Scanning electron microscopy of damage caused by Mesocyclops thermocyclopoides ( Copepoda : Cyclopoidea ) on larvae of the Dengue fever vector Aedes aegypti ( Diptera : Culicidae )

Dengue fever is a mosquito-borne viral disease, whose main biological vector is Aedes aegypti. This mosquito colonizes tropical areas where the disease is endemic. The most obvious action against dengue is attacking its vector. Biological control appears to be an alternative approach, using natural enemies of the mosquitoes, such as predatory copepods. Thus, the morphological study of the damage caused by copepods is important to understand its predatory capacity. Twenty-five A. aegypti larvae were exposed to the copepod Mesocyclops thermocyclopoides and the damage caused by the copepods was evaluated using scanning electron microscopy. The larvae showed damage mainly at the anal segment, the siphon and the abdomen; only three attacks to the head were observed. The size of the siphon might be of importance in determining whether or not a copepod will attack a mosquito larva. Rev. Biol. Trop. 54 (3): 843-846. Epub 2006 Sept. 29.

Dengue and dengue hemorrhagic fever are mosquito-borne viral diseases, which are endemic in more than 100 countries in the tropical and sub-tropical territories of the world, causing an estimated 100 million cases annually (Gubler 1998, Keating 2001).Since there are neither antiviral drugs available to eliminate this virus nor an effective vaccine, the logical approach against these diseases is controlling the mosquito vector (Gubler 1998).During the 1950s, chemicals such as DDT were the main weapons used in this battle (Breakley et al. 1984), and whose apparent success was evident 30 years ago with the eradication of Aedes aegypti from 18 countries of the American continent (Kuno 1995, Guzman andKouri 2002).At the present time, the mosquito has once again colonized these territories and dengue has re-emerged as an endemic disease.There are many causes that explain the re-emergence of dengue, such as the natural selection of strains resistant to insecticides, as well as global changes, such as an increase in international flight, the migration of people from rural to urban areas and uncontrolled re-urbanization (Guzman and Kouri 2002).The peak of the plastics industry around the world could be another factor associated with the re-emergence of dengue, because of the huge quantity of discarded plastic containers that provide an infinite number of artificial breeding grounds for the mosquitoes.
Biological control, using the natural enemies of Aedes, appears to be an alternative approach to the systematic failure of use of insecticides (Lardeux et al. 2002).There is a vast array of agents used in the biological control of mosquitoes, including copepods.These agents are microcrustacea, present in fresh water worldwide.Mesocyclops is one of the genus of copepods that has most been studied as an antagonist of mosquito larvae and whose effectiveness has been demonstrated in different countries, including the United States (Marten 1990), Honduras (Marten et al. 1994), vietnam (Nam et al. 1998), and the French Polynesia (Lardeux et al. 2002).
Mesocyclops thermocyclopoides is a very common species in Costa Rica (Collado et al. 1984, Hernández-Chavarría andSchaper 2000) and was evaluated as a biological control agent against Aedes.This copepod feeds on the 1 st and 2 nd instars of the mosquito larvae, fatally wounding about seven individuals per day (Shaper and Hernández 1998).Although there has been considerable work done on the effects of this and other species of copepods on the populations of A. aegypti and A. albopictus (Brown et al. 1991, 1996, Marten et al. 1994, Tietze et al. 1994, Schaper and Hernández 1998, Dieng et al. 2002), more detailed studies of the effects on the individual seem to be necessary.So far, little is known about the morphological damage caused by the copepod.It is assumed that an evaluation of the injury caused by the copepod might help to explain why some species of mosquitoes are attacked (such as Anopheles and Aedes spp.) and others, such as Culex spp., are not.
Over the span of three days, five A. aegypti larvae at the 1 st instar stage were placed in plastic capsules with 0.5 ml of water and one adult female M. thermocyclopoides.After 5 h of incubation at room temperature, the organisms inside the capsule were fixed with 0.1 ml of 25 % gluteraldehyde for at least 2 h at 4 °C.Then the samples were washed in 0.1 mM phosphate buffer three times and post fixed in 1 % OsO 4 for 1 h, washed again and dehydrated in ethanol (30 to 100 %), dried in a critical point dryer, mounted on aluminum studs, covered with gold and analyzed under a Hitachi S-570 scanning electron microscope.
Adult copepods (M.thermocyclopoides) and the 1 st instars of A. Aegypti are almost the same size, as is shown in Fig. 1.The majority (76 %) of the larvae attacked showed damage on the anal segment, the siphon and the last abdominal segments (19 of 25 cases) as is shown in Fig. 1 and 2. In two cases (8 %), the thorax was also eaten, so that only the head capsule remained (Fig. 1).In three (12 %) cases an attack to the head was observed (Fig. 3).Three (12 %) individuals showed lateral bites on the thorax and abdomen.In the 1 st instars larvae, the siphon is not strongly sclerotized as in the later instars.Apart from the smaller size, this might make the 1 st instars more susceptible to copepod attacks.
If the copepod bites the larvae at the anal segment and the siphon, the consequences are obviously fatal for A. aegypti because the siphon is essential for breathing.Also, the lateral damage detected on the larvae could be the result of primary attacks, as several times the copepod was observed to strike the larva several times to immobilize it before starting to feed on it.
The small size of the siphon of a mosquito larva might be important to predict if a copepod can attack the larva or not. A. aegypti is susceptible to copepod predation but long siphoned species like Culex quinquefasciatus suffer only insignificant attacks by M. thermocyclopoides.Anopheles albimanus, which does not have a siphon at all, is also susceptible to copepod predation (S.Schaper, pers.obs.).
As Nam et al. (1998) demonstrated in vietnam, a combination of both biological control using copepods and active community participation proved the most effective intervention to eradicate Aedes from an infested territory.An environmental education campaign raised awareness, while recycling programs were established to recycle materials such as used tires, glass bottles, aluminum cans and some plastic recipients.In the experience of Nam et al. (1998), copepods were used for biological control, but any isolated intervention would have been effective to eradicate this vector.Nonetheless, it has been determined that an integrated use of all strategies is best in the battle against dengue.A low cost and highly efficient strategy to control Aedes and Dengue should include four elements: 1) A combined vertical and horizontal approach that depends on community understanding, 2) Prioritized control according to the larval productivity of major habitat types, 3) Use of copepods (Mesocyclops) as biological control agent, 4) Community activities of health volunteers in schools and for the public.As a result of such a strategy, Aedes was eradicated from 32 of 37 communities (population 309 730) where no dengue cases have been detected since 2002 (F.H.-C., unp.data).

ACKNOwLEDGMENTS
The authors are grateful to Jorge D. García and Belinda Dick for their suggestions and critical review of the manuscript.Thanks to Leda Muñoz for her support.This study was funded by the vicerrectoría de Acción Social, vicerrectoría de Investigación, Universidad de Costa Rica, and PNUD.