J. Bio. Env. Sci. 2016

Journal of Biodiversity and Environmental Sciences (JBES) ISSN: 2220-6663 (Print) 2222-3045 (Online) Vol. 8, No. 1, p. 192-224, 2016 http://www.innspub.net

RESEARCH PAPER OPEN ACCESS

Varroa destructor in (2013-2015) and mating behavior under laboratory and contamination level of bees colonies with Varroa destructor in Karajs apiaries

Shakib Vaziritabar1*, Assadolah Aghamirkarimi2, Sayed Mehdi Esmaeilzade3

1Department of Animal Science, Islamic Azad University Varamin, Pishva Branch, Tehran, 2Department of Entomology Science, Islamic Azad University Varamin, Pishva Branch, Tehran, Iran 3The Professional Instructor of Culturing Honey Bee, Number 2, Zibadasht, , Iran Article published on January 26, 2016

Key words: Varroa destructor, Mating behavior, contamination, Alborz.

Abstract The present study aimed to evaluation of the contamination level of bees᾽ colonies with Varroa destructor in different Karaj᾽s apiaries and analyze the mites’ mating behavior under laboratory. In order to analyze the mites’ mating behavior and the involved cues, a new bioassay was designed and the male behavior towards different female stages compared. This bioassay represents a simple tool for behavioral observations of Varroa destructor in the laboratory, showing that Reproduction of female Varroa destructor happens within the sealed brood cell of the honeybee host. The mating represents the last step of the reproductive cycle and is usually performed between the mature male offspring and one or more daughter mites. In a laboratory bioassay, we observed interactions among offspring of a Varroa female at 11 days after host cell capping in the absence and in the presence of the sex pheromone and clearly demonstrated that male mites were not able to distinguish between receptive daughters and either older or immature. We first present a general view on the biology of the Varroa mite with special emphasis on host–parasite interactions during reproduction of the female mite. The pathology section describes host damage at the individual and colony level including the problem of transmission of secondary infections by the mite. Knowledge of both the biology and the pathology of Varroa mites are essential for understanding possible tolerance mechanisms in the honey bee host. Average apiaries infection to Varroa destructor 29.7% and most infections were seen in spring season. *Corresponding Author: Shakib Vaziritabar  [email protected]

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Introduction bee mite Varroa destructor is still the greatest threat The present study aimed to evaluation of the for apiculture. No other pathogen has had a contamination level of bees colonies with Varroa comparable impact on both beekeeping and honey destructor in different Karaj᾽s apiaries and analyze bee research during the long history of apiculture. the mites’ mating behavior under laboratory. In order There are several reasons for this unique status of to analyze the mites’ mating behavior and the Varroa mites: (a). Varroa destructor is a new parasite involved cues, a new bioassay was designed and the of the honey bee A. mellifera. Therefore, a balanced male behavior towards different female stages host–parasite relationship is lacking and beekeepers compared. This bioassay represents a simple tool for do not have long-term experience in dealing with this behavioral observations of Varroa destructor in the pest, (b). Without periodic treatment, most of the laboratory, showing that Reproduction of female honey bee colonies in temperate climates would Varroa destructor happens within the sealed brood collapse within a 2–3 year period, (c). Regular cell of the honeybee host. The aim of this survey was, treatments increase the costs for beekeeping and the therefore, to investigate the presence of the two most risk of chemical residues in bee products, (d). The economically important honeybee virus and parasite Varroa mite is considered a crucial factor in the to determine their distribution in 16 cities in Karaj᾽s decreasing numbers of beekeepers and honey bee apiaries. Within the scope of the present research, colonies in Europe and Asia; together with the three objectives were targeted: (a). Evaluation of worldwide decline of natural pollinators, the Varroa contamination level of bees colonies with Varroa mite may exacerbate future problems for pollination destructor in different Karaj᾽s apiaries in Iranian (De la Rua et al., 2009). beehives, (b).Biology and control Varroa destructor and analyze the mites’ mating behavior under Using sustainable methods to control or even laboratory and field conditions, (c). Census of natural eradicate this parasite will re- establish wild and feral mortality rate caused by parasite in each colony, pollinator populations, ease the plight of beekeepers, separately. promote economically important pollination dependent agriculture and benefit natural Honeybees are an extremely important part of the ecosystems. For this ideal to be realized, however, a natural ecosystem because they help to maintain strong and sustained research effort is needed to biodiversity and enhance agricultural productivity by produce the understanding necessary for an efficient providing valuable pollination services, based on the and sustainable control strategy against this most ecological principal of mutual interactions between important of honey bee parasites. pollinated plants and pollinators (Delaplane and Mayer, 2000, Gallai et al., 2008). Factors that may The varroa mite (Varroa destructor) is the most contribute to honeybee diseases and mortality include serious pest of honey bee colonies worldwide. This pathogenic microorganisms (Cox-Foster et al., 2007, parasite was first detected in North Carolina in 1990. De Miranda and Genersch, 2010). Parasite diseases Since the contact between varroa and A. mellifera, such as varroa mites of honeybees are found serious problems have been experienced worldwide in worldwide and may result in significant economic apiculture. Varroa destructor is considered to be the losses, manifested by colony losses, reduced yields of major pest of honey bees since it spread to A. honey and bee products, and reduced pollination mellifera. Recent studies have confirmed its efficiency, resulting in poor quality and reduced yields substantial contribution to honey bee losses across of agricultural products. Little is known about and the Northern hemisphere (Brodschneider et al., 2010, there is no literature pertaining to the prevalence of Chauzat et al., 2010, Dahle, 2010, Genersch et al., honeybee mites in Alborz. The hemophagous honey 2010, Guzman-Novoa et al., 2010, Topolska et al.,

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2010, Vanengelsdorp et al., 2011). No satisfactory research is a challenge for all scientists working in the solution for its control has, however, yet been found, fields of apiculture, insect pathology and acarology. and it has become clear that the development of We will present a general view on the biology of the enduring sustainable control measures will not Varroa mite with special emphasis on recent results happen until we have a better understanding of the on host parasite interactions, breeding honey bees for fundamental biology of the parasite. Such solutions tolerance, and treatment for Varroa infestation. are necessary to ensure the future of the economically most viable pollinator species in a context of The Varroa mite, Varroa destructor, is one of the worldwide pollinator decline. threats to managed honey bee colonies. Elevated colony losses reported from the USA, Europe, the This mite is one of the factors associated with the Middle East and Japan are related to high mite unprecedented loss of honey bee colonies recently infection (Neumann and Carreck, 2010). V. experienced in parts of Europe and North America destructor, the new species is represented by mites of (Vanengelsdorp et al., 2008 and Rosenkranz et al., the Japan/Thailand- Vietnam clade. Mites of Korean 2003). Recent studies suggest that V. destructor may haplotype parasitize A. mellifera worldwide, and are be so harmful to honey bees not only because of its significantly larger and reproductively isolated from feeding on the bee's hemolymph, but also because it the V. jacobsoni haplotypes (Anderson and Trueman, transmits and favors the multiplication of honey bee 2000). The Korean type has worldwide spread on A. viruses. Increases in the incidence and levels of melllifera, while the Japanese/Thailand type has only several honey bee viruses have been observed with V. been reported from A. mellifera colonies in Japan, destructor as an inducer/or vector of several viruses Thailand and North- and South-America (Anderson (Genersch et al., 2010). Since then, commercial and Trueman, 2000, De Guzman et al., 1998, Garrido transportation of colonies and natural spread has et al., 2003; Muñoz et al., 2008). Therefore, the only resulted in a cosmopolitan distribution of Varroa mite of economic importance is V. destructor, which destructor, which has had dramatic consequences for successfully shifted from the original host, A. cerana both managed and wild populations of A. mellifera. to the Western honey bee, A. mellifera. The details of In contrast, V. destructor can reproduce on both male the host shift are unclear. Most likely this shift and female brood of A. mellifera, thus attaining a occurred when A. mellifera colonies were transported longer reproductive season and larger mite to Eastern Russia or the Far East in the first half of populations. The most promising options are based the past century which led to a sympatric distribution on biological control using pheromones, hormones, of both honey bee species (Oldroyd, 1999) and might pathogens, predators or antagonists (Rosenkranz et have allowed the parasite to infest the new host. al., 2010, Meikle et al., 2012). In addition, methods Varroa mites were found in the eastern coastal region that do not involve the application of chemicals or of the USSR (1952), in Pakistan (1955), Japan (1958), other agents into the hives are of particular interest. China (1959), Bulgaria (1967), South-America Such methods do not involve the hurdles, expense (Paraguay, 1971), Germany (1977: Ruttner and Ritter, and delay of registering new compounds or agents, 1980) and the first record for the United States the risk of developing resistance by the parasites originates from 1987 (De Guzman and Rinderer, against compounds or agents, or the accumulation of 1999), Iran (1993: Mossadegh and Bahreini, 1994). residues in hive products. sustainable control Today, V. destructor is almost cosmopolitan, but has methods is still inadequate, we propose solutions to not yet been found in Australia (Fig. 1). The present acquire this missing knowledge and, given the study aimed to evaluation of the contamination level complexity of the task to solve the varroa problem, of bees colonies with Varroa destructor in different promote a collaborative approach. Therefore, Varroa Karaj᾽s apiaries and analyze the mites’ mating

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behavior under laboratory. In order to analyze the the artificial test conditions in a laboratory mites’ mating behavior and the involved cues, a new temperature gradient (Dillier et al., 2006). Without bioassay was designed and the male behavior towards doubt, chemical orientation plays the crucial role different female stages compared. This bioassay during all parts of the Varroa life cycle. This becomes represents a simple tool for behavioral observations obvious in the preference behavior of female mites for of Varroa destructor in the laboratory, showing that certain host stages. As a parasite without a free living Reproduction of female Varroa destructor happens phase, the Varroa mite sticks either to the adult bees within the sealed brood cell of the honeybee host. The or stays within a brood cell. For the reproductive aim of this survey was, therefore, to investigate the success of the Varroa females, two host stages are of presence of the two most economically important crucial importance: the suitable adult bee which honeybee virus and parasite to determine their transports the mite to non-sealed brood cells and the distribution in 16 cities in Karaj᾽s apiaries. Within the 5th instar larvae in brood cells shortly before cell scope of the present research, three objectives were capping. In the case of adult bees Varroa females are targeted: (a). Evaluation of contamination level of obviously able to recognize the age and/or function of bees colonies with Varroa destructor in different the adult bee. Shortly after leaving the brood cell on a Karaj᾽s apiaries in Iranian beehives, (b).Biology and young bee, the mites preferentially infest nurse bees control Varroa destructor and analyze the mites’ for transport to the brood cells (Kraus, 1993, Kuenen mating behavior under laboratory and field and Calderone, 1997). Freshly hatched infested bees conditions, (c). Census of natural mortality rate are less attractive than older ones and the middle age caused by parasite in each colony, separately. These nurse bees are the most infested group of adult bees findings should stimulate further research with the in breeding colonies (Kraus et al., 1986). This may be objective of disturbing the male’s mating behavior as an adaptive strategy for the Varroa females to a first approach for a biological Varroa control. increase their reproductive success. It is assumed that age and/or task specific patterns of cuticular Except for distribution by swarms or foragers the hydrocarbons of the adult bee are used for the Varroa mites spend their whole life within the dark selection of the optimal host. Age dependent patterns honey bee nest, preferably within sealed brood cells have been described for drones (Wakonigg et al., (Boot et al., 1993). Varroa mites are able to perceive 2000). However, confirmation that mites use the light and vibration (Kirchner, 1993). However, there hydrocarbon pattern of the bee’s cuticle for host are no indications that these senses are used for selection is lacking. Additional details concerning the orientation or host finding. This may also be true for recognition of a suitable larval host are known: A long sensitivity to temperature. Depending on the test established phenomenon is the 8–10 fold higher system Varroa mites prefer temperatures between 26 infestation rates of drone brood compared to worker and 33 °C (Le Conte and Arnold, 1987, 1988, brood (Boot et al., 1995b, Calderone and Kuenen, Rosenkranz, 1988), which are significantly lower than 2001). Several reasons for this unequal distribution the normal temperature in the brood nest of have been discussed. Within the honey bee colony, approximately 34.5–35 °C (Becher and Moritz, 2009; worker larvae are infested 15–20 h prior to cell Rosenkranz and Engels, 1994). Varroa mites are able capping, and drone brood 40–50 h (Boot et al., 1992, to discriminate temperature differences of about 1°C Ifantidis et al., 1988), which may be one of the (Le Conte and Arnold, 1987). It was assumed that reasons for the higher invasion rate into drone brood Varroa females preferably invade colder brood cells at cells. Another fact that contributes to the higher the periphery of the brood nest; however, there is no infestation of drone larvae is a more frequent and solid evidence for this hypothesis and the preference intensive tending of the drone larvae with the for low temperature may, at least in part, be due to consequence that mites on nurse bees have a

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significantly more opportunity to reach a 5th instar brood cells, i.e. brood cells with a shorter distance drone brood cell than a worker cell (Calderone and between the larva and the cell rim, are more Kuenen, 2003). The attraction of Varroa females to frequently infested than artificially elongated brood several fractions of the extract from the larval cuticle cells. This effect was confirmed by the use of different has been confirmed in different bioassays. Le Conte et methods with manipulated brood cells inside the al., (1989) first described a kairomonal effect of three honey bee colony (Boot et al., 1995a, De Ruijter and methyl- and ethyl esters in an olfactometer, which Calis, 1988, Goetz and Koeniger, 1993). Also the size was confirmed by further experiments (Le Conte et of the cell and the relative larval size to cell size affect al., 1994, Trouiller et al., 1992). The secretion of the the invasion behavior of the mites. Brood cells of esters by the honey bee larvae shows a clear European honey bees are, in general more highly ontogenetic pattern with a distinct maximum at the infested than slightly smaller brood cells of 5th instar during the time of cell capping (Trouiller et Africanized bees within the same colony (Message al., 1991). Drone larvae produce slightly higher and Gonçalves, 1995; Piccirillo and De Jong, 2003). quantities over a longer time period, which also However, drone brood cells containing smaller supports the preferred infestation of drone brood (Le worker larvae are less infected than normal worker Conte et al., 1989). However, the pheromonal effect of brood cells, probably an effect of too much space these substances is more apparent than the between the larva and cell wall (Calderone and kairomonal effect. After application of the esters to Kuenen, 2001). Older brood cells containing cocoons dummies, (Zetlmeisl and Rosenkranz, 1994) from several brood cycles are also more highly confirmed the pheromonal but not the kairomonal infested than newly built comb (Piccirillo and De effect. By the use of a servo sphere, which measures Jong, 2004). the specific movements of the mites during the application of volatile substances, Rickli et al., (1994) We can summarize, that the host-finding behavior of found only weak reactions of mites toward methyl Varroa females is triggered by a variety of factors palmitate, but a strong response toward palmitic acid. including physical parameters, but the suitable host Using another approach, even simple odd-numbered stage is finally recognized by chemical volatile signals hydrocarbons from C- 19 to C-29, the major part of of the host larva and the adult bee. The biological the non-polar fraction of the cuticle extract, induced activity of many chemical blends has been strong arrestment behavior of the mites when applied demonstrated in laboratory bioassays including on a running surface (Rickli et al., 1994) revealed compounds of several fractions from the extracts of differences in the cuticular hydrocarbon profile larval cuticle and larval food. However, we still have between parasitized and non-parasitized hosts but not identified the real ‘‘host odor” of the 5th instar could not answer the question of whether these larva of the honey bee: not a single experiment differences are the reason for the infestation or the succeeded in luring the Varroa female from the adult effect of the parasitation. Aumeier et al., (2002) bees to a dummy containing a certain blend, neither confirmed that the hydrocarbon pattern of 4th and in the laboratory (Kraus, 1994) nor within the colony 5th instar larvae differ significantly and could, (reviewed in Dillier et al., 2006). In all experimental therefore, also be used for the chemotactic setups, adult bees were always more attractive than recognition of the suitable host stage. any larval stage (LeDoux et al., 2000, Zetlmeisl and Rosenkranz, 1994). This contradicts the natural However, the invasion of Varroa females is also situation in the honey bee colony, where generally influenced by some additional non-chemical factors. significantly more mites are within sealed brood cells A strong effect has been demonstrated for the size, than on adult bees (Boot et al., 1993, Martin et al., height and age of the brood cell itself. Shortened 1998). During the summer, up to 90% of the mite

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population can be within the brood (Rosenkranz and honeybees with high and low grooming behavior Renz, 2003). We must conclude that as long as under different treatment combinations involving Varroa research fails to elicit the shift of Varroa different temperatures (10, 25, 34°C) and humidities females from bees to brood under controlled (low, medium, high). Mite mortality rates were then conditions, the development of a Varroa trap seems quantified. Bee grooming behavior was affected by unrealistic. Some recent work in Iran such as Kimiaei such environmental conditions and the most et al., (2014) Showed that the Varroa mite infestation promising results were obtained at low humidities reduce foraging behavior and increase aggressive with mite mortality greater in the higher grooming behavior and this phenomone finally effect on hive groups at the higher temperatures. power and cause colony collapse. Hoseinian, (2014) found that the natural mortality rate caused by V. Surveys of bee colonies in Iran for A. woodi destructor is in direct relation with the mite (Mossadegh & Bahreini, 1994) demonstrated that the population in the colony and this significant relation mite was found in 19 of the 139 apiaries sampled. The is due to high initial contamination which keeps two external species, A. externus and A. dorsalis were increasing, gradually. However the results obtained found in eight . Mossadegh, (1990) from statistical analysis failed to denote a significant has studied the development and life-cycle of E. relation among the natural mortality caused by the sinhai Delfinado and Baker, on A. mellifera worker mite and the total number of V. destructor in the brood on which the mites fed and reproduced as an colony. Rahimi and Vahedi, (2014) found smaller alternative host to A. florae. Development from egg to capping period cause Varroa mite not to be able to adult took five days for males and six to seven days complete its developmental period and as a result its for females, with female mites normally producing population goes down. Mahmoodi et al., (2011), from one to eight eggs. Elmi, (2010), observed mite found that adult᾽s infection 35.8% and infants 47.2% infestation rate of adult bees and pupas gradually in spring and in summer adult᾽s infection 30.2% and increased from May to December 2007. The Pearson infants 41.5%. Average apiaries infection to Varroa R coefficient was calculated to estimate association destructor 17.3%. Percentages of infected colonies to between specific variable. There is significantly Varroa destructor statistically, there was no negative correlation between number of pupas and significant relationship (P>0.05). their infestation values to Varroa (r = - 0.325, P<0.006) demonstrating that the lower the number Workers in Iran have been researching potential of pupas the higher the infestation rate of each pupa. control methods for Varroa using chemicals, natural This relationship was not found between adults and substances and the behavioral features of bees. These their infestation rate (r = - 0.031, P<0.801) and include studies on different acaricides and their effect infestation rate of adult bees and pupas have had a in controlling the parasite, including formic acid significantly positive relationship (r = 0.355, (Bahreini et al., 2004a, b) and Apistan and P<0.003) and the results showed there were no comparisons between their efficacy. They have also correlation between the number of adult bees and examined grooming behavior as a natural means of infestation rate of pupas and vice versa (r = - 0.089, controlling the Varroa mite (Currie & Tahmasbi, P<0.464; r = - 0.216, P<0.072). 2008). A number of essential plant oils have been evaluated also, as a potential control method for Therefore, Varroa research is a challenge for all Varroa destructor (Ariana et al., 2002). The results scientists working in the fields of apiculture, insect of these researches can be summarized. The grooming pathology and acarology. We will present a general behavior of bees could be a possible lead to Varroa view on the biology of the Varroa mite with special control. Currie and Tahmasbi, (2008) examined emphasis on recent results on host- parasite

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interactions, breeding honey bees for tolerance, and application within a colony could not achieve a treatment for Varroa infestation. The pathology complete mating disruption of Varroa mites. section describes host damage at the individual and However, our results represent a promising road to colony level including the problem of transmission of the first biological control method based on secondary infections by the mite. Knowledge of both pheromones and will hopefully encourage further the biology and the pathology of Varroa mites are investigations in this direction. essential for understanding possible tolerance mechanisms in the honey bee host. To summarize the Materials and methods efforts we can state that we have significantly A survey of honey bee colonies was conducted from increased our knowledge on mite distribution, December 2013 to November 2015 for the presence of pathogenesis, host parasite interactions and effective both internal and external Varroa destructor mite in use of certain treatments. In most countries the 16 cities (Karaj, , Garmdarreh, Kamalshahr, Varroa situation is stable; the beekeepers have , Meshkindasht, Mohammadshahr, learned to ‘‘live with the mite” and most of them do Zibadasht, Kalak & hesar, Azimiyeh, Aderan, not know beekeeping without Varroosis. Our here Hesarak, Baghestan, Mehrshahr, Nesa and presented first approach of a sex pheromone Heydarabad) in Alborz .

Fig. 1. The Varroa mite has spread around the wordwide. (Photograph by Bernd Grünewald, University of Frankfurt).

The study was conducted in the Karaj district in containers with Powdered Sugar Shake Method. Some central Alborz province (Fig. 2). Ten colonies were samples were also taken from feral colonies of A. randomly selected in each of 16 apiaries from each mellifera in mountainous areas. division for inclusion in this survey. For the internal mite, Varroa destructor, samples of 50 adult Study area bees/hive were taken from the hive entrance and The study was conducted in the Karaj district in when available 50 dead bees from the ground in front central Alborz province (Fig. 2). Alborz province is of the same hive. Sample was stored in separate one of the 31 provinces of Iran, centered in Karaj. containers in 75% ethanol and some sample separated After the Parliamentary approval on June 23, 2010,

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and was introduced as 31st province of Iran. In 2014 it Meshkindasht, Mohammadshahr, Zibadasht, Kalak & was placed in Region. The county has sixteen cities: hesar, Azimiyeh, Aderan, Hesarak, Baghestan, Karaj, Asara, Garmdarreh, Kamalshahr, Mahdasht, Mehrshahr, Nesa and Heydarabad.

Fig. 2. Map of Iran and the study area of Karaj Districts in Alborz province.

The state of Karaj (35° 50′ 8″ N, 51° 0′ 37″ E) have been developed in the past (Fries et al., 1994, represents one of the most important beekeeping area Martin et al., 2002). These differ in the range of in Iran (Fig. 3). included parameters, but converge in their general conclusions. Modelling approaches The present study aimed to evaluation of the Collection of the mite parasite of honeybees in hive contamination level of bees' colonies with Varroa For external mites, samples of 10 adult bees/hive for destructor in 16 cities in Alborz counties and analyze each Karaj᾽s county (16 zones) in Alborz province the mites’ mating behavior under laboratory. The (Fig. 3), during (2013-2015) were collected in a treatment of a 160 colonies from 16 apiaries took polyethylene bag and transported in an ice box to our place in different regions in Alborz province, Iran. In laboratory, where they were refrigerated for further each apiary 10 colonies were selected randomly. studies. The bees were examined singly under a Population development within host colonies is a binocular microscope. Each group of bees was central factor influencing the virulence of varroa powdered sugar shaken method (Fig. 4) and washed parasites. It is driven by the parasite’s reproduction, in 75% ethanol after microscopic examination, and and methods to reduce parasite fertility are therefore the alcohol wash method was examined for the of central importance. Other factors affecting presence of external mites. population growth are experimentally difficult, if not impossible, to assess. Modelling offers the possibility Mite biological control methods and behavior of identifying behaviors or processes of bees or mites Biological control methods could overcome some of that potentially affect population growth and could the problems generated by chemical and alternative therefore be candidates for control methods. Several control options (residues, resistance, non-target population growth modelling tools for V. destructor effects, Meikle et al., 2012). These methods can

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involve the use of antagonists, pathogens or predators towards the mite, results of field tests have been of the pest. The behavior and physiology of the pest mixed, with some research groups reporting a can also be influenced with pheromones or hormones measure of success and other groups reporting no to the point where it disturbs its reproduction and effect (Meikle et al., 2007). This could simplify future population growth in the host. So far, among the registration procedures. At present, little is known of pathogens and predators of varroa, only either the ecology of entomopathogenic fungi in bee entomopathogenic fungi have the desired hives or the most effective formulation or application characteristics of a control agent (Chandler et al., method. 2001). Despite the fact that they show specificity

Fig. 3. Map of the area showing the location of the potential distribution V. destructor and deformed wing virus (DWV) in Karaj᾽s County (16 zones) in Alborz province during (2013-2015).

Collection of V. destructor mites, under laboratory Metzingen), with the male separated from the Male and female V. destructor were obtained from females. As the female mites were used for three tests, heavily infested Apis mellifera colonies at the Karajʾs additional males were collected. The mites were kept Districts apiary (16 zones) in Alborz province, Iran at 28–30 °C for a maximum of 2 h in order to prevent (Fig. 3). Brood combs 10–11 days after host cell decline in their vitality and mobility. capping were removed from the colonies and taken to the laboratory. The suitable combs were identified by Synthetic pheromone solutions the age of the bee pupae which have a dark grayish- We used different applications for the laboratory brown head and thorax at this stage. The brood cells bioassay and colony test, respectively. For the were opened with tweezers and examined for entire laboratory bioassay, we used all the so far identified Varroa families consisting of mother mite, two components of the Varroa sex pheromone mature daughters (i.e. first daughter and second (Ziegelmann et al., 2013b): oleic acid, stearic, acid daughter is equal freshly molted), one female and palmitic acid, and the respective ethyl esters deutochrysalis and one deutonymph as well as one (Sigma Aldrich ≥ 99.7 %). These compounds were adult male (Fig. 5). The mites were transferred into solved in equal proportions in diethyl ether (Roth, GC queen cell cups (Nicot system®, Karl Jenter, grade ≥ 99.8 %, stabilized with ~ 1 % ethanol). From a

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pheromone stock solution with a concentration of attempted to ensure the test conditions were as 10,000 ng/μL for each of the 6 components, 4 natural as possible. The filter paper was creased in the dilutions with concentrations of 1, 10, 100 and 1000 middle and placed at the edge of the plastic cell cup ng component/μL were prepared and stored at -20 °C (Nicot system®, Karl Jenter, Metzingen) containing until use. For the test on brood combs we only used the female individuals of the Varroa family, so that the main component oleic acid (Sigma Aldrich, ≥ 99.7 the treated end of the filter paper reached into the cell %), applied as an alcoholic solution with a cup without touching the bottom or the sides. concentration of 1000 ng/μL in (ethanol 96 %). Afterwards the male was added and the cell cup was sealed with a cover slip to prevent the mites from Data analysis escaping. The behavior of the male was then observed The data sets were analyzed using the SPSS 20 and recorded over a period of 10 min using the statistics software. As most data sets were not Observer 2.0 software (Noldus Information normally distributed, the non-parametric Kruskal– Technology). The male responses toward the different Wallis one-way analysis of variance with a Dunn– female stages were categorized in (1) unspecific Bonferroni correction procedure was used to evaluate contacts including movement towards/around the the male behavior toward the females in the absence female, palpating it and mounting the dorsal shield of and in the presence of synthetic pheromone. The the female and (2) copulation attempts on the venter number of spermatozoa in spermathecae of daughter of the female. The tests were carried out on a mites was compared using Mann–Whitney test. hotplate, with temperatures of 28-30 °C at the bottom Differences between groups with P<0.05 were of the cell cup which is slightly lower than the considered statistically significant. temperature of the honeybee brood of 34-35°C. However, mites showed an optimal activity in the Chemical analyses laboratory bioassays at the lower temperature range Analyses were performed in two laboratories: that we subsequently employed (Le Conte and Department of Honeybees, Animal Sciences Research Arnold, 1988, Rosenkranz, 1988). Institute Karaj (ASRI) laboratory, Alborz Province Iran, and Department of Entomology, Animal Pheromone application under colony conditions Sciences Research Varamin-Pishva branch Islamic For the application on brood combs, the alcoholic Azad University (I.A.U) laboratory, Tehran, Iran. oleic acid solution (1000 ng/μL) was sprayed evenly on empty combs which had already been used for one Laboratory bioassay brood cycle within the colony. Each side of the comb Male responses toward the different mature and was sprayed several times, until a total dose of 5 ml immature females were observed and recorded per comb side was achieved. The comb was then according to the modified ‘‘mating bioassay’’ placed in the middle of the brood nest of a bee colony described in Ziegelmann et al., (2013a, b). For each with the queen trapped on the comb. The queen was test, 1μL of the synthetic pheromone solutions was released 2 days after she had started egg-laying on the applied to a piece of filter paper (size: 1.5 ˟ 15 mm) comb. On the day of host cell capping, brood cells with a 10-μL Hamilton syringe. According to the prior to capping were marked on an overhead different solutions described in 2.2 this resulted in projector film. Marked cells that were sealed during doses of 1, 10, 100 and 1000 ng. For each dose 60 the following 5 h were opened with a scalpel and repetitions were performed. Control tests were artificially infested with one phoretic mite female that performed in the absence of additional pheromone or was directly collected from an adult bee. The Varroa solvent as former tests did not reveal any influence of females’ dorsa were marked with paint to distinguish the solvent (Ziegelmann et al., 2013a) and as we them later from their adult daughters. The artificially

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infested host cells (under laboratory condition) were such as traps, repellents or mating disruption by opened 11 days after capping and the adult daughter certain pheromones. The control of reproduction of a mites as well as the foundress, but not the son, were parasite is, in general, a crucial point for the stability transferred into a plastic thimble and kept on a bee of a host parasite relationship (Walter and Procter, pupa for four more days in the incubator at 28°C and 1999), which, obviously, is also the case in the honey approximately 50 % humidity to ensure that all bee Varroa arms race (Fries et al., 1994). Therefore, spermatozoa will have migrated from the rami to the knowledge of factors that trigger the mite’s spermatheca of the daughter mites, if mated. The reproduction might help for selective breeding of spermathecae of the daughter mites were then tolerant honey bees and One honey bee virus dissected in PBS buffer and the number of Deformed wing virus (DWV), and percentage of spermatozoa within the spermathecae was counted infection to pupa (V. destructor) were detected in the using a light microscope. In a separate control colony, investigated apiaries (16 zones) in Alborz province, the same procedure was undertaken with a brood Iranian bee samples (Table 2, Fig. 3). comb treated with the solvent ethanol only. Life cycle Mite parasite cycle phases V. destructor is closely linked to its honey bee host Varroa mite parasites cycle through four temporal and lacks a free living stage. There are two distinct phases. In some locations, there is one cycle per year phases in the life cycle of V. destructor females: A and, in other locations, more than one cycle. The phoretic phase on adult bees and a reproductive phases are: (a). dormant, (b). population increase, (c). phase within the sealed drone and worker brood cells population peak, (d). population decrease. Varroa (Figs. 6-8). Males and nymphal stages of the mite are mite populations increase and decrease in synchrony short lived and can only be found within the sealed with the seasonal pattern of honey bee development. brood cells. On the adult bees the Varroa females are Mite populations reach their highest levels soon after transported to brood cells for their reproduction or the brood and adult honey bee populations reach spread by foraging and swarming bees (Kuenen and their peak, when there are more brood bees on which Calderone, 1997). On the adult bees the Varroa female Varroa reproduce. When the bee population and the usually is hidden under the sternites of the bee amount of bee brood decline, the phoretic mite (Fernandez et al., 1993). The mites suck substantial numbers drastically increase on the adult bees as the amounts of hemolymph from both the adult bees and amount of bee brood decreases. Eventually, Varroa from the preimaginal host stages within the sealed numbers decrease, along with the adult bee brood cells (De D’Aubeterre et al., 1999; Donzé and population. The size of the mite population at the Guerin, 1994, Garedew et al., 2004). Considering start of population decrease is critical because the mortality in brood cells and improper matings, the colony needs to be healthy enough to rear sufficient average foundress mite produces about one offspring numbers of bees to survive the dormant phase (Fig. per worker cell she enters, and about two offspring 16). per drone cell. Drones take longer to develop so more mites are produced in drone cells. Therefore, in the Mite biology and behavior average temperate climate, mite populations can The host finding and reproductive behavior of V. increase 12-fold in colonies having brood half of the destructor is essential for understanding the year and 800-fold in colonies having brood year- population dynamics of the parasite, but it is also of round. This makes the mite very difficult to control, particular significance for the beekeeping practice. especially in warmer climates where colonies Certain cues for the orientation of the mites could be maintain brood year-round. The phoretic period of used for development of biological control methods the mite appears to contribute to the mite's

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reproductive ability. Although mites artificially adult daughter mites (Fig. 9, control) with a clear transferred to brood cells immediately after they preference for the second (equal younger) daughter. mature are able to reproduce, their reproductive rate The mean duration of contacts with this second is lower than that of mites undergoing a phoretic daughter mite was significantly longer compared to period. The phoretic period may last 4.5 to 11 days contacts with all nymphal stages, including the when brood is present in the hive or as long as five to foundress (P<0.05, n=60, Kruskal– Wallis). Specific six months during the winter when no brood is mating attempts by males on the females’ venter present in the hive. Consequently, female mites living could almost exclusively be observed with the second when brood is present in the colony have an average (equal youngest, freshly molted) adult daughter (Fig. life expectancy of 27 days, yet in the absence of brood, 10, control) and only in a few cases with the first they may live for many months. The varroa mite, V. (equal older) daughter mite. The difference of the destructor, the most insidious pest of honey bee mean duration of the copulation attempts between colonies. The varroa mite is an external parasite that both daughter mites was highly significant (P<0.001, attacks both adult bees and the developing honey bee n=60, Kruskal–Wallis). Interestingly, mounting the larvae (Figs. 8 and 15). The mites can also harm the second daughters’ dorsum led in all cases to mating bees indirectly. In addition to the obvious effects of attempts expressed by the typical tip over behavior mites feeding on developing and adult bees, the mites and probing the gonophores. When males were can also serve as transmitters of several viruses that exposed to 1 and 10 ng of the synthetic pheromone, can kill bees. their preference behavior was not significantly different from the control tests (Figs. 9, 10). Again, Results males had on average the longest contacts with the Male responses towards females of synthetic second adult daughter and the copulation attempts pheromone with this female lasted significantly longer compared In control tests without synthetic pheromone, male to the first daughter and all other female stages mites had long-term contacts exclusively with the two (P<0.05, n=60, Kruskal- Wallis).

Table 1. Statistical comparisons of the copulatory responses of V. destructor males towards the 2nd daughter mite in the absence (control) and in the presence of synthetic pheromone. Dose n Copulation attempts with 2nd daughter Mean [s] ± SD Significance 1 ng 10 ng 100 ng 1000 ng Control 60 362.5 ± 124.3 0.564 0.464 <0.001 *** 0.004 ** 1 ng 60 273.3 ± 220.7 - 1.000 0.048 * 1.000 10 ng 60 340.3 ± 149.7 - - 0.004 ** 1.000 100 ng 60 93.5 ± 89.9 - - - 0.375 1000 ng 60 157.4 ± 125.3 - - - - Note: presented is the mean duration of copulation attempts for applications of 1, 10 100 and 1000 ng of sex pheromone on a filter paper and the corresponding P values. P values marked with asterisks were considered statistically significant (Kruskal–Wallis one-way analysis of variance) with * P<0.05, ** P<0.005, *** P<0.001.

The effect on copulation attempts was even more occurred, in addition, less often. In contrast, the pronounced: the average duration of copulation number and duration of copulation attempts with the attempts with the second daughter was reduced to immature deutonymphs and deutochrysalis about 22 % compared to the control (Table 1) and increased. As a consequence, there were no

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significant differences in the duration of the significantly different from the 100 ng applications copulation attempts among all female stages (P<0.05, but still highly significant different from the control n=60, Kruskal–Wallis) except significant shorter (Table 1). Therefore, applications of 100 and 1000 ng copulation attempts with the foundress (P<0.05, had, under laboratory conditions, a clear and n=60, Kruskal - Wallis; Fig. 9). In the presence of significant effect on both the preference behavior of 1000 ng/substance, the mean duration of copulation the males and the duration of the copulation attempts with the second daughter was not attempts.

Table 2. Number of investigated samples and percentage of infection to pupa (V. destructor) and detected virus Deformed Wing Virus (DWV) in the various of Alborz counties in Iran. No. Name of different Alborz apiaries in (Iran) No. of samples (hive) Percentage of positive samples V. destructor DWV 1. Karaj 10 91 100 2. Asara 10 - 30 3. Garmdarreh 10 - 30 4. Kamalshahr 10 33 100 5. Mahdasht 10 - 50 6. Meshkindasht 10 - 50 7. Mohammadshahr 10 36 100 8. Zibadasht 10 - 100 9. Kalak & hesar 10 48 100 10. Azimiyeh 10 26 35 11. Aderan 10 68 100 12. Hesarak 10 30 26 13. Baghestan 10 54 100 14. Mehrshahr 10 - 40 15. Nesa 10 46 100 16. Heydarabad 10 44 46 Source: Field survey, 2015.

Observation of the mating behavior with cell capping the larvae has consumed the rest of the microscopy larval food (Ifantidis et al., 1988) and the mite sucks With this bioassay, the distinct behaviors of the male hemolymph from the larva. Within a few hours mites could be clearly distinguished when attractive oogenesis starts, followed by vitellogenesis (Garrido females were offered. The course of mating can be et al., 2000; Steiner et al., 1994) and approximately described as a behavioral cascade with four steps: (1) 70 h after the cell capping the first egg is laid attraction, (2) mounting the female dorsum (Fig. 11 (Ifantidis, 1983, Steiner et al., 1994). This first egg is left), (3) movement to the venter (Fig. 11 right), and normally unfertilized and due to the haplo-diploid sex (4) transfer of sperm. determination system it develops into a haploid male, while subsequent female eggs are fertilized and laid in Reproduction Varroa mites 30 h intervals (Ifantidis, 1990; Martin, 1994, Rehm After entering a brood cell with a 5th instar larva the and Ritter, 1989). Up to five eggs in worker brood and Varroa female passes between the larva and the cell up to six eggs in drone brood are considered as the wall to the bottom of the cell and becomes stuck normal ‘‘reproductive program” (Garrido and within the larval food. This behavior may be an Rosenkranz, 2003, Martin, 1994, 1995a). The normal adaptation of the mite to avoid detection and removal sequence of a reproductive cycle is shown in (Fig. 7). by hygienic bees. Respiration takes place by the The mite larva develops within the egg during the first peritreme, the common respiratory organ of Gamasid hours after oviposition. From hatching out of the egg mites (Richard et al., 1990). Approximately 5 h after until the adult molt the mite offspring pass through

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protonymph and deutonymph stages; the into a mobile and an immobile pharate phase just developmental time is about 5.8 and 6.6 days for before the molt (Donzé and Guerin, 1994, Ifantidis, female and male mites, respectively (Donzé and 1983). Males are smaller than females throughout the Guerin, 1994, Ifantidis, 1990, Martin, 1994, Rehm whole ontogenetic development and have longer legs and Ritter, 1989). Both nymphal stages are divided in relation to the body size.

Fig. 4. The powdered sugar shake method is non-lethal for estimating mite levels.

The female mites change during their development starts on the periphery of the opisthosoma and shifts from an oblong to a transversely elliptical body shape; to a reddish- brown color after the molt of the female. the deutochrysalis already exhibiting the final body In contrast, the male’s definite body shape is shape. In the deutochrysalis stage the coloration triangular with a light brown color (Fig. 5).

Fig. 5. Composition of the offspring of a Varroa female approximately 11 days after host cell capping. From top left to bottom right: female deutonymph, female deutochrysalis, 2nd mature daughter, 1st mature daughter, foundress (mother) and adult male. (Photograph by Vaziritabar et al, University of Varamin-Pishva in Iran).

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The mother mite creates a hole in the cuticle of the care” and necessary because the soft chelicerae of the pupa for the nymphs to feed through. This single nymphal stages cannot perforate the pupal cuticle ‘‘feeding zone” is generally located on the 5th segment and the male’s chelicerae are modified for sperm on the bee pupa and near to the so called ‘‘fecal transfer. After feeding, the mites return to the fecal accumulation site” (Donzé and Guerin, 1994, Kanbar accumulation site (Donzé and Guerin, 1994). and Engels, 2003). This behavior is part of ‘‘parental

Fig. 6. Simplified life cycle of the Varroa mite. Varroa females switch between a phoretic phase on adult bees and a reproductive phase within the sealed honey bee brood cells. The nymphal stages and the males are short lived without a phoretic phase outside the brood cells. (Photograph by Vaziritabar et al, 2015).

Fig. 7. The reproductive cycle of Varroa destructor within the sealed honey bee worker brood cell, with the normal sequence of the sexes of mite offspring. A female mite enters the brood cell shortly before capping; approximately 3 days later the first male egg is laid followed by up to four female eggs. Depending on the post- capping period, one or two mature daughter mites will leave the brood cell together with the mother mite and the hatching bee. The numbers on the arrow correspond to the days after cell capping. (Photograph by Vaziritabar et al, University of Varamin-Pishva in Iran).

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Varroa mites become sexually mature immediately copulation starts the male cleans his chelicerae. At the after the last molt. Males reach maturity before the beginning of the mating, the male touches the female females and stay at the fecal accumulation site, with his first pair of legs and ascend her dorsum. He waiting for the first adult female which molts to then examines the frontal margin of the female’s adulthood some 20 h later. As reproduction can only dorsum and slips to the ventral side. Young females occur inside the brood cell, males start mating as soon facilitate this action by lifting their bodies. as the female arrives (Donzé et al., 1996). Before

Fig. 8. A phoretic female Varroa mite on the thorax of a hive bee. The honey bee is under threat from a formidable array of pathogens, including the Varroa mite seen here. (Photograph by Vaziritabar et al, University of Varamin-Pishva in Iran).

Fig. 9. Mean duration and standard deviation of unspecific contacts (walking around females and mounting the females’ dorsum) with mature and immature females of a Varroa family (foundress, two receptive daughters and two early instar daughters) 11 days after cell capping in the absence (control) and in the presence of synthetic pheromone. The synthetic pheromone was applied in dosages of 1, 10, 100 and 1000 ng per pheromone component (n = 60 for control and each dose).

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In the venterto-venter position the male searches for prospermatozoa migrate into the spermatheca and the gonopores (which are separated from the genital change to a fusiform shape. Multiple mating is opening where the eggs are delivered) of the female common until the next daughter female is mature and which are located transversely between the third and arrives on the fecal accumulation site. To fill the fourth pair of legs. Then he takes the spermatophore spermatheca with up to 35 spermatozoa several out of his genital opening and transfers it into the matings are needed (Donzé and Guerin, 1994, Donzé gonopore of the female by means of the chelicerae. et al., 1996). Within 2 days after insemination, the roundish

Fig. 10. Mean duration and standard deviation of copulation attempts with different mature and immature females of a Varroa family (foundress, two receptive daughters and two early instar daughters) 11 days after cell capping in the absence (control) and in the presence of synthetic pheromone. The synthetic pheromone was applied in dosages of 1, 10, 100 and 1000 ng per pheromone component.

The mating behavior is initiated by female sex A. cerana (Rath, 1999). The two haplotypes of V. pheromones (Ziegelmann et al., 2008). Due to these destructor that are capable of reproducing on A. volatile pheromones, young freshly molted females mellifera (Muñoz et al., 2008) can reproduce in both, are significantly more attractive than older females or drone and worker brood. However, a certain deutochrysalis (Fahle and Rosenkranz, 2005), which percentage of Varroa females which have entered a ensures that the male copulates with the youngest drone or worker brood cell do not lay any egg at all. female until the next deutochrysalis molts to the adult This percentage of non-reproducing mites is slightly stage. variable according to the species or subspecies of the host and climatic conditions and might, therefore, Infertility and low reproductive rates of Varroa contribute to differences in the host tolerance of females European bees (Fries et al., 1994, Rosenkranz, 1999). In the original host A. cerana the reproduction of In European honey bee subspecies about 5–20 % of Varroa mites (V. jacobsoni and V. destructor) is the mites remain infertile after invading worker or limited to drone brood for yet unknown reasons drone brood cells (Al Aattal et al., 2006, Garrido et (Anderson, 2000, Boot et al., 1996, Garrido, 2004, al., 2003, Martin, 1994, Martin et al., 1997, Rath, 1999). This phenomenon is considered a crucial Rosenkranz, 1999). A long-term example of higher point for the balanced host–parasite relationship in infertility rates in A. mellifera is confined to the

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Africanized honey bees of Brazil: Over a period of point, the distance between the larva and the rim of more than 15 years an average rate of infertile mites the brood cell decreases (Boot et al., 1992). They of about 50% in worker brood was confirmed; mite invade cells based on several different factors, fertility in drone brood, however, did not show any especially olfactory and mechanical cues. Aumeier et particularities (reviewed in Rosenkranz, 1999). al., (2002) found stage-specific odor compounds found in larval cuticles including five methyl esters Cell Invasion and 14 hydrocarbons (kairomones) such as methyl Mated female mites enter worker cells 15-20 hours palmitate, ethyl- palmitate, and heneicosane, all of prior to cell capping in workers, and approximately which are thought to be attractive to female mites 40-50 hours prior to cell capping in drones (Boot et (but see Nazzi et al., 2001), who suggested that al., 1992). Mites enter larval cells when the larvae are methyl palmitate is probably not an attractant to large enough to cover the entire cell bottom; past this mites).

Fig. 11. Photograph of mating behavior V. destructor. Typical steps of the mating behavior of Varroa males before the transfer of the spermatophore: mounting of the female’s dorsum (left) and movement to the venter (right). For details, see text. (Photograph by Vaziritabar et al, University of Varamin-Pishva in Iran).

They found that the composition of larval odors were transferred artificially to other newly sealed changes significantly between the fourth and fifth brood cells, were still able to reproduce (Weller, instars, with levels of methyl- alkanes significantly 2008). This suggests that temporary infertility of decreasing and levels of alkenes and esters Varroa females is induced by host factors. The significantly increasing. Transfer of Varroa mites production of one adult viable daughter requires at among different honey bee subspecies at a Brazilian least the maturation of one male and one female study site confirmed that low mite fertility was more a offspring including mating. Therefore, female mites host than a parasite trait (Rosenkranz, 1999), producing only one egg, no males or with delayed independent of the possible presence of different mite start of oviposition may not contribute to the growth haplotypes with possible different reproductive of the Varroa population. For example, in 11–21% of abilities. Two observations contradict these the brood cells the male is lacking (Donzé et al., 1996, hypotheses: In the phoretic mite populations on adult Martin et al., 1997). The mortality of mite offspring bees nearly all mites have filled spermatheca seems to be a main factor for differences in the (Garrido, 2004) and ‘‘infertile” brood mites, which reproductive rate and varies according to climate,

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season and honey bee subspecies (Ifantidis et al., immature mite on pupa and worker brood cells (Fig. 1999, Mondragón et al., 2005, 2006). Additionally, 12) the reproductive rate per female mite is the reproductive rate depends on the infestation of a significantly reduced (Martin and Medina, 2004, single brood cell; in multiply invaded drone and Mondragón et al., 2006).

Fig. 12. Recently capped drone brood cells with 5th instar larvae multiply infested By Varroa mites and reproductive rate depends on the infestation of a single brood cell; in multiply invaded drone and immature mite on pupa and worker brood cells. (Photograph by Vaziritabar et al, University of Varamin-Pishva in Iran).

Triggering of Varroa reproduction reproducing mites and less offspring in multiply The reproduction of Varroa mites is closely infested cells compared to singly infested cells (Donzé synchronized with the brood development of the host. et al., 1996, Nazzi and Milani, 1996). In artificial Even the sequence of male and female eggs of the brood cells treated with a hexane extract from Varroa mite depends on signals from the multiply infested brood cells the mean number of corresponding stage of the host larvae and pupae, Varroa offspring was significantly reduced (Nazzi and respectively (Garrido and Rosenkranz, 2003). The Milani, 1996). Nazzi et al., (2004) identified the reproductive success of a single Varroa female can be active substance from infested brood cells as (Z)-8- defined by the number of adult, mated and viable heptadecene. In a laboratory test they showed that daughter mites produced during a reproductive cycle this particular isomer significantly affects the within the honey bee brood cell. Therefore, the fecundity of the Varroa female. When (Z)-8- duration of brood development is a limiting factor for heptadecene was applied to freshly capped brood cells the development of the mite. This creates a strong in the colony it reduced the number of Varroa selective pressure on reproducing mites for rapid offspring and the number of viable adult Varroa oogenesis and preimaginal development. The phoretic daughters (Milani et al., 2004). The authors assumed mites are already well prepared for reproduction by that (Z)-8-heptadecene is produced under the stress the presence of a large terminal oocyte which will condition of multiple infestations, however, proof is develop into the first egg (Steiner et al., 1994), lacking that this carbohydrate is produced by infested Certain proteins of the host hemolymph are stored larvae. The relevance of the phoretic phase for the directly in the ovary of the mite (Steiner et al., 1994). reproductive cycle of Varroa mites is not clear. Under There are also inhibitors of mite reproduction within laboratory conditions, Varroa females can a brood cell. Several authors described fewer successfully be transferred from brood cell to brood

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cell without a phoretic phase (De Ruijter, 1987), most important honeybee virus and parasite such as under field conditions, older mites seem to invade Deformed wing virus (DWV) and varroa mite (V. brood cells more rapidly than nulliparous Varroa destructor). The distribution of honeybee virus and females (Fries and Rosenkranz, 1996). It seems that (V. destructor) infections was studied in all 16 especially under unfavorable conditions, the phoretic districts of Alborz province in Iran (Table 2, Fig. 3). phase may have negative effects on the reproductive Bee samples were collected in 160 apiaries, mainly capacity of Varroa mites: After a long phoretic phase from bee colonies with various health problems like of 5 weeks or more, or after a starvation period of 7– depopulation, weakness, and high colony mortality. 18 h the number of infertile mites was two–three-fold Samples of winter losses (from January and February higher than in the control (Rosenkranz and 2013) were collected from the bottoms of beehives Bartalszky, 1996). during the first spring inspection of honeybee colonies. After the first infestation of a new honey bee Population dynamics colony, Varroa mites are able to build up huge In this section manuscript we investigated the populations within a few years (Fries et al., 2003). prevalence and regional distribution patterns of two

Fig. 13. A young bee with crippled wings and shortened abdomen, presumably due to Varroa and Deformed Wing Virus (DWV) infections during the ontogenetic development. (Photograph by Vaziritabar et al, University of Varamin-Pishva in Iran).

The population growth is highly variable and depends authors tried to extract the most important factors by on all of the traits of the host and the parasite that the use of multifactorial analyses (Harris et al., may influence the reproductive rate and the mortality 2003). They confirmed significant correlations of the mite (Calis et al., 1999b). Some of the host between the amount of brood and/or the fertility of features that influence mite population growth are the mites and population growth; however, prediction additionally triggered by ambient factors such as to what extent a starting mite population in the spring climate and nectar flow (Currie and Tahmasbi, 2008). will increase until autumn is still not possible. The The exact impacts of the individual parameters on the results of the present shown that, to avoid the population dynamics are not known. Additionally, probable damages, a precise evaluation of the initial most of these parameters are mutually influenced and contamination rate of the colony during the period part of complex multifactorial interactions. Some March 2013 thru August 2015 is a must, and any

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attempt in term of V. destructor fighting, if necessary, representing the most sensitive host stages. First, the should be taken into account such alternatives like loss of hemolymph during the ontogenetic how to use, which drug to use and when. development within the brood cell significantly decreases the weight of the hatching bee. The weight Pathology loss depends on the number of mother mites and the Damage at the individual level Alborz province amount of mite reproduction, but even a single apiaries infestation results in an average loss of body weight of The individual honey bee is damaged in a variety of 7% for the hatching bee (Schatton-Gadelmayer and ways, with the developing larvae and pupae clearly Engels, 1988).

Fig. 14. A brood cell containing a pupa damaged through Varroa infestation was opened by hygienic bees; the Varroa female is escaping from the opened brood cell. (Photograph by Vaziritabar et al, University of Varamin- Pishva in Iran).

This has also been proven for parasitized drones, these brood cells (Fig. 15). which lose 18-27% of their body weight depending of infestation rate (Duay et al., 2003), which led to Varroa mites and honey bee viruses decreased flight performance (Duay et al., 2002). V. destructor is a vector for various honey bee viruses. Worker bees which were parasitized during their So far, about 18 different viruses have been isolated development, start earlier with foraging and have a from honey bees (Chen and Siede, 2007) and many of significantly reduced life span (Amdam et al., 2004). them can be vectored by Varroa mites. This has been The parasitized foragers display a decreased proven for Kashmir bee virus (KBV), Sacbrood virus capability of non-associated learning, prolonged (SBV), Acute bee paralysis virus (ABPV) and absences from the colony and a lower rate of return to Deformed wing virus (DWV) Boecking and Genersch, the colony (Kralj and Fuchs, 2006, Kralj et al., 2007), (2008). Before the occurrence of Varroa mites, bee which may be due to a reduced ability to navigate. viruses have been considered a minor problem to Adult female varroa mites can be found either on honey bee health (Allen et al., 1986,Yue and adult or immature honey bees. They must, however, Genersch, 2005). Obviously, the direct injection of reproduce on honey bee brood (developing larvae or virus particles into the hemocoel of honey bee pupae pupae). Immature varroa can be found only on and activation of latent virus infections through the capped brood and male varroa mites will never leave additional injection of foreign salivary proteins of the

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mite provoke typical disease symptoms. Best known social behavior like hygienic behavior and queen is the DWV infection causing the typical symptoms of attendance, as well as rapid loss of bee population, is crippled wings and shortened abdomen in heavily an effect of virus infections rather than the effect of infested honey bee colonies (Fig. 13). Some of the direct parasitation through the Varroa mites. For viruses are transmitted horizontally and vertically instance, a two year monitoring of about 2037 honey (Boecking and Genersch, 2008) supporting covert bee colonies in Iran (Alborz province) 2013-2015 infections in managed honey bee populations. revealed a significant correlation of colony winter Additionally, Varroa mites may induce losses with (i) Varroa infestation and (ii) with the immunosuppression in parasitized pupae and, thus, prevalence of DWV (Monitoring Project, 2013-2015). activate these covert virus infections (Yang and Cox- However, the correlation between virus detection, Foster, 2007). It is assumed that the final breakdown Varroa-infestation level and colony mortality is not as of a honey bee colony with the typical ‘‘damage clear as expected and demonstrates the need of a symptoms”, such as scattered brood nest, crippled standardized quantitative virus analysis under field bees (Figs. 13 and 14), eventual loss of coordinated conditions with a definition of damage thresholds.

Fig. 15. Photograph of adult female varroa mites can be found either on adult or immature honey bees (right illustration) and Varroa damage (left illustration): normal bee (on left), deformed bee (on right). For details, see text. (Photograph by Vaziritabar et al, University of Varamin-Pishva in Iran).

Control of mite reproduction methods. A selection of beekeepers often occurs The control of mite reproduction is considered the during the first 10 years, based on whether they are most effective tool for the host to prevent the growth willing or able to include Varroa treatment into their of a Varroa population within the colony (Fries et al., management practice. Beekeepers that do not do this 1994). Various approaches of the honey bee host can will lose their colonies. After several years the limit the reproductive success of Varroa mites: The beekeeping situation normally stabilizes. However, first ‘‘official” detection of Varroa mites in a country is periodic high losses of 30% or more of the hived usually followed by intensive activities of the honey bee population, mostly during overwintering, responsible bee scientists, extension services and are still common and seem to be inevitable veterinary authorities in order to control the spread of (Monitoring Project, 2013-2015). Varroa mites seem the mite and prevent collapse of honey bee colonies. to be the crucial driver for these periodic losses. It is During the first phase, damage and losses of colonies assumed that Varroa is also involved in the recent are common due to the lack of knowledge in control substantial losses of honey bee colonies across the

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United States and several European countries (CCD: well as the final mating attempt. In all control tests, Faucon and Chauzat, 2008, Vanengelsdorp et al., males showed an unmistakable preference for the 2008a). This link is supported by Nguyen et al., youngest adult daughter, whereas other females were (2009) who explored recent colony losses and found less attractive or even not attractive at all. This exactly acaricides to be either prohibited or ineffective resembles the observations made by Donze´ et al., against V. destructor. He concluded that the common (1996) in tests with artificial polystyrene cells treatment methods used by the beekeepers often are containing naturally reared host brood and mites and inadequate for mite control. Without any doubt, most also matches the results of our former investigation of the colonies of A. mellifera in temperate climates on the preference behavior of male mites in plastic will be damaged or even collapse within a few years if queen cell cups (Ziegelmann et al., 2013a). Most no control or inappropriate control methods are used likely, freshly molted yet unmated female mites elicit (Boecking and Genersch, 2008; Rademacher and higher amounts of the sex pheromone. After about 24 Harz, 2006). Nowadays, beekeepers utilize a wide h the attractiveness of these females decreases, range of different chemical substances, application resulting in fewer and shorter copulation attempts by techniques and methods to keep mite populations males. Therefore, the most recently molted daughter under control. mite has a greater chance to mate with the male before the host bee emerges from its brood cell. As Discussion each Varroa foundress usually produces only one The identification of the Varroa sex pheromone male, we consider this a crucial and adaptive behavior (Ziegelmann et al., 2013b) offers for the first time a to ensure sufficient sperm transfer to all adult realistic option for a biological approach to reduce the daughter mites. This important sequence of the male reproductive success of the mite. In this study we mating behavior and preference can be disturbed by determined whether the mating behavior of V. the application of an overdose of the female sex destructor can be disturbed by the use of the female pheromone. The application of 1 and 10 ng of sex pheromone (i) under laboratory conditions and synthetic sex pheromone already resulted in a (ii) by targeted application within the colony. In order reduced duration of mating attempts with the to evaluate the effect of the sex pheromone, we youngest adult daughter and a slight increase of compared male responses toward mature and contacts and mating attempts with other females. An immature females in the absence and in the presence obvious and highly significant change in male of synthetic pheromone solutions. For this approach behavior was seen with the application of 100 and we mimicked the composition of a natural Varroa 1000 ng of the sex pheromone. These amounts are family 11 days after cell capping in our bioassay. At considerably higher compared to the results of the this stage, an infested host brood cell generally quantitative analysis of mite extracts (Ziegelmann et contains the foundress, one single adult son, two al., 2013b) and confirm that a huge overdose of the mature daughters and several immature female stages pheromone is required for significant effects. Under (Ifantidis, 1983). Therefore, the male has the choice these conditions males were confused and seemed for contacts and copulatory attempts with several unable to distinguish whether females were receptive female stages. In V. destructor, the course of mating or not. The average duration of the copulation is characterized by a distinct sequence of behaviors attempts with the youngest adult daughter was (Donze´ et al., 1996, Ziegelmann et al., 2013a) with reduced to about 24 %, even though males had the tip-over of the male to the female’s venter as the contacted these females and mounted their dorsum. crucial step in copulation. In our bioassay we were Males often did not show any attempt to copulate but able to quantify the general attractiveness of certain alternated between the ventral and dorsal side of a female stages (expressed as unspecific contacts) as female or even descended from the dorsum and

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departed the female mite. In contrast, intense females. These results again confirm that the volatile copulation attempts were performed with immature sex pheromone is the crucial signal for the triggering female deutonymphs and deutochrysalis, which are of the mating behavior (Ziegelmann et al., 2013b), completely unattractive under normal conditions. and it confirms that this behavior can be disturbed This is surprising as deutonymphs are much smaller solely by an overdose of pheromone. and also their body shape differs from mature

Fig. 16. Honey bee and Varroa mite seasonal phases, bee population (orange line) and mite population (blue line). see text. (Photograph by Vaziritabar et al, University of Varamin-Pishva in Iran).

The oleic acid solution was sprayed on a brood comb mating under colony conditions, which leads to a prior to egg-laying and Varroa infestation. Counting reduction of transferred spermatozoa and, the number of spermatozoa in spermatozoa of remarkably, 20 % of the daughters without any daughter mites taken from this comb showed that spermatozoa at all. The entire reproductive cycle of V. indeed the number of transferred spermatozoa was destructor is triggered by chemical communication, significantly reduced. This result supports the from host finding (Le Conte et al., 1994) via the suggestion that the application of pheromone disturbs activation of female oogenesis (Garrido and the mating process and leads to a reduced number of Rosenkranz, 2004, Frey et al., 2013) through to copulations and thus spermatozoa transfer. As the mating (Ziegelmann et al., 2013b). It is surprising brood cells were opened on day 11 after host cell that the dependence of the mite’s reproductive capping, the time span for mating was shorter than success on volatile compounds has so far not been under normal conditions (cells normally remain used for the development of a biological control capped for about 12 days, Rosenkranz and Engels, method. Our here presented first approach of a sex 1994). Thus, the mean number of spermatozoa in pheromone application within a colony could not females taken from the control comb was somewhat achieve a complete mating disruption of Varroa lower than the mean number of spermatozoa given in mites. However, our results represent a promising the literature (Donze´ et al., 1996). For an absolute road to the first biological control method based on quantification of the effect of the comb treatment on pheromones and will hopefully encourage further sperm transfer, the comb should be kept in an investigations in this direction. An important factor incubator prior to hatching of the bee. Nevertheless, for honey bee resistance to the Varroa mite (Varroa our results highlight the possibility to disturb Varroa destructor) is shortening the capping period of sealed

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brood cells that the mite cannot complete its quantity, combined with other components of the sex metamorphosis cycle and reproduction and decrease pheromone and/or application methods. Although V. its population and the smaller capping period cause destructor is not the sole cause of colony losses varroa mite not to be able to complete its experienced worldwide in recent years, a consensus developmental period and as a result its population emerges that it represents the key factor (Neumann goes down. and Carreck, 2010). Removing V. destructor from the complex equation of honey bee health would reduce Conclusion the pressure on the honey bee’s extensive natural In conclusion, this bioassay provides a possibility for defense mechanisms (Evans and Spivak, 2010) observing and quantifying the mating behavior of against the many environmental health challenges. mites in the laboratory with reproducible results, and Using sustainable methods to control or even also a basis for the investigation of the involved cues. eradicate this parasite will re-establish wild and feral In choice tests with living and dead females, males pollinator populations, ease the plight of beekeepers, mated preferably with young, freshly molted females promote economically important pollination regardless of whether the females were alive or dead. dependent agriculture and benefit natural The mating of the young female mites within the ecosystems. For this ideal to be realized, however, a sealed brood cells represents a crucial phase of the strong and sustained research effort is needed to reproductive cycle of (V. destructor) Female eggs are produce the understanding necessary for an efficient laid at 30-h intervals, and therefore, young adult and sustainable control strategy against this most female mites appear at similar intervals. Depending important of honey bee parasites. Our results clearly on the capping period of the brood cells, only one to indicate that freshly molted females are most three adult female mites can reach maturity attractive to males, and that this attractiveness is (Rosenkranz et al., 2010). However, the development presumably based on a female sex pheromone being of a successful biological Varroa control treatment released during the process of molting. Older females with sex pheromone under beekeeping conditions lose at least in part their attractiveness by yet requires further improvement of the application unknown mechanisms. These findings should technique. The effect of the pheromone should stimulate further research with the objective of preferably last over a longer period and several brood disturbing the male’s mating behavior as a first cycles. The normal development of the bee brood in approach for a biological Varroa control. our treated combs indicates that side effects on bees and brood are unlikely; however, this aspect needs to Conflicts of interest be considered in further experiments. From the The authors declare that they have no conflicts of perspective of food safety and residues in the human interest. food chain, we do not see insurmountable obstacles in the application of the component of the sex List of symbols and abbreviations pheromone we used because fatty acids (and their Kashmir bee virus (KBV) esters) are commonly of low toxicity and widespread Sacbrood virus (SBV) in animal and food material. Finally, we expect that Acute bee paralysis virus (ABPV). the efficacy revealed in our experiments could be increased. In this first experiment we only used one Deformed wing virus (DWV) application method with one dosage of the main Acknowledgements component of the Varroa sex pheromone (Ziegelmann I would like to thank Dr. Assadolah Aghamirkarimi et al., 2013b). It is likely that mating disruption could for providing helpful comment to the manuscript and be increased by modifications of the pheromone I also thank to Sayed Mehdi Esmaeilzade for help

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during field work. We would like to thank Apicultural Research 49, 23-30. Department of Honeybees, Animal Sciences Research Institute Karaj of Iran (ASRI) and Department of Becher MA, Moritz RFA. 2009. A new device for Entomology and Animal Science Research Varamin- continuous temperature measurement in brood cells Pishva University for their cooperation and excellent of honeybees (Apis mellifera). Apidologie 40, 221- facilitation during study period. I would like to 230. express my deepest gratitude to all beekeepers who contributed to this study and we are also grateful to Boecking O, Genersch E. 2008. Varroosis – the two anonymous reviewers for their insightful ongoing crisis in beekeeping. Journal of Consumer comments. Protected Food Safety 3, 221–228.

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