Journal of Insect Physiology 58 (2012) 1548–1555 Contents lists available at SciVerse ScienceDirect Journal of Insect Physiology journal homepage: www.elsevier.com/locate/jinsphys How does the mite Varroa destructor kill the honeybee Apis mellifera? Alteration of cuticular hydrcarbons and water loss in infested honeybees ⇑ Desiderato Annoscia, Fabio Del Piccolo, Francesco Nazzi Dipartimento di Scienze Agrarie e Ambientali, Università degli Studi di Udine, Via delle Scienze 208, 33100 Udine, Italy article info abstract Article history: Several factors threaten the health of honeybees; among them the parasitic mite Varroa destructor and Received 24 May 2012 the Deformed Wing Virus play a major role. Recently, the dangerous interplay between the mite and Received in revised form 20 September the virus was studied in detail and the transition, triggered by mite feeding, from a benign covert infec- 2012 tion to a devastating viral outbreak, characterized by an intense viral replication, associated with some Accepted 22 September 2012 characteristic symptoms, was described. In order to gain insight into the events preceding that crucial Available online 3 October 2012 transition we carried out standardized lab experiments aiming at studying the effects of parasitization in asymptomatic bees to establish a relationship between such effects and bee mortality. It appears that Keywords: parasitization alters the capacity of the honeybee to regulate water exchange; this, in turn, has severe Apis mellifera Cuticular hydrocarbons effects on bee survival. Varroa destructor These results are discussed in light of possible novel strategies aiming at mitigating the impact of the Water loss parasite on honeybee health. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction 2012), so that, during the Summer, all bees in a colony can be re- garded as DWV infected by different number of viral particles The mite Varroa destructor Anderson & Trueman is the most (Nazzi et al., 2012). important parasite of the western honeybee Apis mellifera L. In a previous study we showed that collapse in mite infested (Sammataro et al., 2000). Since it appeared in the Western World colonies occurs, at the end of the active season, because of a sud- it has caused huge damage to the beekeeping industry and has been den increase of bee mortality caused by lethal numbers of DWV linked to widespread losses of honeybee colonies recorded in the US genome copies resulting from the viral replication triggered by and Europe since 2006 (Le Conte et al., 2010). Recent studies mite infestation (Nazzi et al., 2012). However, the effects of mite showed that the Varroa mite plays a critical role in the collapse of infestation on individual honeybees, before the virus reaches lethal honeybee colonies. In particular, it has been shown that the mite levels and the symptoms of active viral replication (i.e. deformed is related to dramatic changes in strain diversity within Deformed wings) are noted, have not been investigated in detail yet. Wing Virus (DWV) that are likely related to increased prevalence Several studies have been carried out so far dealing with the and viral load (Martin et al., 2012). Other authors, investigating consequences of mite infestation on honeybee physiology and the dynamics of collapse leading interactions, showed that the mite health (Yang and Cox-Foster, 2007 and citations therein) but, due can destabilize the within-host dynamics of DWV with dramatic to the close relationship between the Varroa mite and DWV, the consequences on honeybee mortality (Nazzi et al., 2012). distinction of the causal agent of the observed effects is very diffi- DWV is a widespread honeybee virus normally causing benign cult. However, apart from its biological interest, the study of the ef- covert infections that, in presence of the mite, can turn into devas- fects of mite infestation in the absence of viral infection and tating viral outbreaks (de Miranda and Genersch, 2010). Overt viceversa is probably of limited value under a practical point of infections are characterized by high loads of viral genome copies view, given the ubiquitous distribution of both V. destructor and and some classic symptoms including crippled wings and short- DWV. Instead, the study of the effects of mite infestation on appar- ened abdomen (Möckel et al., 2011). Several studies demonstrated ently healthy, albeit infected bees and, moreover, the elucidation of that, in Europe, most honeybee colonies are infected (see Ribière the biological phenomena underlying the observed effects would et al., 2008 for a review) and prevalence increases throughout be vital in view of developing intervention strategies aiming at the year (Tentcheva et al., 2004; Martin et al., 2010; Dainat et al., mitigating the effects of mite infestation. The most apparent effect of mite infestation at the individual le- vel appears to be weight loss (Yang and Cox-Foster, 2007 and cita- ⇑ Corresponding author. Tel.: +390432558513; fax: +390432558501. E-mail address: [email protected] (F. Nazzi). tions therein). Changes in the content of sugars and proteins 0022-1910/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2012.09.008 D. Annoscia et al. / Journal of Insect Physiology 58 (2012) 1548–1555 1549 (Bowen-Walker and Gunn, 2001) have been reported in infested The experiment was repeated 6 times using in total 77, 68 and bees as well as alterations of the cuticular hydrocarbons (Salvy 52 bees that had been infested during the pupal stage with none, et al., 2001) but the very cause of the reduced weight still remains one or three mites respectively. For each replicate, new sets of cells unknown. infested by none, one or three mites were prepared; the figures re- In order to fill this gap, we carried out a study aiming at inves- ported above refer to the sum of cells of each type that were used. tigating in detail both the effects of Varroa parasitization on indi- vidual asymptomatic honeybees and the underlying causes. To 2.4. Influence of weight at the eclosion on the longevity of adult bees keep under control possible confounding factors that can affect field data, we opted for a lab study in which infested bee larvae In order to verify the potential influence of the weight on the were maintained under controlled conditions in artificial rearing longevity of adult honeybees, we studied the survival of individual cells. This method was used throughout the present study both bees that had been weighted at the eclosion. To do so, bee larvae to confirm and integrate available data on the outcome of mite were infested with none, one or three Varroa mites as described infestation and for a thorough investigation on the possible causes above, then, after 12 days, cells were opened and the emerging of the observed effects. adult bees were weighted using an electronic precision balance We believe that, besides the obvious biological interest in (Sartorius CP2P, maximum loading capacity 2000 mg, readability unraveling this aspect of the host-parasite interaction, a precise 0.001/0.002/0.005 mg), marked with a queen marker kit, consisting description of such effects is vital in view of developing an inte- of colored-numbered tags (2 mm Ø) applied on the thorax with a grated strategy to sustain honeybee health. resin type glue, and transferred into plastic cages as already described. At the emergence, symptoms of DWV infection such as de- 2. Materials and methods formed wings were noted if present; during the experiments, daily controls were carried out to note and remove any dead bee. 2.1. Biological material The experiment was repeated three times using in total 35, 28 and 22 bees that had been infested during the pupal stage with The biological material (larvae and adult females of V. destruc- none, one or three mites respectively. tor) was obtained from an experimental apiary located in Udine (North-eastern Italy). Previous studies indicated that local colonies 2.5. Honeybee water content are hybrids between Apis mellifera ligustica Spinola and Apis melli- fera carnica Pollman (Comparini and Biasiolo, 1991; Nazzi, 1992). To assess water content of eclosing bees infested or not by one The apiary was set up in March and was left untreated for the mite, capsules used for rearing were opened, after 12 days, and the duration of the experiments; the biological material was collected honeybee fresh weight recorded, using an electronic precision bal- from April to September. ance, after removing the infesting mites and their offspring. Mites and the bee larvae were collected from brood cells capped Then samples were maintained at 80 °C for 24 h on a Petri dish 0–15 h previously (Nazzi et al., 2012). (Sage, 1982) and the dry weight of each honeybee registered again Previous studies showed that, in the area, the prevalence of after 30 min at room temperature. DWV is about 15% at the beginning of the Spring but it raises stea- The experiment was replicated 4 times using 50 honeybees for dily to about 100% by the middle of the Summer (Nazzi et al., each experimental group. 2012). Thus, in all cases, the vast majority, if not the total, of bees used in this work were infected by the virus as confirmed by PCR 2.6. Dynamics of weight-loss during pupation tests conducted in the framework of a parallel study. Larvae not infested or infested with one mite, obtained as de- 2.2. Artificial infestation of bee pupae scribed above, were transferred into gelatin capsules (Assing, 6.5 mm Ø), weighted and then maintained in a climatic chamber Larvae obtained as above were transferred into gelatin capsules (34 °C, 75% R.H., dark) for 12 days. The weight of each larva was (Assing, 6.5 mm Ø) with none, one or three mites and maintained noted before and after encapsulation, in order to obtain the weight in a climatic chamber (34 °C, 75% R.H., dark) until the day of the of the empty capsule and the Varroa mite; then the weight of the eclosion, 12 days later (Nazzi and Milani, 1994).