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  1. 8 points
    A honey bee nest and its enclosure provides a rich and stable range of ecosystems where we might expect an abundance of microbe populations to thrive, constantly replenished through its interface with the phyllosphere that surrounds it. We know a great deal about the harmful micro-organisms that cause disease; foulbrood bacteria, chalkbrood and nosema, even virus infections, but very little about beneficial micro-organisms that maintain health. Despite a contemporary obsession with prophylactic ‘probiotics’ and gut health there is actually not much known about the contribution from microbes to honeybee fitness, and still less about what the effects of antibiotics and fungicides from pollutants or veterinary treatments might be on the microbiotic environment ‘bees share. There are two ‘communities’ of bacteria here that are particularly interesting, that associated with the pollen collected and stored for food, and that inhabiting the ‘bee gut itself. Pollen provides the bulk of proteins, vitamins and lipids for colony growth. It is also a warm, moist and sugar rich source of food, albeit acidic, and arrives with its own collection of microorganisms, some well able to exploit it for their own purpose. While it has always been believed pollen stored as ‘bee-bread’ is a pre-digested store of food (similar to honey) closer study show this is probably not true. We find ‘bees prefer pollen less than three days old, that is, they weren’t waiting for some process (akin to fermentation) to occur before eating it. In addition the bacteria counts from collected pollen were low and declined rather than increased. That’s not what we’d expect if the store was supporting the microbes. The populations were so low that it was implausible there were enough to have any effect; the estimate was one microbe for every 2,500 pollen grains. Using microscopy the mostly intact grains did not appear to have been altered in any way by storage, and the bacteria found did not have the characteristics of a stable microbiome but were adapted for survival in acidic, antimicrobial environments. The bacteria in stored pollen are very different from those in the hind gut where pollen is being digested, and where they are present in hundreds or thousands per grain of pollen. It appears then that stored pollen is in a state for preservation rather than conversion, and that actually the true ‘store’ of nutrients is the ‘bees themselves. We often talk about pollen (fresh or stored) being digested in a ‘communal stomach’. What we mean is that pollen is eaten by a fairly limited group of young bees and fed to everyone else in the colony. These ‘nurse’ bees consume pollen and from glands in their head then secrete a semi-liquid food, This can be pooled in honeycomb cells to feed larvae or shared directly, ‘mouth to mouth’, with the queen and other members of the colony. As nurse bees age and become guard or forager bees they gradually lose the ability to digest pollen and have to be fed by a new generation of nurses. How these ‘bees extract nutrients from pollen grains is not fully understood, but it’s possible microbes play their part. Scanning electron microscope observations of pollen passing through the digestive tract show a range of states, depending on far along the gut you look and, presumably, on the type of pollen grain and its construction. Some are fully ruptured, perhaps because of osmotic shock, others seem more or less intact but empty, the contents removed gradually through the germination pores in the grain. Another interesting line of research has considered the inevitability of bees consuming the yeasts, bacteria, and fungi that naturally associate with pollen (or nectar) and suggest that this would mean bees are actually omnivorous. Using native (solitary) bees and examining isotopes of carbon and nitrogen in the amino acids glutamic acid and phenylalanine that were consumed the research was able to show the bees were assimilating both microbial and plant proteins. In another study they demonstrated a decline in biomass and increased development time in larvae fed a diet progressively increasing in sterilised pollen, indicating the microbes were an essential part of the diet. This is analogous to leaf-cutter ants growing and consuming fungi grown on the leaf substrate they collect, rather than eating the leaves. We talk about the bee ‘gut’ as though it was all one homogenous organ, but in reality it is a series of blended zones or organs each with distinct functions and correspondingly different environments and inhabitants. Looking at the microbes in these ‘zones’ is tricky. Older methods rely on culturing bacteria on a growth media, but it possible to miss or underestimate microbes that are unexpected or difficult to culture. Later methods use PCR amplification and genetic makers, but that too has some limitations so that at the moment the most accurate information comes from studies that use and compare both methods, what’s called culture-dependent and culture-independent methods. Working out where these microbes belong is also tricky, some, not adapted to live in a particular environment, may just be passing though and do not form the stable biofilms bacteria flourish in. Newly emerged ‘bees have no, or only a couple of types, of gut bacteria. ‘Bees acquire bacteria that inhabit their gut from their foraging environment, but also from each other when food is shared. Plainly bacteria in the phyllosphere are present, mostly in the crop, but this is an inhospitable temporary haunt because its contents change so often. It seems unlikely there is a permanent ‘core’ group of bacteria associated with this region, and little evidence for one. The mid-gut and hind-gut do have lasting ‘core’ groups of bacteria and the highest bacterial counts are found here, with most actively growing and reproducing biofilms in the hind gut. The communities have quite different inhabitants from those found in pollen, ‘beebread’ or honey and remain consistent across seasons and geographical regions. When we look closely enough we find that although a gene sequence can indicate a close physiological relationship to other organisms, when it comes to bacteria even closely related species can display remarkable differences in their functional genes. Perhaps surprisingly this simple set of perhaps eight or so core bacteria types are found only amongst eusocial bees, and appear to have diversified to occupy particular niches and perform functions that only apply in groups of honeybees or bumblebees, and that are conserved by their sociality. Long-standing co-evolution has produced functionally unique strains adapted to a symbiotic existence with each other and their bee family, possibly unique even at colony level. L. M. Klungness, Ying-Shin Peng, Scanning electron microscope observations of pollen food bolus in the alimentary canal of honeybees (Apis mellifera L.) Canadian Journal of Zoology, 1984, Vol. 62, No. 7 : pp. 1316-1319 https://doi.org/10.1139/z84-189 Anderson KE, Sheehan TH, Mott BM, Maes P, Snyder L, et al. (2013) Microbial Ecology of the Hive and Pollination Landscape: Bacterial Associates from Floral Nectar, the Alimentary Tract and Stored Food of Honey Bees (Apis mellifera). PLoS ONE 8(12): e83125. doi:10.1371/journal.pone.0083125 Anderson KE, Carroll MJ, Sheehan T, Mott BM, Maes P, Corby-Harris V. 2014 Hive-stored pollen of honey bees: many lines of evidence are consistent with pollen preservation, not nutrient conversion. Mol. Ecol. 23, 5904–5917. (doi:10.1111/mec.12966) Steffan S, Dharampal P, Danforth B, Gaines-Day H, Takizawa Y, Chikaraishi Y. (2019) Omnivory in bees: elevated trophic positions among all major bee families. Am. Nat.vol. 194, no. 3. doi:10.1086/704281 Dharampal PS, Carlson C, Currie CR, Steffan SA. 2019 Pollen-borne microbes shape bee fitness. Proc. R. Soc. B 286: 20182894. http://dx.doi.org/10.1098/rspb.2018.2894 Philipp Engel, Vincent G. Martinson, and Nancy A. Moran Functional diversity within the simple gut microbiota of the honey bee. 11002–11007 PNAS July 3, 2012 vol. 109 no. 27. https://www.pnas.org/content/109/27/11002
  2. 7 points
    Everyone knows honeybee females (queens) mate at the beginning of their adult life and are then unable to mate again. A queen mates with many males (drones), often on a single occasion but sometimes after multiple flights in successive days. The mating is very quick, not more than 5 seconds and perhaps no more than one or two seconds, after which the male is paralysed and dies. Competition between males in a mating congregation occurs, mostly as a result of size and power, and some selection operates seemingly on the basis of flight altitude, different strains favouring different heights. A single drone congregation area might contain more than 20,000 drones from potentially hundreds of colonies, and the chance of an individual male being able to mate more than once would be very low. In honeybees therefore it’s not surprising a male expends his entire effort mating with a single queen, and not surprising that probably his best chance of improving his reproductive success is posthumous sperm competition. Queens, at least during mating, appear to have very little ability to choose the paternity of her offspring, but there are good reasons to suppose her interest is in being able to produce a sizeable range of genetic characteristics. Variation is good for managing conflict in a social group, protecting the colony from diseases and environmental change, and providing progressive, adaptable worker performance. Especially in honeybees, as she can never mate again, she has a particular interest in actually opposing or counteracting individual males’ reproductive success, negating sperm competition and choosing diversity. This ‘choice’ is said to be ‘cryptic’, because it is hidden from the male (Eberhard, 1996). Queens are estimated to lay about 200,000 eggs each year; something like 1.0 – 1.6 million fertilised eggs in her 5 – 8 year lifetime. Some hymenoptera (species of ants) do better by a significant margin, producing 8 million workers fertilised by sperm stored for decades. Much more sperm are stored in the spermatheca (in the order of 5 million) than will be needed. Drones will produce between 2 million and 12 million spermatozoa each, and at the end of a mating flight a queen might contain 200 million or so temporarily housed in her oviducts, vagina, and bursa copulatrix. Only around 2.5% of the sperm she acquires during mating is stored, and even less are actually used (think an average two per egg over her lifetime). Spermatozoa can be stored for many years and retain viability. Paternity studies have shown it is completely mixed and used equitably. Discovered in 1905 the key to this remarkable economy of sperm use is something called a Bresslau sperm pump. This structure sits between the spermathecal and the spermathecal duct, a valve in muscular tissue that, if you like, ‘reaches in’ and grabs a constant volume of spermathecal fluid (containing sperm) and transports it out to the eggs. (After mating it ‘pumps’ in the opposite way, filling the spermatheca). While the fluid volume is replaced and always stays the same, the density of spermatozoa it contains gradually declines. The Bresslau sperm pump is also found in ants. With the instruments available nowadays it’s actually possible to count sperm on eggs. Just how bees, wasps and any are able to keep spermatozoa alive for so long eludes a complete explanation, but in short, by an extreme conservation of energy and reduction in oxidative stress. Both seminal fluid and spermathecal fluid must have a role in providing a habitat that nourishing the cells, reduces oxidative stress, and protects them from pathogens, but it’s most likely spermathecal fluid evolved to maximise their long-term viability. Studying spermathecal fluid from virgins and mated queens shows they do differ, but also have some functional similarity with some elements in seminal fluid. Drones too store sperm, although not for as long. In a process that takes at least 40 hours it appears that the storage ‘environment’ is gradually changed from semen to a receptive queen’s spermathecal fluid, to a mated queen’s spermatheca. Spermatozoa in the spermathecal fluid ‘acclimatise’ to their new environment and begin to metabolise very, very slowly, essentially ‘outsourcing’ some of their vital functions to the female environment. In particular, while spermatozoa are able to metabolise aerobically, in storage there is evidence to suggest they switch to anaerobic energy production using a partly metabolised product in spermathecal fluid to limit the release of damaging Reactive Oxygen Species (ROS). As well, the spermatheca is a bead-like organ with two spermathecal glands situated outside a hard sclerotized wall impervious to oxygen. By comparison to other organs the spermatheca has significantly lower oxygen concentrations inside. Spermathecal fluid is also known to contain many highly-active antioxidant enzymes, and these increase if we compare virgins with mated queens. It's become well documented in many species that males don't just transfer spermatozoa during copulation but include a complex mixture of molecules, anti-oxidants, ions and cells other than spermatozoa, including sometimes pathogenic micro-organisms. These male compounds have a variety of functions. Some directly affect the sperm’s survival in the female’s reproductive tract, providing nutrition, pH and osmotic buffering, and defences against oxidizing agents. Other products have important effects on the physiology and behaviour of the female, such as promotion of sperm transport, and inducing ovulation or oviposition. We are now beginning to realise that seminal fluid contains molecules that have a demonstrable effect on gene expression, and that a number of proteins cross the vaginal wall into haemolymph where they can bind to receptors on neurons directly affecting nerve signalling. A somewhat surprising example, consistent with other insect studies and earlier work, establishes a (short-term) loss of visual ability in queens linked to a peptide transferred in male semen. The effect is that queens are less inclined to undertake further mating flights (because they can’t see properly), but with the consequence that the queen tries to fly earlier if she can, before the loss becomes too debilitating. Males have no interest in queens flying to mate with more males. The scientists used RNA-sequencing to look at the changes in gene expression following artificial insemination, comparing them with naturally inseminated queens and queens inseminated with a saline control. They were able to identify the changed genes as ones known to be associated with functions that mostly enable vision. They then carried out a similar exercise, but this time measured the actual performance of the eyes (all of them!), things like their response to different light frequencies, and sensitivity to visual contrast. Last, they used RFID tags to monitor natural flight activity (and queen loss) after the same set of treatments (insemination, mock insemination etc). Each set of experiments indicated that queen’s visual performance deteriorated 24 – 48 hrs after receiving seminal fluid, and they were more likely to be lost on subsequent mating flights. The same effect has been observed in other studies of fruit flies, a parasitoid wasp, and in the bumble bee Bombus terrestris, suggesting that this ability to manipulate female mating using components of seminal fluid could be widespread or even universal amongst Hymenoptera and perhaps all insects. Further reading Boris Baer, Sexual selection in Apis bees. Apidologie 36 (2005) 187–200, INRA/DIB-AGIB/ EDP Sciences. DOI: 10.1051/apido:2005013 Boris Baer, Jason Collins, Kristiina Maalaps & Susanne P. A. den Boer. Sperm use economy of honeybee (Apis mellifera) queens. Ecology and Evolution 2016; 6(9): 2877–2885 doi: 10.1002/ece3.2075 Laura M. Brutscher, Boris Baer, and Elina L. Niño. Putative Drone Copulation Factors Regulating Honey Bee (Apis mellifera) Queen Reproduction and Health: A Review. Insects 2019, 10, 8; doi:10.3390/insects10010008 G Koeniger et al (1988) Assortative mating in a mixed population of European honeybee Apis mellifera ligustica and Apis mellifera carnnica. Insectes Sociaux, Paris Vol36, No2, pp.129-138 Liberti et al. Seminal fluid compromises visual perception in honeybee queens reducing their survival during additional mating flights eLife 2019;8:e45009. DOI: https://doi.org/10.7554/eLife.45009 Boris Baer et al, Insights into female sperm storage from the spermathecal fluid proteome of the honeybee Apis mellifera. Genome Biology 2009, 10:R67 (doi:10.1186/gb-2009-10-6-r67) Ellen Paynter, A. Harvey Millar, Mat Welch, Barbara Baer-Imhoof, Danyang Cao & Boris Baer. Insights into the molecular basis of long-term storage and survival of sperm in the honeybee (Apis mellifera). Scientific Reports, (2017) 7:40236, doi: 10.1038/srep40236 Niño EL, Malka O, Hefetz A, Tarpy DR, Grozinger CM (2013) Chemical Profiles of Two Pheromone Glands Are Deferentially Regulated by Distinct Mating Factors in Honey Bee Queens (Apis mellifera L.). PLoS ONE 8(11): e78637. doi:10.1371/journal.pone.0078637 J. Woyke (1983) Dynamics of Entry of Spermatozoa into the Spermatheca of Instrumentally inseminated Queen Honeybees, Journal of Apicultural Research, 22:3, 150-154, DOI: 10.1080/00218839.1983.11100579 Aldo Poiani, Complexity of seminal fluid: a review. Behav Ecol Sociobiol (2006) 60: 289–310. DOI 10.1007/s00265-006-0178-0 Eberhard, William. (1996) Female Control: Sexual Selection by Cryptic Female Choice. Princeton University Press, ISBN 0-691-01084-6
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