# Three gene markers correlated with Varroa resistance in Saskatraz﻿ bees.



## squarepeg (Jul 9, 2010)

looks like a good one jwchestnut, thanks for sharing it.


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## 1102009 (Jul 31, 2015)

> Thirdly, the synergistic relationship between miticides, parasitic varroa mites and associated pathogens should be further investigated. In reality, honey bees have to encounter not only in hive miticides to destroy varroa mites, but also pesticides sprayed in the agricultural fields for broad-spectrum pest control. The concurrent occurrence between pathogenic virus and varroa mites has been demonstrated among the bee colonies in this study, suggesting the mite can spread viruses among bee colonies and the virus may interfere with the host’s NF-κB signaling (nuclear factor kappa-light-chain-enhancer of activated B cells) and humoral and cellular
> 92
> immune responses, which in turn can facilitate reproduction of the parasitic mite (Di Prisco et al. 2016). In addition, the causal link between insecticide application and virus infection has also been demonstrated, indicating that the increased viral loads in the susceptible bees are possible results of the honey bees being exposed to miticides (Doublet et al. 2015; Locke et al. 2012; Smart et al. 2016). Exposure of honey bees to the neonicotinoid insecticide can compromise the host’s immune-competence and promotion of the viral infection in healthy honey bees (Di Prisco et al. 2013). Thus, further evaluation of the immune-competences among varroa mite-tolerant and mite susceptible honey bees may give new insight into the defensive mechanism of honey bees against the mite.


Very interesting, thanks, JWC


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## Eduardo Gomes (Nov 10, 2014)

"Apivar® and Apistan® are two synthetic miticides and possess strong influences on honey bee behaviors. The treatments drastically increased the virus [DWV] loads in the colony (>160,000 times)." p. 64 

Or I did not understand anything or this data is very intriguing.


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## FlowerPlanter (Aug 3, 2011)

There's other studies showing synergy effects of virus and pesticides. 

http://www.beesource.com/forums/sho...iral-Pathogenicity-and-Mortality-in-Honey-Bee

Very interesting study JW thanks for posting.


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## Juhani Lunden (Oct 3, 2013)

JWChesnut said:


> A sophisticated MsS thesis by Jin Wang published 12/2016 identifies three gene sequences highly correlated with mite resistance in the Saskatraz breeding line of bees. Full document is downloadable at: https://ecommons.usask.ca/bitstream/handle/10388/7651/WANG-THESIS-2016.pdf?sequence=1&isAllowed=y
> *
> Jin Wang, 2016, Identification of potential biomarker genes for selecting varroa tolerant honey bees (Apis mellifera) and biochemical characterization of a differentially expressed carboxylesterase gene in response to mite infestation*


 From the summary: "Most insect cytochrome P450 genes are found to *be expressed* in the digestive and immune systems, such as the midgut and fat body, rather than the head (Huang et al. 2013). The expression patterns of these genes implied their potential functions as detoxification enzymes in the corresponding tissues." "Out of ten selected genes, AmCbE E4, AmApoD and AmCYP6A1 *showed relatively constant, higher expression *levels in dark eyed stage 4 pupae from varroa tolerant colonies in the presence of varroa than those in the absence of the mite. In contrast, significantly* lower expression *was found in varroa susceptible colonies in the presence of varroa than in the absence of the varroa."
"Collectively, this data suggested that the tolerant bees are less susceptible not only to varroa parasitism and DWV infection, but also to miticide applications, probably because they *could stimulate higher expression *of defensive genes involved in detoxification processes and lipid metabolism to minimize the damage caused by mites, viruses, and miticides. "

Is this to be understood so that all the bees in this study (resistant or susceptible) had the same genes, but the bees used their genes differently?


"The virus load in the susceptible colony infested with varroa mites was significantly higher than that without mite infestation and* the degree of the virus infection among the colonies infested with the mite dramatically increased with susceptibility of the bees to varroa. *

In other words: varroa resistance and virus resistance are the two sides of the same coin?


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## Kuro (Jun 18, 2015)

Thanks for the paper, I learned a lot today.

<Is this to be understood so that all the bees in this study (resistant or susceptible) had the same genes, but the bees used their genes differently?

Yes, all bees have the same “cytochrome P450 genes” as DNA. The author says bees from varroa-resistant colonies are capable of expressing three of these genes more efficiently (= making more mRNA transcripts that probably results in more proteins), in response to varroa infestation (Fig.4.4, 4.5, 4.6, 4.7)

<In other words: varroa resistance and virus resistance are the two sides of the same coin?

I’m not sure. Long-lived colonies have fewer mites than short-lived colonies (Figure 4.1). In addition, when individual varroa-infested pupae were examined, those from long-lived colonies showed much less DMV than the short-lived one (Table 5.1, center column. Larger the number less DMV). So I’m guessing at least two different mechanisms (hygienic behavior and resistance to the virus) contribute to colony survival in the presence of varroa.


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## Juhani Lunden (Oct 3, 2013)

double post


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## Juhani Lunden (Oct 3, 2013)

Kuro said:


> Yes, all bees have the same “cytochrome P450 genes” as DNA. The author says bees from varroa-resistant colonies are capable of expressing three of these genes more efficiently (= making more mRNA transcripts that probably results in more proteins), in response to varroa infestation (Fig.4.4, 4.5, 4.6, 4.7)


Same genes but different alleles?


Gene differencies have also been found in this study:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3487727/

Differencies in gene expression: 
From the study:"Differentially expressed genes between bees exhibiting high and low VSH were identified with microarrays [89]. The high VSH stocks were from the same general population that we used for this QTL study. The microarrays revealed 39 genes that were differentially regulated in the brains of 14-day-old worker bees of low- and high-VSH lines."

"Differentially regulated" , the same genes *but different alleles*?

Here one again:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2447852/

"We identified a set of 148 genes with significantly different patterns of expression: 32 varied with the presence of Varroa, 116 varied with bee genotype, and 2 with both. "


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## Kuro (Jun 18, 2015)

Thanks for sharing interesting papers. I’m learning more and more! 



Juhani Lunden said:


> "Differentially regulated" , the same genes *but different alleles*?
> Here one again:
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2447852/


They probably do not know the answer to your question yet (neither Wang nor Navajas et al. mentioned it in their papers). Varroa resistant vs susceptible bees may show differential expression of gene X because (1) they have different alleles of gene X, one of which has more efficient promoter/enhancer, (2) they have different alleles of gene Y, one of which is more efficient in stimulating expression of gene X, (3) they have different DNA sequences somewhere which affect three dimensional structure around gene X, and one of them have more “open” structure, (4) numerous other scenarios. I’m sure they will figure that out once they believe gene X to be a part of the mechanism for varroa resistance (not just association). 



Juhani Lunden said:


> Gene differencies have also been found in this study:
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3487727/


Yes, in this study they took a different approach (analyzed DNA) and mapped a chromosome region associated with the VSH phenotype.


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## Juhani Lunden (Oct 3, 2013)

Kuro said:


> I’m learning more and more!
> 
> 
> They probably do not know the answer to your question yet (neither Wang nor Navajas et al. mentioned it in their papers). Varroa resistant vs susceptible bees may show differential expression of gene X because (1) they have different alleles of gene X, one of which has more efficient promoter/enhancer, (2) they have different alleles of gene Y, one of which is more efficient in stimulating expression of gene X, (3) they have different DNA sequences somewhere which affect three dimensional structure around gene X, and one of them have more “open” structure, (4) numerous other scenarios. I’m sure they will figure that out once they believe gene X to be a part of the mechanism for varroa resistance (not just association).


Thanks for this, very informative, have you some kind of education in genetics?


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## BadBeeKeeper (Jan 24, 2015)

Juhani Lunden said:


> From the summary: "Most insect cytochrome P450 genes are found to *be expressed* in the digestive and immune systems, such as the midgut and fat body, rather than the head (Huang et al. 2013). The expression patterns of these genes implied their potential functions as detoxification enzymes in the corresponding tissues." "Out of ten selected genes, AmCbE E4, AmApoD and AmCYP6A1 *showed relatively constant, higher expression *levels in dark eyed stage 4 pupae from varroa tolerant colonies in the presence of varroa than those in the absence of the mite. In contrast, significantly* lower expression *was found in varroa susceptible colonies in the presence of varroa than in the absence of the varroa."
> "Collectively, this data suggested that the tolerant bees are less susceptible not only to varroa parasitism and DWV infection, but also to miticide applications, probably because they *could stimulate higher expression *of defensive genes involved in detoxification processes and lipid metabolism to minimize the damage caused by mites, viruses, and miticides. "
> 
> *Is this to be understood so that all the bees in this study (resistant or susceptible) had the same genes, but the bees used their genes differently?*


No, it is not that simple. To be clear, CYP450 is not a gene, it is a protein, an enzyme- more accurately a -group- of enzymes that participate in oxidase reactions. There are more than 200k different CYP450 enzymes currently known. The genes they are speaking of code for variations of the CYP450 enzymes.

In humans, there are six specific variations of CYP450 enzymes that affect 90% of all drug metabolism. Variations of the enzymes are the reason why the same drug can affect two people differently.

It is similar for bees. The bees do not 'use' the genes, it is the particular genes they have that code for particular variations of the enzymes, the coding is fixed in the gene and cannot be changed by the bee. The idea is to determine which enzyme acts in a desired manner, and then to select for the gene that codes for the specific variation of the enzyme that is desirable.


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## lharder (Mar 21, 2015)

This is a thought provoking paper JW, judging from the comments, thank you. 

I haven't finished reading it, but its nice his supervisor gave him the latitude to do a fairly thorough job setting the context up. Makes understanding so much easier. 

My initial stock was mostly Saskatraz bees and I'm still around 3 winters later. I should probably get another injection of them as they have quite a few queen lines.


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## Juhani Lunden (Oct 3, 2013)

BadBeeKeeper said:


> No, it is not that simple. To be clear, CYP450 is not a gene, it is a protein, an enzyme- more accurately a -group- of enzymes that participate in oxidase reactions. There are more than 200k different CYP450 enzymes currently known. The genes they are speaking of code for variations of the CYP450 enzymes.
> 
> In humans, there are six specific variations of CYP450 enzymes that affect 90% of all drug metabolism. Variations of the enzymes are the reason why the same drug can affect two people differently.
> 
> It is similar for bees. The bees do not 'use' the genes, it is the particular genes they have that code for particular variations of the enzymes, the coding is fixed in the gene and cannot be changed by the bee. The idea is to determine which enzyme acts in a desired manner, and then to select for the gene that codes for the specific variation of the enzyme that is desirable.


Thanks. Do you have some education in biology?



Kuro said:


> Yes, in this study they took a different approach (analyzed DNA) and mapped a chromosome region associated with the VSH phenotype.



So there seems to be two different types of studies: the ones which study the differencies in DNA and the ones which study the differencies in proteins (coded by DNA) in varroa resistant versus varroa susceptible bees. 



Kuro said:


> They probably do not know the answer to your question yet (neither Wang nor Navajas et al. mentioned it in their papers). Varroa resistant vs susceptible bees may show differential expression of gene X because (1) they have different alleles of gene X, one of which has more efficient promoter/enhancer, (2) they have different alleles of gene Y, one of which is more efficient in stimulating expression of gene X, (3) they have different DNA sequences somewhere which affect three dimensional structure around gene X, and one of them have more “open” structure, (4) numerous other scenarios. I’m sure they will figure that out once they believe gene X to be a part of the mechanism for varroa resistance (not just association).


So, if Kuro is right, studying proteins is not giving straight answers of gene differencies. Why not study directly DNA, it is more expensive, or are there other reasons for two approaches?
Studying proteins may give idea of the mechanisms behind varroa resistance?


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## lharder (Mar 21, 2015)

I believe studying the DNA and protein expression brings clarity. One could have the gene for something, but it isn't activated for whatever reason. Its not just a plus minus situation either. As we have seen with insects, pesticide resistance often is a result in massive amplification of specific detoxification pathways. It can happen quickly, so something interesting is happening with regard to genetic mechanisms in the face of new challenges. Also, it looks like protein expression is not one to one with the genetic code. There seems to be some mixing and matching going on (my poor choice of words) that leads to a more diverse protein mix, than the actual genes coding for them. 

The research group sampling my bees is specifically interested in identification of proteins for purposes of selection.


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## JRG13 (May 11, 2012)

Studying genes is kind of studying the proteins as genes code for proteins or peptides that lead to proteins. Protein expression and function is a separate area though, more molecular biology stuff than genetics. When you start talking about enzymes though, it gets even more complicated as catalysts are typically involved and then there can be drastic shifts in your energy curves which really change the equations on potential activity.


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## BadBeeKeeper (Jan 24, 2015)

Juhani Lunden said:


> Thanks. Do you have some education in biology?


More in Organic Chemistry, I flunked out of Biology when we had to start cutting critters open to look at their insides. My last job partly involved a major pharmaceutical distributor, and I read -a lot- of information and studies on the metabolization of a variety of different drugs and the role that the CYP450 enzymes play in it.


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## Juhani Lunden (Oct 3, 2013)

BadBeeKeeper said:


> More in Organic Chemistry, I flunked out of Biology when we had to start cutting critters open to look at their insides. My last job partly involved a major pharmaceutical distributor, and I read -a lot- of information and studies on the metabolization of a variety of different drugs and the role that the CYP450 enzymes play in it.


Thanks, sounds great. Beesource is great but sometimes hard to know whos input is worth reading.
(although beekeeping was my main subject in Helsinki University, there were not many cources about genes and heredity or biochemistry, mostly self educated afterwards in these)


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## Richard Cryberg (May 24, 2013)

BadBeeKeeper said:


> More in Organic Chemistry, I flunked out of Biology when we had to start cutting critters open to look at their insides. My last job partly involved a major pharmaceutical distributor, and I read -a lot- of information and studies on the metabolization of a variety of different drugs and the role that the CYP450 enzymes play in it.


Background is just fine. You can not start to understand genetics without at least a modest background in organic chemistry these days. And, you can not start to understand organic chemistry without physical chemistry for the last 60 years. If you have those two things understanding genetics is pretty easy. Biology is pretty much irrelevant in understanding modern genetics. Genetics should not even be part of the biology department althou that is going to take a few more years to happen. Hard to overcome history.


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## JRG13 (May 11, 2012)

I agree Richard, although genetics is part of the biological system, when it actually comes down to understanding biology we know very little but genetics is very straightforward for the most part.


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## Kuro (Jun 18, 2015)

Juhani Lunden said:


> So there seems to be two different types of studies: the ones which study the differencies in DNA and the ones which study the differencies in proteins (coded by DNA) in varroa resistant versus varroa susceptible bees. ..... Why not study directly DNA, it is more expensive, or are there other reasons for two approaches?


I agree, if the goal is to find the gene(s) responsible for a hereditary trait, starting with gene mapping by analyzing genomic DNA is probably the most straightforward. With recent technologies, such as “interval mapping of quantitative trait loci”, they do not have to do too many back-crossings. Once they narrow down the area in the genome that is associated with the trait, they will find many genes there, some of which might be irrelevant. From there, they need to begin functional studies on each gene, such as the function of the encoded protein, how mRNA expression is regulated, and so on, and finally come up with logical explanation why those genes are important for the trait. 

Going back to the Jin Wang’s paper the OP posted, gene mapping was not the author’s primary goal. It was a follow up study of this paper (I only have the abstract https://www.ncbi.nlm.nih.gov/pubmed/26919127), where they compared varroa sensitive/resistant lines for mRNA expression, using “DNA microarray analysis”. DNA microarray analysis is one of the technologies to measure expression of large numbers of genes simultaneously, and gives researchers a rough idea of what’s going on. They found differential expression of >100 genes, some of which are already known to be important for biological functions, including olfaction, signal transduction, detoxification processes, protein and lipid metabolisms, and exoskeleton formation. Wang focused on the detox genes using more accurate “quantitative RT-PCR analysis”. Such a study would nicely complement gene mapping studies to understand the mechanism of varroa-resistance. In addition, as the OP said, it may also serve a practical purpose (i.e., looking gene expression patterns to pre-screen potential breeder colonies).


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## Juhani Lunden (Oct 3, 2013)

Kuro said:


> IGoing back to the Jin Wang’s paper the OP posted, gene mapping was not the author’s primary goal. It was a follow up study of this paper (I only have the abstract https://www.ncbi.nlm.nih.gov/pubmed/26919127), where they compared varroa sensitive/resistant lines for mRNA expression, using “DNA microarray analysis”. DNA microarray analysis is one of the technologies to measure expression of large numbers of genes simultaneously, and gives researchers a rough idea of what’s going on. They found differential expression of >100 genes, some of which are already known to be important for biological functions, including olfaction, signal transduction, detoxification processes, protein and lipid metabolisms, and exoskeleton formation. Wang focused on the detox genes using more accurate “quantitative RT-PCR analysis”. Such a study would nicely complement gene mapping studies to understand the mechanism of varroa-resistance. In addition, as the OP said, it may also serve a practical purpose (i.e.,* looking gene expression patterns to pre-screen potential breeder colonies*).


Very informative, thank you, I suppose you know what you are talking about, although you did not answer my question about education, no offense...

The highlighted last sentence: So gene expression patterns might be as good a criteria for selection as genes themselves ?

RT-PCR reverse transcriptase - polymerase chain reaction


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## Kuro (Jun 18, 2015)

If they already know exactly which allele of what genes are necessary and sufficient for the trait they want, carrying such DNA sequences would be the best selection criteria in their breeding program. Unfortunately, in the case of varroa resistant honey bees, they do not have enough information. They have to wait until each colony shows resistance. But if they can develop a quick laboratory test that predicts how each colony will behave, they can save lots of time and effort. Wang says expression of the three genes he/she studied (over-expressed in varroa-resistant lines) may be used for that purpose. My background is something like 20% veterinary medicine, 30% virology and 50% molecular biology and I worked for biotechs and a pharmaceutical on vaccines and antivirals. I’m quite rusty and not familiar with honey bee research, so it was not easy to answer your questions.


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## Juhani Lunden (Oct 3, 2013)

Kuro said:


> If they already know exactly which allele of what genes are necessary and sufficient for the trait they want, carrying such DNA sequences would be the best selection criteria in their breeding program. Unfortunately, in the case of varroa resistant honey bees, they do not have enough information. They have to wait until each colony shows resistance. But if they can develop a quick laboratory test that predicts how each colony will behave, they can save lots of time and effort. Wang says expression of the three genes he/she studied (over-expressed in varroa-resistant lines) may be used for that purpose. My background is something like 20% veterinary medicine, 30% virology and 50% molecular biology and I worked for biotechs and a pharmaceutical on vaccines and antivirals. I’m quite rusty and not familiar with honey bee research, so it was not easy to answer your questions.


:thumbsup:


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## lharder (Mar 21, 2015)

Here is a link to the UBC group

http://www.chibi.ubc.ca/faculty/leonard-foster/foster-lab/bee-ipm/

The bee works goals are 

1) protein markers to facilitate the selection of honey bee stocks with natural disease resistance, 2) RNAi-based treatments for bee diseases, and 3) best-practices guidelines for IPM.

I tried to find an explanation for the focus on proteins but was unable to find any. I'll get a chance to find out in the near future. 

There is a bunch of extra links to data and publications if people are interested.


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