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Vigilancia y analisis genotipico del acaro Varroa destructor (Acari: Varroidae) parasito de Apis mellifera jemenitica (Hymenoptera: Apidae) en Arabia Saudi.

Surveillance and genotyping of Varroa destructor (Acari: Varroidae) parasitizing Apis mellifera jemenitica (Hymenoptera: Apidae) in Saudi Arabia

Introduction

The mite, Varroa destructor (Anderson y Trueman, 2000) is one of the most destructive pests of the honeybee (Apis mellifera L.) worldwide (Rosenkranz et al. 2010). Varroa mite has two haplotypes that are able to reproduce successfully on A. mellifera; the Japan-type is relatively non-virulent whereas the Korea-type, is extremely virulent (Anderson and Trueman 1998; Anderson 2000). In Saudi Arabia, the mite was first reported in 1987 (Al-Ghamdi 1990), perhaps through a shipment of introduced honeybees from Egypt, the usual package bee supplier of Saudi Arabia. Later, several studies reported on the occurrence and impact of Varroa mite throughout the country (Al-Ghamdi 1990; Al-Ghamdi 2002). Yet, general infestation levels and genotyping of V. destructor in Saudi Arabia is still needed.

Beekeeping in Saudi Arabia is based on native as well as on the exotic honeybee subspecies, A. mellifera (Alattal et al. 2014 and Alqarni et al. 2011). About 70 % of these colonies are kept in log hives colonized by native honeybee, A. m. jemenitica (Al-Ghamdi et al. 2013). Disease monitoring is nominal or absent and control of Varroa mite species is set up on calendar bases with almost consistent high colony losses. In addition to Varroa mite species, adverse climatic conditions in Saudi Arabia are the most obvious causes of colony losses in the country (Al-Ghamdi et al. 2013; Alqarni et al. 2011).

In temperate areas, infestation levels are critical and honeybee colonies with no annual treatment against Varroa mite will collapse within 2-3 years (Rosenkranz et al. 2010), but in the majority of tropical climates the infestation levels remain under the injury threshold (Ritter and De Jong 1984). Apparently, it is not well explained whether the bee types of the tropics or climate contribute more to the documented stable honeybee-Varroa species relationships (Rosenkranz 1999; Carneiro et al. 2007; Rosenkranz et al. 2010).

Ruttner (1988) described the native honeybee of Saudi Arabia as the smallest and the most heat and drought tolerant honeybee subspecies of A. mellifera. Drought and high average ambient temperatures are dominant in Saudi Arabia. Consequently, the infestation levels and buildup of Varroa species population density under Saudi Arabia conditions may be different than that in the temperate regions where abundant flows of nectar and pollen lead to profoundly populated colonies causing an exponential population growth of the parasite (Renz 2003). This study focuses on mite haplotyping and on assessment of general infestation levels of Varroa mite species in Saudi Arabia.

Materials and methods

Determination of infestation levels. Samples were taken from eight locations representing beekeeping areas within Saudi Arabia. Samples were collected shortly after the summer season during September and October 2013. Samples colonies were last treated with acaricides eight months before sample collection. Some related metereological parameters of the sampled locations are shown in Table 1. Varroa species infestation levels of adult worker bees were anticipated by sampling 100 adult workers from brood combs using plastic jars (450 [cm.sup.3]) and were then stored at -20[grados]C. To determine the number of adult Varroa mite species in each sample, the jars were filled with water and detergent and shaken for 2-3 min. and were then given into a double-layer honey sieve where adult bees were separated from Varroa mite species by a jet of water and the infestation level was calculated as number of Varroa mites/ number of adult bees and expressed in percentage.

Brood infestation rates were estimated by inspecting about 100 capped worker brood cells. After being frozen, the capping's of the frozen brood samples were cut and the brood with the Varroa mite were rinsed with water in the double-layer honey sieve to separate Varroa mites that were then counted to determine brood infestation. Then, relative infestation ratio = brood infestation rates/worker infestation rates (Renz 2003) was calculated.

Comparation of means were computed based on ANOVA and LSM performed on data after transformation using arc-sin function (SAS 1989).

V. destructor haplotype identification. A number of female adult mites isolated from worker or brood samples were collected (n = 10 mites per apiary). Collected mites were placed in 1.5 micro-centrifuge tubes containing 96% ethanol, labeled and stored at -20[grados]C until DNA was extracted. Total genomic DNA was extracted from individual female mites by chelex resin[TM] according to Cano et al. (1993). After that extracted DNA was used to amplify the cytochrome oxidase subunit one (CO-I) region of the mtDNA with the primers (COXF) 5'-GG(A/G)GG(A/T)GA(C/T)CC(A/T)ATT(C/T) T(A/T)TATCAAC-3' and (COXRa) 5'-CCTGT(A/T)A(A/t) AATA GCAAATAC-3' using PCR (Saiki et al. 1988) following the method described by Anderson and Fuchs (1998). PCR products were then separated by electrophoresis, stained with ethidium bromide, visualized with UV light (260 nm) and photographed. Amplified CO-I region was then used as a template for the RFLP reaction. The restriction enzymes Sac-I, which distinguish the G/AGCTC sequence and Xol-I which distinguish the C/TCGAG sequence were added separately each to 12.5 [micron]l of the PCR product as described by Anderson and Fuchs (1998). The mixture was then incubated at 37[grados]C for about one hour and was then run in a 1.5% agarose gel. The resulting bands were then visualized with UV light and photographed. One amplified CO-I region from each apiary were then sequenced using an automated 96 capillary AbI 3730XI DNA genetic analyzer (Applied Biosystem). Sequences were manually checked and assembled using GeneBank database (NCBI). Sequences were then exposed to two procedures. First, sequences were aligned using CLUSTALW software (Thompson et al. 1994), BLASTed and compared with other sequences available on the GenBank database (NCBI). Sequences were finally submitted to Genebank data base (NCBI).

Results

Results revealed that all colonies sampled in this survey (n = 72) were infested with Varroa mite species. The general average infestation levels were 5.5 [+ o -] 4.8% and 13.1 [+ o -] 10.6% on adult worker bees and in capped brood cells, respectively. Differences in infestation levels of adult worker bees were not significant among sampling locations (F = 1.5, P = 0.19, Figure 1), however, infestation levels in capped brood cells were highly significant among locations (F = 5.2, P < 0.001, Figure 2) the highest brood infestation levels were in Jazan then Abha. Generally, relative infestation of capped worker brood was about 4.7 [+ o -] 6.3 times higher than the infestation on adult bees, and differences between locations were significant (P < 0.001, Figure 3). The highest value being in Jazan.

The banding pattern of the RFLP reaction and sequence analysis of the mtDNA revealed that all mite samples were of V. destructor Korean haplotype. Fragment digestion occurred only when using Xol-I and resulted bands had a fragment size between 200 and 300 bp. Sequences were 99 to 100% identical to that of Korean type of V. destructor previously described by Anderson and Trueman (2000) (GeneBank accession number AF106899). In all Varroa mite samples analysed, two genotypes were detected with more than 99% similarity. The genotypes were deposited in the NBCI GenBank under the accession numbers KJ507740 and KJ507741.

Discussion

The survey demonstrated that V. destructor Korean haplotype is distributed in all beekeeping areas within Saudi Arabia; results also indicated that relative infestation levels may reach higher levels within few months without Varroa treatment. However these infestation levels are lower than what was described for other V. destructor-Apis mellifera systems with different subspecies such as A. m. carnica in central Europe (Rosenkranz et al. 2010), A. m. syriaca system in Jordan (Alattal et al. 2005) or A. m. lamarckii in Egypt (Ismail et al. 2006). This epidemic spread of the mite within Saudi Arabia could be mainly related to mass importation of Varroa species infested package bees from neighboring countries mainly Egypt. In addition to poor management practices in Varroa mite control, an un-controlled migration of bee colonies among locations increases the contact between highly infested colonies, where Varroa mite species can build up more quickly such as Jazan and Abha, and colonies with low infestation. When highly infested colonies die, its remaining bees with the Varroa mites will enter new colonies. Even that damage and colony losses were not visible during the assessment; increased colony mortality due to Varrroa associated viruses is probable after the end of spring and till the end of winter. Although, several locations showed relative infestation levels of adult bees below 15%, a proposed economic threshold on adult worker bees in temperate regions (Toscano 1996), we believe that the impact of Varroa infestation under arid conditions with very limited nectar and pollen resources, such as Saudi Arabia, would be also dramatic if colonies were not treated.

Although the significant differences in the infestation of worker brood cells among different locations might indicates real spatial differences in the course of colony development in these areas, it was not possible to classify locations according to their climatic zones or climatic parameters in predicting high or low infestation rates.

This demonstrates that spatial variations in infestation levels are rather a result of interaction among various factors including bee type, Varroa mite species genotype, and other ecological factors. Since only the highly virulent Korean genotype has been reported in all sampling areas within Saudi Arabia, variation in infestation levels may be related to other factors. On the other hand, nearby colonies in the same apiary may have clearly uneven relative infestation rates, which might be related to the variations in the patterns of colony build-up and colony population growth rate during the season. Consequently, Varroa mite population increased rapidly in some colonies than in others.

The infestation ratio is a sign of the relative number of reproducing mites in the honeybee colony. The maximum ratio took place in Abha and Jazan, which demonstrates that the increase in Varroa mite population is higher in these locations compared to other locations of this study. Consequently, colony losses due to higher infestation levels of adult workers at the time the brood area is reduced could be high. For that reason, monitoring of infestation levels by sampling of brood cells and adult bees is an essential practice to prevent late treatment or multiple treatments.

On the other hand, results indicated an extremely low divergence in the CO-I of Varroa mite in an extensive geographical area and all of these Varroa mite species were of a Korean haplotype identity, this may also suggest the presence of the same genotype in neighboring countries. The Varroa mite species may have a common origin in the investigated populations as also mentioned by Solignac et al. (2005) for the European Varroa mites.

Acknowledgements

This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award number (11-BIO1743-02). We are grateful to the beekeepers who allowed us to sample their hives and James Montoya Lerma (Editor in charge Revista Colombiana de Entomologia) for writing Spanish "resumen" and editing the manuscript.

Received: 16-Dec-2014 * Accepted: 6-Nov-2015

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YEHYA ALATTAL (1,2), MOHAMMAD JAVED ANSARI (1,3), AHMAD AL-GHAMDI (1,4) and AREF SINGLE (1,5)

(1) Ph. D. Chair of Engineer Abdullah Ahmad Bagshan for Bee Research, Department of Plant Protection, College of Food and Agriculture Sciences, King Saud University, PO Box 2460, Riyadh 11451, Kingdom of Saudi Arabia. (2) yalattal@ksu.edu.sa. (3) mjavedansari@gmail.com. Corresponding author. (4) aalkhazim@gmail.com. (5) asingl67@gmail.com.

Leyenda: Figure 1. Varroa destructor infestation levels of adult worker honeybees at different survey locations. Mean separation based on ANOVA and LSM, DF = 7 and P < 0.05, F = 1.5, P = 0.19.

Leyenda: Figure 2. Varroa destructor infestation levels of capped brood cells of the honeybee at different survey locations. Mean separation based on ANOVA and LSM, DF = 7 and P < 0.05, F = 5.2, P < 0.001. Means sharing same letters are not significantly different.

Leyenda: Figure 3. Varroa mite infestation ratio (brood infestation/worker infestation) of the honey bee at different survey locations. Mean separation based on ANOVA and LSM, DF = 7 and P < 0.05, F = 5.2, P < 0.001. Means sharing same letters are not significantly different.
Table 1. Name, latitude, climatic zone, annual maximum and minimum
temperatures and average annual precipitation of sampled locations
(PoMIP, 2007).

Location                                Latitude
           Climatic zone *

Riyadh          Arid         24.63[grados]N, 46.71[grados]E
Al-Hasa         Arid         25[grados]25'N, 49[grados]37'E
Tabuk           Arid          28[grados]23'N 36[grados]34'E
Jazan         Semi-Arid       16[grados]53'N 42[grados]33'E
Najran        Semi-Arid       17[grados]29'N 44[grados]7'E
Taif          Semi-Arid       21[grados]16'N 40[grados]25'E
AlBahah       Semi-Arid       20[grados]00'N 41[grados]27'E
Abha          Dry -Med        18[grados]13'N 42[grados]30'E

           Average Annual Temp. ([grados]C) **

Location                 Rainfall
           Min.   Max.   (mm) **

Riyadh      15    35.5      10
Al-Hasa     10     42       65
Tabuk       -6    30.6      46
Jazan       9      48       23
Najran     14.6   30.9      83
Taif       13.7   30.9     208
AlBahah     12     23      250
Abha        1      40      600

(* Koppen climate classification BWh), ** (PoMEP, 2007).
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Title Annotation:Seccion Control
Author:Alattal, Yehya; Ansari, Mohammad Javed; Al-Ghamdi, Ahmad; Single, Aref
Publication:Revista Colombiana de Entomologia
Article Type:Ensayo
Date:Jul 1, 2015
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