New association between Cecidochares connexa (M.) (Diptera: Tephritidae) and local parasitoids: Revisiting classical biological control

Asosiasi baru antara Cecidochares connexa (M) (Diptera: Tephritidae) dan parasitoid lokal: Sebuah tinjauan terhadap pengendalian hayati klasik

Abstract

The gall fly Cecidochares connexa (L.) (Diptera: Tephritidae) was introduced in Bantimurung, Polewali Mandar Regency,  South Sulawesi in 1999, to control the Siam weed Chromolaena odorata, an invasive weed from Florida, United States. However, some previous studies have shown that C. connexa can be attacked by local predators and parasitoids, which can affect the effectiveness of C. connexa in the field. The objective of the research is to study the new interaction and association between  the introduced C. connexa, and local parasitoids. The research was conducted in April-December 2021 at two habitats namely open fields and cocoa plantations, in four villages of Polewali Mandar Regency. Purposive sampling was conducted to collect C. odorata infested by the gall flies. Thirty C. odorata samples were collected from three different sites at each habitat and each village. The galls were grouped into two different categories i.e. those with and with out holes. The galls with holes were then reared until the insects emerged and the insect specimens were identified. The research results showed that the gall numbers, healthy and parasitized C. connexa at opened areas were significantly higher as compared to cocoa plantations. Parasitism phenomenon indicated the new interaction and association between the C. connexa with the local parasitoids. The parasitization rate of C. connexa in the opened fields (23.24%) was not significantly with in the cocoa plantations (28.84%). On the parasitized C. connexa were found four families of parasitoids i.e. Eulophidae, Braconidae, Eupelmidae, and Ormyridae. The dominant parasitoid found was Quadrastichus sp.1, with the parasitization rate of 14% in open fields and 10% in cocoa plantations.

INTRODUCTION

Biological control is defined as the activity of parasitoids, predators, or diseases in maintaining the other organism’s population density at a lower average than would occur in their absence (DeBach, 1964). Classical biological control involves the introduction of a biocontrol agent to a new habitat to control pests that have spread beyond their original habitat. According to (Caltagirone, 1981), classical biological control is the regulation of exotic pest populations (insects, mites, mammals, weeds, pathogens) by exotic natural enemies due to the lack of local natural enemies that can suppress the exotic pests, thus creating a favorable situation for invasive species to spread. It works on the premise that old associations between the host (pests) and its biocontrol agent have already been established (co-evolved), and thus should be an effective control agent (Hokkanen & Pimentel, 1989)

The most famous classical biological control was the introduction of the vedalia beetle predator Rodolia cardinalis (Coleoptera: Coccinellidae) to control the cottony cushion scale Icerya purchasi (Hemiptera: Margarodidae) in California(Source Title, 1978)(Caltagirone & Doutt, 1989) The introduction of R. cardinalis is regarded as the beginning of classical biological control. The use of biological agents for weed management started in 1795 when the cochineal insect Dactylopius ceylonicus from Brazil was introduced to India to control the invasive weed Opuntia monacantha (Willd.) Haw. (Cactaceae) (Winston et al., 2014) By 2012, a recorded number of 1,555 cases of biological control of weeds were reported in 90 countries in which 468 biocontrol agents were introduced to manage 175 species of weeds in 48 plant groups (Schwarzlander et al., 2018) However, only 982 (63.2%) introductions demonstrated stability, with a total of 332 (70.9%) biological agents used (Schwarzlander et al., 2018)

In Indonesia, Cecidochares connexa (M.) (Diptera: Tephritidae) is a gall fly that has been used as a biocontrol agent to suppress the population of the invasive species Chromolaena odorata (L) R.M. King and H. ob (Asteraceae). C. odorata is a fast-growing invasive species that entered Indonesia in 1934 at the Lubuk Pakan tobacco plantation in North Sumatra and spread rapidly to other major Indonesian islands(Tjitrosoedirdjo, 2005)(Setyawati et al., 2015)(Padmanaba et al., 2017) C. connexa has been established in several countries, including the Philippines, India, Guam, and Papua New Guinea(Aterrado & Bachiller, 2002)(Bhumannavar & Ramani, 2006)(Reddy et al., 2010)(Day et al., 2013) The success of its establishment was one of the reasons for its introduction to Indonesia. (McFadyen et al., 2003) reported that C. connexa causes galls on the terminal and axillary vegetative meristems of C. odorata, and can decrease seed production by 50% and reduce C. odorata population by up to 37,2% within two years (Tjitrosemito, 1999)

C. connexa was first introduced in Indonesia in 1993 from Colombia, USA (Tjitrosemito, 1999) In 1995-2001, releases were carried out in several areas, including eastern Indonesia, one of which was South Sulawesi. The first release of C. connexa in Sulawesi was conducted in the Bantimurung sub-district (60 km northeast of Makassar), South Sulawesi, in March 1999, with a total of 240 galls. A second release was carried out in February 2000 at two different locations, namely the Bantimurung sub-district (60 km northeast of Makassar) with a total of 300 galls and Camba sub-district (25 km northeast of Bantimurung) with 232 galls (Wilson & Widayanto, 2004) In April 2002, the establishment of C. connexa was evaluated at release sites. C. odorata infested by C. connexa was found approximately 10 km from the release point (Wilson & Widayanto, 2004)

Since 2002, no studies have evaluated the effectiveness C. connexa in Bantimurung, South Sulawesi, nor have any studies been conducted to see the changes in the food web interaction due to the introduction. As mentioned by (Pearson & R.M, 2005), biocontrol agents introduced in new areas could indirectly change the interactions in the ecosystem and attack non- targets through food web subsidies or host change. This interaction can change the overall food web and interactions of many species in an ecosystem. One example of research related to food web subsidies is the introduction of Urophora affinis and U. quadrifasciata to control Centaurea maculosa and C. diffusa in western North America (Winston et al., 2014) Their research showed that the two gall flies that were introduced became an additional food source for populations of generalist predators, deer mouse Peromyscus maniculatus(Pearson et al., 2000)(Ortega et al., 2004)(Winston et al., 2014) Another study on ecosystem changes was conducted by (Jaya, 2006)and (Lukvitasari et al., 2021) who showed that C. connexa could be attacked by local parasitoids and predators. Several types of parasitoids have been found to attack C. connexa including those from the Families Ormyridae, Eupelmidae, Bethylidae, Braconidae, Ichneumonidae, Encyrtidae, Eucoilidae, Eulophidae, Eurytomidae, Pteromalidae, and Torymidae(McFadyen et al., 2003)(Safi’i, 2006)(Buchori et al., 2020)(Lukvitasari et al., 2021) These findings show that there is a new association between C. connexa and local parasitoids, which invokes the old hypothesis of (Hokkanen & Pimentel, 1989) regarding new associations that can be formed between biological control agents and local natural enemies. Thus, biological agents introduced in new areas can potentially become new hosts for natural enemies.

The aim of this research is to study community interactions and possible new associations between C. connexa and local parasitoids in Polewali Mandar, West Sulawesi. Does the establishment of the bioagent C. connexa in West Sulawesi cause an array of new interactions with local parasitoid communities? Does the complex of host parasite interactions similar as was found in the Western part of Indonesia? These questions are important since Sulawesi is located on the Eastern side of the Wallace line, thus may have different biodiversity compared to the western part of Indonesia. These are among the questions raised to understand the impact of C connexa released in the field at Polewali Mandar, West Sulawesi.

MATERIAL AND METHOD

Time and location of research

The research was conducted from April to December 2021. Sampling and rearing were conducted at Polewali Mandar Regency, West Sulawesi Province Figure 1 while the specimens were identified at the Biological Control Laboratory, Department of Plant Protection, Faculty of Agriculture, IPB University.

Sampling was conducted using a purposive sampling method, namely infestation of C. odorata by gall-made flies. Sampling locations were in two different habitats, that is, the open fields at three fillages and the cocoa plantations in three villages in Polewali Mandar Regency, West Sulawesi Table 1Figure 1

Sampling, rearing, and observation of Cecidochares connexa galls

galls Galls were collected from 30 C. odorata sample plants that were purposively selected from each of the three sampling locations. Each gall was grouped into two categories: those with and without holesFigure 2. All collected sample galls were labelled and brought to the laboratory for further culture and identification of insect specimens. Galls with bigger holes, about 1.45 mm to 1.6 mm in diameterFigure 3(Figure 3A) were counted as C. connexa emergence, whereas galls with small holes, about 0.4 mm to 0.6 mm in diameter Figure 3.(Figure 3B) were counted as parasitoid emergence. Galls without exit holesFigure 2. were observed every two days for emerging insects (either C. connexa or its parasitoids). After 30 days, each gall without holes reared in the laboratory was dissected to observe the internal conditions. The number of C. connexa and parasitization rates were calculated for each replicate per habitat. Each insect that came out was placed into a 1.5 ml microtube containing 70% alcohol for identification.

Figure 1.Research location.

Table 1.Locations of galls sampling at open field and cocoa plantation habitats in Polewali Mandar Regency, West Sulawesi Province

Sampling locations	Habitat types	Coordinate points (latitude and longitude)	Altitude (m asl)
Mirring	Open field	3.46019°S 119.42999°E	±89
Rea Timur 1	Open field	3.44482°S 119.37697°E	±64
Rea Timur 2	Open field	3.45604°S 119.39420°E	±67
Batetangnga 1	Cocoa plantation	3.38692°S 119.41557°E	±640
Batetangnga 2	Cocoa plantation	3.39664°S 119.40894°E	±375
Kaleok	Cocoa plantation	3.40793°S 119.45488°E	±705

Identification

Identification was performed using a stereo microscope (Olympus SZ61). Identification keys were found in the Insect of Australia Vol 1-2 (Naumann et al., 1991) Manual of Nearctic Diptera Vol 1-2 (McAlpine et al. 1987), and Hymenoptera of The World: An Identification Guides to Families (Goulet & Huber, 1993) at the Laboratory of Biological Control, Department of Plant Protection, Faculty of Agriculture, Bogor Agricultural University.

Data analysis

The parasitization rate was calculated from emerged parasitoids in the laboratory, and small exit holes were found in the field. For every parasitoid exit hole found in the field, we assumed that the parasitoid was solitary and that one C. connexa was parasitized.

The parasitization rate on gall is calculated using the formula:

\documentclass{article} \usepackage{amsmath} \begin{document} \displaystyle \text{Parasitization} = \frac{\text{Number of gall parasitized}}{\text{Total number of gall}} \times 100\% \end{document}

The percentage of parasitization on C. connexa was calculated using the formula:

\documentclass{article} \usepackage{amsmath} \begin{document} \displaystyle \text{Parasitization of } C.\, \text{connexa} = \frac{\text{Number of } C.\, \text{connexa } A}{\text{Number of } C.\, \text{connexa } A + C.\, \text{connexa } B} \times 100\% \end{document}

The percentage of parasitization of each parasitoid species on C. connexa from the results of closed-gall rearing was calculated using the following formula:

\documentclass{article} \usepackage{amsmath} \begin{document} \displaystyle \text{Parasitization of parasitoid} = \frac{\text{Number of } C.\ connexa\ C}{\text{Number of } C.\ connexa\ D} \times 100\% \end{document}

wich in A: parasitized; B: not parasitized; C: parasitized by 1 type of parasitoid; D: parasitized by all types of parasitoids The data obtained were tabulated using Microsoft Excel 2013 software. Data on the number of galls, parasitization of galls, and parasitization of C. connexa were processed by t-test analysis using Minitab 16.0. Data on parasitization of each parasitoid type on C. connexa were processed by analysis of variance (ANOVA) and further tested by Tukey’s significant difference test at the 5% level using Minitab 16.0. Box plots were generated using the package ggplot2 (Wickham 2005) with R-Studio software version 4.2.2 (R Core Team 2022).

Figure 2.A: Galls formed by Cecidochares connexa; B: gall with parasitoid hole; C: gall without hole; D: gall with C. connexa hole; E: parasitoid; F: C. connexa.

Figure 3.Gall with a small insect exit hole of Cecidochares connexa (A) and gall with a big insect hole of parasitoids (B).

RESULTS

The presence of the gall fly C. connexa

The results showed that C. connexa can be wich in A: parasitized; B: not parasitized; C: parasitized by 1 type of parasitoid; D: parasitized by all types of parasitoids found in diverse habitats (open fields and cacao plantations) where the studies were conducted. This shows that the gall flies were dispersed in Polewali Mandar, West Sulawesi. These findings suggest that the gall fly C. connexa can disperse at a distance of more than 250 km from the release point (assuming the release point is Bantimurung) within 20 years or less. This is the first report of the current presence of C. connexa in Polewali Mandar.

Establishment of C. connexa in two different habitats

The gall fly C. connexa attacks C. odorata in different habitats. On average, approximately 1-10 galls per plant were found in C odorata in the open field and cacao plantations; however, the number can vary from 1 to 80 galls per plant Figure 4 Two plants were found with more than 90 galls per plant in the open field. Compared to the galls found in C. odorata from cocoa plantations, galls from open fields exhibited more variance. More than one C. connexa individuals can found in each gallTable 2 in both habitats. There are even galls that can house up to nine individuals, although this is a very rare case.

Based on the results of the T-test at the 5% level, the type of habitat had a significant effect on the average number of galls (P-value = 0.0001) Figure 5(Figure 5A) and C. connexa (P-value = 0.0001)Figure 5 (Figure 5B) per C. odorata with the average gall C. connexa in open fields being higher than that in cocoa plantations.

Figure 4.The number of galls per Chromolaena odorata sample plant in two different habitats (N = 90); N: number of C. odorata per habitat

Table 2.The number of Cecidochares connexa per gall from Chromolaena odorata plants collected at opened field and cocoa plantation habitats

Number of C. connexa per gall	n (locations)		Total numbers of gall from 3 locations		Galls per location (x̅ ± SD)		%
	OF	CP	OF	CP	OF	CP	OF	CP
1	3	3	2,285	614	761,67 ± 347,00	204,67 ± 36,35	89,57	90,96
2	3	3	191	56	63,67 ± 11,06	18,67 ± 1,53	7,49	8,30
3	3	3	60	5	20,00 ± 3,61	1,67 ± 1,15	2,35	0,74
4	3	3	10	0	3,33 ± 2,52	0	0,39	0
5	3	3	4	0	1,33 ± 0,58	0	0,16	0
6	3	3	0	0	0	0	0	0
7	3	3	0	0	0	0	0	0
8	3	3	0	0	0	0	0	0
9	3	3	1	0	0,33 ± 0,58	0	0,04	0
Total	-	-	2,551	675	-	-	100	100

New association between C. connexa and local parasitoids

The presence of C. connexa in C. odorata triggered the formation of a “new association” with local parasitoid insects Figure 6 C. connexa is parasitized by several species of parasitoids. The parasitoids found were from the order Hymenoptera, consisting of four families: Eulophidae, Braconidae, Ormyridae, and Eupelidae. Six morphospecies of parasitoids were found in the open field: Aprostocetus sp.1 (Eulophidae), Aprostocetus sp.2 (Eulophidae), Doryctobracon sp. (Braconidae), Ormyrus sp.1 (Ormyridae), Ormyrus sp.2, and Eupelmus sp. (Eupelidae) Figure 6(Figure 6A). In the cocoa plantations, only three morphospecies of parasitoids were found: Aprostocetus sp.1 (Eulophidae), Doryctobracon sp. (Braconidae), and Ormyrus sp.1 (Ormyridae) Figure 6(Figure 6B).

Habitat differences had a significant effect, based on a t-test at the 5% level, on the average number of parasitized galls (P-value = 0.0027) and C. connexa (P-value = 0.002) Figure 7 The average number of galls and C. connexa parasitized in open fields was also higher than that in the cocoa plantations. However, habitat did not significantly affect the parasitism rates of gall (P-value = 0.2404) and C. connexa (P-value = 0.865) Figure 8.

As many as 319 out of a total of 1.799 C. connexa individuals found in galls (reared in the laboratory) obtained from open fields were parasitized (17,89%). Meanwhile, the parasitization rate in cocoa plantations was higher, with 100 out of 461 C. connexa individuals (21,69%). Compared to cocoa plantations, open fields included more types of parasitoid species with the number of C. connexa parasitized by Aprostocetus sp.1 being more than the other parasitoids in both habitats, whereas the lowest parasitoid species in the open field was Ormyrus sp.2 and in cocoa plantations was Ormyrus sp.1. The parasitization rate of C. connexa in the cocoa plantations was higher than that in the open fieldTable 3.

Analyais of Variance (ANOVA) revealed that the type of parasitoid had a significant effect (P-value = 0.002) on the average number of parasitized C. connexa, as well as the parasitization of C. connexa (P-value = 0.003) in the open field habitat. However, in cocoa plantations, it did not significantly affect the average number of parasitized C. connexa (P-value= 0.131) or parasitization rate (P-value= 0.521)Table 3.

Figure 5.Average number of gall (A) and Cecidochares connexa (B) per sample plant of Chromolaena odorata(N = 90) in different habitat. N: number of C. odorata per habitat.

DISCUSSION

Our result showed that C. connexa are already established in West Sulawesi and can be found in open fields and cocoa plantations. This study is the first to report that C. connexa are able to adapt and become established in Polewali Mandar, West Sulawesi. This finding is in line with (Tjitrosemito, 2002)which stated that C. connexa has been established on most of the larger Indonesian islands and that C. connexa can spread widely over short periods of time. According to (Harjaka & Mangoendihardjo, 2010) C. connexa can spread over a radius of more than 200 km from the release site in Gunungkidul, Yogyakarta, to several areas in East Java within 10 years. (Day et al., 2013) also reported that C. connexa had spread more than 100 km from most of the release points in Papua New Guinea within seven years. It is likely that C. connexa can also be found in areas more than 250 km from the release point. However, the introduction of C. connexa did not seem to reduce the population of C. odorata, as indicated by its abundant population at the study site. (Kenis et al., 2019) reported that the success of biological control is classically characterized by a decrease of the target population. However, our observation found C. odorata to be abundant in the field, hence raising the question of C. connexa effectiveness in as biological control agent. Field observations showed that the attacked branches do not instantly die but continue to develop and it even create new branches. Thus attack of C. odorata seems to increase the branch production.

Our observation showed that C. connexa could lay more than one egg on each shoot of C. odorata. In addition, we also found more than one C. connexa larvae in one gall that can emerge as imago. (McFadyen et al., 2003) reported that C. connexa could lay up to 16 eggs per shoot on the shoots of C. odorata. However, the number of larvae that can survive to become imago is between 2-4 individuals.

Compared to open fields, cocoa plantations have fewer galls and C. connexa. This is because fewer C. odorata plants may be found in cocoa plantations, which leads to fewer galls being discovered. Pruning and spraying of herbicides routinely on C. odorata around cocoa plantations may affect to the population of C. odorata, resulting in a decrease in the number of galls formed. The presence of shade is also believed to be a factor that slows the growth of C. odorata in addition to pruning and spraying. This has an impact on C. connexa which can parasitize parasitoids.

Overall we found six species of parasitoids associated with C. connexa with Aprostocetus sp 1 being the most abundant . The parasitoid complex is different than the community of parasiotids found in West Java(Buchori et al., 2020) Quadrastichus sp was the most abundant parasitoid found in Bogor and Lampung. The difference may seem be due to the different diversity that are present in the island of Java, Sumatra and Sulawesi. This study however, strengthens the findings of(Buchori et al., 2020) (Lukvitasari et al., 2021) (Jaya, 2006) and (Pahlevi, 2006) that new associations have been formed from exotic bioagents introduced to new habitats. The fact that C. connexa from different islands are attacked by different parasitoid complex has shown that adaptation of local parasitoids to invasive/exotic pests can happen. (McFadyen et al., 2003) also reported the attack of C. connexa by parasitoid insects five years after release and the first report of a new association between C. connexa and a parasitoid. (Boughton et al., 2012) also reported that Neomusotima conspurcatalis Warren (Lepidoptera: Crambidae) was introduced to Florida in 2008 to control Lygodium microphyllum (Schizaeales: Lygodiaceae) and are then attacked by local parasitoids in just a few years. These findinga are interesting because it reveals the local adapatation of local parasitoids with exotic herbivore species, thus strengthening (Hokkanen & Pimentel, 1989) argument to use local parasitoids as bioontrol agent, since new associates can happen quite rapdily in the field. Our findings raise other questions e.g. does the presence of C. connexa change the interaction dynamics of the parasitoid with its original hosts? Are there shifts in the preference of the parasitoid from its original hosts, and if there is a shift, what are the implications for the original hosts? Will there be a natural enemy-free space that results in the explosion of new pests? Further investigation is required to corroborate some of the assertions made above because there is a shortage of knowledge on the subject.

One important question that can be raised from this study is whether C. connexa only functions as an additional host for parasitoids or actually functions as a subsidy that can increase the population of parasitoid insects and can indirectly increase the role of these parasitoids to attack other hosts. The parasitoids found attacking C. connexa in this study are most likely generalist parasitoids that attack a variety of gall-forming insects in several habitats in the tropics. Thus, when C. connexa attacks C. odorata and form galls, the local parasitoids can shift and atttack C. connexa as a new host. Parasitoids of the Genus Aprostocetus were the most common and had the highest parasitization rate. This is different from what was previously reported by (Buchori et al., 2020) and (Lukvitasari et al., 2021)that the dominant parasitoid found was Quadrastichus sp. Aprostocetus parasitoids are likely common parasitoids that commonly attack other insects such as Orseolia javanica (Diptera: Cecidomyiidae) on rice in Bogor, West Java (Maqsalina, 2021) Ophelimus eucalypti (Hymenoptera: Eulophidae) on eucalyptus plants Medan, North Sumatra (Anisa et al., 2023)

Parasitoids are generally considered beneficial for pest control as they parasitize insect pests. In this case, they have a detrimental impact, as they attack the biological control agent of C. connexa which is used to control C. odorata populations. Overall, the parasitoid discovered in the cocoa plantations was also present in the open fields. The parasitoid observed to attack C. connexa in this study is most likely a generalist parasitoid that targets different forming-gall insects in several habitats in tropics. The introduced C. connexa which was established in release areas to control C. odorata weed, is a new host for local parasitoids. According to (Pearson & R.M, 2005) biological agents introduced into new areas to control invasive pests have the potential to become hosts for local natural enemies. If they are unable to control the population of invasive weeds, it results in an abundance of weed populations.

One reason for the ineffectiveness of C. connexa in suppressing C. odorata populations is the presence of parasitoids that parasitize C. connexa. Although the level of parasitization is still relatively low, the presence of parasitoids on C. odorata can reduce attacks from C. connexa. (Abdala-Roberts et al., 2019) stated that one of the indirect effects of the presence of natural enemies is that they can reduce herbivore populations and indirectly affect the plants that are attacked.

Classical biological control using C. connexa to suppress populations of the invasive weed C. odorata has been unsuccessful. Although C. connexa is widespread and established, its presence is insufficient to control C. odorata because it is attacked by a variety of parasitoids in a variety of habitats, including industrial plantations, forests, open fields in Bogor(Jaya, 2006)(Safi’i, 2006) palm oil plantations and open fields in Bogor and Lampung (Lukvitasari et al., 2021)cocoa plantations, and open fields in Polewali Mandar. Consequently, its effectiveness is reduced. In addition, C. odorata endured C. connexa attacks by producing additional branches in response to the attack.

Figure 6.Interactions between Chromolaena odorata, Cecidochares connexa, and parasitoids in two different habitats. A: open field; B: cocoa plantation. The green line indicates herbivore insects associated with C. odorata. Red lines indicate parasitoids associated with C. connexa.

Figure 7.The percentage parasitization of galls (A) and Cecidochares connexa (B) per habitat (N = 90). N: number of Chromolaena odorata per habitat.

Figure 8.The percentage parasitizationof galls (A) and Cecidochares connexa (B) per habitat(N = 90). N: number of Chromolaena odorata per habitat.

Table 3.The average number of Cecidochare connexa parasitized and percentage parasitization by each type of parasitoid per habitat (N1 = 1799; N2 = 461)

Parasitoid Famili Species	Open field			Cocoa plantation
	X̅ ± SD	N	% parasitization	X̅ ± SD	N	% parasitization
Braconidae Doryctobracon sp.	11,33 ± 13,32 b	34	1,91 b	12,67 ± 13,58 a	38	8,24 a
Eulophidae Aprostocetus sp.1	80,67 ± 46,06 a	242	13,45 a	16,33 ± 14,57 a	49	10,63 a
Aprostocetus sp.2	3,33 ± 5,77 b	10	0,56 b	0,00 ± 0,00	0	0,00
Eupelmidae Eupelmus spp.	3,33 ± 5,77 b	10	0,56 b	0,00 ± 0,00	0	0,00
Ormyridae Ormyrus sp.1	6,33 ± 5,51 b	19	1,06 b	4,33 ± 5,13 a	13	2,82 a
Ormyrus sp.2	1,33 ± 2,31 b	4	0,22 b	0,00 ± 0,00	0	0,00
Total		319	17,89		100	21,69
Mean		53,67	2,96		16,66	3,64

CONCLUSION

The gall fly C. connexa was found in open fields and cocoa plantations in Polewali Mandar, West Sulawesi, more than 250 km from the release point. This is the first report of C. connexa in Polewali Mandar, West Sulawesi. The abundance of C. connexa in C. odorata was influenced by habitat type. There is a new association between C. connexa and local parasitoids with the discovery of six types of parasitoid morpho-species from four different families. Aprostocetus sp.1 was the dominant parasitoid, with a parasitization percentage of 13.57% in open land and 10.63% in cocoa plantations.

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