Selection and reproduction

by Giacomo Acerbi

Hygienic behavior in bees

We, we are the last in line
We are lands never seen before, only us
We are the anchor and not the sail
We are the amen of a prayer, we are us

Tiki Bom Bom – Levant

After devoting several articles to the ethological, reproductive and behavioral characteristics of kelpies and queen bees, I now propose to focus attention on the hygienic behavior of the hive. This basic attitude for the health of bees is, in fact, the most important character to breed and select more robust, productive colonies and to try to counteract the multiple pitfalls that weigh on the survival of bees and beekeeping today.

Complex social organisms hinder pathogens with individual innate immune responses and behaviors or through social immunity, attitudes that limit the transmission of pathogens. Bees express two different and synergistic immune systems, a “classic” one is characterized by a series of proteins that recognize pathogens and that determine a set of connected reactions to counteract the pathogenic threat; the second is a system of “social immunity” that determines different prophylactic behaviors. Hygienic behavior, for example, is a complex mechanism of social immunity of bees against pathogens that affect the hive superorganism and is present and found in many other social insects (Pull, 2018). Today we have come to a relatively detailed understanding of the genetics and evolution of the innate immune system of animals, but we know very little about social immunity. Important studies have therefore been carried out, with remarkable scientific acquisitions, on the hygienic behavior of the honey bee; it is, in fact, an excellent model for studying and trying to understand the genetics and evolution of social immunity in animals.

Giuseppe Pellizza da Volpedo – Country idyll in the meadows of the parish church in Volpedo (Il girotondo)

In the hive superorganism, as well as in other complex social organisms, to limit the transmission of pathogens are active different and synergistic immune systems, innate individual or social. A “classic” immune system and a system of “social immunity” that determine different prophylactic behaviors

Il girotondo - Giuseppe Pelizza da Volpedo

The complex system of social immunity of the hive

Social immune behavioral mechanisms are considered: secretions that prevent the growth of fungi and bacteria, self-exclusion or exclusion of other bees from the rest of the colony or only from some parts of it, removal or cannibalism against infected or dead workers, cleaning activities, detection and removal of infected or dead brood from the colony.
A set of activities and behaviors that determine a remarkable effectiveness in the prevention of epidemics.

Photo natural hygienic behavior

Natural hygienic behavior (not induced by pin test): bees recognize and
they uncover the brood damaged by the viruses carried by Varroa. Hygienic behavior involves a negative selective pressure on the most virulent varroa strains: hygienic bees recognize thanks to olfactory stimuli the chemical signals released by the brood affected and damaged by viruses

Photo by Giacomo Acerbi

Specifically the hygienic behavior of bees put in place, inside the nest, by bees aged between 15 and 20 days (Arathi, 2000), which recognize and remove brood affected by bacterial infections (Paenibacillus larvae) and fungal diseases (Ascosphaera apis), before the disease becomes endemic and spreads in the hive via sporulation (Rothenbuhler, 1964). In addition, colonies that exhibit a high hygienic behavior can also recognize, uncap and remove the brood infested with the ectoparasite Varroa destructor, reducing the reproduction rate and the spread of viral infections carried by the mite in the colony (Spivak, 1996). The bees selected for hygiene, recognize and remove the brood affected by American plague around the fifth day of life of the larva, so as to avoid the transmission and proliferation of spores.

Pupae of different stages of development, affected by DWV viruses, which show damage and malformations that would not allow them to become active members of the colony

Photo by Giacomo Acerbi

Pupe di diversi stadi

The beginning of the removal of varroa-infested brood, on the other hand, occurs after 60 hours from the operculation, at the pupal stage (Spivak, 1996), after the beginning of the oviposition by the mite (Donzè, 1996).
Therefore the removal of the pupa at this stage, also guarantees the elimination of the progeny of varroa.

The genetic basis of hygienic behavior is complex and the result of the interaction of several associated genes, which determine this attitudinal characteristic. To date, seven specific QLTs (quantitative traits loci) have been identified through genetic mapping and DNA region sequencing (1), each of which controls 9 to 15% of phenotypic variance (2) (Lapidge, 2002), but the complete gene architecture of this trait is still partly unknown. A quantitative character is determined by the sum of the action of several genes expressing an additive phenomenon. As a result, multiple QTLs, which can also be found on different chromosomes, are associated with a single character. The number of QTLs involved in a trait provides information about the genetic characteristics of the trait. Hygienic behavior is therefore a quantitative character, in other words determined by the sum of the action of several genes that determine and participate in its phenotypic manifestation. As a quantitative character, this behavior manifests a phenotypic variability due to genetic differences between individuals and the influence of the environment. Evolution over time modifies the frequency and distribution of genetic characteristics and consequent phenotypes. For example, the speed and efficiency in removing the damaged brood may be affected by harvest conditions (Thompson, 1964) and the percentage of bees in the colony capable of performing this task (Arathi, 2001).

Polygenic qualitative character

An example of a polygenic quantitative character: it varies continuously, within a certain range (for example height) and occurs according to a continuum of phenotypic variability. The inheritance and expression of the phenotype depends on multiple genes distributed in different loci, each of which contributes additively to the expression of the phenotype

Taken from:
Science fiction in high school – Polygenic or multifactorial inheritance

As far back as 1964 Walter Rothenbuhler, geneticist and bee scholar, demonstrated, with crosses between a female line that manifested a high level of hygiene and a male one that did not present this character, the way in which this behavior manifests itself and is inherited in the offspring: recessive homozygosity is necessary (3). This means, for example, that in order to verify in the behavior of the daughter bees of a given cross the aptitude to uncap the damaged brood, the two alleles (one maternal and one paternal) present in the chromosomal locus (4) that encodes that character, must both be recessive. Only the recessive homozygous genotype (aa) manifests the behavior of the opening of the damaged brood; heterozygous individuals, that is, those who have a dominant and a recessive allele, do not manifest it. The components of hygienic behavior are controlled by independent chromosomal loci, which synergistically provide bees with the set of genetic architecture necessary for the manifestation of this behavior: recognizing, opening and removing damaged brood. So the character must be homozygous recessive in more than one locus at once. This means that in a selection line we must try to intensify the pressure on all the behaviors that determine hygiene and that we can observe from the study of the phenotype: open and clean the damaged brood. Hygiene, which is one of the best examples of how a complex behavior of an organism such as bees is under the strict control of a simple Mendelian law.
This means that if within a mating program you want to select and intensify the behavior that can counteract some diseases, it is necessary that in both breeders, there is the presence of this character in the phenotype. If, for example, we fertilize a female line that manifests this behavior with drones from a colony that does not manifest this character, in the result of the crossing (F1), most likely, we will have bees that do not express this behavior.

In the selection of behavior, as for almost all hereditary characteristics related to the health of bees, it is therefore essential to work both maternally and paternally: the selection of male lines and the control of matings is the key to intensify and ensure that this characteristic is inherited in the offspring.
If this aspect is neglected, even the best female line crossed with the wrong drones, can lose this character, within two generations. The selection of male lines with the desired phenotype allows you to increase the presence and content of this character in an additive and synergistic way.

How can bees recognize the damaged brood?

From various scientific researches it has emerged that the ability and speed of detection of the diseased brood, infested with varroa or damaged by viruses carried by the mite, is determined and mediated by stimuli of an olfactory nature.
Definable hygienic bees have a greater predisposition to recognize the smell of damaged brood and this behavior may depend on the ability of bees to detect precise olfactory signals and their response threshold and attention in removing the agents that emit these chemical signals. The genetic predisposition to sensitivity in the detection of abnormal odors in the brood can facilitate the expression of hygienic behavior: for example, it has been shown that definable hygienic bees have a greater olfactory capacity in the detection of pupae infected with very low concentrations of spores of Ascosphaera apis (Materman, 2001). On the other hand, the same brood parasitized by varroa plays an active role in triggering the hygienic behavior of the colony: this character has been associated both with single brood cells infested with varroa (Cheruyot, 2018) and with brood infested with a greater number of mites (Harbo and Harris, 2005). But the hygienic behavior is not influenced either by the perception of movements of the varroa under operculum (Aumeier, 2001), nor by the smell of the mite, since it is able to imitate the smells of the host (passive camouflage of Varroa. Martin, 2001). The olfactory signals that trigger hygienic behavior come directly from the mite-infested brood (Mondet, 2016), thanks to a higher production of cuticular hydrocarbons (Nazzi, 2018) and brood ester pheromones (Mondet, 2016). It is the larval signaling that completes the sum of the hygienic characteristics inherited in adult bees: brood from hygienic colonies is removed better and faster than that coming from unhygienic families. Brood signaling improves and complements the communication system in the bees.

The burial of the victims of the Black Death in Tournai, Belgium – miniature circa 1350

The olfactory signals that trigger hygienic behavior come directly from the varroa infested brood; so much so that brood from hygienic colonies is removed better and faster than that from unhygienic families

Burial of Black Death victims

A singular selective capacity

But there is more and much more interesting in the origin, functionality and consequences in the detection and selective pressure of hygiene in bees and the correlation between signals from brood and characteristics of adult bees: selective hygienic behavior. From a publication by researchers from the University of Berlin (Schoning, Gisder, 2011), then supported by evidence and field tests, it emerges that the extent of the damage to the brood caused by more virulent mites capable of inducing viral infections potentially fatal to the pupae and the colony, rather than the mere presence of varroa in the brood, is the true origin of hygienic behavior. Bees express hygienic behavior as a function of the damage caused by varroa to pupae, reacting to the signals of the brood affected by pathologies, as well as for other pathologies of the brood. Varroa acts as a biological vector of a series of different viral strains with a high replicating and mutation capacity: the DWV deformed wing virus (in its variants A-B-C), the acute paralysis virus ABPV… which are among the worst enemies, subtle and lethal, for the health and survival of bees. The mechanisms of spread and contagion of the bees of these viruses are countless, however the most dangerous and epidemic spread is that through infected mites that parasitize the pupae. It is the mite that acts as an intermediate host in the transmission of increasingly virulent variants of DWV, which cause difficulty in movement (ataxia), brain infections, paralysis, abdominal impairments… and that can lead the colony to collapse. Hygienic bees recognize, uncap and remove more frequently parasitized brood by virulent mites, recognizing their peculiar and distinctive chemical signals, determining a negative selective pressure for the varroa genotypes most favorable to the replication of viral diseases. Unlike other characteristics responsible for resistance to varroa, hygienic behavior is really useful because it saves pupae infested by the mite, but which have not suffered damage and can become active members of the colony and avoids an unnecessary reduction in the breeding efficiency of the brood.

Hygienic behavior could be the main character and founded for a new selective model linked to the health of bees, which responds to today’s needs and criticalities of beekeeping, with a crucial role both in the population dynamics of varroa, and of the pathogens that it transmits.

In the next article we will begin to address the selective techniques and tools to identify hygienic bees in your breeding and we will see how the hereditary characteristics related to the health of the bees if balanced with others, as part of a selection program, can make the difference for production and performance of our hives.


(1) QLT (quantitative traits loci): it is a region of DNA associated with a particular quantitative trait. QLT is closely associated with a gene that determines the phenotypic character in question or participates in its determination.

(2) Phenotypic variance: represents the result of the sum of genetic variance and environmental variance with respect to a trait in a population.

(3) Recessive homozygosity: homozygosis is the condition in which each of the two or more alleles of the same gene, present in each homologous chromosome, encode identically. We speak of recessive homozygote when an individual possesses, for a single gene, two equal recessive alleles. A recessive homozygote has a unique phenotype and is therefore immediately detectable.

(4) Chromosomal locus (plural loci): designates the location of a gene or other significant sequence within a chromosome.

A very effective selective pressure for genetic worsening: lame bees!

In the raging of discussions and instrumental controversies on the impoverishment of beekeeping genetic biodiversity, for some determined solely by the use of strains and characters from non-native subspecies, someone (with a little more common sense) tries to point out the recent and dramatic phenomena that have caused and continue to determine the impoverishment of the genetic biodiversity of bees: the multiple sickles of hives from varroa and from repeated phenomena of resistance to acaricides, the standardization with monocultures of the rural landscape, the structural modifications of the agro-environmental context (such as from overbuilding or varietal selection of seeds), the extremization and climatic upheavals, the increasing, perverse and subtle effects of pesticides, the tendency of recurrence of variegated stress of plant species.
But one aspect is often underestimated if not completely forgotten: the “beekeeping /health” practice of administering antibiotics, spread since the post-war period worldwide, for “prevention and treatment”. Beyond the possible interference and damage to the microbiome of bees and the microbial flora of the hive, the enormous success and custom of this breeding health practice – proposed until recently by public authorities, researchers and veterinarians, as the best prophylaxis of prevention and treatment of brood pathologies – has carried out for decades and decades, and in many parts of the world continues to play, an exceptional and very effective nefarious role in hiding the pathological manifestations of the brood and to guarantee an exceptional affirmation and ubiquitous diffusion of genetic strains of bees with limited if not zero hygienic characteristics. The drones of genetic strains that would not have been able to survive in any way have thus not only been able to endure, but have also and above all been able to spread in a ubiquitous way their limited attitude to the social immunity of the hive. Fortunately, on the one hand, the refinement of analytical techniques for the search for residuality in beekeeping products, on the other a different awareness and management / health capacity of breeding by beekeepers, have strongly contributed to abandoning this deleterious practice, such as to be defined and under various profiles: aberrant. From now on we therefore have ample room for improvement to repair genetic inheritance, for the worst we have done in the past!

Bruegel the Elder - Parable of the Czechs

Bruegel the Elder – Parableof the Blind

Thanks to the widespread practice of administering antibiotics to hives, drones of unhygienic genetic strains have ubiquitously spread this serious lack of social immunity of bees.