"Mystery Spores" in Bee Macerates

Once way apiculturists assess their bee colonies for the relatively common microsporidian gut parasite Nosema and the need to treat their apiary against this pathogen or not (with fumagillin), is by doing a Nosema spore count from macerated honey bee abdomens.  If you've done this and looked at enough bee macerate samples, you may have seen the occasional "Mystery Spore".  You may have felt pretty certain it wasn't Nosema, pollen, or bee cellular debris but beyond that you weren't quite sure what it was you were looking at.  Often, I've heard people use the term "fried egg" cells as a general descriptor for such mystery spores.

"Mystery Spores" can be an important issue for at least 3 reasons:  

1) Getting an accurate Nosema spore count for research or treatment purposes can potentially be wildly incorrect if you confuse the sometimes ubiquitous (e.g. see "Mystery Spores #1") mystery spores for Nosema.

2) If they are fungal spores, some species of Aspergillus mold can be pathogenic to bees including A. flavus, A. nomius and A. phoenicis (Foley et al. 2014).

3)  If they are yeast spores, yeast communities in honey bees are poorly characterized and are a potentially diverse group of organisms associated with bee colonies that could use more research.


Below are two examples of "Mystery Spores" from honey bees that colleagues have sent me this spring.

Mystery Spores #1:  (image credit: Jim Burritt)

Background on Mystery Spores #1
Geographic location:  Wisconsin, USA.
Sample preparation:  fresh and in buffer.
Tissue source:  midgut specifically (carefully dissected out).
Associated with winter collapsed colonies (no, not CCD just dead-outs).
Cultivation:  unsuccessful growth on Sab Dex medium.
Notes:  Very abundant in source bees.  Lots of variation in size of nucleus.  


Mystery Spores #2:  (image credit: Michael Peirson and Carlos Castillo)

Background on Mystery Spores #2
Geographic location:  Alberta, Canada.
Sample preparation:  ethanol preserved or fresh frozen then suspended in PBS buffer.
Tissue source:  whole bee macerate.
Other source:  similar looking organisms were seen in pollen patties.
Notes:  Associated with bees fed with pollen patties; colonies without pollen patties did not seem to have these organisms.  No association with colony collapses.

Have some insight or a guess?

If you have some insight you'd like to share that may help determine what these are, we would appreciate if you Post a Comment below or send me an e-mail through the "Have something you want to share here???" link.
 
My Two Cents
There are a couple of candidates you may want to consider:  fungi/yeasts, trypanosomatids, Malpighamoeba, gregarines (e.g. Apicystis).  Based on prevalence alone, the first two candidates, fungi/yeasts and trypanosomatids are the most likely "mystery spore" you will encounter.  None of the above are trypanosomatid spheroids, I guarantee you (please see my previous post describing these cool but potentially problematic critters).  So, my best guess is that both Mystery spore #1 and #2 are yeast (Candida spp., Rhodotorula spp. etc.) or mold (Aspergillus spp.) spores.  What if it's Beauveria spp., common entomopathogenic fungi used as biocontrol agents in some areas that the bees are picking up?  These can probably (theoretically) be putatively identified to species by cultivating them and/or by purification and DNA amplification using universal fungal rRNA primers to generate sequence data that can be compared to cataloged species in GenBank.

The last two candidates, amoeba and gregarines, are apparently quite rare.  However, this may be due in part to difficulty discerning them and so they are under-reported.  Malpighamoeba mellificae can only be reliably identified during dissection and examination of intact Malpighian tubules, which is the tissue they infest creating diagnostic impactions (see Evans and Schwarz 2011).  Once they are disrupted from the tubules, they simply look like the nondescript "fried egg" cell and can't be reliably identified by any honey bee pathologist I know of currently (please speak up if you are an amoeba expert and disagree!).  There is no molecular data for Malpighamoeba mellificae yet so until that time, molecular diagnostics of this species is unavailable.  Some diverse gregarine taxa have been described from honey bees historically, particularly in honey bee colonies living in tropical environments (see Evans and Schwarz 2011) but recently only cross infection of honey bees with the bumble bee gregarine Apicystis bombi, have been reported (see Plischuk et al. 2011 and Ravoet et al. 2013).

Finally, I want to mention that cells such as these may be environmentally acquired and not truly infectious to bees, thus they may simply be 'passing through' inertly.  Only indisputable evidence of replication in the bees or a clear cellular/molecular level pathology would confirm organisms are infectious and pathogenic to bees, respectively.

References
Evans JD and Schwarz RS. 2011. Bees brought to their knees: microbes affecting honey bee health. Trends in Microbiology 19:614-620.

Foley K, Fazio G, Jensen AB, and Hughes WOH. 2014. The distribution of Aspergillus spp. opportunistic parasites in hives and their pathogenicity to honey bees.  Veterinary Microbiology 169:203-210.

Plischuk S, Meeus I, Smagghe G, and Lange CE. 2011. Apicystis bombi (Apicomplexa: Neogregarinorida) parasitizing Apis mellifera and Bombus terrestris (Hymenoptera: Apidae) in Argentina. Environmental Microbiology Reports 3:565-568.

Ravoet J, Maharramov J, Meeus I, De Smet L, Wenseleers T, Smagghe G, and de Graaf DC. 2013. Comprehensive bee pathogen screening in Belgium reveals Crithidia mellificae as a new contributory factor to winter mortality. PLoS ONE 8.

Emerging dangers: Deadly effects of an emergent parasite in a new pollinator host

Abstract and link to a research article about the impacts of Nosema ceranae in bumble bees (Bombus terrestris) published in the Journal of Invertebrate Pathology by P. Graystock et al. 2013 (apologies for my late administrative duties):

Abstract

There is growing concern about the threats facing many pollinator populations. Emergent diseases are one of the major threats to biodiversity and a microsporidian parasite, Nosema ceranae, has recently jumped host from the Asian to the Western honeybee, spreading rapidly worldwide, and contributing to dramatic colony losses. Bumblebees are ecologically and economically important pollinators of conservation concern, which are likely exposed to N. ceranae by sharing flowers with honeybees. Whilst a further intergeneric jump by N. ceranae to infect bumblebees would be potentially serious, its capacity to do this is unknown. Here we investigate the prevalence of N. ceranae in wild bumblebees in the UK and determine the infectivity of the parasite under controlled conditions. We found N. ceranae in all seven wild bumblebee species sampled, and at multiple sites, with many of the bees having spores from this parasite in their guts. When we fed N. ceranaespores to bumblebees under controlled conditions, we confirmed that the parasite can infect bumblebees. Infections spread from the midgut to other tissues, reduced bumblebee survival by 48% and had sub-lethal effects on behaviour. Although spore production appeared lower in bumblebees than in honeybees, virulence was greater. The parasite N. ceranae therefore represents a real and emerging threat to bumblebees, with the potential to have devastating consequences for their already vulnerable populations.

Link:  doi:10.1016/j.jip.2013.06.005

Can you identify bee guts infected with Nosema?

Last week I needed to collect a fresh Nosema ceranae sample from a honey bee to use in an experiment.  We generally have a high prevalence of Nosema ceranae in the colonies here at the Bee Research Lab in Maryland, and a heavily infected individual is not too difficult to locate.  But, how do I tell if a bee is infected or not?  Some people say they can tell by looking at the midgut region of the bee's digestive tract whether or not they are infected by Nosema.  The midgut is the specific region of the honey bee gut that both species of Nosema (Nosema apis and Nosema ceranae) infect.

Do you think you can identify a Nosema infection by looking at the midgut?

Here's the test

• Adult workers were collected from an apparently healthy colony on July 3rd (that's right; no sign of dysentery, nosemosis, excessive bee poop on the porch, etc.) and brought to the lab.

• Bees were cold anesthetized on ice.

• Digestive tracts were then removed from 9 individual bees, shown below with both light (left) and dark (right) background.

Can you identify which of the 9 midguts shown are infected by Nosema ceranae?

 

Hints 

1.  At least one midgut shown is heavily infected with about 50 million Nosema spores.

2.  At least one midgut shown has no detectable Nosema spores.

Got your answer?  Give up???  Scroll down for the answers.

Here are light microscope images (400x) of the contents from each midgut shown above:

Only midgut #2 is infected with Nosema ceranae.  In some (#1, #4, #8, #9) large pollen grains are visible.  Some cellular debris (including what look to be lipid spheres) can be seen in the remaining images.  If you answered this correctly you were either a) lucky or b) know something I don't and were lucky.

What does this mean?

Gross examination of the midgut itself is not a reliable way to identify infected individuals.  I can never tell with any measure of confidence whether or not a midgut is infected, and I've looked at a lot of midguts!  It has been suggested that a field test can be used to diagnose Nosema infected midguts by visual inspection, looking for a light color and swelling.  This may be true for pure Nosema apis infections (with which I don't have experience), but is not true for the now most prevalent species N. ceranae and is also probably not true for mixed species infections.  As shown above, the lightest colored midguts (#5, #8, #9) were not infected and midguts equally large (#1, #7, #9) as the infected one (#2) were not infected.

How do I know this is Nosema ceranae?  

Because I extracted DNA from this sample and used a molecular diagnostic test called polymerase chain reaction (PCR) with empirically determined species-specific primers that can differentiate Nosema ceranae from Nosema apis. (See Table S1 below from R. Schwarz and J. Evans. 2013. Single and mixed-species trypanosome and microsporidia infections elicit distinct, ephemeral cellular and humoral immune responses in honey bees.  Developmental and Comparative Immunology 40, 300-310. http://dx.doi.org/10.1016/j.dci.2013.03.010 ).  Although N. ceranae spores are slightly smaller than N. apis, molecular diagnostics are the only way Nosema species identification can be reliably made in my opinion.