Nosema ceranae and Nosema apis: Nosemosis, The Danger to Honey Bees and Agricultural Pollination
By [Andrew Parmelee]
Apis mellifera and Apis cerana, the European and Asian honey bees respectively, not only sustain biodiversity but are of particular agricultural interest. As essential pollinators of human maintained crops, these species of bees have become the subjects of many recent studies. The health of agricultural ecosystems is reliant on these critters’ well-being as studies have shown honey bees account for 90% of commercial pollination . Originally introduced by Europeans for crop management, large populations of honey bees are kept in close proximity to high agricultural areas today. Consequently, honey bee health is strongly linked to agricultural success and sustainability. Major emphasis is placed on pathogenic research to assure good health of the honeybee. Despite new advances, populations all over the world, from France to the United States are disappearing. If current decline in populations persists, large scale environmental crisis may grip many agriculturally developed nations. Such a disaster would only expand the world’s hunger epidemic if not properly dealt with. Researchers, acquiring grants from agricultural based economies and large environmental firms have been feverishly working on unearthing why honey bee colonies are failing all over the world.
Honey bee colonies fail for a multitude of reasons; availability of resources, predation, illness and environmental factors are all notable stressors. For example, fluctuation in nest temperature destabilizes brood rearing conditions, which has the potential to decrease organism count and biodiversity within a colony . Any of the fore mentioned burdens could play pivotal roles in facilitating total hive collapse. An area of interest for slowing the extraordinary loss of world honey bee populations is the identification and comprehension of the fungal pathogens, Nosema ceranae and Nosema apis. Pathogenic factors are a health concern for any colony and illness within the hive can be caused by infection of microscopic organisms. Nosema ceranae and Nosema apis, microsporidia, cause nosemosis, a degenerative gut disease. Nosemosis causes organism death which can expedite colony loss. Research being conducted on both parasitic fungi is relatively new and novel. Nosema ceranae, thought to be more virulent than N. apis has immense agricultural value. Shifting virulence from its original host A. cerana, to A. mellifera, dual infection of Nosema ceranae illustrates a challenge in infectious disease control. If rapid changeover in infective targets, continues to occur into all species of bees, colony collapse on a global scale could come to fruition. A. mellifera, the European bee is the more commonly used species in commercial agriculture. The move toward more generalized infection by Nosema ceranae, particularly of A. mellifera could amount in millions of agricultural industry dollars lost. There are limited techniques formulated to combat the parasitic fungi, yet methods are continuously improving. It is essential to develop a comprehensive analysis of said organisms to properly conserve bee populations.
Organismal Structure and Development
Nosema spp. are parasitic microsporidia. Generally these intracellular parasitic fungi infect specific host tissues, relying heavily on opportunistic relationships for proliferation. Nosema ceranae forms spores which are oval or rod shaped, consisting of 3.9-5.3µm in length and 2.0-2.5µm in width. Nosema apis produces spores that are typically 6µm in length and 3.0µm in width. As a generalized mechanism of replication, the microsporidia uses a polar filament to inject sporoplasm from a mature spore into a host cell's cytoplasm, where it reproduces . Further organism qualities are demonstrated in figure 3 shown below from Chen et al. . In conserved mechanisms, both parasitic infections are developmentally complex, maturing sequentially as follows. During early development, the microsporidia contain free ribosomes and a poorly developed rough endoplasmic reticulum in its cytoplasm. These structures form junctions with the host, integrating itself within a host cell. As a meront forms, connection with the organismal and host cytoplasm continues and a diplokarytic two nuclei is introduced into the host. Meronts replicate multiple times, and eventually give rise to the generation of oval sporonts, which are of similar size to merozoites but have a thicker plasma membrane. These sporonts eventually divide into sporoblasts, which divide and grow into mature spores . Matured spores incorporate a dense exospore, an anteriorly positioned anchoring disc and centralized diplokaryoticly arranged nuclei . A posteriorly placed polar filament coils, and the amount of coils is widely used as a determination of species type between Nosema ceranae and Nosema apis . Reproductive phases of the organism are schematically represented in the figure 2. Microsporidia lack proper machinery to autogenously produce energy. Utilizing host stores as supplemental material, the parasitic fungi are able to metabolize energy . Genomically, Nosema ceranae encompasses 2,614 protein coding genes, and contains AT biased repetitive regions increasing likelihood of genetic mutation due to error; N. apis possess smaller yet similar regions of repeats. With higher probability or mutation, these dangerous fungi are difficult to qualify. Despite correlation in function Nosema ceranae is genetically most similar to the wasp parasite N. vespula, as N. apis and N. bombi more closely related . Evolutionary branching differences often
Virulent spores which exist in food, water and fecal matter, are an essential elements to Nosema proliferation. Mature Nosema spores are able to exist outside a host for variable amounts of time, given conditions. Metabolically inactive yet viable spores on average can persist about 25-50 days outside a host. Research has even cited preservation of spore viability for more than a year in some cases. Spore health is contingent on delivery method into the host, concentration of virulent units and temperature at which administration occurs. Spores transmitted through syrup as opposed to honey display stronger virulence, producing larger densities of spores within hosts. However, delivery methods are usually dependent on where the inoculation occurs. Often, laboratory inoculations take place in favorable conditions as efforts are geared toward success. Furthermore, as storage temperature decreases, infection densities decrease . Spore health is considered sensitive up to -20oC, however germination of the spores was significantly hindered when exposed to temperature 4oC for 4 days . A hive, usually kept warm through activity and insulation maintains an ideal temperature range for infection. Not only is a hive environment suitable for infection, but Nosema ceranae and Nosema apis operate ideally at host temperature. Typically higher spore concentration ingested leads to more confluent spore growth within the organism. Higher probability of death from infection has been linked with dense fungal growth. Once ingested, spores go through replication in similar steps as covered previously .
N. apis sporulation occurs only in the host honey bee’s midgut epithelium, while Nosema ceranae was found to infect alimentary canals, malpighian tubules, hypopharyngeal glands, salivary glands and fat body . Nosema ceranae is potentially made more dangerous by the ability to infect multiple cell types. Once localized within specific cells, spore germination occurs. The microsporidium injects sporoplasm into the host cell through a polar tube, implanting virulent machinery into the cytoplasm of the host. The proliferation and sporogonic phase follows suit, using the timeline issued previously, and illustrated in the corresponding figure. Complete reproduction of a host cell within Microsporidia infection grows within tissue types until symptoms appear. These symptoms are discussed further below, but become more prevalent given higher growth.
Research has shown seasonality could be a factor in parasitic infection by both N. apis and Nosema ceranae. A 5 year German study released in 2010 analyzed incidences of infection by season. The highest prevalence of infection observed happened within the spring months. During springtime, the hive is going through individual cycling as older bees are replaced by new bees. Sampled bees contained a proportion of infection (22.4% - 35.4%) in the spring while fall months accounted for a smaller frequency (5.2% - 12.7%). It should be discussed however that a higher spring time density of Nemosa spp. did not correlate with colony loss during subsequent seasons . Although a promising study, more research needs to be conducted to determine a reliance on the seasons for infection. Additionally, the parasite is often transmitted in times of greater confinement. Therefore colony collapse which is linked to Nemosa infection has also been noted to occur more over the winter months. During the winter the bees are often found inside the hive, increasing contact with infected individuals .
Although a virulent entity, Nosema ceranae has not conclusively been linked to colony loss. Studies have correlated the parasitic infection with deteriorating health of a colony, eventually ending in collapse, and others have dismissed Nosema ceranae as colony threatening. For instance, in a small scale study done on controlled colonies, N. ceranae infection was found to cause colony collapse . However in other cases, it appears that there has been no correlation between colony loss and Nosema ceranae infection. In many areas where the parasite has been introduced, colony health remains constant, or not significantly affected. As pointed out, external or other effects may be coupling with infection, and ultimately causing complications . It is also quite possible that certain strains of bees or even fungi affect each other with different potency.
Interestingly, one of those “external factors” may be Fipronil as it was determined to enhance virulence of Nosema ceranaeinfection through synergistic modification. Fipronil is a general insecticide which targets harmful pests. In agriculture such an agent is sprayed on plants to avoid crop destruction; however it has been cited for a potential colony collapse instigator in bees . In their study, both stressors were cited for having a detrimental effect on bee health; however a combination of the two proved even more lethal. The mortality rate in bees exposed to a normal commercially used level of Fipronil and then being infected by Nosema ceranae increased dramatically. After the same amount of time, 84% of co-exposed bees passed away in comparison a mortality rate of 39% in singularly burdened samples. Studies in the past using different organisms have shown a similar synergistic relationship between insecticides, outside stressors and a microbial or fungal infection. Aufauvre et al. hypothesized that exposure to alternative stressors may make it easier for microbial or fungal infections to take hold; a similar concept to the human idea of being immune compromised . In the context of colony collapse, a synergistic effect between a commonly used insecticide and a growing infection is worrisome. Anytime a pesticide is used, non-target species and organisms are always in trouble. The crops in which the honeybee is responsible for pollinating may be covered with something that can increase incidence of death. This fact raises economic issues as well as the farmer now has to choose between natural pollination of crop or potential crop destruction. Artificial pollination does exist but it is not as effective as honeybee pollination.
N. apis infection has been potentially linked to death, but much like other types of infections, its virulence could depend such things as time of infection or dosing concentrations. Similarly Nosema ceranae infection is equally ambiguous. Given a limited database of research, experts find it hard to predict the exact implications of a Nosema ceranae infection. For instance, cage experiments conducted in 2010 produced promising results as inoculated organisms died within 8 days. Despite this however, many studies analyzing wild bees have been proved inconclusive . Further research needs to be conducted on the growing virulence of N. apis and Nosema ceranae.
Studies looking to mediate hive collapse for ecological and monetary gain focus on the symptoms of Nosema spp. It is important to make the distinction between an individual's health when subjected to infection, and how a particular virulent agent can affect an entire colonies health. Individuals obviously make up the colony, however a common cold does not kill the entire state. Bee hives are consisted of worker bees, larva and the queens. Infection may reach a subset of the hive but not all bees come into contact with each other. Infectious roulette is in practice when determining which organism has a higher probability of infection. Complicating matters, it is difficult to properly identify nosemosis. Initial analysis may consist of recognition of sick or dead bees around the hive or brown fecal marks in the combs. Initial qualitative analysis is usually done as a preliminary study. Too often do bee keepers try and remedy the sickness causing pesticide contamination of the hive . In order to fully determine the pathogen at work on any given “sick” colony, especially when dealing with Nosema, microscopy is required. Common laboratory microscopy technique allows for the elucidation of infection by identifying spore growth within target tissues. Much of the microscopy is seen in the figure seen above. Light microscopy and Electron microscopy are commonly used to study samples.
The downfall of individuals does not always mean the demise of a colony . N. apis and Nosema ceranae, are hypothesized to affect colony levels through individuals by atrophy of the hypopharyngeal glands. Nonetheless, despite sharing hosts, these species do not always share the same symptoms of infection. On a case by case basis, N. apis infection is not often coupled with visual signs of disease, but suffering bees may become whitish and swollen. Studies struggle to phenotypically quantify Nosema ceranae infection, displaying little to no outward symptoms. It seems the organisms get sick and die quickly.
In the context of hive health, N. apis infection constitutes an increase in dysentery within the population, which as previously stated could cause higher incidence of infection. Conversely, adverse effects are more widely debated within Nosema ceranae, as it has recently transitioned from A. cerana to Apis mellifera and research is limited. Aforementioned decreases in honey production, and higher death rates among workers are both commonly thought of as large hive encompassing responses to infection when focusing on Nosema ceranae . It should be noted that experimental application of symptom study within the organisms has determined similar growth rates across the Nosema species of interest, however, germination rates could be different . This is an important concept when analyzing the spread of Nosema species. Early detection methods would allow beekeepers to save colonies by separating healthy parts of the hive from sickly subsets.
Movement and Crossover
Much of the danger in the Nosema genus, particularly Nosema ceranae is expansion of virulence into new species of bees. Shipments and movements of goods around the world support world markets, international trade and global commerce, however provide a medium for transmission of parasites across before impassable boundaries. It is clear to researchers that Nosema ceranae has moved from solely infecting Eastern honey bees, to European honey bees as well. In 2007, a study investigated infectious crossover of Nosema ceranae from its native Asian host, Apis cerana, to the more pervasive Apis mellifera, within Finland. Researchers determined the ability for Nosema ceranae to disease European bees actually emerged in 1998, previously thought to be more recent and has quickly become more potent than N. apis. As seen in Figure 4 the study showed a much heavier spore count associated with Nosema ceranae infection than to that of N. apis, while a combination of the parasites an intermediate sporulation (1-158 x106, 1-34 x106, 1-83 x106 respectively). Mortality rate differed largely between species as well, Nosema ceranae accounting for 44% mortality, conversely N. apis only 8% mortality 15 days post inoculation . As mentioned before, studies have shown a higher density of infection perhaps leads to higher mortality. It appears virulent forms of Nosema ceranae when introduced into European bee populations induces individualized failure, eventually destabilizing colonies.
Infectious crossover has not only occurred in Europe, but within the United States as well. The United States is home to Apis mellifera, and is experiencing similar transition as European based bees. Much like the work done by Paxton et al., the Chen et al. research team concluded that Nosema ceranae had become adapted to infect European bees well before previously thought [14,2]. Infection of these bees were found across 12 states and analyzed throughout time (1995-2007) . Parasitic infectious agents are an extremely real problem, and the idea that they have spread throughout all populations is troubling.
Control of Disease
Given the global exchange of goods, a ubiquitous mechanism of disease control is closer surveillance of shipped goods.
Many virulent effectors travel along product routes on the backs of desired goods. This shipment process can be from overseas to cross county exchanges. It is important for beekeepers, farmers and naturalists to monitor honeybee populations, to make sure they are free of Nosema infection before shipment of goods. Compounding the issue of containment is the movement between bee strains of specified fungi as addressed earlier. As with most infectious disease, containment and suppression of dispersion is a broad scale but significant.
Smaller scale efforts can be made to combat the virulent nature of N. apis and Nosema ceranae. In many instances, given the importance of the Western honeybee Apis mellifera, the maintenance of colonial health is crucial. In organismal medicine fungal infections are often matched with an antibiotic, however given the recent discovery of Nosema ceranae research on possible antibiotic treatments of diseased bees is limited. Fumagilin-B or fumagillin dicyclohexylammonium, an antibiotic which is derived from the microbe Aspergillus fumigatus . This microbial produced antibiotic disrupts the DNA replication of the target parasite stopping its ability to properly reproduce. Inoculation methodology must progress as the medicine needs to be ingested by the organisms in order to take effect. In N. apis Fumagilli-B has been shown to work effectively given proper drug administration . However in Nosema ceranae results are more variable. In a study done in 2010, the variability was attributed to intake methodology, and lack of knowledge about Nosema ceranae infection. Under the qualifications of the Williams et al. experiment, Nosema ceranae was not a key factor in colony health either . It has become readily apparent that more research needs to be done into finding a workable antibiotic for Nosema ceranae infection of Apis mellifera.
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