Testing for Varroa sensitive hygiene – VSH

RNSBB research protocol                                                                                                             Version 2014-07-14


Testing for Varroa sensitive hygiene – VSH


author: Ralph Büchler, Kirchhain, with the support of RNSBB members and approval of USDA Baton Rouge lab


pictures: USDA and Ralph Büchler                                    file download: pdf





In the 1990s, honey bee scientists Harbo and Harris working in the USDA honey bee laboratory in Baton Rouge, observed some colonies with one drone inseminated queens showing high rates of non

 reproducing mites in their worker brood. They proved that non reproduction of the mite is a heritable character of worker bees and named it “Suppression of mite reproduction - SMR”.


Harbo and Harris initiated a selection program by testing colonies for the rate of infertile mites in capped brood. Later on, studies on colonies thus selected showed that they performed well in hygienic behavior tests and specific experiments suggested that the low proportion of fertile mites mostly was due to preferential removal of reproducing mites by worker bees, so the trait was renamed “Varroa sensitive hygiene - VSH”.


Although the exact mechanisms of how VSH effects the mites (preference for reproducing mites, disturbance by uncapping and recapping the brood cell, mite invasion to temporary uncapped pupae etc.) are not yet well understood, the final reduction of mite propagation is well confirmed. Strong VSH behavior causes high Varroa resistance of colonies and eliminates or reduces the need for mite treatments. VSH stock is meanwhile successfully used in some beekeeping operations and further breeding is being conducted to improve available stocks.


Bob Danka, Baton Rouge, evaluating

brood cells under the microscope 



Testing for VSH behavior can either be done directly by following the removal and uncapping/recapping activities on infested cells or indirectly according to the original Harbo investigation for non reproducing mites.


The recent standard procedure in the Baton Rouge laboratory is to use highly infested brood combs (> 10% of cells infested) produced in separate mite donor colonies and to check 100-200 cells 3-4 days post capping and another 200 cells of the same age cohort 7 days later (10-11 days post capping) for mite infestation. In between, the combs are exposed to the central broodnest of test colonies. The difference between infestation rate before and after exposure to the test colony is used to estimate the removal rate of infested pupae. During the final brood examination the cell caps are carefully opened to identify recapped cells, marked by the absence of the pupal cocoon, and to determine the reproductive success of remaining mites. However, this method requires high technical ability and thus is limited to a small number of experimental colonies.


For larger field studies, the measurement of mite reproduction in older brood cells (7 – 12 days post capping) is the most suitable VSH test method. At least 20, if possible 30-50 infested cells are needed to reliably estimate the average rate of non reproduction. Higher infestation makes it easier to achieve these numbers. If the test colonies themselves have low infestation, combs recently capped in highly infested mite donor colonies can be used instead. The highest infestation is usually achieved in “trapping combs” from colonies with no or only limited brood resources (i.e. a single comb with Varroa-receptive brood is present in the colony).


As the origin of brood has only minor effects on the VSH behavior different brood sources can be used in parallel. However, the history of brood needs to be documented and should be regarded as an independent factor in subsequent statistical analysis.


The (introduced) combs remain for 7-9 days after capping in the test colonies. If naturally present combs are used pupae at the purple-black eyes stage (corresponding to 7-12 days post capping) should be chosen. After removal from the hives, the combs can be stored in the freezer (-18°C) if they are not examined immediately. Thus, the time consuming examination for mite reproduction can easily be done during offseason and can be locally independent from the test apiary. This allows private beekeepers who lack examination facilities to participate in larger screening programs, where the examination is performed by technicians in different places.


For the investigation of mite reproduction only single infested brood cells (one foundress mite) in the age stage from “purple eyes” (7 days post capping) to “pupal moult completed” (12 days post capping) are evaluated. In the purple eye stage (7-9 days post capping), normally reproducing mites have at least one deutonymph. On pupae with black eyes (10-12 days post capping), normally reproducing mites have at least one adult daughter mite. Infested pupae with no or only younger stages of Varroa offspring are therefore counted as containing non-reproductive mites. If fresh broodcombs are examined, dead mites are also scored as non reproductive.


Examining brood cells requires some care and attention: a stereomicroscope or a magnification lamp (5X is ideal) can support identification of the presence of Varroa mites and their offspring. Fine forceps, a scalpel, and a small paintbrush can aid investigation of the brood cell, together with LED illumination.


The scoring form (see appendix) should comprise the following data:

> Total number of investigated cells

> Total number of infested cells

> Number of single infested cells (one foundress) at least 7 days after capping;

          For each of those cells:

          - Stage of brood

          - Oldest female Varroa offspring
          - Reproductive/non reproductive


 From these raw data the following parameters are calculated:

> brood infestation rate (total number of infested cells/total number of investigated cells)

> rate of non reproduction (non reproductive mites/number of single infested cells)


According to experience from Baton Rouge, non reproduction rates:

> greater 25 % indicate the presence of some VSH alleles,

> about 33 % indicate the presence of about half of the VSH alleles

> greater 50 % indicate high presence of VSH alleles.



Stages of honey bee brood and mite offspring for normally reproducing Varroa




More details on developmental stages of bees and mites can be found in the Bee Book chapters by Human et al., 2013 (Miscellaneous methods) and Dietemann et al., 2013 (Varroa research).