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STUDIES ON THE CONTROL, BEHAVIOR AND MOLECULAR MARKERS OF THE TRACHEAL MITE (ACARAPIS WOODI [RENNIE]) OF HONEY BEES (HYMENOFTERA: APIDAE)
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of
Philosophy in the Graduate School of The Ohio State University
B y
Diana Sammataro, B.S„ M. S.
a |c % $ $ $ af: afe % ifc % $
The Ohio State University
1995
Dissertation Committee: pgpov G. R. Needham
B. H. Smith
P. G. Parker Advisor Department of Entomology
(/£ ' l/S'-tQStexs'------UMI Number: 9612270
Copyright 1995 by Sammataro, Diana All rights reserved.
UMI Microform 9612270 Copyright 1996r by UMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17* United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 C opyright by
Diana Sammataro
1995 DEDICATION
To my husband, P. Kim Flottum for his patience. ACKNOWLEDGMENTS
I express sincere appreciation to Dr. Glen R. Needham and Dr. Brian H.
Smith, my co-advisors, for their help, insight and inspiration throughout this research. Thanks also go to the other members of my advisory committee, Dr. Patricia Parker as well as Dr. David L. Denlinger, the departmental chair, who offered encouragement, suggestions and comments. Gratitude is also extended to those faculty members in the Department of Entomology and elsewhere, who were particularly helpful including, but not limited to, Dr. Donald E. Johnston (1934-1994), Drs. W. Fred Hink, Richard W. Hall, John W.
W enzel, D avid J. H orn, Dana L. W rensch, H arald E. F. Vaessin, Kirsten Bremer and John D. Briggs.
Appreciation is also recognized to the staff of the Entomology Department,
Dr. Holly Ferguson and Ms. Susan Cobey, as well as Ms. Rena L. Peters, Ms.
Betty J. Hetrick and Ms. Linda D. Montano who made my life a little easier.
Heather Bailey, Rob Day and Jos£ G. Diaz, staff in the Zoology Department, have been exceeding generous with their time in taking photos for me. Thanks also go to my fellow graduate students, including George D. Keeney,
Seetha Bhagavan, Beverly Gerdeman, Sherry-Johnson, Keith Phillips and Jennifer Fain-Thornton for all their help and friendship.
To Walter and Claire Rothenbuhler, my special thanks.
Many acarology and beekeeping professionals shared invaluable experience and information, and I thank them all including Mr. Kerry Clark, Mr. Gordon Rudloff and staff at the USDA Honey Bee Laboratories throughout the United States, and participants in the Acarology Summer
Programs at The Ohio State University.
I was also reassured and solaced by Mrs. Dorothy D. and Dr. Susan O.
Kennedy, Gwen and the Drs. John, Jeff and Leslie Nystuen who have helped me greatly throughout my life. For my genetic beekeeping tendencies, I would like to recognize my maternal grandfather (George Weber) and his two brothers, who introduced me to the bees at a tender age. My materal aunts,
Mrs. Vera Campion and Paul and Lois LaRue, have been an ever-present guide since I was a younster.
And last but not in the least are my parents, Nelva Weber Sammataro
(1908-1991) and Joseph Michael Sammataro; you were always there for me.
Thank you all. VITA
April 13,1948 ...... Born—New York City
1970 ...... B.S., U niversity of M ichigan, A nn A rbor, MI
1977 ...... M.S., University of Michigan Ann Arbor, MI
1977—1980 ...... Peace Corps Volunteer, Honey Bee Specialist, Philippines.
1981— 1983 ...... Research Assistant, USDA Honey Bee Research Lab., Madison, WI.
1985 ...... Technical Lab Assistant, University of Connecticut, Waterbury, CT
1988— 1991 ...... Bee Supply Sales Manager, The A.I. Root Co., Medina, OH
1991—present ...... Teaching/Research Associate, The Ohio State University, Columbus, OH
PUBLICATIONS
Flottum, P.K. & D. Sammataro. 1988. The New Starting Right with Bees. The A.I. Root Co., Medina OH.
Robacker, D.C., P.K. Flottum, D. Sammataro & E.H. Erickson, Jr. 1983. Effects of climatic and edaphic factors on soybean flowers and on the subsequent attractiveness of the plants to honey bees. Field Crops Res. 6: 267-78.
Sammataro, D. & A. Avitabile. 1978. Beekeepers Handbook. Peach Mt. Press, Charlevoix, MI. Revised 2nd ed. Ch. Scribner's Sons, NYC, 1986.
v Sammataro, D, & G.R, Needham. 1995. Host-seeking behaviour of tracheal mites (Acari: Tarsonemidae) on honey bees (Hymenoptera: Apidae). J. Experimental & Applied Acarology. In press.
Sammataro, D. & R. Smith. 1994. Dissecting mites using the tracheal pull method. Video. Vesta Video Productions.
Sammataro, D. & R. Smith. 1995. Host-seeking behavior of tracheal mites on honey bees. Video. Vesta Video Productions.
Sammataro, D. Chapter 8; Insects: Diptera (Flies). 1994. For 3rd edition of R.A. Morse, ed. Honeybee Pests, Predators and Diseases. The A.I. Root Co., Medina OH. In press.
Sammataro, D„ E.H. Erickson, Jr. & M.B. Garment. 1983. Intervarietal structural differences of sunflower ( Helianthus spp.) florets and their importance to honey bee visitation. Proceedings Sunflower Research Workshop, Minot, ND.
Sammataro, D., E.H. Erickson, Jr. & M.B. Garment. 1985. Ultrastructure of the sunflower (Helianthus) nectary. ]. Apicultural Research 24(3): 150- 160.
Sammataro, D., P. Parker, & G. R. Needham. In press. Using PCR-based RAPDs (Random Amplified Polymorphic DNA) to determine differences in tracheal mite Acarapis woodi (Rennie) (Acari: Tarsonemidae) populations. Proceedings IX International Congress of Acarology, 1994; Columbus, OH,
Sammataro, D., P.K. Flottum & E.H. Erickson. 1984. Factors contributing to honey bee preferences in sunflower varieties. Proceedings Sunflower Research Workshop. Bismark, ND.
Sammataro, D., S. Cobey, B.H. Smith & G.R. Needham. 1994. Controlling tracheal mites (Acari: Tarsonemidae) in honey bees (Hymenoptera: Apidae) with vegetable oil. /. Economic Entomology 57(4): 910-916
Sammataro, Dv S. Cobey, B.H. Smith & G.R. Needham. In press. Vegetable- Shortening Patties Control Tracheal Mites (Acari: Tarsonemidae) in Honey Bees (Hymenoptera: Apidae). Proceedings IX International Congress of Acarology, 1994; Columbus, OH, BEE CULTURE Articles by D. Sammataro
1979 Sept. Vol. 114, pp. 458-9: Beekeeping in the Philippines. 1986 May pp. 251-253: Landscaping for home and hive; Aug. pp. 422: Landscaping for home and hive, II; Sept. pp. 461-2: Collecting Honey Bee Stamps; Nov. p. 584: Early Winters: Antique Equipment.
1987 Jan. Vol. 115, pp. 42: Bee Flora: The Milkweeds.
1988 March Vol. 116, pp. 139-141: Apiphilately; Dec. pp. 700-1,709,722: Research Review.
1989 Jan. Vol. 117, pp. 10-15,54: May the Forest Be With You; Wax Flowers (20-21); Feb. pp. 108-11: Package Primer (w / K. Flottum); March pp. 160- 163: Package Primer II; April pp. 226-7: Package Primer III; May pp. 297, 303: Raising Waxies; July pp. 406-7: Duct Tales, (w / K. Flottum); Aug. pp. 477-479: Simply Wax; Sept. pp. 532-3: Making Molded Candles.
1990 Jan. Vol. 118, pp. 20-6: Deserts, Droughts and the Drying of the American West; April pp. 220-22: Ukrainian Easter; Tracking Tracheal Mites (206-8); May pp. 284-6: Long Live the Queen; Aug. p. 493: Stamps in the News; Oct. pp. 596-9: Making Craftwax and Foundation Candles; Nov. pp. 663-5: Honey Candy.
1991 Jan. Vol. 119, pp. 32-38: Erosion.
1992 July Vol. 120, pp. 393,396-400: Conducting a honey bee emergency demonstration (made into a video).
1993 July Vol. 121, pp. 393-5: Perfect rounds.
1994 Jan. Vol. 122, pp. 30-39: Races. w/PK Flottum
1995 Feb. Vol. 123, pp. 80-81: 9th International Congress of Acarology: A honey bee mite round table, w / E. Sugden & K. Williams
American Bee Journal Articles Sammataro, D. etal. Why Soybeans Attract Honey Bees. Vol. 122 (7): 481, 1982.
Sammataro, D. Beekeeping in Developing Countries. Vol. 122 (ll):757-9,762- 4.1982.
HELDS OF STUDY
Major Field: Entomology Studies in: Apiculture and Acarology, Landscape Architecture, Urban Forestry
vii TABLE OF CONTENTS
DEDICATION...... ii
ACKNOWLEDGMENTS...... iii
VITA ...... v
LIST OF TABLES...... xi
LIST OF FIGURES...... xii
LIST OF PLATES...... xiii
CHAPTER...... PAGE
I. TRACHEAL MITES, AN OVERVIEW...... 1
Background on Endoparasites ...... 1 History of Acarapis in North America ...... 3 Current Studies of Acarapis ...... 7 List of References...... 8
H. CONTROLLING TRACHEAL MITES (ACARAPIS WOODI [RENNIE]) WITH VEGETABLE OIL...... 10
Introduction ...... 10 Materials and M ethods ...... 12 R esu lts ...... 16 v iii Discussion ...... 25 List of References ...... 27 m. HOST-SEEKING BEHAVIOUR OF TRACHEAL MITES CACARAPIS WOOD/ [RENNIE]) ON THREE DIFFERENT TREATMENTS OF HONEY BEES (APIS MELLIFERA L .)...... 30
Introduction ...... 30 Materials and M ethods ...... 34 Results...... 41 Discussion and Observations ...... 45 List of References ...... 50
VI. PRELIMINARY DOCUMENTATION OF POLYMORPHISMS OF THE TRACHEAL MITE (ACARI: TARSONEMIDAE) USING RAPDs (RANDOM AMPLIFIED POLYMORPHIC D N A ;...... 55
Introduction ...... 55 Materials and M ethods ...... 60 PCR Protocols ...... 64 Scoring Analyses ...... 67 R esu lts ...... 68 Discussion ...... 84 List of References ...... 86
BIBLIOGRAPHY...... 92
APPENDICES
A. Data Relative to Chapter II...... 104
1. Mite Infestation rates ...... 105
2. Hive Weights over a season, by treatment ...... 106
3. Number of Frames of bees by treatment ...... 107 4. Patty weight (grams) of both sugar/oil and TM 108
5. Hive and patty weights by treatment ...... 109
ix B. Data Relative to Chapter III...... 110
1. Measurements of four tracheal tubes ...... I l l 2. Volumetric measurements of tracheal mites...... 112
3. Main left first thoracic tracheal trunk...... 113 4. Six examples of DAMA movements ...... 114-119 5. Proportions of time spent of behavioral traits...... 120
C. Data Relative to Chapter IV...... 121
1. Sex Ratios of tracheal mites, August to October ...... 122
2. Sexes and Sex Ratios of tracheal mites in the sample set from which DNA was extracted...... 123-125
x LIST OF TABLES
TABLE PAGE
1.1 A list of endoparasitic mites on arthropods ...... 2
2.1 Percent mite infestations at Site 1, Prison Yard (PY) A piary, O hio ...... 23
2.2 Percent mite infestations at Site 2, Rings Road (RR) Apiary, Ohio ...... 24
3.1 Tracheal mite body measurements by different authors (|im)..32
3.2 Average weights (ICHmg) of live tracheal mites ...... 44
3.3 Average weights (ItHmg) of varroa mites and braula flies ...... 44
4.1 Percentages of colony loss in selected states, from first, second, third, subsequent years, to present (1995) .....56
4.2 Mite subsamples for RAPDs study ...... 63
4.3 Reaction components of the three protocols used ...... 66
4.4 Operon Primers ...... 69
4.5 Amount of honey bee DNA in dilution series ...... 82
xi LIST OF FIGURES
1.1 Map showing spread of tracheal mites since 1984 ...... 5
2.1 Average infestation rates of the 1991 vegetable oil patty study where the patties were fed twice during the summer ...... 17
2.2 Average infestation rates, by treatment, at Site 1,1992 ...... 19
2.3 Average infestation rates, by treatment, at Site 2, 1992 ...... 20
3.1 Proportion of time of each behavioral trait on the three bee treatments and on the total average of all treatments ...... 42-43
3.2 Behavioral choices that tracheal mites make when moving externally or dispersing on bees ...... 48
4.1 Test run of sample set ...... 71
4.2 Agarose gel illustrating types of water ...... 72
4.3 Agarose gel from two different PCR machines ...... 73
4.4 Agarose gel illustrating variability in mite samples ...... 75
4.5 Agarose gel illustrating variability in mite samples ...... 76
4.6 One complete sample run over two days ...... 77
4.7 One complete sample run over two days, cant'd ...... 78
4.8 Agarose gel illustrating spurious bands in primers with and without DNA template ...... 80
4.9 Agarose gel using different dilutions of the same bee ...... 83
xii LIST OF PLATES
PLATES PAGE
I. Tracheal M ite D raw ing ...... 6
n. Tracheal tube filled with all stages of tracheal m ites...... 33
in. Illustration of questing female tracheal mite on honey bee seta...... 37
IV. Thorax of honey bee showing suture lines, grooves, fissures and other features ...... 39 CHAPTER I
TRACHEAL MITES, AN OVERVIEW
Background on Endoparasites Mites in tracheal systems are apparently rare in arthropods and not well studied. Wehrle and Welch (1925) did preliminary work on Locustacarus trachealis living inside the grasshopper Hippiscus apiculatus H arris
(Acrididae: Oedipodinae) (See Table 1.1). They found these mites frequently and in large numbers, inhabiting not only the tracheal system of adult
females, but the abdominal air sacs as well. More recent work by Husband and Sinha (1970) summarized the life cycle of Locustacarus (=Bombacaraus ) spp. on Bombus spp. They found the mite actually overwinters with the
queen Bombus, then lays eggs in the spring, which hatch into larviform
females that migrate to infest the trachea of the queen's workers.
About the same time as Wehrle and Welch's investigations, a tracheal
mite was discovered in honey bees, Apis mellifera L., by Dr. John Rennie. Clark (1985) reports that earlier in the century, bees from the Isle of Wight
were dying from an unknown disease. At first it was thought to be a bacterial
disease, later identified by Zander (1909) as Nosema apis, a protozoan parasite of the bee's alimentary tract. The report of bees crawling out of their colonies
with disjointed wings, (called K-wings) and dying reached epidemic levels between 1904 and 1919.
1 2 Table 1.1. A list of endoparasitic mites on arthropods.
MITES INHABITING TRACHEAL SYSTEMS M ite Host H abitat Heterostigmata Podapolipidae, Locustacarus spp. Tracheal parasites L. trachealis: Orthoptera: Acrididae Hymenoptera: Apidae, Bombinae Coleoptera Bombacarus (=Locust.) buchtieri Bombinae, Psithyrus Coccipolipus hippodamiae* Hippodamia convergens Near Spiracles, may (=Tetrapolipus) Coletoptera: Scarabidae be external only Tarsonemidae, Acarapis spp. A. woodi Apis mellifera honey bees Tracheal parasites Mesostigmata Aceodromus Orthoptera Respiratory system Otopheidomenidae Katydiseius: Orthoptera Respiratory system Prostigmata Ereynetidae Hydraneies tropistcrnus'* Coleoptera: Hydraphilidae Subelytra, spiracles Tropisternus quadristriatus H. citrinensis T. lateralis numbatus Subelytra, spiracles
N o n -T r a c h e a l ENDOPARASITES Heterostigmata Podapolipidae Ovacarus spp. Coleoptera Reproductive tracts Stigmacarus lukoschusi*** Coleoptera: Curculionidae Stigmata cavity Hylobius abietis Podapolipus komareki H. abietis a Tarsonemidae Coreitarsoneminae Hemiptera: Coreidae Scent glands Eutarsopolis stammeri Pterostichus melattarius Genital tract or P. vulgaris (hemocoel; oviduct) Mesostigmata Laelapidae Dicrocheles phalaenodectes Hadeninae (Noctuidae) tympanicole Otopheidomenidae Otopheidomenis zalelcstes Lepidoptera: Noctudiae tympanicole Data supplied by Drs. John Kcthley, Robert Husband, J.W. Krantz and Barry OConnor; *McDaniel & Morrill 1969; **Kethley 1971, M*Feldman-Musham & Havivi 1977. 3 As the disease spread throughout western Europe, (Phillips 1922), Rennie and his associates began to look more closely at infected bees. They soon
discovered a mite living in the tracheal tubes and named it Tarsonemus
woodi (Rennie 1921), but it was later renamed Acarapis (from Acarus o r m ite, and apis or bee) by Hirst (1921), illustrated on Plate 1. After the discovery of the mite, the disease was called Acarine disease, or Isle of Wight disease. While Nosema and other diseases are sometimes
present in bees infested with these mites, the relationship was, and is still
unclear. Nevertheless, the identification and correlation of the Acarapis m ite
in Europe with the death of bee colonies led the United States in 1922 to
prohibit importation of all bees into this country by an Act of Congress (summarized by Phillips 1923) and to examine for the presence of the mite in bees sampled from apiaries in many states.
History of Acarapis in North America
Honey bees originally came to the New World with European colonists,
and if these insects had been infested, the mites would be already present in
the United States. However, this does not seem to be the case, as the results of
the 1922 survey and others conducted in the 1960's and 1980's were negative
(E. Mussen, pers. comm.). It does not mean the mites were not present, as they could have been missed in the bee samples, especially if infestation levels were low or sampling was taken during the active (summer) bee season when mite levels naturally decline.
O ther Acarapis species were found (A. externis, A. dorsalis) during this time as a result of intensive sampling conducted by the USDA Honey Bee 4 Labs (USDA Quarterly Reports/1960 to 1962), Although the genus was well- represented, why A. woodi was not present then remains a mystery. South America did allow importation of bees during this time, and in 1980 the tracheal mite was first reported in Colombia (Menapace, 1980). By 1984, it had reached Texas and in that year alone, was reported in FL, LA, ND, NE,
NY, SD & WI (See Fig. 1.1). Because of the extensive trucking of bee colonies from southern states northward to orchards for pollination, and for sale as package bees and queens to other beekeepers, the mite was spread quickly. In 1985, CA, DE, MA, MN, MS, NH, NJ, NM, OH, OR, PA, & SC reported Acarapis, spreading a year later into IN, KS, MD, MO, TN, & WA, as well as in the Canadian province of Manitoba. By 1989 to 1991, AK, IA, KY, MT, & UT and the Canadian provinces of New Brunswick, Nova Scotia and Ontario reported these mites (data obtained from a 1995 survey conducted by E. Mussen, personal communication). The only state uninfested to date is Hawaii; New Zealand and Australia are the only countries Acarapis-free.
The fast spread of this mite is attributed to the practice of mass transportation of bees into virgin territory. Beekeepers, by exchanging bees and queens, have successfully infected most of the Old and New World.
The direct loss of honey bees can only be approximated, but in 1994, $7.5 million in colony losses was postulated by Mussen (pers. comm.) with a total of $100 million in the past decade. The loss of colonies does not reflect indirect losses, such as reduced pollination of major agricultural crops, the elimination of feral bee colonies and the loss of beekeepers, the latter declining 25 to 40% in some states (Mussen, pers. comm.). Pattern of Tracheal Mite Infestation in the United States 5
Map by D. Sammataro© from 1984 1985 1986 E S S 1989-91 no data d*ta Euppliod by K MuMcn
Figure 1.1. Map showing spread of tracheal mites since 1984.
Added to this the loss of money to beekeepers from pollination fees, sales of bees and bee products, the total could well be over $180 million
(Robinson et al. 1980). With the introduction of the ectoparasitic mite,
Varroa jacobsoni Oudemans into the U.S. in 1986, the problem of bee death from parasitic mites has been exacerbated and has augmented the dollars lost.
The first studies of the effects of Acarapis on North American bees were done in Florida (E. Mussen, pers. comm.) and it was shown that even with highly infested bees, colonies could survive and produce honey. However, when heavy bee colony losses were reported in northern states between 1986 6 PLATE I. Adult male tracheal mite (a) and female (b), ventral view; and egg (c). Approx. 400x. Drawings by D. Sammataro.
a. 7 and 1989, it was discovered that where bees duster together over winter, infestation levels increased sharply and bees died. The correlation between infestation levels and bee death is still uncertain, as are the specific factors causing colony loss.
Current Studies of Acarapis
To better understand this mite, experiments to curb the mites and allow wax and honey products to remain uncontaminated, were undertaken by myself and colleagues. Reports of solid vegetable shortening and sugar patties controlling tracheal mites (Mussen, pers. comm.) led us to hypothesize that vegetable oil might be contributing to reduced mite levels in a bee colony, either through suffocation, toxic factors or some other mechanism. The research protocol and summary of our findings, that these patties fed continuously to bees controlled tracheal mite populations, is discussed in Chapter II, "Controlling tracheal mites ( Acarapis woodi [Rennie]) with vegetable oil," previously published in Journal of Economic Entomology (Sammataro et al., 1994).
Once it was shown that vegetable oil helps reduce mite levels in bees when fed in a patty form, the next question was what mechanism was responsible. It was hypothesized that the oil either kills the mites outright or interferes with their normal behavioral patterns. This work is examined in
Chapter III, entitled "Host-seeking behavior of tracheal mites (Acarapis woodi
[Rennie]) on three treatments of honey bees ( Apis mellifera)." Data obtained from Dr. Mussen cataloging colony losses since the tracheal mite's introduction show an interesting trend. Initially, bee losses accumulate quickly, reaching 25% to over 50% in some states. Then, eight to 8 ten years after the first introduction, the mite population seems either to disappear or to be at such low levels that colony losses are not significant.
Beekeepers speculated that the bees were becoming resistant to the mites, but a more likely hypothesis is that the more pathogenic mite populations diminished (killing their bee hosts), while the less lethal populations flourished.
To test this hypothesis, we decided to look for genetic markers from mites collected from colonies still alive and to compare them with markers from mites gathered from hives that died. The technique employed was RAPDs, and the results are explained in Chapter IV, "Preliminary work to document polymorphisms of the tracheal mite (Acari: Tarsonemidae) using RAPDs
(Random Amplified Polymorphic DNA)."
In conclusion, there is much yet to learn of these mites, including their life history and population fluctuations over the life cycle of a bee colony, as well as sex determination/ ratios and the evolution of the mite. This research has attempted to answer some questions and to give beekeepers a reliable control to preserve their bees.
References
Clark, K, J. 1985. Mites (Acari) associated withthe honey bee, Apis mellifera L. (Hymenoptera: Apidae), with emphasis on British Columbia. Master's thesis. Simon Fraser University, British Columbia, Canada.
Husband, R. W. & R. N. Sinha. 1970. A revision of the genus Locustacarus with a key to genera of the family Podapolipidae (Acarinea). Annals Entomol. Soc. Amer. 63: 1152-1162.
Kethley, J. 1971. Hydranetes, a new genus of Ereynetidae from hydrophilid beetles (Prostigmata: Ereynetidae). J. Georgia Entomol. Soc. 6(3): 176-184. 9 McDaniel, B. & W. Morrill. 1969. A new species of Tetrapolipus from Hippodamia convergens from South Dakota (Acarina: Podapolipidae). Ann. Entomol. Soc. Amer. 62: 1456-1458.
Menapace, D. M. & W .T. Wilson. 1980. Acarapis woodi mites found in honey bees from Colombia. Am. Bee J. 120 (11), 761-762.
Feldman-Muhsam B. & Y. Havivi. 1977. Stigmacarus lukoschusi, new genus and new species (Acarina, Podapolipidae) from beetles in Italy. Acarologia, 18 (3): 545-552.
Phillips, E. F. 1922. The occurrence of diseases of adult bees. USD A Circular #218,15 ppg.
Phillips, E. F. 1923. The occurrence of diseases of adult bees, II. USD A Circular #287,34 ppg.
Robinson, W. S., R. Nowogrodzki & R. M. Morse. 1989. The value of honey bees as pollinators of US crops. Am. Bee J. 129(6):411-423, 7: 477-487.
Sammataro, D., S. Cobey, B. H. Smith & G. R. Needham. 1994. Controlling tracheal mites (Acari: Tarsonemidae) in honey bees (Hymenoptera: Apidae) with vegetable oil. J. Econ. Entomol. 87: 910-916.
USDA Quarterly Reports. 1960. Entomology Research Division, Bee Culture Research Investigations. Madison, WI.
USDA Quarterly Reports. 1960 to 1962; 1970. Entomology Research Division, Bee Culture Research Investigations. Laramie, WY.
USDA Quarterly Reports. 1960 to 1961 Entomology Research Division, Bee Culture Research Investigations, Beltsville, MD.
Wehrle, L. P. & P. S. Welch. 1925. The occurrence of mites in the tracheal system of certain Orthoptera. Annals Entomol. Soc. Amer. 18 (1): 35-44.
Zander, E. 1909. Tierische parasiten als krankheitserreger bei der Biene. In Leipsiger Bienenzeitung, Jahrg. 24, heft 10: 147-150 and heft 11:164-166. (Also in Miinchener Bienenzeitung, 1090, heft 9.) CHAPTER II
CONTROLLING TRACHEAL MITES (ACARAP1S WOOD1 [RENNIE]) WITH VEGETABLE OIL
Introduction
Since 1984, when the endoparasitic tracheal mite Acarapis woodi (Rennie) was discovered in honey bees, Apis mellifera L., in Texas, efforts to control it have been intense. Migratory beekeeping practices and sale of package bees and queens rapidly spread the mite throughout the United States, and by 1988, it had reached Canada. Normal wintering losses vary widely but average *11% in most states; colony losses attributed to tracheal mites have been reported >31% in Pennsylvania (Tomasko et al. 1993). Nationally, >$165 million worth of pollination services, bees, and honey has been lost since the mites' detection, jeopardizing a significant component of U.S. agriculture (Robinson et al. 1989).
In general, mite populations flux out of synchrony with bees during the year. That is, as bee numbers increase in spring and reach their height during the summer nectar flow, mite numbers are at their lowest (Dawicke et al. 1992). This decreased proportion of bees with mites is likely due to a dilution effect from the rapid emergence of large numbers of young, uninfested bees.
In addition, the spread of mites may be hindered because infested forager bees would have less regular contact with uninfested younger bees in the months when outside flight activity is highest. During peak mite populations,
10 11 colonies with moderate to heavy mite infestation levels rear less brood
(Eischen 1987), have fewer bees, do not form tight winter dusters, and have increased honey consumption relative to uninfested colonies (Bailey & Ball 1991; Otis & Scott-Dupree 1992).
Control of tracheal mites has been difficult because of the location of their protected environment inside bee trachea. Initially, depopulation of bee colonies was used until it became impractical. Currently, the only registered treatment, Mite-A-Thol® (Mann Lake Supply, Hackensack, MN) or menthol crystals, an extract from the plant Mentha arvensis, is only partially effective since its benefidal qualities are temperature dependent. The other pesticide Miticur (amitraz, Hoechst-Roussel Agri-Vet, Somerville, NJ), has been withdrawn recently from the market. Formic add, a potentially effective agent, is not registered for this use. The need for expedient and bee-safe controls for these mites is imperative.
Vegetable oil has long been used in its solid form (shortening) mixed with granulated sugar as a carrier for antibiotics to treat colonies for bee diseases
(Wilson et al. 1970). Beekeepers are seeking products that can be used inside colonies without contaminating honey, pollen or wax. Serendipitously, vegetable oil was discovered to keep bees mite-free in laboratory experiments (Gary & Page 1987, Smith et al. 1991b). Also it has shown potential for controlling mites under field conditions (Delaplane 1992a, Calderone &
Shimanuki 1995). However, researchers treating bees with vegetable oils or combinations of oil and other ingredients, such as menthol (Delaplane 1992a), have found variable success. As a result, it was not clear (in 1991-1992) whether oil provides satisfactory mite control. The resolution of this inconsistency was the main thrust of our research. Studies were designed to 12 test whether control of mites could be obtained using vegetable oil-sugar patties and to test if exposure conditions were important. Additionally, the antibiotic Terramydn (TM) (Pfizer, New York) was
investigated more thoroughly because mite infested colonies often have assodated bacteria and other infections (Bailey & Ball 1991; Otis 1991). Terramydn commonly is applied to colonies for American foulbrood disease control and now is registered for this use.
Materials and Methods
Summer Application of Oil Patties. The first study, conducted in 1991, was designed to measure mite infestations throughout a summer season and to determine whether midseason oil patty treatment would prevent mite levels from increasing in the fall. Twenty honey bee colonies from The Ohio State University Rothenbuhler Honey Bee Laboratory were selected at random from two apiary sites =20 miles apart around the Columbus metropolitan area. Colonies were divided randomly into two treatments of 10, with each treatment consisting of two deep brood chambers and each containing nine frames. Each colony received either an oil-sugar patty or a sugar-only patty.
The 300g oil-sugar patty was made of 1:2 Crisco vegetable shortening:white sugar; the sugar-only patty was made with granulated sugar and corn syrup.
Treatments were administered on 6 June and 23 July on top of the frames (top bars) between the two broodnest chambers to ensure adequate exposure of bees to the treatment. When colonies collected surplus amounts of honey, supers with comb were added on top.
Bees were sampled from the inner covers of the honey supers and at the hive entrance to obtain older bees that usually have more mites, thus 13 facilitating detection. Gees infested with mites are not distributed randomly in a colony (Calderone & Shimanuki 1992). Samples were gathered on several occasions prior to treatment: 25 March; 1,11 April; 7, 30 May; 6 June; and 23 July. After treatments began, three samples were collected on 13
August; 17 September, and 18 October. A modified, hand-held vacuum was used to draw bees into a glass vial, after which they were stored in 70% ethyl alcohol in glass vials for later examination. The potassium hydroxide (KOH) method (Shimanuki & Knox 1991) was used to determine if mites were present. Briefly, the proximal end of the bee's thorax behind the first spiracle was sliced with a single-edge razor blade to obtain a disk of muscle and tracheal tissue. The disks were incubated overnight in a 7% KOH solution, rinsed with water, and the trachea examined for mites with a binocular dissecting microscope. Thirty bees were sampled from each colony. Occasionally, a single 100-bee sample was collected in addition to the 30-bee sample to confirm the accuracy of the mite population estimate from each colony. Trachea were scored as positive or negative, and the percentage of infested bees per colony sample was determined.
Continuous Exposure to Patties. In the second study (1992-1993) mite infestations were recorded in colonies exposed continuously to oil patties compared with no patty (control) from spring 1992 through spring 1993. For this study, we chose two apiary sites =30 mi apart in central Ohio. To ensure similar genetic lines between treatments, colonies were divided, equalized and requeened with Carniolan —Apis mellifera carnica—queens selected at random from a closed population (Page & Laidlaw 1985) maintained cooperatively between The Ohio State University and the California Bee 14 Breeders Association. To equalize bee populations/ each colony was assessed initially for population by counting frames of brood and bees. In total, 33 colonies were established by June 1992, and were assigned each treatment randomly, divided between two apiary sites; i.e., six colonies per treatment at site 1 and five per treatment at site 2 .
The Terramydn oil patty was included as a treatment to determine if antibiotic therapy influenced mite control compared to oil treatment alone. The oil patties were made from 1.35kg (31b.) Crisco vegetable shortening and 2.7kg (61b.) granulated white sugar; an addition of lOOg TM 50 was used for the TM patties (Wilson et al. 1970). Weighs of each patty placed in the colonies was recorded (see Appendix A).
To control Nosema disease at site 1, =3.8L (1 gallon) of 1:1 (by wt. water: sugar) syrup plus Fumidil-B (fumagillin [Al], lOOmg/gallon syrup; Mid-
Continental Marketing, Overland Park, KS) was fed to each colony on 24 March 1992 (before the study began) and again on 10 October 1992. In addition, these colonies were treated on 4 November 1992 with two Apistan strips (fluvalinate, 10% [Al]; Zoecon, Dallas, TX) per broodnest chamber to control varroa mites, Varroa jacobsoni. On 16 April 1993, colonies were given a supplemental feed of powdered sugar and syrup.
At site 2, Fumidil-B was fed to colonies on 14 April 1992 (before initiation of the experiment) and on 17 October 1992. Apistan strips were inserted on 23
October 1992 and in April 1993, these colonies were fed also a powered sugar patty supplement.
Bees were sampled by the same methods and from the same locations within colonies as in the prior study at = 1 mo intervals. Specimens were collected in glass or plastic vials and placed in a portable cooler for transport 15 to the lab freezer (-20°C) for storage. All colonies were treated equally, and normal hive management practices were followed. To ensure continuous bee exposure to the treatments, a new patty was provided when more than three quarters of a patty was gone. We also recorded any noticeable changes between treated or untreated colonies. Also, colonies were weighed at each sampling period using a hand-held scale (Hanson, Model 8920, Shubuta, MS,
The Viking, capacity 2001b.) to monitor bee population growth and changes in honey storage (Brimhall 1991).
Bees were thawed for 1 min at room temperature, after which the prothoracic tracheal tubes were pulled through the spiracle opening (Smith & Needham 1988). This technique proved faster than the KOH method. We examined each trachea with a binocular dissecting microscope and re examined them at higher magnification on a glass slide if no mites were visible initially. The percentage of infested bees per sample was calculated, and the mite loads were expressed as low, medium and high infestation levels. Queens recovered from site 2 in June 1993 were inspected for mites as well. The number of bees inspected varied, depending on the infestation level. If we detected mites within the first five bees sampled, we dissected at least 25 bees; if mites were not visible until after the 1 0 th bee, we examined up to 50 bees.
Statistical Analysis. Because the infestation rates were measured as percentages, an arcsine (square root of the proportion) transformation on all data was performed to normalize the distributions for analysis of variance
(ANOVA) (Little & Hills 1978; Sokal & Rohlf 1981). This transformation prevents the variance from being a function of the mean. 16 Data were analyzed from each site separately using SYSTAT (Wilkinson
1989), with the transformed responses as the dependent variable against treatment and date. Also examined were the treatments oil patty and TM patty and hive weights as dependent variables, against the response and dates.
R esults
Summer Application of Oil Patties. A three-way ANOVA was performed for treatment, date and site effects. A two-time application of oil patties (treatment effect) during the summer of 1991 had no effect on the mite population (F = 0.37; df = 1,172; P > 0.05) at either site (Fig. 2.1). Mite infestation levels in oil-treated colonies were not different from control colonies. Thus, well-populated, established colonies already infested with mites gained no protection from oil patties when fed twice at peak bee populations. Infestation levels ranged between 10 and 50% from March to April for treatments at both sites. In May, the mite levels fell until August, then rose in September. Since mite levels already were decreasing when the patties were applied in June and July, as seen in the control colonies, the beneficial effects of oil could not be established clearly.
Mite levels varied significantly among dates (F = 8 .8 ; df = 8,144; P < 0.05) and sites (F= 17.05; df = 1,144; P < 0.05). Therefore, mite levels changed considerably over time, and differences between apiary locations greatly affected colony conditions.
Interactions were never significant. If oil patty treatments had kept mite levels from rising in the fall, then, given our experimental design, the interaction terms should have been significant; that is, mite populations would have behaved equivalently in both groups of colonies before oil 17
t 1---- 1---- 1----1----1---- 1---- 1---- 1---- r Site 1, Treated Site 2, Treated n=3 n=7
40 J-i J?
20 O O •M° 10*V I M.
i i i i i i I i i i
3 60 Site 1, Untreated Site 2, Untreated n=4 n=6 50
30
20
3/25 4/1 4/11 5/7 5/30 6/6 7/23 8/13 9/1710/18 3/25 4/1 4/11 5/7 5/30 6/6 7/23 8/13 9/1710/18
Figure 2 .1 . Average infestation rates of the 1991 vegetable oil patty study where the patties were fed twice in the summer. Colony number (n) treated with oil/sugar patties (O), had no significant decrease in mite infestation levels at either site (F = 0.37; df = 1,172; P > 0.05) compared to those colonies treated with sugar-only patties (S). 18 treatment of one group. However, if oil had affected the mites, then mite loads in the oil-treated colonies should not have increased significantly in the
fall compared to control colonies. Continuous Exposure to Patties. For the second study (1992-1993), we
hypothesized that a continuous treatment of the patties may be required to keep mites at low levels throughout the season. Yearly mite population fluctuations, as observed in the 1991 study, led to the supposition that
autumn may be the crucial time to treat bees. Perhaps overwintering colonies
exposed to oil patties could reduce the mite levels. Two-way ANOVAs were performed on transformed percent infestation to test for effects of treatment,
date, and site in the two yards. Treatment was significant at both sites: Site 1, F = 14.95; df = 2,165; P < 0.001. Site 2, F = 5.541; df = 2,96; P < 0.001. Oil- and
TM-treated colonies had significantly lower mite loads than control colonies. Interaction terms were not significant.
The maintenance of low levels of mite infestation (<14%) was dramatic in oil- and TM-treated colonies at both sites (Figs. 2.2 and 2.3). In comparison, mite populations in the control colonies peaked between November and
February to >30% before dropping in April when the food resources and bee populations increased. When examining the average infestation rates over 8 mo at site 2 (Fig. 2.3), the oil- and TM-treated colonies rarely exceeded 10%, compared to 4-36% in control colonies. Again, mite populations are at their greatest between August and February. If colonies survive the winter infestations, mite levels appear to decline naturally (see Appendix A).
Mite infestation levels were significantly different between sites (F 2, 301= 16.8; P<0.05). The two apiaries were established two mo apart, and weather conditions, forage, and soil conditions were somewhat different. Site 1, Prison Yard, OH 1992-1993. 19
TM n=5 F=14.95, df=2,165; P<0.001
0 1 i x X r ± i1 1 1 i
OIL n=6
50 ' "I ""'"I- T r I 1 r CONTROL n=6 40
30
20 10 liliiilllii 4/92 5/92 6/92 7/92 8/92 9/92 11/92 12/92 2/93 4/93 5/93
Figure 2.2. Average percentage infestation rates by treatment at Site 1, 1992 to 1993 over 11 months with a continuous treatment of patties. Colonies (n=5) treated with TM patties (antibiotic and shortening/sugar) and shortening/sugar-only patties (n=6) had significantly lower mite infestations (F-14.95; df=2,165; PcO.001) over the season than the control hives (n=6). 20
Site 2— Rings Road, OH 1992-1993.
50
40 F=5.541, df=2,96; P<0.001
30
20
10
0 50 1------1------1------1------1------1------1 r OIL n=5 d 40 ■xi 8 30
2 20 d UQJ 5j io I 0 50 I I CONTROL 40
30
20
10
0 6/92 7/92 8/92 9/92 1/93 4/93 5/93 6/93
Figure 2.3. Average percentage infestation rates at Site 2, over 8 months. The colonies treated with TM-oil patties (n=5) and oil/sugar patties (»=5) had significantly lower mite infestations (F=5.541; df=2, 96; PcO.001) over the season than the control hives (n=5). 21 Hive weights did not change much in 1992, and honey production was below normal. The weights of the colonies varied little and were unaffected by treatment 2.84;P> 0.05 at site 1. F2t60=0.35;P>0.05; at site 2, see Appendix A). Frames of bees was counted in Site 1, Prison Yard (PY) before experiment began, and one year later (see Appendix A, data not analyzed) and at both sites, the amount of sugar/oil and TM patties was recorded. To determine if TM was more effective than oil, a two-way ANOVA was performed with treatment as the dependent variable. There was no statistical difference between the two patties at either site (Fj >101 = 0.18; P>0.05 at site 1.
F1(64 = 0.107; P>0.05 at site 2).
General Observations. Colony responses to mite infestations fluctuated widely, probably influenced by sampling techniques, ages of bees collected, queen supersedure, bees drifting from other colonies, or genetic resistance. It is difficult, despite using genetically similar lines, to account for all variations in a field situation and to make generalizations about colony performance in honey production, winter survival, and mite levels. However, there were some trends and noticeable changes in some colonies that are reported here.
Control colony mortality (four of six) was greatest at site 1. One colony died of starvation during the winter. Three of those four contained the highest infestation rates at that site prior to death (Table 2.1), All of the dead hives at this site were stained heavily with fecal material, and Nosema a n d dysentery may have been associated with these deaths. We did not observe fecal staining in the two remaining controls nor in the other treatments. In addition, 1992 had a poor honeyflow (below average honey collected) in this location. Thus, colonies were more stressed than during a good honeyflow, which may have contributed to the poor overwintering survivorship. 22 The oil-treated hives had the lowest mite populations of all treatments only after August. This may have been caused by the gradual replacement of all infested bees with younger bees protected by the oil patty. In one TM-treated colony the queen was lost, and this colony was united with another hive in May 1993. Another colony superseded its introduced queen in May 1992 and was removed from the study. Colonies treated with TM at site 1 appeared healthier and were, in general, more populous by the spring of 1993 than those treated with oil-only patties. Although these results are not statistically significant, this observation warrants further study.
At site 2, low mite levels were found in two of the control colonies throughout the year (Table 2.2), possibly as a result of the later start-up manipulations, such as colony splitting and requeening, which were completed 2 mo later than at site 1. Two other control colonies survived the winter despite early high levels of mites (51% - 92%, respectively), which dropped to <10% by spring.
Fecal staining on hive bodies was not apparent in site 2, where only one control colony expired. One TM colony died by spring 1993 after having >50% infestation the previous winter. Cause of death for this colony was not obvious because no common microbial diseases were evident and adequate honey stores were present. Also, most of the queens were dissected from site 2, and a total of four queens were superseded (two in the TM treatment, and one each in the oil and control colonies). One TM-superseded queen had a light mite infestation. Some queens’ trachea (two in the control, one in the TM treatments) were heavily scarred and blackened on one side indicating an old infestation. All of the other queens had no mites (one in TM, three in oil and one in control). 23 Table 2.1. Percentage of mite infestations at Site 1, Prison Yard (PY) Apiary.
Dates: £22___ £22__ £22___ Z22__ £22____222___ 1122___ 1222__ 223__ £23__ £21 TM Treatment Hive 1 11.8 33 21.4 35.5 0 5 19.2 17.5 14.1 11. t 10 Hive 4 0 0 0 0 0 0 0 0 0 0 0 Hive 7 0 0 0 0 0 2.5 3B.2 20 24 83 23.8 Hive 13 15.4 0 0 0 0 0 0 0 0 0 0 Hive 16 4.8 11.1 0 0 0 2,5 0 0 0 — 1.4 Avg, 6.4 8.8 24.3 7.1 0 2.0 11.5 7.5 7.6 4.9 7.0 SE 3.1 6.4 4.3 7.1 0 0.9 7.6 4.6 4.9 2.4 4.6
OIL Treatment Hive 2 11.8 17.8 5 6.7 0 0 0 0 0 0 3.3 Hive 5 8.7 6.7 0 0 2.5 0 0 0 0 2.2 2.2 Hive 8 0 0 0 7.7 0 1.1 4 6.7 11.1 2 1.2 Hive 11 5.3 0 0 0 0 0 0 2 0 0 0 Hive 14 0 4 3.3 0 5 5 0 7.9 2.2 2.2 6.0 Hive 17 60 23.1 50 64.5 0 5 0 0 1.8 0 0
Avg. 14.3 8.6 9.7 13.1 1.3 1.9 0.7 2.8 2.5 1.1 2.1 SE 9.34 3.9 8.1 10.4 0.1 1.0 0.7 1.5 1.8 0.5 0.9
CONTROL Treatment Hive 3 8.7 0 0 5.3 0 0.8 17.4 2.5 5 dead - ■■ Hive 6 27.3 44 80 46.2 15 25 23.6 15.1 35 dead __ Hive 9 7.7 11.1 34.6 48,4 20 30 65.4 75 78.6 dead Hive 12 50 26.7 18.5 3.2 0 17.5 2.2 0 0 0 1.2 Hive 15 0 3.7 0 5.1 7.5 7.5 13.9 8 15 13 Hive 18 6.7 16.7 5 22.9 45 40 48.6 75 71.4 dead — Avg. 16.7 17.0 23.0 21.8 14.6 20.1 26.2 30.2 33 7.5 7.1 SE 7.6 6.6 12.6 5.6 6.9 5.9 11.3 14.4 14.2 2.0 2.0
Note: SE = Standard Error. QUEEN'S condition: Hive #16, dead; #12, clean. Numbers are percentage of sampled bees with mites. Rows are individual colonies of bees. Collection Dates are: 4/29/92; 5/15/92; 6/9/92; 7/22/92; 8/21/92; 9/14/92; 11/12/92; 12/30/92; 2/9/93; 4/8/93; 5/4/93. 24 T able 2.2. Percent Mite infestation levels at Site 2, Rings Road (RR) Apiary.
______06-92 07-92 ___ 08-92 09-92 01-93 04-93 05-93 06-93 Queen Condition TM Treatment Hive 1 0 4 16.2 21.2 55.6 11.1 dead — dead Hive 4 0 0 2.1 2.1 2.2 15.6 4 8.9 lite, SS Hive 7 14.3 17.1 6.5 12.5 2 2 15 5 SS ? infest Hive 12 0 0 0 0 0 0 0 2.2 clean Hive 15 0 16.67 9.1 4 0 0 0 0 clean
Avg. 2.9 7.6 6.8 8 12 5.7 3.8 3.2 SE 2.9 3.9 2.9 3.9 11 3.2 3.5 1.9
O IL T reatm ent Hive 2 4.2 0 0 0 2.5 0 0 0 clean/amoeba HiveS 0 4 0 0 2.2 2.5 0 0 ? Hive 8 32 36.4 10 17.5 10.3 15 0 2.5 SS, 1-sidc black Hive 10 0 0 2 2.4 12.5 12.5 30 16.7 clean Hive 110 0 0 0 0 2 2.5 0 clean
Avg. 7.2 8.1 2.4 4 5.5 6.4 6.5 3.8 SE 6.2 7.1 1.9 3.4 2.5 3.1 5.9 3.3
CONTROL Treatment Hive 3 9.5 0 5.4 2.1 4 4 4 0 1-side black Hive 6 0 0 0 2 2.5 6 2 5 clean/amoeba Hive 9 8 11.1 10.3 15 36.4 5 dead — dead Hive 13 4 0 51 51 51 4.4 4 0 1-side black Hive 14 0 57.7 75 91.7 84 42.6 5.6 2.2 SS, clean
Avg. 4.3 13,8 28,4 32.4 35.6 12.4 3.1 1.4 SE 2 11,2 14.7 17.3 15.3 7,6 0.7 1.2
Note: SE is Standard Error. SS indicates queen was superseded by another queen; 1-side indicates tracheal tube had unilateral infestation by mites; black represents a tracheal tube that was very old, infested and no longer habitable. Two queens had amoeba (Malpighamoeba melltficae Prell) in their malpighian tubules. Numbers are percentage of sampled bees with mites. Rows are individual colonies of bees. Collection Dates are: 06-01-92, 07-20-92,08-21-92, 09-14-92, 01-28-93, 04-06-93, 05-11-93, 06-01-93. 25 D iscussion
In our initial study/ we placed oil patties in colonies during the summer to determine whether mite infestations could be reduced to non-threatening levels by fall. Summer treatments failed to prevent mite populations from rebounding when bees clustered during inclement weather. However/ mite levels were restricted by an uninterrupted application of oil, never reaching fatal populations. Although the deleterious effects of this mite are questioned by some (Bailey & Perry 1982), there is no doubt that this mite has had a significant impact on honey bee survival in some areas (Delfinado-Baker
1988; Otis 1990), especially in northern climates where bees are confined for several months. Distinguishing mite-infested colonies from mite-free ones without dissecting bees is impossible. Visible symptoms are unreliable even for highly infested bees but are reported to include bees crawling on the ground in front of the colony, K-winged bees (bees with hind wings held forward of forewings, making a 'K'), and dead hives with large amounts of remaining honey stores in the spring. Some colonies are abandoned outright in midseason when infested bees crawl out, leaving behind brood and food stores (D. S., personal observation; Thoenes & Buchmann 1992).
Additionally, a correlation of Nosema with mite infested hives has been reported by Jadczak (1990), but not found by others (Otis et al. 1992).
The greatest challenge to controlling this parasite is that they spend virtually all their lives within honey bee tracheal tubes. Following development and mating, females exit the trachea in search of new hosts.
They climb onto plumose setae and assume an ambush position (Morse &
Nowogrodzki 1990). Within 24 h, emigrating mites attach themselves to callow bees, <4 days old. Young bees are selected by the detection of cuticular 26 lipids not abundant in older bees (Phelan et al. 1991). Once a host is found,
mites enter and lay eggs. After =16 d, gravid females again emerge (Bailey &
Ball 1991; Pettis 1990) to continue the cycle. A single mite-laden bee can infest an entire mite-free colony within a short time.
Mite populations decline naturally, due to several factors. An interruption
in the brood cyde by swarming reduces infestation levels (Royce et al. 1991).
Similar reductions are found when older field bees (Delaplane 1992b; Eischen et al. 1988) and drones are driven from the colony (Dawicke et al. 1992; Royce
& Rossignol 1991). The cause of colony death remains to be determined but various factors have been suggested, including microbial diseases vectored by mites, stress, or blocking air flow in the tracheal tubes. Our data suggest that bee health, stress, and interactions between mites may contribute to colony demise. Spiro- plasmas or other bacterial or viral pathogens may cause bee death when heavily infested with mites (Bailey et al. 1980; Clark 1977). The addition of an antibiotic appears to be controlling some bacteria vectored by or the result of mites. The effect of spiroplasmas and other pathogens must still be tested in a rigorous manner.
Our study shows that oil treatment interferes with one or more aspects of the mite’s life cycle. The continuous presence of an oil patty with or without
TM helped lower tracheal mite populations and increased colony survivorship. The application of oil and TM treatments, combined with conventional management practices, may significantly suppress mite populations and thereby benefit all aspects of the beekeeping industry. 27 Acknowledgments
We thank Pat Radloff (Ohio State Beekeeper's Association), Daniel Mancoba Nkhanbule, and Aaron Gallagher (Ohio State University) for examining bees. Brian Burrell (Ohio State University) assisted with the 1991
study. In addition, we thank the California State Beekeeping Association, the
Ohio State and Northeast Indiana State Beekeepers Associations for financial support.
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Wilkinson, L. 1989. SYSTAT: the system for statistics. SAS Institute, Inc., Evanston, IL.
Wilson, W., J. R. Elliott & J. J. Lackett. 1970. Antibiotic treatments last longer. Am. Bee J. 110 (9): 348; 351.
Zander, E. 1909. Tierische parasiten als krankheitserreger bei der Biene. In Leipsiger Bienenzeitung, Jahrg. 24, heft 10:147-150 and heft 11:164-166. (Also in Miinchener Bienenzeitung, 1090, heft 9.) CHAPTER III
HOST-SEEKING BEHAVIOR OF TRACHEAL MITES (ACARAPIS WOODI
[RENNIE]) ON THREE DIFFERENT TREATMENTS OF HONEY BEES (APIS MELLIFERA L.)
Introduction
Camin (1963) separated parasitic mites by habitat and activity patterns, into several groups: host-, nest-, and field- or food-dwelling. The endoparasitic tracheal mite Acarapis woodi (Rennie) on honey bees is in the first group, spending their entire lives and several generations in a single host (Bailey &
Ball 1991; Giordani 1967). The fact that endoparasites interrupt the normal functions of their hosts biochemically, physiologically and behaviorally is not new (Brodeur & Vet 1994) and tracheal mites are no exception. Heavy mite loads result in diminished brood production (Eischen 1987), decreased bee populations, looser winter clusters, increased honey consumption (Bailey &
Ball 1991; Otis & Scott-Dupree 1992) and ultimately colony demise. First reported in the United States in honey bees from Texas apiaries in 1984, this mite is responsible for significant colony losses throughout North America
(Delfinado-Baker 1988). Prior to mite detection, 11% of colonies perished over the winter in most northern states, but in Pennsylvania, those losses increased to 31% due to tracheal mite infestation (Tomasko et al. 1993). In general, mite populations increase when bees are confined to their hive during the winter, and decrease in the summer when bee populations
30 31 are highest (Dawicke et al. 1992). As yet, the relationship between mite-
infested colonies and colony death is unclear. The evolutionary history of
tracheal mites is still unknown. Only a few mites inhabit the tracheal systems
of arthropods (Husband and OConnor, pers. comm.) and Eickwort (1993) speculated that Acarapis evolved from saprophagous or predatory mites
althougth many mites are phoretic on honey bees picked up on flowers (Seeman & Walter 1995). One reason for their appearance in bee hives may be the nesting behaviour of Apinae: two species (Apis mellifera and A ,
cerana) evolved to nest in cavities, which also provide habitats for such mites. While the whole-colony effects of tracheal mites have been studied by
many, not yet understood is how minute, eyeless mites leave their original hosts, negotiate the terrain of their old hosts and locate an oviposition site in a new bee's trachea.
Studying mite behaviour on bees in the laboratory is a challenge because of their small size. Females range from 120-189^ long, and males 96-102^ (see
Table 3.1). Giordani (1965a, 1967) had some success in keeping mites alive on
honey bee pre-pupae (11 d maximum), yet the life cycle was not completed in the lab. Mites feed on bee hemolymph obtained by piercing the trachea with
hollow but closed-ended, sharply-pointed stylets (0.2-0.26 mm long), which
are moved by internal chitinous levers (Hirschfelder & Sachs 1952). Once
pierced, the mouth opening, just below the stylets, is pressed to the puncture
wound and fluid is sucked up through the short tube (made up by the epistome, hypostome and two lateral palps) into the pharynx. Reports verify
that mites and eggs have been found in the air sacs of the abdomen and head, and on the wing bases of bees (Giordani 1965a, b). The state of those externally 32 Table 3.1, Tracheal mite body measurements by different authors (|im).
Fem ales______M ales______Eggs ______Larvae Length Width Length Width Length Width Length Ref. 108-134 156-210 Sachs, 1958 190 150 Rennie, 1923 123-180 76-100 96-102 62 110-128 54-67 Hirst, 1921 150.7±2.2 85.6±1.4 115.8±2 70±1.1 Eckert, 1961 143-174 77-81 125-136 60-77 Delfinado- Baker, 1982
laid eggs was not reported. Usually, all mite stages (eggs, larvae, adults) live entirely within the trachea except when females disperse to search for new oviposition sites (Smith et al. 1991a).
Phelan et al. (1991) showed that female mites are attracted to particular hydrocarbons from the cuticle of callow bees (<4 d old). Once a suitable host is found, the female enters the new trachea and lays =21-25 progeny (See Plate II) per oviposition period of 25 to 30 days (Pettis & Wilson 1995). Some daughter mites also disperse when the host bee is =13 d old, peaking sometime between
15 and 25 days. Mites searching for the prothoracic tracheae are vulnerable to desiccation or starvation (Gary et al. 1989; Smith et al. 1991a). Hirschfelder and Sachs (1952) reported that mites die after a few hours of questing on a bee.
The length of time for this to occur depends on the temperature and humidity or state of nourishment of the mite.
The outcome of this host/parasite interaction is difficult to predict. Mites afflict different ages of bees (hive bees and foragers) at different infestation thresholds (Royce & Rossignol 1990). Beekeepers and inspectors confirm that each colony reacts uniquely to mite infestations (Giordani 1977; Sammataro 33 Plate II. Tracheal tube filled with all stages of tracheal mites. E = egg; L = larva; A = adult. Microphotograph by D. Sammataro xlOO. 34 pers. obs.; Smith et al. 1991a). Response differences may reflect tolerance or resistance of bees (Gary & Page 1987), shifts in mite virulence or possible pathogenic factors. The male:female ratio appears to be female biased, but can change in response to seasonal differences or the host's age (Pettis & Wilson 1995; Smith, 1991).
Vegetable oil reduced mite levels in some preliminary lab and field experiments (Gary & Page 1987; Smith et al. 1991b), Subsequently, we found that feeding colonies continuously with a 2:1 white sugar:vegetable shortening patty (grease patty) reduced tracheal mite buildup in the apiary (Sammataro et al. 1994). We suggested that oil was interfering with the transfer of females to new host bees, either killing the mites outright, inhibiting the mite's ability to select callow bees, masking the attractiveness of young bees, or altering mite behavior in some way.
Knowing that colonies given grease patties during the fall and winter remained less infested, we wanted to document how adult mites reacted to oil-exposed bees compared to live and dead bees. To observe this behavior, we dissected mites from live bees, weighed them and then videotaped their movements on bee thoraces.
Materials and Methods
Mite Collection. Two hundred honey bees were gathered at random at the Rothenbuhler Honey Bee Laboratory (The Ohio State University) queen apiary using a hand-held modified vacuum (Sammataro et al., 1994) once a week during September, 1994. To increase the probability of collecting older and therefore more highly infested bees, we gathered drones and returning foragers laden with pollen from hive entrances. Groups of 25 - 30 bees were 35 placed in wooden, hardware doth (wire mesh) and glass cages and given water and fed 1:1 sugar:water, before taking them to the Acarology Laboratory.
These bees were the source of live tracheal mites used in the videotaping.
Our supply of callow bees was obtained from frames of capped bee brood, which were incubated at 34°C and 50% RH until edosing bees were evident.
Once hatched, the bees were placed in cages and fed water, pollen and syrup, and maintained in an incubator until needed. Specimen Preparation. Each older forager or drone was dissected for tracheal mites (Smith & Needham 1988; Smith et al. 1987) and when at least five infested tracheal tubes were accumulated on wet filter paper, a callow bee was selected and its head and abdomen removed. Live bees, even when secured to the microscope stage, moved too much for adequate observation and filming. Bees found dead (>24 h) in the bottom of the bee cages were the source of dead bee thoraces. Caged callow bees, allowed to feed on a «1.5 cm square piece of the vegetable shortening and sugar patty for 24 h were the source of oil-treated thoraces.
Each thorax was secured with Modeling Clay (Alex: Englewood, NJ) onto a piece of white foam board and placed under a Wild Heerbrugg MSA dissecting microscope with an attached Panasonic 3240 Color Video camera. Mite behaviour was recorded for 26 h during September and October. Videos were subsequently time-coded to facilitate analysis of time spent in different behavioural states.
Behavioral Analysis. Mite activity was categorized and recorded on thoraces with and without oil treatment and on thoraces from dead bees.
Mites moved vertically on bee setae and around the hair base. Time spent at any behavioral state (Altmann 1974) was recorded for each mite by noting the 36 time code at the beginning and ending of each activity. Speed and distance were recorded using the Dynamic Animal Movement Analyzer or DAMA software for the Macintosh (Electronic Learning Systems, Inc., Gainesville, FL, data in Appendix B). Host-seeking, feeding and moving behaviors of mites have been studied most in ticks (see Waladde & Rice 1982). For our purposes, we divided tracheal mite activities into five categories. The term "Wandering" describes mites moving purposefully. Wandering mites moved mostly on the setae, rarely touching the surface of the thorax. "Circling" mites did not move in a constant direction, but turned in circles or if stationary, legs were in constant motion. Circling mites moved mostly on the setae but some would circle on the thoracic surface. "Stopping" describes cessation of movement; specimens were probed occasionally to ascertain viability. "Questing" activity is used specifically for host acquisition (Camin 1963). First identified in tracheal mites as the "ambush" position (Hirschfelder & Sachs 1952), the mite crawls up on a bee setae, holds on with one of the "hind" legs, and extends all other legs in an apparent attempt to grasp a moving object. We observed the mites holding on with the fourth leg (See Plate III), Hirschfelder and Sachs (1952) illustrate a mite using the third leg. Questing mites were more easily transferred with a minutin-tipped or eyelash probe than mites exhibiting other behaviors. "Habitat Seeking" describes movement that emphasized niche location on the bee thorax, rather than transferring, circling or stopping.
These mites investigated and explored suture lines, the spiracle flap, grooves or fissures on the thoracic surface and were rarely found on setae (see Plate
IV). Once they located a suitable hiding place, most mites did not leave the habitat, remained still or left the field of view. 37 Plate III. Illustration of questing female tracheal mite on honey bee seta, x
=400. Drawing by D. Sammataro. Plate IV. Thorax of honey bee showing suture lines, grooves, fissures and other features, b. Close-up of spiracle lobe and tegula. Abbreviations: 2is, 2is, 3is, first, second and third intersegmental grooves; IT, propodeal tergum; cx 2, coxa 1; Epm, epimeron; N, notum; ns, external notal sulcus; pis, pleural sulcus; Set 2, scutellum; sf, scutal fissure; slobe, spiracle lobe of pronotum; sp, spiracle; tg, tegula; Drawings by D. Sammataro after Erickson et al. 1986.
38 39
Plate IV.
a
N2 ? Scl 2 n* A N3 X v 3 is
IT Hi N1
/ slobc Abdomen
cxl Head
t
■lobe 40 Statistical Analysis. Because mite movement was measured as percentages of total time, an arcsine transformation was performed on all data to normalize the distribution for ANOVA (Little & Hills 1978; Sokal & Rohlf 1981). This transformation prevents uneven grouping of data at one or the other end of the scale between 0 and 1. A total of 12 bee thoraces from the live bees and four from each of the other two treatments was used. We compared the results of the oil and dead treatments with the results from 26 combinations of four live thoraces, applying the t-crideal confidence interval
(P=0,05 3,3 ) around the means of the 12 thoraces for the questing and habitat seeking behaviors. Only two of the 26 combinations for habitat seeking and four for questing did not lie along the mean. Therefore, we performed a two- way ANOVA for each thoracic treatment despite the different sample sizes.
To determine if the sex of the mites influenced their behavior, we did a two way ANOVA for the questing and habitat seeking on live and oily thoraces. A post-hoc test, using Fisher's least-significant-difference test was also applied to verify the dissimilarity between treatments. Data from each treatment were analyzed separately using SYSTAT® for Macintosh with the transformed responses as the dependent variable against thoracic treatment.
Measurements. All live mite stages were weighed using a Mettler UMT2 micro-balance. Five to 10 live individuals were placed on a 0.5 cm square tared aluminum foil "boat" and moved to the balance. We wanted to discern if heavy life stage masses provided reservoirs for loss of water and energy reserves. Volumetric measurments of tracheal tubes were calculated by first measuring tracheal tubes under a light microscope on a 2 mm micrometer slide (American Optical Co., Buffalo NY). Mites were measured the same way
(See Appendix B #1-3). 41 R esults ' — * Videorecording. Dead bee thoraces (n=4) were used as platforms to continuously monitor nine mites over 6.7 hours. Of these, an average of 2 2 % of the time was spent wandering, 12% circling, 27% stopping, 34% questing and 6 % habitat seeking (see Fig. 3). Oil-treated bee thoraces (n=4) were used with 26 mites over 8.3 hours. Mites on oily bees averaged 13% of their time wandering, 25% circling, 13% stopping, 47% questing and 3% habitat seeking.
For live bees, twelve models were used for 39 mites over 15.4 hours, during which mites wandered 6 % of the time, 9% circled, 19% stopped, 13% quested, and spent 54% habitat seeking. Questing and habitat seeking are readily identified and were therefore more closely analyzed. The former is always on setal tips, the latter on the thorax surface. The former activity suggests that the mite is attempting to transfer, while the latter behavior implies acceptance of the host. Both questing and seeking behaviors were significantly different on each of the thoraces (F 2& =7.88, P < 0.001 and F 2 66 = 21.28, P < 0.001 respectively).
The post-hoc test demonstrated that for questing behavior, all the thoraces were significantly different (P < 0.05), but in the seeking behavior, dead and oily were not different, and dead and live were different at the P= 0.06 level. Weight Measurements. Mite weights are compared to two other ectoparasites (see Table 3.2 a & b). Live females weighed over twice as much as males, averaging 5.5 x 10 "4 mg and 2.6 x lO^mg for males. Larvae weighed even more than females, 6.47 x 10 4 mg while eggs were 3.74 x lO^mg, 42 a.
Mite Activity (n=9) on Dead Bee Thoraces
£ 250 & “ 200 X g 150.. 'o li oa 100 + a a 50 £ 0 W ander Circle Stop Q uest H Seek
B Dead Tx 1 a Dead Tx 2 □ Dead Tx 3 E3 Dead Tx 4 b.
Mite Activity (n=26) on Oily Bee Thoraces
250 T
Q>c ex. cn 200 at ill B m H 150 m O g 100 .. x> S. 50 ■■ s Ph FTTTTTTrrrrrn o I ' 1 Wander Circle Stop Quest HSeek H OIL Tx 1 H OIL Tx 2 E OIL Tx 3 BOIL Tx4
Figure 3.1 a & b. Proportions of time of each behavioral traits on the three bee treatments and on the total average of all treatments. 43 c.
Mite Activity (n=39) on Twelve Live Bee Thoraces
1 0 0
Wander Circle Quest HSeekStop d.
Average Percent Time Spent of Behavioral Traits on all three Treatments
1 0 0 T 90 ..
d Dead d Oil □ Live
Figure 3.1 c &: d. Proportions of time of each behavioral traits on the three bee treatments and on the total average of all treatments. 44 Table 3.2. Average weights (ltHmg) of live tracheal mites.
Females Males Eggs Larvae 3.40 (n=5) 1.00 (n=4) 5.00 (n=l) 5.40 (n=5)
4.20 (n=5) 1.80 (n= 1 0 ) 7.00 (n=l) 6.00 (n=5)
3.63 (n= 8 ) 2.29 (n=7) 1.00 (n-4) 8.67 (n=3) 6.00 (n=5) 4.00 (n=2) 7.67 (n=3) 0.105 (n=4)
0 . 1 0 (n= 2 ) 1.60 (n=5) 4.00 (n=l) 5.00 (n=5) 5.00 (n=2) 1.25 (n=4) 4.40 (n=l) 2.50 (n=2) 5.33 (n=3) 1.50 (n=2) A verage 5.50 2.61 3.74 6.47
Table 3.3. Average weights (mg) of varroa mites (Varroa jacobsoni) and braula fly (Braula caeca), ectoparasites of bees.
Varroa Mites Braula Flies in buffer in alcohol n=ll n=10 n=10 0.14 0.39 0.30 0.13 0.24 0.29 0.20 0.20 0.16 0.13 0.38 0.29 0.15 0.26 0.29 0.13 0.25 0.25 0.14 0.35 0.36 0.14 0.29 0.24 0.13 0.41 0.30 0.12 0.44 0.27 0.14 A verages: 0.14 0.32 0.28 45 Since female mites are two times larger than males, we propose this greater size gives her an advantage against desiccation when transferring to a new host. Additionally, the egg mass was about half the female's weight; thus the presence or absence of an egg within the female likely accounts for their wide weight range.
Other measurements included recording mite movements on the DAM A software. Because the mites were so small and they were walking in a field of tall setae, the tracking system of the computer was compromised, making it difficult to follow the mites. In addition, the system could only monitor x and y movements, not those in the z dimension. Examples are presented in Appendix B #4. Our measurements, therefore, concluded with the proportion of time spent at any of the behavioral traits, rather than distance m oved. Sex differences in mite behavior. Mite questing behaviour was not altered between males and females on live or oily bees (Fi >22 =0.25, P < 0.62.
However, habitat seeking was significantly different between the sexes (F lr22 = 7.42, P < 0.012 ). Data on dead bees were insufficient to do an analysis
(Appendix B 5). Female mites were active longer than males, due perhaps to their greater body mass. We observed questing and habitat seeking behavior in male mites, but these specimens had been collected from tracheae and their proclivity to leave this locale to seek out a new host is unknown (Giordani 1977).
Discussion and Observations
The first question we asked, did oil kill mites outright, has been answered.
Some oils can be toxic to mites (Agnello & Reissig 1994; Pless & Deyton 1995) 46 but vegetable shortening did not kill tracheal mite females for exposures of » 2 h. Sachs (1958) claimed that mites in petroleum jelly and water lived >48 h
and surmised they starved to death. Answers to the questions about the effect of oil, masking callow bee odor and altering mite behavior, have also been proposed. The two behaviors we observed—habitat seeking and questing, changed significantly depending upon the treatment, and we suggest that the oil concealed callow bee odor, forcing a change in the mite's behavior. In other words, mites transferred to oily bees would not seek out the tracheal tube but would quest or wander to find a more acceptable host. This process placed the mite at risk to exposure and desiccation, thus causing it to spend its energy reserves searching for another suitable bee.
Questing and wandering behavior is interpreted as a method of moving off undesirable hosts. For ticks, these are active strategies for leaving a protected rehydration niche to find new hosts (Sonenshine 1993). Questing ticks cue in on odor, heat, vibrations and carbon dioxide. Once contact is made, the acarine attaches to the host and if suitable, stays and feeds. Other work has shown that questing activity is influenced by ambient relative humidity and temperature (reviewed by Needham & Teel 1986).
Though blind, questing tracheal mites readily followed an eyelash probe moved above them, perhaps detecting air currents or infrared heat. Once a probe came within range, the mites would quickly grasp onto it by one of the hind legs, allowing the other clawed legs to fan out in anticipation of grasping onto another seta. However, mites had to be in close contact with appropriate, slow moving hosts or they could become dislodged or lost. Pettis et al. (1992) found mites dispersed more to new hosts during the nighttime 47 than during the daytime. From our earlier work we found mite populations built up in the winter months, when bees are confined and clustered together for warmth (Sammataro et al. 1994).
Once on the new host, the mite has several choices (see Fig, 3.2). It can attempt to abandon an inappropriate host (too old or oil-coated) by wandering, stopping (and dying) or questing, or it can recognize an acceptable bee host and habitat-seek to find a tracheal tube. Differentiation between living or unattractive bees required that the mites explore their new host. Acarines have no real antennae, their sensory structures, usually located on the legs or gnathosoma, include olfactory chemoreceptors, as well as gustatory, mechano-, photo-, thermo-, hygro-, osmo- and radiant-heat receptors (Evans 1992; Waladde & Rice 1982). A sensory cluster of setae on tibia I of adult tracheal mites is reported by Lindquist (1986) and Sachs (1953). In laboratory experiments, Hirschfelder and Sachs (1952) described mites being attracted to an intermittent expiratory air stream or to wing vibrations, especially if the mites were closely surrounded by a solid surface (positive thigmotaxis). Furthermore, they observed that the mites oriented by contact rather than odor, as the mites showed no preference to the scent of old or young bees. Phelan et al. (1991) reported that female mites were attracted to filter paper containing extracted cuticular hydrocarbons of callow bees. The females preferred the cuticular hydrocarbons, which contained saturated hydrocarbons rather than unsaturated compounds, over hexane controls.
Our observations demonstrated that mites could discern the different thoracic conditions only if placed on the host.
Parameters we could not control were the age, sex and the satiation level of each mite removed and placed on the different bee thoraces. We realized 48 Behavior Choices for Dispersing Tracheal Mites
Emigrating female
m ite from old host p✓ bee tracheal tube s . \ I ' ) Vj W anders
Q uests
Unacceptable I Acceptable; Bee Host Callow uee Host: I Wanders^ ■ ) Stops H abitat Seeking Behavior
' ( p ) ft
May enter a new bee host i trachea, a tracheal tube already occupied, or may quest or wander to find an unoccupied trachea. Dies
D.Sammalaro 1995©
Figure 3.2. Behavioral choices that tracheal mites make when moving externally or dispersing on bees. 49 that newly-mated female mites may behave differently from virgins or older females. Nevertheless/ the habitat seeking behavior was so distinct that many times the condition of the thorax could be discerned correctly by simply observing the mite's behavior.
Of concern to beekeepers is how long tracheal mites can live off their host. Mites in our study survived ®4 h on a bee thorax under the fiber optic (cold) light source before becoming torpid/ presumably through desiccation. Even though the relative humidity is likely to be higher in a colony than in our experimental conditions/ Hirschfelder and Sachs (1952) often found desiccated questing mites, which died in that position, after several hours. Giordani
(1967) recorded that well-fed mites lived up to 72 h off the host and mites reared on bee pupae, at a temperature of 28°C and RH of 70%, had the highest survival rates. In addition, it was reported that mites can survive longer outside the host by feeding at the wing base, under the tegula or in similar niches as well as through the thorax (Hirschfelder & Sachs 1952; Sachs 1951,
1953). We conclude that unprotected bees are at risk for up to 72 h but that oil-fed bees were protected from tracheal mites by becoming unpalatable hosts. The result is that at least some mites die of exposure while seeking another host.
Protecting honey bees from mite infestation is a difficult task. Production of honey, beeswax and certain crops that require bee pollination is in jeopardy because of the loss of bees to these parasitic mites. Furthermore, consumers continue to demand pure, uncontaminated hive products and acaricides used currently in the United States and elsewhere, are cause for concern. Various chemical controls of tracheal mites have been tried and residues have been detected in the honey and wax. Moreover, resistance by other parasitic mites 50 to some chemicals is also beginning to occur (Sugden et al. 1995; Imdorf et al.). Vegetable oil has been shown to be an effective, inexpensive, non-toxic treatment for controlling tracheal mites and now we better understand why this is so.
ACKNOWLEDGMENTS Special thanks go to the Eastern Apiculture Society of North America which supported this work, P. Kim Flottum for his comments and
equanimity and Mohammed Selim and others in the Acarology Laboratory for their understanding and patience with loose bees. Dr. Patricia Parker
from the Zoology Department, Dr. Brian H. Smith from the Entomology
Department and Dr. Bill Bruce from the USD A Beltsville Honey Bee Laboratory, who critically reviewed the paper, were most helpful. Thanks go to the support staff at the Rothenbuhler Honey Bee Research Laboratory, and especially to Robert D. Smith, President Vesta Video Productions, who spent many hours processing the videotapes.
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Pless, C. D. & D. E. Deyton. 1995. Control of San Jose scale, terrapin scale, and European red mite on dormant fruit trees with soybean oil. HortScience, 30: 94-7.
Rennie, J. 1923. Acarine disease explained. North Scotland College of Agric. Bull. No. 6 . 50pp.
Rennie, J., P. B. White & E. J. Harvey. 1921. Isle of Wight disease in hive bees. Trans. Royal Soc. Edinb. 52: 737-739.
Royce, L. A. & P. A. Rossignol. 1990. Epidemiology of honey bee parasites. Parasitology Today, 6:348-353.
Sachs, H. 1951. Zur morphologie von Acarapis woodi. I. Bau und funktion der mundwerkzeuge der Tracheenmilbe, Acarapis woodi (Rennie), 1921. [On the morphology of Acarapis woodi. I. Structure and function of mouth-parts of the tracheal mite]. Z. Bienenforsch. 1: 103-112.
Sachs, H. 1953. Zur morphologie von Acarapis. I. Bau und funktion der Biene von Acarapis woodi. [Structure and function of the bee mite Acarapis woodi]. Z. Bienenforsch. 2: 1-7.
Sachs, H. 1958. Versuch zur Ziichtung der Tracheenmilbe, Acarapis woodi woodi. [Experiments on rearing the tracheal mite Acarapis woodi woodi]. Z. Bienenforsch. 4: 107-113.
Sammataro, D., S. Cobey, B. H. Smith & G. R. Needham. 1994. Controlling tracheal mites (Acari: Tarsonemidae) in honey bees (Hymenoptera: Apidae) with vegetable oil. J. Econ. Entomol. 87: 910-916.
Seeman, O.D. & D. E. Walter. 1995. Life history of Afrocypholaelaps africana (Evans) (Acari: Ameroseiidae), a mite inhabiting mangrove flowers and phoretic on honeybees. J. Aust. ent. Soc. 34: 45-50.
Smith, A. W. & G. R. Needham. 1988. A new technique for the rapid removal of tracheal mites from honey bees for biological studies and diagnosis, pp. 530-534. In:: G. R. N eedham , R. E. Page, Jr., M. Delfinado- 54 Baker & C. E. Bowman, [eds], Africanized honey bees and bee mites, pp. 493-497. Chichester: Ellis Horwood, Ltd.
Smith, A. W. 1991. Population dynamics and chemical ecology of the honey bee tracheal mite Acarapis woodi (Acari: Tarsonemidae). Ph.D. dissertation. The Ohio State University, Columbus, OH.
Smith, A. W., G. R. Needham & R. E. Page, Jr. & M, Kim Fondrk. 1991a. Dispersal of the honey-bee tracheal mite, Acarapis woodi (Acari: Tarsonemidae) to old winter bees. BeeSdence, 1 (2): 95-99.
Smith, A. W., G. R. Needham & R.E. Page, Jr. 1987. A method for the detection and study of live honey bee tracheal mites ( Acarapis woodi Rennie). Am. Bee J. 127 (6): 433-434.
Smith, A. W., R. E. Page, Jr., & G. R. Needham. 1991b. Vegetable oil disrupts the dispersal of tracheal mites, Acarapis woodi (Rennie), to young host bees. Am er. Bee J. 131 (1): 44-46.
Snodgrass, R. E. 1956. Anatomy of the honey bee. Ithaca, NY: Comstock Publishing.
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Sonenshine, D. E. 1993. Biology of ticks, Vol. 2. New York: Oxford University Press.
Sugden, E. A., K. R. Williams & D. Sammataro. 1995. IXth International Congress of Acarology: a honey bee mite round table. Bee Culture 123 (2): 80-81.
Tomasko, M., J. Finley, W. Harkness & E. Rajotte. 1993. A sequential sampling scheme for detecting the presence of tracheal mite ( Acarapis woodi) infestations in honey bee ( Apis mellifera L.) colonies. Bulletin No. 871, Penn State Ag. Exper. Sta., University Park PA.
Waladde, S. M. & J. J. Rice. 1982. The sensory basis of tick feeding behaviour. In: F. D. O benchain & R, G alun [eds], Physiology of ticks., pp. 71-118. New York: Pergamon Press. CHAPTER IV
PRELIMINARY DOCUMENTATION OF POLYMORPHISMS OF THE TRACHEAL MITE (ACARI: TARSONEMIDAE) USING RAPDS (RANDOM
AMPLIFIED POLYMORPHIC DNA)
Introduction
The endoparasitic tracheal mite has left a path of destruction worldwide since the beginning of this century. First reported in 1906 by Rennie (1922),
this mite has been dispersed worldwide through the commercial exchange
and sale of queens and bees. The harmful effects of this mite were questioned by some researchers (Bailey 1965), but others have reported a significant decrease in honey bee survival (Delfinado-Baker 1988), especially in northern climates where bees are confined for several months (Sammataro et al. 1994). The causes of colony death are not clearly understood, but may include stress on the colony, microbial diseases or toxins introduced by mites. Within an apiary, mites are spread from colony to colony by drifting bees (Sammataro, personal observation).
The pathology, economic threshold level and ecology of this mite are still not defined, despite the efforts of many investigators. Since mites spend almost their entire lives within honey bee tracheal tubes, observing them is extremely difficult and destructive if samples or inspections are needed.
55 56 Consequently, no one has successfully maintained them in the laboratory for more than a few weeks (Giordani 1965 a, 1967).
Mite infestation rates fluctuate widely between honey bee colonies, rising to over 90% in some and never reaching ~10% in others (P.K.Flottum, pers.
comm.). After the initial year or two, a few colonies are lost, then many colonies are lost for the several years, followed by small losses again (see Table 4.1).
Table 4,1. Percentages of colony loss in selected states, from first, second, third, subsequent years to present (1995). The cause of colony death is attributed to tracheal mite infestations (not any other mite or pathogen), from when the mite was first reported, in the Date column.
State Date Total #Hives 1st year 2nd year 3rd year Subsq Present CA 1985 500K 2% 11.6% 20% 10% 10% DE 1985 2.5K 25 40 20 20 20 IA 1988 n/a 5 15 10 40 20 KS 1984 45K 0.44 1.1 2.2 0,3 0.3 MA 1985 n/a 10 18 35 15 8 MO 1986 24K 0 19 31 21 21 MS 1985 80K 15-20 30-40 40 30 20 OR 1985 70K 0 n/a 7.1 8.6 10 PA 1985 40K 10 30-100 20 10 10 SC 1986 30K 3.3 17 33 17 7 VA 1987 n/a 23 50 50 15 10 WA 1986 75K 1 1.3 6.6 10 15
Note: Data supplied by E, Mussen, personal communication. 57 One researcher (K. Delaplane, pers. comm.) reports that mites seem to disappear after 5-10 years. This has lead many beekeepers to believe that the bees are becoming mite-resistant, but this may not be the case since resistance mechanisms involved for any host-parasite interaction are complicated.
Definitions of Resistance. Using the classic examples of plants and insect pests (Schmidt & Roberts 1989), there are three types of resistance. 1. Non preference, or antixenosis—literally, against the stranger—means that the insect pest will not feed or lay eggs on a host plant if it lacks a particular characteristic or element. In other words, the insect will not prefer that plant when presented with a choice between different plants of the same species, if one of the plants contains certain chemicals distasteful to the insect, or possess a physical barrier (e.g. hairy stems or leaves) to discourage feeding or oviposition by the pest. The insect will choose the plant that has fewer defenses. When referring to mites and bees, Phelan et al. (1991) reported that tracheal mites prefer young bees, and it has been proposed that resistant bee strains had cuticular hydrocarbons that conferred a smell or taste of older bees, i.e. antixenosis (Smith 1991).
2. Antibiosis (against life) means the host plant has physical or chemical attributes that are harmful to the pest; e.g. nicotine (from Nicotiana spp.) and pyrethrum (from Old World Chrysanthemum spp.). No such example is reported in the honey bees. 3. Tolerance allows the host to accommodate the pest, yet continue to live and to produce a crop of seeds or fruits (i.e. to reproduce). There are races of bees, Buckfast and some of the Carniolan subspecies, that have lower 58 tracheal mite levels (Cobey 1995; Danka et al. 1995). Whether or not this is tolerance or some other form of resistance, has yet to be demonstrated. All of these forms of resistance place selection pressure on the host as
well as the parasite, and some parasites can change quickly to overcome defenses produced by the host. Resistance may take many generations of constant selection pressure before its genetic consequences are experienced
(see Futuyma 1979), especially if reproduction rates are low or if the relative fitness of the surviving individuals is expressed as a recessive trait. When referring to bees, a genetically recessive trait called hygienic behavior (removing diseased brood) was identified by Rothenbuhler (1964a & b) and Rinderer & Collins (1986), but is sustained only by careful breeding manipulations and instrumental inseminations of queen honey bees.
Then there is the question of thelevel of resistance. Resistance should be
quantified and compared to a control. This has not been done in the beekeeping industry when referring to mite-resistant bees. Viewed from an acarological perspective, when beekeepers claim to have
resistant bees, the first question should be—against which mites are these bees resistant: the tracheal mite or the ectoparasite Varroa jacobsoni? It is
unlikely that any one strain of bees can be resistant to both mites at the same
time. If bee breeders could select for resistance to one parasite, it may well be at the expense of resistance to the other mite or to the detriment of other traits (e.g. gentleness, honey production, winter survival). Tracheal mites and honey bees share a long evolutionary history
(Eickwort 1993) and have had a long time to adjust to any genetic changes in bees. Endoparasites are very rare in insects and are highly host-specific. For example, tracheal mites infect only Apis mellifera and A. cerana bees, live 59 most of their lives inside the trachea and are thought to be highly inbred. However, studies of trends in the lethality of parasitic bee mites have been largely overlooked by bee researchers.
Trends in tracheal mite virulence. Parasites represent extremes in specialized resource exploitation: they exhibit small size, loss of mobility in some life stages, and are adapted to rapid reproduction (Price 1980). They also exist in non-equilibrium conditions, and under ideal circumstances can out-produce their hosts (Pettis & Wilson 1995). Tracheal mites may have other advantages as well. E. Lindquist (pers. comm.) reports they have two chromosomes, n=2 (2w=4) compared to bees n=16 (2h=32). This small number, the arrhenotokous system of reproduction and the conservation of genes by inverted meiosis (Wrensch 1994) may give these acari further advantages over bees.
If a bee colony is infected by a single foundress mite, little mite recombinant genetic material will be carried into the next generation from that colony (Price 1980). Furthermore, when a strain of "virulent" tracheal mites invades a colony, both host and parasite are destined to perish. It is therefore evolutionary suicide for tracheal mites to kill their host colony.
For the bee-tracheal mite relationship to continue, one or both organisms must change.
Recently, the lethal effect of these acarines has been reported by several researchers to abate after 10 to 20 years of initial infection (K. Delaplane, pers. comm.). After many colonies have died off, the bees in the remaining colonies appear to be more tolerant of mites. But, not only are many 60 colonies lost in the process, especially in northern climates, but this trend
may be weather-dependent, cyclic, or have other unknown associations. I hypothesize that mite populations may be shifting or modifying in some way. To record this shift, I chose to look for genetic markers of mites,
to reflect this change in lethality, if it exists. It was important to determine if mite banding patterns were present that would segregate virulent and
surviving mites. The molecular technique chosen was RAPDs or Random
Amplified Polymorphic DNA (Williams 1990). In other words, could DNA markers for mites be detected and correlated with the shift in colony losses? The null hypothesis states that all mites are
the same genetically with respect to virulence, and any variation is due to seasonal (geographic) or beekeeping management differences. If the null hypothesis is not correct, then the geographical and management differences or other factors may not be reducing virulence. To test this, I expected to cluster the results in three ways. First, to construct a phylogeny of mites onto
which virulence and geographic regions could be mapped. Second, to
ascertain if geographical origin made a difference (were northern mites as
lethal as southern mites?), separating the ecological effects from the genetic causes. And third, to see which of these groupings died and which did not.
Materials and Methods
Mite Collection. Bees were gathered at random in selected apiaries from several cooperators in many states. At the Rothenbuhler Honey Bee
Laboratory, I used bees stored in the freezer (-20°C) from the 1992-1993 experiment (Sammataro 1994) as well as other bees collected in 1994. Bees were separated into high or low infestation levels and by colony survival. 61 Cooperators from several states also sent me bees from the following locations. Samples of heavily infested bees stored in ethyl alcohol came from South Dakota, sent in by Bob Reiners at the SD Department of Agriculture. Dr. Gloria Grandi-Hoffman sent frozen bees and data sheets from the Tucson Honey Bee Research Lab in Arizona. Dr. Jerry Bromenshek supplied data and frozen bee samples from University of Montana, Missoula. Bees also came from the Honey Bee Lab in Weslaco TX collected by Mr. James Baxter (see Table 4.2).
DNA Extraction Protocol. No set protocols have been developed for mites but several methods were tried. Much DNA is lost in a regular phenol:chloroform extraction (Hunt & Page 1992, 1995), so a method used for Drosophila (Pirrotta 1986) modified by Dr. H. Ferguson, proved successful. The bee trachea first were examined (Sammataro 1990; Smith 1988) to determine the infestation level and then placed in lOOfil of lysis buffer in an
Eppendorf tube (lOOmM Tris pH 8.0, lOOmM EDTA {Ethylenediaminetetra- acetic acid), lOmM NaCl, and 0.5% SDS (Sambrook 1989; Longmire 1988)).
Samples were pooled from 10 bees per colony, choosing as treatments two colonies each that either died or survived mite infestations in the same apiary over one winter. Then three tracheae were picked at random from the pooled sample and mites from each tube became a sub-sample, totaling six subsamples per treatment per yard (see Table 4.2), for a total of 816 mites. To obtain bee-free mites, the tracheal tubes were placed in a drop of glycerin on a glass slide and teased apart to release the mites, which were counted, sexed (see Appendix C for sexes of all tracheal tubes examined) and then stored in a clean Eppendorf tube in a drop of the buffer. Mites were Table 4.2. Mite subsamples. The sample label indicates apiary or state of origin (e.g. PY = Prison Yard, OH; RR = Rings Road, OH; B, G or YG, G, R or W identifies queen types from the Rothenbuler Bee Lab in Ohio). AZ, MT, TX, LA all refer to states from which samples were sent. Dead/Alive indicates condition of the hive at end of one year. The column of percent infested (% Infested) numbers, in parenthesis, indicates infestation level of bees for the subsample of the DNA test followed by initial mite count found in bees sampled. Total number of mites maintains the amount of material from which DNA was extracted for each subsample.
62 63 Table 4,2. Mites subsamples for RAPDs study.
No. Sample Dead/ % Infested Total # No, Sample Dead/ % Infested Total # Alive (in sample) mites Alive mites
1 PY18-2.2 OH D (40)66 15 31 AZ10-2 (13) 41 16 2 PY 18- 2 OH D (40)66 22 32 AZ 10 -3 (13) 41 5 3 PY 18- 3 OH D (40)66 22 33 TX1 A 100 18 4 PY 6-1 OH D (15) 12.5 17 34 TX2 A 100 4 5 P Y 6 -2 0 H D (15) 12.5 11 35 TX3 A 100 13 6 PY 15 -1 OH A (75) 13.5 7 36 SD 894-1 (64) 43.8 16 7 PY 15 -2 OH A (75) 13.5 25 37 SD 894 -2 (64) 43.8 27 8 PY 15-3 OH A (75) 13.5 5 38 SD 894 - 3 (64) 43.8 18 9 PY 12-1 OH A (0) 5.88 6 39 SD 879-1 (32) 15.4 12 10 PY 121.2 OH A (0) 5.88 2 40 SD 879 - 2 (32) 15.4 13 11 PY 12-2-2 OH A (0) 5.88 12 41 SD 879 - 3 (32) 15.4 15 12 RR 14 -1 OH A (75) 70.8 14 42 MT 244-1 A (8) 12.5 22 13 RR14-2 OH A (75) 70.8 22 43 MT 244 -3 A (8) 12.5 20 14 RR 14 -3 OH A (75) 70.8 22 44 MT 244 - 4 A (8) 12.5 22 15 RR 9-1 OH D (10)25 13 45 MT 252-1 A (69) 55 4 16 RR 9-2 OH D (10)25 20 46 MT 252 - 2 A (69) 55 13 17 RR 9 -3 OH D (10)25 6 47 MT 252 - 3 A (69) 55 11 18 35-1,G54 OH A 2 7 48 MT 252 - 4 A (69) 55 8 19 B35,YG-1 OH A (16) 14.3 30 49 MT 240-1 D (22) 41 21 20 B35,YG-2 OH A (16) 14.3 16 50 MT 240 -2 D (22) 41 17 21 6,W26 -1 OH A 8 16 51 MT 240 -3 D (22) 41 2 22 8.95, G71 OH A 0 52 MT 240 -4 D (22) 41 6 23 7.95, G51 OH A 1 53 MT 277-1 D (30) 18 22 24 5.95, B4 OH A 5 54 MT 277 -2 D (30) 18 6 25 2.95, R3 OH A 6 55 MT 277 -3 D (30) 18 5 26 A Z25-1 (30) 90 24 56 MT 202a -1 D (51) 8.5 16 27 PY 18,1.2 OH D (66) 40 13 57 MT 202a -2 D (51) 8.5 19 28 AZ25-3 (30) 90 19 58 MT 202a -3 D (51) 8.5 6 29 AZ 25 -4 (30) 90 36 59 G54.3,94 OH A 1 1 30 A Z 10-1 (13) 41 15 60 MT 273 -2 D (2)4 21 64 microwaved in =10|il of the lysis buffer for 4 min at 50% power then brought to the final volume of 400pl. A 1:100 dilution of Proteinase K (10 mg/mL)
(=4pl) was then added into each tube, which was gently agitated. Tubes were incubated at 65°C for 1-2 h, then 56pl of 8M potassium acetate (KOAc) was added and the tubes were placed on ice for 30 min to bring down cellular debris. Tubes were then centrifuged at 13,000rpm for 10 min. Using a disposable plastic transfer pipette, the top portion of the liquid was removed and transferred into a newly-labeled tube, after which 0.5 vol. isopropanol (=250pl) was added; tubes were held at room temperature (RT) for 8 min. The tubes were centrifuged again for 8 min, then decanted and the precipitate washed with 70% cold ethanol (-20°C). After a final 10 min run in the centrifuge, the pellet was dried overnight and resuspended in 30pl TE (lOmM Tris pH 7.6, ImM EDTA) overnight (O/N). To control DNAase, the sample was heated for 5 min at 65°C. DNA samples were stored in a 4°C refrigerator.
Bee DNA (from the thorax) was extracted by the same protocol, except 4|il
RNAase was added at the end of the procedure. Bee DNA was used as a positive control in all gels.
Another crude DNA extraction comprised of merely boiling the mites for
=4 min in a small quantity of TE. This was used in some cases as a comparison to the regular extraction protocol.
PCR Protocols
To minimize contamination, all PCR reactions were performed at a separate location from the genomic DNA extraction. All open autoclaved
Eppendorf tubes, aerosol pipette tips, and pipettors were subjected to at least 65 one hour of germicidal UV light. Originally I used autoclaved, double distilled water subjected to germicidal UV light, but when reactions began to fail, the water source was changed. HPLC Reagent ('Baker Analyzed'®, 4218-
03) water and Abbott Lab #4044 10 mL USP sterile water from glass ampules worked consistently. Three different protocols were used to optimize banding patterns.
Protocol #1 was modified from techniques developed in Dr. Patricia Parker's lab. Protocol #2 was altered from Tom Mullins' work for Haig et al. (1994). Protocol #3, adjusted to fit our requirements, came from Dr. Paul A. Fuerst, The Ohio State University, Department of Molecular Genetics. All reactants were removed from the freezer and kept on ice. Manipulations were conducted under a laminar-flow hood at first, but later abandoned as unnecessary. Components for the three protocols are summarized elsewhere (Table 4,3).
Protocol One (PI): Reactants included — HPLC Reagent dH20; 1.25mM
MgCl2,10x Buffer (GibcoBRL 200mM Tris-HCl (pH 8.4), 500 mM KC1); lOOpM dNTP's (Boehringer-Mannheim dNTP + Li salt); 400—800nM Primer; 0.5 U
Taq polymerase (GibcoBRL); l-5ng DNA. PCR set up was 45 cycles at 92°C 1 min, 35°C 1 min, 72°C for 2 min and stored at 4°C.
Protocol Two (P2): HPLC dH20 , 25mM MgCl^ 10X Buffer (identical to Protocol One), 1.25mM dNTP's, AmpliTaq (0.5 U/reaction), 10|iM primer, and lOng/reaction of DNA. PCR Parameters: 94°C 5 min, then 93°C 1 min;
38-45°C 1 min; 72°C 2 min for 45 cycles, ending with 72°C 5 min.
Protocol Three (P3): HPLC dH20; RAPDs Buffer (670|il 1M Tris pH 8.8, 67(il 1M MgCl2; 83^1 2M Ammonium Sulfate; 4|il 14M Beta-Mercapto- 66 Ethanol; 150pl glycerol; 23p.l ddH jO ); lOOjiM dN TP’s; 400 - 800nM Prim er;
0.5U Taq; 5—25ng DNA. PCR parameters were 1 cycle at 92° for 5 min, then
45 cycles at 92°C 30s, 35°C 1 min, 72°C for 2 min and stored at 4°C. All reactants and DNA were kept on ice. Into each 0.5mL PCR tube, 24pl of reaction mix were combined (except for P2, where 21|ol was aliquotted), to which 1—5pl DNA template was added, except for the negative control tube, where water was added. One drop (=100^1) of PCR grade Mineral Oil (Sigma M-5904) was placed on top (unless the Biometra was used, as no oil was required) and spun down. The samples were then placed in either the Perkin Elmer-Cetus 480 DNA Thermal Cycler or Biometra Uno Thermoblock (Biometra Inc.) that was prewarmed for 15 min.
Table 4,3. Reaction components of the three protocols used.
Protocol 1 M-l/rxn Protocol 2 |il/rxn Protocol 3 p.l/rxn h 2o 13.5 sam e 7.4 h 2o 15.9
IOx Buffer 2.5 2.5 RAPDs Buffer 2.5 dNTPs dilute 3.5 4 dNTPs dilute 2.5
P rim er 1 . 1.5 P rim er 3 Taq 0.2 0.1 Taq 0.1 M gCl2 25mM 2.5 4.5 DNA 1 5 D N A 1
Minigels. Agarose gels at 1.2% were made by microwaving an
Erlenmeyer flask containing 3.6g agarose with 300mL lx TBE buffer (89mM
Tris base pH 7.8; 89mM Boric acid; 2mM EDTA Na+2 x 2-H20) until just boiling and completely dissolved. After boiling, =3|il ethidium bromide 67 (l|ig/m L ethidium bromide) was added; the flask was then run under cold tap water until the temperature reached 50°C, and poured into a gel tray pre cleaned with 75% ethyl-alcohol. Loading wells were made with combs and the gel was allowed to solidify at room temperature for =1 hour. To run samples in the gel, 3pl Blue Juice (0.25% bromophenol blue, 0.25% xylene cyanol, 15% Ficoll in water, at room temperature) was added to each tube. The gel was then placed in the electrophoresis tank and filled with sufficient buffer (lx TBE) to just cover the well tops. Standard DNA ladders (100 bp and 1 Kb, GibcoBRL) were added (lpl ladder, 21jil 0.5x TBE, 3p.l BJ) to the end of the gels, and =»20pl of sample DNA was loaded into each well. The gel was run at 4 volts per cm length of gel. To visualize the bands, if ethidium was not added to the agarose mixture, the gel was stained with ethidium bromide (0.2g ethidium bromide in 20mL dHaO) for 10 min, rinsed in dH20 for 3 min and photographed under UV light.
Primers. Fifty-seven oligonucleotide primers from Operon Technologies, Inc. (Alameda, CA) were used. The primers are listed, with their sequence and molecular weight, in Table 4.4. They had to be screened first to choose those which would work with mites giving good, clear bands. Some primers had bands even when run without DNA, and were eliminated. Those that worked best were S-9, H-7, S-7 and AE-19.
Scoring analyses
Several month's effort were required to perfect the extraction technique, and to optimize PCR reactants (Black 1993; Kaliszewski 1992; MacPherson
1993; Williams 1993). To score gels, Dice’s index of similarity S= 2NAD / (2NAB 68 + NA + Nb) was used for the initial gels (Lynch 1990; Wetton 1987) to measure genetic similarity among individuals. The variable S represents the proportion of bands shared, while NA and NB are the number of bands scored unique to individuals A and B respectively, and NAB are the number of bands A and B have in common. However, later gels were scored using Black's equation (Black 1993) for the proportion of matches, M = NAB / NT, since bandsharing is not as important as the presence or absence of alleles when using RAPDs. The variable NAB is the total number of matched bands in individuals A and B, (both bands present or absent), and NT is the total number of possible bands of both individuals. This was done on some later gels until the method of scoring discussed by Haig et al. (1994) was discovered. Here one distinct band is chosen that is intense and reproducible. The presence or absence of that band (locus) is scored across populations. This last procedure was selected to score the rest of the mite gels.
R esults
There were many problems in obtaining distinct, reproducible results and before these were attained, the mite subsamples were used up. Therefore, no consensus of mite bands could be made to answer the original question and other techniques need to be explored.
Three different techniques were tried and currently are being modified, since no previous reaction protocols for mites existed. RAPDs is highly sensitive, which caused numerous difficulties. Even when all parameters were optimized and new reagents used, I failed to obtain reproducible banding patterns. The final conclusion was that too little mite DNA was 69 Table 4.4. Operon Primers.
Primer 5'______3' Mol. W t P rim e r 5 ' 3' Mol. Wt.
A3 AGTCAGCCAC 2988 Z8 GGGTGGGTAA 3139 A13 CAGCACCCAC 2933 Z9 CACCCCAGTC 2924 Z10 CCGACAAACC 2957 C9 CTCACCGTCC 2915 Z ll CTCAGTCGCA 2979 Z12 TCAACGGGAC 3028 G4 AGCGTGTCTG 3050 Z13 GACTAAGCCC 2988 G17 ACGACCGACA 2997 Z14 TCGGAGGTTC 3050 Z15 CAGGGCTTTC 3010 H 7 CTGCATCGTG 3010 Z16 TCCCCATCAC 2899 H13 GACGCCACAC 2973 Z17 CCTTCCCACT 2890 Z18 AGGGTCTGTG 3090 L12 GGGCGGTACT 3075 Z19 GTGCGAGCAA 3068 L16 AGGTTGCAGG 3099 Z20 ACTTTGGCGG 3050 K15 CTCCTGCCAA 2939 AE 1 TGAGGGCCGT 3075 AE 2 TCGTTCACCC 2930 Q9 GGCTAACCGA 3028 AE 3 CATAGAGCGG 2930 Q16 AGTGCAGCCA 3028 AE4 CCAGCACTTC 2939 AES CCTGTCAGTG 3010 S9 TCCTGGTCCC 2946 AE6 GGGGAAGACA 3117 S7 TCCGATGCTG 3010 AE7 GTGTCAGTGG 3090 S14 AAAGGGGTCC 3068 AE8 CTGGCTCAGA 3019 AE9 TGCCACGAGG 3044 T5 GGGTTTGGCA 3090 AE 10 CTGAAGCGCA 3028 AE 11 AAGACCGGGA 3077 Y14 GGTCGATCTG 3050 AE12 CCGAGCAATC 2988 AE 13 TGTGGACTGG 3090 Z1 TCTGTGCCAC 2970 AE 14 GAGAGGCTCC 3044 Z2 CCTACGGGGA 3044 AE 15 TGCCTGGACC 2995 Z3 CAGCACCGCA 2973 AE 16 TCCGTGCTGA 3010 Z4 AGGCTGTGCT 3050 AE 17 GGCAGGTTCA 3059 Z5 TCCCATGCTG 2970 AE 18 CTGGTGCTGA 3050 Z6 GTGCCGTTCA 3010 AE 19 GACAGTCCCT 2979 Z7 CCAGGAGGAC 3053 AE 20 TTGACCCCAG 2979 70 present in the reaction mixtures. Both problems and some of the solutions,
are segregated into four parts: Protocols, Negative Controls, Primers and DNA Dilutions. I have included in each section background, solutions tried
and an example of typical gels.
Protocols. The first challenge was to optimize the protocols. When mites
were first examined, they were within the bee trachea, but some bee DNA would carry over and trachea-free mites made up the remainder of the
experiment (Sammataro et al. in press). Early on, when evaluating if RAPDs was useful for bees and mites, there
was good success with PI (Fig 4.1). Then after about six to eight months, there was a period when no bands were showing up on the gels at all. By
changing to new primers, the bands returned. To optimize the results, P3 was tried and later, when other problems arose with this protocol, P2 was tested.
Protocol types gave different results in the number and amount of banding patterns, so choosing which one to use was critical to get superior results. The last two protocols seemed to work the best for mites, but much more experimentation and optimization is needed to get dependable results.
While good patterns were showing up and the information they depicted was coherent, it was never reproducible. Some gels showed that bands in the mite lanes were very different from each other. As illustrated in Fig. 4.1, mites from three states, OH, AZ and TX were examined but no geographic determination can be detected, exemplified in the diverse patterns in lanes
10 and 11 of mites from TX. In trying to replicate the same banding patterns in subsequent gels, I could never get consistent results. 71
10
* -.,1
Figure 4.1. Test run of sample set. Primer: H-7. PCR machine: Perkin Elmer-Cetus; Protocol: PI. Lanes 1-11: Sample # 24-34 (see Table 2.4 for complete information). Lane 1, mites from Ohio bee strains resistant to mites (Camiolan x Buckfast); Lane 2, mites from Ohio bee strains somewhat resistant to mites (Camiolan x Camiolan); Lane 3, mites from a highly infested bee from AZ; Lane 4, mites from a highly infested bee from Ohio; Lanes 5-6, mites from highly infested bees from AZ; Lane 7-9, mites from moderately infested bees from AZ; Lanes 10-11, mites from highly infested bees from TX; Lane 12, Negative Control (NC); last lane is lkb ladder. 72
Figure 4.2. Agarose gel illustrating types of water. Primer: L-16 #2; PCR: Perkin Elmer-Cetus; Protocol: P3. First and last lanes, ladder. Top gel, HPLC water, both gels ran at the same time. Lanes 1-3, same mite DNA (from a Buckfast x Camiolan bee, regular extraction protocol), replicated three times; Lanes 4-6, two female mite larvae boiled in TE (Crude extract); Lanes 7-9, Bee 198 (Hunt phenol/chloroform extraction); Lanes 10-15, Cordovan bees (regular extraction). Bottom gel same as above, except the water used was from the sterile ampule, Abbott Labs. Figure 4.3. Agarose gel from two different PCR machines. Left gel used Protocol 1 in the Perkin Elmer-Cetus PCR Thermocycler with oil overlay. Right gel used same sequence of lanes but samples were placed in the Biometra Uno without oil. Lane 1, lkb ladder; Lane 2, NC; Lane 3, Sample W24 #1, mites from moderately infested bees (Buckfast x Camiolan, regular extraction protocol); Lane 4, W24 #4, mites (same bees and extraction protocol as in Lane 3); Lane 5, W26 #2, mites (same bees and extraction protocol as in Lane 3); Lane 6, Bee. 74 Once good bands were achieved within a particular protocol, other problems, such as the condition, handling and age of the reactants, made a huge difference. In the attempt to keep equipment clean, everything was subjected to germicidal UV light. But a recent article (Dohner 1995) suggested that UV treatment may bring about reaction problems, such as causing bands to disappear; so the practice of UVing the water and oil stopped. This greatly improved the consistent visualization of the bands, and other types of non-UV'ed water were tested, including sterile water from glass ampules (USP) and HPLC water (see Fig. 4.2).
While the intensity of the bands changed, depending on the water used, other problems became evident. In order to solve these, different PCR thermalcyciers were tested to help optimize the gels and bands. Figure 4,3 illustrates that the same reactants and DNA template change according to the PCR machine used.
Negative Controls. Throughout this time, bands in the negative control lanes would appear and disappear with no apparent sequence. Significant effort was undertaken to resolve this problem. All pipettors were cleaned, wiped with bleach, rinsed with HPLC water and UV'd overnight. All tubes and pipettor tips were then treated with UV light and one researcher suggested that the DNA extractions be moved to another room, which was done. For a time, all reactions were mixed under a laminar flow hood.
Different labs were even tried, using their equipment, pipettors, reactants and w ater. 75
Figure 4.4. Agarose gel illustrating variability in mite samples. Primer: S-7; Water: HPLC; PCR: Perkin Elmer-Cetus; Protocol: PI. First and last lanes: Ikb ladder. Lanes 1-3, Sample #1-3, mites from OH, highly infested bees; Lanes 4-6, Sample #4-6, mites from OH, moderately infested bees; Lane 7, Sample #60, mites from MT bees with low infestation rates; Lane 8, Sample #59, mites from OH Buckfast x Buckfast bee, very low mite infestation; Lanes 9-11, Sample #58-56, mites from MT bees moderately infested; Lanes 12-14, Sample #41-39, mites from SD bees moderately infested; Lane 15, Sample #38, mites from SD bees highly infested; Lanes 16-17, Sample #35-34, mites from TX bees 100% infested; Lane 18, Sample #32, mites from AZ bees moderately infested. 76
1 5 10 15
Figure 4.5. Agarose gel depicting variability in mite samples. Primer: G-17; Water: HPLC; PCR: Perkin Elmer-Cetus; Protocol: P3. Lanes 1-4, regular extraction of 1 female mite from MT bees, highly infested (C-25 collected Jan. 1994); Lanes 5-6, same extraction, different mite; Lanes 7-10, mixed mites same protocol (no bands except 10); Lanes 11-14, Bee, same extraction protocol; Lanes 15-16, mite from crude extract; Lane 17, NC. 77
Figure 4.6. One complete sample run (samples 1 to 60) over two days. Primer: L- 16. PCR machine: Biometra; Protocol: P3, annealing temperature 37°C. Top Gel, first and last lanes: lkb ladder. Lane 1, NC; Lanes 2-17, Sample #1-17 see Table 4.2 for complete details. Samples 1-25, 27, 59 from Ohio bees; samples 26, 28- 32 from AZ; samples 33-35 from TX; samples 36-41 from SD and samples 42-58, 60 from MT. Bottom Gel, first/last lanes lkb ladder; Lanes 1-18, Samples #18-35. 78
Figure 4.7, One complete sample run (1 to 60) continued. Primer: L-16. PCR machine: Biometra; Protocol: P3, annealing temperature 37°C. Top Gel: first and last lanes, lkb ladder. Lanes 1-17, Sample #36-52 (see Table 4.2 for complete details). Samples 1-25, 27, 59 from Ohio bees; samples 26, 28-32 from AZ; samples 33-35 from TX; samples 36-41 from SD and samples 42-58, 60 from MT. Lane 18, NC. Bottom Gel, first/last lanes lkb ladder; Lanes 1-8, Sample #53-60; Lane 9, Bee; Lane 11, Sample #1; Lanes 12-18, Samples #3-9 repeated. 79 In some gels, the bands lined up with the samples, and other times they did not. It was difficult to score gels with any confidence when there were numerous bands in the negative control lanes. Initially, it was believed the reactants were contaminated from carry-over DNA in the pipettors. By switching to aerosol tips, this alleviated the problem for a time, but bands in the NC later returned. (Simultaneously, I conducted tests for contamination of primers; see Primers, below).
Similarity of bands was especially evident after several complete experiments were conducted, using all 60 subsamples (see Figs. 4.6 and 4.7). If the bands in the negative control lanes were few, there was much variability between the mite samples. But if bands in the NC were many, some lined up with bands in the mite sample. In cases where the NC bands were similar to the rest of the lanes, contamination was suspected and needed to be eliminated.
In those gels where the NC was clean, lack of similarity among the mite bands was evident. The samples contained the same species of mites, only from different colonies (see Figure 4.4 and 4.5). It may have been that these were the true banding patterns of the mites, the result of the different mite compositions or DNA concentrations within each subsample. This latter case was eventually revealed in the last experiments (see DNA Dilution).
To solve the NC problem, fresh reactants were obtained and primers were screened to clean up the contamination problems. In addition, by increasing the annealing temperatures from 35° to 45° this helped clean up the NC lane, (as suggested in Protocol 2). Incremental increases in the temperature quickly demonstrated which temperature was optimal for eliminating bands. 80
1 5 10
Figure 4.8. Agarose gel illustrating spurious bands in primers with and without DNA template. Water: HPLC; PCR: Perkin Elmer-Cetus; Protocol: PI. DNA template included two mite samples, extracted by the regular protocol, from OH bees (Buckfast x Camiolan lineage having low mite levels). In each case below, the first mite sample was W24, and the second W26. First and last lanes: lkb ladder. Lane 1, Primer Z-10 —(minus) DNA template; Lanes 2-3, Z-10 + the two mite DNA samples; Lane 4, Primer S7#2 —DNA; Lanes 5-6, +DNA; Lanes 7-9, Primer AE-6 —DNA, then +DNA; Lanes 10-12, Primer AE-16 —DNA, then +DNA. 81 Primers. Many of the primers had spurious banding patterns when run as negative controls. To test whether the primers were contaminated or just priming on themselves, gels were run with primers only, (i.e. all negative controls). Those that had few or no bands were chosen for subsequent experiments. When used with mite DNA, the contaminated primers could be easily identified and thus were eliminated from future reactions. By selecting those primers that had no bands, I also had to test which of these primers would be useful in discriminating between mite populations (see
Fig. 4.8). Some test runs showed that not all primers were useful for mites and eventually, those clean primers that gave distinct banding patterns and had few spurious bands, were chosen.
DNA Dilution. Despite using clean primers there was still too much variation between the mite gels. I could not get consistent results between similar gels. The final assumption was that the mite DNA was just too dilute for reliable results. Each sample contained either a single mite or multiple mites, as well as various mite life stages: adult, larvae, eggs (see Table 4.2). Each life stage may have different DNA components and concentrations, which could be responsible for the variations seen in the patterns. First, I decided to test the mite DNA extraction protocols (crude and salt extract) compared to the bee DNA template, in multiple, simultaneous reactions. I needed to ascertain if the bee lanes gave similar variations within the same template. The result (Figure 4.5) illustrated that crude as well as regular extracts of mites varied between lanes; the bee DNA varied m uch less. 82 Then a second experiment was conducted with a bee dilution series. Two drone thoraces were crushed together in lysis buffer and later aliquotted into three different tubes for DNA extraction. The standard extraction method was used, and each aliquot was further separated into separate tubes by three sets of dilutions, 1:50,1:100,1:500,1:1000 and 1:5000. Each tube was read on a spectrophotometer and two sets were chosen to run in the PCR, using P2. The amount of DNA in each sample is recorded in Table 4.5. The amount of DNA in the 1:5000 dilution was too low to read on the spectrophotometer, an occurrence I often encountered when trying to quantify mite DNA. No other method of quantifying DNA was tried.
T able 4.5. Amount of Honey Bee DNA in Dilution Series. Original amount of bee DNA added to dilutions was =0,19 pg/pl.
260 O.D. Dilution DNA added to Final [DNA] in make 100 0 |ll sample, ng/pl Bee Aliquot #1 0.000 1:5000 0.1 pi 0.038 0.0014 1:1000 lp l 0.19 0.0113 1:500 2|il 0.38 0.0332 1:100 lOpl 1.9 0.072 1:50 20|ll 3.8 Rpf* Aliijunt #2 0.000 1:5000 O.lpl 0.038 0.0014 1:1000 lp l 0.19 0.0109 1:500 2pl 0.38 0.034 1:100 lOpl 1.9 0.0682 1:50 20pl 3.8
The results (see Fig. 4.9), confirmed my suspicion that extremely low concentrations of DNA caused more and random bands to appear in the gel. 83
10 15
Figure 4.9. Agarose gel showing different dilutions of the two drone honey bees mixed together in the same extraction protocol. Primer: AE-9; PCR: Perkin Elmer-Cetus; Protocol P2 using sterile USP water and an annealing temperature of 39°C. Aliquot #1 of bee. Lane 1, NC; Lanes 2-3,3.8ng/pl DNA; Lanes 4-5,1.9ng/pl; Lanes 6-7 0.38ng/p.l; Lanes 8-9, 0.19ng/pl; Lanes 10-11, could not measure on spectrophotometer, but *4).04ng/|il. Next lanes used second aliquot of same drones. Lanes 12-13, 3.8ng/pl DNA (first lane blank); Lanes 14-15, 1.9ng/pl; Lanes 16-17, 0.38ng/jxl; Lanes 18-19, 0.19ng/pl; Lane 20, could not measure on spectrophotometer, but *=0.04ng/pl. 84 Since the PCR reaction protocols called for l-20ng of material, I am not surprised that I had trouble getting reproducible bands. I am assuming that mite DNA concentrations were similar to the 1:5000 bee dilution, yielding
>0.038ng/(xl. This would explain why the banding patterns of tracheal mites were so inconsistent—too little mite DNA in the samples.
D iscussion
No final results of the RAPDs tests on the tracheal mite Acarapis zvoodi (Rennie) could be made as I ran out of sample DNA by the time the technical problems were solved. The end result was that the mite DNA concentration was too low to quantify and too low for the PCR reaction to work reliably.
Each sample had between one and 22 mites (average was 13.9) and therefore, varying amounts of DNA. All the protocols called for at least lng of template DNA, which I never achieved in my samples. It has been proposed that each time a PCR reaction took place on each of my mite samples, different priming sites were recognized and amplified each time because of the low DNA concentrations, resulting in the variable banding patterns within a template.
The RAPDs technique was chosen because it showed great potential in answering questions such as shifts in genetic characterizations or distinctions between closely related species. Researchers have successfully examined insects (Apostol et al. 1993; Black et al. 1992; Cenis et al. 1993; Fondrk et al. 1993; H adrys 1992; H all 1990; H aym er 1995; H u nt & Page 1992; Kozol et al. 1994; Mendel et al 1994; Scott & Williams 1993; Shoemaker et al. 1994), plants
(Carlson et al. 1991; Russell et al. 1993; Weeden et al. 1993), fungi (Mozes- 85 Koch et al. 1995), the parasitic varroa bee mite (Kraus & Hunt 1995) and other
invertebrates (Jones et al. 1994; Levitan & Grosberg 1993), using this method. Other molecular methods need to be explored, including trying different DNA extraction protocols (Gang & Weber 1995; Hoy 1994; Meunier & Grimont 1993; Micheli et al. 1994; Phillips & Simon 1995; Schweder et al.
1995) to make tracheal mite PCR work correctly and reliably.
Another challenge associated with the RAPDs technique is that different primers can give different results. Thus, many primers must be used to get an adequate representation of the polymorphisms between samples. Molecular methods, such as using specific primers made from mites, arachnids or other organisms, will be essential to test the original hypothesis
proposed in the beginning of this chapter. Haymer et al. (1995) successfully
made DNA probes from Tephritid flies that could differentiate between species; this technique may be adapted to distinguish tracheal mite strains.
Future investigations are still necessary to address questions concerned with the long-range effect of mites on honey bee survival. For example, is
the fluctuation of bee survival or demise a sign of shifting bee tolerance and mite resistance, or of decreasing mite virulence? Selection pressures must be
taking place because if mites become too virulent, the host (colony) may perish and the parasitism was not successful. It is natural for the eventual moderation in this kind of parasitism (Price 1980).
Examinations into the interactions of bee resistance and changes in mite populations need to be explored. The decline in bee populations may also be part of the natural oscillation of the predator—prey population cycles (Lotka
1920). We may be looking at infestation levels only at a certain time, either at the upswing of mite populations overcoming the bees, or visa versa. The 86 eventual goal is to sequence and manufacture a mite primer that may be used to identify the degree of mite infestation in a colony, to determine not only the characterizations of mite lethality, but mite subspecies and even
Acarapis ancestry.
Acknowledgments
The following people provided invaluable assistance: Dr. Patricia Parker and her graduate students and staff; Dr. Greg Hunt; staff and students of Dr.
Paul Fuerst's lab, Tom Mullins lab, and all the people who responded to queries on the RAPDs and Acarology internet usegroups. Drs. Harald
Vaessin and Kirsten Bremer helped translate some German articles. Thanks go also to Mr. Dave Dennis, for the reproductions of the gels.
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Data Relevant to Chapter II
104 105 Appendix A, Chapter II.
Percent Infestation Rates at Both Sites for all Three Treatm ents
m CONTROL a TM Patty □ OIL Patty
A. 1. Mite infestation rates. 106 Appendix A. Chapter IX.
2, Hive weights over season by treatment, (in kg) and date of manipulation, for Prison Yard Apiary (PY), OH.
Queen# Hive # 06-09-92 07-22-92 08-21-92 09-14-92 12-30-92 04-08-93 05-04-93 WB5/ 1 58.97 67.22 76.66 74.30 71.94 66.23 44.23 W97 W97NM 4 40.82 57.15 50.80 52.62 51.71 44.45 47.63 W66 7 67.50 91.45 77.29 94.71 71.85 59.88 51.17 B56 13 48.35 61.92 68.40 73.12 67.22 53.07 48.35 W 57/ 16 39.19 54.43 62.60 55.52 60.96 55.52 0.00 W95 TM Avg 50.97 66.43 67.15 70.05 64.74 55.83 38.27
W97 2 38.10 56.25 48.99 60.78 49.90 44.45 47.63 W 64/ 5 40.10 63.69 76.66 68.40 0.00 51.89 49.53 W66 W 31/ 35.93 38.10 48.44 48.99 52.25 43.55 39.19 W97 8 W 59/ 11 45.36 74.84 60.33 69.85 58.06 44.45 42.64 W45 W 59/ 14 50.71 67.22 66.04 77.84 68.40 57.61 57.79 W45 W 62/ 17 39.01 44.00 15.88 44.45 49.90 49.90 46.27 W66 OIL Avg 42.22 57.57 53.47 61.91 45.72 49.48 47.08
W 66/ 3 63.10 107.32 91.40 113.22 90.81 0.00 0.00 W64 B4 6 37.20 49.90 54.43 55.34 42.64 0.00 0.00 B56/ 9 72.39 89.27 76.20 80.56 72.94 0.00 0.00 90 NM WC4/ 12 39.01 58.97 54.43 58.97 46.27 44.45 41.73 W66 supcrscd 15 36.74 35.38 48.08 31.75 44.45 42.18 43.55 Y72/Y7 18 48.08 68.95 60.78 68.04 69.40 0.00 0.00 B3 CON 49.42 68.30 64.22 67.98 61.08 14.44 14,21 Avg 107 Appendix A. Chapter II.
3. Number of Frames of Bees by Treatment after one year. Prison Yard (PY) Apiary, Ohio.
TM-Patty Hive # April 1992 April 1993 Hive 1 4.50 9.00 Hive 4 4.00 12.00 Hive 7 12.00 9.00 H ive 13 3.00 6.00 H ive 16 2.00 3.00 TM A verage 5.10 7.80
OIL-Patty Hive 2 5.50 7.00 Hive 5 6.00 3.00 Hive 8 5.00 3.00 H ive 11 8.50 11.00 H ive 14 6.00 9.00 H ive 17 10.00 5.00 OIL Average 6.83 6.33
CONTROL Hive 3 8.50 0.00 H ive 6 5.00 0.00 Hive 9 11.00 0.00 H ive 12 6.50 12.00 H ive 15 8.00 7.00 H ive 18 11.00 0.00 Control Average 8.33 3.17 108 Appendix A. Chapter II.
4. Fatty weights (grams) of both sugar/oil and TM (sugar, oil and Terramydn antibiotic) patties, and date of manipulation, for PY.
H ive No. 5/15 6/92 97/92 9/92 10/92 12/92 04/93 5/93 TM 1 159 142 167 330.1 275 — 316 4 108 210 247 270 295 318 216 7 202 204 229 323 275 326 374 13 210 213 150 289 — — — 16 140 112.5 186 301.5 292 297 OIL 2 192 225 203 300 321 142 235 5 158 269 202 300 300 —— 8 437 191 210 300 310 — 230 11 279 252.4 209 300 220 — 279 14 181 182.3 177 268.2 330 — 234 17 203 259 138.5 294 246 100 ---- 109 Appendix A. Chapter II.
5. Hive and patty weights by treatment and date of manipulation, Rings Road (RR) Apiary, Ohio.
Hive Weights RR, kg Patty Weights, grams Queen H ive # 06-92 07-92 09-92 01-93 06-93 08-92 07-92 09-92 01-93 B6 Hive 2 44.00 62.60 53.07 55.34 0.00 146 282 296 302 B1 HiveS 50.80 62.60 57.15 37.20 74.39 183 290 266.4 301 NM W60- Hive 8 46.27 63.50 61.69 54.43 68.49 166 262 281.3 380 G60 B9 Hive 10 46.27 62.60 58.97 44.45 55.34 211 313 298 299 W71/ Hive 11 58.06 53.07 49.90 54.43 77.11 158 282 298 277 Y9 T M A vg. 49.08 60.87 56.16 49.17 55.07 172.8 285.8 287.9 311.8
B6 Hive 2 48.99 57.15 64.41 56.51 68.04 196 235 300 260 B1 HiveS 39.92 57.15 50.80 46.27 80.74 178 183 300 255 NM W60- Hive 8 37.20 44.45 46.27 45.36 48.08 158 209 300 328 G60 B9 Hive 10 46.27 58.06 56.25 48.99 46.27 239 229 300 300 W71/ Hive 11 33.57 45.36 45.81 40.82 53.98 234 278 300 363 Y9 OIL 41.19 52.44 52.71 47.99 59.42 201.0 226.8 300.0 301.2 Avg.
RICK Hive 3 41.73 56.25 57.15 48.08 74.84 B4 Hive 6 45.36 61.24 52.62 43.55 97.52 W60/ Hive 9 43.55 58.06 44.45 39.01 0.00 G60 W19/ Hive 13 38.10 55.79 47.17 46.27 63.50 W55 W50 Hive 14 58.06 67.13 62.60 43.55 54.43 CON 45.36 59.69 52.80 44.09 58.06 Avg. APPENDIX B
Data Relevant to Chapter III
110 I ll Appendix B, Chapter III.
1. Measurements of four tracheal tubes giving volumetric information, in m m 3. Tube D D1 d d2 h Dd D*+Dd+d2 V o lu m e 1 0.17 0.029 0.16 0.03 1.40 0.027 0.082 0.0299 1 0.13 0.017 0.10 0.01 0.63 0.013 0.02 0.0066 T otal 0.0365 2 0.16 0.026 0.12 0.01 1.21 0.019 0.059 0.0188 2 0.18 0.032 0.15 0.02 1.35 0.027 0.082 0.0289 Total 0.0477 3 0.17 0.029 0.12 0.01 0.50 0.020 0.064 0.0083 0.13 0.018 0.12 0.01 0.45 0.016 0.048 0.006 Total 0.014 4 0.19 0.17 1.70 0.032 0.032 0.0144 4 0.18 0.16 2.30 0.029 0.029 0.0173 4 0.17 0.15 1.73 0.026 0.026 0.0115 4 0.15 0.13 1.81 0.02 0.02 0.0092 avg 0.17 0.03 0.15 0.02 1.89 0.026 0.079 0.0391 sides 4.1 0.14 0.02 0.17 0.03 0.45 0.024 0.07 0.0085 4.2 0.13 0.02 0.12 0.01 0.80 0.016 0.05 0.0098 4.3 0.13 0.02 0.10 0.01 0.20 0.013 0.04 0.0021 4.4 0.50 0.25 0.50 0.25 0.46 0.25 0.75 0.0903 0.2024 Total 0.2415
Equations from Oberg & Jones 1966 Volume of Frustrum of Cone, V = 1.0472 h (R^Rr+r2) = 0.2618/i (C^+Dd+d2)
R 112 Appendix B, Chapter III.
2. Volumetric measurements of tracheal mites, x ItHmm3. See Table 3.1 for references of the female and male mite measurements. Current column indicates measurements taken by Sammataro from buffer-stored specimens. Average volume for females = 5.74 xlCHmm3; males = 2,89 x 10*4.
Females L (a) L/2 W (b) W /2 b2 V o lu m e 0.19 0.095 0.076 0.038 0.001444 5.75 0.123 0.0615 0.0842 0.0421 0.001772 4.57 0.1507 0.07535 0.081 0.0405 0.001640 5.18 0.143 0.0715 0.1 0.05 0.0025 7.49 C u rren t A d u lt 0.15 0.075 0.05 0.025 0.000625 1.96 A d u lt 0.12 0.06 0.05 0.025 0.000625 1.57 A d u lt 0.13 0.1 0.08 0.035 0.001225 5.13 Fem aleL rv 0.20 0.1 0.07 0.035 0.001225 5.13 0.18 0.09 0.087 0.0435 0.001892 7.13
M ales 0.15 0.075 0.062 0.031 0.000961 3.02 0.096 0.048 0.07 0.035 0.001225 2.46 0.1158 0.0579 0.06 0.03 0.0009 2.18 0.125 0.0625 0.077 0.0385 0.001482 3.88
Equations from Oberg & Jones 1966 Volume of Spheroid (Ellipsoid) V= 4.1888 ab2 113
0.2Gmm
Spiracle To 1st Legs 0.14mm
To W ing M uscles
To Posterior Ventral Thoracic air sacs
B. 3. Main left first thoracic tracheal trunk of a honey bee. Drawing by D. Sammataro =400x. 114 Appendix B, Chapter III.
4. Six examples of DAMA movements of tracheal mites on three treatments of bee thoraces. X-axis is vertical movement on a horizontal plane, in pixels; Y-axis is horizontal movement, in pixels.
DAMA Mite Movement of Live Callow Bee # n
4W
'JAA
1g K I1
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Appendix B, Chapter III.
4. ContU
320 ■ - DAMA Mite Movement of Live Bee #1.4
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4. Cont'd.
DAMA Movement ofMite on Dead Bee#1.5 —
i i n i J 1 U ^
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11 > t i w n — ™ C 5 C a c > C > C > C ) c > c > « r3 s ? 3 E% 0 ( 3 i S 3 r - « r s r u ? 5 * o c \ s X g i 5 5i 5 *- 4 r - 4 r - 4 r - ■* r - 4 1 -■4 r - 4 r - 4 1 - 4 f - 4 r>4 Appendix B, Chapter III.
4. Corn'd.
DAMA Mite MovementonDead Bee #5.' *
300 —Jf c * ll m r pi 240 - S 230 —4 Fm
160
ci c • c £ ; 5 £ 8 S ■< r i o 5 d d d d s 1• f* < r-< p H r* *\ p -i i- r - * i-i* rs! 118
Appendix B, Chapter III.
4. Cont'd.
320 ' ■ DAMA Mite Movement on Oily/Live Bee Thorax #6.2a ■ j 310 ' i
300 ■
290 - i
280-
270'
260 '
250'
240 '
230 ■
220 ■
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2 0 0 - % m■► 190 ■
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170 ■
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119
Appendix B, Chapter III.
4. Cont'd.
DAMA Mite Movement onOily Bee #6.3 r 3 2 0 h
J w
5 7 H _ I ■ 9 1
J»
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1 «7V C 3 CJ c> c > e > C i d 3 C 1 C 1 c 3 T"* r 4 C * J L3 53 S. o<3 § E3 i - H CH c< i £n . a §i E3 H T-h r* >4 T™4 4 I - 4 *i - 4 ^ 4 1“ 4 r t--i i- f- 4 120 Appendix B, Chapter III.
Percent Time Spent by Sex of Mite
Other Movements HSeek
DLive{M)n=8 alive(F)n=7 aOil(M)n=5 aOil{F)n=10 oDead{M)n=2 nDead(F)n=l
5. Proportions of time of behavioral traits on the three bee treatments, separated by sex of mites. APPENDIX C
Data Relevant to Chapter IV
121 122 Appendix C, Chapter IV.
Sexes of Mites from Various States by M onth
1 0 0 OH mt August August 1992 1992
Avg £ Avg £ Avg £ Avg £ Avg £
E l M a le E) Female BTotal Egg/Larvae
100 ■, „:i.
?o -W ii& B ffX SSSSS 60 .. OH OH TX + LA Jan-Fcb M arch M arch 1994 1994+5 1994+5
Avg £ Avg £ Avg £ Avg £ B M a le a Female BTotal Egg/Larvae
1. Sex ratios of tracheal mites for five states over nine months. Data not analyzed. Appendix C, Chapter IV 2. Sexes and Sex ratio of mites in the sample set from which DNA was extracted. Column % infest indicates percent of bees in original sample infested with mites; number in parenthesis is percent of infested bees in subsample. August samples.
Date Sample # D/A % infest Date Sample # D/A % infest ? Lrv Egg FLrv MLrv X Lav U E g 1 I F/M Lv /E r ratio 09-14-92 PY12, old 1 3 2 0 2 6 3 9-94 #35-1, G54 2% 1 1 3 2 5 6 7 0 09-14-92 PY 18-1 D <40%)66 1 3 4 4 4 8 3 09-14-92 PY18 12 0 8 5 0 5 13 0 ivtvPttS-U i v X- .■X-J'H ViV ,vKv. v.viv/.v.v.Vi. 1 .vlv.v .vj-.v, ■yp: 7 m u : 0 8 8 15 0 09-14-92 PY18-2 2... 4 16 16 16 22 2 09-14-92 PY18-3 1 8 13 13 13 22 8 09-14-92 PY15-1 A (7.5)135 2 3 2 3 5 7 0 09-14-92 PY15-2 1 11 .... 6 2 ...... 5 ...... 7 13 25 11 iBwwpsi: PY1S-3 1 - a .. 1 3 4 5 . -0 ... W ^ 9 l PYlS-4 2 8 .... * " '2 ' " .....0 ...... 2 ..... 12 09-14-92 PY12-1 A (0) 5.88 1 3 1 1 1 2 6 3 09-14-92 PY12-1 2 1 1 2 2 2 0 10-29-94 SD894.1 (64),43.8 3 11 1 1 1 2 22 2 10-29-94 SD 894 -25 13 0 9 9 2 0 10-29-94 SD894-3 4 14 0 0 ...... 4 0 10-29-94 SD879-1 (32),15.4 6 6 0 6 13 0.423 10-29-94 SD879-2 1 6 2 4 2 6 11 5 10-29-94 SD879-3 6 8 1 0 1 8 0.75 AZ25-1 i I £ S f J:JAW^vTv/i'ViV(Wi'WV)V(,HVA'',-V)V,i,,VWi,*VV.V. ‘.WAVAV.V.VJV.V.V.V.W.'(3O),90% 3 7 i p i l f l l il5- rj; ![:■ i ^; i S: ^ ^ g 0 14 14 ' 24' 2.33 1 ^ 3 ^ D ate Sample # D/A % infest er £ Lrv Egg FLrv MLrv £ Lav D Eg I I F/M Lv/Eg ratio 1-94 #35-3, G54 A 1% 1 0 0 1 0 02-09-95 PY 12 - 2-2 5 6 1 1 1 12 1.2 02-18-94 #6, W26 -1 8 % 4 0 12 12 16 0 02-18-94 #6,W26 - 2 8 % 2 10 0 4 4 16 5 02-18-94 8 % ■ 3 " 9 4 4 ■ 16 a :- " 3-94 i - #35-2,G54 ' 1% 2 2 ""8 3 ...... 8 11 15 3-95 TX1 A 100% 5 9 1 3 1 4 18 1.8 3-95 TX2 1 2 1 1 1 4 2 3-95 TX3 2 3 0 8 8 13 1.5 '.'.V.'AV.V.'.y ft- r'.V.VjV ftv 03-17-94 f s v ^ u i ib% 1 2 1111111 7 7 10 2 03-17-94 #6W26-2.2 ■ 1 0 % ' ...... 2 0 '2...... 17 6.5 03-17-94 #36W24 -1 4% 2 4 8 0 8 14 2 03-17-94 #36W24 -2 4% 2 7 0 0 9 3.5 03-17-94 #36,W24-3 4% 4 7 2 0 2 13 1.75 03-17*94 yiJiV.V.V.V.V.’iVWiV.Vft V v - ft .■ ■ ■ - .7 ■■ #36,W24-4 4% 2 4 3 3 . V. v- .V.’1 ’.V.’, Vi'/.V.'.VlV.'.W. .V.V.'.VV.'J.V.V.ViV.V'V'V.'.*''-' r.v v-'ft v ft' ft' ft-ft1 ft1 ft v.'I1. v .'.v i-. 3-95 B35, YG-1 A 143(16) 19 7..... 1 7 8 30 633 3-95 B35, YG -2 5 4 7 7 7 16 0.8 3-95 B35, YG -3 5 . 1 1 0 1 7 0.2 3-95 LA-ldrone A 1 tube 16 9 1 9 10 30 4 .A ':"ftvW ft:>ftv:^ l i l i i i i ...... / ...... 04-22-94 ' #5,R3 255 6% 0 0 0 04-22-94 ^ft'ftvftv. vft'ftv. •'ft' ft’ft V. Vft v!'ft S'.vt #25, B4 5.95 1 ’ * 5% ^ 0 0 o ...... 0 04-22-94 #4,G51 7.95 1% 0 0 0 0 04-22-94 #15,G71 855 0 % 3 0 3 6 3 n-22 X 2.88 6.47 4.86 Z82 1.48 7.75 4.17 11.82 2.07 (I) (49) (123) (34) (31) (34) (31) (96) (272 125