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Nosema Apis a Parasite of Honey Bees

Nosema apis (Zander) is a microsporidian, it is a small, single celled parasite affecting honey bees. It causes nosemosis, mainly known by the term nosema. Although parasitic, it is often thought of and talked about as a disease. A single spore can cause infection, but the mean infective dose is generally reported to be between 20 and 90 spores per bee.

The dormant spore stage of nosema is resistant to extremes of temperature and dehydration and the spores are long lived.

Symptoms evident

The symptoms can be confused with other honey bee problems, but they are genarally...

dysentery

Shorter life of worker bees

Lack of industry in the colony (slowness of development)

Lower than normal propensity to sting

Crawling bees

Disjointed wings

Distended abdomens

High propensity to supersede

I have included disjointed wings, because many other sources quote this symptom, but I have not recognised this feature myself, maybe the effect is less severe than the 'K' wing associated with Acarine infestation.

Bees crawling in front of the hive unable to fly may be infected by nosema but there are other possible causes of this. Crawling bees, trembling wings and dislocated wings are associated with Chronic Bee Paralysis Virus (CBPV).

Transmission and spread

Spores need be ingested by a bee for the infection to be effected. Spores germinate quickly after entering the ventriculus, and the epithelial cells of the ventriculus are infected when the vegetative stage is introduced by way of the extended tubular polar filament allowing inoculation of the sporoplasm into the host cell. Once inside a cell, the vegetative stage increases in size and multiplies, effecting an apparent reduction of RNA synthesis in the host cell. In 6-10 days the infected host epithelial cell becomes filled with new spores. Epithelial cells are shed into the ventriculus where they burst - normally releasing digestive enzymes. The infected cells are shed similarly, but they release 30-50 million infective spores when they burst. With such a rapid amplification of spores, infection is rapidly spread.

Effects on the hive

Queens may become infected from various sources after they emerge from the queen cell. When the disease is severe, colony populations may become depleted and eventually reduce to a handful of bees and a queen. This is often known as "spring dwindling". Colonies that are only mildly affected can recover.

Nosema spores spread easily to other bees by spores in feaces that are voided inside the hive. The disease disrupts the digestion of pollen and shortens the life of the individual bee. A greater proportion of worker bees become infected than drones or queens, likely because of natural comb cleaning activities of young bees in which drones and queens do not participate. Nosema infected bees do not attend or feed the queen to the same extent as healthy bees, which helps the queen to escape infection. When the queen does become infected her ovaries degenerate and her egg laying rate is reduced due to atrophy of the oocytes. Queens that become infected by the parasite during the brood rearing season are likely to be superseded by the bees.

Nosema infection causes earlier worker orientation and commencement of foraging, along with earlier degeneration of hypopharyngeal glands.

There is a seasonal trend to infection, low levels during summer, a small peak during Autumn, and a slow rise of infection during the Winter. In early spring the level of infection increases rapidly when brood rearing starts and flight is limited due to low temperatures. Nosema spores, appearance under microscope

Diagnosis

You can look for the long lived sausage shaped or rice grain shaped spores using a microscope. These spores are 4 to 6µm long and 2 to 4µm wide. They can be seen with a with a magnification between 400x to 1000x (600x prefered). Prepare your sample by macerating 10 or 15 bees under water. Use an opaque medium like Indian (Cuttlefish) ink on the slide to provide contrast.

But nosema can be detected without a microscope... Place your finger nail firmly on the sting and pull the head off exposing the mid gut. The midgut is normally tan and has a distinctive ringed appearance (similar to the segmented body of an earthworm). A midgut that is infected with nosema is off white and is a little swollen and appears slightly less segmented in appearance. This crude field test should be confirmed by microscopy if nosema is suspected to be present.

Treatment

There are two schools of thought on this... My personal preference is not to treat and thus lose the susceptible bees from my collection of stock. This helps to weed out genetic weakness and promotes bees that are capable of withstanding the conditions that they are expected to work in.

Treatment can be given using the antibiotic Fumidil 'B' which is prepared from Aspergillus fumigatus, which is the causative agent of Stone Brood. Fumidil 'B' inhibits the reproduction of spores, but the antibiotic does not kill the spores themselves. Heat treatment of infected equipment, at 49° C for 24 hours or more, can be used to kill the spores.

This medication should not be fed to colonies when there is danger of contaminating the honey crop.

Life cycle of Nosema apis

This can be identified as several separate stages within two phases, the first of these stages is the spore itself, which has a twin nucleus as well as sporoplasm. The filament ranges from 0.1 to 0.2µm in diameter and 50 to 500µm in length. Anteriorly, the coiled polar filament passes through the polaroplast and attaches to an anchoring disc at the apex of the cell.

Stage two On entering the intestine the tubule becomes extruded by eversion, the dense glycoprotein core becomes an outer protective layer. The free end of the tube inserts through the membrane of the host epithelial cell. Nosema Apis after extension of polar filiament

Nosema spore injecting sporoplasm into epithelial cell

The sporoplasm is injected into the along the lumen of the polar filament into the host cell in a short time (15 to 500 msec ). This is part of merogony phase and the infectious element is known as the meront. Nosema spore injecting sporoplasm into epithelial cell

Stage four (merogony)at left is the start of a progressive process of manufacturing more spores

In the host cells they grow and repeatedly divide asexually. The mature parasites (trophozoites) eventually give rise to sexually produced zygotes that produce new spores. However, the honey bee does not secrete digestive juices into the ventriculus. Under normal conditions honey bee epithelial cells shed into the ventriculus (stomach), burst, and release their contents including digestive juices. However, when the cells are infected with N. apis the parasite develops and multiplies in the cytoplasm and form after about 5 days. The spore-filled cells are shed into the lumen. Some cells pass into the rectum and are voided. The spore-filled cells burst and release infective spores rather than digestive juices. If the cells burst in the lumen they may release spores that quickly germinate, infecting additional epithelial cells (Morse and Nowogrodzki, 1990). Spore germination is initiated by a signal from the species' environment; the exact nature of these signals is not known for certain. Upon receiving this signal, the spore swells and the sporoplasmic membranes break down, culminating in the rupture of the anchoring disk. The polar tube is basically shot out of the spore, propelled by the internal pressure. After the polar tube is completely expelled, the sporoplasm enters the tube More spores are produced through sporogony and eventually take over the host cell. At infection, the polar filament will very quickly elongate the polar tube (up to many times the length of the spore) [FK01]. The polar tube or filament (the filament is simply an extension of the tube) is composed of membrane and glycoprotein layers [KF02] and appears to have considerable internal structure, as shown in the image of Tuzetia [C+02]. There is some physical association between the end of the polar filament and the posterior vacuole, but the precise nature and function of this contact are currently speculative The spore extrudes the polar tube by eversion. That is, it turns inside-out with the dense glycoprotein core becoming an outer protective layer [KF02]. The free end of the tube inserts through the cell membrane of the host. The polar tube serves as a pliable hose through which the infectious sporoplasm is pumped into the host cell in 15 to 500 msec The sporoplasm is probably forced through the polar tube by osmotic pressure. The spores are permeable to water and, as a result of high solute concentrations, presumably have high turgor pressures even in the inactive state. At activation, microporidians use various means to increase this turgor, and its effectiveness. In one, well-studied system, the glucose disaccharide, trehalose, is rapidly hydrolyzed to glucose. Since osmotic pressure depends on the number of solute molecules, and not their mass, this results in a sharp spike in pressure. Likewise, other species may rapidly flood the cytoplasm with calcium ions, which has precisely the same effect. The sporoplasm consists of the nuclei and surrounding cytoplasm. Microsporidian ribosomes are a large component of the sporoplasm; and these ribosomes promote a very high rate of protein synthesis during the initial infective cycle [B+02]. When the sporoplasm emerges from the tube, it has somehow already acquired a new cell membrane. This is thought to derive from elements of the polaroplast which precede the sporoplasm through the tube [KF02]. Inside the host cell, the nuclear material in the sporoplasm replicates extensively, either in direct contact with the host cytoplasm or inside a parasitophorous vacuole. Although there is considerable variation, a typical microsporidian may replicate by merogony for some initial period, once it is inside the host cell. During this period, in some cases, the nuclei may proliferate with or without division into individual cells [C+02]. Within the first 24-48 hours after the sporoplasm has reached the host cell, several rounds of division have occurred. Synthesis of the spore coats and sporogony then begin [B+02]. In species which reproduce within a vacuole, the initial steps in replication take place in close association with the inner membrane of the vacuole. The transition to sporogony is marked by release of the developing spore into the lumen of the vacuole [B+00] and the accumulation of electron dense material near the periphery of the cell [H+01]. Both merozonts and sporozonts show little internal organization [H+01] (see also images from [B+00]). In at least two, widely divergent systems, electron-dense extracellular tubules have been observed surrounding developing spores during sporogony [B+00] [C+02]. When the spores completely fill the host cell cytoplasm, the cell lyses and releases the spores to the surroundings. In some systems, the microsporidian infestation may cause the development of xenomas.

References

* The Hive and the honeybee, Chapter 21 T.A. Gochauer, B. Furgala, H. Shimanuki, 1975 published by Dadant * [1] University of Georgia Honeybee Program * [2] University of Florida IFAS Extension, Malcolm t. Sanford Bailey, L. (1963), Infectious Diseases of the Honey Bee * [3] Swedish University of Agricultural Sciences Ingemar Fries, 1993 Morganthaler, Bailey 1963

Inside the intestine the tubule becomes extruded and penetrates the peritrophic membrane thus entering an intestinal cell. The sporoplasm is injected into the epithelial cell along the lumen of the polar filament. 4�12 The sporoplasm (9) grows and asexually divides via quadrinucleate stages in its host cell (). Finally, (i.e. spore formation) is initiated from a diplokaryon stage (10) via a final division (). 13 When mature spores are present, host cells are disrupted and release the infectious spores into the lumen, which are voided with the feces (or infect neighboring cells). At the end of summer, development of Nosema apis may become reduced () and starts again in spring. As well as the intestine, all organs of bees become parasitized. CW, cyst wall; EN, encystation; HC, host cell; N, nucleus; NH, nucleus of host cell; PP, SP, sporoplasm; TI, tubule-injected; TU, tubule (polar filament)

Written... Summer 2000, Upgraded... 20 September 2006,