Bachelor Thesis, 2008
38 Pages, Grade: 1,3
3. Materials and Methods
3.1. Mosquito fauna in the district of Chulucanas
3.1.1. Larvae collection, larval density, biotic & abiotic data
3.1.2. Larvae identification
3.2. Residual effect test of Bti - and Temephos-based larvicides
3.2.1. Residual effect tests in large water volumes
3.2.2. Residual effect tests in small water volumes
3.3. Efficacy test in houses and interview regarding community participation
3.3.1. Efficacy test
4.1. Mosquito fauna in the district of Chulucanas
4.1.1. Breeding site in Chulucanas city, Sector Nr. 20
4.1.2. Breeding site in Chulucanas city, Sector Nr. 4
4.1.3. Breeding site in Yapatera town
4.1.4. Breeding site in Campanas town
4.1.5. Breeding site in Charanal town
4.1.6. Breeding site in Paccha town.
4.2. Residual effect test of Bti - and Temephos-based larvicides
4.2.1. Residual effect tests in large water volumes.
4.2.2. Residual effect tests in small water volumes
4.3. Efficacy test in houses and interview regarding community participation
4.3.1. Efficacy test
5.1. Mosquito fauna in the district of Chulucanas
5.2. Residual effect test of Bti - and Temephos-based larvicides
5.3. Efficacy test in houses and interview regarding community participation
Mosquito (Culicidae) fauna, biological methods to control Ae. aegypti larvae and community participation regarding the present dengue situation and vector control program were evaluated in Chulucanas district, Piura Department, Peru. The study included collection and identification of mosquito larvae in surrounding towns of and in Chulucanas city. Following mosquito species were found: Ae. aegypti, Ae. scapularis, Ae. serratus, An. albimanus, An. pseudopunctipennis, Cx. nigripalpus and Cx. pipiens quinquefasciatus. Two comparative residual effect tests with Bti -based Culinex Tab plus® and Temephos-based Temefar® 1%G were performed in large and small water volumes under laboratory conditions. In the tests with large water volumes, Temefar® 1%G and Culinex Tab plus® showed a residual effect of 9 weeks (100% and 75% mortality, respectively), and, in tests with small water volumes, a residual effect of 7 weeks (100% mortality for both larvicides). Three efficacy tests performed with Culinex Tab plus® at three houses in Chulucanas city under field conditions showed 100% mortality after 24 h of larvicide application. In connection with this tests, an adult person living in each house was interviewed regarding Bti - and Temephos-based larvicides, the present vector control program and community participation. Considering these interviews, a personal testimonial, other statistical social data such as poverty levels, socioeconomic indicators and one survey concerning approval of the present vector control program, it is concluded that lack of knowledge of handling with larvicides and low acceptation regarding present vector control activities may be the main causes for the dengue outbreak in June, 2008.
Whereas cardio- and cerebrovascular diseases (heart attack and stroke) and cancer constitute more than 50% of death cause in developed countries, in developing countries 40% of total death are due to infectious and parasitic diseases (Nentwig, 2005). Of this group, three important mosquito-borne diseases affected and continue in doing so in tropical and subtropical countries worldwide: malaria, yellow fever (YF) and dengue. Yellow fever has been fought successfully since the introduction of an effective vaccine in the 1930s, but thousands of cases are still reported annually -including deaths-, and according to recent studies there is an increasing risk of urban YF in Africa and South America (Barrett and Higgs, 2007). Against malaria prophylactic medicaments are available, such as the effective Malarone™ (Looareesuwan et al., 1999). In case of infection with one Plasmodium spp. -the protozoan parasites causing malaria-, artemisinin-based combination therapies (ACTs) are still effective against the most deadly malaria parasite, P. falciparum. Nevertheless, annually more than 500 million people become severely ill with malaria and 40% of the world population, the majority of them living in the poorest countries, is at risk of malaria (WHO, 2007).
Dengue fever and its severe form, dengue hemorrhagic fever (DHF), is nowadays the most relevant viral mosquito-borne disease worldwide. Every year, 50-100 millions of dengue illnesses, including 250 000-500 000 DHF cases and 24 000 deaths -due to this letal manifestation of dengue- are registered throughout the world (Gibbons and Vaughn, 2002). It is known that the number of dengue cases is under-reported, either since dengue symptoms are often confused with influenza (grippe) or because dengue cases are not reported in many countries (Gibbons and Vaughn, 2002; Kyle and Harris, 2008). Efforts on development of anti-dengue vaccines still continue (WHO, 2008). The existence of four virus serotypes highly virulent to humans and the fact that DHF occurs by second infection with another serotype different of the one in the first infection (Gubler, 1998; Gibbons and Vaughn, 2002), demands a tetravalent vaccine against the four virus serotypes for effective immunisation.
In the absence of an effective vaccine against this viral pathogen, efforts have been focused in vector control, and if possible, vector eradication of this disease: Aedes aegypti mosquito (Family: Culicidae).
In view of the vector emergence in Latin America and the Caribbean, and epidemics in the 1950s, the PAHO (Panamerican Health Organization) executed an eradication plan in the Americas, being successful in most countries, except United States and other Caribbean countries (Monath, 1994). This residual foci, and other circumstantial factors such as social and demographic changes, changes in the public health policies together with the lack or resources for prevention of vector-borne diseases, have promoted increasing of epidemic dengue activity, hyperendemicity (presence of numerous serotypes of dengue virus co-circulating in a location) and, as a consequence of the latter, the emergence of epidemic DHF (Gibbons and Vaughn, 2002).
Since eradication of Ae. aegypti seems, at least in the short- and middle-term, quite improbable, the indispensable tools in order to control dengue are: entomologic surveillance programs together with sustainable development of vector control programs (Gibbons and Vaughn, 2002), either vertical ones in its design and implementation (usually government-led) or horizontal ones (led by the affected community), based primarily on community participation. An ideal model proposes integration of these two types of mosquito control and entomological surveillance, a model relying on the following prerequisites for its successful implementation: entomological studies delivering entomological and ecological data; precise mapping of significant breeding sites; selection of appropriate tools and applications systems for fighting against the mosquito; assessment of the effective dosage to guarantee a cost-effective operation; design of the control strategy; training of the field staff; fulfilling of governmental application requirements for the use of control agents; intensive public relations work to promote public acceptance of the control programme; and community participation (Becker et al., 2003).
Among this requisites, community participation in vector control programs of Ae. aegypti is essential, since the adult flying mosquito is endophilic (flies in the houses) and endophagic (feeds on human blood in the houses) (Kyle and Harris, 2008). Besides, the habitats of its aquatic larval stages are man-made containers for water storage and other discarded recipients which can collect water (e.g. rainwater), objects found inside and around human dwellings (WHO, 2008). Thus, in unplanned urban areas which lack of water pipelines and sanitation, Ae. aegypti populations are extremely benefited to promote dengue epidemics (Monath, 1994). Therefore, active community cooperation is important to facilitate entomological surveillance and fumigation campaigns for vector control in case of dengue outbreaks.
Respectively, selection of appropriate tools implies the election of the suitable mosquito control agent. Recent history of insecticides against mosquitoes was initiated with DTT (1939), continued with the development of organophosphates (e.g. Temephos, used as flea and mosquito larvicide and toxicologically approved by the WHO in 1991), carbamates (created in the early 1950s), photostable pyrethroids (1960-1970s) and the discovery of the larvicidal effects of bacterial spores of Bacillus thuringiensis israelensis (Bti) and its application up to the 1980s (Becker et al., 2003). In safety tests, this biological larvicide has been proven to be harmless for plants, Mammalia, Amphibia, Pisces, Mollusca, Coleoptera and many other representative aquatic organisms (Becker and Margalit, 1993). Its toxicity is restricted to mosquitoes and few nematocerous families of the Order Diptera (Colbo and Undeen, 1980; Miura et al., 1980; Ali, 1981; Garcia et al., 1981; Mulla et al., 1982; WHO/IPCS, 1999). Bti -based larvicides have thus the advantage of not polluting the environment and of securing ecological balance and permanence of biodiversity in the zone of application. The absence of developed mosquito resistance to Bti -based larvicides in mosquito’s species to whom it was applied (Mittal, 2003), and the previous features mentioned above, make Bti -based larvicides and future development of new formulations a long-term promise. At present, Bti products are used in Germany successfully and in combination with other larvicides and insecticides in other European countries (Becker et al., 2003).
As for other insecticides, many chemical insecticides like DDT have been prohibited by many countries due to their negative effect on the environment and non-target organisms. Meanwhile, use of insecticides that protect human health as well as the environment is intended.
Lastly, entomological studies are essential in order to create a mosquito control program. This means collecting data about existent mosquito fauna in order to identify mosquito species having vector capacity and/or nuisance. In addition, data of the ecology of the most important mosquito species: biotic factors such as climate, dry and rainy seasons, floodwater periods; and biotic factors (e.g. natural predators of mosquito larvae in the location).
Considering this three aspects in mosquito control programmes, the research for the present Bachelor thesis was performed in the district of Chulucanas, from the Province Morropón, belonging to the northern Department of Piura, in Peru (See Map 1). As a tropical and subtropical country, Peru is a large habitat for various mosquito vectors responsible for dengue, malaria, yellow fever, other less popular arboviral fevers occurring in the Amazon basin (rarely in the northwest coastal regions) and rainforest region such as Eastern equine encephalitis (EEE), Mayaro, Una (Mayaro-like), Western equine encephalitis (WEE), Venezuelan equine encephalitis (VEE), Ilheus, St. Louis encephalitis (SLE), Oropouche, among others (AFPMB, 1998). Vaccines for these arboviral diseases are not available; the only prevention measures are mosquito control and use of repellents and bednets.
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Map 1: Overview map. The Department of Piura is green-bordered. The red balloon “A” points the city of Chulucanas, located in the Piuran yunga (valley).
The district of Chulucanas (located in latitude 05°05’ south of the equatorial line) has a population of 77749 inhabitants in an area of 871 km² (INEI, 2005) and comprehends the city of Chulucanas (9030 dwellings) and many towns scattered along the district. It is located in a even, valley-like region (known as yunga), crossed by brooks and streams, the Río Piura passing near Chulucanas city (at the bridge “Ñácara”). The main economic activities are agriculture (cultivation of lemon, mango, maize, coconut, American Carob, cotton), cattle breeding (porcine, bovine, caprine and equine cattle) and artisan ceramics (performed by families of the indigenous “Tacllan” clan) (MPMCh, 2008). The temperature range oscillates between 18° C and 38° C throughout the year divided in a dry and a rainy (December-March) season. With regard to socioeconomic aspects, 60% percent of the total population lives in rural and a 40% in urban zones, 40,2% lives in poverty conditions (RdSMCH, 2000), 53,41% lacks of tap water pipelines and 82,20% of sewer pipe systems (RdSMCH, 2002). Only 39,3% of the population finished primary education (Anonymous, 2008).
Due to the favourable climate and socioeconomic conditions mentioned above, the district of Chulucanas (including the city of Chulucanas) and surrounding districts are affected by various arthropod-borne diseases such as malaria, dengue, Bartonellosis, Leishmaniasis and Chagas disease. In the last three years, a total of 1350 dengue cases have been reported, strongly related with increasing Ae. aegypti populations in the rainy seasons (Benavides, 9th June 2008). The last dengue outbreak occurred during the period of this research, with its focus in Chulucanas city, where more than 900 possible dengue cases were reported (Benavides, 9th June 2008).
Considering the three previous aspects concerning mosquito control programs, the present Bachelor thesis has focused on three experiments: Determination of existent mosquito (family: Culicidae) fauna in the district of Chulucanas; comparative residual effect tests of Temephos-based larvicide Temefar® 1% G and the biological Bti -based formulation Culinex Tab plus®; and an efficacy test with the same Bti -formulation in some houses of Chulucanas city, in combination with a brief interview in view of their opinion about the current vector control program, larvicides and community participation.
In this context, the present Bachelor thesis shall be a contribution to the expansion of knowledge regarding mosquito control in this Peruvian region and other tropical countries.
In order to evaluate the mosquito (Family: Culicidae) fauna in Chulucanas, it was aimed to collect and identify the existent mosquito species in its larval stages and determine their corresponding larval densities, as well as collect biotic and abiotic data that helps describing the habitat where they live. For this purpose, search of potential breeding sites was carried out in lagoons, canals, ponds, puddles, swamps, river shores, vats and other recipients in human dwellings, rice field, etc.
Breeding site evaluation set: The standard pint dipper with 350 ml capacity (Dixon and Brust, 1972; Lemenager et al., 1986) and 3 ml plastic pipettes were used for larvae collection, disposable 1 l recipients covered with gauze-like tissue served for transporting samples to the laboratory (located in the health care institution “Red de Salud Morropón-Chulucanas” in Chulucanas city).
Identification set: A field stereomicroscope with a zoom range from 4,8x to 56x, 70% alcohol, sample dishes, clean 3 ml plastic pipettes, featherweight forceps and dissecting needle for larvae manipulation. Identification keys of Lane (1953), Cova García (1966), and Gorham et al. (1973) were used for larvae identification.
3.1.1. Larvae collection, larval density, biotic & abiotic data: Larvae collection was carried out in six different breeding sites, representative of the district of Chulucanas. It took place in the months of May and June 2008. Collected larvae were transported to the laboratory in 1-liter plastic recipients, filled with approx. 200 ml water of the evaluated breeding site and covered with gauze-like tissue. Then, larvae were transferred to small, properly labelled glass recipients with 70% alcohol for its later identification through stereomicroscope.
In order to evaluate larval density, the media of collected larvae per dip was used (pupae were not counted). The media corresponded to 5 dips with the standard pint dipper (filled with water to the 250 ml-mark) performed in the evaluated breeding site, without considering differences in the area of each breeding site since they were no larger than 10 m². Dipping was carried out randomly in the border of the breeding site and considered larvae in the larvae counting were second, third and fourth instar larvae. The larval density index used followed the specifications of Becker (1984 and 1988) for determining larval density:
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The following abiotic parameters of the breeding sites were measured: geographical coordinates and altitude of the breeding site (through a GPS device), air temperature and relative humidity (through an electronic hygrometer), name of location and date. Biotic data is also mentioned, e.g. presence of other noticeable arthropod or vertebrate species and the common name of some plants in the locality. None of this species were identified with identification keys.
3.1.2. Larvae identification: It was performed by means of the identification keys listed in page (see References), using the stereomicroscope. The number of identified larvae for each evaluated breeding site corresponds to the media number of larvae per dip obtained in the larvae collection. Late third and fourth instar larvae were selected, being dead in 70% alcohol at the time of identification.
Of the sampling taken in each breeding site, which contained more larvae than the media larvae number per dip, the media larvae number were identified, taking them randomly of the sampling recipient brought from the breeding site. If the average larvae number per dip was greater than 25, 25 larvae were identified as representative for the greater average larvae number. Then, the corresponding factor was multiplied by the representative number of identified larvae, in order to amplify it to the real, average number of larvae obtained per dip.
Mosquito larvae: Ae. aegypti larvae of late third and, less often, fourth instars were used. The larvae had been collected from the houses in Chulucanas city during entomological surveillance. They had been fed one day with hamster or fish food (Purina and Sera®, respectively). Other larvae used had been cultured at the laboratory. The culture method has been performed by the laboratory technicians since 2001. In this method, mosquito eggs are laid by female adult Ae. aegypti (previously being fed with rabbit blood) located in cages made of Styrofoam and fine mesh to allow mosquito respiration. The egg-filled filter paper stripes are then dried in extended form and stored in a flat box for posterior larvae culturing. The female adult Ae. aegypti had been also collected in the district of Chulucanas.
Larvicides: Bti tablet formulation Culinex Tab plus® (2250 ITU/mg, Valent BioSciences Corp. Libertyville, Illinois – USA; based on Bti serotype H-14) and Temefar® 1% G (1% of active ingredient Temephos, toxicity oral LD50 of active ingredient for male rat: > 1086 mg/kg (kg of body weight), produced in FARMEX S.A., Lima 27 - Peru) were tested. Dosage used: 0,2 g/50 liters of water for Culinex Tab plus® and 12,5 g/50 l for Temefar® 1%G. Culinex Tab plus® tablets were pulverised manually or divided into parts for application. Temefar® 1% G is a granulate formulation and was applied either as loose granulate or enveloped in a gauze bag called in the location as “mina”.
Recipients: For the residual effect test with large water volumes, 15 l plastic buckets (radius on the top: 40 cm, height 30 cm) and 30 l plastic recipients (radius on the top: 55 cm, height 35 cm) were used. For the tests with small water volumes, 1 l plastic recipients were used. In all tests, tap water stored for one day (for chloride evaporation) was used. During the tests all recipients remained covered with gauze-like mesh (for the buckets and 30 l recipients) and gauze-like tissue (for 1 l recipients).
3.2.1. Residual effect tests in large water volumes: Two tests were performed. In the first test, three 15 l plastic buckets were filled with 12,5 l tap water. In one bucket the recommended dosage mentioned above (Becker, pers. comm.) of pulverised Culinex Tab plus® was applied: 0,05 g. In the second bucket, 3,125 g of Temefar® 1% G was applied as loose granules. The third bucket served as control and no larvicide was applied. The buckets were covered. In order to assess residual effect, 25 larvae were introduced in each bucket, and mortality rates were recorded after 24 h. Residual effect was proven on days 1, 2, 9, 10, 30 and 64 after application of larvicides. Dead larvae were considered those that could not be induced to move when they were probed with the dissecting needle in the siphon or the cervical region.
The second test followed the same procedure as in the first test, with following variations: three 30 l plastic recipients were filled with 25 l tap water. In one recipient a half tablet of Culinex Tab plus® (0,1 g) was applied, in the second recipient 6,25 g of Temefar® 1% G in the “mina” formulation, and in the third recipient no larvicide was introduced (control). In order to assess residual effect, 50 larvae were introduced in each recipient, mortality rates were also recorded after 24 h. Residual effect was proven on the same days as in the first test.
3.2.2. Residual effect tests in small water volumes: Two very similar tests were performed. Three 1 l plastic recipients were filled with 1 l tap water for the first test. In the first recipient 0,004 g of pulverised Culinex Tab plus® was applied, in the second 0,5 g of Temefar® 1% G as loose granules, and in the third, no larvicide (control). In order to assess residual effect, 25 larvae were introduced in each bucket, and mortality rates were recorded after 24 h. Residual effect was proven on days 1, 30 and 48 after application of larvicides.
The second test followed the exact procedure as in the first test, but instead of 1 liter, 900 ml tap water was applied to the recipients. The purpose was to give more air space between tissue cover and water surface for larvae respiration.
Both residual effect tests with small and large water volumes were performed in laboratory conditions. Air temperature and relative humidity were registered by using an electronic thermometer and hygrometer, respectively, during the days of residual effect evaluation.
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