Nosema ceranae, a new parasite in Thai honeybees

Adult workers of Apis cerana, Apis florea and Apis mellifera from colonies heavily infected with Nosema ceranae were selected for molecular analyses of the parasite. PCR-specific 16S rRNA primers were designed, cloned, sequenced and compared to GenBank entries. The sequenced products corresponded to N. ceranae. We then infected A. cerana with N. ceranae spores isolated from A. florea workers. Newly emerged bees from healthy colonies were fed 10,000, 20,000 and 40,000 spores/bee. There were significant dosage dependent differences in bee infection and survival rates. The ratio of infected cells to non-infected cells increased at 6, 10 and 14 d post infection. In addition, hypopharyngeal glands of bees from the control group had significantly higher protein concentrations than infected groups. Bees infected with 40,000 spores/bee had the lowest protein concentrations. Thus, N. ceranae isolated from A. florea is capable of infecting another bee species, impairing hypopharyngeal gland protein production and reducing bee survival in A. cerana.     

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.     

Descriptive study of antennation during trophallactic unloading contacts in honeybees <Emphasis Type="Italic">Apis mellifera carnica</Emphasis>

Summary  Trophallaxis, the mouth-to-mouth transfer of food, is a widespread behavior occurring between individuals of eusocial insect societies. Antennal movements during food transfer are, in honeybees, too rapid to be characterized using standard video recordings. Using a high-speed camera (200 frames/s), we recorded nectar unloading performed by forager honeybees (Apis mellifera carnica Spinola) within the hive once they returned from collecting sugar solution at a feeder that delivered nectar at a variable rate. Frequency patterns attained a mean value of 13 Hz. Antennation intensity showed a tendency to increase with the reward rate recently exploited by the food donor. This raises the question whether or not antennation intensity is a reliable parameter providing modulatory information related to food-source profitability.Received 12 September 2002; revised 21 March 2002; accepted 24 April 2003.     

Nosema ceranae, a new microsporidian parasite in honeybees in Europe

Twelve samples of adult honey bees from different regions of Spain from colonies with clear signs of population depletion, positive to microsporidian spores using light microscopy (1% of total positive samples analysed), were selected for molecular diagnosis. PCR specific primers for a region of the 16S rRNA gene of Microsporidia were developed and the PCR products were sequenced and compared to GenBank entries. The sequenced products of 11 out of the 12 samples were identical to the corresponding Nosema ceranae sequence. This is the first report of N. ceranae in colonies of Apis mellifera in Europe. The suggested link of the infections to clinical disease symptoms makes imperative a study of the virulence of N. ceranae in European races of honey bees.     

Dopamine enhances locomotor activity for mating in male honeybees (Apis mellifera L.)

Dopamine plays multiple roles in the regulation of reproduction in female honeybees where it appears to act independently of juvenile hormone (JH). In males the role of dopamine and its relationship to JH control have not been elucidated. In the present study we determined hemolymph levels of dopamine and its metabolite (N-acetyldopamine) in males at post-emergence days 0-16. The development of locomotor and flight activities were recorded over the same period. Hemolymph levels of dopamine and N-acetyldopamine were found to increase at the time of onset of mating flight activity and those of dopamine decreased thereafter. Both locomotor and flight activities increased in parallel with hemolymph dopamine levels but the increased activity levels were maintained following decline of dopamine levels. Brain and meso-metathoracic ganglia levels of dopamine showed a similar developmental profile to hemolymph dopamine levels. Locomotor activities were temporarily inhibited by injection of a dopamine-receptor antagonist (cis(Z)-flupenthixol) into the thorax, and were enhanced by injection of a dopamine-receptor agonist (6,7-ADTN). These results suggest that dopamine regulates locomotor activities for mating and plays a role downstream of JH in premature males in honeybees.     

Comparing injection, feeding and topical application methods for treatment of honeybees with octopamine

Entomologists have used a range of techniques to treat insects with neuroactive compounds, but it is not always clear whether different treatment methods are equally effective in delivering a compound to a target organ. Here, we used five different techniques to treat honeybees with 3H-octopamine (3H-OA), and analysed the distribution of the 3H radiolabelled compound within different tissues and how it changed over time. All treatment methods, including injection of the median ocellus, resulted in 3H-OA detection in all parts of the honeybee. Injection through the median ocellus was the most effective method for delivering 3H-OA to the brain. Topical application of 3H-OA dissolved in dimethylformamide (dMF) to the thorax was as effective as thoracic injections of 3H-OA in delivering 3H-OA to the brain, but topical applications to the abdomen were less so. Most of the 3H-OA applied topically remained associated with the cuticle and the tissues of the body segment to which it had been applied. For all treatment methods, 3H-OA was rapidly lost from the brain and head capsule, and accumulated in the abdomen. Our findings demonstrate the value of thoracic topical treatment with compounds dissolved in dMF as an effective non-invasive method for short-term, systemic pharmacological treatments.     

Influence of carbon dioxide on Nosema apis infection of honeybees (Apis mellifera)

Young workers of the honeybee Apis mellifera carnica were individually inoculated with Nosema apis spores subjected to carbon dioxide (CO(2)) treatment. The spores were kept in a CO(2) atmosphere for 30, 35 and 40 h. The course of the infection was evaluated on the basis of the survival rate of bee workers and the number of N. apis spores in their digestive tracts. CO(2) treatment of N. apis spores resulted in faster proliferation of the parasite as well as higher mortality among workers infected with spores kept in CO(2) for 30 and 35 h.     

Enhanced cholinergic transmission promotes recall in honeybees

The involvement of the cholinergic system in learning and memory in honeybees has been well established using olfactory conditioning. We examined the effect of Methyl Parathion (MeP), an acetylcholinesterase inhibitor of the organo-phosphate family, on the learning and recall of visual and olfactory discrimination tasks in honeybees. One of our expectations was to observe the effects induced by both the nicotinic and muscarinic systems, as the blocking of acetylcholinesterase should induce an increase in the activity of both systems. We were also interested in knowing whether the type of tasks could influence the results. The visual tasks involved learning to discriminate the orientation of gratings in a Y-maze; the olfactory task involved learning to discriminate odours in a proboscis extension reflex (PER) paradigm. The results indicate that MeP treatment enhances recall of learned tasks in the visual and olfactory domains, but it does not affect the acquisition phase in either domain. Surprisingly, MeP treatment led to muscarinic-like effects but failed to mimic the nicotinic-like effects already described in relation to learning phases in honeybees. Implications for the role of cholinergic pathways in learning and memory and the nature of their involvement are discussed, and a hypothesis relating to the organisation of the cholinergic system and the relationship between the nicotinic and muscarinic systems in honeybees is proposed. The results are also discussed in terms of their ecotoxicological consequences.     

Presence of Nosema ceranae in honeybees (Apis mellifera) in Uruguay

The microsporidium Nosema ceranae is an emergent pathogen of European honeybees Apis mellifera. Using a PCR-RFLP diagnosis, 29 samples of infected honeybees obtained in 2007-2008 (N = 26), 2004 (N = 2) and before 1990 (N = 1) were analyzed for the presence of Nosema apis and N. ceranae. Only N. ceranae was found in all samples, indicating that this species dispersed to Uruguay (and likely the region) at some time before 1990. The presence of N. ceranae in Uruguay is not associated with an increase of Nosemosis, and its role in colony loss seems to be irrelevant.     

Experimental infection of Apis mellifera honeybees with Nosema ceranae (Microsporidia)

In this report, an experimental infection of Apis mellifera by Nosema ceranae, a newly reported microsporidian in this host is described. Nosema free honeybees were inoculated with 125,000 N. ceranae spores, isolated from heavily infected bees. The parasite species was identified by amplification and sequencing the SSUrRNA gene of the administered spores. Three replicate cages of 20 honeybees each were prepared, along with one control cage (n=20) supplied with sugar syrup only. The infection rate was 100% at the dosage administered. The presence of Nosema inside ventricular cells was confirmed in the samples using ultrathin sectioning and transmission electron microscopy. By day 3 p.i. a few cells (4.4%+/-1.2) were observed to be parasitized, whereas by 6 days p.i. more than half of the counted cells (66.4%+/-6) showed different parasite stages, this value increasing on day 7 p.i. (81.5%+/-14.8). Only one control bee died on day 7 p.i. In the infected groups, mortality was not observed until day 6 p.i. (66.7%+/-5.6). Total mortality on day 7 p.i. was 94.1% in the three infected replicates and by day 8 p.i. no infected bee was alive. After the infection, the parasites invaded both the tip of folds and the basal cells of the epithelium and the autoinfective capacity of the spores seemed to spread the infection rapidly between epithelial cells. On day 3 p.i., mature spores could be seen inside host cell tissue implying that the developmental cycle had been completed. The large number of parasitized cells, even the regenerative ones, the presence of autoinfective spores and the high mortality rate demonstrate that N. ceranae is highly pathogenic to Apis mellifera. Possible relation with bee depopulation syndrome is discussed by authors.     

Virus infection causes specific learning deficits in honeybee foragers

In both mammals and invertebrates, virus infections can impair a broad spectrum of physiological functions including learning and memory formation. In contrast to the knowledge on the conserved mechanisms underlying learning, the effects of virus infection on different aspects of learning are barely known. We use the honeybee (Apis mellifera), a well-established model system for studying learning, to investigate the impact of deformed wing virus (DWV) on learning. Injection of DWV into the haemolymph of forager leads to a RT-PCR detectable DWV signal after 3 days. The detailed behavioural analysis of DWV-infected honeybees shows an increased responsiveness to water and low sucrose concentrations, an impaired associative learning and memory formation, but intact non-associative learning like sensitization and habituation. This contradicts all present studies in non-infected bees, where increased sucrose responsiveness is linked to improved associative learning and to changes in non-associative learning. Thus, DWV seems to interfere with molecular mechanism of learning by yet unknown processes that may include viral effects on the immune system and on gene expression.     

Genetic parameters for five traits in Africanized honeybees using Bayesian inference

Heritability and genetic correlations for honey (HP) and propolis production (PP), hygienic behavior (HB), syrup-collection rate (SCR) and percentage of mites on adult bees (PMAB) of a population of Africanized honeybees were estimated. Data from 110 queen bees over three generations were evaluated. Single and multi-trait models were analyzed by Bayesian Inference using MTGSAM. The localization of the hive was significant for SCR and HB and highly significant for PP. Season-year was highly significant only for SCR. The number of frames with bees was significant for HP and PP, including SCR. The heritability estimates were 0.16 for HP, 0.23 for SCR, 0.52 for HB, 0.66 for PP, and 0.13 for PMAB. The genetic correlations were positive among productive traits (PP, HP and SCR) and negative between productive traits and HB, except between PP and HB. Genetic correlations between PMAB and other traits, in general, were negative, except with PP. The study permitted to identify honeybees for improved propolis and honey production. Hygienic behavior may be improved as a consequence of selecting for improved propolis production. The rate of syrup consumption and propolis production may be included in a selection index to enhance honeybee traits.     

Nest-mate recognition template of guard honeybees (Apis mellifera) is modified by wax comb transfer

In recognition, discriminators use sensory information to make decisions. For example, honeybee (Apis mellifera) entrance guards discriminate between nest-mates and intruders by comparing their odours with a template of the colony odour. Comb wax plays a major role in honeybee recognition. We measured the rejection rates of nest-mate and non-nest-mate worker bees by entrance guards before and after a unidirectional transfer of wax comb from a 'comb donor' hive to a 'comb receiver' hive. Our results showed a significant effect that occurred in one direction. Guards in the comb receiver hive became more accepting of non-nest-mates from the comb donor hive (rejection decreased from 70 to 47%); however, guards in the comb donor hive did not become more accepting of bees from the comb receiver hive. These data strongly support the hypothesis that the transfer of wax comb increases the acceptance of non-nest-mates not by changing the odour of the bees, but by changing the template used by guards.