Enhancement of Some Protein-Based Baits for Attracting Bactrocera zonata ( Diptera : Tephritidae ) by Adding Ammonium Compounds

Citation: Egypt. Acad. J. Biolog. Sci. (A. Entomology) Vol. 10(6)pp: 153166(2017) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Entomology Journal publishes original research papers and reviews from any entomological discipline or from directly allied fields in ecology, behavioral biology, physiology, biochemistry, development, genetics, systematics, morphology, evolution, control of insects, arachnids, and general entomology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
Fruit flies (Diptera: Tephritidae) stand out as one of the most economically important groups of insect pests attacking fruits and vegetables grown in temperate, tropical, and subtropical areas of the world (White and Elson-Harris, 1992;Hashem et al., 2001;Amro &Abdel-Galil, 2008 andGhanim &Moustafa, 2009).Economically, these pests instigate quarantine restrictions on infested areas, requiring that commercial fruits undergo protective and quarantine treatment prior to export (Vargas et al., 2008).The peach fruit fly, Bactrocera zonata (Saunders) is native to tropical Asia, but it was spread to other regions of the world including the Middle East region (Agarwal et al., 1999 andEl-Minshawy et al., 1999).B. zonata is a polyphagus insect which attack more than 50 species of fruit and vegetable crops as well as wild host plants.In Egypt, B. zonata became a serious pest; however, it attacks a wide range of fruits that differ in their ripening time stage all over the year (White and Elson-Harris, 1992).
Dietary sources of nitrogen have a strong influence on the physiology and behaviour of tephritid flies (Kaspi et al., 2000 andYuval et al., 2007).This behaviorally-based tactic targets female fruit flies primarily based on the female's need for protein for adequate egg production (Epsky et al., 2014 andPinero et al., 2015).Consequently, efforts to suppress pestiferous fruit fly populations around the world have relied heavily on the application of proteinaceous bait sprays mixed with toxicants (Vargas et al., 2001;Moreno andMangan, 2002 andBarry et al., 2003).
To have effective suppression against fruit fly populations, protein-based bait formulations must induce good levels of attraction to the source, and stimulate flies to ingest a lethal dose of the toxicant upon contact (Mangan, 2009 and2014).Ammonia-releasing substances play an important role in fruit fly attraction to food sources (Epsky andHeath, 1998 andHull andCribb, 2001).This information has led to the development of effective ammonia-containing lures.The level of attraction of fruit flies to ammonia-based compounds is known to be dose-dependent, and the range of unattractiveness is very narrow, while the range of rejection (repellence) is much wider (Bateman andMorton, 1981 andMazor et al., 2002).Application of 1% (wt:vol) ammonium acetate to protein baits elicited an adequate female response (Pinero et al., 2015).This amount corresponds to the amount of ammonium acetate present in GF-120 and protein baits that is very effective against fruit flies (Moreno and Mangan, 2002;Mangan and Moreno, 2004;Vargas et al., 2008 andMangan, 2014).Overall, the effects of ammonium acetate added to protein baits were more important for females than males.This finding highlights the importance of adding this compound to commercial baits or local products to enhance their effectiveness for monitoring and suppression the populations of fruit flies (Pinero et al., 2015).
So, the purpose of this study is to enhance attractability of Buminal, Agrinal and Amadene (commercially available protein-based baits in Egypt) for B. zonata by using ammonium acetate and di-ammonium phosphate which are of effective attractants for both females and males of B. zonata (Hanafy et al., 2001;Amin, 2003;Saafan, 2005 andAbd El-Kareim et al., 2008) under the field conditions of a mango orchard.

Field experiment:
To evaluate the efficacy of ammonium acetate or di-ammonium phosphate for enhancing attractability of Buminal, Agrinal and Amadene for B. zonata, an experiment was conducted from the 22 nd of August till the 19 th of September 2017.An area of about 5.5 feddans cultivated with different varieties of mango trees of more than 20 years old were selected for the present study.This orchard located at the Agricultural Experiment Station of Cairo University at Giza district, Egypt.Glass McPhail traps (McPhail, 1939) were used in this experiment by putting about 200 ml of each bait in the trap.Each treatment was replicated three times.The traps were hung in a shady side of the trees at about two meters in height.Traps were distributed at about 20 meters apart between every two adjacent traps in a completely randomized design.Traps were inspected weekly twice (after 4 and 7 days).Captured females and males of B. zonata in each trap were counted.The weekly total numbers of flies were recorded as FTD (No. of Flies/Trap/Day).The solutions were renewed every week and the experiment was repeated four times during four successive weeks.

Statistical analysis:
Data were analyzed using analysis of variance (ANOVA) followed by Least Significant Difference (LSD).Probability of 0.05 or less was considered significant.All statistical analysis was done with CoHort Software (2004).

Enhancement of Buminal: 1.By using ammonium acetate:
Data presented in Table (1) show that Buminal alone is significantly differed with the other mixtures during the first week; while, during the second one, both mixtures of AA.2+Bu.1 and AA.1+Bu.2 were significantly high attractive to B. zonata.During the third and fourth weeks, the numbers of captured B. zonata flies by AA.1+Bu.1 and AA.2+Bu.1 were significantly higher than that of Buminal alone.
Figure (1) showed that the grand mean of attracted B. zonata flies was significantly higher in case of ammonium acetate alone, but the other tested treatments insignificantly differed.The mixture of AA.1+Bu.1 increased the attractiveness to B. zonata by 1.7 times of Buminal alone.On another hand, all tested treatments attracted females more than males (Fig. ,  1).The highest mean number of females per a male was recorded with AA.2+Bu.1 (5.99 females); while, the lowest was recorded with AA.1+Bu.1 (2.63 females).

By using di-ammonium phosphate:
As shown in Table (2), according to the total captured flies Buminal alone was significantly the lowest attractable for B. zonata adults comparedto the other tested treatments all over the four weeks.On contrary, during the first and fourth weeks DAP.1+Bu.1 was significantly the highest in attracting B. zonata flies; while, DAP.2+Bu.1 was the highest during the second week with significantly differences with the other tested treatments.DAP alone significantly surpassed the other treatments during the 3 rd week.The grand mean of attracted B. zonata flies was significantly lower in Buminal alone which significantly between the rests of the tested treatments (Fig. , 2).Also, it can be noticed that adding di-ammonium phosphate to Buminal with a ratio of 1:1 increased Buminal's attractability 3.4 times.zonata adults to Buminal either alone or with added di-ammonium phosphate at different ratios all over the four weeks in mango orchard at Giza governorate, Egypt.
With respect to sex ratio (Fig. , 2), the highest number of females per one male was recorded at DAP.2+Bu.1 (10.05 females); while, the lowest value was recorded with Buminal alone (4.06 females).

Enhancement of Agrinal: 2.1. By using ammonium acetate:
Both of Agrinal alone and AA.1+Ag.2exhibited the lowest attractabillity of B. zonata during the second, third and fourth weeks showing insignificantly differences between them; while, during the first week AA.1+Ag.2only was significantly the lowest (Table,3).AA.1+Ag.1 had a relatively moderate rank during the four weeks of the study.As shown in Fig.
(3), ammonium acetate alone was the best attractant all over the four weeks.AA.1+Ag.1 ranked the second one followed by AA.2+Ag.1.While, both of Agrinal alone and AA.1+Ag.2 were insignificantly the lowest attractable treatments for B. zonata flies.Also, data in  zonata adults to Agrinal either alone or with added ammonium acetate at different ratios all over the four weeks in mango orchard at Giza governorate, Egypt.
The highest number of females per one male was obtained with ammonium acetate alone (4.50 females) followed by Agrinal alone (4.17 females).While, the lowest number of females per one male (2.17 females) was obtained with AA.2+Ag.1 treatment (Fig. , 3).

By using di-ammonium phosphate:
Agrinal alone was significantly the lowest attractive to B. zonata flies in comparison with the other tested treatments all over the four weeks.On contrary, the mixture of DAP.1+Ag.1 was significantly the highest treatment in attracting B. zonata flies during the first week.But, DAP.2+Ag.1 significantly recorded the highest value of FTD of B. zonata during the second, third and fourth weeks (Table,4).In general, Agrinal alone was the lowest treatment for attracting B. zonata flies which significantly differed with the other tested treatments (Fig. , 4).All of the rest treatments significantly super passed Agrinal alone, but they were insignificantly differed.The mixture of DAP.1+Ag.1 increased the attractability of Agrinal to B. zonata by 4.9 times.The highest number of females per one male was obtained with DAP.1+Ag.1 (8.75 females); while, the lowest was obtained with Agrinal alone (4.17 females) (Fig. , 4).

Enhancement of Amadene: 3.1. By using ammonium acetate:
Data presented in Table (5) show that Amadene alone exhibited the lowest mean numbers of attracted B. zonata flies represented as FTD values during the four weeks, which insignificantly varied with the mixture of AA.1+Am.2during the second and fourth weeks.The mixture of AA.1+Am.1 was significantly more attractive to B. zonata than that of attracted by Amadene alone during the four weeks of investigation.5) show that the grand mean of attracted B. zonata flies was insignificantly higher with AA.1+Am.1 and ammonium acetate alone, but they significantly differed with Amadene alone, which was the lowest attractable for B. zonata flies.So, it can be noticed that adding ammonium acetate to Amadene with a ratio of 1:1 increased the efficiency of Amadene in attracting B. zonata flies by 3.0 times.
On the other hand, all of the tested treatment attracted females more than males (Fig. , 5).The highest mean number of females per one male was recorded with AA.1+Am.2(7.38 females); while, the lowest value was recorded with Amadene alone (3.40 females).zonata adults to Amadene either alone or with added ammonium acetate at different ratios all over the four weeks in mango orchard at Giza governorate, Egypt.

By using di-ammonium phosphate:
As shown in Table ( 6), Amadene alone was significantly the lowest attractive to B. zonata flies in comparison with the other tested treatments all over the four weeks.On contrary, during the first and second weeks DAP.2+Am.1 significantly recorded the highest mean numbers of attracted B. zonata flies, whereas the mixtures DAP.1+Am.2 and DAP.1+Am.1 were the most attractive during the third and fourth weeks for the first and second mixtures, respectively.The differences between them and the other tested treatments were significant.The grand mean of attracted B. zonata flies was significantly lower with Amadene alone.The mixtures of DAP.1+Am.1,DAP.2+Am.1 and DAP.1+Am.2 were insignificantly the most attractable for B. zonata flies, where they significantly increased the attractability than that recorded with Amadene or Di-ammonium phosphate (Fig. , 6).Also, it can be noticed from this figure that the mixture of DAP.1+Am.1 increased Amadene's attractiveness to B. zonata by 6.8 times.zonata adults to Amadene either alone or with added di-ammonium phosphat at different ratios all over the four weeks in mango orchard at Giza governorate, Egypt.
With respect to sex ratio (Fig. , 6), the highest mean number of females per one male was recorded with DAP.1+Am.2(12.54 females); while, the lowest value was recorded with Amadene alone (3.40 females).

DISCUSSION
According to Kaspi et al. (2000) and Yuval et al. (2007), dietary sources of nitrogen have a strong influence on the physiology and behaviour of tephritid flies.Heath et al. (2004) and Leblanc et al. (2010) added that a variety of baits take advantage of the key role that ammonia plays in fruit fly attraction by including ammonia in their formulations.For example, ammonium acetate has been shown to be the most attractive component of Biolure for C. capitata.Also, Pinero et al. (2011) reported a significant effect of adding ammonium acetate to GF-120, regardless of the amount added, for Bactrocera cucurbitae (Coquillet) and Bactrocera dorsalis (Hendel), and a significant positive relationship was obtained between relative amounts of ammonium acetate in the bait and the numbers of C. capitata females responding.
The present results showed that mixing di-ammonium phosphate (3% w/v) to the commercially protein-based baits of Buminal, Agrinal or Amadene (5% v/v) with a ratio of 1:1 significantly increased their attractability to B. zonata flies by ranges of 3.4 to 6.8 times.Also, adding ammonium acetate (3% w/v) to Agrinal or Amadene (5% v/v) with the same ratio significantly increased their attractability for B. zonata flies by 2.5 and 3.0 times, respectively.These results are in agreement with those obtained by Pinero et al. (2015) who documented a significant increment in response of females of C. capitata to some protein-baits tested when ammonium acetate was added to the bait.Bateman and Morton (1981) demonstrated that increases in ammonia produced as a consequence of bacterial degradation of protein were associated with increases in bait attractiveness to females of fruit flies.Also, Mazor (2009) documented low attractiveness of proteinaceous baits that were associated with low release rates of ammonia, whereas other sources of ammonia such as fertilizers (e.g., ammonium nitrate) and manure were found to release much higher rates of gaseous ammonia and this resulted in much higher attractiveness to female C. capitata under laboratory conditions.
With respect to the mixtures between Buminal and ammonium acetate, the present results indicated that there were no significant differences between them and Buminal alone.Also, Pinero et al. (2015) reported that the addition of ammonium carbonate to some protein-baits resulted no significant increase in bait attractiveness to females of C. capitata.Also, Rhagoletis cerasi L. did not significantly attract to ammonium carbonate (Katsoyannos et al., 2000).Even within the same fly species, there was some variability regarding the attractiveness of ammonia-releasing substances.For example, whereas eastern populations of Rhagoletis pomonella (Walsh) showed a low response to ammonium carbonate (Reynolds andProkopy, 1997 andRull andProkopy, 2000), western populations of the same species were found to have the same or a greater level of response to the same ammonium compound compared with fruit-based lures (Yee et al., 2014).The variations between the present results and the others may be attributed to the variation of the tested ammonium compound and/or fruit fly species.
The present study conclusively documents that adding di-ammonium phosphate or ammonium acetate increased the numbers of attracted females in comparison with attracted males especially with Buminal and Amadene.These findings supported by Pinero et al. (2015); who mentioned that adding ammonium acetate to a variety of protein baits and materials, resulting in enhanced C. capitata response, especially for females.The same authors added that ammonium carbonate increased C. capitata female response to the commercial protein-bait of DacGel and showed no significant effects in the other tested commercial protein-baits including Buminal in Hawaii.
In conclusion, the commercial protein-based baits of Agrinal or Amadene can be enhanced by mixing any of them with di-ammonium phosphate or ammonium acetate at a ratio of 1:1; while, Buminal can be enhanced by mixing it with diammonium phosphate at the same ratio.These enhancements resulting higher significant attractability for B. zonata flies especially females.

Fig. ( 1
Fig. (1).Daily mean of sex ratio (famles/1 male) and daily grand mean of attracted B. zonata adults to Buminal either alone or added with ammonium acetate at different ratios all over the four weeks in mango orchard at Giza governorate, Egypt.

Fig
Fig. (2).Daily mean of sex ratio (famles/1 male) and daily grand mean of attracted B.zonata adults to Buminal either alone or with added di-ammonium phosphate at different ratios all over the four weeks in mango orchard at Giza governorate, Egypt.

Fig
Fig. (3).Daily mean of sex ratio (famles/1 male) and daily grand mean of attracted B.zonata adults to Agrinal either alone or with added ammonium acetate at different ratios all over the four weeks in mango orchard at Giza governorate, Egypt.

Fig. ( 4
Fig. (4).Daily mean of sex ratio (famles/1 male) and daily grand mean of attracted B.zonata adults to Agrinal either alone or with added di-ammonium phosphate at different ratios all over the four weeks in mango orchard at Giza governorate, Egypt.

Fig. ( 5
Fig. (5).Daily mean of sex ratio (famles/1 male) and daily grand mean of attracted B.zonata adults to Amadene either alone or with added ammonium acetate at different ratios all over the four weeks in mango orchard at Giza governorate, Egypt.

Fig
Fig. (6).Daily mean of sex ratio (famles/1 male) and daily grand mean of attracted B.zonata adults to Amadene either alone or with added di-ammonium phosphat at different ratios all over the four weeks in mango orchard at Giza governorate, Egypt.

Table ( 1
). FTD values of B. zonata adults to Buminal either alone or added with ammonium acetate at different ratios in mango orchard at Giza governorate, Egypt.In each column, means have the same letter did not significantly differ at probability of 5%.

Table ( 2
). FTD values of B. zonata adults to Buminal either alone or with added diammonium phosphte at different ratios in mango orchard at Giza governorate, Egypt.

Table ( 3). Mean number of daily attracted B. zonata adults to Agrinal either alone or with added ammonium acetate at different ratios in mango orchard at Giza governorate, Egypt.
During the four weeks of investigation, AA significantly recorded the highest values of attracted adults of B. zonata.Unfortunately, adding Ammonium Acetate to Agrinal did not enhance attractability for B. zonata adults.

Table ( 4
). FTD values of B. zonata adults to Agrinal either alone or with added diammonium phosphate at different ratios in mango orchard at Giza governorate, Egypt.

Table ( 5
). FTD values of B. zonata adults to Amadene either alone or with added ammonium acetate at different ratios in mango orchard at Giza governorate, Egypt.

Table ( 6
). FTD values of B. zonata adults to Amadene either alone or with added diammonium phosphate at different ratios in mango orchard at Giza governorate, Egypt.