Life history and control of the parasitic bee mite, Tropilaelaps mercedesae Anderson and Morgan (Acari: Laelapidae): A Review

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1 Basic Research Journal of Agricultural Science and Review ISSN Vol. 5(3) pp March 2016 Available online http// Copyright 2015 Basic Research Journal Review Life history and control of the parasitic bee mite, Tropilaelaps mercedesae Anderson and Morgan (Acari: Laelapidae): A Review Boonmee Kavinseksan 1 * and Siriwat Wongsiri 2 1 Biology Program, Department of Science, Faculty of Science and Technology, Bansomdejchaopraya Rajabhat University, Isaraphab Rd., Dhonburi, Bangkok 10600, Thailand 2 Agriculture Interdisciplinary Program, Graduate School, Maejo University, San Sai, Chiang Mai 50290, Thailand *Corresponding author boonmee47@yahoo.com Accepted 08 April, 2016 ABTRACT Tropilaelaps mercedesae Anderson and Morgan (Acari: Laelapidae) feeds on haemolymph of bees. This mite is a natural parasite of Apis dorsata Fabricius but not considered to be a severe pest of this bee species because of the effective defense mechanisms of the host bee. When A. mellifera Linnaeus was introduced into tropical Asia, T. mercedesae successfully switched from indigenous A. dorsata host to be A. mellifera and has become the most economically important mite pest in beehives; causing serious damage to A. mellifera beekeeping industry in tropical Asia. Chemical, physical, biotechnical and combinations of chemical and biotechnical methods were used to control T. mercedesae in colonies of A. mellifera. Some of these methods are either high cost of labor and materials, reduce bee populations, high chance of contaminating bee products with undesirable chemicals, and high risk of the mite developing resistance to acaricides. The use of resistant A. mellifera stocks to T. mercedesae has been thought to be a better solution to the Tropilaelaps problem. Keywords: Tropilaelaps mercedesae, Apis dorsata, Apis mellifera, mite control, acaricide, defense mechanisms INTRODUCTION Mites in the genus Tropilaelaps are brood parasites of honey bees (Apis spp.). The first species, T. clareae, in this genus was first discovered and originally described by Delfinado and Baker in 1961 from a collection of dead A. mellifera and from field rats nesting near a beehive in the Philippines (Delfinado-Baker and Baker, 1961). The second species in the genus was T. koenigerum that was morphologically described by Delfinado-Baker and Baker (1982) and the genetic difference between T. koenigerum and T. clareae was reported by Tangjingjai et al. (2003). From genetic and morphological studies by Anderson and Morgan in (2007), it was found that the genus Tropilaelaps contains four species; T. clareae, T. koenigerum, T. mercedesae (Figure 1) and T. thaii. The taxonomy of the Tropilaelaps genus has been revised as follows (Anderson and Morgan, 2007; Lindquist et al., 2009): Kingdom: Animalia Phylum: Arthropoda Class: Arachnida Subclass: Acari Superorder: Parasitiformes Order: Mesostigmata Family: Laelapidae

2 57. Boonmee and Siriwat Figure 1. Scanning electron micrographs of adult female T. mercedesae (Bar = 100 µm) (Left) ventral view (Central) dorsal view (Right) lateral view (Photo by Boonmee Kavinseksan) Table 1. Tropilaelaps mites and their primary host bees Mite species Primary host bees References T. clareae T. koenigerum T. mercedesae T. thaii A. dorsata breviligula A. dorsata binghami A. dorsata, A. laboriosa A. dorsata, A. laboriosa A. laboriosa Laigo and Morse, 1968; Anderson and Morgan, 2007; Anderson and Roberts, 2013 Bharadwaj, 1968; Delfinado-Baker et al., 1985 Anderson and Morgan, 2007; Anderson and Roberts, 2013 Anderson and Morgan, 2007; Anderson and Roberts, 2013 Figure 2. A colony of A. dorsata (Photo by Boonmee Kavinseksan) Genus: Tropilaelaps Species: T. clareae Delfinado and Baker (1961) T. koenigerum Delfinado-Baker and Baker (1982) T. mercedesae Anderson and Morgan (2007) T. thaii Anderson and Morgan (2007) Tropilaelaps mites are common natural parasites of giant honey bees (Table 1) distributed throughout Asia (Anderson and Roberts, 2013). A. dorsata breviligula in the Philippines (except on Palawan Island) and A. dorsata binghami on Sulawesi Island are the primary hosts of T. clareae. A. laboriosa in mountainous region of Mainland Asia is the natural host of T. thaii. A. dorsata is the primary host of T. mercedesae and T. koenigerum throughout mainland Asia and in Indonesia (except Sulawesi Island) including Palawan Island (Morse and Laigo, 1969; Delfinado-Baker and Baker, 1982, 1983; Tangjingjai et al., 2003; Anderson and Morgan, 2007; Anderson and Roberts, 2013). T. mercedesae and T. clareae have infested A. mellifera colonies which have been introduced into regions of Asia where bee brood is reared continuously through year. However, especial A. mellifera in the Philippines (except on Palawan Island) and on Sulawesi Island has been infested by T. clareae while T. mercedesae has infested A. mellifera throughout mainland Asia, in Indonesian (except Sulawesi Island), Palawan Island, Kenya and New Guinea (Kumar et al., 1993; Baker et al., 2005; Anderson and Morgan, 2007). Therefore, we focus on T. mercedesae since it is a dangerous pest of A. mellifera in many regions. Distributions and infestations of T. mercedesae T. mercedesae is a natural parasite of A. dorsata (Figure 2). This bee mite species has been found infesting different species of honey bees in Asia. Delfinado-Baker (1982) reported T. mercedesae (formally, T. clareae) infestation in brood combs of A. cerana indica in Pakistan

3 Basic Res. J. Agric. Sci. Rev. 58 Figure 3. Distribution of T. mercedesae (Ellis and Munn, 2005) Table 2. Distributions of T. mercedesae and the infested honey bees from countries Countries Infested bee species References Thailand Myanmar Hong Kong India Indonesia Malaysia A. mellifera A. dorsata A. cerana A. cerana A. mellifera A. dorsata A. mellifera A. dorsata A. mellifera A. florea, A. cerana A. laboriosa A. cerana A. mellifera, A. dorsata A. cerana A. dorsata Akratanakul, 1979; Kavinseksan, 2012 Burgett and Kitprasert, 1989; Kavinseksan, 2011a Wongsiri et al., 1989; Anderson and Morgan, 2007 Delfinado-Baker and Baker, 1982 Nixon, 1983 Nyein, 1984 Delfinado-Baker, 1963 Bharadwaj, 1968 Stephen, 1968 Aggarwal, 1988 Delfinado-Baker et al., 1985 Delfinado-Baker, 1963 Anderson and Morgan, 2007 Delfinado-Baker, 1963 Koeniger and Koeniger, 1980 Pakistan A. cerana Delfinado-Baker and Baker, 1982 Afghanistan A. mellifera Woyke, 1984 Vietnam A. mellifera Woyke, 1985a A. dorsata Anderson and Morgan, 2007 South Korea A. mellifera Woo and Lee, 2001 Taiwan A. mellifera McDonald, 1971 Philippines A. mellifera Delfinado-Baker and Baker, 1961 A. dorsata Laigo and Morse, 1968 China A. mellifera Chen, 1993; Dainat et al., 2009; Luo et al., 2011 Kenya A. mellifera Kumar et al., 1993 Nepal A. laboriosa Underwood, 1986 New Guinea A. mellifera Baker et al., 2005; Anderson and Morgan, 2007 and Myanmar. Also, the single adult female T. mercedesae and two white nymphs were found in a capped worker brood cell of an A. cerana indica colony near Chiang Mai in northern Thailand (Anderson and Morgan, 2007). In India, T. mercedesae was found associated with five species of honey bees: A. dorsata, A. laboriosa, A. mellifera, A. florea and A. cerana (Aggarwal, 1988). T. mercedesae cannot survive more than 3 days without feeding on bee brood because their chelicerae (mouthparts) are not specialized for feeding on adult bees (Kitprasert,1984; Woyke, 1984; Koeniger and Muzaffar, 1988; Tangkanasing et al., 1988; Delfinado-Baker et al., 1992; Rinderer et al., 1994). Consequently, T. mercedesae is thought to be limited to the tropical zones because it cannot survive long broodless periods in A. mellifera colonies that happen during winter in the temperate zones (Woyke, 1985a). The geographical distribution of T. mercedesae seems to be limited in tropical Asia and coincides with the indigenous areas of A. dorsata (Crane, 1968; Burgett et al., 1983; Burgett and Akratanakul, 1985; Delfinado-Baker and Aggarwal, 1987; Delfinado-Baker et al., 1989). However, infestation of this bee mite in A. mellifera brood cells was observed in Afghanistan, South Korea, China, New Guinea and Kenya (Figure 3), which are outside the range of A. dorsata (Table 2) (Woyke, 1984; Chen, 1993; Kumar et al., 1993; Anderson, 1994; Woo and Lee, 2001; Baker et al., 2005;

4 59. Boonmee and Siriwat Figure 4. Adult females and males of T. mercedesae (Kavinseksan et al., 2016a) (A) ventral view of T. mercedesae female (B) dorsal view of T. mercedesae female (C) ventral view of T. mercedesae male (D) dorsal view of T. mercedesae male Figure 5. Variation in the shape of the epigynial and anal plates of adult female T. mercedesae (Bars = 50 µm) (Anderson and Morgan, 2007) Anderson and Morgan, 2007; Dainat et al., 2009; Luo et al., 2011; Anderson and Roberts, 2013). The levels of T. mercedesae infestation vary with honey bee species, brood gender and location of the colonies. In A. dorsata colonies, brood infestation of 5-30 mites per colony was observed in India (Bharadwaj, 1968). High infestations of the mite were observed from March to May in this area (Aggarwal and Kapil, 1989). In the Philippines, Laigo and Morse (1968) found mite infestations in seven out of eight A. dorsata nests examined. Brood infestations less than 10% were reported from Nepal (3 to 6%) (Underwood, 1986) and Thailand (0.2 to 9%) (Burgett and Kitprasert, 1989; Kavinseksan, 2003). T. mercedesae infestation in A. dorsata worker brood (4.3%) was higher than that of drone brood (1.2 %) (Underwood, 1986; Burgett and Kitprasert, 1989). In Malaysia, Koeniger et al. (2002) reported that the percentage of infested worker (20.0%) and drone (20.8%) brood cells did not differ, nor did the number of mites per cell (6 in worker brood and 6.1 in drone brood). The mite infestation rates on A. dorsata adult bees ranged from 0.3 to 11.1% (Thapa, 1998; Kavinseksan, 2003). Infestation of T. mercedesae on adult workers of A. mellifera ( %) was less than in brood ( %) (Woyke, 1984; Kavinseksan, 2003). seen without magnification. The mites can walk rapidly on the comb surfaces and are difficult to collect. Morphology of T. mercedesae is similar to T. clareae, but larger. Males are slightly smaller than females (Figure 4). On average, T. mercedesae adult males are 920 µm long and 523 µm wide while females are about µm long and µm wide (Anderson and Morgan, 2007). For females, the color of adult mites is light reddish-brown, and entire body is covered with short setae. Through a strong magnifying glass, a red streak running logitudinally on the ventral surface of the adult female, the fusion of her epgynial and anal shields, can be seen (Akratanakul, 1987). The shape of the apex of the epigynial plate varies from bluntly pointed to sharply pointed (Figure 5) (T. clareae is always bluntly pointed). A small subapical tooth is found on the movable chela of adult female chelicerae (Figure 6-A) (Anderson and Morgan, 2007). Adult males of T. mercedesae do not feed because their chelicerae (the organs originally used for piercing the bees integument) have been modified for the sperm transfer organ or spermatodactyl (Akratanakul, 1987). The sperm transfer organ of T. mercedesae male is long and attenuate with spirally coiled apex (Figure 6-B) (Anderson and Morgan, 2007). The anal plates of males appear to be pear-shaped (Delfinado-Baker and Baker, 1982). Morphology T. mercedesae is smaller than Varroa mites but can be Life cycle of T. mercedesae on A. mellifera Because of the difficulties of studying A. dorsata, most

5 Basic Res. J. Agric. Sci. Rev. 60 Figure 6. Chelicerae and spermatodactyl of T. mercedesae (A) adult female chelicerae of T. mercedesae (light microscopy, 800) (B) scanning electron micrographs of the sperm transfer organ of T. mercedesae male (Bar = 10 µm) (Anderson and Roberts, 2013) Figure 7. Stages in the development of T. mercedesae (A) egg (B) larva (C) protonymph (D) deutonymph (E) adult female (Photo by Boonmee Kavinseksan) information about the life cycle of T. mercedesae comes from infested brood of A. mellifera. The life cycle of T. mercedesae is similar to that of V. destructor (formally, V. jacobsoni) in A. mellifera brood cells (Atwal and Goyal, 1971; Burgett et al., 1983). However, a detailed life history of T. mercedesae, especially in the male mite, has not yet to be published. The life cycle of T. mercedesae is well synchronized with that of the host bee. In all its immature stages, the mites live within the brood cells of the bees, feeding on the brood s haemolymph. One or several female mites enter an open brood cell containing a late instar larva of A. mellifera, and most have begun feeding on larvae by the time the cells are sealed. Both worker and drone brood serve as hosts. The adult mites firmly attach themselves to the prepupae with their mouthparts and feed (Ritter and Schneider-Ritter, 1988). Females swell to twice their thickness while feeding, and most egg laying is apparently associated with this feeding period (Ritter and Schneider-Ritter, 1988; Woyke, 1989). In cells with older pupae, mites become thin again and egg laying ceases (Woyke, 1987a, 1989). Oviposition frequently occurs on the cuticle of the host larva (Burgett and Krantz, 1984). The stages of development of the mite are as follows: egg, six-legged larva, protonymph, deutonymph, adult (Figure 7). Studying worker cells of A. mellifera, Woyke (1987b) found that eggs were not laid immediately after the cells were sealed because the first T. mercedesae eggs and larvae were detected in sealed cells containing spinning larvae (5 th larva instar after sealing), i.e., on brood 9 or 10 days of age (from egg laying). The highest percentage of bee brood with mite eggs occurred among cells containing prepupae (prepupa stage or 11 and 12 days after egg-laying). The first eggs are usually females, while the second are males (Rath et al.,1991). The first protonymphs of T. mercedesae were found on prepupae (Woyke, 1987a). Ritter and Schneider-Ritter (1988) found 39% of protonymphs in the prepupa stage. The first deutonymphs appeared on pupa with white eyes (13 days after egg laying) (Woyke, 1987a; Rembold et al., 1980; Ritter and Schneider-Ritter, 1988). The first young adults were found on pupa with dark brown eyes (16 days from egg-laying) and the first adult males and females of the offspring were found on pupa with red eyes and on pupa with dark brown eyes (Figure 8) (Ritter and Schneider-Ritter, 1988).

6 61. Boonmee and Siriwat Figure 8. Life cycle of T. mercedesae (Kavinseksan et al., 2016a) Table 3. Time (hours) of developmental stages of T. mercedesae after bee brood cell capping. The numbers in brackets represent development time (days) of A. mellifera brood (Modified from Anderson and Roberts, 2013) Places Time of mite developmental stages (hours) References Egg Larva Protonymph Deutonymph Young adult New Guinea 72 (10-11) 96 (11-12) 96 (11-12) 168 (14-15) 216 (16-17) Saleu, 1994 Thailand 96 (12) 96 (12) 192 (16) 216 (17) 312 (22) Kiprasert, 1984 Afghanistan Woyke, 1984, 1985a and Vietnam (9-10) (9-10) (11-12) (13) (16) India 96 (12) 96 (12) 192 (16) 192 (16) 298 (20) Kumar et al., 1993 Woyke (1987a) reported the last stages of T. mercedesae development on honey bee pupae; the last eggs on pupa with pink eyes (14 days from egg-laying), the last larvae on pupa with red eyes (15 days from egg-laying), the last protonymphs on pupa with dark brown eyes and the last deutonymphs on pupa with dark brown eyes and dark thorax (19 days old from egg-laying). Only mite adults were present on brood older than 19 days. Ritter and Schneider-Ritter (1988) reported that 99% of nymphs developed to be adult mites before the emergence of the adult bees. The average developmental period from eggs to adult mites was 8.8 days under laboratory conditions (Kitprasert, 1984) while Woyke (1987a) reported a development period of 6 days in worker brood cells in bee colony conditions (Table 3). Fan and Li (1988) provided an even shorter estimate of 5 days. The egg and larval stages are very brief (0.4 and 0.6 days respectively). Sex ratios The ratio of male to female T. mercedesae varies

7 Basic Res. J. Agric. Sci. Rev. 62 considerably. Under laboratory conditions, the ratio of male to female T. mercedesae was nearly 1:5 (Rath et al., 1991). A sex ratio in debris from A. mellifera colonies in Afghanistan and Thailand was 1:3 to 1:4 (male: female) (Woyke, 1990; Rath et al., 1991; Kavinseksan, 2012). In Thailand, sex ratios (male to female) of T. mercedesae adults in brood cells (1:1.5 to 1:22, average 1:5), on adult bees (1:1 to 1:8, average 1:2.4) and in debris (1:5.6 to 1:8) of A. dorsata colonies favored females (Burgett and Kitprasert, 1989; Kavinseksan, 2003). A higher estimate of 1:29 was reported in India in A. dorsata brood (Aggarwal and Kapil, 1989). The unbalanced sex ratio of T. mercedesae adults seems to be influenced by the much shorter life span of the males as compared to that of females. T. mercedesae males do not survive more than 5 days while the life span of females is about 28 days (Rath and Delfinado-Baker, 1990). However, the unequal sex ratio of T. mercedesae adults also may be attributed to differences in the proportion of initial male to female eggs and differences in mortality between sexes of immatures. Environment, location and time period are also likely to influence sex ratios. T. mercedesae mates by podospermy and can be observed inside or outside of the brood cells (Rath et al., 1991). Multiple mating was observed in both males and females (Woyke, 1994a). In cells infested by a single female T. mercedesae, male offsprings mate with their sisters. Since one male of T. mercedesae can easily serve a number of females (Woyke, 1994a), a foundress mite can increase her reproductive rate by producing more female offsprings than male offsprings. Thus, the biased sex ratio in favor of females would increase the high total number of offspring produced. A few females (2%) produce all male progeny (Ritter and Schneider- Ritter, 1988). These are probably unmated females from cells in which no male offspring were produced. When they attempt to reproduce, they can lay only unfertilized eggs which develop into males. After mating with a son, they can produce a normal mixed-sex progeny in subsequent reproductive cycle (Otis and Kralj, 2001). Mite growth The actual reproductive rate for Tropilaelaps was calculated from reproductive mites (Kavinseksan et al., 2016a). In A. dorsata colonies, multiple infestations of T. mercedesae females in worker brood cells had lower rates of reproduction per foundress (0.4 daughters per foundress) when compared to the rates found in cells having only one reproductive foundress mite (1.3 daughters per foundress). The maximum number of progeny in worker brood of A. dorsata was 3. About 71.7% of reproductive mites had produced 1, 24.2% had produced 2 and 4.1% had produced 3 progeny. The number of progeny produced by reproductive Tropilaelaps mites (or fecundity) was not correlated with the number of non-reproductive mites in colonies of A. dorsata (Kavinseksan and Wongsiri, in press). Similarly, the number of progeny produced by reproductive Varroa mites was independent of the frequency of nonreproductive mites in an A. mellifera colony (Rosenkranz and Engels, 1994; Martin, 1995). In A. mellifera colonies, the percentage of reproductive T. mercedesae was % (Woyke, 1987b). The maximum number of progeny produced by a single T. mercedesae foundress was found to be 4 (Woyke, 1987a). The fecundity has been estimated to be 1.4 to 1.9 progeny per foundress (Woyke, 1987b). In Thailand, Ritter and Schneider-Ritter (1988) reported that 64% of T. mercedesae females had produced 1, 33% had produced 2 and 3% had produced 3 progeny. Nowadays, there are no reports about how many reproductive cycle by individual mites, some Tropilaelaps mites removed from pupae are able to reproducing again (Woyke, 1994b). Mites reproduce on drone brood as well, but their relative contribution to the reproducing rate is unclear. From these informations, it is difficult to estimate the actual rate of growth of T. mercedesae populations. However, T. mercedesae populations can grow rapidly in A. mellifera colonies, and this mite is a more serious pest than V. destructor in tropical Asia (Anderson, 1994; Wongsiri et al., 1987a). One biological factor that differs between T. mercedesae and V. destructor is the length of the phoretic period. T. mercedesae adults stay outside sealed brood cells only for short periods of time. The maximum longevity of T. mercedesae on adult bees is days, and most live for less than two days (Akratanakul, 1984; Kitprasert, 1984; Woyke, 1984, 1987c; Koeniger and Muzaffar, 1988; Luong et al., 1993; Tran et al., 1993; Rinderer et al., 1994). Therefore, the mites are usually phoretic on adult bees for 1-2 days (Woyke, 1987c; Kavinseksan, 2003), in comparison to a much longer phoretic phase (average 27 days or more) of Varroa mites (Ruijter, 1987; Rath, 1991; Calatayud and Verdu, 1995). This shows that T. mercedesae has faster reproductive cycles than V. destructor. Therefore, the population growth rate of T. mercedesae within a bee colony is much higher than that of V. destructor (Anderson and Roberts, 2013), about 25:1 in favour of T. mercedesae (DEFRA, 2005). Symptoms and injuries T. mercedesae feeds on haemolymph of bees (Kitprasert, 1984; Delfinado-Baker et al., 1992; Kavinseksan, 2012; Anderson and Roberts, 2013) and is a much more serious pest of A. mellifera but not considered to be a severe pest of its original host A. dorsata (Wongsiri et al.,

8 63. Boonmee and Siriwat Figure 9. T. mercedesae females feed on haemolymph of A. mellifera brood causing of reduced size abdomen and malformed wings of A. mellifera adult bees (Photo by Denis Anderson) Figure 10. Sealed brood cells infested with T. mercedesae were uncapped by worker bees of A. mellifera. The pupa in the opened cells was either undamaged or eaten so that only the abdomen or parts of the pupa remained in the cells (Photo by Boonmee Kavinseksan) 1989). In A. dorsata colonies, T. mercedesae is present in most colonies and infest brood cells of both drones and workers, causing deformities and mortality similar to that for A. mellifera (Laigo and Morse, 1968; Woyke, 1984; Wongsiri et al., 1989). The damage caused to colonies of A. mellifera by T. mercedesae infestation is similar to that brought by V. destructor, and the injuries on individual bees and bee brood are essentially the same (Atwal and Goyal, 1971, Burgett et al., 1983; Burgett and Akratanakul, 1985; Ritter and Schneider-Ritter, 1988). Lightly infested brood usually complete development. Surviving A. mellifera adult bees from mite infestation in pupal stages are often malformed and frequently have stubby wings, deformed or missing legs, reduced size abdomen (Figure 9) and a shorter life-span than healthy bees (Akratanakul, 1987; Anderson and Roberts, 2013). Adult bees with deformed wings are often removed by their nestmates and can be seen on the comb surfaces, near the hive entrance and on the ground in front of hives. Brood cells that remain capped after surrounding bees have emerged are usually dead and frequently infested heavily with mites (Woyke, 1984) if larvae are infested with three or more mites each (Woyke, 1987b). The cells infested with T. mercedesae (especially in the pupal stages) are recognized by the worker bees of A. mellifera. They attempt to remove the parasitized brood and the mites (Boecking et al., 1992; Kavinseksan, 2003). The detection of mites and subsequent removal of infested brood may have been enhanced by the release of honey bee volatiles in response to nymphal feeding. Likewise, the presence of mites feces inside the brood cells may have assisted worker bees in locating the infested cells. Also, the movement of nymphs inside the brood cells may have assisted detection of infested brood (Khongphinitbunjong et al., 2014). In Thailand, a large number of infested brood cells with T. mercedesae in heavily infested colonies was opened by the worker bees (Ritter and Schneider-Ritter, 1988; Kavinseksan, 2003). The pupa in the opened cells were either undamaged or eaten so that only the abdomen or parts of the pupa remained in the cells (Figure 10), and 94% of the opened cells showed a heavy infestation with T. mercedesae.

9 Basic Res. J. Agric. Sci. Rev. 64 Table 4. Chemicals and applications for controlling T. mercedesae in A. mellifera colonies Trade names Apistan Mavrik Apitol Perizin Folbex-VA Formic acid Sulphur with Naphthalene Asuntol Mitac Folbex Bayvarol CheckMite+ Thymol Active ingredients Fluvalinate Fluvalinate Cymiazole Coumaphos Bromopropylate Formic acid Sulphur with naphthalene Coumaphos Amitraz Chlorobenzilate Flumethrin Coumaphos Thymol Types of products Plastic strip Plastic strip Powder Aqueous solution Paper strip Liquid Powder Aqueous solution Aqueous solution Paper strip Plastic strip Plastic strip Liquid Application methods Suspend Suspend Dissolve in syrup Trickled on Fumigation Evaporation Evaporation Spray Spray Fumigation Suspend Suspend Evaporation References Lensky et al., 2001 Lubinevski et al., 1988 Ritter and Schneider-Ritter, 1988 Wongsiri et al., 1987b Akratanakul, 1987 Rajesh et al., 1984 Hoppe et al., 1989 Raffique et al., 2012 Wongsiri et al., 1987b Wongsiri et al., 1987b Wongsiri et al., 1987b Woyke, 1987b Pichai et al., 2008 Pichai et al., 2008 Raffique et al., 2012 Infested A. mellifera pupae show darkly colored spots mainly on their extremities (Ritter and Schneider-Ritter, 1988). In populous colonies, discarded larvae and pupae accumulate at the vicinity of the hive entrance, frequently with live mites still on them. The mite infestation can lead to weakening of the colony and secondary infestation by wax moths (Laigo and Morse, 1968). Severe infestations in which 90% of pupae are infested are possible. Up to 50% of bee larvae and numerous pupae die in severely infested colonies, resulting in an irregular brood pattern and brood cells with perforated cappings (Otis and Kralj, 2001). Such colonies have diminishing amounts of brood and adult bees causing collapses of the colonies. T. mercedesae can kill untreated colonies of A. mellifera within a few months of infestation (Laigo and Morse, 1968; Crane, 1990). T. mercedesae controls in A. mellifera colonies T. mercedesae populations can not grow in A. dorsata colonies to a dangerous level (Wongsiri et al., 1989; Kavinseksan, 2003), but grow quickly in colonies of A. mellifera. In tropical Asia, T. mercedesae is a more severe pest than V. destructor (Wongsiri et al., 1989; DEFRA, 2005; Anderson and Roberts, 2013). The life cycle of T. mercedesae is similar to that of V. destructor. Therefore, several methods for controlling the two species of mites are similar. However, some methods that were effective in decreasing Varroa populations were not effective in regulating T. mercedesae populations in A. mellifera colonies (Atwal and Goyal, 1971). Chemical control In A. mellifera colonies, Tropilaelaps is usually controlled by using chemicals since chemicals are simple to use, readily available and oftentimes highly effective (Table 4). On the other hand, some chemicals are expensive, can kill honey bees when used improperly and contaminate bee products. Formic acid is a highly volatile compound. This chemical interferes with basic metabolic and respiratory processes. The most widely recommended application rate is to apply 2 ml of 60% formic acid for each comb. The liquid is applied to pads or plates and placed above or below the brood nest. At high concentration, formic acid will kill mites in sealed brood cells. Three to four treatments are usually required to achieve a high degree of control (Ellis, 2001). The highly effective T. mercedesae control were found by the use of continuous fumigation of 65% formic acid (5 cm 3 per day) for 3 weeks (Garg et al., 1984; Rajesh et al., 1984; Hoppe et al., 1989). Formic acid is water soluble, and residues are more likely to occur in honey than bee wax. The residues tend to dissipate with time and exposure to heat. Formic acid liquid and vapors can severely injure mammals, and it should be handled with care (Ellis, 2001). Flumethrin is a synthetic pyrethroid chemical. This chemical is packaged for beekeepers in a miticide impregnated strip, Bayvarol (Ellis, 2001). In Thailand, Bayvarol was found effective for controlling T. mercedesae (Pichai et al., 2008). The target site for flumethrin is the axonal transmission of nerve impulse (Ellis, 2001). For treating colonies, one strip of ten percent concentrations should be applied for every five frames of bees and brood. The strips should be suspended in the brood nest for 6-8 weeks. Flumethrin is a contact toxin. Thus, It is essential that strips are located in contact with the bee cluster. Flumethrin is distributed in the hive by bee to bee contact. Improper placement can result in poor control. Bayvarol strips can be used at any time except

10 65. Boonmee and Siriwat when bees are storing surplus honey for harvest. Flumethrin is a lipophilic molecule. If residues are detected in bee products, they are more likely to be found in bee wax than in honey (Ellis, 2001). Fluvalinate is another synthetic pyrethroid chemical. This chemical is packaged for beekeepers in a miticide impregnated strip, Apistan (Ellis, 2001; Lensky et al., 2001) and Mavrik (Lubinevski et al., 1988). Fluvalinate is similar to flumethrin in its mode of action and chemical properties. It is essential that strips are located in contact with the bee cluster. Apistan and Mavrik strips can be used at any time except when bees are storing surplus honey for harvest (Lubinevski et al., 1988; Ellis, 2001). Fluvalinate is a lipophilic molecule. If residues are detected in bee products, they are more likely to be found in bee wax than in honey (Ellis, 2001). Cymiazole is a modified heterocyclic compound, cymiazole hydrochloride. It is available as a powder that is dissolved in syrup and fed to bees, Apitol (Ritter and Schneider-Ritter, 1988; Ellis, 2001). The chemical is a systemic compound that is spread in the colony by food sharing. Cymiazole is absorbed by mites when they pierce and feed on haemolymph of adult bees. This chemical is a water soluble molecule. If residues are detected in bee products, they are more likely to be found in honey than in bee wax (Ellis, 2001). Toxicity to brood can be a problem if fed to the bees over a long time (Dietz et al., 1987). Also, residues in honey can be a persistent problem (Cabras et al., 1994). Amitraz is a behavior-affecting compound in the formamidine group of chemicals. The target site for Amitraz is the octopamine receptor. Octopamine serves as a fight or flight neurohormone in arthropods (Ellis, 2001). Mortality of mites can be expected to occur over several days following treatment. Amitraz is packaged for beekeepers in a miticide impregnated strip, Mitac (Wongsiri et al., 1987b) and Apivar (Ellis, 2001). Strips of Mitac or Apivar must be positioned in contact with the bee cluster. The active ingredient is spread within the colony by direct contact with strips and by bees contacting other bees with the miticide on their bodies. In Thailand, Mitac was found effective for controlling T. mercedesae. However, resistance to Mitac by T. mercedesae had already been detected (Wongsiri et al., 1987b). Amitraz is a lipophilic molecule. If residues are detected in bee products, they are more likely to be found in bee wax than in honey (Ellis, 2001). Folbex-VA (bromopropylate) is a diphenylcarbinol acaricide. This chemical is packaged for beekeepers in a paper strip which contains potassium nitrate and bromopropylate. Strips are ignited and left to smolder in hives. Bromopropylate is a persistent lipophilic molecule. If residues are detected in bee products, they are more likely to be found in bee wax than in honey (Ellis, 2001). Folbex (chlorobenzilate), Thymol (essential oil) and a mixture powder of sulphur and naphthalene (inexpensive) were found effective in controlling T. mercedesae (Wongsiri et al., 1987b; Woyke, 1987b; Pichai et al., 2008; Raffique et al., 2012). Coumaphos is an organophosphate compound. This chemical has been used in an emulsion in water that trickled over the bees, Perizin (Ellis, 2001) and Asuntol (Wongsiri et al., 1987b). In Thailand, Perizin and Asuntol were found not to be effective in controlling T. mercedesae (Wongsiri et al., 1987b). Currently, coumaphos strips are available, CheckMite+ (Pichai et al., 2008). The target site for coumaphos is synaptic transmission of nerve impulses. Coumaphos inhibits the production of acetyl cholinesterase, a chemical that breaks down the neurotransmitter, acetylcholine. Coumaphos is a lipophilic molecule. If residues are detected in bee products, they are more likely to be found in bee wax than in honey (Ellis, 2001). The ineffectiveness of chemical treatment is due to the short phoretic period of adult mites and the restriction of reproductive mites in the brood cells. T. mercedesae adults stay outside sealed brood cells about 2-3 days. Most mites have re-entered the cells before the next treatment is applied. Thus, the use of chemicals with prolonged continuous action is suggested to effectively controlling T. mercedesae (Woyke, 1987b). Integrated control The use of chemicals can be minimized by using them in combination with biotechnical methods (Ritter, 1993). In Thailand, Tangkanasing et al. (1988) reported that 95% of Tropilaelaps populations in A. mellifera colonies were decreased when organophosphate (Asuntol, 0.8 mg/l of water) was applied twice in 3 days while the queens were caged for 9-12 days. In Myanmar, Nyein and Zmarlicki (1982) caged the queens for at least one complete brood cycle (21 days), uncapping the dead brood to facilitate removal by worker bees and the inclusion of a fumigation regimen (in this case phenothiazine). However, the population of T. mercedesae was not reduced to an economically satisfactory level and the methods were labor-intensive. For a differing technique, beekeepers prefer to combine chemical treatment with the brooddeprivation technique. In this approach, all sealed brood is removed from the infested colonies, which are then fumigated. The most adult mites, having no capped brood cells in which to hide, are killed by the fumigant. Only one chemical treatment is required in this technique instead of three or four (Akratanakul, 1987). However, an integrated control seems to be complicated, time-consuming, and bee products can still be contaminated by the chemicals (Ritter, 1993).

11 Basic Res. J. Agric. Sci. Rev. 66 Non-chemical control Since T. mercedesae survives on adult bees for about 2-3 days, Woyke (1984) developed a non-chemical method to control the mite by caging queens for 3 weeks until all brood emerges. Removal of all brood without caging the queen was also suggested to deprive the mites from their food (sealed and unsealed brood). In both conditions, the last mites died within 3 days after all the bees emerged from the brood combs. However, colonies will suffer due to a significant decline in bee populations when using these methods and queen cells must be removed from colonies at least one and perhaps two occasions. These methods have been modified and refined subsequently (Woyke, 1985a, 1985b, 1993; Tangkanasing et al., 1988; Tran et al., 1993; Nguyen et al., 1995, 1997). One simple technique is to separate the colony. All brood with adhering bees is placed in a new hive, while the remaining bees and the queen remain in the original hive. The queenless colony will produce a new queen, but the resulting interruption in brood production will eliminate the Tropilaelaps mites. Likewise, in the queenright colony the mites also die since there is no brood to infest. At the end of four weeks, the two hives can be reunited (Woyke, 1993; Otis and Kralj, 2001). Honey production and colony strength are not affected if this method is done at the end of the honey flow (Otis and Kralj, 2001). In Vietnam, biotechnical methods were used by commercial beekeepers in controlling Tropilaelaps mites in colonies of A. mellifera with a high degree of achievement (Nguyen et al., 1995, 1997). Natural control A selection of strains of A. mellifera having resistance may be a solution to the Tropilaelaps problem in A. mellifera, which would be of great value to beekeepers throughout the world. At present, very few studies have been done to determine the potential resistance of A. mellifera to T. mercedesae. Kavinseksan (2011b) found that A. mellifera of Far-Eastern Russia or ARS Primorsky honey bee showed some degree of resistance to T. mercedesae, and had more resistance against the mite than Thai A. mellifera (Italian hybrid A. mellifera in Thailand). Also, the Primorsky bee was proven to be resistant to V. destructor and Acarapis woodi (Danka et al., 1995; Rinderer et al., 1997, 1999, 2000, 2001a, 2001b, 2003, 2010; De Guzman et al., 2001, 2007, 2008; Kavinseksan et al., 2016b). Other A. mellifera stocks resistant to V. destructor have been documented by several researchers. The following stocks are shown to have some degree of resistance to Varroa mites: hybrids of A. mellifera monticola in Kenya and A. mellifera ligustica in Sweden (Thrybom and Fries, 1991), A. mellifera in Gotland city, Sweden (Lattorff et al., 2015), A. mellifera carnica or native Austrian bee (Ruttner and Hanel, 1992), A. mellifera carnica from Yugoslavia (ARS- Y-C-1) (Rinderer et al., 1993; De Guzman et al., 1996), A. mellifera capensis and A. mellifera scutellata in South Africa (Moritz and Hanel, 1984; Strauss et al., 2015), A. mellifera in Uruguay (Rosenkranz, 1999), A. mellifera intermissa in Tunisia (Ritter, 1990) and A. mellifera in France (Le Conte et al., 2007). Possibly, some of these A. mellifera stocks may be resistant to T. mercedesae. Defense mechanisms of honey bees against T. mercedesae Known defense mechanisms of honey bees to T. mercedesae include non-reproduction of mites, colony migrations, grooming behavior, hygienic behavior, broodless periods and post-capping duration of the host bees (Wongsiri et al., 1989; Buchler and Drescher, 1990; Spivak and Reuter, 1998; Burgett et al., 1990; Rath and Delfinado-Baker, 1990; Koeniger et al.,1993, 2002; Harbo and Harris, 1999; Harris and Harbo, 2001; De Guzman et al., 2001; Kavinseksan et al., 2003, 2006; Woyke et al., 2004; Kavinseksan, 2011a; Khongphinitbunjong et al., 2012). Defense mechanisms of A. dorsata T. mercedesae feeds mainly on brood and can survive on adult bees of A. dorsata and A. mellifera no longer than 48 hours (Kitprasert,1984; Woyke, 1984, 1985b; Koeniger and Muzaffar, 1988; Tangkanasing et al., 1988; Delfinado-Baker et al., 1992; Rinderer et al., 1994; Kavinseksan, 2003). Therefore, new A. dorsata colonies start with uninfested bees because broodless periods during the swarming and absconding, and the longdistance migration interrupt the life cycle of T. mercedesae (Laigo and Morse, 1968; Koeniger and Koeniger, 1980; Thapa, 1998; Kavinseksan et al., 2003). Populations of T. mercedesae gradually build up in new A. dorsata nests after re-infestation from other Apis species or nests of A. dorsata present in the area during phoretic phase of the mites (Aggarwal, 1988; Kavinseksan et al., 2003). Infested workers or drones may join another colony in error in a process known as drifting or infested workers may deliberately enter in order to rob of stored food (Cook, 1987; Rath et al., 1991; Fries and Camazine, 2001; Koeniger et al., 2002; Oldroyd and Wongsiri, 2006). Also, mites may exchange hosts when conspecifics forage together in the same flower (Oldroyd et al., 1992). Moreover, aggregated colonies of A. dorsata are sometimes so close together that Tropilaelaps could easily scurry between colonies (Oldroyd and Wongsiri,

12 67. Boonmee and Siriwat 2006). However, the mite populations can not grow to a dangerous level in A. dorsata colonies since nonreproduction of T. mercedesae (64.4%), post-capping duration (a physiological factor), hygienic behavior (93%) and grooming behavior ( %) of A. dorsata can limit the population growth of this mite (Wongsiri et al.,1989 Burgett et al., 1990; Rath and Delfinado-Baker, 1990; Rath, 1991; Buchler et al., 1992; Koeniger et al., 1993, 2002; Kavinseksan, 2003, 2011a, 2012, 2013; Kavinseksan and Wongsiri, in press; Woyke et al., 2004; Khongphinitbunjong et al., 2014). Post-capping duration is a significant factor in limiting mite reproduction (Moritz, 1985; Moritz and Mautz, 1990; Le Conte et al., 1994; Spivak, 1996; Wilkinson and Smith, 2002). The postcapping duration of A. dorsata worker pupae (10.9 days) was shorter than that of A. mellifera worker pupae (12 days) about 10% (26 hours) (Qayyum and Nabi, 1968; Moritz and Southwick, 1992), which may result in lower growth of T. mercedesae populations in A. dorsata colonies. When the mite population increases beyond the hygienic and grooming capacities, then A. dorsata colonies migrate in order to minimize the mite populations by absconding their nests leaving the mites died in their deserted combs (Wongsiri et al., 1989; Koeniger et al., 1993; Woyke et al., 2000, 2004; Kavinseksan, 2003). Defense mechanisms of A. mellifera When A. mellifera was introduced to Asia, T. mercedesae has infested this honey bee species and become a serious problem to A. mellifera colonies due to lack defensive mechanisms necessary to regulate the mite populations (Wongsiri et al., 1989). Some studies of nonreproduction by T. mercedesae infesting A. mellifera have been reported. Non-reproduction of T. mercedesae in A. mellifera colonies was 18.3% in Vietnam, 7.3% in Afghanistan (Woyke, 1990) and % in Thailand (Ritter and Schneider-Ritter, 1988; Kavinseksan, 2003; Khongphinitbunjong et al., 2014). A. mellifera workers removed 91.3% of T. mercedesae mites from their bodies and killed 19.5% of the mites within 48 hours (Khongphinitbunjong et al., 2012). The average percentage of injured T. mercedesae in hive debris from colonies of Far-Eastern Russia A. mellifera and Thai A. mellifera was 71-77% (Kavinseksan, 2012). Hygienic behavior has been shown to help limit the population growth of both V. destructor (Boecking et al., 1992; Spivak and Reuter, 1998; De Guzman et al., 2002) and T. mercedesae in A. mellifera colonies (Ritter and Schneider-Ritter, 1988; Boecking and Drescher, 1990; Boecking et al., 1992). A. mellifera in Thailand detected and removed 52.6% of the worker pupae experimentally infested with T. mercedesae (Khongphinitbunjong et al., 2014). Far-Eastern Russia A. mellifera and Thai A. mellifera detected and removed 50% of freeze-killed brood (Kavinseksan et al., 2004). Defense mechanisms of A. cerana Worker bees of A. cerana were able to detect and remove T. mercedesae and V. destructor encountered in brood cells (98.8%) during nursing activities (Rath and Drescher, 1990). The bees removed mites from the colonies or chewed them with their mandibles (Wongsiri et al., 1989). In cage tests, A. cerana has more effient cleaning behavior to remove T. mercedesae from its body than A. dorsata and A. mellifera. Within 24 hours, 93.3% of the mites were removed from bodies of A. cerana, and the percentage of damaged T. mercedesae was 38.3% (Khongphinitbunjong et al., 2012). CONCLUSION T. mercedesae is a natural parasite of A. dorsata which is a native honey bee species of South-East Asia. This mite distribution seems to be limited in tropical Asia and coincides with the indigenous areas of A. dorsata. When A. mellifera was introduced into tropical Asia, T. mercedesae successfully switched from indigenous A. dorsata host to be A. mellifera and has become a serious problem to A. mellifera colonies in the region. The commercial movement of A. mellifera introduced T. mercedesae to areas where are outside the range of A. dorsata such as Afghanistan, South Korea, China and New Guinea. Thus, chemical, physical, biotechnical and combinations of chemical and biotechnical methods have been conducted to control T. mercedesae in colonies of A. mellifera but nothing offer complete control. The use of resistant A. mellifera stocks to T. mercedesae has been thought to be a better solution to the Tropilaelaps problem. The benefits of using resistant honey bees to parasitic mites include: less chance of contaminating bee products with undesirable chemicals, low cost of labor and materials and less risk of the mite developing resistance to acaricides. However, an A. mellifera stock resistant to T. mercedesae has not yet been reported. Based on the success of A. dorsata in controlling Tropilaelaps mites and the inability of A. mellifera to do so, the most promising avenue is the transfer of the behavioral resistance adaptations of A. dorsata to A. mellifera or the use of selection for resistant stocks and breeding programs to increase the level of resistant genetics in A. mellifera. For the transfer of resistant genes from A. dorsata to A. mellifera, this would be through the use of biotechnology since fertile hybrids between these two species can not be made. There are two major obstacles to such a transfer at present. The first obstacle

13 Basic Res. J. Agric. Sci. Rev. 68 is that the knowledge of the genetic basis in A. dorsata for this mite resistance is not yet know. Obtaining such knowledge will take a large amount of work over several years. The second obstacle is that the technology to make such transfers in honey bees is not yet available. Similarly, this will require much work and time. For selections, A. mellifera that has coexisted with T. mercedesae for the longest time should be tested for resistance to the mite because A. mellifera may have genetic traites which impart resistance to T. mercedesae which have already been selected for some degree in such populations. After that, breeding programs should be used to produce resistant hybrids or to increase the level of resistant genetics in A. mellifera to T. mercedesae. REFERENCES Aggarwal K (1988). Incidence of Tropilaelaps clareae on three Apis species in Hisar (India). In Africanized Honey Bees and Bee Mites, G.R. Needham et al.. (Eds.). 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