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1 OPTIMIZATION OF BAIT COMPONENTS FOR N ylanderia pubens (FOREL) By JODI MICHELLE SCOTT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MAS TER OF SCIENCE UNIVERSITY OF FLORIDA 2012
2 2012 Jodi Michelle Scott
3 To my family and friends who have helped me through this process along with my professors and my lab mates for their humor and all the good times, and to my ants who taught me what crazy feels like
4 ACKNOWLEDGMENTS My deepest gratitude goes to Dr. Koehler and Dr. Pereira for the opportunity to be program, for believing in me, giving me support and guidance along the way. I would like to thank Liz Pereira fo r her care of the insects around the laboratory, without her we would be lost, Tiny Willis, seeing his smiling face and hearing his stories made the Urban Laboratory like a second hom e the entomology department; she is a n amazing blessing to the department. I would like to thank my family in the Urban Laboratory, with special thanks to Stephanie Hill, Corraine McN eil l Ma rk Mitola, and Ephraim Raga sa. I thank them for all the laughs, all of the good memories, crape myrtl e times and the adventures we had. I would also like to thank the undergrads who helped me in this process: Holly Beard, Josh Westin, and Cory Goeltzenleuchter I would also like to thank my friends and family for all their love and support though this pro cess, my family: Patricia Bower, Jake Smith, Nichole Bower, Lionel Jaques ( & team Jaques), Arianne Archer my friends:, Gabe Falcon, R oger Granados, Chris Wilson Jason Scott and Jason Degan without these people I would have been lost. Lastly, t hanks go out to
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 PRELUDE ................................ ................................ ................................ ............... 12 2 REVIEW OF LITER ATURE ................................ ................................ .................... 15 Family ................................ ................................ ................................ ..................... 15 Classification ................................ ................................ ................................ ........... 16 Origin and Distribution ................................ ................................ ............................ 17 Colony Structure ................................ ................................ ................................ ..... 17 Description ................................ ................................ ................................ .............. 18 Foraging and Feeding ................................ ................................ ............................. 19 Pest Status ................................ ................................ ................................ ............. 20 Control ................................ ................................ ................................ .................... 20 3 CHOICE BASED EXPERIMENTS OF GRANULAR BAIT COMPONENTS ............ 22 Introduction ................................ ................................ ................................ ............. 22 Materials and Methods ................................ ................................ ............................ 22 Insects ................................ ................................ ................................ .............. 22 Granular Size ................................ ................................ ................................ ... 24 Additives ................................ ................................ ................................ ........... 24 Matrices ................................ ................................ ................................ ............ 24 Insects ................................ ................................ ................................ .............. 25 Cricket Slurry ................................ ................................ ................................ .... 25 Foraging Arenas ................................ ................................ ............................... 25 Bioassay ................................ ................................ ................................ ........... 26 Analysis ................................ ................................ ................................ ............ 27 Results ................................ ................................ ................................ .................... 28 Discussion ................................ ................................ ................................ .............. 30 4 GRANULAR BAIT MATRIX WITH ADDITIVES AND ACTIVE INGREDIENTS ....... 47 Introduction ................................ ................................ ................................ ............. 47 Materials and Methods ................................ ................................ ............................ 48
6 Insects ................................ ................................ ................................ .............. 48 Granular Bait Formulation ................................ ................................ ................ 49 Active Ingredients ................................ ................................ ............................. 49 Bioassay ................................ ................................ ................................ ........... 50 Analysis ................................ ................................ ................................ ............ 52 Results ................................ ................................ ................................ .................... 52 Discussion ................................ ................................ ................................ .............. 53 5 CONCLUSION ................................ ................................ ................................ ........ 61 LIST OF RE FERENCES ................................ ................................ ............................... 72 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 78
7 LIST OF TABLES Table page 3 1 Proteins contents a nd other characteristics of products used in matrices experiments with N. pubens. ................................ ................................ .............. 33 4 1 Products used in bait formulations used in laboratory choice and efficacy experiments against N. pubens colonies. ................................ ........................... 57
8 LIST OF FIGURES Figure page 3 1 Percent number and percent weights of different size granules of dog food removed by Nylanderia pubens in laboratory experiments ................................ 34 3 2 Percent number and percent weight of different size granules of dog food removed by Nylanderia pubens in the field experiments ................................ .... 35 3 3 Percent number and percent weight of different food matrices removed by Nylanderia pubens in laboratory experiments ................................ .................... 36 3 4 Percent number and percent wei ghts of different food matrices removed by Nylanderia pubens in field experiments ................................ .............................. 37 3 5 Percent number and percent weight of different dog food granular formulations plus additives which were removed by Nylanderia pubens in the laboratory experiments ................................ ................................ ....................... 38 3 6 Percent number and percent weight of different dog food granular formulations plus additives which were removed by Nylanderia pubens in the field experiments ................................ ................................ ................................ 39 3 7 Percent numbers and weights of the 3 forms of crickets removed by Nylanderia pubens in the laboratory experiments ................................ ............... 40 3 8 Percent numbers and weights of the 3 forms of crickets removed by Nylanderia pubens in the field experiments ................................ ........................ 41 3 9 Percent numbers and percent weights of gran ules containing macerated slurry crickets removed by Nylanderia pubens in laboratory experiments .......... 42 3 10 Percent numbers and percent weights of granules containing macerated crickets slurry re moved by Nylanderia pubens in field experiments ................... 43 3 11 Workers head, Nylanderia pubens in comparison with dog food granules used in size preference experiments. ................................ ................................ 44 3 12 Nylanderia pubens foraging in a laboratory setting on 1.00 mm dog food granules used in size preference experiments ................................ ................... 45 3 13 Nylanderia pubens foragin g in a laboratory setting on 1.40 mm dog food granules used in size preference experiments ................................ ................... 46 4 1 Testing arena used for experiments on Nylanderia pubens using granular bait matrix applied wit h active ingredient. ................................ ........................... 58
9 4 2 Percent removal by Nylanderia pubens colonies of granular bait with different active ingredients in laboratory experiments ................................ ...................... 59 4 3 Cumulative percent mortality of Nylanderia pubens from laboratory colony fragments, after consumption of granular bait wit h different active ingredients 60
10 Abstract of Thesis Presented to t he Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science OPTIMIZATION OF BAIT COMPONENTS FOR Nylanderia pubens (FOREL) By Jodi Michelle Scott August 2012 Chair: Philip Koehler Major: Entomology and Nematology Components for granular bait for Nylanderia pubens (Forel) were assessed through choice experiments in laboratory and field environments. Experimental colonies consisted of ants collect in field environments and mai ntained in the laboratory. The components tested where: granular size, matrices, additives (sugar, oils and insect tissue), insect tissue preferences, and three insecticidal active ingredients. The preferences of N. pubens were determined by removal by num ber and by weight of the bait components from the foraging arenas. Differences in preferences between laboratory and field environments that were observed were attributed to environmental factors and col ony developmental differences. N. pubens chose granul ar bait sizes that corresponded with their head and body sizes (0.850 1.00 mm). The matrix chosen for the carrier of the bait was a carbohydrate proteinaceous carrier that was easily spreadable and absorbent enough for the addition of additives (dog food). Insect tissue experiments indicated that N. pubens prefers live forms of cricket tissue (53% by weight removed) which led to the addition of cricket tissue, to enhanc e the attractiveness of the carrier. Fipronil, imidacloprid and indoxacarb were added top ically to the formulated bait matrix. The rate and percent of ant mortality was assessed. Fipronil caused faster and
11 higher percent mortality through day s 6 14. LD 50 for fipronil was about 4, whereas ceeded 35% over the 14 days the experiments were conducted.
12 CHAPTER 1 PRELUDE In 2004, the European Environmental Agency defined the term invasive species as any non native species which threatens ecosystems, habitats or other species (Frank et al. 2004) Among invasive species, ants are the most destructive to ecosystems once established (Kenis et al. 2009). The impacts to the ecosystems occur when the invaders disrupt the previously established balance among other species. Instances of this disruption t o the ecosystem can be seen in many polygynous ant invasive species such as the Argentine ant, Linepithema humile (Mayr) and Solenopsis invicta Buren L. humile disrupts native ant species, along with many mammals, avians and reptiles (Kenis et al. 2009). its ability to form numerically large, ecologically dominant colonies, a trait that L. humile shares with Nylanderia pubens (Forel) (Tsutsui et al. 2003). Nylanderia pubens is referred to as a nuisa nce pest; however the economic impact of this invasive species is not yet completely known (MacGown et al. 2010). Millions of these ants accumulate in electrical equipment causing them to fail and short circuit (Drees et al. 2009). Additionally, these ants have caused tens of thousands of dollars in damage to property and remedial costs (Nester et al. 2010). In the Jacksonville, FL Zoo, the numbers of N. pubens got so high that their sheer amount caused the zoo train to halt because of the safety hazard pos ed by the ants occluding the train track (Calibeo Hayes et al. 2010). N. pubens also poses a threat to apiculture business. In Texas, N. pubens invaded at least 100 bee hives in 2009 to raid the brood and colonize the hive (Harmon 2009). In St. Croix, N. p ubens was blamed for crop
13 destruction due to mealy bug tending on fruit trees and aphid tending on coconut trees (Wetter et al. 2008). Nylanderia pubens is especially hard to kil l because of their high numbers, their foraging strategies and patterns, and t heir weak foraging on most traditional ant baits. The current control method for this pest species has been insecticidal sprays that appear to decrease the numbers of ants for short periods of time but do not solve the overall problem. The application of i nsecticidal sprays has been recommended for nest areas and along foragin g trails (Warner et al. 2010), but with this approach N. pubens simply uses their fallen comrades as a bridge, thus burring the insecticide under the fallen ant bodies and making it us eless (Drees et al. 2009) Granular baits, however, may provide a longer term and more thorough solution. B aits can be broadcast over larger areas which takes advantage of the scatter pattern of foraging that N. pubens displays. Baits work by taking advan tage of ant biology such as social grooming and trophallaxis. Once the bait is discovered the foraging ants pick of the bait and take it back to the colony where it can reach the brood and queen, the brood digests the bait and transfer the toxicant to the rest of the colony. The use of granular baits in the control of pest ant species provides a mechanism for using very little insecticide thereby reducing the amount of insecticide in the environment (Hooper Bi et al. 2000). Components of granular baits con sist of an attractant, carrier and active ingredient (Stanely 2004) Optimal granular bait should: 1) display delayed toxicity, 2) be transferred easily from one ant to the next, 3) use an active ingredient that is non repellent on the bait matrix and 4) be formul ated to the ant species that needs to be controlled (Stringer et al. 1964, Hooper Bi et al. 2000).
14 The optimal bait for N. pubens with the added active ingredients should be when finished: easy to carry, widely accepted by the foraging ants, a nd slow acting enough so the active ingredient can be spread throughout the entire colony, making it to the queens. The objectives of this study were intertwined: 1) to tests components of granular bait, in order to formulate carrier size, carrier and addi tive for enhancement of the carrier and 2) the addition of insecticidal active ingredients to the carrier, to include their attractiveness to N. pubens rate of mortality and over all percent mortality for possible control of N. pubens
15 CHAPTER 2 REVIEW OF LITERATURE Family Nylanderia pubens (Forel) belongs to the sub family of ants known as Formici d ae. The subfamily Formicid ae is a very common and widespread group (Triplehorn et al. 2005). The major traits of the family Formicidae which separates them fro m the other insects in the order of Hymenoptera is the formation of the pedicel of the metasoma, which can be one or two segmented and have an upright lobe appearance, elbowed antennae and eusociality (Triplehorn et al. 2005). Almost all insects in the f amily Formicidae have a caste system, with the exception of some lesser ants, the morphology of the castes can be classified as: monomorphi sm (workers all the same si ze), Monophasic allometry, ( non isometric growth, two sizes connected by median class size ) Diphasic allometry (increase in size leads to larger major class), Triphasic and tetraphasic allometry and complete di morphism (two very distinct size groups) (Hlldobler et al. 1990). These castes do have a similar trait s among them, they are all ruled by a single queen (monogyny), although in some species there can be multiple queens (polygyny). Insects in the family Formicidae are found almost anywhere on earth T hey have the unique ability to adapt quickly to their environments, and build nests in alm ost anything. The location of the nesting site does depend on the area they reside in and the species of ants in that area. Common nest locations include logs, plant cavities leaf litter, under potted plants, in housing structures and in the ground.
16 Clas sification Nylanderia pubens can be a difficult species to acquire literature on due to the numerous proposed common names and the recent reclassification into a new genus. N. pubens was initially described by Forel as Paratrechina pubens in 1893 and ident ified in Florida by Deyrup et al. in 2000. In 2002, a potentially similar, if not the same species, was identified in Texas as Paratrechina near pubens (Meyer and Gold 2008). In 2010, a new description transferred P. pubens into the genus Nylanderia (LaPol la et al. 2010, 2011) T he new recognized scientific name became Nylanderia pubens (Carlton et al. 2012). In some literature, N. pubens has been described as N. fulva which was originally thought to be a subspecies of N. pubens but was raised to the statu s of a species by Trager in 1984 (Trager 1984, Carlton et al. 2012). The confusion between the names N. pubens and N. fulva arose because Forel misidentified the American specimen as P. pubens when it should have been P. fulva Creighton (1950) made a taxo nomic listing of the current ant species in North America, where he identifie d this mistake and corrected it. LaPolla University is currently working on the taxonomic status of N. pubens to see whether or not it is truly N. fulva (Car lton et al. 2012). There is still some debate on the valid scientific name of N. pubens For the purpose of this work Nylanderia pubens will be used. Common names used for N. pubens vary as much, if not more than the scientific names. Nylanderia pubens a re called crazy ants in some regions due to their erratic movements once they are disturbed. Another name comes about because course hairs cover the thorax of N. pubens T his identifying factor for the species has led some to use the common name hairy craz y ants (Wetterer et al. 2008). Other proposed common names include: Caribbean crazy ant, due to the belief that these ant originate in the
17 Caribbean (Warner et al. 2010 ), and Rasberry crazy ant, after the pest control operator that first observed the ants in Texas (Carlton et al. 2012). Because there has been no ESA approved common name, the local names used for this ant species will likely vary throughout the country. Origin and Distribution Nylanderia pubens is an invasive species that is quick spreading and hard to eliminate once established in an area. N. pubens was believed to either originate from the St. Vincent, Lesser Antilles or in South America (Trager 1984, Meyer 2008). The first recorded introduction of N. pubens to the United States was in Mia mi, Florida in 1953 (Trager 1984). The next mention of this species was in 1990 in a hospital and at two other locations in around Miami, Florida (Klotz et al 1995). In 2000 N. pubens was spotted at the U niversity of Miami running up and down trees. From 2000 to now these ants have spread up the coasts of Florida. Seven years a fter the first observation in Harris County Texas in 2002, N. pubens had spread to isolated spots in 14 counties in Texas (Drees et al 2009). In 2009 Hancock County, Mississipp i, reported large numbers of the Texas variety of N. pubens (MacGown et al 2010). In 2010, N. pubens was discovered in Port Allen, Louisiana, which represented the first record of this species in Louisiana. In 2011, N. pubens was identified in Calcasieu Pa rish, Louisiana (Hooper Bui et al. 2010, Carlton et al. 2012). Within a period of less than 10 years, N. pubens colonies have been established in four states and are continuing to spread throughout the southern United States. Colony Structure Unicoloniali ty, or super colonies, are characterized by many wide spread but interconnected colonies that may contain one or many queens (Tsutsui et al. 2003).
18 Super colonies are formed by budding, which occurs when one or many queens abandon the original colony to fo rm a separate one. The original colony can be the origin of many colonies in a super colony; ants from the separate colonies do not display aggression toward the ants in other colonies in the same area (MacGown et al. 2010). The lack of aggression appears to be a resource allocation tactic by ant s that form super colonies. I f no energy is wasted in defending the nest from their sister colonies, more resources can be dedicated to defense from other ants, colony growth, and foraging (Tsutsui et al. 2003). N. pubens fits this model because they are a polygynous species with anywhere from 8 40 queens in their colonies, they form large super colonies in areas where they have been found and they show a lack of aggression toward other colonies of N. pubens in thei r area ( Tsutsui et al. 2003, Warner et al. 2010). Nests sites for the colonies can be found in multiple outdoor locations and occasionally indoor locations. N. pubens prefer moist areas and will nest under and in almost anything. They have been found outd oors in soil, rotting wood, in and under potted plants, in vehicles, and in various outdoor structures (MacGown et al. 2010). Inside building s, N. pubens have been seen to occupy places such as inside computers and moist areas. Following the model of L hu mile another polydomous ant species, N. pubens may follow a nest dispersion model referred to as central place, which occurs when a colony of ants places its nest s ites closer to a food source to save on resources (Holway et al. 2000). Description N. pu bens q ueens are 4.0 mm or longer, males are 2.4 2.7 mm, which is not much larger than the workers, which are 2.0 2.4 mm and monomorphic. All castes are reddish
19 brown in color, their thoracic region is covered in thick pubescence N. pubens have one petiola r segment and they do not sting but can spray formic acid (Warner et al. 2010, Hooper B i et al. 2010, MacGown et al. 2010, LaPolla et al. 2011). Distinguishing characteristics of this species are the striped light and dark appearance of gaster after feedi ng has taken place and the length of their antennal scape which is nearly twice the width of their head with 12 segments on the antennae and no clubs (Warner et al. 2010). Foraging and Feeding Nylanderia pubens have an omnivorous diet. In nature, they ca n be seen tending to hemipterous insects and in nectaries of plants, along with foraging for insect tissue (Creighton 1950, MacGown et al. 2010). Like other ant species the foragin g adults cannot eat solid foods; they must return to the nest and place the solid food on the brood to be digested and redelivered to the workers through trophallaxis. In other invasive ant species foraging adults have been observed to allocate proteinaceous food to the larvae and the queens (Cassill et al. 1995). Food granules h ave been observed in the nest cells of N. pubens not located directly next to the brood (personal observation) T his may indicate that they store food for later use. Most ant species that store food use seeds, although protein storage has been a observed in the ant species Solenopsis invicta (Buren) (Gayahan et al. 2008) N. pubens is a tropical ant species which means weather may have a strong effect on foraging behaviors. N. pubens have not been observed foraging heavily in weather colder Hayes et al. 2010).
20 Pest Status Nylanderia pubens falls under the European Environmental ition of an invasive species (Frank et al. 2004). N. pubens is similar to other invasive ant species in having numerically large, ecologically dominant colonies, with multiple queens which causes them to out compete native species for resources (Tsutsui et al. 2003). Damages reported thus far include economic losses due to the disruption of business such as the t rain stopping in the Jacksonville, FL Zoo destruction of electrical equipment and p roperty damage such as damage to livestock, death of rabbits in Little F ountain, St Croix, crop destruction as a side effect of insect tending and the destruction of 100 bee hives in Texas (Wetterer et al. 2008, Calibeo Hayes et al. 2010, Drees et al. 2009, Harmon 2009 and Nester et al. 2010). Control Controlling N. pubens is difficult due to their high numbers and weak foraging on most traditional ant baits. Therefore, p est control operators say that typical control methods for a number of ant species will not work on N. pubens (Nester et al. 2010). Control methods previously tried include sprays, which reduce the numbers of ants but fail to completely control the ants. I n Texas, the growth inhibitor Esteem 0.86% was applied by spraying it in an area, but the study indicated that this application decreased the ants seen in the treatment area, but not all of N. pubens in the area were killed (Nester et al. 2010). Drees et al ( 2009 ) suggested using Termidor SC Termiticide/Insecticide (9.1% fipronil) sprayed on the outside perimeter of a building infested with N. pubens
21 Insecticide (0.0143% fipronil), and for ants enter ing the house use of Phantom Termiticide Insecticide (21.45% chlorfenapyr). Louisiana scientists suggested using an overall integrated pest management approach for N. pubens (Hooper Bui et al. 2010). Their a pproach includes six parts: 1) m onitoring f or the ants, 2) sanitation practices, 3) d isrupt ion of foragi ng on trees and structures, 4) d estr uction of visible nests, 5) u se of small particle baits, and 6) r epeat ing the entire process after 12 weeks. In step five, they indicated that in Texas N. puben s prefer s Whitmire Advance Carpenter Ant Bait (abamectin B1 0.011%) in small particles. This bait along with liquid bait stations that have been tested f or palatability can be effective. T he authors did not include information on the e fficacy of th e baits in controlling the ants.
22 CHAPTER 3 CHOICE BASED EXPERIM ENTS OF GRANULAR BAI T COMPONENTS Introduction Nylanderia pubens has been established in Florida since the 1950 s ( Trager 1984). In recent years this invasive ant species has quickly risen to the st atus of pest in many areas in and around Florida. Colonies of N. pubens can be in the thousands if not millions. T his characteristic of their biology causes them to be hard to control and allow them to out compete native species and push them out of their environment (Warner and Scheffrah n 2010). The current approach for the control of this ant species is to use insecticidal sprays. This method has proven to be not very effect ive due to the sheer numbers of these ants Because of the lack of effectiveness in controlling this ant species, a different method of control should be developed, such as granular bait. There is little information on the food preferences of N. pubens Creighton observed that these ants preferred honey dew, plant nectar and insect tissue. The purpose of this stud y was to develop a granular bait matrix with the idea that an active ingredient could be applied to it and used for N. pubens control This knowledge could be used in the future to develop a more effective contro l method for this invasive species of ant. Materials and Methods Insects Colonies of N. pubens were collected in from three sites in Gainesville, FL: 4821 Northwest 6th Street (The Rancher), between SW 5 th St and SW 3 rd ST (Depot) Moistened corrugated ca rdboard nest cells (25.4 cm x 15 cm) were placed in areas with
23 high numbers of foraging ant trails. Nest cells were collected after 2 wk, and ants were shaken off the cells into a tray Collected ants were placed in gardening trays (13 cm H x 39 cm W x 52 cm L) with the inner sides lined with Insect a SLIP (BioQuip Products, Rancho Dominguez, CA) to prevent ant escape. Each tray, depending on numbers of ants, contained 1 5 nest cells (Petri dishes [100 mm x 15 mm] with plaster on the bottom, and the lid co vered with yellow cellophane). Food for the ants consisted of fresh orange slices, honey, ground cat food (Purina cat chow naturals plus vitamins & minerals, Nestle Purina, St. Louis, MI ), li ve insects, water and 10% sugar water supplied weekly. Live insec ts consisted of crickets ( Acheta domesticus ( Linnaeus) ), American cockroaches ( Periplaneta americana, ( Linnaeus) ), and mealworms ( Tenebrio molitor Linnaeus ). Satellite colonies were established by placing 1.5 g of N. pubens (3 queens, brood and workers) into a (33.5 cm x 24 cm) container (RubberMaid take along, RubberMaid, Fairlawn, Ohio) with the inner sides coated with Insect a SLIP One nest cell, a water vial, a s ugar water vial, four food trays, and a 60 ml souffl container for waste disposal from colony were placed in each container. A w aste container was utilized to clear the satellite colonies of food waste s. Because ants may be on top of spent food items, these items were placed into the waste container and ants were allowed to move out before waste was removed from the satellite colonies. The diet for the satellite colonies was identical to the diet used for parent colonies. Each satellite colony was allowed to acclimate for a minimum of 72 h before use in experiments and then starved for 24 h by removing all food. Healthy satellite colonies were randomly selected for experiments.
24 Granular Size Dog food (Purina One healthy puppy food, Nestle Purina pet care company) was baked at 100C for one hour and allowed to cool for 30 minutes to eliminate potential insect or mite infestations. Dog food was ground into small granules with a coffee grind er and sieved into four sizes. Stacked soil sieves (2.0 mm, 1.40 mm, 1.18 mm, 1.00 mm and 0. 850 mm openings [Fisher Scientific Inc., Pittsburg, Pa.]) were us ed to separate granules that passed through the larger and were retained by the smaller size sieves. Granules were given designations for the sieve size which retained them. Granule sieve sizes used were 1.40 mm, 1.18 mm, 1.00 mm and 0. 850 mm Additives A dditives used were soy bean oil (Eden Organic, Clinton, Michigan ), 25% corn syrup (ACH Food Companies, Memphis, TN ) in water solution and cricket slurry Additives (0.2mL) were pipetted onto sieved (1.00 mm) dog food granules (1 g) placed into a souffl cup ( 30 ml ). Granules were shaken until the additive was evenly distributed an d absorbed onto the dog food. Granules were refrigerated and stored for 24 h before use in experiments. Matrices The s ix different food products were tested along w ith two dif ferent matrices type s : Bird feed (Zupreem fruit blend flavor, Premium nutritional products, Shawnee, KS ) with 14% protein, reptile feed (Juvenile iguana food growth formula Rep Cal research labs, Los Gatos, CA ) with 24% protein, Dog Food (Purina one heal thy puppy formula, Nestle Purina) with 28% protein, Cat Food (Purina cat chow Naturals plus vitamins & minerals, Nestle Purina) with Anderson, SC ) with 65% protein, Tast E bait (Endres
25 Processing, LLC., Rosemount, MN ) with unknown protein content and r oot watering crystals (Agrosoke international, Arlington, TX) protein content N/A. The matrices choices were ground to size in a coffee grinder and granules of size 1.00 mm were used (Tab le 3 1) Insects Lab reared 3 rd and 4 th instar common house crickets, ( Acheta domesticus (Linnaeus )), were fed to N. pubens as im mobilized live, dried or freeze killed insects. All the insects were immobilized by removing their legs using a razor blade to cut the legs at the trochanter. Dried crickets were prepared by baking them at 100C for an hour and then allowing them to cool for 30 minutes before use. Freeze killed crickets were held in a freezer for 24 hours and were allowed to thaw for 30 minutes p rior to the experiment. Non insect controls were dog food granules ( 1.00 mm granules) which were weighed out to match the weight of the live crickets used in experiments. Cricket Slurry Laboratory reared crickets were ground into a pulp material in a Cuisi nart food processor (Cuisinart, East Windson NJ) then macerated using a 2 ml Pyrex tissue grinde r (Cardinal Health, Dublin, OH). W ater (0.5 ml) was added for every 1 g of cricket in the grinding process. T he product of the grinding was cricket slurry and applied immedia tely to the dog food granules. A 0.2 ml aliquot every 1 gram of 1.00 mm sieved dog foo d granules and mixed using a 30 ml souffl container until the granules were saturated. Foraging Arenas Either 4 way or 6 way foraging arenas were constructed. Foraging arenas consist ed of a Petri dish lid (4 way: 100 mm x 15 mm, 6 way: 150 mm x 25 mm)
26 mounted on three 2.5 cm vial cap lids in a stable triangle base design for the 4 way arenas, or supported by a another Petri dish hot glued to the foraging arena for the 6 way arenas Four 1.5 cm holes were drilled around outer walls of the Pe t r i dish base in a compass fashion (N, E, S, W ) this allowed entry and access t o the wooden applicator. A 0.5 cm hole was drilled into the center o f the foraging arena, and a 2.5 cm wooden applicator was hot glued to the bottom of the foraging arena at the opening of the 0.5 cm drilled hole. The outside of the foraging arena was coated with Insect a SLIP This only allowed for one entry on to the foraging arena. A nts entered arena from the center by climbing the wood applicator and chose from the four or six choices of food given. The choice position opposite to where the applicator tip connected to the arena, w as designated as position o ne. The following positions (2 4 or 2 6) were designated in a clock wise fashion around the arena. Before arenas were reused for different experiments they were thoroughly washed and wiped with isopropyl alcohol to eliminate any traces of food items or pro ducts tested previous ly and any ant trail pheromone. Bioassay For the laboratory experiments, satellite colonies were chosen at random from those prepared previously. Parafilm (American National Can, Greenwich, CT) squares were folded so the opposite edges (1 mm) were perpendicular to the central part forming small trays. Food choice items were individually counted out and placed onto the parafilm trays. The trays were then placed on to the foraging arena. In each experiment, the food choices were shifted i n a clockwise manner for different replications and each food choice was tested in each foraging tray position twice. The ants were given 60 minutes to forage, after which the foraging arenas were removed
27 and the remaining granules were counted. The experi ments were monitored in order to determine food choices were not depleted before the 60 minute foraging time limit. E ight replications were run for 4 way tests and 12 replications were run for 6 way tests The field bioassay s w ere conducted at the three l ocations where the ants were collected. Granules of each food choice wer e counted and placed into a 1.5 mL centrifuge snap cap vial. Each snap cap via l was labeled and color coded. A set of four or six food choices were considered a repetition and four rep etitions were run in a location at a time. At each location, strong foraging trails with at least three ants wide w ere identified ; a flag was placed to mark the trail. Foraging trails were followed to make sure that each trail was unique and not a branch o f another trail. The vials with the food choices were placed along the trails. For each replicate the marker flag was placed on the opposite side of the foraging trail from the observer. To the left of the marker flag were spot one, and spot two. To the r ight of the marker f lag were spot three, and four. The distance between the snap cap vials, was 2.5 cm. Each food choice was in every position (1 4) in each field location, twice. Ants in the field were allowed a shorter time (10 min) to forage d ue to the large number of ants. After the experiment was completed the snap cap vials containing the food choices were capped and the vials were returned to the laboratory and placed into a refrigerator freezer ( were counted after 24 hours when ants picked up in the snap cap vials were dead. Analysis To compare the food choices by percent number of granules removed and percent weight of the granules removed, the data was arcsin square root transformed and
28 ANOVAs were run in the statistical soft ware JMP (SAS Inst., Cary, NC). Student Tukeys tests were used to compare means. Results The number and weight of different sizes of granules removed in the granular size experiments were significantly different both in the laboratory ( Number: F = 13.7923 P < .0001 ; and Weights: F = 9.1400 P: <. 0003 ) an d in the field ( Number: F = 4.1 830 P= 0.008 1 ; and Weights: F = 28.5255 P <.0001 respect ively) In the laboratory, 33% of the removed dog food pieces were 1.00 mm the granule size with the greatest removal, but there was no statistical difference in removal between granules of sizes 1.00 mm, 1.18 mm and 0.850 mm (Fig 3 1). Based on weights of the granules in laboratory experiments, the dog food size 1.40 mm was the most removed, but there was no sign ificant difference between 1.40 mm and 1.18 mm granules The field data for number of granules removed showed t hat the ants removed more 0.850 mm dog food pieces (35% removed), but considering the weights of the varying sizes of dog food pieces removed, the ants p referred the granule size 1.40 mm with 52% removed (Fig 3 2). In t he experiment with different animal food granules the number and weights of granules removed in the laboratory experiments we re not significantly different ( Number: F = 1.4493 P= 0 2306; and Weights: F = 2. 0021 P= 0. 1018 ) In field experiments, b oth in term s of the numbers and weights of granules preference for the different granules was statistically significant ( F = 58.7638 P < .00 0 1 and F = 72.9351 P < 0 001, respectively) In laborato ry experiments there was no significant difference in the number or weights of the different animal food removed (Fig 3 3). The field experiments
29 preference was shown toward dog treat with 53% removal of dog treat pieces or 61% based on the weight of dog treats removed (Fig 3 4). When dog food granules containing additives were tested in the laboratory neither the numbers nor weights of different granules removed were significant ly different among the removed treatments ( F = 2.6967 P= 0.0 684 ; and F = 2.2 5598 P= 0.0 787 respectively ) (Fig 3 5). In the field experiment, however the numbers and weights of the different granules with additive removed were significantly different ( F = 9. 7876 P < .0001 ; and F = 9. 2595 P < .0001 respectively ) (Fig 3 6). In experiments with different forms of cricket, no significant differences were observed in the numbers of crickets removed ; however, in the weights removed significant differences were observed ( F = 1.3 788 P = 0.2 738 ; and F = 57.2410 P < .000 1, respectivel y) I n the field both number and weights of crickets removed were significantly different among treatments ( F = 39.5317 P < .0001 ; and F = 79.7471 P < .0001 respectively ) Nylanderia pubens preferred live and freeze killed cricket forms with 47% and 41% of the weight removed in the laboratory experiments (Fig 3 7) and live cricket with 53% removal in the field (Fig 3 8). In matrices experiments with cricket slurry additive, there were significant differences among the treatments both when number of gra nule and weight of granules were considered both in the laboratory ( F = 7.5359 P= 0 .000 4 ; and F = 5.5559 P= 0.0028 respectively ) and in the field ( F = 15.1999 P < .0001 ; and F = 14.8248 and P < .0001 respectively ) The laboratory experiments with differen t matrices containing cricket slurry additive showed that the ants preferred dog food matrix wit h cricket slurry additive with 53% removal (Fig 3 9) of the granules whereas the field experiments the
30 plain dog food by weight removed, was the most prefer red with 50% removal (Fig 3 10) Discussion The purpose of this study was to gain a better understanding of the foraging and food preferences of N pubens that would allow us to formulate bait for the control of this invasive pest species. Based on fiel d and laboratory observations, N. pubens forage on a vari ety of foods, but prefer nectar based foods and insect tissue (Creighton 1950). Although N. pubens have an erratic foraging behavior in both the field and laboratory setting, when an acceptable food source is found limited if any, recruitment is seen when foraging trails are established to the source. The foraging trails disappear once the food source is depleted; this was observed in both the laboratory and field settings. Foraging behaviors in the lab and field settings were noticeably different in my experiments. T his can be due to a number of factors such as different colony needs based on time of year, the significantly larger number of ants in the field than the lab colonies and potential ly different stage of colony development for laboratory and field colonies. The National Pest Control Association describes ideal granular bait as containing granules of similar size that can be labor saving and easily ap plied to areas when needed. The autho rs also go on to describe the carrier of the active ingredient as being the most important part of the granular formulation because both the particle size and the materials and components used determine the spreading characteristics, the effectiveness of r ecruitment and removal of the bait and the residual life of the active ingredient ( NPMA 1965).
31 Based on food particle size preference experiments d one by Hooper et al. (2002), smaller ant species preferred smaller particle sizes to larger ones when given a choice. If the particle size could be matched to the ant species, this could increase the efficacy of the granular bait by providing greater opportunity for more bait to be taken into the colony (Hooper et al. 2002). My data indicates that the 1.00 mm gr anular size should be used with N. pubens Because the 1.00 mm particle is similar to the size of head of foraging workers of N. pubens this size granule can be easily carried by the ants (Fig. 3 11 & 3 12 ). This particle size is also easier to work with when adding additives to the dog food matrix Based on the weights of the removed pieces of dog food matrix, the granular size 1.40 mm was the most removed. 1.40 mm was not the chosen size of the final granul ar matrix because, although it was the most wei ght removed both in the lab and in the field T he ants were observed to have difficulty carrying the larger pieces of dog food, which appeared to cause constraints for the ants removing the larger pieces from the foraging arenas and constraints in trying t o bring the 1.40 mm pieces into the small openings of the Petri dish nest cells. This observed difficulty could be due to the small size of this ant species, and their heads (Fig 3 13 ). The 1.40 mm dog food particle is approximately 0.40 mm larger compare d to the head size of this monomorphic species, whereas the granule size 1.00 mm is closer to the size the head size of N. pubens workers. However more active ingredient can be added to a larger particle size allowing more active ingredient to be introduce d into the colony. Nevertheless the ease in transport by the ants navigating the larger granular bait into the multiple nest sites does
32 not seem feasible due this ant specie and the locations of their nest sites can have very small o penings. The matrix chosen for the bait formulation was the dog food matrix. In the matri x choice lab experiment, there was no difference between dog treats and dog food or cat food in terms of number removed. Although d og treat was prefer red by ants in t he lab and the field experiments d og treats were not the choice for the final experiment because the dog treats are hard to sieve out to the uniform size, and are not a good porous carrier for active ingredient s Dog food is a preferred bait matrix becaus e it fulfills ant nutrient requirements, is easy to prepare in a uniform granular size an d it readily absorbs additives, although this experiment was not designed to for the effects of seasons. No sugar based or oil based additive was chosen to be added t o the dog food matrix; because my experiments indicated that there was no significant advantage in adding these ingredients to the plain dog food matrix. Because ants showed preference to live crickets, cricket slurry was added on to different matrices. D og food with the addition of cricket slurry was the decided on bait matrix not only because the worker ants removed more of this formulation then the other formulations but because this formulation also allows the addition of active ingredients.
33 Table 3 1. P roteins contents and other characteristics of products used in matrices experiments with N. pubens N/A = not applicable for this product. Unk= unknown. Food type Crude protein Crude fat Crude fiber Moisture Carbohydrates/ minerals Bird food 14.0% 4. 0% 3.5% 10.0% 68.5% Iguana food 24.0% 1.0% 16.0% 12.0% 47.0% Dog food 28.0% 16.0% 3.0% 12.0% 41.0% Cat food 38.0% 13.0% 5.0% 12.0% 32.0% Dog treat 65.0% 1.0% 0.5% 16.0% 17.5% Root Crystals N/A N/A N/A N/A N/A Tast E Bait Unk Unk Unk Unk Unk
34 Figure 3 1. Percent number and percent weights of different size granules of dog food removed by Nylanderia pubens in laboratory experiments. Means with the same letter are not signific antly different. Error bars = SEM.
35 Figure 3 2. Percent number and percent weight of different size granules of dog food removed by Nylanderia pubens in the field experiments. Means with the same letter are not signific antly different. Error bars = SEM.
36 Figure 3 3. Percent number and percent weight of different food matrices removed by Nylanderia pubens in laboratory experiments. Means with the same letter are not significa ntly different. Error bars = SEM.
37 Figure 3 4. P ercent number and percent weights of different food matrices removed by Nylanderia pubens in field experiments. Means with the same letter are not significa ntly different. Error bars = SEM.
38 Figure 3 5. Percent number and p ercen t weight of different dog food granular formulations plus additives which were removed by Nylanderia pubens in the laboratory experiments. Means with the same letter are not significantly diffe rent. Error bars = SEM.
39 Figu re 3 6. Percent number and percent weight of different dog food granular formulations plus additives which were removed by Nylanderia pubens in the field experiments. Means with the same letter are not significa ntly diffe rent. Error bars = SEM.
40 Figure 3 7. Percent numbers and weights of the 3 forms of crickets removed by Nylanderia pubens in the laboratory experiments. Dog food was used as a standard treatment but not included in statistics it consists of small granules relativ e to the size of the crickets. Means with the same letter are not signific antly different. Error bars = SEM.
41 Figure 3 8. Percent numbers and weights of the 3 forms of crickets removed by Nylanderia pubens in the field experime nts. Dog food was used as a standard treatment but not included in statistics it consists of small granules relative to the size of the crickets. Means with the same letter are not significa ntly different. Error bars = SEM.
42 Fi gure 3 9. Percent numbers and percent weights of granules containing macerated slurry crickets removed by Nylanderia pubens in laboratory experiments. Means with the same letter are not significa ntly different. Error bars = SEM.
43 Figure 3 10 Percent numbers and percent weights of granules containing macerated crickets slurry removed by Nylanderia pubens in field experiments. Means with the same letter are not significa ntly different. Er ror bars = SEM.
44 Figure 3 11 Workers head, Nylanderia pubens in comparison with dog food granules used in size preference experiments.
45 Figure 3 12 Nylanderia pubens foraging in a laboratory setting on 1.00 mm dog food granules used i n size preference experiments.
46 Figure 3 13 Nylanderia pubens foragin g in a laboratory setting on 1.4 0 mm dog food granules used in size preference experiments.
47 CHAPTER 4 GRANULAR BAIT MATRIX WITH ADDITIVES AND ACTIVE INGRED IENTS Introduction Baits are one of the most effective means of urban pest management because they are easy to work with, can contain little active ingredient and they capitalize on social ant behaviors such as foraging and trophallaxis (Silverman et al. 2003). Current granular baits on the market are formulated for a number of invasive ant species, but so far none seem to be effective against N pubens T his is because these baits are not specifically formulated for this ant species. For the development o f a bait formulation that targets N. pubens ( chapter 3 ); active ingredients must be added and the final product tested for its efficacy in controlling this N. pubens The active ingredients to be applied to the fo rmulated granular bait include indoxacarb, imidacloprid and f ipronil. Indoxacarb, S) methyl 7 chloro 2,5 dihydro 2[[(methoxycarbonyl) [4(trifluoromethoxy)phenyl]amino] carbonyl]indeno[1,2 e][1,3,4]oxadia zine 4a (3H) carboxylate, is cons idered to be an organophosphate replacement by the EPA and bel ongs to the chemical family of oxadiazine s action blocks the sodium channels in the insect nervous system which affects many of the inse cts systems such as the digestive system Indoxacarb is considered to be a reduced risk pest icide, which makes it an optimal choice for application to a formulated bait matrix Imidacloprid, 1 [(6 Chloro 3 pyridinyl) N nitro 2 imidazolidinimine, is a neonicotinoid insecticide in the chloronicotinyl nitroguanidine family (Gervais et al. 2010). Imid affects the central nervous system through several types of post synaptic nicotinic acetylcholin e receptors. This causes nerve impulses to be spontaneously released and subsequently failure of the neuron to
48 propagate any si gnal (Gervais et al. 2010). Imidacloprid can be extremely toxic to fish, but application in ant baits should offer little risk to fish Fipronil, 5 amino 1 (2,6 dichloro 4 (trifluoromethyl)phenyl) 4 ((1,R,S) (trifluoromethyl)sulfinyl) 1 H Pyrazole 3 carbo nitrile, is considered a broad spectrum is due to the blockage of the essential GABA A gated chloride channels in th e insects central nervous system. T his prevents the uptake of chloride ions which leads to excess neuronal stimulation and eventually death (Jackson et al. 2009). Fipronil is considered to be a moderate to low risk insecticide, with some environmental concerns because it can be highly toxic to some species of bir ds and highly toxic to fresh water fish. Termidor which contains fipronil, has been utilized to control populations of N. pubens populations (Meyer 2008). The recent increase, spread, and destruction caused by N. pubens have pushed the limits of traditi onal ant control. B ecause of large numbers of ants and multiple queens this pest species becomes a neighborhood problem, not just an individual property problem, and one that has not been successfully controlled by the traditional spray and bait control methods The purpose of this study is to evaluate a granule bait formulation that targets this pest speci es with the addition of a slow acting insecticidal active ingredient to allow for distribution of the a.i. within the ant colonies Materials and Meth ods Insects Insects used in the experiment were collected, reared, and handled as explained in Chapter 3.
49 Granular Bait Formulation The granular bait used to apply the active ingredient was a formulation based on choice experiments in both field and labo ratory settings as described in chapter 3 D og food (Purina One healthy puppy food, Nestle Purina pet care company) was baked at 100C for one hour and allowed to cool for 30 minutes to eliminate potential insect or mite infestations. The d og food was then ground into small granules with a coffee grinder. The ground up dog food pieces were sieved using different sieve sizes (Thermo Fisher Scientific, Rochester, NY) and the pieces that remained in the 1.00 mm sieve were used in the formulation. Laboratory r eared crickets were ground in to a pulp material in a Cuisinart food processor (Cuisinart, East Windson, NJ) then macerated using a 2 m l Pyrex tissue grinde r (Cardinal Health, Dublin, OH). W ater (0.5 ml) was added for every 1 g of cricket in the grinding p applied immediately to the dog food granules. An aliquot of 0.2 ml of cricket slurry was added to every 1 gram of 1 .00 mm sieved dog food granules and mixed using a 30 ml souffl containe r until the granules were saturated. Active Ingredients Active ingredients applied to the formulated bait matrix were chosen from current ant baits on the market that ta rget other ant invasive species. T he active ingredients that were chosen for these ex periments were: indoxacarb, fipronil, and imidacloprid ( Table 4 2 ) T he amount s of active ingredient to be added to the formulated bait matrix were based on commercial granular ant baits containing the same active ingredient but further dilution of the act ive ingredients was necessary to obtain maximum foraging by N. pubens in preliminary experiments. Indoxacarb (
50 diluted with water to a final concentration of 0.0225 % in the formulate bait. T his dilution was based on Advion Fi DE) granular ant bait on the market. Imidacloprid (Bayer Environmental Science, Research Triangle Park, NC) was diluted with water, to the concentration of 0.015% in the formulated bait, based on Maxforce Quantum Ant Bait, which contains 0.03% active ingredient (Bayer Environmental Science, Research Triangle Park, NC). Fipronil (BASF Corporation, Research Triangle Park, NC) was diluted with water, to a final concentration of 0.000225%, based on Ma xforce FC Fire Ant Bait, ( 0.00045% active ingredient ) (Bayer Environmental Science, Research Triangle Park, NC) The control was formulated granu lar ant bait 0.00288% water added. To apply the active ingredient to the formulated granular bait matrix, for every 1 g of matrix, weighed out in a 60 ml souffl container and 0.20 ml aliquots of the diluted active ingredient s w ere pipetted o nto the matrices in the souffl container T he mixture s were shaken until saturation of the bait matrix occu rred. The form ulated bait matrices with active ingredient s w ere placed into the refrigerator until were used. Bioassay Individual test colonies were prepared by pulling 300 worker ants, two queens and approximately 100 pieces of brood were pulled from laboratory coloni es of N pubens The ants were pulled using an aspirator and included similar numbers of foraging and nurse workers ( 150 of each ) Nurse ants were taken from the nest cells in the laboratory colonies, and most of the nurse ants pulled were carrying brood. An attempt was made to get a minimum amount of 100 pieces of brood per individual test colony Once the ants were pulled from the originals colonies they were placed into a (14.5 cm x 2.5 cm) Petri dish testing arena s The Petri dish lid had a 2.0 cm hol e drilled
51 into its center which was covered with a 2.5 cm stainless steel 40 wire cloth dish (Small Parts I nc, Miami, FL) hot glued to cover the hole on the lid. T his hole allowed for air flow into the arenas. Each Petri dish testi ng arena included two 1. 5 mL centrifuge snap cap vials (Thermo Fisher Scientific, Rochester, NY), which had the lids removed with scissors. T he lidless centrifuge snap caps served as water vial and sugar water vials which were held in place by folded pieces (1 cm X 5 cm) of Paraf ilm (American Nati onal Can, Greenwich, CT). The lids of the snap cap vi als were used as dishes for soy bean oil ( Eden Organic, Clinton, Michigan ) and clover honey (Wal Mart Stores, Bentonville, AR) also offered to each experimental colony To avoid unnecess ary deaths in these liquids, cheese cloth was cut and placed over both the soy bean oil and honey snap cap s Also included in the testing arenas was a 3 cm X 9 cm lid from a plastic snap cap vial (Thornton Plastics, Salt Lake City, UT) containing ground cat food (Purina cat chow naturals plus vitamins & minerals, Nestle Purina St. Louis, MI ) (Fig 4 1). The ants were given a 4 cm X 1 cm Petri dish con taining a plaster bottom. T he lid of the Petri dish was cover ed with yellow cellophane, and this provided a darkened nest cell for the ants. The ants were allowed to acclimate to the testing arenas for 72 hours before the experiments After the acclimation period the hon ey, soy bean oil, cat food and sugar water were removed from the testing arenas for two days After two days, the formulated bait matrices with active ingredient w ere taken out of the fridge and allowed to reach room temperature before being placed in the experimental colonies The dead ants in the test arenas were counted and replaced before the experiment was started Once at room temperature 90 granules were counted out and weighed on a (41 x 41 x 8 mm)
52 polystyrene weigh boat (Thermo Fisher Scientific, Rochester, NY). The weighed granules were then placed into the testing arenas. Ants were all owed one hour to forage on the granules. After one hour the bait granules remaining on the dishes were removed from the testing arenas, weighed and counted. The original food that had been removed was refreshed if any food was low and it was then repla ced into the testing arenas. Sugar water and water were added and the nest cell were moistened every third day afterwards The ants were obser ved every other day for 14 days. N umbers of dead ants were recorded and the dead ants were removed from the testing a renas at that time At the end of the 14 days the remaining live ants were freeze killed T he live ants in the control arenas were placed back into the colonies they were originally pulled from. Analysis Experimental ant colonies that removed less than 10 % of the granules provided, were no included in the analysis of the results. This was done to eliminate colonies that did not show typical foraging behavior. To compare the added active ingredients by consumption and percent mortality the data was arcsin square root transformed and ANOVAs run were in the statistical software JMP (SAS Inst., Cary, NC). Student Tukeys tests were used to compare means Results There was no significant difference in the consumption of baits ( F = 1.3 691 P = 0.2 824 ) (Fig. 4 2) Because all baits were consumed evenly, there was no correlation between the amount of bait taken and ant mortality rate. T here are significant differences amog the different active ingredient formulations ( F =3.5069, P= 0.0353; F = 4.4 851, P= 0.0153; F =4.3135, P= 0.0176; F =4.4610, P= 0.0156; F = 4.8206,
53 P= 0.0116) with fipronil causing significantly greater mortality than the other treatments (Students T test: = 0.05, t = 2.0930) (Fig. 4 3) than indoxacarb, i midacloprid and the control baits M ortality over the 14 day tria l period caused a decrease in the numbers of N. pubens in all active ingredients treatments The total percent mortalities for indoxacarb and imidacloprid, over the 14 day experimental period, were 37% and 26%, respectively. These treatments caused less than half the mortality caused by the of fipronil bait, which caused percent ant mortality of 86% over the 14 day experimental period. Discuss ion There is much debate about an effective control method for N. pubens Meyers (2008) tested a variety of insecticidal control methods on this species, but no effective formulation was found. Due to the limited knowledge on nutritional preferences of N. pubens a n efficient form of control has not been found. The most common means of controls are insecticidal sprays, other ant granular and liquid baits, which have had limited success in controlling this invasive pest ant species. Because of the inefficie n cy of current methods of control, a new control method is needed. Research was conducted on including different active ingredient s o n a bait matrix. The active ingredients chosen had been successful against similar ant species: S invicta and L humile Stringer et al. (1964) proposed that a bait active ingredient should have delayed toxicity, be able to be transferred from one ant to another, and be non repellent to the foraging ants. Fipronil fits this model by displaying delayed toxicity, was transferr ed from one ant to another either through trophallaxis or by social grooming, and, as with all of the active ingredients in this experiment, did not discourage for aging from the ants (Fig.
54 4 2). In Texas, Drees et al. (2009) suggested using Termidor SC (9 .1% fipronil) as an outdoor spray for control of N. pubens because they have observed high mortality rates with this product. Fipronil has also proven to give high rates of mortality in formulations for L. humile (Hooper Bi et al. 2000). The results fo r i ndoxacarb and imidacloprid did not confirm past success rates of these active ingredients (Rust et al. 2002, Barr 2003, Oi et al. 2006 ). The low rate of mortality with indoxacarb bait was surprising because experiments on S. invicta offered Advion fire ant bait (0.0045% indoxacarb) showed a reduction to colony size of 95% by day five (Oi et al. 2006). Over the fourteen day trial period the total percent of mortality of N. pubens in my experiment did not exceed 37%. These results could be due to the lower pe rcent of active ingredient (0.00255%) applied to my experimental granular bait. With further dilution the active ingredient through trophallaxis most of the ants N. pubens may not have acquired a lethal dose. F ire ant bait is an lipid place on top of a d efatted corn cob which could cause the active ingredient to react differently as seen in deactivation of an active ingredient, from binding to a bait too closely ( Cress 1990, Stanley 2004) Deactivation of the active ingredient, indoxacarb, could have bee n a contributing factor in the ineffectiveness I observed with my experiments Indoxacarb has been proven to suppress foraging in fire ant experiments, in 48h in fie ld experiments (Barr 2003). The lack of foraging suppression I observed suggests that the d osage of indoxacarb I applied to the bait matrix may have been too low Klotz et al. ( 2004) observed L. humile actively forag ing on a bait but with no reduction on ant numbers, as it was seen in my experiments the authors concluded that the A.I. concentrat ion was too low More trials s hould be conducted with different
55 concentrations of indoxacarb in the baits which containing materials the ants forage readily. Experiments with L. humile proved imidacloprid to be successfully in the suppression of ants in a field setting. I midacloprid is photosensitive and needs to be protected from the sun in order to be effective in suppressing ants in a field setting (Daane et al. 2008) Shielding for bait stations maybe required for high efficacy. The photo degradation of hours. My experiments with the same active ingredients were run in a laborato ry setting, under a temperature and light controlled environment where photo degradation should have been at a m inimum. More experiments should be conducted on the rate of degradation of imidacloprid and its photo sensitivity. In studies with L. humile the rate of colony decline was 50% with dosages of imidacloprid between 0.0 005% 0.005% (Rust et al. 2002), but thi s A.I. was applied at 0.0015% to the granular bait matrix in my experiment. The lack of success in controlling N. pubens could have been due to the baiting method. The high numbers of L. humile have been controlled by using bait stations with liquid imidac loprid whereas I used a granular bait with imidacloprid. In previous experiments liquid bait stations, although impractical control of a large infested area, are very attractive to L. humile This is because they L. humile prefer, in some studies, liquid sugary food sources offered as observed by Rust et al. (2002). Several factors may have caused the low bait efficacy I observed: including t he low concentration of the active ingredients in the baits possible deactivation of the active ingredients, and i nsufficient time for the delayed toxic effect to be expressed with only a 14 day experimental period. These experiments should be observed over greater
56 amount of time, as seen in experiments by Rust et al. (2002) and Daane et al. (2008) with L. humile an d Collins et al. (1998) with S. invicta A longer observation would provide information on whether or not the diluted active ingredients caused an additional delay in toxicity. Since these experiments took place in a controlled laboratory environment, furt her research on the effectiveness of the formulated granular ant bait with fipronil should be done in a field environment. This will test the efficacy of this bait in a non controlled setting thus providing information on the possible limitations of this b ait formulation.
57 Table 4 1 Products used in bait formulations used in laboratory choice and efficacy experiments against N. pubens colonies Product names Trade name Chemical name % Active ingredient in commerical product % Active used Insecti cide Indoxacarb (S) methyl 7 chloro 2,5 dihydro 2[[(methoxycarbonyl)[4(trifluoromethoxy)phenyl]amino] carbonyl]indeno[1,2 e][1,3,4]oxadiazine 4a (3H) carboxylate 20.0 0.02250 Premise 2 Imidacloprid 1 [(6 Chloro 3 pyridinyl) N nitro 2 imidazolidinimine 21. 4 0.01500 Termidor SC Fipronil 5 amino 1 (2,6 dichloro 4 (trifluoromethyl)phenyl) 4 ((1,R,S) (trifluoromethyl)sulfinyl) 1 H Pyrazole 3 carbonitrile 9.1 0.00023
58 Figure 4 1. Testing arena used for experiments on Nylanderia pubens using granular bait m atrix applied with active ingredient.
59 Figure 4 2. Pe rcent removal by Nylanderia pubens colonies of granular bait with different active ingredients in laboratory experiments Means with the same letter are not significantly di fferent. Error Bars= SEM.
60 Figure 4 3. Cumulative p ercent mortality of Nylanderia pubens from laboratory colony fragments, after consumption of granular bait with different active ingredients. Error Bars= SEM.
61 CHAPTER 5 C ONCLUSION Nylanderia pubens placed in arenas or located in field situations exhibited preferences for certain particles sizes and compositions of food. These preferences were based on both the efficient retrieval of resources and nutritional needs of the c olony. In my experiments foraging ants: 1) located the food through random foraging, 2) picked up or fed on food particles, 3) returned to the nest, laying down a pheromone trail to recruit other ants. Nylanderia pubens did not follow trails precisely in order to recruit large numbers of ants to overwhelm the food source. N. pubens appears to follow the foraging strategy outlined by Oster et al. (1978) as trunk trail foraging rather than mass recruitment. Trunk trail foraging is when a pheromone scent tra il is laid down, but the ants following the trail deviate to forage from the main trail in scattered patterns looking for food (Oster et al. 1978). In my experiments N. pubens depleted a variety of particle sizes, they showed a lack of cooperation in remov al of food particles, and massive numbers of ants were never observed in an area with high concentrations of preferred food size particles. In my laboratory and field experiments, N. pubens foraged on granule sizes 0.850 mm 1.18 mm, the percentages of foo d particles removed were 28 33% in the lab and 21 35 % in the field, which indicates both scattered pattern of foraging and a lack of mass recruitment to a food source. N. pubens style of foraging contrasts the foraging strategy observed in L. humile whic h employs mass recruitment as their foraging strategy (Roulston et al. 2002). Foragers of L. humile have been observed cooperating with other foragers, recruiting large numbers of their foragers to a food source, and depleting preferred food sizes first. I n my experiments N. pubens was observed not to
62 follow the foraging strategy employed by L. humile the removal of varying sizes of granules in both the field and laboratory along with their scattered foraging pattern followed what is described as trunk tra il foraging. In my experiments, N. pubens trunk trail foraging behavior appears to not only follow the optimal foraging theory but also follows the head width and body size in relation to food particle theories as well. On top of foraging strategies, ants also follow patterns based on the food choices they make, those patterns are called foraging theories. In my experiments the foraging strategies that N. pubens followed was the : optimal foraging theory (Nonacs et al. 1990, Roulston et al. 2002) .It was al so clear that the head width and the overall size of the ants dictates the size particle they pick up (Traniello 1989, Hooper Bi et al. 2002). Ant foraging normally fits what has been described as fitting the optimal foraging theory (Nonacs et al. 1990, R oulston et al. 2002). The optimal foraging theory states that ants should take the biggest pieces of food particles that they can carry, in order to increase their net energy intake per unit of effor t (Roulston et al. 2002). In my experiments with granular size removal in the laboratory and in the field, N. pubens seemd to fit the optimal foraging theory, because the ants were taking the most amount of calories by returning with the greatest mass of food particle, while spending the least amount of energy. N. pubens removed the largest sizes of food particles by weight thus decreasing the number of trips to the food source, which follows the optimal foraging theory. N ylanderia pubens did not cooperate with one another to remove a food source. This lack of c ooperation indicates that although these ants follow the optimal foraging theory for weights of granules removed, more important reasons for their foraging
63 choices need to be examined to elucidate factors associated with their moving an optimal amount of f ood back to the colony. In the theories discussed by Hooper Bi et al. (2002) and Traniello (1989) the preferences of ant species not only are, governed by the foraging ant head width, but also determin ed by the over all size of the foraging ant in relati on to the food particles. Nylanderia pubens actively fo raged on dog food pieces 0.850 1.18 mm in the laboratory, and 0.850 mm predominately in the field. Nylanderia pubens has a relatively small head width (0.55 0.64 mm) and body size (2.5 3.0 mm) (Trager 1984, Meyers 2008) which determines the preferred size of food they will remove. Traniello (1989) and Hooper Bi et al. (2002), used similar ants species and measured head widths and body sizes in relation to food particle preferences. The head widths and body size of N. pubens are similar to the head width and body size of L. humile (0.66 mm and 2.0 3. 0 mm, respectively) (Wild 2004). B oth species preferred granular sizes, 0.840 1.00 mm, whereas the larger, polymorphic ants species S. invicta (head widths: 0.45 1.50 mm, body sizes: 2.00 6.0 mm) preferred the granular sizes: > 2.00 mm (Wood et al. 1981, Tschinkel et al. 2003). N. pubens is a monomorphic species with an overall size of 2.5 3.0 mm, so the particles that the workers can carry are limited to spe cific sizes, unlike S. invicta that can carry much larger range of particles due to polymorphic nature of the colonies In analyzing the experiments of Traniello (1989), Roulston et al. (2002), and Hooper Bi et al. (2002) on foraging theories, in conjunct ion with the observations in my experiments, N. pubens is small size ant and small head width causes the optimal size food granule for this species to be between 0.850 1.00 mm.
64 There is a need for the development of insecticidal controls for N. pubens. Cu rrent methods of control include insecticidal barrier sprays, which have been less than effective in controlling of this species. Baits have the ability to offer a level of control that has not yet been obtained by sprays. The current types of baits out on the market for ant control include gel baits, liquid baits, and solid granular baits. A liquid or gel bait is usually one that requires a bait station and constant reapplication due to the elements. Liquid baits are developed for ants that display mass re cruitment to food sources; N. pubens lacks mass recruitment strategy, so liquid baits will fail to control this pest species. Granular baits, which can be scattered on the landscape take advantage of the trunk trail foraging of N. pubens and of their for aging strategy optimizing the chance that the bait will be located and foraged upon. The potential for broadcast application of g ranular bait s makes this formulation ideal for large scale ant control. I n some cases there is little distinction between liqu id and solid baits, as in the instance of popular fire ant baits. Fire ant ba i ts consist of oil placed on a carrier (de fatted corn cob) (Loggren et al. 1963, Stanley 2004). This method of baiting targets foraging ants, which can only feed from liquid base d foods. With fire ant bait s workers forage on and remove the oil off the bait, so this bait looks like a solid granular bait, but behaves like a liquid. This behavior does not force the foraging ants to return to the nest with the bait particle, so the a ctive ingredient will enter the colony as a liquid On the other hand the experimental components that I tested were solid foods, which would have to be taken back to the colony in order for them to be broken down by the larvae before they could be utiliz ed by the adult ants Solid bait matrices I chose were based
65 on the foraging strategies of N. pubens and previous research on ideal components of solid baits. Components of baits consist of an attractant, carrier and active ingredient (Stanely 2004). Trad itional baits for S. invicta consist of oil (attractant) on de fatted corn grit matrix (carrier) and multiple active ingredients have been applied to these components (Loggren et al. 1963, Stanley 2004). This method of control is ideal for S. invicta beca use, it capitalize s on the recruitment foraging strategy, and on the food preferences this ant species has toward lipids (Stanley 2004). N. pubens was not determined to be lipid feeders, nor are they mass recruitment ants, as described previously, so traditional baits that work for S. invicta do not work for N. pubens On the other hand baits that contain proteins and carbohydrates are very attractive to species such as L. humile and Paratrechina spp. that are not attracted to the lipid based fire a nt baits (Stanley 2004). In order to enhance a carrier, it is important to know the ant species food preferences, based on field observations, laboratory experiments, and by studying the literature on other similar ant species. I have observed N. pubens t o be feeding from insect tissu e, and to be attracted to honey in field and laboratory environments. In other observations, N. pubens foraged on honey dew from aphids, plant nectaries and insect tissue (Creighton 1950). N. pubens has been blamed for crop de struction from tending to aphids and mealy bugs (Wetterer et al. 2008). N. pubens prefers carbohydrate powders 2:1 to protein powders (Cook et al. 2012). In similar ant species, such as Rhytidoponera metallica (Smith), Dussutour et al. (2009) found that th e ants also preferred carbohydrates to proteins in a 2:1 ratio. Petralia et al. (1980) suggests, that a solid proteinaceous food which has to be digested
66 by the brood would be ideal bait. With the findings of Cook (2012), and Dussutour (2009), the observat ions of Wetterer (2008), Creighton (1950), in conjunction with N. pubens foraging theories and strategies, and my own personal observations, I ch ose to test a number of animal foods based on their protein and carbohydrate/minerals as possible granular bait matrices along with additive for possible enhancement to the carriers. Because N. pubens foraging follows the optimal foraging theory, field colonies of ants displayed a preference to dog treats, the highest protein matri x choice. In the laboratory, given the l imited number of ants and brood per satellite colony, along with the close proximity of the nest cell, ants foraged on most food matrix choices evenly. The laboratory results also point to the optimal foraging theory as the predominant force in N. pu bens foraging. N. pubens actively foraged on all foods available thus increasing the amount of calories returned to the nest cell. T he relative composition of essential elements in the bait matrices may be important in maxim izing foraging by specific ant s pecies (Stanley 2004). Although dog treat was more readily foraged upon in the field experiments, dog food had the close st carbohydrate to protein ratio (1.5: 1) as prescribed by Cook et al. (2012), compared with other materials used. Dog food was an opti mal carrier component, not only because of its ca rbohydrate/ protein composition and spread ability to take advantage of N. pubens foraging patterns, but also because it was readily accepted by the ants and could be easily enhanced by addition of other mat erials. The enhancements made to the granular bait were based on a compilation of knowledge obtained from experiments done on food preferences of: S. invicta (Vogt et
67 al. 2002) Anoplolepis gracilipes (Smith) (Harris et al. 2012) and Paractrechina longic ornis (Latreille) (Kenne et al. 2005) S. invicta and P. longicornis have different foraging characteristics but are similar to N. pubens in at least one as pect of their nutritional needs, whereas A. gracilipes has both a similar foraging strategy and diet to N. pubens S. invicta is an omnivore which actively mass recruits and forages on a broad range of liquid materials, seeds, and arthropods, including: plant sap, plant nectars, and honeydew from hemipteran tending (Vogt et al. 2002). A. gracilipes has a broad diet and displays scatter pattern foraging. A. gracilipes is described as a scavenging predator that preys on a variety of insects, isopods and their diet can include larger animals such as birds and reptiles (Harris et al. 2012). They also actively forage on carbohydrate rich foods including: honey dew from aphids, plant exudates, and fruit particles (Haines et al. 2008). Paratrechina longicornis are opportunistic omnivores, which employ the foraging strategy of mass recruitment and group hunting (K enne et al. 2005). They thrive on live and dead insects, honeydew, fruits, plant exudates, and foods from around human dwellings (Pagad 2010). N. pubens is somewhat similar to the se three ant species, based on observed dietary habits such as aphid tending, scavenging and foraging on plant nectars. The information learned from these ant species led me to try oils, sugars, and insects applied to the dog food carrier, as possible optimal enhancements. T he oil and sugar bait enhancements I chose were based on other ant preferences and baits used in their control, but they did not significantly increase foraging by N. pubens However, the addition of live cricket tissue, resulted in increased foraging, as
68 was the case in a study by Williams et al. (1990), who us ed live fly pupae as a carrier for bait. Although the use of live insects as bait worked in experiments done by Williams et al. (1990), live insects are currently impractical as commercial bait However, i nsects can be incorporated into baits as macerated tissue added to the baits. In my experiments N. pubens actively foraged well on the dog food matrix with added cricket tissue, but field colonies did not differentiate between plain dog food and dog food with the cricket additive. The active ingredients us ed in my experiments were chosen for their relatively fast act ion despite being slow enough to allow transfer through the colony by social grooming and trophallaxis. Slow acting ant toxicants are preferred because if the toxicant ants too quickly for in stance by paralyzing the mouthparts of the ants or killing the foraging ants ; th at can prevent the toxicant from reach ing the entire colony, such as paralyzing the mouth parts of the ants or killing the foraging ant. Because of the limited foraging of the baits with the commercially available label rates, the active ingredients were diluted by 50% which caused an increase in foraging. In my experiments baits with all three active ingredients were foraged upon evenly, indicating that the active ingredients applied to the bait matrix did not discourage foraging. In my laboratory experiments, fipronil displayed faster mortality and higher rate of mortality than the other tested active ingredients. Fipronil has been effective in control of many ant species men tioned previously : A. gracilipes L. humile (Stanley 2004, Wiltz et al. 2010b), and S. invicta (Wiltz et al. 2010a). T he reason for the success of fipronil in my laboratory experiments was probably its high rate of horizontal transfer within a colony besid es transfer by trophallaxis The horizontal transfer can be from social
69 grooming or the removal of dead ants that are contaminated with fipronil, as seen in experiments by Soeprono et al. (2004), Choe et al. (2008), Wiltz et al. (2009), and Wiltz et al (2 010a,b). Choe et al. (2008) suggest that the high rate of horizontal transfer seen is due to the chemical nature of fipronil that give this active ingredient a high affinity for the lipids found on the wax layer of insect cuticles. Although fipronil outper formed the indoxacarb and imidacloprid in the laboratory experiments, further research should be conducted to explore the performance of all of these insecticidal act ive ingredients in a field environment. M y observations and those of other scientist (Vogt et al. 2003, Challet et al 2005 and Wiltz et al. 2010b) point to noticeable differences in the preference and behaviors between laboratory and field insects. Differences between controlled environment in the laboratory and more variable environment in th e field lead to differences in foraging behaviors preference to bait components (Traniello et al. 1983, Vogt et al. 2003, Challet et al. 2005). D ifference s in the presence and proportion of different developmental life stages in the colony (Traniello 1989) can also be important factors in determining difference between laboratory and field results. Temperature also can play an important role in the foraging activities of N. pubens Calibeo Hayes et al. (2010) described limited foraging by N. pubens when the temperature was ature also plays important role in the shape and structure of ant nesting areas (Challet et al. 2005. In my experiments, the difference in food choices between the laboratory and field colonies could have resulted from to the varying ranges of temperatures (23.9 26.6 C) and microclimates in the field location used (Vogt et al. 2003, Challet et al. 2005 and Wiltz et al. 2010b).
70 Another reason for the differences seen between results from laboratory and field environments could be attributed to the developmental differences between the colonies. Selection of food sources is done at a colony level, and can be affected by age of foragers, their life expectancy and their foraging ability (Traniello 1989). The average life of a nt foragers in the field is wrought with dangers from predators, and other factors, but in the laboratory the controlled condition s allow for a longer life span Foragers of Cataglyphis bicolor ( Fabricius) have a life expectancy of 6.1 days under field co nditions, whereas in a laboratory setting they can live up to months (T raniello 1989). Life stages in ant colonies also play an important role in the foraging preferences and the fitness of the colony The amount of brood in the colonies, in the laboratory and field, could be responsible for the foraging preference differences observed. Fourth instar larvae do the most of the digestion of the protein digestion in the ant colon (Petralia et al. 1980, Weeks et al. 2004), and their presence in a colony can determine the colonies foraging on proteinaceous materials. The development of bait that may work at different times of the year, when numbers of the 4 th instars may be at very different levels, may require use of formulation that is not affected by the an t population composition (Weeks et al. 2004). Bait matrices choices in the laboratory could have been caused by the absence of 4 th instar larvae in the colonies (Petralia et al. 1980). In my laboratory colonies choices of components removed could have bee n a reflection of their inability to digest materials with high protein content. This was not the case in the field colonies, which probably had relatively more brood than in the laboratory colonies, given the time of year the experiments were conducted an d the high ant populations
71 observed. Temperature and colony developmental stages, I believe are the reasons for the foraging and preference differences that I observed. Even with the differences between in the laboratory and field experiments my observat ions can help In the design of future strategic control of N. pubens My experimental results provide a better understanding of potential components for a granular bait to be used with N. pubens Future experiments should include other active ingredients b eyond those I applied to the formulated bait matrix Other future experiments should include longer laboratory studies on the active ingredients I tested, as explained in chapter 4, to explore the possibility of horizontal transfers of active ingredients. My results can serve as a basis for future development of baits for Nylanderia pubens
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78 BIOGRAPHICAL SKETCH Jodi Michelle Scott, daughter of Patricia and Robert Bower was born in Orlando Florida. She was raised in Orlando, Florida, with her older sister, Nichole Bower. She graduated from Colonial high school in 1998. She attended Valencia community college, eas t campus and gradua ted with an Associate of Arts degree in 2001. She then join ed the United States National Guard from June 2001 to June 2007. While in the Guard she attended the University of Central Florida, earning the degree of Bachelor of Science in 2008. She then became a laboratory technician in the physiological department a t the University of Florida. She entered the graduate program in the Department of Entomology and Nematology at the University of Florida specializing in the urban entomology under Dr. Philip Koehler in 2011.