**4. Discussion**

When used at sublethal doses applied via a diffuser-mediated spatial treatment, the pyrethroid prallethrin affected the fitness of laboratory-reared *Cx. pipiens* and *Ae. albopictus* adult mosquitoes. The insecticide influenced short- and long-term mosquito mortality, physical status, and egg laying. As a result of reduced mosquito fitness, the size of the F1 population declined in the three prallethrin groups in both species. The mosquitoes' behaviour was also altered. Biting was completely inhibited in as little as 15 min, offering 100% protection to potential human hosts. The modelling revealed that lower doses pose less risk to human and environmental health.

More than 50% of female mosquitoes were still alive 24 h after exposure to the 0.4 and 0.8 mg/h prallethrin doses; this figure was 28.8% for the 1.6 mg/h prallethrin dose. Although technically alive, these mosquitoes nonetheless suffered severe damage to their locomotor systems (e.g., they were missing up to five legs; Figure 4). Previous studies have also observed this phenomenon in response to insecticide exposure [38,39]. Leg loss could theoretically have a major impact because mosquitoes use their legs for a wide variety of functions, including locomotion, mechanical support (e.g., remaining on the water surface, laying eggs), chemical communication, sensory perception of the environment, and protection from desiccation [40,41]. However, other work found that insecticide-induced leg loss did not significantly affect the success of blood feeding or egg laying [38]—mosquitoes with fewer legs were still able to bite humans and reproduce, maintaining their life cycle. The mortality of adult mosquitoes increased in the days following prallethrin exposure, a pattern that may have been due, entirely or in part, to the insecticide's irreversible effects on the nervous system. For example, the mosquitoes may have been unable to metabolise the AS [42], or they may have struggled to seek out and/or acquire food [43]. Furthermore, female *Cx. pipiens* were found dead in the water when eggs were counted at 48 h post-treatment. It may be that, having lost legs, they were unable to remain on the water surface when laying eggs [38,44]. The combined percentage of dead and affected mosquitoes exceeded 90% for almost all groups at 24 h into the post-treatment period. The only exception was the female *Cx. pipiens* exposed to the 0.4 mg/h prallethrin dose (24 h: 41.6% and 48 h: 75.20%). According to European efficacy guidelines, for an AS/BP to be officially classified as an insecticide useable in spatial treatments, it must kill 90% of females within 24 h of exposure [30]. None of the doses tested in this study would meet the minimum requirements allowing insecticide authorisation; repellent use would also be prohibited because the compound is not authorised for that purpose. It should be noted that the 24-h window of observation means that authorisation decisions are based solely on "immediate" mosquito mortality. Therefore, the long-term mortality observed in this study would not be taken into account for authorisation purposes, even if the mosquitoes were to be "moribund/affected" at 24 h and then finally die at 48 h [30]. OECD guidelines provide specific instructions for such situations: "*Insects in [a] supine position and those [in a] ventral position without [the] ability to move forward and exhibiting uncoordinated or sluggish movements of legs are classified as moribund. Moribund test organisms are counted as dead, if they die within the test duration*" [32].

Looking at the long-term mortality, starting at 1 week into the post-treatment period, total mortality (females and males) for both species for all the prallethrin doses was 80–95%. The lowest level of LTM, 82.4%, was seen in the *Cx. pipiens* exposed to the 0.4 mg/h prallethrin dose. The highest level of LTM, 94.8%, also occurred in *Cx. pipiens*, in the mosquitoes exposed to the 1.6 mg/h prallethrin dose. In contrast, in the controls, total LTM was lower than 30% for both species. At the end of the first experiment (i.e., 4 weeks into the post-treatment period), even doubling the dose from 0.4 to 0.8 mg/h did not significantly increase LTM, regardless of species or sex. However, LTM did climb when tripling the dose from 0.4 to 1.6 mg/h. It should be noted that the mosquitoes in all the prallethrin groups had significatively higher LTM than the mosquitoes in all the control groups (Figure 1); there was no difference in LTM between the untreated and negative controls. Additionally, the first experiment showed that females were less susceptible than males to prallethrin (Figure 5). Sex-specific differences in susceptibility to insecticides have been seen before in laboratory populations [45] and field populations [46]. In both cases, males were found to be more susceptible than females. It is hypothesised that this difference is related to the males' smaller size and/or greater physiological susceptibility [47,48]. Nevertheless, it should be noted that, in all treatments, females survived significantly longer than did males. Consequently, biological factors appear to also influence mosquito mortality and survival.

Prallethrin exposure caused a marked decline in the size of the F1 population. The higher the dose, the larger the decline, which reached a maximum of 80–90% for both species. The above pattern likely stemmed from the higher mortality in exposed mosquitoes. The insecticide did not appear to affect female fertility in *Ae. albopictus*, given that, across treatment groups, there was consistency in the ratio of larvae to females (see Table 6). Additionally, because eggs could be accurately counted in this species, it was possible to confirm that the percentage of eggs that developed into third/fourth instar larvae was also fairly consistent (43.36% in the negative control and 53.8% for mosquitoes exposed to the 0.4 mg/h prallethrin dose), although it was rather low for the group exposed to the 0.8 mg/h prallethrin dose. For *Cx. pipiens*, it was hypothesised that insecticide exposure could affect egg viability via its impacts on raft assemblage (Figure 7) [37]. This hypothesis was based on the results of previous research. For example, Bibbs et al. [22] discovered that sublethal doses of the pyrethroid transfluthrin could cause chorion collapse in *Ae. aegypti* eggs, rendering them non-viable. In this study, the eggs of *Ae. albopictus* did not show any external signs of damage that could sugges<sup>t</sup> issues with their viability. However, no clear conclusions could be drawn from the ratio of larvae to females, which ranged between 35.27 for the untreated control and 42.16 for the mosquitoes exposed to the 0.8 mg/h prallethrin dose.

Other studies have shown that exposure to pyrethroid vapours (i.e., those of metofluthrin or transfluthrin) at sublethal doses can affect female fertility and egg laying by causing declines in egg viability [22,24] and larval survivorship [24]. However, in those studies, the mosquitoes were placed in small containers (<500 cm3), not in a large chamber as in this study (30 m3). Room size and/or the distance of the mosquitoes from the source of the insecticide could influence treatment efficacy. Another factor that could have an influence on the results is whether the mosquitoes were free flying or in cages. For example, any equipment used to constrain the mosquitoes could restrict the aerial diffusion of the AS [15,23,49]. Here, mosquitos could fly freely within a large chamber. As a result, it was impossible to control mosquito distance from the diffuser, but such a design probably better replicates AS use in real life and their influence on mosquitoes. Thus, returning to this study's results, the testing conditions used did not allow clear conclusions to be made about the effect of sublethal prallethrin doses on mosquito fertility. Further research is needed to determine whether more prolonged prallethrin exposure (i.e., longer than 90 min) could yield more definitive results.

With regards to biting behaviour, even the lowest dose of prallethrin, 0.4 mg/h, reduced the host-seeking efficiency of mosquitoes, resulting in 100% protection and 80–100% KD after 15 min. However, it was not necessary to reach 80% KD to greatly inhibit biting (Figure 8). In fact, even when just 10% of the population was knocked down, the level of protection against mosquito bites was approximately 90% (Figure 8). This result can be explained by prallethrin's effects. At low doses/exposure times, the insecticide causes mosquitoes to become disoriented. At higher doses/exposure times, the effects on the nervous system are more pronounced. Certain mosquitoes are knocked down, while others experience a dramatic impairment of their host-seeking abilities [50,51]. Although the importance of modifying vector behaviour has been recognised for decades, the utility of this tool remains greatly underestimated from the standpoints of both BP authorisation and disease control efforts.

When assessing an AS, it is also crucial to consider any risks to human and environmental health. The toxicological results showed that only the lowest dose (0.4 mg/h) would allow 24-h insecticide use by adults and children indoors while also limiting the environmental risks. However, such a low dose would not be authorised in this context of use under current EU requirements for insecticides, which only focus on immediate mortality and do not consider additional data such as LTM and/or beneficial behavioural modifications. Further studies are needed to define how much longer exposure would need to last at low doses for the compound to meet European efficacy requirements (i.e., 90% mortality within 24 h).

Worldwide, pyrethroids are commonly used to control insects, both at the individual level and the environmental level; for example, they are frequently part of IVM programmes [52]. Extensive research has been carried out to assess the effects of sublethal pyrethroid doses on mosquito fitness [22,24,49] and behaviour [23,53,54]. Although pyrethroids are used as insecticides, they can also function as repellents when certain doses or exposure times are used. If insecticides have appropriate levels of volatility, they can be used in space treatments at sublethal doses. Examples of such insecticides include metofluthrin [24,49], transfluthrin [22,55], d-allethrin [25], or prallethrin, the compound studied here [54]. Less volatile insecticides such as permethrin or deltamethrin function better as contact repellents [26,56,57]. For the latter group to be effective, mosquitoes must come into direct contact with the AS, which is possible when insecticides are applied to netting, for example [58,59]. In the case of space treatments, mosquitoes can detect the airborne compounds and avoid entering the treated area [18,60,61]. Multiple studies have demonstrated the efficacy of these insecticides at low doses and their potential benefits for

public health and mosquito control efforts [22–25,49,60]. However, in Europe, they are only authorised for use as insecticides, which greatly limits their potential utility [11].

This study found that sublethal prallethrin doses applied indoors via a spatial treatment had a significant effect on mosquito mortality and biting behaviour. This approach could thus potentially be used to reduce the vector capacity of mosquitoes and, consequently, public health risks. Although the research results presented here are promising, more studies on this complex topic are obviously needed. First, this study utilised two mosquito strains that have been bred exclusively in the laboratory for several years. As a result, it is unknown how well the above findings may reflect the reality in wild mosquito populations. Further studies addressing this issue should be performed. There are other directions that future research can take to explore the benefits and/or limitations of using sublethal doses of pyrethroids in mosquito control efforts. A logical tack to take is to further examine the usefulness of sublethal pyrethroid doses in IVM programmes by evaluating how compounds used as spatial treatments operate under field conditions. Although the concentration of the AS in the air is much lower, the environmental risks could be greater. When considering outdoor applications, an important factor to examine is the development of resistance in mosquito populations via continuous exposure to sublethal pyrethroid doses. Potential shifts in vector sensitivity or susceptibility under such conditions must be explored to assess the likelihood of this potential side effect [62–64].

It is essential to remember that, in the future, a major constraint will be the costs associated with justifying the use of, evaluating the efficacy of, and registering new compounds or compound uses under the BPR [65]. By utilising new evaluation parameters and/or adopting new authorisation paradigms (i.e., LTM and mosquito biting behaviour), it should be possible to exploit currently authorised compounds in new ways [66]. As a result, it may be possible to eliminate the above barrier to innovation and thus help ensure the continued availability of compounds that can effectively control mosquitoes while limiting risks to human and environmental health.

**Author Contributions:** Conceptualization, M.M.-G.; methodology, M.M.-G.; validation, M.M.-G., R.B.-M., and M.A.M.; formal analysis, M.M.-G.; investigation, M.M.-G.; resources, M.M.-G.; data curation, M.M.-G.; writing—original draft preparation, M.M.-G.; writing—review and editing, M.M.- G., R.B.-M., and M.A.M.; visualization, M.M.-G., R.B.-M., and M.A.M.; supervision, R.B.-M. and M.A.M.; project administration, M.M.-G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The work conducted herein was approved by the ethics committee of Henkel AG & Co. KGaA. It meets the company's corporate standards, which ensure health, safety, and respect for the environment, as well as the protection and ethical treatment of all study participants. The study was also conducted in accordance with the ethical principles of the Declaration of Helsinki. Participants were recruited and signed a written informed consent form that explained the study's purpose and procedures as well as the participants' roles and responsibilities; the form also notified participants of their right to withdraw or refuse to take part in the study at any point.

**Data Availability Statement:** The datasets generated during and/or analysed during the study are available from the corresponding author upon reasonable request.

**Acknowledgments:** We thank S.A. of the University of Girona for her assistance with the statistical analysis and J.P.-D. for her diligent proofreading of the manuscript. We are also grateful to all the study participants who volunteered for this research. We want to express our grea<sup>t</sup> appreciation for the work done by Henkel R&D staff: L.L., who was an essential assistant in the laboratory; J.I.C., who provided his valuable expertise about toxicological risk assessments; our colleagues in the chemical team; F.S. and J.C., who made this research possible by formulating the insecticides; and E.M., who facilitated this research overall.

**Conflicts of Interest:** The authors declare no conflict of interest.
