Research Article

Efficacy of Essential Oils and Formic Acid in the Management of Tropilaelaps clareae in Apis mellifera Linnaeus Colonies in Relation to Honey Production

Noor Islam1, Muhammad Amjad2, Ehsan ul Haq3, Elizabeth Stephen1 and Falak Naz4

1Honeybee Research Institute, National Agriculture Research Centre (NARC), Islamabad; 2Plant Sciences Division, Pakistan Agriculture Research Council, Islamabad; 3Insect Pest Management Programme, NARC, Islamabad; 4Coordination, Pakistan Agriculture Research Council, Islamabad, Pakistan.

Received | December 23, 2016; Accepted | April 04, 2017; Published | June 29, 2017

*Correspondence | Noor Islam, Honeybee Research Institute, National Agricultural Research Centre, Islamabad, Pakistan; Email: khattakni@gmail.com

Citation | Islam, N., M. Amjad, E. Haq, E. Stephen and F. Naz. 2017. Efficacy of essential oils and formic acid in the management of Tropilaelaps clareae in Apis mellifera Linnaeus colonies in relation to honey production. Pakistan Journal of Agricultural Research, 30(2): 194-201.

DOI | http://dx.doi.org/10.17582/journal.pjar/2017/30.2.194.201

Keywords | Apis mellifera, Essential oils, Formic acid, Mite mortality, Tropilaelaps clareae

Introduction

 

Different pests and predators attack the honey bee colonies as they live together in a tightly knit social group. Among pests, honey bee mites Varroa destructor and Tropilaelaps clareae are considered as the major limiting factor in world apiculture (Hosamani et al., 2006; Zhou et al., 2007). These mites can be categorized as parasitic, predatory and phoretic. Amongst these, parasitic mites are quite harmful to honey bees causing brood mortality; reduced colony strength and increase in the number of deformed bees (Kapil et al., 1985). Tropilaelaps can cause 50-100% loss of bee colonies (Hosamani et al., 2006). Kiprasert (1984) reported that T. clareae infests as much as 90% of the brood of A. mellifera colonies but smaller brood infestation levels of 3-6% have been consistently reported from A. dorsata colonies (Underwood, 1986).

 

T. clareae was observed in 1981 for the first time in Pakistan and spread to European honey bee, A. mellifera in 1991 throughout the country by commercial migratory beekeepers as they shift their bee colonies in different areas, where good honey flora is available for the production of different types of honey. At present T. clareae is causing major economic losses and damage to beekeeping industry in Pakistan with A. mellifera. The highest infestation of T. clareae occurred in February, March and April with a decline from May to August (Camphor et al., 2005). Burgett and Akratanakul (1985) predicted that in the near future T. clareae would play a major role in the destruction of commercial honey bee colonies far greater than that caused by Acarapis woodi and Varroa mites. Poor management of bee colonies, hive microclimate and weak colony has increased the incidence of T. clareae in bee colonies (Mahavir and Gupta, 1999). T. clareae caused 30-70% colony loss of A. mellifera with reduced honey production (Woo and Lee, 1997).

 

Beekeepers are using low costs/quality synthetic acaricides such as sulphur, flumethrin, fluvalinate strips, coumaphos, amitraz, tobacco, fluvalinate injections and formic acid in order to control T. clareae and Varroa destructor mites in Pakistan. Camphor et al. (2005) observed that T. clareae mite has developed resistance to sulphur, tobacco and naphthalene because in spite of regularly treating A. mellifera colonies with these chemicals many colonies were still found infested. Honey bee mites are highly resistant to chemical treatments, therefore, even if necessary, the repeated applications of the same chemical may be avoided. Elzen et al. (2000) indicated that these mites are not only resistant to fluvalinate, but also to the commonly used amitraz. Moreover, the wide spread use of synthetic acaricides has lead to the accumulation of residues in bees wax, propolis and honey (Martel and Zeggane, 2002; Mullin et al., 2010). These concerns have provided considerable incentive to develop new strategies that minimize the potential for acaricides resistance and the accumulation of such residues. The use of pyrethroid and fluvalinate has also made the honey bees more susceptible to pesticides sprayed on crops and block the detoxification mechanisms for some fungicides in bees (Pilling et al., 1995). Another study demonstrated that the combination of some pesticides can increase toxicity in honey bees 1,000 folds (Iwasa et al., 2004). Due to the severity of the residue problems national and international food regulations were established for the consumption and trade of honey bee products. These concerns have led to beekeepers looking for alternative acaricides, such as essential oils, to control T. clareae in light of the large colony decline. Essential oils are natural, safer alternatives to chemical control measures. Essential oils are non toxic to non target species and have less resistance problems as to other pesticides (Imdorf et al., 1999).

 

It is well known that many essential oils and their compounds exhibit acaricidal activity (Lee et al., 1997). Different components of essential oils were tested for their activity against A. woodi. Recently, research has shown that several essential oils and individual compounds of essential oils have high acaricidal activity against V. jacobsoni. Calderone et al. (1997) suggested that they may be useful in maintaining mite infestation rates below economic injury level. More than 150 essential oils have been used for the control of insect pests and mite; few remain viable at controlling mites without affecting honey bees (Imdorf et al., 1999). Essential oils kill the mites when they come in contact within a few minutes and they also impair their reproduction. If the oil is strong enough, the females are unable to lay eggs. If the oils are used at lower concentration, eggs are laid but development of immature mites is delayed. Young mites do not reach maturity before the bees emerge from the cell. Consequently, the immature mites die (Allam et al., 2003).

 

Very little work has been carried out by using essential oils to control T. clareae in Pakistan. Therefore, the aim of the present study is to investigate the efficacy of essential oils of thyme, lemon grass, lemon, oregano and basil against T. clareae in honey bee colonies and their affect on honey production.

 

Material and Method

 

The experiment was carried out at the apiary of Honeybee Research Institute, National Agricultural Research Centre, Islamabad during June 2014 to October 2014.

Experimental procedure

Selection of honey bee colonies: Before starting the experiment, 300 honey bee colonies in the apiary were monitored to estimate the consistency of the bee population and mite infestation levels in order to obtain homogenous experimental groups (six for treatment and one for control). Sixty three honey bee Apis mellifera colonies in Langstroth standard bee hives naturally infested with T. clareae, with a normal brood pattern (eggs, larvae and pupae) and removable bottom inserts were used in this experiment. Each bee colony had 10-11 full depth combs of worker bees and 3-5 brood frames. Honey bee colonies were randomly allocated to seven treatment groups. Three colonies were used for each concentration of essential oils and formic acid and three untreated colonies served as control (Ali et al., 2002; Satta et al., 2005).

 

Chemicals and concentrations used: Five essential oils of lemongrass (Cymbopagon citrate), lemon (Citrus lemon), origano (Origanium vulgare), basil (Ocimum basilicum), thyme (Thymus linearus) and formic acid (65%) at three different concentrations of 25%, 50% and 100% were used for conducting this experiment.

 

Plants identification and essential oils extraction

Plants were identified from the National Herbarium Programme, NARC, Islamabad. These plants were dried in the shade at room temperature and chopped into small pieces with electric grinder. The essential oils were extracted by the process of water steam distillation using a clevenger-type apparatus (Richard et al., 1992) within three hours. An average of 100 g plant material was used for each extraction and several distillations were made until obtaining an appropriate volume for all the trials. After oil extraction, anhydrous sodium sulphate was used for the removal of excess water. These oils were transferred to dark brown glass vials with aluminum foil and stored in refrigerator at 40C until used in the experiment.

 

Percent infestation of Tropilaelaps clareae

The ectoparasitic bee mite, T. clareae infestation in worker brood was determined before and after treatment. The level in worker brood was recorded by randomly choosing fifty sealed worker brood cells from three combs per hive (Satta et al., 2005; Rashid et al., 2011; Islam et al., 2016). The capped brood cells were unsealed and then the pupae were withdrawn from the cells with the help of a forceps and picked up in petri dishes. Samples were transferred to the laboratory and the body surfaces of the brood were inspected for Tropilaelaps infestation under stereomicroscope, which were counted and recorded.

 

Mortality of Tropilaelaps clareae

The number of dead/fallen mites per hive from treated and control colonies were counted on a weekly basis using a white formica sheet under mesh at the bottom of each hive (Floris et al., 2001; Gregoric and Planincy, 2005). The metal sheet was covered by a wire screen to prevent access by bees (Calderone and Lin, 2003; Gregoric and Planincy, 2005). On the sampling dates, the number of dead mites was recorded and the sheets were emptied (Gregorc and Smodis Skirl, 2007). T. clareae mite mortality was observed at different times a week before the treatment, at one week intervals during the entire period of the experiment and finally after the treatment.

 

Honey yield

It is known that there is a strong correlation between a colony’s strength and food supply and honey yield provides a good indication of this relationship (Jevtić, et al., 2009). By measuring the total honey yield from each colony, we can determine whether the treatments had any long term effects.

 

Data analysis

The efficacy of the treatments was calculated on the basis of the percentage of T. clareae mite mortality (Floris et al., 2001; Satta et al., 2005) using the following equation:

 

Where Bt and At are the respective percentages of infestations in the treated colonies before (Bt) and after treatment (At) and Bc and Ac the respective parameters measured in the control colonies. Percent mite mortality in A. mellifera colonies was determined by using the formula (Pawar, 2008; Islam et al., 2016):

 

Analysis of variance (ANOVA) was carried out by using F- test and least significant difference (LSD) test at 1% probability level was used to compare between the different treatments (Fisher, 1950; Snedecor and Cochran, 1972).

Results and Discussion

 

Plant essential oils of Basil, Thyme, Lemongrass, Lemon, Oregano and organic acid formic acid were evaluated at three different concentrations to determine their effect on the reduction percentages infestation of Tropilaelaps clareae, number of dead/fallen Tropilaelaps mites on white formica sheet, percent mite mortality and honey yield (Figure 1 to 10).

 

 

 

 

Percent infestation of Tropilaelaps clareae

The data indicated that the percent infestation of sealed worker brood before application of tested oils was 34.5 to 37.6% at 25%, 34.7 to 38.5% at 50% and 35.8 to 39.0% at 100% concentrations in the experimental honey bee colonies. After treatment infestation decreased gradually to the lowest infestation %age observed after 4th treatment application. The results showed that at 25% the highest mean percent reduction infestation in worker brood by T. clareae was 82.67% (Formic acid), 81.66% (Lemongrass), 75.81% (Thyme), 74.07% (Lemon), 72.10% (Basil) and 72.06% (Oregano), respectively. At 50%, the highest mean percent reduction infestation in worker brood by T. clareae was 89.42% (Formic acid), 86.43% (Lemongrass), 83.72% (Thyme), 81.73% (Lemon), 80.51% (Basil) and 78.94% (Oregano), respectively. Whereas at 100% concentration, the highest mean percent reduction infestation in worker brood by T. clareae was 93.47% (Formic acid), 91.44% (Lemongrass), 88.70% (Thyme), 86.39% (Lemon), 86.24% (Basil) and 83.94% (Oregano), respectively. While this percent infestation in worker brood was increased from 40.2% to 47.4% in the control colonies. The analysis of variance indicated that throughout the first three applications, all the essential oils and formic acid were significantly more effective against Tropilaelaps mites as compared to the untreated colonies. The highest concentration (100%) of all the essential oils and formic acid was highly efficient in reducing the mean infestation percentage of Tropilaelaps on worker brood. The results also showed that mean percentage of Tropilaelaps infestation on the worker brood reduced to 100% after the fourth application with formic acid, lemon, thyme and basil. These results were in agreement with the findings of Allam et al. (2003) who found that formic acid caused 91.7% mortality of the Varroa mites. Abd El-Halim et al. (2006) observed significant differences in mean infestation percentages of Varroa in worker brood after 1st, 2nd, 3rd and 4th week of treatments between the sour orange, lemongrass and citronella oils at three different concentrations (25, 50 and 100%) and control. Abd El-Wahab et al. (2012) recorded highest reduction infestation percentage in adult worker bees at the highest concentration (100%) of Formic acid, Thyme, Cinnamon, Lemongrass and Anise whereas more than 96% reduction infestation percentage of worker brood with Formic acid, Lemon grass, Thyme and Anise oils were recorded against Varroa mites.

 

Mortality of Tropilaelaps clarea

The results showed that highest number of fallen/dead Tropilaelaps mites was recorded after 1st and 2nd treatments, particularly at 100% concentration of the essential oils and Formic acid in comparison with control colonies. It gradually decreased from 2nd to 4th treatment. These results were in accordance with those of Shoreit and Hussein (1994) who determined that maximum number of dead mites was observed after the first treatment with Coriander extract in both of winter and spring feedings which steadily decreased during 2nd and 3rd week of treatments. The results showed that the total maximum number of dead T. clareae mites/hive fallen on formica sheet after treatment of four application was 352, 472, and 761 (formic acid), 220, 316 and 445 (lemongrass), 172, 264 and 349 (thyme) 134, 227 and 298 (lemon) 133, 200 and 283 (basil) and 99, 155 and 250 (oregano) at 25%, 50% and 100% concentrations, respectively as compared to control with 62 dead mites/hive. There were highly significant differences between formic acid, essential oils and control in the total number of dead/fallen Tropilaelaps mites between the treatments during the experimental period. These results also indicated that the number of dead/ fallen Tropilaelaps mites was higher after 1st application than 2nd and 3rd application whereas the 4th application resulted in the lowest number of mites. These results are in agreement with Islam et al., (2016) who found that the highest total number of dead Varroa mites (323) fallen on the sheet was recorded by formic acid followed by lemon grass (306), thyme (263), mint (204) and rosemary (188) respectively. This reduction in the number of fallen mites could be attributed to either the reduced rate of infestation or the reduced efficiency of the control agents (Shalaby et al., 1996).

 

 

Percent mite mortality data (Figure 7, 8 and 9) showed that among seven treatments, formic acid and lemongrass were significantly better in reducing the mite’s population. Formic acid treatment gave maximum percent mite mortality (93.33%) at 1st application which was reduced to 90.15%, 86.47% 3rd application and 67.37% after 2nd, 3rd and 4th application represented with overall mean mortality of 84.33%. Similarly, lemon grass oil treatment recorded 91.42% mortality after 1st application which was dropped to 83.88% after 2nd application, 76.69% after 3rd application and further to 61.17% after 4th application withan overall mean mortality of 78.29%. On the basis of present study, it was apparent that the equally successful in producing desired and intended control of Tropilaelaps mites’ infestation especially when applied at 100% concentration (100%).

 

 

 

 

Honey Yield

Regarding the results of honey yield, the honey bee colonies treated with essential oils and Formic acid yielded higher honey yield in comparison with control colonies during experimental period. In control (untreated) colonies 3.8 kg honey per hive was extracted due to the increased mite infestation on bees causing low honey yield. Formic acid gave higher honey yield with a mean value of 9.3 kg per hive followed by lemon grass 8.8 kg, thyme 8.5 kg, lemon 8.5 kg, basil 7.7 kg and oraegano 7.6 kg honey per hive. Rashid et al. (2011), Abd El-Wahab et al. (2012) and Islam et al. (2016) also recorded that formic acid treated colonies produced more honey as compared to the essential oils treated and control colonies.

 

 

 

 

Authors Contribution

 

ES designed the study. NI collected and analysed data while FN helped him. NI conducted reseach and wrote the article. FN contributed in graph prepration.MA supervised the work, evaluated and editted the manuscript. EH did review and critical revision of the article.ES contributed in finalization of “Results and Discussion”.

 

References

 

Abd El-Halim, M. Ismail, A. Helmy, Ghoniemy and A. Owayss, 2006. Combating honey bee Varroa mites by plant oils alone or in an IPM program. The 2nd Conference of Farm Integrated Pest Management, 16-18 January, Fac. Agric. Fayoum Univ. pp. 172-185.

Abd El-Wahab, T.E., I.M.A. Ebadah and E.W. Zidan. 2012. Control of Varroa mite by essential oils and formic acid with their effects on grooming behav­iour of honey bee colonies. J. Basic Appl. Sci. Res. 2 (8): 7674–7680.

Ali, M.A., M.D. Ellis, J.R. Coats and J. Grodnitzky. 2002. Laboratory evaluation of 17 monoterpenoids and field evaluation of two monoterpenoids and two registered acaricides for the control of Varroa destructor Anderson and Truemean (Acari: Varroidae). Am. Bee J. 142: 50-53.

Allam, S.F., M.F. Hassan, M.A. Rizk and A.U. Zaki. 2003. Utilization of essential oils and chemical substances alone or in combination against Varroa mite (Varroa destructor), a parasite of honeybees. Insect Pathogens and Insect parasitic Nematodes IOBC wprs Bulletin. 26: 273-274.

Burgett, M. and P. Akratanakul. 1985. Tropilaelaps clareae, the little known honey bee brood mite. Am. Bee J. 125: 112-114.

Calderone, N.W. and S. Lin. 2003. Rapid determination of the numbers of Varroa destructor, a parasitic mite of the honey bee, Apis mellifera, on sticky-board collection devices. Apidologie. 34: 11–17. https://doi.org/10.1051/apido:2002042

Calderone, N.W., W.T. Wilson and M. Spivak. 1997. Plant extracts used for control of the parasitic mites Varroa jacobsoni (Acari: Varroidae) and Acarapsi woodi (Acari: Tarsonemidae) in colonies of Apis mellifera (Hymenoptera: Apidae). J. Econ. Entomol. 90: 1080-1086. https://doi.org/10.1093/jee/90.5.1080

Camphor, E.S.W., A.A. Hashmi, W. Ritter, and I.D. Bowen. 2005. Seasonal changes in mite (Tropilaelaps clareae) and honey bees (Apis mellifera) populations in Apistan treated and untreated colonies. Apiacta. 40: 34-44.

Elzen, P.J., J.R. Baxter, G.W. El Zen, R. River, and W.T. Wilson. 2000. Evaluation of grapefruit essential oils for controlling Varroa jacobsoni and Acarapis woodi. Am. Bee J. 35: 666-668.

Floris, I., A. Satta, V.L. Garau, M. Melis, P. Cabras and N. Aloul. 2001. Effectiveness, persistence, and residue of amitraz plastic strips in the apiary control of Varroa destructor. Apidologie. 32: 577–585. https://doi.org/10.1051/apido:2001145

Fisher, R.A. 1950. Statistical methods for research workers II. (Rev. Ed Oliverand Boyed, London).

Gregorc, A. and I. Planincy. 2005. The control of Varroa destructor in honey bee colonies using the thymol-based acaricide, Apiguard. Am. Bee J. 145: 672–675.

Gregorc, A. and M.I. Smodis Skirl. 2007. Combating Varroa destructor in honeybee colonies using flumethrin or fluvalinate. Acta Veterianaria Brunensis. 76: 309–314. https://doi.org/10.2754/avb200776020309

Hosamani, R.K., G. Rachna and S.K. Sharma. 2006. Bioecology and management of honey bee mite, Tropilaelaps clareae, Delfinado and Baker. A review. Agric. Rev. 27: 197-199.

Imdorf, A., S. Bogdanov, R.I. Ocha and N.W. Calderone. 1999. Use of essential oils for the control of Varroa jacobsoni Oud. In honey bee colonies. Apidologie. 30: 209-228. https://doi.org/10.1051/apido:19990210

Islam, N., M. Amjad, E. Ul-Haq, E. Stephen and F. Naz. 2016. Management of Varroa destructor by essential oils and formic acid in Apis mellifera Linn. colonies. J. Ento. Zool. Stud. 4 (6): 97-104.

Iwasa, T., N.Motoyama, J.T. Ambrose and R.M. Roe. 2004. Mechanism for the differential toxicity of neonicotinoid insecticides in the honey bee, Apis mellifera. Crop Protect. 23: 371–378. https://doi.org/10.1016/j.cropro.2003.08.018

Jevtić, G., M. Mladenović, B. Anđelković, N. Nedić, D. Sokolović, and R. Štrbanović. 2009. The correlation between colony strength, food supply, and honey yield in honey bee colonies. Biotechnol. Anim. Husbandry. 25 (5-6): 1141-1147.

Kapil, R.P., B.N. Putatunda and K. Agarwal. 1985. Studies on ectoparasitic mites of Apis species. Fifth Annual Report, Department of Zoology, HAU, Hisar, India. P. 188.

Kiprasert, C. 1984. Biology and systematic of the parasitic bee mite Tropilaelaps clareae Delfinado and Baker (Acarina: Laelapidae). M. Sc Thesis, Kasetsart University, Bangkok, Thailand.

Lee, S., R. Tsao, C. Peterrson and R. Coats. 1997. Insecticidal activity of monoterpenoids to Westeron corn rootworm (Coleoptera: Chrysomelidae), twospotted spider mite (Acari: Tetranychidae) and house fly (Diptera: Muscidae). 90: 883-892.

Mahavir, G. and M. Gupta. 1999. Infestation of Apis mellifera L. colonies with ectoparasitic mites. Annl. Biol. 15: 227-229.

Martel, A.C. and S. Zeggane. 2002. Determination of acaricides in honey by high performance liquid chromatography with photodiode array detection. J. Chromatogr. A. 2: 173-180. https://doi.org/10.1016/S0021-9673(02)00126-7

Mullin, C.A., M. Frazier, J.L. Frazier, S. Ashcraft and R. Simonds. 2010. High levels of miticides and agrochemicals in North American apiaries: Implications for Honey bee health. PLoS ONE. 5(3): e9754. https://doi.org/10.1371/journal.pone.0009754

Pawar, S.B. 2008. Efficacy and persistence of some plant products and chemicals against Varroa jacobsoni (Oudemans) in Apis mellifera L. colonies and their impact on brood development and honey production. M. Sc Thesis G. B. Plant University Agriculture and Technology, Pantnagar.

Pilling, E.D., K.A.C. Bromley-Challenor, C.H. Walker and P.C. Jepson. 1995. Mechanism of synergism between the pyrethroid insecticide λ-cyhalothrin and the imidazole fungicide orochloraz in the honeybee (Apis mellifera L.). Pestic. Biochem. Physiol. 51: 1-11. https://doi.org/10.1006/pest.1995.1001

Rashid, M.E.S., Wagchoure, S. Raja, G. Sarwar and M. Aslam. 2011. Effect of thymol and formic acid against ectoparasitic brood mite Tropilaelaps clareae in Apis mellifera colonies. Pak. J. Zool. 43: 91-95.

Richard, H.J., I. Nolean and P. Giampoli. 1992. Techniques of analysis of the spices and aromatics. In: Richard H (ed) Epices et Aromates. Tec and Doc, Lavoisier, Paris. P. 191-211.

Satta, A., I. Floris, M. Eguaras, P. Cabras, V.L. Garau and M. Marinella. 2005. Formic acid- based treatments for control of Varroa destructor in a Mediterranean area. J. Econ. Entomol. 98: 267–273. https://doi.org/10.1603/0022-0493-98.2.267

Shalaby, A.A., S.L. Yousif-Khalil, S.M. El-Shakaa and E.Z. Matter. 1996. Efficiency of some control agents against Varroa mites infesting honey bee colonies. Zag. J. Agric. Res. 23 (6): 113-130.

Shoreit, M.N. and M.H. Hussein. 1994. Field trials for the control of Varroa disease of honeybees by using coriander seeds extract. Zagazig J. agric. Res. 21: 279-288.

Snedecor, G.W. and W.G. Cochran. 1972. Statistical methods. Iowa State Univ. Press, Ames, Iowa.

Underwood, B.A. 1986. The natural history of Apis laboriosa Smith in Nepal. M. Sc Thesis, Cornell University; Ithaca, NY, USA.

Woo, K.S. and J.H. Lee. 1997. Current status of honeybee mites in Korea. Honeybee Sci. 18 (4): 175-177.

Zhou, T., Q. Wang and J. Yao. 2007. The progress on Varroa destructor in China. J. Apic. China. 58 (2): 5-7.