂cross, designated as F1♀(TK♀×WX♂) and F1♂(tk♀×WX♂).These hybrids will encounter TK♀, TK♂and WX♂, resulting in six possible mating combinations. Three extra mating combinations WX♀×F1♂(TK♀×WX♂), WX♀×WX♂and WX♀×TK♂that would not accompany this population suppression measure were also included in this set to provide more information about the crossing type of F1♂(TK♀×WX♂).These nine mating combinations were compared for fecundity. Our results show, the mating combinations F1♀(TK♀×WX♂)×F1♂(TK♀×WX♂) and F1♀(TK♀×WX♂)×TK♂had comparably high hatching rates, while F1♀(TK♀×WX♂)×WX♂produced no larva. These indicate F1♀(TK♀×WX♂) maintained the crossing type of TK♀. On the other hand, the average hatching rates of TK♀×F1♂(TK♀×WX♂) and TK♀×TK♂were comparably high, while the hatching rates in WX♀×F1♂(TK♀×WX♂) and WX♀×TK♂crosses were comparably low, indicating that F1♂(TK♀×WX♂) maintained the crossing type of TK♂. These results demonstrate that in TK♀×TK♂cross, both male and female F1offspring maintained their maternal crossing type.Reciprocally, the strategy of using TK♂to suppress WX population was tested for sustainability. To test the crossing type of F1♀(WX♀×TK♂) and F1♂(WX♀×TK♂) generated from the cross between WX♀and TK♂, nine possible mating combinations were compared for hatching rate. The average hatching rates of F1♀(WX♀×TK♂)×F1♂(WX♀×TK♂) and F1♀(WX♀×TK♂)×WX♂were comparably high, while the hatching rate was low for F1♀(WX♀×TK♂)×TK♂group. These indicate F1♀(WX♀×TK♂) maintained the crossing type of WX♀. On the other hand, the average hatching rates of WX♀×F1♂(WX♀×TK♂) and WX♀×WX♂were comparably high, while the average hatching rates of TK♀×WX♂and TK♀×F1♂(WX♀×TK♂) were comparably low. These indicate F1♂(WX♀×TK♂) maintained the crossing type of WX♂. In WX㏕x TK♂cross, both male and female F1offspring maintained their maternal crossing type.Taken together, when using incompatible males to suppress a target Cx. pipiens pallens population, hybrid offspring may be generated if the incompatibility is not absolute. However, these hybrids maintain their maternal crossing types. This phenomenon indicates that both original target population and inadvertently generated hybrids are subject to suppression by the released incompatible males, suggesting that this control strategy is sustainable.6. The three Cx. pipiens pallens populations are all infected with WolbachiaA fragment around500bp was amplified from all three populations using the wPip strains-specific wBpipF-wR primer pair.These results are consistent with previous reports that most mosquito-infecting Wolbachia are wPip strains of supergroup B.PCR and sequencing analysis of ank2gene revealed that NJ population was infected by Wolbachia of wPip-Ⅲ group (GenBank accession number JX050182), while WX and TK populations were both infected by Wolbachia of wPip-IV group (GenBank accession numbers JX050183and JX050184). The ank2genes from WX and TK populations are identical.7. The incompatibilities between different Cx. pipiens pallens populations are caused by WolbachiaTo confirm that the observed incompatibilities were caused by Wolbachia, the mosquitoes were treated with tetracycline. The elimination of Wolbachia was checked by Hoechst33342staining. Eggs from untreated mosquitoes had strong fluorescence at both anterior and posterior ends, indicating Wolbachia were concentrated at these poles. In contrast, eggs from tetracycline-treated mosquitoes had even distribution of background fluorescence. No strong fluorescence was observed around the micropyle or at the posterior end. These results indicate that Wolbachia were removed from these mosquito populations. Crossing experiments were carried out using both Wolbachia-positive and Wolbachia-negative populations. WX females were crossed with Wolbachia-positive TK males (TK(?)), Wolbachia-negative TK males (TKtet(?)) and WX males. Similarly, TK females were crossed with Wolbachia-positive WX males (WX(?)), Wolbachia-negative WX males (WXtet(?)) and TK males. The hatching rate of WX♀x TKtet(?) cross was not significantly different from that of WX♀x WX(?) cross, but was significantly higher than that of WX♀x TK(?) cross. The hatching rate of TK♀x WXtet(?) cross was not significantly different from that of TK♀x TK(?)cross, but was significantly higher than that of TK♀x WX(?) cross. These results indicate that the incompatibilities between TK and WX populations are dependent on the presence of Wolbachia, i.e. they are Wolbachia-induced CI.