Norberto Milani

Reinfestation of an acaricide-treated apiary by Varroa jacobsoni Oud.

The sources of reinfestation of a treated apiary byVarroa jacobsoni Oud. were studied in Friuli (North-Eastern Italy), in an area with a high density of colonies. Ten colonies, initiallyVarroa-free, were treated with Apistan or Bayvarol strips and mites killed by these treatments were counted twice a week for 1 year. Five hives were provided with “drone excluders”, to avoid the entrance of drones from other apiaries. Nectar and honeydew flow were monitored.The reinfestation rate was low during spring, varied between 1.6–13.7 mites/day/colony during June, July and first week of August, and rose impressively during September and October (up to a mean of 75.6 mites/day/colony); it was relatively high when nectar flow was scarce. The presence of drone excluders did not help to limit the number of mites imported: drones did not seem to be the main cause of reinfestation. The coincidence between the increase in the reinfestation rate and the scarcity of nectar flow and the massive importation of mites, observed especially in September and October, suggest that reinfestation was mainly caused by robbing of infested colonies (mostly feral swarms) by the bees of treated colonies.

Selection of Apis mellifera workers by the parasitic mite Varroa destructor using host cuticular hydrocarbons.

The parasitic mite, Varroa destructor, is the most important threat for apiculture in most bee-keeping areas of the world. The mite is carried to the bee brood cell, where it reproduces, by a nurse bee; therefore the selection of the bee stage by the parasite could influence its reproductive success. This study investigates the role of the cuticular hydrocarbons of the European honeybee (Apis mellifera) in host-selection by the mite. Preliminary laboratory bioassays confirmed the preference of the varroa mite for nurse bees over pollen foragers. GC-MS analysis of nurse and pollen bees revealed differences in the cuticular hydrocarbons of the two stages; in particular, it appeared that pollen bees have more (Z)-8-heptadecene than nurse bees. Laboratory experiments showed that treatment of nurse bees with 100 ng of the pure compound makes them repellent to the varroa mite. These results suggest that the mite can exploit the differences in the cuticular composition of its host for a refined selection that allows it to reach a brood cell and start reproduction. The biological activity of the alkene encourages further investigations for the development of novel control techniques based on this compound.

Octanoic acid confers to royal jelly varroa-repellent properties

The mite Varroa destructor Anderson & Trueman is a parasite of the honeybee Apis mellifera L. and represents a major threat for apiculture in the Western world. Reproduction takes place only inside bee brood cells that are invaded just before sealing; drone cells are preferred over worker cells, whereas queen cells are not normally invaded. Lower incidence of mites in queen cells is at least partly due to the deterrent activity of royal jelly. In this study, the repellent properties of royal jelly were investigated using a lab bioassay. Chemical analysis showed that octanoic acid is a major volatile component of royal jelly; by contrast, the concentration is much lower in drone and worker larval food. Bioassays, carried out under lab conditions, demonstrated that octanoic acid is repellent to the mite. Field studies in bee colonies confirmed that the compound may interfere with the process of cell invasion by the mite.

(Z)-8-Heptadecene from infested cells reduces the reproduction of Varroa destructor under laboratory conditions

The parasitic mite Varroa destructor Anderson and Trueman, the most serious threat to apiculture in many countries of the world, reproduces inside honeybee brood cells. Previous research, using artificial cells for rearing the mite on an Apis mellifera larva, indicated that semiochemicals affecting the reproduction of Varroa destructor are released into such cells. In order to isolate these semiochemicals, infested artificial cells were extracted with hexane and the extract fractionated twice. Several unsaturated hydrocarbons were identified in the active fraction; some of them were released in higher amounts in case of infestation and were, therefore, bioassayed for their effect on the mites reproduction. Of five alkenes tested under laboratory conditions, (Z)-8-heptadecene, caused a 30% reduction in the mean number of offspring of mites reared in cells treated with this compound.

The presence of inhibitors of the reproduction of Varroa jacobsoni Oud. (Gamasida: Varroidae) in infested cells

The mite Varroa jacobsoni was reared in artificial gelatin cells under laboratory conditions and the possible presence of factors inhibiting Varroa reproduction was studied. In cells infested with three mites, the mean offspring per female was reduced to 75% of that in singly infested cells. When gelatin cells were used for two successive rearing cycles, both the proportion of reproducing females and the offspring per reproducing female were significantly lower in cells that had contained an infested larva during the first rearing cycle than in those with an uninfested larva. The mean reduction of the offspring per female was 48%; this suggests that inhibitors of the reproduction are released into infested cells. Treatment of gelatin cells with the hexane extract of cells in which an infested bee pupa had developed caused a 21% reduction in the mean offspring per female, with a difference close to the significance level (p=0.07).

Determination of the LC50 of chlorfenvinphos in Varroa destructor

Chlorfenvinphos is an organophosphorus insecticide ((EZ)-2-chloro-1(2, 4dichlorophenyl) vinyl diethyl phosphate) authorised in some countries for agricultural and veterinary uses but not for apiculture. Nevertheless, in recent years it has been increasingly employed to control Varroa destructor Anderson & Trueman, as indicated by the growing percentage of wax samples found to be positive for residues (17% in 2003: Costa et al., 2006). A possible lack of efficacy of treatments with chlorfenvinphos has been claimed as a cause of bee losses. We developed a bioassay to evaluate the toxicity of this organophosphorus acaricide to V. destructor, to provide a technique for the early detection of resistance. A technique similar to that described in Milani and Della Vedova (1996) was used. Adult females of V. destructor were sampled in three areas of Northern Italy: in Udine, in an apiary never treated with organophosphorus acaricides; and in two districts of Emilia, from heavily infested colonies, in an area where chlorfenvinphos had been widely and repeatedly used. Mites were taken from brood cells and assayed within 90 min. Mites from brood of different ages: spinning larvae (l5); white eyed pupae (pw); pupae with dark eyes and white or pale body (pd) were assayed separately. Those from pupae with pigmented bodies and adult bees were not used (Milani, 1995). Shallow capsules, consisting of two glass discs (62 mm dia.) and two stainless steel rings (56 mm inner dia.) were prepared. Their interior, including the rings, was coated with paraffin wax (Merck 7151; melting point 46-48°C), containing chlorfenvinphos (Riedel-de Haën 36551), as described in Milani and Della Vedova (1996). The concentrations used were: 0 (control); 0.02; 0.05; 0.1; 0.2; 0.5; 1; 2; 5 mg/kg. The capsules were used within 12 days of preparation. About 15 females mites were introduced into each capsule. After 4 h they were transferred to a clean glass Petri dish (60 mm dia.) with three worker larvae taken from cells 0-24 h after capping. The mites were observed under a dissecting microscope 4 h (when transferred to the Petri dish), 24 and 48 h after the introduction into the capsule, and classified as mobile, paralysed (knocked down) or dead (as in Milani, 1995). The assays were carried out at 33°C and 70% RH; a series of tests was carried out at 26°C. As a rule, the assay was repeated 2 or 3 times per brood stage and origin of the mites, so that 30-45 mites per concentration were assayed. As in previous work, more mites were assayed at concentrations around the median lethal concentration. The data were analysed using probit transformation, according to Finney (1971). As in previous work, observations at 48 h gave the most reliable results, as paralysed mites were less than 0.5%, while natural mortality did not exceed 2%. The results of the replications were consistent and a good fit using the probit regression was found. The variance of the lethal concentrations was low, resulting in steep

A bioassay to assess the activity of repellent substances on Ixodes ricinus nymphs

A simple laboratory assay was developed to evaluate substances and extracts for their repellent effect on the tick Ixodes ricinus L. The bioassay involved testing the locomotory activity of I. ricinus nymphs in a circular glass arena. The stimulus to be tested was applied onto the arena outside a circle line (4 cm diameter). One field-collected I. ricinus nymph was placed in the centre of the arena and the time spent before entering the treated area was compared with that recorded in suitable control experiments where no stimulus or the solvent alone was used. Apart from a DEETbased product that was used as a positive control, extracts of basil leaves (Ocimum basilicum L.) and leaves of various grasses were tested. The commercial DEET-product proved to be active and so was an acetone extract of basil leaves; the hexane extract of basil leaves and the acetone extract from leaves of mixed grasses had very little or no activity. The bioassay appeared to be suitable to assess the repellent activity of natural products. Basil seems to contain substances that are repellent to I. ricinus nymphs.