Corrigendum: Detection of nosemosis in European honeybees (Apis mellifera) on honeybees farm at Kanchanaburi, Thailand (2019 IOP Conf. Ser.: Mater Sci Eng. 639 012048)

Abstract

Description of corrigendum e,g, Page 1: In the Abstract section, the following text appears: This study was aimed to the detection of Nosema in European honeybees at Kanchanaburi Province and Identify species of Nosema by Polymerase Chain Reaction technique. The ventriculus of bees was individually checked to verify the presence of Nosema spores under light microscope. The number of spores per bee were quantify on a haemocytometer for infectivity. It was studied for three periods of the year. The first period was studied between October 2015 to January 2016, the second period from February to May 2016 and the third period from June to September 2016. The results showed that the highest infection rate in June-September 2019 was 100 and the lowest infection rate was 40 in October 2015 and January 2019. The average number of Nosema spores infection level in an individual was infectivity. The highest infectivity was 2.47x106 spores/bee in February to May 2016 and the lowest infectivity was 0.04x106 spores/bee in October 2015 to January 2016. Nosema ceranae was found in this study by Polymerase Chain Reaction technique. This should read: This study was aimed to the detection of Nosema in European honeybees at Kanchanaburi Province and identify species of Nosema by Polymerase Chain Reaction technique. The ventriculus of bees was individually checked to verify the presence of Nosema spores under light microscope. The number of spores per bee were quantify on a haemocytometer for infectivity. It was studied for three periods of the year. The first period was studied between October 2015 to January 2016, the second period from February to May 2016 and the third period from June to September 2016. The results showed that the highest infection rate in June to September 2016 was 100 and the lowest infection rate was 40 in October 2015 to January 2016. The average number of Nosema spores infection level in an individual was infectivity. The highest infectivity was 2.47x106 spores/bee in February to May 2016 and the lowest infectivity was 0.04x106 spores/bee in October 2015 to January 2016. Nosema ceranae was found in this study by Polymerase Chain Reaction technique. Page 2: In the Materials and Method section, the following text appears: 2.1 DNA extraction, amplification and sequencing Genomic DNA was extracted from a whole Nosema spores from the A. mellifera colonies at apiaries of Wanna Bee Farm using DNA Extraction Kit (Qiagen) according to the manufacturer’s instructions. The 16S rRNA gene were amplified by polymerase chain reaction (PCR) using the following primers: Noce239L (5’–AGGGGCGAAACT TGACCTAT-3’) and Noce950R, (5’–GGGCATAACKG ACCTGTTTA-3’) [9]. PCR was carried out using 20 μl volumes containing 5 units of Taq DNA polymerase (biotechrabbit™), 2 μl of 10X PCR reaction buffer, 0.6 μl of 50 mM MgCl2, 0.4 μl of 10 mM of each dNTP, 30 pmol of each primer, and 2 μl of the extracted DNA. The amplification profile comprised initial denaturation at 94°C for 3 min, 35 cycles at 94°C for 50 s, 55°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. The amplicons were then run on a 1% agarose gel, stained with ethidium bromide (0.5 lg/ml) and visualized with a UV illuminator (AB1500 Printgraph and AE 6905H Image Saver HR, ATTO, Tokyo, Japan). PCR products of 16S rRNA gene were purified with the PCR Purification Extraction Kit (Qiagen) and sent for sequencing using Sanger method (Macrogen, South Korea). The revealed sequences were verified by Bioedit software (version 7.0.5; Ibis Therapeutics, Carlsbad, USA). 2.2 Sequence and phylogenetic analysis Sequences of 16S rRNA gene were searched against the GenBank nucleotide database (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) for gene homology Nosema sequences and their highest homologies obtained from GenBank, including N. apis (accession number. FJ789796). The sequences were aligned using the CLUSTALX multiple alignment program [10]. Gap sites were excluded from the following analysis. Genetic distances were estimated with the Kimura two-parameter method [11]. Construction of neighbor-joining trees [12] and the bootstrap test with 1,000 replications were conducted with the MEGA version 7 programs [13]. 2.3 Detection of Nosema infection The presence of nosemosis in honeybee colonies was investigated in three different seasons in Thailand. The first period was done on winter season, during October 2015 - January 2016 February, second period on dry season (February - May 2016) and the third period on raining season (June - September 2016). The honey bees used in this study were collected from the Apis mellifera colonies maintained at apiaries of Wanna Bee Farm, located at Kaeng Sian Subdistrict, Mueang District, Kanchanaburi Province, Thailand. Honeybee was randomly collected from 20 colonies, 50 bees from each colony in different season to verify the presence of Nosema spp. spores and the number of spores per individual bee. For this, the abdomens were individually separated, placed into a 1.5 ml Eppendorf tube and then homogenized thoroughly in 200 ml of deionized H2O. Ten microliters of the homogenate were loaded onto a hemocytometer and the number of spores counted. This should read: 2.1 DNA extraction, amplification and sequencing Genomic DNA was extracted from a whole Nosema spores from the A. mellifera colonies at apiaries of Wanna Bee Farm using DNA Extraction Kit (Qiagen) according to the manufacturer’s instructions. The 16S rRNA gene were amplified by polymerase chain reaction (PCR) using the following primers: Noce239L (5’–AGGGGCGAAACTTGACCTAT-3’) and Noce950R, (5’–GGGCATAACKG ACCTGTTTA-3’) [9]. PCR was carried out using 20 μl volumes containing 5 units of Taq DNA polymerase (biotechrabbit™), 2 μl of 10X PCR reaction buffer, 0.6 μl of 50 mM MgCl2, 0.4 μl of 10 mM of each dNTP, 30 pmol of each primer, and 2 μl of the extracted DNA. The amplification profile comprised initial denaturation at 94°C for 3 min, 35 cycles at 94°C for 50 s, 55°C for 30 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. The amplicons were then run on a 1% agarose gel, stained with ethidium bromide (0.5 lg/ml) and visualized with a UV illuminator (AB1500 Printgraph and AE 6905H Image Saver HR, ATTO, Tokyo, Japan). PCR products of 16S rRNA gene were purified with the PCR Purification Extraction Kit (Qiagen) and sent for sequencing using Sanger method (Macrogen, South Korea). The revealed sequences were verified by Bioedit software (version 7.0.5; Ibis Therapeutics, Carlsbad, USA). 2.2 Sequence and phylogenetic analysis Sequences of 16S rRNA gene were searched against the GenBank nucleotide database (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) for gene homology Nosema sequences and their highest homologies obtained from GenBank, including N. apis (accession number. FJ789796). The sequences were aligned using the CLUSTALX multiple alignment program [10]. Gap sites were excluded from the following analysis. Genetic distances were estimated with the Kimura two-parameter method [11]. Construction of neighbor-joining trees [12] and the bootstrap test with 1,000 replications were conducted with the MEGA version 7 programs [13]. 2.3 Detection of Nosema infection The presence of nosemosis in honeybee colonies was investigated in three different seasons in Thailand. The first period was done on winter season, during October 2015 - January 2016 February, second period on dry season (February - May 2016) and the third period on raining season (June - September 2016). The honey bees used in this study were collected from the Apis mellifera colonies maintained at apiaries of Wanna Bee Farm, located at Kaeng Sian Subdistrict, Mueang District, Kanchanaburi Province, Thailand. Honeybee was randomly collected from 20 colonies, 50 bees from each colony in different season to verify the presence of Nosema spp. spores and the number of spores per individual bee. For this, the abdomens were individually separated, placed into a 1.5 ml Eppendorf tube and then homogenized thoroughly in 200 μl of deionized H2O. Ten microliters of the homogenate were loaded onto a hemocytometer and the number of spores counted. Page 3: In the 2.4 section, the following text appears: 2.4 Nosema spore morphological identification using light microscopy The mid gut of 50 bees was macerate in 1.5 ml Eppendorf containing 1,000 μl deionized water, the suspension was centrifuged at 6000g for ten minutes and the homogenate examined under the light microscope using × 400 magnification and photographed. Measurement is present in micrometers and data are expressed as the mean followed by the range in parentheses. This should read: 2.4 Nosema spore morphological identification using light microscopy The mid gut of 50 bees was macerate in 1.5 ml Eppendorf containing 1,000 μl deionized water, the suspension was centrifuged at 6000g for ten minutes and the homogenate examined under the light microscope using 400x magnification and photographed. Measurement is present in micrometers and data are expressed as the mean followed by the range in parentheses. Page 3: In the Results and discussion section, the following text appears: Results and discussion 3.1 Nosema species determination The amplified product of 16S rRNA gene from two Nosema spores strains was successfully amplified by PCR “Fig.1” using Noce239L (5’–AGGGGCGAAACTTGACC TAT-3’) and Noce950R (5’– GGGCATAACKGACCTGTT TA-3’) described by Reference [9]. These primers generate PCR products approximately 710 and 685 bp for N. ceranae and N. apis, respectively. The results showed that the 710 bp DNA fragments were successfully obtained in one PCR reaction. While after PCR products were purified and sequenced, slight differences were found at five nucleotide positions (positions 240, 312, 350, 423, and 425; Fig. 6 shows only the 672-bp aligned sequences). One gap was present among these aligned sequences and the GenBank retrieved sequences (N. ceranae (DQ673615), N. ceranae France (DQ374655), N. ceranae Germany (DQ374656), N. ceranae (XR_002966746). These base differences may be due to recombination or mutation. When comparing the Nosema species to the N. apis 16S rRNA sequences obtained from GenBank (N. apis (FJ789796)) there were 32 additional positional differences and 21 gaps. These results indicate that Nosema strain tested in this study were N. ceranae. The phylogeny shows that a N. ceranae Noce950R strain cluster together with the GenBank N. ceranae strains and are clearly separated from N. apis ‘Figs. 6 and 2”. 3.2 Infection rate and infectivity All 20 colonies of A. mellifera were monitor throughout the 3 different season period for Nosema infection level; there were significant differences in infection levels between periods. We observed the infections level were significantly lower in October 2015 - January 2016, a 40% of colonies was infection by Nosema. The infections level was increased in February-May 2016 with 80% of colonies were infected, while during June-September 2016 a 100% of colonies were infected with Nosema “Fig.3”. However, a total number of Nosema spores counted from individual honeybee were not corresponding with the infection level. The highest number spores counted per bee was found in bees sampled during the warmer months were collected in February-May 2016 with 2.47 x106 ± 4.27 spores per bee. The lower number spores counted per bee were showed in bees sampled in June-September 2016 with represent the average number of spores counted 0.27 x106 ± 0.43 spores per bee, whereas it was the lowest in bees collected in February - May 2016 with 0.04 x106 ± 0.08 spores per bee. There was a significant difference in number of spores counted in different periods sampled (Chi2 = 24.98, P<0.05), as expected “Fig.4”. This should read: Results and discussion 3.1 Nosema species determination The amplified product of 16S rRNA gene from two Nosema spores strains was successfully amplified by PCR “Fig.1” using Noce239L (5’–AGGGGCGAAACTTGACCTAT-3’) and Noce950R (5’– GGGCATAACKGACCTGTTTA-3’) described by Reference [9]. These primers generate PCR products approximately 710 and 685 bp for N. ceranae and N. apis, respectively. The results showed that the 710 bp DNA fragments were successfully obtained in one PCR reaction. While after PCR products were purified and sequenced, slight differences were found at five nucleotide positions (positions 240, 312, 350, 423, and 425; Fig. 6 shows only the 672-bp aligned sequences). One gap was present among these aligned sequences and the GenBank retrieved sequences (N. ceranae (DQ673615), N. ceranae France (DQ374655), N. ceranae Germany (DQ374656), N. ceranae (XR_002966746). These base differences may be due to recombination or mutation. When comparing the Nosema species to the N. apis 16S rRNA sequences obtained from GenBank (N. apis (FJ789796)) there were 32 additional positional differences and 21 gaps. These results indicate that Nosema strain tested in this study were N. ceranae. The phylogeny shows that a N. ceranae Noce950R strain cluster together with the GenBank N. ceranae strains and are clearly separated from N. apis ‘Figs. 6 and 2”. 3.2 Infection rate and infectivity All 20 colonies of A. mellifera were monitor throughout the 3 different season period for Nosema infection level; there were significant differences in infection levels between periods. We observed the infections level were significantly lower in October 2015 - January 2016, a 40% of colonies was infection by Nosema. The infections level was increased in February-May 2016 with 80% of colonies were infected, while during June-September 2016 a 100% of colonies were infected with Nosema “Fig.3”. However, a total number of Nosema spores counted from individual honeybee were not corresponding with the infection level. The highest number spores counted per bee was found in bees sampled during the warmer months were collected in February-May 2016 with 2.47 x106 ± 4.27 spores per bee. The lower number spores counted per bee were showed in bees sampled in June-September 2016 with represent the average number of spores counted 0.27 x106 ± 0.43 spores per bee, whereas it was the lowest in bees collected in February - May 2016 with 0.04 x106 ± 0.08 spores per bee. There was a significant difference in number of spores counted in different periods sampled (Chi-Square = 24.98, P<0.05), as expected “Fig.4”. Page 5: In the description on figure 4, the following text appears: Figure 4. The infectivity of European honeybees on honeybees farm at Kanchanaburi Province (Chi2 = 24.98, P<0.05) This should read: Figure 4. The infectivity of European honeybees on honeybees farm at Kanchanaburi Province (Chi-Square = 24.98, P<0.05)