Effect of natural attractant essential oil from Ocimum gratisimum from Timor Island against fruit flies

Ocimum gratisimum, well-adapted to arid regions in East Nusa Tenggara, has essential oil that attracts fruit flies, pest important affecting crops like mango and jackfruit. This study aims to assess the impact of O. gratisimum essential oil on the diversity of fruit flies on these plants. The essential oil, source from leaves of O. gratisimum, was obtained from the Bioscience Laboratory of the University Nusa Cendana, East Nusa Tenggara, Kupang, Indonesia. It was applied to mango and jackfruit plants in Baumata Village, Taebenu District, Kupang Regency, East Nusa Tenggara. For experiment, total of 0.2 µL of O. gratisimum essential oil was carefully placed on a cotton swab, which was subsequently positioned within traps constructed using 1.5-L mineral bottles. The results of this study indicated that the essential oil extracted from O. gratisimum leaves had the capacity to attract Bacrtocera umbrosa and Bactrocera dorsalis flies when applied on mango plants. In case of jackfruit plants, the traps successfully lured B. umbrosa, B. dorsalis, and Bactrocera musae. This attraction contains methyl eugenol in the oil derived from O. gratisimum.


Introduction
Fruit flies belonging the Diptera Family Thripitidae are not only pests important but also as a serious biosecurity threat in international trade.This threat is particularly pronounced in various horticultural crops [1][2][3][4].If leaf uncontrolled, these pests causes crop damage up to 100% if not controlled.The resulting yield losses have a substantial impact on economic growth, amounting trillions of rupiah [5].Fruit flies fall into the quarantine category of pest [6,7], which means that fruit infested by these flies cannot be exported.
To curb the spread of these pests, several countries have implemented regulation through their respective Department of Plant Quarantine.For example, the Department of Plant Quarantine in Japan, Europe, and the United States required horticultural commodities, both imported and exported, must be free of fruit flies and pesticide residues in global trade [8].This stringent requirement is applicable to all agriculture commodity products to ensure that they are free fruit fly infestations and pesticide residues.For example, Japan, Australia, and South Korea do not accept mangoes from Indonesia due to the absences of fruit fly species form Indonesia in these countries.
Fruit flies have many species and are polyphagous.More than 500 species of fruit flies in the world have been identified from genera Anastrepha, Bactrocera, Ceratitis, Dacus, Rhagoletis, and Toxotrypana; and 35% of them are important pests [9].The fruit fly of the Genus Bactrocera has species more than other genera, with more than 500 species spread in the tropics and subtropics [10].In Indonesia, 44 species of fruit flies from Genus have been identified as damaging 45 species of host plants [11].
Control of fruit flies involves the integrations of methods, including collection and destruction of infested fruit to disrupt the life cycle of larvae and pupae cycle, the utilization of sex pheromones, plant repellent, and attractant.The three control techniques are more effective, economical, environmentally friendly, and safe for health.In general, fruit flies control with sex pheromone use methyl eugenol compounds found in the sex pheromone glands of fruit flies [12,13].Methyl eugenol, chemically know as 4-ally 1-1,2-dimethoxy-benzene-carboxylate, serve as a synthetic compound to attract males fruit flies [3] and can also be naturally present in various plants containing essential oils [14,15].
One of the plant genera, Ocimum is rich and possesses properties that make it suitable for use as a pesticide, attractant, anti-inflammatory, and anti-carcinogenic [13].Due to its volatile nature, Ocimum plants can effectively function as attractant for fruit flies [16)].When used as pesticide, all parts of Ocimum sanctum can attract fruit flies, specifically Bactrocera cucurbitae on mango and guava [17].Essential oils extracted from basil have been demonstrated to attract species fruit flies [13].
In dryland regions, particularly in Timor Island, East Nusa Tenggara Province, a local variety of basil known by its scientific name, Ocimum gratissimum, is abundant.This plant grows naturally, is easy to cultivate, an is known for its rich flavour.Given its affiliation with the Ocimum genus, there is a likelihood that it may serve as an attractant for fruit flies as well.Plants containing essential oils have been collected since 2019 and have been experimented against several pathogens.Research on the use of essential oil from O. gratissimum has not been conducted before, so it needs to be experimented against fruit flies in 2021 and re-experimented in 2022 and 2023 on other hosts.To assess this potential, the study aimed to evaluate the effectiveness of O. gratissimum essential oil in attracting fruit flies on Mangifera indica and Artocarpus heterophyllus Lam, both of which are important crops in East Nusa Tenggara, frequently affected by fruit fly damaged.The research also aimed to identify the diversity of fruit fly species attracted to this essential oil.

Materials and Methods
Essential oil O. gratisimum was collected from the Bioscience Laboratory of the University of Nusa Cendana, East Nusa Tenggara, Kupang.It was applied to jackfruit and mango plants in Baumata Village Taebenu District, Kupang Regency, East Nusa Tenggara (Figure 1).Treatment is applied to mango and jackfruit plants that are currently bearing fruit.This research was conducted from February to December 2021.

Essential oil distillation
Leave O. gratissimum collected from island Timor and then dried at room temperature for approximately two weeks.The dried leaves of O. gratissimum were mashed using a blender until became flour.The flour was taken as much as 500 gr by steam-water distillation for three hours.The first distillate produced was immediately separated from the oil using a separator funnel.Furthermore, the distillate was extracted using diethyl ether as a solvent, thus forming two layers, namely to the top layer which was the organic phase and the bottom layer was the aqueous phase.The organic layer was then separated using a separator funnel and removed the water content using anhydrous Na 2 SO 4 .The separated organic layer is then filtered and the solvent is evaporated.

Application of essential oil to Mangifera indica and Artocarpus heterophyllus
Traps made a 1.5 L mineral water bottle.The mouth bottle was cut and put back in an inverted position to make it easier for fruit flies to enter the traps.The bottle cap is removed as a door for fruit flies to enter the trap.One side of the bottle in the middle is perforated to insert a wire measuring 3 cm with a length of 20-30 cm.The base of the wire contained in the bottle is given of cotton, while the other end made placed to hang the plant.
A total of 0.2 µL of O. gratisimum essential oil was absorbed into a cotton swab.In the traps, 0.05 µL of deltamethrin insecticide was given so that the fruit flies that entered the trap fainted or died.Traps are installed in a horizontal position, at a height of 2-3 meters from the ground.Traps placed horizontally.Applications are given once during the experiment to see the durability of O. gratisimum essential oils.Observations were made on the number and species of fruit flies trapped every two days.The observed variable is the number of trapped fruit flies, the number of trapped male and female fruit flies, and the number of trapped species.
Fruit flies that entered the trap at each observation were removed from the traps with a brush and put into a plastic box measuring 10 cm x 15 cm x 5 cm.The sex and species were fruit flies identified.In general, the sex of fruit flies is observed at the tip of the abdomen.Male flies have a blunt abdomen, while female flies have a pointed abdomen.Based on these characteristics, all species of male fruit flies that enter the trap come from mango and jackfruit plants.

Data analysis
Data on each species and quantity of fruit flies obtained from mango and jackfruit plants are calculated for species abundance.An abundance of the diversity of fruit flies trapped using the formula percentage abundance [18]. (1)

The effect of Ocimum gratisimum local basil oil on fruit flies catching.
When O. gratissimum essential oil traps were placed on mango plants, there were no observable signs of fruit fly damage on mango fruit.In contrast, jackfruit exhibited clear symptoms of fruit fly infestation.These symptoms on jackfruit included necrotic spots with enlarging holes, often accompanied by the presence of black pathogenic mycelium in the vicinity.The necrotic spots, resulting from punctures made by fruit fly ovipositors, created favorable conditions for secondary pathogens to thrive and expedited the deterioration of the fruit.To confirm the involvement of fruit flies in these symptoms, jackfruit was dissected, revealing fruit fly larvae responsible for the damage.
The observed symptoms and the presence of fruit fly larvae on host plants were the outcomes of the communication process or semi chemistry between fruit flies and secondary metabolites or ∑ individual species trapped Abundance (A) = X 100% ∑ sum of all species trapped phytochemical compounds produced by the host.This suggests that female fruit flies recognize jackfruit as a suitable host and deposit their eggs on the fruit's surface using an ovipositor.
The study results indicated that a higher number of fruit flies were captured in the O. gratissimum essential oil traps placed on mango plants compared to those placed on jackfruit plants (Table 1).The average population of fruit flies trapped in mango was 16.83%, while on jackfruit plants, it was 15.16%.On mango plants, the peak population of fruit flies entering the traps occurred on the second day after the treatment (38 fruit flies).Conversely, on jackfruit plants, fruit flies began entering the traps between the fourth and sixth day after treatment (DAT).Subsequently, the number of fruit flies started to decrease from the 10 th to the 12 th DAT.By the 14 th to 16 th DAT, no fruit flies were observed entering the traps.This suggests that from the 2 nd to the th DAT, the concentration started to diminish by the 10 th DAT.Consequently, male fruit flies could no longer recognize the sex pheromone compounds.Leading to a decline in trap entries.This decline in scent detection was also experienced by the research team during their observations.From the 2 nd to 8 th DAT, it began to wane at the 10 th DAT and could no longer be detected by the researcher's sense of smell by the 12 th to 14 th DAT.
The results showed that O. gratissimum essential oil was applied to mango and jackfruit plants, and it effectively attracted fruit flies.A total of 192 fruit flies were captured from two traps, one placed on each type of plant, using a 0.2µL dose.Remarkably on the 2 nd DAT (during the initial observation), male fruit flies were already present in the traps located on the both mango and jackfruit plants.In particular, in the case of jackfruit plants, within one hour of setting up the O. gratissimum essential oil trap, several fruit flies were observed hovering around the trap, although they had not yet entered it.This observation suggests that the antenna of male fruit flies is capable of recognizing O. gratissimum essential oil as a sex pheromone compound released by females to facilitate copulation, even at a dose as low as 0.2µl.Fruit flies trapped in each observation on mango plants are dominated by B. dorsalis compered to B. umbrosa.The number of B. dorsalis fruit flies on the second day of observation after application reached 37 individuals.The population then decreased over time, reaching its lowest point on the 12 th DAT.B. umbrosa fruit flies were recorded as 4 individuals, trapped on the 2 nd , 4 th , and 10 th DAT.However, on the 12 th and 14 th days, no B. umbrosa were trapped (Figure 2).
On A. heterophyllus plants, the number of B. dorsalis fruit flies trapped on the 2 nd , 4 th , and 6 th DAT continued to increase.On the 8 th observation, the population began to decrease.Meanwhile, the population of B. umbrosa trapped on the A. heterophyllus increased from the 2 nd to the 8 th DAT.The number of trapped insects did not exceed 10 individuals.The same occurred with B. umbrosa fruit flies, with the population starting to decreased on the A. heterophyllus plants (Figure 3).

The abundance of fruit fly species trapped in Ocimum gratisimum oil
The results revealed that the O. gratissimum essential oil traps, placed on both mango and jackfruit plants, captured three distinct species of fruit flies.On mango plants, the traps captured two fruit fly species, namely B. dorsalis and B. umbrosa.These same two species were also found in traps set on jackfruit plants, along with the addition of B. musae.
The abundance of these fruit fly species trapped in the essential oil O. gratissimum varied between mango and jackfruit plants.The abundance of B. umbrosa on jackfruit plants is 19.78%, whereas on mango plants, it was 3.96%.In contrast, the fruit fly species B. musae was exclusively found on jackfruit plants, with an abundance of 1.10% (Table 2).This indicates that B. dorsalis is the primary host for both mango and jackfruit plants, while B. umbrosa primarily targets jackfruit.Bactrocera musae, although not a primary host for jackfruit, possibly migrate from other hosts in the surrounding ecosystem and stop jackfruit.This study identified three fruit fly species that are associated with mango and jackfruit plants.Among the mango plants, two fruit fly species, namely B. dorsalis and B. umbrosa.On the other hand, in the case of jackfruit plants, three fruit fly species were detected B. dorsalis, B. umbrosa, and B. musae.It's worth noting that all the fruit flies captured in the traps were male, as the O. gratissimum attractant specifically lured male flies for mating with females.However, it is important to recognize that within this ecosystem, female flies also visit jackfruit fly infestations observed on jackfruit plants, where female flies lay their eggs on the host's surface, leading to larval development and subsequent damage to the host.The presence of fruit flies on these host plants can be attributed the composition of primary and secondary metabolism in the host plants, which contribute to the fruit flies' survival [19].In theory, plants produce secondary metabolites as a defense mechanism against potential threats, which can serve as signals to attract other species to from associations and act as natural enemies to protect the plants.Secondary metabolites, in this context, also function as volatile compounds emitted by various plant organs, serving as cues for species that pose a threat to the plants or as kairomones.Volatile compounds produced during fruit ripening play a crucial in the selection and host-searching behavior of male and female fruit flies [20].
The outcomes of the experiment assessing the attractant properties of O. gratissimum essential oil demonstrated its ability to lure three distinct fruit fly species, namely B. dorsalis, B. umbrosa, and B. musae, from both M. indica and A. heterophyllus plants when used at a dosage of 0.2 µL/trap.Notably, fruit flies were observed in each trap as early as the 2 nd DAT.This suggests that the 0.2 µL dosage of O. gratissimum essential oil contains an attractant compound in the form of methyl eugenol, which is perceptible to the olfactory sense of male fruit flies.The results of the GC-MS analysis showed that one of the secondary metabolites present in O. gratissimum essential oil is indeed methyl eugenol.Additionally, other studies reported that O. gratissimum essential oil contains around 54% methyl eugenol [13,21,22].Methyl eugenol serves various roles, including its involvement in sexual communication and aggregation [23], source of food for male fruit flies [24], its power attracts male flies [25], increases copulation of male flies, source of protein, and reduces stored female sperm (due to exposure to male flies contains methyl eugenol [26,27].
In our study, we opted for vegetative leaves since they were more abundant than leaves in the generative phase.The vegetative phase is characterized by higher concentrations of methyl eugenol, especially within organs leaf and trichomes [28,29].
The essential oil derived from basil on Timor Island contains methyl eugenol, which effectively attracts three fruit fly species for a period of 12 th DAT when applied to mango and jackfruit host plants.However, by the 14 th DAT, no fruit flies were found in the trap.It is suspected that the attractant compounds within O. gartissimum essential oil diminish due to exposure to sunlight.Consequently, it become undetectable to male fruit fly's olfactory senses.Methyl eugenol tends to evaporate rapidly when subjected to prolonged exposure to UV-B radiation and high temperatures [29].The essential oil's shelf life for attracting fruit flies is notably short compared to synthetic methyl eugenol, making it essential oil to incorporate specific proteins that can extend the durability of methyl eugenol.
The male B. dorsalis captured in the trap is a result of semi chemical interactions facilitated by its olfactory sense, which respond to sex pheromone compounds present in the O. gratissimum essential oil.This interaction is possible because the antenna of B. dorsalis likely contain an array of proteins that play crucial role in sensing external stimuli, including compounds that attract male flies.The antenna of the B. dorsalis fruit fly has been found to contain various types of proteins, including 20 candidate smell-binding proteins (OBH), 5 candidate chemosensory proteins (CSP), 35 odor receptors (OR), 12 ionotropic receptors (IR), and 4 sensory neuron membrane protein (SNMPs) [27].The interplay of these smell-binding proteins, chemosensory proteins, smell-receptors, ionotropic receptors, and sensory neuron proteins within the antenna of B. dorsalis allows for detection of the male-attracting compounds present in the O. gratissimum essential oil.
In addition to the essential oil in O. gratissimum, there are also various types of other secondary metabolite such as alkaloids, steroids, and others.These compounds have shown the ability to impede the growth Colletotrichum musae causing anthracnose disease in plant bananas [30].Moreover, the essential oil exhibits toxicity towards Artemia salina larvae and inhibitory effects against the development of several disease-causing agents in humans, such as Candida albicans, Escherichia coli, and Staphylococcus aureus [31].Additionally, it has been observed to deter egg-laying and the emergence of adults of Callosubruchus maculatus [29].Thus, O. gratissimum is recommended as a natural attractant for monitoring the progress and containment of the fruit fly population, as well as for the identification and trapping of these insects.The essential oil of O. gratissimum attracts B. dorsalis, B. umbrosa, and B. musae.The durability of the essential oil is 8 th -10 th DAT.Furthermore, Ocimum grastissimum represents an environmentally friendly alternative for pest control.

Conclusion
The leaves of O. grastissimum contain essential oil, which includes methyl eugenol, a secondary metabolite compound that plays a role in attracting male fruit flies.The application of essential oil from grastissimum leaves on mango and jackfruit plans has successfully captured three species of flies, namely B. dorsalis, B. umbrosa, and B. musae.Essential oil exhibit volatile properties that enable fruit flies to detect them within 10 to 12 days.

Figure 1 .
Figure 1.Map of Baumata Village, research location

Figure 2 .Figure 3 .
Figure 2. The development of fruit fly populations trapped on Mangifera indica

Table 1 .
The population of fruit flies trapped in oil trap Ocimum gratisimum on Mangifera indica and

Table 2 .
The abundance of species of fruit flies caught in traps