Ovicidal, pupicidal and bactericidal effects of aminopyridinium-based ionic liquids on Culex pipiens and certain human pathogenic bacteria

Culex pipiens (Diptera: Culicidae) is one of the most prevalent mosquitoes particularly in the Al-Madinah Al-Munawwarah region, Kingdom of Saudi Arabia, transmitting numerous diseases such as arboviruses, avian malaria, elephantiasis, dirofilariasis, and encephalitis. Worldwide, Culex pipiens has developed resistance to numerous insecticides; therefore, considerable efforts have been done to introduce new mosquito control candidates. Due to their unique physical, biological, and eco-friendly properties, ionic liquids (ILs) have been recently considered as promising agents in controlling a variety of organisms. Six aminopyridinium-based ionic liquids (IL1–IL6) were assayed against eggs and pupae of C. pipiens. Percentages of non-hatched eggs, non-emerged pupae, the lethal concentrations (LC50 and LC90) of both C. pipiens eggs and pupae were recorded. after exposure, to different ILs. The effects of ILs against eggs and pupae were concentration and IL-dependant. The percentages of egg unhatchability were much higher than those of pupal mortality, as the percentage of egg unhatchability reached 99% after treatment with IL6 at 0.5 g/L, while the percentage of pupal mortality reached 40.8% after treatment with IL4 at 0.5 g/L. The LC50 and LC90 of ILs against pupae were much higher than those of ILs against eggs. Effects of ILs on Staphylococcus aureus (Bacillales: Staphylococcaceae) and Escherichia coli (Entrobacterales: Entrobacteriaceae) were also studied; some ILs showed a considerable effect on both bacteria species. This is the first study to show the ovicidal, pupicidal, and bactericidal effects of aminopyridinium-based ILs in controlling C. pipiens, S. aureus, and E. coli.


Introduction
Mosquito-borne diseases are widely distributed among human populations and transmitted to more than 700 million people per year [1][2][3][4][5][6][7]. Culex pipiens are mainly distributed in temperate regions and represent crucial vectors of many diseases [8,9]. For example, C. pipiens can transmit St. Louis encephalitis, West Nile viruses, avian malaria, western equine encephalitis, Rift Valley fever, and bancroftian filariasis [10][11][12]. Culex pipiens has a flight range from 1.15 to 2.48 Km, which allows females to lay their eggs within a wide geographical range. This flight range also enables females to distribute their eggs in several spots and in turn dispersing C. pipiens broadly [13].
Controlling mosquitoes is a keystone to prevent mosquito-borne diseases. Insecticides have been recommended for years, but their continuous usage, many damaging consequences have appeared, including, toxicity to other living organisms, irreversible destruction to the ecosystem, and non/low-degradability nature of most insecticides [7]. Several mosquito species including C. pipiens have developed resistance to a variety of insecticides such as organophosphates and pyrethroids [9,[14][15][16][17][18]. Due to increased insecticide resistance, considerable efforts have been made to develop other alternative eco-friendly strategies [19][20][21]. Vector control is still considered an effective strategy against mosquito-borne diseases as no effective treatments or vaccines relieving these diseases [22].
Currently, there is an expanding demand for dependable, innovative, and inexpensive mosquito control methods [23][24][25][26]. Alternatively, eco-friendly, and safe sustainable approaches should be established that can affect a wide range of different mosquito species to diminish the existing dependence on controlling mosquitoes with insecticides.
In addition to mosquito immatures, domestic wastewater (DWW) harbours several important other microorganisms. Both Staphylococcus aureus and Escherichia coli are among the most abundant species of Gram-positive and Gram-negative bacteria in DWW, respectively [27,28]. Up to 65% of S. aureus release enterotoxins that target human intestines, causing food poisoning and are considered one of the most threatening bacteria in the globe [29,30]. On the other hand, E. coli could be either commensal in the intestinal tract or pathogenic. According to their virulence factors, E. coli are classified into six pathotypes [31]. Both S. aureus and E. coli can be detected in DWW, particularly coliforms bacteria (lactose fermenters) [27] Microbial multidrug resistance is a major global issue. Hence, scientists investigate antimicrobial activity through screening natural products that might lead to a promise inhibiting level [32]. The relationship between chemical compounds and their biological activity against bacteria has been documented previously as the cell wall and plasma membrane disruptors, inhibitor of nucleic acid synthesis and negatively affecting enzymatic proteins structurally [33,34]. The bacterial ability to resist several antibiotics is a worldwide concern. Therefore, investigation of new chemical compounds challenging microbial pathogens is a key need. A variety of chemical compounds have been extracted from plants such as flavonoids, quinones, terpenoids, tannins, coumarins, saponins, steroids, glycosides and alkaloids can affect bacterial infections and/or also as antibacterial agents [35][36][37][38][39][40][41].
Numerous mosquito surveys [ex. [57][58][59] were performed in some Saudi Arabia regions, demonstrating various mosquito species (Aedes caspius, Anopheles multicolor, Culex perexiguus, Culex pipiens, Culex pusillus, Culiseta langiareolata, and Culiseta subochrea). Culex pipiens considered among the most abundant mosquitoes in these studies. However, a previous study by [56] determined the efficacy of novel aminopyridinium-based ILs on C. pipiens larvae. The present hypothesis is to evaluate the ovicidal, pupicidal effects of these novel compounds as useful mosquitocidal agents. Lethal concentrations (LC 50 and LC 90 ) of ILs against eggs and pupae will be determined as well. Eventually, the bactericidal effects of the same IL derivatives against E. coli (Gram-negative) and S. aureus (Gram-positive) were also assessed in Al-Madinah Al-Munawwarah, Saudi Arabia.

Ovicidal and pupicidal bioassay
Pupae alongside larvae of C. pipiens were collected from the DWW drain area of Saad Al-Ghab [56]. Numerous water samples were collected and poured into 5L containers, The sampled water with larvae and pupae was then transferred into plastic trays (25 × 25 × 15 cm) which were 2/3 filled and kept at 24 ± 2°C for further experiments.
Larvae, pupae, and adults of C. pipiens were identified according to [60,61]. Larvae and pupae were reared in the laboratory; larvae were fed on fish food. Fieldcollected pupae and pupae transformed from fieldcollected larvae were kept in cages containing quail birds. Each cage was divided into two chambers separated by a net that allows emerged female mosquitoes to move through for blood-feeding on quail birds. Feathers on the quail's back were removed to facilitate blood-feeding. Adults were also allowed to feed on supplied 20% sucrose solution through soaked cotton pads placed in Petri dishes and on top of the cage nets. Daily fresh laid eggs were collected using a fine brush from white plastic cups that were halffilled with water. Collected egg rafts (about 100-200 eggs/raft) were counted and used directly in the experiments.

Aminopyridinium-based ILs
Derivatives of 4-dimethylaminopyridinium-based ionic liquids (Table 1) were synthesized in the Department of Chemistry, College of Science, Taibah University. The synthesis process was based on three criteria, firstly, keeping the same cationic nucleus for all tested ILs: secondly, the introduction of different length alkyl chains containing two to six carbons as in the case of IL1-IL4 or a phenyl group as in IL5. Thirdly, introduction a different functional group such as ketone for IL6

Screening of aminopyridinium-based ILs for ovicidal and pupicidal activity
Based on [56], ILs concentrations, 0.0156, 0.0312, 0.0625, 0.125, 0.25, 0.5 g/L, were tested for their effect against eggs. Concentrations, 0.125, 0.25 and 0.5 g/L, showed an effect against C. pipiens eggs, so they were selected for ovicidal bioassay. However, all those and higher IL concentrations had no effect against pupae, therefore, much higher concentrations (10, 15, and 20 g/L) were used to test the pupicidal efficacy of ILs. The ovicidal and pupicidal effects of six synthesized ILs (Table 1) were monitored daily for 4 days posttreatment to determine egg un-hatchability and pupal mortality.

Toxicity of selected aminopyridinium-based ILs on eggs and pupae
Aminopyridinium-based ILs were weighed up, then added separately into 250 ml glass beakers and dissolved in 100 ml of DWW to simulate the natural environmental conditions faced by C. pipiens eggs and pupae in the study area. Concentrations of 0.125, 0.25, 0.5, 10, 15 and 20 g/L for each IL were prepared. The bioassays were conducted in triplicates by using C. pipiens eggs (one egg raft/replicate) and pupae (25/replicate) at 24 ± 2˚C in the insectary. Both egg unhatchability and pupal mortality were recorded daily.
For each concentration, one egg raft and 25 untreated pupae served as controls. Culex pipiens eggs were considered un-hatched when they did not transform into larvae within a 4-day period. Pupae were considered dead when they settled motionless at the beakers' bottoms and did not respond to visual or mechanical stimuli, consequently failed to emerge to adults in a 4-days period. According to [62,63] eggs and pupae of C. pipiens transformed to the next stages in about 1.1 and 2.7 days respectively at ∼ 28˚C. Similar observations were noticed for hatching and eclosion of eggs and pupae respectively; so, a 4-days period was adopted to completely guarantee the unhatchability and non-emergence of eggs and pupae, respectively.

Bacterial growth
Both S. aureus and E. coli were clinical isolates, which were kindly obtained from King Fahad hospital. Both bacterial species were also abundant in DWW of Saad Al-Ghab. Gram-positive S. aureus was grown on nutrient agar and Gram-negative E. coli was grown on MacConkey agar before incubating for at least 16 h at 37°C.

Bactericidal activity of ILs
The biological activity of ILs against both bacterial species was tested using agar disc diffusion methods. The 6 mm discs filter paper was soaked with 0.25 g/L of different ILs solutions (10 µl each) before mounting on the agar surface. All plates were incubated for at least 16 h at 37°C. Then, the inhibition zones were measured by calculating the diameter in (mm) to determine the bactericidal activities of ILs.

Data analysis
Minitab R software 17 (a trial version) was used to analyze data. The mean percentages of un-hatched eggs and non-emerged pupae of C. pipiens were analyzed using one-way ANOVA to determine the differences among concentrations of each IL and the differences among ILs for each concentration. When significant differences were existing, this was followed by a Tukey's test for pairwise comparisons of means. The alpha value was set at 5%. Probit analysis [64] was used to determine LC 50 and LC 90 values of different ILs used against eggs and pupae of C. pipiens.

Alkyl side chain
In the present study, C. pipiens eggs and pupae were exposed to five synthesized ILs (IL1-IL5) that contained an alkyl chain with carbon atoms or a phenyl group (Table 1) to identify their effect on eggs and pupae. Different concentrations of ILs showed significant differences in the toxicity to C. pipiens eggs and pupae (P < 0.05). We noticed that the length of the alkyl side chain was directly proportional to the efficacy of ILs. The data showed that the longer the side chain, the greater the effect against C. pipiens eggs and pupae ( Table 2). For example, IL4 (side chain with six carbon atoms) caused high significant egg un-hatchability (96.7%) if compared to lower egg un-hatchability (88%) of IL1 (side chain with two carbon atoms) at 0.5 g/L (P < 0.05). Pupal mortality was significantly higher when exposed to IL4 (23.7%) than to IL1 (13%) at 15 g/L (P < 0.05).
Concerning IL1, 76.8% of eggs were un-hatched at 0.125 g/L and this percentage increased gradually reaching 83.4% egg un-hatchability at 0.25 g/L and the highest egg un-hatchability was 88%, which achieved at 0.5 g/L (Table 2 and Figure 1A). For pupal mortality, IL1 showed 7.4% mortality at 10 g/L. This effect increased to 13% and 20% mortality at 15 and 20 g/L, respectively (Table 2 and Figure 2A).
In the case of IL2, un-hatched eggs represented 80.3% at 0.125 g/L, while at 0.25 g/L, un-hatched eggs recorded 85.4%. At the highest concentration (0.5 g/L), 92.6% of eggs did not hatch (Table 2 and Figure 1B). At 10 g/L, un-emerged pupae attained 10.7% and this percentage increased to 18.3% at 15 g/L and finally reached 26.4% at 20 g/L (Table 2 and Figure 2B).
The IL5 showed a higher ovicidal efficiency as more than 90% of eggs did not hatch at 0.125 g/L and 98% did not hatch at 0.5 g/L (Table 2 and Figure 1E). Concerning pupal mortality, at 10, 15, and 20 g/L of IL5 recalled in 15.3, 21.5, and 31.9% mortality, respectively (Table 2 and Figure 2E).

Halides containing an acyl group
Among the ILs tested, only IL6 had a functional group (acyl group) on its side chain. At concentrations of 0.125, 0.25, and 0.5 g/L of IL6, the ovicidal activity ranged from 93.4% to 99% (Table 2 and Figure 1F). On the other hand, at 10, 15, and 20 g/L, pupicidal activity of IL6 ranged from 16.1% to 35.7% (Table 2 and Figure 2F). From the previous results, both ovicidal and pupicidal activities of tested ILs increased proportionally with concentrations ( Table 2 and Figures 1 & 2). For egg un-hatchability, significant differences among ILs were reported at each concentration tested (P < 0.05). At 0.125 g/L, the percentages of egg un-hatchability of IL1 and IL2 were significantly lower than those of IL3 -IL6. It was also reported that IL3 was significantly lower than IL6. At 0.25 g/L, the percentages of egg un-hatchability of IL1, IL2, and IL3 were significantly lower than those of IL4, IL5, and IL6. At 0.5 g/L, the percentages of egg unhatchability of IL1 were significantly lower than those of IL3, IL4, IL5, and 6. It was also found that the percentages of egg un-hatchability at the same concentration of IL2 were significantly lower than those of IL5 and IL6.
For the pupal mortality percentages, significant differences among ILs were also recorded at each concentration used (p < 0.05). At 10 g/L, for IL1, the mortality percentages were significantly lower than those of IL3 -IL6. At the same concentration, it was also reported that the percentages of pupal mortality of IL2 were significantly lower than those of IL3, IL4, and IL6. A similar trend was also recorded when comparing IL1 with IL3-IL6 at 15 g/L concentration. At the same concentration, the mortality percentages of IL2 were significantly lower than those of IL6. At the highest concentration used in the present study (20 g/L), the percentages of pupal mortalities of IL1 were significantly lower than those of IL3 -IL6. The percentages of mortality of IL2 were significantly lower than those of IL4 and IL6. The mortality percentages of IL3 were significantly lower than those of IL4. Eventually, the percentages of mortality of IL4 were significantly higher than those of IL5. From these results, it can be concluded that IL3-IL6 at most concentrations are more effective against eggs and pupae.    lowest effect on both bacteria. On the other hand, IL3, IL4, and IL5 had no effect on both bacteria (Table 3). Inhibition zones of IL1, IL2, and IL6 are illustrated in Figure 4. Furthermore, it was shown that IL3, IL4, and IL5 had no biological effect on the investigated bacteria ( Figure 4). It was found that the Gram-positive bacteria, S. aureus, were more significantly resistant than Gramnegative bacteria, E. coli, (p < 0.05) to ILs used against both bacteria.

Discussion
Insecticides are well recognized for their instant action but have major drawbacks in their application as they are non-selective and may negatively affect other inhabitants in the environment [65][66][67][68]. Mosquitoes have become resistant to several categories of insecticides [ex. [69][70][71][72][73][74]. Consequently, searching for new safe and degradable compounds should be encouraged for controlling mosquitoes. This study highlights the promising potential of ILs as practical alternatives to conventional insecticides [56]. Similar recent studies showed that numerous plant extracts and Eucalyptus oil have a potent effect at lower concentrations against larvae and pupae of different mosquito species [75,76].
Recently, larvicidal and pupicidal activities of Eucalyptus and neem oils against Aedes aegypti and Aedes albopictus were evaluated by [75] as they found that Eucalyptus oil was more effective against larvae and pupae. In the current study, ovicidal and pupicidal efficacies of ILs depended on volume rather than the surface area of water as compared to the application of Eucalyptus oil. On the other hand, the high ovicidal efficacy of ILs could be attributed to higher permeability of eggshells through de-waxing and disruption of endochorion tanning.
Comparable trends of ovicidal and pupicidal efficacy of ILs in the present investigation were observed, as high egg un-hatchability and moderate pupal mortality when using leaf extract of Annona senegalensis against C. quinquefasciatus were observed [77]. Cassia fistula fruit extract, carvacrol, thymol, and Anacardium occidentale nutshell extract against C. pipiens eggs, larvae, and pupae were effective [78][79][80]. It was also reported by [81] that the un-hatchability of freshly laid C. pipiens eggs significantly increased when treated with diflubenzuron, pyriproxyfen, and azadirachtin when compared to the effects of the same compounds on embryonated eggs.
El-Sheikh et al. [82] studied the effect of different concentrations of selected heavy metals' salts against immature and mature stages of C. pipiens and they found that the potential survival of second instar larvae was highly affected by these salts. The number of eggs laid by females resulted from treated larvae decreased significantly and therefore, a lower fecundity was recorded for these females compared with control mosquitoes. Consequently, egg hatchability was significantly decreased compared to control eggs. The presence of such heavy metals in the ecosystem of C. pipiens could contribute to mosquito breeding reduction [82].
Comparing with synthetic products that exert commonly neurotoxic effects and promote the development of cross-resistance in insects, ILs may have some modes of action, such as the generation of reactive oxygen species and disruption of embryonic and pupal development. A recent study by [83] showed that buprofezin and azadirachtin affected embryonic development and egg hatchability through hormonal alterations. The current study showed that egg unhatchability and pupal mortality were ILs concentration dependent. The same trend was observed in the response of freshly laid eggs of C. pipiens when treated with different insect growth regulators [81].
The toxic effects of ILs were tested against C. pipiens larvae and a specific cell line of the fall armyworm, Spodoptera frugiperda [56,84]. They showed that the ILs caused death in the treated larvae and cells; the toxicity of ILs was dependent on concentration, structure, and exposure time [56,84]. Goellner et al. [55] obtained comparable findings but against Aedes aegypti larvae with aqueous solutions of two imidazolium salts, recording up to 90% mortality after 48 h exposure. These results are like those of the present study, as the ovicidal effect was around 90% at a higher concentration (0.5 g/L).
Based on the obtained results by [56] it can be clearly conclude that alkyl chain length played a major role in the toxicity caused by different ILs and that the efficiency of the tested ILs increased when the length of the attached alkyl chain increased. The concentrations of tested IL contributed to their toxicity. Accordingly, a possible correlation between the chemical structure of ILs and their ovicidal and pupicidal effects may be present.
It was recorded in the present work that the percentages of mortalities in treated eggs were higher than their counterparts in treated pupae and this was also expressed in the LC 50 and LC 90 values. These values were much higher for ILs used for treating pupae than those of ILs used for treating eggs; this indicates the higher susceptibility of eggs ( ∼ 77-99% un-hatchability) treated with different ILs. In contrast, pupae can highly tolerate the efficacy of ILs. Similar results for the un-hatchability of eggs were obtained by [85] when they treated eggs of C. quinquefasciatus, Aedes aegypti, and Anopheles stephensi with methanol extract of Asparagus racemosus. It was also reported by [86] that the ovicidal activity of ethanol extract of Gliricidia sepium reached 100% in Anopheles stephensi. They also reported the pupicidal activity against the same mosquito species and they found moderate pupal mortality of about 40%. It was also recorded by [79] that LC 50 of phenolic lipid products from Anacardium occidentale against pupae of C. quinquefasciatus was very high if compared to that of the same products against larvae. The same trend was also observed by [77], as high egg un-hatchability and moderate pupal mortality recorded when using leaf extract of Annona senegalensis against C. quinquefasciatus. This difference of tolerance between eggs and pupae may be due to the presence of special structures/modifications found in both non-feeding stages (eggs and pupae). It was concluded by [56,87] that the effects of ILs on C. pipiens larvae and adults occurred after the ingestion of ILs; and they also attributed the larval mortality to the damage of the intestinal epithelium. [88] noted in the early embryogenesis of different mosquito species including C. quinquefasciatus, that the water passes freely through the transparent eggshell, which at this moment is composed of exochorion and endochorion layers. Concerning, the pupal stage, it was reported by [89], the presence of a ring of hydrofuge cuticle encircles the spiracular aperture which resists the water entrance inside the pupal body. These explanations may reveal the cover of why eggs were much susceptible to ILs than pupae. This resistance of pupae to ILs could be also due to 1) pupal stage is preceded by a feeding stage (larvae) which could possibly gain cross-resistance throughout exposure to other relevant insecticides and/or chemicals and 2) stored fats in pupae may enhance their resistance to different pupicidal substances.
Concerning the bactericidal effect of ILs in the present study, we noticed that the treated water had both bacterial species (E. coli and S. aureus); so it seemed that the water may be contaminated after discharging in the valley (study area). Human pathogens have been found in the water body of the study region (data not shown) indicating pollution with sewage was merged at some points. The illegal discharge could be one of the reasons.
It was found that E. coli, were more significantly susceptible than S. aureus. This finding parallels the effects of previous antibacterial compounds' activity against S. aureus and E. coli [90]. The difference in the cell wall structure could differentiate between Gram-positive and Gram-negative bacteria. The resistance of S. aureus to ILS may be due to the presence of peptidoglycans, which are 70-100 layers in the cell wall of Gram-positive bacteria. Another reason for the resistance of Grampositive bacteria is the structure of peptidoglycans, as they are consisting of two types of polysaccharides, Nacetyl-muramic acid, and N-acetyl-glucosamine crosslinked by cross bridges and peptide side chains. The difference of resistance against ILs may be also attributed to the differences in modifications of fatty acid composition in Gram-positive and Gram-negative bacteria [91]. The Gram-negative bacteria can resist some of the antibiotics such as penicillin by the lactamase enzyme secretion which exists in the periplasmic space between the cytoplasmic membrane and the thin outer membrane [90].

Conclusion
Overall, our findings show that the ovicidal and pupicidal effects of ILs are concentration dependent. Percentages of egg un-hatchability and pupal mortality are proportionally increased with concentrations of different ILs. Generally, the pupae are more tolerant to ILs than eggs, as the pupal mortalities reached 40.8% at the maximum, while egg un-hatchability reached 99%. Concerning the bactericidal effect of ILs, E. coli was more significantly susceptible than S. aureus. This is the first study to display the ovicidal and pupicidal effects of aminopyridinium-based ILs in controlling C. pipiens; as well as their effects against certain human pathogenic bacteria. Further studies are required to test the applications of ILs under field circumstances and to characterize their probable fate, residual traces, and side effects against C. pipiens and other non-target inhabitants.

Disclosure statement
No potential conflict of interest was reported by the author(s).