Assessment of geraniol-incorporated polymers to control Aedes albopictus (Diptera: culicidae)

Effective control of mosquito borne diseases has proven extremely difficult with both vector and pathogen remaining entrenched and expanding in many disease endemic areas. When lacking an effective vaccine, vector control methods targeting both larval habitats and adult mosquito populations remain the primary strategy for reducing risk. Aedes albopictus from Thailand was used as a reference baseline for evaluation of natural insecticides incorporated in polymer disks and pellets and tested both in laboratory and field conditions. In laboratory and field tests, the highest larval mortality was obtained with disks or pellets containing IKHC (Insect Killer Highly Concentrate) from Fulltec AG Company. This product is reputed to contain geraniol as an active ingredient. With pellets, high mortality of Ae. albopictus larvae (92%) was observed in presence of 1 g of pellets per 500 ml of water at day 1st, and the mortality was 100% at day 1st for larvae in presence of 5 or 10 g of pellets. Fulltec AG Company has not accepted to give us the exact composition of their IKHC product. Therefore, we cannot recommend it, but the principle of using monoterpenes like geraniol, incorporated into polymer disks or pellets as natural larvicide needs more attention as it could be considered as a powerful alternative in mosquito vector control.

S ince the implementation of the European directive 98/8/EC, known as the "biocide directive", most of the old effective, inexpensive but polluting biocides used for vector control (Rozendaal, 1997) have been withdrawn from the European market. Now, assessment of unwanted side-effects is compulsory for any active biocide substance and, eventually, for any formulated biocide product that contains it, prior to acquiring marketing authorization. Considerations of cost, acceptability, safety and, more recently, respect for the environment, have led companies, research institutes and international agencies to offer new compounds such as insecticides of biological origin, like Bacillus thuringiensis ser. israelensis (Bti), Bacillus sphaericus, Spinosad and growth regulators mimicking insect hormones (Fontenille et al., 2009). Certain active pyrethroid substances show the advantage of being active at low doses and having low persistence (no residual effect), but have low selectivity. Some trials are also under way on natural substances such as essential oils or plant extracts, like geraniol (Khallaayoune et al., 2009;George et al., 2009) or other monoterpenes. Most compounds used for vector control are implemented by trained specialized teams, but after the epidemics of chikungunya in La Reunion Island (2005Island ( -2006 and dengue in Martinique and Guadeloupe, personal protection against vectors (PPAV) revealed to be also very important (PPAV Working Group, 2011). The main dengue and chikungunya vectors like Aedes aegypti, Ae. albopictus (Guzman et al., 2010) and also nuisance caused by Culex quinquefasciatus, could be partly controlled by mechanical elimination of breeding sites around houses and in gardens, and/or by larvicide treatment and spatial spraying with insecticides. To implement a larvicide treatment by people, some companies are developing monoterpenes-incorporated polymer disks or pellets to be used in gardens or terraces. The efficiency of some of these products has been assessed in Thailand at the Entomology Research Laboratory of Kasetsart University, Bangkok, in laboratory conditions where mosquito colonies are maintained, as well as in field conditions where Ae. albopictus is prevalent.

AEDES ALBOPICTUS COLONY
A e. albopictus MOPH colony (Thailand) was used as a reference baseline for these tests. This colony was originally collected from Nonthaburi Province and has been maintained in a colony at Vector Borne Disease Bureau, Disease Control Deparment, Ministry of Public Health (MOPH, Thailand) for approximately ten years (Chuaycharoensuk et al., 2011). Mosquito colony was carefully segregated to prevent contamination from cross-mating and reared under identical laboratory-controlled condition at 25 ± 5 °C and relative humidity 80 ± 10 % in the Department of Entomology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand (Kongmee et al., 2004). Adult mosquitoes were allowed to mate and females permitted to blood feed on restrained live guinea-pigs beginning on the fourth day postemergence. Moistened oviposition substrates were provided to the females for depositing their eggs. Following conditioning, eggs were re-hydrated and allowed to hatch in pans reserved for each strain. In the present experiment, only stage 3 mosquito larvae were available in sufficient numbers at the time of the test and used.

GERANIOL INCORPORATED DISKS AND PELLETS
Polymer disks and pellets were sent to us under sealed aluminium bags by Valcoplast SA (4, Avenue Bordaberri, 64990 Mouguerre, France) without any indication on their composition for evaluation in blind conditions. The composition was revealed after the tests were done. The disks were made of polymer with incorporation of different products (Table I). IKHC was presented by Fulltec AG (Zug, Switzerland) as a watery concentrate with geraniol (CAS 106-24-1) from natural origin as an active ingredient. Geraniol is extracted by chromatography from palmarosa essential oil (Java Lemongrass, Cymbopogon martinii). The final concentration of the products incorporated in the polymer disks was around 10 %. The pellets of 0.5 cm of diameter were made of a mixture of polymer and wood fibers impregnated with IKHC.

IN THE LABORATORY
S tage 3 mosquito larvae were tested in plastic saucers. In the Experiment A, 50 larvae, 500 ml of filtered water, two tablets of fish food and one polymer disk were added in each saucer. In this experiment, three replicates were done for each of the polymer disk lot. The number of dead larvae was counted every day during one week (day 6 for practical reasons). Afterwards, the polymer disks were kept dry at room temperature and experiment was repeated after one and two months to evaluate the remanence of the effect. A similar experiment (Experiment B) was performed at the same time with polymer disks replaced by IKHC incorporated pellets. One, two or five grams of pellets were added to each saucer containing 500 ml of filtered water. As in Experiment A, 50 stage 3 larvae and two tablets of fish food were added in each saucer. A saucer without pellets was incorporated as a control. Two replicates were done. The number of dead larvae was counted every day until all larvae were dead in all saucers with pellets.

IN THE FIELD
In this experiment saucers were placed in the garden near the laboratory and protected from rain (Experiment C). Wild populations of Ae. albopictus are abundant in this area. The saucers were filled in with 500 ml of filtered water and two tablets of fish food. There were two replicates for each polymer disk lot. until Day 18 (D18). This experiment was also repeated after one and two months like Experiment A.

RESULTS
F or Experiments A and B, results are expressed as larval reduction rates of stage 3 Ae. albopictus for each of the polymer disk or pellet lots, with indication of standard deviation. For Experiment C, results are expressed as presence and abundance of larvae after exposure of the saucers in the garden.
In Experiment A (Table II), a very low larval mortality was noticed in lot 1903/1 (control) until D6. A very low mortality was also observed for lots 1903/2 and /3 with 2.7 % and 4.7 % respectively at D6. On the opposite, a strong mortality was observed with lots 1903/4, /5 and "Juillet2010" with 99 %, 94 % and 100 % respectively at D3 and 100 % afterwards. For lot 1903/6, the mortality was low until D3 and increased dramatically onwards to 50 % at D4 and 91 % at D6.
In Experiment B (Table III), there was a very low mortality (2 % at D3) in the control saucer (without any pellet), but a very high mortality in presence of pellets. With 1 g of pellets in the saucers, the larval mortality was 92 % at D1, 98 % at D2 and 100 % at D3. With 5 and 10 g of pellets, the mortality was 100 % as soon as D1. One and two months later (Tables III bis, III ter), the mortality stays quite high with 84 % and 83 % at D1 for 1g pellets, and 100 % at D1 for both 5 g and 10 g pellets.

DISCUSSION AND CONCLUSION
P olymer disks or pellets, impregnated with different geraniol based products, were tested in laboratory and in open field conditions. The main objective of this study was to check if those products could be used on an individual basis to control Ae. albopictus or other anthropophilic mosquito populations in gardens or terraces.
The Ae. albopictus MOPH colony of the Department of Entomology (Faculty of Agriculture, Kasetsart University, Bangkok, Thailand) was used for laboratory tests where stage 3 larval reduction rates were calculated using different polymer disks or pellets in saucers.
For all tests, the mortality in the controls was very low, thus indicating that colonized mosquitoes used in this study were healthy at time of testing.
With the polymer disks in saucers in presence of 50 stage 3 larvae, we observed a strong mortality with disks impregnated with IKHC. This mortality was maximum with disks lot named "Juillet2010" impregnated with IKHC only, reaching 100 % from D3 onwards. The mortality decreases when IKHC is diluted with different synthetic geraniol. With pure synthetic geraniol (lots 1903/2 and /3), we observed a very low mortality. That means that the geraniol alone is probably not the active larvicide ingredient of IKHC. This test has been repeated after one and two months, using the same disks kept dry at room temperature. The larval reduction rate decreased dramatically from the first month, even with lot "Juillet2010" where the mortality dropped from 100 % to 19 % on day 6. That shows that the disks could not be used several times. Research note Parasite, 2012, 19, 427-432 However, the duration of the effect during the first test (Table II) has not been evaluated beyond six days.
The efficiency of the IKHC is confirmed in experiment B using pellets. The mortality reached 100 % with 1 g of pellets in 500 ml of water at D3 (Table III) and this efficiency was conserved with pellets kept at room temperature in their original bag after one and two months (Tables III bis and III   tests respectively (Tables IV, IV bis and IV ter). In the saucers with IKHC impregnated disks, no larva was observed until the end of the test at D18 in all 3 tests for lot "Juillet2010" (Tables IV, IV bis and IV ter). There are two possibilities for this absence of larvae during the experiment; either the product is a repellent for females Ae. albopictus avoiding egg-laying in those saucers, or the product is also ovicide and all eggs have been killed before the emergence of larvae. These two hypotheses need now to be checked. Finally, these polymer disks or pellets impregnated with natural products with insecticide activity could be an efficient tool to control mosquito populations in gardens, terraces or greenhouses. The pellets made of polymer and wood fibers seem especially practical for an individual usage. Our results showed that IKHC presents a very strong larvicide activity and, on the contrary, synthetic geraniol has no larvicide activity. As we do not know the active ingredient(s) of this product because the manufacturer refused to give us the complete and exact composition of the product, we therefore cannot recommend it for the moment. The way is opened to discover a natural product, probably a mixture of monoterpenes with larvicide activity which could be used with those slow release polymers or wood fiber systems. The advantage of using natural product against mosquito vectors is that they do not develop resistance and they are non-toxic to fishes, mammals and most other non-target organisms in the environment. As larvicide in mosquito control, insect growth regulator, biopesticides (Rozendaal, 1997), essential oil and natural product like mixture of monoterpenes (Ben Marzoug et al., 2011;Guerrini et al., 2011;Mkaddem et al., 2009;Sacchetti et al., 2004) represent alternative products for mosquito vector control.