Research Article |
Corresponding author: Pia Addison ( pia@sun.ac.za ) Academic editor: Vera Silva
© 2024 Welma Pieterse, Marc De Meyer, Massimiliano Virgillio, Pia Addison.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Pieterse W, De Meyer M, Virgilio M, Addison P (2024) Development of a multi-entry identification key for economically important fruit fly larvae (Diptera, Tephritidae, Dacinae). ZooKeys 1197: 127-136. https://doi.org/10.3897/zookeys.1197.116887
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Identification of fruit fly larvae is difficult due to the limited morphological characteristics present. However, this is the stage at which fruit flies are intercepted at ports of entry through horticultural imports. Molecular tools are useful but are time-consuming and expensive compared to morphological identifications. This project aims to use available information from the literature and our own research to build a multi-entry identification key for thirteen tephritid species and species groups that are of economic concern for the European Union. Third-instar larvae were obtained from different regions and hosts. Thirteen species or representatives of species groups were obtained, including Ceratitis, Dacus, Bactrocera and Zeugodacus spp. The cephalopharyngeal skeletons were dissected out, cleared in a 10% NaOH solution, dehydrated and mounted in Euparal on glass slides. Images of at least 20 larvae/species were captured using a compound microscope fitted with a camera. Measurements were taken of the mounted mandibles and the number of tubules and their position in the anterior spiracles in relation to the cephalic skeleton were noted. Differences between morphometric parameters were tested via ANOVA and verified using discriminant function analysis. A matrix was compiled including nine characters for which significant inter-specific differentiation was preliminarily detected. The key was converted into a mobile application by LucID.
Identification tool, interactive key, larvae, LucID, mandible
The family Tephritidae has more than 4000 species distributed globally (
The majority of the thirteen species studied here are at risk of being intercepted when entering Europe through imported fruit, namely, Ceratitis rosa s.l. Karsch (Natal fruit fly), Ceratitis cosyra (Walker) (Mango fruit fly), Bactrocera dorsalis (Hendel) (Oriental fruit fly), Zeugodacus cucurbitae (Coquillett) (Melon fruit fly) and Bactrocera zonata (Saunders) (Peach fruit fly). All are considered potential quarantine pests for the European Union (EU) and are listed as such in the Commission Implementing Regulation 2019/2072 and amending Implementing Regulation 2021/2285. In addition, B. dorsalis and B. zonata have also been included in the list of priority pests in EU regulation 2019/1702. Zeugodacus cucurbitae, Bactrocera minax (Enderlein) (Chinese citrus fly), Bactrocera tryoni (Froggatt) (Queensland fruit fly), B. dorsalis and C. rosa are listed as A1 quarantine pests in the European and Mediterranean Plant Protection Organization (EPPO) countries (
Being able to make a correct identification of insect species in the shortest possible period of time is essential to comply with international biosecurity measures, since not all species are of quarantine importance in all countries (
The presence or absence of a preapical tooth on the mandible can be used as a distinguishing characteristic in a taxonomic key, but since the characteristics of the mouthparts of tephtritid species are not known for all species, it could be a controversial character to use.
According to
Lucid® (https://keys.lucidcentral.org/search/) was developed at the University of Queensland (
Multi-entry keys have several benefits over molecular identification tools, namely, they are more accessible and cost-effective; they can be used anywhere without specialised equipment, and the answer is obtained quickly. The key developed here is converted into a mobile application by LucID, for both Android and Apple devices, making it freely available. Multi-entry keys can, furthermore, be used on specimens that are too degraded to be used for DNA analysis. Larval mouth hooks are heavily sclerotized and can still be used even when a specimen is degraded. The aim of the key is to provide a practical identification tool for third-instar fruit fly larvae that are commonly intercepted in the EU.
Preparation of slides. Larvae from 13 species were obtained from colonies from various laboratories around the world and were preserved in 70% ethanol. The heads of the larvae were cut off and cleared by heating in 10% NaOH. The cephalopharyngeal skeletons (Fig.
Cephalopharyngeal skeleton of 3rd instar larva, lateral view. Region of interest circled. Abbreviations used in LucID key: AT = Apical Tooth; DA = Dorsal Apodeme; DS = Dental Sclerite; MD = Mandible; MN = Mandibular Neck; PT = Preapical Tooth; VA = Ventral Apodeme (from:
Fig.
A, B Images of a typical tephritid mandible indicating the areas measured. Image (A) indicates the measurements for the mandibles without a preapical tooth and (B) indicates the measurements for the mandibles where the preapical tooth is present. Measurements are as follows for A: (a) the distance between the dorsal apodeme and the ventral apodeme; (b) the ventral angle between the apical tooth and the ventral apodeme; (c) the distance between the ventral apodeme and the apical tooth; (d) the distance between the dorsal apodeme and the apical tooth. Measurements are as follows for B: (a) the distance between the dorsal apodeme and the ventral apodeme; b) the distance between the ventral apodeme and the preapical tooth; (c) the distance between the apical tooth and the preapical tooth; (d) the distance between the ventral apodeme and the apical tooth; (e) the distance between the dorsal apodeme and the apical tooth. Measurements were recorded in µm (distances) and degrees (angles).
Images of the cephaloskeleton and anterior spiracles of ten larvae per species were taken in the same way. The position of the anterior spiracles in relation to the cornua and the number of tubules were recorded for each species (Fig.
Discriminant function analysis with classification functions was carried out to statistically allocate the specimens to the species studied using the recorded mandible measurements in Statistica v. 14.1.0 (TIBCO Software Inc, Palo Alto, CA, USA). Measurements for all the specimens of the same species were pooled for these analyses.
A total of 873 mandibles from thirteen species were mounted on slides and examined (Table
Species, origin, and sample size of mandibles used to develop a multi-entry key of tephritid larvae of economic importance to the European Union.
Species | Origin | Number of mandibles measured |
---|---|---|
Bactrocera correcta | IAEA Vienna colony | 51 |
Bactrocera dorsalis | Quarantine Station Stellenbosch colony | 18 |
Bactrocera dorsalis | Atomic Energy Research Establishment Baipayl, Bangladesh colony | 13 |
Bactrocera dorsalis | IAEA Vienna colony | 24 |
Bactrocera dorsalis | CRI Nelspruit South Africa colony | 24 |
Bactrocera dorsalis | University of Pretoria colony | 20 |
Bactrocera minax | Changsa, Hunan, China colony | 17 |
Bactrocera oleae | IAEA Vienna, colony | 29 |
Bactrocera oleae | Madrid, Spain, olives | 18 |
Zeugodacus tau | IAEA Vienna, colony | 20 |
Zeugodacus tau | Atomic Energy Research Establishment Baipayl, Bangladesh, colony | 22 |
Bactrocera tryoni | Queensland University of Technology, Brisbane, Australia, colony | 16 |
Bactrocera tryoni | IAEA Vienna, colony | 37 |
Bactrocera zonata | The “Israel Cohen” Institute for Biological Control, Yehud-Monosson, Israel, colony | 24 |
Bactrocera zonata | Atomic Energy Research Establishment Baipayl, Bangladesh, colony | 18 |
Bactrocera zonata | IAEA Vienna, colony | 14 |
Bactrocera zonata | CIRAD La Réunion, colony | 45 |
Ceratitis capitata | The “Israel Cohen” Institute for Biological Control, Yehud-Monosson, Israel, colony | 19 |
Ceratitis capitata | Plant Quarantine Station, Stellenbosch, South Africa, colony | 21 |
Ceratitis capitata | CRI Nelspruit South Africa, colony | 11 |
Ceratitis capitata | Citrus, Plant Quarantine Station, Stellenbosch, South Africa | 4 |
Ceratitis capitata | Nectarine, Plant Quarantine Station, Stellenbosch, South Africa | 8 |
Ceratitis capitata | CIRAD La Réunion colony | 19 |
Ceratitis capitata | Greece, Bitter orange | 11 |
Ceratitis capitata | CIRAD La Réunion, wild host | 18 |
Ceratitis cosyra | CRI Nelspruit South Africa colony | 74 |
Ceratitis quilicii | CRI Nelspruit South Africa colony | 63 |
Ceratitis rosa | CRI Nelspruit South Africa colony | 67 |
Dacus ciliatus | Piketberg, South Africa, Pumpkin | 19 |
Dacus ciliatus | Eduardo Mondlane University, Maputu, Mozambique, Cucumber | 20 |
Zeugodacus cucurbitae | Atomic Energy Research Establishment Baipayl, Bangladesh colony | 30 |
Zeugodacus cucurbitae | IAEA Vienna colony | 20 |
Zeugodacus cucurbitae | CIRAD La Réunion colony | 14 |
We did not see a prominent preapical tooth in any of the mandibles of C. capitata third-instar larvae that we examined, so we used the presence/absence of the preapical tooth as one of the distinguishing characteristics in the key. The characteristics and measurements (Table
Average, minimum, and maximum distances (µm) and angles (°) that were used to compile the LucID key for thirteen species of fruit fly larvae of economic importance to the European Union.
Prominent preapical tooth absent | ||||
---|---|---|---|---|
Bactrocera dorsalis | Bactrocera zonata | Ceratitis capitata | Bactrocera oleae | |
Distance a (µm) | 168 (144–195) | 156 (131–171) | 141 (130–154) | 133 (113–151) |
Angle b (°) | 102 (87–117) | 107 (96–129) | 103 (94–116) | 103 (93–120) |
Distance c (µm) | 174 (146–214) | 152 (128–180) | 130 (113–151) | 115 (96–132) |
Distance d (µm) | 279 (241–326) | 252 (203–329) | 213 (186–236) | 194 (152–215) |
Bactrocera tryoni | Bactrocera correcta | Bactrocera minax | ||
Distance a (µm) | 156 (139–169) | 148 (132–161) | 278 (253–301) | |
Angle b (°) | 107 (96–116) | 106 (77–120) | 110 (101–121) | |
Distance c (µm) | 156 (144–172) | 157 (134–188) | 282 (265–303) | |
Distance d (µm) | 242 (222–262) | 257 (232–285) | 424 (398–456) | |
Ceratitis rosa | Ceratitis quilicii | Ceratitis cosyra | Zeugodacus cucurbitae | |
Distance a (µm) | 149 (134–167) | 161 (147–179) | 151 (138–168) | 194 (163–223) |
Distance b (µm) | 107 (96–119) | 98 (86–110) | 96 (82–106) | 135 (105–169) |
Distance c (µm) | 73 (65–84) | 74 (64– 79) | 79 (69–90) | 85 (64–102) |
Distance d (µm) | 149 (132–171) | 148 (127–160) | 149 (132–146) | 186 (150–225) |
Distance e (µm) | 233 (202–257) | 241 (221–265) | 239 (212–263) | 299 (245–340) |
Dacus ciliatus | Zeugodacus tau | |||
Distance a (µm) | 169 (153–188) | 198 (169–224) | ||
Distance b (µm) | 109 (93–121) | 139 (112–160) | ||
Distance c (µm) | 47 (40–60) | 92 (83–101) | ||
Distance d (µm) | 148 (129–164) | 190 (167–211) | ||
Distance e (µm) | 250 (230–267) | 315 (279–357) |
Bactrocera zonata, B. tryoni and B. correcta could not be identified reliably without including distribution data as well as the position of the spiracle, indicating a percentage correct identification of below 61% based on the discriminant function analysis (Table
Classification matrix of the species where a secondary tooth is absent on the mandibles. Rows: Observed classifications; Columns: Predicted classifications.
Species | Percent | p-value | Bd | Cc | Bz | Bo | Bt | Bc | Bm |
---|---|---|---|---|---|---|---|---|---|
Correct | |||||||||
Bactrocera dorsalis (Bd) | 93.12 | 0.2985 | 149 | 0 | 4 | 0 | 0 | 7 | 0 |
Ceratitis capitata (Cc) | 97.19 | 0.1996 | 0 | 104 | 1 | 1 | 0 | 1 | 0 |
Bactrocera zonata (Bz) | 56.43 | 0.1884 | 6 | 10 | 57 | 0 | 14 | 14 | 0 |
Bactrocera oleae (Bo) | 80.85 | 0.0877 | 0 | 9 | 0 | 38 | 0 | 0 | 0 |
Bactrocera tryoni (Bt) | 49.06 | 0.0989 | 0 | 1 | 25 | 0 | 26 | 1 | 0 |
Bactrocera correcta (Bc) | 60.78 | 0.0951 | 5 | 1 | 14 | 0 | 0 | 31 | 0 |
Bactrocera minax (Bm) | 100 | 0.0317 | 0 | 0 | 0 | 0 | 0 | 0 | 17 |
Total | 78.73 | 160 | 125 | 101 | 39 | 40 | 54 | 17 |
Classification matrix of the species where a secondary tooth is present on the mandibles. Rows: Observed classifications Columns: Predicted classifications.
Species | Percent | P-value | Cr | Cq | Cc | Zc | Dc | Bt |
---|---|---|---|---|---|---|---|---|
Correct | ||||||||
Ceratitis rosa (Cr) | 94.03 | 0.1988 | 63 | 3 | 1 | 0 | 0 | 0 |
Ceratitis quilicii (Cq) | 79.36 | 0.1869 | 6 | 50 | 7 | 0 | 0 | 0 |
Ceratitis cosyra (Cc) | 66.13 | 0.1840 | 9 | 12 | 41 | 0 | 0 | 0 |
Zeugodacus cucurbitae (Zc) | 75 | 0.1899 | 0 | 3 | 0 | 48 | 0 | 13 |
Dacus ciliatus (Dc) | 100 | 0.1157 | 0 | 0 | 0 | 0 | 39 | 0 |
Zeugodacus tau (Zt) | 76.19 | 0.1246 | 0 | 0 | 0 | 10 | 0 | 32 |
Total | 81.01 | 78 | 68 | 49 | 58 | 39 | 45 |
The LucID key was transformed to an app that can be downloaded from Google Play store (for Android) or Apple App store (for Apple phones). https://play.google.com/store/apps/details?id=com.lucidcentral.mobile.lucid.fruit_fly_larvae&hl=en-US&ah=RArn8-TSJV3KC1m-QBBa0Vfcz7s.
This is the first time a multi-entry key for tephritid larvae of economic significance has been developed in app format. While the characters rely mostly on measurements, it does require some knowledge of how to prepare the mouthparts so that measurements of specific distances can be made. However, it will be a valuable tool for enabling non-molecular identifications of fruit fly larval pests in fruit.
We are grateful to Anthony Clarke, Australia; Domingos Cugala Mozambique; Hélène Delatte, CIRAD, Reunion; Marc de Meyer, Africa Museum, Belgium; Yoav Gazit, Israel; Manuel González Núñez, Spain; Minette Karsten, Pretoria, South Africa Mahfuza Khan, Bangladesh; Aruna Manrakhan, Nelspruit, South Africa, Rui Pereira, IAEA Vienna, Ronald Ramukhesa and Saadiek Rosenberg, DALRRD, South Africa who sent us the larval specimens used in this project. Thank you to the Entomological Society of Israel for permission to use the image of the cephalopharyngeal skeleton in Fig.
The authors have declared that no competing interests exist.
No ethical statement was reported.
This study was funded by the European Union’s Horizon 2020 Research and Innovation Program FFIPM (grant agreement No 818184) and the Standards and Trade Development Facility’s project Fruit Fly Free STDF/PG/567.
Conceptualization: MDM, PA. Data curation: WP. Formal analysis: WP. Funding acquisition: MV. Methodology: MDM, PA. Software: MV. Writing - original draft: WP. Writing - review and editing: MDM, MV, PA.
Welma Pieterse https://orcid.org/0000-0002-1290-8636
Marc De Meyer https://orcid.org/0000-0003-0755-2898
Massimiliano Virgillio https://orcid.org/0000-0002-1323-6886
Pia Addison https://orcid.org/0000-0002-8227-339X
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Raw data of all measurements
Data type: xlsx