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02 May 1017 - Case studies
Worldwide, the appearance and proliferation of invasive mosquito species and the threat of mosquito-borne diseases are being observed. Global warming indicates that we are at serious risk of the appearance of a range of invasive species not only in Europe but also in Latin America. In recent years, the rapid south-to-north spread of the tiger mosquito (Aedes albopictus) has been noticed. The spread of the Zika virus is causing concern in many countries, and significant efforts and financial resources have been allocated to find solutions to stop its spread.
Insect Growth Regulators (IGRs) are the most important larvicides with strong potential to suppress mosquito larvae infestations. It is widely recognized that some IGR-based formulations are suitable for pre-emergent applications, which allow for timely control.
Methoprene is an IGR active against a variety of insect species, including mosquitoes. The EPA (Environmental Protection Agency – USA) has classified Methoprene as a biochemical or biorational pesticide. Today, the use of S-methoprene is more common.
S-methoprene is an insect growth regulator that mimics the natural juvenile hormone (JH) in mosquitoes. Juvenile hormones are responsible for growth during the late larval stages. The amount of juvenile hormone decreases with each larval stage, and during the fourth stage, just before the molt from larva to pupa, the JH level becomes almost zero. The effect of S-methoprene during these sensitive stages produces morphological deformities, abnormalities that lead to death by drowning. S-methoprene has minimal or no control effects on mosquitoes that have already reached the pupal or adult stages. The most susceptible species are Aedes, Anopheles, and Ochlerotatus, while Culex are slightly less sensitive. S-methoprene has favorable toxicological and environmental characteristics.
During the formulation development phases, different mosquito species, habitats, water retention systems, applications, and end users were considered. Several formulations based on S-methoprene were then developed and studied.
Larvicol Tablets contain 0.5% S-methoprene and are typically used against Aedes aegypti, Aedes albopictus, and Culex pipiens. Typical treatment areas include rainwater drainage systems and cisterns in cities, ditches, areas with excessive standing water, flooded areas, etc. The 2.0-gram tablet is suitable for treating 1000-2000 liters of water. The use of tablets is highly recommended, especially where Aedes albopictus is present in smaller water retention systems.
Studies with IGRs in general require a different approach compared to studies conducted with traditional larvicides. Instead of lethal effects, the inhibition of adult emergence is observed. IGRs have no direct lethal effect. By applying S-methoprene, the increase in hormone levels blocks the development of insects. Mortality occurs due to suffocation and drowning.
Basic efficacy studies on Aedes aegypti and Culex pipiens species were conducted at the Babolna Bio laboratory. Some of these studies on the same species and at similar concentrations of S-methoprene were repeated at the independent T.E.C. Laboratory in France. Studies on Aedes albopictus were carried out at the T.E.C. Laboratory.
120 hours before the test, 17 containers are filled with tap water. The water temperature is allowed to stabilize with the ambient temperature, and chlorine evaporates. The ambient temperature is 23-26ºC, with relative humidity at 40-50%. Natural light conditions are maintained. To minimize evaporation and algae presence, the containers are covered with fine mesh. Granulated rabbit food is added to each container as nutrition.
Working solutions are prepared. Three repetitions were performed for each dose. With a glass pipette, 50 L3-L4 mosquito larvae are selected in each container. The control setup is the same, but instead of larvicides, tap water is used.
Test containers are examined every 24 hours. Dead larvae and, occasionally, dead pupae are removed, and their numbers are recorded. Subsequently, the surviving larvae are collected and placed in a plastic container with 500 ml of clean, non-fresh tap water. The containers are covered with fine mesh. The number of live, flying imaginal stages (which are then killed by cooling) is counted daily. Larvae unable to molt and emerge properly, or those that could not leave the water surface and drowned, are considered dead and grouped together. The rapid or immediate hatching test usually lasts 7-9 days.
The residual tests are the same as those described above. After every 8-10 days, a new batch of L3-L4 larvae is placed in the treated water to demonstrate the impact of the formulation. The process is terminated when each stage in the treated containers and controls is dead, drowned, or incapable of leaving the water surface or emerging as a live adult.
The following formula is used to determine the percentage of inhibition of emergence:
Adult Development Inhibition (%) = 100 – (T/C x 100)
Where:
T = % development in treated samples
C = % development in control group
The efficacy of a larvicide is considered acceptable if, at the end of the test, emergence inhibition remains above 90%. In general, tests are terminated when the inhibition falls below 70%. According to WHO guidelines, if the adult emergence in the control group is below 80%, the test should be discarded. If the inhibition percentage is between 80 and 95%, the data should be corrected using the Abbott formula. The result obtained indicates the inhibition of emergence as a percentage.
The study was conducted between August 31 and October 4, 2011, with Culex pipiens in the drainage ditch area of the AES Tisza power plant in Tiszaújváros (an area of 1400 m x 2 m). Three plots were treated, and three untreated control areas were maintained. Traps for emerging mosquito larvae were used.
Floating traps were used to retain L3 and L4 larvae. After treatment, the floating traps with larvae are placed in the water. Depending on weather conditions, usually after 7-14 days, the traps are completely emptied into a clean container full of untreated water for further observations. A new group of larvae is introduced into the floating traps to establish the residual capacity of the larvicide. From here onward, the same procedure is repeated as described above.