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Dave Black

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About Dave Black

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  1. Traditional beekeeping lore (law) going back eons is that a swarm is lost as soon as it is no longer in sight. Lost as in, no longer 'captive' , no longer 'owned'. Land ownership was never part of the equation as bees were husbanded by commoners and not the land-owning classes.
  2. NZBF Mixing apivar and bayvarol strips

    That was the short answer. I've put the long answer here:
  3. This article was originally published in 2015 Everybody needs to look over the fence once in a while, especially beekeepers. Something that caught my eye recently was a study looking at weeds and glyphosate resistance, a study which itself took a glance over the palings at antibiotic resistance in hospitals. Resistance is not a phenomenon unique to beekeeping, it is universal and, at its simplest, just about how organisms adapt and evolve in their environment. From our point of view, when we think about resistance, the aspect that concerns us most often is varroa mites not disappearing after we have treated the hive with a substance that usually kills them, and usually we’re talking about the efficacy of the two synthetic pyrethroids used to treat the pest, Apistan and Bayvarol. Pyrethroids have a long history and it was never in doubt that varroa mites would adapt to the treatment. Pyrethroids are copies of naturally occurring esters from Chrysanthemum flowers. What we call pyrethrins were first used as insecticides in the first century in China. They were used in Europe more than 200 years ago and in commercial production by the mid-19th century. Although effective, they were expensive to produce and quickly degraded by light and air, so by 1924 chemists began making ‘synthetic’ copies which were more stable and better suited to one pest or another, or to different ‘delivery systems’. By the 60s and 70s many variants had been produced, including deltamethrin, at the time the most active insecticide ever produced. Apistan and Bayvarol are different pyrethroids, called tau-fluvalinate and flumethrin respectively, which differ in the construction of one end of the molecule. One of life’s ironies (one of my favourites) finds that the ‘natural’ pyrethrins, the synthetic pyrethroids, and the environmentalist’s bête noire, DDT, are all much the same when it comes to their effect on arthropod pests. All of them affect the normal transmission of nerve impulses along the nervous system by stopping the exchange of sodium and potassium ions across an impermeable membrane through a special ‘channel’. (If you’re interested look up ‘voltage gated sodium channels’). What resistant pests have done is to modify the sodium-channel proteins so the insecticide can’t bind to the site. Different pests have modified different proteins, but resistance is extraordinarily common; whitefly, cockroaches, fleas, lice, mosquitos, flies, aphids, thrips, and many others have all evolved resistance to pyrethroids and DDT, to a greater or lesser extent. Known as ‘knockdown’ resistance (knockdown being the excited but paralysed state caused by the nerve disruption) it was first recognised in 1951 in house flies. When we add a treatment to our hives we alter the environment in the hive, hoping that the bees can still flourish but that the things we don’t want die or can’t reproduce. In this sense varroa mites are analogous to the weeds I mentioned at the start. Weeds, and mites, adapt to pesticides in various ways. Besides changing their behaviour (avoiding the chemical for instance) pests can slow or stop the chemical getting into their body, or store it up where it can’t do any damage; they can use enzymes to destroy the active ingredient, or they can change the site or process the chemical is able to disrupt. It’s important to realise these changes come at a cost, sometimes a very high cost, sometimes a very low cost, and rarely, sometimes with an added advantage. For example, a bacteria may construct a thicker ‘skin’, but it will have to use more resources to do so. As long as it faces a threat from the chemical it’s worth doing that, but in an environment where the threat no longer exists if there is extra ‘effort’ involved it’s wasted, and puts the ‘resistant’ organism at a disadvantage compared with an organism that doesn’t bother. For any particular adaption it’s important to understand the cost of that adaption if we want to understand how long-lived and pervasive that adaption will become. Complex, costly changes are easily lost when they are unnecessary; simple, cheap alterations are likely to remain. The science looking at pyrethroid resistant mites has been able to describe in pretty good detail what genes are involved and what they do to allow make mites less sensitive to the chemical. This type of chemical and it’s mode of action is pretty well understood as it been in widespread use for a long time. Around four amino acid substitutions have been proposed in the case of varroa’s pyrethroid resistance. So far, it looks as though the metabolic cost of this adaption to pyrethroids comes at a very low cost for mites, and no differences in ‘fitness’ (for their environment) has been observed between resistant and non-resistant mites. So far, there is very little evidence that regression to a predominately non-resistant strain occurs within a reasonable period of time if pyrethroid treatments are suspended, and that also suggests the cost of carrying the adaption is relatively low. There is not a lot of data about the incidence and spread of resistant mites. Beekeepers aren’t looking for resistance, even though it is simple to do so, and often the only clue is the unexplained loss of treated colonies. Ascribing the cause of the collapse to resistant mites is a long bow to draw however and so usually the cause is hidden by the many plausible reasons for a colony dying. On the large scale however, we do have a picture, and wherever we have looked we can see that the spread of ‘resistance’ replicates the same route that the original mite invasion took; a slow local spread accompanied by an occasional ‘long-distance’ hop. That’s no surprise, mites can’t survive and travel without a bee host, and so the spread of the resistant variety relies on the movement of bees and their colonies, and the same ineffective phytosanitary regimes that permitted the original spread of the pest. It’s also no surprise because that is what happened with other examples of resistance. We will have to wait for more information about the genes involved in providing resistance, but so far it does not look as though it appears spontaneously from multiple origins, it emerges once or twice, by chance, and the mites carrying the attribute spread. Pyrethroid resistant mites have been reported all over Europe, the US, Israel, and parts of S. America. Resistance to coumaphos has been reported in Italy, and resistance to amitraz has been reported in Croatia, France, and the USA. In October 2009, not quite ten years after varroa was first discovered, resistant mites were reported from a hive in Auckland. In the ensuing discussion speculation suggested resistance was likely present in Northland, Waikato, and the Bay of Plenty, and maybe further south. We do not know if the necessary genetic change arose here and was selected by the prevailing pyrethroid use at the time, or whether we managed to import a resistant strain as easily as the original import had occurred. As we don’t know how varroa entered the country it’s possible the ‘gate was never closed’. However it arose, we know it will gradually spread, just as the non-resistant type(s) have. While the available evidence suggest pyrethroid resistance is not widespread amongst varroa mites in New Zealand we have to add the word ‘yet’. Resistance isn’t anyone’s fault, it’s just the way Nature works, and it was not caused by the injudicious use of the chemicals used to control it; the fact is that the clock was always ticking and at some point luck ran out. Inappropriate pesticide use will ‘fix’ the problem in place though. The challenge is to understand its incidence and find the best strategy to manage resistant mites. That’s why I’m interested in looking over the fence. Everywhere we are faced with similar challenges; super-resistant weeds and antibiotic resistant hospital bacteria are two sides of the same coin. The paper studying glyphosate resistance in Ilinois (Evans et al) used a lot of data to look at the outcome of different management practices, broadly, rotating herbicides with different modes of action (MOA) or mixing different herbicides together. These two divergent strategies are also used to manage bacterial resistance to antibiotics in public hospitals, and in both cases the finding is that mixing, not rotation, is the better (but more expensive) strategy. This appears to be true when the fitness cost of a resistant trait is low, in which case the adaption is fixed even in the absence of the selective agent (the pesticide). Mixing strategies are not reliant on the cost of fitness driving depletion. Instead, mixing depletes any resistance alleles by decreasing survival probabilities of all individuals carrying the relevant alleles. It is an expensive strategy as the pesticides for each MOA must be effective. Ineffective, low-dose mixtures can potentially increase the risk of non-target-site resistance and cross-resistance evolution. I can see very little justification for adopting the strategy as a prophylactic treatment; besides the extra cost it only increases the chance of eventually selecting for cross-resistance. In the conclusion the paper points out; ”Herbicide mixtures are not a permanent solution to the problem of target-site resistance; herbicidal mixtures may delay evolution of resistance, but they do not prevent it…long-term, cost-effective, environmentally sound weed management will require truly diversified management practices… Combining chemical, cultural, physical, and biological tactics can provide cost-effective weed management while reducing reliance on herbicides.” For Evans, “We will encounter resistance evolution repeatedly in natural systems managed for human benefit. Sustainable stewardship of these systems will depend on recognising that we are always applying selective pressures, and that management responses need to grow from our understanding of applied evolution” Can’t help feeling there is a lesson for beekeepers in there. Evans, Jeffrey A., et al, (2015) Managing the evolution of herbicide resistance. Pest Manag Sci, (wileyonlinelibrary.com) DOI 10.1002/ps4009 Martin, Stephen, J., (2004) Acaracide (pyrethroid) resistance in Varroa destructor. BeeWorld 85(4): 67-69. Davis, T.G.E., et al (2007) DDT, Pyrethrins, Pyrethroids, and Insect Sodium Channels. IUBMB Life, 59: 151-162. Lagator, Mato et al (2013) Herbicide mixtures at high doses slow the evolution of resistance in Chlamydomonas reinhardii New Phytologist Vol 198(3): 938-945. DOI 10.1111/nph.12195
  4. NZBF Mixing apivar and bayvarol strips

    Alcohol wash? Yes. Always. Use both drugs? Yes, but only at the specified dose.
  5. Some new research on Varroa

    It isn't the case that everyone simply assumed haemolymph was the food source; there have been a few studies that suggest that is the case. In 2004 for example Garedew looked at the energy and nutritional demand and studied mite respiration and calorimetry, measuring oxygen uptake and heat production, and the loss of weight for the various castes and their larvae. DeJong, Bailey and Ball, Kovac and Crailsheim and others studied the chemical composition of the haemolymph and found lower protein content in parasitized pupae (circa 1982 - 1991). Mites have been found to have few enzymes for digesting proteins, supporting the idea that they get their amino acids from bee haemolymph instead. It is the case that we really know very little about mite nutrition though. Mites use different food sources at different times, foundress mites live in brood food, phoretic mites probably have different needs winter to summer, and mites feed on larvae that have very little in the way of distinct organs. What we all refer to as 'the fat body' is not a 'thing'. In larvae the 'fat body' is a very loose association of specialised cells, so loose that as the development stages progress through to pupation they can be free-floating in the haemolymph or completely reabsorbed. In adults most of the 'fat body', which by now is more like a proper 'organ', is found along the dorsal (top) ceiling of the abdomenal cavity, with some on the abdomen floor, and sometimes extending a little up the sides close to the wax glands. That only roughly coincides with the diagram in the video. I'd expect that any trend in the protein and lipid status of the haemolymph would be mirrored in the fat body and vice versa. Bowen-Walker did find a distinct preference for feeding site in adult over-wintering bees, limited by the number of mites trying to feed. The preference was clearly for a posi between the 3rd and 4th tergites, avoiding the wax mirrors, on the left-hand side of the abdomen. 85% of the 77% of the mites on the abdomen were there, 66% of them preferred the left side. 99% were found between tergites in preference to the sternites where the most significant 'fat body' would be. I don't think we have seen a similarly emphatic preference in larvae or pupae. In this case they suggested the choice of site was due to the proximity of the curve of the mid-gut (especially when the rectum was full) that would mean it would be the most concentrated region of haemolymph. Given what we know I would have some questions about the idea expressed in the video (it may be simplified account...). I'm not convinced we can jump straight to filling the 'fat body' with anti-varroa meds. That said using a fluorescence trace is an interesting idea, an alternative would be using radioisotopes of carbon to work out what came from where, because we aren't sure. I'm guessing this is a PhD thesis in the making, Ramsey was working in Dennis vanEnglesdorp's lab, but so far the only reference to the work is in a presentation to a 2016 conference. Maybe one day it will get published and peer reviewed. Garedew, Schmolz, and Lamprecht (2004) The energy and nutritional demand of the parasitic life of the mite Varroa destructor. Apidologie 35 pp419-430 doi: 10.1051/apido:2004032 Bowen-Walker, P. L., Martin, S. J., & Gunn, a. (1997). Preferential distribution of the parasitic mite, Varroa jacobsoni Oud. on overwintering honeybee (Apis mellifera L.) workers and changes in the level of parasitism. Parasitology, 114(2), 151–157. https://doi.org/10.1017/S0031182096008323
  6. Some new research on Varroa

    As you've mentioned it I'll have a look when I get to a computer. I don't watch unsolicited videos with no synopsis, especially on the phone!
  7. Contaminated Honey?

    Here’s some context for the numbers in the study. The measurements are listed in nanograms per gram (ng/g), the equivalent of parts per billion (ppb). Their average value, across all five compounds, ranged from around 0.3 to 2ppb; the range in all the tests was between 0.07ppm and 46.8ppb. The Limits of Quantification (LOQ) were in hundredths, or thousandths of 1ppb (0.002 – 0.03ppb). The New Zealand values ranged from 0.018 – 0.337ppb. All but two were less than 0.07ppb. In a US study of residues in wax and pollen samples collected in 2007 and 2008 they published values for 121 pesticides and their metabolites found. These included (in wax) fluvalinate (2.0 - 204,000.0ppb; median 3595.0), amitraz (9.2 – 43,000ppb; median 228.0), thiacloprid (1.9 – 7.8ppb; median 5.9), imidacloprid (2.4 - 13.6ppb; median 8.0), and acetamiprid (14 – 134ppb; median 57.0). In Spain a study looking at Bayvarol residues in honey (2009) had a detection limit of around 7.9ppb – 11.5ppb and couldn’t find anything. An earlier Swiss study (1998) had a detection limit for flumethrin (Bayvarol) of 25ppb in wax and couldn’t detect any. The detection limit for flumethrin in honey was 3ppm and there wasn’t any. In this study the Limit of Detection was between 0.001 and 0.01ppb. The Swiss MRL (1999) was 50ppb for fluvalinate and 5ppb for flumethrin. Germany was finding fluvalinate at 2 – 7ppb (7% of samples). It not clear what kind of levels have effects on honey bees. It depends on the active ingredient, the length and route of exposure, and on any synergy with other immunological challenges. Some chronic effects on neurology and behaviour have been reported at 1 or 2ppb, occasionally at a fraction of 1ppb. Effects on reproduction are being reported at 5ppb, and increased mortality by exposure to 50ppb or more. The New Zealand values in the new study are mostly hundreds or thousands times lower. The study authors have this to say; “… to some extent, our results illustrate that the ever-increasing analytical sensitivity allows detecting traces of pesticides where they previously were not detectable. But given the increasing use of neonicotinoid pesticides in the different regions of the world, despite partial bans such as the one implemented in the EU, it is also reasonable to expect contamination to have increased over time.” A little ironic maybe, given they use the increased sensitivity to demonstrate the increased use, but if the compounds weren’t there, they couldn’t be detected, whatever the sensitivity. It’s plainly pollution. Mullin CA, Frazier M, Frazier JL, Ashcraft S, Simonds R, et al. (2010) High Levels of Miticides and Agrochemicals in North American Apiaries: Implications for Honey Bee Health. PLoS ONE 5(3): e9754. doi:10.1371/journal.pone.0009754 Bogdanov, S., Kilchenmann, V., Imdorf, A. (1998) Acaricide residues in some bee products. Journal of Apicultural Research 37(2): 57–67 Wallner, Klaus. Varroacides and their residues in bee products. Apidologie, Springer Verlag, 1999, 30 (2-3), pp.235-248.
  8. Contaminated Honey?

    All fair points. We have of course no idea if the samples went through an extraction plant - that's one of the problems with the study!
  9. Contaminated Honey?

    A common sentiment I'm sure @Alastairbut no less depressing. I don't think its the sensitivity of the testing. Perhaps it surprises me because I suspect it's nectar that is not from food crops, so the contamination is indirect. I have in mind NZ honey crops, kamahi and so on. We are not treating manuka seed are we?
  10. Contaminated Honey?

    A couple of days ago the results of a global survey began to hit the headlines; A worldwide survey of neonicotinoids in honey, Mitchell et al., Science 358, 109–111 (2017) 6 October 2017. The study was based on a collection of samples from 'local producers' all around the world donated by colleagues, friends, and relatives connected the the University and Botanic Gardens of Neuchtel in Switzerland. Of the 300 received, gathered from apiaries, markets, and commercial sources, 198 were selected, in theory representing a broad range of environments but also avoiding an over-representation of European-sourced samples. Most but not all the EU samples were collected before the 'ban' on neonicotinoid use on bee-visited crops. Only 7% had the name of the beekeeper provided. The researchers point out this 'citizen science' aspect meant they had only a hazy (or no idea) about the honey samples and their provenance, whether they were blended, what nectar sources might be present, or the time period involved. Nor did they have any idea of the potential pesticides the sample was exposed to, or how the sample was stored prior to them receiving it. They are limited in what they can conclude about the potential environmental contamination by their ignorance about the behavioural and ecological aspects that many have affected the sample. There are four New Zealand samples in the data. One from near Marsden Point for thiamethoxam. Two from near the Korowai-Torlesse Tusockland Park outside Springfield for imidacloprid. One between the Tekapo military camp and Mt Cook, positive for imidacloprid, thiamethoxam, thiacloprid, and acetamiprid. I've had a look and acetamiprid is not in the current ACVM list as registered for use in New Zealand. Imidacloprid has bee registered since 1992, thiamethoxam and thiacloprid since 2000, and clothianidin from 2003. The seven Australian samples presented a simpler picture, one with no detection, four for imidacloprid (one including thiacloprid), one with only thiacloprid, and one with only thiamethoxam. Of the 198 samples tested, 149 had a measurable amount of at least one compound - 75%. Several compounds were found in 60% of samples. It's curious that three tests, for Italy, Portugal, and Spain, list thiamethoxam as 'Not detected' (ND, rather than >LOD - Limit of Detection). In the other 987 tests results are reported as an amount or as below the limit of quantification (<LOQ). If the distinction is correctly made, in 990 tests 987 must positive (but not quantifiable), or 99.7% This is not the claim being made. They do say that 48% of their samples exceeded a 'biologically relevant dose. The biologically relevant dose they chose has been derived mainly from a study on bumble bee brains reporting harm to neurons after several days exposure. I'm not really able to say if their choice was a valid one, the evaluation of the scientific literature on this is very difficult. My only observation would be that there are good reasons to think, behaviourally and ecologically, bumble bees, honey bees, and other pollinators are not the same, and neither are the five neonicotinoids. In the New Zealand results, most are very low and seemingly not significant. The two thiamethoxam results are relatively high, but it's difficult to draw the conclusion they are significant from the table of concentrations given in the Supplementary Data. Overall I'd say this is an interesting attempt at answering some important questions about the use of these pesticides. That they have become so ubiquitous in (for NZ) the last 25 years is startling, and from samples in, to me, pretty unlikely places. The claim neonicotinoids have been found in 75% of their samples isn't helpful, in that newspapers and consumers just hear "contaminated honey". Science magazine reported the study with the headline "Nerve agents in honey". Whether or not we can say it is harming pollinating insects (it isn't harming people!), it is actually an indictment of the way in which we are managing our landscapes, and the fact that so many samples had two or more of the compounds says something about the persistent and pervasive nature of these pesticides. There is a case to be made for a well designed long term monitoring programme, even in New Zealand.
  11. Breeder queens

    Like here? https://www.nzbees.net/topic/99-international-yearly-queen-bee-colouring-standards/?tab=comments#comment-6175
  12. Another look at American Foul Brood

    True, and not their only defence. Distinct from larvae with a blind gut - open only at the 'in' end. Is defecation 'voluntary'?
  13. Another look at American Foul Brood

    The bacterial brood disease American Foul Brood (AFB) occurs worldwide and leads to significant losses of honey bee colonies every year. In several countries, as in New Zealand, AFB is a notifiable disease and infected bee colonies have to be burned to contain the disease. Although it has been under investigation now for more than a century, the underlying characteristics of the host–pathogen interactions on larval level remain elusive. An effective treatment of AFB does still not exist, partly since the progression of the disease following ingestion of spores has only been described superficially. The bacteria that cause AFB belong to a large group of similar bacteria that can be both extremely useful and highly damaging. In 1988 scientists began to divide the main 'bacillus' classification into smaller, more comparable sub groups based on genetic, phylogenic principles rather than morphology, and one of the initial five sub-groups was 'Paenibacillus'. Literally, 'almost' bacillus. The species known for AFB became Paenibacillus larvae. The continual re-evaluation of the groups has led to the re-naming of some new species and the re-classification of existing species so that the classification continues to grow, and the genus currently has about 200 species. Paenibacillus is still expected to undergo significant taxonomic subdivision as these useful or otherwise relevant Paenibacillus continue to be studied, more are added, and the groups get unreasonably large. Many species of Paenibacillus produce antimicrobial compounds that are useful to fields like medicine, agriculture, industrial chemistry or bioremediation. Another well-known aspect of Paenibacillus is its role in the spoilage of milk and other dairy products, where its enzymes and metabolic products negatively impact the flavour or texture of dairy products, in, for example, the milk curdling caused by proteases. One of the challenges is that Paenibacillus are 'psychrotolerant' spore-forming bacteria. They form hardy spores, and function well at low temperature. Many Paenibacillus strains grow well in refrigerated temperatures used for storing milk. They represent more than 95% of bacteria in raw milk after 10 days of refrigerated shelf life, while spoilage of pasteurized milk due to Paenibacillus is delayed by the germination process of spores and usually occurs after 17–21 days. Paenibacillus species have also been used as pesticides to kill agricultural pests in a variety of ways. The chitinase enzymes they produce hydrolyse chitin, which is a structural polysaccharide of insect exoskeletons and gut linings. Like other bacteria, Paenibacillus species compete with other micro-organisms through the production of a wide range of antimicrobial compounds. All these general properties of Paenibacillus - curdling proteases, cool temperature range, chitin-destroying enzymes, persistent spores, and antimicrobial activity, are apparent in the symptoms produced by honey bee colonies infected by Paenibacillus. For beekeepers, we know AFB is caused by strains of Paenibacillus larvae, each identity named after particular gene sequences known as ERIC (Enterobacterial Repetitive Intergenic Consensus) sequences. ERIC I and II are the types typically isolated from hives; ERIC III and IV are not considered to be significant. ERIC II is the most virulent strain, but ERIC I is more prevalent globally, not just because it can infect all honeybee subspecies, whereas ERIC II has been confined to to certain subspecies, but also because, being less virulent, it is more likely to be found. ERIC II will kill larvae in just 6 to 7 days. This strain produces a unique protein which facilitates attachment of the bacteria to part of the honeybee’s midgut. By contrast, ERIC I takes up to 12 days to kill infected larvae. The earlier larvae die the more effectively they can be removed as part of the social immune response of honey bees. By removing the dead larvae quickly during normal brood hygiene fewer bacterial spores are produced and spread in the hive. As a worker larval stage lasts six days ERIC I strains tend to die as pupae, while ERIC II strains can have deaths in open brood which are easily detected and removed by the bees. In one study only around 5% of ERIC II infected larvae died after cell capping, while 27% of the ERIC I infected larvae died after capping. Reports about the variable behaviour of AFB infections, and of the disappearance of infections in some circumstances, are presumably explained by the different expression of symptoms for each strain, and the variation between different strains and races of honey bee. For example, different species of honeybees produce subtly different royal jelly proteins, and this seems to affect their vulnerability to P. larvae infections. Apis mellifera ligustica’s royal jelly proteins are known to differ from those produced by Apis cerana. Interestingly, this difference in activity may provide Apis mellifera ligustica faster metabolic function and enhanced immune activity, and so the strain is a little more resistant to P.larvae (and A.apis) than is Apis cerana in that larval exposure to the spores results in infection less frequently. This may also help to explain the specificity observed in P. larvae ERIC II infections. Until recently it had been assumed ERIC II was restricted to northern and central Europe, Argentina and Uruguay, but examples have now (2014) been isolated in Canada and New Zealand (Schäfer 2014). Honeybee larvae are most susceptible to infection by the bacteria's spores within the first 36 hours after egg hatching when larvae are fed with accidentally contaminated food. After metamorphosis bees are not susceptible to infection by the spores. As pupa they are not fed, and as adults they are able to void any spores they ingest. Foul brood eventually kills all the colony's offspring so there are no replacement workers to replace aging and dying individuals and the colony will collapse, often succumbing to robbers that spread the infectious spores in the process. Only a few spores are needed to initiate infection, but the number depends on the age, caste, and genetics of each larvae. As a rule, very young larvae are fed with brood food produced by nurse bee head glands, but as they age this food is progressively diluted by adding some of the honey or nectar content regurgitated by the nurses, increasing the risk of contamination. This is mitigated to an extent by the nurse's proventriculus filtering out spores, and with increasing age the larva's gut environment becomes progressively more hostile to the bacteria. By 48 hours after egg hatching most larvae are no longer vulnerable to the bacteria. The phospholipid lysophosphatidylcholine (LPC) has been identified as an antibacterial compound in honeybee midguts in both naturally developed and artificially reared honeybees. The concentration of LPC in royal jelly was below the limit of quantification, which probably indicates that it is not produced from the head glands. On the other hand, feeding experiments using fluorescent-labelled LPC indicate that it is delivered to young larvae with regurgitated honey stomach content to larvae. Fluorescence was found in middle-aged and older larvae to a higher extent than in young larvae, which corroborates that the delivery route via honey stomach becomes increasingly important with larval age. In older larvae and adults, in addition to a fully-formed peritrophic membrane, LPC is present in honeybee midguts, coexisting with the normal gut microbiota, as a permanently active immune trait representing a first line of defence against AFB and other infections. A healthy (lipid-rich) pollen supply is partly responsible for enabling LPC production. Spores that have been ingested germinate rapidly in the midgut of infected larvae. Soon after ingestion vegetative bacteria can be found randomly distributed in the midgut cavity or 'lumen' and proliferate in the following days. Four to five days post infection the guts of larvae can be filled with P. larvae, while no bacteria are detected in the body, the midgut epithelium remains intact, and the 'bolus' containing the bacteria is still contained with the insect's peritrophic membrane, protecting the gut lining. This membrane, common to most insects, begins to form in larvae after about 8 hours and is thought to have evolved from the mucus lining the gut cells. It will in time form a barrier to protect the bees from mechanical abrasion and invasion by micro-organisms. As the bacteria multiply in the gut cavity they release compounds that have antibacterial and antifungal properties that kill any and all other species that might compete in the space. Metabolic fingerprinting (tracing carbon use) of P. larvae has revealed that this bacterium is able to use different sugars and sugar derivatives as a carbon source, so that the larval food and chitin containing larval structures can serve as food during this phase of infection. Both strains use chitinases that will degrade the peritrophic membrane for nourishment and once degraded they have access to the midgut lining. They eventually penetrate the midgut epithelium and migrate through the spaces between the cells into the haemocoel using an extensive arsenal of enzymes that destroy the proteins and collagen that bind the cells together, or bind the cells to extracellular matrices. At this point he host larvae dies and the toxins and enzymes produced continue to destroy and homogenise the body contents. When the larva dies a pure culture of P.larvae is present, a mix of both vegetative bacteria and bacterial spores. While we have always thought that the bacteria begins to form long-lived spores as a response to the immanent consumption of its food supply it is now clear that spore formation begins immediately and continues through the entire infection process. This article is available as a Portable Document (.pdf) here; The bacterial brood disease American Foul Brood.pdf Photographs 1. An infected and normal pupa, cell cap removed, both 13 days old. The infected lava shows no body differentiation or segmentation and gravity makes it slump or ‘melt’ into the bottom of the cell. (Genersch 2010) 2. An advanced infection, with ragged and perforated and ‘wet’ cell caps, ooze through the cap, and scales forming on the cell floor. Note the brown colour of the exposed pupa. (Bee Informed Partnership) 3. Top/bottom row. Fluorescent markers added to the bacteria highlight the bacteria green against the yellow (or red) body parts. In B the large arrow points to a break in the gut wall. The images in the bottom row are of an uninfected larva. (Yue et al 2008) 4. Electron micrograph of a single P.larvae spore. The germ cell of the spore itself starts at position ‘GW’. The outer coats make it extremely resistant to desiccation, direct heat, chemicals, and antibiotics. (de Guzman et al 2011) References and Reading Current knowledge and perspectives of Paenibacillus: a review, (2016) Grady et al. Microbial Cell Factories 15:203 DOI 10.1186/s12934-016-0603-7 Reclassification, genotypes and virulence of Paenibacillus larvae, the etiological agent of American foulbrood in honeybees - a review, (2006) Ainura Ashiralieva, Elke Genersch Apidologie, 37 (4), pp.411-420. Strain and Genotype-Specific Differences in Virulence of Paenibacillus larvae subsp. larvae, a Bacterial Pathogen Causing American Foulbrood Disease in Honeybees, (2005) Elke Genersch, Ainura Ashiralieva, and Ingemar Fries Applied and Environmental Microbiology, Vol. 71, No. 11 p. 7551–7555. doi:10.1128/AEM.71.11.7551–7555.2005 Rapid identification of differentially virulent genotypes of Paenibacillus larvae, the causative organism of American foulbrood of honey bees, by whole cell MALDI-TOF mass spectrometry. (2014) Marc Oliver Schäfer, Elke Genersch, Anne Fünfhaus, Lena Popping, Noreen Formell, Barbara Bettin, Axel Karger. Veterinary Microbiology Volume 170, Issues 3–4, Pages 291-297 https://doi.org/10.1016/j.vetmic.2014.02.006 How to Kill the Honey Bee Larva: Genomic Potential and Virulence Mechanisms of Paenibacillus larvae, (2014) Djukic M, Brzuszkiewicz E, Funfhaus A, Voss J, Gollnow K, et al. PLoS ONE 9(3): e90914. doi:10.1371/journal.pone.0090914 Paenibacillus larvae Chitin-Degrading Protein PlCBP49 Is a Key Virulence Factor in American Foulbrood of Honey Bees, (2014) Garcia-Gonzalez E, Poppinga L, Funfhaus A, Hertlein G, Hedtke K, et al. PLoS Pathog 10(7): e1004284. doi:10.1371/journal.ppat.1004284 Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood of honeybees (Apis mellifera), (2008) Dominique Yue, Marcel Nordhoff, Lothar H. Wieler, and Elke Genersch. Environmental Microbiology 10(6), 1612–1620 doi:10.1111/j.1462-2920.2008.01579.x Lysophosphatidylcholine acts in the constitutive immune defence against American foulbrood in adult honeybees, (2016) Ulrike Riessberger-Gallé, Javier Hernández-López, Gerald Rechberger, Karl Crailsheim & Wolfgang Schuehly. www.nature.com/scientificreports/ Scientific Reports | 6:30699 | DOI: 10.1038/srep30699. Honey bee age-dependent resistance against American foulbrood (2001) Karl Crailsheim, Ulrike Riessberger-Galle. Apidologie 32 (2001) 91–103 91. Response of in-vitro reared honey bee larvae to various doses of Paenibacillus larvae larvae spores, (1998) Camilla J. Brødsgaard, Wolfgang Ritter, Henrik Hansen Apidologie, 29 (6), pp.569-578. Involvement of secondary metabolites in the pathogenesis of the American foulbrood of honey bees caused by Paenibacillus larvae, (2015) Sebastian Muller, Eva Garcia-Gonzalez, Elke Genersch and Roderich D. Sussmuth The Royal Society of Chemistry Nat. Prod. Rep., 2015, 32, 765. Radiation inactivation of Paenbacillus larvae and sterilization of American Foul Brood (AFB) infected hives using Co-60 gamma rays, (2011) De Guzman, Z.M., et al Applied Radiation and Isotopes, 69 pp1374-1379 doi:10.1016/j.apradiso.2011.05.032 American Foulbrood in honeybees and its causative agent, Paenibacillus larvae, Elke Genersch, (2010) Journal of Invertebrate Pathology Volume 103, Supplement Pages S10-S19
  14. Varroa controlled by sound

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