A Comparative Trial of the Pollen Subs 2020: Half 2

A COMPARATIVE TRIAL OF THE POLLEN SUBS 2020

Half 2

First revealed in ABJ August 2021

Vincent Ricigliano^a and Randy Oliver^b
aUSDA-ARS, ^bScientificBeekeeping.com

Final month I described how we arrange our comparative trial of a number of pollen subs. Now it’s time to usher in my collaborator, Dr. Vincent Ricigliano, to point out the outcomes of a few of his laboratory analyses of the bee samples taken on the midpoint of the trial, because the colonies went into winter cluster.

A notice from the authors: Since Randy began scripting this sequence within the first particular person, we’re going to proceed alongside that vein, though a lot of this text might be about Vince’s findings. Since beekeepers have gotten increasingly dependent upon supplemental feeding of their colonies, for the good thing about the beekeeping business we’re doing a deep dive into extracting as a lot usable data as we are able to from the information gathered throughout this area trial of synthetic diets.

That stated, this text presents a variety of data from the midpoint of the trial — each area measurements and molecular analyses. The midpoint findings offered on this article assist us to know synthetic diets on the whole, and can probably be of particular curiosity to these serious about growing higher diets for honey bees. Subsequent month we’ll current the endpoint outcomes, which can actually get right down to the nitty-gritty of {dollars} and cents, sensible utility to beekeepers curious concerning the pollen subs in the marketplace.

EFFECT OF DIET TYPE UPON COLONY STRENGTH AT THE MIDPOINT ASSESSMENT

After a feeding interval of 84 days — throughout which the colonies depended upon the examined pollen subs and sugar syrup as their important sources of diet — the hives went broodless in November because the climate cooled. We graded them for energy on a chilly morning, when the clusters have been very tight (however which allowed for correct and constant grading).

The primary query to reply was whether or not midpoint colony strengths mirrored their beginning strengths, since I’d deliberately begun the trial with an equal vary of beginning cluster sizes for every weight loss program kind. A scattergram (Determine 1, wherein most of the 144 knowledge factors overlap) signifies that there was no profit from beginning with a bigger cluster measurement.

Fig. 1 Change in cluster measurement from August to November; colonies beneath the No Change line shrunk in measurement. The median beginning cluster measurement (in hot-weather grading) was 7.5 frames of bees (FOB), by November (with cold-weather grading); most colonies had shrunk in measurement (to a median 5.3 FOB). The information above point out that there was no profit to beginning with a bigger cluster measurement in August, for the reason that bigger colonies tended to lose extra energy than the smaller ones (as evidenced by the higher vertical drop of the information factors from the No Change line from left to proper).

Sensible utility: Being stronger in August didn’t imply {that a} colony can be stronger in November. This will likely have been an artifact of the clusters of the sturdy colonies being extra expanded throughout hot-weather grading, greater than precise bee rely.

Since beginning cluster measurement was not that predictive of midpoint measurement, and since all weight loss program teams began with an equal vary of cluster sizes, which means that we are able to evaluate the typical colony strengths in November by weight loss program kind with out bearing in mind beginning colony strengths (Determine 2).

Fig. 2 The bigger cluster sizes of the colonies fed the higher subs, relative to the Sugar Controls, signifies the optimistic profit from feeding pollen sub (a t-test p-value < 0.00001 for World vs. Management, thus validating the experimental design). The comparatively small normal errors point out that the variations in efficiency relative to weight loss program kind are significant. The above knowledge matched our area observations so far as colony desire for (consumption sensible), and efficiency in response to, weight loss program kind — with the blue, pink, and silver therapy teams being standouts (notice that solely two of these three diets contained pure pollen).

Sensible utility: As we’ll see subsequent month, the cluster measurement of a colony going into winter made a giant distinction on how they construct up for almond pollination. Maintain the above rating of efficiency of the subs in thoughts.

OK, now that you just’ve bought the rating of how the weight loss program kind affected colony efficiency so far as cluster measurement on the midpoint, let’s see how weight loss program affected the biology of the bees.

VINCE’S LABORATORY ANALYSES AT THE MIDPOINT

We put a variety of area work into this comparative trial of the diets. However this wasn’t only a check to see which subs have been “greatest,” however moderately a chance to be taught why some diets carry out higher than others. On the grading in early November, I froze samples of staff vacuumed from the highest bars of the broodnests. Vince and his technician Steven Tyler Williams carried out weight and molecular analyses on the sampled bees, in addition to sending samples of the patties out for dietary evaluation.

On the USDA lab, they eliminated and dried the heads and thoraces of 15 bees from every pattern, and weighed them as a bunch. This enables us to check particular person bee glandular and muscular growth to colony cluster measurement on account of being fed every weight loss program (Determine 3).

Fig. 3 Common particular person bee mass (weight) carefully correlated with colony energy (R² = 0.80 [[1]]), indicating that what’s nutritionally good for the person bee is sweet for the colony as a complete.

Sensible utility: Head and thorax weights are sometimes used because the metric for laboratory research on bee weight loss program high quality; the information above assist that assumption. I’ll focus on this in higher depth in a subsequent article.

PATTY CONSUMPTION

Since a synthetic weight loss program is of little worth if the bees aren’t stimulated to eat it, a query to reply is whether or not the bees handled the provided patties as meals, or as junk to be faraway from the hive. The one weight loss program kind that we noticed being discarded to any extent was the Wholesome Bee with thyme oil. Conversely, it was no shock that the 2 patty varieties containing pure pollen have been consumed rapidly and utterly. Nonetheless, notice that the opposite diets weren’t far behind (Determine 4).

Fig. 4 The bees most well-liked some formulations over others, however not less than 90% of all patty varieties have been consumed throughout this feeding interval. World and Homebrew, probably attributable to being 15-20% pure pollen, have been essentially the most rapidly and utterly consumed, however the others weren’t far behind. There have been no considerable yard-to-yard variations in general patty consumption — 95% and 96% on the E and T yards; and 97% on the H yard (which was fed 2 lbs. extra).

Sensible utility: Though the bees consumed some subs extra eagerly than others, it doesn’t seem that there was a large enough distinction in consumption between weight loss program varieties to account for the variations in colony efficiency on the midpoint, and diminished consumption could have partially been a perform of colony measurement.

WHY WERE SOME SUBS MORE (OR LESS) PHAGOSTIMULATORY?

The scientific time period for exciting an organism to eat is “phagostimulation” — “phago” which means “to eat.” So what makes a synthetic weight loss program phagostimulatory to the nurse bees (the employees that truly eat the patties)?

BACKGROUND

Though some researchers have proposed that nurse bees can differentiate diets by dietary high quality, I discover their arguments unconvincing. Then again, in a highly-informative examine, Vanessa Corby-Harris [[2]] carried out a sequence of alternative assays of pure and nutritionally-supplemented pollens, and got here to the conclusion:

 … that dietary worth performs a minimal position in nurse bee weight loss program alternative … although sure pollens present a higher profit per unit of consumption, honey bee nurses both can’t or don’t discriminate amongst pollens based mostly on diet.

Sensible utility: I’m not satisfied that bees can inform whether or not a weight loss program is nutritious or not by style or scent. However you will need to make any synthetic weight loss program scent or style “good” to the patty-consuming nurse bees, with a view to encourage them to eat it.

Facet notice: Having just lately misplaced all sense of style for some months attributable to radiation therapy of my tongue, I can attest to the significance of sensory phagostimulation to induce what we’d name “the need to eat” any specific meals alternative.

I and others have noticed that bees clearly have preferences for some pure pollens over others. Every of the patty varieties smelled completely different to my human nostril, so I don’t doubt that odor was a think about nurse feeding desire for the examined subs.

NATURAL POLLEN AS A COMPONENT

 Again in 1973, USDA researcher Lonnie Standifer discovered, not surprisingly, that the addition of 5% pure pollen to their examined synthetic weight loss program improved consumption and the quantity of brood reared, however that there was not a lot achieve from growing the proportion to twenty% [[3]]. Standifer concluded that:

The higher efficiency of colonies fed rations containing pollen could happen due to feeding stimulants current within the pollen moderately than as a result of the rations containing no pollen lack vitamins.

Schmidt and Hanna [[4]] later decided that there are a collection of phagostimulatory compounds in pollen. Not surprisingly, on this trial the bees eagerly consumed the 2 merchandise containing pure pollen, however a few of the utterly synthetic diets have been additionally rapidly consumed by the bees. The World patties are marketed as containing 15% pure pollen (irradiated towards pathogens); the Homebrew patties contained 20% pollen by dry weight. The addition of pure pollen to these merchandise implies that they have been technically “pollen complement” or “pollen extender” formulations, moderately than pollen substitutes (utterly “synthetic diets”).

It’s well-known that colonies reply nicely to patties of trapped pollen moistened with sugar syrup. However pure pollen is pricey, could comprise agricultural chemical substances, can fluctuate significantly in dietary and phagostimulatory worth, and if not sterilized, can transmit the complete spectrum of bee pathogens.

Sensible utility: I’ve bought nothing towards utilizing trapped pollen for feeding bees, however true “pollen substitutes” are synthetic diets that don’t comprise bee-collected pollen as an ingredient, which permits them to be extra standardized and cheaper. Among the examined synthetic diets carried out almost in addition to merchandise containing pure pollen, which could be very encouraging.

FEEDING ATTRACTANTS OR DETERRENTS

 Among the subs contained lemongrass oil, which accommodates one of many elements of honey bee orientation pheromone (citral). I’m not clear as as to whether lemongrass oil is phagostimulatory or merely attracts the nurses to the proximity of the patty. At the least one sub additionally contained spearmint oil and thymol.

One have to be cautious with important oils in diets, since bees are strongly repelled by some fragrant important oils. The Wholesome Bee patties that I examined have been fairly fragrant with thyme oil, which can have sadly acted as a feeding deterrent to the bees (and in addition to Small Hive Beetle). I confirmed a photograph in my earlier article of how the bees dragged Wholesome Bee patty out of the hive.

MOISTURE AND TEXTURE

 The bees appeared to choose moist patties over drier patties. The examined subs diverse extensively in texture and moisture content material. (I strongly counsel to the producers that they wrap their subs in plastic baggage, in order that they don’t dry out throughout storage.)

Vince additionally had a business lab analyze the patties for protein, amino acids, lipids, and sugars. Let’s see whether or not the quantities of these parts affected how nicely the bees “preferred” the patties.

SUGAR CONTENT

 Everyone knows that bees like sugar. And if there’s both a pure pollen move occurring, or if the bees don’t like one thing about a synthetic weight loss program, they may usually simply suck the sugar out of a patty, and take away the rest as trash. So how a lot sugar was within the examined subs (Determine 5)?

Fig. 5 I ordered the subs from left to proper by consumption desire as within the earlier graph. The patties ranged from 30% to 60% sugar content material. Word that the sugar content material didn’t correlate with consumption desire. Regardless of AP23 and MegaBee each having low sugar contents, they have been nonetheless nicely consumed. Apparently, a excessive sugar content material just isn’t essential for a synthetic weight loss program, nor does it assure speedy consumption.

The breakdown of the sugars signifies that almost all formulations used high-fructose corn syrup (consisting of fructose and glucose) as the primary sweetener, probably not solely attributable to its low value, however as a result of it makes for a patty that maintains its mushy texture. Most additionally contained a lesser quantity of sucrose.

PROTEIN CONTENT

 The primary objective for feeding pollen sub is to offer protein to the nurse bees. However protein content material didn’t correlate with how nicely the patties have been consumed or carried out (Determine 6).

Fig. 6 The diets are ranked from left to proper by consumption (from Determine 4). The protein focus didn’t correlate with consumption, nor (as we’ll see additional alongside) with efficiency.

Sensible utility: The colonies clearly didn’t eat patties in accordance with their protein contents. A powerful shot of protein could permit a burst of colony progress [[5]], however as we’ll see, over the long run patties with solely 15% protein can carry out in addition to these with over 20%. That is necessary to know, since protein is often the most costly element of pollen subs. However as I’ll elaborate upon in a subsequent installment, our outcomes counsel that extra necessary than complete protein is the stability of the important amino acids.

LIPID CONTENT (INCLUDING ESSENTIAL FATTY ACIDS AND STEROLS)

 Pure pollens fluctuate of their lipid contents, however typically fall throughout the 3-10% complete lipid content material vary, with the pollen of the bee-attractive and nutritionally full clovers and mustards falling into the 6-10% vary [[6]^{, [7]}]. So how necessary was lipid content material so far as consumption? See Determine 7.

Fig. 7 Once more proven so as of consumption. Cleary lipid content material didn’t outline how phagostimulatory the subs have been. Most shocking to us was that World, which was probably the most rapidly consumed patties, had by far the bottom lipid content material.

Australian researcher Rob Manning examined various vegetable oils as phagostimulants [[8]], and my very own observations are that canola and corn oil look like engaging to bees. Vegetable oil might also be added to enhance the feel of a sub. The chart above signifies that the producers don’t agree upon how a lot oil in a weight loss program is optimum.

Sensible utility of the above findings: Properly, it positive didn’t look like the sugar, lipid, or protein proportions of the patties that outlined how phagostimulatory the formulations have been. So based mostly upon these outcomes, it could be exhausting to provide you with any guidelines of thumb for easy methods to formulate a synthetic weight loss program with a view to stimulate consumption, apart from having sufficient sugar, and a few scent or style elements which are phagostimulatory to the nurses.

MOLECULAR ANALYSIS OF THE GUT MICROBIOME

We’ve heard loads about how necessary the intestine microbiome is to our well being and that of our bees. Vince centered on the “core” microbiome bacterial varieties since these represent 90+% of all micro organism within the intestine and have useful significances linked to physiology and well being (Determine 8).

Fig. 8 Of curiosity is that one may count on to see the best quantity of core intestine micro organism within the two teams fed patties containing pure pollen, however that was solely the case with World. Surprisingly, bees fed Homebrew, with even a higher proportion of pollen, had solely a 3rd the quantity of micro organism current in World — and even lower than the sugar controls. The thyme oil in Wholesome Bee could not have been pleasant to the intestine microbiome — a outcome that Vince is at the moment following up on with laboratory experiments.

The final statement is of curiosity, since Vince has beforehand proven that spirulina has nice potential as a element of synthetic diets [[9]]:

Spirulina diets produced biomarker profiles (thorax weight, head protein content material, and helpful intestine micro organism abundance) that have been indicative of elevated dietary states, assembly or exceeding the opposite diets in some metrics regardless of diminished consumption.

Sensible utility: We strongly suspect that it was the added thyme oil in Wholesome Bee that negatively affected consumption, head and thorax weight, the intestine microbiome, in addition to general colony efficiency. The very last thing that we wish to do is to trash spirulina as a possible bee feed element. I’ve been in common contact with the producer of Wholesome Bee, who knowledgeable me as I used to be typing these phrases that they now provide a Wholesome Bee formulation freed from thyme oil [[10]].

Though the 2 extremes within the above graph correlate with colony efficiency, that’s hardly the case for these diets mendacity between. Word, nonetheless, it doesn’t matter what the weight loss program kind or whether or not it contained pure pollen, the relative proportions of the core bacterial teams remained constant.

Sensible utility: The core microbial intestine group seems to have the ability to preserve proportional homeostasis regardless of the weight loss program formulation or the presence of pure pollen. That’s excellent news for the event of synthetic diets. Why the bees consuming Homebrew had solely a 3rd as many core micro organism as did World stays an open query, however because it didn’t hamper Homebrew’s efficiency, maybe the quantity of micro organism within the intestine just isn’t that crucial. You will need to remember the fact that intestine microbiome dynamics are complicated, with their relationship to foraging surroundings and meals sources nonetheless poorly understood.

NOSEMA CERANAE

The bee midgut (the place pollen sub is definitely digested) can also be the place Nosema ceranae reproduces. Within the sampled bees, there was nice yard-to-yard and sample-to-sample variation, which prevents us from drawing agency conclusions as to weight loss program kind and nosema. Nonetheless, it occurred to me that since nosema an infection depth typically displays the quantity of pollen that the bees are consuming [[11]^,[12]], that the quantity of nosema within the sampled bees may correlate with the protein focus of the weight loss program that they’d been consuming. That seems to certainly be the case (Determine 9).

Fig. 9. The trial was not set as much as particularly reply this query, however the knowledge definitely means that the an infection depth of Nosema ceranae correlated with the quantity of protein within the weight loss program.

Sensible notice: Beekeepers freak out about nosema spore counts, not understanding that spore counts go up relative to the quantity of pollen that the bees are consuming. On the identical time, the elevated diet from the pollen largely offsets the damaging results of nosema an infection within the bees. This probably additionally applies to the feeding of pollen sub — spore rely will go up, however the bees will nonetheless profit from the supplemental weight loss program.

THE EFFECT OF OXALIC ACID TREATMENT

Not being one to overlook a chance to squeeze some additional data out of a knowledge set, it occurred to me that every one the colonies within the trial had been handled regularly with extended-release oxalic acid at a low degree [[13]] — that’s, aside from eight potential breeder colonies unfold all through the yards and fed the identical diets, however by no means having obtained an oxalic therapy. Aha, I assumed, maybe we are able to achieve some perception into the query of whether or not prolonged publicity to oxalic acid impacts the intestine microbiome (Determine 10).

Fig. 10 The trial was not set as much as reply this query, and there weren’t sufficient samples within the untreated group to attract a agency conclusion, however the above knowledge counsel that prolonged publicity to a low degree of oxalic acid doesn’t adversely have an effect on the intestine microbiome of the handled bees.

Sensible utility: Excellent news!

OK, since I’m on a roll, Vince additionally carried out qPCR to find out the relative abundance of DWV-A in the identical bee samples (low-dose OA-treated vs. untreated near-zero-mite breeders). We often monitored the OA-treated hives for varroa, and mite wash counts not often exceeded a 2% infestation fee. Regardless of these low infestation charges, the variations of their relative ranges of DWV-A in November is putting (Determine 11).

Fig. 11 We have been shocked by the large variations in DWV-A degree between the low-mite OA-treated hives, and the few potential breeders that stored mite counts to zero by themselves. Because the DWV ranges of the E- and H-yard breeders are too low to point out on this non-logarithmic graph, I simply put the relative expression numbers above the place their blue columns can be. Qualifier: Once more, there have been solely 8 mite-resistant colonies, however their common virus expression ranges have been wherever from eleven to over 1,000,000 instances decrease than the averages of the analyzed different hives in the identical yards.

Sensible utility: The employees within the varroa-resistant colonies had strikingly decrease Deformed Wing Virus ranges than did these within the low-mite handled hives. We gotta get critical about choosing for varroa-resistant bee inventory!

Molecular analyses equivalent to these above could assist us to know why and the way completely different diets may have an effect on employee physiology and their microbiomes, and the way that pertains to colony efficiency.

TAKE HOMES AT THE MIDPOINT

So what have we realized to this point?

• The bees consumed many of the diets nicely, though some have been a bit extra phagostimulatory than others. Though the bees clearly “preferred” pure pollen, it wasn’t a essential element of a profitable weight loss program.
• Complete protein within the 15-20% vary seems to be ample, however notice that researchers on the Tucson lab discovered {that a} 23-30% protein weight loss program labored higher than 10% or 50% [[14]].
• Most diets contained sugars within the 50% vary (moist weight), however colonies consumed MegaBee nicely regardless of it containing solely 30% sugars.
• One factor that involves thoughts is the mandatory protein to carbohydrate (P:C) ratio in a bee weight loss program, the carbohydrate portion consisting of sugars. Royal jelly, the only real weight loss program of younger larvae or the queen, has a P:C ratio between 1:1 and 1:2 [[15]^{, [16]}]. For newly-emerged staff or nurse bees producing jelly (crucial for colony buildup), it’s probably within the vary of 1:2 – 1:5. However for foragers (which solely require tiny quantity of protein for physique upkeep), it jumps to one thing within the neighborhood of 1:250. So if one is feeding pollen sub for colony buildup or preparation for winter, the P:C ratios of present pollen subs seems to be in the appropriate ballpark (18% protein and 50% sugar is a 1:2.8 ratio), with the bees in a position to stability any further want for sugar by consuming honey or fed sugar syrup.
• HFCS is often used as the primary sugar supply, because it maintains patty texture. The optimum quantity of sucrose just isn’t clear (whether it is essential in any respect). So I contemplate the best sugar formulation of a sub to stay an open query.
• It was extraordinarily shocking to us how eagerly the colonies consumed, and the way nicely they carried out, on World, regardless of it containing solely 2% lipids.
• One necessary query was whether or not there’s a want for pollen exines as a prebiotic for the core bacterial intestine microbiome — or do the fiber and sophisticated carbohydrates of the soy meal, corn gluten meal, or brewer’s yeast sometimes used as protein sources of a weight loss program serve the aim? Our evaluation signifies that the core bacterial microbiome construction remained constant regardless of the presence or absence of pollen, and even with the completely different synthetic weight loss program formulations, which is probably going excellent news. As well as, bacterial abundance didn’t correlate with colony efficiency, thus making us query whether or not the quantity of micro organism is that necessary.
• Going into winter, the colonies fed the higher subs had clusters half once more bigger than the Controls, consisting of staff with better-developed heads and thoraces (regrettably, we didn’t measure fats physique growth). As we’ll see, this gave them an necessary head begin after they started late-winter broodrearing for spring buildup.

TO BE CONTINUED…

OK, you’ve seen the outcomes as much as the midpoint, however don’t draw any conclusions but! After winter break, the “winter bees” reared on these synthetic diets then needed to construct up once we resumed feeding the identical diets, previous to going to almonds. That is when the rubber hit the highway, and the true efficiency of the diets grew to become obvious. I’ll additionally carry out a cost-benefit evaluation, after which in a following installment counsel why some subs have been in a position to outperform others.

NOTES AND CITATIONS

[1] Not proven is that I ran a linear regression of the information.

[2] Corby-Harris, V, et al (2018) Honey bee (Apis mellifera) nurses don’t eat pollens based mostly on their dietary high quality. PLoS ONE 13(1): e0191050.

[3] Standifer, L, et al (1973) Affect of pollen in synthetic diets on meals consumption and brood manufacturing in honey bee colonies. Am. Bee J. 113: 94-95.

[4] Schmidt,J & A Hanna (2006) Chemical nature of phagostimulants in pollen engaging to honeybees. J Insect Behav 19: 521–532.

[5] http://scientificbeekeeping.com/a-comparative-test-of-the-pollen-sub/

[6] Dong, J, et al (2015) Fatty acid profiles of 20 species of monofloral bee pollen from China. Journal of Apicultural Analysis 54(5): 503-511.

[7] Manning, R (2001) Fatty acids in pollen: a assessment of their significance for honey bees, Bee World, 82(2): 60-75.

[8] Manning, R, et al (20100 Added vegetable and fish oils to low-fat pollen diets: impact on honey bee (Apis mellifera L.) consumption. Australian Journal of Entomology 49: 182–189.

[9] Ricigliano, V & M Simone-Finstrom (2020) Dietary and prebiotic efficacy of the microalga Arthrospira platensis (spirulina) in honey bees. Apidologie 51: 898–910.

[10] https://www.healthybeesllc.com/submit/back-to-the-roots-of-nutrition-our-new-formula

Subject testing of our product with out important oils started round a yr in the past with business beekeepers. To date, the suggestions has been nice. Even higher royal jelly manufacturing than the unique, higher consumption and “fluffier bees”. Which I usually correlate to good particular person physiology attributable to diet…So now we’re proud to supply a strictly dietary model of our product, formulated to offer superior intestine well being together with diet.

[11] Zheng, H, et al (2014) Spore masses is probably not used alone as a direct indicator of the severity of Nosema ceranae an infection in honey bees Apis mellifera (Hymenoptera:Apidae). J. Econ. Entomol. 107(6): 2037-2044.

[12] Jack, C, et al (2016) Results of pollen dilution on an infection of Nosema ceranae in honey bees. Journal of Insect Physiology 87:12-19.

[13] I had a Pesticide Analysis Authorization to take action.

[14] Herbert E, H Shimanuki, D Caron (1977) Optimum protein ranges required by honeybees (Hymenoptera, Apidae) to provoke and preserve brood rearing, Apidologie 8, 141–146.

[15] Garcia-Amoedo L & L de Almeida-Muradian (2007) Physicochemical composition of pure and adulterated royal jelly. Quim Nova 30(2):257–259

[16] Helm, BR, et al (2017) The geometric framework for diet reveals interactions between protein and carbohydrate throughout larval progress in honey bees. Biol Open 6(6): 872–880.