Citation/ Atıf: Usta, M. 2021. Determination of Honey Bee (Apis Mellifera) Bacterial Flora, Cry Gene Analysis and Honey Bee Health, (Bal Arısı (Apis mellifera) Bakteri Florasının Belirlenmesi, Cry Geni Analizi ve Bal Arısı Sağlığı). U. Arı. D./U. Bee J. 21(2):157-167, DOI: 10.31467/uluaricilik.954479 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE DETERMINATION OF HONEY BEE (Apis mellifera) BACTERIAL FLORA, CRY GENE ANALYSIS AND HONEY BEE HEALTH Bal Arısı (Apis mellifera) Bakteri Florasının Belirlenmesi, Cry Geni Analizi ve Bal Arısı Sağlığı Mehtap USTA Trabzon University, Tonya Vocational School, Tonya, Trabzon, TURKEY, ORCID No: 0000-0001-7656-5655 Yazışma Yazarı/Corresponding author E-mail: mehtapyakupoglu@trabzon.edu.tr Geliş Tarihi / Received: 18.06.2021 Kabul Tarihi / Accepted: 06.09.2021 DOI: 10.31467/uluaricilik.954479 ABSTRACT Beekeeping provides important contributions to the agricultural economy and crop production through pollination both in Turkey and the world. It is evaluated that without bees, the plant production can decrease by 47%. Many factors affect honey production negatively. Among these reasons, besides diseases caused by microorganisms, diseases and dangers originating from organisms are at the forefront. Today, many methods are used in the control these pests and yet they are still unavoidable. Among these methods, the biological control method is not used commonly. The aim of the study is to create a basis for the development of biopesticides to control bee diseases. In this context, as a result of the study, 16 bacteria were isolated from honey bees. While, 12 bacteria belonging to the genus Bacillus, two bacteria belonging to the genus Lysinibacillus, one bacterium belonging to the genus Paenibacillus and one bacterium belonging to the genus Pantoea were obtained. Molecular and biochemical identifications of these bacteria were done and registered in GenBank and their accession numbers were obtained. cry gene analyzes of 15 bacteria belonging to the genus Bacillus were performed. As it is known, cry genes have the potential to be used against pests. In the future, these bacteria and their genes will have the potential to be used as biopesticides. According to these results, the cry1 gene was observed in 8 bacteria and the cry3 gene was observed in 3 bacteria. cry2 and cry4 genes could not be detected in these bacteria. Bacteria that including cry genes are of great importance for honey bee health. Bacteria have the potential to be developed as internal biopesticides and used against different bee diseases to improve honey bee health. Keywords: Apis mellifera, Bacteria, Microbiology, Honey bee health, cry genes ÖZ Arıcılık gerek Türkiye’de gerekse dünyada tarım ekonomisine ve tozlaşma yoluyla bitkisel üretime önemli katkılar sağlar. Arıların olmadığı bir ortamda bitkisel üretimin %47 oranında azalabileceği değerlendirilmektedir. Arıcılık sektöründe birçok etken de bal üretimini olumsuz yönde etkilemektedir. Bu sebepler arasında mikroorganizma sebepli hastalıkların yanı sıra, organizma kaynaklı hastalıklar ve tehlikeler de ön sıralarda yer almaktadır. Günümüzde bu zararlılarla mücadelede birçok yöntem kullanılmakta olup halen önüne geçilememiş durumdadır. Bu yöntemler arasında biyolojik mücadele yöntemi kullanılmamaktadır. Buradan yola çıkarak, çalışmanın amacı, bal arılarının sağlığını korumak için biyolojik bir etmen kullanılarak hastalık ve zararlı organizmalarla mücadele konusunda biyopestisit geliştirilmesinde taban oluşturmaktır. Bu bağlamda çalışma sonucunda bal arılarından 16 adet bakteri izolasyonu gerçekleştirilmiştir. Elde edilen bakterilerden 12 tanesi Bacillus cinsine, iki tanesi Lysinibacillus cinsine, bir tanesi Paenibacillus cinsine ait iken bir tanesi de Pantoea cinsine aittir. Bu bakterilerin moleküler ve biyokimyasal tanımlamaları yapılarak GenBank a kayıt yaptırılmış ve kayıt 157 U.Arı D. – U.Bee J. 2021, 21 (2): 157-167 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE numaraları alınmıştır. On beş adet Bacillus, Paenibacillus ve Lysinibacillus cinslerine ait bakterinin cry gen analizleri yapılmıştır. Bilindiği üzere cry genleri hem zararlılara karşı kullanılma potansiyeline sahiptir hem de ileride bu bakteri ve genleri geliştirilerek biyopestisit kullanılma potansiyeli olabilecektir. Bu sonuçlara göre cry1 geni 8 bakteride ve cry3 geni de 3 bakteride gözlemlenmiştir. cry2 ve cry4 genleri bu bakterilerde tespit edilememiştir. Bu genleri taşıyan bakteriler bal arısı sağlığı açısından büyük önem taşımaktadır. Bakteriler biyopestisit olarak geliştirilerek başta organizma gibi zaralılar olmak üzere farklı arı hastalıklarına karşı kullanılma potansiyeline sahiptirler. Anahtar Kelimeler: Apis mellifera, Bakteri, Mikrobiyoloji, Bal arısı sağlığı, cry genleri GENİŞLETİLMİŞ ÖZET Lysinibacillus cinsine, bir tanesi Paenibacillus cinsine ait iken bir tanesi de Pantoea cinsine aittir. Amaç: Bal arıları ekonomik ve biyolojik yönden Bu bakterilerin moleküler ve biyokimyasal oldukça önemlidir. Bal arıları bal, propolis, arı sütü, tanımlamaları yapılarak GenBank a kayıt yaptırılmış polen, bal mumu, arı zehri gibi arı ürünleri sayesinde ve kayıt numaraları alınmıştır. On beş adet Bacillus, dünya pazarında önemli yer almaktadır. Arıcılık, Paenibacillus ve Lysinibacillus cinslerine ait Dünya’nın hemen hemen her yerinde yapılan bakterinin cry gen analizleri yapılmıştır. Bilindiği tarımsal bir faaliyettir. Arıcılık faaliyetlerini üzere cry genleri hem zararlılara karşı kullanılma engelleyen en önemli nedenlerden biri de arı zararlı potansiyeline sahiptir hem de ileride bu bakteri ve ve hastalıklarıdır. Bu nedenle de arı sağlığını genleri geliştirilerek biyopestisit kullanılma korumak büyük önem arz etmektedir. Günümüzde potansiyeli olabilecektir. Bu sonuçlara göre cry1 geni bu zararlılarla mücadelede birçok yöntem 8 bakteride ve cry3 geni de 3 bakteride kullanılmakta olup halen önüne geçilememiş gözlemlenmiştir. cry2 ve cry4 genleri bu bakterilerde durumdadır. Bu yöntemler arasında biyolojik tespit edilememiştir. mücadele yöntemi kullanılmamaktadır. Çalışmanın amacı, bal arılarının sağlığını korumak için biyolojik bir etmen kullanılarak hastalık ve zararlı INTRODUCTION organizmalarla mücadele konusunda biyopestisit geliştirilmesinde taban oluşturmaktır. According to the 2020 data of the Ministry of Food, Agriculture and Livestock, Directorate of Beekeeping Yöntem: Çalışma için gerekli olan bal arısı örnekleri Research Institute, Turkey ranks at the forefront of Gümüşhane ili Kürtün ilçesinden elde edilmiştir. the beekeeping sector in the world with an average İlçedeki belirlenen sağlıklı arılık ve kovanlardan of 8 million 128 thousand hives and 109,330 tons of ortalama 20 şer arı toplanmıştır. Bu arılar steril honey production. Beekeeping provides important taşıma kaplarına alınarak laboratuvar ortamına contributions to the agricultural economy and crop getirilmiştir. Laboratuvara getirilen arı örnekleri production through pollination both in Turkey and in öncelikle %70’lik alkol ile yüzey strelizasyonu the world. So honey bees (Apis mellifera) play a very sağlanmıştır. Yüzey sterilizasyonunun ardından important role in pollination in natural ecosystem and 500µl Nutrient broth besiyeri içerisinde arıların agricultural field (Evans et al. 2010). In recent years, ezilerek parçalanması sağlanmıştır. Artık parçaları bee population and colony losses have increased in ortamdan uzaklaştırmak için steril filtre ile süzme the world. Pathogens (parasites, fungi, viruses and işlemi gerçekleştirilip, geri kalan sıvı Nutrient agar bacteria) and abiotic stress factors can adversely besiyerine ekim yapılarak 30°C’ de 2-3 gün affect colony health. All these factors are affecting inkübasyona bırakılmıştır. Bu süre sonunda the bee ecosystem (Brutscher et al. 2015, Li et al. besiyerinde oluşan bakterilerin saflaştırılması için 2018, Larsen et al. 2019). Over time, insects have ayrı ayrı besiyerilerine ekimleri yapılmıştır. Bu developed a strong and effective immune system. bakterilerin tanımlanması için moleküler The immune system of insects combats various karakterizayonu, biyokimyasal testleri, fiziksel ve pathogens and conseuqently has become one of the morfolojik analizleri yapılarak belirlenmiştir ve most diverse and successful immune systems in the bakteriler GenBank’a kayıt ettirilmiştir. world. Insects defense mechanisms include cellular Sonuç: Çalışma sonucunda bal arılarından 16 adet and humoral immunity (Evans and Armstrong 2005, bakteri izolasyonu gerçekleştirilmiştir. Elde edilen Schmid et al. 2008, Wilson-Rich et al. 2008). bakterilerden 12 tanesi Bacillus cinsine, iki tanesi Uludağ Arıcılık Dergisi – Uludag Bee Journal 2021, 21 (2): 157-167 158 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE Epidemic infection risks in honey bee colonies is proteins produced during spore formation constitute reduced by individual and social immunity (Ilyasov et approximately 30% of the total protein content of the al. 2012). Both the type of immunities together at bacteria (Höfte and Whiteley, 1989; Aronson, 1993). various levels protects the bees from diseases and ICPs are found undissolved under normal ectoparasites (DeGrandi-Hoffman and Chen 2015, conditions. Therefore, they do not pose a risk to Larsen et al. 2019). humans and other vertebrate organisms. On the other hand, their soluble properties at pH 9.5. Thus, Pathogens, acaricides, fungicides, herbicides and the ICPs in the structure of this protein dissolve in other insecticides affect the bee immune system and the insect gut and turn into protoxin. Then, protoxins thus affects bee health (Ilyasov et al. 2021). The are broken down by intestinal enzymes and active social immune defense strategy, individual bees, toxins are formed. Active toxins cling to the receptors reduces the pressure on the immune system (Larsen of intestinal epithelial cells, paralyze the intestinal et al. 2019). wall of the insect and destroy it, forming pores. The Like every living creature, honey bees are also have poisoned insect can die immediately due to toxin a microflora (Gilliam 1997, Olofsson and Vásquez activity and die as a result of blood poisoning within 2008). These bacteria can play a positive or negative 2-3 days (Knowles, 1994). In this context, cry gene role on honey bee health and it is also important of analyses were performed because 15 of the bacteria rbutrient aquisition from a healthy diet (Evans and obtained belonged to the genus Bacillus. These Spivak 2010). Bacterial microflora in bees, can bacteria will pave the way for developing inhibit lime disease (Reynaldi et al. 2004) and biopesticides for organisms and microorganisms American foulbrood (Evans and Armstrong 2005, that affect bee health. The ground will be prepared Evans et al. 2006). With a healthy microbiome, the for development for different agricultural or forest reproduction of beneficial microorganisms is pests. In this context, it is aimed to obtain bacterial supported and the reproduction of pathogens is flora by collecting healthy honey bees. Bacteria prevented. (Evans et al. 2006). Honey bees both at obtained from bacterial flora will have the potential to the individual and colony level is capable of immunity be used against organisms that cause harm to bees. defense. Beekeepers use antibiotics to control pathogens and parasites, so pesticide applications are done frequently. This situation results in residues MATERIAL AND METHODS in hive products and hive equipment leading to an Sampling increased antibiotic resistance problem. Honey bee samples used in this study were collected One of the biggest problems in beekeeping is from hives where are located in Gümüşhane attempting to treat honeybee diseases with chemical province, distinct from Kürtün (It is located at latitude treatments. Limited success is achieved after 40.748302 and longitude 38.984703). No chemical chemical treatment, and there are problems, such as application has been made on the hives that a danger to human health with chemical residues in samples were taken. The collected Apis mellifera the honey (Barganska et al. 2011). Problems are were placed in sterile tubes and brought to the experienced in export markets for honey from laboratory. The samples were collected twice a year, treated bees, so attempts are made to sell this honey in the spring (June) and autumn (September) of in the domestic market. As a result, the products 2020. cannot be sold easily and at their proper value. For this reason, biological control methods gain Isolation of Bacteria importance. Generally, for biological control Bacillus Before bacterial isolation, honey bees were surface genus bacteria are used. B. thuringiensis, which was sterilized with 70% alcohol to remove possible first isolated from diseased silkworm larvae by contamination and then washed in sterile distilled Japanese researcher Ishiwata (Ishiwata, 1901) in water. The bee bodies were homogenized in 0.5 ml 1901, is the most widely used microbial control agent of sterilized Nutrient Broth (NB) using a glass tissue (Lacey et al. 2001). The insecticidal activity of B. grinder and filtered. After preparing the homogenate thuringiensis is carried out by toxins in protein for bacterial isolation, suspensions were diluted to structure called insecticidal crystal proteins (ICP, cry 1×10-5 (Thiery and Frachon 1997) and 0.1 ml were proteins). These proteins are encoded by the genes spread on nutrient agar (Thiery and Frachon 1997). (cry) located on the plasmids. These insecticidal Plates were incubated at 30°C for 2-3 days. Isolates 159 U.Arı D. – U.Bee J. 2021, 21 (2): 157-167 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE were determined based on the color and morphology consisting of denaturation at 94°C for 3 min, of the colonies. Individual colonies were isolated, annealing at 55°C for 60 s and extension at 72°C for sub-cultured twice to ensure purity and then stored 3 min), followed by a final extension step at 72°C for in 15% sterilized glycerol at -80°C for further studies. 5 min, by using a thermal cycler (BioRad). Each Pure cultures of bacterial colonies were identified by experiment was associated with negative (without their morphology, biochemical, physiological and DNA template) controls. PCR products were molecular characteristics (16S rRNA). analyzed on a 1.2% agarose gel. Sequence analysis of 16S rRNA products samples were Morphological, Physiological and Biochemical performed using 16S universal primers by Characterisations of Isolates SenteBiolab (Ankara/TURKEY). Using the NCBI Bacterial strains were selected based on GenBank database, determined sequences were morphological biochemical, physiological features used to perform BLAST searches (Altschul et al. according to Bergey’s Manual of Systematic 1990). Comparison of approximately 1.400 bp Bacteriology (Sneath et al. 1986). Phenotypic fragments of 16S rRNA gene sequences of each characteristics of the strains include cell and colony isolate with other 16S rRNA sequences in the NCBI shape on NA. Optimum pH was determined, after 16 GenBank database (Altschul et al. 1990) were h incubation at 30°C by measuring the densities performed. using a spectrophotometer (Spectramax M2) at Optimum pH Range of All Bacteria OD600 (Ben-Dov et al. 1995). Biochemical panel test system API 20E (bioMerieux, France) was In order to determine the pH range in which the handled according to the manufacturer’s bacteria grows optimally, media at different pH instructions. Then the panels were incubated for 18- values (pH3, pH4, pH5, pH6, pH7, pH8, pH9 and 24 h at 30°C. The results of the tests were performed pH10) were prepared and incubated at 30°C for 24 by referring to the API 20E reading table. hours. 16S rRNA Gene Sequence Analysis Determination of cry Genes of Bacteria Genomic DNA from all samples was extracted by PCR amplification of cry genes was performed of the phenol/chloroform procedures (Sambrook et al. toxin genes using cry1, cry2, cry3 and cry4 primers 1989). PCR amplification of 16S rRNA genes of (Table 1). PCR was constructed according to the bacterial isolates was performed with the following following conditions: pre-amplification 94°C for 3 min universal primers (William et al. 1991); UNI 16S-L: followed by 30 cycles of denaturation at 94°C for 30 5_-ATTCTAGAGTTTGATCATGGCTCA-3_ as s, annealing at 52-59°C for 60 s, and elongation at forward and UNI 16S-R: 72°C for 3 min. Reactions were totally in 50 μl; 1 μl 5_ATGGTACCGTGTGTGACGGGCGGTG TGTA- of template DNA was mixed with 5 μl reaction buffer, 3_ as the reverse. PCR conditions were adjusted 0.2 mM of each deoxynucleoside triphosphate according to William et al. (1991). Reactions were (dNTP), 0.5 μM (each) with primer and 0.5 U with totally in 50 μl; 1 μl of template DNA was mixed with Taq DNA polymerase. PCR products were analyzed 5 μl reaction buffer, 0.2 mM of each deoxynucleoside on a 1% agarose gel. triphosphate (dNTP), 0.5 μM (each) with primer and 0.5 U with Taq DNA polymerase. Amplification was performed with 30 cycle program (each cycle Table 1. Cry gene primers Primer Primer sequence Sequence lenght Tm (°C) Name (PCR amplification) cry1Fw CATGATTCATGCGGCAGATAAC cry1Rv TTGTGACACTTCTGCTTCCCATT 277bp 55 cry2Fw GTTATTCTTAATGCAGATGAATGGG cry2Rv CGGATAAAATAATCTGGGAAATAGT 701bp 52 cry3Fw CGTTATCGCAGAGAGATGACATTAAC cry3Rv CATCTGTTGTTTCTGGAGGCAAT 604bp 54 cry4Fw GCATATGATGTAGCGAAACAAGCC cry4Rv GCGTGACATACCCATTTCCAGGTCC 439bp 59 Uludağ Arıcılık Dergisi – Uludag Bee Journal 2021, 21 (2): 157-167 160 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE RESULTS flexus (GAP14), Bacillus paramycoides (GAP15) and Lysinibacillus sphaericus (GAP16) from Apis In this study, 16 bacterial isolates; Bacillus cereus mellifera were isolated and characterized. The (GAP1), Bacillus subtilis (GAP2), Bacillus optimum pH range in which bacteria can grow was wiedmannii (GAP3), Bacillus megaterium (GAP4), also determined (Table 2). When the pH results of Pantoea rodasii (GAP5), Bacillus nakamurai the bacteria obtained in the study in Genbank are (GAP6), Bacillus mobilis (GAP7), Bacillus pacificus examined, it is seen that they confirm each other (GAP8), Bacillus thuringiensis (GAP9), (https://www.ncbi.nlm.nih.gov/genbank/).These Lysinibacillus fusiformis (GAP10), Bacillus results are used to confirm the accuracy of the vallismontis (GAP11), Paenibacillus odorifer bacteria. (GAP12), Bacillus velezensis (GAP13), Bacillus Table 2. Optimum pH range of bacteria. pH GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 pH3 - - - - - - - - - - - - - - - - pH4 - - - - - - - - - - - - - - - - pH5 - - - + + - - - - - - - - - - - pH6 + + + + + + + + + + + + + + + + pH7 + + + + + + + + ++ + + + + + + + pH8 + + + + + + + + + + + + + + + + pH9 + + + + + + + + + + + + + + + + pH10 + + + + + + + + + + + + + + + + Some biochemical characteristics (API20E) of including which growth medium is suitable for their bacterial isolates are summarized in Table 3, growth. Table 3: Biochemical characteristics of bacteria (API20E). API20E GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP GAP Tests 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 GEL - - + + - + - - + + - + - - + - GLU + - + - - - + - + - - - + - - - MAN + - - - - - + - - - - - + - - - INO + - - - - - + - - - - - + - - - SOR - - - - - - - - - - - - - - - - RHA + - - - - - + - - - - - + - - - SAC + - + - - - + - + - - - + - - - MEL + - - - + - + - - - + - + - - + AMY + - + - - - + - + - - - + - - - ARA + - - - - - + - - - - - + - - - ONPG + - - - - - + - - - - - + - - - ADH - - - + + - - - - + + - - - + + LDC - - - - + - - - - - + - - - - + ODC - - - - - - - - - - - - - - - - CIT + - - - + - + - - - + - + - - + H2S - - - - - - - - - - - - - - - - URE - - - - + - - - - - + - - - - + TDA - - - - + - - - - - + - - - - + IND - - - - - - - - - - - - - - - - VP + - - - - - + - - - - - + - - - 161 U.Arı D. – U.Bee J. 2021, 21 (2): 157-167 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE When the biochemical test results of the bacteria As seen in Figure 1, cry gene analyzes were made obtained in the study in Genbank are examined, it is and cry 1 gene was only found in 8 bacteria, and cry seen that they confirm each other 3 gene in 3 bacteria. cry 2 and cry 4 genes were not (https://www.ncbi.nlm.nih.gov/genbank/).These observed in any bacteria. cry 1 gene has 277 bp and results are used to confirm the accuracy of the cry 3 gene has 604 bp. In the first lane (M) there is bacteria. marker (1kb) and the bands are 250 bp, 500 bp, 750 bp, 1000bp, 1500 bp, 2000 bp and 2500 bp, 16S rRNA gene sequence analysis results of respectively (from the bottom to top). The bands isolates are given in Table 4. The 16S rRNA partial under the marker bands, which are seen in all four gene sequences generated in this study have gel images (A, B, C and D), are the bands belonging accession numbers MZ097346, MZ097347, to the primers, and their appearance indicates the MZ097348, MZ097349, MZ097350, MZ097351, quality of the gel. According to these results and MZ097352, MZ097353, MZ097354, MZ097355, looking at the marker, the bands corresponding to MZ097356, MZ097357, MZ097358, MZ097359, around 250 bp in A shows the presence of cry1 MZ097360 and MZ097361, respectively. In the gene. According to the results, bands seen around Table 4, the isolate name is the code that we gave 500 bp in the B also indicates the presence of cry3 in this study, the bacterium name is from the 16S gene. In addition, one sample of each of these bands identifiation, accession numbers are provided from was sent to the sequence and were confirmed. On GenBank and the similarity score is presented from the other hand, the bands seen in the gel images of GenBank for each strain identification. cry 2 and cry 4 genes belong to the primers. As can be seen, these bands are below the marker bands. Table 4. 16S results of bacteria. Isolate name Bacterium name Accession number Similarity GAP1 Bacillus cereus MZ097346 99% GAP2 Bacillus subtilis MZ097347 99% GAP3 Bacillus wiedmannii MZ097348 99% GAP4 Bacillus megaterium MZ097349 99% GAP5 Pantoea rodasii MZ097350 99% GAP6 Bacillus nakamurai MZ097351 99% GAP7 Bacillus mobilis MZ097352 99% GAP8 Bacillus pacificus MZ097353 99% GAP9 Bacillus thuringiensis MZ097354 99% GAP10 Lysinibacillus fusiformis MZ097355 99% GAP11 Bacillus vallismontis MZ097356 99% GAP12 Paenibacillus odorifer MZ097357 99% GAP13 Bacillus velezensis MZ097358 99% GAP14 Bacillus flexus MZ097359 99% GAP15 Bacillus paramycoides MZ097360 99% GAP16 Lysinibacillus sphaericus MZ097361 99% Uludağ Arıcılık Dergisi – Uludag Bee Journal 2021, 21 (2): 157-167 162 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE M M A B M M C D Figure 1: cry gene analysis of bacteria. A: cry 1 gene PCR results, B: cry 3 gene PCR results, C: cry 2 gene PCR results, D: cry 4 gene PCR results. DISCUSSION each other according to their oxygen needs. Species belonging to the genus Bacillus are aerobic or As a result of the study, 12 bacteria belonging to the facultative anaerobic, while species belonging to the genus Bacillus, two bacteria belonging to the genus genus Clostridium are anaerobic. Both genera have Lysinibacillus, one bacterium belonging to the genus rod-shaped cells that form chains (Tanada and Kaya Paenibacillus and one bacterium belonging to the 1993). Insect pathogens known in the genus genus Pantoea were obtained. Pantoea species, Clostridium reproduce only in the insect gut and which are common in plants and soil, belong to the cause disease and never pass into insect hemocele. Enterobacteriaceae family. Pantoea species are The genus Bacillus contains important insect members of the normal flora of the human pathogenic species. The most important of these is gastrointestinal tract and can be found in water, Bacillus thuringiensis bacteria, which is in the wastewater, soil, plants and foods such as Bacillus cereus group. B. thuringiensis is a spore- fruit/vegetables. Many Pantoea species are known forming soil bacterium that produces toxin in as plant disease agents and are used as crystalline structure and has an insecticidal effect biopesticides in the agricultural industry (Cruz et al. mostly against insects in Lepidoptera, Diptera and 2007). Pantoea species, known as plant pathogens, Coleoptera groups (Beegle and Yamamoto 1992). are microorganisms that develop following injuries According to studies conducted in recent years, it with plant spines, and the most frequently isolated has been found to be lethal among the insect groups species is Pantoea rodasii, which was also identified of Hymenoptera, Homoptera, Orthoptera and from the GAP5 isolate (Kurşun et al. 2012). Mallophaga, as well as on nematodes, ticks and The Paenibacillus genus includes some strains protozoa (Feitelson et al. 1992, Feitelson 1993). The previously found in the Bacillus and Clostridium fact that insecticidal products obtained from B. genera. Paenibacillus odorifer bacteria identified in thuringiensis bacteria do not cause infection on GAP12 isolate are found in soil, water and various humans, non-target organisms and beneficial foods (Beno et al. 2020). insects has increased the effective use of these products in the control harmful insects (Lacey et al. Members of the Bacillaceae family, which are 2001, Seigel 2001). B. thuringiensis-derived important in terms of biological control, are Gram- products constitute 95% of the world biopesticide positive, motile or non-motile rod-shaped bacteria market. Many commercial companies have that produce endospores. Bacillus and Clostridium introduced products from B. thuringiensis. According genera in this family contain important insect to 1998 figures, more than 200 products of B. pathogen species and are mostly separated from thuringiensis origin are used against pests only in the 163 U.Arı D. – U.Bee J. 2021, 21 (2): 157-167 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE USA (Schnepf et al. 1998). In addition, many B. resistance against other bee pathogens (Forsgren et thuringiensis-derived products are susceptible to al. 2010). synthetic chemical pesticides, obtained at a lower In this study, it was aimed to determine the general cost. Some other species belonging to the genus bacterial flora of honey bees and the optimal growth Bacillus are also used in the control of harmful conditions. We found that bacteria can be partially insects. Bacillus popilliae (Dutky) is used in the detected by using selective growth media. control of some species belonging to the Scarabaeidae family, while Bacillus sphaericus Both similar and different bacteria were obtained Neide is used in the control of mosquito larvae (Klein previously from the honey bee intestinal flora, so we and Jackson 1992). B. popilliae needs to be suspect that there could be regional differences of produced in vivo, and lower than expected levels of honey bee gut bacteria based on their floral diet infection in many field applications reduces the selection (Yarılgaç, 2016). For future research, we potential of this bacterium to be used in large areas aim to obtain and characterize local isolates to (Klein and Kaya 1995). Compared to B. determine their biological significance in terms of thuringiensis, B. sphaericus is resistant to polluted improving honey bee health. habitats and environmentally friendly. Although it is Conclusion more resistant to various factors, its biggest disadvantage is that the host spectrum is very As a result, considering the definitions of the bacteria narrow (Lacey and Undeen 1986, Charles et al. obtained in the study, 8 bacteria with the cry 1 gene 1996, Nicolas et al. 1994). However, some fly and 3 bacteria with the cry 3 gene have the potential species are resistant to this bacterium. It has been to be used in future biopesticide development reported (Rao et al. 1995, Nielsen-Leroux et al. studies. GAP6 (Bacillus nakamurai) bacteria, in 1997). which both cry genes (cry 1 and cry 3) are common, should be studied in more detail. It is thought that it The most important factor in Bacillus species are may have the potential to be a more potent crystal (cry genes) genes. In the study, 16 Bacillus biopesticide with the activity of both cry genes. genus bacteria were obtained and cry gene analyzes were performed. In this context, the bacteria According to isolated bacteria and gene analyses, obtained are of great importance. According to the these bacteria should be tested primarily for use in results of cry gene analysis 8 bacteria have cry1 maintaining honey bee health. Previously, some of genes and 3 bacteria have cry3 genes were these bacteria have been effective for manageing obtained in bacteria. cry2 and cry4 genes could not Varroa mites and especially Galleria mellonella be obtained in these bacteria. The most common cry pests. In addition to honey bee pests, future gene types in nature are cry1 and cry3. According to research can be carried out to develop bacterial these results, the potential of using these bacteria biopesticides for different agricultural and forest against bee pests seems high. pests in general. For the honeybees to be healthy, under the chemical Financial Resource: Financial resource for this treatment practices, antibiotics are frequently used study was provided by Trabzon University Scientific against bacterial diseases (Mutinelli, 2003). Research Projects Coordination Unit. Antibiotic use weakens the immune system of the bees and leads to antibiotic-resistant bacterial Ethics Certificate: Ethics certificate is not required pathogens (Doğaroğlu and Samancı, 2006; for this study. Barganska et al. 2011). Unfortunately, the fight against these bacteria is self-defeating. In previous Acknowledgements studies, positive effects have been shown by the This work has been supported by Trabzon University resistance of the bacterial flora in the bodies of bees Scientific Research Projects Coordination Unit under against disease (Gilliam, 1997).Thus, the idea arises grant number was 21HZP00159. that if the naturally occurring microbial flora in the bodies of the bees are supported, the bees may be more resistant to disease (Tajabadi et al. 2013) In particular, the bacteria with probiotic properties found in the honey stomachs or intestines of honeybees have been observed to provide Uludağ Arıcılık Dergisi – Uludag Bee Journal 2021, 21 (2): 157-167 164 ARAŞTIRMA MAKALESİ / RESEARCH ARTICLE REFERENCES Curr Opin Insect Sci. 10:170–176., doi: 10.1016/j.cois.2015.05.007. Altschul, SF., Gish, W., Miller, W., Myers, EW., Lipman, DJ. (1990). Basic local alignment Doğaroğlu, M., Samancı, T. (2006). Balda Yorelere search tool. J Mol Biol. Oct 5;215(3):403-10. 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