Black queen cell virus and Nosema ceranae coinfection in Africanized honey bees from southern Brazil / Coinfecção por vírus da realeira negra (BQCV) e Nosema ceranae em abelhas africanizadas no sul do Brasil

Bees are fundamental in several aspects, especially in relation to plant biodiversity and pollination. Recently, immense losses are being faced in the number of Brazilian colonies, mainly in southern states of the country, which has a strong beekeeping activity. There are indications that, among the reasons for the losses, pathogens that affect the health of bees may be involved. Among them, the microsporidium Nosema and the black queen cell virus (BQCV) stand out for their prevalence. In this study, 92 colonies of 17 apiaries from southern Brazil were evaluated for infection by Nosema ceranae, Nosema apis and BQCV. Nucleic acid extractions and cDNA synthesis were performed from adult bee samples, followed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and multiplex PCR. Eight BQCV positive samples were subjected to sequencing. The results showed that N. ceranae and BQCV are circulating in the Southern region of the country, which may be the reason for the loss of colonies. N. apis was not found. N. ceranae was found in 57.6% (53/92) of the colonies and BQCV in 32.6% (30/92). Co-infection was found in 25% (23/92) of the colonies studied, a factor that is suggested to be reducing the hosts' longevity due to the synergistic action of the pathogens. The samples submitted to sequencing indicated similarity of 96.8 to 100% between them, in addition to strong similarity with sequences from Asia, United States, Germany and Peru. This study reports the circulation of N. ceranae and BQCV in apiaries in southern Brazil, in addition to being the first phylogenetic analysis of the Brazilian BQCV sequence.(AU)

As abelhas mostram-se fundamentais em diversos aspectos, especialmente com relação à biodiversidade de plantas e polinização. Recentemente, estão sendo enfrentadas imensas perdas no número de colônias brasileiras, principalmente nos estados do sul do país, com forte atividade apícola. Há indicativos de que, dentre as razões para as perdas, possam estar envolvidos patógenos que afetam a saúde das abelhas. Dentre eles, o microsporídio Nosema e o vírus da realeira negra (BQCV) destacam-se pela prevalência. Neste estudo, foram avaliadas 92 colônias, de 17 apiários do sul do Brasil, a respeito da infecção por Nosema ceranae, Nosema apis e BQCV. Foram realizadas extrações de ácidos nucleicos e síntese de cDNA a partir de amostras de abelhas adultas, seguidos de Reação em Cadeia da Polimerase-Transcriptase Reversa (RT-PCR). Oito amostras positivas para BQCV foram submetidas a sequenciamento. Os resultados mostraram que N. ceranae e BQCV estão circulando na região sul do país, podendo ser a razão para as perdas de colônias. N. apis não foi encontrado. N. ceranae foi encontrado em 57.6% (53/92) das colônias e BQCV em 32.6% (30/92). Foi encontrada coinfecção por ambos em 25% (23/92) das colônias estudadas, fator que sugere a diminuição da longevidade do hospedeiro por ação sinérgica dos patógenos. As amostras submetidas ao sequenciamento indicaram similaridade de 96.8 a 100% entre elas, além de forte similaridade com sequências da Ásia, Estados Unidos, Alemanha e Peru. Este estudo relata a circulação de N. ceranae e BQCV nos apiários do sul do Brasil, além de ser a primeira análise filogenética da sequência do BQCV brasileiro.(AU)

Expression of Polar Tube Protein 1 (NbPTP1) from Nosema bombycis in Drosophila S2 cell lines and its glycosylation / 生物工程学报

The polar tube protein is the major component of polar tube, and can specifically locate on the polar tube of microsporidia and plays an important role in invasion host cell. In this study, we analyzed the potential O- and Nglycosylation sites in polar tube protein 1 from Nosema bombycis. NbPTP1 was successfully cloned to eukaryotic expression vector pMT/Bip/V5-His A, involved V5 and His tags. After transfection, NbPTP1 gene could be efficiently expressed in Drosophila S2 cells. In addition, Lectin blotting and beta elimination analysis showed that NbPTP1 expressed in Drosophila S2 cells was O-glycosylation. These studies provided a basis for understanding the relationship between glycosylation and function of NbPTP1, helped us to reveal the infection mechanism of microsporidia and established effective diagnosis and prevention methods for microsporidia.

Molecular identification of Nosema species in provinces of Fars, Chaharmahal and Bakhtiari and Isfahan (Southwestern Iran)

Objective: To molecularly identify Nosema species in provinces of Isfahan, Fars, Chaharmahal and Bakhtiari. Methods: One hundred and eighty adult honey bees suspected with nosemosis from provinces of Fars (different counties), Isfahan, and Chaharmahal and Bakhtiari were tested. In order to determine the species of Nosema, previously developed PCR and primers based on 16S rRNA gene were used. PCR products were purified and sent to the Korean company of Macrogen for sequencing. Results: Only Nosema ceranae was determined in all samples based on their molecular profile. Sequences of the 16S rRNA gene were sent to GenBank/NCBI (samples acces-sion numbers KP318660–KP318663). Conclusions: This species currently exists in European honeybee apiaries of Apis mel-lifera in the studied provinces.

Efficient Method for the Rapid Purification of Nosema ceranae Spores

Nosema ceranae is an obligate intracellular fungal parasite that causes mortality in honey bees and enhances the susceptibility of honey bees to other pathogens. Efficient purification of Nosema spores from the midgut of infected honey bees is very important because Nosema is non-culturable and only seasonably available. To achieve a higher yield of spores from honey bees, in this study, we considered that the initial release of spores from the midgut tissues was the most critical step. The use of 2 mm beads along with enzymatic treatment with collagenase and trypsin enhanced the homogenization of tissues and the yield of released spores by approximately 2.95 times compared with the use of common 3 mm beads alone. The optimal time for the enzyme treatment was determined to be 1 hr as measured by the yield and viability of the spores. A one-step filtration using a filter paper with an 8–11 µm pore size was sufficient for removing cell debris. This method may be useful to purify not only N. ceranae spores but also other Nosema spp. spores.

Molecular identification of Nosema species in provinces of Fars, Chaharmahal and Bakhtiari and Isfahan (Southwestern Iran)

Objective To molecularly identify Nosema species in provinces of Isfahan, Fars, Chaharmahal and Bakhtiari. Methods One hundred and eighty adult honey bees suspected with nosemosis from provinces of Fars (different counties), Isfahan, and Chaharmahal and Bakhtiari were tested. In order to determine the species of Nosema, previously developed PCR and primers based on 16S rRNA gene were used. PCR products were purified and sent to the Korean company of Macrogen for sequencing. Results Only Nosema ceranae was determined in all samples based on their molecular profile. Sequences of the 16S rRNA gene were sent to GenBank/NCBI (samples accession numbers KP318660–KP318663). Conclusions This species currently exists in European honeybee apiaries of Apis mellifera in the studied provinces.

Study on impact of parasite (Nosema species) on characters of tropical tasar silkworm Anthereae mylitta drury.

Infection of the pebrine disease has been found to be highly virulent and harm the cocoon yield as well as characters of silkworm Anthereae mylitta. Therefore, an attempt was made to evaluate the impact of parasite Nosema species on the ecorace (Sukinda) of A.mylitta in respect of transovarial transmitted (T1), secondary infection (T2) and healthy silkworm (T3). In comparison to T3, the number of larval mortality was 16 and 11 in T1 and T2 respectively; whereas as number of pupal mortality was 6 and 5 in T1 and T2 respectively.The larval weight, number of moths emerged, number of eggs laid and percent hatchability were reduced in T1 and T2 in comparison to T3.The infected layings were high in T1(51%) and T2(42%) as against T3(0%). Similarly, the infected moths were 34% in T1 and 15% in T2 as against 0 percent in T3.All the characteristics parameters of cocoon were reduced in T1 and T2 against T3. The study explains that there was no significant variation between T1 and T2 on different parameters of larva, pupa and cocoon characters.

Prevalence of Nosema and Virus in Honey Bee (Apis mellifera L.) Colonies on Flowering Period of Acacia in Korea

Honey production from approximately 1.6 million colonies owned by about 199,000 Korean beekeepers was almost 23,000 metric tons in 2009. Nosema causes significant losses in honey production and the virus decreases population size. We initiated a survey of honey bee colonies on the blooming period of Acacia to determine the prevalence of Nosema and virus in 2011. Most Korean beekeepers have moved from the south to north of Korea to get Acacia nectar for 2 mon. This provided a valuable opportunity to sample bees originating from diverse areas in one location. Twenty hives owned by 18 beekeepers were sampled in this year. Nosema spore counts ranged from zero to 1,710,000 spores per bee. The average number of nosema spores per bee was 580,000. Approximately 95% of the colonies were infected with Nosema, based on the presence of spores in the flowering period of Acacia. This indicates that Nosema is the predominant species affecting honeybee colonies. Also, the seven most important honeybee viruses were investigated by reverse transcription-PCR. Among them, four different viruses were detected in samples. Black queen cell virus was present in all samples. Chronic bee paralysis virus was detected in 10% of samples. Deformed wing virus was present in only 5% of the samples. Prevalence of Sacbrood virus was 15%. However, Cloudy wing virus, Israel acute paralysis virus and kashmir bee virus were not detected in any of samples.

Research Progress on the Technology of Microsporidian Detection / 中国寄生虫学与寄生虫病杂志

The microsporidia are obligate intracellular eukaryotic parasites.They have been paid more attention as being the emerging pathogen of human, so it is important to control microsporidiosis using fast and precise detecting technology.In order to provide a reference for controlling microsporidian infection effectively, this paper reviews the progress of studying on the detecting technology from the microscopic staining methods, immunological and molecular biology.