Abstract:
The invention relates to a specific attractant for male and female  Anastrepha obliqua  fruit flies. The inventive attractant comprises a mixture of identified, isolated synthetic compounds which have been formulated according to the proportions in which they were found in the  Spondia mombin  volatiles.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention is related to the development of a fruity attractant for detecting, monitoring and controlling the fruit fly  Anastrepha obliqua.  All the components of the attractant are commercially available. 
       BACKGROUND OF THE INVENTION 
       [0002]    Fruit flies from the family Tephritidae represent a major problem for fruit growing in general, due to the damage caused when in larval stage when feeding off the fruits and also because of the restrictions established by countries and international organizations on fruit commercialization. For instance, in Mexico, there are about 170,000 hectares of mango crops ( Mangifera indica ), with a production of 1.4 millions of tons, from which about 196,000 tons are for exportation. This crop is attacked mainly by  Anastrepha oblique  and  Anastrepha ludens  (Aluja et al., J. Econ. Entomol. 89:654-667, 1996). Also, these fly species attack other commercial fruits as well and are considered as species subject to quarantine in the US and Europe, which constitutes a limiting factor for fruit exportation to those countries&#39; markets. 
         [0003]    Traditionally, flies from genera  Anastrepha  are monitored using McPhail traps baited with liquid attractants such as protein hydrolyzate+borax as microbial inhibitor (López and Hernández-Becerril, J. Econ. Entomol. 60:136-140, 1967) and/or Torula yeast (López et al., Fla. Entomol. 64: 1541-1543, 1971). However, there are some disadvantages in using liquid attractants including low selectivity since they attract other insects besides fruit flies. The fact that some non-target insects are being captured in the traps increases the checking time of the same. On the other hand, during the change of attractant in the traps, some of the attractant may be spilled and the spilled bait becomes a food source for flies outside the trap, thereby decreasing the trap efficiency (Epsky et al., Fla. Entomol. 76: 626-635, 1993; Thomas et al., Fla. Entomol. 84: 344-351, 2001). The above-described disadvantages had led to seek out solid attractants from different sources including volatile substances produced by bacteria, feces and bird feathers (Demilo et al., J. Entomol. Sci. 32: 245-256, 1997; Epsky et al., Fla. Entomol. 80: 270-276, 1997; Robacker et al., Fla. Entomol. 81: 497-508, 1998; Robacker et al, J. Chem. EcoL., 26: 1849-1867, 2000). By example, ammonium salts and putrescine (1,4-diaminobutane) have been shown to be selective attractants both for males and females from  Anastrepha ludens  (U.S. Pat. Nos. 576,667 and 5,907,923). However, the catches using ammonium acetate, putrescine and trimethylamines depend on the season of the year (Heath et al., J. Econ. Entomol. 90: 1584-1589, 1997). In the draught season, traps baited with these compounds captured significantly less  Anastrepha ludens  than the traps baited with liquid protein. The opposite is seen in the rain season, when traps lured with ammonium acetate, putrescine and trimethylamines captured significantly more flies than traps lured with protein (Heath et al., J. Econ. Entomol. 90: 1584-1589, 1997). An attractant based on the combination of 2,5-dimethylpirazine with ammonium salts and putrescine resulted more effective in capturing  Anastrepha obliqua  than the combination of ammonium salts and putrescine (Patent GB 2356141). 
         [0004]    Volatile compounds released by host fruits for fruit fly constitute other potential source of attractants. Volatiles from fruits could be used by fruit flies to locate food and ovoposition sources, and to find and mate a partner (Jang &amp; Light, olfactory semiochemicals, pp. 73-90, in: Fruit fly pest, a world assessment of their biology and management, MacPheron, B. A. &amp; G. J. Steck (eds), St. Lucie Press, Delray Beach, Fla., 1996). Regarding attractants obtained from native host for  Anastrepha  species, the most relevant work has been done with  Anastrepha ludens  (Robacker et al., J. Chem. EcoL., 16: 2799-2815, 1990; J. Chem. EcoL., 18: 1239-1254, 1992). These researchers have isolated and identified four volatile compounds attractive to  Anastrepha ludens  from fruits of its native host ( Sargentia greggii ). Compounds were identified as 1,8-cinneole, ethyl hexanoate, hexanol, and ethyl octanoate, which when formulated together resulted more attractive than Torula in laboratory trials. However, in filed trials, this volatiles formulation did not show a better performance than Torula yeast (Robacker &amp; Heath 1996). Traps lured with ethanol extracts of  Spondias purpurea  captured more  Anastrepha obliqua  adults than traps lured with protein hydrolysate in a mango orchard (Ortega-Zaleta and Cabrera-Mireles, Agric. Téc. Méx 22: 63-75, 1996). None of the compounds was identified as responsible of the attractancy of the extracts. 
         [0005]    Furthermore, the attractants that were found could be used for controlling fruit flies through the attraction-killing approach. With this approach, captured insects are killed by means of an insecticide, a pathogen agent, or an sterilant. 
         [0006]    We have developed a specific attractant for detecting, monitoring and controlling males and females from the fruit fly  A. obliqua  using a mixture made up of synthetic compounds from a native fruit from Chiapas, Mexico, locally known as jobo de pava,  Spondias mombin.    
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0007]    Next, the extraction, electroantennographic detection, chemical identification, formulation and assessment of the attractant for both adult males and females from fruit fly  Anastrepha obliqua  are described. 
         [0008]    Evaluation of Biological Activity of Jobo Fruit in Attracting  Anastrepha obliqua    
         [0009]    Firstly, an experiment was developed to evaluate whether the  Anastrepha obliqua  adults are attracted to the jobo fruit using a flight tunnel under laboratory conditions (Rojas et al., Entomología Mexicana, Vol. 2, 690-694, 2004). Treatments were evaluated in non-choice trials. One ripe fruit from  Spondias mombin  was used for each repeat, which was placed inside the tunnel in the opposite end from an air extractor at a height of 15 cm above the floor. An orange colored styrofoam sphere mimicking the color of the fruit (artificial fruit) was used as control. For each repeat, 25 males or females were released into the tunnel and observed for 10 min. Two parameters were recorded: attraction and landing. Attraction is defined as ⅔ the distance traveled by the flies from the point of release to the sample. Landing involves that the insect steps on the fruit. Results from the bioassays demonstrated that both females and males were more attracted to the jobo fruits than to the control. Both sexes also landed more frequently on the jobo fruits than on the artificial fruit. 
         [0010]    Collecting and Evaluation of Biologically Activity of Volatiles from Jobo de Pava Fuit 
         [0011]    The next step is to collect volatile compounds released by the jobo the pava fruit and to evaluate the extracts activity. Healthy ripe fruits and infection-free from flies or others insects were directly obtained from trees located in the vicinity of Tapachula, Chiapas, Mexico. The volatile compounds were collected using the dynamic aeration technique (Malo et al., Entomología Mexicana, Vol. 3, 115-118, 2003). Volatiles were trapped using 1 g of the adsorbent Porapak Q, and 16 hours after collecting, volatiles were eluted from the adsorbent using 200 μl of anhydrous ether. 
         [0012]    Extracts were evaluated in the flight tunnel, in the following manner: 100 μl of the extract were loaded in a rubber septum, previously washed with hexane. The septum was placed on an artificial fruit. As control an artificial fruit impregnated with 100 μl of anhydrous ether was used. Results from this test show that males and females were more attracted to the jobo&#39;s volatiles extracts than to control. Both sexes also landed more frequently on the artificial fruit lured with the extract than on the artificial fruit lured with solvent alone. 
         [0013]    Gas Chromatography-Electroantennography (GC-EAD) Analysis of  Anastrepha obliqua  to Jobo de Pava&#39;s Volatiles 
         [0014]    The following step is to determine the chemical compound(s) responsible for the attractancy and landing of the insect. To this, a technique known as gas chromatography coupled to electroantennography (Arn et al., Z. Naturforsch. 30 C: 722-725, 1975) was used. The advantage of this technique is that it includes the high sensitivity of the insect antennae, whereby only the compounds showing electrophysiological activity are identified. A minimum of 16 different antennae from males and females were used. During the analysis by GC-EAD 9 antennally active peaks were found, both in males and females of  Anastrepha obliqua.    
         [0015]    Chemical Identification 
         [0016]    Once that the antennally active compounds were determined, the chemical identification of said compounds were performed using a gas chromatography unit coupled to a mass spectrometer, according to a previously described procedure (Malo et al., Entomología Mexicana, Vol. 3, 115-118, 2003; Rojas et al., Entomología Mexicana, Vol. 2, 690-694, 2004). Identifications were confirmed comparing both retention times and spectra data to authentic standards obtained from commercial sources. Compounds were identified as esters and alcohols. 
         [0017]    Attractant Evaluation in the Lab 
         [0018]    Once that the compounds were identified the making of the mixture of synthetic compounds, and its evaluation in the flight tunnel took place. In this test 1 μl of the blend of the nine synthetic compounds, made according to the ratio in which they were found in the jobo extracts, was used. The compounds blend was loaded into a rubber septum. The septum was placed onto the artificial fruit. An artificial fruit having a septum loaded with 1 μl of hexane was used as control. Results indicated that both sexes were significantly more attracted towards the mixture of the nine components than to control. Males and females landed more frequently onto the artificial fruit lured with the mixture of the nine components that in the control fruits. 
         [0019]    Evaluation of the Attractant in Field Cages 
         [0020]    Finally, we proceed to evaluate the attraction of the synthetic mixture of the components comparing it versus the protein hydrolysate and water as control in non-choice and triple choice trials in field cages. The tests were performed in field cages of 2.85 m in diameter by 2 m high. In non-choice trials one coffee tree ( Coffea arabica ) and one mango tree ( Mangifera indica ) of 1.20 meters high were placed centrally inside the cage. In triple choice trials, 5 coffee trees and 6 mango trees were placed inside the cage, one tree from each species in each of the cardinal points and two trees (one in the case of coffee) in the center of the cage. In non-choice trials one Multilure trap was placed 50 cm above the tree. In triple choice trials, 3 Multilure traps were placed at 30 cm in the periphery of the cage, with a separation of about 185 cm between each trap. Traps were hung at 10 cm from the cage. The mixture of synthetic compounds was prepared according to the ratio in which they were found in the jobo&#39;s volatiles extracts, and 100 mg of this mixture were loaded in rubber septa. Protein hydrolysate Captor 300 was prepared using 5 g of borax mixed with 10 ml of protein hydrolysate dissolved in 235 ml of water per trap. Water plus Tween 80 were used to keep the flies captured in traps lured with jobo&#39;s volatiles and water. Twenty five (12 or 13 of each sex, in non-choice trials) and 150 flies (75 of each sex, in triple choice trials) subjected to a 15 hours fasten were released in the center of the cage during the early hours of the morning. Baits were placed in each trap 15 minutes before flies were released in the cage. Traps were placed at 08:00 a.m. and the number of captured insects in each trap was counted 24 hrs later. The position of each trap was re-distributed on a daily basis to avoid any effect due to position. In the trials, temperature fluctuated from 23-31° C. and relative humidity fluctuated from 60 to 95%. Each experiment was repeated 12 times. 
         [0021]    Results from the field tests show that traps lured with the mixture of jobo&#39;s synthetic volatiles captured significantly more  Anastrepha obliqua  than the traps lured with protein hydrolysate and than the control.