Potent targeted larvicidal activities of marine-derived Bacillus sp. bacterial extracts on mosquito vectors (2025)

Mosquitoes serve as potent vectors for a multitude of diseases, notably malaria, dengue, chikungunya, filariasis, leishmaniasis, and japanese encephalitis. The burden of mosquito borne diseases is a global health problem and affects more than 40% of the world’s population¹. Around half of the globe’s population is at risk of dengue infection, with WHO estimating 100–400 million dengue cases each year². According to world malaria report 2023, there were an estimated 249 million malaria cases, globally and in the past one decade, 88 countries have reported insecticide resistance amongst the vector populations³. Successful vector control strategies employ long lasting insecticide-treated bed-nets (LLINs) and indoor-residual spray (IRS), where pyrethroids are most widely used for treating LLINs and for IRS^4,5. However, continuous application of pyrethroids has resulted in emergence of resistance among mosquito vector populations⁶. Thus, necessitating the development of novel insecticidal molecules or strategies to combat the looming challenge of resistance. Moreover, these approaches encounter additional obstacles, such as elevated costs and concerns about toxicity, environmental hazard, etc.

Unlike synthetic insecticides, majority of bio-insecticides are derived from microbial sources and have proven to be an effective alternative strategy for vector control^4,7,8. Bio-insecticides can include plant-incorporated protectants, microbial pesticides, phytochemicals, and pheromones. These alternatives exhibit lower toxicity, greater specificity, biodegradability, require lesser quantities, and are less prone to develop resistance by insects^9,10. Bacillus thuringiensis israelensis (Bti) and Lysinibacillus sphaer extracts were found to effectively kill the larvae of multiple mosquito species¹¹. Similarly, larvivorous fishes in aquatic water bodies reduced the larval population, thereby abolishing the development of adult mosquitoes^12,13. Furthermore, certain bacterial extracts and isolated compounds have demonstrated larvicidal activity. Notably, spinosyn A and spinosyn D, derived from the bacterium Saccharopolyspora spinosa from Actinomycetes, were found effective against various mosquito species including An. gambiae and An. funestus¹⁴, An. dirus, An. minimus¹⁵. Field-tested formulations of spinosad have been found to be effective against Culex quinquefasciatus, a vector of filariasis in India¹⁶. Essential oils have also shown promise as a valuable source of bio-larvicides against Ae. aegypti^17,18,19. Crude leaf extracts of Momordica foetida, Calpurnia aurea and Zehneria scabra displayed low IC₅₀ values of 35ppm against An. stephensi²⁰. Additionally, Saussurea costus extract exhibited significant activity against three major mosquito vectors (Aedes, Anopheles and Culex), with an IC₅₀ value ~ 8ppm against An. stephensi²¹. Numerous ethnobotanical plants have demonstrated larvicidal and adulticidal properties against An. arabiensis with notable examples including Ocimum lamiifolium, Ocimum americanum, Azadirachta indica, Moringa olifeira leaf and seed species²².

Previous studies have showed bacterium Bacillus and its isolates as efficient bio-larvicides against mosquito vectors. For example, Bacillus thuringiensisvar.israelensisandBacillus sphaericus have been popularly used as an effective larvicide for a long time, with minimum non-target effects on other organisms²³. A recent study showed the supernatants of Bacillus safensisBac I67 andBacillus paranthracisC21 efficiently killed the Aedes aegypti larvae from a cohort of 254 different bacterial extracts²⁴. Similarly, Bis-(2-ethylhexyl) phthalate fromLactiplantibacillus plantarum was reported to have anti-larval activity against Culex sp²⁵. Metabolites isolated from Nocardia and Streptomyces were tested against the fourth stage larvae of Anopheles stages and were found to be effective with LC₅₀ ranging from 300 to 600 ppm²⁵. All these studies indicated that bacteria-derived larvicides are effective and could be exploited for mosquito larval control. In context of the larvicidal activities from marine bacteria have not gained much attention. Crude extracts of the marine bacteria Streptomyces sp. exhibited antilarval activity against three different disease vectors (An. stephensi, Culex tritaeniorhynchus and Rhipicephalus microplus). One of the purified marine actinobacterial compound DMBPO had displayed LC₅₀ values of 88.97ppm against the An. stephensi²⁶. In the present exploratory study, we investigated the larvicidal activities of the extracts from culture supernatants of various marine bacteria (denoted as ‘extracts’ further on in the study) collected from various locations across Indian peninsula.

Larvicidal activities of extracts from marine bacteria on mosquito vectors

We examined the larvicidal activities of the 55 marine bacterial extracts against the larvae of An. stephensi (Fig.1A). Amongst 55 extracts, 12 extracts (NIO 97, 116, 124, 132, 258, 276, 311, 701, 706, 707, 710, 718) were found effective against An. stephensi larvae at concentrations (250 and 125ppm) (Fig.1A). Further, the effective extracts were assessed for their dose-dependent larvicidal activities against An. stephensi larvae (250–7.5ppm) (Fig.1B). Except NIO 132 and 707; all extracts exhibited significant larval mortality (> 50%) at 62ppm compared to control. Using PROBIT analysis, the LC₅₀ values and fiducial limits of 12 extracts were calculated and summarized in Table 1. Unfortunately, estimation of the larvicidal activities required large amounts of extracts; during the course of experiments, some of the extracts exhausted and could not be continued for further investigation. Therefore, extracts NIO 97, 124, 276, 701, 707 and 710 were tested for their larvicidal activities against Ae. aegypti larvae at two concentrations (500 and 250ppm) (Fig.2A). Only NIO 707 extract demonstrated > 70% mortality for Ae. aegypti larvae at 500ppm (Fig.2A). To check the effectiveness of the extracts in a semi-field setting, An. subpictus larvae were collected from the NIMR field unit at Mewat, Haryana, India. Available effective extracts (NIO 97, 124, 276, 701 and 706) were used at a concentration of 125ppm against the field collected larvae of An. subpictus. Among the tested extracts, only NIO 706, showed > 80% mortality at 125ppm concentration in 24h. (Fig.2B).

Reduced egg hatching in Anopheles

The eggs of An. stephensi and An. culicifacies were incubated with the extracts to check their action on the hatchability of the eggs. Reduced transformation of the eggs to the larvae was observed in both the Anopheles vectors (Fig.3A,B). In case of An. stephensi, extracts NIO 701, 706 and 707 found to reduce the hatchability of the eggs at 500ppm, compared to control. However, for An. culicifacies, all extracts at 500ppm (NIO 97, 124, 276, 701, 706, 707 and 710) were effective in reducing the number of eggs transformed to larvae, compared to control.

Phylogenetic analysis of the larvicidal extracts

Phylogenetic analysis of the extracts subjected to antilarval activities against different mosquito vectors and reduction in egg hatching was performed. 16S rRNA identification for NIO 97, 124, 276, 701, 706, 707 and 710 was performed and a phylogenetic tree was constructed (Fig.4). The locations of the collection sites of marine bacteria included in the phylogenetic analysis; their closest strains and sequence identities are summarized in Table 2. Phylogenetic analysis revealed that most of the marine bacteria were isolates of Bacillus sp. (NIO 97, 124, 701, 706 and 707). However, we observed that the larvicidal activity of NIO 276 (Enterococcus casseliflavus) extract was a peculiar finding of this study.

Assessment of the antiparasitic activity of the extracts

To examine the non-specific activity of selected marine extracts, we performed P. falciparum parasite growth inhibition assay. Notably, except NIO 97, none of the tested extracts showed any significant detrimental effect on parasite growth at concentration of 50µg/ml (Fig.5A). The treated parasites were observed to have similar growth pattern as untreated and methanol exposed control parasites. The parasites treated with the extracts displayed similar parasitemia and morphology as compared to control (Fig.5B). We used chloroquine (CQ) as positive control in the growth inhibition assay with very few or no parasites after CQ treatment after 24h. of exposure.

The emergence of resistance against the conventional chemical insecticides by mosquitoes and the associated public health risks have spurred the search for safer, eco-friendly, and more economical options. For mosquito vector control, microbes-derived larvicides have proven as promising alternatives to conventional insecticides. In this study, we explored An. stephensi larvicidal activities of extracts from the culture supernatants of marine bacteria (n = 55) collected from different Indian coastal regions. 12 extracts were found to possess larvicidal activities. Unfortunately, due to requirement of large quantities of extracts in larvicidal screening assays, many extracts exhausted and could not be continued further for investigation. Subsequently, the available extracts were further subjected to larvicidal assays in other mosquito vectors such as Ae. aegypti, An. subpictus in semi-field setting; and impact on egg hatching in An. stephensi and An. culicifacies, respectively. Interestingly, there was no significant anti-parasitic activities observed in the extracts against P. falciparum.

It was interesting to observe that extracts (NIO 97, 124, 276, 701 and 710) were effective against An. stephensi larvae, but except NIO 707 none of the effective extracts demonstrated larvicidal activity against Ae. aegypti. Moreover, when the effective extracts were tested in semi-field setting on An. subpictus larvae, only NIO 706 was found effective. The difference in larvicidal activities on lab reared larvae and field collected larvae might be attributed to the physiological adaptation/resistance, because later larvae dwell in pond/natural water bodies along with other bacteria and organisms. The LC₅₀ values indicating the larvicidal activity against An. stephensi are in the range of 44–83ppm, which is considered potent for whole extracts. Further, the active chemical constituents upon characterization might be more effective compared to whole extracts. These results point towards targeted action of the chemical constituents present in different extracts. Such differential larvicidal activities of extracts against different mosquito species warrant further elucidation in future studies. The effective extracts were tested for their potential to reduce eggs hatching in An. stephensi and An. culicifacies. Interestingly, for An. stephensi, only extracts NIO 701, 706 and 707 impacted egg hatching, while in An. culicifacies, all tested extracts significantly reduced egg hatching; pointing towards selective action of the chemical constituents in the extracts, warranting further elucidation.

Phylogenetic analysis of the extracts tested for larvicidal as well as egg hatching impact, demonstrated that majority of the effective extracts belonged to Bacillus sp. Although, the elucidation of the chemical constituents imparting larvicidal activities needs mechanistic characterization, which is beyond the scope of current study. Interestingly, one extract NIO 276 having larvicidal activity against An. stephensi larvae, belonged to Enterococcus genera.

Importantly, for any larvicidal product to be useful for ground implementation, a detailed characterization with respect to its ecological impact and toxicological profiles have to be assessed thoroughly, along with its chemical characterization. Novel formulations with other currently used insecticides or other potent bio-larvicides should be empirically determined and validated in field settings. The present study provided concrete evidence for the larvicidal activities of bacterial extracts collected from marine bodies of India and warrants further exploration for more bacterial species.

Preparation of the extracts of culture supernatants from marine bacteria

Bacteria were collected from different locations of marine bodies across Indian peninsula, namely-Gujarat, Maharashtra, Karnataka, Goa, Tamil Nadu and Lakshadweep (Kavaratti) (Table 2). The bacterial isolates were collected from marine invertebrates and sediments etc. The isolates were sub-cultured on Zobell Marine Agar till pure colonies were obtained. A loopful of the grown bacterial culture was inoculated in Zobell Marine Broth and incubated at 35°C and 100rpm for 96h. The culture was then centrifuged at 10,000rpm for 15min to obtain the supernatant and pellet. The chemical constituents from the supernatant were extracted thrice with Ethyl acetate (A.R Grade). The solvent fractions were pooled together and concentrated on a rotary vacuum evaporator to obtain the final crude extract.

Phylogenetic analysis

The genomic DNA of bacterial isolates for 16S rRNA gene sequencing was extracted using Himedia HiPurA^® Bacterial Genomic DNA Purification Kit. Universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used. Polymerase Chain Reaction (PCR) was carried out in Takara Thermal cycler Dice TP600 Gradient PCR. The reaction mixture consisted of 12.5µl of Promega GoTaq Green Master Mix (2×), 1µl of forward primer, 1µl of reverse primer, 1µl of 100ng DNA template, 9.5µl of Nuclease free water, making a total reaction volume of 25µl. The PCR conditions were as follows: Denaturation at 95°C for 3min, followed by 95°C for 1min; annealing at 52°C for 30s; extension at 72°C for 2min (30 cycles) and final extension at 72°C for 10min. The amplified products were cleaned using Promega Wizard^® SV Gel and PCR Clean-Up System. The PCR purified products/samples were sent to Barcode Biosciences (Bangalore, India) for sequencing. The forward and reverse sequence of 16s rRNA were trimmed manually by using Bioedit Sequence Alignment Editor Software (Freeware, CA, USA)²⁷. Basic Local Alignment Search Tool [BLAST] from National Centre for Biotechnology Information (NCBI) was used to find the identities of the isolates. A phylogenetic tree was constructed by MEGA 11 software²⁸ by neighbour joining method using 1000 bootstrap replicates.

Mosquito rearing

The mosquito strains used in this study were reared and maintained at the insectariums of the ICMR-National Institute of Malaria Research, New Delhi, India. The mosquito strains for the semi-field study were collected from Mewat, Haryana, India. The larvae were maintained at a temperature of 27 ± 2°C and with a relative humidity of 78 ± 2%. The larvae were fed with a mixture of dog food (Pet Lovers Crunch Veg) and fish food (Tetra Bits Complete fish food). The larvae were fed and reared under the above-mentioned conditions until they turned stage three instars (L3) and were taken for screening as per WHO guidelines²⁹ and previously reported studies^30,31.

Dose-dependent larval bioassay

The larvicidal activity of the extracts were assessed by exposing L3 larvae of the Anopheles stephensi to the marine bacterial culture supernatant extracts. The bioassays were conducted at 27 ± 3°C, 80 ± 3% relative humidity (RH), with six replicates per extract, including a positive control (temephos) and negative control (water). The % mortality were counted at the concentrations of 250ppm and 125ppm of extracts^32,33. Selected hits from the preliminary screening were taken for further dose-dependent bioassays with lower concentrations starting from 125 to 7.5ppm. LC₅₀ and fiducial limits of hits were calculated according to the PROBIT statistical analysis.

Egg hatchability

Selected extracts (NIO 97, 124, 276, 701, 706, 707, 710) at 500ppm were added to the water in a bowl with 100 eggs of An. stephensi. The number of eggs were counted at the beginning and at the end of the experiment, number of live larvae in treated and control groups were counted. The percent egg hatchability was calculated as number of live larvae divided by total number of eggs exposed^33,34.

In vitro P. falciparum growth inhibition assay

P. falciparum strain 3D7 was cultured in fresh A + human erythrocytes at 5% hematocrit with 10.4g/l RPMI 1640 (Gibco), pH 7.2, 0.5% AlbuMAX II (Gibco), 5% sodium bicarbonate (Sigma) supplemented with 50mg/ml gentamycin and 50mg/l hypoxanthine and incubated at 5% CO₂ and 37°C. For general maintenance of culture, parasites were grown in a non-synchronous manner with parasitemia between 0.2 to 10% and monitored regularly. P. falciparum 3D7 cultures were synchronized using 5% sorbitol and parasitemia was assessed. Parasites with synchronized ring stages at 0.5% parasitemia were then added to 96-well plates with 5% hematocrit. Different concentrations of the extracts (50 µg/mL and 1 µg/mL) were incubated with the culture, as mentioned above for 24h. Parasite growth was assessed by reading thin blood smears made by fixing in 100% methanol and stained for 20–30min in 10% Giemsa stain solution. Chloroquine and Methanol were used as negative and positive controls, respectively. Experiment was performed in duplicates with two different biological replicates^35,36,37.

Authors and Affiliations

1. ICMR-National Institute of Malaria Research, New Delhi, India

   Cherish Prashar,Vandana Vandana,Madhavinadha P. Kona,Om P. Singh,Ram Das,Kapil Vashisht&Kailash C. Pandey

2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

   Cherish Prashar,Heena Devkar,Ram Das&Kailash C. Pandey

3. CSIR-National Institute of Oceanography, Dona Paula, Goa, India

   Heena Devkar&Narsinh Thakur

4. HeteroChem InnoTech, Hansraj College, University of Delhi, New Delhi, India

   Kapil Vashisht

Contributions

Conceived the study-K.C.P., K.V., N.T. Data generation, interpretation-C.P., H.D., V.V., M.P.K., K.V. Manuscript writing and review-C.P., K.V., O.P.S., N.T., K.C.P., R.D.

Corresponding authors

Correspondence to Kapil Vashisht, Narsinh Thakur or Kailash C. Pandey.

Competing interests

The authors declare no competing interests.

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