Selection and breeding

by Giacomo Acerbi

The other half of the sky. Spermatozoids with wings… underestimated… misunderstood…

Drones play a crucial role in the well-being, performance, survival, reproduction of the hive super organism: the well-bred males, in full reproductive health, with robust and viable offspring, represent an essential component of the future of the colony.
A summary of the aspects that may or may not determine the health and quality of drones, about important new knowledge and about the recent pitfalls of this harmonic link indispensable to the complex life cycle of the hive.

Humanity has been in mutual interaction with bees since ancient times, it has conditioned their life cycle a lot and in different ways and has spread their presence territorially, but in thousands and thousands of years the human being has understood little or nothing about bees and their life cycle! On the other hand, the understanding of the “wonders and mysteries” of nature has evolved in our history easier in what regards the perceptible phenomena, better if they are perceived with the naked eye. For millennia, human being has been able to understand the invisible, the numerous, the indispensable vital forms, which are too tiny to be noticeable.
Besides the fact that they are tiny and with a life cycle hidden in their built nest, bees are, among all grown living species, one of the most alienated, different, and “distinct” from us humans. When the detachable and observable honeycomb was invented, it allowed us to begin – finally! – to understand something that we considered (with the usual assumption!) that we fully understood! But those simplistic initial and primitive “explanations” of the basic phenomena, did nothing but open us the chest of mysteries of the hive. How many of us have learned from textbooks that applied the “scientific truth” that the colony would be ruled only by the queen’s pheromone?
In a strange way, despite the fact that almost all human civilizations relied on the rigid subordination of the female to the male, in the case of bees it was opposite: to the male of the bee it was assigned value only in relation to the queen and the reproductive act. Suddenly an image of substantial futility was built and transmitted, including with the development of practices, procedures, and tools of “modern beekeeping technique” to variously counteract the laying, reproduction, if not even the survival in the hive.

I suggest instead that the drone be should be considered differently: an indispensable and determining link in the life cycle of the colony

The drone has, in fact, an equal, if not greater, importance than the female component, both as an element of fundamental quality (in capacity of reproducer) for the well-being and performance of the hive, both from genetic point of view but ultimately selectively: a super organism with complex, distinct, and definitely “specific” characteristics compared to most living species, where, for example, kinship between different subfamilies of female workers from the colony is determined by the male sperm sample with which the queen has fertilized her own eggs. Not just a single male (bull, stallion, or wild boar), but a number which ranges from 6 to 25 drones (15 on average) determines the genetic inheritance of a colony. In recent years it grow bigger and bigger a serious trend of losing the longevity and fertility of queens, increasingly “regular” and repeated replacements, reduced fertility, which often has as a result the lack of fitness (1) and performance of the entire colony, with high consequences and additional costs for the breeder. And we, the beekeepers, what did we do? A lot of things have been said and improvements have been tried on this issue, but attention has focused only on the queen, starting from her biology and reproductive behaviour, to the manipulation by the beekeeper. Our focus was on: the quality or the lack of quality of the fertilization depending on the spermatheca, adequate temperature and nutrition to facilitate sperm migration through oviducts, size of fertilization nuclei, synchronization for correct homeostasis (2), possible damage caused by handling, caging and transport.

And drones, to what extent what is their importance in this recent and dramatic deterioration of bee vitality and fecundity?

Recent studies, confirmed by a wide range of experiments in the field, show that the health of drones and their reproductive value (3), the quality and vitality of their spermatozoids, are crucial for the physiology, efficiency and longevity of queen bees, with important consequences for the overall performance of the entire colony.
There are many factors that can influence the reproductive quality of drones: environmental causes (nutrition, temperature, seasonal trends), exposure to contamination, pathologies, genetics of origin, age, management of beekeeping. In this regard, the new scientific achievements are notable and varied, well highlighted in the review of knowledge, recently proposed (2019) by Rangel and Fisher . Here are some clues about the ethology of the drones, crucial for understanding the reproductive quality of the male component of the hive.

It matters more than the act itself…the foreplay!

The drone has a development from egg laying to birth of about 24 days, this process can vary depending on haplotype (4) (DeGrandi, Hoffman 1998), temperature (Bienkowsha 2011) and general conditions of the colony (Winston 1987, Collison 2004) such as size, availability of food resources, etc.
Spermatogenesis, that is, the process of sperm formation, begins during the larval stage (Bishop 1920) and ends in the pupal stage; sexual maturation is instead completed in the first week after birth, due to the migration of sperm into the seminal vesicles (Snodgrass 1956), through the formation of two mucous glands that protect against pathogens and nourish spermatozoids (Woyke 1983, Rhodes 2008). The vitality and longevity of spermatozoids are determined by the quality and composition of proteins contained in seminal fluid (Baer 2009, King 2011). It is interesting to note how the composition of these proteins is connected actually to the immunoreaction to the Nosema apis spores of the still hatched drone (Grassi 2017).
Most defects of a morphological nature (size, weight, wing length) and reproductive quality (number, motility (5) and vitality of spermatozoids), are determined actually in its prenatal phase.

In other words: the quality of the drone is determined and played mainly “under the operculum”

The drone reaches sexual maturity around after 6/8 days (Bishop 1920), but can reach to 10/12 days (Woyke and Ruttner 1958) up to a maximum of 16 days after birth (Rhodes 2008). In the first days of life, young males interact with bees and are fed, warmed and cared for. They feed only with honey or honeydew, either by trophylaxis or by direct sucking, but only if the cells in which the supplies are stored are not covered and within its reach. Participates from birth in thermal control and to the ventilation of the colony thanks to an exceptional muscular apparatus.
Orientation flights of the young drone, to locate the reference points and the exact location of the nest, start around the fifth/eighth day after birth (Galindo – Cardona 2015).

After establishing the position, its life cycle culminates with flights to the drone congregation area (“DCA”), where an average of 11,000 drone congregate to look for young virgins. It is not easy to identify a congregation area: males make numerous flights of different duration and frequency, only to scatter the congregation after only a few minutes (Holm 2010). Strategic device to avoid predators such as robins from discovering where to eat; all the more so since the large number of males reduces the risk of predation of the virgin queens (Holm 2010). The drone can ejaculate a volume of seminal fluid ranging from 0.91 to 1.7 microlitres (Rousseau 2015) containing 3.6 to 12 million of spermatozoids, all genetically identical (Collins, Pettis 2001).
Contrary to popular beliefs and prejudices, it is important to know that even though drones can fly an average of 30 km/h (1 km in two minutes) and cover great distances (Holm 2010), they prefer to search for and create congregation zones (DCA) a few hundred meters from their own hive/apiary of origin, in order to maximize the stay time and chances of fertilization (Koeniger 2005) and to maintain adequate sperm vigour and vitality.
In fact, the drone migrates away from its original hive only for emergencies (search for food and a family to greet it) often caused by the stress of its own colony, but almost all drones that cover long distances are exposed to heat stress, malnutrition, leading to an inability to compete in a congregation area (DCA) where physical vigour and size are crucial to take precedence in the clutter (Berg 1997, Tarpy 2019).

Some factors that can compromise the reproductive health of the drone

It is difficult to try to list and indicate in order of importance and priority, the possible causes that can cause the loss of male fertility of bees. Globally, I assume that hecatombs and the fragility of hives caused by the almost ubiquitous spread of the varroa parasite and related diseases can be a determining and priority factor. But I have some reason to assume that the specific context of the evolution of the environment in our country may have brought – even more than the decimation due to the parasite varroa – in the foreground, the set of effects on the environment. This hypothesis is based on the observation of the combination and contemporaneity of several factors, such as: the progressive and incessant loss of food resources (for example: drastic decrease in nectariferous exudations from rapeseed or sunflower; decrease of the resources from the barre due to impact with parasites – eucalyptus and chestnut etc …); the constant growth of monocultures, combined with the largest spread in Europe of biocides for “phytosanitary protection”, with relative and subtle consequences (for example: maize, vines, hazelnuts, fruit trees, etc.) and finally – precisely in order not to deprive us of anything – all seasoned with the special exposure of our country to the increasingly frequent phenomena of climate extremes, with relatively serious consequences both on the possibility of supply and on the thermoregulation and health of bee colonies. Moreover, even the Italian people themselves face serious and growing criticism for reproductive sterility, especially for men, with a worrying and significant increase, scientifically attributed mainly to the interaction of various environmental factors.

Temperature

Under optimal conditions, drones are cared for and thermoregulated inside the hive at a constant temperature of 33 to 35 degrees. The reproductive quality of males can be severely compromised even by modest temperature variations, both in development and after birth.
For example, Jaycox (1961) observed how the sexual maturation of drones is compromised when they are raised at 31.1 degrees, while at 28.33 degrees their total sterility is determined.
The same drastic consequences due to excess temperature: drones exposed even for a few minutes at a temperature of 40 degrees have a spermatozoids mortality rate of more than 40% (Czekonska 2013).
In the pupal stage, in which the complete formation of spermatozoids occurs, drones are extremely sensitive to small temperature variations: at 32 degrees they have larger testicles, more developed seminal vesicles and more extensive mucous glands compared to being raised at a temperature of 35 degrees (Czekonska 2013). Therefore, if we cannot glimpse, at least we can try to imagine the possible effect of sudden deaths and depopulation of hives, especially in spring, but also due to the extreme increase of summer temperatures. In fact, a sudden and maximum change in temperature outside the hive can completely compromise the quality of drones, which are still in the pupal stage: they will be born, cared for by bees and mate with queens, but with very poor fertility and very weak results (low motility (5) and high spermatozoids mortality), which will affect the reproductive value (3) and longevity of the queen and therefore the performance of the colony.

Nutrition

Various scientific studies have confirmed the phenomenon that we have been witnessing for years as a production sector: the impact of the quality and quantity of food availability in the morphology, fecundity, and quality of drones. Czekonka (2015) tested how forced deprivation of pollen with traps placed outside the hive during the larval period, causes a decrease in the size and weight of the drone, a low volume of sperm and a difficulty in ejaculation at the adult male. On the other hand, Rousseau and Giovenazzo (2015) found that additional nutrition with pollen and syrup substitutes during the spring period greatly influences the reproductive quality of drones: larger male size, better sperm vitality, increased number of spermatozoids.

Contamination from biocidal products

The bibliography regarding the lethal and sub-lethal effects of bees’ exposure to the various agro-biocides is already known in size and results, but scientific studies on the effects on the reproductive quality of the male are very limited, if not rare. The few researches available, which distinguish between artificial conditions in which drones are given incremental doses of pesticides and situations of contamination in the field, highlight the serious effects on: morphology, vitality and male fertility rate. The neonicotinoid family is the most studied – especially: imidacloprid, thiamethoxam, clothianidin – and has demonstrated lethal effects on the reproductive capacity of drones, both if molecules are administered and if they are present in infinitesimal concentrations in hive’s matrices (wax, honey, pollen) (Di Prisco 2013, Williams 2015, Straub 2016). In addition, is very interesting a study of males’ exposure to the insecticide Fipronil (Straub 2016), which highlights a clear correlation between the molecule and a strong increase in spores and aggressiveness to Nosema ceranae infection. This is the interpretation to reflect on the most: we are often in the situation of facing the ubiquitous influence of a multitude of residual molecules that, acting in a subtle and synergistic way between them and with other contributing causes (pathologies, lack of food resources, etc.), all these can cause both mortality and less known, but perhaps even more serious, total senescence (6) of drones..

Various scientific publications have shown how even the simple contamination of the nest wax with various acaricides (often combined one with the other) causes a drastic decrease in the laying period of the queen and strongly reduces its attractiveness for bees (Rangel and Tarpy 2016).
Effects on reproductive physiology of drones, on the number of spermatozoids and their vitality were highlighted by the presence, either individually or in combination, of acaricides. In fact, it is more than disturbing to note that even the biological accumulation of various acaricides in very low concentrations in the honeycomb wax from the male e – of amitraz, fluvalinate and coumaphos, as well as pesticides, such as chlorpyrifos, and chlorothalonil – determines the vitality of a very low sperm count, almost the absence of spermatozoids motility (5) as well as a drastic decrease in their number (Fisher & Rangel 2018). Moreover, a large amount of amitraz and its metabolites was also found in the wax of almost all the families examined and numerous damage to bee health was observed due to this bioaccumulation (Boncristiani 2012), namely: low queen fertility and low spermatozoids vitality in spermatheca (Range and Tarpy 2016). On the other hand, organic acids are no exception: in addition to affecting the spermatogenesis of drones both quantitatively and qualitatively, they also have an effect on the physiology of the adult male. Direct exposure to oxalic acid and formic acid (as well as thymol and eucalyptol) affects smaller drones, with short wing lengths and a decrease in spermatozoids count (Shoukry 2013).

Pathology

A strong infestation with the varroa parasite under the operculum causes the birth of drones of low quality and with less physical vigour: sub-standard size (Hrassningg and Moritz 2010), reduced wing force (Metz and Tarpy 2019) and consequently a poor flight capacity (Slone 2012). The few males that manage to develop and be born from a cell infested with 20 mites, can have half their normal body weight (Duay 2003). But even an infestation of one to six varroe female parasites under the operculum, causes both in the development phase and at the exit, a considerable reduction in the weight and size of the male (Engels 2001). There is very limited literature on the effects of viral load on male reproductive health and vertical transmission of infections to the queen and offspring. But recent studies have shown the presence of a viral genome with five different viruses (DWV, ABPV, BQCV, SBV, AmFV) in sperm ejaculated by drones apparently in excellent health (Titera 2019), with evidence of a subsequent venereal infection of queens inseminated with that sperm. But even more important and dangerous is the demonstration of transovarial transmission (7) of a strong viral load from infected sperm of drones to eggs laid by queens (Titera 2019). The tendency of colonies from these queens inseminated with samples of viralinfected infected semen was also monitored and symptomatic pathogens were observed, such as brood’s virus in the sac, mortality of new queens in the actual replacement cells (black cell) and, in general, loss of fitness (1) and performance of apparently asymptomatic colonies (Titera 2019).

Age

Many studies have shown that the age of drones can influence: viscosity (Cobey 2007), Volume (Locke, Peng 1993), motility (5) and sperm viability (Locke, Peng 1993). Sperm becomes darker and more viscous the more advanced the age of the drone is: males over 21 days old have too high viscosity of sperm, with the consequence of the difficulty for queens to expel excess from the oviducts (Czekonska 2013). Locke and Peng (1993) also showed how spermatozoids motility (5) decreases by about 80% in drones over 20 days of life.
Therefore mating with excessively “mature” males can lead to loss of longevity and poor performance of queens. I hope with these first examples that I have stimulated and awakened new ones: curiosity, attention and interest with regards to “the other half of the sky” of the bee colony.. I hope that soon, I will return to the same topic about what the beekeeper, breeder and the reproducer of bee queens can try to achieve to improve the bee colonies. If we want to have healthy and productive bee colonies, queens that have a long live and high-performance, we cannot continue to neglect the strengths and fragility of drones and therefore not to invest some of our assets: commitment, effort and work for their health, reproduction and selection. Thanks to the appropriate techniques for the preparation of colonies used for male lines, for maintaining the vitality and fecundity of drones, for genealogical selection, more than interesting results can be obtained in terms of the physical condition (1) of the hives and the application of a widespread, rapid and ubiquitous genetic improvement in the bee colonies can be attempted.