By: Clarence Collison
IMPACT OFNOSEMA DISEASE ON PRODUCTIVE CASTES
The biological impact of Nosema disease has mostly been associated with worker bees, whereas drones and queens are generally considered to be more resistant or less susceptible to infection and the dangers to them have been somewhat underestimated.
Microsporidiosis of adult honey bees caused by Nosema apis and Nosema ceranae is a common worldwide disease with negative impacts on colony strength and productivity. The role of the queen in bee population renewal and the replacement of bee losses due to Nosema infection is vital to maintain colony homeostasis. Younger queens have a greater egg-laying potential and they produce a greater proportion of uninfected newly eclosed bees to compensate for adult bee losses. Botias et al. (2012) performed a field study to determine the effect of induced queen replacement on Nosema infection in honey bee colonies, focusing on colony strength and honey production. In addition, the impact of long-term Nosema infection of a colony on the ovaries and ventriculus (midgut) of the queen was evaluated. Queen replacement resulted in a remarkable decrease in the rates of Nosema infection, comparable with that induced by fumagillin treatment. However, detrimental effects on the overall colony state were observed due to the combined effects of stressors such as the queenless condition, lack of brood and high infection rates.
The biological impacts of Nosema disease has mostly been associated with worker bees, whereas drones and queens are generally considered to be more resistant or less susceptible to infection and the dangers to them have been somewhat underestimated (Loskotova et al. 1980). Queens can be infected by both N. apis and N. ceranae (Traver and Fell 2012; Webster et al. 2004) and N. ceranae has even been detected in larval queens (Traver and Fell 2012). Most transmission of Nosema spp., presumably occurs during the adult stage, including mating, although antimicrobial molecules in drones’ semen are able to kill N. apis spores and reduce the risk of disease transmission during mating (Peng et al. 2016). Nosemosis in queens causes aberrant physiology, as well as similar gut lesions and metabolic costs as in workers (Alaux et al. 2011: Higes et al. 2009). In addition, queens infected by N. apis start oviposition later than healthy ones (Hassanein 1951; Loskotova et al. 1980), display changed pheromone production (Alaux et al. 2011) and in extreme cases their oocytes degenerate leading to infertility (Liu 1992). N. apis infection may severely reduce queen lifespan to an average of nearly 50 days, resulting in queen supersedure (Loskotova et al. 1980). Compensatory increases in the level of vitellogenin and other antioxidant enzymes occur in infected queens (Alaux et al. 2011). These counterintuitive changes may be protective mechanisms that are too costly in the long-term for the infected queen to survive (Amiri et al. 2017).
When a queen honey bee becomes infected with Nosema apis, the result can be very serious indeed for her colony. The metabolic processes are disturbed by the damage done by the parasite to the epithelial cells of the mid-gut, and this apparently leads to severe damage to the ovaries, at first by the production of a high proportion of eggs that fail to hatch, and ultimately by complete cessation of oviposition and suppression or death of the queen. Although large numbers of eggs, larvae, and pupae produced by infected queen honey bees were examined, none was found to be infected with any stage of Nosema apis (Hassanein 1951).
Terminal oocytes (egg production) containing yolk in both healthy and nosema infected queen honey bees were studied (Liu 1992). In the healthy queens the terminal oocytes exhibited a layer of follicular cells which were covered by a smooth-surfaced ovariole sheath. In the ooplasm were numerous electrodense yolk granules and lipid yolk droplets. The electron-dense yolk granules exhibited crystalline structure. Stacks of endoplasmic reticulum were observed in the yolk granules throughout the ooplasm. Numerous mitochondria possessing well defined cristae were also observed. Oocytes in the ovary of queen honey bees appeared degenerated after seven days of infection by Nosema apis. The ovariole sheath was wrinkled. In the ooplasm, yolk granules were broken down into small spheres and granular substances. Numerous ribosomes without stacks of endoplasmic reticulate were observed. Lysosomes were abundant and numerous electron-dense materials surrounded by a membrane were detected. The oocytes appeared to be extensively autolysed (cell destruction through the action of its own enzymes).
Queen honey bees were inoculated with known numbers of Nosema apis Zander spores and introduced into frame nuclei. Inoculations with as few as 1000 spores resulted in supersedure. All superseded queens recovered were found infected. The degree of infection, and the time which elaspsed before supersedure resulted, varied within a given dosage level. Some inoculated queens survived and were found free of spores, suggesting that selection for resistance to Nosema disease is possible (Furgala 1962).
Worker and queen honey bees were fed individually with N. apis spores in sucrose solution and then returned to cages containing several hundred of their worker bee nestmates. After three to seven days, the workers and queens that had been fed spores were sacrificed. Worker and queen ventriculi (midguts) were removed and examined for spores by light microscopy, and DNA was extracted. The DNA was subjected to amplification with polymerase chain reaction (PCR), using primer sequences specific to N. apis DNA. The PCR analysis was more sensitive than examination for spores by light microscopy, in detecting N. apis infection. Worker bees and queen bees were infected at similar rates by the inoculation procedure (Webster et al. 2004).
Traver and Fell (2012) also investigated whether queens from colonies with a known N. ceranae infection can become naturally infected and, if so, whether immature queens are also infected. They were also interested in determining whether N. ceranae could infect other tissues which might be involved in vertical transmission, such as the ovaries and/or spermatheca. Queens were analyzed using real-time PCR and included larval queens, newly emerged and older mated queens. Overall, they found that all tissues examined were infected with N. ceranae at low levels but no samples were infected with Nosema apis.
Nosemosis in queens causes aberrant physiology, as well as similar gut lesions and metabolic costs as in workers.
Larval queens and newly emerged queens were analyzed to determine whether N. ceranae can be transmitted to developing queens, i.e. through brood food. Larval queens were infected at low levels and N. ceranae DNA was detected in royal jelly; however they could not rule out the possibility that this was due to contamination since royal jelly samples were not decontaminated with bleach. A subsample of royal jelly was used for spore counting, but spores were not observed in any sample. Because N. ceranae was detected in royal jelly samples, brood food could provide a mechanism for the horizontal transmission of N. ceranae to all developing bees; however, infectivity studies for royal jelly are still needed (Traver and Fell 2012).
In newly emerged queens, Traver and Fell (2012) found abdomens, thoraces, heads, and ovaries to be infected with low levels of N. ceranae. Overall trends indicated that abdomens (minus the reproductive organs from the same queens) tend to have higher levels of infections compared to other tissues; though in some cases, ovaries were found to have a higher level of infection than the other tissues examined. The analysis of mated, laying queens also indicated the presence of N. ceranae and these infections had spread to other tissues such as the ovaries. Low levels of N. ceranae in the ovaries and spermatheca suggest that vertical transmission could be involved in transmitting N. ceranae.
Alaux et al. (2011) analyzed the impact of Nosema ceranae on queen physiology. They found that infection by N. ceranae did not affect the fat body content (an indicator of energy stores) but did alter the vitellogenin titer (an indicator of fertility and longevity), the total antioxidant capacity and the queen mandibular pheromones, which surprisingly were all significantly increased in Nosema-infected queens. Thus, such physiological changes may impact queen health, leading to changes in pheromone production, that could explain Nosema-induced supersedure (queen replacement).
Since the prevalence of drone infection by N. ceranae was unknown, Traver and Fell (2011) set out to determine whether drones are naturally infected with N. ceranae and at what levels. Drones were analyzed for N. ceranae infections using quantitative real-time PCR with species specific primers and probes. Drone pupae were collected from capped cells at the purple eye stage (with body pigmentation) and were approximately 17-23 days old. In-hive and flying drones were also sampled. They found that both immature and mature drones are infected with N. ceranae at low levels. No N. apis infections were detected in drones of any age. Average spore counts were 9,436 and 13,839 spores per bee for in-hive and flying drones, respectively. Only 19.6% of in-hive drones had sufficient spore numbers to count. Drone pupae were infected at low levels and most frequently in May and June and this is the first report that has detected N. ceranae in immature bees. If pupae are infected before emergence, they may be infected through brood food or contamination in their cells. The low level infections found in pupae could be due to developmental changes associated with pupation and reorganization of the alimentary canal. In-hive drones had the highest infections in June. For both drone pupae and in-hive drones, the highest levels of infection coincided with high levels of infection in the sampled hives. Mature, flying drones were also infected, but generally at lower levels and may be due to flying drones not returning to the hive or because heavily infected drones do not survive as long. Because drones are known to drift from their parent hives to other hives, they could provide a means for disease spread within and between apiaries.
When a queen honey bee becomes infected with Nosema apis, the result can be very serious indeed for her colony.
Peng et al. (2015) was able to show that Nosema apis is able to infect drones and that these infections built up to a point where they induced significant costs for males. They found a reduction in fertility and life span as drones aged, as well as ejaculate of infected drones becoming contaminated with spores. Honey bee drones become sexually mature about 12 days after emergence and consequently maintain their maximal fertility potential over a time period of approximately 10 days. During that time, they participate in nuptial flights in order to find and mate with virgin queens. The phenotypic expression of a N. apis infection such as spores in the ejaculate and a reduction of drone fertility and survival therefore affects these drones during their main reproductive period.
These findings indicate that drones which become infected shortly after hatching will eventually face substantial fitness costs and pose an infection threat to virgin queens in case they mate. More research is required to quantify the risk of vertical transmission posed by males infected with N. apis.
Although their visual inspections both morphologically as well as histologically, did not reveal any obvious signs of N. apis infections in reproductive tissues or their products, DNA of N. apis can be detected in both accessory glands and testes. Even though they found that N. apis is able to establish low levels of infections in reproductive tissues, no spores were found which would indicate that the pathogen seems unable to complete its reproductive cycle or to build up an infection. Therefore, even though N. apis is able to infect drone testes and accessory glands, the drones seem able to slow down or prevent this parasite from producing spores within their reproductive tissue, thereby reducing the risk of sexual transmission. This is an interesting finding, because N. apis infections are already known to spread to different honey bee organs such as the fat body, the Malpigian tubules or the hemolymph (Webster et al. 2004). In comparison, Nosema ceranae has been reported to infect drones at the pupal stage (Traver and Fell 2011), to reduce drone body weight and life span and to induce physiological changes in honey bee queens (Alaux et al. 2011).
Nosema apis has been reported to be present in ejaculates of honey bee drones, and artificial insemination experiments have confirmed that this pathogen can in principle be transmitted during matings (Roberts et al. 2015). They conducted a series of experiments to unravel the effects of a N. apis infection on a male’s somatic (relating to the body) tissue, by inspecting infected somatic tissues of drones at different ages and comparing the survival of infected and uninfected drones. They also investigated whether drones are able to protect their reproductive organs and their ejaculates from N. apis infections to minimize the risk of transmitting infective spores during copulation.
Nosema was present in 69% of the drone semen samples examined. Semen sampled in 2011 had both N. apis and N. ceranae, but with much lower intensities of N. apis, while semen sampled in 2012 had only N. ceranae. No Nosema was detected in any of the tissue samples (spermatheca, ovary, gut) from any of the control queens that had been inseminated with sterile semen diluent. Ten of the 13 queens inseminated with Nosema spores were found to subsequently be positive for N. apis and/or N. ceranae. The prevalence and intensities of infections differed significantly between N. apis and N. ceranae. Infections of N. ceranae were far more prevalent and intense than those of N. apis and were found in all tissues, whereas those of N. apis were found only in the gut. Queens inseminated with Nosema-infected semen were subsequently found to be positive for Nosema but at a much lower frequency than found in the experiment when the queens were inseminated with Nosema spores. None of the 400 eggs laid by queens that were either naturally infected with Nosema or had been inseminated with semen containing Nosema, were found to carry the parasite (vertical transmission).
Alaux, C., M. Folschweiller, C. McDonnell, D. Beslay, M. Cousin, C. Dussaubat, J.-L. Brunet and Y. Le Conte 2011. Pathological effects of the microsporidium Nosema ceranae on honey bee queen physiology (Apis mellifera). J. Invertebr. Pathol. 106: 380-385.
Amiri, E., M.K. Strand, O. Rueppell and D.R. Tarpy 2017. Queen quality and the impact of honey bee diseases on queen health: potential for interactions between two major threats to colony health. Insects 8: 48; doi: 10.3390/insects8020648.
Botías, C., R. Martín-Hernández, J. Días, P. García-Palencia, M. Matabuena, Á. Juarranz, L. Barrios, A. Meana, A. Nanetti and M. Higes 2012. The effect of induced queen replacement on Nosema spp. infection in honey bee (Apis mellifera iberiensis) colonies. Environ. Microbiol. 14: 845-859.
Furgala, B. 1962. The effect of the intensity of nosema inoculum on queen supersedure in the honey bee Apis mellifera Linnaeus. J. Insect Pathol. 4: 429-432.
Hassanein, M.H. 1951. Studies on the effect of infection with Nosema apis on the physiology of the queen honey-bee. Q. J. Microsc. Sci. 92: 225-231.
Higes, M., R. Martín-Hemández, P. Garcia-Palencia, P. Marin and A. Meana 2009. Horizontal transmission of Nosema ceranae (Microsporidia) from worker honeybees to queens (Apis mellifera). Environ. Microbiol. Reports 1: 495-498.
Liu, T.P. 1992. Oocytes degeneration in the queen honey bee after infection by Nosema apis. Tissue Cell 24: 131-136.
Loskotova, J., M. Peroutka and V. Vesely 1980. Nosema disease of honeybee queens (Apis mellifica L.) Apidologie 11: 153-161.
Peng, Y., B. Baer-Imhoof, A.H. Millar and B. Baer 2015. Consequences of Nosema apis infection for male honey bees and their fertility. Sci. Rep. 5:10565; doi: 10.1038/srep10565.
Peng, Y., J. Grassl, A.H. Millar, and B. Baer 2016. Seminal fluid of honeybees contains multiple mechanisms to combat infections of the sexually transmitted pathogen Nosema apis. Proc. R. Soc. B Sci. 283: 20151785.
Roberts, K.E., S.E.F. Evison, B. Baer and W.O.H. Hughes 2015. The cost of promiscuity: sexual transmission of Nosema microsporidian parasites in polyandrous honey bees. Sci. Rep. 5: 10982, doi: 10.1038/srep10982.
Traver, B.E. and R.D. Fell 2011. Nosema ceranae in drone honey bees (Apis mellifera). J. Invertebr. Pathol. 107: 234-236.
Traver, B.E. and R.D. Fell 2012. Low natural levels of Nosema ceranae in Apis mellifera queens. J. Invertebr. Pathol. 110: 408-410.
Webster, T.C., K.W. Pomper, G. Hunt, E.M. Thacker and S.C. Jones 2004. Nosema apis infection in worker and queen Apis mellifera. Apidologie 35: 49-54.
Clarence Collison is an Emeritus Professor of Entomology and Department Head Emeritus of Entomology and Plant Pathology at Mississippi State University, Mississippi State, MS.