Veeranan Chaimanee

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.

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.

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.

Differential physiological effects of neonicotinoid insecticides on honey bees: A comparison between Apis mellifera and Apis cerana

Acute toxicities (LD50s) of imidacloprid and clothianidin to Apis mellifera and A. cerana were investigated. Changing patterns of immune-related gene expressions and the activities of four enzymes between the two bee species were compared and analyzed after exposure to sublethal doses of insecticides. Results indicated that A. cerana was more sensitive to imidacloprid and clothianidin than A. mellifera. The acute oral LD50 values of imidacloprid and clothianidin for A. mellifera were 8.6 and 2.0ng/bee, respectively, whereas the corresponding values for A. cerana were 2.7 and 0.5ng/bee. The two bee species possessed distinct abilities to mount innate immune response against neonicotinoids. After 48h of imidacloprid treatment, carboxylesterase (CCE), prophenol oxidase (PPO), and acetylcholinesterase (AChE) activities were significantly downregulated in A. mellifera but were upregulated in A. cerana. Glutathione-S-transferase (GST) activity was significantly elevated in A. mellifera at 48h after exposure to imidacloprid, but no significant change was observed in A. cerana. AChE was downregulated in both bee species at three different time points during clothianidin exposure, and GST activities were upregulated in both species exposed to clothianidin. Different patterns of immune-related gene expression and enzymatic activities implied distinct detoxification and immune responses of A. cerana and A. mellifera to imidacloprid and clothianidin.

Antimicrobial activity of plant extracts against the honeybee pathogens, Paenibacillus larvae and Ascosphaera apis and their topical toxicity to Apis mellifera adults

To explore alternative nonchemical control measures against two honeybee pathogens, Paenibacillus larvae and Ascosphaera apis, 37 plant species were screened for antimicrobial activity.

Infectivity of Nosema ceranae isolates from different hosts and immune response in honey bees Apis mellifera and Apis cerana

The microsporidium, Nosema ceranae, was first discovered in Apis cerana. Currently, N. ceranae is a highly prevalent parasite in A. mellifera colonies worldwide. The pathology of N. ceranae and how bees defend themselves from this pathogen have been reported. Here, we investigated the infectivity of two N. ceranae isolates from different honey bee species, A. mellifera and A. cerana. Our results showed that the N. ceranae isolate from A. mellifera had a significantly higher infectivity than another isolate from A. cerana in all experimental cages of A. mellifera and A. cerana. Moreover, when comparing the infectivity of each N. ceranae isolate between the original and new hosts, it seems that no significant difference was obtained. The immunity-related enzymes in honey bees following N. ceranae inoculation were also analyzed at day six after spore feeding. Phenol oxidase (PO) transcript levels were up-regulated in A. cerana when inoculated by both N. ceranae isolates, but were not significantly different. However, the transcript level for PO in one experimental cage of A. mellifera was significantly down-regulated when inoculated with N. ceranae. Significant glucose dehydrogenase suppression occurred in only one cage of each honey bee species.

Chemical and cultural control of Tropilaelaps mercedesae mites in honeybee (Apis mellifera) colonies in Northern Thailand

At least two parasitic mites have moved from Asian species of honeybees to infest Apis mellifera. Of these two, Varroa destructor is more widespread globally while Tropilaelaps mercedesae has remained largely in Asia. Tropilaelaps mites are most problematic when A. mellifera is managed outside its native range in contact with Asian species of Apis. In areas where this occurs, beekeepers of A. mellifera treat aggressively for Tropilaelaps and Varroa is either outcompeted or is controlled as a result of the aggressive treatment regime used against Tropilaelaps. Many mite control products used worldwide may in fact control both mites but environmental conditions differ globally and thus a control product that works well in one area may be less or ineffective in other areas. This is especially true of volatile compounds. In the current research we tested several commercial products known to control Varroa and powdered sulfur for efficacy against Tropilaelaps. Additionally, we tested the cultural control method of making a hive division to reduce Tropilaelaps growth in both the parent and offspring colony. Making a split or nucleus colony significantly reduced mite population in both the parent and nucleus colony when compared to un-manipulated control colonies. The formic acid product, Mite-Away Quick Strips®, was the only commercial product that significantly reduced mite population 8 weeks after initiation of treatment without side effects. Sulfur also reduced mite populations but both sulfur and Hopguard® significantly impacted colony growth by reducing adult bee populations. Apivar® (amitraz) strips had no effect on mite or adult bee populations under the conditions tested.