From the Sea to Mosquito Control: The Potential of Halymenia dilatata Marine Alga as an Eco-Friendly Mosquitocidal Agent

Abstract

A marine-derived red alga, along with their active constituents, were actively screened for their biocontrol potential against major insect pests. The current study explored the larvicidal activity of crude methanolic extracts of the red alga Halymenia dilatata (Mx-Hd) against Aedes aegypti (Linn.), and their enzyme inhibition, repellent activity, and non-target toxicity was screened against non-target species. The toxicity of Mx-Hd to exposed larvae was dose-dependent, and it was significant at the maximum concentration of 100 ppm (parts per million) across the second, third, and fourth instars of Ae. aegypti. The LC₅₀ and LC₉₀ Mx-Hd concentrations for second-instar larvae were 37 and 93 ppm, respectively. Moreover, the level of major detoxifying enzymes was altered in response to the Mx-Hd treatment. The repellent activity of Mx-Hd showed maximum repellent protection at 100 ppm dosage for up to 210 min. The toxicity against non-target species showed that Mx-Hd was safe or less toxic at the prominent dosage (1000 ppm). The photomicrography results provided a prominent damage rate in fourth-instar midgut cells and tissues treated with Mx-Hd. Overall, the present study delivered an insect toxicological screening study of bioactive red alga extracts against a dengue mosquito vector, as well as a baseline for better commercialization of bioactive insecticides. Also, the bioeconomy of algal-based pesticides in managing mosquito larvae presents an exciting avenue for sustainable and eco-friendly pest control.

1. Introduction

The yellow fever mosquito Aedes aegypti (Linn.) can pose a significant threat to human health, as it is the primary vector responsible for transmitting the dengue virus to humans. The key arthropod vector Aedes aegypti is known to transmit the dengue virus to humans through its bite and causes severe flu-like symptoms, including severe headache, high fever, muscle and joint pain, and rash, leading to lethal impacts [1]. This dengue vector is recognized as a major global health concern, with approximately three hundred and ninety million infections due to dengue predicted to occur worldwide annually [2,3]. Aedes aegypti is responsible for transmitting all four serotypes of the dengue virus, and its widespread distribution in many tropical and subtropical regions has contributed to the increasing incidence of dengue outbreaks and epidemics [4,5]. Dengue outbreaks can have significant economic consequences, including increased healthcare costs, loss of productivity due to illness and hospitalization, and reduced tourism and trade in developing nations [6,7]. Dengue can also impose a heavy economic burden on families due to medical expenses and loss of income. Moreover, dengue vectors are highly adapted to urban environments and are known to thrive in warmer climates [8]. With climate change potentially leading to increased temperatures and altered precipitation patterns, there are concerns that the distribution and abundance of Ae. aegypti mosquitoes may expand, leading to increased dengue transmission in previously unaffected areas [9]. Therefore, increased attention is being paid to controlling populations of Ae. aegypti and preventing viral transmission to protect public health and reduce the burden of dengue disease. Traditional mosquito control methods use different types of insecticides to kill mosquitoes. By reducing or eliminating mosquito breeding sites, as well as using microbiological larvicides, ovicides, and pupicides, an eco-friendly method has been followed [10]. Vector control at the larval stage is preferred over other mosquito lifecycle stages. A vital tool for controlling mosquito larvae is the application of chemical insecticides, including organophosphate and organochloride compounds [11]. Reducing the number of mosquito larvae is a valuable approach to reducing the abundance of arthropod diseases [7]. Eco-friendly replacements are widely accepted to decrease the selective pressure created by insecticides due to their resistance.

Naturally derived pesticides can be effective in the management of Ae. aegypti and its viral transmission. Improving bioactive compounds for managing and controlling mosquito larvae and the transmission of disease is indeed a promising approach [10]. They are generally safe for non-target species, including humans, and are utilized as part of integrated vector management strategies that also include other measures, such as source reduction, insecticide spraying, and community engagement [11]. Marine-derived red algae, along with their active constituents, have been specifically studied as potential biocontrol agents against major insect pests, including Ae. aegypti mosquitoes. Extracts or products derived from red algae were found to possess larvicidal properties against mosquito larvae [12,13]. These extracts contain bioactive compounds that can interrupt the growth and progress of mosquito larvae, leading to their mortality.

Red-algae-derived larvicides are used in stagnant water bodies, such as water storage containers, flowerpots, and drains, where mosquito larvae breed to reduce their populations [14]. Halymenia dilatata is a red marine alga that is found on the surface of the water in the Mediterranean and Atlantic Oceans. It has been traditionally used as food and medicine in several countries across Europe and Asia [15]. Recently, there has been a growing interest in investigating the pharmacological proportions of the chief derivatives of H. dilatata with antioxidant activity, including carotenoids, phycobiliproteins, and phenolic compounds [16,17]. In addition, algal extracts are potentially safe, effective, and low-cost alternatives to synthetic pesticides, which adversely affect the environment and human health. Several active compounds in them are capable of acting as bio-pesticides for the control of pests, contributing to the development of an eco-friendly society [14]. The use of algae-derived bio-insecticides for mosquito control has been identified as a safe and cost-effective alternative compared with synthetic pesticides [16]. The major metabolites of H. dilatata, including terpenes and sulfated polysaccharides, showed antimicrobial activity [18].

Manilal et al. [19] illustrated that marine-derived algae synthesize diverse phytocompounds in response to the selective pressures due to the microbes and herbivores that are considered major insecticides. The different red algae belonging to the Halymenia genus are part of the Rhodophyta phylum, species of which have a distinctive red color and are widely distributed in the seas of the tropical zone, including India, Kenya, and Sri Lanka [20]. Major phytocompounds present in red algae include alkaloids, saponins, tannins, flavonoids, and triterpenoids [21]. Halymenia dilatata is considered an important food source for herbivorous marine animals, such as sea urchins and certain species of fish. It also has several potential commercial applications, including as a source of carrageenan, which is a polysaccharide that is widely used in the food and cosmetic industries [22]. Phytocompounds from red algae deliver different biological actions, such as the hypo-cholesterolemic properties of common red algae harvested from the coastal waters of the Philippines [23]. Red algae that develop in high-salinity situations develop different biological actions, including anticoagulant, antibacterial, anti-inflammatory, antioxidant, anti-plasmodial, antidiabetic, and cytotoxic activities [24,25].

The present research investigated the insecticidal properties of crude methanolic extracts of Halymenia dilatata (Mx-Hd), including (a) larvicidal toxicity; (b) lethal dosage determination (LC₅₀ and LC₉₀); (c) inhibition activity against major digestive and detoxifying enzymes, including α-esterase, β-esterase, glutathione S-transferase (GST), cytochrome P450 (CYP450), alkaline phosphatase (ALP), and acid phosphatase (ACP); (d) repellent activity; (e) non-target toxicity toward the aquatic predators Alpheus bouvieri, Toxorhynchites splendens, and Deinococcus indicus; and (f) photomicrography of larvae of the dengue mosquito Aedes aegypti.

2. Methodology

2.1. Red Alga Extraction and Harvesting

Halymenia dilatata was harvested during a morning session across the Rameswaram coastal regions, Tamil Nadu, India (Figure 1A,B). Further, the samples were fixed to sheets of the herbarium at the Department of Biotechnology, St. Peters Institute of Higher Education and Research, Tamil Nadu, India. The alga was rinsed well with sterile distilled water and dried under shade at room temperature. Further, it was chopped finely, prepared into a fine residue, and then placed in an airtight container. The dried specimen was further dissolved using methanol, and extraction and dissolution were performed at room temperature; after this, methanolic crude extracts (Mx-Hd) were obtained and preserved in a tight container without exposure to any light at −4 °C for use in further toxicity assays.

2.2. Mosquito Rearing

Ae. aegypti larvae have been maintained in the Bio-pesticides and Environmental Toxicology Laboratory (BET Lab, Vicksburg, MS, USA), SPK Centre for Excellence in Environmental Sciences, since at least 2007, without exposure to pesticides. The cultures were conserved, and all the experiments were carried out in our laboratory at 27 ± 2 °C and 75–85% RH under a 14:10 L/D photoperiod, without exposure to any chemical insecticides. Brewer’s yeast, dog biscuits (Choo Stix Biskies), and the alga collected from pools in a ratio of 3:2 were fed to the larvae as a diet. Pupae that emerged from the larvae were transferred to a plastic cup (round, 250 mL capacity) containing tap water that was located in a breeding cage (60 × 60 × 60 cm dimensions) for adult emergence. Wet raisins (dried grapes) and 10% sucrose solution soaked in cotton were fed to the adults. The adult females were destitute of sucrose from 6 h, and then a mouse was placed in the breeding cage overnight for blood feeding. Adult mosquitoes were maintained under similar conditions to those of the larvae. The time intervals for the development of mosquito larvae varied for the second instar (24 to 48 h), followed by the third and fourth instars (24 to 72 h). The first-generation larvae were used for the experiment. The complete insect-rearing procedure was performed as per the standard protocol [26].

2.3. Chemicals

Temephos (organophosphate larvicide), which was used as a positive control and procured from SIGMA-ALDRICH (Burlington, MA, USA), (Pestanal ^®, Analytical Standard), was utilized for the non-target toxicity against aquatic predators.

2.4. Larvicidal Assay

The Mx-Hd larvicidal activity was assessed using the standard protocol of the World Health Organization [26] with minor modifications [14]. The second, third, and fourth instars (25 larvae each) were kept in 250 mL one-use plastic dishware, covering the samples with 25 mL of Mx-Hd at different lethal dosages (20, 40, 60, 80, and 100 ppm), with a ratio of 24.5 mL of chlorinated water and 0.5 mL of Mx-Hd herbal extracts, and then they were reared at 26 ± 5 °C. During the treatment period, larval food was added to each test cup, especially if high mortality was noted in the control.

Larvae were considered dead when they were unable to reach the surface of the solution when the cups were disturbed. The larvae were considered moribund if, at the end of a 24 h period, they showed no sign of swimming movements, even after gentle touching with a glass rod. The dead and moribund larvae were recorded after 24 h as larval mortality. The whole setup was kept undisturbed for another 24 h, and mortality counts were recorded again after 48 h. The number of dead larvae was determined at the start of the experiment (0, 24, and 48 h). The larvicidal tests with more than 20% mortality in the controls and pupae formation were discarded and repeated. The treatments were replicated five times, and each replicate set contained one control. Methanol (0.5%) was employed as the negative control. The entire assay was carried out according to the Finney [27] probit method. The percentage mortality (1) in the treatments was corrected when necessary for mortality in the controls using the Abbott [28] formula (Equation (2)):

$$\mathrm{Percentage} \mathrm{of} \mathrm{mortality}=\frac{\mathrm{Number} \mathrm{of} \mathrm{dead} \mathrm{larvae}}{\mathrm{Number} \mathrm{of} \mathrm{larvae} \mathrm{introduced}}\times 100$$

(1)

$$\mathrm{Corrected} \mathrm{percentage} \mathrm{of} \mathrm{mortality}=(1-\frac{n \mathrm{in} \mathrm{T} \mathrm{after} \mathrm{treatment}}{n \mathrm{in} \mathrm{C} \mathrm{after} \mathrm{treatment}})\times 100$$

(2)

(n—number, T—treated, C—control).

2.5. Enzyme Assay

The Ae. aegypti larvae of the second to fourth instars (n = 25) were washed with double-distilled water, and the adhering water was totally removed from the surface by blotting with tissue paper. The larvae were separately homogenized in an Eppendorf tube using a hand homogenizer in 500 μL of ice-cold sodium phosphate buffer (20 mM, pH 7.0) to assess the enzyme activity. The homogenates were centrifuged (8000× g at 4 °C) for 20 min, and the supernatants were used for further analyses. The final homogenates were held on ice until being used in the assays. Carboxylesterase (α and β), alkaline and acid phosphatase, and glutathione S-transferase activities were measured. Larval extracts were prepared in 0.1 M phosphate potassium, pH 7.2 (20 μL, 84 μg of protein), containing 1% acetone, which was mixed with 500 μL of a solution containing 0.3 mM α- or β-naphthyl acetate. The reaction mixture was incubated for 20 min at 30 °C. Then, 0.1 mL of a mixture containing 0.3% Fast Blue B and 3.3% sodium dodecyl sulfate (SDS) was added. After centrifugation (3000× g, 28 °C), the supernatant absorbance at 590 nm was recorded. One unit of enzyme activity was defined as the amount of enzyme required to generate 1 μmol of α- or β-naphthol per minute and expressed as U/mg protein. For the GST and cytochrome P450 action (CYP450), protein concentrations were verified using an albumin standard and bicinchoninic acid protein assay kit (Catalog TP0l00, Sigma Aldrich, Bangalore). The activities of GST and CYP450 were expressed in U/mg protein. The activity was measured using our previously modified methodology [29].

2.6. Repellent Assay

Different dosages of Mx-Hd (20, 40, 60, 80, and 100 ppm) were evaluated for their ability to repel Ae. aegypti adult mosquitoes with the adapted protocol [30]. One hundred gravid female adults (mated, 5–7 days post-emergence) that were starved for 24 h without a blood meal but previously fed on 10% sucrose solution were kept in net cages (40 cm × 30 cm × 40 cm). An uncovered area of 3 × 10 cm on each forearm of three human volunteers was marked out with a permanent marker, with the remaining area covered with a paper sleeve, and the humans had no previous contact with lotions or perfumed soaps on the day of the assay. Each volunteer exposed both forearms to mosquitoes. One forearm was untreated and the other was treated. The treatments were a blank control (methanol 0.5%), MX-Hd 20 ppm, MX-Hd 40 ppm, MX-Hd 60 ppm, MX-Hd 80 ppm, or MX-Hd 100 ppm. Assays were performed during the daytime from 08.00 to 16.00 h. The control and treated arms were introduced simultaneously into the mosquito cage; by gently tapping the sides of the experimental cages, the mosquitoes were activated. Each test concentration was repeated three times. The volunteers conducted the test for each concentration by inserting the treated and control arms into the cages at the same time for 1 min every 5 min. The mosquitoes that landed on the hand were recorded and then shaken off before blood imbibition. The percentage of repellency was calculated using the following formula (Equation (3)):

% Repellency = [(Ta − Tb)/Ta] × 100

(3)

where Ta is the number of mosquitoes in the control group and Tb is the number of mosquitoes in the treated group.

2.7. Non-Target Toxicity

The screening of Mx-Hd on the non-target species was performed with the modified protocol of Thanigaivel et al. [31]. The aquatic predator species Alpheus bouvieri, Toxorhynchites splendens, and Deinococcus indicus were field-collected and separately maintained in cement tanks (80 cm diameter and 25 cm depth) containing water at 26 ± 2 °C with a relative humidity of 85%, and authentication was provided by the zoologist at Manonmaniam Sundaranar University, India. Second- and third-instar larvae of Ae. aegypti were supplied as food and maintained at 26 ± 2 °C and 85% RH. The treatment dosages of Mx-Hd (250, 500, 750, and 1000 ppm) were considerably higher than the concentrations tested on the mosquito larvae compared with the commercial pesticide temephos (1.0 ppm). Fifteen replicates (n = 25) were performed for each concentration, along with five replicates for the untreated controls. After 24 h, the mortality rate was recorded.

2.8. Photomicrography Assay

Fourth-instar larvae were subjected to the major morphological changes caused by the treatment dosage of Mx-Hd (55 ppm). The treated fourth-instar larvae and controls were transferred to sterile glass slides (n = 25) after being fixed via ethanol dehydration at 40–70% for 30 min at 25 °C for each group. Finally, the sections were examined under 40× magnification using a light microscope (make: Optika Microscopes, Ponteranica, Italy, Optika vision lite 2.0).

2.9. Data Analysis

The statistical values from the experiments were subjected to analysis of variance (ANOVA of arcsine, logarithmic, and square-root-transformed percentages). Differences between the treatments were determined using the Tukey–Kramer HSD test (p = 0.05). Sigma Plot 11 in the Microcal software, 15.0.0.13 (x86), was employed to measure the enzyme activity. The Minitab^®17 program was used to perform a probit analysis to determine the lethal dosages of Mx-Hd needed to kill 50% of the larvae (LC₅₀) and 90% of the larvae (LC₉₀) within 24 h.

3. Results

3.1. Larval Toxicity of Mx-Hd

The larval toxicity of Mx-Hd was examined for Ae. aegypti (second, third, and fourth instars), finding a dosage-dependent mortality rate across the second, third, and fourth instars. The mortality rate was observed to be prominent in the second instar compared with the third and fourth instars. The young larvae (second instar) were more susceptible to the Mx-Hd dosages compared with the third and fourth instars (Figure 2). The mortality rate was the highest at the 100 ppm concentration in the second instar (96.81%, F_4,20 = 19.67, p ≤ 0.001), followed by the third (92.90%, F_4,20 = 12.17, p ≤ 0.001) and fourth instars (91.33%, F_4,20 = 19.33, p ≤ 0.001). In addition, the lowest-dosage treatment (Mx-Hd 20 ppm) also displayed a significant difference with the control in the second (8.21%, F_4,20 = 14.88, p ≤ 0.001), third (7.45%, F_4,20 = 12.66, p ≤ 0.001), and fourth instars (6.79%, F_4,20 = 17.45, p ≤ 0.001). For all the instars, the control displayed no significant values for the second (4.21%, F_4,20 = 13.11, p ≤ 0.001), third (4.06%, F_4,20 = 12.12, p ≤ 0.001), and fourth instars (4.02%, F_4,20 = 12.41, p ≤ 0.001).

The lethal concentrations of Mx-Hd were significant for the second instar, with an LC₅₀ of 37 ppm (chi-square = 60.6684) and LC₉₀ of 93 ppm (Figure 3A). Correspondingly, for the third instar, an LC₅₀ of 43 ppm (chi-square = 68.626) and an LC₉₀ of 96 ppm were found (Figure 3B). Additionally, the LC₅₀ and LC₉₀ values for the fourth instar were 52 ppm (chi-square = 72.84) and 97 ppm, respectively (Figure 3C).

3.2. Enzyme Inhibition of Mx-Hd

In Ae. aegypti, there was a significant dose-dependent difference in enzyme inhibition. The digestive enzyme α-esterase showed a significant reduction at the concentration of 55 ppm in the second instar (0.4432 mg/protein), third instar (0.4213 mg/protein), and fourth instar (0.4113 mg/protein) (Figure 4A). All treatment dosages were significantly different from the lowest-dosage treatment of 10 ppm in the second (F_4,20 = 11.45, p ≤ 0.001), third (F_4,20 = 13.88, p ≤ 0.001), and fourth (F_4,20 = 18.22, p ≤ 0.001) instars.

Correspondingly, dose-dependent enzyme inhibition activity was also shown for β-esterase, and the inhibition rate was prominent at 55 ppm in the second instar (0.6021 mg/protein), third instar (0.6130 mg/protein), and fourth instar (0.8760 mg/protein). Additionally, this activity was significantly different from the lowest dosage of 10 ppm in the second (F_4,20 = 14.66, p ≤ 0.001), third (F_4,20 = 12.31, p ≤ 0.001), and fourth instar (F_4,20 = 17.89, p ≤ 0.001) (Figure 4B).

However, the Mx-Hd dosage (55 ppm) increased GST-detoxifying enzymes across the second (0.5921 mm/mg), third (0.5791 mm/mg), and fourth instars (0.5131 mm/mg), and these results were significantly different from those of the lowest dosage (10 ppm) in the second (F_4,20 = 16.21, p ≤ 0.001), third (F_4,20 = 17.55, p ≤ 0.001), and fourth (F_4,20 = 18.22, p ≤ 0.001) instars (Figure 4C). A similar trend was observed for CYP450, as the enzyme rate was increased in the Mx-Hd treatment (55 ppm) across the second instar (8.330 mmol 7-OH/mg), third instar (7.8321 mmol 7-OH/mg), and fourth instar (7.4100 mmol 7-OH/mg). All the dosages produced results that were significantly different from those of the lowest Mx-Hd treatment dosage (10 ppm) in the second instar (F_4,20 = 12.11, p ≤ 0.001), third instar (F_4,20 = 10.44, p ≤ 0.001), and fourth instar (F_4,20 = 11.60, p ≤ 0.001) (Figure 4D). Enzyme inhibition was higher in a concentration-dependent manner for alkaline phosphatase (ALP), with the maximum at the 55 ppm dosage of Mx-Hd in the second instar (0.2854 mM/mg), third instar (0.3012 mM/mg), and fourth instar (0.3314 mM/mg) (Figure 4E). Similarly, acid phosphatase (ACP) also declined in a dose-dependent manner (55 ppm Mx-Hd) across the second instar (0.2312 mM/mg), third instar (0.2410 mM/mg), and fourth instar (0.2410 mM/mg) (Figure 4F).

3.3. Repellent Activity of Mx-Hd

The repellent actions of Mx-Hd were concentration-dependent, and the repellency percentage was prominent at the dosage of 100 ppm of Mx-Hd at the maximum repellent time of up to 210 min. The repellent time was directly proportional to the repellency percentage as the repellent time (15–210 min) increased, in turn decreasing the repellency percentage. The repellency percentage was significant at the maximum repellent time (210 min) with the Mx-Hd treatment dosages of 20 ppm (46.8%, F_4,20 = 10.44, p ≤ 0.001), 40 ppm (50.4%, F_4,20 = 14.21, p ≤ 0.001), 60 ppm (54.3%, F_4,20 = 13.89, p ≤ 0.001), 80 ppm (77.8%, F_4,20 = 11.43, p ≤ 0.001), and 100 ppm (83.2%, F_4,20 = 7.19, p ≤ 0.001) (Figure 5).

3.4. Toxicity of Mx-Hd against Aquatic Predators

The aquatic predator species A. bouvieri, Tx. Splendens, and D. indicus were field-collected and maintained in separate containers. Mx-Hd and the commercial insecticide temephos led to significant dose-dependent mortality across all the non-target species. The treatment dosages of Mx-Hd (250, 500, 750, and 1000 ppm) displayed less toxicity compared with the commercial pesticide (1 ppm) (Figure 6). The maximum mortality rate was 11.23% (F_4,20 = 13.43, p ≤ 0.0001), 16.78% (F_4,20 = 17.75, p ≤ 0.0001), and 18.43% (F_4,20 = 12.41, p ≤ 0.0001) under the highest concentration of Mx-Hd (1000 ppm) in A. bouvieri, Tx. splendens, and D. indicus, respectively. The mortality rate after the temephos treatment (1.0 ppm) was 95.46% (F_4,20 = 18.44, p ≤ 0.001) in A. bouvieri, 93.21% (F_4,20 = 15.23, p ≤ 0.001) in Tx. splendens, and 91.23% (F_4,20 = 12.25, p ≤ 0.001) in D. indicus. The mortality rate of A. bouvieri was observed to be dose-dependent across the Mx-Hd treatments and it was higher in the 1000 ppm (11.20%, p ≤ 0.0001), 750 ppm (9.43%, p ≤ 0.0001), 500 ppm (8.32%, p ≤ 0.0001), 250 ppm (6.54%, p ≤ 0.0001) treatments compared with the control (4.53%, p ≤ 0.0001). The same trends were observed in the Tx. splendens and D. indicus, i.e., as the dosage increased, the mortality rate increased.

3.5. Photomicrography of Ae. aegypti Larvae

Fourth-instar larvae exposed to Mx-Hd (55 ppm) dosage displayed morphology dysregulation, along with changes in an anal segment (AS), epithelial layer (EL), segment (S), and gut lumen (GL). There were clear, well-defined cells in the control larvae (Figure 7). There was a prominent damage rate in the Mx-Hd treatment compared with the control.

4. Discussion

Methanolic crude extracts of Halymenia dilatata (Mx-Hd) led to concentration-dependent mortality across second-, third-, and fourth-instar larvae of the dengue vector Ae. aegypti. Similarly, different biocontrol tactics target diverse mosquito lifecycle stages while being harmless to the environment [32,33,34,35,36]. Algae share a similar environmental niche to Ae. aegypti populations and most algae are considered a major food source for mosquitos and other aquatic organisms [29,30].

In addition, methanolic extracts of H. palmata (HPMe) and their active fractions showed potential larvicidal actions against the dengue mosquito Ae. aegypti [37]. Previously, Yu et al. [38] illustrated that crude extracts of the red alga Laurencia dendroidea in the family Rhodomelaceae displayed significant larvicidal actions against Ae. aegypti. Similarly, Bianco et al. [39] investigated the larval mortality of fifteen different marine algae from the northeastern Brazil region, showing >50% larvicidal activity in Canistrocarpus cervicornis (Kuetzing), Hypnea musciformis (commonly known as Crozier weed), and Chaetomorpha antenna (Bory) at a high concentration of 300 ppm. A high mortality rate (>91%) was found in Laurencia dendroidea (J. Agardh) compared with other natural seaweeds. In addition, the LC₅₀ value was observed to be 10.7 ppm for L. dendroidea. Similarly, the LC₅₀ of Mx-Hd was found to be 37 ppm against the second-instar larvae of the dengue vector. Correspondingly, Galaxaura elongate methanol extract showed the highest LC₅₀ rate (31.13 ppm) for third-instar Culex pipiens L. mosquito larvae compared with other red and green algae, such as Jania rubens, Dictyota dichotoma, Padina boryana, Sargassum dentifolium, Ulva intestinales, and Codium tomentosum [40]. In parallel, Deepak et al. [37] showed that red algae, along with their active compounds alkyl halides and carboxylic acid, are highly responsible for larval toxicity in Ae. aegypti. The detoxifying enzymes of mosquito larvae are the vital components of their antioxidant defense system against toxic compounds [40,41].

The enzyme inhibition activity of major biomarker enzymes in Ae. aegypti upon treatment with different dosages of the Mx-Hd showed significant inhibition of digestive marker enzyme levels, including α-esterase and β-esterase. Despite this, the Mx-Hd dosages increased the major marker detoxifying enzyme levels of CYP450 and GST. Correspondingly, Haleem et al. [40] illustrated that the different dosages of red and green algal extracts (G. elongate, J. rubens, C. tomentosum, and U. intestinalis) significantly affected the major oxidative marker enzymes, such as catalase, superoxide dismutase, and glutathione peroxidase, of C. pipiens larvae in a concentration-dependent manner. Yu et al. [42] illustrated that the existence of different phytochemicals, like flavonoids, phenolics, polysaccharides, alkaloids, saponins, halogenated compounds, and terpenoids, from marine red algae led to significant larvicidal and enzyme inhibition actions on different mosquito vectors. In addition, Blunt et al. [43] found that red algae are more active in targeting mosquito larvae and other biological activities compared with brown and green algae, as they are enriched in terpenes and polyphenolic compounds, which, in turn, block the mechanisms of major detoxifying enzymes of mosquito vectors.

The repellents derived from bioactive extracts and their major compounds do not have a harmful impact on non-targets, including human beings, as they are biodegradable, in parallel with commercial pesticides or repellents [25]. There were no prominent reactions including rashes, swelling, and irritation in the applied regions of the volunteers during the entire period of this research. In addition, bioactive extracts derived from different algae are considered a vital tool in the effective management of medically challenging mosquito vectors [44]. Our repellent activity results showed that Mx-Hd (100 ppm) had the highest repellent activity at the maximum repellent time (210 min) on the dengue mosquito compared with the other treatment dosages. In parallel, Yogarajalakshmi et al. [14] showed that ethanolic crude extracts of the marine macroalga Champia parvula had the highest repellent activity (>97%) on the dengue arthropod vector Ae. aegypti. Xu et al. [45] illustrated that controlling or managing blood-sucking pests with chemical pesticide treatment is harmful, as it delivers an adverse effect on beneficial or aquatic species that share ecological regions with mosquitoes.

Our results showed that Mx-Hd is ecologically safer (<10%) against the beneficial predators of mosquitoes, even at the two-fold higher dosages (1000 ppm) used for the larvicidal activity, compared with the commercial pesticide temephos (1.0 ppm), which had a >90% mortality rate on the aquatic predators. In addition, the selected aquatic predators’ A. bouvieri, Tx. Splendens, and D. indicus coexistence with mosquito larvae is an example of the natural balance and dynamics within ecosystems. All these predators play an important ecological role by regulating the population sizes of their prey species and they can help keep mosquito populations in check, which is beneficial for controlling the spread of mosquito-borne diseases [46]. Alpheus bouvieri is a species of snapping shrimp, also known as pistol shrimp. Snapping shrimps are known for their unique predatory behavior and specialized physiology. These shrimps have one enlarged claw that is used as a weapon to stun or kill their prey like mosquito larvae [4]. In addition, Tx. splendens is a species of mosquito commonly known as the “elephant mosquito” or “mosquito eater.” Unlike most mosquitoes, Tx. splendens does not feed on blood. Instead, it exhibits predatory behavior toward other mosquito larvae. The hatched larvae of this species then feed on the larvae of other mosquito species present in the same water source. This predatory behavior is an effective means of controlling mosquito populations [29]. Also, D. indicus is a species known for its remarkable resistance to extreme environmental conditions, such as radiation and desiccation, and its predatory actions are specific to the larval gut, as reported earlier [14]. Additionally, Yu et al. [42] and Vasantha-Srinivasan et al. [47,48] stated that mosquitocides are recognized as safer and more effective, as they target only harmful pests and mosquitoes and have a harmless or less toxic impact on non-target species. It seems that algae toxins derived from H. dilatata are selective to Ae. aegypti larvae. This may be due to the differences in the physiology and biochemistry between the two species. Tx. splendens is greater in size (approx. 18 mm), and it may have different specific receptors and biochemical pathways that are unresponsive to the algal toxins [10]. Further detailed research is required for a better understanding. The active constituents of algal extracts affect the midgut epithelium of mosquito larvae and may be the pivotal reason for the decrease in the metabolic ratio and key marker enzymes. The gut lumen may block parasites’ growth in mosquitoes by developing a powered barrier. Our toxicological screening was found to agree well with the photomicrography assays, as it displayed significant alterations in the midgut morphology of Ae. aegypti parallel to the control. Correspondingly, fourth-instar Ae. aegypti larvae showed significant morphological dysregulation in the midgut cells post-treatment with ethanolic crude extracts of the macroalga (Ex-Cp) Champia parvula [14]. In addition, the terminal spiracles and anal papillae of Ae. aegypti displayed significant aberrations upon exposure to a lethal dosage of seaweed extracts of Bryopsis pennata [38].

Overall, the findings of the present study demonstrate that Mx-Hd exhibited larvicidal activity against the dengue mosquito species Ae. aegypti. This extract contained bioactive compounds that are toxic to mosquito larvae and could even inhibit their midgut enzyme regulation. What makes H. dilatata especially promising as a mosquitocidal agent is that it is also harmless to aquatic predators of mosquitoes, making it a target-specific and ecologically safe alternative to traditional insecticides. From a sustainability point of view, the use of red marine algae in mosquito control is consistent with the principles of sustainable development. This approach recognizes the need to protect the environment while simultaneously promoting public health and socio-economic development. In contrast to traditional insecticides that are often associated with negative environmental impacts, such as killing non-target species, disrupting ecosystems, and contaminating water sources, red algae do not harm non-target species and are not associated with environmental pollution. Moreover, their use promotes socio-economic development by generating income and employment opportunities for local communities involved in their harvesting and processing [48,49,50]. The bioeconomy of algal-based pesticides in managing mosquitoes involves utilizing algae-derived compounds and products as alternatives to conventional chemical pesticides for mosquito larvae control. Algae are diverse photosynthetic microorganisms that can produce a wide range of biologically active compounds with potential larvicidal properties. Moreover, marine algae are renewable resources that can be continuously cultivated and harvested, making them a sustainable option for pest management. In addition, algal-based pesticides break down naturally in the environment, reducing their persistence and potential for bioaccumulation. The present approach aligns with the principles of sustainable development and the switching of synthetic compounds with biological resources, the cascading usage of biomass, and the minimization of biowaste [51].

5. Conclusions

The present research delivers a baseline toxicological assessment of the marine red alga Halymenia dilatata (Mx-Hd) against the arthropod vector Ae. aegypti. The larvicidal actions showed that Mx-Hd produced a strong mortality rate in the second, third, and fourth instars at the maximum dosage (100 ppm). Similarly, the Mx-Hd dosage exhibited a decreased enzyme rate in the alpha and beta esterase activity, while the enzyme rate was increased for GST and CYP450. The repellent activity of Mx-Hd showed the highest repellent actions for up to 210 min at the maximum dosage of 100 ppm. The non-target toxicity of Mx-Hd against aquatic predators showed minimal toxicity compared with the commercial pesticide temephos. In addition, Mx-Hd caused major physiological changes in the larval midgut compared with the control.

Developing sustainable and cost-effective methods for the large-scale production of algal bio-pesticides is essential for their widespread implementation. This includes optimizing cultivation techniques, exploring innovative production systems (such as photo-bioreactors), and establishing efficient supply chains to ensure availability and affordability. As an endnote, algal mosquitocides offer a promising alternative for managing Aedes aegypti populations. While there are limitations to address, ongoing research, integrated pest management approaches, community engagement, and sustainable production methods can contribute to the future success and wider adoption of algal bio-pesticides in mosquito control efforts. The efficacy of algal-based pesticides may vary depending on the algal species, extraction methods, and formulation. In addition, scaling up production and improving algal extraction techniques can help to reduce costs, and obtaining approval from regulatory authorities to ensure their safety and efficacy are the key challenges for market acceptance. Overall, research and development efforts are needed to optimize the potency of these algal-based bio-pesticides for a sustainable environment.