ATMOSPHERIC PRESSURE CHANGES ARE ASSOCIATED WITH DIFFERENCES IN CALLING, MATE SEEKING, AND LIGHT ATTRACTION BEHAVIORS OF DIAPHORINA CITRI (HEMIPTERA: LIVIIDAE) By NINA ZAGVAZDINA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR TH E DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014
Â© 2014 Nina Zagvazdina
To women in science. Immanuel Kant
4 ACKNOWLEDGMENTS I would like to start by thanking Dr. Susan Halbert for all of my experiences at DPI, where I got my first real taste of entomology as an applied science. Also, t hank you to Charlotte Campana, Mark Rothschild , and Dr. Gary Steck for your support and positivity. Thank you to Dr. Richard Mankin for allowing me to be a part of a great workplace environment, and being flexible with me while working together on this project. Thank you to my co workers, Everett Foreman , Betty Weaver , Larry Pitts, and Heidi Burnside, as well as Neha Ainpudi, our SSTP student , for all of your help . Special thanks to Dr. Sandra Allan, Thomson Paris , and Bradley Udell, who designed and collected data for the light response assays, and were very supportive of me looking at their observations from a different angle. Another special thank you goes to Dr. Dan Hahn, for taking the time to help me with this project and for being such an amazing teacher. I consider myself very fortunate with my committee. nk Dr. Larry Winner and James Colee for helping me address statistical challenges. Thank you, John Mocko for maintaining the Physics Weather Station and making the data sets I needed available to me. I also want to thank Brett Lackey and Lauren Olesky for generously taking time to come to the CMAVE and answer my meteorology related questions. Also, special recognition goes to Milda Stanislauskas, Dr. Seth McNei l l, Baruk h Rohde, Avraham Brun Kestler, Mechael Brun Kestler, Daniel Fialkovsky, and Ruth Rohde fo r pioneering the psyllid caller project. Thank you to Dr. Stephanie Webster and all of the awesome consultants from the Public Speaking Lab who patiently helped me prepa re my oral defense presentation. I also want to recognize Dr. Heather McAuslane and Rut h Brumbaugh for helping me with administrative
5 concerns, and Nancy Sanders, Elena Alyanaya, and Steve Lasley for all of your behind the scenes help. Dr. Ia Zagvazdina. and Dr. Alex Zagvazdin. Finally I thank Ana Silva, Michelle Garcia, Bevin McCormick, Bob Aldridge, and Erin Sherwood for their friendship and priceless support .
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 4 LIST OF TABLES ................................ ................................ ................................ ........................... 8 LIST OF FIGURES ................................ ................................ ................................ ......................... 9 ABSTRACT ................................ ................................ ................................ ................................ ... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 12 Atmospheric Pressure ................................ ................................ ................................ ............. 12 Effects of Atmospheric Pressure on Animal Behavior ................................ ........................... 14 Effects on Flight and Dispersal ................................ ................................ ....................... 14 Effects on Olfaction and Feeding ................................ ................................ .................... 16 Effects on Courship and Mating ................................ ................................ ...................... 18 Effects on Oviposition, Eclosion, and Other Behaviors ................................ .................. 20 Effects on Overall Locomotor Activity ................................ ................................ ........... 21 Summary of Effects of Atmospheric Pressure on Animal Behavior ................................ ...... 22 Potential Biological Mechanisms for Sensing Atmospheric Pressure ................................ .... 24 2 EFFECTS OF ATMOSPHERIC PRESSURE ON DIAPHORINA CITRI BEHAVIOR ....... 25 Diaphorina citri Biology ................................ ................................ ................................ ........ 25 Vibr ational Communication ................................ ................................ ................................ ... 27 Summary of Economic and Evolutionary Context of Experiment ................................ ......... 30 Materials and Methods ................................ ................................ ................................ ........... 32 Atmospheric Pressure ................................ ................................ ................................ ...... 32 Calling and Searching Behavior Study ................................ ................................ ............ 33 Light Response Study ................................ ................................ ................................ ...... 35 Results ................................ ................................ ................................ ................................ ..... 35 Atmospheric Pressure Measurements ................................ ................................ .............. 36 Calling, Searching, and Light Attraction Bioassays ................................ ....................... 37 3 DISCUSSION ................................ ................................ ................................ ......................... 41 Atmospheric Pressure Changes and Psyllid Mating Behavior ................................ ............... 41 Atmospheric Pressure Changes and Psyllid Movement ................................ ......................... 43 Challenges in Atmospheric Pressure Research ................................ ................................ ....... 44
7 LIST OF REFERENCES ................................ ................................ ................................ ............... 46 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 53
8 LIST OF TABLES Table page 2 1 Means and standard deviations of absolute pressure at the time of trial and changes in pressure from 3 48 h prior to trial (time lag). ................................ ................................ 37 2 2 Contingency analysis of effects on calling and searchi ng behavior of males exposed to low/falling, steady, or high/rising barometric conditions measured at different times (h) before trials (time lag). ................................ ................................ ....................... 38 2 3 Analysis of variance of effects on light attraction of males exposed to low/falling, steady, or high/rising barometric conditions measured at different time (h) before trials. ................................ ................................ ................................ ................................ ... 39
9 LIST OF FIGURES Figure page 2 1 Pressure values from indoor logger. ................................ ................................ .................. 36 2 2 Mean percentages of responsive psyllids in acoustic and light attraction assays. ............. 40
10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ATMOSPHERIC PRESSURE CHANGES ARE ASSOCIATED WITH DIFFERENCES IN CALLING, MATE SEEKING, AND LIGHT ATTRACTION BEHAVIORS OF DIAPHORINA CITRI (HEMIPTERA: LIVIIDAE) By Nina Zagvazdina December 2014 Chair: Richard Mankin Major: Entomology and Nematology Many insects and other animals exhibit behavioral changes in response to shifts in atmospheric pressure, a physical cue that is correlated with impending windy and rainy weather conditions. We examined the effects of absolute pressure and pressure changes on time scales from 3 48 h prior to observation of two behaviors of the Asian citrus psyllid, Diaphorina citri . Bioassays were designed to evaluate calling and mate seeking behavior and light attraction. T he proportions of psyllids responding in calling, mate seeking, and light attraction bioassays varied significantly with prior exposure to different conditions where the barometer was rising, steady, or falling during different time periods before the bioassays . L ower proportion s of male psyllids activel y called and searched for females when the pressure rose by more than one standard deviation of the mean values measured 24 h before bioassays . H igher proportion s of psyllids responded to light when pressure fell by more than one standard deviation of the mean values measured 24 h before bioassays . Such results are consistent with a hypothesis that t here may be survival or fitness benefits to focus ing energy on flight rather than mating when large pressure changes occur over a 24 hour period. In addition , s ignificant increases were found in the proportions of psyllids attracted to light when pressure decreased by more than one standard
11 deviation over a 9 h or 12 h period before bioassay s . More knowledge about the effects of different environmental changes on behavior, including rising or falling barometric pressure , can lead to improved models of psyllid movement and improved timing of pest management activities .
12 CHAPTER 1 INTRODUCTION Atmospheric Pressure Atmospheric pressure is the force exerted by air pa rticles on a given area. gravitational pull serves as the source of this force, holding surface. C omposed primarily of nitrogen and oxygen , t he atmosphere is a 30 km high gaseous envelope that also includes a highly variable quantity of water vapor, a key driver in shaping weather and weather patterns. When r adiant energy from masses, part of the energy is transferred to the surrounding air. Thus, air molecules are in a stat e constant flux and air is essentially an unevenly distributed compressible fluid. The amount of air particles in a given space and the speed at which they are moving affect atmospheric pressure (Williams, 1997). Atmospheric pressure is measured by evaluat ing the force exerted by a column of air (Lowry and Lowry, 1989). Pressure can be described by many units of measure, including the Pascal ( the International System unit ) , and the millibar (mbar) , which is the unit preferred by meteorologists globally (Bur ch, 2009). Air pressure is measured with a barometer, which can be mercury, aneroid, or electronic. Basic mercury barometers are glass tubes which display the level of liquid mercury supported by ambient air pressure. Aneroid barometers incorporate compre ssible metal bellows, while modern electronic barometers are built with pressure sensors. Since its inception nearly 400 years ago, the barometer has been used as a tool to help forecast the weather . L ow pressure weather systems are characterized by stron g winds, cloud cover, and precipitation. lthough there is an approximate worldwide average sea level pressure (1013.2 relative terms and are not defined by specific pressure values. An air system that is considerably
13 Air density is highly dependent on temperature. Higher temperatures result in faster moving air molecules and subsequently more frequent collisions that ca use air to rise. Lows are associated with clouds and rain because the water vapor in the warm rising air mass begins to cool at higher elevations. The water vapor condenses and liquid water precipitates out. In addition to precipitation, another potentiall y adverse condition associated with lows is heavy wind. Wind is the result of air movement from areas of high density to areas of low density. Wind intensity is dependent on t he pressure gradient. In contrast to Lows , Highs are often cooler and are associa ted with clear, dry skies and light wind or relative stillness . This is in part because cold air is denser than warm air, with a higher quantity of air particles moving and colliding with less energy (Burch, 2013). Highs often follow a passing Low. Barome ters are excellent tools that aid in weather forecasting, but it is important to can be misleading because they depend on a single variable, the pressure value at a single moment, to predict a complex set of atmospheric interactions. The actual weather is better predicted by the overall shape of the pressure pattern as it changes over time instead of the specific pressure value barometer as a 1.6 to 3.5 mb ar change over a 3 hour time period. A falling barometer may suggest the approach of inclement wea ther , while a rising barometer suggests potential improvement. Generally, the barometer will not help forecast imminent events but enables the user to make predictions about the period 12 to 24 h into the future (Burch, 2009).
14 Effects of Atmospheric Pressure on Animal Behavior For many animals, w eather conditions that include heavy winds and precipitation can directly cause mortality or can affect other aspects of life such as foraging success or the ability to locate mates ( Cornell and Hawkins, 1995 ) . Because pressure fluctuations are associated with other, potentially unfavorable weather events, atmospheric pressure is an abiotic factor that animals may cue in on. Studies on the effects of pressure on animal behavior have been conducted for over 30 organisms, including mammals, reptiles, birds, fish, and insects. This section will focus on pressure variations of natural magnitudes and will not include effects of extremely high pressures or near vacuum states on living things. Effects on Flight and D ispersal Studies on the effects of pressure or pressure variation on flight have frequently focused on Diptera. Haufe (1954) conducted an extensive study on the effects of subtle pressure variation on Aedes aegypti , addressing directionality, amount, and r ate of pressure change. No change in flight activity was observed at steady pressure levels between 733 and 1066 mbar, but there was an effect of pressure change. At starting pressures below 979 mbar, rising pressure resulted in higher number s of mosquitos that spontaneously took flight. However below 979 mbar, the trend was reversed, and flight activity increased with falling pressure . Haufe also varied rates of pressure change and determined that for rising pressures, smaller rates appeared to have a stim ulatory effect, while larger rates of change were more stimulatory with falling pressures. Haufe noted that there was less behavioral variability in response to pressure when mosquitos from the same brood were tested compared with mixed groups. Wellington (1946) conducted studies on the pressure effects on species in the following fly families: Calliphoridae, Muscidae, Sarcophagidae and Tachinidae. Wellington showed that the aristae were important in sensing small, localized increases in pressure (0.3 mbar) such as
15 those created by waving flies away with a hand. He demonstrated that it was the movement of air rather than the visual stimulus that triggered escape flight . When s ubjecting the flies to small, rapid (2 10 mbar) pressure fluctuations at starting pressures of 1000 mbar in a controlled chamber, t he flies responded with a marked increase in activity . Wellington also conducted fast (5s) and more gradual (2 min) 50 mbar pressure drops and reported that flies exhibited a noticeable increase in flight ac tivity at the maintained low pressures but that the behavior became normal again when the jar was returned to the original pressure. Wellington was interested in explaining but did not include quantification of flight beha vior in the study (i.e. percent response, amount of time in flight), making it difficult for future readers to interpret the results . Edwards (1961) subjected two Calliphora species to gradual (1 mbar per hour for 3 hours) and stepwise (1 mbar per 0.25 hou r, with 1 hour intervals in between) pressure changes. For C. vicina, flight activity (measured with a probe/electrometer set up) increased when pressure deceased, but increasing pressure had no effect. However, no pressure changes influenced C. vomitoria behavior, though the two species are closely related. Forunier et al. (2005) tested 6 different atmospheric pressure regimens on Trichogramma pretiosum and Trichogramma evanescens females, including steady pressures ( 1025 mbar and 975 mbar ) and rapid (1 h) or gradual (6 h) changes between those two pressure values. Neither wasp had a behavioral change during stable conditions n or slow changes but rapid increases or decreases were significantly associated with reduced flight initiation (p<0.05) . A number of studies did not directly test effects of pressure on behavior in an experimental chamber, but found that pressure was correlat ed with flight or dispersal in the field or lab oratory . Climatic factors that influenced flight activity in field cages of a Bra conid wasp ( Fopius arisanus ) included humidity ( P < 10 10 ) , temperature ( P < 10 5 ) , and atmospheric
16 pressure ( P < 10 3 ) (Rousse et al. 2009) . Increased flight acti vity was anecdotally noted for a Tortricid moth (Henson, 1951) and a Dytiscid diving beetle (Lytle and White 2007) , both in relationship to the onset of inclement weather. Unable to explain high variability in dispersal rates in a laboratory study using two spotted spider mite, Li and Margolies (1994) began tracking atmospheric pressure and found that pressure values did not help to explain behavior, but changes in pressure did. Faster flight initiation was associated with a rising barometer. A study relating bat activity to atmospheric pressure was conducted on the Eastern Pipistrelle, Pipistrellus subflavus (Paige 1995) . Cave dwelling bats are particularly interesting test subjects because cave interiors are relatively stable environments, wi th very little change in temperature, relative humidity, and marked by complete absence of photoperiod . Atmospheric pressure, however, does fluctuate in caves and may be one of relatively few environmental cues available to organisms . A strong negative rel ationship was found between number of bats exiting the cave at dusk and pressure (r 2 =0.87, P <0.0003). Effects on Olfaction and Feeding Effects of atmospheric pressure on olfactory responses of bark beetles to aggregation attractants were tested experimen tally by subjecting Scolytus multistriatus and Ips pini to fluctuating pressure (up to 33 mbar) for 30 minutes (Lanier and Burns 1978) . In the case of S. multistriatus , the mean percent response in an olfactometer assay was 37.3 Â± 2.48% for individuals subjected to pressure change compared with the control group at 6 5.3 Â± 1.64%. The fluctuating pressure had a greater effect than a single increase or decrease in pressure, though these also reduced responsiveness. Increasing in pressure did not affect olfactometer I. pini response rate, but fluctuating pressures and a single decrease in pressure reduced responsiveness. Data from correlational studies were consistent with the experimental data. There was no relationship between response rates of S. multistriatus in laboratory olfactometer studies and
17 atmospheric pressure reading s, but there was a difference in response rate based on the rate of change. Changing pressure was correlated with a lower response rate. N o trend in I. pini behavior was observed. Lanier and Burns (1978) commented on the size differences between th e two bark beetles, suggesting that I. pini would not be as susceptible to weather challenges due to its larger size. Gast et al. (1993) excluded atmospheric pressure as a significant factor when explaining female I. pini attraction to male pheromone or ho st odor in the field. Atmospheric pressure was considered as a possible explanation for variation in odor discrimination by adult plum curculios, Conotrachelus nenuphar (Leskey and Pro kop y 2003) . Female beetles were subjected to a dual choice still air system study over the course of thre e years . L ower pressures correspond ed to lower response for two of the three years ( P = 0.03, 0.05, and 0.31). N o relationship was found between atmospheric pressure and feeding behavior in field cages. The authors discussed the possibility that pressure c hanges over an unknown time frame prior to testing may have influenced results. Atmospheric pressure changes over the course of 12 hours affected the locomotory activity of Diabrotica speciosa males in response to female pheromone extracts in a Y tube olf actometer (df=2, 2 =13.38, p<0.001), with significantly lower response when pressure decreased (Pellegrino et al. 2013) . Steinberg et al. ( 1992 ) found that the response of Cotesia glomerata to volatile infochemicals was higher when atmospheric pressure increased over the course of the trial . V ariation in odor discrimination by Mallophora ruficanda in olfactometer studies was partly explained by an interaction between atmospheric pressure change and initial pressure (Crespo and Castello, 2012). Host orientation occurred if pressure decrease was less than 0.85 mbar / 90 min or if pressure rose. A steeper decrease than 0.85 mbar at an initial pressure of less
18 than 1014.8 resulted in larvae orienting at rand om. The authors suggested that pressure may influence larval physiology that increases fitnes s when detrimental weather conditions arise, but it is not clear how postponing host searching is advantageous. It is also interesting to note that M. ruficanda larvae live underground. In addition to olfaction, feeding behavior in response to pressure has been an area of interest . Simuliid flies fed more actively during periods where pressure was low or rapidly falling (Wellington 1944) . In an experimental pressure chamber, Breuner et al. (2013) evaluated the effects of pressure on metabolic rate, stress physiology , and food intake of white crowned sparrow ( Zonotrichia leucophrys ). There was no clear relationship between pressure and metabolic rate or stress physiology, but after 3 hours of a steady drop in pressure (20 mbar), foraging increas ed significantly ( P <0.05) . Effects on Courship and Mating Ankney (1984) found that lower atmospheric pressure was associated with reduced mating activity in Drosophila pseudoobscura in the laboratory (R 2 = 0.29, P < 0.025. Austin et al. (2014) conducted a direct test on the effects of pressure on male courtship behavior and female receptivity of several strains of Drosophila melanogaster in no choice mating assays. Flies held at a 943 mbar base pressure were subjected to extreme pressure change ( 33 35 mbar/30 minutes) which represented low or high pressure weather fronts. Individual strain (x 2 = 33.8, df = 5, 582, P <0.001), experimental pressure (x 2 = 8.8, df = 2, 587, P <0.05), and an interaction between individual s train and experimental pressure (x 2 = 42.6, df = 12,570, P <0.001) were found to be significant predictors of courtship , but not copulation (x 2 = 5.6, df = 2, 224, P <0.061). Due to a high degree of variation in male responses, the authors suggested that t here is no single, universal response in mating behavior to atmospheric pressure changes and
19 that the relationship between strain or genetic differences within a single interbreeding population and pressure is complex. Pellegrino et al. (2013) showed that changes in atmospheric pressure affected sexual behavior in three unrelated insects: Diabrotica speciosa , the cucurbit beetle, Pseudaletia unipuncta , the armyworm moth, and Macrosiphum euphorbiae , the potato aphid. Decreasing pressure decreased male preco pulatory behavior in cucurbit beetles and also decreased female calling in armyworm moths. As pressure decreased, time spent on male precopulatory behavior decreased in the cucurbit beetle, and time spent on female calling also decreased with decreasing p ressure in the armyworm moth. Mating rate in P. unipuncta and both calling and mating rate in M. euphorbiae increased when pressure was stable. Male Diabrotica speciosa spent significantly less time performing precopulatory behavior when interacting with females under falling pressure conditions (5.2 Â± 1.2 min. vs. 16.2 Â± 3.1 min, p = 0.001). In a pressure chamber, Pseudaletia unipuncta and M . euphorbiae were subjected to stable and changing (Â±5 mbar / 6 h) conditions. Signi ficantly f ewer P . unipuncta female s called (exposed pheromone glands) over a two hour time frame when pressure was lowered compared to the stable or increasing pressure conditions. Mating in P. unipuncta was also affected ; a significantly higher proportion of pairs mated under stable conditions than either of the two fluctuating conditions . Stable pressure also increased M . euphorbiae mating success (df = 2, x 2 = 43.75, P <0.001) and frequency of female calling behavior (df = 1, x 2 = 36.25, P < 0.01). Pete rson (1972) studied the relationship between various meteorological factors and spawning activity of rainbow trout in Wyoming . Atmospheric pressure was more strongly correlated with spawning activity than water temperature or cloud cover . Over a three year study period, spikes in spawning trap catch numbers correlated with sudden drops in atmospheric
20 pressure. Fish are an interesting study subject because fishing barometers some times include dial ( http://www.basspro.com/TRAC Fishing Barometer/product/10200632/ ), suggesting that there is a very strong relationship between outside pressure an d fish activity. Yet , pressure under water is probably affected more by the depth at which the organism is swimming than it is by changes above the water (Eddy and Handy 2012) . Mate searching behavior of Aphidius nigripes, an aphid parasitoid was associa ted with atmospheric pressure change over the 24 hours prior to testing (Marchand and Mcneil 2000) . C ompared to stable conditions, there was a significant decrease in the percentage of males entering the pheromone plume under large variations in pr essure (x 2 = 0.1573, df = 1, P = 0.0006). The percentage of males completing upwind flight and landing on the pheromone source was significantly smaller under a slight pressure increase (36.7%) than a slight decrease (81.22%) (x 2 = 11.1, df = 1, P = 0.0009) . Decreasing pressure may indicate reduced life expectancy if it precede s poor conditions , which could cause a change in male behavior . However, a difference in female pheromone production cannot be discounted because synthetic pheromones were not availabl e . Effects on Oviposition, Eclosion, and O ther B ehaviors Roitberg et al. (1993) suggested that atmospheric pressure change can serve as a reliable indicator of lower life expectancy for small organisms because falling pressures are associated with approac hing storms. Leptopilina heterotoma were subjected to either steady pressure or decreasing pressure and then released into a high quality habitat (containing only unparasitized fruit fly larvae) or a poor quality habitat (containing only parasitized fruit fly larvae).There was no difference in host searching behavior and superparasitism frequency in the two different habitats for the parasitoids that were exposed to steady press ure. However, wasps subjected to
21 decreasing pressure conditions searched longer p P < 0.02) and superparasitized more often ( P < 0.00014) in the poor quality habitat than in the high quality habitat. Correlational studies have been conducted on air pressur e influence on oviposition behavior with Pieris rapae and Eudonia sabulosela . Cowley (1987) evaluated the effects of meteorological variables on ovipositing female E. sabulosella in field cages. More eggs were laid under conditions of light rain ( P <0.001 ) , which corresponded with a high, falling barometer. Stephen and Bird (1949) subjected ovipositing Pieris rapae females to low or high pressures in sealed jars with access to a cabbage leaf. T he number of eggs laid per female in low pressure conditions wer e significantly fewer than number of eggs laid pre fem ale in high pressure conditions . Pieris rapae was also used in Koyler and Palmer (1968) study on the effects of atmospheric pressure, both ambient and experimental, on eclosion. Pupae were subjected t o alternating cycles of low (926.25 983.25 mbar) and high (1023.2 1059.9 mbar) pressure. Pressure fluctuation did not appear to affect eclosion, and no correlational evidence was evident after analyzing previous laboratory eclosions . Southwick and Moritz (1987) analyzed the effects of meteorological factors on the defensive behavior of A. mellifera . All of the weather variables were highly intercorrelated. The strongest, single factor predictor of defensive behavior was temperature (r 2 = 0.29), but wind sp eed, solar radiation, relative humidity, and atmospheric pressure were also influential. Multiple regression analysis which incorporated all of the weather variables was able to explain 92% of the variation of defensive behavior in closely related hives. E ffects on Overall Locomot o r Activity Some studies on the effects of atmospheric pressure on insect behavior consider overall activity rather than a specific behavior. Witter et al. (2012) developed weather based indices to monitor mosquito and fly harassment of caribou in northern Canada. In building a multinomial
22 logistic regression model, atmospheric pressure, along with temperature, wind speed, lig ht, and relative humidity were found to be significant factors in determining activity level based on trap catches. For mosquitos, the relationship between atmospheric pressure values and activity levels was positive and significant ( P < 0.05). The model s uggested a negative association between pressure levels and black fly activity, but the relationship was not significant ( P > 0.05). Freeden and Mason (1991) were also interested in analyzing meteorological factors that influenced activity of Simulium lugg eri . The most important limiting factors were wind speeds greater than 15 km / h and temperature less than 20 C, and outside these thresholds, humidity, light intensity, and atmospheric pressure became influential. In contrast to the Freeden and Mason (199 1) black fly data, S. luggeri activity was positively correlated with atmospheric pressure, but only when temperatures exceeded 20 C. It was not possible to establish clear thresholds for most of the meteorological parameters because their varied interacti ons tended to confound apparent trends. A study on Encarsia formosa activity on tomato leaflets showed that many of the parasitoids became inactive when pressure levels dropped but were active at stable or increasing pressures (Roermund and Lenteren, 1995 ) . The significant effect of atmospheric pressure (p<0.05) became apparent only when the time period 12 hours prior to observations were considered. A study on the sn ow surface activity of Collembo la demonstrated that gradual changes (2.2 mb ar / h) in pres sure in either direction were positively correlated with snow surface activity of Isotoma hiemalis (Zettel 1984) . Sudden, extreme pressure changes (i.e. 50 mbar), did not have stimulatory effects. For all snow surface activity, temperature was still the limiting factor. Summary of Effects of Atmospheric Pressure on Animal Behavior Because atmospheric pressure is a dynamic variable, there are many ways to approach studies considering the effects of p ressure on animal behavior and there is currently no standard
23 in the literature. Studies describing correlations between behavioral change and pressure or pressure trends have involved over 20 species of arthropods with differing results. When considering pressure values only, the strongest relationship was the negative association between pressure and bat activity (Paige, 1995). In contrast, low pressure values were correlated wi th reduced overall locomoto r y activity and fewer matings in D. pseudoobscura ( Ankney , 1984) and reduced black fly activity (Freeden and Mason, 1991) . C. nenuphar had a lower olfactory response rate during low pressure conditions (Leskey and Prokop y 2003) , but pressure did not affect olfaction in I. pini (Gast et al. 1993) . Additionally, there was no relationship between pressure and oviposition in P. rapae (Stephen and Bird, 1949). Multiple studies considered pressure change rather than pressure values at the time of the trials, also with mixed results. A d ecreasing pressure trend resulted in reduced olfactory response in C. glomerata (Steinberg et al. 1992) and D. speciosa (Pellegrino et al. 2013) and less movement in E. formosa (Roermund and Lenteren, 1995). Behavior (flight and olfactory response) was inhibited in A. nigripes by large pressure changes in either direction. In contrast, E. sabulosella spent more time flying and ovipositing under a falling barometer (Cowley 1987). Crespo and Castello (2012) found an interaction between initial pressure and pressure change when lo oking at host orientation by M. ruficanda . If pressure decreases were sharp and occurred at a low starting pressure, larvae oriented at random. Because of the complexities of these analyses and differences in approach, it is difficult to compare the result s of the studies. Direct studies using experimental chambers have been conducted on over 20 insect species, also with a wide variety of approaches. Rapid increases in pressure reduced flight activity of T. pretiosum and T. evanescens (Fournier et al. 2005 ). However, flight activity in over nine species of Diptera was inhibited by pressure drops (Wellington, 1944). Both M. euphorbiae
24 and P. unipuncta called and mated more often under steady pressure conditions (Pellegrino et al. 2013) . Steady conditions als o yielded highest olfactometer responses in I. pini and S. multistriatus (Lanier and Burns 1978) . Lowering pressure resulted in higher rates of feeding for the white crowned sparrow ( Breuner et al . 2013) and higher rates of oviposition by L. heterotoma (Roitberg et al. 1993). Based on the variations in experimental a pproach, differences in study species, and the dynamic nature of pressure as an explanatory variable, it is very difficult to identify patterns in starting p ressure, and pressure change over time. Potential Biological Mechanisms for Sensing Atmospheric Pressure There is very little literature that describes a proximate analysis of the effects of pressure on insects; most focuses on the question from an ultima te perspective. Wellington (1944) hypothesized that because sound is also air movement, any auditory organ is probably also able to sense pressure changes. While this appears to be true for the inner ear of mammals (Funakubo et al. 2010), similar physiolog ical studies on insect auditory organs are not available. However, Tichy and Kallina (2010) showed that antennal hygroreceptors respond to continuous changes in humidity and air pressure. They also demonstrated variation in response; Caurausius morosus was more sensitive to air pressure changes than Periplaneta americana .
25 CHAPTER 2 EFFECTS OF ATMOSPHERIC PRESSURE ON DIAPHORINA CITRI BEHAVIOR Diaphorina citri Biology Diaphorina citri , the Asian citrus psyllid , has received significant attention from the scientific community following its invasion into the Caribbean and southeastern United States in 1998 (Halbert and Manjunath, 2004) and the subsequent discovery of citrus greening disease in Florida in 2005 ( Bo vÃ© , 2006). The offici al name for citrus greening is H uanglongbing, and is probably caused by three phloem limited bacterial agents, Candidatus Liberibacter asiaticus, Candidatus Liberibacter africanus, or Candidatus Liberibacter americanus (Wang and Trived i, the bacteria (Sechler et al. 2008). Because of its status as a disease vector, Diaphorina citri is regarded as a serious pest and a threat to the citrus indus try. The disease is very harsh on citrus, causing a distinct leaf mottling, stunted shoot growth, fruit of an unmarketable quality, and eventual tree deat h (Gottwald et al., 2007) . In Asia, D. citri and Huanglongbing have been a documented problem since the early 20 th century (Husain and Nath, 1927). Citrus greening has been very difficult to manage. The time between infection and the emergence of visible symptoms could be as long as 6 years (Shen et al. 2013), meaning that seemingly healthy trees could b e serving as inoculum sources for more psyllids. New and surprising information on psyllid transmission mechanisms continues to emerge, including information on trans ovarial and sexual transmission, as well as data on disease acquisition by nymphs (Pelz S telinski et al. 2010, Mann et al. 2011). Although D. citri can feed on and infect all known commercial and privately grown citrus cultivars as well as other non citrus Rutaceous plants (Wang and Trivedi, 2013), the Asian citrus psyllid likely evolved on Be rgera (Rutaceae) on the Indian subcontinent (Beattie, 2008).
26 The Florida citrus industry has been hit very hard by the greening epidemic. The total estimated cost of HLB to the citrus industry is about $4.5 billion, including employment impacts (Hodges an d Spreen 2011). Since H uanglongbing was first documented in Florida, over 200,000 acres have gone out of production (USDA Census of Agriculture 2012, USDA Commercial Citrus Inventory Preliminary Report), resulting in the lowest bearing acreage since the e arly well as the intense winter in 2010 probably also contributed to the decline, but the primary driver is still considered citrus greening spread. The disease i s very expensive to manage because growers must adjust to significantly higher insecticide costs and application labor, more scout employment, and problems from other diseases and pests that tend to target weakened trees. The growers who remain in business have experienced a 16% revenue drop for the fruit they continue to produce, despite higher fruit market value . Although growers have been incorporating cultural practices such as using clean nursery stock and applying nutritional supplements to mitigate d isease severity, they continue to rely heavily on vector management. Diaphorina citri is a phloem feeding pest in the family Liviidae. There are at least 7 psyllid species documented as citrus pests. Of these, two are disease vectors, Diaphorina citri and Trioza erytrae , which has not been known to occur outside the African continent (Burckhardt, 1994). Diaphorina citri is documented to have up to 16 generations per year and has a wide geographic distribution (Mead, 1977). Although its range is restricted t o tropical and subtropical areas, both adults and nymphs can survive low temperature extremes for short durations , down to 6Â° C., and eggs can remain viable after exposure to still lower temperatures (Hall et al. 2010). D. citri is also distributed in area s with high relative humidity as well as arid regions (Mead, 1977).
27 After eggs are laid on new flush, the first instar nymphs hatch between 2.6 and 7.7 days. It takes between 9.4 to 35.8 days to develop through the 5 immature stages (Nava et al. 2007). Af ter eclosion into adulthood, psyllids are usually sexually mature in 2 3 days. Multiple matings allow females to lay more eggs, and a single female can lay up to 800 eggs (Grafton Cardwell et al. 2103). Vibrational Communication Diaphorina citri use subst rate borne vibrational signals for mate location. Psyllids move their wings up and down rapidly to pro duce vibrations (Tishechkin 2006 ). D. citri produce simple, low amplitude calls between 170 250 Hz, lasting between 140 to 700 ms. There is minimal sexual dimorphism in this species and there is muc h overlap in signal spectra as well as temporal properties of calls, so distinguishing by sex is not always possible (Wenninger et al. 2009 a ). Males call at relatively even intervals, and receptive females respon d to male calls within 0.3 1.2 seconds. This duetting behavior continues as the male moves along the plant, using the vibrational cue s for orientation (Cocroft 2005 ) while the female remains stationary. When the male reaches the female, possibly with the aid of visual or olfactory cues, the pair mate, remaining in copula for 15 98 minutes (Wenninger and Hall 2007). Neither the presence of odors from females or from host plants stimulated males to call more frequently in an olfactometer, but latency to init iate calling was shorter in the absence of odors. However, calling rate did appear to increase with male age (Wenninger et al. 2009 a ). Vibrational communication may be important in several ways. In addition to mate location, psyllids may call to locate gro ups of conspecifics, but this has not been studied for D . citri . Females occasional call spontaneously, or respond to other females, but it is unclear why. Because they did not move, it is possible that background noise or the other female calls, being vir tually indistinguishable from male calls, were misinterpreted.
28 Because vibrational communication plays an important role in D. citri mate location, it has become the subject of applied research, with possibilities in trapping or mate disruption. Rohde et al. (2013) synthesized artificial calls which elicited responses from male and female psyllids when the signal passed through the host plant . No single pitch low frequency signal was as successful as signals that included multiple harmonics of their wingbe at frequency. Both sexes responded well to multiple harmonics of 200 Hz frequency chirps, up to 1400 Hz. Electropiezo buzzers programmed to emit the synthetic signals were clamped directly to plants in experimental arenas, and stimulated males to engage in regular duetting behavior (Rohde et al. 2013). This prompted further research for the development of low cost, automated systems that could detect and emit D. citri calls for possible use in trapping (Mankin et al. 2013). Many groups within the class Ins ecta use some form of acoustic communication. There is tremendous diversity in mechanisms, signal characteristics, and the presumed functions of calls. Unlike airborne sounds produced by some groups that tend to be high pitched, vibrational signals that pa ss through a solid or semi solid substrate (i.e. plant matter, soil) tend to be low frequency sounds (Bailey, 1991). Orders known to use substrate borne signals include Coleoptera, Psocoptera, Isoptera, Hemiptera, Orthoptera, Hymenoptera, Plecoptera, and N europtera. Mechanisms include striking the substrate with the head or legs (percussion), unspecialized vibration (tremulation), using specialized structures (stridulation), or any combination (Claridge 2006). Within the order Hemiptera, many families use vibrational communication under different, sometimes very unique, behavioral contexts. Membracids, some of which live in social groups, use substrate borne signals for mate location, recruitment to new feeding sites, and for alarm notification. For example , Umbonia crassicornis engage in parent offspring interactions
29 where nymphs under attack by a parasitic wasp or predator vibrate in chorus, which prompts their mother to locate and attempt to physically ward off the threat (Cocroft, 2001). Within Membracid ae, many species have also been observed to perform courtship vibratory duetting, sometimes using a call unique to this behavior, and using calls with different properties for other social contexts ( Cocroft and McNett 2006). Although substrate transmitted courtship signaling appear to be ubiquitous or nearly so among studied Auchenorrhynchans, this does not appear to be the case for groups within Sternorrhyncha. Scale insects are not known to engage in vibrational communication, while aphids have been shown to use vibration as an alarm indicator (Clegg and Barlow, 1982), but not for locating mates. While male calls are prevalent among whiteflies (Kan miya , 1996), female replies are not as common (6 out of 32 studied species), though this may be because such b ehavior is rarely the subject of research within Aleyrodidae ( Kanmiya 2006). However reciprocal, species specific mate signaling is common in Psylloidea worldwide. Many species of psyllids engage in duetting behavior much like that observed in D . citri , th ough call specificity by sex is often more pronounced in other genera. Vibrational behavior appears to be subject to sexual selection and is a driver of sympatric speciation (Percy et al. 2006). Terrestrial Hemiptera are able to sense substrate borne infor mation with leg receptors, including the subg enual organ on the tibia and ch o r dotonal organs on the femur, tibia, and tarsi ( et al. 2006). Localization of the vibration source is difficult due to substrate frequency distortion and signal degradation b y distance (Cocroft et al. 2006). Tishechkin (2013) showed that rain drops, wind, and other mechanical activities that induced higher frequency vibrations create conditions where substrate borne communication becomes distorted and indistinguishable, and su ggested that this is the reason why small Hemipterans emit signals during gaps between wind spells.
30 Summary of Economic and Evolutionary Context of Experiment Small organisms such as insects are vulnerable to harsh weather conditions and are known to use multiple types of cues to res pond to meteorological changes (Cowley 1987 , Southwick and Moritz 1987 , Fredeen and Mason 1991) . Atmospheric pressure is a physical factor that can serve as a tool for forecasting local weather conditions 12 24 hours into the future because of the association b etween pressure shifts and other changing weather variables (Burch, 2013). When colder, heavier air comes in contact with lighter, warmer air, the often turbulent collisions are accompanied by precipitation and high winds. A barometer can help predict such an occurrence because decreasing pressure often precedes an influx of denser air which can result in a storm (Williams, 1997). The ability of insects or other animals to respond behaviorally to approaching storms has interested researchers for several cen turies (Noverre Press, 1834). Numerous correlative studies describe relationships between atmospheric pressure and the behavior of insects or other animals in the field or laboratory, including feeding behavior (Leskey and Pro k o p y 2003) , flight initiation (Fournier et al. 2005) , and olfactory response (Steinberg et al. 1992) . Because atmospheric pressure is a dynamic variable, there are potentially many approaches to evaluating its effect s on behavior. Some studies have focus ed on immediate pressure values at the time of the study or a short term pressure change (Kolyer and Palmer 1968, Paige 1995, Witter et al . 2012) , and others have considered longer term pressure shifts (P ellegrino et al. 2013) . Due to the variation in study organisms, target behaviors, and approaches to analysis, the effects of atmospheric pressure on insect behavior remain only partly characterized, and further study is required for better understandin g. The Asian citrus psyllid ( Diaphorina citri Kuwayama; Hemiptera: Liviidae) has recently received significant attention from the scientific community following its invasion into the
31 Caribbean and southeastern United States in 1998 (Halbert and Manjunath, 2004) and the subsequent discovery of citrus greening disease in Florida in 2005 ( Bov Ã©, 2006) . As the primary vector of citrus greening disease, D . citri has become subject of considerable behavioral research (Grafton Cardwell et al. 2013). Anecdotal accou nts by researchers who frequently work with D. citri suggest that unfavorable weather conditions may affect behavior in laboratory settings, but formal studies on the effects of weather on behavior are lacking . Atmospheric pressure variation has explained erratic laboratory behavior in other insects (Ankney 1984, Crespo and Castelo 2012) . Here, we test for correlations between atmospheric pressure and pressure changes and D. citri behavioral responsiveness to vibrational mating signals and light . During courtship, m ate seeking D. citri produce vibrational signals in a call and reply system (duetting) during which males search for females located on the host plant or other substrate (Wenninger et al. 2009 a ). Because the acoustic duetting and searching behavior is important for successful psyllid mating, there is much potential for its application in pest management. For instance, we have been testing a recently developed synthetic mi mic of female vibrational signals in numerous bioassays (Rohde et al. 2013 ). The mimic consists of a piezoelectric buzzer powered by an open source microc ontroller platform (Arduino Uno) and could be used as a low cost tool to monitor or trap males searchi ng for females on branches within citrus tree canopies. In addition to acoustic behavior , vision is an important sensory modality for D. citri , especially for host plant orientation (Wenninger et al. 2009b). Mangan and Chapa (2013) showed that the additio n of LED lights to sticky traps greatly increased capture rates . We have been evaluating the propensity of psyllids to fly or jump towards a light stimulus in a two choice
32 vertical arena. Both acoustics and vision are important sensory modalities, and more research in these areas are needed to improve trapping methods. Observations that variability in D. citri responsiveness frequently occurred when indoor laboratory bioassays of acoustic mating behavior or responses to light were conducted during periods o f inclement outdoor weather suggested that psyllids may be responding to atmospheric pressure changes. To explore this possibility, we conducted analyses on the correlation between atmospheric pressure trends and behavioral responsiveness. It was our goal in particular to assess behavioral changes during periods when atmospheric pressure or pressure change values had deviated from values expected for normal diurnal pressure fluctuation. Pressure and pressure/change values in normal ranges would not be expec ted to elicit significant changes in behavior, but values outside the normal ranges might serve as cues that drive behavioral responses if they result in increased survival or reproductive fitness . Materials and Methods Atmospheric Pressure Pressure data w ere obtained from a weather station (model WRL 25. Texas Weather Instruments, East Pilot Point, TX) located 1.32 km from the United States Department of Agriculture, Agricultural Research Service, Center for Medical, Agricultural, and Veterinary Entomology (CMAVE) laboratory where mate seeking and light response bioassays were conducted (see below). The pressure at the beginning of each trial, the changes in pressure from 3, 6, 9, 12, 24, 36, and 48 h before the trial, and the means and standard deviations (SD) for each bioassay type . To check the assumption that pressure change indoors is comparable to pressure change outdoors in the same general location, pressure data logged by the weather station were compared with logged indoors by a handheld logging d evice (model B1100 1, Gulf Coast Data Concepts, Waveland MS). Two 72 h data sets logged at 60 s intervals were obtained at different
33 times from the anechoic chamber and the greenhouse where the psyllids were reared. The measurements were compared by regres sion analysis in Excel (Microsoft Office 2013) with data obtained from the same time frame as from the weather station. Calling and Searching Behavior Study Behavioral observations of D. citri calling and searching behavior were conducted on 70 d over a 400 d period, and a total of 131 individuals were assayed. The psyllids used in these studies were obtained from a colony maintained in the CMAVE greenhouse. Late instar nymphs were isolated and placed onto individual Citrus sp. seedlings, as described in Paris et al. (2013). After eclosion, adults were sexed, and unmated males between 4 14 days post eclosion were tested. Bioassays were performed inside a vibration shielded anechoic chamber (Mankin et al. 1996), between 8:00 am and 6:00 pm. Psyllids were pl aced individually on a leaf of a potted, 30 cm height Murraya exotica (L.) Jack (Sapindales: Rutaceae) plant. A psyllid calling buzzer system similar to that described in Mankin et al. (2013) was used to emit recorded or synthetic mimics of female acoustic responses psyllid. A microcontroller platform (Arduino Uno, Arduino Inc., Italy) and a small amplifier circuit board were connected together, forming a single unit (6 by 5.4 by 2 cm). This controlled the sound producing piezoelectric buzzer (9S3164, Taiyo Yuden, Tokyo, Japan) which was attached directly to the plant using alligator and/or binder clips. The piezo output produced multiple harmonics between 0.2 and 2 kHz, which have previously been shown to elicit D. citri responses (Rohde et al 2012). Signal s were triggered manually using software written for the microcontroller (Bug Phone 2.2 Â©2013). Trial length and signal control varied, but generally the trials were approximately 30 min in duration and signals were emitted immediately following a male cal l, or between 1 and 5 min in absence of calling. Light was supplied by three 60 W floodlamps ca. 1 m above the plant. To reduce
34 background noise, the chamber door was closed after the psyllid was placed on the plant, and visual observations took place on a monitor outside of the anechoic chamber from footage captured by a video camera (model HDR SR1, Sony Corp., New York, NY) which was focused on the plant. An accelerometer (model 4371, BrÃ¼el & KjÃ¦r [B&K], Naerum, Denmark) attached to the base of the plant by an alligator clip system (Mankin et al. 2004) was used to continuously monitor vibrations. Vibrational signals were amplified 30dB with a charge amplifier (model NEXUS 2692, B&K or similar), band pass filtered between 500 HZ and 10kHz, recorded at a rat e of 44.1 kHz (model HD P2, TASCAM, TEAC America, United States), and the signals were sent directly to a computer located outside the anechoic chamber where spectrograms and periodograms were constructed using Raven 1.4 software (Charif et al. 2008). Two aspects of the mating/duetting behavior, calling activity and searching activity, were recorded and categorized into discrete classes. For calling activity, individuals that were unresponsive or called at a mean rate of < 3 calls min 1 were scored as 0, w hereas individuals 1 over the course of the trial were scored as 1. Searching activity was scored as follows: no movement or a mean distance of less than 9 mm covered per min, 0; a mean distance of 9 m m or more (approximately three or more psyllid body lengths) covered per min, 1. For each test of correlation between calling or searching activity and pressure or pressure change category, the trials were subdivided into three categories. Trials that occu rred when the pressure or pressure change values were within one standard deviation of the mean value over the time period of all bioassays were categorized as occurring under normal conditions. Those that occurred when the pressure or pressure change valu es fell below the mean 1 SD were categorized as occurring under reduced pressure/pressure change conditions, and trials when the
35 pressure or pressure change values exceeded one standard deviation above the mean were categorized as occurring under elevated conditions. Contingency analysis (Pearson 2 ) was performed for each correlation using JMP (SAS Institute 2013) to test the hypothesis that the rate of calling or searching activity was the same for each pressure or pressure change condition. Light Respon se Study The light response arena consisted of a black PVC pipe (30.5 x 17 cm) with a release chamber (33 ml glass tube) at its base. At the top of the PVC tube was a polystyrene petri dish (150 x 15 mm) coated with TanglefootÂ® applied using an aerosol can . Two choice tests were conducted using different combinations of light filters (Yellow 4530, Medium Yellow #10, Green 89, Moss Green 88, Light Green 88, Polarized, UV blocking) placed directly on the petri tray. Each replicate consisted of a mixed cohort of 20 40 adults that were dark adapted for 20 minutes prior to the experimental assay, for a total of 50 replicates. A total of 1423 D. citri (49% male) were tested during the study, with a mean of 28.48 per replicate. Diaphorina citri were placed in the r elease vial and allowed 45 min to walk or fly towards the filtered light targets at the top of the arena. At the end of the trial, responsive and unresponsive individuals were counted and sexed. Results In this study, changes in D. citri bioassay responsi veness were correlated significantly with rising or falling pressure conditions that exceeded the standard deviation (SD) of the changes observed in 3 48 h time periods preceding the bioassays. For this reason, it is worthwhile to first consider the pressu re changes in an ecological context.
36 Atmospheric Pressure Measurements The weather station was near enough to the bioassay arenas that the pressure changed at the same rate as a logger placed in both the anechoic chamber and greenhouse (Fig. 1C), though in itial values differed slightly (Fig. 1A B). The 72 h pressure changes in Fig. 1A B represent natural diurnal atmospheric pressure fluctuation during the Florida summer and we did not expect this type of variation to affect behavior. Therefore, we estimated the natural variability during the period of bioassays by identifying trials where the pressure and pressure change values exceeded or fell below one standard deviation from the mean (Table 1). Fig ure 2 1 . Pressure values from indoor logger in A) anechoic chamber (dash dotted line); B) greenhouse (dashed line) compared with weather station output for two 72 h periods (solid line); and C) simple regression showing relationship between pressure values obtained from the weather station and indoor pres sure logger (y=0.9955x+1.144, x=weather station output, y=indoor pressure logger output, residual mean square error=0.0561).
37 Table 2 1. Means and standard deviations of absolute pressure at the time of trial and changes in pressure from 3 48 h prior t o trial (time lag) . Time lag (h) Calling/Searching (n=131) Light Response (n=50) Mean (mbar) SD (mbar) Mean (mbar) SD (mbar) 0 1016.58 4.57 1013.85 2.19 3 1.0 1.15 0.603 1.16 6 0.5868 1.999 0.101 2.04 9 0.16 1.84 0.169 2.34 12 0.1318 1.94 0.345 2.64 24 0.2 3.74 1.341 3.59 36 0.14 5.12 0.87 4.03 48 0.14 5.76 0.93 4.47 Calling, Searching, and Light Attraction Bioassays The percentages of D. citri performing calling, searching, or light attraction responses under conditions of reduced, normal, or elevated pressure change are shown in Fig. 2. Contingency analyses (Table 2) revealed significant differences in percentages of calling and searching beha viors of males exposed to falling, steady, and rising barometric conditions 24 h before the calling and searching behavior trials. There also were significant differences in percentages of calling males exposed to falling, steady, and rising barometric con ditions 12 h before trials. Finally, there were significant differences in the percentages of males searching when the barometer low, steady, or high at the time of trials. Analysis of variance (Table 3) revealed significant differences in percentages of l ight attraction behaviors of psyllids exposed to falling, steady and rising barometric conditions 9, 12, 24, and 48 h before the trials.
38 Table 2 2 . Contingency analysis of effects on calling and searching behavior of males exposed to low/falling , steady , or high/rising barometric conditions measured at different times (h) before trials (time lag) 2 tests for significant differences among response percentages for each condition, and P indicates the probability of obtaining by chance alone a va lue of 2 greater than computed. Time lag (h) No. trials in condition category Calling Searching Low/Falling Steady High/Rising 2 P 2 P 0 20 92 19 4.696 0.09 8.92 0.01* 3 19 90 22 0.327 0.85 0.137 0.93 6 25 87 19 1.433 0.49 1.331 0.51 9 24 86 21 1.507 0.47 1.805 0.41 12 12 103 16 7.154 0.03* 3.820 0.15 24 19 87 25 10.520 0.005* 7.144 0.03* 36 20 93 18 1.928 0.38 4.317 0.12 48 18 93 20 3.823 0.15 3.770 0.15 Values of P < 0.05 are marked by asterisk.
39 Table 2 3 . Analysis of variance of effects on light attraction of males exposed to low/falling , steady , or high/rising barometric conditions measured at different time (h) before trials. Time lag (h) No. trials in condition category Light attraction Low/Falling Steady High/Rising F P 0 8 32 10 0.526 0.59 3 8 32 10 01.54 0.22 6 10 30 10 2.68 0.08 9 10 34 6 5.22 0.009* 12 7 35 8 4.24 0.02* 24 9 32 9 3.98 0.03* 36 13 28 9 2.24 0.12 48 12 29 10 3.31 0.045 a Values of P < 0.05 are marked by asterisk. a Although the F test showed significant differences between groups at the 48 Hour time interval ,
40 Fig ure 2 2 . Mean percentages of responsive psyllids in acoustic and light attraction assay s. Percentages of psyllids performing A) calling, B) searching, or C) light attraction responses under conditions of a steady (solid line), falling ( dashed line), or rising (dotted line) barometer 3 48 h before trial. Circles indicate responses to steady (solid), low (dashed), or high (dotted) pressure at time of trial. Dash dotted boxes designate contingency tests where calling, searching, or light responses of psyllids exposed to steady (solid line), falling (dashed line), or rising (dotted line) barometric conditions had significantly different values. A binomial test of equal proportions with a for multiple comparisons was conducted on the distributions that were found to be significant by the 2 was conducted on the distributions found to be significant by the F test. Different letters designate means for each condition that were significantly different from each other.
41 CHAPTER 3 DISCUSSION Knowledge gained about environmental factors affecting behavior of an important pest such as D . citri has relevance in both fundamental and applied contexts. In this study , we show that large ( >1 SD) changes in barometric pressure levels during the 24 h intervals preceding trials are associated with significa nt differences in the percentages of D. citri responding in acoustic, searching, and light attraction bioassays. From a fundamental perspective, th is result is consistent with a hypothesis that there may be survival benefits to focus ing energy on flight ra ther than mating when large pressure changes occur over a 24 h period. Also, because atmospheric pressure measurements are easily measured or retrieved from reliable sources, a better understanding of how pressure changes are associated with trends in D. c itri mating and movement behaviors can help pest managers make more informed decisions about the timing of control activities. Atmospheric Pressure Changes and Psyllid Mating Behavior The observed reduction in D. citri mating activity after 24 h periods wh en pressure rose or fell is consistent with the hypothesis offered by Pellegrino et al. (2013) that lowered activity might reduce the risk to survival from exposure to strong winds and rain. When compared with steady pressure conditions, a large increase i n pressure was strongly associated with fewer male psyllids responding to synthetic acoustic cues. Similarly, large decreases in pressure also were associated with lower proportions of response. Marchand and McNeil (2000) found a similar pattern when asses sing effects of 24 hour pressure changes on attraction of male Aphidius nigripes to female volatiles. In their study, steady pressure conditions resulted in the highest percentage of responsive males, while large increases in pressure resulted in the lowest. The mating system for A. nigripes is pheromone mediated while that of D. citri relies on vibrational
42 cues, but engagement levels for the respective sexual behaviors was affected by pressure in a similar manner for both species. It is possible that any deviation from steady pressure conditions results in reduction in behavior for some insect species, but directionality may pl ay a significant role for others (Lanier and Burns 1977, Pellegrino et al. 2013). Alternatively, it is possible that in natural settings, fewer psyllids engage in vibrational communication when physical factors inhibit its effectiveness. Cocroft and Rodr iguez (2005) characterized plant vibrations induced by wind as low frequency patterns, while the frequencies produced by rain are much more variable and depend on the position of the droplet as it impacts the leaf. In field trials, Tishechkin (2013) observ ed that small Hemipterans emit signals during gaps between wind spells, perhaps because wind or other mechanical activities that induced interfering vibrations create conditions where substrate borne communication becomes distorted and indistinguishable. B ecause the pressure changes that psyllids experience in the laboratory are not followed by physical factors such a precipitation, the reduced levels of mating behavior associated with such pressure changes may be an evolved rather than a learned response. Field studies on the vibrational communication behavior of D. citri in citrus groves have recently began, and it will take time to gain information on wild psyllids and their calling patterns. We propose a focus on the environmental variables that affect t he vibration aided mating behavior of D. citri . Insect acoustics, while previously an understudied area of biology, is now a more active area of research, and the exploitation of vibrational signals hold promise for new methods in pest management (Eriksson et al. 2012). Understanding when psyllids are m ost willing to engage in vibrational communication is important since field monitoring of psyllids using vibrational techniques is currently a research focus (Mankin et al. 2013). Consequently, it was of interest to
43 us that the highest rate of response fro m male psyllids was elicited when pressure decreased significantly over a 12 hour period. This observation contrasted with the findings of Pellegrino et al. (2013), where gradual 12 hour drops in pressure suppressed mating behavior in three unrelated insec ts. It is possible that the shape of the pressure change curve influences behavior; however, we were not able to account for this in our analysis. There may be other important considerations that could affect how pressure influences behavior. For example, Crespo and Castelo (2012) found that there was interaction between magnitude of pressure change and absolute pressure when they examined effects of pressure on olfactory orientation of M. ruficauda . Atmospheric Pressure Changes and Psyllid Movement Decrea sing pressure conditions were associated with higher proportions of D. citri flying or hopping to a light source in our study. This is consistent with the findings of Wellington (1946), who showed that several species of Diptera increased flight activity d uring controlled drops in pressure. Haufe (1954) described a pressure threshold for Aedes aegypti ; whereby above the pressure threshold, flight activity was stimulated by lowering pressure, but below the threshold, the effect was achieved by raising pressu re. If such a pressure threshold exists for D. citri , it would provide an improved framework for evaluating the effects of pressure on behavior. It may also be useful to consider interactions between atmospheric humidity and pressure change because water b alance is generally a concern to small bodied organisms, especially as they expend energy on flight. Air pressure experiments with Aedes sp. in an experimental chamber showed that very high humidity inhibited activity, even if the air pressure treatment it self had previously stimulated activity (Haufe, 1964). Interestingly, one of the proposed mechanisms that insects may use to detect pressure changes was described by Tichy and Kallina (2010) as an
44 antennal hygroreceptor, which responds to continuous change s in both relative humidity and changes in air pressure. The occurrence of a 24 h period with large decreases in pressure is potentially an indicator of increasing D. citri populations in subsequent weeks. Egg laying by D. citri females is strongly depend ent on the availability of flush (e.g., Hall and Albrigo 2007), which is dependent on the age of the tree, pruning, storm damage, and environmental factors such as temperature and rainfall (Spiegel Roy and Goldschmidt 1996, Chen 1998). D. citri attraction to light was also higher for 24 h periods with large increases in pressure, though this trend was not significant. Sometimes, rising pressure is indicative of dry sunny weather with low wind levels, so this observation is consistent with previous findings that D. citri flight and hopping activity, measured by sticky trap capture rate are greatest on windless sunny afternoons (Aubert and Xia 1990). Understanding psyllid movement is of huge importance for optimizing control strategies. For example, in Florida , psyllid scouting takes place on an area wide basis and on relatively fixed time scales. Better estimation of when psyllids are most likely to move, perhaps through predictions of computer generated models, may help improve the efficiency of scouting and trapping activities. Challenges in Atmospheric Pressure Research During this study, we made the assumption that small pressure changes are unlikely to affect behavior because even in absence of significant meteorological events, pressure fluctuates in a r elatively predictable pattern semi diurnally (Harris 1954). Gaining a better understanding of the complex relationship between pressure and other factors such as wind and rain (Burch 2009) will be important for future studies. Important considerations incl ude addressing the shape of the pressure change curve and the starting and ending pressure. It should be noted here that this study was conducted in Florida, which frequently has convectional rain not associated with
45 weather fronts that are preceded by a c lear pressure drop. Nevertheless, based on the results of this study, we suggest that pressure change over time be considered a covariate in future analyses of D. citri laboratory behavioral bioassays. To confirm the results from this correlative study, we suggest a direct test on the effects of pressure on acoustic or light response bioassays of D . citri .
46 LIST OF REFERENCES Ankney, P. 1984. A note on barometric pressure and behavior in Drosophila pseudoobscura . Behav. Genet. 14: 315 317. Aubert , B ., and X . Y. Hua . 1990 . Monitoring flight activity of Diaphorina citri on Citrus and Murraya canopies , pp. 181 187 . In Proceedings, 4 th International Asia Pacific Conference on Citrus Rehabilitation , 4 19 February 1990 , Chiang Mai, Thailand . FAO UNDP . Austin, C. J., C. G. Guglielmo, and A. J. Moehring. 2014. A direct test of the effects of changing atmospheric pressure on the mating behavior of Drosophila melanogaster . Evol. Ecol. 28:535 544 Bailey, W. J. 1991. Acoustic behavior of insects: an evolution ary perspective. Chapman & Hall, London, England. Beattie, G.A.C, P. Holford, D.J. Mabberley, A.M. Haigh, R. Bayer and P. Broadbent. 2008. Origins of Citrus, huanglongbing and the Asiatic citrus psyllid, pp. 820 834. In Proceedings of the Sixth Vi e tnam Con ference on Entomology, 9 10 May 2008, HÃ N e i, Vi e t Nam. Bert, P. 1944 . Barometric Pressure. Researches in Experimental Physiology. College Book Company, Columbus, OH. BovÃ© , J . M. 2006. Huanglongbing: a destructive, newly emerging, century old disease of c itrus. J. Plant Pathol. 88:7 37 B urch, B. 2013 . Modern marine weather, 2 nd ed. Starpath Publications, Seattle, WA. B urch, B. 2009 . The b arometer h andbook : a modern look at barometers and applications of barometric pressure . Starpath Publications , Seattle, WA . Burckhardt, D . 1994. Psylloid pests of temperate and subtropical crop and ornamental plants (Hemiptera, Psylloidea): A Review. Trends in Agricultural Sciences, Entomology 2: 173 186. Breuner, C. W., R. S. Sprague, S. H. Patterson, and H. A. Woods. 2013 . Environment, behavior and physiology: do birds use barometric pressure to predict storms? J. Exp. Biol. 216: 1982 90. Chadwick, L. E., and C. M. Williams. 1949. The effects of atmospheric pressure and composition on the flight of Drosophila. Biol. Bull. 97: 115 137. Claridge, M. 2006 . Insect sounds and communication an introduction , pp. 3 1 0. In S. Drosopoulos and M. F. Claridge, Insect sounds and communication: physiology, behavior, ecology, and evolution. Taylor & Francis, Boca Raton, FL.
47 C legg, J. M., and Barlow, C. A. 1982 . Escape behaviour of the pea aphid Acyrthosiphon pisum (Harris) in response to alarm pheromone and vibration. Can. J. Zool. 60: 2245 2252 . Cocroft, R . B . , H. J. Shugart, K. T. Konrad, and K. Tibbs. 2006. Variation in plant substrate s and its consequences for insect vibrational communication. Ethology 112: 779 789. Cocroft, R . B . , and G. D. McNett. 2006 . Vibrational communication in treehoppers (Hemiptera: Membracidae) , pp. 305 317 . In S. Drosopoulos and M. F. Claridge, Insect sounds and communication: physiology, behavior, ecology, and evolution. Taylor & Francis, Boca Raton, FL. Cocroft, R.B., and R.L. RodrÃguez. 2005. The behavioral ecology of insect vibrational communication. BioScience 55 : 323 334. Cocroft, RB. 2001. Vibrational communication and the ecology of group living, herbivorous insects. Am . Zool . 41: 1215 1221. . 2006 . Sense organs involved in vibratory communication of bugs, pp. 71 80 . In S. Drosopoulos and M. F. C laridge, Insect sounds and communication: physiology, behavior, ecology, and evolution. Taylor & Francis, Boca Raton, FL. Cornell, H . V., and B. A. Hawkins. 1995. Survival patterns and mortality sources of herbivorous insects: some demographic trends. Amer . Nat. 145: 563 593 Cowley, J. M. 1987. Oviposition site selection and effect of meteorological conditions on flight of Eudonia sabulosella (Lepidoptera: Scopariinae) with implications for pasture damage. New Zeal. J. Zool. 14: 527 533. Crespo, J. E., and M. K. Castelo. 2012. Barometric pressure influences host orientation behavior in the larva of a dipteran ectoparasitoid. J. Insect Physiol. 58: 1562 7. Dillon, M. E., and M. R. Frazier. 2006. Drosophila melanogaster locomotion in cold thin air. J. Exp. Bio l. 209: 364 371. Eddy, F. B., and R. D. Handy. 2012. Ecological and envi ronmental physiology of fishes. Oxford University Press . Oxford, England. Edwards , D . K . 1961 . Activity of two species of Calliphora (Diptera) during barometric pressure changes of natural magnitude . Can. J. Zool. 3 9: 623 635 . Eriksson, A., G. Anfora, A. Lucchi , F. Lanzo, M. Virant Doberiet, and V. Mazzoni . 2012 . Exploitation of insect vibrational signals reveals a new method of pest management. PLoS One. 7 : e 32954 . Fournier, F., D. Pelletier, C. Vigneault, and G. Boivin. 2005. Effect of Barometric Pressure on Flight Initiation by Trichogramma pretiosum and Trichogramma evanescens ( ) Hymenop. Environ. Entomol. 34: 1534 1540.
48 Fredeen, F. J. H., and P. G. Mason. 1991. Meteorological factors influencing host seeking activity of female Simulium luggeri (Diptera: Simuliidae). J. Med. Entomol. 28: 831 840. Funakubo, M., J. Sato, T. Honda, K. Mizumurua. 2010. The inner ear is involved in the aggregation of nocic eptive behavior induced by lowering barometric pressure of nerve injured rats. Eur. J. Pain 14:32 39 Gast, S., M. Stock, and M. Furniss. 1993. Physiological factors affecting attraction of Ips pini (Coleoptera: Scolytidae ) to host odor or natural male pheromone in Idaho. Ann. Entomol. 86: 417 422 Gottwald, T. R . , J. V. da Graca, and R. B. Bassanezi. 2007. Citrus H uanglongbing: the pathogen and its impact. Plant Health Progress. doi:10.1094/PHP 2007 0906 01 RV Grafton Cardwe ll, E. E., L. L. Stelinski, and P. A. Stansly. 2013. Biology and management of Asian citrus psyllid, vector of the Huanglongbing pathogens. Annu . Rev . Entomol . 58: 413 432 Halbert , S . E . , and K. L. Manjunath. 2004. Asian citrus psyllid (Sternorryncha: Psy llidae) and greening disease of citrus: a literature review and assessment of risk in Florida. Fla. Entomol. 87:330 53 Hall , D . G . , E. J. Wenninger , and M. G. Hentz . 2011. Temperature studies with the Asian citrus psyllid, Diaphorina citri : cold hardiness and temperature thresholds for oviposition. J. Insect Sci. 11:1 15 Hall, D. G., S. L. Lapointe, and E. J. Wenninger. 2007. Effects of particle film on biology and behavior of Diaphorina citri (Hemiptera: Psyllidae) and its infestation in c itrus. J. Econ. Entomol. 100: 847 854 Harris , M . F. 1954 . Pressure change theory and the daily barometric wave . J. Meteor . 1 2: 394 404 . Husain, M. A. and D. Nath. 1927. The citrus psylla ( Diaphorina citri , Kuw.) [Psyllidae: Homoptera]. Memoirs of the Depar tment of Agriculture in India, Entomological Series 10: 1 27. Haufe, W . 19 64 . Quantitative measurements of activity of Aedes aegypti (L.) (Culicidae: Diptera) in response to changes in the hygrothermal environment . Int. J. Biometeor. 7 : 245 264 . Haufe, W. 1954. The effects of atmospheric pressure on the flight responses of Aedes aegypti (L.). Bull. Entomol. Res. 45: 507 525. Henson, W. R. 1951. Mass Flights of the Spruce Budworm. Can. Entomol. 240: 1945. Hodges, A. W., and T. H. Spreen. 2012. Economic impac ts of citrus greening (HLB) in Florida, 2006/07 2010/11. http://edis.ifas.ufl.edu/fe903
49 Kanmiya , K . 2006 . Mating behavior and vibratory signals in whiteflies (Hemiptera: Aleyrodidae) , pp. 365 379. In S. Drosop oulos and M. F. Claridge, Insect sounds and communication: physiology, behavior, ecology, and evolution. Taylor & Francis, Boca Raton, FL. Kanmiya, K. 1996. Discovery of male acoustic signals in the greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Homoptera: Aleyrodidae). Appl. Entomol. Zool. 31: 255 262. Kolyer, J. M., and H. B. Palmer. 1968. The effect of barometric pressure and other factors on eclosion of tile cabbage butterfly Pieris rapae (Pieridae). J. Lepid. Soc. 22: 211 225. La nier, G. N., and B. W. Burns. 1978. Barometric flux: Effects on the responsiveness of bark beetles to aggregation attractants. J. Chem. Ecol. 4: 139 147. Leskey, T. C., and R. J. Pro kop y. 2003. Influence of barometric pressure on odor discrimination and ov iposition by adult plum curculios (Coleoptera: Curculionidae). Eur. J. Entomolo. 100: 517 520. Li, J., and D. Margolies. 1994. Barometric pressure influences initiation of aerial dispersal in the two spotted spider mite. J. Kansas Entomol. Soc. 67: 386 393 . Lowry, W. P. 2009 . The Barometer Handbook: a modern look at barometers and applications of barometric pressure . Starpath Publications, Seattle, WA. Lytle, D. , and N. J. White. 2007. Rainfall Cues and Flash Flood Escape in Desert Stream Insects. J. Insect Behav. 20: 413 423. Mangan, R. L., and D. L. Chapa. 2013. Evaluation of the effects of light source and plant materials in psyllid trapping levels in the traps for citrus shipping containers. Fl a. Entomol . 96 : 104 111. Mankin, R. W., B. B. Rohde, S. A. Mc Neil, N. Y. Zagvazdina, and S. Greenfeder. 2013. Diaphorina citri ( buzzer communication signals of potential use in vibration traps. Florida Entomol. 96: 1546 1555. Mankin, R. W., D. Shuman, and J. A. Cof felt. 1996. Noise shielding of acoustic devices for insect detection. Journal of Economic Entomology 89: 1301 1308 Mann, R. S., K. Pelz Stelinski, S. L. Hermann, S. Tiwari, L. L. Stelinski. 2011. Sexual transmission of a plant pathogenic bacterium, Candidatus Liberibacter asiaticus, between conspecific insect vectors during mating. PLoS O ne. 6 : e29197 Marchand, D., and J. N. Mcneil. 2000. Effects of Wind Speed and Atmospheric Pressure on Mate Searching Behavior in the Aphid Parasitoid Aphidius nigrip es Aphidiidae ). J. Insect Behav. 13: 187 199.
50 Mead, F. W. 1977. The Asciatic citrus psyllid, Diaphorina citri Kuwayama (Homoptera: Psyllidae). Entomology Circular 180. Florida Department of Agriculture and Consumer Services, Division of Plan t Industry. 4 pp. Nava, D. E., G. M. L. Torres, M. D. L. Rodrigues, J. M. S. Bento, and J. R. P. Parra. 2007. Biology of Diaphorina citri (Hem., Psyllidae) on different host plants at different temperatures. J. Appl. Entomol. 131:709 15 Noverre Press. 1834 . The pocket barometer and weather guide. Hampshire, England. Paige, K. N. 1995. from the roost. Funct. Ecol. 9: 463 467. Paris, T. M., B.B. Rohde, S.A. Allan, R. W. Mankin, and P.A. Stansly. 2013. Synchronized rearing of mated and unmated Diaphorina citri (Hemiptera: Liviidae) of a known age. Florida Entomol. 96: 1631 1634 Pellegrino, A. C., M. F. G. V. PeÃ±aflor, C. Nardi, W. Bezner Kerr, C. G. Guglielmo, J. M. S. Bento, and J. N. McNeil. 2013. Weather forecasting by insects: modified sexual behaviour in response to atmospheric pressure changes. PLoS One. 8: e75004. Pelz Stelinski, K. S., R. H. Brlansk, T. A. Ebert, and M. E. Rogers. 2010. Transmission parameters for Candidatus L iberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). J. Econ. Entomol. 103:1531 41 . Percy, D. M., G. S. Taylor, M. Kennedy. 2006. Psyllid communication: acoustic diversity, mate recognition and phylogenetic signal. Invertebr. Syst. 20: 431 445. Peterson, D. A. 1972. Barometric Pressure and its Effect on Spawning Activities of Rainbow Trout. Progress. Fish Culturist. 34: 110 112. Raguso, R. , T. Ojeda Avila, S. Desai, M. Jurkiewicz, and H. A. Woods. 2007. The influence of larval diet on ad ult feeding behaviour in the tobacco hornworm moth, Manduca sexta . J. Insect Physiol. 53: 923 932. van Roermund, H. J. W., and J. C. van Lenteren. 1995 . Effects of m eteorological f actors on d efensive b ehaviour of h oney b ees. J. Appl. Ent. 119: 465 475. Roh de, B., T. M. Paris, E. M. Heatherington, D.G. Hall, and R. W. Mankin. 2013. Responses of Diaphorina citri (Hemiptera: Psyllidae) to conspecific vibrational signals and synthetic mimics. Ann. Entomol. Soc. America 106: 392 299 Roitberg, B. D., J. Sircom, C . A. Roitberg, J. J. M. van Alphen, and M. Mangel. 1993. Life expectancy and reproduction. Nature. 364: 108. Rousse, P., F. Gourdon, M. Roubaud, F. Chiroleu, and S. Quilici. 2009. Biotic and abiotic factors affecting the flight activity of Fopius arisanus , an egg pupal parasitoid of fruit fly pests. Environ. Entomol. 38: 896 903.
51 Sechler, A., E. L. Schuenzal, P. Cooke, S. Donnua, N. Thaveechai, E. Postnikova, A. L. Stone, W. L. Schneider, V. D. Damsteegt, and N. W. Schaad. 2008. Cultivation of Candidatus L Ca Ca . L. americanus' associated with Huanglongbing. Phytopathology. 99: 480 486 Shen, W., S. E. Halbert, E. Dickstein, K. L. Manjunath, M. M. Shimwela, and A. H. C. van Bruggen. 2013. Occurrence and in grove di stribution of citrus huanglongbing in north central Florida. J. Plant Pathol. 95:361 371. Southwick, E. E., and R. F. A. Moritz. 1987. Effects of m eteorological f actors on d efensive b ehaviour of h oney b ees. Int. J. Biometeor. 31: 259 265. Steinberg, S., M. Dicke, and L. E. M. VetR. Wanningen. 1992. Response of the braconid parasitoid Cotesia (=Apanteles) glomerata to volatile infochemicals: effects of bioassay set up, parasitoid age and experience and barometric flux. Entomol. Exp. Appl. 63 : 163 175. S tephen, W. P., and R. D. Bird. 1949 . The effect of barometric pressure upon oviposition of the imported cabbageworm, Pieris rapae (L.) . Can . Entomol . 81 : 132 132 . Tichy, H., and W. Kallina. 2010. Insect hygroreceptor responses to continuous cha nges in humidity and air pressure. J. Neurophysiol. 103: 3274 86. Tishechkin , D . Y. 2013 . Vibrational background noise in herbaceous plants and its impact on acoustic communication of small Auchenorrhyncha and Psyllinea (Homoptera). Entomol. Rev. 93: 548 5 58. Tishechkin , D . Y. 2 006 . Vibratory communication in Psylloidea (Hemiptera) , pp. 357 363 . In S. Drosopoulos and M. F. Claridge, Insect sounds and communicationL physiology, behavior, ecology, and evolution. Taylor & Francis, Boca Raton, FL. U.S. Department of Agriculture. 2012. USDA Commercial Citrus Inventory Preliminary Report , Beltsville, MD. Wang , N . , and P. Trivedi . 2013. Citrus huanglongbing: a newly relevant disease presents unprecedented challenges. Phytopathology . 7 :652 665. Wellington, W . 1946 . The effects of variations in atmospheric pressure upon insects. Can. J. Res. Sect. D. 24: 51 70. Wellington, W. 1944. Barotaxis in Diptera, and its possible significance to economic entomology. Nature. 154: 671 672. Wenninger E . , D. G. Hall , and R. W. Mankin . 2009 a . Vibrational communication between the sexes in Diaphorina citri (Hemiptera: Psyllidae). Ann. Entomol. Soc. Am. 102:547 55 .
52 Wenninger E . , L. L. Stelinski, and D. G. Hall. 2009 b . Role of olfactory cues, visual cues and mating status in orientation of Diaphorina citri Kuwayama (Hemiptera: Psyllidae) to four different host plants. Environ. Entomol. 38:225 34 . Wenninger , E . J . , and D. G. Hall . 2007. Daily timing and age at reproducti ve maturity in Diaphorina citri (Hemiptera: Psyllidae). Fla. Entomol. 90:715 22 Williams, J. 1997 . The weather book: an easy to . 2 nd ed. Vintage Books, New York , NY . Winsberg, M . D. 2003. Florida weather, 2 nd ed. University Press of Florida. Gainesville, FL. Witter, L. a., C. J. Johnson, B. Croft, A. Gunn, and L. M. Poirier. 2012 . Gauging climate change effects at local scales: weather based indices to monitor insect harassment in caribou. Ecol. Appl. 22: 1838 1851. Zettel, J. 1984. The significance of temperature and barometric pressure changes for the snow surface activity of Isotoma hiemai ls (Collembol a). Experientia. 40: 1369 1372.
53 BIOGRAPHICAL SKETCH Born in Leningrad, USSR, Nina Zagvazdina spent most of her childhood in Memphis, TN until her family moved to Cooper City, FL, where she completed high school in 2007. After graduating from the University of Florida in 2010 with a B.A. in anthropology , she worked at the McGuire Center for Lepido ptera and Biodiversity. After completing a Clinical Trials internship with the Plant Medicine Program, Nina enrolled as a student in the program in January 2011. Nina began working on a research project working on experimental traps at the Division of Plan t Industry under the supervision of Dr. Susan Halbert, and started to pursue an M.S. in e ntomology and nematology . She decided to focus on this degree route and began working under the al, and Veterinary Entomology.