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

THE INVESTIGATION OF HONEY BEE DISEASES AFTER COLONY LOSSES IN HATAY AND PROVINCES OF

TÜRKİYE, ADANA-HATAY İLLERİNDEKİ KOLONİ KAYIPLARININ ARDINDAN BAL ARISI HASTALIKLARININ İNCELENMESİ

Aygün Yalçınkaya* • Nevin *

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 Nosemo- sis. “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 deb- ris 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 yöntem- leri bir arada uygulanmıştır. Amerikan ve Avrupa yavru çürüklüğü hastalıklarının belirlenmesi amacıy- la 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, Aca- rapiasis, Koloni Kayıpları

*Hacettepe University, Department of Biology, Bee Health Lab., Beytepe Campus, 06800, /Turkey e-mail: [email protected] 25

Introduction The situation of Nosemosis still is not clear worldwide Honey bees have important roles not only in the pro- and further researches are necessary. duction of honey and bee products, but also pollina- ting of agricultural crops. Colony depletion and dise- American foulbrood (AFB) is caused by the bacterium ase outbreak in managed honey bees are not unusual. larvae and European foulbrood (EFB) However, serious colony losses and decline in bee po- is caused by the bacterium Melissococcus pluton pulations have been reported in recent years and the (Bailey,1983). Both species are Gram-positive bacte- term CCD (Colony Collapse Disorder) was first used ria. Melissococcus pluton is a chain, single or double all over the world in the year of 2006 (vanEngelsdorp formed coccus. Several other like Acromo- et al., 2007). This decline in pollinators is a global bacter euridae, Enterococcus faecalis, Paenibacillus threat to biodiversity. Therefore honey bee health has alvei, Brevibacillus laterosporus maybe associated a great impact on economy and biodiversity worldwi- with EFB (Forsgren, 2010). Paenibacillus larvae is a de (Jaffé et al., 2009). spore forming, Gram-positive rod-shaped bacterium. AFB is common bacterial disease affecting apiculture There are a lot of parasites and pathogen microor- worldwide. It is included in the Notifiable Disease list ganisms associated with honey bees affecting adult by the World Organization for Animal Health (OIE). bees and brood. Parasitic mite Varroa destructor is AFB is difficult to be managed by apiculturists be- the most damaging honey bee parasite in the world cause the pathogen produces environmentally stable (Rosenkronz et al, 2010). Mite infection which causes spores which are very virulent and infectious after dramatic colony losses have repeatedly occurred in decades, as well as resistant to heat, to desiccation affected countries. Adult Varroa mites feed on adult and chemical disinfectants and it spreads easily and bees, but reproduction depends on bee brood. Both the rapidly within a colony, among colonies in an api- adult female and her offspring feed on pupae, where ary and between apiaries (Ashiralieva and Genersch, they can cause damage by infestation of hemolymph, 2006; Fries et al. 2006) resulting in severe nutritional deficits for the develo- ping bee (Bowen-Walker and Gunn, 2001, Duay et al., It is aimed to search all kinds of honey bee diseases 2003, Garedew 2004). Varroa mites can transmit se- in Hatay and Adana provinces in this study. Because, veral viruses which cause severe damages on bees and Hatay and Adana provinces serve as a resource for cit- colony losses (Chen et al., 2006). Acarapis woodi is rus and cotton honey, and have important role in Tur- another mite related to honey bees. Tracheal mite Aca- kish Beekeeping as wintering location for migratory rapis woodi damages tracheal walls mechanically and beekeepers. Colony losses in these provinces caused causes blockage on tracheas by its metabolic products great impact on Turkish Beekeeping industry. (Erickson et al.,1999)

Nosema apis and Nosema ceranae are intracellular Material and Methods microsporidian parasites infecting the midgut epit- Collection of Samples helial cells of adult honeybees (Forsgren and Fries, In this study adult and brood honey bee samples were 2010). Nosema ceranae is more virulent than N. apis collected from Adana and Hatay provinces at early and it is replacing with Nosema apis in most count- spring and late fall of 2006-2007 season, in coopera- ries and Nosemosis is worldwide (Higes et al., 2007, tion with Adana and Hatay Beekeeping Associations Klee et al., 2007, Paxton et al., 2007). It is known to (Table 1). All samples were examined for parasitic and be correlated with reduced lifespan of individual bees, microbial diseases. Field studies were carried out in reduced performance of colonies and increased winter Aladağ, , İmamoğlu, Karataş, Karaisalı, Ko- mortality (Fries, 1993). On the other hand, some pub- zan, Pozantı, , , , Yumur- lications emphasized that Nosema ceranae is not rep- talık districts of and , Belen, lacing with Nosema apis and their virulence is not so Dörtyol, Erzin, Hassa, İskenderun, Kırıkhan, Rey- different in some countries (Forsgren and Fries, 2010). hanlı, Samandağ, Yayladağ districts of Hatay provin- MELLIFERA 26

ce. Live adult bee samples and brood were collected 0.2x0.2x0.1=0.004 mm cube 9x0.004= 0.036mm cube directly from colonies and brought to the laboratories A/0.036= mm cube x 1000= number of spore/ml in cold conditions (4 0C). Examination of Brood Honey Bee Samples Examination of Adult Honey Bee Samples Brood foundation was examined for clinical signs. All adult bees were firstly investigated under dissec- Diseased larva appears moist and darkened, cell cap- tion method for Varroasis and other symptoms like ping is concave and possibly punctured in foulbrood swollen abdomen and hair losses. Varroa mites have diseases. For isolation of bacteria, larval samples were been detected on adult bees and also in debris. For inoculated firstly to Nutrient Broth, Brain Heart In- the detection of the tracheal mite Acarapis woodi, two fusion Broth and then from broth cultures to Brain different methods were used: Dissection method and Heart Infusion Agar with Nalidixic acid and MYPG Guanin visualization method. Dissection method was Agar (Mueller-Hinton broth, yeast extract, potassi- applied due to OIE manual. Adult bees were secured um phosphate, glucose and pyruvate). Cultures were on their backs. Their heads and forelegs were remo- incubated at 37 0C for 48 hours. Colony morphology ved and the prothoracic circle was cut in front of the was examined and biochemical analyses were run for middle pair of legs with razorblade. Thin disks were identification of AFB and EFB pathogens. All bacte- heated in a solution of 8% potassium hydroxide for rial cultures were investigated under light microscope clearing the muscle tissue. The first pair of tracheae by using Gram Staining Method (Hornitzky and Wil- was examined under microscope. Thin layer chroma- son, 1989, OIE Manuel, 2000, Shimanuki and Knox, tography technique was used in Guanin visualization 2000). method. In this method 5 adult bee thoraxes were kept in 0,1M sodium hydroxide solution to clean and soften Statistical Analyses tissues for homogenization. Homogenized thorax tis- All collected data were analyzed by ANOVA and chi- sues were centrifugated at 16.000 rpm for 20 minutes. square tests statistically. Supernatants were carried over to new tubes and cent- rifugated at 16.000 rpm for 2 minutes to clean tissues properly. Samples and Guanine marker (Sigma) were Results loaded to thin layer and the layer was put in to run- All honey bee samples were investigated for adult ning buffer (5% Ammonium sulfate, 13M Ammonia and brood honey bee diseases. Varroa destructor was an 1-Propanole in the ratio of 60:30:10). After running observed in all samples. Varroa mites were found on the procedure thin layers were examined under UV adult bees, pupae in brood combs and debris. Cont- (Koch and Gerson, 1997, OIE Manual, 2000). rary to Varroa mite, tracheal mite Acarapis woodi was For the detection of Nosemosis, at least 30 honeybee not found in any sample (Table 2). samples were examined from each colony. Abdomens Nosema spores were found in all adult honey bee of honeybees were cut by scissors. All abdomens were samples. Nosemosis levels have been graded accor- homogenized by mortar and pestle. Homogenates were ding to Nosema spp. spore numbers. Infection levels diluted with 10 ml of saline solution. The solution was were classified as “low” between 0-500.000 spores, filtered through cotton fabric. The homogenates were “medium” between 500.000-1.000.000 spores and centrifuged for 10 minutes at 800 g. The supernatants “high” more than 1.000.000 spores. High Nosemosis were poured out. Saline solution was added 1 ml per level rate was 12,97%, medium level was 35,67% and bee. Final homogenate was mixed properly by vortex. low level was 51,36% for all samples. All results are 0.1 ml of the solution was put on heamocytometer and presented in Table 3-6 and Figure 1. Nosema spp. spores were counted under the light mic- Foulbrood diseases were detected 48% percent of all roscope. The number of Nosema spores per bee was samples. All results are presented in Table 7-8 and Fi- calculated. Totally 9 squares were counted for spores gure 2-4. (Shimanuki and Knox, 2000; Yalçınkaya et al., 2009). Spore count formula: 27

Table 1. The number of adult and brood honeybee Table 5. Comparison of Nosema spp. spore density in samples collected from Adana and Hatay provinces Adana and Hatay provinces in Spring. Sample Number/ Sample Number/ Avarage Prov- Hive Fall Spring Spore sd df P ince Number Adult 1600 1920 Number ADANA Brood 40 48 Adana 54 408518,52 263219,60 Adult 1720 2160 100 0,003 HATAY Hatay 48 568541,67 257243,90 Brood 43 54 sd: Standard deviation df: degrees of freedom P<0,05: Difference between provinces is significant. Table 2. Mite infestation results Sample Number/ Sample Number/ Provinces Fall Spring

Varroa Adana 100% 100% destructor Hatay 100% 100% Table 6. Comparison of Nosema spp. spore density in Adana and Hatay provinces in Fall. Acarapis Adana 0% 0% woodi Hatay 0% 0% Avarage Prov- Hive Spore sd df P ince Number Number

Table 3. Comparison of Nosema spp. spore density in Adana 43 617209,30 349157,66 two seasons in Adana Province 81 0,138 Avarage Hive Hatay 40 749250,00 451319,36 Season Spore sd df P Number Number sd: Standard deviation df: degrees of freedom

Spring 54 408518,52 263219,60 P>0,05: Difference between provinces is not significant. 95 0,001 Fall 43 617209,30 349157,66 Table 7. Comparison of foulbrood diseases percenta- sd: Standard deviation df: degrees of freedom P<0,05: Difference between seasons is significant. ges in provinces chi-square P Spring 0,228 0,892* Table 4. Comparison of Nosema spp. spore density in Fall 1,981 0,371** two seasons in *P>0,05 Difference between Adana and Hatay is not signi- Avarage Hive ficant in Spring. Season Spore sd df P Number **P>0,05 Difference between Adana and Hatay is not sig- Number nificant in Fall. Spring 48 568541,67 257243,90 59,365 0,028 Fall 40 749250,00 451319,36 sd:Standard deviation df:degrees of freedom P<0,05: Difference between seasons is significant. MELLIFERA 28

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 29

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

Table 8. Comparison of foulbrood diseases percenta- Varroa mite to bees and these pathogens affect colony ges in seasons directly. In this study, viruses were not directly exa- chi-square P mined but the relationship between Varroa mite and Adana 7,810 0,020* viruses is obvious (Boecking and Genersch, 2008, Hatay 2,228 0,328** Bogdanov, 2006, Wallner, 1999). Total 8,223 0,016*** The other honey bee mite Acarapis woodi was not de- *P<0,05 Difference between Fall and Spring is significant tected in any of the samples. In Turkey there are a few for Adana province studies about Acarapis woodi (Çakmak et al., 2003, **P>0,05 Difference between Fall and Spring is not signifi- Keskin and Başer, 1996). Previous studies declared cant for Hatay province that Acarapis woodi was found in Turkey by Guanine ***P<0,05 Difference between Fall and Spring is signifi- visualization with thin layer chromatography method cant for all samples. (Özkırım and Keskin, 2005a). Therefore, honey bees should be investigated for tracheal mite infestation as Discussion a precaution and wintering locations should be kept Severe colony losses have been occurring since the under control for possible infestations. year of 2006 in the world. There are many factors that affect colony collapse. Pathogens and pesticides are Nosemosis was one of the most common honey bee major effects of these losses (vanEngelsdorp and Me- diseases in Adana and Hatay provinces. Nosema spp. ixner, 2010). spores were found in all samples. Nosemosis level was graded according to spore numbers. Infection The results of our study showed that all apiaries were level was classified as high infection over 1.000.000 infested with Varroa destructor. Beekeepers notice spores. There was no significant difference between the Varroa mite clearly, but using excessive and imp- the towns studied. Comparison for provinces revea- roper use of drugs leads to the formation of high che- led that there was no significant difference between mical resistance. Reduced life span of Varroa infes- Adana and Hatay provinces for fall season, but dif- ted honey bees causes colony collapse. On the other ference in spring was significant (Table 5-6). When hand viruses, bacteria and fungi are transported by all results were compared in terms of seasons, infec- MELLIFERA 30

tion level was found higher in fall (Table 3-4). The This research showed that honey bee diseases consti- number of young honey bees is less in fall than spring tute a very important part of factors that cause colony and decrease of air temperature and increase of rain losses. On the other hand there are a lot of factors be- fall cause decline in resistance to Nosema diseases. sides diseases. The combination of diseases with other Honey bees also remain close into the hive, in rainy factors raises colony losses. Consequently, knowledge days more than sunny days. This situation constitutes of the biology and epidemiology of these infections is convenient environment for Nosema spp. infection. urgently needed for prevention of diseases outbreaks Also proflactive and/or over use of Fumagillin effects and colony losses. colony. In this study each colony was found infected with Nosema spores. This situation indicates how contagious and infective Nosema spp. spores are. In Acknowledgement order to prevent losses caused by Nosemosis, drugs This study was financially supported by Hacettepe should be used in correct manner according to pros- University (Project Number: 06D09601001). Special pectus advice (Fries, 1993, Hornitzky, 2005, Wyborn thanks to Dr. Aslı ÖZKIRIM for her contributions to and Mccutchan, 1987). this study. We are also grateful for supports of Hatay and Adana Beekeeping Associations. Foulbrood diseases were found in very high rates. In Adana province, infected colony rate was found 37% References in spring and 64,1% in fall (Figure 2). In Hatay pro- Ashiralieva A., Genersch E., 2006. Reclassification, geno- vince, infected colony rate was found 41,7% in spring types and virulence of Paenibacillus larvae, the and 52,5% in fall (Figure 3). When the provinces were etiological agent of American foulbrood in honey- compared, the difference was not found significant bees-a review. Apidologie, 37, 411–420. for foulbrood diseases (Table 7). This result was ex- Bailey L.,1983. Melissococcus pluton, the cause of Europe- pected because these two neighbour provinces are an foulbrood of honey bees (Apis spp.). Journal of located in the same geographical region and climatic Applied Bacteriology 55, 65-69. zone. However, disease level comparison for seasons Boecking O., Genersch E., 2008. Varroosis – the Ongoing represented significant difference (Table 8). In spring, Crisis in Bee Keeping. Journal für Verbrauch- foulbrood diseases were found in lower rates. Since, erschutz und Lebensmittelsicherheit, 3:2, 221-228 nectar flow increases in spring season, climatic con- Bogdanov S., 2006. Contaminants of bee products. Apidolo- ditions change for the better and colonies gain power. gie, 37, 1–18. Also beekeepers are more careful for colony mainte- Bowen-Walker P. L., Gunn A., 2001. The effect of the ec- nance with the higher honey yield expectation from toparasitic mite, Varroa destructor on adult work- the new season. Conversely, colonies have less power er honeybee (Apis mellifera) emergence weights, in the fall, flight time decreases and maintenance is water, protein, carbohydrate, and lipid levels. Entomologia Experimentalis et Applicata, 101, not always enough. Results indicated that American 207–217. Foulbrood was more prevalent than European Foulb- rood in both seasons. Paenibacillus larvae (the causal Chen Y., Evans J., Feldlaufer M., 2006. Horizontal and verti- cal transmission of viruses in the honey bee (Apis agent of AFB), is more resistant than Melissococcus mellifera). Journal of Invertebrate Pathology, 92, pluton (the causal agent of EFB). Paenibacillus larvae 152–159. spores could stay infective more than decades and it is Çakmak İ., Aydin L., Gulegen E., Wells H., 2003. Varroa known that 10 bacteria per larvae are enough for in- (Varroa destructor) and tracheal mite (Acarapis fection (Ashiralieva and Genersch, 2006). AFB, could woodi) incidence in the Republic of Turkey. Jour- transmit vertically and horizontally between bees, co- nal of Apicultural Research 42:4, 57–60 lonies and apiaries. Moreover prepared comb founda- Duay P., De Jong D., Engels W., 2003. Weight loss in drone tions and beekeeping equipment could carry P. larvae pupae (Apis mellifera) multiply infested by Varroa spores for long terms. (Bogdanov, 2006, Fries, 2006, destructor mites. Apidologie, 34, 61–65. Özkırım and Keskin, 2005b). 31

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