Multiplex PCR in determination of Opiinae parasitoids of fruit flies, Bactrocera sp., infesting star fruit and guava

Abstract

Malaysia is a tropical country that produces commercial fruits, including star fruits, Averrhoa carambola L. (Oxalidales: Oxalidaceae), and guavas, Psidium guajava L. (Myrtales: Myrtaceae). There is a high demand for these fruits, and they are planted for both local consumption and export purposes. Unfortunately, there has been a gradual reduction of these fruits, which has been shown to be related to fruit fly infestation, especially from the Bactrocera species. Most parasitic wasps (Hymenoptera: Braconidae: Opiinae) are known as parasitoids of fruit fly larvae. In this study, star fruits and guavas infested by fruit fry larvae were collected from the Malaysian Agricultural Research and Development Institute. The parasitized larvae were reared under laboratory conditions until the emergence of adult parasitoids. Multiplex PCR was performed to determine the braconid species using two mitochondrial DNA markers, namely cytochrome oxidase subunit I and cytochrome b . Two benefits of using multiplex PCR are the targeted bands can be amplified simultaneously using the same reaction and the identification process of the braconid species can be done accurately and rapidly. The species of fruit flies were confirmed using the COI marker. The results obtained from our study show that Diachasmimorpha longicaudata (Ashmead) (Hymenoptera: Braconidae), Fopius arisanus (Sonan), and Pysttalia incisi (Silvestri) were parasitoids associated with Bactrocera carambolae (Drew and Hancock) (Diptera: Tephritidae) infested star fruits. Fopius arisanus was also the parasitoid associated with Bactrocera papayae (Drew and Hancock) infested guavas. Maximum parsimony was been constructed in Opiinae species to compare tree resolution between these two genes in differentiating among closely related species. The confirmation of the relationship between braconids and fruit fly species is very important, recognized as preliminary data, and highly necessary in biological control programs.

Prevalence and evolutionary history of endosymbiont Wolbachia (Rickettsiales: Anaplasmataceae) in parasitoids (Hymenoptera: Braconidae) associated with Bactrocera fruit flies (Diptera: Tephritidae) infesting carambola

“…), Fopius arisanus (Sonan, 1932), F. vandenboschi (Fullaway, 1952) (Chua & Khoo ), Psyttalia incisi (Silvestri, 1916) (Shariff et al . ), P. makii (Sonan, 1932) and P. fletcheri (Silvestri, 1916) (Chinajariyawong et al . ).…”

“…Carambola Averrhoa carambola L. (Oxalidales: Oxalidaceae) and guava Psidium guajava L. (Myrtales: Myrtaceae) are among the tropical fruits that have been highly produced in Malaysia (Shariff et al 2014). However, the most critical carambola pests are the Bactrocera fruit flies, particularly B. papayae Drew & Hancock, 1994and B. carambolae Drew & Hancock, 1994(Chua 1994Chua & Khoo 1995;Clarke et al 2001;Chua et al 2010).…”

“…In order to control the damage caused by fruit flies, researchers and entomologists have developed a biological control strategy involving endoparasitoids. There are several braconid endoparasitoids that parasitize the Bactrocera fruit flies that infest carambola, such as Diachasmimorpha longicaudata (Ashmead, 1905) (Ibrahim et al 1994), Fopius arisanus (Sonan, 1932), F. vandenboschi (Fullaway, 1952 (Chua & Khoo 1995), Psyttalia incisi (Silvestri, 1916) (Shariff et al 2014), P. makii (Sonan, 1932 and P. fletcheri (Silvestri, 1916) (Chinajariyawong et al 2000). In previous studies, F. arisanus, F. vandenboschi, Psyttalia sp., P. fletcheri, P. incisi and D. longicaudata have been proven to harbor Wolbachia (Sintupachee et al 2006;Morrow et al 2014;Mohammed et al 2015).…”

Species Composition and DNA Barcoding of Hemipteran Assemblages Throughout Paddy Growing Seasons

“…The PCR process was carried out in a total of 25 μL reaction comprising of 2 μL DNA template (10-15 ng/μL), 8.50 μL ddH 2 O, 0.5 μL 10 mM dNTPs, 2.5 μL PCR buffer 10× (Vivantis, United Kingdom), 1.30 μL 50 mM magnesium chloride (MgCl 2 ), 1.0 μL forward and reverse primers (10 pmol/μL), 0.2 μL Taq DNA polymerase (5 U/μL) (Vivantis, United Kingdom) and analysed using MyGene MG96G thermal cycler (China). The PCR condition followed by Halim et al (2017Halim et al ( , 2018 and Shariff et al (2014); 1 cycle 3 min at 95 °C (denaturation), 40 cycles under 30 s at 95 °C (anneal), 30 s at 47 °C (extension), 1 min at 72 °C for 10 min (final extension). Then, the PCR product underwent electrophoresis for 30 min at 90V using 1.5% agarose gel.…”

Molecular Identification and Species Richness of Flies (Diptera) and Their Associated Bovidae Hosts at Cattle Farms in Selangor, Malaysia

“…Polymerase Chain Reaction (PCR) and DNA Purification. DNA extracted from different fly samples was amplified using the GeneAmp PCR System 2400 (Perkin Elmer, USA) and cytochrome oxidase subunit I (COI) mitochondrial DNA based on Hebert et al (2003) and Shariff et al (2014) profiles. The amplification product of forwarding primer, COI-LCO1490 5'GGT CAA CAA ATC ATA AAG ATA TTG G 3' and reverse primer COI-HCO22198 5'TAA ACT TCA GGG TGA CCA AAA AAT CA 3' was 750 bp, in reference to Folmer et al (1994).…”