Role of trap colors and exposure time of pheromone on trapping efficacy of males of the tomato leafminer , Tuta absoluta ( Meyrick , 1917 ) ( Lepidoptera : Gelechiidae )

Field studies were conducted in two sites (Saheline and Chott-Mariem) in the Centre-East of Tunisia in 2012 and 2013 to evaluate the influence of different colors of pan water traps and age of pheromone dispenser on capture of males of the tomato leafminer, Tuta absoluta. Three experiments were setup using colored traps (white, yellow, orange, red and green) in a randomized block design with four replicates. The first trial was conducted during the period from November, 2012 to February, 2013, the second between December, 2012 and April, 2013 and the third during April to May, 2013. Results indicate, in all experiments, that there is no significant difference in male capture according to trap color. However, in the third trial, green colored traps captured, respectively, more than 5-fold, 4-fold, 4fold and 2-fold for red, orange, yellow and white colored traps. The experiments aimed to determine the effect of pheromone aging on male trapping, results revealed a significant difference between aged lures and control. Fresh lures captured from 2 to 13-more moths than weathered pheromone capsules.


INTRODUCTION
The tomato leafminer, Tuta absoluta (Lepidoptera: Gelechiidae) is the most important constraint to tomato production in Tunisia (Photo 1) where this crop plays an important role in farming production both in greenhouses and open fields.In spite of health hazards, occurrence of resistant biotypes of the pest, high cost and destruction of population of natural enemies, insecticides are routinely applied as sole control strategy against this pest (Braham et al., 2012).
Lepidoptera pheromones have been successfully used for insect monitoring and mating disruption of insects (Wyatt, 1998).Virgin female of tomato leafminer releases a sex pheromone that strongly attracts males (Quiroz, 1978) which is identified by Attygalle et al. (1996) as (3E, 8Z, 11Z)-3,8,11-tetradecatrien-1-xyl acetate.Pheromones are used to monitor population of insects to determine the correct timing to apply control strategies such as introduction of natural enemies, intensification of mass trapping and applying insecticides.Complementary strategies of pest management through an efficient E-mail: braham.mohamed@gmail.commonitoring of pest population need to be determined.including the use of monitoring pheromones to correct timing of pesticide application and other means of control To our knowledge, in Tunisia, the only study relating to the use of T. absoluta pheromone lure is made by Abbes and Chermiti (2011) in which two marks of pheromone capsules were compared for their efficiency in trapping males under tomato greenhouse.
The objectives of this study are: (1) to test the attractiveness of pheromone traps differently colored to T. absoluta males since farmers use these kind of traps usually self made recyclable buckets, (2) to test the effectiveness of capsule pheromone traps following field aging.Farmers are advised to replace pheromone capsules every 5 to 6 weeks, but little information is available on factors influencing the longevity of field-aged capsules.These parameters are important to define in order to improve trapping technique either for monitoring or for mass trapping.

Effect of Trap colour on attractiveness of males of Tuta absoluta
For all experiments, tomato plants, Lycopersicon esculentum Mill (cv Rio Grande) were sown in climatic chamber.Seeds were deposited in cells in a rectangular polyester tray of 60 × 40 × 5 cm filled with peat (Potgrond, Germany).
In Chott-Mariem locality, tomato seedlings were planted in a sample row (1 m between rows and 0.4 m on the row) on November 26, 2012, while in Saheline locality, two crops were established one in autumn, the other in spring.The autumn crop, seeds were sown on 16 August 2012 and seedlings were transplanted on 12 September 2012 in a plot of about 2400 m 2 (60 × 40 m).Plants were spaced as mentioned above to get a density of about 25000 plants/ha.In the spring crop, sowing was done on 17 February 2013 and plantation on 22 March 2013.The plot of about 4000 m 2 (65 × 62 m) was planted with tomato in 1600 m 2 (28 rows, 14 in each side) and 36 rows of potato in the middle to obtain large area and increase the inter-traps distance.Traps were setup in a randomized block (Figure 1).In all experiments, to avoid trap interference, the distances between traps and between blocks were more than 15 m (Bacca et al., 2006).Pan traps made of plastic were purchased from a local store, originally colored in light red (height from bottom to top = 14 cm, diameter = 35 cm).
Each trap was perforated with two holes parallel on the edge of the trap.Each plug of pheromone was placed inside a plastic cylindrical container (7 cm length and 3 cm diameter) perforated by many holes.The pheromone container was fixed on the top middle of the trap using metal wire tied to the body of the trap.The container is used to provide shelter and avoiding direct exposure of pheromone to sunlight.The inside and outside of the bucket trap were painted with the commercially available paints using a brush.The colours were selected based on reported evaluations of trap colours influencing the attraction of various Lepidoptera species (Hendrix and Showers, 1990;Bloem et al., 2005) and on our observations on what is used by farmers.Each trap was filled with approximately 4 L of tap water then traps were setup in a randomized block (Figure 1) and the water renewed at every inspection date.
In all experiments, to avoid trap interference, the distances between traps and between blocks were more than 15 m (Bacca et al., 2006).Caught insects were removed with a piece of wood and moths of T. absoluta were counted.Captured insects were removed with a piece of wood and T. absoluta moths counted.In Chott-Mariem, traps were setup in the field on 27 November 2012, while in Saheline on 9 September 2012 and on 25 April 2013, respectively for autumn and spring tomato.

Effect of age of pheromone on attraction males of Tuta absoluta
The objective of the study is to determine the attractive period of pheromone lure in the field.Two sets of 24 pheromone capsules were setup in pan traps without water in the field in 2012 and one set in 2013.The first set was put on 7 March 2012 in the edge of uncultivated plot in Saheline region and 4 pheromones capsules were removed at weekly interval (on 14 March, 22 March, 29 March, 13 April and 20 April 2012) wrapped in aluminum foil and put in the freezer at 0°C until use.Aged capsules (from 1 to 6 weeks) and fresh ones were deployed in traps in tomato crop on 27 April 2012.
The second set was put on in the same place on 20 April 2012 and 4 pheromone capsules were collected at weekly interval (on 27 April, 4 May, 11 May, 17 May, 24 May and on 31 May) and stocked in the freezer.Aged capsule and 4 fresh were setup in a tomato field on 4 June 2012.The second trial lasted from 4 June 2012 to 11 July 2012.Tomato field planted with Rio Grande cultivar having approximately 2000 m 2 (50 × 40 m in length).Traps were set up in a randomized block design at the edge of the plots and spaced at about 10 to 12 m.
The third set was put in the field on 17 October 2012; four pheromone capsules were collected at weekly interval then put in a freezer (on 24 October, 01 November, 9 November, 15 November and 23 November 2012).Traps with aged capsules (from 1 to 5 weeks) were deployed in the tomato field as described above on 1 March 2013.Data on the minimum, maximum and average monthly temperatures for the Saheline were reported from meteorological station at about 12 km from the site.Meteorological data of Chott-Mariem were not very different from Saheline locality, both located at the border of Mediterranean Sea (about 25 km from each other).

Statistical analysis
The mean number of captured T. absoluta males was analyzed statistically using one way analysis of variance (ANOVA).When ANOVA indicates that significant differences were found, (P < 0.05) means were separated by least significant difference test (LSD).Tests were run with Minitab software (13.0).

Chott-Mariem trial
The result revealed that, the mean number of male captured varied from 3 adults in late February 2013 to 17 in December 2012 (Figure 2).In the region, January and February are the coldest months of the year (Figure 3).The ANOVA of the average number of captured males according to inspection dates, shows no significant difference between different tested colored traps on   attraction males of T. absoluta (F 4.115 = 1.45;P = 0.22) (Table 1).

Saheline trial
Autumn crop: As presented in Figure 4, for autumn tomato cultivation, statistical analysis shows no significant difference between different colored traps tested on attracting males of T. absoluta (F 4.54 = 0.53; P = 0.71) (Table 2).

Spring crop:
For spring cultivation, the ANOVA of the average number of captured males according to inspection dates, shows no significant difference between colored traps (F 4.29 = 2.69; P = 0.054; Table 3).The mean number of captured males varies between 3 and 17 (Figure 5).

Effect of age of pheromone on attraction males of Tuta absoluta
For the first trial (from April 2012 to June 2012), significant difference was observed between aged and fresh lures (ANOVA, one factor F 6.63 = 6.06;P = 0.001).Fresh lures captured from 2-fold to 4-fold T. absoluta males (Table 4).Surprisingly, lures aged 5 weeks captured numerically more moths than capsules aged 1 week.
Concerning the third trial, there is no significant difference regarding d capsules and fresh ones probably due to low adult population (ANOVA, 1 factor F 5.30 = 1.12;P = 0.73) (Table 6 and Figure 8).

DISCUSSION
The tomato Tomato leafminer, T. absoluta can be  considered as a potential pest owing to its presence a year round in the coastal region of Tunisia (Centre-East) in an open tomato fields.Although originates from hot regions of South American countries (Miranda et al., 1998), the insect is able to survive winter under Mediterranean climate and causes serious damage to winter tomato crop which indicated by male captures in January-February, the coldest moths of the year.The author did not evaluate level of infestation on tomato leaves and fruits, but low damage was noticed.
Trap color may be important to increase the effectiveness of a pheromone monitoring or mass trapping system.The parameters that make one color more effective than another are poorly understood.Trap color has been shown to improve trapping efficiency in other economically important Lepidoptera (Knight and Miliczky, 2003).Similar numbers of male tomato leafminer were captured in bucket traps at different colors demonstrating    perceived over large distances hiding the trap color, (2) the distance between traps and blocks (15 m) may be relatively short resulting in maximum interference and competition among the pheromone plumes of neighboring traps (Elkinton and Cardé, 2008) and (3) color may be hidden by plant canopies; bucket trap are deployed on the ground.In spring tomato trial, though not shown statistically, green traps performed better.It may be due to the presence of tomato and potato in the same plot.It is known that T. absoluta attacks potato (Solanum tuberosom) leaves but no studies relating to the preference of the insect when potato and tomato cultivations are mixed or planted side by side which is common in the Centre-East region of Tunisia.Accordingly, Knight and Miliczky (2003) evaluated different trap colors for attraction of males of codling moth Cydia pomonella (Lepidoptera: Tortricidae), honeybees   and non-target insects reported a significantly high number of males of codling moth were caught in green delta traps versus the unpainted white ones.
To our knowledge, this is the first study aiming to demonstrate the effect of trap color on the attraction of T. absoluta males; we hypothesize that trap color may enhance the effectiveness of pheromone baited-trap.Roubos and Liburd (2008) reported a significant difference regarding the effect of bucket trap color on the male capture of the grape root borer, Vitacea polistiformis (Harris) (Lepidoptera:Sesiidae) using synthetic sex pheromone.Green and yellow colored traps caught more grape root borer males than white or blue.It was suggested that males do not distinguish between green and yellow colors which have similar reflectance.
Concerning the effect of weather factors on the decrease of pheromone attraction, the tests of the two experiments of 2012 (April and June), fresh lures captured from 2 to 13-fold more T. absoluta than aged lures (from 1 to 6 weeks).In spring, fresh lures can be attractive for 4 weeks (Figure 6) and in summer, this period is about 2 weeks (Figure 7).Indeed, in Tunisia, Abbes and Chermiti (2011) evaluated the efficiency of two marks of sex pheromone dispensers used for trapping T. absoluta males under greenhouse showed that the number of trapped males peaked the second week following lure installation to decrease afterwards until its renewal.
In a study relating the effect of aging lures on the attractiveness of male codling moths C. pomonella, Kehat et al. (1994) demonstrated that captures in traps were negatively correlated with the aging of lure.

Conclusion
Among five different colors used in bucket pheromone traps to capture T. absoluta males, no preference was noticed.However, green-colored traps colored are more suitable.Capsule pheromone lures for the monitoring and/or mass trapping of T. absoluta males should be replaced after 4 weeks in spring and 2 weeks in the summer at the most.Because of high temperature, the effect of aging of dispenser was more marked during summer than during spring.More research on the design of the tube used to host pheromone capsule providing a shelter is needed.

Photo 1 .
Photo 1. Symptoms of infestation by Tuta absoluta on tomato leaves.

Figure 4 .
Figure 4. Average number of captured males per trap (mean of 20 colored traps) during the study period in tomato autumn crop.

Figure 5 .
Figure 5. Mean number of captured males according to trap inspection in spring tomato cultivation (Saheline, 2013).

Figure 6 .
Figure 6.Average number of captured Tuta absoluta males (Effect of weather factor on attractiveness of pheromones, first trial).

Figure 7 .
Figure 7. Mean number of males captured in traps in weathering capsule tests (second trial).

Figure 8 .
Figure 8. Mean number of Tuta absoluta captured in bucket traps according to weathering capsules (third trial).

Table 3 .
Mean numbers (±SD) of captured Tuta absoluta males according to color trap (spring tomato cultivation).
*Means with the same letter are not statistically different at P = 0.05.N = number of inspection dates.
*Means with the same letter are not statistically different at P = 0.05.N = number of inspection dates.
the superiority of pheromone attraction over color.Two hypothesis may explain this: (1) pheromone plumes are

Table 6 .
Mean numbers (±SD) of captured males in different aged lure traps (third trial, June 2013)