The Effects of Nosema apis and Nosema ceranae Infection on Survival and Phenoloxidase Gene Expression in Galleria mellonella (Lepidoptera: Galleriidae) Compared to Apismellifera

The study aims to prove the possibility of colonization of N. apis and N. ceranae to the intestine of the greater wax moth, detect the differences of greater wax moth based on the presence of Nosema species and examine the effect of Nosema species on the phenoloxidase level of greater wax moth compared with honeybees. Each group was fed on the 1st day of the experiment with its appropriate diet containing 106 Nosema spores per insect. Each group was checked daily, and dead insects were counted. Furthermore, changes in the level of expression of the phenoloxidase-related gene after Nosema spp. treatment on the 6th, 9th and 12th days, which was detected by Q-PCR, and the mRNA level of phenoloxidase gene were measured in all experiment groups with the CFX Connect Real-Time PCR Detection System. This study shows that Apis mellifera L. has a 66.7% mortality rate in mixed Nosema infections, a 50% mortality rate in N. ceranae infection, a 40% mortality rate in N. apis infection, while there is no death in G. mellonella. A significant difference was found in the mixed Nosema infection group compared to the single Nosema infection groups by means of A. mellifera and G. mellonella (Duncan, p < 0.05). G. mellonella histopathology also shows that Nosema spores multiply in the epithelial cells of greater wax moth without causing any death. The increase in the mRNA level of Phenoloxidase gene in A. mellifera was detected (Kruskal–Wallis, p < 0.05), while the mRNA level of the Phenoloxidase gene did not change in G. mellonella (Kruskal–Wallis, p > 0.05). These findings prove that the Nosema species can colonize into the greater wax moth, which contributes to the dissemination of these Nosema species between beehives.

Variation in the Distribution of Nosema Species in Honeybees (Apis mellifera Linnaeus) between the Neighboring Countries Estonia and Latvia

The unicellular spore-forming parasites Nosema apis and Nosema ceranae are considered to be one of the causes of increased honey bee mortality in recent years. These pathogens attack their honey bee hosts through their gut, causing changes in behavioral stress responses and possibly resulting in decreased honey yield and increased honey bee mortality. The present study aimed to determine the prevalence of Nosema spp. (nosemosis) in Estonia and Latvia, as well as the persistence of the disease in previously infected hives. Currently, N. ceranae is considered the most virulent species and is predominant worldwide. However, in some regions, usually with colder climates, N. apis is still prevalent. To achieve better disease control, it is important to determine the species distribution. For this purpose, we selected 30 apiaries in Estonia and 60 in Latvia that were positive for Nosema spp. in the EPILOBEE (2012–2014) study, which was 5 years prior to the present study. The results show that, while both species are present in Estonia and Latvia, N. apis is dominant in Estonia (43%), and N. ceranae is dominant in Latvia (47%). We also found that the pathogens are very persistent, since 5 years later, only 33% of infected apiaries in Estonia and 20% of infected apiaries in Latvia, we could not detect any pathogens at the time of sampling.

Nosema ceranae - a new threat to honey bees (Apis mellifera)?

Back in 1900 already, the Microsporidium Nosema apis was described inApis mellifera. Thereby the Nosemosis remains without symptoms in the beehive to a certain degree. Studies indicate that infected bees have a shortened lifespan, due to a series of changes in physiological parameters. The consequence of these changes are diarrheal symptoms and the spread of infectious spores in thehive. There is also a seasonal infection course observed, which has its peak in spring time (April, May). Colloquially, the Nosemosis is therefore also known as spring shrinking craze. More recently, a new Nosema species in the European honey bee has been described, where a host-switch from the Asian honey bee A. cerana to A. mellifera has occurred. N. ceranae is blamed for colony losses in the south of Spain, many general colony losses during wintertime in Europe and has also a contribution to the Colony Collapse Disorder (CCD) in the U.S. It seems likely that the original Nosema species (N. apis) is displaced more and more by N. ceranae for unknown reasons. Within the EU project „BEE DOC“, monitoring studies on colonies in southern Germany, Switzerland, southern France, Sweden and Finland were performed. Although the high prevalence of N. ceranae could be confirmed, no increased colony mortality due to Nosemosis was recorded. This was also observed by other colleagues and thus the „new threat“ is open to debate.

Nosema apis and Nosema ceranae Tissue Tropism in Worker Honey Bees (Apis mellifera)

The microsporidia Nosema apis and Nosema ceranae are major honey bee pathogens that possess different characteristics in terms of the signs they produce, as well as disease development and transmission. Although the ventricular epithelium is generally considered the target tissue, indirect observations led to speculation that N. ceranae may also target other structures, possibly explaining at least some of the differences between these 2 species. To investigate the tropism of Nosema for honey bee tissues, we performed controlled laboratory infections by orally administering doses of 50 000 or 100 000 fresh mature spores of either species. The fat body was isolated from the infected bees, as well as organs from the digestive (esophagus, ventriculus, ileum, rectum), excretory (Malpighian tubules), circulatory (aorta, heart), respiratory (thoracic tracheas), exocrine (hypopharyngeal, mandibular and labial, cephalic, thoracic salivary glands), and sensory/nervous (brain, eyes and associated nerve structures, thoracic nerve ganglia) systems. Tissues were examined by light and electron microscopy at 7, 10, and 15 days postinfection. Both Nosema species were found to infect epithelial cells and clusters of regenerative cells in the ventriculus, and while the ileum and rectum contained spores of the microsporidia in the lumen, these structures did not show overt lesions. No stages of the parasites or cellular lesions were detected in the other organs tested, confirming the high tropism of both species for the ventricular epithelium cells. Thus, these direct histopathological observations indicate that neither of these 2 Nosema species exhibit tropism for honey bee organs other than the ventriculus.

First Detection of Nosema Ceranae and Nosema Apis in Greater Wax Moth Galleria Mellonella

The greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae), one of the pests of honey bee (Apis mellifera L.) colonies, has spread almost all over the world. Although the G. mellonella is often reported to infest weak honey bee colonies that are exposed to pesticides and diseases, it is also a threat for healthy colonies. Therefore, there is a fairly high probability of transmission of both microflora-specific bacteria and pathogen microorganisms, especially Nosema species, between these organisms (Moth and bees). The aim of this study was to investigate the presence of Nosema species in greater wax moth G. mellonella collected from apiaries as well as grown in laboratory conditions. Adults and late instar larva of wax moth were used for detecting Nosema apis and Nosema ceranae. Real-time PCR amplification studies were performed and specific ITS regions were targeted to distinguish Nosema species. Real-time PCR results showed that N. apis and N. ceranae were found in both phases of G. mellonella. This is the first study to confirm that N. apis and N. ceranae are present in greater wax moth collected from apiaries and grown at laboratories in Turkey.

Determination of spore longevity and viability of Nosema apis and Nosema ceranae according to storage conditions

Honey bee colonies are often infected with Nosema apis and Nosema ceranae which cause adult honey bee disease called nosemosis. All honey bee colony members can be infected with these species. In addition, it is claimed to be the main cause of honey bee winter losses in many countries. Nosema spores are expected to resistant the environmental conditions and their infectivity continues for a long time because of long-term durability of fungal spores. In this study, the viability of Nosema spores were investigated in terms of storage situations under laboratory conditions. Honey bee samples that were collected from apiaries in 2011 were investigated to detect the presence of Nosema species with real-time PCR amplification studies. After determination of Nosema species, each sample was divided in two groups. One of these groups was used to find Nosema spore concentration. Nosema solutions were divided and stored at both -20°C and +4°C. The spore concentration was measured every year in the period 2011-2015. Other group of honey bee samples was also stored at -20°C and every year was used for Nosema spore counting. Furthermore, it was examined the infectivity of Nosema spores with sugar solutions which obtained each sample using cage experiment techniques. According to results, when we compare the solutions annually, there is no change at Nosema spore concentration of the solution in -20°C and honeybee samples in -20°C. But reduction was seen at Nosema spore concentration of the solution in +4°C. Nosema spore infectivity tests revealed that infectivity of Nosema spores has not changed significantly between 2011 and 2015. This is the first time mixed Nosema spores found more infective than one-type spore after prolonged exposure to different conditions.