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National Fruit Fly Diagnostic Protocol

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National Fruit Fly Diagnostic Protocol 6 Identification 6.1 Overview The National Handbook for the Identification of Fruit Flies in Australia (overview presented in Figure 3 and Figure 4) proposes that primary identification is undertaken using conventional taxonomy with the support of molecular genetic techniques for some species. The diagnostic methods available for each species are presented in Table 2 and covered in greater detail in sections 6.2. (Morphological), 6.3.1. (PCR amplification), 6.3.2 (DNA barcoding) and 6.4 (Allozyme Electrophoresis). These techniques are currently in use in Australia and form the basis of this national protocol. Section 7 contains data sheets with the specific morphological and molecular diagnostic information for each species. Molecular techniques are best used to support or augment morphological identification. They can be used to identify early larval stages (which are hard to identify reliably on morphological features) and eggs. They can also be used for incomplete adults that may be missing specific anatomical features required for morphological keys, or specimens that have not fully developed their features (especially colour patterns). It should be recognised, however, that the success of a molecular diagnosis can be impacted by factors such as life stage, specimen quality or any delays in processing. As a result, the suitability of each method has been identified. The molecular protocols require a laboratory to be set up for molecular diagnostics, but can be carried out by almost any laboratory so equipped. Access to published sequences is required for whichever protocol is being used1. Most molecular techniques presented in this standard involve the amplification of particular region(s) of the fly genome using a polymerase chain reaction (PCR). Often the target is the internal transcribed spacer region of the ribosomal RNA operon referred to as ITS1. Many species can be identified by the size of the ITS1 alone although similar species often produce fragments of the same size. In this case, restriction digestion of the ITS1 PCR product can be performed, using each of up to six different restriction enzymes. This approach is referred to as restriction fragment length polymorphisms (RFLP) analysis. This does not necessarily eliminate non-economic fruit flies but will identify if the restriction pattern produced conforms to that produced by a reference fly from an economically important species. If the species is still not identified, more comprehensive information can be obtained by undertaking nucleotide sequence analysis. DNA barcoding, focusing on analysis of the mitochondrial gene for cytochrome oxidase subunit I (COI) is now available as an alternative to ITS-based techniques. This technology sometimes provides more accurate and consistent results than analysis of the ITS region, with less confusing overlap between taxa; however, inconsistencies and anomalies can still arise, particularly among closely related species complexes. This national protocol is presented on the premise that ITS analysis and DNA barcoding are used alongside morphological methods. Most species can generally be resolved using traditional or molecular taxonomy without ambiguity. However, more difficult cases will only yield to a combination of both morphological and one or more molecular approaches. 1 Many of the DNA barcoding sequences were obtained by the CBOL tephritid fruit fly project (www.dnabarcodes.org/pa/ge/boli_projects), which examined all economically important tephritid fruit fly species known to be agricultural pests as well as many closely related species. 14 Figure 3. Overview of fruit fly diagnostic procedures (adult specimens) Inspection of material, Allocation of samples Fruit fly sample Start quality check and sample to staff according to receipt preparation experience REFERENCES RESOURCES MORPHOLOGICAL National protocol and IDENTIFICATION Targeted species list reference specimens, see also Section 8 for Microscope procedures See Table 2 further references Reassessment by No Confident of Yes “in house” second diagnosis? entomologist Notification as required Confident of Yes diagnosis? GENETIC Report as: No DETERMINATION (PCR-RFLP) • Target species Separation of material See Table 2 • Endemic for molecular genetic • Exotic testing • Seeded Referral to network PCR-DNA laboratory BARCODING See Table 2 Databased Yes Confident of Option diagnosis? No ALLOZYME ELECTROPHORESIS Specimen Referral to a See Table 2 of value? national authority Yes No RESOURCES Pinned as part Specimen of regional disposed of Victoria, NSW and SA and/or national protocols collection 15 Figure 4. Overview of fruit fly diagnostic procedures (larval specimens) Receipt of Inspection of Allocation of samples to Preparation of larvae for fruit fly material, quality staff according to morphological Start larvae check experience examination RESOURCES MORPHOLOGICAL REFERENCES Targeted species list IDENTIFICATION National protocol, see also Section 8 for further Microscope procedures See Table 2 references Reassessment by “in No Yes house” second Confident of diagnosis? entomologist Notification as required Yes Confident of diagnosis? GENETIC Report as: DETERMINATION No (PCR-RFLP) • Target species See Table 2 • Endemic Separation of material for molecular genetic testing • Exotic • Seeded Referral to network PCR-DNA laboratory BARCODING See Table 2 Databased Yes Confident of Option diagnosis? No ALLOZYME ELECTROPHORESIS Specimen of value? Referral to a See Table 2 national authority Yes No Specimen RESOURCES Alcohol preserved as disposed of Victoria, NSW and SA part of regional and/or protocols national collection 16 Table 2. Diagnostic methods used to identify fruit fly species Scientific name Morphological PCR-RFLP PCR-DNA Allozyme description (6.2) (6.3.1) Barcoding4 (6.3.2) Electrophoresis (6.4) Anastrepha fraterculus (14) Anastrepha ludens (10) Anastrepha obliqua (16) Anastrepha serpentina (13) Anastrepha striata (14) Anastrepha suspensa (7) Bactrocera albistrigata (1) Bactrocera aquilonis 1 1 (36) Bactrocera atrisetosa (0) 5 Bactrocera bryoniae (10) Bactrocera carambolae (10) Bactrocera caryeae (1) Bactrocera correcta (15) Bactrocera cucumis (8) Bactrocera cucurbitae (72) Bactrocera curvipennis (2) Bactrocera decipiens (0) 5 Bactrocera dorsalis 2 2 (29) Bactrocera facialis (1) Bactrocera frauenfeldi (16) Bactrocera jarvisi (6) Bactrocera kandiensis (10) Bactrocera kirki (5) Bactrocera latifrons (20) Bactrocera melanotus (3) Bactrocera musae 3 (5) Bactrocera neohumeralis 1 1 (4) Bactrocera occipitalis (5) Bactrocera papayae (11) Bactrocera passiflorae (1) Bactrocera philippinensis 2 2 (9) Bactrocera psidii (2) Bactrocera tau (5) Bactrocera trilineola (2) 17 Scientific name Morphological PCR-RFLP PCR-DNA Allozyme description (6.2) (6.3.1) Barcoding4 (6.3.2) Electrophoresis (6.4) Bactrocera trivialis (3) Bactrocera tryoni 1 1 (12) Bactrocera umbrosa (9) Bactrocera xanthodes (7) Bactrocera zonata (22) Ceratitis capitata (120) Ceratitis rosa (24) Dirioxa pornia (3) Rhagoletis completa (0) 5 Rhagoletis fausta (1) Rhagoletis indifferens (0) 5 1 Species cannot be distinguished from each other at the ITS or COI region 2 Species cannot be distinguished from each other at the ITS or COI region 3 Requires full ITS sequencing to split B. musae from the B. philippinensis, B. dorsalis group 4 Numbers in brackets refer to the number of individuals of that species with (COI) DNA barcodes of >500 bp on the Barcode of Life website (www.boldsystems.org/views/taxbrowser.php?taxid=439; as of 23 August 2011). 5 DNA barcodes (COI) are available for other species in these genera. There are 86 species of Bactrocera, 65 species of Dacus, and 19 species of Rhagoletis that do have barcodes available (as of 23 August 2011). 6.2 Morphological identification Approximately 90% of the dacine pest species can be identified accurately, and quickly, by microscopic examination of the adult. For these species there is no need for supporting evidence. The remaining 10% (mainly some dorsalis complex species) can be identified with this same method but require expert examination and may require additional supporting evidence such as the molecular diagnosis or host association records. Only morphological diagnostic procedures and information for adult fruit flies are contained in this document. Aside from molecular techniques, larval diagnosis has been excluded from this protocol. 6.2.1 Procedure The following apparatus and procedures should be used to prepare the specimen for identification (adapted from QDPIF 2002): Apparatus: • Stereoscopic microscope or Stereomicroscope with magnification range of 7X to 35X. • Light source • 90mm diameter petri dishes • Forceps (Inox #4) 18 Preparation procedure: 1) Ensure the workstation is clean and clear of all flies before commencing. 2) Adjust chair height and microscope, and turn on the light source (refer to specific operating procedures for the microscope in use). 3) If applicable, record the lure and trap type or host material in which the specimen was found. 4) Carefully place the fruit fly into a plastic petri dish. If examining more than one fly at once ensure there is a single layer of flies only, with room to move flies from one side of the dish to the other. 5) While looking through the microscope check each fly individually. Manipulate them with the forceps so that diagnostic features are visible. 6.2.2 Identification Key features (Figure
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