Development and evaluation of a real-time PCR assay for the detection of Acarapis woodi (tracheal mites) in Apis mellifera

Abstract

-The tracheal mite, Acarapis woodi , is an obligate endoparasite of honeybees and a regulated pest in countries where these mites are absent. This work describes the development of a real-time PCR method for detecting tracheal mites in honeybees. The real-time PCR was evaluated for specificity, sensitivity and speed to detect A. woodi , compared to the standard manual thoracic disc method (TDM). The assay detected A. woodi down to a 1 % incidence level in bees and 1000 copies of the target DNA when using plasmid standards. Initial testing showed no cross-reaction with the other two Acarapis species from different geographical regions or with other species of mites associated with bees. However, during extensive testing of bees, a rare population of Acarapis externus mite was identified that did cross-react with the assay. Despite this cross-reaction, the assay has been shown to be a useful screening tool and results are reliable if the TDM is used as a backup to screen hives where a positive signal is obtained.honeybee / tracheal bee mite / Acarapis / automated DNA extraction / real-time PCR

Molecular Detection of Acarapis woodi Using Hive Debris as Innovative and Non-Invasive Matrix

“…Another factor discouraging the diagnosis of A. woodi in Italian apiaries is the timeconsuming official method applied for its diagnosis. In fact, the conventional diagnostic method used in the surveillance program for detection of tracheal mites is the thoracic disk methods (TDM), which involves cutting a thoracic disc containing the prothoracic trachea for slide mounting and microscopic examination of the bee's tracheae [12,13]. Although TMD has a good sensitivity, allowing for the detection of even low levels of infestation (below 5%), this method is very laborious and requires a high level of skill and concentration of the laboratory staff [13].…”

Prevalence of the tracheal mite Acarapis woodi (Rennie) in Chile

“…A similar situation occurs with the molecular diagnostic technique for this disease, which is primarily used in the scientific field. These diagnostics require a high level of specialization and technology and are thus more expensive (Sammataro et al, 2013;Delmiglio et al, 2015). In Chile, there are approximately 10 institutions in the entire country that perform the direct diagnosis of the parasite, and to date, there are no institutions that have implemented and validated the PCR technique for their molecular diagnosis.…”

“…It is well documented in the literature that beekeepers usually do not perform the diagnosis of this endoparasite (Maggi et al, 2016). The reason for this is that diagnosis requires scientific qualifications and because it is a laborious and complex process (Sammataro et al, 2013) involving molecular techniques that require large numbers of bees, such as polymerase chain reaction (PCR) (Sammataro et al, 2013;Delmiglio et al, 2015) or involving enzymatic techniques such as enzyme-linked immunosorbent assay (ELISA), which have also been show effectiveness as a diagnoses method (Sammataro et al, 2013).…”

Estudio preliminar sobre la presencia de enfermedades en enjambres de abejas africanizadas (Apis mellifera) en diferentes zonas de Costa Rica

“…Acariosis: Padecimiento provocado por el ácaro Acarapis woodi, el cual es un parásito microscópico que afecta únicamente a las abejas adultas, parasitando la parte pro-torácica de la tráquea (Cepero et al 2015). Este ácaro perfora las paredes de la tráquea y se alimenta de la hemolinfa (Delmiglio et al 2015). Su efecto patógeno en las abejas individuales depende del número de parásitos dentro de la tráquea (Sakamoto et al 2017).…”

“…Algunos síntomas que podrían relacionarse con acariosis son abejas con las alas distendidas, abanicándolas sin poder volar, abejas muertas o moribundas en frente de la colmena, entre otros (Bailey & Ball 1991;Ritter 2001). La presencia de ácaros traqueales en las abejas puede afectar la recolección de néctar y polen, así como la producción de miel (Delmiglio et al 2015).…”