|Ahead of print publication
Laboratory evaluation of spinosad as a potential larvicide against immature forms of Aedes aegypti
Nikhil Sisodiya1, Rina Tilak2, Anmol Sharma3, Arti Sarin4
1 Medical Officer, Military Hospital Pithoragarh, Uttarakhand, India
2 Department of Community Medicine, Armed Forces Medical College, Pune, Maharashtra, India
3 Medical Officer, Eastern Naval Command, Visakhapatnam, Andhra Pradesh, India
4 Commanding Officer, INHS Asvini, Mumbai, India
|Date of Submission||30-Aug-2020|
|Date of Decision||07-Oct-2020|
|Date of Acceptance||05-Feb-2021|
|Date of Web Publication||10-May-2021|
Medical Officer, INS Circars, Naval Base, Visakhapatnam, 530 014, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Background and Objectives: Aedes mosquito has been recognized as a global emerging threat with its potential to transmit fatal diseases of international public health importance such as dengue, chikungunya, and yellow fever. Prior attempts to manage the vector with various synthetic larvicides have resulted in emergence of resistance, thus necessitating search for a safer and effective alternative. The study was an experimental laboratory-based study to screen the recent World Health Organization (WHO)-approved insecticide spinosad for larvicidal activity and compare the efficacy of the same with other currently used larvicides, namely temephos and Bacillus thuringiensis var. israelensis (Bti). Methodology: An experimental setup was done as per the WHO Pesticide Evaluation Scheme to assess the larvicidal activity of the spinosad at varying concentrations along with a positive and negative control. A preliminary screening to assess the larvicidal property of the spinosad was undertaken with 0.5 ppm concentration as recommended by the WHO. The mortality was checked after 24 h and results were statistically analyzed and LC50 and LC90 values were calculated. Results: Spinosad brought about 100% larval mortality at the recommended dosage (0.5 ppm) as well as at a lower dosage of 0.1 ppm. The comparison with the other two commonly used larvicides, namely temephos and Bti, reveals 100% and 90% mortality, respectively, in wild Aedes larvae. Conclusion: The study concludes that spinosad is a promising larvicide that can be used in rotation with temephos against Aedes aegypti in potable water and may be used at a lower dosage of 0.1 ppm. However, large-scale field trials are required to ascertain the effectiveness of the larvicide in field conditions.
Keywords: Aedes aegypti, dengue, spinosad
| Introduction|| |
Dengue is a globally recognized rapidly advancing vector-borne disease. 2.5 billion people or roughly one-third of the world's population is estimated to be living in dengue-endemic areas, hence it poses a major challenge to public health, especially in populated countries of South-East Asia. Annually over 50 million cases of dengue fever are recorded in Asian countries, with a case fatality rate of 3%–5%. The sharp increase in global prevalence of dengue has been facilitated by increased air travel, global urbanization, and population growth facilitating the creation of ideal breeding grounds which, when coupled with insufficient mosquito control programs, poses as an ideal pandemic recipe. Dengue fever is caused by the four serotypes transmitted by bites of female Aedes aegypti and Aedes albopictus mosquito species (day biters).
In the absence of vaccines and preventive drugs, controlling vector breeding is an effective method to control viral transmission. Chemical and biological agents thus remain the most significant components of dengue vector control programs, like Bacillus thuringiensis var. israelensis (Bti), a Gram-positive, spore-forming entomopathogenic bacterium, and temephos, an organophosphorus compound which is currently the gold standard larvicide against A. aegypti. However, with their widespread use, resistance has set in which has led to the shift of the World Health Organization (WHO) priority toward the advancement of new and effective biological or biorational larvicides.
Spinosad qualifies as a novel candidate biorational larvicide which is produced during the fermentation of actinomycete (Saccharopolyspora spinosa). The molecules are neurotoxic to a restricted range of insects, particularly Diptera, Lepidoptera, and Thysanoptera by causing excitation of postsynaptic nicotinic, acetylcholine, and GABA receptors leading to paralysis and death.[7 The action results primarily after ingestion, as well as by contact with the active form. Spinosad is classified as a Group 5 insecticide by the Insecticide Resistance Action Committee.,, The unique mode of action ensures no cross-resistance with other chemicals. Spinosad is considered to be one of the most selective products available for the conservation of insect predators in agriculture as it provides the right balance of efficacy with environmental stewardship owing to its selective mortality to specific genus.
Limited studies have been conducted to assess the efficacy or effectiveness of spinosad as a larvicide targeted at control of Aedes, especially in this part of the world along with estimation of dose–response relationship to ascertain effective dose of larvicide. The development of resistance among mosquitoes to currently used larvicides temephos and Bti has necessitated the evaluation of alternative larvicides for the control of mosquitoes.,,, The study was undertaken to evaluate the effectiveness of this novel larvicide under Indian settings at various dosages and to establish a dose–response relationship by estimating larval mortality. Large-scale field evaluations of the same at further decreasing dosages are to be undertaken to conclusively determine its effectiveness and the optimum dosage under Indian settings.
| Methodology|| |
A laboratory evaluation was carried out in the entomology laboratory of a government medical college over a period of 8 months (July 2016–February 2017) as per the WHO Pesticide Evaluation Scheme (WHOPES) Protocol (WHO/CDS/WHOPES/GCDPP/2005-13). Institutional ethical committee clearance was obtained prior to the commencement of the study.
Rearing of Aedes aegypti in laboratory
Bioassay of the spinosad was tested vis-à-vis Bti and temephos against the third larval instar (immature) of wild and laboratory-bred A. aegypti. The wild larvae were collected using a larval pipette and 0.05 mm mesh size sieve from their breeding sites and were transferred to the laboratory. Both laboratory-bred and wild larvae were segregated into separate batches and were stored in ceramic bowls containing dechlorinated tap water and were fed yeast tablets. All active Aedes third instar larvae were included in the study whereas those with no or sluggish movement prior to the experiment were excluded. Bioassay was performed at a temperature of 27°C–32°C and relative humidity of 60%–80%.
Investigation of larvicidal potential
The test chemicals spinosad direct application, Bti (12AS), and temephos 50% EC were procured from open market and were diluted to the required concentrations. Twenty-five third instar larvae of laboratory-reared and wild A. aegypti each were placed in test containers (plastic cups) with holding capacity of 200 ml of dechlorinated tap water. A total of 100 third instar A. aegypti larvae (4 replicates) were exposed to the respective chemicals initially at the recommended dosages. Concurrent positive control with temephos/Bti at the recommended concentration of 1 ppm and negative controls with normal tap water were set up. A total of 50 larvae (2 replicates) of negative and positive controls were set up for each treatment. Post initial testing with spinosad at the recommended dosage of 0.5 ppm, fresh lots of mosquito larvae (wild and laboratory-reared separately) were exposed to four decreasing concentrations, i.e., 0.25, 0.1, 0.05, and 0.025 ppm with similar laboratory setup and procedure. All the experiments were repeated three times. Larval mortality was monitored after 24 h by checking for active and passive movements of the larvae. The observations were focused on the wriggling speed of larvae, aggregation behavior at corners horizontal and vertical movements, and larval knockdown as a marker of treatment effect on larvae.
The tests which recorded 20% mortality in control assays and over 20% pupae formed were rejected and reconducted. The control mortality that ranged between 5% and 20% was corrected by Abbott's formula. One-way analysis of variance was carried out to determine the significant difference in the insecticidal property of spinosad and the positive controls temephos and Bti.
| Results|| |
The potential larvicidal property of spinosad was investigated under laboratory conditions against larval forms of A. aegypti. The preliminary comparison was done with temephos and Bti at 1 ppm concentration against wild and laboratory-bred larvae, respectively. The result recorded a lesser mortality percent of Bti as a larvicide vis-à-vis temephos (positive control) which recorded 100% mortality, as shown in [Table 1]. Spinosad recorded a consistent 100% mortality in both wild and laboratory-bred larvae. The lowest concentration with 100% mortality in case of spinosad was noted at 0.01 ppm for laboratory-bred and at 0.05 ppm for wild Aedes larvae, respectively, as reflected in [Table 2]. No alteration of behavior was noticed during immediate exposure of the larvae to spinosad and also no instant or rapid mortality was noted. The larval mortality with spinosad at 0.1 ppm reflected a significant mortality equivalent to temephos. LC50 value for wild larvae was calculated to be 0.0557. Since 100 % mortality was recorded at the lowest concentration of 0.05ppm, thus LC 50/90 values could not be calculated for laboratory bred larvae.
|Table 1: Percentage mortality of Aedes aegypti larvae with Bacillus thuringiensis var. israelensis and temephos (positive controls)|
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|Table 2: Concentration-mortality relationship of wild and laboratory-bred Aedes aegypti larvae exposed to spinosad direct application|
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| Discussion|| |
Controlling mosquito-borne illnesses is an emergent health issue. Aedes mosquito is known to transmit multiple diseases such as dengue, chikungunya, Zika, and yellow fever (in endemic countries), and it is a glaring health issue. Various biochemical, phytochemical as well as synthetic chemicals have been studied over the time in search of a perfect larvicide. The use of chemical larvicides in the past now has proven to be detrimental majorly due to increasing resistance, environmental toxicity, and economic burdens posing as the key factors negating their exploitation for continuing their use in the future. Studies conducted across various parts of India reported moderate-to-severe resistance to majority of insecticides including temephos among field-caught A. aegypti, with metabolic detoxification being the underlying phenomenon.,,,
Results found by us were similar to those documented earlier about the larvicidal effect of Bti on Aedes and Culex species., Temephos currently is the gold standard, owing to its low-cost, high-efficacy, and low-mammalian toxicity, and is used for treating standing surface, water in tanks, and other natural or man-made containers in developing countries including India.
Spinosad is a relatively new insecticide and has not been studied extensively in the Indian subcontinent. Laboratory analysis conducted in West to test its larvicidal properties using 0.05–0.5 ppm demonstrated over 95% control of Culex spp. larvae for a period of 7–35 days., Spinosad acts like a contact poison and affects the nervous system causing paralysis and death. Natular™, a larvicide product, being marketed in the USA and Mexico uses spinosad at concentrations up to 1.6 ppm for community control of mosquito-borne illnesses. Evaluation done by Kovendan et al. reflected a good potency of spinosad against all forms of A. aegypti with 31% mortality among the first larval instar with spinosad at 20 ppm, and the mortality increased to 87% at 100 ppm. Pupal mortality was found to be 12% at 20 ppm which increased to 64% at 100 ppm.
A study by Darriet et al. concluded lethality of spinosad against Aedes larvae with LC50 recorded as 0.35.
Nontarget effects with spinosad have been reported to be lower as compared to other marketed chemical and biological larvicides. Spinosad is, however, toxic to aquatic invertebrates such as Plecoptera, Ephemeroptera, and Odonata order.,, The WHOPES in the year 2013 approved a granular formulation, a tablet, and an emulsifiable concentrate of spinosad for field testing. The recommended dose for field application is 0.1 ppm, however, the present study found spinosad effective at 0.05 ppm with 100% larval mortality. Further studies and large-scale field evaluations need to be conducted to conclusively arrive at the effectiveness of this dosage against Aedes.
| Conclusion|| |
The result reveals a noteworthy potential of spinosad as a prospective control and management agent of A. aegypti. Its mode of action with its low toxicity and cost poses an effective, safe, and cost-effective larvicide and is found efficacious against A. aegypti. The authors recommend that combinations of various larvicides can be tested for augmentation of the effects of insecticides and minimization of environmental toxicity which may also delay the development of resistance. Spinosad can be used in rotation with temephos against A. aegypti in potable water and may be used at a lower dosage of 0.1 ppm.
It is crucial that further studies and field trials (Phase 2 and 3) in various ecological regions be conducted to establish spinosad's effectiveness across geographical variation, different mosquito populations, and species.
We would like to thank the Department of Community Medicine, AFMC, Pune.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]