Abstract
Plasmodium falciparum is primarily transmitted by Anopheles gambiae. Malaria caused by Plasmodium falciparum is a major public health issue in western Kenya and sub-Saharan Africa, accounting for 90% of malaria deaths. The primary methods of malaria prevention are indoor residual spraying and the use of insecticide-treated nets. These tools face challenges such as mosquito resistance to insecticides as well as their toxic effect to the non-target organism, therefore this study aims to explore the application of lichen secondary metabolites as potential oral biological insecticides by assessing mosquito mortality in varying concentrations. Lichen secondary metabolites were extracted from Cladonia foliacea thalli. Bioassay experiments were conducted on A. gambiae Kisumu strain mosquitoes. Mortality rates were measured after ingesting sugar bait and lichen extracts in different concentrations. Three test replicates and negative control were used, with mortality measured after 4, 24, 48, and 72 hours. Analysis using three-way analysis of variance with twoway interactions was performed using R program to determine the effect of different lichen extract concentrations, time of exposures and mosquito sex on mortality. Our results showed that the ingestion of C. foliacea extract at 50 mg/ml and a post-exposure period of 24 to 48 hours had a maximum effect on the mortality rate of targeted male and female A. gambiae. No statistical difference was found between male and female mosquitoes in mortality. Our study confirms firstly that the extract of C. foliacea is a promising oral toxic agent against adult malaria vector A. gambiae.
INTRODUCTION
The mosquito Anopheles gambiae Giles, 1902 is among the main malaria vectors in Kenya. Molecular studies on mosquitoes have confirmed the invasion of Anopheles stephensi Liston, 1901 as a new distribution record of the species in Kenya that may threaten the efforts to eliminate malaria transmission in urban and rural areas in Kenya (Ochomo et al. 2023, Robi et al. 2010, Stevenson et al. 2012).
The success in the reduction of malaria burden in Africa has been attributed to the application of conventional vector control tools such as long-lasting insecticide-treated nets (LLITNs)* and indoor residual spraying (IRS) (Bhatt et al. 2015). These methods target the adult indoor resting males and females as well as indoor feeder female mosquitoes, considering that only female mosquitoes bite and transmit malaria, and lessening of males reduces the reproduction capabilities. However, their full potential as control tools has been limited due to the resistance of target mosquitoes to the chemical agents found in their formulation and other human factors such as non-adherence to regular use of bed nets (Lindsay et al. 2021, Owuor et al. 2021). Effective multiple vector control strategies have proved to overcome the limitation of individual control tools when used alone (Kiware et al. 2017). However, the sugar-feeding behaviour of both males and females did not receive much interest and attention so that oral toxic bait could be applied and control mosquitoes in the past, but currently, it has received more attention as a potentially vulnerable point for vector control (Müller et al. 2010 b, Revay et al. 2015, Sissoko et al. 2019). It is known that male and female mosquitoes naturally obtain their carbohydrate energy sources to survive. The sugars are obtained from flowering plant nectary, honeydew, and varieties of ripe rotting fruits (Foster 1995, Gouagna et al. 2010). A mixture of toxic agents to the sugar and deliberate offering to the target mosquitoes is the basis of the novel toxic sugar bait intervention tool for vector control.
The concept of sugar-baited insecticide started about five decades ago by Lea who used malathion sugar solution to control Aedes aegypti (Lea 1965). This concept has gained wide interest and application as a new intervention method against mosquitoes and other vectors of human diseases (Fiorenzano et al. 2017, Kumar et al. 2024, Rochlin et al. 2022).
As a result of the laboratory efficacy of attractive targeted sugar bait studies (Allan 2011, Sippy et al. 2020, Stewart et al. 2013), field trials have been tested to evaluate their insecticidal potential. Toxic synthetic insecticides (Diarra et al. 2021, Maia et al. 2018, Müller et al. 2010 a, 2010 b, Qualls et al. 2015, Stewart et al. 2013, Traore et al. 2020) and toxic biological insecticide agents (Revay et al. 2015, Schlein and Müller 2015, Schlein and Pener 1990) are spiked in sugar solution and their impact has been associated with reducing the population of mosquitoes and malaria transmission in treated habitats. The application of TSBs using boric acid and fipronil as toxic agents reduced the landing rates of mosquitoes on humans in an attempt to probe and feed (Xue et al. 2008). Initiation and sustained feeding by the mosquitoes are determined by the attractiveness of the sugar bait, and it has been confirmed that guava juice is the most attractant formulation among other competing fruit juices that are attractive to the mosquitoes (Kumar et al. 2022).
Recent reviews of lichen secondary metabolites have highlighted their insecticidal properties on the various stages of mosquitoes (Araújo et al. 2015, Cocchietto et al. 2002). Since 2000, limited literature on the insecticidal activity of usnic acid against mosquito life stages has emerged (Araújo et al. 2015, Cocchietto et al. 2002, Nimis and Skert 2006). However, the promising results regarding the potential of usnic acid against larval stages have not been exploited. This is possibly due to the toxicity of usnic acid on Artemia salina and therefore its safety might have raised environmental safety concerns (Bomfim et al. 2009). Mosquitoes can still develop resistance to chemicals in synthetic insecticides when administered orally. However, a recent review from 2023 by Njoroge et al. suggests that using oral toxic sugar baits is a safe option for non-target organisms (Njoroge et al. 2023, Stewart et al. 2013).
A recent review of the insecticidal properties of LSMs by Muhoro and Farkas (2021) indicates that lichen secondary metabolites have been tested mostly for their insecticidal property in the larval stages of mosquitoes. Equally and notably, recent studies have explored the potential of LSMs against mosquito larvae but have not investigated their impact as oral poison on adult male and female mosquitoes (da Silva et al. 2023, Koc et al. 2021).
Synthetic insecticides in aqueous and dried forms have been tested on adult mosquitoes in the form of toxic sugar bait (TSB) (Allan 2011, Sippy et al. 2020). Comparably, an attempt to use biological agent (Bacillus sphaericus spores) together with sucrose/dye as a bait has been evaluated by Schlein and Pener (1990) to control Culex pipiens L.
Biological control methods have been emphasized and gained more attention due to their promising results. They are valued for being safe to non-target organisms, having a lower risk of resistance and for being biodegradable and safe for the environment (Benelli et al. 2016).
Lichens are among organisms that can be used for biocontrol. They are a self-sustained ecosystem consisting of fungi, algae, and other microorganisms living together (Hawksworth and Grube 2020). The importance of the primary fungus and photosynthetic partner is emphasized by Sanders (2024) in his recent comment on the definition of lichens. Lichens are known to produce over 1000 secondary metabolites (Goga et al. 2018) with a wide range of biological properties, including insecticidal potential (Huneck 1999, Huneck and Yoshimura 1996, Kosanić et al. 2018). Little is known about the use of LSMs as an oral poison against adult male and female A. gambiae mosquitoes.
The lichen Cladonia foliacea (Huds.) Willd. is widely distributed over Europe and relatively frequent in Hungarian lowlands and low elevation mountain rocky grasslands (Wirth et al. 2013) (Fig. 1). It contains two major LSMs (usnic acid and fumarprotocetraric acid) in known concentrations (for usnic acid ranging from 6.88 to 34.27 mg/g dry weight and fumarprotocetraric acid from 1.44 to 9.87 mg/g dry weight – Farkas et al. 2020, 2024). These metabolites protect the photobionts against harmful UV- and solar radiation (Nguyen et al. 2013), furthermore they have various bioactive roles (Molnár and Farkas 2010), e.g. regarded as insecticides (Cetin et al. 2008, Emmerich et al. 1993). Usnic acid occurs in nature in two optical isomers (enantiomers) (Kinoshita et al. 1997). The larvicidal activity of the isomers against mosquitoes (Culex pipiens L.) has been evaluated, and a not significant dose-dependent outcome of the two isomers was reported (Cetin et al. 2008).
The lichen Cladonia foliacea extracted for applying in sugar bait (photo by E. É. Farkas)
Citation: Acta Botanica Hungarica 66, 3-4; 10.1556/034.66.2024.3-4.7
However, Emmerich et al. (1993) found significantly different toxicity and antifeedant activity in the larvae of a herbivorous insect (Spodoptera littoralis Boisduval), since the (–)-usnic acid was found to be almost 10-times more effective than the (+)-usnic acid. It was known from 2004 (Bézivin et al. 2004), that C. foliacea produces (–)-usnic acid and it was confirmed that the Hungarian populations also contain this isomer (Farkas et al. 2024) in additionally to fumarprotocetraric acid and 9′-(O-methyl)protocetraric acid. In agreement with Galanty et al. (2019) the study of the usnic acid enantiomers, consequently the (–)-usnic acid enantiomer containing lichen C. foliacea seems to be promising.
This study aims to investigate the effect of crude extract of Cladonia foliacea on adult mosquitoes (Anopheles gambiae) as biological oral poison in the form of toxic sugar bait (TSB). It was aimed to compare the effects 1) of different concentrations of TSB, and 2) of exposure time, and 3) in male and female specimens. The results are expected to enhance existing vector control tools within integrated vector management through this novel application for malaria control.
MATERIAL AND METHODS
Set up of the study and laboratory experiments
Bioassay laboratory experimental tests were conducted at Kenya Medical Research Institute (KEMRI-CDC) laboratories in Kenya according to Allan (2011) and Stewart et al. (2013) with slight modifications as it is described below. Adult mosquitoes (Anopheles gambiae – Kisumu strain) whose susceptibility to pyrethroids is known were used in the experiments. All experiments were performed at 27 ± 2 °C and 75% ± 10% relative humidity and the insectary standard conditions were maintained at 27 ± 2 °C and 75% ± 10% relative humidity and 12 : 12 hours (L:D) light periods. Adult females were reared in 20 × 20 × 20 cm cages. They were fed on bovine blood using a membrane-feeding machine, and 10% sugar solution (brown sugar Mumias brand) soaked in cotton wool. Non-chlorinated water (rainwater) was used to prepare sugar solution and for breeding larval and pupal stages. Adult males and females were maintained and fed 10% sugar solution ad libitum.
They were starved by depriving them of sugar solution and blood for 8 hours to eliminate confounding factors and ensuring physiological uniformity to ensure minimized variation before bioassay experiments. Only mosquitoes with the ability to fly and affinity to feed on sugar were used for the bio-assay. All pieces of equipment and aspirators were free of any contamination with insecticides and the insectary staff did not use volatile perfumes during the handling of the mosquitoes.
Collection of Cladonia foliacea lichen material
Well-developed thalli of Cladonia foliacea (Huds.) Willd. (Cladoniaceae, lichenized Ascomycota), were collected from Hungary [Pest County, Vácrátót, Tece, along the ’red line’ tourist route (Ág-dűlő), in open sandy grassland from the soil. Lat.: 47.702358° N; Long.: 19.224312° E] and identified by the authors (AMM and EÉF). A voucher specimen was deposited in Lichen Her-barium VBI (Vácrátót, Hungary) (abbreviation follows Thiers 2024, continuously updated). The lichen specimens were air-dried at room temperature.
Extraction and concentration of lichen secondary metabolites from Cladonia foliacea
Lichen thalli of Cladonia foliacea were carefully checked using a stereo microscope (Olympus SZX9, Tokyo, Japan) for mixed species and any unwanted flora that grow together or near the lichen and soil. They were pulverized and stored at 4 °C in falcon tubes. A coarse texture was maintained to prevent clumping of the lichen powder in the thimble.
Extraction was performed according to the method described by Mohammadi et al. (2020) at Egerton University, Department of Biochemistry and molecular Biology. The lichen powder was placed in the thimble, acetone was added to a round-bottomed flask, and the apparatus was heated at 60 °C for 6 hours. Lichen secondary metabolites are known to be limitedly soluble in water (Elix and Stocker-Wörgötter 2008, Popovici et al. 2022, Rundel 1978), therefore, acetone was used to dissolve and extract the metabolite from C. foliacea. According to Emsen et al. (2012) acetone as solvent extracted 9.65% and 8.42% (w/w) of lichen substances from C. foliacea and Flavoparmelia caperata (L.) Hale) respectively. Similarly, Kosanić et al. (2018) used acetone to extract secondary metabolites from C. foliacea.
The extract in the round-bottomed flask from the Soxhlet apparatus was carefully removed and fixed into the rotary evaporator machine. The mouth was well fixed to avoid leakage by applying grease. The water bath was set at 60 °C and allowed to be in contact with the round-bottomed flask containing the extract. After total evaporation of acetone, the extract obtained was carefully scoped off and placed in a clean glass bottle and kept at room temperature with the lid open to ensure complete drying and stored at 4 °C.
Bioassay experiment
Newly hatched adult Anopheles gambiae mosquitoes 3–4 days old were starved for 8 hours (that consuming of the sugar bait could be justified through visualising the added food dye) before the start of the experiment, 5 males and 5 females were used by treatments (i.e. concentrations). Using 2% food dye and observing the abdominal status with the help of the light microscope (Allan 2011) was applied. However, in our experiment, the volume was increased to 5 ml, which could be sustained for up to 72 hours (Allan 2011). Mixing 5.5 ml 10% sugar solution, 0.5 ml 2% food dye solution and 0.8 ml acetone was used as a negative control.
Different and increasing concentrations between 10 to 90 mg/ml were prepared (10, 20, 30, 40, 50, 70, 80, and 90 mg/ml) by dissolving the lichen extract in acetone and diluted in 10% sugar solution that contained 2% food dye initially prepared using rainwater.
The role of the red food dye (Erythrosine NaCl, dye content 9%) was to act as a marker to visualize the abdomen of an engorged mosquito and confirm that it had swallowed the poison. A mixture of 2% dye, 10% sugar solution, and acetone served as negative control for all three replicate tests performed. Five male and five female A. gambiae mosquitoes were aspirated and blown gently in disposable coffee cups (100 ml) and covered with an insecticide-free net at the top. They were allowed to acclimatize to the new environment and shock after aspiration. Five ml of the test toxic sugar bait was dispensed on 2 g of cotton wool to avoid leakage that could wet the mosquitoes during feeding and placed on the top of the net of each cup.
Knockdown and mortality effects were monitored after 4, 24, 48, and 72 hours post-exposure; any knocked-down mosquitoes were considered dead due to the moribund effect. Dead mosquitoes were sorted by sex and abdomen engorgement with the help of a dissecting microscope. After all the experiments, the live mosquitoes were humanely killed by freezing.
Statistics
The estimated marginal means (EMMs) of mortality for various concentration levels and exposure times were analysed. For statistical and subsequent statistical analysis, the data obtained from the experiment were reorganized using the dplyr R package (v1.1.4; Wickham et al. 2023)., Three-way factorial ANOVA with two-way interactions was conducted using the aov() function in base R, assessing the effects of independent variables: concentration (Conc.), time of exposure (Time), and mosquito sex (Sex) on mortality; this was decided because of the size of our data and the kind of combinations we wanted to observe. Following ANOVA, residual normality was evaluated using the Shapiro-Wilk test (shapiro.test() function in base R), revealing a significant deviation from normality (W = 0.94656, p = 0.0041). Homogeneity of variances across groups was examined with the leveneTest function from the car package (v3.1.2; Fox and Weisberg 2018), which indicated a violation for concentration (Conc.: F(8, 63) = 3.64, p = 0.0015) and a marginal violation for time (F(3, 63) = 2.68, p = 0.05), while no significant variance heterogeneity was observed for sex (F(1, 63) = 0.40, p = 0.486). Despite these violations, we proceeded with ANOVA due to the small sample size and the lack of non-parametric alternatives capable of analysing the interaction effects of interest. To substantiate the robustness of our ANOVA analysis, we checked effectsizes (η2) of the independent variables used in our selected model using the effectsize(v0.8.9; Ben-Shachar et al. 2020) R package. Post-hoc comparisons were across significant groups and interactions were performed using the emmeans() and cld() function to group the significant differences in the levels of our variables from the emmeans (v1.10.3; Lenth 2024) and multcompView (v0.1-10; Graves et al. 2024) packages, respectively. Estimated marginal means (EMMs) were calculated to provide the average total mortality adjusted for other covariates in the model. Visualization of results was completed using the ggplot2 R package (v3.5.1; Wickham 2016). All statistical analyses were conducted in R Statistical Software (v4.4.0; R Core Team, 2024).
RESULTS AND DISCUSSION
The effects of concentration and time of exposure on Anopheles gambiae mortality were found to be statistically significant, with concentration showing a large effect size (F(8,36) = 5.837, p < 0.0001, η2 = 0.56) and time also indicating a substantial effect (F(3,36) = 4.611, p = 0.00787, η2 = 0.28). As sex did not significantly influence mortality (F(1,24)=0.889, p = 0.3614), it was excluded from the model. The interaction between concentration and time (Conc:Time) was statistically significant with a large effect size (F(24,36) = 2.559, p = 0.00526, η2 = 0.63), while other two-way interactions lacked significance (Conc:Sex (F(8,24) = 0.531, p = 0.8215); Time:Sex (F(3,24) = 1.984, p = 0.1432) and were therefore removed from the further analysis (in the new model).
According to the three-way ANOVA results, concentration and exposure time have statistically significant effects on the total mortality of adult Anopheles gambiae, indicating that mortality levels vary significantly with changes in concentration and exposure duration. However, sex does not have a statistically significant effect on mortality, suggesting that male and female A. gambiae show similar susceptibility to the Cladonia foliacea extract.
Exposure durations of 24 hours (M = 1.444, 95% CI = [0.952, 1.937]) and 48 hours (M = 1.222, 95% CI = [0.729, 1.715]) had significantly higher mean mortality compared to other exposure times and were marked with the letter “b,” with a slightly higher mortality observed at 24 hours than at 48 hours (Fig. 2, Table 1).
Mean mortality after exposure to Cladonia foliacea extract from 4 hours to 72 hours. Similar letters signal no statistical difference between the groups at 95% confidence
Citation: Acta Botanica Hungarica 66, 3-4; 10.1556/034.66.2024.3-4.7
Results of analysis of variance (ANOVA) followed by Tukey-HSD post-hoc-test on mortality effect between 4 and 72 hours after Anopheles gambiae ingested Cladonia foliacea extract sugar bait. Results are averaged over the levels of concentration and sex. 95% of confidence level was used
| Exposure time (hours) | emmean | SE | df | Lower CL | Upper CL | Group |
|---|---|---|---|---|---|---|
| 4 | 0.278 | 0.239 | 24 | –0.215 | 0.771 | a |
| 72 | 0.944 | 0.239 | 24 | 0.452 | 1.437 | ab |
| 48 | 1.222 | 0.239 | 24 | 0.729 | 1.715 | b |
| 24 | 1.444 | 0.239 | 24 | 0.952 | 1.937 | b |
Mortality of A. gambiae was observed in the range between 10 mg/ml to 90 mg/ml. The EMM of mortality at 50 mg/ml (M = 2.375, 95% CI = [1.658, 3.092]) was significantly higher than at other concentration levels (Fig. 3, Table 2). These results indicate dose and time-dependent mortality response.
Mean separation of the significant effects of Cladonia foliacea extract concentration on the average total Anopheles gambiae mortality. (Concentrations sharing similar letter are not significantly different in terms of total mortality)
Citation: Acta Botanica Hungarica 66, 3-4; 10.1556/034.66.2024.3-4.7
Results of the analysis of variance (ANOVA) followed by Tukey-HSD post-hoc test to test the effect of different concentrations of Cladonia foliacea extract after oral administration to Anopheles gambiae. Results are averaged over the levels of exposure time and sex. 95% of confidence level was used
| Concentration (mg/ml) | emmean | SE | df | Lower CL | Upper CL | Group |
|---|---|---|---|---|---|---|
| 0 (negative control) | 0.125 | 0.358 | 24 | –0.6143 | 0.864 | a |
| 10 | 0.125 | 0.358 | 24 | –0.6143 | 0.864 | a |
| 20 | 0.375 | 0.358 | 24 | –0.3643 | 1.114 | a |
| 30 | 0.125 | 0.358 | 24 | –0.6143 | 0.864 | a |
| 40 | 0.75 | 0.358 | 24 | 0.0107 | 1.489 | ab |
| 50 | 2.375 | 0.358 | 24 | 1.6357 | 3.114 | b |
| 70 | 1.5 | 0.358 | 24 | 0.7607 | 2.239 | ab |
| 80 | 1.625 | 0.358 | 24 | 0.8857 | 2.364 | ab |
| 90 | 1.75 | 0.358 | 24 | 1.0107 | 2.489 | ab |
The interaction between a concentration of 50 mg/ml and 24 hours of exposure showed the highest mean mortality (M = 6.0, 95% CI = [4.52, 7.48]) (Fig. 4, Table 2).
Effect of concentration of Cladonia foliacea extract on mortality of male and female mosquitoes: Estimated marginal means after 4, 24, 48, and 72 hours. (Concentrations by exposure time sharing similar letter are not significantly different regarding average total mortality)
Citation: Acta Botanica Hungarica 66, 3-4; 10.1556/034.66.2024.3-4.7
The findings of this laboratory study indicate that adult male and female A. gambiae are susceptible to C. foliacea lichen extract because of higher mortality observed in the test experiment compared to the control. This observation can be explained by the mosquitoes’ innate behaviour to search for sugar after emergence, indicating that oral poison in a 10% sugar solution can be used as bait to kill both male and female mosquitoes. Male and female mosquitoes have been observed searching and feeding on flower nectar in their natural bait, and field studies have investigated this behaviour of mosquitoes to meet their nutritional needs and confirmed various sugars in their abdomens (Gouagna et al. 2010). A study in western Kenya found that sugar-fed males outnumbered females and newly emerged mosquitoes fed rather on sugar than blood-fed females, and that the local Anopheles mosquitoes were more attracted to Mangifera indica than to other sugar sources (Omondi et al. 2022, Yalla et al. 2023).
Using 2- to 4-day-old adult mosquitoes in this bioassay was appropriate because newly emerged males and females seek sugar for energy compared to the blood-fed females. This important concept has been successfully applied in the field targeting to kill mosquitoes by use of attractive toxic sugar bait (Müller et al. 2010 a, 2010 b). A study in Israel has demonstrated that the availability of sugar sources affects population dynamics and the vectorial capacity of A. sergentii (Gu et al. 2011). Therefore, the availability of sugar during the initial and subsequent life of a mosquito is an important factor in their life cycle.
The bioassay results showed a dose-related mortality response, ingested C. foliacea extract exhibited a significantly higher lethal effect on male and female mosquitoes at concentrations above 30 mg/ml to 90 mg/ml, however, a significantly higher effect was observed at 50 mg/ml compared to controls. Equally, increasing the exposure time at concentrations between 10 to 30 mg/ ml has no significant effect on mortality, therefore, when considering the most toxic concentration to kill more mosquitoes, a dose that yields maximum mortality of test mosquitoes should be selected and the concentration that kills bigger number within a short period would also be an important factor to consider. Therefore, if this concept were applied, acute toxicity to the target mosquitoes means the mosquitoes will be killed sooner and their lifespan and transmission of the malaria parasite will be reduced. In other studies, the effect of (–)-usnic acid on larval stages of Aedes aegypti exhibited a dose-response relationship. Exposure time and mortality were positively correlated (Koc et al. 2021). Oral toxicity of extracts of C. foliacea on Sitophilus granaries has also demonstrated dose and time response mortality effects where high concentration and longer exposure time resulted in a significantly higher mortality rate (Emsen et al. 2012). These findings are in agreement with the current study.
Both male and female mosquitoes readily ingested the extract despite the extract being known to contain antiherbivore substances. This was denoted by the distended abdomen with the red dye hence the food dye can effectively indicate the feeding rate when advanced methods like fluorescence microscopy are inaccessible. Results according to Sandra A. Allan also agree with our study by using 2% food dye and observing the abdominal status with the help of the light microscope (Allan 2011). Their study used 1 ml of the toxic solution, in our experiment, the volume was increased to 5 ml which could be sustained for up to 72 h limiting any possibility of recording false positive deaths (Allan 2011).
The current study did not focus on measuring the amount of the secondary metabolites in C. foliacea since it was initially determined by Farkas et al. (2020, 2024) from samples obtained from the same location of Tece, Vácrátót, Hungary. Use of acetone and 2% food dye as negative control did not cause high mortalities of mosquitoes to warrant discarding any of the test results during the bioassay tests. Therefore, our study recommends that the following negative controls are safe for mosquitoes and can be made by mixing 5.5 ml 10% sugar solution, 0.5 ml 2% food dye solution and 0.8 ml acetone as a negative control for bioassay studies using organic plant-based metabolites as an oral insecticide.
This study was the first attempt to determine the mortality effects of LSMs on adult A. gambiae. We have demonstrated that the acetone extract of C. foliacea that is known to contain (–)-usnic acid with insecticidal potential is toxic to newly emerged male and female adult of Anopheles gambiae when used as an oral poison in toxic sugar bait. Fumarprotocetraric acid and 9′-(O-methyl) protocetraric acid are also present in the crude extract, and may add to the effect of (–)-usnic acid. Both male and female A. gambiae showed a dose-related response at a concentration of up to 90 mg/ml and the mortality rate was time-dependent for up to 72 h. The ingestion of C. foliacea extract at 50 mg/ml and a post-exposure period of 24 to 48 h had a maximum effect on the mortality rate of targeted male and female A. gambiae. Therefore, it was shown that the C. foliacea extract when used as oral toxic sugar bait effectively killed the target mosquitoes and had insecticidal potential. Therefore, it can be recommended as an alternative biological toxic agent in the current trials and future studies on attractive targeted sugar bait as a vector control tool in malaria-endemic regions, where A. gambiae is the main vector of malaria transmission.
To enhance existing vector control tools within integrated vector management, our novel findings may open an avenue for the application of lichen secondary metabolites with insecticidal properties as oral poison in the form of toxic sugar bait (TSB).
Acknowledgements –
The authors thank Johnson Onguka for insectary maintenance, and Joan Wanjiku and Diana Wagura for their technical help. Abigael Onyango is responsible for quality control, Polo Brian is responsible for corresponding with the ethics review team, and Eric Ochomo is thanked for his suggestions at the initial stages of developing the proposal for this study. The National Commission for Science, Technology and Innovation is acknowledged for the permission to carry out this study. Stipendium Hungaricum Scholarship (2020–2024) support funded laboratory work and travelling cost. Procurement of reagents was funded by the National Research Development and Innovation Fund (grant number NKFI K 124341). The sponsors had no role in the study design, collection, analysis and interpretation of data. This study was approved by the scientific and ethics review unit of KEMRI protocol number SERU04-06-423/4610.
Abbreviations: ATSB = attractive toxic sugar bait; CI = Confidence Interval; IRS = indoor residual spraying; KEMRI = Kenya Medical Research Institute; LLITNs = long-lasting insecticide-treated nets; M = the average mortality of insects at 24 hours of exposure to the extract (an actual value); TSB = toxic sugar bait
REFERENCES
Allan, S. A. (2011): Susceptibility of adult mosquitoes to insecticides in aqueous sucrose baits.–J. Vector Ecol. 36(1): 59–67.
Araújo, A. A. S., de Melo, M. G. D., Rabelo, T. K., Nunes, P. S., Santos, S. L., Serafini, M. R., Santos, M. R. V., Quintans-Júnior, L. J. and Gelain, D. P. (2015): Review of the biological properties and toxicity of usnic acid.–Nat. Prod. Res. 29(23): 2167–2180.
Benelli, G., Jeffries, C. L. and Walker, T. (2016): Biological control of mosquito vectors: past, present, and future. – Insects 7(4), 52.
Ben-Shachar, M., Lüdecke, D., and Makowski, D. (2020): effectsize: estimation of effect size indices and standardized parameters. – J. Open Source Software 5: 2815.
Bézivin, C., Tomasi, S., Rouaud, I., Delcros, J.-G. and Boustie, J. (2004): Cytotoxic activity of compounds from the lichen: Cladonia convoluta.–Planta Med. 70 9: 874–877.
Bhatt, S., Weiss, D. J., Cameron, E., Bisanzio, D., Mappin, B., Dalrymple, U., Battle, K., Moyes, C. L., Henry, A., Eckhoff, P. A., Wenger, E. A., Briët, O., Penny, M. A., Smith, T. A., Bennett, A., Yukich, J., Eisele, T. P., Griffin, J. T., Fergus, C. A., Lynch, M., Lind-gren, F., Cohen, J. M., Murray, C. L. J., Smith, D. L., Hay, S. I., Cibulskis, R. E. and Gething, P. W. (2015): The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015.–Nature 526(7572): 207–211.
Bomfim, R. R., Araújo, A. A. S., Cuadros-Orellana, S., Melo, M. G. D., Quintans-Júnior, L. J. and Cavalcanti, S. C. H. (2009): Larvicidal activity of Cladonia substellata extract and usnic acid against Aedes aegypti and Artemia salina.–Lat. Am. J. Pharm. 28(4): 580–584.
Cetin, H., Tufan-Cetin, O., Ozdemir Turk, A., Tay, T., Candan, M., Yanikoglu, A. and Sumbul, H. (2008): Insecticidal activity of major lichen compounds, (–)-and (+)-usnic acid, against the larvae of house mosquito, Culex pipiens L.–Parasitol. res. 102: 1277–1279.
Cocchietto, M., Skert, N., Nimis, P. L., and Sava, G. (2002): A review on usnic acid, an interesting natural compound.–Naturwissenschaften 89(4): 137–146.
Da Silva, A. S., de Oliveira Farias de Aguiar, J. C. R., da Silva Nascimento, J., Costa, E. C. S., dos Santos, F. H. G., Andrade de Araújo, H. D., da Silva, N. H., Pereira, E. C., Martins, M. C., Falcão, E. P. S., Scotti, L., Scotti, M. T. and do Amaral Ferraz Navarro, D. M. (2023): Larvicidal activity and docking study of Ramalina complanata and Cladonia verticillaris extracts and secondary metabolites against Aedes aegypti. – Industrial Crops and Products 195: 116425.
Diarra, R. A., Traore, M. M., Junnila, A., Traore, S. F., Doumbia, S., Revay, E. E., Kravchenko, V. D., Schlein, Y., Arheart, K. L., Gergely, P., Hausmann, A., Beck, R., Xue, R.-D., Prozorov, A. M., Kone, A. S., Majambere, S., Vontas, J., Beier, J. C. and Müller, G. C. (2021): Testing configurations of attractive toxic sugar bait (ATSB) stations in Mali, West Africa, for improving the control of malaria parasite transmission by vector mosquitoes and minimizing their effect on non-target insects. – Malaria Journal 20(1): 184.
Elix J. A. and Stocker-Wörgötter E. (2008): Biochemistry and secondary metabolites. – In: Nash, T. H. III (ed.): Lichen Biology. 2nd ed. Cambridge University Press, Cambridge, pp. 104–133.
Emmerich, R., Giez, I., Lange, O. L. and Proksch, P. (1993): Toxicity and antifeedant activity of lichen compounds against the polyphagous herbivorous insect Spodoptera littoralis.–Phytochemistry 33(6): 1389–1394.
Emsen, B., Yildirim, E., Aslan, A., Anar, M. and Ercisli, S. (2012): Insecticidal effect of the extracts of Cladonia foliacea (Huds.) Willd. and Flavoparmelia caperata (L.) Hale against adults of the grain weevil, Sitophilus granarius (L.) (Coleoptera Curculionidae).–Egypt. J. Pest Control 22: 145–149.
Farkas, E., Biró, B., Szabó, K., Veres, K., Csintalan, Z. and Engel, R. (2020): The amount of lichen secondary metabolites in Cladonia foliacea (Cladoniaceae, lichenised Ascomycota).–Acta Bot. Hung. 62: 33–48.
Farkas, E., Xu, M., Muhoro, A. M., Szabó, K., Lengyel, A., Heiðmarsson, S., Viktorsson, E. Ö. and Ólafsdóttir, E. S. (2024): The algal partnership is associated with quantitative variation of lichen specific metabolites in Cladonia foliacea from Central and Southern Europe.–Symbiosis 92: 403–419.
Fiorenzano, J. M., Koehler, P. G. and Xue, R.-D. (2017): Attractive toxic sugar bait (ATSB) for control of mosquitoes and its impact on non-target organisms: a review. – Int. J. Environ. Res. Public Health 14(4): 398.
Foster, W. A. (1995): Mosquito sugar feeding and reproductive energetics.–Annu. Rev. Entomol. 40: 443–474.
Fox, J. and Weisberg, S. (2018): An R Companion to Applied Regression. – SAGE Publications Inc., London, 608 pp.
Galanty, A., Paśko, P. and Podolak, I. (2019): Enantioselective activity of usnic acid: a comprehensive review and future perspectives.–Phytochem. Reviews 18(2): 527–548.
Goga, M., Elečko, J., Marcinčinová, M., Ručová, D., Bačkorová, M. and Bačkor, M. (2018): Lichen metabolites: an overview of some secondary metabolites and their biological potential. – In: Merillon, J.-M. and Ramawat, K. G. (eds): Co-evolution of secondary metabolites. Reference series in phytochemistry. Springer International Publishing, Cham, pp. 1–36.
Gouagna, L.-C., Poueme, R. S., Dabiré, K. R., Ouédraogo, J.-B., Fontenille, D. and Simard, F. (2010): Patterns of sugar feeding and host plant preferences in adult males of An. gambiae (Diptera: Culicidae).–J. Vector Ecol. 35(2): 267–276.
Graves, S., Piepho, H.-P., Selzer, L. and Dorai-Raj, S. (2024): _multcompView: Visualizations of Paired Comparisons_. – R package version 0.1-10. https://CRAN.R-project.org/package=multcompView (accessed 10 July 2024).
Gu, W., Müller, G., Schlein, Y., Novak, R. J. and Beier, J. C. (2011): Natural plant sugar sources of Anopheles mosquitoes strongly impact malaria transmission potential. – PLoS ONE 6(1): e15996.
Hawksworth, D. L. and Grube, M. (2020): Lichens redefined as complex ecosystems.–New Phytol. 227 5: 1281–1283.
Huneck, S. (1999): The significance of lichens and their metabolites.–Naturwissenschaften 86(12): 559–570.
Huneck, S. and Yoshimura, I. (1996): Identification of lichen substances. – Springer Verlag, Berlin, Heidelberg, 493 pp.
Kinoshita, Y., Yamamoto, Y., Yoshimura, I., Kurokawa, T. and Huneck, S. (1997): Distribution of optical isomers of usnic and isousnic acids analyzed by high performance liquid chromatography.–J. Hattori Bot. Lab. 83: 173–178.
Kiware, S. S., Chitnis, N., Tatarsky, A., Wu, S., Castellanos, H. M. S., Gosling, R., Smith, D. and Marshall, J. M. (2017): Attacking the mosquito on multiple fronts: insights from the Vector Control Optimization Model (VCOM) for malaria elimination. – PLoS ONE 12(12): e0187680.
Koc, S., Tufan Cetin, O., Candan, M., Turk, A. and Cetin, H. (2021): Larvicidal activity of lichen secondary metabolites atranorin and (-)-usnic acid against the yellow fever mosquito Aedes aegypti.–Fresenius Environ. Bull. 30: 11938–11941.
Kosanić, M., Ristić, S., Stanojković, T., Manojlović, N. and Ranković, B. (2018): Extracts of five Cladonia lichens as sources of biologically active compounds.–Farmacia 66(4): 644–651.
Kumar, S., Sharma, A., Samal, R. R., Kumar, M., Verma, V., Sagar, R. K., Singh, S. P. and Raghavendra, K. (2022): Attractive sugar bait formulation for development of attractive toxic sugar bait for control of Aedes aegypti (Linnaeus).–J. Tropical Med. 2977454: 1–10.
Kumar, S., Sharma, A., Samal, R. R., Verma, V., Sagar, R. K., Singh, S. P. and Raghavendra, K. (2024): Development of deltamethrin-laced attractive toxic sugar bait to control Aedes aegypti (Linnaeus) population.–J. Tropical Med. 6966205: 1–9.
Lea, A. O. (1965): Sugar-baited insecticide residues against mosquitoes.–Mosquito News 25(1): 65–66.
Lenth, R. (2024): _emmeans: estimated marginal means, aka least-squares means_. – R package version 1.10.3. https://CRAN.R-project.org/package=emmeans (accessed 16 October 2024).
Lindsay, S. W., Thomas, M. B. and Kleinschmidt, I. (2021): Threats to the effectiveness of insecticide-treated bednets for malaria control: thinking beyond insecticide resistance. – The Lancet Global Health 9(9): e1325–e1331.
Maia, M. F., Tenywa, F. C., Nelson, H., Kambagha, A., Ashura, A., Bakari, I., Mruah, D., Simba, A. and Bedford, A. (2018): Attractive toxic sugar baits for controlling mosquitoes: a qualitative study in Bagamoyo, Tanzania. – Malaria J. 17: 22.
Mohammadi, M., Zambare, V., Malek, L., Gottardo, C., Suntres, Z. and Christopher, L. (2020): Lichenochemicals: extraction, purification, characterization, and application as potential anticancer agents.–Expert Opinion on Drug Discovery 15(5): 575–601.
Molnár, K. and Farkas, E. (2010): Current results on biological activities of lichen secondary metabolites: a review.–Zeitschr. f. Naturforsch. C 65(3–4): 157–173.
Muhoro, A. M. and Farkas, E. É. (2021): Insecticidal and antiprotozoal properties of lichen secondary metabolites on insect vectors and their transmitted protozoal diseases to humans. – Diversity 13(8): 342.
Müller, G. C., Junnila, A., and Schlein, Y. (2010 a): Effective control of adult Culex pipiens by spraying an attractive toxic sugar bait solution in the vegetation near larval habitats.–J. Med. Entomol. 47: 63–66.
Müller, G. C., Beier, J. C., Traore, S. F., Toure, M. B., Traore, M. M., Bah, S., Doumbia, S. and Schlein, Y. (2010b): Successful field trial of attractive toxic sugar bait (ATSB) plant-spraying methods against malaria vectors in the Anopheles gambiae complex in Mali, West Africa. – Malaria J. 9: 210.
Nguyen, K.-H., Chollet-Krugler, M., Gouault, N. and Tomasi, S. (2013): UV-protectant metabolites from lichens and their symbiotic partners.–Nat. Prod. Rep. 30(12): 1490–1508.
Nimis, P. L. and Skert, N. (2006): Lichen chemistry and selective grazing by the coleopteran Lasioderma serricorne.–Environ. Exp. Bot. 55: 175–182.
Njoroge, T. M., Hamid-Adiamoh, M. and Duman-Scheel, M. (2023): Maximizing the potential of attractive targeted sugar baits (ATSBs) for integrated vector management. – Insects 14(7): 585.
Ochomo, E. O., Milanoi, S., Abong’o, B., Onyango, B., Muchoki, M., Omoke, D., Olanga, E., Njoroge, L., Juma, E. O., Otieno, J. D., Matoke-Muhia, D., Kamau, L., Rafferty, C., Gimnig, J. E., Shieshia, M., Wacira, D., Mwangangi, J., Maia, M., Chege, C., Omar, A., Rono, M. K., Abel, L., O’Meara, W. P., Obala, A., Mbogo, C. and Kariuki, L. (2023): Detection of Anopheles stephensi mosquitoes by molecular surveillance, Kenya.–Emerging Infectious Diseases 29(12): 2498–2508.
Omondi, S., Kosgei, J., Agumba, S., Polo, B., Yalla, N., Moshi, V., Abong’o, B., Ombok, M., McDermott, D. P., Entwistle, J., Samuels, A. M., Ter Kuile, F. O., Gimnig, J. E. and Ochomo, E. (2022): Natural sugar feeding rates of Anopheles mosquitoes collected by different methods in western Kenya. – Scientific Rep. 12(1): 20596.
Owuor, K. O., Machani, M. G., Mukabana, W. R., Munga, S. O., Yan, G., Ochomo, E. and Afrane, Y. A. (2021): Insecticide resistance status of indoor and outdoor resting malaria vectors in a highland and lowland site in Western Kenya. – PLoS ONE 16(3): e0240771.
Popovici, V., Bucur, L., Gîrd, C. E., Popescu, A., Matei, E., Cozaru, G. C., Schröder, V., Ozon, E. A., Fita, A. C., Lupuliasa, D., Aschie, M., Caraiane, A., Botnarciuc, M. and Badea, V. (2022): Phenolic secondary metabolites and antiradical and antibacterial activities of different extracts of Usnea barbata (L.) Weber ex F. H. Wigg from Călimani Mountains, Romania. – Pharmaceuticals 15: 829.
Qualls, W. A., Müller, G. C., Traore, S. F., Traore, M. M., Arheart, K. L., Doumbia, S., Schlein, Y., Kravchenko, V. D., Xue, R.-D. and Beier, J. C. (2015): Indoor use of attractive toxic sugar bait (ATSB) to effectively control malaria vectors in Mali, West Africa. – Malaria J. 14: 301.
R Core Team (2024): R: a language and environment for statistical computing. – R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/. (accessed 10 July 2024).
Revay, E. E., Schlein, Y., Tsabari, O., Kravchenko, V., Qualls, W. A., Xue, R.-D., Beier, J. C., Traore, S. F., Doumbia, S., Hausmann, A. and Müller, G. C. (2015): Formulation of attractive toxic sugar bait (ATSB) with safe EPA-exempt substance significantly diminishes the Anopheles sergentii population in a desert oasis.–Acta Tropica 150: 29–34.
Robi, M., Sinka, M., Minakawa, N., Mbogo, C., Hay, S. and Snow, R. (2010): Distribution of the main malaria vectors in Kenya. – Malaria J. 9: 69.
Rochlin, I., White, G., Reissen, N., Swanson, D., Cohnstaedt, L., Chura, M., Healy, K. and Faraji, A. (2022): Laboratory evaluation of sugar alcohols for control of mosquitoes and other medically important flies. – Scientific Rep. 12(1): 13763.
Rundel, P. (1978): The ecological role of secondary lichen substances.–Biochem. Syst. Ecol. 6(3): 157–170.
Sanders, W. B. (2024): The disadvantages of current proposals to redefine lichens.–New Phytol. 241: 969–971.
Schlein, Y. and Müller, G. C. (2015): Decrease of larval and subsequent adult Anopheles sergentii populations following feeding of adult mosquitoes from Bacillus sphaericus-containing attractive sugar baits. – Parasites & Vectors 8: 244.
Schlein, Y. and Pener, H. (1990): Bait-fed adult Culex pipiens carry the larvicide Bacillus sphaericus to the larval habitat.–Med. Veterin. Entomol. 4(3): 283–288.
Sippy, R., Rivera, G. E., Sanchez, V., Heras, F., Morejón, B., Beltrán, E., Hikida, R. S., LópezLatorre, M. A., Aguirre, A., Stewart-Ibarra, A. M., Larsen, D. A. and Neira, M. (2020): Ingested insecticide to control Aedes aegypti: developing a novel dried attractive toxic sugar bait device for intra-domiciliary control. – Parasites & Vectors 13(1): 78.
Sissoko, F., Junnila, A., Traore, M. M., Traore, S. F., Doumbia, S., Dembele, S. M., Schlein, Y., Traore, A. S., Gergely, P., Xue, R.-D., Arheart, K. L., Revay, E. E., Kravchenko, V. D., Beier, J. C. and Müller, G. C. (2019): Frequent sugar feeding behavior by Aedes aegypti in Bamako, Mali makes them ideal candidates for control with attractive toxic sugar baits (ATSB). – PLoS ONE 14(6): e0214170.
Stevenson, J., St. Laurent, B., Lobo, N. F., Cooke, M. K., Kahindi, S. C., Oriango, R. M., Harbach, R. E., Cox, J. and Drakeley, C. (2012): Novel vectors of malaria parasite in the Western Highlands of Kenya.–Emerging Infectious Diseases 18(9): 1547–1549.
Stewart, Z. P., Oxborough, R. M., Tungu, P. K., Kirby, M. J., Rowland, M. W. and Irish, S. R. (2013): Indoor application of attractive toxic sugar bait (ATSB) in combination with mosquito nets for control of pyrethroid-resistant mosquitoes. – PLoS ONE 8(12): e84168.
Thiers, B. (2024, continuously updated): Index Herbariorum: a global directory of public herbaria and associated staff. New York Botanical Garden’s Virtual Herbarium. – Available online: http://sweetgum.nybg.org/science/ih/ (accessed 25 July 2024).
Traore, M. M., Junnila, A., Traore, S. F., Doumbia, S., Revay, E. E., Kravchenko, V. D., Schlein, Y., Arheart, K. L., Petrányi, G., Xue, R.-D., Hausmann, A., Beck, R., Prozorov, A., Diarra, R. A., Kone, A. S., Majambere, S., Bradley, J., Vontas, J., Beier, J. C. and Muller, G. C. (2020): Large-scale field trial of attractive toxic sugar baits (ATSB) for the control of malaria vector mosquitoes in Mali, West Africa. – Malaria J. 19: 72.
Wickham, H. (2016): Ggplot2: elegant graphics for data analysis. – Springer-Verlag GmbH, Heidelberg, 255 pp. (accessed 10 July 2024).
Wickham, H., François, R., Henry L., Müller K. and Vaughan D. (2023): dplyr: a grammar of data manipulation. R package version 1.1.4. https://github.com/tidyverse/dplyr, https://dplyr.tidyverse.org (accessed 10 July 2024).
Wirth, V., Hauck, M. and Schultz, M. (2013): Die Flechten Deutschlands. – Ulmer Verlag, Stuttgart, 1244 pp.
Xue, R.-D., Ali, A., Kline, D. and Barnard, D. (2008): Field evaluation of boric acid-and fipronil-based bait stations against adult mosquitoes.–J. Amer. Mosquito Control Assoc. 24: 415–418.
Yalla, N., Polo, B., McDermott, D. P., Kosgei, J., Omondi, S., Agumba, S., Moshi, V., Abong’o, B., Gimnig, J. E., Harris, A. F., Entwistle, J., Long, P. R. and Ochomo, E. (2023): A comparison of the attractiveness of flowering plant blossoms versus attractive targeted sugar baits (ATSBs) in western Kenya. – PloS ONE 18(6): e0286679.
