Reliability of Morphological and PCR Methods for the Official Diagnosis of Aethina tumida (Coleoptera: Nitidulidae): A European Inter-Laboratory Comparison
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Participating Laboratories
2.2. Reference Methods for Panel Sample Characterisation
2.3. Selection of Inter-Comparison Samples
- The fact that the coleopteran species belong to the Nitidulidae family (i.e., same family as A. tumida), and therefore have similar morphological and molecular characteristics, and/or,
- The fact that the species were likely to be found in the honeybee hive environment, and/or,
- The fact that the species presented morphological features close to A. tumida, and,
- The availability of specimens in sufficient numbers to constitute the panels and to carry out homogeneity and stability tests.
2.4. Homogeneity and Stability Tests
- The specimens from batches A-POS, A-NEG1, L-NEG1, A-NEG4 were produced artificially in a controlled environment;
- The specimens from batches L-NEG3 and A-NEG3 were provided and previously analysed by specialised entomologists;
- The EURL produced the G. mellonella larvae (L-NEG2) artificially from combs coming from the ANSES apiary in Sophia-Antipolis (territory officially free from A. tumida at sampling date in 2019);
- The results of the homogeneity tests of a preliminary study carried out in 2018 were satisfactory for A. tumida specimens (A-POS) and T. molitor specimens (A-NEG1 and L-NEG1), of the same origin;
- The specimens were visually controlled when the tubes were prepared.
2.5. Process for the Interlaboratory Comparison
- Sensitivity, i.e., the ability of the laboratory to give a positive result for a positive sample [30];
- Specificity, i.e., the ability of the laboratory to give a negative result for a negative sample [30];
- Accuracy, i.e., the closeness of agreement between the obtained results and the assigned values, definition adapted from international standard NF EN ISO 16140 [31].
2.6. Results Evaluation
2.7. Information on Analytical Methods Employed by the Participants
3. Results
3.1. Individual Laboratory Performance for the Morphological Identification of A. tumida
3.2. Individual Laboratory Performance for PCR Identification of A. tumida
3.3. Individual Laboratory Performance for Expressing Opinions
3.4. Overall Performance of the Participants
3.5. Performance of the Methods Used for the Official Diagnosis of A. tumida
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Acknowledgments
Conflicts of Interest
Appendix A. Information on Homogeneity Tests
Code | Species | Number of Samples Prepared | Number of Samples Tested | Results |
---|---|---|---|---|
A-POS | Aethina tumida | 115 | 10 | 100% Pos. |
A-NEG1 | Tenebrio molitor | 60 | 7 | 100% Neg. |
A-NEG2 | Alphitobius diaperinus | 60 | 10 | 100% Neg. |
A-NEG3 | Epuraea luteola | 50 | 5 | 100% Neg. |
A-NEG4 | Cryptolaemus montrouzieri | 60 | 7 | 100% Neg. |
L-POS | Aethina tumida | 115 | 10 | 100% Pos. |
L-NEG1 | Tenebrio molitor | 75 | 7 | 100% Neg. |
L-NEG2 | Galleria mellonella | 60 | 7 | 100% Neg. |
L-NEG3 | Carpophilus dimidiatus | 60 | 7 | 100% Neg. |
Appendix B. Scoring Individual Performance
Step 1–Scoring of Each Single Results, According to the Following Process | |
Score of “0” | Results that matched the assigned values exactly:
|
Score of “1” | Results that did not match the assigned values exactly:
|
Score of “2” | Results that did not match the assigned values:
|
Step 2–Assessment of the three criteria for individual performance | |
Sensitivity | Sum of the scores obtained for all the positive samples of the panel. |
Specificity | Sum of the scores obtained for all the negative samples of the panel. |
Accuracy | Sum of the scores obtained for all the samples of the panel (positive and negative). |
Step 3–Final performance evaluation | |
For each performance criterion:
|
Appendix C. Detailed Results of the Participants
Morphology | PCR | |||||
---|---|---|---|---|---|---|
Lab Code | Acc 1 | Se 2 | Sp 3 | Acc 1 | Se 2 | Sp 3 |
1 | 0 | 0 | 0 | 0 | 0 | 0 |
2 | 0 | 0 | 0 | 0 | 0 | 0 |
3 | 0 | 0 | 0 | 0 | 0 | 0 |
4 | 0 | 0 | 0 | 0 | 0 | 0 |
5 | 0 | 0 | 0 | 0 | 0 | 0 |
6 | 1 | 0 | 1 | 0 | 0 | 0 |
7 | 0 | 0 | 0 | NP | NP | NP |
8 | 0 | 0 | 0 | NP | NP | NP |
9 | 0 | 0 | 0 | 0 | 0 | 0 |
10 | 0 | 0 | 0 | 0 | 0 | 0 |
11 | 2 | 0 | 2 | 8 | 0 | 8 |
12 | 0 | 0 | 0 | NP | NP | NP |
13 | 0 | 0 | 0 | 0 | 0 | 0 |
14 | 0 | 0 | 0 | 0 | 0 | 0 |
15 | 0 | 0 | 0 | 0 | 0 | 0 |
16 | 0 | 0 | 0 | 0 | 0 | 0 |
17 | 0 | 0 | 0 | NP | NP | NP |
18 | 0 | 0 | 0 | 0 | 0 | 0 |
19 | 0 | 0 | 0 | 0 | 0 | 0 |
20 | 0 | 0 | 0 | NP | NP | NP |
21 | 0 | 0 | 0 | 0 | 0 | 0 |
22 | 0 | 0 | 0 | NP | NP | NP |
Appendix D. Members of the ILC Consortium
Name | Institute | |
---|---|---|
Rosen Aleksandrov | National Diagnostic and Research Veterinary Medical Institute, Aquaculture, Fish and Bee Diseases, NRL for Bee Diseases, 1040 Sofia, Bulgaria | [email protected] |
Gabriela Chioveanu | Institute for Diagnosis and Animal Health, NRL for Animal Health, 50557 Bucharest, Romania | [email protected] |
Mary Coffey | Department of Agriculture, Food and the Marine, Plant Health Laboratories, W23 X3PH Celbridge, Ireland | [email protected] |
Benjamin Dainat | Agroscope Liebefeld, Swiss Bee Research Centre, 3003 Bern, Switzerland | [email protected] |
María Pilar Fernández Somalo | Laboratorio Central de Veterinaria, Ministerio de Agricultura, Pesca y Alimentación (Gobierno de España), D.G. Sanidad de la Producción Primaria, S.D.G. Sanidad e Higiene Animal y Trazabilidad, 28110 Algete (Madrid), Spain | [email protected] |
Miriam Filipova | Department of Molecular Biology and Epizootiology, Veterinary and Food Institute in Dolny Kubin, State Veterinary and Food Institute, NRL for Honeybee Health, 026 01 Dolny Kubin, Slovakia | [email protected] |
Heather Graham | Wageningen Bioveterinary Research, Wageningen University and Research, NRL for Bee Diseases, 8221 RA Lelystad, The Netherlands | [email protected] |
Anna Granato | Istituto Zooprofilattico Sperimentale delle Venezie, NRL for Honey Bee Health, 35020 Legnaro (PD), Italy | [email protected] |
Sirpa Heinikainen | Kuopio laboratory, Veterinary Bacteriology and Pathology Unit, Laboratory and Research Division, Finnish Food Authority, 70210 Kuopio, Finland | [email protected] |
Hemma Köglberger | Department for Apiculture and Bee protection, Ages-Austrian Agency for Health and Food Safety, 1220 Vienna, Austria | [email protected] |
Nóra Krejczinger | National Food Chain Safety Office, Food Chain Safety Laboratory Directorate, NRL of Parasitology, Fish and Bee Diseases, 1095 Budapest, Hungary | [email protected] |
Merle Kuus | Veterinary and Food Laboratory, 51006 Tartu, Estonia | [email protected] |
Severine Matthijs | Sciensano, Enzootic, Vector-borne and Bee Diseases, 1180 Brussels, Belgium | [email protected] |
Konstantinos Oureilidis | Veterinary Laboratory of Kavala, Directorate of Veterinary Center of Thessaloniki, Ministry of Rural Development and Food, 64012 Kavala, Greece | [email protected] |
Zanda Ozolina | Parasitology group, Laboratory of Microbiology and Pathology, Institute of Food Safety, Animal Health and Environment “BIOR”, LV-1076, Riga, Latvia | [email protected] |
Metka Pislak Ocepek | National Veterinary Institute, Veterinary Faculty, University of Ljubljana, Laboratory for Bee Health Care, Gerbičeva 60, 1000 Ljubljana, Slovenia | [email protected] |
Marc Oliver Schäfer | Friedrich Loeffler Institut-Germany, NRL for Bee Diseases, 17493 Greifswald-Insel Riems, Germany | [email protected] |
Emilia Semberg | Swedish University of Agricultural Sciences, Ecology, NRL for Honey Bee Health, 75007 Uppsala, Sweden | [email protected] |
Ivana Tlak Gajger | Laboratory for Honeybee Diseases APISlab, Department for Biology and Pathology of Fish and Bees, Faculty of Veterinary Medicine, University of Zagreb, 10 000 Zagreb, Croatia | [email protected] |
Maria José Valério | Patologia Apícola, Patologia, Instituto Nacional de Investigacao Agraria e Veterinaria, I. P., 2780-157 Oeiras, Portugal | [email protected] |
Sarka Vyroubalova | Department of Pathological Morphology, State Veterinary Institute Olomouc, NRL for Honeybee Health, 77900 Olomouc, Czech Republic | [email protected] |
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Criteria | Adult Form |
---|---|
1 | Body divided into three parts: head, thorax and abdomen. |
2 | Three pairs of legs. |
3 | Presence of elytra 1. |
4 | Elytra not covering the entire abdomen: some abdominal segments are apparent in dorsal view. |
5 | Overall uniform body colour (no spots), light brown to black 2. |
6 | Antenna tips with compact, almost rounded club ends. The three terminal articles of the antennae, corresponding to the “clubs” 3, are narrowed between them, and their length is almost equal to their width. |
7 | Sharp latero-posterior tips of the pronotum 4. |
8 | Dimensions. Length: 4 to 7 mm (±1 mm). Width: 3 mm (±1 mm). |
Larval form | |
1 | Three pairs of legs, one on each of the anterior segments, corresponding to the larva thorax. |
2 | All of the abdominal segments are bare and have no false legs (also called pseudopods) on their ventral part. |
3 | From the mesothorax 5, presence on each segment, of two dorsal tubercles on either side of the midline. These tubercles are finished with a short fine seta. They look like “spines”. |
Sample Code | Species | Origin | Criteria Evaluated | ||
---|---|---|---|---|---|
ADULTS | |||||
A-POS | Aethina tumida (Murray, 1867) | | Coleoptera Nitidulidae Nitidulinae | Specimens obtained experimentally in the confined laboratory of Fera Science, Ltd. in 2019 | Se, Ac |
A-NEG1 | Tenebrio molitor (Linnaeus, 1758) “Mealworm Beetle” | | Coleoptera Tenebrionidae Tenebrioninae | Specimens obtained in 2018 by the company MICRONUTRIS providing insects for human consumption | Sp, Ac |
A-NEG2 | Alphitobius diaperinus (Panzer, 1797) | | Coleoptera Tenebrionidae Tenebrioninae | Specimens collected in 2018 on the frame lid of honeybee hives located near a rabbit farm in Vendée (France) | Sp, Ac |
A-NEG3 | Epuraea luteola (Erichson, 1843) | | Coleoptera Nitidulidae Epuraeinae | Specimens collected in 2018 on Citrus sinensis in Corsica (France) and provided by the Plant Health Laboratory (ANSES, Montpellier, France) | Sp, Ac |
A-NEG4 | Cryptolaemus montrouzieri (Mulsant, 1853) “Ladybug” | | Coleoptera Coccinellidae Coccinellidae | Specimens obtained in 2019 by the company BIOLINE AGROSCIENCES providing insects for biological control in the plant field | Sp, Ac |
LARVAE | |||||
L-POS | Aethina tumida (Murray, 1867) | | Coleoptera Nitidulidae Nitidulinae | Specimens collected in 2013 in Maryland (United States) and provided by J.S. Pettis | Se, Ac |
L-NEG1 | Tenebrio molitor (Linnaeus, 1758) “Mealworm beetle” | | Coleoptera Tenebrionidae Tenebrioninae | Specimens obtained in 2018 by the company MICRONUTRIS providing insects for human consumption | Sp, Ac |
L-NEG2 | Galleria mellonella(Linnaeus, 1758) “Wax moth” | | Lepidoptera Pyralidae Galleriinae | Specimens obtained experimentally in 2019 by the EURL, from hive frames coming from the EURL apiary and naturally infested by wax moth | Sp, Ac |
L-NEG3 | Carpophilus dimidiatus (Fabricius, 1792) “Sap Beetle” | | Coleoptera Nitidulidae Carpophilinae | Specimens collected in 2018 on crops (Prunus dulcis) in Côte-d’Or (France), provided by the Plant Health Laboratory (ANSES, Montpellier, France) | Sp, Ac |
Morphology Result | PCR Result | |||
---|---|---|---|---|
Analysis Not Performed | Positive | Negative | Inhibited | |
ADULT | ||||
Positive | (1) | (1) | (3) | (1) |
Negative | (2) | (3) | (2) | (2) |
Inconclusive | (4) | (1) | (2) | (4) |
LARVA | ||||
Suspicion | (3) | (1) | (2) | (4) |
Negative | (2) | (3) | (2) | (2) |
Inconclusive | (4) | (1) | (2) | (4) |
Opinion | ||||
(1) Positive identification of the Small Hive Beetle, A. tumida. (2) Negative identification of the Small Hive Beetle, A. tumida. (3) Suspected identification of the Small Hive Beetle, A. tumida. Further analysis is required to ascertain the identification. (4) Inconclusive result of A. tumida identification. |
Predicted Result | Morphology | PCR | Analytical Conclusion | |||
---|---|---|---|---|---|---|
Compliant Results | Non-Compliant Results | Compliant Results | Non-Compliant Results | Compliant Results | Non-Compliant Results | |
Positive | 88 | 0 | 88 | 0 | 88 | 82 |
Negative | 152 | 2 | 150 | 4 | 148 | 6 |
Sensitivity | 100% (88/88) | 100% (88/88) | 93.2% (82/88) | |||
Specificity | 98.7% (152/154) | 97.4% (150/154) | 96.1% (148/154) | |||
Accuracy | 99.2% ((88 + 152)/(88 + 154)) | 98.3% (88 + 150)/(88 + 154) | 95.0% (88 + 148)/(88 + 154) |
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Share and Cite
Franco, S.; Cougoule, N.; Tison, A.; Del Cont, A.; Gastaldi, C.; Consortium, I.; Duquesne, V. Reliability of Morphological and PCR Methods for the Official Diagnosis of Aethina tumida (Coleoptera: Nitidulidae): A European Inter-Laboratory Comparison. Insects 2022, 13, 33. https://doi.org/10.3390/insects13010033
Franco S, Cougoule N, Tison A, Del Cont A, Gastaldi C, Consortium I, Duquesne V. Reliability of Morphological and PCR Methods for the Official Diagnosis of Aethina tumida (Coleoptera: Nitidulidae): A European Inter-Laboratory Comparison. Insects. 2022; 13(1):33. https://doi.org/10.3390/insects13010033
Chicago/Turabian StyleFranco, Stéphanie, Nicolas Cougoule, Amandine Tison, Aurélie Del Cont, Cristina Gastaldi, ILC Consortium, and Véronique Duquesne. 2022. "Reliability of Morphological and PCR Methods for the Official Diagnosis of Aethina tumida (Coleoptera: Nitidulidae): A European Inter-Laboratory Comparison" Insects 13, no. 1: 33. https://doi.org/10.3390/insects13010033