7
Identification
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 or immature stages. The diagnostic
methods available for each species are presented in Table 3 and covered in greater detail in sections
7.2 (Morphological), 7.3 (Molecular) and Section 8 (data sheets with the specific morphological and
molecular diagnostic information for each species). These techniques are currently in use in Australia
and form the basis of this national protocol.
Tephritid fruit fly adult specimens are primarily identified through an examination of diagnostic
morphological characters. Other life stages are more problematic, with only third instar larvae (and
sometimes pupae) of some species usually identified through visual examination (e.g. White & ElsonHarris 1992). Identification of earlier life stages (early instars, eggs), and adults of morphologically
ambiguous species, generally requires the use of molecular techniques.
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 also can 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 (See Section 7).
The molecular protocols require a laboratory to be set up for molecular diagnostics, but can be
conducted by almost any laboratory so equipped. Access to on-line published sequences is required
for the DNA barcoding protocol.
Most molecular techniques presented in this standard involve the amplification of particular region(s)
of the fly genome using a polymerase chain reaction (PCR), while the other technique covered
(allozymes) examines protein variation. The PCR target is either the mitochondrial gene for
cytochrome oxidase subunit I (COI), known as the DNA barcode region, or a region of the ribosomal
RNA operon, either just the first internal transcribed spacer (ITS1) or part of the 18S subunit plus the
ITS1.
DNA barcoding is now available in this manual as an alternative to the original ITS-based techniques.
This technology, in contrast, incorporates population variation and has an international, independently
growing reference dataset. However, difficulties can still arise among closely related species within
species complexes.
For the ITS1, the size of the PCR amplicon is useful for identification of a few species. However,
restriction digestion of the ITS1 PCR amplicon, which denotes the actual sequence in defined regions
of the amplicon, is recommended for all analyses as a more robust method of identification. This is
referred to as restriction fragment length polymorphisms (RFLP) analysis. Reference data has been
developed for the economically important species. However, the RFLP does not necessarily eliminate
non-economic fruit flies for which reference data have not been developed.
This national protocol is presented on the premise that most species can generally be resolved using
traditional taxonomy without ambiguity. The molecular methods described here are recommended for
use alongside morphological methods where there is any ambiguity; although they may be equally
ambiguous in the case of species complexes. They can be used on their own for morphologically
cryptic immature life stages, or when only part of a specimen is available, or when access to the
appropriate taxonomic expertise is lacking.
57
Figure 3. Overview of fruit fly diagnostic procedures (adult specimens).
58
Figure 4. Overview of fruit fly diagnostic procedures (larval specimens).
59
Table 3. Diagnostic methods used to identify fruit fly species
Scientific name
Morphological
description
Anastrepha fraterculus

PCRRFLP1
PCRRFLP 2
PCR- DNA
Barcoding4

49/141e*
0/27e*
Anastrepha distincta

3/35

b
68/174e*



52/133e*
Anastrepha serpentina



8/52*
Anastrepha striata


18/59
Anastrepha suspensa

b
5/76e*
Bactrocera albistrigata


Bactrocera aquilonis

a
Bactrocera atrisetosa

Bactrocera bryoniae


Bactrocera carambolae


Bactrocera caryeae

1/1g
Bactrocera correcta

54/54
Bactrocera cucumis



2/11
Bactrocera cucurbitae



244/289*
Bactrocera curvipennis



2/2
Bactrocera decipiens

Bactrocera depressa

Bactrocera dorsalis s.s. 12



>1000/1000g
Bactrocera facialis



1/1
Bactrocera frauenfeldi


d
2/16
Bactrocera jarvisi



3/7
Bactrocera kandiensis

Bactrocera kirki13

Anastrepha grandis

Anastrepha ludens

Anastrepha obliqua
Bactrocera kraussi
11
Allozyme
qPCR11
electrophoresis
24/24
c
3/38f

1/11

110/111

17/17g

d,3
5/5
3/3
Real-time PCR
Includes B. papayae, philippinensis and invadens since their synonymisation with dorsalis sensu stricto as per Schutze et al.
(2015). This is a taxonomic update of the previous list as opposed to an omission of these species.
12
13
B. kirki can be distinguished from B. frauenfeldi and B. trilineola using an additional enzyme Acc I
60
Scientific name
Morphological
description
PCRRFLP1
PCRRFLP 2
PCR- DNA
Barcoding4
Bactrocera latifrons



82/86
Bactrocera melanotus


3/3
Allozyme
qPCR11
electrophoresis
58/58
Bactrocera minax
Bactrocera musae14



21/255
Bactrocera neohumeralis

a
c
2/4f

Bactrocera obliqua
Bactrocera occipitalis

Bactrocera oleae

Bactrocera passiflorae

Bactrocera psidii

31/34g

109/114


0/1


2/2
Bactrocera pyrifoliae
85/100*
Bactrocera tau

Bactrocera trilineola

d
21/21
Bactrocera trivialis


3/3
Bactrocera tryoni

c
48/55f
a
Bactrocera tsuneonis
21/21
Bactrocera tuberculata
5/5
Bactrocera umbrosa



39/39
Bactrocera xanthodes



6/7
Bactrocera zonata



36/61
Ceratitis capitata



208/292
Ceratitis rosa



46/50*


2/2
Dacus longicornis

Dacus solomonensis
Dirioxa pornia




3/3

The 17 species within the B. musae complex (Drew et al. 2011) have not been tested by the RFLP method, but DNA
barcodes will distinguish B. musae s.s. from at least the non-pest species B. conterminal, B. prolixa, B. rufivitta, and B.
tinomiscii; adults of all species can be distinguished morphologically, see section 7.2.
14
61
Scientific name
Morphological
description
PCRRFLP1
PCRRFLP 2
PCR- DNA
Barcoding4
24/29
Rhagoletis cingulata
Rhagoletis completa

Rhagoletis fausta

Rhagoletis indifferens


21/21*
1/1
Rhagoletis mendax
2/2h
Rhagoletis cerasi
17/29
Rhagoletis pomonella
Allozyme
qPCR11
electrophoresis

23/24h
1
Species with the same superscript letter cannot be distinguished from each other with this test.
2
This test is best used when the unknown samples may be suspected as one for which the PCR-RFLP test 1 has not been
developed; all RFLP patterns available in Armstrong & Cameron (1998) are listed in Appendices 3 and 4, and includes other
species not listed in this table, A. bistrigata, A. sorocula, A. zenildae and B. quadriestosa.
3
Numbers in brackets (x/y) refer to the x number of individuals of that species with publically accessible DNA (COI) barcodes
on the Barcode of Life (BOLD) website and y number of total sequences available if using the BOLD identification engine; the
difference represents private barcodes unavailable for download
(www.boldsystems.org/views/taxbrowser.php?taxid=439) as of 18th September 2015.
Where there are no specific DNA barcodes available, others for other species within the genus could be used to at least
determine genus-level identification; as of 18th September 2015, there are 203 species of Bactrocera, 66 of Ceratitis, 82 of
Dacus, 28 species of Rhagoletis and 1 of Toxotrypana that have barcodes available.
Species with the same superscript letter cannot be distinguished from each other with DNA barcodes, or can only be
identified to the level of a species complex.
Species with * are not well resolved amongst a number of other species in BOLD, although low divergence clades may be
apparent. Used in conjunction with other information about the specimen this may still be sufficient to make a confident
identification. Complimentary identification by ITS1 PCR-RFLP could also be considered.
62
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. Methods here are updated according to Drew & Romig (2013).
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.
For routine morphological identification:
 Collect flies in dry traps.
 When clearing traps collect samples into a tissue. Put tissue in a small box with collection
details on the outside. Samples in tissue can also be collected into a vial though this is less
preferred particularly in the tropics as samples can sweat causing specimens to deteriorate. If
collecting into vial a pencil or permanent pen label (not biro as it runs) should be put inside as
writing on the outside can rub off.
 Store samples in freezer until ready for identification.
 Sort dry specimens in a petri dish under a binocular microscope.
 If keeping specimens after identification store in freezer to prevent deterioration.
 Do not store specimens in ethanol/alcohol/propylene glycol unless being kept for DNA
analysis. They leach diagnostic colours and patterns necessary for morphological
identification.
For suspect specimens requiring further identification
 Store the specimen in a small vial with tissue to protect it until ready to pin or ship. Add a
pencil or permanent ink label (not biro as it runs) detailing collection location, collection date,
collection method, collector, tentative identification and identifier.
 As manipulating loose specimens with forceps tends to damage them, suspect specimens
should ideally be pinned to keep them as intact as possible. If the specimen is a suspect
exotic and needs to be shipped to a specialist ASAP they can be sent unpinned in a tube as
above.
For pinning:
 Put specimen in relaxing chamber with thymol (to prevent mould growth) for 6-12 hours.
 Using a micropin, pin through RHS of scutum. Mount micropinned specimen on a pith stage
on a pin.
 Add label to pin.
 Store pinned specimens in reference collection conditions i.e. 21 OC and 50% RH.
SAMPLE PREPARATION
7.2.1.1
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
63
Forceps (Inox #4)

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.
7.2.2
Identification
Key features (Figure 5, Figure 6, Figure 7 and Figure 8) used for the morphological diagnosis of adult
fruit flies include:

Overall colour and colour patterning

Wing morphology and infuscation

Presence, shape and colour of thoracic vittae. A vitta is a band or stripe of colour.

Presence or absence of various setae, and relative setal size (for Trypetinae). (Note:
chaetotaxy, the practice of setal taxonomy, is not as important for Dacinae, i.e Bactrocera and
Dacus)
Use the morphological diagnostic key and descriptions contained in Section 8 to identify the species of
fruit fly under microscopic examination.
If identification cannot be made using this diagnostic procedure and/or the specimen is suspected to
be of quarantine concern, it should be referred to either a State or National authority (see section 9.1
Key contacts and facilities). If the specimen is identified as an exotic fruit fly, it should be referred to a
National Authority within 24 hours and the appropriate National Authority notified as required in
PLANTPLAN.
64
Figure 5. Adult morphology; head (top) and wing (bottom) (White and Elson-Harris 1992).
ar – arista
comp eye – compound eye
fc – face
flgm 1 – 1st flagellomere
fr – frons
fr s – frontal setae
gn – gena (plural: genae)
gn grv – genal groove
g ns – genal seta
i vt s – inner vertical seta
lun – lunule
oc – ocellus
oc s – ocellar seta
o vt s – outer vertical seta
orb s – orbital setae
pafc – arafacial area
ped – pedicel
poc s – postocellar seta
pocl s – postcular setae
ptil fis – ptilinal fissure
scp – scape
vrt – vertex
65
Figure 6. Adult morphology, Thorax; Dorsal features (White and Elson-Harris 1992).
a npl s – anterior notopleural seta
hlt – halter or haltere
prepst – propisternum
a sctl s – apical scutellar seta
ial s – intra-alar seta
presut area – presutural area
a spal s – anterior supra-alar seta
kepst – katepisternum
a spr – anterior spiracle
kepst s – katepisternal seta
presut spal s – preutural supraalar seta
anatg – anatergite
ktg – katatergite
anepm – anepimeron
npl – notopleuron
anepst – anepisternum
p npl s – posterior notopleural
seta
anepst s – upper anepisternal
seta
b sctl s – basal scutellar seta
cx – coax
dc s – dorsocentral seta
psctl acr s – prescutellar
acrostichal seta
psut sct – postcutural scutum
sbsctl – subscutellum
p spal s – posterior supra-alar
seta
scape – scapula setae
p spr – posterior spiracle
trn sut – transverse scuture
sctl – scutellum
pprn lb – postpronotal lobe
pprn s – postporontal seta
66
Figure 7. Adult morphology, thorax; lateral features (White and Elson-Harris 1992).
See Figure 5 for abbreviations.
Figure 8. Adult morphology, abdomen; male with features of typical dacini (left), Female, with extended ovipositor
(right) (White and Elson-Harris 1992).
acul – aculeus
ovsc – oviscape
ev ovp sh – eversible ovipositer
sheath
st – sternites numbered 1-5 in
the male and 1-6 in the female
tg – tergites where 1+2 are fused to
form syntergosternite 1+2, followed
by tergites 3-5 in the male and 3-6 in
the female
67
Molecular identification
Three methods are presented here, all of which can use a common sample storage and handling
technique (Section 6), DNA extraction method (Section 7.3.1), and are based on PCR (Polymerase
Chain Reaction) analysis.
Choice of method: The DNA barcoding method (Section 7.3.2) is generally recommended over the PCRRFLP (restriction fragment length polymorphism) methods (Section 7.3.3) because:
(a) DNA barcoding can produce better resolution between species as it utilises variation in the
complete sequence amplified. PCR-RFLP is limited to variation at just a few or several 4-6 bp
restriction sites within the amplicon, the suite of which are dependent on the nature of the
restriction enzymes used
(b) DNA barcoding uses a very large reference sequence database. This is international, publicly
accessible and constantly being added-to by unrelated institutions and projects. Consequently
there is greater inclusion of taxonomically comparative species and population data to improve
confidence in identification. PCR-RFLP generally relies on in-house developed reference
restriction patterns; therefore comparative species and population data are incorporated at a
significantly reduced rate without contributions from independent and international laboratories
(c) DNA barcoding is quantifiable and accessible to bioinformatics analyses. PCR-RFLP is
essentially qualitative, relying on visual inspection against control samples and molecular weight
markers.
If access to a laboratory with DNA sequencing equipment is difficult, PCR-RFLP is a useful alternative
for the majority of species. There are also some species for which identification is ambiguous with DNA
barcoding but not for PCR-RFLP (Table 3); however, while this may be a function of the different gene
regions used, it may also be a result of the many more species and populations included in the DNA
barcode database covering more of the variation.
Gene regions used: DNA barcoding utilises a mitochondrial locus within the cytochrome oxidase I (COI)
gene, while the RFLP methods both utilise a ribosomal DNA (rDNA) gene region that includes the first
internal transcribed spacer (ITS1). Both gene regions have been chosen for the suitability of their
sequences to be distinct between species (Jinbo et al 2011; Wang 2015).
Preparation of general reagents is provided in Appendix 2.
68
7.3.1
DNA extraction for adults and immature stages
The preferred method presented here uses the DNeasy® Blood and Tissue kit (Qiagen). This produces
DNA of very high quality suitable for archiving, should any future work be needed. Other cheaper and
more rapid methods, such as prepGEM (ZyGEM, AKL, NZ) and ZR Tissue & Insect DNA MicroPrep
(Zymo Research, CA, USA) kits, are also highly suitable for diagnostic purposes, but the DNA is of
poorer quality and not so well suited to long-term archiving.
DNA should ideally be obtained using relatively non-destructive techniques, to ensure that a voucher
specimen is available for future morphological examination (Floyd et al. 2010). For fruit fly adults a leg
or the head of a specimen can be used, therefore retaining many other valuable morphological features
of the specimen (such as wings, thorax and abdomen). For larvae, anterior and posterior sections can
be sectioned off and retained, preserving the morphologically valuable mouthparts and spiracles.
Alternative even less destructive methods, involving Proteinase K digestion of internal tissues, are also
very effective (e.g. Gilbert et al. 2007).
DNA should be extracted in a room or a bench separated from PCR set up, and from post-PCR product
analysis, i.e. electrophoresis, sequencing or RFLP tests (Sections 7.3.4 and 7.3.3).
Suitable DNA can be obtained from dry, frozen or ethanol / propylene glycol preserved specimens,
(see Section 6.4.1).
Equipment and/or material needed
•
Blotting paper or Kimwipe tissues
•
Scalpel blades (if sub-sampling each sample)
•
Benchtop Microcentrifuge
•
Microcentrifuge tubes (1.5 mL), (certified DNA free, or autoclaved)
•
QIAGEN extraction kit (DNeasy® Blood and Tissue kit)
•
Heating block or water bath to 56°C
•
Forceps
•
Sterile micro pestle
•
Vortexer
•
Bead-Mill with 3 mm solid glass beads, or plastic micropestle and matching microcentrifuge tube,
(certified DNA free, or autoclaved)
•
QIAGEN DNeasy® Blood and Tissue Kit Handbook, July 2006 (for reference if required)
METHOD
The DNA is extracted by following the manufacturer’s instruction in the DNeasy Blood and Tissue kit
handbook (July 2006) for animal tissue (spin column). Slight modifications are highlighted here, with
additional notes, for DNA used in the real-time or quantitative PCR (qPCR) assay.
Prior to starting the extraction:
 Check if the ATL and AL buffers have precipitated during storage and, if so, warm them until
the precipitate has fully dissolved.
 Ensure ethanol has been added to the AW1 and AW2 buffers as specified on the reagent
bottles.
 Heat the heating block or water bath to 56°C
 Pipette 100 µl x (n+1) of AE buffer into a clean 1.5 mL Eppendorf tube, and place the tube in
the 56°C heating block. (n=the number of samples to be extracted). Eluting the DNA with pre69
warmed AE buffer can increase DNA yield and this is recommended for use with the real-time
PCR protocol.
Starting the extraction:
1)
Allow several hours for processing (Proteinase K digest time dependent).
2)
If using a “Bead-Mill” to homogenise the tissue add two 3 mm solid glass beads (acid-washed in
10 % HCl prior to use) to a clean microcentrifuge tube and add 20 µL of proteinase K. If using a
micro pestle to homogenise the tissue, add 20 µL of Proteinase K directly to the paired microfuge
tube.
3)
Remove samples (e.g. larva, adult head or leg) from ethanol and dry on blotting “tissue” until
ethanol evaporates (approximately 1 min). If samples were “dry” frozen or air dry omit this step.
Eggs can be used, but usually require multiple eggs (>5) to be processed together to obtain enough
DNA (K. Armstrong pers. comm.). Note: 1-2 eggs are sufficient for use in the real-time PCR assay.
4)
Add sample to the Proteinase K tube, cleaning forceps (ethanol wipe) in between samples to
prevent cross-contamination. Homogenise the specimen, either in a Bead-Mill (1 min @ 30 MHz)
or with plastic micro pestle in a microfuge tube.
5)
Quick-spin in centrifuge (up to 10,000 x g / rpm).
6)
If processing large numbers of specimens, rotate previously “outer” samples to “inner” position of
the Bead-Mill (i.e. swap the inner microcentrifuge tube insert around). Repeat Bead-Mill shaking (1
min @ 30 MHz).
7)
Repeat Bead-Mill and centrifuge steps until samples contain no large visible fragments. If using a
plastic pestle, initially grind until this point is reached.
8)
Add 180 µL of Buffer ATL and vortex. Note: the tissue can alternatively be homogenised in 180 µl
of ATL buffer plus the 20 µl of proteinase K together.
9)
Incubate for 0.5-1 ½ h at 56oC, or possibly overnight for complete digestion. For empty pupal cases,
or aged and dried samples, an overnight incubation is recommended.
10) Vortex, quick-spin to remove liquid from inner lid of microcentrifuge tubes.
11) Add 200 µL of Buffer AL and 200 µL of ethanol (Buffer AL and ethanol can be premixed in a large
tube for multiple samples, and then dispensed [400 µL] to each sample). Vortex.
12) Pipette mixture to QIAGEN kit column to bind DNA and centrifuge at ~6000 x g for 1 min. Discard
lower collection tube.
13) Place column into a new collection tube. Add 500 µL of Buffer AW1 to wash. Centrifuge at ~6,000
x g for 1 min. Discard lower collection tube.
14) Place column into a new collection tube. Add 500 µL of Buffer AW2 to wash. Centrifuge at ~20,000
x g (17,000 x g is acceptable) for 3 min. Discard lower collection tube (making sure no AW2 Buffer
splashes onto the base of column).
15) Label the top and side of clean 1.5 mL microcentrifuge tube with the sample’s identification /
database number.
16) Place column into the new microcentrifuge tube. Add 100 µL of AE buffer (this buffer must come
into direct contact with the column filter). Incubate at room temperature for 1 min, then centrifuge
for 1 min at 6000 x g to elute the DNA from the column. Note: 50 µl of pre-warmed AE buffer should
be used for DNA to be used in the real-time PCR protocol.
17) Repeat elution a second time.
18) Discard column and retain microcentrifuge tube containing ~200 µL (or ~100 µL) of DNA in AE
Buffer.
20) Store DNA in -20°C or -80oC freezer. Ideally, DNA can be aliquoted for long-term storage.
70
7.3.2
DNA Barcoding
This test was developed by Mark Blacket, Linda Semeraro and Mali Malipatil, Victorian Department of
Economic Development, Jobs, Transport and Resources (Blacket et al. 2012). This section (7.3.2) was
written by Mark Blacket and Karen Armstrong.
INTRODUCTION
Tephritid fruit fly adult specimens are primarily identified through an examination of diagnostic
morphological characters (Table 2). Other life stages are more problematic, with only third instar
larvae (and sometimes pupae) of some species usually identified through visual examination (e.g.
White & Elson-Harris 1992). Identification of earlier life stages (early instars, eggs), and adults of
morphologically ambiguous species, generally requires the use of molecular techniques.
DNA barcoding is a diagnostic molecular method that is routinely applied at the DEDJTR (Vic.) and
Ministry for Primary Industries (MPI) laboratories (NZ) to identify morphologically problematic
specimens and confirm new fruit fly incursions. The Australian Department of Agriculture and Water
Resources (DAWR) also regularly uses it to screen immature stages (e.g. eggs) collected from fruit to
exclude the non-tephritid exotic pest Drosophila suzukii (A. Broadley pers. comm.). DNA barcoding of
an unknown insect specimen involves obtaining a DNA sequence of a specific region of the
mitochondrial Cytochrome Oxidase I (COI) gene, and then comparing it with a database of sequences
from positively identified reference specimens.
There are currently many reference DNA barcoding sequences available. The Barcode of Life Data
Systems (BOLD) (http://www.boldsystems.org, accessed June 2015) holds 833 x Tephritidae,
including 198 x Bactrocera “species with barcodes”. This also includes data from NCBI GenBank and
of regional studies covering a broad range of endemic or intercepted tephritid species – e.g. 60
species from Oceania (Armstrong and Ball 2005), 153 species from Africa (Virgilio et al. 2012), 135
species from Europe (Smit et al. 2013). However, there is no single peer-reviewed published DNA
barcoding laboratory protocol that covers all of the targeted tephritid species listed in Table 3.
DNA barcoding should ideally obtain DNA using relatively non-destructive techniques, to ensure that a
voucher specimen is available for future morphological examination (Floyd et al. 2010). For fruit fly
adults a leg or the head of a specimen can be used, therefore retaining many other valuable
morphological features of the specimen (such as wings, thorax and abdomen). For larvae, anterior
and posterior sections can be sectioned off and retained, preserving the morphologically valuable
mouthparts and spiracles. Alternative even less destructive methods, involving Proteinase K digestion
of internal tissues, are also very effective (e.g. Gilbert et al. 2007).
There are a number of potential issues that should be taken into consideration when applying a DNA
barcoding approach to fruit fly species identification:
1) Many studies have not used the same gene region rendering the data unusable (Boykin et al.
2012);
2) Some commonly applied combinations of standard polymerase chain reaction (PCR) primers
(e.g. LCO/HCO) may amplify a nuclear copy (a numt pseudogene) of the COI barcode region
in some fruit flies (Blacket et al. 2012);
3) The presence of fruit fly species complexes can limit precise species identification (e.g.
Blacket et al. 2012, Jiang et al. 2014), enabling identification only to the complex level (e.g. B.
tryoni complex);
4) The confidence of assigning an unknown sequence to a reference species can be problematic
if all relevant potential taxa are not able to be included in the reference dataset (e.g. Virgilio et
al. 2012). The degree of identification uncertainty is dependent upon a number of factors,
71
including the accuracy of the taxonomic reconstruction (Lou & Goulding 2010), how recently
the sister species diverged (e.g. van Velzen et al. 2012), and the geographic scale of
reference samples (Bergsten et al. 2012).
Other applications of DNA barcoding data in addition to species identification are beginning to be
explored. For example, in a biosecurity context the DNA sequence data generated can also be used to
trace lineages (Blacket 2011) and possible source populations (Barr et al. 2014).
AIM
This test aims to identify fruit fly species, from any life stage, through DNA sequencing and
comparison with reference sequences of the DNA barcoding region using the publicly available
reference information in BOLD.
TARGETS
Almost all of the relevant species of fruit flies from the Australasian region are represented in BOLD
(Table 3). The small number of species that have not been sequenced to date belong to genera where
many other species have been examined; this allows a DNA barcoding approach to at least place
these species to the appropriate genus. Note: for some species complexes COI or ITS DNA
sequencing is unable to differentiate individual species (see Table 3).
PROCEDURE
This document provides supporting information for a three-step DNA barcoding process involving:
1) DNA extraction for fruit flies (adults and immature stages)
2) PCR of the mitochondrial COI barcode gene region from the fruit fly DNA.
3) Data analysis of the COI barcode gene region for species assignment using BOLD.
7.3.2.1
PCR OF THE MITOCHONDRIAL COI BARCODE GENE REGION FROM FRUIT FLY
DNA
The DNA barcode region of the COI gene is PCR amplified using a fruit fly-specific priming system,
producing a ~550 bp amplicon for sequencing. The PCR Master Mix reagents and set up must be in a
separate storage and at a separate bench (respectively) to the DNA extraction and post-PCR product
analysis (electrophoresis or RFLP, Sections 7.3.3 and 7.3.4).
Equipment and/or material needed

Primers:
o Forward: FruitFlyCOI-F (FFCOI-F) 5’-GGAGCATTAATYGGRGAYG-3’ (Blacket et al.
201215)
o Reverse: HCO 5’- TAAACTTCAGGGTGACCAAAAATCA-3’ (Folmer et al. 1994)
This primer is a fly-specific primer that was initially successfully tested on Bactrocera, Ceratitis (Tephritidae) and Calliphora
(Calliphoridae) species.
15
72

PCR Master Mix:
o BSA [1X] (diluted with distilled H2O from 100X BSA stock)
o NEB 10X Buffer (Cat# M0267S)
o dNTP’s [2.5 mM]
o MgCl2 [25 mM]
o Primers [10 µM] (working primer concentration is 10 µM, store stocks at 100 µm, -20oC)
o NEB Taq DNA Polymerase (Cat# M0267S)
o QIAGEN QIAquick Spin Handbook, March 2008 (for reference if required)

DNA template of unknown specimen(s) (see Section 7.3.1)

Fruit fly DNA to use as a positive control (to confirm that the PCR amplification is working)

AE Buffer (or TE) from the DNA extraction kit can be used to replace DNA as a negative
control (to confirm that no PCR reagents are contaminated).

Thermal cycler, e.g. “T800”, Eppendorf (epgradient S) Thermocycler

Additional equipment / materials: Submerged gel electrophoresis power pack, gel tank, TBE
buffer, agarose etc.
Methods
1) Extract fruit fly DNA for use as template (see Section 7.3.1).
2) Set up Master mix (see below), keeping all reagents on ice during setup and adding the DNA
template last.
3) The Master Mix consists of 23 µL reaction volume for each sample (plus a negative and
positive control). Note, if dealing with large numbers of samples (e.g. >10) an extra reaction
may be required to account for retention of liquid in pipette tips:
MasterMix
Reagent
x 1 reaction (µL)
x 3 reactions (µL)
1X BSA
15.3
45.9
10X Buffer
2.5
7.5
dNTP’s
2
6
MgCl2
0.5
1.5
FFCOI-F
1.25
3.75
HCO
1.25
3.75
NEB Taq
0.2
0.6
DNA Template
2
-
TOTAL:
25 µL
69 µl (x 23 µL per reaction)
73
4) PCR Conditions (“T800”, Eppendorf (epgradient S) Thermocycler):
1 x cycle
94 °C, 2 min
40 x cycles
94 °C, 30 s
52 °C, 30 s
72 °C, 30 s
1 x cycles
72 °C, 2 min
1 x hold
15 °C, indefinitely
5) After PCR is complete, load 5 µL of the PCR product (plus 2 µL loading dye) onto a 2%
agarose checking gel (use 5 µL of SYBR Safe [Cat# S33102] per 50 mL liquid gel mix, before
casting gel). Mix PCR product and dye together in plastic “gel loading” plate, using a new
pipette tip for each sample. Run agarose gel at 100 V, for 30 min. Visualise and photograph
gel on a UV light box. Estimate PCR product concentration for DNA sequencing from the
agarose gel photo (weak PCR ~20 ng µL-1, strong PCR >100 ng µL-1).
6) If necessary, clean successful PCR products using QIAquick PCR Purification Kit (QIAGEN,
Cat# 28104), elute final volume in 30 µL of EB Buffer (some external sequencing companies,
e.g. Macrogen do this step).
7) Send to external facility (e.g. Micromon, Monash University or Macrogen, Korea) for DNA
sequencing in both directions using the PCR primers above (Note, depending on each
laboratories procedures, and quality of the sequence obtained therein, sequencing may be
conducted in one direction only).
8) After high quality DNA sequences have been obtained (preferably with a QV or Phred score of
greater than 20), and with PCR primer sequence removed and the forward and reverse
consensus sequence determined (if sequencing both directions), they can be compared with a
public database (i.e. BOLD, Barcode of Life website16) to identify species as outlined in
Section 7.3.2.2.
16
http://www.boldsystems.org
74
7.3.2.2
DATA ANALYSIS – DNA BARCODING SPECIES IDENTIFICATION
The DNA sequence data for each specimen is compared with other publicly available reference
sequences for species identification. Some species, such as Bactrocera tryoni, are members of a
species complex, where the very closely related species within the complex cannot be distinguished
by their DNA barcode sequence; these specimens are thus reported as being identified to a species
group (e.g. B. tryoni complex, rather than B. tryoni).
Method
1) Go to the Barcode of Life website (http://www.boldsystems.org/).
2) Click the “Identification” tab (http://www.boldsystems.org/index.php/IDS_OpenIdEngine).
3) Paste the DNA sequence (use only the high quality section of the DNA sequence) into the
“Enter sequences in fasta format:” box (the sequence can just be entered as simple text and
does not need to be in FASTA format).
4) Select the “Species Level Barcode Records” (default); this selects reference sequences that
are >500 bp from named (identified) specimens, but some species may be represented by
only one or two specimens or be included with interim taxonomy.
5) Click the “Submit” button.
6) The top 20 matches are displayed (default), 99 best matches are available, together with the
“Specimen Similarity” score (as a percentage).
7) The matches with the highest percentage similarity (listed from highest to lowest) are the
reference sequences that best match the unknown specimen being identified. The best
matches are listed under “Search Results” and in a “Identification Summary” table together
with the percentage “Probability of Placement” (Figure 9).
8) The matches should also be viewed as a phylogenetic tree using the “Tree based
Identification” button.
9) Click “View Tree” to view a PDF of the phylogenetic tree.
10) On the tree the specimen being identified is referred to as the “Unknown Specimen” (written in
red) on the tree (indicated with arrows in Figure 10 and Figure 11), and is shown closest to the
reference specimens that it best matches.
11) The specimen can now be assigned to the species that it is most similar to, with three caveats:
a. Some specimens can only be assigned to a species group (i.e. a closely related
complex of species, Figure 11), where the species within it are unable / too similar to
be distinguished using DNA barcoding. The indications for this are when different
specimens of the same species are mixed among other species and there is no clear
single-species clade (Figure 11); this is known to occur for the complexes of B. tryoni,
B. dorsalis and Anastrepha fraterculus.
b. The assignments made by BOLD are based on a generic <2% within-species
divergence (i.e BINs, Ratnasingham & Hebert 2013). This may not be appropriate for
all of the species being analysed. For example Figure 12 shows divergences within
the B. tryoni complex are very similar (<2%) to another closely related distinct species,
B. curvipennis.
c.
The closest match is not always with the correct species. BOLD recommends that an
“identification is solid unless there is a very closely allied congeneric species that has
not yet been analysed”. While most pest fruit fly species of interest are represented in
BOLD (e.g. Table 2), closely related congeners may not. Indications for this are
difficult to observe and this caveat is dependent on knowledge of the local taxonomy.
75
Figure 9. BOLD generated identification results for the closest matches for an Island Fly (Dirioxa pornia) COI
sequence (accessed June 2015).
76
Figure 10. Specimen confidently assigned to species (Lamprolonchaea brouniana), (BOLD tree June 2015).
77
Figure 11. Specimen only confidently assigned to species group (Bactrocera tryoni complex), due to three closely
related species (B. tryoni, B. aquilonis, B. neohumeralis) being “mixed together” (i.e. non-monophyletic) on the
phylogenetic tree (BOLD tree June 2015).
78
Figure 12. Closely related species (B. curvipennis) genetically distinct from the species complex (B. tryoni) but
within the <2% assignment limit set by BOLD (BOLD July 2015).
79
7.3.3
PCR-RFLP
OVERVIEW
Two tests are described. Both are based on fruit fly-specific PCR of the rDNA ITS region (Figure 13).
Differences in method details (sections 7.3.3.1 and 7.3.3.2) reflect subtle differences in the standard
operating procedures of the original laboratories.
Test 1, developed by McKenzie et al. (2004), utilises a 0.6 to 1.2 kb PCR fragment of the ITS1 only
(Figure 13). As the ITS1 can be variable in length, for some species the size of the PCR fragment can
be an additional diagnostic character. Separate aliquots of the PCR amplicon are digested with each
of up to six different restriction enzymes to produce a species-specific RFLP profile. This number may
be reduced if the potential species could be narrowed down by knowledge of likely origin and/or host,
and by reference to Table 4 which illustrates which enzymes distinguish which species. This test can
be used for identification of 26 fruit fly species (Table 3).
Test 2, originally developed by Armstrong and Cameron (1998), is similar to Test 1 but amplifies a
larger 1.5-1.8 kb DNA fragment, encompassing the 18S and the ITS1 gene regions (Figure 13).
Because of this, the variability in PCR fragment length is not so discernible and is therefore not useful
as an additional character. Various combinations of three or four out of 10 restriction enzymes are
recommended to produce a species-specific RFLP profile. The specific suite will depend on the likely
species identity if it can be narrowed down by likely country of origin or the fruit. This test can be used
for identification of 33 fruit fly species (Table 3); details are available in Armstrong & Cameron (1998).
In the specific circumstance to distinguish just B. tryoni complex from C. capitata the enzymes AluI,
DdeI, RsaI and SspI are diagnostic.
Figure 13. Part of the ribosomal RNA operon with the location of primer positions for Tests 1 and 2
ITS1
18S
baITS1f
5.8S
baITS1r
ITS2
28S
Test 1
Test 2
NS15
ITS6
The general workflow is presented in Figure 14.
80
Figure 14. Workflow of PCR-RFLP procedures for fruit fly identification
1. Choose standards to
run in conjunction with
unknown and up to four
restriction enzymes for
diagnosis
Start
10. Set up fresh reactions;
incubate for longer
No
2. DNA extraction from
unknown sample
Section 7.3.3
8. Test 1 & 2
Restriction analysis.
Section 7.3.3.1 or
7.3.3.2
9. Have restriction
reactions gone to
completion?
Yes
3. PCR amplification
Section 7.3.3.1 or 7.3.3.2
5. Electrophoresis test for
amplified DNA
11. Compare fragment
number and length with
reference data
13. Consider additional
enzymes, or conclude it
is a different species to
any of the reference
species
Test 1 Table 5
Test 2 Appendix 4
12. Document fruit fly
identification
No
6. Has DNA been
amplified?
Yes
No
7. Test 1. Record if ITS1
fragment length is
diagnostic (Table 4).
9. Is identity consistent
across enzymes; does
identity make
sense re
Yes
other information on
fruit host or origin?
Yes
End
81
SPECIES TARGETS
Test 1 can distinguish 25 of 27 target species within the current list of 59 species (Table 1); three species
within the Bactrocera tryoni group (including B. tryoni, B. neohumeralis and B. aquilonis) cannot be
distinguished from each other.
Test 2 can distinguish 24 of 33 target species within the current list of 59 species (Table 1); nine species
form four species groups.
Test 2a distinguishes B. tryoni complex from C. capitata only.
All tests should be considered in terms of the host fruit (for immature life stage samples) and likely
country/place of origin to (a) narrow the possible species at the outset for choice of restriction enzymes
and (b) to check the results for reliability. Host records (Section 7) for the target taxa may assist in the
elimination of possible non-target species.
CHOICE OF RFLP TEST
Test 1 is more rapid, the RFLP patterns less complex to interpret and the variation in size of the ITS1
region easier to detect than Test 2. Therefore this test is recommended when the potential species is
within that listed for this test (Table 3).
Test 2 takes slightly longer, the RFLP patterns are sometimes more complex to interpret and the
variation in size of the ITS1 region not as easy to detect is in Test 1. However, a slightly modified version
(Semeraro & Malipatil 2005) provided below, is recommended instead of Test 1 specifically for the
diagnosis of the high-risk pest species B. tryoni (although indistinguishable from B. neohumeralis and
B. aquilonis within the species complex) and Ceratitis capitata. The original method provided in
Armstrong and Cameron (1998) (not included in this Handbook) is also recommended for a further 11
species not included in Test 1 but within the now extended species list in this version of the Handbook
(Table 3); the diagnostic data for Test 2 is however provided in Appendices 3 and 4.
Potential for misidentification:
 False positives are possible if other less economically important species, not included during
development of the tests, have the same RFLP pattern. This scenario is reduced by increasing
the number of restriction sites screened by using several restriction enzymes for an
identification. It is also unlikely for exotic fruit fly interceptions given the low potential for nonpest species to reach Australia. For Australian methyl eugenol or cue lure trapped specimens,
the range of taxonomically close local non-target species has not been included in development
of either test. Therefore care is needed in interpretation of the results in light of other information
about the sample, e.g. data on host or geographic origin (Section 8).
 False negatives could arise if a non-conforming RFLP pattern is produced by a population of
a species that has an aberrant polymorphism at a diagnostic restriction site. This is unlikely
given the range of populations included during the development of these tests. Also, the ITS1
is generally not useful for detecting population-level variation (cf COI barcodes, Section 7.3.3).
False negatives could also arise if the restriction fails or fails to go to completion. DNA from a
positive control (known species) should therefore be included to detect this.
CHOICE OF STANDARDS
All analyses should incorporate at least one standard, i.e. DNA from a known species. This can include
one of the anticipated species for identity of the unknown specimen, which is useful as a positive control
to help with sizing the restriction fragments of the unknown. Alternatively, it can be from a completely
unrelated species, which is useful to minimise any question of contamination or mix up. Standards are
essential to provide confidence that the PCR and restriction steps are operating correctly.
82
7.3.3.1
PCR-RFLP TEST 1
A) PCR
Equipment
 Pipettors and tips
 Sterile disposable microcentrifuge tubes
 Microcentrifuge
 Gel tank and power pack
 Latex or Nitrile gloves
 Microwave
 UV transilluminator with camera
 Thermocycler
 Personal protective equipment including lab coat, eye protection, gloves
Reagents (see APPENDIX 2 for reagent compositions)
 Manufacturer’s polymerase enzyme buffer 10X
 Taq polymerase enzyme (5U μL-1)
 Primer (McKenzie et al. 1999) are:
o baITS1f 5’ GGA AGG ATC ATT ATT GTG TTC C 3’, 10 μM
o baITS1r 5’ ATG AGC CGA GTG ATC CAC C 3’, 10 μM
 dNTP’s (2 mM)
 MgCl2 (50 mM)
 Sterile water
 1X TBE buffer
 1% (w/v) agarose gel: 1 g DNA grade agarose per 100 mL 1X TBE
 6X Loading dye
 DNA molecular weight marker (aka 100 bp ladder)
 Gel staining solution, e.g. Ethidium bromide (final concentration 800 ng/μL), or non-toxic options
such as Syber Safe, or Red Safe according to manufacturer’s instructions
Method
In a pre-PCR cabinet:
1) label sterile 0.2 ml PCR tubes
2) make a Reagent Master Mix for the total number of samples to be analysed according to the
table below, all reagents except for the polymerase (note, recommend adding an extra volume
for more than 10 samples to allow for any loss of solution via adherence to the outside of the
pipette tip):
Reagent
Final concentration
Vol per reaction (μl)
Manufacturer’s reaction buffer (10X)
1X
5
MgCl2 (50 mM)
1.5 mM
1.5
dNTP’s (2 mM)
200 μM
5
Forward primer (10 μM)
1 μM
5
Reverse primer (10 μM)
1 μM
5
H2O
20.25
Taq polymerase enzyme (5U μL-1)
0.25
Total volume
42
83
3) Store Master Mix on ice in sterile 1.5 mL centrifuge tube.
4) Add 8 μL of sterile distilled H2O or DNA buffer to the first negative control tube.
In a laminar flow hood or PCR cabinet:
5) Add the Taq polymerase to the Master Mix
6) Aliquot 42 μL Taq Master Mix to each PCR tube
7) Add 8 μL of DNA extract/control to each sample tube as appropriate
8) PCR amplify the DNA in a thermal cycler using the following program:

Cycle 1
- denature 94°C 2 min

Cycles 2 to 35 - denature 94°C 1 min, anneal 60°C 1 min, extend 72°C 1 min

Cycle 36
- extend 72°C 5 min
9)
10)
11)
12)
13)
Place reaction products on ice or freeze until ready to analyse.
Mix 3 μL of each PCR sample with 2 μL loading dye.
Load samples and 100 bp DNA ladder onto separate wells of 1% (w/v) agarose gel in 1X TBE.
Electrophorese in 1X TBE buffer at 100 V for around 40 min.
Stain the gel in ethidium bromide or other stain, according to local Standard Operation
Procedure.
14) Visualise bands and capture image using the Gel Documentation System.
Diagnostic use of ITS1 amplicon size
The expected size of the amplified product is between 500 and 1000 bp, depending on the species
(Table 4). For some species the size can be an additional diagnostic character. However, relying on
the resolution available on a gel to distinguish the majority of these amplicon sizes is not advisable as
the only diagnostic character, but is useful in conjunction with restriction analysis as described below.
Flies producing fragments of less than 700 bp or greater than 900 bp are segregated and then
restriction enzymes are used in series to differentiate the species. Sizes are given as a range to reflect
that sizing is approximate when using low-resolution gel electrophoresis systems as here.
84
Table 4. Approximate size of the ITS1 amplicon for each species. Potential diagnostic size ranges are contained
in a block
Species Fragment range
ITS1 amplicon ~size range (bp)
Potential species
500-520
D. pornia
590-610
B. cucurbitae
640-680
A. ludens
650-690
A. obliqua
670-700
B. xanthodes
740-760
A. serpentina
740-780
R. pomonella
750-780
B. umbrosa, B. facialis
760-770
B. cucumis
760-780
B. latifrons
770-790
B. musae
770-800
B. endiandrae
780-800
B. psidii
790-840
B. bryoniae, B. tryoni sp. complex
800-820
B. moluccensis
800-840
B. dorsalis sp., B. jarvisi
810-840
B. passiflorae
820-850
B. zonata
830-860
B. carambolae, B. curvipennis, B. frauenfeldi
840-860
B. albistrigata, B. kirki
890-900
C. capitata
1000-1040
C. rosa
85
B) RESTRICTION DIGESTION OF PCR PRODUCT
The use of a combination of enzymes, in series, allows definitive identification of the majority of the
species. This also eliminates the reliance on discrete restriction sites and minimises the likelihood of
false negatives that may arise through a rare recombination event.
Restriction endonucleases used are VspI, HhaI, SspI, HinfI, BsrI, SnaBI and/or Sau3aI. During the
development of this test standard enzymes purchased from New England Biolabs were used but other
brands would work equally well. Enzymes were also selected based on the requirement for differences
in fragment sizes to be easily detected by visual examination of an agarose gel.
If the likely species can be narrowed down, e.g. by fruit or geographic region, then a reduced number
of enzymes could be used. However, for robust diagnoses, it is recommended that at least four enzymes
are used, even if there is a diagnostic pattern for one enzyme that distinguishes a species from all
others.
Equipment
 Pipettors and tips
 Sterile disposable microcentrifuge tubes
 Microcentrifuge
 Dry heating block, waterbath or similar
 Gel tank and power pack
 Latex or nitrile gloves
 Microwave
 UV transilluminator with camera and image capture and analysis software
 Personal protective equipment including lab coat, eye protection, gloves
Reagents
 Sterile distilled water
 Bovine serum albumin (BSA, 10 μg/μL) (comes supplied with NEB enzymes)
 Restriction enzymes VspI, HhaI, SspI, HinfI, BsrI, SnaBI, and Sau3aI
 Restriction buffer supplied with enzyme
 Ethidium bromide solution, 800 ng/μL final concentration, or non-toxic options such as Syber
Safe, or Red Safe according to manufacturer’s instructions
Method
1) Label microcentrifuge tubes, including one for the positive control.
2) To each centrifuge tube add:
Water
2.3 μL
10X buffer
2 μL
BSA (10 ug/μL)
0.2 μL
PCR product
5 μL
Restriction enzyme
0.5 μL
3) Mix reagents and place tubes in a water bath preheated to 37oC for 2 h.
4) Store tubes on ice or at -20oC until ready to load on agarose gel.
5) Add 3 μL of 6X loading buffer to each tube.
6) Load the entire volume of each sample (23 μL) into a lane of a 2% (w/v) high resolution agarose gel.
7) Load 100 bp DNA molecular weight marker into one or two wells of the gel.
8) Analyse products by electrophoresis at 100 V for 50 min.
86
9) Stain the gel with ethidium bromide, or alternative non-toxic stain.
10) Visualise fragments using a UV transilluminator.
11) Capture gel image using gel documentation system.
Analysis of RFLP products
For diagnostic purposes, RFLP bands under 100 bp and over 1500 bp in size are disregarded due to
difficulty in accurate sizing. The molecular weights of the restriction fragments are estimated with
reference to the DNA molecular weight standard loaded on the same gel.
Tables 4 and 5 summarise the expected fragment lengths for the six restriction enzymes used in this
method. All species listed can be differentiated from each other, with the exception of those within the
B. tryoni complex. Care should be taken with fragments produced for B. albistrigata and B. kirki,
which are very similar for all six restriction enzymes and potentially difficult to confidently distinguish on
an electrophoresis gel.
Species
87
Table 5. Analysis of RFLP products from ITS1 fragments from fruit flies
ITS1*
Species
˂700
700-900
HinfI
˃900
DNC
VspI
Cuts
DNC
Hhal
Cuts
DNC
A. ludens
X
550
550
X
A. obliqua
X
450, 270
550
X
420, 250
X
A. serpentina
X
X
B. albistrigata
X
X
B. aquilonis
X
B. bryoniae
X
B. carambolae
X
X
B. cucumis
X
X
B. cucurbitae
X
Cuts
SspI
DNC
BsrI
Cuts
DNC
X
550, 150
X
Cuts
SnaBI
DNC
Sau3aI
Cuts
DNC
X
X
X
450, 200
X
X
530, 200
X
X
450, 400
X
X
670, 180
620, 180
770
X
640, 190
570, 180
600, 200
X
415
760
X
620, 200
560, 180
600, 230
X
400
680, 200
X
X
550, 180
X
X
X
X
X
X
400, 180
X
X
X
480, 350
X
X
X
620, 170
X
650, 190
X
650, 260
600, 180
X
600, 200
B. curvipennis
B. dorsalis sp.
complex
X
B. facialis
X
X
X
B. frauenfeldi
X
X
X
B. jarvisi
X
B. kirki
X
X
770
770
650, 250
550, 200
620, 180
570, 250
X
600, 250
530, 350
450, 400
X
420
540, 320
X
X
390
X
450, 400
X
420
X
450, 400
X
640, 180
700
X
X
680, 190
620, 180
B. latifrons
X
X
X
600, 190
X
600, 200
B. musae
X
X
X
635, 220
X
600, 250
B. neohumeralis
X
770
X
640, 190
570, 180
600, 200
X
B. passiflorae
X
770
X
650, 190
750
650, 270
X
X
B. psidii
X
X
640, 190
570, 250
X
X
X
Cuts
X
X
X
X
X
520, 320
X
420
88
Species
ITS1*
˂700
700-900
HinfI
DNC
770
X
640, 190
570, 180
B. umbrosa
X
730
X
600, 190
680
B. xanthodes
X
680
X
670, 200
380, 250
B. zonata
X
X
680, 190
750
X
X
X
C. rosa
X
800, 200
X
X
DNC
Cuts
DNC
BsrI
Cuts
X
Cuts
SspI
X
C. capitata
DNC
Hhal
B. tryoni
D. pornia
˃900
VspI
Cuts
DNC
SnaBI
Cuts
600, 200
DNC
Cuts
Sau3aI
DNC
Cuts
X
420
X
X
380
X
X
600, 200
X
535, 330
X
650, 200
X
520, 160
X
X
X
600, 300
X
570, 480
X
X
X
X
300, 220
X
X
X
The length of the ITS1 fragment and the response of each to seven restriction enzymes (HinfI, VspI, HhaI, SspI, BsrI, SnaBI, Sau3aI) are indicated for each of the target
species. ITS1 fragment length is scored as one of three classes (approximate length in bp). Enzyme responses are measured in two classes - either does not cut (DNC) or
cuts.
(Cuts – this column shows the length of each fragment in bp). Highlighted boxes denote diagnostic RFLP patterns, which are more than 20 bp different to other fragment produced
by that restriction enzyme.
89
7.3.3.2
PCR-RFLP TEST 2
A) PCR
Materials and equipment
As for PCR-RFLP Test 1 Section 7.3.3.1
Reagents
As for PCR-RFLP Test 1 Section 7.3.3.1 with the exception of primers, which are here:
 NS15 5’ CAATTGGGTGTAGCTACTAC 3’
 ITS6 5’ AGCCGAGTGATCCACCGCT 3’
Method
In a PCR (laminar flow or clean bench) cabinet:
1) label sterile 0.2 ml PCR tubes
2) make a Reagent Master Mix for the total number of samples to be analysed according to the
table below, adding the polymerase last (note, recommend adding an extra volume for more
than 10 samples to allow for any loss of solution via adherence to the outside of the pipette tip):
Reagent
Final concentration
Double-distilled H2O
Vol per reaction (μl)
30.6
Expand High Fidelity polymerase buffer
1X
5
dNTPs (2.5 μM)
200 μM
4
Forward primer 10 μM (NS16)
0.5 μM
2.5
Reverse primer 10 μM (ITS6)
0.5 μM
2.5
Expand Hi Fidelity Taq Polymerase
2 Units
0.4
Total
45
3) Store Master Mix on ice.
4) Vortex DNA extractions for 5 s.
5) Aliquot 5 μL of each unknown DNA template to labelled 0.2 mL tubes, plus at least one positive
control DNA and one negative control (water or the buffer in which the DNA is suspended).
6) Aliquot 45 μL of master mix to each 0.2 mL tubes (containing 5 μL of template DNA).
7) Mix product and reagents well (or vortex briefly) and centrifuge for 3-5 s.
8) Place samples in PCR machine and program the following temperature profile (based on
Armstrong and Cameron 1998):
•
Cycle 1
- denature 94°C 2 min
•
Cycles 2 to 35 - denature 94°C 15 s, anneal 60°C 30 s, extend 68°C 2 min
•
Cycle
- extend 72°C 5 min
9) Add 1 μL of loading dye to 5 μL of PCR product
10) Load onto a 1.5% agarose gel, together with a 100 bp molecular ladder at either side and
electrophorese according to Appendix 2. If product visible at 1.5-1.8 kb then proceed to Section
B) – restriction digestion.
90
B) RESTRICTION DIGESTION OF PCR PRODUCT
Equipment
See 7.3.3.1 B
Reagents
 Restriction enzymes, on ice.
o NB. Choose at least four of the enzymes listed in Appendix 3 according to which combination
will provide the best discrimination for the potential species; the letters represent RFLP
patterns in Appendix 4
o Note: In the specific circumstance of distinguishing only B. tryoni complex and C. capitata the
enzyme combination of AluI, DdeI and RsaI (10 U/μl) and SspI (5U/μl) is diagnostic.
 Restriction enzyme buffers, 10X
 Sterile nuclease free H2O
Method
1) Prepare master mix (following recipe below) for each enzyme; multiplying volumes plus one
for the number of reactions required.
Adapted from Armstrong & Cameron (1998)
Final concentration
Double-distilled H2O
Vol per reaction (μl)
5.6*
10X Buffer
1X
1.0
Enzyme†
4U
0.4
Total
7.0
* volume of water can be varied to accommodate any change to DNA volumes added, see below
† Standard stock concentrations are usually 10 U/µl; if not, this volume will change accordingly
2) Aliquot 7 µL of each master mix into labelled tubes
3) Add 2-3 μL (100-200 ng) PCR product into each tube depending on how strong or weak the
PCR products are; adjust water in reaction mix accordingly
4) Flick to mix reagents and PCR product, centrifuge briefly for 3-5 s.
5) Place samples in incubator at 37°C 2-3 h unless otherwise recommended by the enzyme
manufacturer
6) Prepare a 2-3% agarose gel according to Appendix 2, at least 10 cm in length, to visualise
fragment pattern and use a 100 bp ladder for determining fragment sizes
7) Compare results with positive controls and fragment patterns in Appendix 4
8) Species diagnosis is considered positive if all four enzyme patterns agree.
91
7.3.4
Real-time PCR detection of Bactrocera tryoni complex
This test was developed and validated by Dhami et al., (2015), further validation was conducted by
David Waite and Dongmei Li during 2014-2015 at the Plant Health and Environment Laboratory
(PHEL), Ministry for Primary Industries (MPI), New Zealand. This section (7.3.4) was written by
Dongmei Li, David Waite and Disna Gunawardana.
INTRODUCTION
Genetically similar but morphologically different species have been reported in several fruit fly species
(Clarke et al., 2005; Krosch et al., 2012). Such species are considered as belonging to “species
complexes” (Drew, 2004). The commonly called ‘Queensland fruit fly’ (Q-fly) is one such species
complex, consisting of four species; B. tryoni, B. aquilonis, B. neohumeralis and B. melas (Drew, 1989).
However, while B. melas exists in historic documents, researchers now consider it a cryptic form of B.
tryoni (Clarke et al., 2011).
Analysis of microsatellite markers has shown that there is only intraspecies level of differentiation
present between B. tryoni and B. aquilonis (Wang et al., 2003). Those analyses also reflect that B.
neohumeralis and B. tryoni represent separate species (Clarke et al., 2005; Wang et al., 2003). The
closely related species B. curvipennis, however, is difficult to distinguish from B. tryoni complex based
upon DNA sequencing of the cytochrome c oxidase subunit 1 (COI) gene (Armstrong & Ball, 2005),
although it is morphologically distinct. More comprehensive molecular analysis has similarly indicated
that B. curvipennis has a paraphyletic relationship with the B. tryoni complex and is currently considered
very close to, but not within, the B. tryoni complex sensu Drew 1989 (Blacket et al., 2012; Jiang et al.,
2014).
DNA barcoding relies on PCR of predetermined marker genes, DNA sequencing and comparison of
those sequences to a database of reference sequences (Armstrong & Ball, 2005). Currently, DNA
barcoding of the COI gene is the most commonly used technique for the molecular identification of fruit
flies (Armstrong & Ball, 2005; Blacket et al., 2012). This process is still relatively time consuming, with
the quickest possible identification requiring a full day. In contrast, real-time PCR, or quantitative PCR
(qPCR), drastically reduces the end-to-end time of analysis. This technique is based on the amplification
of DNA monitored in real time and therefore without the need of post-PCR processing (e.g. gel
electrophoresis), thus it enables identification within a few hours. The premise for this technology is that
highly species-specific PCR primers or probes are designed to give positive reactions only with DNA of
the target species. A positive identification is determined by the measurably lower number of PCR cycles
taken for the amplicon to reach a given concentration, and for fluorescence to be detected (the
quantitation cycle, Cq), than would occur for a negative or suboptimal amplification, as would be
expected from DNA of the wrong species. The TaqMan chemistry has the potential for greater specificity
through the incorporation of a third oligonucleotide in the reaction. The method has previously been
applied to other fruit fly species on the list in this manual (Burgher-MacLellan et al., 2009; Yu et al.,
2005; Yu et al., 2004).
Here, a real-time PCR assay for the B. tryoni complex has been developed, validated and applied by
PHEL in the routine diagnostics of intercepted fruit fly material at borders, post borders and in recent
New Zealand Q-fly responses.
AIM
This assay aims to provide a rapid method for the identification of the B. tryoni complex, from any life
stage, using the real-time TaqMan technique.
TARGET
This assay can be used identify B. tryoni complex, (B. tryoni, B. aquilonis, and B. neohumeralis), and
the closely related species B. curvipennis.
92
PROCEDURE
This document provides all the supporting information for conducting the real-time PCR assay and
analysis of the results, including:
1. DNA extraction and subsequent PCR from a range of life stages of the fruit flies including eggs,
part of an adult (one leg is commonly used in diagnostics), part of a larva, part of a pupa, empty
pupal case
2. PCR competency test
3. Real-time PCR assay for the B. tryoni complex (FAM probe)
4. Results - analysis of Real-time PCR assays.
7.3.4.1
DNA Extraction protocols
See section 7.3.1.
7.3.4.2
Real-time PCR assay for B. tryoni complex
Set up the PCR assay in a PCR workstation. The assay is described as run on the CFX1000 TM realtime PCR system (BioRad), with the data analysed in the CFX manager 3.0 analysis software
(BioRad).
Equipment and material
 Primers and Probe (Dhami et al., 2015)
o Forward primer: Btry2F, 5’-AATTGTAACAGCCCATGC-3’
o Reverse primer: Btry1R, 5’- GTGGGAATGCTATATCGG-3’
o Probe: Btry2PL: 6FAM-AG[+C]CA[+G]TTTCC[+G]AA[+A]CC-BBQ (or BHQ1)
 Real-time PCR mastermix
o SsoAdvanced Universal Probe Supermix (Cat#1725280, Biorad), 2x qPCR mix,
containing dNTPs, Sso7d fusion polymerase, MgCl2, stabilizers, ROX normalization
dyes.
o Primers/probe, working concentration of 5 µM (primer stock concentration 100 µM,
stored at -20°C in dark)
o BSA (Bovine serum albumin, working concentration of 10ng/µl) (Sigma Cat# A78850g)
 DNA samples of unknown Tephritidae species extracted as in section 7.3.1.
 Internal positive control to test unknown DNA samples for PCR competency to avoid false
negative results
o Conventional PCR with Folmer primers LCO1490 and HCO2198 (Folmer et al., 1994)
o TaqMan ribosomal RNA control reagents (VIC probe) (Applied Biosystems, cat #
4308329), see Appendix 1 for details.
 Controls for real-time PCR setup:
o Positive control to monitor the performance of the real-time PCR
 DNA samples of B. tryoni complex or
 Plasmid DNA of COI insert of B. tryoni complex (available from MPI PHEL,
NZ on request)
o Non-template control
 Sterile water or PCR-clean water
 PCR work station or DNA/RNA-free area for PCR setup
 Thermal cycler: CFX96 or CFX1000 Touch Real-time PCR (BioRad). Other brands of realtime thermal cycler can be used.
 Additional equipment: centrifuge, pipettes, plugged PCR tips, PCR tube, PCR plates, plate
seals.
Method
1. PCR competency test: It is recommended prior to any qPCR analyses to test all the DNA
extractions for PCR competency, using either conventional PCR or 18S internal control
TaqMan assay (see Appendix 1 for details)
93
2. Set up the real-time PCR Mastermix in a PCR work station or clean area, see below for the
compositions (Table 6). Include enough volume to run the samples in duplicate or triplicate
plus positive and non-template controls. It can also be run at 20 µl or 10 µl volume.
a. For 20 µl volume, aliquot 18 µl of master mix to each PCR tube
b. For 10 µl volume, aliquot 9 µl of master mix to each PCR tube
Table 6. TaqMan real-time PCR for B. tryoni complex protocol using SsoAdvanced Universal Probe Supermix
(BioRad)
Reagents
1 x reaction (µl)a
10x reactions (µl) b
Sterile distilled H2O
2.2
22
2 x Probe Supermix
10.0
100
5 µM Btry2F (400 nM)
1.6
16
5 µM Btry1R (400 nM)
1.6
16
5 µM Btry2PL(250 nM)
1.0
10
BSA (10mg/ml)
1.6
16
DNA template
2.0
Note: aThe compositions for 20 µl are listed in this table, halve the volumes for each reagent if using in 10 µl volume. bUsing 10x
reaction as an example, calculate the volumes of each reagents using the number of reactions you are going to test when
conducting the assay; note, one or two extra reaction volumes should be included to account for loss due to retention of liquid in
pipette tips.
3. Set up the Mastermix, mix well and spin briefly, aliquot 18 µl of the Mastermix to each labelled
PCR tube or well of a plate.
4. Add the appropriate DNA template or control solution to the tubes or the wells of a plate with
the aliquoted Mastermix, close the tubes or seal the plate, mix well and spin the tubes or plate
briefly
5. Put the tubes/plate into real-time PCR machine and run the program below:

PCR cycling parameter (CFX 1000TM Thermal cycler)
1 x cycle
95 °C, 2 min
40 x cycles
95 °C, 15 sec
65 °C, 60 sec
Plate read after each cycle
6. Open CFX manager 3.0 program, set up the protocol and run the real-time PCR assay.
7. Name the assay and save the data in a folder.
8. Once the run is finished, the data can be opened in the CFX manager 3.0 and analysed.
94
7.3.4.3
Analysis of Real-time PCR results
The real-time PCR results are analysed here with the CFX Manager 3.0 (BioRad). If different real-time
PCR systems are used, analysis of the amplification curves will need to be carried out according to
that manufacturers’ manual.
1. Open BioRad CFX Manager 3.0.
2. Click file, open the data file, and choose the real-time PCR data you have saved for the assay.
3. Click on quantification tab, the amplification curve will appear on the screen, see Figure 15 for
an example.
4. Set up the baseline for 50.
5. Report: Click Tools tab and choose reports, an analysis report for the real-time PCR including
amplification curves and Cq values will be generated. It can be saved as pdf format or print it
out.
6. Interpretation of the results - 18S internal control:
a. If Cq values ≤30 cycles, the DNA extraction is PCR competent and the samples can
be used for real-time PCR assay against B. tryoni complex.
b. If Cq values >30 cycles, this indicates that inhibitors are in the DNA sample or there is
insufficient DNA. Re-extraction of the samples will be needed.
7. Interpretation of the results - B. tryoni complex assay (use the PCR competent DNA samples
only):
a. For Cq values ≤30 cycles, the sample is considered as positive for B. tryoni complex,
and possible for closely related B. curvipennis (Cq values between 25-30 cycles)
i. DNA barcoding needs to be conducted to confirm the species if the Cq values
>25 cycles.
ii. The origin of the specimen might assist in distinguishing B. tryoni and B.
curvipennis (e.g. B. curvipennis is endemic to New Caledonia and not present
in Australia).
b. Samples with Cq values between 30-35 cycles could be interpreted as either a
different species with suboptimal match to the primers or that there is a very low copy
number of the target species DNA. These should be considered as questionable and
require further investigation by either DNA barcoding or re-extraction of DNA.
c. The negative threshold for the assay is Cq values >35, at which point the samples
should be considered as negative for B. tryoni complex, and identification by another
method is necessary.
8. Validation of the real-time PCR assay for B. tryoni complex used DNA extracted from fresh
and aged (up to 2 year olds) samples. This included B. tryoni from NSW, VIC, QLD (n>80)
and B. neohumeralis from QLD (n>20). The following results were observed;
a. Cq values ≤25 cycles were obtained with the DNA extracted from the B. tryoni
complex except those with empty pupal cases.
b. Cq values between 25-35 cycles were observed for the DNA extracted from B.
curvipennis samples. DNA barcode sequencing confirmed the identification.
c. Negative results (Cq >35) were obtained for all other Tephritidae species tested (see
Appendix 2).
95
Sample tested
B. tryoni
Positive control
Water control
Figure 15. Amplification curves of real-time PCR assay for B. tryoni complex. The sample tested positive for B.
tryoni complex, DNA sequences has further confirmed the results.
96
7.3.5
Allozyme electrophoresis
This section was written by Mark Adams.
AIM
Allozyme electrophoresis provides a method for the rapid molecular identification of various species of
fruit fly.
TARGETS
Routinely targets Bactrocera tryoni, Ceratitis capitata, Dirioxia pornia, Bactrocera dorsalis s.s. and
Bactrocera jarvisi (Table 3). Additional species can be incorporated where suitable reference
material is provided as freshly-frozen specimens (either as larvae or adults).
SUITABILITY
Suitable for the comparison of genetic profiles, as expressed in a range of soluble proteins, from
live, recently-dead, or freshly-frozen larvae or adults. The service is currently routinely provided
by the South Australia Museum's Evolutionary Biology Unit laboratory. The procedures take 2-3
hours to complete for a single screen of up to 20 specimens for 10 different genes.
Given the comparative nature of the technique and its continued reliance on reference samples, it is
important to note that additional species can only be identified as "new" (i.e. not one of the five
reference species) unless suitable, known-identity samples can also be provided for a putative
match. Moreover, the incorporation of additional species into the routine screening procedure may
also require a re-evaluation of which enzyme markers are diagnostic for these additional species, in
order to satisfy the minimum requirement of three diagnostic genetic differences between every pair
of species.
PROCEDURE OVERVIEW
Crude extracts of soluble protein from live, recently-dead, or freshly-frozen larvae or adults
are compared electrophoretically against known-identity extracts representing these five
species.
Test samples are readily identifiable by their comparative allozyme profile (i.e. relative band mobility)
at a suite of at least six enzyme markers, encoded by a minimum of 10 independent genes, and
together able to unambiguously diagnose the five reference species from one another at a minimum
of three genes.
Neither B. aquilonis nor B. neohumeralis are listed in the five target species, so this method is
not designed to differentiate between B. tryoni and these other two, genetically very similar
species.
7.3.5.1
Specimen preparation
Test specimens

Need to be supplied either (a) alive, (b) freshly dead and kept cool and moist, or (c) frozen
when alive and not allowed to thaw until tested (dry ice required for transport; ice is not
suitable)

Can represent any life history stage
97
Reference specimens

Frozen specimens representing the species requiring discrimination must be available. When
kept at -70oC, these remain suitable for use as controls for at least 10 years.

A single homogenized larva provides enough homogenate to act as a reference specimen on
up to eight separate occasions. Once prepared, these reference homogenates can be stored
at -20 oC as separate ~5 µL aliquots inside glass capillary tubes. Thus reference specimens
for any test run are usually available as pre-prepared homogenates straight from the freezer.
Specimen Preparation (ideally in cold room at 4oC)

Specimens are hand-homogenized in an equal volume of a simple homogenizing solution
(0.02 M Tris-HCl pH 7.4 containing 2 g PVP-40, 0.5 mL 2-mercaptoethanol and 20 mg NADP
per 100 mL).

~0.5 µL of homogenate loaded directly onto each gel.

The remaining homogenate is transferred as a series of ~5 µL aliquots into individual glass
capillary tubes and stored at -20oC. These samples can either be subjected to further
allozyme analysis if doubt remains as to species identity, or used as fresh reference material
for the species thus identified (activity declines over a 12 month period at -20oC).
7.3.5.2
Electrophoresis (ideally in cold room at 4oC)
Allozyme analyses are conducted on cellulose acetate gels (CellogelTM) according to the principles
and procedures of Richardson et al. (1986). Table 7 indicates the suite of enzymes most commonly
used for fruit-fly genetic identifications and details the electrophoretic conditions employed for each.
7.3.5.3
Gel interpretation
The interpretation of allozyme gels requires some expertise; the intensity of allozyme bands changes
over time after a gel is stained, plus banding patterns can be affected by the “freshness” of the
specimen and by what type of gut contents are present (some plants contain compounds which affect
fruit fly enzymes once the sample is homogenised). Richardson et al. (1986) devote an entire section
to the interpretation of allozyme gels, but there is no substitute for experience.
7.3.5.4
Recording of results
All gels are routinely scanned several times over the time course of stain incubation and the resultant
JPG files archived as a permanent record.
98
Table 7. Enzymes most commonly used for fruit-fly genetic identifications using allozyme electrophoresis.
Enzyme
Abbr
E.C.
No.
No. of
genes
Buffer1 Run time
Stain
Aconitase
hydratase
ACON
4.2.1.3
2
I17
Richardson
et al. (1986)
C. capitata vs B. tryoni vs
B. jarvisi vs B. dorsalis vs
D. pornia
Aminoacylase
ACYC
Manchenko
(1994)
C. capitata vs B. tryoni
vs B. jarvisi / B. dorsalis
vs D. pornia
Richardson
et al. (1986)
C. capitata vs B. tryoni vs
B. jarvisi vs D. pornia
method 3;
Manchenko
(1994)
C. capitata vs B. tryoni /
B. dorsalis vs B. jarvisi
vs D. pornia
Richardson
et al. (1986)
C. capitata vs B. tryoni /
B. jarvisi / B. dorsalis vs
D. pornia
Richardson
et al. (1986)
C. capitata vs B. tryoni /
B. dorsalis vs D. pornia
Richardson
et al. (1986)
C. capitata vs B. tryoni /
B. jarvisi vs B. papaya vs
D. pornia
1.2 h
@ 250 V
3.5.1.14 1
C
1.5 h
@ 250 V
Alcohol
dehydrogenase
ADH
Aspartate
aminotransferase
GOT
Glycerol-3phosphate
dehyrogenase
GPD
Glucose-6phosphate
isomerase
GPI
3Hydroxybutyrate
dehydrogenase
HBDH
Isocitrate
dehydrogenase
IDH
Malate
dehydrogenase
MDH
Dipeptidase
PEPA
1.1.1.1
2
C
1.5 h
@ 250 V
2.6.1.1
2
B
1.5 h
@ 250 V
1.1.1.8
1
C
1.5 h
@ 250 V
5.3.1.9
1
B
1.5 h
@ 250 V
1.1.1.30 1
B
1.5 h
@ 250 V
1.1.1.42 2
B
1.5 h
@ 250 V
1.1.1.37 2
C
1.5 h
@ 250 V
3.4.13
2
B
1.3 h
@ 250 V
1Code
17
Species delineated
Richardson
et al. (1986)
B. tryoni vs B. dorsalis
Richardson
et al. (1986)
C. capitata vs B. tryoni /
B. dorsalis / B. jarvisi vs
D. pornia
Richardson
et al. (1986)
C. capitata vs B. tryoni /
B. jarvisi / B. dorsalis vs
D. pornia
for buffers follows Richardson et al. (1986).
Sample origin is placed in the centre of the gel
99