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10-20:24-31 (2010) HARUM RESEARCH ARTICLE

THE INVESTIGATION OF HONEY BEE DISEASES AFTER COLONY LOSSES IN HATAY AND ADANA PROVINCES OF TURKEY TÜRKİYE, ADANA-HATAY İLLERİNDEKİ KOLONİ KAYIPLARININ ARDINDAN BAL ARISI HASTALIKLARININ İNCELENMESİ

Aygün Yalçınkaya* • Nevin Keskin* Summary: Sudden colony losses occurred in Turkey, mostly in Hatay and Adana region in the year of 2007. After this event, all of our laboratory experiments were focused on this region. The aim of the study was to investigate the situation of honey bee diseases in Hatay- Adana region and to determine probable causes of colony losses. In this research, 97 and 88 honey bee brood combs were collected from Adana and Hatay during the spring and autumn field works. The total numbers of investigated adult honey bee were 3880 from Adana and 3520 from Hatay. All debris and adult honey bee samples were investigated under dissection microscope for diagnosis of Varroasis. Spore counting method was used for Nosemosis. “Guanine visualization” and “dissection” methods were used for diagnosis of Acarapiasis. Larval samples were inoculated to selective media for determination of American and European Foulbrood. For bacterial identification, biochemical tests used and Gram- stained slides were investigated under light microscope. As a result of laboratory analyses, Varroasis was determined in the ratio of 98% and high level Nosemosis was determined in the ratio of 12.97%. As a result of diagnostic tests that were applied to all honey bee samples, American Foulbrood and European Foulbrood were detected at the rate of 29% an 19% respectively. The ratio of the healthy bees was 52%. Consequently, we saw that parasitic and microbial diseases exist and they are prevalent in this region but the level of diseases showed that not only a single disease but also the combination of diseases and other factors could cause severe colony losses. Keywords: American Foulbrood, European Foulbrood, Varroasis, Nosemosis, Acarapiasis, Colony Losses Özet: Türkiye’de 2007 yılında, çoğunlukla Hatay ve Adana bölgesinde ani koloni kayıpları meydana gelmiştir. Bu kayıplardan sonra laboratuar çalışmalarımız bu bölge üzerinde yoğunlaşmıştır. Çalışmanın amacı, Adana-Hatay bölgesindeki arı hastalıklarını araştırmak ve koloni kayıplarının olası sebeplerini belirlemektir. Bu çalışmada, ilkbahar ve sonbahar arazi çalışmalarında Adana ve Hatay’dan 97 ve 88 yavrulu petek toplanmıştır. Adana’dan 3880, Hatay’dan 3520 adet ergin bal arısı incelenmiştir. Tüm debris ve ergin bal arısı örnekleri Varroasis tanısı için diseksiyon mikroskobu altında incelenmiş; Nosemosis için spor sayma yöntemi kullanılmıştır Acarapiasis tanısı için Guanin görüntüleme ve diseksiyon yöntemleri bir arada uygulanmıştır. Amerikan ve Avrupa yavru çürüklüğü hastalıklarının belirlenmesi amacıyla larva örneklerinden, seçici-ayırdedici besiyerlerine inokulasyonu yapılmıştır. Bakteriyel tanımlama yapılması amacıyla biyokimyasal testler kullanılmış; Gram boyama yapılan preparatlar ışık mikroskobu altında incelenmiştir. Laboratuar analizleri sonucunda Varroasis % 98, yüksek seviyedeki Nosema % 12,97 oranında tespit edilmiştir. Tüm bal arısı örneklerine uygulanan tanısal testler sonucunda Amerikan Yavru Çürüklüğü % 29 ve Avrupa Yavru Çürüklüğü % 19 olarak saptanmıştır. Sağlıklı arıların oranı ise % 52 olarak tespit edilmiştir. Sonuç olarak, bölgede paraziter ve mikrobiyal hastalıkların yaygın olarak bulunduğu görülmüş ancak hiç bir hastalığın tek başına büyük çaplı kayba neden olmayacağı, koloni koyıplarında arı hastalıkları ve diğer faktörlerin bir arada etkili olduğu kanaatine varılmıştır. Anahtar kelimeler: Amerikan Yavru Çürüklüğü, Avrupa Yavru Çürüklüğü, Varroasis, Nosemosis, Acarapiasis, Koloni Kayıpları *Hacettepe University, Department of Biology, Bee Health Lab., Beytepe Campus, 06800, Ankara/Turkey e-mail: [email protected]

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Introduction

Honey bees have important roles not only in the production of honey and bee products, but also pollinating of agricultural crops. Colony depletion and disease outbreak in managed honey bees are not unusual. However, serious colony losses and decline in bee populations have been reported in recent years and the term CCD (Colony Collapse Disorder) was first used all over the world in the year of 2006 (vanEngelsdorp et al., 2007). This decline in pollinators is a global threat to biodiversity. Therefore honey bee health has a great impact on economy and biodiversity worldwide (Jaffé et al., 2009). There are a lot of parasites and pathogen microorganisms associated with honey bees affecting adult bees and brood. Parasitic mite Varroa destructor is the most damaging honey bee parasite in the world (Rosenkronz et al, 2010). Mite infection which causes dramatic colony losses have repeatedly occurred in affected countries. Adult Varroa mites feed on adult bees, but reproduction depends on bee brood. Both the adult female and her offspring feed on pupae, where they can cause damage by infestation of hemolymph, resulting in severe nutritional deficits for the developing bee (Bowen-Walker and Gunn, 2001, Duay et al., 2003, Garedew 2004). Varroa mites can transmit several viruses which cause severe damages on bees and colony losses (Chen et al., 2006). Acarapis woodi is another mite related to honey bees. Tracheal mite Acarapis woodi damages tracheal walls mechanically and causes blockage on tracheas by its metabolic products (Erickson et al.,1999) Nosema apis and Nosema ceranae are intracellular microsporidian parasites infecting the midgut epithelial cells of adult honeybees (Forsgren and Fries, 2010). Nosema ceranae is more virulent than N. apis and it is replacing with Nosema apis in most countries and Nosemosis is worldwide (Higes et al., 2007, Klee et al., 2007, Paxton et al., 2007). It is known to be correlated with reduced lifespan of individual bees, reduced performance of colonies and increased winter mortality (Fries, 1993). On the other hand, some publications emphasized that Nosema ceranae is not replacing with Nosema apis and their virulence is not so different in some countries (Forsgren and Fries, 2010).

The situation of Nosemosis still is not clear worldwide and further researches are necessary. American foulbrood (AFB) is caused by the bacterium Paenibacillus larvae and European foulbrood (EFB) is caused by the bacterium Melissococcus pluton (Bailey,1983). Both species are Gram-positive bacteria. Melissococcus pluton is a chain, single or double formed coccus. Several other bacteria like Acromobacter euridae, Enterococcus faecalis, Paenibacillus alvei, Brevibacillus laterosporus maybe associated with EFB (Forsgren, 2010). Paenibacillus larvae is a spore forming, Gram-positive rod-shaped bacterium. AFB is common bacterial disease affecting apiculture worldwide. It is included in the Notifiable Disease list by the World Organization for Animal Health (OIE). AFB is difficult to be managed by apiculturists because the pathogen produces environmentally stable spores which are very virulent and infectious after decades, as well as resistant to heat, to desiccation and chemical disinfectants and it spreads easily and rapidly within a colony, among colonies in an apiary and between apiaries (Ashiralieva and Genersch, 2006; Fries et al. 2006) It is aimed to search all kinds of honey bee diseases in Hatay and Adana provinces in this study. Because, Hatay and Adana provinces serve as a resource for citrus and cotton honey, and have important role in Turkish Beekeeping as wintering location for migratory beekeepers. Colony losses in these provinces caused great impact on Turkish Beekeeping industry.

Material and Methods

Collection of Samples In this study adult and brood honey bee samples were collected from Adana and Hatay provinces at early spring and late fall of 2006-2007 season, in cooperation with Adana and Hatay Beekeeping Associations (Table 1). All samples were examined for parasitic and microbial diseases. Field studies were carried out in Aladağ, Ceyhan, İmamoğlu, Karataş, Karaisalı, Kozan, Pozantı, Saimbeyli, Seyhan, Tufanbeyli, Yumurtalık districts of Adana province and Antakya, Belen, Dörtyol, Erzin, Hassa, İskenderun, Kırıkhan, Reyhanlı, Samandağ, Yayladağ districts of Hatay provin-

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ce. Live adult bee samples and brood were collected directly from colonies and brought to the laboratories in cold conditions (4 0C). Examination of Adult Honey Bee Samples All adult bees were firstly investigated under dissection method for Varroasis and other symptoms like swollen abdomen and hair losses. Varroa mites have been detected on adult bees and also in debris. For the detection of the tracheal mite Acarapis woodi, two different methods were used: Dissection method and Guanin visualization method. Dissection method was applied due to OIE manual. Adult bees were secured on their backs. Their heads and forelegs were removed and the prothoracic circle was cut in front of the middle pair of legs with razorblade. Thin disks were heated in a solution of 8% potassium hydroxide for clearing the muscle tissue. The first pair of tracheae was examined under microscope. Thin layer chromatography technique was used in Guanin visualization method. In this method 5 adult bee thoraxes were kept in 0,1M sodium hydroxide solution to clean and soften tissues for homogenization. Homogenized thorax tissues were centrifugated at 16.000 rpm for 20 minutes. Supernatants were carried over to new tubes and centrifugated at 16.000 rpm for 2 minutes to clean tissues properly. Samples and Guanine marker (Sigma) were loaded to thin layer and the layer was put in to running buffer (5% Ammonium sulfate, 13M Ammonia an 1-Propanole in the ratio of 60:30:10). After running the procedure thin layers were examined under UV (Koch and Gerson, 1997, OIE Manual, 2000). For the detection of Nosemosis, at least 30 honeybee samples were examined from each colony. Abdomens of honeybees were cut by scissors. All abdomens were homogenized by mortar and pestle. Homogenates were diluted with 10 ml of saline solution. The solution was filtered through cotton fabric. The homogenates were centrifuged for 10 minutes at 800 g. The supernatants were poured out. Saline solution was added 1 ml per bee. Final homogenate was mixed properly by vortex. 0.1 ml of the solution was put on heamocytometer and Nosema spp. spores were counted under the light microscope. The number of Nosema spores per bee was calculated. Totally 9 squares were counted for spores (Shimanuki and Knox, 2000; Yalçınkaya et al., 2009). Spore count formula:

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0.2x0.2x0.1=0.004 mm cube 9x0.004= 0.036mm cube A/0.036= mm cube x 1000= number of spore/ml Examination of Brood Honey Bee Samples Brood foundation was examined for clinical signs. Diseased larva appears moist and darkened, cell capping is concave and possibly punctured in foulbrood diseases. For isolation of bacteria, larval samples were inoculated firstly to Nutrient Broth, Brain Heart Infusion Broth and then from broth cultures to Brain Heart Infusion Agar with Nalidixic acid and MYPG Agar (Mueller-Hinton broth, yeast extract, potassium phosphate, glucose and pyruvate). Cultures were incubated at 37 0C for 48 hours. Colony morphology was examined and biochemical analyses were run for identification of AFB and EFB pathogens. All bacterial cultures were investigated under light microscope by using Gram Staining Method (Hornitzky and Wilson, 1989, OIE Manuel, 2000, Shimanuki and Knox, 2000). Statistical Analyses All collected data were analyzed by ANOVA and chisquare tests statistically.

Results

All honey bee samples were investigated for adult and brood honey bee diseases. Varroa destructor was observed in all samples. Varroa mites were found on adult bees, pupae in brood combs and debris. Contrary to Varroa mite, tracheal mite Acarapis woodi was not found in any sample (Table 2). Nosema spores were found in all adult honey bee samples. Nosemosis levels have been graded according to Nosema spp. spore numbers. Infection levels were classified as “low” between 0-500.000 spores, “medium” between 500.000-1.000.000 spores and “high” more than 1.000.000 spores. High Nosemosis level rate was 12,97%, medium level was 35,67% and low level was 51,36% for all samples. All results are presented in Table 3-6 and Figure 1. Foulbrood diseases were detected 48% percent of all samples. All results are presented in Table 7-8 and Figure 2-4.

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Table 1. The number of adult and brood honeybee samples collected from Adana and Hatay provinces

ADANA

HATAY

Sample Number/ Fall

Sample Number/ Spring

Adult

1600

1920

Brood

40

48

Adult

1720

2160

Brood

43

54

Provinces

Sample Number/ Fall

Sample Number/ Spring

Varroa destructor

Adana

100%

100%

Hatay

100%

100%

Acarapis woodi

Adana

0%

0%

Hatay

0%

0%

Hive Number

Avarage Spore Number

sd

Adana

54

408518,52

263219,60

Hatay

48

568541,67

257243,90

df

P

100

0,003

Table 6. Comparison of Nosema spp. spore density in Adana and Hatay provinces in Fall.

Table 3. Comparison of Nosema spp. spore density in two seasons in Adana Province Season

Province

sd: Standard deviation df: degrees of freedom P0,05 Difference between Adana and Hatay is not significant in Fall.

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Figure 1. Nosema levels in the hives located in Adana and Hatay provinces for Spring and Fall

Figure 2. Foulbrood diseases rates in Adana Province

Figure 3. Foulbrood diseases rates in Hatay Province

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Figure 4. Annual percentages of foulbrood diseases in the period of 2006-2007

Table 8. Comparison of foulbrood diseases percentages in seasons chi-square

P

Adana

7,810

0,020*

Hatay

2,228

0,328**

Total

8,223

0,016***

*P0,05 Difference between Fall and Spring is not significant for Hatay province ***P