brood cells in each colony were estimated according to the Liebefeld method (modified) before and after the treatment periods. No significant differences were recorded between treated and control groups in each trial. No external reaction to the treatment was detected. Small groups of bees were fed 60% sugar syrup, 60% sugar syrup and ethanol (19%), ethanol (19%), water, Apedin and a 1:2 solution of Apedin. The 60% syrup uptake was 81.5 mg/bee over a period of 28 h. The presence of ethanol seemed to considerably decrease the syrup palatability (the uptake was only 19.4 mg). 2.3 mg, 2.9 mg, 2.7 mg and 2.7 mg of water, ethanol, pure and diluted Apedin were removed, respectively and 84.6%, 80.0%, 71.4%, 74.5% of the bees were found dead or showed clear symptoms of starvation. Furthermore, respectively 0% and 37.5% of the bees in the groups fed plain syrup and syrup to which ethanol had added were dead or nonreactively lying on the bottom of the cages because of the insufficient feeding. The possible side effects of lactose as an Apedin Vapor component are discussed.

Keywords

Acaricide Apis mellifera Control Efficacy Varroa jacobsoni.

Introduction

Considerable research has been devoted to the control of the mite Varroa destructor Anderson & Trueman for many years. Surveys have been conducted for decades in several countries by researchers, often collaborating with beekeeper associations. The efforts made several control concepts being set-up but at the moment the parasite eradication still remains an unreachable objective. Remarkable progress has been achieved recently using natural active ingredients belonging to very different chemical groups. Problems with efficacy and practical application of these chemicals have been solved, making possible environment- and product-safe techniques being adopted by several beekeepers. Some

A. NANETTI E ALTRI

VETERINARIA ITALIANA
volte, con un intervallo di alcuni minuti, sulle api presenti al di sopra dei favi al momento della rimozione del coprifavo. Le varroe cadute sono state recuperate attraverso fogli adesivi collocati nel fondo diagnostico e rinnovati generalmente una o due volte la settimana. Il trattamento di controllo, necessario per recuperare le varroe sopravvissute stato eseguito con acido ossalico.

ecological methods to control the mites have been studied more than others, but the lack of adequate and careful research did not prevent the commercial successes of some of the lasts. Scanty and sometimes conflicting information is available about Apedin Vapor, produced by Istituto Fitofarmaceutico Euganeo Srl, Rubano, Padua, (Figure 1) which seems to place this easily obtainable product amongst group of formulations that have not been submitted to a sufficient body of research. The composition of the product is given on label (water, ethanol, lactose, hydro-alcoholic extracts of Echinacea angustifolia, Thuya occidentalis, Spiraea ulmaria and Oxalis acetosella) but, apart from the alcohol (19% vol.), concentrations are not indicated. Moreover, information about the use is not given on the packages (200-ml flasks) but only on leaflets separately issued by the manufacturer. The above directions classify Apedin Vapor as a feed, but pinpoint its efficacy against Varroa destructor, the causative agent of varroosis. In addition, they state that the product strengthens, helping in the varroa treatment and that the maximum efficacy is reached two weeks after the second treatment and lasts for one month at least. The product description continues: it is not toxic; it does not destabilise the swarm; it does not affect the queen fertility; it does not leave residues nor jeopardises the honey quality; it can be used in presence of super boxes and brood. The published information exclusively covers the use of Apedin Vapor in the control of varroosis (often experiences relevant to the practical use rather than real tests are reported), while no references about the nutritional qualities of the product are available. This paper aims to acquaint readers with work on this product, that the Authors have considered as both an acaricide and feed, as stated by the notice provided by the manufacturer.

Materials and methods

Field tests
Two different treatment concepts
Tabella 1: Trattamento prolungato: consistenza iniziale e finale delle colonie (differenze fra gruppi non significative). Table 1: Size of colonies before and after long-term treatment (no significant difference was recorded between groups). Controllo del 21 maggio 2002 Control on 21 May 2002 Api Celle di covata Bees Brood cells 15,433 14,650 33,040 37,760 Controllo del 15 ottobre 2002 Control on 15 October 2002 Api Celle di covata Colonie senza covata Bees Brood cells Colonies without broods* 15,386 15,2,215 63.6% 53.8%

Gruppo/Group TRT/Treated CTR/Control * Test
Tabella 2: Trattamento di breve termine: consistenza iniziale e finale delle colonie (differenze fra gruppi non significative). Table 2: Size of colonies before and after short-term treatment (no significant difference was recorded between groups). Controllo del 18 maggio 2002 Control on 18 May 2002 Gruppo/Group Api/Bees TRT1/Treated 1 TRT2/Control 1 19,063 19,833 Facciate di favo con covata Comb sides with brood 12.3 12.7 Controllo del 25 agosto 2002 Control on 25 August 2002 Api/Bees 20,750 20,625 Facciate di favo con covata Comb sides with brood 11.5% 11.2%
Tabella 3: Trattamento prolungato: cadute corrispondenti ad ogni intervento con Apedin Vapor (differenze fra gruppi non significative). Table 3: Long-term treatment: mite fall after each treatment with Apedin Vapor (no signifcant difference was recorded between groups). Somministrazioni TRT/Treated group Media/Mean No. 2.0 1.3 2.4 8.5 11.1 29.9 30.CTR/Control group Media/Mean No. 1.9 1.4 1.9 8.7 14.5 83.8 45.13 13
24 May June July July August September October 2002
using Apedin Vapor were taken into consideration: in one case the most part of the active season was covered by the administrations, but only a twomonth period in the second case. The product belonged to the batch number C02/APV, with an expiry date of March 2004. Efficacy and tolerability of treatments were evaluated by comparing treated colonies with control groups, belonging to the same apiary but receiving water alone. The colonies were of similar strength and, in any case, of normal development if the period of the year is taken as a reference. Each apiary was randomly split into two groups. During each trial period, good rearing practices were followed to keep the colonies. Apedin Vapor solution was prepared by diluting the product with water in ratio 1:2 (v/v). After a vigorous shaking, that is recommended by the manufacturer himself (which refers to the procedure as the dynamisation), it was administered by spraying 25-ml twice, at a few minute interval, on the bees located on the comb top bars. Fallen mites were collected using sticky sheets placed in the bottom boards, that were replaced with clean ones once or twice a week. The control treatments, needed to collect the surviving mites, were performed using oxalic acid.

Long-term treatments

parametrici (U test di MannWhitney; test 2), assumendo la soglia di significativit =0,05 Risultati

Condizioni delle colonie

I dati riportati nelle Tabelle 1 e 2 mostrano una notevole uniformit fra il gruppo trattato e quello di controllo di ciascuna prova per quanto attiene alla forza iniziale delle colonie. A determinare tale favorevole condizione di partenza ha certamente contribuito il metodo utilizzato per formare i gruppi, che si basava sulla casualit. Le colonie della prova di

Mortalit delle varroe

Nella prova di trattamento prolungato con Apedin Vapor sono state registrate modeste
cadute di acari (Tabella 3, Figura 2) cui, evidentemente, hanno corrisposto livelli dinfestazione moderati, grazie ai quali si potuto protrarre il periodo di trattamento fino al termine della stagione attiva senza che le colonie venissero sopraffatte dalle popolazioni dei parassiti. Nelle colonie dei due gruppi non vi sono state differenze significative nel numero di acari di volta in volta recuperati nei fondi diagnostici, nella mortalit totale rilevata nel periodo di trattamento e nel numero di acari caduti con il successivo trattamento di controllo (Tabella 4). In corrispondenza delle diverse somministrazioni non si sono manifestati incrementi di mortalit. L'efficacia complessiva dei sette interventi eseguiti con Apedin Vapor stata del 19,8%. 50
This test was performed in an apiary in the province of Turin divided into two groups (treated 1 and control 1) of 12 colonies each. The first application of Apedin Vapor was made on 18 May 2002 and was followed by two additional applications, 21 and 42 days later. For the control treatment, a solution of oxalic acid (4.4% containing approximately 30% sugar) was administered twice at three-week intervals, according to the dose of 5 ml per comb occupied by the bees. To increase the efficacy of this application, further egg lying was prevented by caging the queens during the first 12 days of the control period. The method for evaluating the colony strength using the sixth part of a DB frame as a unit of measure was used even in this case before (18 May) and after (25 August) the treatment period.

Feeding test

Appropriate feeders allowed to feed groups of approximately 50 to 70 bees confined in lab cages respectively with the following solutions: pure or diluted (1:2, with water) Apedin Vapor, 60% sugar syrup (w/v) with or without ethanol (19% vol.), water, ethanol (19%). The solutions were dyed with methylene blue (2%) in order to trace them in the bee intestines. The amounts taken from each feeder was determined after 28 h as a difference in weight. At the same time, the bees that had died or that were unable to regularly walk were counted, assuming that these bad conditions of the bees were due to insufficient feeding. The statistical analysis of the results was made using non-parametric methods (Mann-Whitney U test; 2 test), taking a significance threshold = 0.05. Results

colours of the intestines.

Discussion

The tests have demonstrated that honeybee colonies have good tolerance of Apedin Vapor . The treatments did not cause any visible behavioural abnormality or significant alteration in the quantity of adult bees or brood in comparison to the controls. The exclusion of some colonies from the experiment was not related to problems with the product tolerability. If the apiary was intended for production instead of the experimental use, the beekeeper could have recovered those colonies without difficulty. The administration of treatments was relatively simple and rapid, but splitting each dose into two aliquots made the intervention times lengthier than the ones needed for other treatments, e.g. the trickling method for oxalic acid administration (8). It is not clear the reason why the manufacturer suggests a beforehand dynamisation of the solution, but it is obvious that such an additional requirement contributes to make the treatments more complicated. In the feeding tests, only very small quantities of Apedin Vapor (pure and diluted) and of substances showing neither attractiveness nor feeding value, were taken by the bees. In these groups, the mortality of bees due to insufficient feeding occurred according to similar times and modes. The sugar syrup, which was assimilated very actively (approximately 80 mg per bee), underwent a noticeable decrease in attractiveness to the bees when ethanol was added at the same alcohol concentration of Apedin Vapor. The manufacturer states that lactose is present in this formula, but no mention is made of the concentration. However, the presence of that sugar may be inappropriate in a product intended for the honeybee feeding. In fact, small quantities of lactose are naturally present in the honey (0.00620.0383%) (12), but they are of no nutritional value for bees, that do not avail of the enzyme needed for its digestion (lactase) (3). Additionally, artificial feeding tests have pointed out a decreasing life span of the bees supplied with feed containing
Non avendo riscontrato i presupposti per utilizzare Apedin Vapor nella lotta alla varroasi o nellalimentazione delle api, la sperimentazione non proseguita con ulteriori indagini sui residui dei trattamenti nel miele e negli altri prodotti dellalveare. Bibliografia/References

1. Accorti M. (1985). Valutazione numerica degli adulti di Apis mellifera L.: variazioni e modifiche al metodo dei sesti. Apicoltura, 1, 63-73. 2. Baek D.H., Goh Y.G. (1999). [Food value of pollen from China, domestic pollen substitute and lactose on the basis of palatability and life span in honey bees]. Korean J. of Apiculture 14 (1), 5-12. 3. Dietz A. (1975). Nutrition of the adult honey bee. In The hive and the honey bee. Dadant & Sons, Hamilton, Illinois, 740 pp. 4. Floris I., Satta A. (1999). Lotta alla varroa. Aspetti sperimentali e pratici della valutazione dellefficacia di trattamenti acaricidi in presenza di covata opercolata. Apitalia, 26 (2-3), 35-44. 5. Gerig L. (1983). Lehrgang zur Erfassung der Volkstaerke. Schweiz. Bienen-Zeitung, 106 (4), 1099. 6. Gianinetto M. (2003). A proposito di Apedin Vapor e per fare un po di chiarezza. Apitalia, 30 (6), 25-28. 7. Grassone U., Allais L. (2002). Prove di efficacia del trattamento tampone. Lapis, 10 (8), 4-8. 8. Nanetti A, Stradi G. (1997). Varroasi trattamento chimico con acido ossalico in sciroppo zuccherino. Lape nostra amica, 19 (5), 6-14. 9. Nanetti A., Arrigoni E., Antonietti S., Bendinelli A., Castiglioni G. (1999) Stecche di legno impregnate con acido ossalico. Lapis VII, (7), 4-7. 10. Niero C. (2001). Varroa: con Apedin-Vapor una risposta tutta naturale. Apitalia, 28 (7-8), 35-37. 11. Persano Oddo L., Marinelli E. (2002). Impiego di prodotti omeopatici nella lotta contro Varroa destructor Anderson & Trueman. In Atti Congresso Internazionale di Entomologia, Catania, 10-15 giugno 2002, 1319-1324. 12. Val A., Huidobro J.F., Snchez M.P., Muniategui S., FernndezMuio M.A., Sancho M.T. (1998). Enzymatic determination of galactose and lactose in honey. J. Agri. Food Chem., 46, 1381- 1385.
increasing concentrations of lactose (2). The inadequacy of Apedin Vapor in the direct nutrition of bees is not only due to the lack in attractiveness and in the presence of potentially negative substances. Above all, it is due to the absence of nutritional substances, such as sugars and proteins. On the other side, no suggestions are provided either by the producer or by other sources supporting the use of the product in combination with syrups, sugar candies, protein integrators or other honeybee feeds. Obviously, the needs of acaricides that are increasingly efficient, versatile and respectful of bee products have focused the greater expectations of beekeepers on the potential use of Apedin Vapor in the varroa control. Actually, the mode of administration proposed by the manufacturer is similar to the indications for other acaricides, but deeply differs from the method usually adopted in the feeding of honeybee colonies, which are supplied with much higher amounts by the means of appropriate feeders. The efficacy of Apedin Vapor was very low and insufficient to control the mite populations, which considerably increase during the active season. In both trials the mite fall gradually increased during the time, testifying that the mite populations were growing, despite of the treatments. As a result and notwithstanding seven administrations and more than four months of treatment, the number of mites still infesting the colonies did not significantly differ in treated and control colonies. This conflicts with the statement of a long lasting acaricidal effect (10). A similar situation occurred in past experiments, showing a substantial inefficacy of wooden strips soaked with an oxalic acid solution. In this case, no peaks of mite mortality were seen after each administration, and the number of mites increased irrespective of the treatment (9). The present investigations also did not show the peak of efficacy that is expected to happen during the second week after the second administration (6). The conclusions of some beekeepers that have published their trials on beekeeping journals seem

unsupportedly optimistic. Often they lack of appropriate control groups, which are badly needed to assess the natural mite mortality and keep from the risk of errors in the efficacy evaluation (Table VI). The present results agree with the findings of other Authors. For instance, a substantial inefficacy was recorded in a two-year experiment in Central Italy, where two summer Apedin Vapor administrations per year at three-week interval (11) were made. Other trials on the summer treatment had been made previously in North Italy (two administrations at three-week interval). They yielded an efficacy of 25 and 21%, that was only slightly above the mortality recorded in the control groups (22 and 18%, respectively) (7).

Conclusions

Several indications coming from the manufacturer of Apedin Vapor could not be confirmed by the present experiments. The lack of attractiveness for the honeybees, the chemical composition and the lack of information about a possible use in combination with other products make Apedin Vapor unsuitable in the colony feeding. Long or short lasting treatments did not show an Apedin Vapor efficacy sufficient to suggest its use against very dangerous parasites, like varroa mites are. Other natural active ingredients may be administered in a similar way (e.g.: oxalic acid trickling), with the expectation of a considerably higher acaricide effect and a good tolerability. However it must be pointed out that formulations allowing a gradual release of the active ingredient are generally more suitable to the brood rearing period, when many mites are prevented from the contact with acaricidal solutions, that are usually removed quickly by the bees themselves. On the contrary, Apedin Vapor did not show side effects on the treated colonies. Since the results do not support the use of Apedin Vapor in the field, the study was not extended further to examine its residues in the honey and in the other bee products.