{"id":6960,"date":"2020-10-24T10:34:26","date_gmt":"2020-10-24T18:34:26","guid":{"rendered":"https:\/\/scientificbeekeeping.com\/?p=6960"},"modified":"2020-10-24T10:50:09","modified_gmt":"2020-10-24T18:50:09","slug":"6960-2","status":"publish","type":"post","link":"https:\/\/scientificbeekeeping.com\/6960-2\/","title":{"rendered":"Refining the Mite Wash: Part 2 – Mite Release"},"content":{"rendered":"

Refining the Mite Wash- Part 2<\/strong><\/h2>\n

Mite Release<\/strong><\/h2>\n

Randy Oliver
\nScientificBeekeeping.com<\/strong><\/p>\n

First Published in ABJ in August 2020<\/h4>\n

The high efficacy of hand dishwashing detergent at getting mites to release their grip on bees bestirred me to investigate this finding more deeply.\u00a0 What I\u2019ve come to realize is that there are four steps involved between dumping the bee sample into the agitation vessel, and the counting of the mites.<\/em><\/strong><\/p>\n

Mite release, dislogement, precipitation, and separation<\/h2>\n

We use the term \u201cmite wash,\u201d but that \u201cwashing\u201d actually involves four discrete steps:<\/p>\n

Step 1: To cause the mites to release their grip on the bees.<\/p>\n

Step 2: To then dislodge the mites from the bees\u2019 bodies.<\/p>\n

Step 3: To then agitate or wash the bee sample enough to allow for the precipitation of the mites through the tangle of bee bodies, and finally<\/p>\n

Step 4: To separate the mite sample from the bee sample, typically by allowing the much-smaller mites to drop through a screen.<\/p>\n

So let\u2019s go through these four steps one at a time, since each is important.<\/p>\n

Getting mites to release their grip<\/h2>\n

As pointed out by Dr. David de Jong back in 1982 [[1]<\/a>], mites attached superficially to a host bee are relatively easy to remove by shaking the bees in a liquid, but mites that are embedded deeply in the intersegmental membranes for feeding may be more difficult to dislodge.\u00a0 Curious to determine exactly how varroa mites hold onto a bee, I spent some time looking closely at them under the \u2018scope.<\/p>\n

Mites moving on the surface of a bee\u2019s \u201chairy\u201d body are able to get a tight grip by inflating sticky pads called \u201cempodia\u201d at the ends of their feet (Figure 1).<\/p>\n

\"\"<\/a><\/p>\n

Figure 1.\u00a0 It\u2019s fascinating to watch a mite walking across glass.\u00a0 At each step the mite inflates a sticky empodium. For the above photo I placed a live mite on its back on a microscope slide, and then dropped a thin glass cover slip over it.\u00a0 The mite will then walk upside down across the glass.\u00a0 The empodium of the uppermost leg in this photo is just starting to unfold; the next one down is fully extended on the glass.<\/p>\n

The connection with oxalic acid treatments<\/h3>\n

I\u2019m very much involved in experimentation on how best to use oxalic acid to control varroa.\u00a0 One poorly understood aspect is the exact mechanism by which the acid gets into the mites\u2019 bodies.\u00a0 It appears that one main pathway is through their feet.\u00a0 The question then is, why would the feet be more permeable to the acid than the rest of the mite\u2019s body?\u00a0 So I searched the literature for clues.<\/p>\n

Of great interest is an important study by Peattie [[2]<\/a>].\u00a0 They found that arachnids (spiders, mites, and their kin) can secrete a thin film of sticky liquid on the surface of their empodia.\u00a0 I strongly suspect that it is this damp surface that allows the acid crystals to dissolve and thus make their way into the mite\u2019s body tissue and\/or hemolymph.<\/p>\n

Practical application: the required stickiness of the empodia may be varroa\u2019s Achille\u2019s Heel, as far as oxalic acid is concerned.\u00a0 This is because in order for the mite to evolve resistance to the acid, it would need to come up with a new mechanism for getting a grip on the bee.<\/strong><\/p>\n

But I digress \u2013 let\u2019s get back to mite washes.<\/p>\n

Practical application:\u00a0 In order to break the adhesive force between the mites\u2019 empodia and the bees\u2019 exoskeletons, any mite wash fluid would need to act as a surfactant.\u00a0 Both detergents and high-proof alcohol act in this way.\u00a0 Powdered sugar also disrupts the grip, but requires the mite to first step on the sugar crystals to gob up its empodia [[3]<\/a>].<\/strong><\/p>\n

It\u2019s not just the feet<\/h2>\n

The empodia are clearly important to allow a mite to walk over the surface of a bee, but more often a mite is more firmly attached to a bee, with its head thrust deeply between the abdominal sclerites (the plates that cover the abdomen).\u00a0 To see this, you need to view bees from the underside (Figure 2).<\/p>\n

\"\"<\/a><\/p>\n

Figure 2.\u00a0 I shook some bees into a clear plastic clamshell food container so that I could view their bellies as they walked inside.\u00a0 Note the mite clearly visible on the underside of the bee to the right. I\u2019ve gently lifted the sclerites on live bees, and am amazed at how deep a protective cavity they provide for a mite.<\/p>\n

An alternative method for varroa monitoring?<\/h2>\n

While looking for attached mites, using the plastic clamshell food container, it occurred to me that this might actually be an alternative method of mite monitoring for those loathe to sacrifice bees in a mite wash (hey, I don\u2019t like to kill bees either).\u00a0 While not as quick or accurate as a mite wash, this method certainly could be used for monitoring the infestation level of the adult bees.<\/p>\n

Practical application: If you\u2019ve got good eyesight and good light, you could dump a scoop of house bees into a clear plastic clamshell food container [[4]<\/a>], and then scan the bellies of the bees to get a rough idea of the infestation rate.\u00a0 \u00a0\u00a0Allow any older bees fly off, close the lid, and give the remaining bees several seconds to calm down and spread out.\u00a0 I have no data as far as accuracy, but was easily able to differentiate an infested hive from a number of others in which I couldn\u2019t find a single mite.\u00a0 As you\u2019re likely used to hearing, more research is needed (any grad students listening?).<\/strong><\/p>\n

The anchoring of feeding mites<\/h2>\n

A bee clearly notices when a mite is walking over their body \u201chairs,\u201d and generally responds by trying to groom it off.\u00a0 But once a mite gets its head under a sclerite, and appears to be firmly anchored in place, the bee seems to ignore it. \u00a0My question then is, is there additional anchoring involved in addition to having sticky feet?<\/p>\n

Ticks, which are related to mites, are often difficult to dislodge once they\u2019ve drilled into your skin (personal observation).\u00a0 This is due to them having saw-like mouthparts (some photos worth viewing at [[5]<\/a>]). Some ticks also secrete a bonding glue [[6]<\/a>].\u00a0 So I wondered what kinds of adaptations varroa has to anchor itself to a bee while feeding.\u00a0 I pulled out my microscopes and used my cell phone to take some crude photos.\u00a0 Figure 3 shows a ventral (bottom) view of a mite, showing its eight walking legs, and its sensory pedipalps between the front legs.<\/p>\n

\"\"<\/a><\/p>\n

Figure 3.\u00a0 A ventral view of a female mite, with its pedipalps visible between the front legs.\u00a0 Between the pedipalps lie the chelicerae (mouthparts), not visible in this photo.<\/p>\n

Let\u2019s take a closer look at those palps, since they have curved, fanglike spikes near their tips (Figure 4).<\/p>\n

\"\"<\/a><\/p>\n

 <\/p>\n

Figure 4.\u00a0 A closer bottom view of the tips of the pedipalps, with the nasty-looking curved claws clearly visible.\u00a0 It\u2019s apparently not yet completely clear just how the mites use those hooks.\u00a0 I wish to thank Dr. Samuel Ramsey for helping me with the identification of mite anatomy and function in these photos.<\/p>\n

Between the pedipalps are the paired chelicerae \u2013 the mite\u2019s mouthparts.\u00a0 There are also spines in the bottom of the chelicerae that appear to help anchor the mite into the feeding wound (Figure 5).<\/p>\n

\"\"<\/a><\/p>\n

Figure 5.\u00a0 Note the two wicked-looking spines on this bottom view of a mite\u2019s chelicerae (the dark pointed ends of the chelicerae are to the left).\u00a0 These spines appear to help to lock the mite\u2019s chelicerae into the feeding wound.<\/p>\n

I wanted to get a side view of the mouthparts, so I tried my hand at dissecting the pedipalps and chelicerae from some mites.\u00a0 I now have the greatest respect for Dr. Lilia De Guzman and other researchers who routinely dissect organs from a mite.\u00a0 My finest forceps and needles looked like blunt shovel handles under the \u2018scope.\u00a0 But I was able to clumsily rip the mouthparts off an alcohol-killed mite and take a picture (Figure 6).<\/p>\n

\"\"<\/a><\/p>\n

Figure 6. A side view of the feeding apparatus of a varroa mite.\u00a0 At the top are the two pedipalps, one with the curved spine partially in focus. Below is the blade-like end of one of the chelicerae (mouthparts) that penetrate the bee\u2019s soft integument (the anchoring cheliceral spines are folded back in this image).<\/p>\n

Ticks are able to alternately extend and retract their toothed cheliceral shafts to pull themselves into the feeding wound, analogous to how the reciprocating lancets of a bee\u2019s stinger drives it through your skin.\u00a0 I\u2019m not clear whether varroa does so, or to what extent it uses its hooks for anchoring.<\/p>\n

It makes me cringe to think of having a parasite the size of a Dungeness Crab rip and stab its feeding apparatus between my ribs, but that\u2019s what varroa does to the poor honey bee.\u00a0 So back to my question, how firmly are feeding mites anchored to the bee?\u00a0 To answer that, I needed to collect infested bees that had mites well-buried between their abdominal sclerites.\u00a0 Using the clamshell container described above, I identified some bees carrying mites.\u00a0 I then prodded,\u00a0 stroked, and tugged on several mites with forceps to see how firmly they were anchored (Figure 7).<\/p>\n

\"\"<\/a><\/p>\n

Figure 7.\u00a0 I\u2019m holding a bee by the wings so that I determine how firmly the mite was attached (yes, the bees were uncooperative and some stingers were shed).\u00a0 Upon first touch, the mites, such as this one, responded by quickly wedging themselves even more deeply under the translucent sclerite.\u00a0 I couldn\u2019t dislodge them by stroking them with forceps, but unlike as with a tick, I could easily pull them from the bee.<\/p>\n

Result: mites are clearly not ticks, and although they strongly resisted being removed, their mouthparts did not appear to be locked in place, and they could be easily pulled from the bee.<\/p>\n

Practical application: I\u2019ve noticed that alcohol rapidly kills bees upon immersion, but not so with varroa \u2013 they can stand several minutes of immersion and walk away.\u00a0 Could it be that alcohol functions as an irritant that gets the mites to release their grip?\u00a0 Or on the other hand, could strong alcohol kill or paralyze the mites so quickly that they would remain locked in their feeding wounds?\u00a0 This question led me to bunch of additional tests.<\/strong><\/p>\n

How much agitation is necessary?<\/h2>\n

It\u2019s commonly assumed that one must vigorously agitate the bees to separate the mites from them (Figure 8).\u00a0 But in order to \u201cthresh\u201d (to separate the grain from the chaff), do we really need to \u00a0\u201cthrash\u201d (to beat mercilessly) the bee carcasses (or live bees in the case of sugar dusting) to do so?<\/p>\n

\"\"<\/a><\/p>\n

Figure 8.\u00a0 The standard recommendation is to vigorously shake the bees to dislodge the mites.\u00a0 My question is just how much agitation is actually necessary for dislodgement.\u00a0 Once dislodged, further up-and-down agitation just keeps stirring the mites back up into the bees.\u00a0 With the above style of hand agitator, I found that around 15% of the mites in a sample get stuck again in the bees\u2019 bodies as the final shake filters back down.<\/p>\n

I performed a small exploratory study to see whether and how quickly mites would release from bees if immersed in 91% isopropyl alcohol, without agitation<\/em><\/strong>.\u00a0 I set up a row of 6 cups of alcohol, placed a single-deep layer<\/em> of bees in a cylinder with a screened bottom, and immersed the cylinder of bees\u00a0 sequentially in each cup for 10 seconds, giving a slight jiggle to allow any loosened mites to drop off as I moved \u00a0the cylinder from cup to cup. After six 10-second immersions, I then mechanically agitated the bee sample to recover any remaining mites.\u00a0 In this small study of 15 samples, on average 83% of the mites dropped off by 60 seconds (Figure 9).<\/p>\n

\"\"<\/a><\/p>\n

Figure 9.\u00a0 Mite release without appreciable agitation after seconds of immersion in 91% isopropyl alcohol.\u00a0 Although the median release by 60 seconds was 83%, please note that there was considerable variation (not shown), ranging from 20% – 100% (there were not enough mites per sample for anything other than suggestive findings).<\/p>\n

The results of this preliminary mite-release data made me wonder how many mites would release of their own accord if I were to extend the immersion period to two minutes?\u00a0 So I fabricated a larger flat-bottom sieve that could hold up to 500 bees in a single layer.<\/em><\/strong>\u00a0\u00a0 I placed the sieve in a white tub containing fresh 91% alcohol, and sampled bees from a collapsing high-mite hive.\u00a0 I first shook the bees from the frames into a dry tub, and then quickly sprinkled them a single layer deep into the alcohol in the sieve, at which point I started the stopwatch.<\/p>\n

Sample size varied since these bees were very flighty and defensive; the first two samples were of about a half cup of bees (~300 bees), the third somewhat less, and the fourth of over a full cup (that final sample consisted largely of lower-mite bees that had previously returned to the hive).<\/p>\n

It was amazing to observe how quickly the mites released from the bees and dropped through the screen of the sieve without any agitation at all.\u00a0 Within seconds there were dozens on the bottom of the tub (Figure 10).\u00a0 After two minutes were up, I very gently lifted the sieve up and down slightly to jiggle any released mites through the screen, and then dumped the bees into cups for final vigorous agitation to recover any remaining adhering mites (Table 1).<\/p>\n

\"\"<\/a><\/p>\n

Figure 10.\u00a0 A view from the top.\u00a0 To the right is the sieve full of bees gently immersed in 91% alcohol.\u00a0 The mites begin dropping en masse several seconds after the bees hit the alcohol \u2013 without any agitation whatsoever.<\/p>\n

Table 1 below shows the results.\u00a0 After 2 minutes immersion in 91% alcohol, most of the mites had already released of their own accord.<\/p>\n

\"\"<\/a><\/p>\n

Practical application: Patience, patience, patience.\u00a0\u00a0 In high-proof alcohol, nearly all of the mites released from the bees of their own accord within two minutes.\u00a0 By allowing the sample to set for a couple of minutes before beginning agitation, you can save your wrist.\u00a0 This does not mean that you don\u2019t need to agitate \u2013 if the bees are deeper than one layer thick, not all mites drop to the bottom (more results to follow).<\/strong><\/p>\n

Next month<\/h2>\n

At this writing, alcohol is still in short supply, and lower proof alcohol doesn\u2019t work very well, so let me jump ahead and present a follow up on last month\u2019s findings re Dawn Ultra dishwashing liquid:\u00a0 the optimal dilution appears to be 2 tablespoons per gallon.\u00a0 More findings on using Dawn and other solutions and methods to follow.<\/p>\n

Citations<\/h2>\n

[1]<\/a> de Jong, D, et al (1982) A comparative analysis of shaking solutions for the detection of Varroa jacobsoni on adult honeybees.\u00a0 Apidologie 13(3): 297-306.<\/p>\n

[2]<\/a> Peattie AM, et al (2011) Arachnids secrete a fluid over their adhesive pads. PLoS ONE 6(5): e20485.<\/p>\n

[3]<\/a> Fakhimzadeh, K. (2001) Effectiveness of confectioner sugar dusting to knock down Varroa destructor<\/em> from adult honey bees in laboratory trials. Apidologie 32 (2): 139-148.<\/p>\n

[4]<\/a> The Genpak AD16S works very nicely.\u00a0 And a headband magnifier.<\/p>\n

[5]<\/a> Richter, D, et al (2013) How ticks get under your skin: insertion mechanics of the feeding apparatus of Ixodes ricinus <\/em>ticks.\u00a0 Proc Biol Sci. 280(1773): 20131758.\u00a0 Open access.<\/em><\/p>\n

[6]<\/a> Medical University of Vienna (2017) “Tick ‘cement’ as a potential bioadhesive for human tissue.” www.sciencedaily.com\/releases\/2017\/02\/170220085116.htm<\/a><\/p>\n

 <\/p>\n","protected":false},"excerpt":{"rendered":"

Refining the Mite Wash- Part 2 Mite Release Randy Oliver ScientificBeekeeping.com First Published in ABJ in August 2020 The high efficacy of hand dishwashing detergent at getting mites to release their grip on bees bestirred me to investigate this finding more deeply.\u00a0 What I\u2019ve come to realize is that there are four steps involved between […]<\/p>\nRead More<\/a><\/span>","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[15,24,3],"tags":[],"acf":[],"yoast_head":"\nRefining the Mite Wash: Part 2 - Mite Release - Scientific Beekeeping<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/scientificbeekeeping.com\/6960-2\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Refining the Mite Wash: Part 2 - 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