1 INVESTIGATION OF BIOLOGICAL AND EDUCATIONAL FACTORS FOR BED BUG MANAGEMENT By CORRAINE ATHOL MCNEILL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012
2 2012 Corraine Athol McNeill
3 To God, who continually strengthens me my husband and my parents, the prime motivators and encouragers through this eventful journey, and the Koehler lab for their welcoming arms, strong support and quirky humor
4 ACKNOWLEDGMENTS Many, many pe ople have made this arduous journey successful. My foremost thanks go to my God, without whom I would never had made it this fa r. I am continually renewed by H is strength and humbled by H is mercies. All my successes are His. I have been exceedingly fortu nate to have met and married my strong est support e r and biggest fan, my husband Dr. Seth McNeill. His love, sacrifices, prayers and amazing friendship have brought me through some almost impossible times. The continuous enthusiasm of my pa rents Stanhope and Sonia Scott has never failed to surprise me. The ir constant mantra of always helped me keep focused whene ver I thought I was going to lo se it. I would also like to thank my brother Derron Scott for his words of wisdom and encouragement. Very special thanks are res erved for my in laws, Margaret McNeill and the late Dr. James (Jim) McNeill for their fantastic support of my love of insects. I am sincerely grateful to have been mentored by my advisor Dr Philip Koehler. His unique wisdom, strong support and st ellar guidance has helped to make the completion of my PhD a reality and a success. He has exposed me to a world outside the walls of academia and shown me how to really help people with the work that I do. He has inspired me to think more creatively, and be a sharper scientist and conscientious educator. I am very privileged to have been under the tutelage of this incredible genius. Special thanks are also extended to my committee members Drs. Roberto Pereira, T. Grady Roberts, and Rebecca Baldwin. Their encouragement and expertise have been invaluable throughout my graduate studies.
5 There are many other individuals who have helped m e survive and have added personal words of wisdom, delightful humor, care, professional advice, and a listening ear when I needed it the most. Most notable are: Tracey Wellington, Steve Willms Lucy Skelley, Glinda Burnett Erin Harlow, Jennifer Leggett, C heryl Hall, Drs. Oscar Liburd, Deirdre Gons alves Jackson, Phil Stansly, Lukasz Stelinski and John Capinera Special thanks are also in order for Drs. Sandra Allan from the CMAVE/ARS/USDA and Dr. Emma Weeks from the University of Florida who were great men tors for ERG studies. Thanks to individuals of other USDA labs and programs such as Kelly Sims, Julia Meredith, Bevin Forgeson, and Dr s Jared Ali, Adrian Deuhl, Jimmy Becnel who have helped shaped my research. Special thanks to Dr. Mihai Girucanu for sta tistical help and Jane Medley for her creative genius and help with the Bed Bug and Book Bags curriculum. Thanks also go all the members of the Jacksonville Bed Bug Task Force and members of the Florida Pest Management Association for being such a great co Many undergraduates have risked their lives and have given of their talents that have tremendously helped to make my experiments easier and more efficient. Therefore special thanks go to Shanel l Fos ter, Zack Warr en, Micheal Yohpe Jade Hillard Gabriel l a Milanes, Casey Parker and Josh Weston. Good friendships are precious especially when they help see one through the whirlwind of a PhD. The following individuals have definitely brought sunshine to my crazy PhD days (whether they know it or not) : Dr. Teresia Nyoike, Jodi Scott (yay yellow ball!) Dr. Nathan McNeill, Ben Hottel, Dr. Elena Rhodes, Dr. Heid i HansPeterson, Dr. Delano Lewis and soon to be Dr. Dadria Lewis, other memb ers of the Urban
6 Entomology Lab, me mbers of my church family and my Sabbath School Juniors. And when mental stress gave way to physical duress, very special thanks are reserved for Pamela Rodriquez who gives the most amazing massages, as well as members of Team Blue and Sue Greishaw for fan tastic health and wellness care! Finally (and this is for you Dr. Koehler) my cats, Askher and Bruiser ; thank you! These furry felines gave me numerous sanity checks and helped to remind me that a good long nap is most often times the answer.
7 TABLE OF CO NTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 ABSTRACT ................................ ................................ ................................ ................... 15 CHAPTER ................................ ................................ ................................ ......................... 1 INTRODUCTION ................................ ................................ ................................ .... 17 2 LITERATURE REVIEW ................................ ................................ .......................... 20 Cimex lectularius Linnaeaus ................................ ................................ ................... 20 Description and Taxonomy of the Common Bed Bug ................................ ....... 20 Distribution and Dispersal of the Bed Bug ................................ ........................ 20 Bed B ug Development and Life History ................................ ............................ 22 Bed bug eggs ................................ ................................ ............................. 22 Bed bug nymphs ................................ ................................ ........................ 23 Bed bug adults ................................ ................................ ........................... 23 Insect Vision ................................ ................................ ................................ ........... 24 Structure and Function of Compound Eyes ................................ ...................... 25 Color Vision ................................ ................................ ................................ ...... 27 Public Health Importance of Bed Bugs ................................ ................................ ... 31 Bed Bug Management Strategies ................................ ................................ ........... 35 Cultural Control ................................ ................................ ................................ 36 Insecticide Control ................................ ................................ ............................ 39 Bed Bug Education in Schools ................................ ................................ ................ 39 3 SP ECTRAL SENSITIVITY OF THE COMMON BED BUG CIMEX LECTULARIUS L. (HEMIPTERA: CIMICIDAE) ................................ ...................... 43 Materials and Methods ................................ ................................ ............................ 46 Insects ................................ ................................ ................................ .............. 46 Automontage System ................................ ................................ ....................... 47 Electroretinogram (ERG) System ................................ ................................ ..... 48 Extracellular ERG Procedure ................................ ................................ ........... 49 Criterion response tests ................................ ................................ ............. 50 Spectral sensitivity tests ................................ ................................ ............. 50 Intensity responses ................................ ................................ .................... 51 Data Analysis ................................ ................................ ................................ ... 52 Results ................................ ................................ ................................ .................... 52 Compound Eye Morphology ................................ ................................ ............. 52
8 ERG Wave forms ................................ ................................ .............................. 53 Spectral Sensitivity ................................ ................................ ........................... 53 Intensity Responses ................................ ................................ ......................... 56 Discussion ................................ ................................ ................................ .............. 57 4 BEHAVIORAL RESPONSE OF CIMEX LECTULARIUS TO VARIOUS WAVELENGTHS OF LIG HT FOR OPTIMAL HARBORAGE CHOICE ................... 88 Materials and Methods ................................ ................................ ............................ 89 Bed Bug Rearing and Maintenance ................................ ................................ .. 89 Visual Arena ................................ ................................ ................................ ..... 90 Adult Visual Bioassays ................................ ................................ ..................... 91 Nymph Visual Bioassays ................................ ................................ .................. 93 Oviposition Bioassays ................................ ................................ ...................... 93 Statistica l Analysis ................................ ................................ ............................ 94 Results ................................ ................................ ................................ .................... 94 Spectrometer Readings ................................ ................................ .................... 94 Behavioral Bioassays ................................ ................................ ....................... 95 Discussion ................................ ................................ ................................ ............ 100 5 DEVELOPMENT AND IMPLEMENTATION OF A BED BUG IPM ENRICHMENT CURRICULUM ................................ ................................ ............. 121 Materials and Methods ................................ ................................ .......................... 124 Curriculum Development ................................ ................................ ................ 124 Step 1: Identification and analysis of a need or problem that could be addressed by a curriculum. ................................ ................................ ... 125 Step 2: Needs assessment of the targeted group that would benefit from the curriculum ................................ ................................ ............. 125 Step 3: Setting goals and objectives. ................................ ....................... 126 Step 4: Development of educational strategies to meet outlined goals and objectives. ................................ ................................ ...................... 126 Step 5: Implementation ................................ ................................ ............ 126 Step 6: Curriculum evaluation ................................ ................................ .. 128 Results ................................ ................................ ................................ .................. 129 Curriculum Development ................................ ................................ ................ 129 Curricul um Implementation ................................ ................................ ............. 131 Discussion ................................ ................................ ................................ ............ 135 6 SUMMARY ................................ ................................ ................................ ........... 162 APPENDIX A BED BUGS AND BOOK BAGS CURRICULUM ................................ ................... 165 B PRE AND POST TEST FOR STUDENTS, TEACHERS AND PARENTS ............ 166 C BED BUGS AND BOOK BAGS EVALUATION FORM ................................ ......... 174
9 LIST OF REFERENCES ................................ ................................ ............................. 176 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 189
10 LIST OF TABLES Table page 3 1 Anatomical characteristics of the compound eye of Cimex lectularius across various life stages. ................................ ................................ .............................. 68 5 1 Timeline of major highlights in the development of the Bed Bugs and Book Bags curriculum for 3rd 5th graders. ................................ .............................. 146 5 2 Reading ease and levels for students associated with various activities in the Bed Bugs and Book Bags curriculum based on Flesch Kincaid model. ........... 150 5 3 Reading ease and levels for teachers associated with various activities in the Bed Bugs and Book Bags curriculum based on Flesch Kincaid model. ........... 151 5 4 Demographic profile of 194 individuals that downloaded the Bed Bugs and Book Bags curriculum (July 2012). ................................ ................................ ... 161
11 LIST OF FIGURES page 3 1 Schematic diagram of the electroretinogram (ERG) apparatus from Dr. Sandra Allen, ARS/CMAVE/USDA. ................................ ................................ .... 66 3 2 Automontage images of different stages of bed bug ( Cimex lectularius ) eyes.. 67 3 4 A sample electrophysiological (ERG) response of a dark adapted female bed bug responding to a 520 nm light flash ................................ ............................... 70 3 5 Spectral sensitivity of dark adapted eyes of adult common bed bugs Cimex lectularius ................................ ................................ ................................ .......... 71 3 6 Spectral sensitivity of red adapted eyes of adult common bed bugs Cimex lectularius ................................ ................................ ................................ .......... 72 3 7 Spectral sensitivity of blue adapted eyes of adult common bed bugs Cimex lectularius ................................ ................................ ................................ .......... 73 3 8 Spectral sensitivity of green adapted eyes of adult common bed bugs Cimex lectularius ................................ ................................ ................................ .......... 74 3 9 Visual template fittings for bed bug spectral sensitivity ................................ ....... 75 3 10 Comparison of Stavenga (SSH) and Govardovskii (GOV) nomogram fittings for bed bug spectral sensitivity. ................................ ................................ .......... 76 3 11 Spectral sensitivit y of dark adapted male and female adult common bed bugs, Cimex lectularius. ................................ ................................ ..................... 77 3 12 Spectral sensitivity of red adapted male and fe male adult common bed bugs, Cimex lectularius. ................................ ................................ ............................... 78 3 13 Spectral sensitivity of blue adapted male and female adult common bed bugs, Cimex lectularius. ................................ ................................ ..................... 79 3 14 Spectral sensitivity of green adapted male and female adult common bed bugs, Cimex lectularius ................................ ................................ ..................... 80 3 15 Spectral sensitivity of adult common bed bugs under different color adaptations. ................................ ................................ ................................ ........ 81 3 16 Intensity response of adult bed bugs ( C. lectularius ) for selected wavelengths and slope cutoff between points a and b. ................................ ........................... 82 3 17 Intensity response of male and female adult bed bugs for green light (520 nm). ................................ ................................ ................................ .................... 83
12 3 18 Intensity response of male and female adult bed bugs for blue light (450 nm). .. 84 3 19 Intensity response of male and female adult bed bugs for yellow light (580 nm). ................................ ................................ ................................ .................... 85 3 20 Intensity response of male and female adult bed bugs for red light (620 nm). ... 86 3 21 Intensity response of male and female adult bed bugs fo r ultraviolet light (365 nm). ................................ ................................ ................................ .................... 87 4 1 Colored harborages for bed bug choice tests. ................................ .................. 106 4 2 Reflectance spectra of colored cardstock, and male and female bed bugs as determined by Ocean Optics Spectrometer (Dunedin, FL). .............................. 107 4 3 Percentage of bed bugs responding to lilac versus white colored harborages. 108 4 4 Percentage of bed bugs responding to violet versus white colored harborages. ................................ ................................ ................................ ...... 108 4 5 Percentage of bed bugs responding to blue versus white colored harborages. 109 4 6 Percentage of bed bugs responding to green versus white colored harborages. ................................ ................................ ................................ ...... 109 4 7 Percentage of bed bugs responding to yellow versus white colored harborages. ................................ ................................ ................................ ...... 110 4 8 Percentage of bed bugs responding to orange versus white colored harborages. ................................ ................................ ................................ ...... 110 4 9 Percentage of bed bugs responding to red versus white colored harborages. 111 4 10 Percentage of bed bugs responding to black versus white colored harborages. ................................ ................................ ................................ ...... 111 4 11 Adult bed bugs response each colored harborage versus white standard harborage A) fed females, B) fed males. ................................ .......................... 112 4 12 Adult bed bugs response each colored harborage versus white standard harborage. ................................ ................................ ................................ ........ 1 13 4 13 Overall bed bug harborage color choice. ................................ .......................... 114 4 14 Influence of gender on harborage color choice. ................................ ................ 114 4 15 Influence of nutritional status on bed bug harborage color choice. ................... 115 4 16 Influence of aggregation on bed bug harborage color choice. .......................... 115
13 4 17 Influence of gender and aggregation on bed bug harborage color choice. ....... 116 4 18 Bed bug two choice bioassay for various nymphal instars. .............................. 117 4 19 Mean percentage of 1st instar bed bug nymphs (never fed) choosing colored harborages in a visual P etri dish arena. ................................ ........................... 118 4 20 Mean percentage of 1 st instar bed bug nymphs (fed but not yet molted) choosing co lored harborages in a visual Petri dish arena. ............................... 118 4 21 Bed bug nymph choices to harborage colors.. ................................ .................. 119 4 22 Bed bug oviposition preference. ................................ ................................ ....... 120 5 1 The six step approach to curriculum development ................................ ........... 144 5 2 Evaluation checklist for successful curricula ................................ ..................... 145 5 3 Lesson activity sections within the Bed Bugs and Book Bags Curriculum that meet 2008 Sunshine State Standards for 3rd graders in Florida schools. ........ 147 5 4 Lesson activity sections within the Bed Bugs and Book Bags Curriculum that meet 2008 Sunshine State Standards for 4th graders in Florida schools. ........ 148 5 5 Lesson activit y sections within the Bed Bugs and Book Bags Curriculum that meet 2008 Sunshine State Standards for 5th graders in Florida schools. ........ 149 5 6 ................................ ................................ ................................ ...... 152 5 7 prevention techniques? ................................ ................................ .................... 152 5 8 4 re you willing to participate in the pilot ................................ ................................ ...................... 153 5 9 4 us use the ................................ ................................ ........... 153 5 10 4 curriculum i ................................ ................................ ........... 154 5 11 4 ................................ ............................. 154 5 12 Pre and post test score comparisons for each group tested. ............................ 155 5 13 Biology pre and post test score comparisons for each group tested. ............... 156
14 5 14 Medical pre and post test score comparisons for each group tested. ............... 157 5 15 Prevention and treatment pre and post test score comparisons for each group tested. ................................ ................................ ................................ ..... 158 5 16 Map of the U.S.A. showing the percentage of curriculum downloads that came from each state (July 2012). ................................ ................................ ... 159 5 17 Map of the U.S.A. representing the projected number of individuals that would be impacted (July 2012). ................................ ................................ ........ 160
15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INVESTIGATION OF BIOLOGICAL AND EDUCATIONAL FACTORS FOR BED BUG MANAGEMENT By Corraine Athol McNeill December 2012 Chair: Philip G. Koehler Major: Entomology and Nematology Bed bugs have resurged and are currently deemed significant public health pest s worldwide. Although they do not transmit disease t o humans, they are of medical and veterinary importance. T he evaluation of bed bug visual stimuli and the development of a bed bug IPM curriculum, are two novel approaches to bed bug management that were addressed. Bed bug visual biology is a promising strategy for bed bug management. Electroretinogram studies showed that bed bugs are di chromatic and have spectral sensitivity peaks in the green (520nm) and yellow green (550 nm) wavelengths based on differe nt chromatic adaptations. Automontage images of the bed bug eye revealed morphological characteristics that differentiate the common bed bug from the tropical bed bug. Two choice and seven choice behavioral color assays indicate that overall bed bugs prefer red and black for optimal harborage choice. Yellow and green harborages seemingly repel bed bugs. H arborage color preferences change according to gender, nutritional status, aggregation and life stage. Female bed bugs prefer harborages with shorter wavelengths ( UV and violet) compared to males, whereas males prefer
16 harborages with longer wavelengths ( red and black ) compared with females. The preference for orange and violet harborages is stronger when bed bugs are fed as oppo sed to when they are hungry. Lone bed bugs prefer to be in black harborages while red harborages appear to be the optimum harborage color for bed bugs naturally occurring mixed aggregations. Bed bu g nymphs prefer different colored harborages at each stage which is indicative of their developing eye structures and pigments. F emale bed bugs oviposit ed more eggs in orange harborages compared to green or violet harborages. Use of visual cues such as specific colors offers great potential for improving bed bug m onitoring tools by increasing trap captures. Bed bug management can also be regulated by reshaping how people are educated. Teachers and 4H agents were willing to pilot test and use the Bed Bugs and Book Bags curriculum in their classrooms. This curriculum which is accessible on the internet, potentially impacts not just 3 rd 5 th grade students but more than 70,000 people from various societal groups throughout the United States, Canada and Saudi Arabia.
17 CHAP TER 1 INTRODUCTION Cimex lectularius L, the common bed bug, has been intimately associated with humans for many thousands of years (Panagiotakopulu and Buckland 1999) Although they are known to transmit diseases to humans, they are of medical and veterinary importance and are considered significant nuisance and public health pests (Mallis 1954, Thomas et al. 2004, Leverkus et al. 2006, Goddard and deShazo 2009, Heymann 2009, Mullen and Durden 2009, Reinhardt et al. 2009, Schwartz and James 2011) that have re emerged Their resurgence in the past decade has been attributed to man y factors such as increased human travel, resistance to various insecticides, chan ges in pest management pr actices, general neglect, improper identification of the insect and its bites lack of bed bug reports by tenants, and reuse of discarded items (Mallis 1954, Maestre 2011, Potter et al. 2011) Bed bugs are normally found in dark cracks and crevices, but will seek out a host based on heat and CO 2 Initial experiments by (Abou l Nasr and Erakey 1969) have shown that bed bugs are also capable of visual perception. They appear to see color and may have color preferences. Several monitoring tools and traps (Wang et al. 2009, Hottel et al. 2011) which currently exist on the market are mainly white in color. ClimbU p Intercept or Bed Bug Traps are available in bl ack, and a small portion of the Verifi active bed bug trap has a dark red area. However, many monitoring tools and traps do not intentionally incorporate the use of color as a visual enhancer Active and passive traps do not always deliver consistent resul ts and c ustomers often have to choose between expense versus accuracy for active versus passive traps. As a nonchemical control strategy, heat (Pereira et al. 2009) is a very effective tool but has significant labor and
18 energy costs. Cultural (sanitation practices) control also has its benefits (Potter 2011) but will result in heavy infestations if people do not spend adequate tim e and effort dealing with this pest. Despite so many websites, news reports, blogs and extension articles addressing bed bug behavior, prevention and treatment measures, bed bug numbers continue to rise Approximately 99% of responding pest control compan ies indicated that their company was asked to treat or has treated bed bugs in 2010 (Potter et al. 2011) Interestingly, the highest increase (26%) in bed bug sightings have been in schools and daycare centers (Potter et al. 2011) Unfortunately, many schools do not have proper procedures for taking care of bed bug infestations, and often times, bed bug control requires the use of very tox ic pesticides in schools. E ducation is a fundamental first step in helping to eliminate this pest, but re shaping how people are educated is equally important. In 2009 at the Bed Bug Summit in Arlington, VA, the EPA recommended the creation of a curriculum as a n educational method of helping with the fight against bed bugs. In lieu of the challenges with managing bed bugs, t he overall goal of my research was to 1) evaluate an underdeveloped area of bed bug biology which involves bed bug spectral sensitivity and color vision and 2) to evaluate a more broad based ap proach for managing bed bugs within schools and the community I hypothesized that 1) bed bugs have trichromatic vision similar to other insects and that they have specific color prefere nces as it relates to harborage choice and oviposition sites, and 2) the creation of the bed bug curriculum will increase bed bug awareness in schools and communities in ways that other types of education cannot.
19 C urrently there are no studies documenting bed bug spectral sensitivity. I characterized the spectral sensitivity of adult bed bugs using ERG techniques using da rk, red, blue and green adapted conditions (C hapter 3). Bed bugs (adults and nypmhs) were exposed to colors within and outside of the visi ble spectrum to determine if colors influence harborage choice and oviposition b ehaviors (C hapter 4). Finally, the Bed Bugs and Book Bags" curriculum was created and implemented to determine if it would be a good strategy of reshaping how bed bug informat ion is absorbed by schools and co mmunities (C hapter 5, Appendix A).
20 CHAPTER 2 LITERATURE REVIEW Cimex lectularius Linnaeaus Description and Taxonomy of the Common Bed Bug The common bed bug, Cimex lectularius L., belongs to order Hemiptera, suborder Heteroptera, family Cimicidae, subfamily Cimicinae. The subfamily Cimicinae has two genera and six species of which Cimex is the largest genus with five species (Maw and Canada 2000) Four species within the genus Cimex are ectoparasitic on humans and include Cimex columbarius Leptocimex (Cimex) boueti C. hemipterus and C. lectularius (Mullen and Durden 2009) Cimex lectularius the common bed bug, was first described by Lin naeus in 1758 (Grozeva et al. 2010) Distribution and Di spersal of the Bed Bug The bed bug is presumed to have originated in the Old World, Southern Europe and the Middle East (Usinger 2010) It was described as living in caves and being closely associated with humans for approximately 4000 years (Reinhardt and Siva Jothy 2007) However as civilization advanced from cav e dwellings to villages to city living, so di d the spread of bed bugs. Reports of b ed bugs showed that they continued to spread from the Mediterranean, moving to Greece in 400 B.C., Italy in 77 A.D., China in 600 A. D., Germany in the 11th century, France in the 13th century, England in the late 16th century and Scandinavia in the 19th century (Pinto et al. 2007, Usinger 2010, Potter 2011) Bed bugs came to the Americas in the 18th century with European colonists. Today, every state in the U.S. has bed bugs. The more heavily infested states are those with the densest populations of people in the temperate (non tropical) parts of the country.
21 Bed bugs are excellent hitchhikers and are able to get around via active migration as well. They are carried around on items such as clothing, furniture and luggage (Pinto et al. 2007) Bed bugs are found in a variety of places including but not limited to homes, apartments, college dorms, hotels, public transportation, movie theaters, cruise ships, prisons, gyms, hospitals, airports, d aycare centers, and restaurants (Pinto et al. 2007) Overall, bed bugs are considered a global, cosmopolitan pest, with marked and increased infestations in the U.K., Australia, Israel, Canada, Denmark, Italy and Africa and Asia (Doggett et al. 2004, Omudu and Kuse 2010, Suchy and Lewis 2011) Although human blood is preferred, bed bugs have been known to feed on the blood of vertebrate hosts such as rats, m ice, rabbits, monkeys, dogs, cats, guinea pigs calves, various species bats and birds, chickens, a gecko, frog and chameleon (Rivnay 1930, Matheson 1950, Mallis 1954, Pinto et al. 2007, Usinger 2010) Therefore, bed bugs are not only found in huma n dwellings, but are found in areas where alternative hosts live. Bat caves, wild bird nests, poultry breeding houses, livestock stalls and pet bedding are also areas that encourage the establishment of bed bugs (Matheson 1950, Smart 1965, Usinger 2010) These alternative homes provide bed bugs with warmth, shelter and an alternate source of food when humans are not available. Bed bugs were largely controlled after World War II by insecticides such as DDT, lindane, ronnel and malathion, as well as impro vements in hygiene and sanitary practices (Pinto et al. 2007) However since the last decade, bed bug populations hav e resurfaced and are spreading rapidly This resurgence in bed bug numbers has been associated wi th 1) increased travel, 2) changes in pest management practices (shift from use of generalize pesticid e to targeted bait applications ) 3) resistance and
22 avoidance of bed bugs to many insecticides, 4) improper identification of bed bugs and bed bug bites by PMPs and health care professionals, 5) re use of discarded, infested furniture and use of infested rental and secondhand furniture and 6) general neglect (Pinto et al. 2007, Cooper and Harlan 2011 Maestre 2011) Bed bug Development and Life History Bed bugs undergo hemimetabolous metamorphosis and have three st ages in the life cycle: egg, nymph and adult. Their survival and reproduction is strongly determined by temperature conditions and the availability of hosts (Herrick 1936, Doggett et al. 2004) 13 C ) can extend the length of the life cycle considerably (up to 2 years) by delaying the hatching period or by preventing hatching (Mallis 1954, Doggett et al. 2004) Under average temperatures ( 23.5 C ) The life cycle of a bed bug can range from less than one month at 30 C to app roximately nine months at 17.8 C (Mullen and Durden 2 009) B ed bug eggs Bed bug eggs are white or yellowish white, oval, elongated, reticulated objects that are ~ 1 mm long and 0.44 mm in diameter (Mallis 1954, Cheng 1969, Mullen and Durden 2009) There is a rim (operculum) around one end (free end) of the egg which the nymph pushes off when it emerges from the egg (Herrick 1936, Mallis 1954) Eggs are laid continuously in batches of 1 12 eggs per day depending on nutritional and environmental conditions (Mallis 1954, Mullen and Durden 2009) and are coated with a quick drying, transparent cement that glues them firmly to various substrates Temperature ranges between (21 C and 27.8 C) are ideal for maximum egg production (Mallis 1954) Duri ng summer months, eggs normally hatch in 4 12 days (~ 9 days at 23.2 C ) (Doggett et al. 2004, Mullen a nd Durden 2009)
23 Bed bug nymphs Nymphs hatch within 8 10 days; th ey ra nge in size from 1 to 6 mm and appear tan (straw colored) (Cheng 1969, Pinto et al. 2007) They are similar to the adults except they lack developed reproductive organs T hey have few er bristles, 2 segmented tarsi, and 3 abdominal scent glands (Cheng 1969, Usinger 2010) It takes approximately eight days to go from one instar to the next. Nymphs must feed in order to move to the next instar and their color gets darker with each molt (Herrick 1936) There are five nymphal stages and after the fifth and f inal molt, the nymph becomes an adult and wing pads become visible (Mallis 1954) Bed bug adults Adult bed bugs are reddish brown, dorso ventrally flattened, oval shaped insects with fine, golden hairs that cover the body. The head is comprised of a 3 segmented be ak and 4 segmented antennae and two prominent, deeply pigmented compound eyes but no occeli (Matheson 1950, Usin ger 2010, Cooper and Harlan 2011 ) Bed bugs completely lack hindwings and the two forewings are great ly reduced scale like pads (Cheng 1969) With reduced wings, bed bugs cannot fly, or jump, but they can crawl relatively quickly ( 0.12m /min ). They are approximately 4 to 6 mm long and 3 mm wide (Matheson 19 50, Doggett et al. 2004, Co oper and Harlan 2011 ) When disturbed, bed bugs often give off a sweet, musty scent that is produced by special stink glands that are located on the ventral side of the last thoracic segment (Mathe son 1950, Cooper and Harlan 2011 ) Male and female bed bugs can be distinguished from each other based on a couple of characteristics. For males, the tip of the abdomen is poi nted and houses the genetalia, whereas for females, the tip of the abdomen is broadly rounded (Cooper and Harlan 2011 ) An additio nal morphological feature for female identification
24 is the presence of a distinctive spermalege groove on the ventral side of the abdomen (Cooper and Harlan 2011 ) Adult bed bugs will feed on average for 10 minutes every 1 7 days depending on temperature; the lower the temperature (18 the C, 90% R.H.) the more they fee d (Usinger 2010) Feeding is necessary for egg production in females and sperm pr oduction in males (Reinhardt and Siva Jothy 2007) After feeding bed bugs will immediately seek out a harborage for mating and egg laying. Feeding is therefore an essential precursor to mating (Reinhardt and Siva Jothy 2007) After feeding, male and female bed bugs mate via traumatic insemination. The m ale mounts the female and injects sperm into an extragenital site (paragenital tract) on the ventral side of the female. Copulations can last up to half an hour, during which time, the sperm travels from its point of injection to w ithin the wall of the oviduct and the base of the ovarioles. Scars from multiple inseminations are permanent and can be observed on the underside of female (Reinhardt and Siva Jothy 2007, Mullen and Durden 2009) Females lay eggs between 3 6 days after mating. Insect Vision Highly developed visual systems are found in most insects because evolutionarily, vision is a very important part of the insect sensory system. Although there are some insects that can only differentiate between ligh t and dark with very little visual acuity, many other insects are able to differentiate small, colored, patterned objects with superb visual acuity (Larsson and Svensson 2005). Insects use visual cues in a wide range of behaviors such as locating and recog nizing hosts, landing and navigation, judging distances, perceiving movement, capturing prey, avoiding predators, finding mates and detecting and differentiating between colors, patterns and shapes (Evans 1984, Nation
25 2002, Hausmann et al. 2004) For most insects, the main visual organs that perceive light include a pair of faceted compound eyes and frontal ocelli. Insects also have secondary means of visual perception which include using light detecting photoreceptors on epidermal (skin) cells, or via stemmata (larval eyes) (Gullan and Cr anston 2010) Structure and Function of Compound Eyes Compound eyes are comprised of individual functional units called ommatidia which can range from as few as 2 in the certain ant species ( Ponera punctatissima ) to as many as 10,000 in dragonflies (Chapman 1998) The ommatidia is an elon gated groupi ng of various cells such as pigment cells and retinula cells. The optical system is comprised of transparent cuticular corneal lens, crystalline cone cells and the rhabdom (photoreceptor) (Evans 1984) Primary and secondary pigment cells surround the crystalline cone cells and act as shield s They are responsible for cutting out the glare of extra light rays that randomly bounce around within the eye. This helps the insect to resolve contrasts (Evans 1984) Retinula cells are nerve cells that have one axon. There are usually eight of these cells in each ommatidium in a circular arrangement. Microvilli that are present at the end of each retinula, collectively form a rhabdomere. Visual ligh t sensitive pigments (rhodopsin molecules) are located in the microvilli of the rhabdomeres. Retinula cells are also surrounded by secondary pigment cells that also prevent glare and separate each ommat idia from neighbor ing ones (Chapman 1998) Many rhabdomeres together make up the rhabdom (photoreceptor ) which lies along the longitudinal axis of the ommatidia. In some insects, the rhabdomeres are packed very closely together to form what ap pears to be a fused rhabdom. A fused rhabdom allows for each reitnula cell in each
26 ommatidia t o have the same field of view. Also individual rhabdomeres laterally filter light that may get to neighboring rhabdomeres (Chittka and Waser 1997) Conversely, if the rhabdomeres are spaced farth er apart, this creates an open rhabdom which means that each retinula cell will have a separate field of view (Evans 1984, Chapman 1998) Having an open rhabodomere system also means there is no filter present, which makes for greater spectral sensitivity (Chittka and Waser 1997) Tiered rhabdomeres have also been documented in insects. Tiered rhabodemeres have distal photore ceptor cells that filter light that would reach the proximal photoreceptor cells. This phenomenon produces less spectral sensitivity (Chittka and Waser 1997) Light enters the eye thro ugh the corneal lens and is guided by crystalline cone cells to photoreceptors which absorb the light. The absorbed light energy changes the structural configuration of the visual pigment rhodopsin. Rhodopsin consists of retinal, an aldehyde of vitamin A and a protein, opsin. When light hits the unstable retinal, it changes the rhodopsin to the active form called metarhodopsin, which activates G protein in the cell membrane. Metarhodopsin then triggers an electrical potential that makes the retinula cell membrane more permeable. The flow of ions causes depolarization in the retinular axon. This conversion of light energy to electrical energy is called phototransduction. The electrical signal travels directly from retinual axons to the optic lobe of the bra in via chemical synapses (Evans 1984, Ch apman 1998, Nation 2002, Gullan and Cranston 2010) In the brain, visual signals of patterns, colors, fields of motion and polarization are translated to behavioral signals which the insect displays (Homberg 2004)
27 There are two main types of ommatidia based on their structure and function: photopic ommatidia and scotopic ommatidia Photopic ommatidia are mainly found in day active insects such as dipterans, mantids, odonates and hymenoptera. The photoreceptors of photopic ommatidia are large and extend from the cone to the basement membrane of the eye. The pigment shielding cells t hat surround the photoreceptors do not shift around, but remain in place to prevent stray light from neighboring ommatidia (Nation 2002) For this reason there is better image resolution, with a constant overlapping field of view (Evans 1984) In contrast, scoptopic ommatidia are usually found in nocturnal insects. The photoreceptor cells are smaller and shorter than in photopic cells, extending only one third of the way from the basement membrane of the eye to the cone cells (Nation 2002) The r emaining space from the cone to the cornea is filled with retinula cells that join to form a crystalline tract. The pigment shielding cells that surround the tract and the photoreceptors are able to migrate, moving especially to the cone cells in low light conditions. This causes the eye to be more sensitive in dim light, but have a lower resolution (Evans 1984, Nation 2002) Color Vision The visible light spectrum can be broken down into the several wavelengths, which correspond to various colors. These approximate wavelengths from short to long include: violet (380 450 nm), blue (450 475 nm), cyan (476 495 nm ), green (495 570 nm), yellow (570 590 nm), orange (590 620 nm), and red (620 750 nm) (Bruno and Svoronos 2005) The concept of color is based on the fact that color (certain wavelength) is comprised of three qualities: hue, saturation and brightness. Brightness refer s to variations along the light dark scale, saturation denotes how close a wavelength is to grey or white, and hue describes any aspect of color not related to
28 brightness or saturation (Kelber et al. 2003) However, there is no documentation that insects can distinguish between these qualities of color as separate entities (Kelber et al. 2003) Nation ( 2002) defines color vision as the ability to distinguish between two wavelengths of light Color vision is important in insects primarily because it allows insects to be able to distinguish objects in their environment and provides the insect with important resource information (Larsson and Svensson 2004, Hoel et al. 2007) Insect eyes are generally sensitive to wavelengths between 250 nm to 600 nm in comparison to humans who are sensitive to wavelengths between 450 nm and 7 00 nm (Borror et al. 1989) The majority of insects includi ng most moths, beetles and honey bees are trichromatic, which means that they have photoreceptors that are able to differentiate between three wavelengths, ultraviolet (~ 360 nm), green (~ 530 nm) and blue ( ~ 460 nm) (Burkhardt 1977, Larsson and Svensson 2004) However, there are other instances in nature where insects are dichromatic or have up to six different photoreceptors. The owl fly Ascalaphus macaronius the American cockroach, Periplaneta americana six beetle species, and several ant species lack blue receptors, and therefore only differentiate between UV and green wavelengths (Chittka and Waser 1997, Hausmann et al. 2004) Insects belonging to the orders Coleoptera, Hymenoptera, Odonata and Lepidoptera possess an additional fourth photoreceptor that allows them to see red wavelengths (Chittka and Waser 1997) whereas certain Diptera have up to six photoreceptors (Larsson and Svensson 2004) A lthough an insect may have certain spectral sensitivi ties as can be determined by electroretinogram (ERG) studies, this does not mean that insects will respond differently to all those
29 wavelengths. In addition to knowing spectral sensitivities of a particular insect, true color vision is further determined b y behavioral bioassays to color choices (Hausmann et al. 2004) When an insect is able to exhibit a certain behavior toward a particular wavelength, true color vision is demonstrated. Insects are not only able to see color but they also exhibit strong color preferences. Insects can prefer colors that look like the color of their food, or that are close to the color of their hosts (Reza and Parween 2006) Insect color preferences can also produce various behaviors (Larsson and Svensson 2004) Flower and foliage color is very important for herbivorous insects especially for host location. Insects that feed on flowers have various visual pigments that enable them to use color to distinguish between host color and the colo r of their surroundings (Yaku and Walter 2007) For hematophagous (blood sucking) insects color is used for host location, seeking shelter and ovipositing (Jones and Schreiber 1994, Steverding and Troscianko 2004, Hoel et al. 2007, Mann et al. 2009) There are many examp les of color preference in hemat ophagous insects and knowledge of the color preferences of these pests can help to control them better. Shades of blue and black are highly attractive to the tsetse fly, Glossina s pp. which is well known for transmitting human sleeping sickness and the nagana disease in livestock. Blue and black are present in daytime shadows and these colors help tsetse flies find hosts and shelter (Steverding and Troscianko 2004, Geden 2006) Research showed that blue traps were attractive and effective at catching tsetse flies, black and red colored areas inside traps optimiz ed trap captures (Green and Cosens 1983, S teverding and Troscianko 2004)
30 Sandflies such as Lutzomyia shannoni (Dyar) and L .vexator (Coquillett) have similar spectral sensitivity to tsetse flies and are similarly important as vectors of diseases that adversely affect humans and livestock (Mann et al. 2009) Although sandflies have similar spectral sensitivity to tsetse flies, this may not correspond with what color wavelength they are attracted to and eve n sandflies in the same genera can have very different color preferences. For example, traps fitted with red L ED lights (660 nm) caught more L shannoni but the fewest L vexator Lutzomyia vexator was more attracted to a combination of colors which may ha ve created shorter wavelengths (Mann et al. 2009) Two predatory mosquitoes Toxorhynchites splendens (Wiedemann) and Toxorhynchites rutilus rutilus (Coquillet) prim arily prefer red to lay their eggs in black containers but would also oviposit in dark red and brown containers (Jones and Schreiber 1994) Various species of mosquitoes such as Aedes albopictus Sku se, and Culex nigripalpus Theobald will feed significantly long er at 500 600 nm wavelengths compared with black. Even within the Aedes genus, A. aegypti (L.) did not show any color preferences at all. Conversely Anopheles quadrimaculatus Say feed longer at black feeding stations than other wavelengths (Burkett and Butler 2005) Both t he Aedes and Mansonia genera show low attraction for wavelengths greater than 600 nm (Browne and Bennett 1981) Infamous animal pests such the cat flea Ctenocephalides felis (Bouch) and the orien tal rat flea Xenopsylla cheopis (Rothschild) prefer red wavelengths around 330 nm and 530 nm, but wavelengths above 630 nm are not particularly attractive (Crum et al. 1974) On the other hand the horse fly Tabanus nigrovittatus Macquart a serious pest
31 of livestock and humans, h as interesting color preferences that vary by gender. The male horse flies are more sensitive to blue light which they may use to distinguish between potential mates and the sky. On the other hand, female horse flies are much more sensitive to green wavele ngths and that allows them to differentiate between their host and their normal yellow green habitat (Alla n et al. 1991) The color preferences of bed bugs were originally studied by Aboul Nasr and Erakey ( 1969) Bed bug color preference was studied by creating colored solutions from various chemical compounds and then simultaneously offering two colors to the insect in a choice test. The alternative experiment involved using four different colors of transparent ce llophane paper mounted over a blackened P etri dish and allowing the insect to choose among the colors (Aboul Nasr and Erakey 1969) The colors tested were yellow, red, violet and bluish green. The authors obser ved that bed bugs showed more of a preference for shorter wavelengths like violet (haematoxylin 0.005%) and bl uish green (fast green 0.001%), least attractive. Also, darker shades of colors such as deep re d (eosin 5% solution) seemed to be more appealing than brighter shades of color such as light violet (haematoxylin 0.001%) (Aboul Nasr and Erakey 1969) Public Health Importance of Bed Bugs It has been well doc umented in the literatu re that the common bed bug, C lectularius is a significant public health pest (Mallis 1954, Thomas et al. 2004, Leverkus et al. 2006, Goddard and deShazo 2009, Heymann 2009, Mullen and Durden 2009, Reinhardt et al. 2009, Schwartz and James 2011) Bed bugs are blood sucking ectopar asites that prefer human blood (Heukelbach and Hengge 2009) to other hosts (birds, bats livestock, pets). They are considered ser ious public health pests not just
32 because of their nuisance biting but also because of the physical and psychological reactions they cause, and their potential to tran smit diseases to different animals (Jupp and McElligott 1979, Quarles 2007) One type of reaction caused by bed bugs include various dermatologi cal reactions. Bed bugs have a piercing sucking mouthpart designed for taking in blood and liquefied epidermal tissue when they feed (Thomas et al. 2004) Bed bugs act as solenophages (vessel feeders) when they insert the tip of the mouthpart directly into the host's peripheral blood vessels, or they can act as telmophages (pool feeders) when they damage the superficial skin tissues and suck up blood that has accumulated in the damaged area (Schwartz and James 2011) When the bed bug pierces the skin, it injects an anticoagulant, a vasodilatant (nitric oxide) (Heymann 2009) an anesthetic compound, a nitrophorin antigen which causes an allergic IgE mediated hyper sensitivity, and other chemicals (kinins, hyaluronidases, proteases) that cause different skin reactions and increased sensitivity (Omudu and Kuse 2010, Schwartz and James 2011) The most common reactions to bed bug bites that require medical attention are pruritic maculopapular erythematous lesions (round, elevated areas of discolored skin) (Goddard and deShazo 2009) Various other skin reactions due to bed bug bites include bullous cimicosis (skin lesions), erythema (reddening of skin due to irr itation of capillaries), wheals (red, swollen marks, usually accompanied by itching), hemorrhaging (bleeding due to ruptured blood vessels), pruritus (severe itching), and formation of liquid or air filled cavities (Thomas et al. 2004, L everkus et al. 2006, Reinhardt et al. 2009) Severe scratching of papules (elevated skin areas) often cause open wounds on
33 the skin surface. This presents an opportunity for bacteria to enter the open wound and cause secondary infections such as ecthyma (skin ulcers), cellulitis (severe inflammation of the skin), folliculitis (inflammation of hair follicules) or lymphangitis (inflammation of lymphatic vessel wall) (Goddard and deShazo 2009, Schwartz and James 2011) Although bed bug bites are initially painless, the papules and inflammed areas that ac company the bites are sometimes painfully swollen and have burning sensations (Liebold et al. 2003) Bed bug bites are usually observed on exposed skin surfaces such as arms, legs, trunk, back, chest, breasts, face and hands (Goddard and deShazo 2009) Reactions to bed bug bites range from an immediate reaction (within a few minutes of being bitten) to symptoms that show up several weeks after the insect bites have occ urred. However, not everyone that is bitten by bed bugs display dermatological reactions. Many individuals may not react at all (Doggett et al. 2004) In addition to the various dermatological reactions, the frequency of bed bug bites and the level of immune response in some individuals can trigger systemic reactions such as asthma or anaphylactic shock due to IgE mediated hypersensitivity (Leverkus e t al. 2006) Bed bug bites also cause psychological reactions in humans. Anxiety, sleeplessness, stress, irritability, and overall discomfort (Doggett et al. 2004, Mullen and Durden 2009) are observed in individuals living in infested areas or are closely acquainted with others that have infestations. Extreme anxiety and fear associated with insects suc h as bed bugs is called "delusory parasitosis". Individuals that exhibit this type of emotional trauma are convinced that they are infested with or are constantly bitten by insects. These individuals usually make rash decisions to get rid of all their
34 clot hes and furniture and are usuall y inconsolable despite any proof that insects are not present (Hinkle 2000 ) Heavy bed bug infestations have also been known to cause chronic blood loss and anemia in adults and malnourished children (Doggett et al. 2004, Mullen and Durden 2009, Omudu and Kuse 2010) For example, repeated bites from a heavy bed bug infestati on drastically decreased a 60 yr r ange of 139 157 g/L to 52 g/ L in just a few weeks (Pritchard and Hwang 2009) When his house was t reated for bed bugs, the man's hematocrit levels resumed to normal levels (Pritchard and Hwang 2009) Bed bugs are also medically important because of their implications as carriers or vectors of disease agents. These blood feeders can be hosts to over 40 disease causing agents, and 27 pathogens are known to survive within the proboscis of the bed bug (Goddard and deShazo 2009, Omudu and Kuse 2010) Bed bugs appear to have the capability and machinery necessary to transmit diseases to humans, but no such cases have been documented (Goddard and deShazo 2009, Omudu and Kuse 2010) (Mallis 1954) stated that bed bugs were able to transmit the follow ing diseases among laboratory animals: anthrax, plague, tularemia, yellow fever, European and African relapsing fevers, typhus, lymphocyt ic choriomeningitis, Minas Gerai s spotted fe ver, leishmaniasis and American trypanosomiasis. However, it has not been proven that bed bugs are able to transmit any of these diseases to man (Mallis 1954, God dard and deShazo 2009) Within recent years, there have been concerns regarding the ability of bed bugs to transfer hepatitis B virus (HBV), human immunodeficiency virus (HIV) or Chagas disease. Sc hwartz and James ( 2011) stated that there is evidence to show that
35 bed bugs are vectors of the Hepatitis B virus. However, (Goddard and deShazo 2009) reported that although HBV can persist in bed bugs and their feces for at least 6 weeks, and the HBV surface antigens were detected in bed bugs even 7 weeks after feeding on infected blood, the HBV DNA cannot replicate in the insect. HBV can however be passed transtadi ally (from one instar to the next) in bed bug nymphs (Blow et al. 2001) Similarly, HIV can persist in bed bugs 8 days after the bu g had fed on infected blood, however, HIV is not able to replicate within the insect (Jupp and Lyons 1987, Goddard and deShazo 2009) and therefore cannot be transmitted to another organism. A lso, there has been reports of bed bugs having the abil ity to host and vector Trypanosoma cruzi the agent of Chagas disease in mice but not in humans (Jrg 1992, Goddard 2011, Schwartz and James 2011) Bed Bug Management Strategies Currently, bed bugs are thought to be the most difficult and expensive urban pests to control in the United States (Pinto et al. 2007) Their cryptic habits, pervasive nature and relatively fast proliferation make them a tough challenge. An effective pest management program for bed bugs involves several methods of control: an integrated pest management approach involving a combination of sanitary practices, education and chemical control. Effective bed bug el imination will depend on several factors including, but not limited to: amount of clutter and harborage sites available, how frequently new bed bugs are rei ntroduced into the area, how co operative people are with control, the commitment to spend time and m oney to solve the problem, the type, condition and age of the infested area and the competency of the pest management professional (Pinto et al. 2007)
36 Cultural C ontrol Early cultural controls included hanging animal parts around the bed, and thorough cleaning of houses, especially beds. Early b e ds were usually made from straw and it was recommended that the straw be frequently changed (Potter 2011) It was also recommended that sleeping quarters and whole buildings be made as unsuitable for bed bugs to harbor in as possible. Elaborately carved wooden beds provided great harborages for bed bugs to hide and develop in. It was recommended that t hese beds be replaced by plain, metal beds, which provide fewer bed bug harborages and were hard er for the bed bugs to climb on (Doggett et al. 2004, Potter 2011) Today ther e are several ways of mechanically controlling bed bugs. Approximately 65% of companies use vacuum cleaners (Potter et al. 2008) which has its advantages and disadvantages. Vacuuming can pick up adults and nymphs, but eggs are usually left be hind. Also vacuuming does not get into the cracks and crevices where bed bugs hide (Pinto et al. 2007) The use of bed encasements for mattresses and box springs are high ly recommended. Encasing these areas prevents the bed bugs from reaching or occupying hard to reach cracks and crevices, and also makes it easy to see and destroy bed bugs (P into et al. 2007) Visual inspections and detections: V isual inspections of cracks and crevices for signs of bed bugs was and still is very important People originally filled these harborage sites with various sealants or soap (Potter 2011) One of the main reasons that eliminating bed bugs is so hard is be cause they can hide anywhere (Potter et al. 2008) Bed bug infestations in the early st ages are the easiest to treat, but the hardest to spot. The reverse is also true; advanced infestations are the easiest the spot, but the most difficult to eradicate. To supplement visual detections, the use of scent is employed.
37 Well trained bed bug dogs are used in many commercial and residential areas to detect bed bugs based on their smell. This is particularly helpf ul in low infestation areas where visual inspections are unsuccessful. Temperature treatments: People would originally put their beds and b elonging in the sun so that the heat would get rid of bed bugs. However this does not work because the bugs can find darkened, protective areas to hide under or in (Doggett et al. 2004, Omudu and Kuse 2010) Nevertheless, heat can be used as an effective tool for bed bug control Approximately 25% of pest management companies use steamers. A low vapor, high temperature steamer is needed to do an effective job (Vail 2006) However if not used properly, the ejection pressure of the steam can blast and spread bed bugs to other areas in the room. Steamers should have broad nuzzles, and be moved slowly and directly across the area to be treated for effective use (Potter et al. 2008) Bed bugs are killed at re latively low temperatures (~ 41 C) depending on the amount of time they are exposed to it (Pereira et al. 2009) A temperature of 49C for one minute is enough to kill all stages of bed bugs (Pereira et al. 2009) Today it is highly recommended that bed bug infested clothing and bedding be bagged and washed in hot water using a standard wash cycle. This wi ll give similar results to putting infested items in a clothes dryer and running the dryer fo r one hour on high heat (~ 80C) (Potter et al. 2008) This length of time takes into account that the dryer has a ramp up and ramp down cycle. If the entire dwelling appears to be infe sted, pest management firms can heat treat the entire house, or t reat infested items by putting most items (couches, toys, furni ture etc.) into a Styrofoam heat chamber that is assembled in a room of the house (Pereira et al 2009) The oven contains heaters, fans, and several thermometer probes
38 for monitoring temperatur e inside the heat chamber Alternatively large heating units can be placed in several rooms of the house, thereby raising the temperature of the whole house to the critical temperature needed to kill the bed bugs. This is in contrast to localized heat tre atments that target only a specific room/area of the house (Pinto et al. 2007) Cold temperatures are also used to manage bed bugs. There has been a lot of controversy abo ut how long bed bugs can tolerate cold temperatures before they die. It is recommended that keeping infested items at 0 C for 4 5 days is likely to kill bed bugs, whereas it only takes 2 hrs for mortality at 1C ( Pinto et al. 2007) The Cryonite system is the only spot cold treatment currently on the market for killing bed bugs. A snow like material formed by the rapid release of compressed CO 2 to freeze the bed bug immed iat ely on contact (temperature 78 C) (Pinto et al. 2007) However, of > 18,000 bed bug treatment cases in Florida, less than 1% of pest management professionals (PMPs) used a freezing method to kill bed bugs (FBBS 2011) Traps: Decades ago before any drastic measures were taken to reduce bed bug infestations, people would put dish pans with kerosene or oil under their bed legs to deter the bugs from reaching the b ed (Potter 2011) Today, there are many types of bed bug traps on the market in c luding active and passive traps, which mimic older control methods but also use a combination of host cues such as CO 2 heat host odors, as well as bed bug pheromones for monitoring bed bug infestations. While these are all helpful, they are not alwa ys r eliable or 100% effective in control ling bed bugs Active traps take advantage of various lures singly or in combination eg. CO 2 CO 2 + heat, heat + pheromones to create super attractive trap s, whereas passive monitors catch
39 bed bugs as the crawl around (Hottel et al. 2011) Active traps are more expensive but more accurate than passive traps (Hottel et al. 2011) Insecticide Control The first report of chemical control for bed bugs was pyrethrum powder (Osborne 1896) So on after, hydrogen cyanide was used in soldier barracks during WWII (Potter 2011) Mercury and arsenic compounds were also used and applied with syringes, feathers or brushes to the infested sites (Potter 2011) Then DDT was discovered and this insecticide k ept bed bugs under control for several years. However bed bug resistance to DDT started to emerge in the mid 1950s. It was then that other pesticides such mal athion, lindane and ronnel were being used as alternative control measures (Pinto et al. 2007; Potter 2011) Other synthetic pesticides such as organophosphates and carbamates (Propoxur) are still used for bed bug control today. Today, pesticides are one of the common treatment methods for bed bugs. These pesticides include a wide range of liquids, dusts, aerosols, fumigants, and foggers (Pin to et al. 2007) The top three rated bed bug pesticides currently include Phantom (chlorfenapyr), Temprid (beta cyfluthrin and imidacloprid) and Bedlam (phenothrin plus synergist) (Potter et al. 2011) Pyrethrum which was used in the mid 19th century to control bed bugs is still being used today. However, as with most other bed bug insecticides, there is the issue of insect resistance and avoidance of this chemical. Bed Bug Education in Schools A recent, rampant resurgence of bed bugs has occurred globally, nationally and locally (Eddy and Jones 2011) Within the past few years, bed bug infestations have dramatically increased in homes and in many public places (schools, hospitals, dorms, airports, nursing homes etc.), and have created major concern for the public and for
40 pest management professionals (PMPs) (Potter et al. 2008) Bed bugs sightings in schools have been on the rise over the past decade. Unfortunatel y the nature of bed bugs makes public places, especially schools, prime opportunistic sites for introductions, re introductions or constant infestations. Communities with high numbers of bed bug infestations often have frequent bed bug introductions within schools in those communities (Green an d Gouge 2009) The grave reality is that children can and do transport bed bugs from infested homes to uninfested schools or vice versa. Bed bugs can be transferred from child to child, child to surround ings, surroundings to staff, etc ( New Jersey Education Association 2010) until both homes and schools are infested. This spread is not difficult because bed bugs are mobile and not easily detected. It is very easy for bed bug s wh ich have found its way unto clothes or a backpack, to fall off in s everal places between home and school (Thompson 2011) Currently, bed bugs are the greatest challenge for the adoption and implementation of school IPM programs nationwide. The introduction of just one bed bug into a classroom by students has resulted in school closings (Richards 2008) to allow pesticide treatments sometimes used illegally, which results in contamination of classrooms (McFarland 2010) A proper bed bug IPM program should complement traditional treatment strategies by targeting the education of the affected population (students) and consequently their parents, as the main tool in preventing infestation s from moving from infested homes to the school buildings The development of a bed bug school curriculum is a proactive way of supplementing traditional school IPM and promoting a wareness of be d bug education. Elementary school children, for example, grades 3 5, provide an excellent starting point because they are old enough to
41 communicate bed bug identification and prevention to their parents, but still young enough for parents t o be intimately involved with their education. Also, students in elementary grades are naturally inquisitive and excited about learning new concepts. They also tend to communicate more eagerly with parents and relatives about the material learned in class compared with older students. The implementation of a curriculum in this manner has excellent potential to spread bed bug awareness from the school population to parents and ultimately the community. As a result, the spread of bed bugs into schools from t he communit y can be reduced and pesticide use in schools can be curtailed through education and prevention programs. Having a bed bug curriculum can be a model for school IPM targeting other pests and can represent an essential element to be incorporated into every school IPM program. Unfortunately many school officials when presented with incidences of bed bugs, overreact, panic, and make drastic and unwise decisions (Thompson 2011) Such rash d ecisions, which are made to reduce damage or complaints, place children at risk for exposure to unnecessary pesticide applications. Pesticide spraying is usually the quick fix method, but as was evidence d by a New York Public School, pest control operator s can contaminate school buildings by inappropriately using pesticides (McFarland 2010) I n this case left pesticide residues on books, chairs, desks and other belongings. The cost to decontaminate (~ $250,000) was more than twice the original cost for bed bug control. Schools in other areas such as New Jersey, Kentucky, Ohio, and Texas have had similar issues. Because bed bugs and bed bugs eggs are resistant to many common insecticides such as pyrethroids, combinations of harsher chemicals are often used in control programs, which can leave t oxic residues behind.
42 Unfortunately, several pesticides that have been approved for bed bug control present serious health risks ( BP 2010) Forty eight common bed bug pesticides registered for use in schools are known carcinogens, sensitizers or irritants, cause birth and reproductive effects, and damage the nervous system, liver and kidneys. Even at low levels, these pesticides can adversely affect neur ological, respiratory, immune and endocrine systems in children (BP 2010) There are a significant number of pesticide related illnesses in schools that were not originally recognized as exposure to pesticide residues (Alarcon et al. 2005) Approximately 69% of pesticide related illnesses were caused due to pesticides used at schools (Alarcon et al. 2005) Proper prevention programs and education of the populations affected in school can diminish the da nger of pesticide poisoning or repeated illnesses, which are the usual trigger for safer pest management strategies (Owen s 2003, Alarcon et al. 2005) Education is paramount for preventing and controlling bed bugs. Prevention strat egies limit the use of potentially harmful pesticides and ultimately contamination of schools. At the 2009 National Bed Bug Summit in Arlington VA, the EPA recommended the development of a bed bug education curriculum for children in school as part of public education (Necochea 2009) This approach is in direct contrast with the current approach for bed bug control in schools that emphasizes the use of pesticides and with other IPM approaches, which tend to be operational in nature and rely on education of only pest managers and school officials.
43 CHAPTER 3 SPECTRAL SENSITIVITY OF THE COMMON BED BUG CIMEX LECTULARIUS L. (HEMIPTERA: CIMICIDAE) Cimex lectularius L. (Hemipera: Heteroptera: Cimicidae) are blood sucking ectoparasites that have been intimately associated with human beings for thousands of years (Panagiotakopulu and Buckland 1999) They are typically most active between midnight and dawn (Mellanby 1939) rel ying on olfactory and thermal cues for finding hosts mates and harborages (Singh et al. 2012) However bed bugs can be observed actively moving around in the day either locating a host or seeking out shel ter. (Usinger 2010) observed that bed bugs that are increasingly starved will bec ome more likely to be found searc hing for a host in the daytime. In daylight visual cues may be used in addition to olfactory and thermal cues for a variety of bed bug activities. (Reisenman and Lazzari 2006) state d that v isual cues are important for both diurnal and nocturnal insects. Especially in many blood feeding insects, vision is important for activation and orientation and is used as an enhancer or used in conjunction with information from other senses for host finding predator avoidance and seeking refuge (Lehane 2005 Reisenman and Lazzari 2006) Although bed bugs are active in the daytime, they have a strong negative phototaxis to light and will actively seek out darker areas (Aboul Nasr and Erakey 1969) (Reisenman et al. 2002) explained that light sensit ivity, as observed in h ematophag o us Triatoma infestans is rhythmically and internally controlled by the shifting of various screening pigments in the rhab dome res of the compound eyes and the ocelli Light sensitivity in the h ematophagous bed bug is most likely sol ely controlled by mechanisms in their compound eyes since there are no ocelli present.
44 There is little documented information on the eye morphology of C. lectularius (Ford and Stokes 2006) but a natomical studies on the compound eye of the tropical bed bug C. hemipterus have revealed the presence of 52 ommatidia in each of the two eyes of the adults (Singh et al. 1996) and five ommatidia with two serrated grooved bristles in each of the two eyes of a 1 st instar nymph (Bastos et al. 2011) T he compound eyes of the common bed bug C. l ectularius should be somewhat similar to its congener C. hemipterus Early studies by Aboul Nasr and Erakey ( 1969) have qualitatively documented bed bug responses to varying inten sities of transmitted light, reactions to uniform light and reflected lights, blinding, shading and adaptation experiments, as well as simple color preferences. Nevertheless, to gain a better understanding of how light and colors influence bed bug behavior s, it is important to first understand the fundamental visual physiology specifically the fu nction of spectral sensitivity (Prokopy and Owens 1983) Spectral sens itivity describes how efficient the eye is at absorb ing a photon of light a t a given wavelength; it also describes the characteristics of various photoreceptor (light sensitive) cells in the eye and how they change based on varying wavelengths of light (Levine 2000, Kelber et al. 2003, Schwartz 2009) Exposing the eye to specific wavelengths of ligh t (chromatic adaptations) allows photoreceptor cells of the adapting color to become less sensitive in relation to photoreceptors of other wavelengths. In dark adaptations the eye is adapted to an environment in which the luminance is less than 0.034 cd/m 2 (Mischle r 2011) The peaks observed in a spectral sensitivity curve provide an indication of how many potential photoreceptors may be present in the eye. The presence of one
45 photoreceptor denotes color blindness, but does not rule out motion and pattern detection (Briscoe and Chittka 2001). Two or more different types of photoreceptors indicate the ability of the insect to see color (discriminate between different wavelengths) regardless of light intensity (Carlson and Chi 1979, Kelber et al. 2003) Spectral sensitivities are mainly influenced by visual pigments present in photoreceptor cells and to a lesser extent by colored screening pigments and corneal filters (Stavenga 2002, Kelber et al. 2003) V isual pigments are composed of an opsin protein (rhodopsin or porphyropsin ) with a carotenoid chromophore (A1: 11 cis retinal or A3: (3R) and (3S) enantiomers of 11 cis 3 hydroxyretinal) (Lythgoe and Partridge 1989, Kelber et al. 2003) (Briscoe and Chittka 2001) Visual pigments ar e characterized by the wavelength at which they absorb the most number of photons (Govardovskii et al. 2000, Stavenga 2010) Visual pigments have absorbance spectra which can be described by a visual pigment template ( nomogram ) Interest ingly, vertebrate and invertebrate visual pigment absorbance spectra have a similar standard sh ape and follow similar spectral rules (Stavenga et al. 1993, Govardovskii et al. 2000, Stavenga 2010) Therefo re the concept of a universal visual pigment template is still upheld, which allows nomograms that were originally developed using vertebrate rhodopsins to be used by scientists doing research on vertebrate and invertebrate organisms such as insects (Govardovskii et al. 2000, Stavenga 2010) Research on other vertebrate ( i.e. geckos, fish, frogs and toads ) visual pigments over the years ( Govardovskii et al. 2000) have improved how well vertebrate nomograms model insect spectral sensitivit y curves (Govardovskii et al. 2000, Stavenga 2010) Both Stavenga and Govardovskii have contending nomograms
46 based on specially selected parameters. Nomograms from both researchers have been shown to fit insect spectral sensitivity curve s best in regions between 400 700 nm (Govardovskii et al. 2000, Stavenga 2010) Unfortunately, their vertebrate based nomograms do not fit well in region s < 400 nm (UV sections). Electroretinography is a reliably robust method for characterizing the spectral sensitivity of insect eyes. This technique involves studying the change in potentials in the sensory and associated ganglion cells of the eye when these cells are stimulated by light (Burkhardt 1977) Spectral sensitivity studies have been accomplished for several h ematophagous insects such as mosquitoes, black flies, tabanids, stable flies, horn flies and tset s e flies (Agee and Patterson 1983, Green and Cosens 1983, Allan et al. 1991, Muir et al. 1992, Mellor et al. 1996, Reisenman and Lazzari 2006) H owever there have been no studies that have documented the spe ctral sensitivity of the bed bug. Based on insufficient documentation on bed bug eye morphology and spectral sensitivity th e aims of this study were to 1 ) use the Automontage technique to acquire enhanced images in order to improve our knowledge of the bed bug eye morphology such as size of eyes and number of ommatidia in nymph and adult bed bug eyes 2) measure the spectral sensitivity of bed bugs eyes using the electroretinography (ERG) technique to physiologically determine the number of photoreceptor s present within the eye. Materials and Methods Insects The bed bugs used in these experiments were the Harlan strain of the common bed bug (Harold Harlan, Armed Forces Pest Management Board, U.S. Department of Defense, Washington, D.C.). These bed bugs were reared at the Urban Entomology
47 Laboratory at the University of Florida (Gainesville, FL) at 23.5 C 0.5 C ~ 50% R H under a 12:12 (L:D) photoperiod using methods similar to Pfiester et al. (2009 ). Bed bugs were main tained in 240 mL pla stic con tainers that were lined with 90 mm Whatman no. 1 filter paper circles (Whatman International Ltd, Maidstone, England) and had several har borages made from folding circular pieces of Whatman no. 1 filter paper in a fan like manner. To prevent escape and facilitate feeding, the top of the plastic holding containers were covered with 90 m mesh screw top lids. Each week, bed bugs were fed on live chickens until they were fully engorged. All colony maintenance was in accordance with the University of Fl orida Institutional Animal Care and Use Committee (UF/IACUC) protocol E876. Automontage S ystem E quipment and t echniques used to captur e images of the bed bug eye were modified from (Obenaue r et al. 2009) A JVC KY F70B 3 CCD digital camera (Cypress, CA), integrated with a dissecting Leica MZ 12.5 stereomicroscope (Leica Microsystems GmbH, Munich, Germany) was used to capture images of the compound eyes of bed bugs. This optical setup also included a Leica motor focus drive on a Leica Z step stand which helped to capture incremental images at multiple focal levels from the top to the bottom of the eye to produce a single well f ocused image. Based on the depth of field and si ze of the eye, no less than four source images were taken. The light source consisted of four fiber optic lights. The image data was processed using the AutoMontage Pro software, version 5.02 ( Syncroscopy, Frederick, MD) and displayed on a Dell Optiplex GX280 desktop co mputer ( Round Rock, TX). Five bed bugs from each life stage (1st instar through to adult including adult males and females) were used to determine the number of ommatidia as well as the
48 length and width of the compound eye. Bed bugs were killed using ethy l acetate and then mounted on a rotating microscope stage As the number of ommatidia across each row of the eye was counted under the microscope, a small pin was used to make a depression in modelling clay (Michaels Stores Inc. Irving, TX ) to represent t he number and position of each ommatidia in modeling clay This helped to ensure counting accuracy. Ele ctroretinogram (ERG) S ystem The stimulating light source used for these experiments was a 300 W Halogen photo optic lamp (EXR 300W/82V, OSRAM) housed in a Kodak Carousel 4600 Projector (Eastman Kodak Company, Rochester, NY). White light was passed through 10 nm narrow bandpass filters (Edmund Industrial Optics, Barrington, NJ) additional neutral density filters ( Wratten gelatin filters; Eastman Kodak Com pany, Rochester, NY ; 0.50: 32% transmittance; 1.00: 10% transmittance; 4.00: 0.01% transmittance ) and a neutral density wheel, which allowed the light in tensity to vary over c.a. seven log units. The resulting monochromatic light had the following wavelengths: 340, 350, 365, 380, 405, 430, 450, 470, 500, 520, 550, 580, 620, 660 and 700 nm. Each of these wavelengths were then passed through a manual shutter (Polaroid 102 camera, Polaroid), and guid ed by a 6.35 mm diameter quartz fiber optic cable (Dolan Jenner Industries, Boxborough, MA), into a lightproof Faraday cage and project ed on to the be d bug eye (Figure 3 1). Bed bugs were positioned such that two ERG micromanipulators with probes holding e lectrodes could be appropriately positioned for recording depolarizations from the compound eye. The recording electrode contained a 10 x amplifier (EAG COMBI 10X, Silvertech, Hilversum, The Netherlands). P rior to
49 experiments, electrodes were electrolytic ally sharpened with 10 M sodium hydroxide and cleaned with distilled water The data acquisition and analysis were managed using a Syntech Autospike IDAC controller system (Syntech, Kirchzarten, Germany ) and the Syntech EAG 2000 ver 2.4 software (Syntech Kirchzarten, Germany ). Electrophysiological responses from light stimuli applied to the eye were amplified via an AC amplifier (10x) (Syntech, The Netherlands), and displayed on a Gateway 2000 desktop computer (Gateway, Irvine, CA). The optical setup use d with the bed bugs was calibrated using a radiometer (StellarNet Inc, Tampa, FL) that measured emitted light ( photons/cm 2 /sec ) from all combinations of band pass and neutral density filte rs. Extracellular ERG Procedure Prior to electrophysiological record i ngs, bed bugs were placed in a P etri dish on top of a frozen icepack for approximately 3 4 minutes to decrease their activity for mounting purposes. The bed bug wa s then mounted (ventral side up ) on a cylindrical, wax coated Plexiglas stage, and immobili zed by gently depressing all legs, antennae and the proboscis into the wax. All this was achieved while e nsuring that the eyes were not obstructed. The stage mounted bed bug was then transferred to the Faraday cage where sharpened r ecording and indifferent electrodes (0.127 mm tungsten wire, A M Systems Inc, Carlsburg, WA), were appropriately positioned. The indifferent electrode was first manipulated into position and inserted into the abdomen of the bed bug, then the recording elec trode was inserted into the center of the left eye. The terminal end of the fiber optic cable that would deliver the stimulus flash was positioned approximately 1 cm from the test eye using a micromanipulator (World Precision Instruments, Sarasota, FL). A test stimulus flash was given to see if the experimental eye was responsive If no
50 depolarization response was observed, a new insect was prepared If the test flash resulted in a depolarization, then the bed bug was adapted for 30 minutes prior to the sta rt of the experiment. Bed bug spectral sensitivity w as explored under dark (no light), red (620 nm), blue (450 nm) and green (520 nm) adapted conditions. Selective adaptation by a particular wavelength desensitizes the visual pigments associated wi th that wavelength, thereby increasing the possibility of revealing the existence of other visual pigme nts that may have been masked (Defrize et al. 2011) All color adapted bed bugs were exposed at a neutral density filter value that produced 40% of the maximum depolarization for that particular wavelength (following dark adaptation) After the adaptation period, each bed bug spectral sensitivity test lasted for about 40 50 minutes Criterion response tests T hree replications of criterion response tests were performed at wavelengths of 340, 350, 365, 380, 405, 520 and 660 nm to ensure that a constant size of ERG response could be obtained across all tested wavelengths by only adjusting light intensity levels. Once the criterion response was selected, it was possible to proceed with the spectral sensitivity experiments. Spectral sensitivity test s For each selective adaptation experiment (including dark adaptat ion) there were 10 replicates (five males, five fema les). Following a particular adaptation, e ach bed bug was exposed to a ll 15 monochromatic wavelengths to produce ERG responses that were close to the designated criterion response. Bed bugs were allowed to recover for one minute following each 0.125 ms sti mulus light flash. Th e number of photons that produced a criterion response at each particular wavelength was calculated from a
51 linear regression of neutral density against photons x 10 12 The inverse of the number of photons giv es spectral sensitivity val ues. The equation S ( i ( ) denotes the sensitivity of the curve where S = spectral sensitivity and i ( ) = the sensitivity of the alpha band at each wavelength (Skorupski and Chittka 2010) For each adaptation spectral sensitivity curves were calculated from the mean of all ten replicates Standard error s of the means were also calculated. To remove differences in absolute sensitivity between individual insects mean spectral curves were full scale normalized (Allan et al. 1991) To f urther solidify the number of visual pigments present in the bed bug eyes t he dark adapted sensitivity curve was comparatively fitted with t wo nomogram s based on coefficient values that were tabulated by (Stavenga et al. 1993) The dark adaptation sp ectral curve was used because it represents the summed responses of all photoreceptors (Kirchner et al. 2005) Intensity response s Intensity re sponse curves were obtained for r ed (620 nm), yellow (580 nm) blue (470 nm), green (520 nm) and ultraviolet (365 nm) light s which were tested at wavelengths between 340 7 00 nm using neutral density filters of 30 increments from 30 350. Bed bugs were dark adapted for 30 minutes before testing. F ive male and five female bed bugs were used for each selected wavelength tested. Voltage vs log of intensity (V log I) curves were plotted to determine how the response varied with increasing light intensity. Bed bugs were exposed to a maximum intensity of 5.2 x 10 15 photons/cm 2 /sec (green 520 nm light at 350 filter), whereas a typical daylight (sunlight) intensity i s 3.6 x10 13 photons/ cm 2 /sec (Forward et al. 2000)
52 Data Analysis A chi square goodness of fit test was used to compare how well fit the Stavenga nomogram was compared with Govardovskii for the dark adapted sensitivity curve. To measure the differences in mean intensity responses between male and female bed bugs as well as differences between male and female sensitivities under selective chromatic adaptation unpaired t tests were performed using JMP software (version 9.0.2 SAS Institute Inc. 2010). Means separation was achieved using the least significant difference (LS D) post hoc test. To determine whether there were difference s in the number and siz e (length and width) of ommatidia for each bed bug life stage, a one way analysis of variance (ANOVA) was performed. Difference was used as the po st hoc test to determine which means were significantly different. Results Compound Eye Morphology The c ompound eyes of bed bugs have a red pigmented area surrounding dark pigmented ommatidia for all nymph and adult stages Also no ocelli were present (Fig ure 3 2) The smooth corneal surface of the compound eye was divided i nto ommaditial facets which varied in number depending on the life stage (Table 3 1). The number of ommatidia increased with life stage and a significant increase in the number of ommati dia were observed between the 3rd and 4th instar and between the 4th and 5th instars ( F = 114.61; df = 6 ; P < 0.0001) The number o f ommatidia found in adult male, female and 5th instar nymph eyes wer e not significantly different. A lso the number of ommatid ia was the same for the right and left eye for each stage. The length and width of each eye also significantly increased with life stage (length: F = 72.89 ; df =
53 6 ; P < 0.0001; width: F =110.54; df = 6 ; P < 0.0001) (Figure 3 2 ). All instars except fifth instars had significantly shorter and narrower eyes than the adults. Specifically, fifth instar nymphs had similar sized eyes (length and width) compared with adult males, but their eyes were significantly smaller (length and width ) than adult females. The length of a dult male and female eyes were not significantly different but females had significantly wider eyes compared with males ( F = 72.89 ; df = 6 ; P < 0.0001). Two i nterommatidial setae were observed protruding from the middl e of the compound eye during the early (1 st to 3 rd ) instars ( Figure 3 3 ). Later instars and adults (4th to adult) had longer setae and these setae protruded from the edges, rat her than in the middle as in the early instars (Figure 3 3 ) In addition to inte rommatidial setae, several additional setae were observed on the outer periphery of the eye that became more prominent in the late instars and into the adult stage. ERG Waveforms Electroretinogram recordings from adult bed bugs in response to short flashes of light typically produced a negative waveform (Figure 3 4 ). In intensity experiments waveforms had a similiar negative waveform Spectral Sensitivity When compound eyes of adult bed bugs were dark adapted one peak with a maximum of 520 n m was observed and a smaller peak in the ultraviolet region at 365 nm was also observed (Figure 3 5 ). With s elective adaptation to red (620 nm) monochromatic light modified the sensitivity curve the maximal peak was shifted to 550 nm and the smaller peak originally in the 365 nm UV region was no longer present A strong shoulder was also observed at the 500 nm region (Figure 3 6 ) Under blue adaptation, the spectral curve was again modified to reveal two peaks; one at 500 nm
54 and the other at 550 nm (Figure 3 7 ), whereas under green (520 nm) adaptation, a peak at 520 nm was observed with a distinct shou lder at 550 nm region (Figure 3 8 ) Nomograms that were modeled by Govardovskii (GOV) (Figure 3 9A) and Stavenga Smits Hoenders (SSH) (Figure 3 9B) fit the long wavelength ( 450 nm 660 nm ) region of the curve well (GOV: 2 = 19.5, SSH: 2 = 12.64) based on the combination of the potential peaks obtained from the selective color adaptation experiments but both nomograms unsatisfactorily fit the curve in the shorter wavelength range (Figure 3 9 ). For both the SSH and the GOV models, a single 520 nm nomogram fit s reasonably well however the addition of a second nomogram at the 550 nm wavelength in a 85% 520 and 15% 550 combination fit the spectral sensitivity curve better than either 520 nm or 550 nm alone (Figure 3 9 ). A simultaneous co mparison of both GOV and SSH in the 450 660 nm region showed that the that the SSH no mogram more closely approximated the mos t points compared with the GOV nomogram (GOV: 2 = 19.5, SSH: 2 = 12.64; smaller chi square means better fit) but again, both nomogram s did not accurately fit the shorter wavelength region (Figure 3 10) A comparison of spectral sensitivities for male and female bed bugs show ed significant gender differenc es for selective color adaptation s in the green (520 nm), yellow green (550 nm) and yellow (580 nm) regions for most adaptations More specifically, u nder dark adaptation (Figure 3 11) males were significantly more sensitive i n the green (520 nm) region whereas females were significantly more sensitive in the yellow (580 nm) region ( t = 2.44 df = 1, P = 0. 0164 ) Under red adaptation (Figure 3 12) males were significantly more sensitive in the yellow green
55 (550 nm) and yellow region s ( t = 3.19, df = 1, P = 0.0019) The reverse was observed under blue adaptation (Figure 3 13) where the females were overall significantly more sensitive in the green (520 nm) and yellow (580 nm) compared with the males ( t = 2.04, df = 1, P = 0.044 2) Finally, under green adaptation (Figure 3 14) females were more sensitive over a wider wavelength range (405 470 n m and 580 nm) compared with males ( t = 4.51, df = 1, P <0.0001). However in the areas where differences had been observed in other adaptations [green (520 nm), yellow green (550 nm) and yellow (580nm) regions ] males and females were similarly sensit ive A comparison of males (Figure 3 15A) under all adaptations show that most of the significant differences were observed with the 520, 550 and 580 nm regions ( F = 11.1052, df = 3, P < 0.0001). Red and blue adaptations significantly dampened the 520 nm peak, thereby revealing a 550 nm peak which differed significantly amo ng three adaptations Also, all the color adaptations significantly affected how sensitive the males wer e to the yellow (580 nm) region Males were less sensitive to yello w light when dark adapted but more sensitive to yellow light under red adaptation (F igure 3 15A ). When females were compared under all adaptations (Figure 3 15B) significant differences also were observed in the 520, 550 and 580 nm regions (as were males) but additional differences were observed in the 450 nm 470 nm region ( F = 11.1052 df = 3, P < 0.0001). Under dark adaptation, females were less sensitiv e to green light than when they were when green adapted. Also under green adaption, females were significantly more sensitive to the 550 nm wavelength than all other adaptations. Red a nd dark adaptati on significantly lowered female sensitivity to yellow (580nm) light, wh ile green and blue adaptation significantly increased female sensitivity to the same
56 wavelength Also under green adaptation females were more sensitive to blue light a t 450 nm, and blue green light at 470 nm than if th ey had been blue or red adapted (Figure 3 15B ). Intensity Responses V log I curves showed that as the light intensity increased, the response produced by bed bug also increased (Figure 3 16 ) Green light (520 nm) gave higher responses than all other wavelengths measured, and ultraviolet light (365 nm) had the lowest responses overall. Between higher intensi ties, i.e 240 350 on the neutral density filter wheel, responses became more varied and a plat eau e ffect was observed after 27 0 for all wavelengths. Overall the s lope of the response curves was not parallel (Figure 3 16 ) The steepness of the c urves did not appear to depend on whether the wavelengths were long or short. Steep curves were observed in the long and short wavelengths regions ( red, green and ultraviolet ) whereas shallow slopes were also observed in the blue (short) and yellow (long) wavelength regions. To more accurately determine intensity differences between males and females, the slope of the curve ( i.e. between points a and b in Figure 3 16 ) was more closely examined. Males were significantly more sensitive to green ( t = 2.57 df = 1, P = 0.0135) (Figure 3 17), blue ( t = 4.87, df = 1, P < 0.001) (Figure 3 18) and red ( t = 4.35, df = 1, P < 0.001) ( Figure 3 20) wavelengths at the selected intensities compared with females However males and females were similarly sensitive to yellow ( t = 1.84, df = 1, P = 0.0708) (Figure 3 19) and ultraviolet ( t = 1.55, df = 1, P = 0.1 272) (Figure 3 21 ) wav elengths Differences in the steepness of the slopes between males and females were most noticeable in blue, red and ultraviolet wavelengths
57 Discussion The aim of this study was to determine the spectral sensitivity and hence the number of photoreceptors present in the eyes of C. lectularius as determined by electrophysiological data Electroretinogram recordings can provide insight into the electrophysiological input to the (Dring and Chittka 2007) Coupled with behavioral bioassays, these electroretinogram results can help us determine what wavelen gths are biologically significant for bed bugs. T he common bed bug, C. lectularius, has at least two spectral sensitivity peaks representing possibly two separate visual pigments ; a primary pigment in the green region (520 nm) and a second ary pigment in the green yellow ( 5 5 0 nm ) region The bed bug spectral curve was most closely fitted in the 400 660 nm range by a combination of 85% 520 nm (green) and 15% 5 50 nm (yellow green) rhodopsins from peaks of theoretical visual templates offered by SSH (Stavenga et al. 1993) and GOV (Govardovskii et al. 2000) the UV region of the spectral curve. Unfortunatel y the vertebrate and invertebrate spectral data that is currently available is insufficient to allow for improvements to the modeling of the UV region at this time (Stavenga 2010) A comparison of the SSH fit versus the GOV fit demonstrated that the SSH nomogram is a better fit for the bed bu g spectral sensitivity curve The dark and red color s of the bed bug eye implies the presence of screening pigments These screening pigments usually found in insects eyes (Stavenga 2002) can affect the spectral sensitivity of the eye by broadening the spectral curve (Carlson and Chi 1979 ) As observed in the SSH n omogram fitting the SSH curve still misses a couple of the data points along the curve and this is primarily due to the presence of the
58 screening pigments. While the GOV nomograms were comparable to SSH, GOV nomograms resulte d in curves that were broader th an what could only be explained by screening pigments visual templates work better on spectral curves that span more than t hree log units in sensitivit y and involve many more parameters (Stavenga 2010) The bed bugs spectral sensitivity values which range from 10 13 to 10 16 is better suited for SSH which uses fewer parameters and is more s uited for spectral values spanning three or less log units (Stavenga 2010) A dult bed bugs do not seem to possess the typical trichromatic sensitivity that is commonly observed in most insects (Chittka and Waser 1997) Electroretinography studies have revealed that most insec ts are trichromatic with spectral sensitivity peaks at ~350 nm (UV), ~440 nm (blue), and ~ 530 nm (green) (Chittka and Waser 1997) Tetrachromacy, the presence of an additional red visual pigment around 565 nm, has been observed for insects in the orders Diptera, Lepidoptera, Hymenoptera, Co leoptera, Odonata and Hempitera (Chittka and Waser 1997, Reisenman and Lazzari 2006) However, other insects have been known to have as few as two types of visual pigments (e.g. owlflies, the American cockroach and several ant species), whereas some Diptera are known to possess up to six visual pigment types (Chittka and Waser 1997) F rom the chosen adaptations, the presence of a green (520 nm) and yellow green (550 nm) visual pigments in the common bed bug are supported. Dark adaptation enhanced s ensitivity across the entire visual spectrum, which allowed for documentation o f the dominant visual pigment with a maximum at 520 nm. Interestingly, when the bed bugs were green light (520 nm) adapted, this did not seem to de sensitize the green visual pigment because a dominant peak was still observed at the 520 nm wavelength.
59 It is possible that the intensity of green light that was used for the adapt ation was not sufficient to diminish the 520 nm peak. Nonetheless, exposing bed bugs to low intensity green light (green adapting) appeared to heighten the sensitivity of the bed bugs eyes to ultraviolet (365 nm) blue (470 nm), yellow green (550 nm) and ye llow (580 nm) wavelengths. This suggests the dominant influence of the green (520 nm) visual pigments of the bed bug. The dominance of green visual pigments in particular has been do cumented for other blood feeding insects (Lehane 2005) Red (620 nm) and blue (450 nm) chromatic adaptations enabled the detection of an additio nal visual pigment at 550 nm. Behavioral color studies with bed bugs ( C hapter 4 ) showed that red was the most attractive color to adult bed bugs. However no peaks were observed in the red region of the spectral sensitivity curve, which means that bed bugs do not have red visual pigments Studies with bees have shown that even t hough an insect may not have red visual pigments it does not mean that the red wavelength is invisible to the insect (Dring and Chittka 2007) Colors of an object that an insect can see depends on the reflectance curve from that object and visual pigments present For example when a red stimulus was provided to the potato aphi d which lacks red visual pigments its green receptors were stimulated (Dring and Chittka 2007) indicating that green visual pigments could assist in see ing red because of the broad absorbance curve of the green receptors A similar phenomenon is perhaps what is being exhibited in the common bed bug where it was observed that red adaptation increased sensitivity in the yellow green (550 nm) region. Rea sons for why bed bugs are so sensitive to green and yellow green wavelength s are unknown. It is possible that green visual pigments are ancestral traits
60 and all insects have them. If there is no selection against having that visual pigment then it would no t be lost. Also, perhaps the green receptors are very convenient for providing contrast for other wavelengths. For example in related blood feeders such as female tabanid flies, their sensitivity to yellow green is biologically important because the salt m arshes in which they search for a blood meal have a yellow green background. I t is important to have this a contrast between the prey and their background (Allan et al. 1991) However, in behavioral studies ( C hapter 4 ) green harborages were the most avoided for both males and female bed bugs. Perhaps green or green yellow wavelengths signify being outdoor s with plants and bright light which is not where C. lectularius are normally found. Their s ensitivity to green may be strong to ensure avoid ance of unsuitable habitats This would also support their strong avoidance of yellow harborages that may indicate brightness or too much light based on their cryptic, nocturnal photonegative behavior (Aboul Nasr and Erakey 1969) These electrophysiological studies have not provided sufficient information to confirm the presence or absence of a UV photoreceptor in bed bugs To date UV photoreceptors have not been confirmed to be absent for all insects studied (Chittka and Waser 1997) Even in the case of some fireflies where UV re ceptors were not detected, researchers concluded that UV receptors were possibly present, but were beyond detectable regions (Cronin et al. 2000) For many flying and terrestrial nocturnal insects, the pre sence of ultraviolet receptors (ultraviolet sensitivity) enhances the detection of UV light which helps in selecting mates, finding food, navigating t heir habitat and orientation (Stark and Tan 1982, Cowan and Gries 2009)(Cruz and Lindner 2012) In m y study, there is no evidenc e of an additional UV receptor but there are
61 several reasons why a UV photoreceptor may not have been detected. It is possible that 1) the UV peak is outside the range of the wavelengths that were measured with our equipment ( i.e. we did not hav e f ilters lower than 340 nm), 2) the number of UV absorbing pigments were not enough to be detected with the selectiv e color adaptations that we completed (Defrize et al. 2011) or 3) the UV produced from our light sour ce was too low. I n the dark adaptation, however beta pigments show absorpti ons in the UV which means that UV light can be detected even with no specific receptor. Intercellular studies on nocturnal arthropods have generated additional evidence for UV abso rbing pigments (Walla et al. 1996, Kelber et al. 2002, Skorupski and Chittka 2 010) In order to fully determine the presen ce or absence of a UV receptor in bed bugs f uture studies should focus on intercellular studies of the adult bed bug eye. Also electrophysiological studies and molecular studies should be carried out on other cim i cid congeners such as C. hemipterus, C. adjunctus etc to compare the presence or absence of UV photoreceptors. Perhaps the UV photoreceptor s were diminished or lost as species evolved from feeding on herbivorous insects to feeding on humans (Usinger 2010) Although the presence of secondary blue photoreceptors has been show n for both blood feeding and nocturnal insects (Green and Cosens 1983, Lehane 2005) bed bugs appear to lack specific b lue photoreceptor s This is rather unique given the typi cal trichomacy (UV, blue and green) in insects. The absence o f these blue photoreceptors was supported by the non significant difference in spectral sensitivity under dark and blue chromatic adaptations This is quite surprising because of the important ro le that blue light plays for other crepuscular insects. As the sun goes below the horizon, the
62 amount of blue wavelengths in the sky increases until blue light is the primary light found in the irradiance spectrum of a twilight sky (Cronin et al. 2006, Johnsen et al. 2006) Nocturnal insects use blue photoreceptors to discern o rientation cues that are present when clear blue twilight skies provide constant polarization patterns (Cronin et al. 2006) B ed bugs may have no need for these photoreceptors since they are able to locate their host quite well from thermal and olfactory cues and the task of avoiding predators is low in human dwelling s Based on behavioral observations ( C hapter 4 ), blue did not appear to be the most attractive color for being used as a harborage unlike the hematophagous tsetse fly (Green and Cosens 1983) However, blue was one of the top choices for oviposition sites for bed bugs ( C hapter 4 ). Possibly be that bed bugs react differently in the presence of blue reflected li ght and blue transmitted light. For example, t setse flies have been known to avoid UV reflected surfaces but ar e attracted to UV light sources (Green and Cosens 1983, Green 1989) B ed bugs may be using their green receptors to see blue wavelen gths because of the broad curve of their dominant green based photoreceptors. Future studies should examine bed bug re action to various light sources to supplement what is just now being discovered concerning their behavior to reflected targets ( C hapter 4 ) Under the selective chromatic adaptations, the sensitivity of male and female bed bugs shifted considerably Overall, in the dark and when exposed to red light, males become more sensitive than females to specific wavelengths. Also under blue and green lights, females were overall more sensitive than males. All chromatic adaptations eliminated UV sensitivities in both genders, specifically using green light with males. These findings suggest that one could manipulate bed bug behavior via disorientation
63 by exposing their eyes to another wavelength. This strategy could be useful when improving traps and monitoring tools. In terms of light intensity, the compound eyes of C. lectularius only respond over a range of one log unit of light intensity. Intensity response data indicate that bed bugs follow a normal biological response T hey can respond to light intensitie s as low as 1.5 x 10 11 photons/cm 2 /sec, which is similar to the light intensity from a moon or starlight (McFarland and Munz 1975, Lythgoe 1979) and t heir eyes get more sensitive as light i ntensity increases. Greater than ~ 1.1 x 10 15 photons/cm 2 /sec ( upper threshold intensity for bed bugs ) the l ight intensity gives a plateau in response and no increase in sensitivity occurred with further increases in intensity This seemingly narrow range of sensitivity to light is surprising give n that the bed bug can be acti ve both in the day and at night when the variation of light intensity ranges between 7 9 log units between moonlight and sunligh t (Reisenman et al. 1998, Dacke et al. 2003) Light intensity studies with another h ematophagous insect, T. infestans has sho wn that this insect is able to respond to over four log units of intensity based on their dusk and dawn activity period (R eisenman et al. 1998, Dacke et al. 2003) Future studies should be focused on more detailed phototaxis studies for the bed bug, especially in the low light intensity regions such as those found in cracks and crevices (0.002 0.004 W/cm 2 ) (~ 6.3 x 10 9 1 .26 x 10 10 photons/cm 2 /sec ) (Reisenman et al. 1998) Bed bug gender specific differences in light intensity were also observed for some wavelengths ( g reen [520nm], blue [470 nm] and red [ 6 20nm] ) but not for others (yellow [580nm] and UV [ 365 nm ] ). For gender related intensity differences, males were overall more sensitive than females as green, blue and red light intensity increased T hese
64 wavelengths may be behaviorally important for reasons other than harbo rage finding (as discussed in C hapter 4) Also, the eye structure arrangement for males or the location of pigments with optimal maxima may be different from that of females This difference would result in electrophysiological differences. For example, female bed bug eyes are significantly wider than male bed bug eyes and this phenomenon may suggest females do not have to be as sensitive to light as the males. For many insects it is usually the males that have larger eyes than females (Land 1989) The findings presented in this study concerning the compound eyes of C. lectularius rep resent s a more detailed documentation of the eye morphology of this species of bed bugs than is currently available The eyes of C lectularius are similar to its congener C. hemipterus in terms of shape and the number of ommatidia present in the 1st instar based on studies by (Reisenman and Lazzari 2006) However one of the major differences between the two species is that the number of ommatidia present in the adult eyes of C. lectularius (~30) are almost half the number found in C. hemipterus eyes (~ 52) (Bastos et al. 2011) Also interommatidial setae are still present in the adults of C. lectularius (Singh et al. 1996) but are absen t in the eyes of adult C. hemipterus (Ford and Stokes 2006) Th ese differences could be used as a dditional morphological characteristic s to differentiate between both species of bed bugs. Future studies should first involve 1) compound eye histological studies to document characteristics such as spatial arrangement of rhabdoms and designation of apposition or superposition eyes and 2) do cument ation of a more detailed ultrastructure of compound eye of C. lectularius using SEM as initiated by (Singh et al. 1996) This technique allows for the study of bed bug eyes in a more natural state without excessive
65 coating. This will help us to better understand vision and visual be havior in C. lectularius. These morphological and ERG studies on the bed bug C. lectularius provides novel information that can practically help to improve control of this urban pest. This study has provided evidence for at least two photoreceptors in the green and yellow green range as supported by Stavenga and Govardovskii nomograms. Clear differences between male and female bed bugs based on sensitivity to chromatic adaptations and increased ligh t sensitivity provi des insight into the fine details of bed bug vision. It appears that males and females have similar peripheral sensory physiology, but differences in the type and level of hormones present in their bodies may cause them to respond differently to various re flected wavelengths as was documented in C hapter 4 Also improved knowledge of eye morphological characteristics can enhance understand ing of the visual behavior of nymphs and adults to environmental wavelength cues.
66 Figure 3 1. Schematic diagram of the electroretinogram (ERG) apparatus from Dr. Sandra Allen, ARS/CMAVE/USDA
67 Figure 3 2. Automontage images of different stages of bed bug ( Cimex lectularius ) eyes. A) 1 st instar, B) 2 nd instar, C) 3 rd instar, D) 4 th instar, E) 5 th instar, F) adult male, G) adult female
68 Table 3 1. Anatomical characteristics of the compound eye of Cimex lectularius across various life stages. Life Stage Mean no. ommatidia SEM Mean length of eye ( m) SEM Mean width of eye( m) SEM 1 st instar 5.2 0.2c 74 5.1d 62 3.7d 2 nd instar 7.8 0.4c 84 2.4d 64 2.5d 3 rd instar 9.0 0.0c 90 3.2d 72 2.0d 4 th instar 21.2 1.6b 154 5.1c 112 3.7c 5 th instar 27.0 1.8a 182 3.7bc 130 3.2b Male adult 30.0 0.9a 192 14.7ab 146 7.5b Female adult 30.4 0.7a 214 4.0a 164 2.5a Means followed by the same letters are not significantly different P = 0.05 (LSD) Ommatidia no: F =114.61; df = 6; P < 0.0001; Length: F =72.89; df = 6; P < 0.0001; Width: F =110.54; df = 6; P < 0.0001 N = 5 bed bugs observed per life stage.
69 Figure 3 3 Interommatidial setae for A) 1st instar and B) 5th instar nymph bed bugs. A B
70 Figure 3 4 A sample electrophysiological (ERG) re sponse of a dark adapted female bed bug responding to a 520 nm light flash with a = baseline, b = stimulus applied, c = amplitude response.
71 Figure 3 5 Spectral sensitivity of dark adapted eyes of adult common bed bugs Cimex lectularius N = 10 individuals. Means standard errors.
72 Figure 3 6 Spectral sensitivity of red adapted eyes of adult common bed bugs Cimex lectularius N = 10 individuals. Means standard errors.
73 Figure 3 7 Spectral sensitivity of blue adapted eyes of adult common bed bugs Cimex lectularius N = 10 individuals. Means standard errors.
74 Figure 3 8 Spectral sensitivity of green adapted eyes of adult common bed bugs Cimex lectularius N = 10 individuals. Means standard errors.
75 Figure 3 9 Visual template fittings for bed bug spectral sensitivity A) Stavenga (SSH) and B) Govardovskii (GOV) A B
76 Figure 3 10 Comparison of Stavenga (SSH) and Govardovskii (GOV) nomogram fitting s for bed bug spectral sensitivity
77 Figure 3 11 Spectral sensitivity of dark adapted male an d female adult common be d bugs, Cimex lectularius N = 5 individuals of each gender. Means standard errors
78 Figure 3 12 Spectral sensitivity of red adapted male and female adult common be d bugs, Cimex lectularius. N = 5 individuals of each gender. Means standard errors.
79 Figure 3 13 Spectral sensitivity of blue adapted male and female adult common be d bugs, Cimex lectularius N = 5 individuals of each gender. Means standard errors.
80 Figure 3 1 4 Spectral sensitivity of green adapted male and female adult common bed bugs, Cimex lectularius N = 5 individuals of each gender. Means standard errors.
81 Figure 3 15 Spectral sensitivity of A) male and B) female adult common be d bugs under different color adaptations N = 5 individuals of each gender for each adaptation. Means standard errors. A B A
82 Figure 3 16 Intensity response of adult be d bugs ( C. lectularius ) for selected wavelengths and slope cutoff between points a and b N = 5 individuals of each gender for each adaptation. Means standard errors.
83 Figure 3 17 Intensity response of male an d female adult bed bugs for green light (520 nm). N = 5 individuals of each gender for each adaptation. Means standard errors.
84 Figure 3 18 Intensity response of male and female adult bed bugs for blue light (450 nm). N = 5 individuals of each gender for each adaptation. Means standard errors.
85 Figure 3 19 Intensity response of male and female adult bed bugs for yellow light (580 nm). N = 5 individuals of each gender for each adaptation. Means standard errors.
86 Figure 3 20 Intensity response of male and female adult bed bugs for red light (620 nm). N = 5 individuals of each gender for each adaptation. Means standard errors.
87 Figure 3 21 Intensity response of male and female adult bed bugs for ultraviolet light (365 nm). N = 5 individuals of each gender for each adaptation. Means standard e rrors.
88 CHAPTER 4 BEHAVIORAL RESPONSE OF CIMEX LECTULARIUS TO VARIOUS WAVELENGTHS OF LIGHT FOR OPTIMAL HARBORAGE CHOICE Insects use visual cues for a wide variety of behaviors including, but not limited to, recognizing hosts, finding mates, ovipositing and seeking shelter. These visual cues are often times based on the perception of color. The ability to distinguish between different wavelengths of light as opposed to different light intensities, is termed color vision (Menzel and Backhaus 1991, Cuthill 2006) Many insects exhibit color vision and color preferences. Color preference is an example of how color vision, as a receptor neural strategy (Menzel and Backhaus 1991) a biologically significant behavioral response. Weiss ( 1943) states that in addition to abiotic factors such as temperature and moisture, and biological factors such as the size and development of th e compound eyes, are important for understanding how insects respond to visual stimuli. The common bed bug, Cimex lectularius, is a nest parasite, which implies that when it is not feeding, it is searching for a crack or crevice to hide in, and spends th e majority of time within that harborage P heromonal components which attra ct bed bugs to harborages are crucial for improving bed bug monitoring tools (Siljander et al. 2008) In addition to pheromones color is also an important clue that haematophagous insects use to seek shelter (Green and Cosens 1983, Steverding and Troscianko 2004) These authors showed that t raps with a blue exterior and black and red interior were very effective at attracting and optimizing tsetse fly trap captures. Early studies by Aboul Nasr and Erakey ( 1969) have documented that the common bed bug is able to distinguish between different wavelengths of light using various chemical solutions. Short wavelength c olors such as violet (haematoxylin
89 0.005%) and bluish green (fast green 0.001%) were preferred compared with the other colors tested R appeared to be the least attrac tive. As documented in C hapter 3, bed bugs appear to be dichromatic due to the presence of photoreceptors i n the green (520 nm) and yellow green ( 550 nm ) wavelengths. These electroretinogram studies, along with previous color studies by Aboul Nasr and Erakey ( 1969) indicate that bed bugs have the mechanism for color vision and are particularly sensitive to certain wavelengths. Because many hematophagous insects use including bed bugs, have color vision, the goal of this research was to determine if bed bugs use their color vision to select harborages Therefore the object ives of this research were to 1 ) determine whether color influence s bed bug harborage choice 2 ) ex amine the effect of gender, nutritional status, aggregation and life stage on harborage color choice, and 3 ) determine whether color influences w here female bed bugs oviposit Results from these objectives will be fundamental in helping us understa nd bed bug behavior so that we can improve monitoring devices and traps used in bed bug management. Materials and Methods Bed Bug Rearing and Maintenance The bed bugs used in these experiments were the Harlan strain of t he common bed bug (Harold Harlan, Armed Forces Pest Management Board, U.S. Department of Defense, Washington, D.C.). These bed bugs were reared at the Urban Entomology Laboratory at the University of Florida (Gainesville, FL) at 23 24 C, ~ 50% R H under a 12:12 (L:D) photoperiod using methods similar to Pfiester et al. (2009). Bed bugs were maintained in 240 ml plastic containers that were lined with 90 mm filter paper circles and had several harborages made from folding strips of Whatman no. 1 fi lter paper
90 (Whatman International Ltd, Maidstone, England) in a fan like manner. To prevent escape and facilitate feeding, the top of the plastic holding containers were covered with 90 m mesh screw top lids. Each week, bed bugs were fed on live chickens until they were fully engorged. All colony maintenance was in accordance with the University of Florida Institutional Animal Care and Use Committee (UF/IACUC) protocol E876. B ed bugs in two nutritional stages were used : fed and starved. Starved bed bugs were those that had not been fed within their norm al once a week feeding regimen. Fed bed bugs were those that had been blood fed 1 2 day before experimentation Visual Arena The visual bioassays were conducted in a Lab Tek, extra deep, Petri dish (150 x 20 mm; ThermoFisher Scienti fic, Pittsburgh, PA). The base of each Petri dish was scourged with 60 grit Aluminum Oxide sandpaper ( Gator Finishing Products Fairborn, O H) to allow bed bugs walk to more easily within the arena. Each Petri dish area was placed within a large Pyrex dish (25 x 33 cm; Anchor Hocking, Co. Lancaster, OH) to further prevent bed bugs from escaping. Each Petri dish arena contained two or seven color choices (depending on the type of experiment) that were arranged as small tent like harborages (Fig ure 4 1 ). The colored tent harborages (2 cm x 1 cm) were made from various color ed cardstock paper (Michaels Stores Inc., Irving, TX). The wavelength of each harborage color was measured using an Ocean Optics USB 2000 Spectrometer with a Sony ILX 511 linear silicon CCD array detector (Ocean Optics, Dunedin, FL) ( Figure 4 2 A ). All colored harborages had a dominant wav elength peak, except t he lilac harborage which had two primary peaks violet and red. The spectral reflectance of three male and three female adult bed bugs were measured usin g the Ocean Optics spectrometer (Figure 4 2B). The
91 spectrometer was held at an angle (~ 23 ) and at a fixed distance (0.5 cm) above the dorsal surface of the bed bug. Two 32 watt 4 Pin CFL bulbs (2040 lumens each) (Phillips Lighting Company, Somerset, NJ), and six 15 W F15T8 D fluorescent daylight bulbs (700 lumens each) ( General Elect r ic, Indonesia) (were suspended above the test areas and provided light for the experiments. The experimental room was maintained at 26 27 C and average RH of 60.5%. All bed bugs were placed in the experimental room for acclimatization 24 hrs prior to the bioassays. As the bed bug harborage color choice experiments were conducted the doors were closed, and no human hosts were present inside the experimental room. Adult Visual Bioassays Two choice assay s. These bioassays were performed to determine whether bed bugs would show significant gender and nutritional status differences in response to various colors when selecting a suitable harborage. Bed bugs were given the choice of each of eight color harborage s : lilac violet, blue, green yellow, orange, red and black against white (standard). Both harborage choices were in the arena c.a. 3 cm apart and 2.5 cm from the perimeter of the Petri dish to prev ent edge effects. A single bed bug was then placed in the middle of the Petri dish arena and was given 10 min to make a choice of going under a particular colored harborage. This was considered to be one replication. After the end of the 10 min period, the harborage under which the bed bug was found was recorded After each replicate, new colored harborages were placed in the arena and the positions of the control and color ed tents were randomized to prevent positional biases. Each colored harborage and each bed bug was used only once. If any fecal spots were seen in any bioassay arena, this arena was washed with soapy water
92 and dried before it was used for another bioassay. This experiment was replica ted 40 times with 7 10 day starved males and females, and 1 2 day fed males and females. Data from the two choice assay determined the harborage colors that were used in the seven choice assay. Seven choice assay s. These bioassays were performed to de termine whether bed bugs would show significant gender, nutritional status, and aggregation differences in response to various wavelengths of reflected light when selecting a suitable harborage. The harborage colors chosen for this experiment were depe ndent on their attractiveness in relation to the white standard. Any color that was marginally significant or significantly less preferred tha n white was not included in the choice of seven colors. Therefore the following s even harborage color choices tes ted simultaneously against each other were : lilac violet, blue green, orange, red and black The colored harborages were arranged in a semi circular manner in the are n a with each harborage approximately 2.5 cm from the edge of the Petri dis h to control for edge effects. Bed bugs were tested either individually (one at a time) or aggregated (groups of 10 at a time). Aggregated bed bugs were either groups of all males, all females or a 1 : 1 ratio of males and females. One replicate consisted of all seven color choices randomly arranged with either one or 10 bed bugs in the center of the arena. Bed bugs were given 10 min s to make a choice for a particular colored harborage. At the end of the 10 min period, the number of bed bugs under each harb orage was counted and recorded. As before, i f any fecal spots were seen in any bioassay arena, this arena was washed with soapy water and dried before it used for another bioassay. There wer e 80 replicates of individually tested bed bugs for each group (7 10 day starved males and
93 females, 1 2 day fed males and females). Also there were 10 replicates of aggregated bed bugs (100 insects in total) for each group (7 10 day starved males and females, 1 2 day fed males and females). No bed bug or colored harborage was used more than once. Nymph Visual Bioassays Two choice bioassay. These bioassays were similar to the two choice bioassays for the adults with the exception that the only harborag e color choice offered was yellow v ersus white. The nymph stages tested with the choice of yellow and white colored harborages included: 1 st stage never fed, 1 st stage fed but not molted, and 2 nd 5 th stages. These bioassays were repli cated 40 times. Seven choice bioassay T hese bioassays setups were similar to the seven choice bioassays for the adults. Individual (one nymph) and aggregated nymphs (groups of 10) were tested as well. The nymph al stages were tested with all seven harborage colors ( lilac violet, blue green, orange, red and black ) included was as in the previous experiment These bioassays were replicated 80 times for each nymph al stage. Oviposition Bioassays These bioassays were conducted to test if female bed bugs prefer to oviposit eggs in harborages of specific colors Seven color harborages were tested simultaneously ( lilac violet, blue green, orange, red and black ). Immediately following the usual feeding regime, female bed bugs were allowed to mate for 1 hour in the testing room (24 26 C, 50 51% R H). Following mating, 10 female bed bugs were placed in a Petri dish arena with seven colored harborages listed above and left for 72 hrs to oviposit. After 72 hrs the number of eggs under each colored harborage was counted. These oviposition bioassay experiments were replicated 10 times (100 females in total).
94 Statistical A nalysis Nominal logisitic regression was used to determine whether gender, nutritional status and aggregation influenced harborage color choice. Two choice and seven choice preference data were analyzed using the 2 analysis (JMP 9.0.2 SAS Institute Inc. 2010). Me an separation was determined by comparing upper and lower 95% confidence interval (CI) limits for overlap. Means with c onfidence interv al limits that overlapped were considered similar while means with CI limits that did not overlap were considered signi ficantly different. Also, adult bed bug data were pooled to determine overall be d bug color preference and these data were also analyzed using 2 analysis and comparison of overlapping 95% confidence limits. One way analysis of variance (ANOVA) was used to determine bed bug oviposition preference and the number of females present in relation to the number of eggs. Total number of eggs in the harborage were square root transformed before statistical analyses. M eans were separated usin g the least significant difference (LSD) post hoc test. Results Spectro meter Readings The colored cardstock used for bed bug visual harborage cues represented the full breadth of the visual spectrum (~ 400 700 nm) The following are the colored harborages at their primary peaks based on their reflectance curves (Figure 4 2): violet max = 457 nm, blue max = 470 nm, green max = 520 nm, yellow max = 578 nm, orange max = 608 nm, red max = 639 nm and lilac max = 43 7 and 664 nm : two primary peaks at violet and red The peak wavelength at which bed bug reflected was in the red region of the spectrum: male max = 685 nm, female max = 679 nm (Figure 4 2B).
95 Behavioral Bioassays Based on nominal logistic regression, be d bug harborage color choice was influenced by all factors tested : gender ( 2 = 49.9, df = 6, P < 0.0001), nutritional status ( 2 = 15.8, df = 6, P = 0.0151), aggregation ( 2 = 50.6, df = 6, P < 0.0001) and an interaction of gender and aggregation ( 2 = 14.28, df = 6, P = 0.0266). Adult t wo color choic e bioassays Starved males and females showed a preference for lilac harborages over white harborages (males: 2 = 12.1, df = 1, P = 0.0 005; females: 2 = 6.4, df = 1, P = 0.0114), whereas fed males and fed females showed marginal preference between these two colored harborages ( 2 = 3.6, df = 1, P = 0.057) (Figure 4 3 ). With the exception of starved males who had a marginal preference for violet over white harborages ( 2 = 3.6, df = 1, P = 0.057) starved females and fed male and females showed no preference for violet harborages over white harborages (Figure 4 4 ). W ith blue harborages starved males ( 2 = 6.4, df = 1, P = 0.0114), sta rved females ( 2 = 4.9, df = 1, P = 0.0269), and fed males ( 2 = 4.9, df = 1, P = 0.0269) significantly preferred blue to white colored harborages (Figure 4 5 ). Fed females were the only group that showed no preference for blue over white colored harborage s ( 2 = 0.4, df = 1, P = 0.5271) (Figure 4 5 ). S tarved males showed a preference for green over white ( 2 = 4.9, df = 1, P = 0.0269 ), whereas starved females showed marginal p reference for green over white harborages ( 2 = 3.6, df =1, P = 0.057) (Figure 4 6 ). Fed males and females showed no preference for green harborages over white harborages (males: 2 = 1.6, df = 1, P = 0.2059; females: 2 = 2.5, df = 1, P = 0.1138) (Figure 4 6 ).
96 Neither starved nor fed males and females show ed any preference for yellow over white harborages (starved males: 2 = 1.6, df = 1, P = 0.2059, starved females 2 = 0.10, df = 1, P = 0.7518, fed males: 2 = 0.10, df = 1, P = 0.7518, fed females 2 = 0.00, df = 1, P = 1.00) (Figure 4 7 ). F ed males ( 2 = 14.4, df = 1, P = 0.0001), f ed females ( 2 = 19.6, df = 1, P < 0.0001) and starved males ( 2 = 8.1, df = 1, P = 0.0044) significantly preferr ed orange harborages over white harborages (Figure 4 8 ). Starved females were the only group that showed no preference for orange or white harb orages ( 2 = 2.5, df = 1, P = 0.1138) (Figure 4 8 ). A ll bed bug groups (starved and fed, males and females) strongly preferred red and black harborages to white harborages (Figure 4 9 and Figure 4 10 ) (RED: starved males 2 = 22.5, df = 1, P < 0.0001, starved females 2 = 22.5, df = 1, P < 0.0001, fed males 2 = 16.9, df = 1, P < 0.0001, fed females 2 = 16.9, df = 1, P < 0.0001; BLACK: starved males 2 = 14.4, df = 1, P = 0.0001, stav ed females 2 = 10.0, df = 1, P = 0.0016, fed males 2 = 19.6, df = 1, P < 0.0001, fed females 2 = 16.9, df = 1, P < 0.0001). Adult t wo choic e bioassays (by nutritional status) Over 80% of f ed male and female bed bugs chose harborages that reflected longe r wavelengths (orange, red, black) over white harborages (Figure 4 11A, B) Although fed females were not particularly interested in shorter wavelength harborages ( lilac violet, green yellow), fed males found lilac harborages more attractive than white harborages. Starved females seemed to be less discriminatory in their color choices (Figure 4 12A) However, with the exception of the yellow harborage, more starved males were found in every harborage color compared with white harborages (Figure 4 12 B)
97 Adult seven choice bioassays When all data were pooled to compare all seven colors for bed bug harborage preference, it was observed that adult bed bugs significantly preferred red (28.5%) and black (23.4%) harborage s compared with all other colored harborages (Figure 4 1 3 ). R ed and black harborages were not significantly different from each other. Orange harborages (17.9%) were less preferred than red or black harborages, but were significantly preferred over lilac violet, blue and green harborages. Blue (10.9%) and lilac (9.3%) harborages were the third most preferred harborages and were not significantly different from each other. Blue harborages were significantly preferred to violet ( 6.8%) and green (2.8% ) harborages. L ilac harborages and were similarly preferred to violet harborages but were both significantly preferred to green harborages. Green colored harborages were the least preferred harborage color of the seven colors test ed Yellow harborages were not included in the choice of multiple colors to be tested because yellow was not significantly different compared with the white standard for any of the bed bug groups tested G ender influences bed bug color harborage choice 2 = 48.68, df = 6, P < 0.0001. Male and female color preferences are significantly different (Figure 4 1 4 ). Male bed bugs significantly prefer black and red compared with female bed bugs, whereas female bed bugs significantly prefer lilac and violet compared to male bed bugs. Nutritional status also influences bed bug color choice. F ed bed bugs prefer orange and violet harborages significantly more when they were starved 2 = 15.21, df = 6, P = 0.0187 (Figure 4 1 5 )
98 Harborage color preferences change depending on how bed bugs are grouped ( 2 = 67.94, df = 12, P < 0.0001). Lone bed bugs prefer black and green significantly more than when they are in a mixed gender group (Figure 4 1 6 ). When male and female bed bugs are together in a group they prefer red and violet significantly more than when t hey are in single sex groups (Figure 4 1 6 ). Also bed bugs groups of the same gender significantly preferred black over groups of bed bugs of mixed gender. Interestingly, bed bugs show no preference for blue, orange and lilac colored harborages whether bed bugs are alone, in same gender aggregations or mixed gender aggr egations (Figure 4 16 ) When males and females were tested individually or as an aggregation of mixe d gender (5 males 5 females), there were no preferences for any colored harborage over the other ( 2 = 2.803, df = 6, P = 0.8331 and 2 = 10.51, df = 6, P < 0.1049 respectively). However, in a same gender aggregation, males preferred red and black compared with the females, while the females preferred lilac and violet compared with the males ( 2 = 57.421, df = 6, P < 0.0001) (Figure 4 1 7 ). Nymph two choice bioassays. First instars (never fed) ( 2 = 14.4 df = 1 P = 0.0001 ) and 5 th instars ( 2 = 19.6 df = 1 P < 0.0001 ) significantly preferred white harborages to yellow harborages. There was no significant differences between yellow or white harborages for, 1 st instars (fed but not molted) ( 2 = 0.4 df = 1 P = 0.527), 2 nd instars ( 2 = 0.4 df = 1 P = 0. 527), 3 rd instars ( 2 = 0.4 df = 1 P = 0.527) or 4 th instars ( 2 = 3.6 df = 1 P = 0.0578) (Figure 4 18 ). Nymph seven choice bioassays. Yellow harborages were excluded from the harborage color choice for the nymphs because yellow harborages were not
99 sig nificantly preferred over white harborages, and in two cases, white was preferred over yellow. When 1 st instar bed bug nymphs that had never been fed were exposed to all seven harborage color choices, none of these harborage colors were significantly prefe rred over any other (Figure 4 1 9 ). However, after these 1 st instar nymphs were allowed to feed some color preferences was observed ( 2 = 14.15 df = 6 P = 0.028). Significantly fewer bed bug nymphs were found under the lilac harborage compared with green, orange and black harborages (Figure 4 20 ). Color harborage preferences were also observed for 2 nd instar nymphs ( 2 = 25.18 df = 6 P = 0.0003), 3 rd instar nymphs ( 2 = 29.02 df = 6 P < 0.0001), 4 th instar nymphs ( 2 = 29.02 df = 6 P < 0.0001), and 5 th instar nymphs ( 2 = 43.2 df = 6 P < 0.0001). Second instar nymphs significantly preferred all colored harborages except lilac (black, green, orange, red, and violet) to blue (Figure 4 21 A). Third instar nymphs significantly prefer green and red harborages to blue, lilac and violet harborages (Figure 4 21 B). Fourth instar nymphs significantly prefer bl ack, blue and red harborages to lilac and violet harborages (Figure 4 21 C). For fifth instar nymphs, black, blue, orange, red and lilac harborages were all significantly preferred to green and violet harborages (Figure 4 21 D). Ovipos i tion bioassay Female bed bugs preferred some colored harborages to others for where eggs were ovipositie d (Figure 4 22 ). For instance, females significantly preferred to oviposit under orange, black and blue harborages compared with green harborages ( F = 2.33 df = 6 P = 0.0427) Also, red lilac and violet harborages were similarly preferred to green harborages.
100 Discussion Both adult and immature bed bugs are able to differentiate between different colors and preferentially select harborages based on co lor specific visual cues. The se harborage color bioassays indicate the important role that light play s for bed bugs as they locate a suitable hiding/nesting area. When bed bugs are exposed to bright light, they are often observed moving away from the light towards darker areas (Aboul Nasr and Erakey 1969) Ha rborages (crack and crevices) are very important to bed bugs because they spend 90% of their time in harborages, and when not in a harborage, they are either actively searching for a host or looking for new harborage sites (Pinto et al. 2007) It has been speculated that a bed bug would go to any harborage in an attempt to escape visibility. However, these color experiments show that bed bugs do not hide in just any harborage; rather they will select a harborage base d on its color. Therefore it is possible that specific color s represent 1) an opportune oviposition site or mating arena or 2) safety due to the presence of other bed bugs and/or a site that limits visi bility by predator s. When the seven most attractive colors were tested simultaneously, bed bugs preferred to hide in red and black colored harborages more than any other color harborage. These findings are similar to those reported for tsetse flies in (Steverding and Troscianko 2004) Black and red interiors on tsetse traps increased the number of tsetse flies that were captured. Also for bed bugs, green harborages were the least preferred th an any other color. This finding is actually contrary to color experiments from Aboul Nasr and Erakey ( 1969) who stated that short bluish green wavelengths were more appreciated b y bed bugs.
101 It has been documented that nutritional status and population density are important factors influencing the search for new habitats in haematophagous triatomines (Minoli and Lazzari 2006) It appears that once b ed bugs are fed, the color of a harborage becomes more important, perhaps because the bed bugs are not interested in finding food, but are more interested in looking for the most suitable nest sites where they would be less disturbed. In this case orange and violet colors appear to represent wavelengths that are more appreciated. The reason for this is unclear, but could be due to hormonal changes from increased nutrition. However for starved bed bugs harborage color appeared to be less important perhaps because hungry bed bugs are more interested in less protected areas where a host is more likely to be found. Nutritional status does also affect bed bug harborage choice in a manner such that fed bed bugs prefer orange and violet harborages more readily than when they are hungry. It may be that orange and violet wavelengths are particular wavelengths that are desensitized when a bed bug is hungry, but become important for harborage finding after a bloo d meal is consumed. Color preference shifts with regards to nutritional status has also been observed in pigeons (Delius 1968) and toads 1999) I n addition to nutritional status and aggregation, harborage color preferences were also influenced by gender M ale bed bugs preferred to hide under red and bl ack (longer wavelength) harborages, whereas female bed bugs preferred to hide under lilac and violet (shorter wavelength) harborages. A similar trend was observed when males and females were in groups of ten according to their gender. Although attractiveness to the shorter wavelengths was observed in my experiments, as was also observed by (Aboul
102 Nasr and Erakey 1969) a greater percentage of females were fo und in red and black harborages compared to the lilac or violet harborages even when the aggregation factor was considered. Additional evidence pointing to the significance of red and black harborages appears from the two choice assays which sh owed that females strongly preferred both colors when each color was offered against a white standard. However males and females were not always keen on choosing lilac or violet when those colors were each offered against white. Apparently bed bugs behave differently when they are in a mixed group of males and females as opposed to being alone or in a same gender group. In mixed aggregations, bed bugs prefer to be in red harborages. The fact that o nly about 12.5% of bed bugs from a mixed aggregation a ctually chose a black harborage was surprising since cracks and crevices are considered black (dark) areas. It appears that red may be the optimum harborage color for bed bugs in their naturally oc curring mixed aggregations. However, when bed bugs are alone or in a same gender aggregation, they much prefer to be in b lack harborages. This information is particularly useful when we think about the behavioral and physiological mechanisms that help bed bugs seek out and colonize new harborages. Although most bed bugs are found heavily aggregated in harborages, within most infestations single, isolated bed bugs have been observed away from the primary harborage site s (Pfiester et al. 2009) For a wanderi ng / isolated bed bug, red harborages may signify highly populated areas where competition for space may be a problem. In that case, lone bed bugs may seek out another harborage such as a black
103 harborage, which may signify a minimally populated area suitab le for colonization and egg laying. Also, it has also been observed that female bed bugs tend to prefer female f emale aggregations over female male aggregations for fear of excessive mating (Pfiester et al. 2009) This may explain, to some degree why black harb orages were more attractive to female bed bugs because this may have been consi dered a safer harborage that provided an area away from the males Y ellow and green were least preferred or somewhat repelle nt to bed bugs. These unattractive visual cues should be taken into consideration when improving monitoring tools or traps for bed bugs. Other examples exist in the literature where either of these colors have been avoided or considered unattractive by blo od sucking insects such as sand flies triatomines and other such insects houseflies, and weevils (Reisenman and Lazzari 2006; Hausmann et al. 2004; Diclaro et al. 2012; Hoel et al. 2007) It should also be noted that yell ow harborages were not preferred over white harborages for any group. Aboul Nasr and Erakey (1969) originally made the observation that yellow appeared to be the least attractive c olor for the colors they tested. In the case of choosing of a harborage, it appears that yellow colored harborages are not seen as suitable harborage by a bed bug under any circumstance. Color vision and preference s are also influenced by life stage. Immature bed bugs do exhibit harborage color preferences and these preferences change between successive instars. In the two choice assay, 1 st instars (never fed) and 5 th instars strongly preferred white to yellow harbo rages, which would imply that yellow is repellent color for bed bug nymphs. When all stages of the nymphs were offered all seven color choices (similar to the adults), it became apparent that 1 st instar nymphs that have
104 never taken a blood meal c ould not differentiate between colors and should theref ore be considered color blind. However once those 1 st instars were fed, but had not yet molted to the next instar, some color preference began to occur; black, green and orange were more attractive than lilac C olor preferences in the 2 nd instar were most different compared to all other instars. More 2 nd instars were found under violet harborages, whereas blue harborages were the least preferred. From the 3 rd inst ar to the 5 th instar, we begin to see color preferences that appeared to be more similar to the adult. By the 5 th instar, all harborage colors except green and violet were similarly attractive This shifting of color preferences may be due to developmental differences in structure, shape and number of ommatidia in the eye in each instar ( as documented in C hapter 3), as well as neurological processes in the brain. It appears that colors influence where female bed bugs will oviposit. Female bed bugs preferred to oviposit in orange, black and blue harborages. Low oviposition rates were found in green and violet harborages. Specific harborage colors do influence where bed bugs choose to deposit their eggs and should be definitely considered when improving bed bu g monitoring tools and traps. Surprisingly, red harborages were not necessary considered to be the best egg laying sites, although we have documented that red harborages are strongly preferred for various stages of bed bugs. Black and blue colors were also found to be the preferred oviposition sites for a couple of other well known insect species (Katsoyannos et al. 1986, Hoel et al. 2011) Results from these studies indicate that bed bugs have different color preferences for their harborage and oviposition sites. Harborage color preferences are influenced by gender, nutritional status and aggregation, and will change with life stage. Red and
105 black harborages, overall, appear to be the most attractive harborages for a wide range of bed bug life stages, whereas yellow and green harborages, overall appear to be the least attractive for most life stages, yellow being definitely repellent for two nymph stages. These findings should prove useful for optimizing trap designs for bed bug management strategies. Future studies should include additional light intensity studies for additional visual cue information, and involve research on a combinatio n of visual and volatile cues.
106 A B Figure 4 1 Colored harborages for bed bug choice tests. A) Two choice bi oassay of harborage colors, B) S even choice bioassay of harborage colors
107 Figure 4 2. Reflectance spectra of A) colored cardstock, B) male and female bed bugs as determined by Ocean Optics Spectrometer (Dunedin, FL) A B
108 Figure 4 3 Percentage of bed bugs responding to lilac versus white colored harborag es Indicated significant difference in 2 test ( P < 0.05; 95% confidence intervals). Figure 4 4 Percentage of bed bugs responding to violet versus white colored harborages No significant differences in 2 test at ( P < 0.05; 95% confidence intervals).
109 Figure 4 5 Percentage of bed bugs responding to blue versus white colored harborages. Indicated significant difference in 2 test ( P < 0.05; 95% confidence intervals). Figure 4 6 Percentage of bed bugs responding to green versus white colored harborages. Indicated significant difference in 2 test ( P < 0.05; 95% confidence intervals).
11 0 Figure 4 7 Percentage of bed bugs responding to yellow versus white colored harborages No significant differences in 2 test at ( P < 0.05; 95% confidence intervals). Figure 4 8 Percentage of bed bugs responding to orange versus white colored harborages Indicated significant difference in 2 test, ( P < 0.05; 95% confidence intervals).
111 Figure 4 9 Percentage of bed bugs responding to red versus white colored harborages Indicated significant difference in 2 test, ( P < 0.05; 95% confidence intervals). Figure 4 10 Percentage of bed bugs responding to black versus white colored harborages Indicated significant difference in 2 test ( P < 0.05; 95% confidence intervals).
112 Figure 4 1 1 Adult bed bugs response each colored harborage versus white standard harborage A) fed females, B) fed males Indicated significant difference in 2 test, ( P < 0.05; 95% confidence intervals). A B
113 Figure 4 1 2 Adult bed bugs response each colored harborage versus white standard harborage A ) S tarved females, B ) S tarved males Indicated significant difference in 2 test, ( P < 0.05; 95% confidence intervals). A B
114 Figure 4 1 3 Overall bed bug harborage color choice. Means topped by different letters are significantly different ( P < 0.05; 95% confidence intervals). Figure 4 1 4 Influence of gender on harborage color choice. Indicated significant difference in 2 test ( P < 0.05; 95% confidence intervals).
115 Figure 4 1 5 Influence of nutritional status on bed bug harborage color choice. Indicated significant difference in 2 test ( P < 0.05; 95% confidence intervals). Figure 4 1 6 Influence of aggregation on bed bug harborage color choice. Means topped by different letters are significantly different ( P < 0.05; 95% confidence intervals).
116 Figure 4 1 7 Influence of gender and aggregation on bed bug harborage color cho ice. Indicated significant difference in 2 test ( P < 0.05; 95% confidence intervals).
117 Figure 4 1 8 Bed bug two choice bioassay for various nymphal instars. Indicated significant difference in 2 test ( P < 0.05; 95% confidence intervals).
118 Figure 4 1 9 Mean percentage of 1st instar bed bug nymphs (never fed) choosing colored harborages in a visual P etri dish arena. (No significant difference detected). Figure 4 20 Mean percentage of 1 st instar bed bug nymphs (fed but not yet molted) ch oosing colored harborages in a visual Petri dish arena. Means topped by different letters are significantly different ( P < 0.05; 95% confidence intervals).
119 Figure 4 21 Bed bug nymph choices to harborage colors A) 2nd instar nymphs B) 3rd instar nymphs C) 4th instar nymphs and D) 5th instar nymphs Means topped by different letters are significantly different ( P < 0.05; 95% confidence intervals). A B C D
120 Figure 4 22 Bed bug oviposition preference. Means topped by different letters are significantly different ( P < 0.05; 95% confidence intervals).
121 CHAPTER 5 DEVELOPMENT AND IMPLEMENTATION OF A BED BUG IPM ENRICHMENT CURRICULUM Bed bugs are the greatest current challenge for the adoption and implementation of school IPM programs nationwide. Schools are prime opportunistic sites for bed bug introductions, re introductions or constant infestations. Communities with high numbers of bed bug infestations often have frequent bed bug introduction s within schools in those communities ( Green and Gouge 2009) Because bed bugs are not easily detected, they can be transported from infested homes to school on a child's belongings such as a book bag (Thompson 2011). At school they can be transferred from students to surroundings and from s urroundings to staff (New Jersey Education Association 2010). Currently there are numerous documents on the Internet about bed bugs ranging from various websites, news reports, and extension publications (Gangloff Kaufmann and Shultz 2003, Lewis et al. 2009, Merchant 2010, Miller 2012) These sources document information concerning the biology, medical significance, prevention and treatment methods for bed bugs, and are aimed at educating the general public. However, regardless of the wealth of information that exists concerning bed bugs, there is still much ignorance, and bed bug s continue to spread. Unfortunately, some PMPs cannot properly identify bed bugs, and occasionally get them confused with secondary pests such as carpet beetles. There are still many of the general public who think that bed bugs a re non existent or microsco pic. Schools that have encountered incidences of bed bugs unfortun ately resort to pesticides as their primary management strategy and in some cases the improper use of pesticides lead to bigger problems such as pesticide related illnesses (McFarland
122 2010, Owens 2003, Alarcon et al. 2005). Fortunately there are available online protocols on how schools can mitigate bed bugs (Miller 2012) The more serious concern is how to educate the public so the transference of bed bug s to schools is greatly reduced. Advancements in school integrated pest management (IPM) depend on continuous involvement wi th teachers, stude nts and staff within the school. Basic building blocks of IPM include education, monitoring, identification, and prevention (sanitation and exclusion). Reduction in bed bug spread can be accomplished by reshaping how we educate different groups of people. Education is the foundation of IPM and is paramount in preventing and controlling bed bugs. Properly educating people on various p revention strategies would reduce pesticide applications and residue contamination of schools. At the 2009 National Bed Bug Su mmit in Arlington, VA, the EPA recommended the development of a bed bug education curriculum for children (Arlington Bed Bug Summit, 2009). Curricula are viewed as the link between schools and society and are useful when they have merit (intrinsic value) and worth (payoff value) (Welch 1969) They are comprehensive learning plan s that inv olve creating guided activities that students can do within or outside the ir school environment These guided activities translate into learning experiences based on predetermined goals (Frey 2011, Kerr 1968) A ny successful curriculum takes into account external standards and local goals (content), and shapes a plan that delivers effective teaching and learning (Wiggins and McTighe 2005) Curriculum develo pment is a cyclical, coordinated set of process es that has six stages as shown in Figure 5 1 (Kern et al. 2009) This developmental process is
123 dep endent on the evaluation of the implement ed curriculum. While m an y curriculum evaluation models exist they all share the following parameters : 1) study of context, 2) determination of client concerns 3) use of qualitative methods 4) assessment of opportunity costs, 5 ) sensitivity to unintended effects and 6 ) development of reports for different audiences (Glatthorn et al. 2011) Curricula need to be evaluated to dete rmine if the planned activities and programs produce desired results ( intended outcomes, achievement targets or performance standards) (Wiggins and McTighe 2005) Figure 5 2 provides a n evaluation checklist (Glatthorn et al. 2011) to aid educational planners in estimating cur riculum success A focus group of public school educators from the D uv al County School (Duval County, FL) district determined that 3rd 5th graders were high priority targets for a bed bug curriculum. According to Piaget's theory of cognitive development, children between age 7 12 go through the concrete operational stage of cognitive development in which 1) they can more competently draw conclusions from interrelationships through logical reasoning, and 2) they learn by interacting with family and the wider social world (Goswami 2001, Santrock 2008) Also, at this stage, their socioemotional processing (child's relationship with other people, changes in emotion a nd personality) increases, they are more likely to be environmentally aware because they have fewer established environmentally harmful behaviors, and they can be effective at influencing their parents to adopt pro environmental behaviors (Leeming et al. 1997, Santrock 2008) These research observations show that 3rd 5th grade rs can be naturally inquisitive an d excited learners who tend to share what they learn in the classroom with their parents and relatives and who can influence parents and relatives concerning issues such as
124 bed bug awareness. Since bed bugs are ectoparasites that children can bring to sch ool in their belongings, the educator focus groups decided that the educational curriculum should be tailored for health education where concepts such as hygiene and health, critical thinking, critical understanding, environmental understanding, communicat ion, creativity, social development, technology, and self concept could be covered. Other 4 H curricula that have targeted elementary aged students by focusing on similar health education concepts, health related concerns or overall healthy living ( e.g. Munchy Adventures and Health Rocks! ) have demonstrated success. For example, M unchy Advent ures which was developed to educate students to think critically about their heath and make better health choices showed that 90% of participants were more active as a result of the implemented curriculum and all students agreed to make healthier choices (Elbert et al. 2009) A similar health based curriculum, Health Rocks! showed that 93% of youth increased knowledge about risky health behaviors, while 95% of youth learned social, personal and/resistance skills ((4 H) 4 H Youth Development 2012) Therefore the goal s of this research were to: 1) develop a bed bug curriculum for 3rd 5th graders, and 2) evaluate the curriculum through knowledge based evaluations for various groups including both youth and adult learners Materials and Methods Curriculum D evelopment After input from the focus group, a curriculum outline was developed according to the six step process outlined by (Kern et al. 2009). Lessons and activities were written between April and June 2011 and were modified through April 2012.
125 Step 1: I dentification and analysis of a need or problem that could be addressed by a curriculum The Duval county UF/IFAS extension office began receiving requests from the Jacksonville public schools for inform ation on school IPM policies about bed bugs in early 2011. Jacksonville is a rapidly growing urban center in the state of Florida with a US Census Bureau population estimate of 827, 908 in 2011, and approximately 33,000 are 3rd 5th graders from Duval cou nty. T he Duval County Health Department also experienced increases in the number of calls concerning bed bug sightings in elementary schools during this time Step 2: N eeds assessment of the targeted group that would benefit from the curric ulum To meet the bed bug educational needs of the community, Ms. Erin Harlow, the Duval county extension agent, established the Jacksonville Bed Bug Task Force (JBBTF) The task force which included 24 members representing the University of Florida, Duval County Publi c Schools, Duval County Health Department, Duval County Extension Office, Florida Department of Agriculture and Consumer Services City o f Jacksonville, ElderSource, Housing and Urban Development (HUD) Florida Pest Management Association (FPMA), and vario us local pest management professionals met on several occasions to consider how an educational intervention for 3rd 5th graders would aid in reduction of bed bug re introductions and stimulate overall bed bug awareness The task force set a list of objec tives including forming a list of crucial information concerning bed bug awareness w as important for educators and students to acquire.
126 Step 3: Setting goals and objectives. One of the primary goals of the JBBTF was to increase student awareness of bed bu gs so friends and relatives ). This was accomplished through the health curriculum developed by the education committee of the JBBTF. Step 4: Development of educational strategies to meet outlined goals and objectives. C ontent (material to be included in the curriculum) and methods (manner in which the content was to be presented) were outlined according to the 4H experiential learning model. The 4 H experiential learning model uti lizes the 5 step model of experiencing, sharing, processing, generalizing and applying the concepts learned (Deim 2001) Based on the teacher focus group list of needs, th e curriculu m lesson activities include d a combi nation of nine learning concepts including hygiene and health, critical thinking, critical understanding, environmental understanding, communication, creativity, social development, technology, and self concept The Florida Department of Education 's 2011 N ext Generation Sunshine State Health Standards and Benchmarks for 3 rd 4 th and 5 th gra de ( F DOE 2012) were correlated with each of the lesson activities. Step 5: I mplementation Curriculum implementation involved measuring knowledge transfer from the curriculum into the community via pilot testing. IRB approval (UF IRB # 2011 U 1267) was obtain ed for this curriculum and its supporting materials to be delivered to educa tors, students, and parents Two phases were involved in pilot testing the curriculum.
127 Phase one: Pilot testing the bed bug curriculum in D uv al County, Florida: The first pilot test was conducted in 3 rd 5 th grade classes in Jacksonville, D uv al County, with the potential of impacting approxim ately 33 ,000 students. This involved onsite teacher instruction, and in service training for extension agents and 4 H personnel to facilitat e implementation of material in D uv al County. Data from pre tests and post tests were collected from these educators to determine how and when they would use the curriculum in the classroom. Pre test and post test questions were then divided into biology, medical, and prevention and detection categories to allow access ing the knowledge in these areas. After the teachers used the curriculum, they were asked to evaluate the curriculum on appropriateness for age group, encouragement of experiential learning, and potential and observed material retention among stud ents. Data from actual students will be forthcoming as the curriculum is implemented. With IRB approval, a follow up survey will be distributed to families to measure knowledge transfer from the class room to the home. Phase 2: Pilot testing bed bug curriculum on a national and global level The secon d phase of this project involved presenting this curriculum to other academic and non academic systems throughout the USA and other countries. This was ach ieved by: 1) creati on of an online webinar for educators so that they could take a 30 minute online training session, which allowed them access to a free version of the downloadable curriculum, provided that they score d over 80% on the quiz following the t raining session, and 2) live presentations of the curriculum and bed bug knowledge to the various groups of people. Live presentations involved giving a bed bug knowledg e pre
128 test, then conducting a 2 hr bed bug training presentation on concepts within the curriculum, followed by a post test (same as the pre test) to evaluate what was learned. Pre and post tests were given to several groups of people: Pest Management Professionals (PMPs), NAVFAC (Navy PMPs), 4 H Agents Master Gardeners D uv al County Pub lic School (DCPS) Teachers Students (Non Entomology Majors and 5 th graders), and a Mixed Group which included a partment managers, housekeepers, janitorial staff, transitional workers with low literacy levels. Once the pre tests and post tests were collect ed, the questions were divided in biology, medical, and prevention and detection categories to allow assess ment of the knowledge in these areas. To measure the differences between pre test and post test scores for each societal group, paired t tests were p erformed using JMP software (version 9.0.2 SAS Institute Inc. 2010). Also, to compare differences among all groups for pre test scores and post test scores a one way ANOVA using JMP software (version 9.0.2 SAS Institute Inc. 2010) was used, while means sep aration was determined by LSD post hoc tests. Step 6: Curriculum e valuation In this step, the knowledge transfer data that was obtained from different societal groups were used to formatively evaluate the curriculum This involved identifying areas of improvement and recommending specific suggestions on how to improve the curriculum for these groups in society An assessment of the reading level and reading ease of the curriculum can be found in Table 5 1. The following formula was used to calculate the reading ease score: 206.835 (1.015 x ASL) (84.6 x ASW) where ASL is the average sentence length (number of words/number of sentences) and ASW is the average number syllables per word (number of syllables divided by number of words) (Kincaid et al. 1975) The
129 Flesch Kincaid Grade Level test was calculated using the following equation: (0.39 x ASL) + (11.8 x ASW) 15.59 (Kincaid et al. 1975) Results Curriculum Development The compl eted cu rriculum is a 103 page document that is composed of 1) a b) three lesson plans which encompass a total of 10 lessons, and c) appendices which include the Next Generation Sunshine State Standards and Benchmarks for 3 rd 4 th and 5 th graders (Figures 5 3 5 4 and 5 5) as well as a teacher feedback form (Appendix A). A timeline of the major events from the inception of the curricul um through development and revision to where it is now is presented in Table 5 1. and prevention and detection techniques for 3 rd 5 th grade teachers, and is separated into various sections so that appropriate sections can be referenced for each lesson taught. The lesson activities are divi ded into nine learning concepts. The first lesson hygiene as well as the medical significance of how bed bugs feed. That lesson focuses on the following learning concepts: communication, health and physical, critical thinking Beyo es on the habitat, growth and development of bed bugs. That lesson has two activities and focuses on critical and environmental understanding learning d on bed bug prevention,
130 detection and control. That lesson has multiple learning concepts including: self concept, creativity, critical thinking, social development and technology. Each lesson activity has the following template: A key concept, objectives, activity introduction with quest Each lesson has one or more of the following hands on activities: crosswords, find a word, matching and mystery games, short compositions, scavenger hunts, drawing and annotating, dances, a nd card games. In preparation for any lesson activity, a rectangular blue box to the right of the page provides pertinent information to the teacher such as: 1) the learning concepts for that particular lesson as recommended by a focus group of certified health educators in Duval county FL 2) the grade level to which the lesson applies, 3) the recommended setting for that lesson, 4) the amount of time that lesson should take, 5) the entomology skill(s) that will be learned, 5) the life skill(s) that will be learned, 6) the next generation sunshine state standards applicable to that lesson, 7) the materials needed for that lesson, and 8) the section of the When the curriculum was evaluated for ease o f reading and grade level based on the Flesch Kincaid scale, heavy text student activities were easy to read (reading e ase average score 82 out of 100 %). Some activities ( Bed Bug Scavenger H unt; 100 out of 100%) were a lot easier to read than others ( Bed B ug Old Maid game; 65 out of 100%) based on the reading ease scores. Also, the average curriculum activity was determined to be at a 4 th grade level and the majority of heavy reading activities in this curriculum were within the 3 rd 5 th grade level based on the Flesch Kincaid Grade Level Scale (Table 5 2 ).
131 For teachers the overall ease of reading the material in the curriculum and on the quiz was on average 70 out of 100% based on the Flesch Kincaid Scale. The majority of the lessons (i ncluding the teacher's guide) was at a 7th grade or higher reading level (Table 5 3 ). Curriculum Implementation Phase 1 : During the onsite training sessions when teachers ( N = 11) were asked if they were willing to participate in the pilot program, and wh ether they would use the curriculum in their classrooms, 100% of the teachers responded yes to both these questions. When they were asked when they would start using the curriculum, 70% said in the next six months, 30 % within the next year (Figure 5 6 ). A s ked when they would start to implement bed bug prevention practices in the classroom, over half (60%) of the teachers wanted to start immediately or within the next six months, where as 40% were not sure (Figure 5 7 ). The 4 H Agents ( N = 14) were also asked a similar series of questions to which 67% of agents were willing to participate in the pilot program (Figure 5 8 ). Of those willing to participate, 87% were planning on using the curricu lum in the classroom (Figure 5 9 ), and 86% were planning on using th e curriculum within 6 months to a year (Figure 5 10 ). W hen the 4 H agents were asked when they would plan on implementing bed bug prevention techniques, 0% r esponded immediately (Figure 5 11 ). Instead, half (50%) planned on implementing prevention techniqu es in the next six months and the other half (50%) were not sure when they would begin implementation (Figure 5 11 ). Phase 2 : When various groups of people were presented with a live presentation of the curriculum concepts and given the pre tests and post tests, there was a significant difference between the amount of knowledge that each group knew before ( F
132 =12.81, df = 7, P < 0.0001) (Figures 5 1 2 ), and after ( F =8.16, df = 7, P < 0.0001) (Figure 5 1 2 ) the tests. Pest management professional (PMPs) had a significantly higher score of 79% in the pre test than all other groups except the 4 H agents ( F =12.81, df = 7, P < 0.0001). The 5 th graders had the lowest score of 53%, but were not significantly lower than the teachers or the mixed group. After the bed bug curriculum pres entation was given and the post test was taken, all groups scored significantly higher than the mixed group ( F =8.16, df = 7, P < 0.0001) (Figure 5 1 2 ). When pre tests and post tests were compared with t tests for each group, all group s scored s ignificantly higher on the post tes t than they had done on the pre test (PMPs: t = 3.87, df = 114, P < 0.0001, 4 H agents: t = 4.28, df = 26, P = 0.0001, Master Gardeners: t = 9.11, df = 94, P < 0.0001, NAVFAC: t = 5.39, df = 66, P < 0.0001, Non Entomology Students: t = 5.17, df = 24, P < 0.0001, Teachers: t = 7.39, df = 20, P < 0.0001, 5 th graders: t = 13.75, df = 166, P < 0.0001, and Mixed group: t = 3.35, df = 64, P = 0.0007) (Figure 5 12 ). The teachers and 5 th graders had the highest percentag e increase in post test versus pre test grades of 34% an d 34.7% respectively (Figure 5 1 2 ). For biology based questions on the pre test and post test, all groups had higher scores on the post test than on the pre test (PMPs: t = 4.04, df = 114, P < 0.0001, 4 H agents: t = 3.25, df = 26, P = 0.002, Master Gardeners: t = 6.19, df = 94, P < 0.0001, NAVFAC: t = 4.18, df = 66, P < 0.0001, Non Entomology Students: t = 3.46, df = 24, P = 0.001, Teachers: t = 9.89, df = 20, P < 0.0001, 5 th graders: t = 12.4 8, df = 166, P < 0.0001, and Mixed group: t = 3.47, df = 64, P = 0.0005) (Figure 5 1 3 ). Again the
133 teachers and 5 th graders had the highest percentage difference between their biology pre test and post test grades of 39% and 36% respectively (Figure 5 1 3 ). For medical significance based questions on the pre test and post test, all groups had higher scores o n the post test than on the pre test (PMPs: t = 3.75, df = 114, P = 0.0002, 4 H agents: t = 2.98, df = 26, P = 0.0031, Master Gardeners: t = 9.94, df = 9 4, P < 0.0001, NAVFAC: t = 4.77, df = 66, P < 0.0001, Non Entomology Students: t = 5.82, df = 24, P < 0.0001, Teachers: t = 7.47, df = 20, P < 0.0001, 5 th graders: t = 9.53, df = 166, P < 0.0001, and Mixed group: t = 5.77, df = 64, P < 0. 0001) (Figure 5 1 4 ). The teachers and non entomology majors had the highest percentage difference (54%) between their medical significance pre test and post test grades (Figure 5 1 4 ). For prevention and treatment based questions on the pre test and post test, all groups had higher scores on the post test than on the pre test, except the mixed group (PMPs: t = 2.43, df = 114, P = 0.0082, 4 H agents: t = 3.67, df = 26, P = 0.0005, Master Gardeners: t = 3.28, df = 94, P = 0.0007, NAVFAC: t = 3.65, df = 66, P = 0.0003, Non Entom ology Students: t = 2.40, df = 24, P = 0.0112, Teachers: t = 2.21, df = 20, P = 0.0196, 5 th graders: t = 8.57, df = 166, P < 0.0001, and Mixed group: t = 0.56, df = 64, P = 0.2858) (Figure 5 1 5 ). The 5 th graders had the highest percentage difference betwee n their prevention and treatment pre and post t est grades of 34.5% (Figure 5 1 5 ). Curriculum demographics. Of the people that have downloaded the online bed bug training module (as of July 2012) 30% were from Florida, 20% were from Indiana, and 10% were from Minnesota, whereas 40% were from a ll the other states (Figure 5 16 ). One individual each from Canada and Saudi Arabia have also downloaded the bed bug training module.
134 Based on information provided by people who have downloaded the curriculum, over 3 9,437 individuals across the U.S., Canada, and Saudi Arabia would be potentially impacted based on the knowledge gained from the curriculum by the poll takers. Figures 5 1 7 prov ides a breakdown of the number of potential impacts by state/province. States w ith over 1000 potential impacts include MN, WI, TX, IN, NC, FL with NJ alone projecting o ver 10,000 impacts (Figure 5 1 7 ) Of the 194 individuals that downloaded the Bed Bugs and Book Bags curriculum, 59% were females and 25% were males ( Table 5 4 ). Over half of these individuals (62%) were Caucasian; the least represented ethnic group being Hispanic American Indian, Indian, and Mixed (each 0.4% of the ethnic mix). Forty seven percent of respondents would be teaching the curriculum to adult learners, where as only 20% of respondent would teach the curriculum to 3 rd 5 th graders. When asked what special groups of people would benefit from the curriculum, 40% of respondents would focus on underprivileged and special needs people, whereas ~8% of respondents woul d focus on the elderly. Thirty one percent of respondents did not have a special group that they would present the curriculum, and only 5% of respondents would present the curric ulum to children under 5 years Twenty three percent of responders would prese nt the curriculum to a group of 11 50 individuals. Approximately 8% of respondents were unsure what size audience they would have when presenting the curriculum. O ne person (0.4% of respond ents) estimated that he/she would be able to get the curriculum c oncepts out to more than 5000 people. Twenty four percent of the respondents were either K 12 teachers or PMPs. Approximately 5% were either 4 H leaders/volunteers,
135 whereas ~ 4% were university faculty. Most of the respondents (52%) had other jobs outside these areas ( Table 5 4 ). Discussion The Bed Bugs and Book Bags IPM curriculum for 3 5th grade students was requested by the D uv al County School Board (Jacksonville, FL), and was created out of a need to heighten bed bug awareness as a result of increased bed bug sightings in Duval County Public Schools and at the same time reduce pesticide usage in schools This curric ulum which has gone through several revisions and pilot testings based on the curriculum timeline, uses the 4 H experiential le arning model, nine learning concepts and hands on activities to encourage students to make healthy choices for themselves and their environment, while providing bed bug prevention strategies if they should encounter bed bugs in their home or at school. The 4 th grade reading level of the curriculum coupled with a reading ease of 82% may indicate that 4 th and 5 th graders are not struggling to read and comprehend the presented activities, but rather they would be more independent in tackling the activities. Fo r the 3 rd grader, a 4 th grade reading level activity means a little more support from the teacher. T he curriculum and most of the act ivities are quite flexible and c ould be adapted for use with a lower or higher grade level. The pre test and post tests whi ch are designed for 3 rd 4 th and 5 th graders was at a 3 rd grade level which means it should be sufficiently appropriate for the targeted grade range for the curriculum. guide and other aspects of the curriculum meant for teachers was at a high 7 th grade level, which is sufficient for reading and understanding given that newspapers and are ta rgeting 9 th to 10 th grade reading leve l ( IIPLS 2005)
136 True education happens when a student develops an understanding of what is being taught, rather than a mere retention of facts for later regurgitation on a test or quiz (Wiggins and McTighe 2005) ; the same is true for learning about bed bug s. In order to determine whether student s have properly understood the material taught in the Bed Bugs and Book Bags curriculum they need to demonstrate what they know and can do based on state content standards (Schmoker 2002) Therefore one of the main characteristics of a successful curriculum is how wel l the content (curriculum), instruction and the assessments (CIA) a re aligned (or agree) with state standards (Schmoker 2002, Aldrich 2007) In other words, (content specified by district or school to be promoted a t a certain grade level) should be align ed (Marzano 2003) T he Bed Bugs and Book Bags curriculum went through the first sta ges of its alignment when the Duval County School H ealth Educational Specialist, Cheryll Hall met with teachers in Duval County and used the Florida Department of Education website and matched the Sunshine State Standards with the lessons in the curriculu m. T he Bed Bugs and Book Bags curriculum was used as an afterschool program in Maine, where educators were teaching kids about pests. Four of the children who had learned about bed bugs during the afterschool program came back the following day saying they had bed bugs, which in fact they did. This is one clear example of successful alignment of Lesson Bed State standards accompanying those lessons encouraged kids to explore and predict how different family and friend traditions and
137 customs influence health behaviors, suggest to and persuade others to make positive health choices, and identify circumstances that help or hinder health decision making Also, the kids were able to learn two of the key learning concepts that teachers wanted for the Bed Bugs and Book Bags curric ulum: self concept (empowerment by learning to take action) and critical understanding (Kern et al. 2009) describes curriculum development as a continuous, on going process with evaluation as one of the key steps Schools are known to provide teachers with opportunities for curriculum review, alignment of instructional strategies and cla ssroom assessments with state standards (DA 2004) T he final appendix of the curriculum contains a teacher evaluation form that encourages feedback This evaluation form includes questions such as which lessons/activities were c ompleted and a comments section where teach ers are free to address how aligned the lessons were with the state standards. Although several models exist that are used for aligning curriculum s with state standar d s (Roach et al. 2008) (Glatthorn et al. 2011) strongly emphasizes the importance of teachers be ing invol ved with curriculum development and that the success of any curriculum program depends on the active involvement and thinking of teachers Therefore a s teachers conclude the pilot testing of the Bed Bugs and Book Bags Curriculum model whic h heavily involves teachers in alignment evaluation should be used as a model for determining how well the Bed Bugs and Book Bags curriculum is aligned with state teaching standards. One of the unique characteristics of this alignment model is that it all ows for an alignment statistic known as an alignment index
138 which produces a score between 0 and 1, (0 being no match and 1.0 being perfect alignment). This model also generates a topographic map that allows for visual display of alignment results (Roach et al. 2008) Another way of measuring curriculum development success is to determine how well the Bed Bugs and Book Bags curriculum compares with other similar curriculum that have been widely used by educators. One of the most successful IPM curricula available is Project Wild (PW 2008) that has been used by over one million educators. Similar to Project Wild, t he Bed Bugs and Book Bags curriculum has been actively sought after by various groups of educators in society (Demographics Figure 5 20), won 1st place State Communications award in the Learning Module category from the Florida Association of County Agriculture Agents (FACAA), and is known nationally and internationally. Also both curricula share similar components such as a background (teacher review) key vocab ulary term s for students in each lesson, answer keys for teachers, stated objectives, and futur e extensions for each activity. The re are two ways in which the Bed Bugs and Book Bags curriculum seems to be superior to Project Wild 1) direct and repeated emphasi s on the 4 H Do Reflect Apply model and 2) accessibility. Both the Bed Bugs and Book Bags curriculum and Project Wild offer training courses before receipt of the curriculum. However the Bed Bug and Book Bags curriculum can be easily downloaded (at no cost) from the University of Florida IFAS website: http://duval.ifas.ufl.edu/Bed_Bugs.shtml whereas the Project Wild curriculum requires travelling to a training site on a specified day to receive the Project Wild curriculum.
139 The Bed Bugs and Book Bags curriculum is an example of a cost effective solutio n to bed bug pest management that is available to the public. In addition to the short training module anyone downloading the Bed Bugs and Book Bags curriculum needs to pass a post quiz and is issued a certificate of being knowledgeable and rightly traine d to deliver proper bed bug information and prevention strategies. There are several other IPM curricula from various universities (University of Michigan State University IPM Pilot Program, Clemson University IPM Teacher Resources, Minnesota Entomology for Kids program) (School IPM Curricula 2005) that are comparable to the Bed Bugs and Book Bags curric ulum in terms of objectives, activity timelines, materials needed, and engaging activities. curriculum most of these curricula do not directly and explicitly present the experiential lea rning Do Reflect Apply Model in their curriculum which is well known for its premise that youth use life skills to learn concepts and that they retain those concepts better when activities are combined with questions (CSREES 1992) Also for many of these curricula, a short background on the activity is g iven which is synonymous to the activity introduction found in the Bed Bugs and Book Bags curriculum. These background sections may not provide a full fact based overview that prepares the educator to teach the class Often, this leads the educator to loo k for fact based overviews at other sources, which may be incomplete or incorrect. With the Bed Bugs and Book Bags curriculum, each lesson specifically points the teacher to specific areas
140 of the t relevant knowledge n eeded to present the lesson. The Bed Bugs and Book Bags c urriculum is in many ways most comparable to the ABC's of Entomology curriculum (Neeley 2002) in terms of direct emphasis on the 4 H experiential model (Do Reflect Apply), and general layout The ABC's of Entomology curriculum even has an informative backgro und basics (similiar to Teacher's Guide in the bed bug curriculum) but the background basics prec edes every major lesson covered. On the other hand, the Bed Bugs and Book Bags curriculum, there is only one large source of background basics at the beginning of the curriculum which forms the Teacher's Guide that is divided in different sections for each lesson for reference. The Bed Bugs and Book Bags curriculum was approved for the 2011 2012 school year by D uv al County Public Schools and is currently being used by te achers and 4 H agents, in D uv al Country as well as within and outside of the US. An important feature of this curriculum is that it not only target students wit hin the classroom but has large scale impact beyond school walls. Based on the demog raphic data collected from online downloads, the majority of people who downloaded the curriculum do not teach within the typical classroom. Their focus is on the general adult population, which implies that there is a real need for this curriculum that wi ll not only be appropriate for 3 5 th grade students but the public at large. Based on observations from delivering the curriculum across Florida, information from the curriculum will be incorporated into informational programs in shelters, churches, and a wide range of community facilities. Based on the targeted audience of 3 5 th grade, once the pilot testing is completed in D uv al County, the curriculum would have potentially impacted as many as 3 3 ,000 students. In addition,
141 the total estimated impact of th e curriculum including Canada and Saudi Arabia will be close to 40,000 individuals, giving a combined potential of about 7 3 ,000 people, who may share the information gained with family and friends This may represent an important step in improving bed bug management in many areas. T he National Center for Healthy Housing suggests that professional educational materials be developed and made easily accessible for multi family housing management (Taisey and Neltner 2010) These materials should communicate the major messages at a t hird grade reading level, or lower, be translated into different languages and Braille and have companion and visual materials for illiterate populations. To date, the Bed Bugs and Book Bags curriculum has been presented to low income families (all of whi ch had bed bug infestations) in five languages (Alabanian, Arabic, Burmese, Nepalese and English). A dditionally, demographic data from the online curriculum downloads show the diversity in ethnic groups that have been exposed to the curriculum. It is benef icial to get the curriculum concepts out to as many groups as possible to make them aware that bed bugs do not discriminate across ethnic groups or places (Pinto et al. 2007) T he teachers and 4 H agents that were trained in the pilot program recognize the importance of the Bed Bugs and Book Bags curriculum and are ready and willing to implement it. The enthusiasm of both teachers and 4 H agents should generate similar excitement in number of 3 5 th graders they educ ate who are most likely to disseminate the information into their homes and community. In the second phase of pilot testing the curriculum, additional evaluation of the Bed Bugs and Book Bags curriculum was achieved through d ata gained from assessing how
142 much people from various groups know about bed bugs This type of data collection for evaluation purposes during curriculum development is known as formative evaluation as described by (Scriven 1967) D uring the curriculum development process, the curriculum has undergone four revisions as a part of the process of curriculum evaluation. Most of the evaluation so far has involved collecting descriptive and critical information on curriculum content, objec tives and methodology which helps in determining the effective ness of the curriculum Although no formal evaluation model (Glatthorn et al. 2011) has been selected for evaluating the curriculum, the use of workshops, focus groups, targeted observations quizzes and questionnaires (Chikumbu and Makamure 2000) have provided sufficient data for formative evaluative p urposes. Based on the evaluation checklist provided by (Glatthorn et al. 2011) the Bed Bugs and Book Bags curriculum can potentially be an effective tool Based on result s from testing various societal groups on different aspects of bed bug information except 4H agents, the pest management professionals (PMPs) knew the most overall about bed bugs before we did the bed bug presentation. The PMPs are the ones who are handli ng resurging bed bug pro blems on a daily basis so it is important that they are largely knowledgeable compared with other groups of people in that area. Also, even though the teachers and the 5 th grade students were among the groups that knew the least abo ut bed bugs, these two groups performed very well based on their experience with the curriculum material. Both groups had the highest overall im provements between pre test and post test Besides the teachers and the 5 th graders, the non entomology students had the 3 rd highest pre to post test score improvement. Even with students outside the 3 5 th grade range, the Bed Bugs and Book
143 Bags curriculum is an effective tool for the traditional teacher student interaction for improving bed bug awareness. R egardle ss of job status (mixed group or PMPs), literacy (mixed group or college level non entomology majors), and age (5 th graders or all other groups), e very group tested was more knowledgeable in area of bed bug biology and the medical significance of bed bugs after they heard the curriculum presentation as opposed to before. However, in the area of bed bug prevention and treatment, only the mixed group did not show increase d learning in this area It is possible that, because of their low literacy level, this m ay have been the hardest portion for them to understand B ed bug prevention and treatment was one area that was expected t o show improvement for all groups, because this is the crucial section of bed bug pest management. As this was not the case, th is subj ect area needs to re thought a nd re taught in a way that different individuals are able to effectively comprehend. The Bed Bugs and Book Bags curriculum currently serves as a model/prototype that can be modified for use for different education levels. The curriculum has also been awarded 1 st place in the Florida Communication Awards in the category of Learning Modules by the Florida Association of County Agriculture Agents (FACAA). This award shows the potential of the curriculum to foster bed bug awarenes s within a wide audience range. Future research for this curriculum should involve a summative evaluation conducted at the end of the curriculum implementation that is aimed at assessing curriculum performance (Chikumbu and Makamure 2000)
144 Figure 5 1. The six step approach to curriculum development (excerpted from Curriculum Development for Medical Education: A Six Step Approach [Kern et al. 2009]).
145 Figure 5 2. Evaluation checklist for successful curricula (excerpted from Chapter 12: Curriculum Evaluation [Glatthorn et al. 2011 ]).
146 Table 5 1 Timeline of major highlights in the development of the Bed Bugs and Book Bags c urriculum for 3rd 5th grade rs Date Accomplishment February 2011 Jacksonville Bed Bug Task Force (JBBTF) formed in response to bed bug incidences in schools March 9 and 24 2011 Meetings at Duval County Public School to discuss bed bug curriculum with health specialist and facilities managers Targeted age range for curriculum agreed on March 16, 2011 Curriculum Development meeting Identification of key concepts for students Identification of the Sunshine State Standards to reinforce those concepts Ideas suggested for curriculum activities March 17, 2011 Curriculum development started March 25, 2011 First JBBTF meeting with all members (with curriculum updates on science standards, teacher's guide) April, 20, 2011 First draft of curriculum completed and curriculum presented to the Duval County School Health Adviso ry Committee April 25, 2011 JBBTF meeting to discuss curriculum development updates May June 2011 Curriculum reviewed by 4 H Curriculum specialist 4 H Regional Specialized agent and Chair of UF Entomology Dept. 1st curriculum revision June 14, 2011 Community workshops started based on concepts from curriculum July 2011 Workshop with inner city missions promoting the curriculum (Jacksonville, FL) July 12, 2011 2nd curriculum revision October 25, 2011 3rd curriculum revision October 28, 2011 Start of teacher trainings with the curriculum November 2011 Extension enhancement grant ($12,000) awarded for pilot testing the curriculum in Duval County December 12, 2011 IRB approval for curriculum December 13, 2011 Bed bug summit (Jacksonville, FL) introduction of wide range of individuals to the curriculum January 12, 2012 Curriculum presented to 4 H extension agents at in service training at the Youth Development Institute (YDI); Ocala, FL January 24 27, 2012 Curriculum introduced to Florida Pest Management Association PMPs February 22, 2012 Introduction of curriculum to Master Gardeners February 28, 2012 4th (and current) curriculum revision March 1, 2012 Curriculum introduced to Navy PMPs March 15, 2012 Curriculum introduced to non entomolo gy majors at the University of Florida March 24, 2012 Curriculum and curriculum training available on the internet March 26, 2012 Curriculum presented to the 7th International IPM symposium; Memphis, TN April 3, 2012 Curriculum wins 1st place state award in category of excellent learning modules by the FACAA April 5, 2012 Curriculum presented to 4 H agents at an in service training in Apopka, FL May 17 and 30, 2012 Curriculum introduced to Z. L. Sung Junior Academy 3rd 5th graders and Pine Ridge Elementary 3rd 5th graders August 2012 Curriculum presented to Extension agents at the Extension Professional Association of Florida meeting October 2012 Curriculum being pilot tested in schools
147 Figure 5 3 Lesson activity sections within the Bed Bugs and Book Bags Curriculum that meet 2008 Sunshine State Standards for 3rd graders in Florida schools.
148 Figure 5 4 Lesson activity sections within the Bed Bugs and Book Bags Curriculum that meet 2008 Sunshine State Standards for 4th graders in Florida schools.
149 Figure 5 5 Lesson activity sections within the Bed Bugs and Book Bags Curriculum that meet 2008 Sunshine State Standards for 5 th graders in Florida schools.
150 Table 5 2 Reading ease and levels for students associated with various ac tivities in the Bed Bugs and Book Bags curriculum based on Flesch Kincaid model. Student Activit ies Flesch Reading Ease (%) Flesch Kincaid Grade Level Bed Bug Scavenger Hunt 100 1 Student Quiz 92 3 Describe the Culprit 88 3 Feeding Crossword 77 5 Feeding Facts 75 6 Field trip Scenario 77 6 Bed Bug Old Maid Game 65 7 Overall Average 82 4 .4
151 Table 5 3 Reading ease and levels for teachers associated with various activities in the Bed Bugs and Book Bags curriculum based on Flesch Kincaid model. Teacher Activities Flesch Reading Ease (%) Flesch Kincaid Grade Level 67 7 55 8 Hygiene and Health 73 6 Eat Like a Bug 80 6 Bed Bug Barracks & Beyond 70 7 Bed Bug Biology 69 8 Bed Bug Game 65 8 Starring Bed Bugs 69 7 Feelings Finder 73 7 Healthy Hand Healthy Home 78 7 It's Getting Hot in Here! 77 6 High Tech Bed Bugs 65 8 Overall Average 70.1 7.6
152 Figure 5 6 Figure 5 7 prevention techniques?
153 Figure 5 8 4 pilot Figure 5 9 4
154 Figure 5 10 4 Figure 5 11 4 you planning on
155 Figure 5 1 2 Pre and post test score comparisons for each group tested. Percentages above groups indicate significant differences in t test 0.05 between groups.
156 Figure 5 1 3 Biology pre and post test score comparisons for each group tested. Percentages above groups indicate significant differences in t test 0.05 between groups.
157 Figure 5 1 4 Medical pre and post test score comparisons for each group tested. Percentages above groups indicate significant differences in t test 0.05 between groups.
158 Figure 5 1 5 Prevention and treatment pre and post test score comparisons for each group tested. Percentages above groups indicate significant differences in t test 0.05 between groups with the exception of the mixed group.
159 Figure 5 16 Map of the U.S.A. showing the percentage of curriculum downloads that came from each state (July 2012)
160 Figure 5 17 Map of the U.S.A. representing the projected number of individuals that would be impacted (July 2012).
161 Table 5 4 Demographic profile of 194 individuals that downloaded the Bed Bugs and Book Bags c urriculum (July 2012). Demographic N % respondents Gender Male 58 25.3 Female 136 59.4 Ethnicity African American 3 1.3 Asian 1 0.4 Black 10 4.4 Caucasian 146 63.8 European 3 1.3 Hispanic 2 0.9 Hispanic American Indian 1 0.4 Non Hispanic 9 3.9 Indian 1 0.4 Mixed 2 0.9 Not Answered 16 7.0 Grades taught College 4 1.7 Grades 6 8 4 1.7 Grades 9 12 4 1.7 Grades 3 5 28 12.2 K 2 47 20.5 Other adults 107 46.7 Groups Served Children under 5 11 4.8 Elderly 18 7.9 Underprivileged 45 19.7 Special Needs 49 21.4 None 71 31.0 Potential Impact > 5000 1 0.4 1001 to 5000 7 3.1 501 to 1000 6 2.6 101 to 500 24 10.5 51 to 100 30 13.1 11 to 50 64 27.9 0 to 10 42 18.3 Not Sure 20 8.7 Job Status 4 H Leader or Volunteer 1 0.4 University Faculty 9 3.9 Extension Agent 10 4.4 K 12 Teacher 28 12.2 Pest Management Professional 28 12.2 Other 118 51.5
162 CHAPTER 6 SUMMARY Effective bed bug control requires an integrated pest management approach This includes investigating underdeveloped areas of bed bug biology such as bed bug vision that can improve effective non chemical control and re shaping bed bug education to the general public. Automontage imag ing of the eye of the common bed bug revealed ommatidial characteristics (smaller number of ommatidia and the presence of interommati dial setae in the adult stage) that can serve as additional morphological defining characteristics between the common bed bug and the tropical bed bug. Electroretinogram techniques were used to better understand adult bed bug vision, specifically their sp ectral sensitivity. Male and female adult bed bugs have at least two different types of photoreceptors (green and yellow green) which is unique compared to most other insects that are sensitive to UV, blue and green Also males and females have different sensitivities towards light intensities. These unique spectral sensitivities are important because it give clues as to what wavelengths of light are biologically significant to these pests. Although bed bugs are largely nocturnal insects, they can be acti ve in the day time and use visual cues as aids for various biological activities. Behavioral bioassays were conducted to determine if bed bug harborages and oviposition sites were influenced by color. Bed bug adults and nymphs prefer specific colored harb orages and these color preferences were influenced by factors such as gender, nutritional status, life stage and whether or not the bed bugs were aggregated Overall, a dult bed bugs preferred red and black harborages the most and green harborages the least When compared yellow harborages were compared to white
163 harborages, bed bugs did not show any preference between the two, indicating that yellow was also an unatt ractive color choice. F emales preferred shorter wavelengths compared with males, while males preferred longer wavelengths compared with females. A shift in nutritional status f rom starved to fed, triggered color based neural integration change s that resulted in bed bugs choosing orange and violet harb orages, colors that were not normally chosen. M ore lone bed bugs chose to go to black harborages while grouped bed bugs preferred to go to red harborages. Ny mph color preferences also chang ed with each successive instar. These changes may be indicative of developmental changes in the number of ommatidia and pigments present in the compound eyes within each instar Color also seems to influence where female bed bugs oviposit. More eggs were deposited in orange harborages as opposed to violet and green harborages. It appears that orange wavelength s are quite important for bed bugs, because it was preferred as a harborage after feeding and also for oviposition site for females. Similarly to adults, nymphs did not prefer yellow harborages compared with white harborage and in two instars (1 st and 5 th ) white was preferred to yellow, indicating a repellency for yellow wavelengths. The creation and implementation of the 3 5 th grade bed bug curriculum wa s received with much enthusiasm from health educators to teachers and the pest management industry within and outside the United States. Teachers and 4 H agents were willing to be a part of the pilot program to use the curriculum in their classrooms and were ready to start implementing prevention methods as soon as they could. From the curriculum we wer e able to garner what various groups of people knew about bed bugs and measure their knowledge gain after they were presented with curriculum
164 material. Teachers and students were the groups that had the highest increases in knowledge gain after being intro duced to the curriculum. This demonst rates that the curriculum is being successful in reaching the audiences it was created for. The curriculum also helped to point out that information about bed bugs as well as prevention and treatment strategies would h ave to be addressed differently for different groups of people. Based on the online availability of the curriculum, current data indicates that the curriculum has the potential to impact approximately 70,000 people across the United States, Canada and Saud i Arabia. The curriculum has been awarded 1 st place in the Florida in the category of learning modules. This demonstrates its potential to be used as a model/prototype for educational bed bug material for high er grades within and outside academia. This all ows community members, shelters, churches and nursing homes access to an effective way of raising bed bug awareness.
165 APPENDIX A BED BUGS AND BOOK BAGS CURRICULUM The Bed Bugs and Book Bags c urriculum was digitally archived and can accessed at the following site: http://ufdc.ufl.edu/AA00011610/
166 APPENDIX B PRE AND POST TEST FO R STUDENTS, TEACHERS AND PARENTS Educator Training Pre and Post Test for Bed Bugs and Book Ba gs Curriculum Information on knowledge gain from educational programs provided to teachers is being gathered by the University of Florida /IFAS Extension. You will be asked to complete a short quiz both before and after an educational program about bed bugs. There are no anticipated risks to you as a participant. Upon completion of the program, you will be provided with a set of classroom curriculum, Bed Bugs and Book Bags. You are free to withdraw your consent to participate and may discontinue your particip ation at any time without consequence. If you have any questions about this research protocol, please contact the Jacksonville Bed Bug Task Force at 904 255 7450. Questions or concerns about your rights as a research participant may be directed to the IRB0 2 Office, University of Florida, Box 112250, Gainesville, FL 32611; (352) 392 0433. Name: _______________________________________________ Date: ________________________ School District: ________________ Grades Taught: ________________ Subjects Taught: _______________ Years of Teaching Service: _______ Ethnicity: Caucasian African American Hispanic Asian Other __________ Please select the most appropriate answer based on your knowledge of the bed bug. 1. Bed bugs have: 1. Flattened bodies with leathery wings 2. Yellowish hairs over oval, flattened bodies 3. Jumping legs and shortened wings 4. Oval bodies and eight legs 2. Bed bugs find a host through which cues? 1. Blood from open sores 2. Snores and body movement 3. Carbon dioxide, heat, and body od or 4. Household clutter 3. Bed bugs feed by: 1. Chewing on the skin 2. Piercing/sucking blood with a proboscis 3. Burrowing under the skin 4. Lapping up pools of blood
167 4. Are bed bugs known to spread disease? 1. Yes 2. No 5. How do bed bugs search for their host? 1. Walking/crawling 2. Flying 3. Jumping 4. Digging 6. Bed bugs are confined to the bedroom. 1. True 2. False 7. When inspecting for bed bugs, you should look for: 1. Live bed bugs 2. Dead bed bugs 3. Bed bug eggs 4. Brown spots 5. All of these 8. People who think they have been bitten, although they have had no real encounter with a hematophagous (blood sucking) insect may have: 1. Entomophobia 2. Arachnophobia 3. Delusory parasitosis 4. Bugophobia 9. Immature bed bugs look like 1. Caterpillars 2. Small adult bed bugs 3. Worms 4. Ants 10. Bed bug infe stations are noted by: 1. Blood or fecal spots and a sweet odor 2. Pepper like droppings 3. Silver scales in cracks and crevices 4. Insects around windows and lights 11. Transporting bed bugs into an area is known as a bed bug? 1. Investigation 2. Inspection 3. Introduction 4. Impact
168 12. Which tools would be useful for bed bug inspection and monitoring? 1. Climb up traps 2. A magnifying glass 3. A flashlight 4. Tweezers and a container 5. All of these 13. Bed bug infestations are a more common occurrence in the US today for all of the follow ing reasons except : 1. Travelling has become more affordable and people are going to more places. 2. People in an effort to save money reuse discarded furniture. 3. 4. Bed bugs only affect large urban areas. 5. People may not have the money to properly treat a bed bug infestation so the problem goes unresolved. 6. Bed bugs are resistant to many pesticides so are not easily controlled. 14. Bed bug bites: 1. Are painful at the time of the bite. 2. Always present themselves on every person the same way. 3. Frequently appear in rows. 4. Are normally seen only on hands and feet of individuals. 15. Important bed bug prevention techniques include all of these except: 1. Avoiding clutter. 2. Not taking home abandoned couc hes, mattresses, and other furniture. 3. Washing and drying clothing on high heat. 4. Travelling with bed bug prevention repellent. 5. Taking precautions when travelling such as inspecting rooms. 16. The most likely way bed bugs will be transferred from one locati on to another is: 1. Hitchhiking on clothing and bags 2. Migration 3. Hitchhiking on cats and dogs 4. Flying from one location to another 17. At what temperature should bed bugs be exposed to for at least 1 minute before they will die? 1. 200 F 2. 120 F 3. 110 F 4. 85 F
169 18. If a student spots an insect they suspect to be a bed bug, they should be instructed to? 1. Alert the teacher 2. Smash the insect 3. Collect the insect 4. Leave the classroom Student Pre and Post Test for Bed Bugs and Book Bags Curriculum Information on knowledge gain from educational programs provided to students is being gathered by the University of Florida /IFAS Extension. You will be asked to complete a short quiz both before and after an educational program about bed bugs. There are no anticipated risks to you as a participant. You are free to withdraw your consent to participate and may discontinue your participation at any time without consequence. If you have any questions about this research protocol, please contact the Jacksonville Bed Bug Tas k Force at 904 255 7450. Questions or concerns about your rights as a research participant may be directed to the IRB02 Office, University of Florida, Box 112250, Gainesville, FL 32611; (352) 392 0433. Student Name _____________________________________ Date _______________ Please circle the best answer for each question. 1. Bed bugs feed mostly on a. Cat blood b. Human blood c. Pizza d. Pillows 2. A bed bug has this many legs. a. 8 b. 4 c. 6 d. 10 3. Bed bugs have what type of mouthpart? a. Chewing b. Piercing sucking c. Rasping 4. The term ectoparasite means: a. An animal that feeds on blood b. An animal that lives outside its host c. An animal that glows in the dark d. An animal that lives underground
170 5. Are bed bugs ectoparasites? a. Yes b. No 6. If you think you see bed bugs at school, what should you do? a. Tell a teacher or school nurse b. Ignore it c. Step on it d. Put it outside 7. If you have bed bugs in your book bag, what is the best way to get rid of them? a. Throw the book bag away b. Give it to your brother or sister c. Put it in the dryer on high heat d. Spray it with bu g spray 8. Bed bugs a. Carry diseases b. Do not carry diseases c. Live only in America d. Live only with poor people 9. Identify the locations where bed bugs may be found: a. Home, library, daycare b. School, airplane, bus c. Movie theatre, clothing store, hotel d. All of the above 10. Which of these is a false statement? a. Bed bugs have no wings b. Bed bugs cannot fly c. Bed bugs can jump d. Bed bug bites are not painful 11. Head lice, mosquitoes, ticks, chiggers, fleas and bed bugs are all a. Ectoparasites b. Insects that fly c. Insects that live exclusively on humans d. Insects that have chewing mouthparts 12. L ooking for bed bugs at home, cleaning up after yourself, and washing your clothes are considered bed bug _______________ techniques. a. Infestation b. Prevention c. Control d. Pesticide
171 13. Bed bugs can cause people to become _________ and not sleep at night. a. Stressed b. Joyous c. Tired d. Happy 14. Bed bugs most likely are spread from one place to another by ______________. a. People unknowingly taking them in their bags b. Animals such as dogs and cats c. Bed bugs walking to another house d. People bringing them home from the store 15. A good way to get rid of bed bugs includes these, except : a. Using the vacuum b. Placing items in the freezer c. Placing items in the dryer d. Giving the item to someone else Parent Survey for Bed Bugs and Book Bags Curriculu m Information on knowledge gain from educational programs provided to teachers is being gathered by the University of Florida /IFAS Extension. There are no anticipated risks to you as a participant. You are free to withdraw your consent to participate an d may discontinue your participation at any time without consequence. If you have any questions about this research protocol, please contact the Jacksonville Bed Bug Task Force at 904 255 7450. Questions or concerns about your rights as a research partic ipant may be directed to the IRB02 Office, University of Florida, Box 112250, Gainesville, FL 32611; (352) 392 0433. Please circle answers where appropriate. My child(ren) are in grades: 1 2 3 4 5 They go to school at ___________________________________________________________ In what school district? _________________________________________________________ 5. On a scale of 1 5 how knowledgeable are you about bed b ugs? (5) very knowledgable (4) knowledgable (3) fairly knowlegable (2) not very knowlegable (1) I have never heard about bed bugs
172 6. Have you discussed with your child(ren) the different locations where bed bugs could be found in the h ome? 5. Yes 6. No 7. Were you aware that your child(ren) had learned about bed bug identification and prevention at school? A. Yes B. No 8. Prior to your child(ren) learning about bed bugs in school, had you ever heard of bed bugs? A. Yes B. No 9. Prior to your child(ren) learning about bed bugs in school, had you ever seen bed bugs in your home? A. Yes B. No 10. Have you spoken with your child about bed bugs as a result of a homework or school assignment? A. Yes B. No 11. Bed bugs have: 5. Flatt ened bodies with leathery wings 6. Yellowish hairs over oval, flattened bodies 7. Jumping legs and shortened wings 8. Oval bodies and eight legs 12. Bed bugs feed by: 3. Chewing on the skin 4. Piercing/sucking blood with a proboscis 5. Burrowing under the skin 6. Lapping up pools of blood 13. Are bed bugs known to spread disease? 5. Yes 6. No 14. How do bed bugs search for their host? 3. Walking/crawling 4. Flying 5. Jumping 6. Digging
173 15. Bed bugs are only found in the bedroom. 6. True 7. False 16. When inspecting for bed bugs, you should look for: 5. Live bed bugs 6. Dead bed bugs 7. Bed bug eggs 8. Dark brown/black spots 9. All of these 17. Bed bug bites: 5. Are painful at the time of the bite. 6. Always present themselves on every person the same way. 7. Frequently appear in rows. 8. Are normally seen on hands and feet of individuals. 18. Impo rtant bed bug prevention techniques include all of these except: 5. Avoiding clutter 6. Not taking home abandoned couches, mattresses, and other furniture 7. Washing and drying clothing on high heat 8. Travelling with bed bug prevention repellents 19. The most likely way bed bugs will be transferred from one location to another is: 6. Hitchhiking on clothing and bags 7. Migration 8. Hitchhiking on cats and dogs 9. Flying from one location to another 20. At what temperature should bed bugs exposed to for at least 1 minute before they wil l die? 7. 200 F 8. 120 F 9. 110 F 10. 85 F Thank you for participating
174 APPENDIX C BED BUGS AND BOOK BA GS EVALUATION FORM Plea se return ungraded pre and post tests to the contact information below. If you would like to score the student pre tests and post tests, please complete the following questions and provide test scores by assigning students to numbers (do not use names), and reporting scores in the following format: # correct/total # (For example, 12/15 means 12 correct answers out of 15 total q uestions). Please mail this class evaluation form to: Erin Harlow University of Florida/IFAS Duval County 1010 N McDuff Ave. Jacksonville, FL 32254 In an effort to ensure ongoing program development, please complete the following questions. Your observ ations and comments are appreciated. 1) Which lessons did you teach? _______________________________________________________ 2) What question(s) do you feel were answered incorrectly on the post tests and should receive more focus? _____________________ _________________________________________________________ ______________________________________________________________________________ ______________________________________________________________________________ 3) What question(s) do you feel were too easily answered by students? Explain why. ______________________________________________________________________________ ______________________________________________________________________________ __________________________________________________________ ____________________ 4) What questions(s) would you like to see included on the pre and post tests? Explain why. ______________________________________________________________________________ _______________________________________________________________ _______________ ______________________________________________________________________________ 5) What question(s) would you like to see deleted from the pre and post tests? Explain why. _____________________________________________________________________ _________ ______________________________________________________________________________ ______________________________________________________________________________
175 Other comments: _________________________________________________________________________ _____ ______________________________________________________________________________ School: _________________________________________________________________ Grade: ___________________________ STUDENT # PRE TEST SCORE (# correct/# on test) POST TEST SCORE (# correct/# on test) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 CLASS AVERAGE
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189 BIOGRAPHICA L SKETCH In 1983 Corraine Athol McNeill (ne Scott) was born to Stanhope and Sonia Scott in Mandeville, Jamaica. After graduating from Glenmuir High School in Clarendon, Jamaica, she earned a Presidential Scholarship to attend Randolph College (formerly Randolph Macon b i ology and had double minors in chemistry and sociology/a nthropology. Following her undergraduate studies, she worked for one year at Virginia Commonwealth University teaching introductory biology l abs, and heading a residential hall a s Resident Director, all while carrying out her graduate studies. She got the opportunity to have some amazing field work experience in organic farming a nd therefore pursued a Master of Science degree at the University of Florida under the supervision of Dr. Oscar Liburd. Corraine continued her graduate studies in the area of chemical ecology but switched to urban e passion is teaching and she will be pursuin g a faculty position at Union College in Lincoln, NE fol lowing the conclusion of her Doctor of Philosophy degree.