Sherly George

Standard methods for tracheal mite research

Summary The honey bee tracheal mite (HBTM) Acarapis woodi (Rennie) (Acari: Tarsonemidae) is an obligate endoparasite of honey bees. First described from the Western (European) honey bee Apis mellifera L., this mite species was initially observed when honey bee colonies on the Isle of Wight, UK were dying between 1904 and 1919 (Rennie, 1921). Since then, this mite has been found in Europe, North and South America and parts of Asia, but its global distribution is not well understood. In this chapter, we outline protocols for collecting, detecting, identifying, diagnosing and measuring the infestation rates of A. woodi. We also describe methods to determine the damage threshold, outline several control measures, and describe methods for studying live mites.

Development and Validation of a Real-Time PCR Assay for Rapid Detection of Two-Spotted Spider Mite, Tetranychus urticae (Acari: Tetranychidae).

Spider mites of the genus Tetranychus are difficult to identify due to their limited diagnostic characters. Many of them are morphologically similar and males are needed for species-level identification. Tetranychus urticae is a common interception and non-regulated pest at New Zealand’s borders, however, most of the intercepted specimens are females and the identification was left at Tetranychus sp. Consequently, the shipments need to be fumigated. DNA sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) protocols could be used to facilitate the accurate identification. However, in the context of border security practiced in New Zealand, insect identifications are required to be provided within four hours of receiving the samples; thus, those molecular methods are not sufficient to meet this requirement. Therefore, a real-time PCR TaqMan assay was developed for identification of T. urticae by amplification of a 142 bp Internal Transcribed Spacer (ITS) 1 sequence. The developed assay is rapid, detects all life stages of T. urticae within three hours, and does not react with closely related species. Plasmid DNA containing ITS1 sequence of T. uritcae was serially diluted and used as standards in the real-time PCR assay. The quantification cycle (Cq) value of the assay depicted a strong linear relationship with T. urticae DNA content, with a regression coefficient of 0.99 and efficiency of 98%. The detection limit was estimated to be ten copies of the T. urticae target region. The assay was validated against a range of T. urticae specimens from various countries and hosts in a blind panel test. Therefore the application of the assay at New Zealand will reduce the unnecessary fumigation and be beneficial to both the importers and exporters. It is expected that the implementation of this real-time PCR assay would have wide applications in diagnostic and research agencies worldwide.

Molecular detection of small hive beetle Aethina tumida Murray (Coleoptera: Nitidulidae): DNA barcoding and development of a real-time PCR assay

Small hive beetle (SHB), Aethina tumida can feed on honey, pollen and brood in honey bee colonies. It was endemic to Africa, but since 1996 has been detected in a number of countries worldwide, including Australia, Brazil, Canada, Italy, Mexico, South Korea, Philippines and the USA where it has had economic effects on local apiculture. To improve SHB identification, we obtained the first reference sequences from the DNA barcoding 5′ COI gene region for SHB and some species of the family Nitidulidae associated with beehives. Phylogenetic analysis of SHB COI sequences (3′ COI) revealed two divergent lineages, with those from Australia and USA being genetically different from the recent detection in Italy. Many countries, including New Zealand, are currently free from SHB, and require a rapid detection method for biosecurity. Here we present the development and validation of a real-time PCR assay for detection of SHB. The assay showed high specificity and sensitivity for detecting SHB, with no cross-reaction observed with closely related species, such as A. concolor. The real-time PCR is sensitive, detecting the target sequences up to 100 copies/µL. This assay should prove a useful biosecurity tool for rapid detection of SHB worldwide.

An improved technique for quantifying infestation level of external mites (Acari) on honey bees

An improved wash method was developed to determine the infestation level of the external parasitic mites on adult honey bees, Apis mellifera. Compared to the old method the improved method detected the similar number of large mites viz. Varroa destructor but nearly 14 times the number of small mites viz. Acarapis externus and Acarapis dorsalis. It was more efficient as it required significantly less time at the post-shaking stage requiring in-person attendance. The sample processing procedure was also simple and practical and required less laboratory space.

Rapid and accurate identification of Xanthomonas citri subspecies citri by fluorescence in situ hybridization

Citrus canker is an economically important disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). This organism targets a wide range of citrus plants, including sweet orange, grapefruit, lemon and lime. As Xcc is spread by environmental factors such as wind and rain, it is difficult to control its movement once the disease has established. In order to facilitate monitoring of citrus canker we sought to design a novel diagnostic protocol based on fluorescence in situ hybridization (FISH) for identification of bacterial cells directly from canker pustules without cultivation or DNA extraction. This method was validated for specificity against a range of Xanthomonas species and strains. We show that our assay is extremely rapid (typically requiring between 2 and 3 h), and possesses a similar specificity to existing PCR diagnostic tools. The sensitivity of the assay is comparable to that of an existing PCR‐based technique and sufficient for identifying Xcc in symptomatic plant material. The method is easily transferable to diagnosticians without prior experience using FISH.

Establishment of Oulenziella gen. nov. for Oulenzia bakeri (Hughes, 1962) (Acari: Winterschmidtiidae).

A new genus, Oulenziella, is proposed for Oulenzia bakeri (Hughes, 1962), a species originally collected on jute (Corchorus sp., Malvaceae) from India. Oulenziella bakeri (Hughes, 1962) comb. nov. is re-described. Oulenzia gossypii Meyer & Rodrigues, 1965 collected from Gossypium sp. in Mozambique is considered a junior synonym of Oulenziella bakeri. The new genus differs from Oulenzia in having hT present on tibiae I and II, kT on tibia IV, la and ra on tarsus II, w and r on tarsus III, e and f on tarsi I-III; and by seta d on tarsi III and IV positioned at level of apical 1/8 to 1/6 of the segments.

Redescription of Oulenzia arboricola (Oudemans, 1928), type species of Oulenzia Radford, 1950 (Acari: Astigmata: Winterschmidtiidae)

Abstract A detailed description and illustration of Oulenzia arboricola (Oudemans, 1928) are provided based on the type specimen collected on Hevea leaves, from Medan, Deli, Sumatra (Indonesia). The definition of the genus Oulenzia is clarified and the taxonomic position of species under Oulenzia is discussed.