Use of micro and macroalgae extracts for the control of vector mosquitoes

Survey methodology

In this study, we conducted an up-to-date literature search covering the years 2010-2023 (until July 2023) on “Potential of micro and macroalgae extracts for larvicidal effect on mosquitoes” using various databases including PubMed, Scopus, Web of Science and Google Scholar. A limited number of pre-2010 studies have also been used in the evaluations, which contain information that supports the up-to-date study results. The keywords used for the search were “freshwater algae”, “microalgae”, “macroalgae”, “transgenic algae”, “mosquito control”, “seaweed”, “nanoparticles” and “larvicides”. The criteria for inclusion in our article were that the articles discussed the use of micro and macroalgae for mosquito control. Although cyanobacteria are known as algae, they are prokaryotic photosynthetic bacteria. Therefore, this group of microorganisms was not included in this research. Articles with calculated lethal concentration values (LC₅₀ and/or LC₉₀) were primarily considered in our approach and evaluations, and these studies are listed in Tables 1 and 2. Studies using the Probit method as described by Finney (1947) were selected.

Mosquito larvicidal activity of microalgae extracts

Microalgae are unicellular photosynthetic microscopic algae, typically found in freshwater and saline environments. Microalgae have been searched for their potential use in various fields including biofuels, bioremediation, and as a source of valuable compounds such as pigments, proteins, and omega-3 fatty acids (Nagi et al., 2021; Parmar et al., 2023; Xu, Miao & Wu, 2006). When extracting compounds from algae, the choice of solvent and extraction method is crucial to ensuring efficient and effective extraction. The choice depends on the type of compounds targeted and their solubility properties. Solvents used in the extraction of algae are methanol, chloroform, ethanol, acetone, hexane, and water. The dried algae biomass is mixed or wetted with the selected solvent so that the solvent comes into contact with the algae cells and the desired compounds are extracted. Various extraction techniques such as maceration, infusion, decoction, and boiling under reflux, a wide range of modern techniques, such as microwave-extraction, ultrasound-extraction or supercritical CO₂ extraction can be used to improve extraction efficiency (Stranska-Zachariasova et al., 2016). More advanced techniques such as column chromatography, high-performance liquid chromatography, and solid-phase extraction can be applied to isolate or purify specific compounds.

Some metabolites synthesized by microalgae can be potential biological insecticides for the control of agricultural pests (Costa et al., 2019). Furthermore, research have shown that some microalgae species have larvicidal activity against mosquitoes and act on target organisms with the toxins they produce. Microalgae can also produce certain substances such as microcystin produced by cyanobacteria that inhibit larval development and delay the development of surviving larvae to the adult stage (Rashed & El-Ayouty, 1991; Rey, Hargraves & O’Connell, 2009). The utilization of microalgae for the control of mosquito larvae has gained significant attention due to their various advantages such as ease of production, cost-effectiveness, biodegradability, and being non-toxic to non-target organisms (Asimakis et al., 2022).

In one of the first remarkable studies on the subject before up-to-date studies (between 2010–2023), the effect of four green algae species, Ankistrodesmus convolutus, Chlorella vulgaris, Chlorococcum sp. and Scenedesmus quadricauda on some vital parameters of Ae. aegypti was investigated. It was found that mosquito larvae fed on C. vulgaris, Chlorococcum sp. and S. quadricauda were delayed in pupation and adults were reduced in size. In addition, at the end of the sixth exposure day, mortality range from 84–100% was detected in larvae fed with microalgae (Ahmad et al., 2001). After that research, the effect of ten green algal isolates from mosquito breeding sites such as metal containers, discarded tires, and empty coconut shells on Ae. aegypti larvae for seven days was evaluated using a feeding test. Of these isolates, all mosquito larvae treated with Chlorella were found to die after seven days. The researchers interpreted the deaths of mosquito larvae as evidence for the resistance of these isolates to digestion. The authors also noted that the shape, size, and cell wall characteristics of algae may have an impact on their digestibility and consumption as food by mosquitoes (Ahmad et al., 2004). In a recent study, Ae. aegypti mosquito larvae fed Chlorella sp. were found to be affected in terms of development time, body size and life span. Larvae fed with the microalgae took longer to develop to pupation and adult stages, but their survival as adults was shorter (Souza et al., 2019).

Up-to-date articles in which microalgae species have been studied and proven to exhibit larvicidal activity against some mosquito species (indicating the solvent used and LC₅₀₋₉₀ values) are listed in Table 1. When the LC₅₀ and LC₉₀ values of mosquito species treated with microalgae extracts prepared with various solvent types in Table 1 are examined, the high lethal power of chloroform extracts is notable. It was reported in this research that when the chloroform extract of microalgae (Chlorella sp.) was purified, the larvicidal effect increased six times (Sigamani et al., 2020). It is seen from Table 1 that the larvicidal effect of chloroform has not been tested for other microalgae species (Amphora coffeaeformis, Chlorococcum sp., Scenedesmus sp., Scenedesmus obliquus) on mosquitoes. In a study using other solvents, the insecticidal activity of acetone, ethanol, and methanol extracts of Chlorococcum sp. and Scenedesmus sp. microalgae against third instar larvae of Ae. aegypti was investigated by Rani & Kumar (2023). The authors reported that extracts of Chlorococcum sp. and Scenedesmus sp. showed larvicidal effect and the ethanol extract of Scenedesmus sp. had the highest larvicidal activity (Table 1).

In a comprehensive study with lethal concentration values indicated, the larvicidal potency of various solvent extracts of a microalgae species (Chlorella sp.) against four larval stages of the Dengue vector Ae. aegypti was examined by Sigamani et al. (2020). Notably, purified fractions of chloroform extracts were found to have strong larvicidal activity against the mosquito larvae tested. The larvicidal activity in the fractions of chloroform extracts was attributed to the presence of n-hexadecanoic acid (palmitic acid), oleic acid, and β-sitosterol acetate compounds. Also in other studies, it has been reported that these substances have larvicidal properties (Ragavendran, Dubey & Natarajan, 2017; Rahuman, Venkatesan & Gopalakrishnan, 2008; Rahuman et al., 2008). Sigamani et al. (2020) emphasized that the high larvicidal activity was due to the particularly high n-hexadecanoic acid content in Chlorella sp. extract. In addition, they reported that third instar larvae of Ae. aegypti showed morphological and behavioral changes when exposed to a purified fraction of 50 ppm concentration of microalgae. In the study conducted using 200–1,000 ppm concentrations of extracts, it was found that younger stages of larvae were more susceptible than older ones.

In another research, Hassan et al. (2021) studied the potential insecticidal activity of two microalgae species: Amphora coffeaeformis and Scenedesmus obliquus against the larvae of Cx. pipiens mosquitoes. They reported that both species showed larvicidal activity, but A. coffeaeformis extract had a higher activity than S. obliquus extract. In the study, the presence of substances such as polygalic acid, taxifolin, cinnamic acid, kaempferol, caffeic acid, methyl gallate, naringenin and syringic acid in the polyphenolic compound contents of these two species was pointed out. It was mentioned that the insect control effects of Gallic acid (toxic effect on many insects), Taxifolin (feeding deterrent), Cinnamic acid (high feeding inhibitor), Kaempferol (insecticidal effect), Caffeic acid (insect growth inhibitor and feeding deterrent), Naringenin (insecticidal effect) have been proven by previous studies.

The toxicity of aqueous extract and lectin purified from Chlorella vulgaris microalgae on Ae. aegypti fourth instar larvae was examined by Cavalcanti et al. (2021). They showed that lectin from C. vulgaris inhibited the activity of trypsin-like enzymes in the larvae’s gut. The LC₅₀ values of C. vulgaris aqueous extract and purified lectin after 72 h were 10.62% and 106.5 µg ml⁻¹, respectively. The authors stated that lectins are carbohydrate-binding proteins with previously proven insecticidal activity (Oliveira et al., 2020) and suggested that the purified lectin and the aqueous extract of C. vulgaris are potential mosquito larvicides. We hope that further research on this topic can be conducted and larvicide formulations can be developed.

Rani & Kumar (2023) reported that quinones and terpenoids detected in ethanol extracts of Scenedesmus sp. and Chlorococcum sp. but not found in other extracts may cause larvicidal effects. Similarly, saponins detected only in ethanol and methanol extracts may be the source of larvicidal activity. The insecticidal effect of quinones on mosquitoes has been proven in the past (Silva et al., 2020). In addition, terpenoid compounds derived from a species of red algae, dibromomertensene and dihydromertensene, have proven insecticidal effects (Argandoña et al., 2000). The mosquito-killing effect of these substances (quinones, terpenoids, saponins, lectins etc.) and their derivatives obtained from algae has not yet been studied and is thought to be the subject of many future studies.

Possible mechanisms of the larvicidal effect of algae on mosquitoes were reported to be based on damage to the gut of mosquito larvae by compounds in algae, inhibition of larval feeding, inhibition of digestive enzymes and processes, inhibition of ATPase production, inhibition of detoxification enzymes such as glutathione-s-transferase and synergistic effects of fatty acids and polyphenolic compounds (Hassan et al., 2021). Similar to this possible explanation of the mechanism of larvicidal activity, Sigamani et al. (2020) reported various histological changes in intestinal epithelial cells of larvae exposed to purified microalgal (Chlorella sp.) fractions of chloroform extracts compared to control groups. It was considered that in larvae exposed to microalgae fraction, compounds that enter the cell due to the damage of the exoskeleton bind to macro-components and cause physiological changes (Farnesi et al., 2012; Dawet, Ikani & Yakubu, 2016). Additionally, in one study, the levels of carboxylesterase (α and β), Glutathione-S-Transferase and Cytochrome P450, the major detoxifying enzymes of Ae. aegypti treated with algal extracts, were examined (Yogarajalakshmi et al., 2020). It was determined that there was a significant decrease in α and β carboxylesterase enzyme levels, whereas Glutathione-S-Transferase and Cytochrome P450 levels were higher in a dose-dependent manner. Thus, algal preparations administered to mosquitoes were found to cause significant physiological alterations by inhibiting or stimulating digestive enzyme activities.

Mosquito larvicidal activity of macroalgae (or seaweed) extracts

Macroalgae, also known as seaweed, are chlorophyll-containing organisms. They are the source of some products such as alginate, carrageenan, and agar, all of which are deemed important in food and cosmetic industries (Morais et al., 2021). Polysaccharides, proteins, fatty acids, flavonoids, carotenoids and polyphenols in macroalgae have antifungal, antiviral, and antibacterial effects against some plant pathogens (Hamed et al., 2018). Many researchers have conducted studies showing that macroalgae have toxic effects on different insects, especially mosquitoes. Yu et al. (2014) compiled studies between 1991 and 2014 in which the effects of macroalgae extracts on some mosquito species were examined. Recent articles investigating the larvicidal activity of macroalgae extracts against some mosquito species are presented in Table 2.

The research in Table 1, Bantoto & Dy (2013) showed that the crude extracts of brown algae Padina minor and Dictyota linearis have larvicidal activity against fourth instar Ae. aegypti larvae. According to LC₅₀ values, P. minor showed significantly more larvicidal activity compared to D. linearis. It was reported that concentration-dependent mortality for both extracts and the mortality of Ae. aegypti larvae increased as the concentration of the extract increased. The researchers found that larvae exposed to the extracts had a longer larval stage than the control groups. It was also reported that the significant larvicidal activity of Padina minor and Dictyota linearis species against Ae. aegypti larvae may be due to their content of terpenoids, brown algal phenolics, unsaturated fatty acids or phlorotannins. Phlorotannins are a substance specific to brown algae and are rarely found in red algae. There is information in the literature that phlorotannins have a larvicidal effect, including on mosquitoes (Negara et al., 2021). Purified phlorotannins showed pesticidal, herbicidal and algicidal effects on some organisms (Artemia salina, Chlorella vulgaris, Daphnia magna, Lactuca sativa) in aquatic environments (Harwanto et al., 2022).

In another study, the insecticidal activity of methanol and chloroform extracts of red algae Porteria hornemannii on the fourth stage larvae of three vector mosquitoes; Culex quinquefasciatus, Ae. aegypti and An. stephensi was investigated and larvicidal effects were detected in all extracts applied to all species (Table 2). Furthermore, the methanol extracts were found to have significantly higher toxicity than the chloroform extracts (Arumugam et al., 2022). According to the results of GC-MS analysis of P. hornemannii methanol extract, it was reported that the substance with 41.88% content was n-hexadecanoic acid, which Sigamani et al. (2020) showed as the source of high larvicidal effect on mosquito.

Acetone extract of green algae Caulerpa scalpelliformis, which live in tropical to subtropical regions and is considered exotic and invasive on the Turkish coast, has been reported to have toxic effects on late second to early third instar larvae of the common mosquito species Culex pipiens (Cetin, Gokoglu & Oz, 2010). In a previous study, Thangam & Kathiresan (1991) reported that the toxicity of acetone extract of the same species on Ae. aegypti larvae was high and the LC₅₀ value was 53.7 ppm. In another study, the larvicidal effects of caulerpin and caulerpinic acid isolated from Caulerpa racemosa on Cx. pipiens mosquito were documented (Alarif et al., 2010). Thus, the larvicidal effect of Caulerpa sp. species on mosquitoes may be attributed to caulerpin and caulerpinic acid.

Yu et al. (2015) reported the mosquitocidal activity of four extracts (chloroform, methanol, n-hexane, and aqueous) of the green algae Bryopsis pennata against two vector mosquitoes, Ae. aegypti and Ae. albopictus. It was pointed out that the larvicidal potential of the chloroform extract was higher than the other extracts (Table 2). The authors also found that the purified chloroform extract of B. pennata was fifteen times more effective. When the LC-MS content analysis results given in the article was examined, it was seen that chloroform extracts of algae contain caulerpinic acid and caulerpin derivatives.

Extracts of brown algae Sargassum wightii and green algae Halimeda gracillis were obtained by Suganya et al. (2019) using acetone, chloroform, ethanol, methanol, and water as solvents. These extracts were found to have very high toxicity on the third instar larvae of three different mosquito species (Anopheles stephensi, Ae. aegypti and Culex tritaeniorhynchus) and the authors reported that the LC₅₀ values of ethanol extract on the larvae of all species were less than 50 ppm.

It was reported that Champia parvula marine algae caused 97% or more mortality on second and third stage larvae of Ae. aegypti at a concentration of 100 ppm and LC₅₀ and LC₉₀ values were 43 ppm and 88 ppm, respectively (Yogarajalakshmi et al., 2020). The authors reported that the applied extracts increased the activities of superoxide dismutase, catalase, and glutathione peroxidase enzymes, which are markers of oxidative stress. The larvicidal activity of methanolic extracts of eight algal species (Jania rubens, Galaxaura elongata, Gelidium latifolium—red algae; Ulva intestinales, Codium tomentosum—green algae; Dictyota dichotoma, Sargassum dentifolium, Padina boryana—brown algae) against third instar larvae of Cx. pipiens was investigated by Haleem et al. (2022). The extracts of all algae showed larvicidal activity (Table 2). Similar to Yogarajalakshmi et al. (2020), Haleem et al. (2022) were also reported that the activities of oxidative stress markers superoxide dismutase, catalase and glutathione peroxidase enzymes increased. They indicated that the lethal effect may be related to oxidative imbalance resulting from these excessive enzymatic increases.

The insecticidal activity of ethanolic extracts from Ulva rigida, Asparagopsis taxiformis, Dictyota dichotoma and Cystoseira barbata algae was tested against Aedes albopictus larvae. Only D. dichotoma showed activity against Ae. albopictus larvae. The LC₅₀ and LC₉₀ values of ethanolic extracts are 44.32 and 85.92 mg/l, respectively.

In addition, in many toxicity studies with macroalgae (seaweeds), one or more solvents were used simultaneously for extraction. Some of these solvent combinations are petroleum ether-acetone, ethanol-water, methanol-acetone, and dichloromethane-methanol. These studies were conducted on different mosquito species (Cx. quinquefasciatus, Ae. aegypti and An. stephensi) and extracts prepared using solvent combinations were found to be more toxic to mosquitoes (Thangam & Kathiresan, 1991; Manilal et al., 2009; Ali, Ravikumar & Beula, 2013; Yu et al., 2014).

Newer approaches