Field assessment of baits for frugivorous flies ( Tephritidae and Lonchaeidae )

Hydrolyzed proteins are used as attractive agents in McPhail traps for monitoring of fruit flies (Tephritidae). There has been no defined role for monitoring of insects belonging to the Lonchaeidae family. Currently, there is a great concern in using most efficient and low-cost attractive materials. Thus, this study aimed at assessing the attractiveness of solid and liquid baits in capturing of frugivorous flies (Tephritidae and Lonchaeidae). Therefore, we carried out four trials in coffee plantations (Coffea arabica L.) during two different periods (May/ 2013 and July/ 2014). A randomized block design with four treatments and five replications was used in the first period, and ten treatments with four replications the following year. Two distinct experiments were carried out, one using baits in solid and another in liquid form, which consisted of applying 10 g for trap (solid) or 200 mL solution of the same compounds diluted in water (5%: p/ v). In 2013, we tested yeast extract with and without sugar, brewer's yeast, citrus pulp and hydrolyzed protein as control. In 2014, five additional baits were tested: açai, plum and grape flours, passion fruit fiber and poultry feces. In all experiments, a 5% hydrolyzed protein solution was used as control. Eight days after being set, traps were assessed. Even the yeast extract, with and without sugar, and the brewer's yeast, in solid form, were as attractive as control treatment in capturing flies. The poultry feces and yeast extract, with and without sugar in solution form, can be used to replace hydrolyzed protein in capturing tephritids. Lonchaeids are barely attracted by the hydrolyzed protein. Additionally, yeast extract and poultry feces can be used for monitoring and biodiversity studies of the Lonchaeidae fauna.


INTRODUCTION
The term frugivorous flies is used to indicate all flies belonging to the Tephritidae and Lonchaeidae families, while fruit flies refers only to the Tephritidae family (Zucchi, 2000).Tephritids are major economic pests of fruit plantations worldwide, being responsible for yield losses due to damages on fruit that make them unavailable for in natura consumption, as well as having importance as quarantine pests (Aluja and Mangan, 2008;Jenkins et al., 2011).The Lonchaeidae family has been long overlooked by researches because of a lack of taxonomic knowledge; however, there has been an increasing interest in studying such insects since lonchaeids have been collected during tephritids' monitoring.In addition, these insects have also been assigned as primary pest in Malpighia punicifolia L. (Araújo and Zucchi, 2002), Citrus reticulada Blanco (Lopes et al., 2008), Passiflora edulis f.Flavicarpa (Aguiar-Menezes et al., 2004) and Manihot esculenta (Lourenção et al., 1996;Gistoli and Prado, 2011).
Coffee plants are considered preferred hosts for fruit flies, mainly for the species C. capitata (Wiedemann, 1824) and Anastrepha fraterculus (Wiedemann, 1830), since this plant provides shelter during periods of low availability of hosts in the field (Montes et al., 2012).The coffee tree has also major importance for the maintenance of frugivorous fly populations, even though these insects are not taken as main pests for this crop.However, these insects may threaten a few fruits commercially grown in natura, becoming their primary pest (Souza Filho et al., 2003).
Fruit fly monitoring is an important tool for decisionmaking in pest control managements and consists of using Jackson's traps based on sexual pheromone as well as McPhail traps, which contain 5% corn hydrolyzed protein (Carvalho, 2005).The use of traps and standard attractive increase monitoring operation costs for small farmers; therefore, alternative materials such as plastic bottles, fruit juice or sugarcane molasses are often employed in samplings.
A capture system has not been established yet for monitoring of lonchaeids, which are usually collected through the same attractive traps used for tephritids (Raga et al., 2006), since there are still few studies on such group of flies.
In recent years, there has been an increased concern to find efficient and low-cost attractive materials.As well, some studies have indicated good prospects for solid baits (Conway and Forrester, 2007;Epsky et al., 2011;Lasa et al., 2014), which have shown some advantages in water use and less time for trap supply, which can be used in monitoring and biodiversity studies.
The current study aimed at assessing the attractiveness of solid and liquid baits in capturing of frugivorous flies (Tephritidae and Lonchaeidae), which may be further indicated in pest management programs and biodiversity studies.

MATERIALS AND METHODS
The studies were carried out in coffee plantations (Coffea arabica L.) of "Catuaí Amarelo" and "Catuaí Vermelho" varieties.The plantations are located in Santa Fé farm (14º 44' 8.7" S; 40º 26' 06" W), in the city of Planalto-BA, Brazil.The treatments consisted of baits in solid and liquid form making two different and sequential experiments.The experiments were performed under a randomized block design.Four treatments (baits) and five replications (traps) were tested in 2013, and 10 treatments with four replications in 2014, in a total of 25 and 40 plots, respectively (Table 1).The plots were composed of McPhail traps with each attractive material, being set on plants at 1.50 m height from soil and 20-m equidistant, with a 10-m border.Ten grams of each product was weighed and then distributed to the base of traps.Hydrolyzed protein was used as a control treatment for all experiments.10 g was used for each trap (solid) or 200 mL solution of the same compounds diluted in water (5%: p/v) (liquid), as per the manufacturer's recommendations.
The compounds were selected based on their commercial composition.Therefore, compounds with a predominance of proteins (yeast extract -Bionis®, brewer's yeast and corn hydrolyzed protein), of carbohydrates and fibers from fruit (açaí, plum and grape flours, citrus pulp and passion fruit fiber), plus poultry feces, which are considered important sources of protein for fruit flies in nature was used (Christenson and Foote, 1960).The evaluations were made eight days after traps were set for all experiments, which is considered a trap attractive refill period when using hydrolyzed protein in orchards under official monitoring.The flies captured in field traps were sorted, counted and divided according to genus and/ or species.
The identification of the C. capitata species was made based on descriptions of Zucchi (2000).For Anastrepha, we have solely used females, identifying them by a apical spine pointing outward, with the aid of a stereomicroscope (40x) and biological microscope (100x), according to the method described by Zucchi (2000).Yet the lonchaeids were identified at genus level by morphological characters in the chest and abdomen.

Statistics
The statistical analysis was carried out for data of the most abundant fly species.The data under non-normal distribution were submitted to non-parametric mean comparisons using the Friedman's test (p> 0.05); the remaining data underwent variance analysis and means were compared by the Tukey's test at 5% probability, using ASSISTAT 7.7 beta.

RESULTS AND DISCUSSION
In 2013, 1.611 flies were caught, being 84 (5.2%) with solid compounds and 1,527 (94.7%) with 5% solutions (Table 2).Tephritids prevailed in all collections, making 98.8% of solid treatments and 94.0% of liquid ones.The most abundant species was C. capitata, representing 95.2 and 96.0% of flies caught in solid and liquid experiments, respectively (Table 2).The genus Anastrepha was represented by the species A. fraterculus (2.4% in solid and 3.2% in liquid) and A. consobrina (Loew, 1873) (1.2%).Among the lonchaeids, only representatives from the genus Neosilba were captured, contributing in 1.2 and 0.8% of flies captured with solid and liquid baits, respectively (Table 2).In absolute terms, hydrolyzed protein was the most attractive treatment for tephritids, being responsible for 39.2% of tephritid capture in solid experiments and 38.3% in liquid ones.For lonchaeids, the product Bionis, with and without sugar, was most attractive, collecting 92.3% of the flies in both experiments, demonstrating thus a low attractiveness of proteins to these flies.
Both Bionis ® (with -T1, and without sugar -T2) and brewer's yeast (T4), in solid form, showed an attractiveness similar to the hydrolyzed protein (control -T5) for female and total (male plus female) captures of the C. capitata, among which citric pulp had the lowest attractiveness (Table 3).Regarding the evaluations of the same products, in liquid form, the results were similar for female collections; however, no significant difference was seen for total captures.Male captures were less expressive compared to female ones for all treatments (Table 3).Adults belonging to the C. capitata species, particularly females at sexual maturity had greater preference for protein-based materials, which was ingested according to the foraging behavior and mating of these insects (Cohen and Voet, 2002).
The population of A. fraterculus captured was low.Moreover, solid treatments did not take any specimen of this species, except for control (Table 4).The Bionis in solution, with and without sugar, did not differ significantly from the hydrolyzed protein to the total number of adults captured.The yeast extract, with and without sugar, and the brewer's yeast were as attractive as the hydrolyzed protein for females.However, these treatments did not differ as to the males, except for citrus pulp that had the worst performance when collecting adults of A. fraterculus.
The hydrolyzed protein has shown to be a highly attractive lure to tephritids.This compound is similar or slightly higher than several others, e.g.guava juice (Azevedo et al., 2012), syrup (Raga et al., 2006), corn steep liquor and sugarcane molasses (Montes and Raga, 2006), 25% grape juice (Scoz et al., 2006), vinegar (Monteiro et al., 2007), hydrolyzed enzymatic protein and ammonium acetate and putrescine (Lasa et al., 2014), and yeast (Santos et al., 2010).Few studies demonstrate lower attractiveness by hydrolyzed protein relative to other baits.For instance, Epsky et al. (2011) carried studies that showed compounds based on mixture of ammonium acetate and putrescines are most attractive.
Some studies have shown that yeasts are attractive lures to tephritids, with higher than or similar to different corn hydrolysate baits (Santos et al., 2008) as observed for 25% grape juice in this study (Monteiro et al., 2007).
An amount of 14,382 flies were caught in 2014, among which 2,109 (14.6%) in solid baits and 12,273 (85.3%) in the same compounds diluted in water at 5% (Table 5).Again, tephritids were predominant, reaching 99.6% in studies with solid baits and 99.7% with liquid ones.The species C. capitata has also prevailed, accounting for 87.9 and 96.6% of all captured flies in solid and liquid baits, respectively (Table 5).The genus Anastrepha was represented for the species A. fraterculus (11.7 and 3.1%) and A. manihoti Lima, 1934 (0.04%).Among lonchaeids, solely specimens belonging to the Neosilba genus were taken, representing 0.4 and 0.3% in studies using solid and liquid baits, respectively (Table 2).
Not all liquid baits differed from hydrolyzed protein, except for grape flour that was less attractive to C. capitata (Table 6).Although not significant, sugarless yeast extract and poultry feces showed the highest  average of captures for C. capitata compared to control.The attractiveness of odor released by the baits to adults of C. capitata may be influenced by its nutritional status, that is, protein-deficient adults may associate the odor as being a protein source by means of olfactory receptors (Manrakhan and Lux, 2008).Among solid baits, the hydrolyzed protein remained most attractive to A. fraterculus.However, yeast extract (T1 and T2), citric pulp (T3), beer yeast (T4), passion fruit fiber (T7) and poultry droppings (T9) had no difference with control for collections of A. fraterculus (Table 7); thus, they still deserve more attention.Studies on protein-based commercial baits have shown a high efficiency in capturing Anastrepha spp.flies (Raga et al., 2006), as well protein is regarded as an important nutrient for fruit flies, directly influencing the insect longevity and sexual performance in adults (Oviedo et al., 2011).
Differently, the results for liquid form baits demon-strated a greater attractiveness of plum flour against other treatments.The release of volatile substances by compounds might have been responsible to attract adults of A. fraterculus.For Kendra et al. (2005), the response of Anastrepha supensa (Loew, 1862) females to the releasing of volatile compounds as ammonia, for example, is related protein intake period.
A fewer specimens of lonchaeids were found   compared to tephritids, with hydrolyzed protein showing low or even null attractiveness to the species Neosilba sp.(Tables 2 and 3).Yeast extract with and without sugar, in both solid and in liquid form, plus the bird feces in liquid form enabled the capturing of the vast majority of insects.These baits should be indicated for biodiversity studies as well as monitoring of this group of flies.The genus Neosilba comprises species of great economic interest; therefore, its population monitoring is of upmost importance, once they affect commercial fruits (Strikis et al., 2011).

Treatments
In solid form, yeast extract and beer yeast have potential as alternatives to the use of hydrolyzed  protein for C. capitata and A. fraterculus.In liquid form, all baits had an increase in attractiveness, except for grape flour, which was less attractive to both species of flies.It is also noteworthy mention that poultry feces were attractive, especially in liquid form, for the capture of both species of flies.Females seem to be showing a greater selectivity for baits than males do.Good catches provided by poultry liquid material might be derived from ammonium release when decaying, being attractive to the flies.Such attractiveness of poultry droppings have been reported in a few species as the case of Rhagoletis pomonella (Wash, 1867) (Prokopy et al., 1993a) and A. suspensa (Epsky et al., 1997).For C. capitata, this attractiveness varies with the source and excrement conditions.Droppings of birds and lizards are most attractive than mammal feces, since the first two show uric acid decomposition that is more attractive than the urea decomposition (Prokopy et al., 1993b).The rates of decomposition and ammonium release to the atmosphere could vary with interactions between biotic and abiotic factors as microbial activities, temperature and rainfall (Mazor, 2009).
From an economic point of view, the use of yeast extract (Bionis, with or without sugar) would be most expensive since this product costs around US$ 8.26 per kilo (Biogirin, 2014), while a liter of hydrolyzed protein (Bio Anastrepha®) costs US$ 2.77, excluding transportation expenses.Poultry feces would probably have much lower costs; however, the lack of suppliers with standardized products could hinder this process.

*
Means followed by the same letter within a column do not differ from each other by the Tukey's test at 5% probability.The data were transformed into log x+1.** Means followed by the same letter do not differ from each other by the non-parametric Friedman's test (p>0.05).
by the same letter do not differ from each other by the non-parametric Friedman's test (p>0.05).

*
Means followed by the same letter within a column do not differ from each other by the Tukey's test at 5% probability.The data were transformed into log x+1.** Means followed by the same letter do not differ from each other by the non-parametric Friedman's test (p>0.05).

Table 1 .
Treatments used in the field experiments on the attractiveness of baits to frugivorous flies.

Table 2 .
Number and percentage (%) of frugivorous flies captured by solid and liquid baits in a coffee plantation in the city of Planalto -BA, Brazil.May of 2013.

Table 3 .
Average number (± standard deviation) of captured C. capitata in solid baits, in a coffee plantation in the city of Planalto -BA, Brazil.May of 2013.

Table 4 .
Average number (± standard deviation) of A. fraterculus captured in solid and liquid baits, in a coffee plantation in the city of Planalto -BA, Brazil.May of 2013.

Table 5 .
Number and percentage (%) of frugivorous flies captured by solid and liquid baits in a coffee plantation in the city of Planalto -BA, Brazil.July of 2014.M = Number of male flies belonging to the genus Anastrepha caught in traps at different treatments. *

Table 6 .
Average number (± standard deviation) of C. capitata captured by solid and liquid baits in a coffee plantation in the city of Planalto -BA, Brazil.July of 2014.Means followed by the same letter within a column do not differ from each other by the Tukey's test at 5% probability.The data were transformed into log x+1. *

Table 7 .
Average number (± standard deviation) of A. fraterculus captured in solid and liquid baits in a coffee plantation in the city of Planalto -BA, Brazil.July of 2014.