Agustin ARIAS MARTINEZ(1), Francisco ARRANZ DEL REY(2), Dra. Pilar DE LA RUA TARIN(3), Iratxe PEREZ COBO(4), Dr. Pedro Pablo RODRIGUEZ RODRIGUEZ(5)
March-June 2001

1. Aula Apicola Coordinator, Azuqueca de Henares, Guadalajara, Spain
2. Public Health Veterinary Officer, Public Health Board, Castilla-La Mancha, Spain
3. Animal Biology Department, School of Veterinary Medicine, Murcia, Spain
4. Research Project, Veterinaran in Charge
5. Coordinator and Technical Adisor, Food Grade Mineral Oil

SUMMARY: The purpose of this study was to study the acaricide effect of food grade mineral oil, 0.86 density, in the form of emulsion and 15 microns vapor. The research was performed in an apiary of the beekeeping school of the municipal government of Azuqueca de Henares, (Guadalajara, Spain). The test was performed with 10 colonies hived in Langstroth type hives equipped with 4mm hardware cloth bottom screens. Test results demonstrate that food grade mineral oil is an efficient, economic and non-contaminating acaricide, especially when integrated with other control methods. High resolution liquid chromatography laboratory analysis (HCPL) showed that food grade mineral oil does not alter the quality of the honey. DNA tests of mites collected during the study identified Varroa destructor as the primary parasitic mite in the apiary. Similarly, DNA tests of the honey bees determined that three of the colonies belonged to African lineage while the rest belonged to Western European lineage.

Key Words: Varroa destructor, Apis mellifera, food grade mineral oil (FGMO)


INTRODUCTION

Varroa infection of honey bees (Apis mellifera) represent a world wide menace to beekeeping (Ellis, 2000) having destroyed the majority of feral colonies and a high percentage of domestic colonies (de Jong, 1977; Sammataro, 1997). The pathogenicty of this plague is very serious, having been demonstrated that untreated colonies die within one or two years (Rodriguez, 1997; Anderson, 2000; Ellis, 2001). Infected colonies die due to pathology caused by the mites, pillage and secondary illnesses. It has been suggested that honey bees are susceptible to bacterial and viral diseases when their tegument is damaged by mites and specifically, that Varroa jacobsoni contributes to the introduction of these diseases acting as a vector for mycotic diseases, (Brodsgaard et al, 2000) and their harmful effects diminish the performance of adult honey bees (Salvachua et al, 1999).

Scientists around the world have dedicated great efforts attempting to find efficient acaricides but harmless to honey bees and will not contaminate honey with toxic residues. Their studies have shown that the number of substances that can be utilized against the mites is small due to multiple reasons:

resistance to acaricide products (Barbero et al., 1997; Baxter et al, 1998; Braunstein, 1998a; Elzen et al, 2000; Ellis, 2000; Faucon et al, 1995; Lodesani et al, 1995; Milani, 1995, 2001; Spreafico et al, 2001). high cost of treatment (Callejo and Iniesta, 2000; De Las Rosas, 2001). toxic residues that contaminate the honey (Anderson, 1994). treatments are limited to certain seasons of the year according to manufacturers recommendations for the acaricides.

Due to difficulties inherent in chemical pesticides, it is evident that there is great need to find integrated alternate methods (Ellis, 2001; Spreafico et al, 2001; Kamran, 2000) to treat these mites. Hence a great number of alternate methods have evolved with a varied degree of acceptance due to their complexity, toxicity, ambient temperature dependency, high cost and low degree of effectiveness.

The following are some of the most commonly found:

genetic selection according to hygienic behavior of the colonies (Buchler, 1994). mite collection in drone cells. metal screen bottom boards (Pettis and Shimanuki, 1999; Ellis J. D.; 2001). aromatic oils (Imdorf et al, 1999). food grade mineral oil (Aguirre, 1999; Rodriguez, 1997a,b, 2000, 2001; Pajuelo, 2001; De las Nieves, 2001; Zola, 2000).

This study is the result of a continued effort in search of more economic and easier methods of application of food grade mineral oil combined with integrated beekeeping practices that have demonstrated to be effective in the control of varroa mites. Among these practices are the utilization of screened bottom boards, selection for hygenic behavior and DNA analysis of the mites, a very important factor since the existence of genetically different varroa mite populations with distinct differences in their virulence (Anderson and Fuchs, 1998; De Guzman et al, 1999).

Food grade mineral oil, 0.86 grams/l is a petroleum derivative that is odorless, colorless, and does not contaminate and is especially utilized for operations requiring a mineral oil exempt from toxicity. It is widely used by industrial nations in the food industry and medicine as a vehicle and as a lubricant. Utilization of food grade mineral oil as an acaricide is considered highly beneficial. Because of its efficacy, it can be utilized at times when there are large numbers of mites and synthetic acaricides can not be used.

The acaricide mechanism of food grade mineral oil is based on various factors:

a) Morphologic and biologic characteristics of the mites.

The body of the Varroa mite is flat offering a large surface/volume relationship that makes it vulnerable to treatment with oils (factor also utilized by Italian investigators (Bee-L archives; Rodriguez, 2001). Varroa mites as well as the honey bees breath through spiracles through which gaseous exchange occur by means of adjustments of their respiratory system (Pugh et al, 1992). Mineral oil blocks the spiracles of the mites causing their death by asphyxia. While honey bees also breath the oil, the size of their spiracles is much larger than that of the mites, thus it is possible to utilize mineral oil as an acaricide without harming the honey bees. Also the body of the mites is covered by pores which the mites utilize to take in moisture for their hydration. These pores are also blocked by mineral oil thus interfering with another biologic process of the mites. Varroa mites cling to the body of the bees while being carried about. During the application of mineral oil, in vapor or emulsion form, a fine film of oil is deposited on the bodies of the bees which interferes with the ability of the mites to cling to the bees (Lujan, 2000; Kamran, 2001), causing the mites to fall off. Sanitary behavior of the honey bees: honey bees begin to remove the emulsion coated cords promptly and in the process their legs become coated with mineral oil that is later transferred to their bodies when they comb themselves.

b) Utilization of screened bottom boards.

Screened bottom boards prevent mites that have fallen off from re-attaching themselves to the bees due to the effect of the mineral oil.

MATERIALS AND METHODS

1. Establishment of the experiment

The research project took place on 13 March to 16 July 2001 at the Azuqueca de Henares school apiary, sponsored by the municipal government. The apiary is located adjacent to the Villanueva de la Torre road, among an olive tree farm with scant flowering vegetation. The hives were Langstroth model and had not been treated since the past autumn at which time they were treated with food grade mineral oil.

Since it is well known that untreated colonies die during the test period or soon thereafter, all colonies were treated during this test because they were on a loan basis and zero financing.

2. Treatment with food grade mineral oil.

The treatment consisted in applying food grade mineral oil vaporized (15 micron size particles) with a Burgess Propane Bug Killer and cotton cords coated with emulsified food grade mineral oil mixed with bees wax and honey.

Dr. Pedro Rodriguez and Veterinarian Iratxe Perez Cobo apply FGMO with Burgess fogger at apiary just outside the city of Azuqueca de Henares, Spain.

2.1. Preparation of the emulsion

The emulsion was prepared according to the formula developed by Dr. Pedro Pablo Rodriguez (developer of the use of food grade mineral oil). Water has been omitted (as described in the original formula) to prevent fermentation of the honey. The ingredients for the emulsion are as follows: 500 mls food grade mineral oil, 225 grams bees wax, 300 grams honey, sixty 500 mm long by 8mm diameter cotton cords. The procedure for making the emulsion is as follows: heat the food grade mineral oil in a metal container, add bees wax and stir to dissolve the wax and prevent it from burning. Remove the container from the heat source and add the honey and cords. Stir with a wooden spoon to allow the cords to soak well. Allow the emulsion to cool.

2.2. Treatment with the emulsion and vaporizer

Two pieces of the emulsion soaked cords were placed on top of the frames, and a stream of vapor (about two seconds per hive) was blown through the hive entrance every 15 days during the duration of the experiment.

Professor Agustin Arias Martinez demonstrates a Varroa free brood with FGMO treatment.

2.3. Application of fine paste boards coated with solid Vaseline on bottom boards

Utilization of hardware cloth screens allowed mites to fall through for subsequent counting every 7 days without interfering with normal hive activities.

Metal screened bottom board with removable tray for mite collection. Tray is lined with FGMO coated paper.

3. Chemical analysis of the honey.

Samples were collected at the end of the experiment and sent for analysis. Tests revealed that the use of food grade mineral oil does not alter the quality of honey.

4. DNA characterization of mites and honey bees.

Honey bee and mite samples were collected from the 10 test hives, preserved in ethane alcohol and sent to the University of Murcia laboratory for testing.

Molecular characterization was based on DNA mitochondrial sequence, according to which are classified the four evolutionary lineages of honey bees present in Spain, African A in the southern peninsula and Western European M in the north. Similarly, the presence or absence of mitochondrial sequences are utilized to determine the species and type of Varroa (destructor or jacobsoni) present in the colonies.

5. Mean temperature.

Temperature data was recorded by the Azuqueca meteorology station located one kilometer away from the apiary. See graph No. 1.


RESULTS AND DISCUSSION

1. Development and condition of the hives

Special emphasis was made on the following aspects of the hives: number of frames with brood, number of frames with bees, honey stores and other observations made on March 13, April 23, and June 25. See graph below.
Key: MB: very good; B: good; B-R: good to regular; R: regular; M: bad; MM: very bad; +: added

TABLE 1. Development of the hives during the experiment

Honey yield by individual hive is reflected on table 2 below. Hive No. 7 did not yield honey because it was utilized for bee package production (4) of which each had a full super by 11 September 2001.

TABLE 2. Honey yield per hive.

Hive No. Net yield kg 1st (16/7/01) Net yield kg 2nd (11/9/01) Net yield kg 3rd (6/11/01) TOTAL 2 13 0 5 18 5 27 16 21 64 6 28 0 14 42 7 0 0 10 10 11 0 10 0 10 15 0 15 0 15 19 27 32 5 64 20 18 30 0 48 26 0 15 12 27 29 0 25 0 25 Total kg 113 143 67 323

2. Degree of infection of the hives. Evaluation of the treatment with food grade mineral oil.

Mite counts were performed weekly to evaluate the efficacy of food grade mineral oil as an acaricide. See table 4.

Evaluation of the degree of infection was obtained by means of counting the number of mites in 100 sealed brood cells (50 female cells and 50 drone cells). Mite counts were made at the start, middle and at the end of the study.

The following formula was utilized to estimate efficiency of the treatment. (see graphs below.)


initial % of varroa - final % of varroa
_____________________________________ X 100

initial % of varroa

TABLE 3. Hive health and efficiency of the treatment

Hive No. 13/March/01 20/May/01 16/July/01 Efficiency (%) 2 5 6 7 11 15 19 20 26 29 11'5 0'55 0 0 1'05 1 2'14 0 No cria 1 44 2 0 0´8 0 0 0 0 0 5 5 0 0 0 0 0 0 0 0 0 56'52 >99 >99 >99 >99 >99 >99 >99 >99 >99

At the start of the study, hive No. 2 showed an infection level of 11 %, while the rest of the hives showed low levels of infection. After 68 days into the study, hive No. 2 showed an increase of infection (44%), hives No. 5, 7, 29 showed a slight increase, while in the rest of the hives, infection showed to be below 1%, and hive No. 2 showed a level of infection of 5%.

BAR GRAPHS: INDIVIDUAL HIVE MITE COUNT

3. Honey Analysis

Honey showed the following analysis: Humidity 14.1 % H.M.F concentration 0.4 mg/Kg. Pollinic analysis showed origin of honey to be of a local leguninous plant (retama).

4. Molecular characterization

Honey Bees

Molecular analysis of samples of bees from the 10 colonies studied was performed according to previously described protocol (De la Rua et al., 2000). Analysis of samples from the Azuqueca de Henares colonies indicate that three of the colonies belong to the African lineage while the rest belong to the Western European lineage. See table 5 below.

TABLE 5. Lineage and haplotypes of the bees in the Azuqueca de Henares apiary.

Hive No. Lineage Haplotype 2 African A2 5 African A3 6 African A2 7 European (M) M4 11 European (M) M4 15 Europeam (M) M4 19 European (M) M4 20 European (M) M4 26 European (M) M4 29 European (M) M4

Varroa

The molecular analysis of the mites in the Azuqueca de Henares apiary was performed according to the protocol by Anderson & Fuchs (1998). See Table 6 below. The analysis reveals that all hives were infected with the destructor species, predominant in Europe, except hive No. 20 that was infected by the jacobsoni species.

TABLE 6. Distribution of varroa by species and haplotype in the studied colonies.

Hive No. Lineage Haplotype 2 destructor Korean 5 destructor Korean 6 destructor Korean 7 destructor Korean 11 destructor Korean 15 destructor Korean 19 destructor Korean 20 jacobsoni Java 26 destructor Korean 29 destructor Korean

5. Mean Temperature

Study of temperature recordings seem to indicate a relationship between the ups and downs in the mean temperature in the area and that of varroa infection in the hives.




6. DISCUSSION

The use of food grade mineral oil has shown to be highly efficient for the control of varroa infection and economic, non-contaminating and gentle to the environment.

Maximum efficiency of food grade mineral oil is obtained when used during the entire biologic cycle of the hive contrary to that of synthetic acaricides that can be utilized only during restricted periods of time. In addition, since food grade mineral oil can be utilized during the entire biologic cycle of the colony, it contributes to maintain low levels of varroa infection. It is strongly recommended as a prophylactic to prevent reinfestations. Honey yield of the colonies of this study can be considered as excellent considering the area to have scant flowering plants.

Next year's study with food grade mineral oil will include other forms of application without sacrificing its efficiency.

Dr. Pedro Rodriguez and Veterinarian Iratxe Perez Cobo demonstrate a hive with 1-1/2 supers full by mid July with FGMO treatment.

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