Panuwan Chantawannakul

Differential expression of immune genes of adult honey bee (Apis mellifera) after inoculated by Nosema ceranae

Nosema ceranae is a microsporidium parasite infecting adult honey bees (Apis mellifera) and is known to affects at both the individual and colony level. In this study, the expression levels were measured for four antimicrobial peptide encoding genes that are associated with bee humoral immunity (defensin, abaecin, apidaecin, and hymenoptaecin), eater gene which is a transmembrane protein involved cellular immunity and gene encoding female-specific protein (vitellogenin) in honey bees when inoculated by N. ceranae. The results showed that four of these genes, defensin, abaecin, apidaecin and hymenoptaecin were significantly down-regulated 3 and 6days after inoculations. Additionally, antimicrobial peptide expressions did not significantly differ between control and inoculated bees after 12days post inoculation. Moreover, our results revealed that the mRNA levels of eater and vitellogenin did not differ significantly following N. ceranae inoculation. Therefore, in this study we reaffirmed that N. ceranae infection induces host immunosuppression.

Infections of Nosema ceranae in four different honeybee species

The microsporidium Nosema ceranae is detected in honeybees in Thailand for the first time. This endoparasite has recently been reported to infect most Apis mellifera honeybee colonies in Europe, the US, and parts of Asia, and is suspected to have displaced the endemic endoparasite species, Nosema apis, from the western A. mellifera. We collected and identified species of microsporidia from the European honeybee (A. mellifera), the cavity nesting Asian honeybee (Apis cerana), the dwarf Asian honeybee (Apis florea) and the giant Asian honeybee (Apis dorsata) from colonies in Northern Thailand. We used multiplex PCR technique with two pairs of primers to differentiate N. ceranae from N. apis. From 80 A. mellifera samples, 62 (77.5%) were positively identified for the presence of the N. ceranae. Amongst 46 feral colonies of Asian honeybees (A. cerana, A. florea and A. dorsata) examined for Nosema infections, only N. ceranae could be detected. No N. apis was found in our samples. N. ceranae is found to be the only microsporidium infesting honeybees in Thailand. Moreover, we found the frequencies of N. ceranae infection in native bees to be less than that of A. mellifera.

T-RFLP analysis of bacterial communities in the midguts of Apis mellifera and Apis cerana honey bees in Thailand

This study investigated bacterial community structures in the midguts of Apis mellifera and Apis cerana in Thailand to understand how bacterial communities develop in Apis species. The bacterial species present in replicate colonies from different locations and life stages were analysed. PCR amplification of bacterial 16S rRNA gene fragments and terminal restriction fragment length polymorphism analyses revealed a total of 16 distinct terminal restriction fragments (T-RFs), 12 of which were shared between A. mellifera and A. cerana populations. The T-RFs were affiliated to Beta- and Gammaproteobacteria, Firmicutes and Actinomycetes. The Gammaproteobacteria were found to be common in all stages of honey bee, but in addition, the Firmicutes group was found to be present in the worker bees. Bacterial community structure showed no difference amongst the replicate colonies, but was affected to some degree by geographical location, life stage and species of honey bees.

Survey of six bee viruses using RT-PCR in Northern Thailand

Six honey bee viruses were surveyed using RT-PCR in Northern Thailand where about 80% of Thai apiaries are located. Tested samples were found to be positive for deformed wing virus (DWV), acute bee paralysis virus (ABPV), sacbrood virus (SBV) and Kashmir bee virus (KBV). In the collected samples, neither chronic bee paralysis virus nor black queen cell virus nucleic acids could be detected. It was found that DWV was the most widespread and ABPV was the second most prevalent. Kashmir bee virus was found only in the Lampang province where high infestation of Varroa destructor mite occurred. Tropilaelaps, European foulbrood, and Chalkbrood diseases were found in some apiaries.

First detection of Kashmir bee virus in the UK using real-time PCR

Kashmir bee virus (KBV) often persists in bees as a covert infection with no apparent symptoms. The virus can switch to become an overt lethal infection, especially in the presence of Varroa mites. Although the virus is distributed worldwide, it was thought to be absent from the UK. A real-time PCR assay was developed for specific detection of KBV. No cross-reaction was observed with other bee viruses. KBV was successfully amplified from different life stages of honey bees and from a wasp and bumble bee. Using the real-time PCR assay, a survey of hives was conducted in England and Wales to investigate the presence and geographical distribution of the virus. KBV was detected within three colonies at two locations. The virus titre in the positive samples was quantified and found to contain similar levels to other bees with covert KBV infection. We conclude that KBV is present in the UK and cannot now be considered an exotic disease. The discovery of KBV in the UK has major significance for import policies.ZusammenfassungWie viele andere Bienenviren ist auch das Kaschmir-Bienenvirus (KBV) in vielen Bienen als latente Infektion ohne offensichtliche Symptome präsent. In der Anwesenheit von Varroamilben kann der Virusbefall sich jedoch zu einer lethalen Infektion entwickeln. Das KBV ist zwar weltweit verbreitet, für Grossbritannien wurde jedoch bislang kein Vorkommen gemeldet.Wir entwickelten ein Protokoll zur spezifischen Detektion von KBV mittels Real-Time-quantitativer PCR. Dieses zeigte keine Kreuzreaktionen mit RNA anderer Bienenviren, wie ABPV, BQCV, SBV, CWV, SPV, DWV, CPV und AIV, und ermöglichte die Ampflifikation von PCR-Produkten aus virenbefallenen Bienenproben der verschiedenen Stadien des Lebenszyklus, ebenso wie von KBV-infizierten Bienen aus verschiedenen geographischen Regionen. PCR-Fragmente konnten auch aus RNA-Proben einer Wespe (Vespula germanica) aus Australien und aus einer aus Hummeln aufgereinigten KBV16 Virusprobe amplifiziert werden. Alle PCR-Produkte wurden kloniert und mittels Sequenzierung als KBV-RNA identifiziert.Mittels dieses Protokolls einer Real-Time-quantitativer PCR analysierten wir Proben aus 458 Völkern aus ganz England und Wales, um die eventuelle Anwesenheit und die geographische Verbreitung des KBV-Virus zu erfassen. Diese Übersichtsstudie zeigte die Präsenz des Virus in drei Völkern von zwei Ständen an. Mittels Real-Time-quantitativer PCR konnten wir zeigen, dass der Virentiter in diesen positiven Proben ähnliche KBV-Werte aufwies wie in Bienenproben aus Australien, die einen klaren Befall gezeigt hatten. Wir schliessen daraus, dass das KBV in Grossbritannien vorkommt, und dass es jetzt nicht länger als eine exotische Krankheit betrachtet werden kann. Die Klärung der Auswirkung des KBV-Befalls auf Völker in Grossbritannien erfordert weitere Untersuchungen, aber es scheint nicht die grosse Bedrohung zu sein, für die es bislang gehalten wurde, und es kann durchaus auch sein, dass das KBV keine grössere Bedrohung darstellt als andere, bereits in Grossbritannien vorkommende Viren. Die Entdeckung des KBV in Grossbritannien ist jedoch von Bedeutung in Hinsicht auf Importregelungen und belegt die Notwendigkeit von genauen Übersichtsdaten über das Vorkommen von pathogenen Agentien für die Erstellung formaler Importrisikoanalysen und für Entscheidungsprozesse innerhalb der Vereinbarungen des Internationalen Büros für Epizootien (OIE) und der WTO.

Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand.

Nosema ceranae was found to infect four different host species including the European honeybee (A. mellifera) and the Asian honeybees (Apis florea, A. cerana and Apis dorsata) collected from apiaries and forests in Northern Thailand. Significant sequence variation in the polar tube protein (PTP1) gene of N. ceranae was observed with N. ceranae isolates from A. mellifera and A. cerana, they clustered into the same phylogenetic lineage. N. ceranae isolates from A. dorsata and A. florea were grouped into two other distinct clades. This study provides the first elucidation of a genetic relationship among N. ceranae strains isolated from different host species and demonstrates that the N. ceranae PTP gene was shown to be a suitable and reliable marker in revealing genetic relationships within species.

The occurrence of Melissococcus plutonius in healthy colonies of Apis mellifera and the efficacy of European foulbrood control measures

European foulbrood (EFB) persists in England and Wales despite current treatment methods, all of which include feeding honey bee colonies with the antibiotic oxytetracycline (OTC). A large-scale field experiment was conducted to monitor a husbandry-based method, using comb replacement (known as Shook swarm), as a drug free EFB control option. The understanding of EFB epidemiology is limited, with little information on the presence of Melissococcus plutonius in disease free colonies. Additional samples were collected from diseased and disease free apiaries to identify symptomless infection. EFB reoccurrence was not significantly different between OTC and husbandry methods and real-time PCR data demonstrated that fewer Shook swarm treated colonies contained M. plutonius carryover to the Spring following treatment. Asymptomatic colonies from diseased apiaries showed an increased risk of testing positive for M. plutonius compared to asymptomatic colonies from disease free apiaries. The probability of a sample being symptomatic increased when a greater quantity of M. plutonius was detected in adult bees and larvae. The possibility of treating EFB as an apiary disease rather than a colony disease and the implications of a control strategy without antibiotics are discussed.

Neonicotinoid insecticides can serve as inadvertent insect contraceptives

There is clear evidence for sublethal effects of neonicotinoid insecticides on non-target ecosystem service-providing insects. However, their possible impact on male insect reproduction is currently unknown, despite the key role of sex. Here, we show that two neonicotinoids (4.5 ppb thiamethoxam and 1.5 ppb clothianidin) significantly reduce the reproductive capacity of male honeybees (drones), Apis mellifera. Drones were obtained from colonies exposed to the neonicotinoid insecticides or controls, and subsequently maintained in laboratory cages until they reached sexual maturity. While no significant effects were observed for male teneral (newly emerged adult) body mass and sperm quantity, the data clearly showed reduced drone lifespan, as well as reduced sperm viability (percentage living versus dead) and living sperm quantity by 39%. Our results demonstrate for the first time that neonicotinoid insecticides can negatively affect male insect reproductive capacity, and provide a possible mechanistic explanation for managed honeybee queen failure and wild insect pollinator decline. The widespread prophylactic use of neonicotinoids may have previously overlooked inadvertent contraceptive effects on non-target insects, thereby limiting conservation efforts.

A novel ascosporogenous yeast species, Zygosaccharomyces siamensis, and the sugar tolerant yeasts associated with raw honey collected in Thailand

Diversity of yeasts in association with bees and their food sources has been explored during the last decade. In Thailand, there has been no study of yeast identification in honey and bees. Hence, a total of 186 yeast strains were isolated from 37 honey samples of 12 different bee species. On the basis of morphological and physiological characteristics, 55 representative strains were chosen and identified by sequence analysis of the 26S rDNA D1/D2 domain and the ITS region. The data were compared with the published sequences and the results showed the occurrence of 19 ascomycetous and 1 basidiomycetous yeast species. Six strains of the new species were isolated. Phylogenetic analysis of the 26S rDNA D1/D2 sequence revealed that they were conspecific and most closely related to Zygosaccharomyces mellis. Based on the ITS sequence, the new species was clustered with the type β and clearly distinguished from the type α. Sequence analysis of combined ITS-26S rDNA D1/D2 showed similar results. The occurrence of these two types, with a divergence of more than 1% in their sequences, and low DNA relatedness among them suggested that members of the type β can be regarded as separate species. An analysis of the morphological and physiological characteristics was performed. Ascospore formation was observed on acetate agar and Gorodkowa agar. The new Zygosaccharomyces species differed physiologically from Z. mellis in 4 assimilation tests. This data supports the hypothesis that the new species, Zygosaccharomyces siamensis, is a novel ascosporogenous yeast. The type strain is JCM 16825T (=CBS 12273T) and a description is given here.

Susceptibility of four different honey bee species to Nosema ceranae.

In this study, we investigated the infectivity of Nosema ceranae and the immune response of the European honey bee, Apis mellifera and the Asian honey bee species, Apis cerana, Apis dorsata and Apis florea when inoculated with two isolates of N. ceranae isolated from different climates (Canada and Thailand), using cage experiments. The results indicated that the local isolate of N. ceranae (Thailand) had high infectivity in A. mellifera, A. cerana and A. dorsata but only a few spores were observed in A. florea. However, we found that only two honey bee species, A. mellifera and A. dorsata became infected when inoculated with N. ceranae isolated from Canada. Finally, our results showed that transcript levels of antimicrobial peptides (AMPs) in Asian honey bees were significantly higher than that of A. mellifera in both the control and N. ceranae inoculated bee groups. Comparing the expression of AMPs between the control and inoculated bees in each species, it was evident that N. ceranae inoculations did not affect the expression level of abaecin in all four honey bees species investigated in this experiment. Nevertheless, we found a significant up-regulation of apidaecin in A. cerana and A. florea when inoculated with N. ceranae (Canadian isolate). Also, the mRNA levels of hymenoptaecin were significantly increased in A. cerana after inoculation by N. ceranae isolated from Canada as compared with the Thai isolate.