Current report of Silba adipata (Diptera: Lonchaeidae) attacking local fig (Ficus variegata) fruits in Bali: Molecular identification, invasion, and preference

Silba adipata is a new pest attacking white cayenne pepper (Capsicum frutescens L.) whose attack symptoms are similar to those of Bactrocera dorsalis. The S. adipata was also reported to attack local fig (Ficus variegata). This study aimed to ascertain the type of black fruit fly on F. variegata, the invasion time of S. adipata attack on F. variegata fruit, and the potential of F. variegata fruit extract as an attractant for the black fruit fly. The methods used for objectives (1), (2), and (3) were CO1 analysis, a survey method, and a preference test, respectively. The results showed that the species of black fruit fly that attacked local figs (F. variegata) in Bali was S. adipata. S. adipata invades fruit at the age of 10 weeks after the formation (WAF) of the ovary; the highest attack occurs on fruit 13 days after the formation of the ovary. Local fig fruit extract effectively attracts black fruit flies. Local fig extract was effective in attracting S. adipata to chili plants. Concentrations of 20%-100% were significantly effective in attracting adult S. adipata compared to the control. The extract concentration of 60% was highly effective in attracting S. adipata adults.


Introduction
Silba adipata is a new pest attacking cayenne pepper fruit in Bali, with attacks reaching 40.31% [1,2].The problem has never been reported before in Bali and even in Indonesia.Farmers assume that the symptoms of the attack are caused by known fruit fly pests from the Tephritidae family, including the species Bactrocera dorsalis complex [3].So, control measures aim to control this type of pest [1].After a search, the pest S. adipata was first reported to attack figs (Ficus carica L.) in the Mediterranean and Middle East [4].It then spread to Iraq [5], Japan [6], and Turkey [7].In 2007, S. adipata was reported to attack figs in South Africa [8].In 2015, S. adipata was first reported as a pest infesting all fig plantations in Tunisia, with infestation reaching 88.17% [9].
Preliminary research found that local figs (F.variegata) are also attacked by a black fly pest morphologically resembling Silba.Pest identification is very important as a basis for plant protection.Identification is classifying an organism to a certain status, either taxon or based on the economic losses it causes.Morphological identification of Silba sp. is distinguished through eye color, abdominal color, and ventral abdominal features [1].The method used to trace the phylogenetic relationship of Silba sp. by looking at the similarity of its mitochondrial DNA.Silba sp.DNA identification uses the cytochrome oxidase subunit I (COI) gene as a marker (genetic barcode).COI gene in molecular studies is one of the genes that can be used as a genetic marker to study the genetic characteristics between species and between individuals [10,11,12].Molecular identification will obtain certainty of the type of fly that attacks F. variegata fruit so that it becomes the basis for conducting further research to trace the invasion and preference of S. adipata on F. variegata fruit.Invasion is the entry of pests into cultivated plants to carry out feeding and nesting activities.Generally, invasion occurs due to resource competition, which results in the insect experiencing stress, such as food shortage and discomfort.To survive, the insect moves to another place [13].Adult S. dipata actively flies at 08.2.00 [14].Silba sp.utilizes the fruit of F. variegata as an alternative host, so it is necessary to explore what age of fruit S. adipata begins to attack and what age of fruit is the highest preference level of Silba sp.towards the age of F. variegata fruit.Preference is the level of preference for food or food [15].With known invasion and preference, it can be assumed that the age of the fruit contains chemical compounds that can be used as volatile compounds to attract S. adipata.
Volatile compounds have been studied in several plant species as attractants for several types of insect pests.Research on attractants derived from nutmeg essential oil effectively attracts B. dorsalis fruit flies [16].Research on dried fig fruit extract (F.carica L.) also attracts S. adipata to F. carica plants [9].Based on these data, a survey of the Silba attack on F. variegata found in Bali was conducted.The plant has not been widely known for its potential as an attractant because research on the plant still needs to be undertaken.Testing the potential of F. variegata fruit extract in controlling S. adipata is important for controlling S. adipata.This study aimed to ascertain the species of black fly pests that attack F. variegata plants, their invasion time, and their preferences and to test the potential of F. variegata as an attractant for S. adipata.

Time and Place
This study was conducted in several locations.Observations to ensure the most interesting age of F. variegata fruit in searching and finding black fly host plants were carried out in Prerenan Village, Mengwi District, Badung Regency, Bali Province, from March 2023 to April 2023.Infested F. variegata fruits were sampled for molecular identification, and F. variegata fruits were sampled for extracts in Keramas Village, Blahbatuh District, Gianyar Regency, Bali Province.The infested fruits were then maintained at the Integrated Pest Management Laboratory, Faculty of Agriculture, Udayana University.from March 2023 to April 2023.After the flies were hatched, they were identified molecularly at Genetics Science (West Jakarta).Observations on the age of infested fruits and the collection of S. adipata infested fruits were carried out in Prerenan Village and Keramas Village because local fig trees (F.variegata) were found in those locations, and after preserving the fallen figs, black fly pests suspected to be S. adipata were found.F. variegata fruit extract was prepared at the Genetics and Molecular Biology Resource Laboratory of Udayana University from April 2023 to May 2023.F. variegata fruit extract was tested on chili plants in Bonyoh Village, Kintamani District, Bangli Regency, Bali Province, Indonesia.

Identification Molecular
Sample preparation for molecular analysis was carried out by purposively collecting infested F. variegata fruits in the field; 50 infested fruits were taken and then maintained in the laboratory by preparing plastic cups (diameter = 8, High = 10.5 cm).Plastic cups were filled with 20 g of sand; the sand was moist using water, and one plastic cup was inserted with one infested F. variegata fruit covered with gauze and labeled with the sampling date, maintained until adult flies appeared.Then, molecular identification was carried out.

Determining the age of F. variegata fruit that most attracts black flies
Observations were made during the forming of F. variegata fruit ovules by marking the fruit ovules with tape (Figure 1).Samples were marked as many as ten fruit ovules with ten replicates.Samples were observed and recorded at the most interesting fruit age in the process of searching and finding black fly host plants.Observations were made every day.

Extract Test F. variegata fruit in the field
The stage of testing compounds that are effective as attractants to attract S. adipata was carried out using F. variegata fruit extract.The trap used was made from a 1.5-liter plastic bottle.The bottle was perforated on four sides; then a clear hose was inserted.The bottle cap was perforated to place the hanger.F. variegata fruit extract (5 g) was placed in a cylindrical bottle with a diameter of 2 cm and a height of 3.5 cm and then hung inside the trap.The traps were then hung at a height of 1 meter on the cayenne pepper plantation (Figure 2).The treatmentments used in this study are listed in (table 1).This research was conducted using the Field Research Method to determine the effectiveness and efficiency of using F. variegata fruit extract attractants.Field research was conducted on cayenne pepper plantations with an area of 3,600 m2.This study used F. variegata fruit extract attractant treatment and vaseline as a control.Traps totaled 24.Traps were set with a distance of 10 m between traps.The basic design used was a group randomized design.
The number of replicate blocks was determined based on the number of treatments (p), thus fulfilling (p-1) (u-1) ³ 15 [17]; for p = 6, then u = 4 (Figure 3).Data were analyzed using variance analysis with SPSS version 24.Observations of the number of catches were made every day from when the trap was set until no flies were trapped.The number of catches was counted and recorded, and data were analyzed.

Identification molecular
DNA isolation is carried out to obtain DNA templates in the amplification process.The PCR process has successfully amplified, which is shown through electrophoresis results in (figure 4).Electrophoresis results indicate the presence of clear and thick DNA bands as evidence of successful amplification [18].The PCR results were sequenced to determine the nucleotide sequence.The sequencing results of Silba sp.based on the COI gene were searched for homology in GenBank using BLAST software, and several nucleic acid sequence data of Silba sp.species were found in GenBank data that had similarities with S. adipata found in F. variegata plants.BLAST results obtained 100 data from GenBank with Query Cover values ≥ 97% and identical levels ≥ 89.93%, as described in (Table 3.1).Silba sp.sequence data in GenBank that have similarity with sequence data obtained in F. variegata plants are S. adipata (OM949837.1),(OM949836.1), (OM949835.1),(OM949834.1),(OM949833.1),(OM949832.1),(OM949831.1),(OM949831.1),and (OM949830.1).with a Query Cover value of 97% and an identical level of 89.93%.Genetic characteristics based on the level of homology of the COI gene DNA sequence and phylogenetic analysis have close genetic characteristics with S. adipata in GenBank data; this is shown by the phylogenetic tree presented in (figure 5).While Merta [1] previously in Bali also identified molecularly the type of pest that attacks cayenne pepper fruit in Bali is S. adipata, which is evidenced by molecular analysis with a Query Cover value ≥ 96% and an identical level ≥ 88.74%.

Invasion and Preference of Silba adipata on F. variegata Pruit
The invasion and preference study of adult S. adipata showed that F. variegata fruit aged 1 to 9 weeks after fruit formation had not been attacked by S. adipata.S. adipata attack began at fruit age 13 to 15 weeks after fruit set.Sequence from 16 to 22 weeks after the fruit set was not attacked by S. adipata (figure 6).The most favorable fruit age for egg laying is 13 weeks after fruit set (figure 7).This can be proven from the number of adult S. adipata attacks; namely, when the fruit was ten weeks after fruit formation, there were three attacked fruits; when the fruit was 11 weeks after fruit formation, there were five attacked fruits; when the fruit was 12 days old there were 12 attacked fruits when the fruit was 13 weeks after fruit formation there were 65 attacked fruits when the fruit was 14 weeks after fruit formation there were three attacked fruits.When the fruit was 15 weeks after fruit formation, there were two attacked fruits (figure 8).Merta [1] also researched the age of cayenne pepper fruit, which was favored as egg-laying by adult females of S. adipata pests.Cayenne pepper fruit was aged 15 days after fruit formation.This is because, at that age, the chili fruit is not too hard, making it easier for adult females to penetrate the ovipositor to lay eggs.Fruit age of 15 days is also preferred because the chili fruit starts to turn yellowish at that age.The color and aroma of the fruit determine fruit fly activity in finding host plants.Fruit flies prefer fruits that are ripe and yellowish compared to other colors.When the yellow color begins to appear on the fruit, the female fruit fly can recognize its host to lay eggs [19].According to Gautama and Redana [14,20], effective traps for detecting and monitoring S. adipata pests are bright yellow adhesive traps.Likewise, yellow attracts other types of pests in searching for hosts, namely Thrips parvispinus Karny [21].
According to the National Aeronautics and Space Administration [22], yellow has a wavelength of 570 nm.Meanwhile, fruit flies can capture light wavelengths ranging from 400 -600 nm [19].This may be related to the nutritional requirements of S. adipata larvae for F. variegata fruit, as the biochemical composition of the fruit changes during the ripening process [23].F. variegata fruit undergoes color changes and possibly changes in biochemical composition according to fruit age (figure 6).This study's results align with those of [24], who found that citrus plants produce certain chemical compounds attractive to insects, and changes in the chemical composition of citrus fruits cause changes in fruit sensitivity to pests [25].Masriany [26]

Extract test F. variegata fruit
The number of S. adipata caught in the 20%-100% treatment differed significantly from the control.The highest number of catches in the 100% treatment amounted to 23.25 individuals but was not significantly different from the 80% (22 individuals) and 60% (21.5 individuals) treatments.The 40% and 20% treatments were 14.75 and 6 fish, respectively (table 3).Plant phytochemical compounds that are very instrumental in searching, recognizing, and matching host plants are generally from the terpenoid group [27].Terpenoids are a group of compounds that provide taste, smell, and color in plants.Terpenoids are usually found in the leaves and fruits of higher plants [28].Hexanol (C6H14O), a natural substance found in essential oils of plants [29], also extracted from dried figs [30], was the most attractive substance for capturing up to 60 flies/trap/day with a 3:1 ratio of females to males.According to the same authors, combining hexanol with ammonium sulfate (2%) increased its attractiveness to S. adipata by three times.

Figure 1 .
Figure 1.Prospective sample fruit F. variegata 2.4 Extract Test F. variegata fruit in the fieldThe stage of testing compounds that are effective as attractants to attract S. adipata was carried out using F. variegata fruit extract.The trap used was made from a 1.5-liter plastic bottle.The bottle was perforated on four sides; then a clear hose was inserted.The bottle cap was perforated to place the hanger.F. variegata fruit extract (5 g) was placed in a cylindrical bottle with a diameter of 2 cm and a height of 3.5 cm and then hung inside the trap.The traps were then hung at a height of 1 meter on the cayenne pepper plantation (Figure2).

Figure 2 .
Figure 2. Form a trap for adult flies of S. adipata with local fig extract attractant

Figure 3 .
Figure 3. Randomization of local fig fruit concentration levels in the experimental area.

Figure 4 .
Figure 4. Agarose Gel Analysis of S. adipata found at 750 bp

Figure 5 .
Figure 5. Tree phylogenetics of the S. adipata COI gene (IclQuery_31829) were compared with data from NCBI with level similarity closest and comparable (Outsider) (Source www.ncbi nim here gov)

Table 2 .
Level of identical data in genbank that has simplicity with data on S. adipata in planting chilli white rawit (Source: www.ncbi.nlm.nih.gov )

Table 3 .
Significance test extract fruit F. variegata Based on molecular analysis, the species attacking local figs (F.variegata) in Bali is S. adipata (query cover value of 97%), which has never been reported to attack F. variegata fruit in Indonesia.The first invasion of S. adipata infested F. variegata occurred ten weeks after the fruit set, with the highest infestation occurring 13 weeks after the fruit set.Local fig extract was effective in attracting S. adipata to chili plants.Concentrations of 20%-100% were effective in attracting adult S. adipata, but the most effective was 60%.