Our study revealed that the implementation of equivalent selective pressure for three successive generations with both DFB and Bti against the form pipiens and molestus of Cx. pipiens induced differential susceptibility levels only in the first case. The selected populations of Cx. pipiens f. molestus to both larvicides exhibited a high fitness cost in terms of reduced winter larval survival rates accompanied with increased larval developmental times. Moreover, the obtained adults of the Bti selected population appeared to suffer an additional cost in terms of shorter lifespan compared to the other two (control and DFB selected). On the other hand, the selection process had no apparent effect on Cx. pipiens f. pipiens female winter survival rates relative to control. Additionally, overwintered females showed similar reproductive parameters among populations. Interestingly, these females, irrespective of population origin, experienced considerable post-overwintering longevity periods.
The EI50 values estimated in the current study for the control populations of both forms were found to be almost identical with that of the Benaki Cx. pipiens f. molestus laboratory reference strain [23], suggesting high susceptibility to DFB. Similarly, earlier studies conducted in different regions of Greece using the WHO diagnostic dose protocols demonstrated high susceptibility of most Cx. pipiens populations tested [37]. Moreover, recent surveys failed to detect any specific mutations in the chitin synthase gene of Cx. pipiens sampled from Greece that are associated with high levels of resistance against DFB. On the contrary, two and three different mutations at amino acid I1043 of the chitin synthase gene have been reported in neighboring Turkey and Italy, respectively with phenotypes exhibiting up to 128 fold Resistance Ratios (RR) relative to the Benaki reference strain [23–26]. The different selection pressure regimes imposed on these populations over the past years relative to the Greek ones, have been proposed as a possible explanation. Interestingly, in our study by imposing the same short term selection pressure with DFB we observed differential response on the susceptibility levels between the form pipiens and molestus, especially as EI50 RR values are concerned. This tendency was found to be even more pronounced after additional generations of continuous selection to DFB (F6, F9 & F12 generation) in the laboratory (Ioannou et al. in preparation). The reasons underlying this differential response to DFB selection of the two forms of Cx. pipiens remain largely unknown. A possible explanation could lay on their different biology. The tendency of the molestus form to reproduce below ground may largely decrease both its exposure to insecticides as well as the gene flow rates among different populations. On the other hand there are no such limitations for the above ground free living pipiens form. Therefore, it is anticipated that the molestus form populations may lack the genetic background for a rapid apparition and evolution of tolerance. This argument is also supported by the fact that in the current study the EI90 value of the control Cx. pipiens f. pipiens population (without selection) was twice as much as of the molestus form. Another possible explanation could be the fact that the DFB selected populations of each form may encounter differential levels of fitness costs in the wild such as winter survival rates as evidenced by the present study.
Sort term selection with Bti had a minor impact on the susceptibility levels of both biotypes. This is an expected outcome since there are no consistent recordings of mosquito resistance development to the full crystal. Despite some sporadic reports describing considerable resistance levels of wild mosquito populations against Bti [27, 42–44], long-term studies under both laboratory and field conditions provide no support to these data [45–51]. The low tolerance against Bti after long periods of applications in natural settings or intensive laboratory selection have been primarily attributed to the synergistic action between the Cry and the Cyt toxins as previously mentioned. In contrast, selection with single, purified Cry toxins can rapidly lead to considerable resistance levels [45, 52, 53]. Another factor that seems to promote the lack of Bti resistance in mosquitoes is the fact that in the absence of selection pressure within only a few generations (3–5), any acquired tolerance disappears almost completely [46, 48]. The high fitness costs following extensive selection to this microbial larvicide have been proposed as the most convincing explanation of this phenomenon.
Both DFB and Bti selection against Cx. pipiens f. molestus conferred a high fitness cost in terms of larvae winter survival as mortality rates relative to control increased more than 50% and 30% respectively. When we repeated the same experimental procedure after three additional generations of selection (F6) under optimum (laboratory) conditions we found no apparent differences in larval survival rates among tested populations. This suggests that the observed costs are manifested only under stressful conditions. Indeed, the mean prevailing temperature during larval development reached the lowest developmental thresholds described for the species [54] shaping a very challenging environment for survival. In general, the fitness costs determined in optimal conditions are not always representative of that experienced in the wild. This is because stressful environments and/or limited resources might be more deleterious for tolerant/resistant individuals. Similarly to our findings, the prolongation of larval developmental times in OPs resistant Cx. pipiens populations was found to emerge only under stressful crowding conditions in natural breeding sites [33]. Therefore, both biotic and abiotic factors as in our case, may shape the expression of fitness costs related to insecticides’ selection. The observed differences on the reduction of larval survival rates between DFB and Bti selected populations suggest deferential levels of fitness costs. A possible explanation may lay on the different mode of action of the two larvicides. An interesting finding that emerged from the current study is the fact that while short selection against Bti has minor effects on the tolerance levels, it may confer a high fitness cost under stressful conditions.
Reduction on winter larvae survival rates following selection against DFB and Bti in Cx. pipiens f. molestus was also accompanied with a discrete increment of their developmental duration relative to control. Interestingly, the longest larvae developmental times were recorded in the Bti selected population. Moreover, the adults obtained exhibited significantly shorter lifespan compared to DFB selected and control population. This is probably because a higher tolerance to Bti has been associated with a modified microbiota of larvae, which may affect their midgut proper function and therefore the nutrients assimilation, developmental processes and ultimately adult performance as it is well documented that nutrition during the mosquito larval stage may shape important fitness elements of the emerging adults [55, 56]. Similarly to our results, Bti selection against Ae. aegypti also resulted in significant prolongation of larvae developmental times relative to control [48]. No effects were found regarding adult survival in the same study. However, it should be stressed that in this case, adults had access to only water and therefore no direct comparisons can be made with our results. A moderate reduction on both male and female longevity following selection with Bti was observed in Cx. pipiens f. pipiens compared to untreated control [46]. The optimal laboratory conditions in this study may account for not detecting significant differences as in our case.
Contrary to Cx. pipiens f. molestus, selection against DFB and Bti had no apparent effects on the winter survival of the pipiens form. The differential overwintering developmental stages (larva vs adult) in these two forms may account for the observed outcome. Mosquito larvae require a minimum amount of nutrition to fully mature and pupate. Furthermore, larval developmental completion takes place within specific time limits which are endogenously defined. This dynamic process may be more prone to selection costs relative to the adult stage where full development have been already attained. In contrast to our findings, resistance of Cx. pipiens f. pipiens against OPs have been associated with reduced overwintering survival [34]. Among others, the differential exhaustion of fat reserves was proposed as the basic proximate cause. Indeed, a later study confirmed that the presence of resistance alleles against OPs in this species is negatively correlated with female lipid reserves [57]. The fact that female winter survival patterns in our study were almost identical between the control and the selected populations suggest no differences on the physiological process of fat reserves accumulation and/or exploitation. But most importantly, it means that these females have equal probabilities of survival as the untreated ones, providing the base for the building of higher tolerance levels in the next year. This argument seems to be supported by the fact that the resistance ratio against DFB of Cx. pipiens field populations in Italy, from 32.5 fold in 2015 reached 128.5 fold in 2016 [23], which backs the idea of the persistence of resistant mosquitoes in the wild from year to year. However, this is something that needs to be demonstrated for the specific populations, as in contrast to our case, they carry mutations of the chitin synthase gene associated with striking DFB resistance levels. Interestingly, further studies in the same country reveled a high focal distribution of DFB resistant Cx. pipiens mosquitoes which was attributed to the differential selection pressure imposed by both agricultural and mosquito control applications with DFB in the tested areas [24]. Since no separation between the two forms of Cx. pipiens took place in this study, the following explanation is possible. The high focal distribution of DFB resistant mosquitoes may only reflect differences in the mosquito populations’ composition regarding the two forms, with the predominance of pipiens form to account for the observed outcome. For instance, analysis of population structure from different areas of Greece, a country with identical climate, revealed extreme variations between the two forms on the composition of Cx. pipiens populations [9]. Nevertheless, as mentioned above, the validity of this hypothesis has to be confirmed for the specific populations.
Winter survival of diapausing Cx. pipiens f. pipiens females may largely vary depending on the hibernacula conditions [3, 58]. It has been observed that females may abandon their overwintering sites and actively search for new ones, a behavior described as an adaptive response associated with increased survival. It seems that the quality of each overwintering site, as experienced by females, is depending on multiple parameters such as the prevailing temperature and humidity levels, predator density, parasite frequency and human disturbance and therefore this quality may change during winter [34]. Under our experimental design, mosquitoes were not able to select the most optimal environment for maximizing their survival as they were forced to overwinter in a given place (warehouse). Nevertheless, this does not diminish the reliability of our results since females from all populations experienced exactly the same conditions. Female survival rates observed in the current study are comparable with that found by Koenraadt et al. [58] considering similar overwintering sites such as unheated house rooms. Interestingly, they found that non-diapausing females kept under the same conditions, died within four days, suggesting that in the absence of nutritional resources such females have a very limited ability to survive but also see Rinehart et al. [59].
Reproductive parameters of overwintered females of Cx. pipiens f. pipiens were similar between the two selected populations and the control. Blood meal acceptance percentages ranged between 34.4–46.4% suggesting that females had partially terminated their reproductive diapause by the time that trials took place. Contrary to other studies that used artificial conditions to terminate diapause in order to induce females to either respond to host stimuli [60] or receive a blood meal [58] we intentionally preferred to simulate as much as possible the natural conditions in an attempt to detect any potential variations in feeding activity. Although, no significant differences were observed regarding both the preoviposition period and the average number of eggs per egg raft, the better performance of the control population in respect to these parameters may reflect an early form of fitness cost in the selected ones. The short term selection to both larvicides may have acted against more pronounced differences as indicated by other studies. For example, Bti selection against Cx. pipiens f. pipiens for 20 generations resulted in 44.8% decrease of female fecundity [46] while Belinato & Valle [61] by applying the same experimental protocol as we, found that DFB selection against Ae. aegypti for 6 generations also conferred a significant reduction on this parameter. Finally, an interesting finding that emerged from the current study is the fact that females, irrespective of population origin (selected or not), experienced extensive post-overwintering longevity periods, which appeared to even exceed the total lifespans of Cx. pipiens f. molestus females (Tables 3 & 5). This observation is in accordance with a previous study documented that the physiological changes that take place during the pre-hibernation transition of these females confer a considerable increase in their longevity potential [62].