A major aim of this project is to develop a cheap, effective and safe method for controlling the parasitic bee mites Varroa destructor and Tropilaelaps clareae on European honey bees (Apis mellifera) in Indonesia and the Philippines. Formic acid (FA) is a cheap and effective means of controlling one of these mites (V. destructor) on A. mellifera in developed countries. It works by producing a vapour which penetrates the inside of bee hives, thereby coming into contact with mites and killing them. However, control relies on using a 65% concentrate, which is dangerous to human health and strongly corrosive to hive equipment, particularly in humid regions. These undesirable side-effects, plus the fact that virtually nothing is known about the effects of FA on T. clareae, has led to a poor uptake of the use of FA by beekeepers in developing countries. Undoubtedly, FA would be more widely used by those beekeepers if its use were made safer (by lowering concentrations) and if it could be shown to effectively control T. clareae.

During the past year, laboratory-based experiments in Indonesia and the Philippines showed that low and high concentrations of FA do not severely effect the longevity of A. mellifera worker bees and that low concentrations of the acid kill both V. destructor and T. clareae, albeit at slightly slower rates than high concentrations. Checks also showed that hive temperatures in tropical regions are not significantly different from those in temperate regions. These combined findings indicate that low concentrations of FA may be effective in controlling V. destructor and T. clareae in hived A. mellifera colonies provided air-flow in the bee hives can be increased (to help disperse the acid vapour) and treatments are prolonged for longer times than treatments using high concentrations of FA. An application method with these properties will be designed and tested over the next 2 years.

Another major aim of this project is to obtain information on the genetic diversity among Asian bees and their parasitic mites together with information on the bee/mite host/parasite relationships. Work carried out during 2 previous ACIAR-funded projects had determined the genetic variation among different population of mites thought to be V. jacobsoni. Those mites were found to be part of a species-complex. Therefore, work during the first year of this project was mostly directed at determining the genetic variation among those mite's natural host, A. cerana. The information generated would then tell whether different genotypes of A. cerana carry different Varroa mites genotypes. Other studies were also directed at determining factors responsible for mite host-specificity. All these studies relied on DNA data obtained from the bees and mites.

The findings showed that there are many genotypes of A. cerana distributed throughout Asia and each carries its own specific genotype of Varroa mite. Only 2 A. cerana genotypes, the Korea and Japan genotypes, carry Varroa genotypes that are harmful to the European honey bee (A. mellifera), the Korea and Japan genotypes of V. destructor respectively. Further, even though these two mite genotypes have the ability to utilise A. mellifera as an alternative host (by being able to reproduce on the brood of that bee), they cannot survive on other genotypes of A. cerana in Asia. These findings have important implications for the worldwide trade in live bees, for quarantine in Australia and for the long term control of Varroa mites in Asia. Studies for the remainder of this project will now focus on the genetic diversity among different populations of the giant Asian honey bee A. dorsata and its parasitic mite, T. clareae.

Other aims of this project are to gain information on pests and diseases of Asian and European honey bees in Indonesia and the Philippines. During the past year, several pathogens, including Nosema apis, Paenibacillus larvae, Ascosphaera apis and the honey bee sacbrood virus were confirmed as present in European honey bee colonies in Indonesia and the Philippines. Serious brood disorders were also observed in Apis cerana colonies in Java and in Apis dorsata colonies in Sumatra. Their cause is not known.

The final aim of this project is to gain information on why it is that only 2 different Varroa mite genotypes have been able to exploit A. mellifera as an alternative host. Bee factors that might be responsible for initiating egg laying in the parasitising mites are being investigated. In the past year blood samples were collected in Java from Apis cerana and Apis mellifera brood and from Varroa mites and these will be analysed during the coming year.

During the second year further progress was made to develop the use of formic acid (FA) as a cheap, effective and safe method for controlling the parasitic bee mites Varroa destructor and Tropilaelaps clareae on European honey bees (Apis mellifera) in Indonesia and the Philippines. FA is used in developed countries to control the varroa mite (Varroa destructor) on A. mellifera, but control relies on using a 65% concentrate, which is dangerous to human health and strongly corrosive to hive equipment, particularly in humid regions. Findings from the first year of this project indicated that it might be possible to use low concentrations of FA to control bee mites in hived A. mellifera colonies provided that air-flow in the bee hives was increased (to improve FA vapour production) and that treatments be applied for longer than treatments that use high concentrations of FA (to allow for longer contact between the FA and mites). Hence, during the past year, studies were directed at developing a hive design for applying low concentrations of FA and planning experiments to test its effectiveness.

The hive design devised for applying low concentrations of FA involved replacing the bottom board of A. mellifera hives with deep wooden trays that could each hold 3 litres of dilute FA solution but maintain a distance of between 5 and 10 cm from the top of the FA solution to the bottom of brood frames. This modification increased airflow within the hives and prolonged exposure of bees to FA. Gauze-covered division boards separated the wooden trays from the brood boxes, thus preventing bees from falling into the FA. The front end of each division board was removed to allow bees to enter their colony. A small raised platform was also built into each tray so that a sticky trap could be attached to the platform to monitor for mite-drop after each treatment. Trays were constructed by each collaborating organization in Indonesia and the Philippines and experiments to test their effectiveness are in progress.

Also during the second year, further information was obtained on the genetic diversity among Asian bees and their parasitic mites, together with information on bee/mite host/parasite relationships. Techniques were developed for determining the levels of genetic variation among populations of Tropilaelaps clareae on their natural bee host, A. dorsata (the giant Asian honey bee) and on their recently acquired bee host, A mellifera. These techniques were also used to examine the genetic variation among A. dorsata.

Samples of T. clareae were collected from A. dorsata and A. mellifera colonies from various parts of Asia along with samples of adult A. dorsata worker bees. All samples were transported to Canberra, where they are currently being identified and typed. To date, several different genotypes of A. dorsata have been found, each carrying its own particular genotype of Tropilaelaps mite. This early finding has important ramifications for beekeeping in Asia and for quarantine in Australia. This work will continue during the third year.

Bee/mite host/parasite relationships were examined to resolve the taxonomy of Varroa mites in the northern Philippine island of Luzon (the Luzon 1 and Luzon 2 genotypes) and for 1 taxonomically unresolved Varroa mite in the southern Philippine island of Mindanao (The Mindanao genotype). The Luzon 1 and 2 genotypes of Varroa were found to be specific parasites of the Luzon 1 and 2 genotypes of A. cerana respectively. Evidence indicated that these two mites couldn't utilise A. mellifera as an alternative host because they lacked the ability to reproduce on that bee. The Mindanao genotype of Varroa was found to be specific to the Mindanao genotype of A. cerana and evidence also indicated that this mite couldn't reproduce on A. mellifera. These findings lend support to the reclassification of these mites as stand-alone species.

Further progress was made in developing the use of low concentrations of formic acid (FA) for controlling the parasitic bee mites Varroa destructor and Tropilaelaps clareae on European honey bees (Apis mellifera) in Indonesia and the Philippines. Initially, delays were encountered in preparing modified bee hives for applying the FA and with getting bee colonies in the hives sufficiently infested with mites. However, experiments are now well under way to test the effectiveness of the modified hives in delivering low concentrations of to control mites. Preliminary results from both Indonesia and the Philippines indicate that low concentrations of FA delivered by the modified hives reduced Varroa and Tropilaelaps mite levels by about 80-90%, compared with 90-95% for high concentrations of FA and 99-100% for commercial chemical miticides. These experiments are continuing.

For the past 2 years studies have also focussed on determining the real and potential pest status of T. clareae. For this, information has been sought on whether there are different genotypes of the mite existing on its primary Asian bee host (A. dorsata), and whether there are different genotypes of A. dorsata. At the same time, further information has also been sought on the identity of T. clareae mites infesting A. mellifera. Specimens assumed to be T. clareae have now been collected from 53 feral A. dorsata colonies, 1 feral A. laboriosa colony (a bee closely related to A. dorsata) and 34 hived A. mellifera colonies from throughout Asia. Specimens of the only other species in the genus (T. koenigerum) have also been collected from A. dorsata colonies at 3 different locations in Asia for comparative studies. DNA sequences are presently being obtained from these mite and bees to determine their identity. Preliminary results have shown that there are different genotypes of Tropilaelaps each hosted by a specific genotype of A. dorsata.

The use of DNA sequences to identify bee and bee mite genotypes has serious drawbacks in that it is time-consuming, expensive and can only be done in well-equipped laboratories. Hence, efforts have been directed at identifying restriction enzyme cutting sites in bee and mite DNA sequences that could be used in most laboratories in conjunction with polymerase chain reaction (PCR) to identify specific genotypes without the need for DNA sequencing. Restriction enzymes have now been identified that can be used in conjunction with PCR to identify the different A. cerana genotypes and their specific Varroa mites. Early results also indicate that it will be possible to identify different A. dorsata and T. clareae genotypes in the same way.

Knowledge of bee pathogens is crucial for bee management. Hence, studies have continued to identifying pests and pathogens of Asian and European honey bees in the Philippines and Indonesia. During the past year high incidences of the serious diseases American foul brood (AFB) and European foulbrood (EFB) were detected in A. mellifera colonies on the Philippines island of Luzon. Other studies in Luzon continued to indicate that the Luzon 1 and 2 genotypes of Varroa are genetically isolated from each other and that each cannot reproduce on A. mellifera, thus supporting the re-classification of these mites as stand-alone species.

Studies have continued on obtaining information on the signals that trigger Varroa mite reproduction on A. mellifera. This work forms part of the current project but is funded by the Australian Rural Industries Research and Development Corporation (RIRDC). During the past year female V. destructor and V. jacobsoni mites were collected from A. mellifera and A cerana broods in Java and used to develop tissue-sectioning techniques that will allow for future studies to develop a conceptual model of the Varroa mite reproduction system.

During the past year experiments were completed on testing formic acid (FA) as a possible inexpensive means of controlling parasitic bee mites on European honeybees (Apis mellifera) in Indonesia and the Philippines. In the lead-up to these experiments laboratory results had indicated that FA was very lethal to bee mites and that it might be possible to use it as an effective control agent at low concentrations (15% or 30%) in entire hived bee colonies, provided that air-flow inside the hives could be improved (to improve FA dispersion) and that the treatments could be prolonged without harming the bees (to allow longer contact between the FA and mites). A system of delivering the FA to bee hives that met these criteria was subsequently developed and used during the past year by a Philippine collaborating organization (DMMMSU) to test the effectiveness of both high and low concentrations of FA in controlling Varroa destructor in hived bee colonies. All concentrations of the acid tested were found to be very effective in ridding colonies of V. destructor, both from the bodies of adult bees and from the bees' capped brood cells. The level of control achieved was also comparable with that obtained using Apistan, a relatively expensive chemical acaricide used for controlling bee mites in Western countries. Nevertheless, only relatively low concentrations of the acid, not high concentrations, were found suitable for use in the new delivery system, as the use of higher concentrations led to some harmful side effects to the bee colonies. Similar results to these were also obtained by one of the Indonesian collaborating organizations (Perum Perhutani) using the same delivery system. Hence, beekeepers in the Philippines and Indonesia are now being encouraged and educated to use FA as part of their mite management practices. The recommended treatment for a single hive with mites is 2 applications, each of 3 litres of 15% FA (in water) delivered to hives via the new delivery system, with the second application given 2 weeks after the first application.

During the past year studies were also completed on the genetic variation among different populations of the small Asian bee Apis cerana and their parasitic Varroa mites, and among different populations of the giant Asian bee Apis dorsata and their parasitic Tropilaelaps mites. While conducting these studies, information was also gathered on the identity of Varroa and Tropilaelaps mites that are harmful to European honeybees. The studies involved analysing DNA sequences obtained from large numbers of bee and mite isolates. In brief, the results showed that the species A. cerana consists of many different genotypes each of which hosts a particular genotype of Varroa mite. Only 2 A. cerana genotypes host Varroa mites that are harmful to the European honeybee. The species A. dorsata also consists of many genotypes each of which hosts a particular genotype of Tropilaelaps mite. Most of these Tropilaelaps genotypes are harmful to the European honeybee. As part of this work, methods were devised that now enable each bee and mite genotype to be easily and cheaply identified.

Most details of the A. cerana/Varroa genetic variation studies were given in previous Annual Reports. However, some significant details of the A. dorsata/Tropilaelaps genetic variation studies have only come to light during the past year. Each A. dorsata genotype detected fell into one of 2 broad genetic groups of bees that corresponded to the preciously recognized 'yellow bees' of Indonesia/Mainland Asia and the 'black-and-white bees' of Philippines/Sulawesi. The black-and-white bees were found to host different genotypes of Tropilaelaps clareae, while the yellow bees hosted Tropilaelaps genotypes that appear different from any previously described species. These yellow bees also carried different genotypes of T. koenigerum, some of which were detected in countries from which the mite had not been previously reported. All genotypes of T. clareae and of the seemingly new Tropilaelaps species from the yellow A. dorsata bees were found to be harmful to European honeybees, while T. koenigerum appears harmless to that bee.

While carrying out the genetic variation studies on A. dorsata and their Tropilaelaps mites, a previously undescribed species of Tropilaelaps was also isolated from the giant Asian rock bee, A. laboriosa. This mite appears to be harmless to European honeybees.