Revised description of the blueberry bud mite, Acalitus vaccinii (Acari: Trombidiformes: Eriophyidae), and a key to all Eriophyoidea on Vaccinium

In 2014 the blueberry bud mite, Acalitus vaccinii (Acari: Trombidformes: Eriophyidae), was detected causing significant damage to cultivated blueberries in the Mpumalanga province of South Africa. This was the first detection of this pest outside of North America, to which it is native. However, its taxonomic description at that time lacked critical detail and omitted characters and life stages important for easy and accurate identification. Using an integrative taxonomic approach, we combined phase contrast light microscopy with low-temperature SEM and DNA barcoding data to revise the description of A. vaccinii using South African specimens. Additional characters not included in previous descriptions but reported here are the h1 (accessory) setae, leg I and II u’ (mesal) setae, and leg II bv (femoral) setae. Detailed descriptions and measurements of all life stages are included, along with a discussion of morphological variation and biology. Two DNA sequences of common barcode regions (nuclear and mitochondrial) are provided to further aid in identification. In addition, a key to all known species of eriophyoid mites present on Vaccinium is provided.


INTRODUCTION
Blueberries, Vaccinium spp.(Ericaceae), have become a rapidly expanding commercial crop in South Africa since the 1980s.Most commercial blueberry plantations are in the Western Cape province, where the longer winters contribute to better berry yield (Meyer and Prinsloo 2003).South African blueberry plantations have been relatively free of pests until 2012 when Acalitus vaccinii (Keifer 1939) (Trombidiformes: Eriophyidae), the blueberry bud mite, was discovered for the first time in South Africa on a farm in the Mpumalanga province.It was identified as such based on the original and subsequent species descriptions, and by comparison to other eriophyoids known on Vaccinium spp., and to other Acalitus spp.known from Africa.The mite caused substantial damage that resulted in an estimated 80% decreased yield within only two years of its detection.Symptoms included red blistering on buds, production of small leaves and fruit, as well as malformed flowers (Craemer 2018).Further surveys by the South African Department of Agriculture, Land Reform and Rural Development (DALRRD) also confirmed blueberry bud mite infestations in other locations within the Mpumalanga and North West provinces (Ngubane-Ndhlovu et al. 2018) Acalitus vaccinii is part of the superfamily Eriophyoidea, casually referred to as eriophyoid mites.Eriophyoidea contains three families, namely Eriophyidae, Diptilomiopidae and Phytophtidae.Eriophyoid mites are highly specialised, plant-feeding and are typically host-specific.These mites are minute, between 100 and 300 µm long with worm-like bodies and two pairs of legs.Many species are of commercial interest as they can cause malformation of buds, can form galls, or cause rust-like symptoms on leaves and fruit.
The lifecycle of A. vaccinii is typical for eriophyoid mites and includes eggs, larvae, nymphs, and adult males and females.Two female forms can be present, a deutogyne hibernating winter form and protogyne sexually active summer form.In A. vaccinii, the presence of a deutogyne has been noted in the colder areas of its native range in North America (Manson and Oldfield 1996;Cromroy and Kuitert 2001).The identification of both forms is important for assigning species identity to eriophyoid mites, where misidentification of the deutogyne is frequent (Zhao 2000;Smith et al. 2010;Guo et al. 2015).As is the case with many eriophyoids, accurate identification of A. vaccinii is hampered by incomplete species descriptions, inaccurate description of some characters and life stages in original descriptions and a lack of identification keys.For example, no comprehensive key to the >90 Acalitus species worldwide or to the nine eriophyoids on Vaccinium spp.(one Diptilomiopidae and eight Eriophyidae) exist.
Acalitus vaccinii was first described by Keifer (1939) as Eriophyes vaccinii, but later moved to Aceria (Keifer 1946) and thereafter to Acalitus (Baker and Neunzig 1970).In this paper we used modern methods to examine A. vaccinii and revise its description, including originally missed characters and all developmental stages.For enhanced clarity, specimens are examined using two imaging techniques, namely phase-contrast light microscopy (PCLM) and low-temperature scanning electron microscopy (LT-SEM) to scrutinise characters on slide-mounted and in situ mites.We also aim to provide diagnostic DNA barcoding sequences including nuclear (28S) and mitochondrial (COI) regions of A. vaccinii to accompany morphological descriptions and to increase accuracy of future identifications of this important pest.Additionally, we provide an identification key to eriophyoid species on Vaccinium worldwide.

Mite collection
Plant material showing symptoms of A. vaccinii infestation was collected from farms near three different towns in the Mpumalanga province in South Africa (Table 1).On each farm at each sampling occasion, 30 samples of 30 cm long shoots were taken at random per cultivar and per block and placed in resealable plastic bags.These shoot samples were kept at 4 °C until examination for the presence of eriophyoids using a stereomicroscope.For traditional microscopic examination using a compound microscope, eriophyoids were collected into a drop of sorbitol and isopropyl-alcohol solution until mounting (de Lillo et al. 2010).For scanning electron microscopy (SEM), mites were kept in situ until preparation.For molecular analysis, eriophyoids were placed into a drop of distilled water on a glass slide and processed immediately.

Phase contrast light microscopy (PCLM)
Collected eriophyoids were mounted on glass slides using F-medium following published protocols (Keifer 1975;de Lillo et al. 2010).Specimens were examined at 1 000× magnification using a Zeiss Axioskop Imager M2 microscope (Zeiss, Germany), equipped with a drawing tube and Zeiss AxioCam Cc5 digital camera.ZEN 2012 software was used for line drawings and capturing of images.Seventy-five characters for females, 69 for males and 68 for immatures were measured using a Leica DM 2500 microscope (Leica Weitzlar, Germany) connected to a Leica digital camera and Leica application suite v 3.1.0software.

Principal component analysis (PCA)
To determine if distinctive clusters of characters were present, we performed a principal component analysis using morphological characters of 11 females collected from different seasons (six in summer and five in winter) (Table S1).From the initial 75 morphological characters measured, only independent and non-repeated characters which showed a standard deviation greater than one were used, resulting in 28 characters used in the PCA.PCA was performed in Rstudio v.1.1.447running R statistical analysis v.3.5.0 (R Core Team 2020; R Studio Team 2020).To visualise the results, the packages ggfortify (Tang et al 2016;Horikoshi and Tang 2018) and ggplot2 (Wickham 2016) were used.Slide-mounted voucher material of all stages were deposited in the mite collection of DALRRD, Plant Quarantine Station in Stellenbosch, South Africa, and in the National Collection of Arachnida -Acari of the Agricultural Research Council -Plant Health and Protection, in Pretoria, South Africa.

Scanning electron microscopy (SEM)
A modified version of the cryo-fixation technique described by Echlin et al. (1970) was used for preparing specimens and studying them with a conventional JEOL JSM 840 SEM with a cryo-stage.Fourteen females, two males, two nymphs and two larvae were mounted on double-sided carbon tape.The tape was attached in a specimen holder with silver paint, which was plunge-frozen in liquid nitrogen slush and then transferred via the pre-chamber of the cryo-system to the pre-cooled cryo-stage in the chamber of the SEM (ca.-170 °C).Here the specimen was etched for ca.30 minutes by increasing the temperature to ca. -80 °C to remove ice crystals.The specimen holder was then transferred back to the pre-chamber and sputter-coated with gold, then returned to the cryo-stage for observation of specimens at an accelerating voltage of 5 kV or 2 kV (to prolong viewing time).Digital images were captured using a frame grabber controlled by Orion ® 6.6.

Revised description of Acalitus vaccinii
Identification was confirmed based on species-specific microscopic characters according to Keifer (1939Keifer ( , 1946) ) and Baker and Neunzig (1970).A revised description of A. vaccinii is presented following the recommendations of Amrine and Manson (1996) andDe Lillo et al. (2010).All measurements are given in micrometers (μm), rounded off to the nearest integer, as a range (minimum to maximum).Measurements refer to the length (not width) of the morphological character unless specified otherwise.Terminology follows that of Lindquist (1996).

DNA extraction
Groups of four to eight live mites were crushed in a small drop of distilled water on a glass slide.Using a micropipette, the drop containing the mites was then transferred into a sterile microcentrifuge tube for DNA extraction.DNA extractions were performed using a Qiagen QIAamp DNA Micro Kit (Qiagen, California, USA) following the manufacturer's instructions with the exception that all reaction volumes were halved to improve DNA concentration.The DNA extractions were eluted into final volumes of 30 µl and stored at -20 °C.

DNA amplification and sequencing
PCR reactions were performed in 25 μl volumes on a Techne Prime Thermal Cycler (Staffordshire, UK).Amplification was performed using 6 μl of DNA extract with half volumes of Promega Corporation (Madison, WI) GoTaq DNA polymerase, following manufacturer's instructions.
Prodorsal shield: (Figure 4) oval, 23-28 long, 31-50 wide; frontal lobe small, thin, anteriorly pointed or slightly rounded.Prodorsal shield with pair of usually obscure admedian lines on posterior ¼ of shield between scapular sc setae, more or less curving outwards from rear, then curving inwards, few granules on the outer side of scapular tubercles, with eye-like structures on their outer side partly margined with single rounded, shallow ridge, band of granules on outer margins of shield and on epicoxal area (sensu Chetverikov and Craemer 2015).Scapular setae sc 20-24, 22-25 apart, projecting posteriad.
Blastx of the COI sequences returned sequence WLI54571.1 of Leipothrix sp.(Eriophyidae) as the best hit (COI, 100% coverage, 76.98% identity) when sorted by either E-value or percent identity.Only two other Acalitus species (A.phloeocoptes and A. rudis) have sequences available on GenBank, neither of which have sequences for regions that overlap with those sequenced in the current study.

DISCUSSION
Acalitus vaccinii occurs on wild and cultivated blueberry, causing significant economic damage on susceptible varieties in its native distribution.The damage seen due to A. vaccinii on South African blueberry was significant, with yield losses ranging from 30-90% (Craemer 2018).When A. vaccinii was first identified in South Africa it was noted that the description of this mite needed revision (Craemer 2018).Available descriptions did not include all life stages, and important morphological features had not been noted or were inadequately described.No comprehensive key to Acalitus species nor eriophyoid species on blueberry was available.Here we rectified these omissions by providing accurate details of key features of multiple life stages.
In the original description of A. vaccinii by Keifer (1939), some key morphological features were omitted in both the drawing and text description.Most importantly these included the h1 (accessory) setae, leg I & II u' (mesal) setae and leg II bv (femoral setae) that are considered taxonomically important as the presence or absence of setae may be an indication of a different species (Amrine and Manson 1996; De Lillo 2010).Keifer measured 33 female and 5 male (without description and drawing) characteristics, as compared to the 75 characters measured for females, 69 for males and 68 for immatures in this study.
Many of the features not included in the original description are minute and may have been missed in original observations.For example, the observation of the setae mentioned above in the current study may largely be due to advancements in microscopy since the original description.The Scanning Electron Microscopy (SEM) technique, specifically Low-temperature SEM (LT-SEM), used here was able to substantially increase visual detail of A. vaccinii including these minute structures.LT-SEM also eliminated uncertainties in the shape of structures, especially when viewing the h1 setae, empodium and other subtle features such as the frontal lobe and shape of microtubercles.
In addition to Keifer (1939), Baker and Neunzing (1970) described the immatures of A. vaccinii.Differences observed between the former and this study is in the presence and arrangement of the opisthosomal microtubercles in immatures.The original description presented the larva without microtubercles and the nymph with microtubercles covering the entire opisthosoma.In the present study, on nymphs, the ventral microtubercles were arranged medially about the width of 3a -3a setae and the dorsal microtubercles were arranged in an hourglass shape medially about the width of sc -sc setae and were more widely spaced than those on the ventral side.On larvae, microtubercles were variously present or absent on either the dorsal, ventral or both surfaces, with variable nonuniform arrangements.These observations may be a result of intraspecific variations due to a limited number of studied specimens in previous studies and advances in microscopy encouraging qualitative and quantitative analysis.Many measurements that are standard for modern descriptions were not presented by Baker and Neunzing (1970) for the immature life stages.Additionally, the 35 measurements presented in that study cannot be used for comparison with current standards and procedures, as measurement techniques were not stipulated.
The presence of all life stages (females, males, immatures and eggs) of A. vaccinii on cultivated blueberries confirmed that the crop is an obligate host.Specimens were collected and studied throughout the year to capture variation and in attempt to detect the presence of a deutogyne, should one exist.A deutogyne is a winter form of eriophyoid mite and was detected in North America for A. vaccinii (Baker et al. 1996;Manson and Oldfield 1996;Cromroy and Kuitert 2001).It is important to establish whether both forms of a species occur in a particular area to avoid future misidentification of the deutogyne as a separate species (or even genus) because of morphological differences (Zhao 2000;Smith et al. 2010;Guo et al. 2015).Although females collected in winter appeared, on average, larger than the summer specimens, this was not uniform and did not form a separate cluster when analysed by PCA (Figure S1 in Supplementary Material).Other characters did not differ between winter and summer specimens.Further, immature life stages and males were collected in all seasons.Thus, morphological differences and biological evidence do not prove without doubt the presence of a deutogyne in South Africa.The absence of deutogynes in SA might be explained by the mild winter conditions of Mpumalanga (8-19 °C) (South African Weather Service, 2018), in comparison to the mite's native range (-1 to -7 °C) (www.usclimatedata.com/climate/united-states/us).The lack of deutogynes and the viability of all life stages through the winter season might have contributed to the increased population size and significant crop injury at the Mpumalanga farm.This also suggests that the mite is likely to be a more serious pest in warmer regions of blueberry production.In addition to the enhanced morphological descriptions added here, sequence information for two DNA regions commonly used in mite species identifications were made available on GenBank to aid future identification.Partial sequences of the COI gene are routinely used for identification of many animal species including mites, and the D2 region of 28S rDNA has shown differences between eriophyid species within a genus (Skoracka and Dabert 2010).In conjunction, these regions have potential for identification of A. vaccinii and other eriophyoid species.It will be of great benefit if more sequences were generated and deposited in GenBank to increase the pool of sequences for molecular identification of eriophyoid mites.
This study supplemented and enhanced the previous descriptions of A. vaccinii to enable more accurate identification and ease of comparison when conducting taxonomic analyses on this important group.Importantly, additional characters (including two DNA barcodes), morphological measurements and some life stages that were not included in previous descriptions are here presented in detail.The use of complementary morphological and molecular techniques greatly enhanced our ability to see and image minute characters and provide additional information and it is recommended that future workers on this group do the same.

Figure 1 .
Figure 1.SEM image of the opisthosoma (body) of A. vaccinii protogyne female, showing the ventral (A) and dorsal (B) aspect.Annuli are the rings around the body, and microtubercles are the protrusions on these rings (more detail can be seen in Figure2).SEM images were cropped to show the region of interest.For sizes of structures, refer to the measurements included in text.

Figure 2 .
Figure 2. Caudal region, viewed by SEM in ventral (A) and lateral (B) view and by PCLM in lateral view (C).Microtubercles and setae are labelled, including the presence of the minute h1 setae (B) which is very difficult to see using PCLM and was previously described as missing.SEM images were cropped to show the region of interest.PCLM images were taken with 100× oil objective.For sizes of structures, refer to the measurements included in the text.

Figure 3 .
Figure 3. SEM image of the gnathosoma, showing the pedipalps with the segments labelled and the small frontal lobe originating from the prodorsal shield.Setae v and ep are labelled.The chelicerae are retracted within the pedipalps and cannot be seen in this image.SEM images were cropped to show the region of interest.PCLM images were taken with 100× oil objective.For sizes of structures, refer to the measurements included in the text.

Figure 5 .
Figure 5. Schematic line drawings of Legs I and II showing the segments and setae names.

Figure 4 .
Figure 4.The prodorsal shield in dorsal (A and B) and lateral (C and D) view, as viewed by SEM (A and C) and PCLM (B and D).Setae sc, admedian lines and the frontal lobe are labelled.The eye-like area can be seen in lateral view with a band of granules on the outer margin.SEM images were cropped to show the region of interest.PCLM images were taken with 100× oil objective.For sizes of structures, refer to the measurements included in the text.

Figure 6 .
Figure 6.Legs I and II (A, C and D) and empodia (B and E) viewed by SEM (A and B) and PCLM (C, D and E).Leg segments are labelled (A), as well as femoral II setae bv which was previously described as missing.The six empodial rays are indicated by arrows (B).In PCLM images, features may only be visible at different focal points (C and D).SEM images were cropped to show the region of interest.PCLM images were taken with 100× oil objective.For sizes of structures, refer to the measurements included in the text.

Figure 7 .
Figure 7.The coxisternal area of the protogyne female, viewed by SEM (A) and PCLM (B and C).External features, including the genital coverflap with ridges in a single rank (row) and setae are labelled (A and B).C shows the shape of the internal genitalia, only visible with PCLM.SEM images were cropped to show the region of interest.PCLM images were taken with 100× oil objective.For sizes of structures, refer to the measurements included in the text.

Figure 8 .
Figure8.The coxisternal area of the male, viewed by SEM (A) and PCLM (B).External features and setal arrangement are as for the female (Figure6), except for the genitalia and genital coverflap, which is not present in the male.SEM images were cropped to show the region of interest.PCLM images were taken with 100× oil objective.

Figure 9 .
Figure 9. Nymph, viewed by SEM (A) and PCLM in dorsal (B) and ventral (C) view.Setal arrangement is as for the adult.Dorsally, microtubercles form an hourglass shape approximately the width of sc -sc (A and B).Ventrally, microtubercles are arranged in a band about the width of 3a -3a (C).SEM images were cropped to show the region of interest.PCLM images were taken with 100× oil objective.For sizes of structures, refer to the measurements included in the text.

Figure 10 .
Figure 10.Larva, viewed by SEM (A) and PCLM (B and C), showing dorsal (A and B) and ventral (C) aspects.SEM images were cropped to show the region of interest.PCLM images were taken with 100× oil objective.For sizes of structures, refer to the measurements included in the text.

Table 1 .
Location and collection information of blueberry plantations from which Acalitus vaccinii was collected from November 2014 to November 2016 *Due to confidentiality of information, sites are named in this study according to the nearest town.GPS coordinates also refer to the nearest town.

Table 2 .
DNA markers, PCR primers and cycling conditions used for amplification and sequencing of Acalitus vaccinii specimens in this study.F/R indicates a forward (F) or reverse (R) primer.Accession numbers for sequences submitted to GenBank are included.Extractions are from the Dullstroom (D) and Lydenburg (E) populations.