Current Research in Insect Science 2 (2022) 100029

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Current Research in Insect Science

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A loop-mediated isothermal amplification (LAMP) assay for rapid

identification of Ceratitis capitata and related species
Wannes Dermauw#, Yannick Van Moerkercke, Negin Ebrahimi, Hans Casteels, Jochem Bonte,
Johan Witters#
Flanders research institute for Agriculture, Fisheries and Food, Plant Sciences Unit, Burg. Van Gansberghelaan 96, B-9820 Merelbeke, Belgium

a r t i c l e i n f o a b s t r a c t

Keywords: True fruit flies (Tephritidae) are among the most destructive agricultural pests in the world, attacking a wide range
Tephritidae of fruits and vegetables. The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous species but, being
LAMP widely established in the Mediterranean region, is not considered as a EU quarantine pest. Hence, it is important to
quarantine organism
discriminate Ceratitis capitata from non-EU tephritid species, present in imported fruit and vegetables, as non-EU
molecular diagnostics
species have a quarantine status. However, morphological identification of tephritid larvae, the most frequently
intercepted stage in non-EU produce, is difficult and an easy-to-use molecular diagnostic tool would be helpful for
rapid species identification. Therefore, a loop-mediated isothermal amplification (LAMP) method was developed
for C. capitata and non-EU tephritids Ceratitis cosyra group1 and Ceratitis species from the FARQ complex, C.
fasciventris, C. anonae, C. rosa and C. quilicii. LAMP assays were run with DNA from ILVO collected specimens
and DNA samples collected during previous research surveys. LAMP primers were species-specific, with LAMP
amplification occurring within 45 minutes for the targeted species. In addition, LAMP assays were successful for
all C. capitata life stages or a limited amount of tissue. To conclude, the LAMP assays developed in this study
were able to distinguish C. capitata from non-EU Tephritidae species and could be a useful tool for the rapid
identification of C. capitata.

1. Introduction the importer, putting great pressure on all parties involved, including
operators, importers, NPPO and NRL.
The Tephritidae is a group of colourful fruit flies and includes The Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae),
approximately 5000 species from 500 genera (Brown et al., 2018; is one of the most destructive pests in the world. It occurs in more
EFSA Panel on Plant Health, 2020). Currently, around 35% of the than 130 countries and has a very wide host range, including impor-
Tephritidae are recognized as pests of commercial fruit and vegetables. tant commercial fruits and vegetables such as citrus, apple, peach, and
According to the European Union (EU) 2016/2031 regulation, the so- mango (White & Elson-Harris, 1992). Although C. capitata originates
called non-EU Tephritidae have a quarantine status and may not be in- from Sub-Saharan Africa (De Meyer et al., 2002), it is now widespread
troduced into the EU. Hence, imported produce needs to be examined in Southern Europe, including Spain, Italy, Greece, Malta, Cyprus and
by each EU member state to prevent the entry, establishment and spread Portugal (EPPO, 2021) and therefore, not considered as a EU quarantine
of these quarantine organisms. In Belgium, these controls are performed pest stricto sensu (Regulation (EU) 2016/2031). Consequentially, it is im-
by the Federal Agency for the Safety of the Food Chain (FASFC) which portant to discriminate C. capitata from non-EU tephritids listed in the
also acts as the National Plant Protection Organisation (NPPO). Sus- new directive for reasons described above. Experts are able to discrim-
pected products are further analyzed by the National Reference Labora- inate between adults of different tephritid species, but morphological
tory (NRL) for Entomology (at the Flanders Research Institute for Agri- identification of larva - the most frequently found stage in intercepted
culture, Fisheries and Food (ILVO)) for conformation of non-EU fruit fruit and vegetables - is difficult even when dealing with the third lar-
flies. During the identification of the fruit fly, the suspected cargo is val instar (Balmès & Mouttet, 2017; EPPO, 2011). Further rearing to
halted until further notice. The longer the cargo - chilled, perishable the adult stage prolongs the identification procedure considerably and,
products - is blocked, the higher the financial consequences may be for in most cases, the success rate is low. Moreover, C. capitata feeds on

#
Corresponding authors: Wannes Dermauw, Johan Witters, Flanders research institute for Agriculture, Fisheries and Food, Plant Sciences Unit, Burg. Van Gans-
berghelaan 96, B-9820 Merelbeke, Belgium.
E-mail addresses: [Link]@[Link] (W. Dermauw), [Link]@[Link] (J. Witters).

[Link]
Received 1 September 2021; Received in revised form 7 January 2022; Accepted 10 January 2022
2666-5158/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
([Link]
W. Dermauw, Y. Van Moerkercke, N. Ebrahimi et al. Current Research in Insect Science 2 (2022) 100029

the same hosts as many non-EU Tephritidae, including Ceratitis cosyra, sium sulphate and 0.67 g potassium phosphate. Once larvae had devel-
Ceratitis rosa and Bactrocera dorsalis, further complicating correct iden- oped into adults, an adult was transferred to a 1.5 mL Eppendorf tube,
tification. As a consequence, molecular diagnostic assays have been rec- subsequently held in liquid nitrogen for five minutes and homogenized
ommended to assist in the identification of tephritid larvae. Different using a pestle. Next, DNA was extracted using the NucleoSpin Plant II
molecular methods for identification of C. capitata have been described Kit (Machery-Nagel, Germany), in an elution volume of 50 μL. In addi-
(PCR-RFLP method based on ITS1 primers (Douglas & Haymer, 2001), tion to DNA from fresh specimens, DNA from tephritid specimens (adult
RFLP-PCR identification method based on the cytochrome oxidase sub- males or females), kindly provided by Dr. Marc De Meyer and Dr. Mas-
unit 1 gene (Barr et al., 2006) and DNA barcoding (Barr et al., 2012; similiano Virgilio (Royal Museum of Central Africa (RMCA), Brussels,
Van Houdt et al., 2010)) to distinguish C. capitata from other Cerati- Belgium), was also used. The Qiagen Blood and Tissue kit (Qiagen, Bel-
tis species. These techniques require a well-equipped laboratory with gium) was used to extract DNA from these RMCA samples, using an AE
expensive equipment and well-trained staff. Moreover, these tests sig- buffer (10 mM Tris-Cl, 0.5 mM EDTA, pH 9.0) elution volume of 70 μL.
nificantly increase the analysis cost and, in general, take a considerable All adult specimens were morphologically identified using taxonomic
amount of time, especially when some steps of the protocol need to be keys of White and Elson-Harris (1992), Virgilio et al. (2014) and/or
outsourced (e.g. sequencing). Therefore, a fast, reliable and inexpensive Carroll et al. (2019).
identification technique to discriminate C. capitata from non-EU Tephri-
tidae would be very useful and accelerate the release of suspected ship- 2.2. PCR of Ceratitis COI region and phylogenetic analysis
ments.
The loop-mediated isothermal amplification (LAMP) method, devel- PCRs were performed to amplify a 710 bp region of the cytochrome
oped by Notomi et al. (2000), is an appropriate alternative for the oxidase subunit 1 (COI) gene using the primers of Folmer et al.
rapid molecular identification of quarantine insects. Currently, LAMP (1994) and MyFiTM -mix (Meridian Bioscience, Belgium). The PCR mix
assays already exist for certain quarantine insects such as Thrips palmi, consisted of 12.5 μL MyFi-mix, 1 μL HCO2198 (10 μM), 1 μL LCO1490
Bemisia tabaci and Liriomyza sp.. These were commercialized by Opti- (10 μM), 2 μL of kit extracted DNA (see above) or 5 μL of a 1/10th di-
gene ([Link] and lution of larva lysis solution (larva homogenized in plant lysis buffer)
are used for species identification at two ports of entry in Switzer- and 8.5 or 5 μL of ultrapure water, respectively. PCR conditions were
land (von Felten, 2017). For a similar purpose, a LAMP kit was de- as follows: 95°C for 1 min followed by 35 cycles of 95°C for 1 min,
veloped for rapid identification of Bactrocera flies (Bactrocera dorsalis 51°C for 30 sec and 72°C for 15 sec. PCR amplicons were purified using
complex, Bactrocera cucurbitae/Bactrocera latifrons and Bactrocera cor- the Smartpure PCR Kit (Eurogentec, Belgium) and sent for sequencing
recta/Bactrocera zonata) (Blaser et al., 2018), while recently a LAMP to Macrogen (The Netherlands), Genewiz (Germany) or Eurofins Sci-
identification method was reported for B. tryoni (Blacket et al., 2020), entific (Luxemburg) using both the forward (HCO2198) and reverse
Dacus ciliatus (Sabahi et al., 2018) and Zeugodacus scutellatus (Kitano primer (LCO1490). COI sequences were aligned with those of a selec-
& Takakura, 2020). In 2009, Huang et al. developed a LAMP assay tion of tephritid species available in the NCBI/EPPO-Q-BANK database
for C. capitata but species-specificity of these primers was not assessed using MAFFT version 7 (Katoh & Standley, 2013) and default settings.
(Huang et al., 2009). In this study, we describe a user-friendly DNA ex- A phylogenetic analysis was subsequently performed at the IQ-TREE
traction method for tephritid species and developed LAMP-assays that web server ([Link] with sequence type set to
could aid in the rapid identification of C. capitata, C. cosyra group1 (ac- “codon”, genetic code set to “Invertebrate mitochondrial”, model set to
cording to Virgilio et al. (2017)) or Ceratitis species belonging to the “AUTO” and with 1000 ultrafast (UF) bootstraps (Nguyen et al., 2014;
FARQ complex (Virgilio et al., 2019). Trifinopoulos et al., 2016).

2. Materials and Methods 2.3. LAMP assays

2.1. Collection of tephritid samples and DNA extraction LAMP primers were designed for C. capitata (Ccap), C. cosyra group1
(Ccos g1) and Ceratitis species belonging to the FARQ complex (C. fas-
Fruit and vegetable samples that arrived at the Diagnostic Centre civentris, C. anonae, C. rosae and C. quilicii) (CFARQ) (Virgilio et al.,
for Plants (DCP) of the Flanders Research Institute for Agriculture, Fish- 2019). For all species, the COI gene was used as template for primer
eries and Food (ILVO, Merelbeke, Belgium) were inspected for the pres- design. Primers were designed to be as specific as possible using the
ence of tephritid larvae. Detected larvae (either L2 or L3 instars) were Primer Premier Software (PREMIER Biosoft, USA) or Primer Explorer
stored in 90% ethanol until DNA extraction or lysis solution prepara- V5 (Eiken Chemical Company, Japan) and taken into account COI se-
tion. Before DNA extraction or lysis preparation, larvae were allowed to quences of the most closely related species available in either the NCBI
dry on filter paper to remove excess of ethanol. The NucleoSpin Plant or EPPO-Q-BANK databases. LAMP assays were performed using the
II Kit (Machery-Nagel, Germany) with lysis buffer PL1, which is based GspSSD Isothermal Mastermix (ISO-001; Optigene, UK) and a Quantstu-
on the CTAB lysis method (a method that has been previously used for dio 5 Real-Time PCR system (Qiagen, Belgium) with the fluorescence
DNA extraction of tephritid specimens, see e.g. Asokan et al. (2011)), reporter set to FAM. The LAMP reaction volume was 20 μL consisting
and an elution volume of 50 μL, was used to extract DNA of larvae. of 10 μL ISO-001, 0.25 μM of F3/B3, 1.25 μM of BIP/FIP and 2 μL of
Alternatively, some fresh intercepted larvae were cut into two pieces sample: 1/10th dilution of larva homogenized in plant lysis buffer or kit
(separating the anterior part from the rest of the body) and the rest of extracted DNA (see Table S1). A 1/10th dilution of the larva lysis so-
the body was again divided into two pieces. These two posterior pieces lution was used as the anneal derivative melting temperature (Ta, see
were transferred to a 1.5 mL Eppendorf tube containing 200 μL plant below) of LAMP products in LAMP assays using this dilution was similar
lysis buffer (EXT-001 Buffer; Optigene, UK) (Witters et al., 2018). Sub- to the Ta of LAMP products in LAMP assays using kit extracted (pure)
sequently, the larva lysis solution was vortexed for 1 min, incubated for DNA as input, while for LAMP products in LAMP assays using undiluted
5 min at room temperature, and 1/10th diluted in water. Lastly, some larva lysis solution the Ta was 3-4°C lower (data not shown). For each
intercepted tephritid larvae were grown on an artificial diet at 22°C. LAMP run, a positive control (LAMP primers with DNA from targeted
The artificial diet was based on those of Lebreton et al. (2014) and species) and a negative control (LAMP primers with water or a 1/10th
Enriquez et al. (2019) and consisted of 1 L water, 15 g agar, 50 g su- dilution of plant lysis buffer) were included. Each LAMP primer set was
crose, 30 g inactive dry brewer’s yeast, 20 g cornmeal, 50 g carrot also tested on Nucleospin Plant II kit extracted DNA (5-10 ng/ μL) of Za-
powder, 1.2 g methyl 4-hydroxybenzoate (Nipagin) dissolved in 12 mL prionus ornatus (Diptera: Drosophilidae). LAMP assays were run at 65°C
ethanol, 2.22 g tartaric acid, 0.89 g ammonium sulphate, 0.22 g magne- for 1 hour, followed by a melt curve analysis from 95°C to 60°C and

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W. Dermauw, Y. Van Moerkercke, N. Ebrahimi et al. Current Research in Insect Science 2 (2022) 100029

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Figure 1. Cladogram of tephritid samples used in LAMP assays. COI sequences of tephritid species were aligned (File S1) using MAFFT (Katoh et al. 2013) and used
in a maximum likelihood phylogenetic analysis. Tephritid samples of the Flanders research institute for Agriculture, Fisheries and Food (ILVO) are indicated in red,
while those of the Royal Musem of Central Africa (RMCA) are indicated in blue font and those of NCBI/QBANK in yellow font (see Table S1 for sample details). “-”
indicates that the country of origin was not known. Only UF bootstrap values above 90 and at phylogenetically important nodes are shown. For the Ceratitis FARQ
complex a letter between brackets indicates to which Ceratitis species of the FARQ complex the specimen was morphologically assigned (A: C. anonae, R: C. rosa, F:
C. fasciventris, Q: C. quilicii).

Table 1
LAMP amplification time (Tp) and anneal derivative temperature (Ta) for different
stages/tissues of C. capitata

Lysis buffer Lysis buffer + pestle

stage Tp1 Ta2 Tp1 Ta2

egg -∗ -∗ 19:10 (02:03)∗∗ 81.12 (0.18)∗∗

L1 -∗ -∗ 19:31 (00:24) 80.82 (0.06)
L2 19:39 (1:42) 81.13 (0.14) nd∗∗∗ nd
L3 19:17 (1:41) 81.07 (0.07) nd nd
adult leg 25:21 (04:11)∗∗ 81.19 (0.06)∗∗ 20:54 (0:55) 81.10 (0.13)
pupa 18:54 (0:36) 81.00 (0.01) nd nd
empty pupa 25:52 (02:15) 81.25 (0.12) nd nd
1
time in mm:ss (standard deviation)
2
anneal derivative temperature in°C (standard deviation)
∗
no LAMP amplification
∗∗
LAMP amplification in only three out of four biological replicates
∗∗∗ nd, not determined,

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W. Dermauw, Y. Van Moerkercke, N. Ebrahimi et al. Current Research in Insect Science 2 (2022) 100029

Primers Primers

g1

g1

Q
AR
ap

os

AR
ap

os
CF
Cc

Cc
FARQ (80.7±0.2°C)

CF
Cc

Cc
ILVO−01 − Ruanda RMCA−47 − South Africa
ILVO−02 − South Africa C. rosa
RMCA−48 − South Africa
ILVO−03 − Egypt
RMCA−50 − South Africa
ILVO−04 − Gambia
RMCA−55 − Tanzania
ILVO−05 − Kenya
ILVO−06 − Brazil C. anonae RMCA−05 − Congo (D.R.)
C. capitata ILVO−07 − Brazil RMCA−06 − Benin
(81.3±0.2°C) ILVO−08 − Ruanda RMCA−08 − Kenya
ILVO−09 − Congo (D.R.) RMCA−60 − Ivory Coast
ILVO−10 − Uganda
[Link] RMCA−49 − South Africa
RMCA−38 − Australia
RMCA−56 − La Reunion
RMCA−51 − South Africa
RMCA−57 − Tanzania
RMCA−52 − Mozambique
ILVO−11 − Ivory Coast RMCA−24 − Mozambique
C. quinaria RMCA−25 − Mali
ILVO−12 − Burkina Faso
ILVO−13 − Ivory Coast RMCA−41 − Sudan
ILVO−14 − Ivory Coast * B. dorsalis
ILVO−24 − Cameroon
C. cosyra ILVO−15 − Burkina Faso ILVO−25 − Ivory Coast
group 1 ILVO−16 − Ivory Coast RMCA−34 − Indonesia
(84.4±0.2°C)
ILVO−17 − Burkina Faso * ILVO−26 − Mali
ILVO−18 − Burkina Faso
B. latifrons RMCA−12 − Japan
ILVO−19 − Mali
ILVO−20 − Burkina Faso RMCA−13 − USA
ILVO−21 − Ivory Coast RMCA−33 − Tanzania
RMCA−39 − Mozambique D. punctatifrons ILVO−27 − Ruanda
RMCA−40 − Sudan RMCA−22 − Mozambique
RMCA−43 − Kenya
RMCA−14 − Mozambique
C. cosyra ILVO−22 − Uganda D. ciliatus RMCA−15 − Nigeria
group 2 ILVO−23 − Uganda RMCA−23 − Namibia
RMCA−42 − Burundi

amplification time (min) stage

≥ 15 & < 25 ≥ 25 & < 35 ≥ 35 & < 45 no amplification adult larva

Figure 2. Heatmap of Tp values for LAMP assays with Ccap, Ccos g1 and CFARQ primers and ILVO and RMCA collected tephritid DNA samples. Each sample name
(ILVO or RMCA) is followed by the country of origin of the sample. See Table S1 for detailed Tp values. All LAMP assays were run on a Quantstudio 5 system. An
asterisk indicates that an atypical amplification curve (with Tp values of 49 and 47 minutes) was observed for these samples (ILVO-14 and ILVO-17, respectively)
with Ccap primers.

analyzed using the Quantstudio Design and Analysis software v 1.4.3 100-fold and 1000-fold and LAMP assays were performed on a Quantstu-
(Qiagen, Belgium). Time to positivity (Tp, expressed in mm:ss) reported dio 5 Real-Time PCR system as described in section 2.3. Four technical
in this study was determined using a deltaRn threshold of 75,000 and replicates were performed for each dilution. An average Tp was calcu-
“Automatic baseline” in the “Ct settings” of the “Analysis settings” of lated when a Tp value could be determined for three out of four repli-
the Quantstudio Design and Analysis software, while the Ta (expressed cates.
in°C) was determined by a melt-curve analysis. A heatmap of LAMP as-
say Tps was generated using ComplexHeatmap package in R (Gu et al., 2.5. LAMP assays with different stages of Ceratitis capitata
2016). In addition to LAMP experiments with the Quantstudio 5 Real-
Time PCR system, we also ran LAMP assays with each LAMP primer set Ceratitis capitata stages - egg, 1st instar (L1), 2nd instar (L2), 3rd instar
and a 1/10th dilution of larva lysis solution of C. capitata (ILVO-10), (L3), pupa, empty pupa and adults - were provided by the International
C. cosyra g1 (ILVO-21) and kit extracted DNA of C. quilicii (RMCA-56, Atomic Energy Agency (Vienna) and stored in 90% ethanol at 4°C (one
with a concentration of 5 ng/ μL) on a Genie II instrument (Optigene, stage/10 mL Falcon tube) until further use. Single specimens were al-
UK), with default settings for Tp and Ta assessment (melt curve analy- lowed to dry on filter paper before being transferred to plant lysis buffer
sis from 70°C to 98°C) and with “Amplification height” set at 5000. A (Optigene, UK). Egg and L1 stages were not manipulated and directly
1/10th dilution of B. dorsalis larva lysis (ILVO-26) solution and a 1/10th transferred to 30 μl lysis buffer while L2 and L3 stages were decapi-
dilution of plant lysis buffer were used as a negative control. Last, we tated and the posterior region (cut in two) was transferred to 200 μl
also performed LAMP assays with the C. capitata primers described in lysis buffer (see also section 2.1). Pupa and empty pupa were cut into
Huang et al. (2009) on the Genie II device but using the LAMP reaction two pieces and both pieces were transferred to 200 μl lysis buffer. For
specifications described in this study. adults, a leg was detached using a forceps and subsequently transferred
to 200 μl lysis buffer. Next, tubes were vortexed for 1 min, incubated
2.4. Sensitivity of Ceratitis LAMP primers for 5 min at room temperature, and 2 μL of a one-tenth dilution (in wa-
ter) of the lysis solution was used in LAMP assays on a Quantstudio 5
The sensitivity of our LAMP assays was assessed by testing serial di- instrument as described above (see section 2.3). Last, another batch of
lutions of DNA with the Ccap, Ccos g1 and CFARQ primers. Around 50 LAMP assays with eggs, L1s and adult legs were performed but differ-
ng/μl DNA from a C. capitata larva (ILVO-09), C. cosyra group1 larva ing from the assays described above by micropestle homogenisation of
(ILVO-17), and a Ceratitis quilicii adult (RMCA-56) was diluted 10-fold, tissue before vortexing the sample for 1 min.

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W. Dermauw, Y. Van Moerkercke, N. Ebrahimi et al. Current Research in Insect Science 2 (2022) 100029

Figure 3. Scheme of a LAMP assay with an L3 larva and a Genie II device. First, the anterior part of the L3 larva is separated from the rest of the body. Next, the
rest of the body is cut in two using a scalpel blade, and both posterior parts are transferred to plant lysis buffer (Optigene, UK), vortexed and then incubated at room
temperature for five minutes. A 1/10th dilution of the larva lysis solution is then used in a LAMP reaction. True positive results are then determined based on Tp and
Ta values. See File S2 for a detailed protocol.

3. Results and Discussion cosyra group1 DNA samples varied between 31 and 41 minutes with a
Ta of 84.4°C (SD, 0.2), while LAMP amplification did not occur with
Ceratitis capitata (Diptera: Tephritidae) is not considered a EU quar- other tephritid samples. CFARQ primers with C. rosa, C. anonae or C.
antine pest and non-EU fruit and vegetables infested with this species quilicii samples resulted in Tp values between 29 and 38 minutes with a
pass import control at the port of entry. The larval stage is the most fre- Ta of 80.7°C (SD, 0.2) and LAMP amplification with CFARQ primers did
quently intercepted stage of Tephritidae in imported produce. However, not occur with other tephritid samples tested (Figure 2, Table S1). LAMP
tephritid larvae are difficult to identify using morphological character- amplification was also not observed with Zaprionus ornatus, an unrelated
istics only and a molecular diagnostic tool might aid in species identi- fruitfly that feeds on plant hosts on which Ceratitis capitata also occurs
fication. In 2009, a LAMP assay, targeting the ITS1-5.8S-ITS2 region, (Poligui et al., 2014) (Johan Witters, pers. communicaton) (Figure S4).
was developed for C. capitata (Huang et al., 2009) but was not highly LAMP assays are prone to false positives, and two false positives (with
specific as these LAMP primers also amplify C. cosyra and B. dorsalis atypical amplification curves) were observed in our study (Figure 2, Fig-
DNA (Figure S1). Hence, the main goal of this study was to develop ure S3). Time-gating has been suggested as a valuable tool to reduce the
a new LAMP assay that is more specific for C. capitata. Therefore, we probability of false positive results. In addition, the Ta might be used to
first collected DNA samples from a selection of tephritid species, origi- discern specific from non-specific amplification (Moehling et al., 2021;
nating from different countries, and subsequently PCR-amplified a COI Rolando et al., 2020). Therefefore, we suggest to use the following Tp
fragment of each sample. A maximum likelihood phylogenetic analy- and Ta thresholds to assess whether the result of our LAMP assays can
sis showed that COI sequences form ILVO or RMCA collected samples be considered as a true positive: Tp < 35 min and Ta of 81±1°C for Ccap
clustered with either Ceratitis sp., Bactrocera sp. or Dacus sp. COI se- LAMP primers, Tp < 45 min and Ta of 84.5±1°C for Ccos g1 primers and
quences from the NCBI database. Of particular note, sequences of C. Tp < 40 min and Ta of 80.5±1 for CFARQ primers. Lastly, our C. capitata
cosyra group2 (as defined by Virgilio et al. (2017)) clustered as a sis- LAMP assay might not be able to discriminate between C. capitata and
ter group of C. quinaria COI sequences. RMCA or ILVO COI sequences the closely related Ceratitis caetrata, as the COI sequence diversity (“bar-
from adult specimens also clustered according to their morphological code gap”) is too low between these species (Barr et al., 2012); 99% iden-
identification (Figure 1, Table S1, File S1). Next, LAMP primers were tity between the LAMP primer region (273 bp) of C. capitata (GenBank
designed for C. capitata (Ccap, Table S2, Figure S2) and tested on all accession AJ242872.1) and C. caetrata (GenBank accession AY788414)).
tephritid samples for which a COI fragment was obtained (Figure 2). Tp However, as C. caetrata has not been reported for the last ten years
values with Ccap primers were below 30 minutes for all C. capitata sam- ([Link] and only occurs
ples (Figure 2, Table S1) and the melting temperature (Ta) was 81.3°C in Kenya on hosts that are not commercially exported (De Meyer et al.,
(SD, 0.2). Ccap primers did not result in LAMP amplification of Cerati- 2002), it is highly unlikely that this species would be present in produce
tis FARQ complex species, B. dorsalis, Dacus sp. samples, while for two imported in the EU.
out of 17 C. cosyra samples (ILVO-14 and ILVO-17) an atypical ampli- LAMP assays with serial dilutions of Ceratitis sp. DNA revealed that
fication curve was observed, with a Tp values of 49 and 47 minutes reliable detection (Tp SD less than 5 min, amplification in all four tech-
(Figure 2, Figure S3). To complement the Ccap primers, we also devel- nical replicates) of C. capitata and Ceratitis species from the FARQ com-
oped LAMP primers for C. cosyra species (Ccos g1) and Ceratitis species plex was possible at 1 ng of input DNA while C. cosyra group1 DNA
from the FARQ complex (CFARQ), which could be run in parallel with could be reliably detected up to 10 pg (Table S3). The sensitivity of the
the C. capitata LAMP assays (Table S2, Figure S2). We also attempted C. capitata LAMP assay is lower than the sensitivity of the recently de-
to design LAMP primers for C. cosyra group2 species but were not suc- veloped Bactrocera tryoni LAMP assay (Blacket et al., 2020) but seems
cessful (data not shown). Tp values for Ccos g1 LAMP primers with C. more than sufficient to reliably identify L3 larvae and most other stages

5
W. Dermauw, Y. Van Moerkercke, N. Ebrahimi et al. Current Research in Insect Science 2 (2022) 100029

Amplification curve
Ccap Anneal derivative melting curve
40.000
80.000

60.000 30.000

40.000
20.000

20.000

10.000
0

-20.000 0
00:05:00 00:15:00 00:25:00 00:35:00 00:45:00 00:55:00 69,00 74,00 79,00 84,00 89,00 94,00

Ccos g1
35.000
60.000
30.000

Fluorescence derivative
40.000 25.000
Fluorescence

20.000

20.000 15.000

10.000

0 5.000

-20.000 -5.000
00:05:00 00:15:00 00:25:00 00:35:00 00:45:00 00:55:00 69,00 74,00 79,00 84,00 89,00 94,00

CFARQ
60.000 30.000

25.000

40.000 20.000

15.000
20.000
10.000

5.000
0

-20.000 -5.000
00:05:00 00:15:00 00:25:00 00:35:00 00:45:00 00:55:00 69,00 74,00 79,00 84,00 89,00 94,00

Time (hh:mm:ss) Temperature (°C)

ILVO-10 (C. capitata) RMCA-56 (C. quilicii)

ILVO-21 (C. cosyra) 1/10th lysis buffer
ILVO-26 (B. dorsalis)

Figure 4. LAMP assays with Ccap, Ccos g1 and CFARQ primers using a Genie II device LAMP assays were run with three primersets - Ccap, Ccos g1 and CFARQ –
and using DNA from four different tephritid species (ILVO-10 (C. capitata larva lysis solution), ILVO-21 (C. cosyra larva lysis solution), ILVO-26 (B. dorsalis larva lysis
solution) and RMCA-56 (C. quilicii kit extracted DNA)). Amplification curves (for Tp determination) are shown on the left, while melting curves (for Ta determination)
are shown on the right. For each primer set, LAMP amplification only occurred for the targeted species.

of C. capitata. LAMP amplification with Ccap primers occurred using signed for B. tryoni (Blacket et al., 2020), B. dorsalis complex/B. latifrons
L2, L3, pupa as template, but did not occur for egg and L1s (Table 1) (Blaser et al., 2018) and Z. scutellatus (Kitano & Takakura, 2020) might
or only for three out of four replicates in case of adult legs. We specu- be run simultaneously, allowing rapid screening of tephritid pests. Such
lated that this difference in amplification success could be related to the screening might also be achieved by PCR amplification and sequenc-
limited amount of DNA that is released during vortexing of eggs, L1s ing of a fragment of the COI gene but, to our knowledge, on-site se-
or adult legs. Hence, we performed an alternative extraction protocol quencing facilities at ports of entry are currently rather exceptional and
(addition of a “pestle homogenisation” step, see section 2.5 in Materials PCR requires more trained personnel and special equipment compared
and Methods) for these stages/samples. LAMP assays using this alter- to LAMP assays (Hsieh et al., 2012). To conclude, in this study, we de-
native extraction method resulted in successful LAMP amplification for veloped LAMP assays for C. capitata and related species that could aid
eggs, L1s and adult legs (Table 1). in the rapid identification of these pests in imported produce.
A set of LAMP assays was also run with larva lysis solution and the
Genie II (Figure 3, Figure 4, File S2), which is a portable device and
can easily be used for in-field diagnostics (see e.g. Blacket et al. (2020)). Declaration of Competing Interest
Noteworthy, for the samples tested on the Genie II, Ta values were 0.5°C
to 1°C less than those obtained using the QuantStudio 5 system, indicat- The authors declare that they have no known competing financial
ing that Ta values can slightly deviate when running LAMP assays on interests or personal relationships that could have appeared to influence
different devices. The LAMP assays developed in this study and those de- the work reported in this paper.

6
W. Dermauw, Y. Van Moerkercke, N. Ebrahimi et al. Current Research in Insect Science 2 (2022) 100029

CRediT authorship contribution statement Enriquez, T., Colinet, H., 2019. Cold acclimation triggers major transcriptional changes
in Drosophila suzukii. BMC. Genomics 20 (1), 413. doi:10.1186/s12864-019-5745-7.
EPPO, 2011. Ceratitis capitata. EPPO Bulletin 41 (3), 340–346.
Wannes Dermauw: Methodology, Formal analysis, Writing – origi- doi:10.1111/j.1365-2338.2011.02519.x.
nal draft. Yannick Van Moerkercke: Investigation, Validation. Negin EPPO, 2021. EPPO Global Database (available online). [Link]
Ebrahimi: Writing – review & editing. Hans Casteels: Funding acqui- Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for am-
plification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan
sition. Jochem Bonte: Conceptualization, Writing – review & editing. invertebrates. Mol. Mar. Biol. Biotechnol. 3 (5), 294–299.
Johan Witters: Conceptualization, Methodology, Formal analysis, Writ- Gu, Z., Eils, R., Schlesner, M., 2016. Complex heatmaps reveal patterns and cor-
ing – original draft. relations in multidimensional genomic data. Bioinformatics 32 (18), 2847–2849.
doi:10.1093/bioinformatics/btw313.
Hsieh, C.-H., Wang, H.-Y., Chen, Y.-F., Ko, C.-C., 2012. Loop-mediated isothermal am-
Acknowledgements plification for rapid identification of biotypes B and Q of the globally invasive pest
Bemisia tabaci, and studying population dynamics. Pest Manag. Sci. 68 (8), 1206–
1213. doi:10.1002/ps.3298.
We would like to thank Dr. Marc De Meyer and Dr. Massimiliano
Huang, C.G., Hsu, J.C., Haymer, D.S., Lin, G.C., Wu, W.J., 2009. Rapid identification of
Virgilio (Royal Museum of Central Africa (RMCA), Brussels, Belgium) the Mediterranean fruit fly (Diptera: Tephritidae) by loop-mediated isothermal am-
for providing Tephritidae samples, Carlos Caceres (International Atomic plification. J. Econ. Entomol. 102 (3), 1239–1246. doi:10.1603/029.102.0350.
Energy Agency, Vienna) and John Vontas (University of Athens, Greece) Katoh, K., Standley, D.M., 2013. MAFFT Multiple Sequence Alignment Software Version
7: Improvements in Performance and Usability. Mol. Biol. Evol. 30 (4), 772–780.
for providing different stages of C. capitata, Joeri Verstraeten (ILVO) for doi:10.1093/molbev/mst010.
help with DNA extraction and LAMP primer design and Margo De Smet Kitano, D., Takakura, K.-I., 2020. Simple and on-site DNA purification for LAMP reaction
for performing a set of LAMP assays. This study was supported by FPS applicable to non-adult tephritid fruit fly (Diptera: Tephiritidae). J. Appl. Entomol.
144 (9), 824–829. doi:10.1111/jen.12820.
Public Health Belgium (RF 19/6332 TEPHRIFAST). Lebreton, S., Witzgall, P., Olsson, M., Becher, P.G., 2014. Dietary glucose regulates yeast
consumption in adult Drosophila males [Original Research]. Front. Phys. 5 (504).
Supplementary materials doi:10.3389/fphys.2014.00504.
Moehling, T.J., Choi, G., Dugan, L.C., Salit, M., Meagher, R.J., 2021. LAMP Diagnostics at
the Point-of-Care: Emerging Trends and Perspectives for the Developer Community.
Supplementary material associated with this article can be found, in Expert. Rev. Mol. Diagn. 21 (1), 43–61. doi:10.1080/14737159.2021.1873769.
the online version, at doi:10.1016/[Link].2022.100029. Nguyen, L.-T., Schmidt, H.A., von Haeseler, A., Minh, B.Q., 2014. IQ-TREE: A Fast and
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