Laboratory Evaluation of Some Botanicals against the Honey Bee Ectoparasitic Mite Varroa Destructor (Acari: Varroidae) and its Impact on Honey Bees (Apis cerana indica)

Honey bees are of great economic importance to agriculture, not only by providing honey but also by pollinating crops. Apart from honey, they also provide bee wax, bee pollen, royal jelly, propolis, etc. However, domesticated honeybees started declining all over the world, due to colony collapse disorder (CCD), a syndrome caused by different types of stress as well as pathogens and pests (Cox-Foster et al., 2007). Among these, the ectoparasite mite Varroa destructor (Mesostigmata:Varroidae) has been considered as the most obnoxious enemy by most honey bee researchers. (Dietemann et al., 2012).
       
Varroa mites cause damage to the honey bee colony directly through feeding on the haemolymph of adults and brood, preferring the later, especially drone brood (Plate 1) and indirectly by transmitting viral diseases such as Kashmir bee virus (KBV), acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), deformed wing virus (DWV), etc. (Boecking and Genersch, 2008). Infested colonies may die or migrate, resulting in economic loss and decreased honey production (Needham, 1988).
 

       
Several acaricides of natural origin are used for the control of Varroa mites in domesticated colonies. (Calderone and Spivak, 1995; Sammataro et al., 1998). Organophosphates such as coumaphos; pyrothroids such as tauflavinate; and formamidine such as amitraz (Miani and LIob,1998, Li Li et al., 2017) were some of the commonly used synthetic acaricides against honey bee ectoparasitic mites. The repeated use of synthetic varroacides results in the development of resistance in the varroa mite to these products (Thomson et al., 2002). Apart from this, the use of synthetic acaricides causes residue hazards as traces of these pesticides are reported in bee products (Wallner, 1999; Howis and Nowakowski, 2009). Therefore, it is very pertinent that beekeepers have rule out new control tactics that is not detrimental to bees as well as contaminate hive products.
       
Plant derived products have shown toxicity, repellence, antifeedant and growth regulatory properties against insect pests and mites (Ntalli et al., 2022; Aivazi and Vijayan 2009; Ciccia et al., 2000; George et al., 2014;) thus can be thought of as a desirable alternative. Therefore, the present work has been designed to screen and evaluate six easily available botanical extracts that have varroicidal action with little or no hazardous effects on honeybees. 

Experimental location
 
The present study was conducted in the Apiary unit located at Tamil Nadu Agricultural University, Coimbatore (11°N, 77°E), Tamil Nadu, India during 2022-2023.
 
Preparation of botanical extracts
 
The plant samples (Table 1) were obtained from the field of germplasm collections maintained by the department of medicinal plants and aromatic crops, TNAU, Coimbatore. The collected plant samples were shade-dried and pulverised using an electrical mixer. Microwave-assisted extraction is used for extracting the samples. 70% ethanol was used as the solvent. The extract thus obtained was filtered with Whatman No.1 filter paper and allowed to dry following evaporation. The crude extractives are then stored in glass vials and are further used for making stock solutions, from which further working solutions of different concentrations are formed.
 

 
Mites and bees
 
The mites (V. destructor) were collected from colonies of Apis mellifera and Apis cerana indica maintained at Tamil Nadu Agricultural University, Coimbatore. To collect Varroa mites from adult bees, bees were collected in a screen capped plastic can and shaked vigorously with powdered sugar to dislodge mites. Mites from broods were collected carefully with the help of forceps and a “00” camel hair brush by opening drone brood cells and removing the larvae or pupae (Plate 2).
 

 
Screening tests
 
The bio-effectiveness of essential oils against Varroa has been determined using a complete exposure method (Lindberg et al., 2000). The experiment was conducted in a completely randomised design under laboratory conditions (28±1°C, 70±5%) with three replications. Each replication consisted of an insect rearing petri dishes (10 cm diameter × 4 cm height) with filter paper treated with botanical extract placed at the bottom. Each filter paper was treated with 500 microliters of plant extract diluted with ethanol and kept for 5-10 minutes to evaporate the ethanol. Another similar container treated with 70% ethanol was used as a check. After complete evaporation of solvent, five adult Varroa mites were released in each container. A pupa of Apis mellifera is kept as a host and the lid is closed. Humidity is maintained by keeping wet filter paper below the lid of the container. Afterwards the mortality of mites was recorded at 1,2,4,6,12 and 24 hours after treatment (HAT). Mites were considered dead when they became immobile and showed no signs of activity even after being stroked with a single hair brush. Similarly, the mortality of honeybees has also been recorded giving the same treatment followed by releasing five adult worker bees in insect rearing container (diameter =10 cm, height=4 cm). The bees present in insect rearing cages was provided with sugar solution and the number of dead bees after 24 hours of treatment was recorded to evaluate relative efficiency of botanicals on honey bees (Plate 3).
 

 
Data analysis
 
The per cent corrected mortality of honeybees was done using Abott’s formula and arc sin transformation is adopted for the data transformation of percentage (Abott,1925). Analysis of variance (ANOVA) was done in IBM SPSS v.20 statistical program and used Tukey’s Honestly Significance difference (HSD) (p<0.05) for the comparison of means.

The enumerated cumulative mortality value recorded at each observation time against the treatments are presented in Table 2. Extracts of sweet basil, turmeric, coleus, sweet flag, tulsi and goat weed showed significant variation in mite mortality from control.  At one HAT, only sweet flag and tulsi plant extracts registered mite mortality, with a mean mortality of 33.33% and 6.67%, respectively. At 2 HAT, the order of efficiency was sweet flag (60%), followed by coleus (33.33%), turmeric (20%), goat weed (13.33%) and tulsi (6.67%). Sweet basil extract didn’t show any mortality and did not varied significantly from control. Likewise, at 4 HAT, sweet flag registered more than 90% mortality (93.33%), followed by coleus with more than 50% mortality (66.67%). At 6 HAT, the order of descending toxicity was: sweet flag (100%), followed by coleus (86.67%), turmeric and tulsi (53.33%), goat weed (40%) and so on. All the treated plant extracts registered more than 50% mortality, whereas coleus and sweet flag showed resulted in to mortality to the tune of 100% at 12 HAT. At 24 HAT, coleus, sweet Flag and tulsi extracts recorded 100% mortality, followed by turmeric (93.33%), sweet Basil (86.67%) and goat weed (80%). There was a noticeable change in mortality with prolonged exposure in all the treatments and no mortality was observed in the untreated control up to 24 HAT.
 

       
Mean per cent mortality of Apis cerana indica workers after exposure to 24 h of botanical extracts have been depicted in Table 3. All the extracts registered <30% mortality of honey bees. The lowest mortality was to the tune of 14.29% in case of turmeric followed by sweet basil and coleus (19. 05%). Goat weed (28.57%), sweet flag (28.57%) and tulsi (23.81%) showed relatively high toxicity to bees.
 

       
The bioassay experiment was conducted through a simple and rapid methodology to rule out some of the botanical extract for their varroicidal activity. High varroicidal activity was recorded in Sweet flag, more than 50% mortality at 2 HAT and also 100% mortality at 3 HAT followed by coleus which registered a 100% mortality at 12 HAT. Perusal of available literatures reveal that, scanty information are available in this direction on the varroicidal action of sweet flag (Acorus calamus and coleus (Coleus aromaticus), though there are numerous studies conducted with the aforesaid ingredients for the management of storage pests (El-Nahal et al., 1989; Singh et al., 2021; Shukla et al., 2016) and plant mites (Pushpa and Nandihalli, 2008; Thevan et al., 2005; Janlaor and Auamcharoen, 2021). Swamy et al., (2006) reported that sweet flag (Acorus calamus) and tulsi (Ocimum sanctum), along with some other botanicals, can be used for the effective management of greater wax moth, which is another serious pest of honey bees. Dusting of sugar powder, sulphur and Acorus calamus as a method to control varroa mites is also stated by Tej et al., (2017). Many researchers reported δ-Asarone as one of the major constituents of Acorus calamus, which has insecticidal and miticidal action (Venskutonis and Dagilyte, 2003; Liu et al., 2013). According to Dutta (1959), Coleus aromaticus, native of India, is rich in carvacrol, thymol, eugenol and chavicol. Thymol is a volatile monoterpenoid and a natural constituent of thyme (Thymus vulgaris). It is one of the most widely used essential oils for varroa mite management (Glavan et al., 2020; Gregorc and Planinc, 2012). Vimla and Khan (2013) reported mite mortality ranging from 56.85 to 75.03 per cent when treated with tulsi oil and turmeric oil in the hive. In one of the experiments, hives treated with garlic oil, turmeric oil, tulsi oil, ajwin oil, cinnamon oil and clove oil showed mite mortality ranging from 65 to 77% with no bee mortality in any of the treatments (Goswami et al., 2014).
       
 The variability of the effects of these botanicals and essential oils on mites and bees in various studies is perhaps due to the change in plant constituents at different locations as well as the age of the plant. Also, differences in concentration and application methods may play a role. (Ariana et al., 2002). In the present experiment, mortality of the mites and bees was the only observation taken, but there is also a chance that some of the constituents of plant extract may also have sub lethal effects on Varroa as well as on honeybees. Colin (1990) and Kraus et al., (1994) reported the inhibitory action of thymol and menthol on the growth and reproduction of insect and mite pests. Further investigations should be done to study these effects on mites and bees.

The present study gave a relative efficiency analysis of some of the plant extracts for the management of varroa and suggests the possibility of using them in the integrated management of varroa mites in honey bee colonies; however, more work need to be done to optimise the correct dose of treatment and application method at field level. Further research should be done to study its effect on bee survival and reproduction for a prolonged exposure.