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Factors affecting host orientation and selection in Nitidulidae (Coleoptera)
Blackmer, Jacquelyn Lee, Ph.D.
The Ohio State University, 1991
UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106
FACTORS AFFECTING HOST ORIENTATION AND SELECTION
IN NITIDULIDAE (COLEOPTERA)
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of the Ohio State University
By
Jacquelyn Lee Blackmer, A.S., B.S., M.S.
*****
The Ohio State University
1991
Dissertation Committee:
L.V. Madden Approved by
L.R. Nault
P.L. Phelan Advisor B.R. Stinner Department of Entomology To my children, Felisa and Bryl an
n• • ACKNOWLEDGMENTS
I would like to express my sincere appreciation to my major
professor, Dr. P. Larry Phelan, for his guidance and encouragement
during my time at Ohio State University. To Dr. Nault, my co-advisor,
I extend a warm thank you for taking time from his very busy schedule
to show concern and for answering my many questions. For statistical
advise, I would like to thank Dr. Larry Madden, and for assistance in
addressing ecological questions I thank Dr. Benjamin Stinner. For
providing space to maintain my fungal cultures I thank Dr. Mike Ellis,
and for helping me keep these cultures going I am indebted to Lee
Wilson. I would like to especially thank Caryn Roelofs and Felisa
Blackmer for rearing my beetles, and Dr. Roger Williams and Dan Fickle
for answering many questions about nitidulid biology and evolution.
For their incredible patience, I thank Mabel Kirchner and Maxine
Johnson for dealing with most of the paperwork that kept my paychecks coming in.
I am especially grateful to my children, Felisa and Brylan, for enduring the hardship of having a mother that was usually preoccupied, and to my family and friends who encouraged me to continue my education. A special thanks is extended to Astri Wayadande, Dr. Susan
Heady and Ty Vaughn who were always there to lif t my spirits when life and research were in disarray, and to Ed Zaborski, Julie Todd, and Andy Chappie who remained dear friends throughout this ordeal. Finally, I thank Athayde Tonhasca Jr. for many stimulating intellectual conversations about research and life, for assisting me with my research when necessary, and for persevering in our challenging relationship. VITA
December 6, 1954...... Born - Parkersburg, W.Va.
1976-197 7 ...... A.S., Science and Math, Niagara County Community College, Sanborn, N.Y.
1977-1979 ...... B.S., Forest Biology: Entomology and Botany, College of Environmental Science and Forestry, State University of New York, Syracuse, N.Y.
1978-1980...... Medical Entomological Assistant, N.Y. State Department of Health, Syracuse, N.Y.
1980-1985 ...... Scientific Aide: Agricultural Entomology, University of Idaho Research and Extension Center, Parma, Idaho
1984-198 6...... M.S., Department of Entomology, University of Idaho, Moscow, Id.
1985-198 7 ...... Scientific Aide: Insect Behavior and Physiology, Department of Entomology, University of Idaho, Moscow, Idaho
1987-1991 ...... Graduate Research Associate/ Fellow, Department of Entomology, OARDC-Ohio State University, Wooster, Ohio
v PUBLICATIONS
Halbert, S., G.W. Bishop, J. Blackmer, J. Connelly, R. Johnston, L. Sandvol and K.S. Pike. 1990. Barley yellow dwarf infectivity of Rhooalosiphum oadi in maize as an estimate of primary inoculum pressure in irrigated winter wheat. In: World Perspectives on Barley Yellow Dwarf. P.A. Burnett ed. CIMMYT, Mexico, D.F. Mexico.
Klowden, M.J., J.L. Blackmer and G.M. Chambers. 1988. Effects of larval nutrition on the host-seeking behavior of adult Aedes aeavpti mosquitoes. Amer. Mosq. Contr. Assoc. 3: 73-75.
Klowden, M.J. and J.L. Blackmer. 1987. Humoral control of pre-oviposition behavior in the mosquito, Aedes aeavpti. J. Insect Physiol. 33: 689-692.
Bishop, G.W., J.L. Blackmer and C.R. Baird. 1984. Observations on the biology of Prionus californicus Mots, on hops, Humulus lupulus L. in Idaho. J. Entomol. Soc. Brit. Columbia 81: 20-24.
Homan, H.W., C.R. Baird, G.W. Bishop, J. Blackmer and D.G. Boltz. 1984. The Western Corn Rootworm. University of Idaho Current Information Series No. 533.
FIELD OF STUDY
Major Field: Entomology TABLE OF CONTENTS
DEDICATION...... ii
ACKNOWLEDGMENTS...... iii
VITA...... v
LIST OF TABLES...... ix
LIST OF FIGURES...... xi
INTRODUCTION...... 1
CHAPTER PAGE
I. BEHAVIOR OF CARPOPHILUS HEMIPTERUS IN A VERTICAL FLIGHT CHAMBER: TRANSITION FROM PHOTOTACTIC TO VEGETATIVE ORIENTATION...... 4
Introduction ...... 4 Materials and Methods ...... 6 Results...... 13 Discussion ...... 32
II. THE EFFECT OF PHYSIOLOGICAL STATE AND FUNGAL INOCULATION ON CHEMICALLY MODULATED HOST-PLANT FINDING BY CARPOPHILUS HEMIPTERUS AND CARPOPHILUS LUGUBRIS...... 37
Introduction ...... 37 Materials and Methods ...... 39 Results...... 45 Discussion ...... 60
III. CHEMICAL 'GENERALISTS' AND BEHAVIORAL 'SPECIALISTS': A COMPARISON OF HOST FINDING BY STELIDOTA GEMINATA AND STELIDOTA OCTOMACULATA...... 65
Introduction ...... 65 Materials and Methods ...... 67 Results...... 72 Discussion ...... 90
vii IV. EFFECT OF HOST VOLATILES, SEASONAL OCCURRENCE AND HABITAT ON HOST ORIENTATION AND SELECTION BY NITIDULIDAE...... 95
Introduction ...... 95 Materials and Methods ...... 97 Results...... 101 Discussion ...... 127
SUMMARY...... 131
LIST OF REFERENCES...... 136 LIST OF TABLES
TABLE PAGE
1. Horizontal and vertical displacement of Caroophilus hemipterus 0-36 min after takeoff in a vertical flight chamber during freeflight ...... 24
2. Rate of climb (cm/s) of Carppphilus hemipterus measured for a 1 min period after takeoff, immediately preceding food-odor introduction, and following food-odor removal for 'landers' and 'non-landers' ...... 29
3. Percent takeoff and flights to source for Caroophilus hemipterus. and percent walks to source for C. luoubris in a wind tunnel, maintained as adults with water, or with artificial diet until 36 h or 2 h before the experiment ...... 51
4. Percent takeoff and flights to source for Caroophilus hemipterus. and percent walks to source for C. luoubris in a wind tunnel, maintained with water or artificial diet as adults and given 0 or 3 h of 1ocomotory opportunity in a vertical flight chamber ...... 53
5. Percent response (+ s.e.) to aseptic and fungal-inoculated substrates, or whole wheat bread dough (WWBD) in a wind tunnel by Caroophilus hemipterus and C. luoubris ...... 59
6. Reported hosts of Stelidota oeminata and S. octomaculata ...... 73
7. Mean percentage (+ s.e.) of Stelidota oeminata and S. octomaculata responding to food odors ...... 83
8. Mean (+ s.e.) rate of locomotion, distance traveled, number of turns > 45°, number of pauses > 5 s, and duration of pauses for Stelidota oeminata walking or flying and for S. octomaculata walking to aseptic banana located 50 cm upwind in a horizontal wind tunnel ...... 86
9. Mean (+ s.e.) number of beetles collected per 2-d trapping period with food substrates and control in 1989 ...... 105 10. Analysis for habitat preference (whole plot) and substrate preference (subplot) and interaction for three locations: Moreland, Ullom and Maurer Farms, 1990...... 119
11. Nitidulid community description for agricultural settings (Ag) and woodlots (Wd) using species total (ST), species abundance (N), and Shannon-Wiener diversity index (H‘) ...... 125
12. Other less common nitidulid species with numbers collected in each habitat, 1990...... 126
x LIST OF FIGURES
FIGURE PAGE
1. Vertical flight chamber...... 9
2. Mean percent takeoff (+ s.e.) of Caroophilus hemipterus in a vertical flight chamber when maintained as adults with water (solid bars) or artificial diet (hatched bars), 1-9 days post-emergence ...... 15
3. Mean percent takeoff (+ s.e.) of Caroophilus hemipterus in a vertical flight chamber when maintained with water or artificial diet, a) from 16-2 h prior to scotophase, or b) from 6 h prior to 1 h after when the onset of scotophase normally would have occurred ...... 17
4. FIight duration in a vertical flight chamber for Caroophilus hemipterus males (solid bars) and females (hatched bars)...... 20
5. Representative strip-chart record for Caroophilus hemipterus flown in a vertical flight chamber ...... 22
6. Mean rate of climb + s.e. for Caroophilus hemipterus maintained as adults with water or artificial diet (P<0.0001, except at 16 min P<0.05, paired t-test, numbers above s.e. bars represent data points compared during each one min period) ...... 26
7. Representative strip-chart records with rate of climb (cm/s) during the free flights of Caroophilus hemipterus with food-odor introduction at 2 (a), 6 (b), 11 (c), and 22 (d) min after takeoff (C=blank control, V=apple cider vinegar) ...... 28
8. Representative strip-chart record with rate of climb (cm/s) during the free flight of Caroophilus hemipterus with multiple food-odor introductions (C=blank control, V=apple cider vinegar) ...... 31 9. Percentage (+ s.e.) of 2- to 10-day-old Caroophilus hemipterus and C. luoubris maintained with water only as adults, taking off (solid line), flying to the source (diagonally-slashed bar), and walking to the source (cross-hatched bar)...... 47
10. Percentage (+ s.e.) of Caroophilus hemipterus and C. luoubris maintained with water only as adults, flying or walking to the source from 16 h prior to scotophase to 2 h after the onset of scotophase ...... 50
11. Percentage (+ s.e.) of Caroophilus hemipterus maintained with water only as adults, that flew or walked to corn, strawberry, tomato, banana, prune, and whole wheat bread dough. Comparisons are among aseptic and inoculated fruit for each substrate; letters that are the same are not significantly different using LSD (P>0.05) ...... 56
12. Percentage (+ s.e.) of Caroophilus luoubris maintained with water only as adults, that flew or walked to corn, strawberry, tomato, banana, prune, and whole wheat bread dough. Comparisons are among aseptic and inoculated fruit for each substrate; letters that are the same are not significantly different using LSD P>0.05) ...... 58
13. Mean percent takeoff (+ s.e.) by Stelidota oeminata in a vertical flight chamber, from 16 h prior to scotophase to 2 h after when the onset of scotophase normally would have occurred ...... 75
14. Percentage (+ s.e.) of Stelidota oeminata (solid line) and S. octomaculata (dashed line) flying and/or walking to the source from 16 h prior to scotophase to 2 h after the onset of scotophase ...... 77
15. Percentage (+ s.e.) of Stelidota oeminata that flew or walked to banana, strawberry, corn, tomato, fig, Candida krusei on YM-agar, Saccharomvces cerevisiae on YM-agar, Ceratocvstis faoacearum on PDA-agar, Oudemansiella radicata on PDA-agar, black-oak acorn or red-oak acorn ...... 80
16. Percentage (+ s.e.) of Stelidota octomaculata that walked to banana, strawberry, corn, tomato, fig, Candida krusei on YM-agar, Saccharomvces cerevisiae on YM-agar, Ceratocvstis faoacearum on PDA-agar, Oudemansiella radicata on PDA-agar, black-oak acorn or red-oak acorn ...... 82
xii 17. Representative tracks for Stelidota octomaculata walking to aseptic banana (O.s.) and J>. oeminata a) walking and b) flying to aseptic banana located 50 cm upwind in a horizontal wind tunnel ...... 85
18. Mean rate of walking (+ s.e.) for Stelidota oeminata (dashed line) and S. octomaculata (solid line), 50 to 0 cm from the odor source ...... 89
19. Total number per trapping period of a) Stelidota oeminata. Carpoohilus luoubris. Glischrochilus fasciatus and b) G. ouadrisionatus collected at Morel and Fruit Farm, Moreland, Ohio, 1989 ...... 103
20. Cumulative number of Glischrochilus ouadrisionatus collected with whole wheat bread dough, corn, banana, strawberry and tomato at Moreland Fruit Farm, Moreland, Ohio, 1989...... 107
21. Proportion of Stelidota oeminata (diagonally-dashed bars), Carpophilus luoubris (solid bars), Glischrochilus fasciatus (cross-hatched bars), and G. ouadrisionatus (dotted bars) collected at the agricultural site and woodlot at Moreland Fruit Farm, Moreland, Ohio, 1989...... 109
22. Total number per trapping period of Stelidota oeminata col 1ected at Moreland, Ullom and Maurer Farms and adjacent woodlots, 1990 ...... 112
23. Total number per trapping period of Carpophilus luoubris collected at Moreland, Ullom and Maurer Farms and adjacent woodlots, 1990 ...... 114
24. Total number per trapping period of Glischrochilus fasciatus collected at Moreland, Ullom and Maurer Farms and adjacent woodlots, 1990 ...... 116
25. Total number per trapping period of G1ischrochilus ouadrisionatus collected at Morel and, Ullom and Maurer Farms and adjacent woodlots, 1990 ...... 118
26. Proportion of Stelidota oeminata (diagonally-slashed bars), Caroophilus luoubris (solid bars), Glischrochilus fasciatus (cross-hatched bars), and G. ouadrisionatus (dotted bars) collected in agricultural sites and woodlots at Morel and, Ullom and Maurer Farms, 1990...... 121
xiii 27. Dendrogram comparing nitidulid communities in agricultural settings and woodlots; based on cluster analysis using the single linkage method and Euclidian distance ...... 124
xiv INTRODUCTION
Considerable effort has been directed towards understanding the mechanisms insects use to locate their hosts. However, the major focus of this work has dealt with close-range response by insects to chemical and visual cues from plants, and has neglected the mechanisms used in long-range orientation. As a result, our understanding of host finding
is limited and our ability to test hypotheses concerning the relationship between ecological parameters and strategies of host location is hindered.
The research presented herein used nitidulid beetles as a model system for testing various hypotheses concerning strategies of host location. It has long been suspected that chemical cues play an important role in the host-locating ability of the Nitidulidae
(Wildman, 1933). Additionally, the plant-pathogenic fungi with which they are associated may enhance host location (Miller & Mrak, 1953;
Dorsey & Leach, 1956). However, until now, little was known about the behavioral mechanisms or ecological factors governing nitidulid host location and selection. As most nitidulid beetles exploit ephemeral resources (i.e., mature to decaying fruits and vegetables) that can be locally abundant, put patchy on a geographic scale, it seems likely that efficient host-locating tactics have evolved to compensate for the transitory nature of their food sources.
1 The host-orientation behaviors of four closely related nitidulid
species were examined herein, using a vertical flight chamber and a
horizontal wind tunnel. The four species (Carpophilus hemipterus.
Carpophilus luoubris. Stelidota oeminata. and Stelidota octomaculatal
have different (although often overlapping) geographical distributions
and host-plant ranges (Williams et a l., 1983). C. hemipterus and S.
oeminata are broad in their response to food substrates, C. luoubris is
somewhat narrower in its breadth of response, and S. octomaculata is
almost exclusively restricted to acorns. Based on these facts and the
few references that implicate fungi and host volatiles as being
important in host location within this group the following hypotheses were addressed: 1) because a period of flight exercise is required in
newly emerged aphids and bark beetles before host-orientation behaviors
are expressed (Kennedy & Booth, 1963b; Choudhury & Kennedy, 1980), I
submit that newly eclosed nitidulid beetles will similarly need a period of flight exercise before exhibiting host orientation, 2) the volatiles from fungus-inoculated food substrates will elicit a greater upwind response than will the volatiles from aseptic food substrates, and 3) nitidulid beetles with a broad host range will exhibit a less specialized orientation to food-substrate odors, whereas nitidulid beetles with a narrower host range will orient to a narrower range of food substrates. Chapters I, II and III focused on these first three hypotheses and also on other parameters (i.e., age, diet, and die! periodicity) that influence the expression of host orientation.
Because insects can partition their resources along at least three dimensions: spatial, temporal, and chemical, additional studies were conducted in the field to determine the relative importance of each dimension for species separation. As the primitive condition in the Nitidulidae is believed to be the association with wood and wood fungi, and the derived condition to be an association with herbaceous plants (Skalbeck, 1976), I hypothesized that 1) spatial differences
(i.e ., habitat preference) would be the most important dimension for separating species; chemical and temporal partitioning would be important within a particular habitat, and 2) since nitidulid beetles have had a longer association with forests, I submit that species diversity will be greater in the wooded habitats than in the agricultural environments. Chapter IV addresses these two hypotheses.
The research contained herein represents the first detailed studies in host orientation and selection in the Nitidulidae. It is hoped that this information will bring about a greater understanding of odor-modulated insect host-finding and that it will also elucidate the role of fungi and rot-causing microorganisms in insect host finding, information that is sorely needed considering the abundance of such relationships in the Insecta. At the applied level, this research was valuable for enhanced control of nitidulid pests and the pathogens they transmit. CHAPTER I BEHAVIOR OF CARPOPHILUS HEMIPTERUS IN A VERTICAL FLIGHT CHAMBER: TRANSITION FROM PHOTOTACTIC TO VEGETATIVE ORIENTATION Introduction
Nitidulidae, commonly referred to as sap beetles, feed on decaying fruit and vegetables (Lindgren & Vincent, 1953; Miller & Mrak,
1953; Windels et a l., 1976), on fungal mats under bark (Neel et a l.,
1967; Hinds, 1972), on carrion (Abbott, 1937), and on a variety of flowers (Gazit et al., 1982; Nagel et al., 1989). Although the majority of nitidulid species perform beneficial roles as detritivores, predators or pollinators, a few species are pests because they have been implicated in the transmission of oak wilt disease (Ceratocvstis faoacearum (Bretz)) (Dorsey & Leach, 1956; Juzwik & French, 1986), corn ear rot (Fusarium sp.) (Windels et a l., 1976), pineapple disease of sugarcane (C. paradoxa (Dade)) (Chang & Jensen, 1974) and cankers of stone fruit and aspens (Ceratocvstis spp.) (Hinds, 1972; Agrios, 1980).
Carpophilus hemipterus. the dried fruit beetle, is a major pest throughout much of the fig- and date-producing regions of the world.
These beetles introduce souring agents and leave behind feces that render fruit inedible (Miller & Mrak, 1953). It has long been suspected that chemical cues play an important role in host finding by
C. hemipterus and other nitidulid species (Wildman, 1933; Miller &
4 5
Mrak, 1953; Dorsey & Leach, 1956; Neel et al., 1967; Windels et al.,
1976; Aim et a l., 1985, 1986), but almost nothing is known about the
underlying mechanisms that aid the insect in host location.
The present study examined the flight behavior of C. hemipterus
from shortly after emergence, and throughout the transition from
phototactic to vegetative orientation. In both aphids and bark
beetles, a gradual switch from phototactic to vegetative orientation
occurs, but only after a period of sustained flight (Kennedy & Booth,
1963b, 1964; Kennedy, 1965, 1966; Kennedy & Ludlow, 1974; Choudhury &
Kennedy, 1980; Borden et a l., 1986 and references therein). An initial phototactic response during which response to vegetative cues are temporarily inhibited, enhances dispersal of individuals even in the presence of favorable habitat conditions. Here I sought to understand the flight responses of C. hemipterus from the perspectives of the underlying behavioral mechanisms, as well as their ecological implications. 6
Materials and Methods
Insects
C. hemipterus has been reared in the laboratory for 7 years, with
annual introductions of feral beetles to maintain genetic variability.
The beetles were reared in bell jars (19 x 18.5 cm) on artificial diet
(Hall et al., 1978) at 25 + 1° C, 65% r .h ., on a L16:D8 light regime.
Prior to each experiment, pupae were separated from the colonies and
sexed (Hinton, 1945), except where mated beetles were tested. Newly
emerged adults were held with water or artificial diet until tests
began.
Vertical Flight Chamber
Behavioral bioassays were conducted in a vertical flight chamber modified from Kennedy and Booth (1963a) (Fig. 1). The chamber was
constructed of 1.9 cm plywood except for the front, which was 6 mm
Plexiglas® and the bottom, which was 1.2 cm2 mesh screen covered with
black cotton cloth. A 38 cm, 1/2 HP Dayton belt-driven blower was mounted to a 81 x 81 x 81 cm plywood box. The light source was positioned above this box and four muslin filters, which smoothed the airflow, were positioned at its base. Below the muslin filters was the
85 x 77 x 92 cm flight chamber. The two side walls adjacent to the
Plexiglas® front were hinged at the top (where they connected to the 81 x 81 x 81 cm box) which enabled me to vary the width of the bottom of the flight chamber from 77 to 163 cm. As the cross section of the bottom of the flight chamber increased, air speed from top to bottom decreased, which provided an automatic dampening of the beetles'
flight. The chamber was painted flat-black and was l i t from above by
a high-pressure sodium lamp (Philips 400W) which had major emission peaks at 490, 565, 580, 590 and 615 nm. The light projected through the series of muslin filte rs which were painted black except for a 40 cm center region, hereafter referred to as the 1ight window, the air speed (0-100 cm/s) was controlled manually by a damper that was positioned in front of the blower. The chamber was equipped with a model 618 flo-multimeter (Sierra In str., Carmel Valley, CA) connected to a strip-chart integrator (Hewlett Packard 3390A), which measured air speed, providing an indirect record of the beetles' rate of climb or photokinetic response. The flo-multimeter produced a nonlinear analog signal, which was calibrated by comparing the air speed to the chart output. The Plexiglas® front enabled me to videorecord flights for later measurement of horizontal and vertical displacement. A RCA TV camera (TC 1005/U9) was connected to a RCA TCI12 video monitor and a
Panasonic (NV-8950) dynamic tracking video cassette recorder which al1 owed single frame (1/60 s) analysis free of picture distortion. An exhaust system mounted below the flight chamber minimized chemical contamination by removing the odor plume from the building. The temperature within the chamber was maintained at 26 + 1° C. Figure 1. Vertical flight chamber.
8 9
High-pressure Sodium Lamp
Fan Fitters
Flo-muftimeter
Strip-chart Recorder
Exhaust
Figure 1. 10
Behavioral Studies
Flight Propensity
The propensity of beetles to fly 1-9 days post-emergence was
investigated using individuals maintained with water or artificial diet. Time to oviposition and mortality rates were monitored until the day of the experiment. Immediately prior to testing, beetles in groups
of 10 were placed in 40-ml vials and held in the wind chamber for 1 h.
After this preconditioning period, the vial containing the beetles was
placed on a flat-black platform 15 cm above the chamber floor, and beetles were allowed 5 min to take flight. Frequency of takeoff was recorded for eight replicates, or 80 beetles per age category and diet regime. All replicates were conducted from 3 h prior to the onset of scotophase to 1 h after when the onset of scotophase normally would have occurred. Beetles were tested only once.
Flight Periodicity
Flight propensity during photophase was determined using 4- to 5- day-old beetles maintained on each diet regime. One study examined flight activity from 16-2 h prior to scotophase at 2 h intervals, and a second study examined the period from 6 h prior to 1 h after when the onset of scotophase normally would have occurred at 1 h intervals.
Beetles were preconditioned as described above and each time interval replicated six times using 10 fresh beetles per diet regime and replicate. Flight Duration
Duration of phototactic flight was determined for 103 males (75 with water, 28 with artificial diet) and 89 females (71 with moisture,
18 with artificial diet) that were 4-5 days old. Beetles were flown individually and maintained in-flight until they landed. If the beetle failed to re-initiate flight within 1 min after landing, the test was terminated.
Light Versus Odor-Modulated Responses
Beetles 4-5 days old that had been maintained with either water or artificial diet were flown in the flight chamber during their period of maximum flight activity (3 h prior to scotophase to 1 h after when the onset of scotophase normally would have occurred). The air speed needed to maintain free flight 20-40 cm below the 1ight window was recorded using the flo-multimeter, strip-chart setup previously described. The moveable side walls of the chamber were piaced perpendicular to the chamber floor, resulting in a constant air speed from top to bottom. To determine the change in rate of climb during an uninterrupted flight, 15 beetles were flown until they landed (minimum flight 10 min). Changes in phototactic response were determined by measuring horizontal deviations from the midpoint of the light window and vertical deviations from the ceiling of the chamber at 4 s increments during a 1 min period every 5 min during a representative 36 min flight. The effect of dietary regime on phototactic response was examined using 12 beetles maintained with water and eight beetles maintained with artificial diet. The change in rate of climb was 12
measured for 1 min periods at 3 min intervals, during flights which
ranged from 10-40 min in duration. Within each 1 min period, 10 points
(at 6 s intervals) on the strip-chart recording were measured and a paired t-te st was used to compare rates of climb between beetles maintained on the two diet regimes at each time interval.
To determine the interaction between light-modulated flight and response to food odors, C. hemipterus was presented food odor after flight periods of varying duration (ranging from 3-25 min, n=25). A blank filte r paper (Whatman #1, 5.5 cm) was positioned directly above the flying beetle for 1 min to control for visual response. Next, the filte r paper was treated with 0.25 ml of apple cider vinegar and then re-introduced for an additional 1 min. During preliminary studies, apple cider vinegar evoked upwind flights to the source in a horizontal wind tunnel (Blackmer, unpublished data). After food-odor introduction the beetle was given 1 min to return to its previous rate of climb or to re-initiate its flight if landing had occurred. In a subsequent study (n=10), vinegar was introduced three to five times (allowing several minutes to elapse between introductions) during each of the flights, to determine if chemical cues would act in a cumulative manner to eventually override phototactic flight. 13
Results
Flight Propensity
Very few flights (<6%) occurred prior to 3 days post-emergence, regardless of diet regime (Fig. 2) or sex. The period of greatest activity (68-88% takeoff response) for beetles maintained with water was 3-6 days post-emergence; the decline in flight propensity after six days coincided with an increase in mortality. Beetles maintained with diet showed their greatest flight propensity (58-71% takeoff) from 4-8 days post-emergence. Only females maintained with artificial diet developed eggs and oviposition began on Day 4. The development of mature eggs did not inhibit the flight propensity of females when compared to males maintained with artificial diet. However, both sexes did show a one-day delay in peak flight propensity when maintained with artificial diet compared to beetles maintained with water.
Flight Periodicity
C. hemioterus showed a bimodal periodicity in flight propensity during the photophase with a small peak occurring 14-10 h prior to scotophase and a large peak occurring 4-2 h prior to scotophase (Fig.
3a). When this second period was examined at one-hour intervals, Figure 2. Mean percent takeoff (+ s.e.) of Carpophilus hemipterus in
a vertical flight chamber when maintained as adults with
water (solid bars) or artificial diet (hatched bars), 1-9
days post-emergence.
14 iue 2. Figure Mean % Takeoff 15 Figure 3. Mean percent takeoff (+ s.e.) of Carpophilus hemipterus in
a vertical flight chamber when maintained with water or
artificial diet, a) from 16-2 h prior to scotophase, or
b) from 6 h prior to 1 h after when the onset of
scotophase normally would have occurred.
16 100
80 water maintained «*- o artificlal diet © 60 m H
1 40 © 2
20
16 14 12 10 8 6 4 2 Time prior to scotophase (h)
100
80
60
§ © 2
20
6 6 4 3 2 1 0 1 Time prior to scotophase (h) Figure 3. 18
maximum activity occurred 3 h prior to scotophase to 1 h after when the
onset of scotophase normally would have occurred (Fig. 3b). Beetles on
both diet regimes showed the same general activity pattern, but beetles
maintained with artificial diet displayed lower flight activity
compared to beetles maintained with water.
Flight Duration
The flight duration of C. hemipterus ranged from 1-100 min with
73% of all males and 85% of all females flying for less than 15 min
(Fig. 4). Females maintained with artificial diet had the greatest
proportion of flights exceeding 35 min (28%) with an average flight duration of 22 + 6 min (mean + s.e.). A considerably smaller proportion of males maintained with diet (4%, average flight duration
10+2 min) or water (5%, average flight duration 12+1 min), and females maintained with water (3%, average flight duration 10 + 1 min) had flights exceeding 35 min.
Light Versus Odor-Modulated Responses
A typical flight, regardless of sex or diet regime, began with a rapid increase in the rate of climb in response to the overhead light.
Initially there was instability in the flight, characterized by rapid fluctuations in the rate of climb but the beetle soon entered into a
'cruising' flight (Kennedy and Booth, 1963a) which was relatively long and stable. After a period of flight, an increase in instability in both the horizontal and vertical dimensions occurred until the beetle ended the flight (Fig. 5). Horizontal and vertical displacement Figure 4 Flight duration in a vertical flight chamber for
Carpophilus hemipterus males (solid bars) and females
(hatched bars).
19 iue 4. Figure Percentage of Beetles 50 5 5- 15 15- 20- 25- 30- >35 5 -3 0 3 0 -3 5 2 6 -2 0 2 0 -2 5 1 5 -1 0 1 0 -1 5 -5 0 uain f lgt (min) flight of Duration Hm. m IH 20 Figure 5. Representative strip-chart record for Carpophilus hemipterus
flown in a vertical flight chamber.
21 22
100 S3 76
o E 6 8 10 12 14 16 Time after takeoff (min) 100-j S 3 76- o 60- ■ s i o S a EC 25- ■ ■ v 1 I I T I 18 20 22 24 26 28 30 32 36 Time after takeoff (min) Figure 5. 23 during flight, defined as the mean deviation from the midpoint of the light window and from the ceiling of the chamber, respectively, increased with time (Table 1). The increase in horizontal and vertical displacement indicated that a decrease in phototactic response and an increase in variability had occurred. With regard to diet effects, beetles maintained with water measured significantly higher rates of climb throughout their flights than those on artificial diet (Fig. 6). Also, beetles maintained with water generally ended their flights by landing on the floor of the chamber (61%) while beetles maintained with diet ended their flights when their horizontal excursions reached the walls of the chamber (78%). When vinegar was introduced, the beetles' rate of climb (43.1 + 2.9 cm/s, mean + s.e.) consistently dropped to 0 cm/s (Fig. 7) and in most cases the beetle actually 1 anded on the floor or wall. This depression of flight was evoked irrespective of the time of odor introduction, sex or diet regime. When vinegar was removed, the beetle usually re initiated flight (if landing had occurred) and its rate of climb was equal to its initial takeoff rate of climb; however, if landing had not occurred the beetle returned to its rate of climb just prior to food- odor introduction (Table 2). When vinegar was introduced repeatedly (up to five times) during a single flight, there was no indication that chemical cues acted in a cumulative manner; the beetle always returned to its previous rate of climb (Fig. 8). 24 Table 1. Horizontal and vertical displacement of Carpophilus hemipterus 0-36 min after takeoff in a vertical flight chamber during freeflight. Displacement (cm) Horizontal Vertical Mean + s.e. Mean + s.e. Time after takeoff (mini ______ 0-1 5.5 + 1.9 16.1 + 1.9 5-6 6.2 + 0.3 16.0 + 1.6 10-11 9.2 + 1.9 26.8 + 3.3 15-16 8.2 + 1.3 20.5 + 1.8 20-21 6.7 + 2.2 25.2 + 3.3 25-26 10.2 + 2.7 21.1 + 3.2 30-31 15.3 + 3.3 37.4 + 1.7 35-36 24.8 + 6.0 40.5 + 8.9 Figure 6. Mean rate of climb + s.e. for Carpophilus hemipterus maintained as adults with water or artificial diet (P<0.0001, except at 16 min P<0.05, paired t-te st, numbers above s.e. bars represent data points compared during each one min period). 25 iue 6. Figure Mean Rate of Climb (cm/s) 30 45 60 15 75 90 0 0 iso iso 70 5 120 40 ie fe tkof (min) takeoff after Time 20 100 0 80 20 SO 40 20 80 25 20 20 artificial diet water water 035 30 20 maintained 20 10 40 26 Figure 7. Representative strip-chart records with rate of climb (cm/s) during the free flights of Carpophilus hemipterus with food-odor introduction at 2 (a), 6 (b), 11 (c), and 22 (d) min after takeoff (C=blank control, V=apple cider vinegar). 27 28 xj 100 x j 100 C J ^ 75- 1 ^ 7 5 O ° • V - w ^ - -5 60 50 o E O w (0 a oc 25 DC 25 i mym 2 4 0 2 4 4 6 Time after takeoff (min) Time after takeoff (min) 1 100 2 5 75 OJ v-* o 50 CO T c 25 0 4 6 8 10 12 Time after takeoff (min) 100i 75- 50- 25- 12 14 Time after takeoff (min) J3 100- J o o e *3 DC 18 20 22 Time after takeoff (min) Figure 7. 29 Table 2. Rate of climb (cm/s) of Carpoohilus hemipterus measured for a 1 min period after takeoff, immediately preceding food-odor introduction, and following food-odor removal for 'landers' and 'hon- landers'1. Mean + s.e. Rate of Climb (cm/s) 'landers'' 'non-landers' At takeoff 58.1 + 4.4 a2 62.8 + 2.7 a Prior to food-odor introduction 43.2 + 3.1 b 53.0 + 5.4 b Following food-odor removal 58.6 + 4.8 a 55.8+4.5b landers - landed on the floor of the chamber when the food odor was introduced, non-landers - showed a depressed rate of climb but landing did not occur. 2Means followed by the same letter within a column are not significantly different (P<0.001 landers, P<0.05 non-landers; one-way ANOVA followed by Fisher's LSD). Figure 8. Representative strip-chart record with rate of climb (cm/s) during the free flight of Carpophilus hemipterus with multiple food-odor introductions (C=blank control, V=apple cider vinegar). 30 75 o E 60 n 4 8 8 Time after takeo ff (min) ja 100 C V g ~ 75 O to - ^ 6 0 L, o S <0 25 cc 0- 14 16 18 20 22 Time after takeoff (min) Figure 8. 32 Discussion The delay in flight activity of C. hemipterus until 3 days post- emergence is indicative of a teneral period during which hardening and darkening of the cuticle takes place, and maturation of enzymes and substrates necessary for flight occurs (Johnson, 1969). Flight activity increased dramatically 3 days after emergence for beetles maintained with water, while a comparable level of activity was not attained until Day 4 for beetles maintained with artificial diet. Dietary influence on the propensity for flight has been demonstrated in Sitona cvlindricollis Fahr. (Hans & Thorsteinson, 1961), Locusta spp. (Ellis & Hoyle, 1954), Phormia reoina (Meigen), and Musca domestica L. (Barton Browne & Evans, 1960), where starvation always led to an increase in locomotion and/or flight activity. Explanations for the influence of dietary regime on locomotion varied considerably, but the ecological implications would be similar; insects with low food reserves would be more likely to disperse. For C. hemipterus. this depression in flight propensity by available food was due to a reduction in the photokinetic response as evidenced by a lower rate of climb (Fig. 6). Egg development in C. hemipterus females did not reduce their propensity for flight as is the case in many migratory species, a phenomenon termed the "oogenesis-flight syndrome" (Rankin et a l., 1986). Our studies indicated that short flights are more likely the 33 norm in C. hemipterus. perhaps because their resources (dates, figs, and other mature fruit) are locally abundant but ephemeral. For nitidulid beetles, the ability to continue flight with a fully developed batch of eggs would be advantageous for exploiting this type of resource. C. hemipterus demonstrated a crepuscular flight periodicity with the major peak corresponding to dusk. Insects that fly at dawn and/or dusk avoid desiccation, harmful radiation or day-flying predators (Johnson, 1969). The timing of flight also is influenced by wind speed, which increases during the day and is comparatively low at dawn and dusk (Johnson, 1969). Insects displaying long-distance migratory flights probably take advantage of the increase in wind speed during the day while insects exhibiting vegetative fl ights may take advantage of lulls in the wind at dawn and dusk to enhance host location. Lewis and Taylor (1964) described the flight periodicities of approximately 400 insect taxa. After grouping these taxa into feeding guilds, they found that most species that feed on decaying organic matter (i.e. nitidulid beetles) fly at dawn and dusk, with a preference for dusk, primarily because of warmer temperatures. The flight behavior of C. hemipterus is similar in many respects to the flight behavior of aphids. Kennedy and Booth (1963a) described a typical flight in aphids as being aerodynamically stable but quite 'wild' at the start. After several minutes, the aphid settled into a relatively steady 'cruising' fl ight that lasted from several minutes to hours. During the 'cruising' flight, the rate of climb gradually declined and occasionally was interrupted by short fluctuations in vertical flight. A third phase was marked by an increase in horizontal excursions ('ranging'), and fluctuations in the rate of climb that were more downward than upward. Although C. hemipterus exhibited much shorter flights in comparison to aphids, the stages from flight initiation to termination were similar. However, when conflicting cues (light vs. food cues) were presented to a beetle, the response was quite different from that described in aphids. Kennedy and Ludlow (1974) demonstrated that early in a phototactic flight, aphids presented with visual host cues (a yellow card) increased their rate of climb to the overhead light, while those presented the yellow card later in flight showed a depression in vertical flight and landed on the card if allowed. These experiments gave rise to the behavioral principles of antagonistic induction and depression, which address how conflicting behavioral systems are resolved in the aphid nervous system. David and Hardie (1988) examined this same conflict using summer (virginoparae) and autumn (gynoparae) forms of Aphis fabae Scopoli. They found that virginoparous aphids approached a host cue (green light) within 30 s after takeoff and continued to approach the light with the same level of intensity irrespective of when the light was presented in the flight. Gynoparae, on the other hand, were inhibited from approaching the light until after a period of sustained flight. While David and Hardie (1988) did not address settling behavior, it appears that not all aphids exhibit antagonistic induction during their phototactic flight. The transition from phototactic to vegetative orientation in certain bark beetles has also been regarded as a physiological consequence of flight activity. Choudhury & Kennedy (1980) demonstrated that flight activity in Scolvtus multistriatus (Marsham) had a 'priming effect' on upwind orientation to host odors. However, Balfour & Paramonov (1962), and Atkins (1966) demonstrated that some bark beetles do not need to fly before host-orientation behavior is evoked. Perhaps more flexibility in response exists at both the individual and population level than was previously thought. Exposure of nitidulid beetles to food odors caused an immediate depression of light-oriented flight, irrespective of when the odor was presented. This depression was virtually absolute as it was almost always followed by a rapid drop in the rate of climb and usually by landing, but when the food odor was removed, takeoff usually followed within a few seconds. If landing occurred with the introduction, the rate of climb after takeoff was equivalent to the initial rate of climb; however, if landing did not occur the rate of climb was equal to the rate of climb just prior to food-odor introduction. The fact that there was no change in the rate of climb in the absence of landing indicates that antagonistic induction or depression were not operating in this behavioral system. The increased rate of climb in beetles that landed is more probably explained by a post-inhibitory rebound rather than recuperation from fatigue as the duration of landing was less than 1.5 min. Thus, while the availability of food (and presumably higher food reserves in the beetle) had a long-lasting effect of reducing photokinetic response, volatile food cues did not. A key element in Kennedy's (1985) behavioral definition of true migration requires the temporary inhibition of vegetative responses, the disinhibition of which occurs very slowly during a bout of locomotion. This is distinct from "foraging," which he defines as "reiterative locomotory activity that is readily interrupted by an encounter with a resource item of one particular kind." The flight of C. hemipterus falls readily into the latter category in which there is no evidence for a reciprocal interaction between phototactically modulated flight and host-finding flight, but rather where phototactic flight is inhibited by the presence of food odor irrespective of the beetles' behavioral history. CHAPTER II EFFECT OF PHYSIOLOGICAL STATE AND FUNGAL INOCULATION ON CHEMICALLY MODULATED HOST-PLANT FINDING BY CARPOPHILUS HEMIPTERUS AND CARPOPHILUS LUGUBRIS Introduction A fundamental question in studies of insect-plant interactions is how insects select their ovipositional and/or feeding sites from an almost uniimited array of potential hosts. Most attempts to answer this question have focused on feeding behaviors and have neglected the role of directed orientation to the plant from a distance. As a result, our understanding of long-range orientation is lacking and our ability to test hypotheses concerning the relationship between ecological parameters and strategies of host location is limited. Both Carpophilus hemipterus and Carpophilus lugubris are associated with mature to rotting fruits and vegetables; however, C. hemipterus has a cosmopolitan distribution and extensive host range, whereas C. lugubris is restricted to the New World and has been recorded on only a few hosts (Williams et a l., 1983, and references cited therein). It is suspected that volatiles released from the hosts of nitidul id beetles play an important role in host finding. Moreover, there is evidence that the rot-causing fungi associated with the decaying fruit may enhance the beetle's orientation. Wildman (1933), trapped more C. hemipterus with fungal-inoculated peaches and figs than with uninoculated fruit, but because no observations of behavior were made it is difficult to determine if the beetles' response was directed by long-range chemical cues. Miller and Mrak (1953), also showed that C. hemipterus was more inclined to respond to inoculated figs than to uninoculated figs, but their experimental design only required that the beetles respond from a distance of approximately 23 cm. The present study examined the effect of food odor in combination with fungal odors on long-range chemo-orientation (Kennedy, 1977) by C. hemipterus and C. lugubris. and also determined the physiological state (i.e ., age, diet, die! activity, and locomotory conditioning) resulting in the greatest host orientation. 39 Materials and Methods Horizontal Wind Tunnel Behavioral studies were conducted in a 2.5-m horizontal wind tunnel (Phelan et a l., in press). The tunnel floor was covered with a white sheet marked with randomly spaced 7.5-cm-diam red circles to provide optomotor cues for the beetles as they flew upwind; the back side of the tunnel was covered with a white sheet to aid observation. Three 60-W incandescent lights were controlled at 80 volts by a rheostat (Superior Elect. Co., Bristol, Conn.) to provide 17 lux measured at the tunnel floor, and windspeed was maintained at 0.4 m/s. Beetle Aoe and Response to Host Odors All studies used laboratory-reared beetles maintained as larvae with artificial diet (Hall et al., 1978; Peng & Williams, 1990) at 25 + 1°C, 65% r.h., on a 16L:8D light regime. To determine how age influenced response to host odors, 2- to 10-day-old C. hemipterus and C. lugubris were tested. From previous studies, apple cider vinegar was known to attract C. hemipterus (Blackmer & Phelan, in press), and whole wheat bread dough (WWBD) was found to be most attractive to C. 1uqubris in the field (Blackmer, unpublished data). Therefore, for this experiment, 0.25 ml of apple cider vinegar was presented to C. hemipterus on a 5.5-cm filte r paper (Whatman #1) held by an alligator clip located in the center of the upwind end of the tunnel 15 cm above the floor. Similarly, 30 g of freshly raised WWBD (24-h-old) placed inside a sterilized jar (240 ml) was positioned on the floor in the center of the upwind end of the tunnel for C. lugubris. Five or ten individuals (number of individuals/vial did not affect propensity for takeoff or upwind response; Blackmer, unpublished data) maintained with water as adults were put in 40-ml vials and preconditioned for 1 h in the wind tunnel. A vial containing beetles was placed in the odor plume 2.5 m downwind from the source, and the beetles were given 10 min to respond. The proportion of beetles taking off, and flying or walking to within 5 cm of the source, were recorded. The test was replicated five times for 2-, 9- and 10-day-old beetles or nine times for 3- to 8-day-old beetles for C. hemipterus. and six times for all age groups for C. lugubris. Fresh beetles were used for each replication and age group. The tunnel was maintained at 28 + 1°C and 29-49% r.h. Data are presented as means + standard error (s .e .). Pearson's product moment coefficient of correlation (r) was calculated for percent takeoff versus percent flights to source for C. hemipterus: r was not calculated for C. lugubris because they rarely flew in the horizontal tunnel. Paired t-tests were used to compare the proportion of beetles walking to the source versus flying to the source (Minitab, 1989). Periodicity of Host Finding Five- to 6-day-old C. hemipterus and 8- to 9-day-old C. lugubris (age when maximum host orientation was exhibited, and before significant mortality was observed) were maintained with water as 41 adults, and their propensity to orient to an odor source (WWBD) was measured at 2-h intervals from 16 h prior to scotophase to 2 h after the onset of scotophase; tests during scotophase were conducted under red light. Ten individuals where placed in 40-ml vials, preconditioned as described in the previous experiment, and given 10 min to respond to the odor source, with each time period replicated six times using fresh beetles. The tunnel was maintained at 28 + 1°C and 42-71% r.h. Data are presented as means + s.e. Pearson's product moment coefficients of correlation were calculated for percent takeoff versus percent flights to source, and for percent flights to source versus percent walks to source for C. hemipterus. Effect of Diet on Takeoff and Flights or Walks to Source Host orientation was measured in 5- to 6-day-old C. hemipterus maintained as adults with water only (n=15 groups of 10 fresh beetles), with artificial diet removed 36 h before the test (n=15 groups), or with diet removed 2 h before the test (n=12 groups), and in 8- to 9- day-old C. lugubris (n=4 groups of 10 beetles for each diet regime). The test protocol was identical to the previous experiment and the tunnel was maintained at 28 + 1°C and 25-56% r.h. Beetles were examined during their die! periods of maximum host-orienting activity (3 to 0 h before scotophase for C. hemipterus and either 14 to 12 h before scotophase or 2 to 0 h before scotophase for C. lugubris). Percentage of takeoff, flights, or walks to source were analyzed using a two-way analysis of variance (ANOVA) after arcsin(/y) transformation, and Fisher's Least Significant Difference (LSD) was used to separate 42 means. Effect of Locomotorv Opportunity on Takeoff and Flights or Walks to Source Beetles were maintained with artificial diet or water until 5- to 6-days of age for C. hemipterus and until 8- to 9-days of age for C. luaubris. Immediately prior to wind-tunnel testing, beetles were divided and one group was placed inside a 24-cm-diam x 53-cm-high Plexiglas® tube for 1 or 3 h, where they increased their locomotory activity in response to an overhead high-pressure sodium lamp. The second group of beetles were kept in 10-cm-diam x 3-cm-high containers and were also placed beneath the high-pressure sodium lamp; however, their locomotion remained restricted. In the first study, the flight behavior of C. hemipterus. maintained with water and having 0, 1 or 3 h of locomotory opportunity was compared. The second study examined the effect of restricting locomotory opportunity using C. hemipterus and C. lugubris maintained with water or artificial diet (36 h before the test). Both species were tested during their diel period of maximum host-orienting activity. A t-test, using arcsin(/y)- transformed values, was used in the first study to compare C. hemipterus having no locomotory opportunity with those having 1 or 3 h of locomotory opportunity. A two-way ANOVA was used in the second test to determine if there were differences in host-orienting propensity between beetles that had been restricted and beetles that had received 3 h of locomotory opportunity and were maintained with artificial diet or water. 43 Response to Aseptic and Inoculated Substrates Six substrates were used to compare the host-finding response of C. hemipterus and C. lugubris and to determine the role of fungi in eliciting host finding: corn, strawberry, tomato, banana, prune, and WWBD. Approximately seven days before the bioassay, the fruits and vegetables were weighed to 30 g, transferred to a laminar-flow clean bench unit (Pure Aire Corp, Calif. #72013-130) where each substrate was placed in 70% alcohol, then transferred to a 10% solution of NaOCl disinfectant, which was followed by a rinse in sterilized, doubly distilled water. Each substrate was immediately placed inside a sterilized 240-ml wide-mouth jar whose lid was equipped with a bacterial air-vent (Gelman #4210), which allowed air exchange, but prevented microbial contamination. Substrates remained aseptic for many weeks in these containers. Inoculated treatments received surface smears of Saccharomvces cerevisiae var Montrochet1 Hansen or Candida krusei2 (Castellani) Berkhout before being sealed. The jars were placed inside an environmental chamber at 25°C with 16L:8D light regime until the beginning of the bioassay. Frozen WWBD was placed in jars approximately 12-24 h before the bioassay and held at 25°C. The two fungi were maintained on yeast-malt agar (Difco #0712-01-8), at 25 + 2°C with a 10L:14D light regime, and were transferred to fresh agar plates every 7-14 days as needed. During the bioassay, substrates were tested individually in a randomized complete-block design, by opening 1Source: J. Gallander, Department of Horticulture, 0ARDC - Ohio State University, Wooster, Ohio, USA. 2Source: American Type Culture Collection, Rockville, Maryland, USA. 44 a jar and placing it in the center of the upwind end of the tunnel. Five- to 6-day-old C. hemipterus and 8- to 9-day-old C. lugubris maintained with water as adults and with restricted locomotory opportunity were placed in 40-ml vials in groups of 10. The bioassay was repeated five times for C. hemipterus and seven times for C. lugubris, and new beetles were used for each replication. Beetles were tested during their die! period of maximum activity using the previously described protocol. The tunnel was maintained at 26 + 1°C and 43-52% r.h. The data were transformed by arcsin(Vy) and analyzed by a two-way ANOVA with substrates (excluding WWBD) as one factor and inoculation treatments as the second factor. To compare substrate preference, means for the six substrates, including WWBD were analyzed by ANOVA and separated by LSD. 45 Results Beetle Age and Response to Host Odor Both species responded to food odors beginning on Day 3 by walking to the source (Fig. 9), and these walks were continual and directed up the center of the odor plume. Flight activity and flights to the odor source increased on Day 4 for C. hemipterus, but C. lugubris rarely flew to the source regardless of age. For C. hemipterus. upwind walking remained at about the same level from Days 3 to 10 and only exceeded flights to source on Day 3, but for C. lugubris there was a steady increase in walking response up through Day 9. C. hemipterus flew to the odor source more frequently than it walked (t=3.06, P<0.01, df=12), whereas C. lugubris almost always walked to the source (t=98.36, PcO.OOl, df=14). Regardless of how the beetles arrived at the source, maximum response occurred from 6 to 9 days after emergence for both species. There was a strong linear correlation (r=0.94, P<0.Q1) between the proportion of C. hemipterus taking off and the proportion flying to the source, with 44% of the takeoffs resulting in flights to the source. Periodicity of Host Finding Both C. hemipterus and C. lugubris displayed bimodality in host- orienting behavior during the photophase. C. hemipterus exhibited a peak in activity from 12-8 h before scotophase and from 2-0 h before Figure 9. Percentage (+ s.e.) of 2- to 10-day-old Carpophilus hemipterus and C. lugubris maintained with water only as adults, taking off (solid line), flying to the source (diagonally-slashed bar), and walking to the source (cross- hatched bar). 46 iue 9. Figure Response (%) Response (%) 100 0 0 1 40 20 60 80 20 40 60 80 0 l s i r b u g u l s ilu h p o p r a C 9 8 7 6 5 4 3 2 l s u r e t p i m e h s ilu h p o p r a C 9 8 7 6 5 4 3 2 g (days) Age 10 10 47 48 scotophase (Fig. 10). A significant linear correlation (r=0.67, P<0.05) existed between the proportion of C. hemipterus taking off and the proportion flying to the source, and between the proportion flying to the source and the proportion walking to the source during photophase (r=0.72, P<0.05). No flights to the odor source were observed after the onset of scotophase. C. lugubris displayed a peak in activity from 14-12 h prior to scotophase and from 2-0 h prior to scotophase (Fig. 10). Both species exhibited a depression in response to host odors during mid- to late day; however, this depression began 4 h earlier for C. lugubris. Effect of Diet on Takeoff and Flights or Walks to Source Takeoff propensity in C. hemipterus maintained with water was not different from that of beetles maintained with artificial diet if the latter were starved for 36 h before the test; however, there was a reduction in takeoff if these beetles were maintained with artificial diet until 2 h before the test (Table 3). C. hemipterus maintained with water flew to the odor source more frequently than did either group of beetles maintained with artificial diet, and beetles starved for 36 h before the test flew to the source more often than did beetles starved for only 2 h. C. lugubris maintained with water walked to the source more frequently than did beetles maintained with artificial diet until 36 h before the test. Figure 10. Percentage (+ s.e.) of Carpophilus hemipterus and C. 1uqubris maintained with water only as adults, flying or walking to the source from 16 h prior to scotophase to 2 h after the onset of scotophase. 49 iue 10. Figure Response to Source (%) Response to Source (%) 20 40 80 20 40 60 80 16 614 16 l s u r e t p i m e h s ilu h p o p r a C us lugubris i r b u g u l s lu N p o p r a C 14 12 12 10 10 ie (h) Time 8 8 6 6 4 4 2 2 0 0 2 2 Table 3. Percent takeoff and flights to source for Carpophilus hemipterus, and percent walks to source for C. lugubris in a wind tunnel, maintained as adults with water, or with artificial diet until 36 h or 2 h before the experiment. C. hemipterus C. lugubris Dietary regime Takeoff (%) Flights to N1 Wal ks to N1 ± s.e. Source {%) + s.e. Source {% ) ± s.e. Water 43.5 + 5.5 a2 23.8 + 6.2 a 15 56.9 + 8.6 a2 4 Artificial diet until 36 h before test 42.7 + 5.7 a 11.5 + 3.4 b 15 2.5 + 2.5 b 4 Artificial diet until 2 h before test 6.2 + 2.4 b 0.9 + 0.9 c 12 ------ 1Groups of 10 beetles each 2Means within a column followed by the same letter are not significantly different (p<0.001 for takeoff, p<0.05 for flights to source for C. hemipterus. and p<0.0001 for C. lugubris; ANOVA followed by LSD). 52 Effect of Locomotory Opportunity on Takeoff and Flights or Walks to Source In the firs t study, where C. hemipterus was allowed 1 or 3 h of locomotory opportunity, no effect was observed in takeoff propensity (t=0.79, P=0.44, df=20 for 0 vs. 1 h [59 + 5% vs. 64 + 6%, respectively], and t= l.87, P=0.07, df=35 for 0 vs. 3 h [34 + 5% vs. 20 + 4%, respectively]) or in flights to source (t= l.05, P=0.3, df=31 for 0 vs. 1 h [46 + 6% vs. 37 + 5%, respectively], and t= l.44, P=0.16, df=35 for 0 vs. 3 h [20 + 3% vs. 12 + 3%, respectively]). In the second study, where movement was restricted or 3 h of locomotory opportunity was allowed for C. hemipterus maintained with artificial diet or water, locomotory opportunity significantly reduced takeoff propensity (F=5.86, P<0.03, df=l,18). However, dietary regime did not significantly affect takeoff (F=l.07, P>0.30, df=l,18), nor was there a significant interaction between locomotory opportunity and dietary regime (Table 4; F=0.95, P>0.30, df=l,18). Flights to the source by C. hemipterus were significantly increased by starvation (F=4.89, P<0.04, df=l,18), but were not significantly affected by locomotory opportunity (F=0.92, P>0.30, df=l,18), nor was there an interaction between these two factors (F=1.44, P>0.25, df=l,18). Starvation strongly increased upwind response to host odors by C. lugubris (F=60.10, P<0.001, df=l,9), whereas locomotory opportunity did not influence upwind response (F=0.66, P>0.45, df=l,9) (Table 4). A significant interaction existed between dietary regime and locomotory opportunity (F=5.97, P<0.04, df=l,9), in that unrestricted (3 h) locomotory opportunity increased the proportion of beetles that walked Table 4. Percent takeoff and flights to source for Carpophilus hemipterus. and percent walks to source for C. lugubris in a wind tunnel, maintained with water or artificial diet as adults and given 0 or 3 h of locomotory opportunity in a vertical flight chamber. C. hemipterus C. lugubris Diet Locomotory Takeoff (%) Flights to Walks to Regime opportunity + s.e. Source (%) + s.e. Source (%) + s.e. Water restricted 63.4 + 7.1 a1 29.7 + 4.6 a 60.0 + 5.8 a Water unrestricted 52.6 + 6.2 ab 31.9 + 4.7 a 47.5 + 6.3 a Diet restricted 63.1 + 6.4 a 20.6 + 6.3 ab 2.5 + 2.5 c Diet unrestricted 36.9 + 5.2 b 6.1 + 2.8 b 14.6 + 5.7 b ^eans within a column with the same letter are not significantly different (p<0.05, n-7 groups of 10 beetles for C. hemipterus. and p<0.05, n=4 groups of 10 beetles for C. lugubris) : two-way ANOVA followed by LSD. cn CO 54 to the source if maintained with artificial diet but not if maintained with water. Response to Asebtic and Inoculated Substrates Fungal inoculation of substrates significantly enhanced upwind response by C. hemipterus to tomato and S. cerevisiae-inoculated corn (Fig. 11); for C. lugubris. upwind response was enhanced by fungal inoculation in every substrate except prune (Fig. 12). When substrates were combined for the three inoculation treatments, substrates inoculated with S. cerevisiae were more attractive to C. hemipterus than were aseptic substrates (51.2 + 9.1 vs. 33.5 + 6.9%; F=4.04, P<0.02, df=2,56), but those inoculated with C. krusei were not significantly different from aseptic treatments ( 43.5 + 9.2 vs. 33.5 + 6.9%, P>0.1), and there was no interaction between these two factors (F=0.96, P>0.40, df=8,56). Both inoculation treatments were more attractive to C. lugubris than was the aseptic treatment (27.4 + 5.3% C. krusei-inoculated. 33.2 + 8.0% S. cerevisiae-inoculated, and 8.6 + 3.2% aseptic; F=42.96, P<0.001, df=2,84), and there was no interaction between the substrates and inoculation treatments (F=1.66, P>0.12, df=8,84). S. cerevisiae inoculations did not differ in attractancy from C. krusei inoculations for either beetle species. C. hemipterus exhibited little preference between substrates, but C. lugubris displayed a preference for WWBD over all other substrates (Table 5). Figure 11. Percentage (+ s.e.) of Carpophilus hemipterus maintained with water only as adults, that flew or walked to corn, strawberry, tomato, banana, prune, and whole wheat bread dough. Comparisons are among aseptic and inoculated fruit for each substrate; letters that are the same are not significantly different using LSD (P>0.05). 55 iue 11. Figure Response to Source (%) 100 20 40 60 80 0 Corn e tic sep A raw- To t Baaa ue ead d a re B rune P anana B ato om T - w a tr S r Dough rry e b krusei e s u r k . C Inoculated - - cerevisiae- e a i s i v e r e c . S noc lated cu o In 4 a T- 56 Figure 12. Percentage (+ s.e.) of Carpophilus lugubris maintained with water only as adults, that flew or walked to corn, strawberry, tomato, banana, prune, and whole wheat bread dough. Comparisons are among aseptic and inoculated fruit for each substrate; letters that are the same are not significantly different using LSD (P>0.05). 57 iue 12. Figure Response to Source (%) 100 40 60 80 20 0 r Staw- mao nn Pr Br d a re B e n ru P anana B ato om T - w tra S orn C e i H tic sep A | r ough D rry e b C.krusei- Inoculated S. cerevisiae S. noc lated cu o In 58 59 Table 5. Percent response (+ s.e.) to aseptic and fungal-inoculated substrates, or whole wheat bread dough (WWBD) in a wind tunnel by Carpophilus hemipterus and C. luaubris. C. hemipterus C. luaubris Substrates Corn 50.6 + 6.9 a1 36.1 + 5.7 b Strawberry 52.2 + 7.4 a 16.8 + 4.1 c Tomato 49.9 + 6.2 a 20.6 + 4.8 c Banana 49.3 + 7.6 a 26.1 + 4.2 be Prune 11.6 + 3.2 b 2.2 + 1.0 d WWBD 56.0 + 8.7 a 67.1 + 9.2 a 1Means within a column with the same letter are not significantly different; ANOVA followed by LSD (P<0.05). Discussion Behavior is often a consequence of the physiological state of an organism. C. hemipterus and C. luaubris displayed noticeable changes in behavior with age, during the diel period, and when maintained with different dietary regimes. Both species were unresponsive to food odors until three days after emergence when walking to the odor source began. Four days after emergence, an increase in flights to source was observed for C. hemipterus. with no noticeable increase in walking response. C. luaubris rarely flew to the source, but did exhibit an increase in walking response until Day 10. It appears that in C. hemipterus the olfactory system matures prior to flight readiness, but once the capacity for flight is realized it is preferred to walking (66% vs. 34%), whereas C. luaubris almost always favored walking (1% vs. 99%) (at least under the conditions of our tests). Although flight can be risky, the benefits of finding a host in less time are obvious. One risk in flight is that the insect may be lifted above the odor plume and lose contact, but C. hemipterus lessens the chance of this by reducing its flight activity during mid-day (Blackmer & Phelan, in press), the period of maximum atmospheric movement (Johnson, 1969, and references cited therein). It is possible that C. luaubris did not exhibit flight in this study because a windspeed of 0.4 m/s inhibits takeoff. The fact that C. luaubris exhibited a longer period of suppressed host-orienting behavior at mid-day in comparison to C. 61 hemipterus is consistent with this idea. Furthermore, in a previous study, C. luaubris had demonstrated a much greater propensity for takeoff (38.3 + 11.1%) when they were tested in a vertical flight chamber where the airspeed was in itially 0 m/s (Blackmer, unpublished data). Lewis & Taylor (1964) found that species that were associated with fungi often flew at dawn and dusk when air was least turbulent and most humid. Both species were less responsive to food odors when supplied with a food source than when starved, and C. hemipterus was even reluctant to takeoff if recently deprived. Because there were no differences in takeoff propensity in C. hemipterus receiving water or artificial diet up to 36 h before the experiment, the reduction in flights to source (or walks to source in the case of C. lugubris) might be related to differences in behavioral threshold levels with regards to host acceptability. In nature, insects with ample food are generally reluctant to disperse and only do so as their resource becomes depleted (Johnson, 1969). During their dispersal flight and until they encounter their next resource, host acceptability changes; hosts that were only minimally attractive when the insect was amply nourished become acceptable (Dethier, 1982). Supposedly this occurs because the internal milieu is changing as a result of food deprivation, which leads to a change in behavioral threshold levels (Miller & Strickler, 1984). Kennedy (1965) and Choudhury & Kennedy (1980) demonstrated that in aphids and bark beetles, respectively, orientation to host cues (either visual or chemical) usually occurred only after a period of phototactic flight. Furthermore, Kennedy & Ludlow (1974) demonstrated that early in a phototactic flight, aphids presented with a yellow card increased their rate of climb to an overhead light, while those presented the yellow card later in flight showed a depression in vertical flight and landed on the card if allowed, phenomena termed antagonistic induction and antagonistic depression, respectively. Using a vertical flight chamber similar to that of Kennedy (1965), we previously found that exposure of C. hemipterus to food odors caused an immediate depression of light-oriented flight, irrespective of when the host odor was introduced (Blackmer & Phelan, in press). In the present study, I expanded this investigation to determine the effect of 1ocomotory exercise on the propensity of beetles to takeoff and to orient to food odors. C. hemipterus did not demonstrate an increased propensity to respond to the odor source after 1ocomotory experience, regardless of diet regime, and C. luqubris only showed an increased propensity to respond to the odor source after 1ocomotory experience if maintained with diet. We are unable to provide an explanation for this difference between the two species. Perhaps differences in rate and form of locomotion during the period of 1ocomotory opportunity affected the response observed. In either case, the resources used by these species, i.e., mature to decaying fruits and vegetables are locally abundant but ephemeral, and it seems plausible that an obiigatory dispersal period might be detrimental as it would take them away from their food source. The role that rot-causing fungi and bacteria play in host orientation has not received much attention in insect-plant interactions. The few cases that have been documented clearly illustrate that these microorganisms can have an important function in host location. The onion fly, Hvlemva antioua (Meigen), displays an increased attraction to onions inoculated with either Fusarium or other bacteria relative to uninoculated onions (Dindonis and Miller, 1980). Similar associations with microorganisms are seen in the seedcorn fly, H. piatura (Meigen), in which oviposition is stimulated by microbial volatiles emitted during germination of the seed (Eckenrode et a l ., 1975). Fungi apparently play an important role in resource location by a number of stored-product beetles such as Cvnaeus anqustus (Leconte), which displayed a greater attraction to corn flour infected with Cladosporium than to uninfected flour (Kao et al., 1984). Similarly, Tribolium confusum Jacquelin duVal, has been found in aggregations associated with Nigrosoora in food stores (Starratt and Loschiavo, 1971), and three species of stored-grain beetles have shown better development on sorghum molded by Aspergillus (Wright and Burroughs, 1983). Conogethes ounctiferal is (Guenee), the yellow peach moth, also displays a significant attraction and ovipositional preference to nonpathogenic and phytopathogenic fungi (Honda, et a l., 1988). The present study represents the first investigation demonstrating a long- range preference to inoculated fruit versus aseptic fruit by nitidulid beetles. Dowd & Van Middlesworth (1989), demonstrated that C. hemipterus larvae were able to metabolize 4-monoacetoxyscirpenol (a class of mycotoxins produced by several species of fungi) at ten times the rate of non-fungus-feeding caterpillars and they suggested that this species may possess a metabolic adaptation for feeding on fungi. Preliminary studies, in our laboratory, indicate that fungal-inoculated fruit may enhance larval growth (H. Lin, unpublished data), suggesting an adaptive function underlying the adult beetle's preference for fungal-inoculated substrates. CHAPTER III CHEMICAL 'GENERALISTS' AND BEHAVIORAL 'SPECIALISTS': A COMPARISON OF HOST FINDING BY STELIDOTA GEMINATA AND STELIDOTA OCTOMACULATA Introduction Nitidulid beetles are capable of causing significant economic damage through feeding and by the transmission of plant-pathogenic fungi. Stelidota geminata (Say) and S. octomaculata (Say) are two important economic pest species, although under different situations. The former species is primarily an agricultural pest (Gertz, 1968), whereas the latter has been implicated in the failure of oak-stand regeneration projects (Galford, 1987). S. geminata is distributed throughout the Neotropical and Nearctic regions and has an extensive host range, whereas S. octomaculata is found only in the Nearctic regions and is restricted in its feeding almost exclusively to acorns (Table 6). L ittle is known about nitidulid beetle host finding, although recently our laboratory has begun to discern the cues that elicit host- finding behaviors in these beetles. For example, we established that for Carpophilus hemipterus and Carpophilus luoubris. certain hosts e lic it greater olfactory response than others, and that host attractancy was usually enhanced by fungal inoculation for both of these species (Blackmer & Phelan, submitted a). Phelan & Lin (in 65 66 press) and Lin & Phelan (submitted) showed that the greater behavioral response to fungal inoculation was due primarily to a quantitative increase in host volatiles, rather than due to a production of unique fungal metabolites. The present study examined the role of fungi in host orientation by S. geminata and S. octomaculata. and compared their olfactory response to a range of food substrates. Behavioral differences in host-finding tactics are also presented and the findings are discussed in relation to the current hypotheses regarding host specialization. 67 Materials and Methods Behavioral studies were conducted in a 2.5-m horizontal wind tunnel (Phelan et a l., in press), or in a vertical flight chamber (Blackmer & Phelan, in press). The floor of the horizontal wind tunnel was covered with a white sheet marked with randomly spaced 7.5-cm-diam red circles to provide optomotor cues for flying beetles; the back side of the tunnel was covered with a white sheet to aid observation. Three 60-W incandescent lights were controlled at 80 volts by a rheostat (Superior Elect. Co., Bristol, Conn.) to provide 17 lux measured at the tunnel floor, and windspeed was maintained at 0.4 m/s. All studies used laboratory-reared beetles maintained as larvae with artificial diet (Hall et al., 1978; Peng & Williams, 1990) at 25 + 1°C, r.h. 65%, on a 16L:8D light regime. Die! Periodicity and Response to Light Quality in a Vertical Flight Chamber Die! periodicity in phototactic-flight activity was determined for 5- to 7-day-old S. geminata and S. octomaculata by measuring flight response at 2-h intervals from 16 h before the onset of scotophase to 2 h after when the onset of scotophase normally would have occurred. Immediately prior to testing, beetles in groups of 10 were placed in 40-ml vials and held in the vertical flight chamber for 1 h. After this preconditioning period, the vial containing the beetles was placed 68 on a flat-black platform 15 cm above the chamber floor, and beetles were allowed 5 min to respond to the overhead light source. A high- pressure sodium lamp (major emission peaks at 490, 560, 580, 595, 615 nm) or a mercury-vapor lamp (major emission peaks at 360, 405, 435, 545, 575 nm) was placed above the flight chamber and the propensity for each species to take off was recorded. Five and four replications were conducted using the high-pressure sodium lamp or the mercury-vapor lamp, respectively; the chamber was maintained at 26 + 2°C, r.h. 39- 71%. A general linear model (GLM) (Minitab, 1989) on arcsin(/y)- transformed values (in which y was the proportion of beetles responding to the overhead light) was used to determine if flight response was affected by light quality or time of day. Diel Periodicity of Host Finding Diel periodicity in host-finding response was determined for 5- to 6-day-old S. geminata and S. octomaculata by measuring flight or walking response at 2-h intervals from 16 h before the onset of scotophase to 2 h after the onset of scotophase; tests during the scotophase were conducted under red light. Ten beetles were placed in 40-ml vials and given 10 min to respond to aseptic banana which was placed in the center of the upwind end of the horizontal wind tunnel. Each time period was replicated four times for each species. The tunnel was maintained at 26 + 2°C and 35-45% r.h. Pearson's product moment coefficient of correlation was calculated for percent takeoff versus percent flights to source for S. geminata. and an analysis of 69 variance (ANOVA) on arcsin(/y)-transformed values was used to determine if response differed because of time of day for either species. Response to Food Odors S. geminata and S. octomaculata orientation to food odors was compared in the horizontal wind tunnel using seven food substrates (banana, strawberry, corn, tomato, fig, germinating red-oak acorns and black-oak acorns) and four fungi on agar (Saccharomvces cerevisiae var Montrochet1 Hansen, Candida krusei2 [Castellani] Berkout, Oudemansiella radicata3 [Relhan ex Fries] Singer, and Ceratocvstis faqacearum4 [Bretz] Hunt). The two yeasts, S. cerevisiae and C. krusei. were maintained on yeast-malt agar (Difco #0712-01-8) and the two wood fungi, C. faqacearum and 0. radicata. were maintained on potato-dextrose agar (Difco #0013-01-4), at 25 + 2°C, 10L:14D light regime. All fungi cultures were transferred to fresh agar plates every 7-14 days as needed. To determine the role of fungi in host finding, food substrates (excluding acorns) were held aseptically or were inoculated with S. cerevisiae or C. krusei. Approximately 4 to 7 days before the experiment, the substrates were surface sterilized (Blackmer & Phelan, 1Source: J. Gallander, Department of Horticulture, 0ARDC - Ohio State University, Wooster, Ohio, USA. 2Source: American Type Culture Collection, Rockville, Maryland, USA. 3Source: E. Dorworth, USDA - Forest Service, Madison, Wisconsin, USA. 4Source: J. Stipes, Department of Plant Pathology and Physiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA. 70 submitted) and placed into 240-ml wide-mouth jars. The jars were kept inside an environmental chamber until the beginning of the bioassay at 25°C with a 16L:8D light regime. During the bioassay, substrates were tested individually in a randomized complete-block design (n=6 for S. octomaculata and n=7 for S. geminata for each fruit or vegetable substrate [aseptic and inoculated]; n=4 for acorns and fungi on agar), by opening a jar and placing it in the center of the upwind end of the tunnel. Five- to 7- day-old S. octomaculata and S. geminata maintained with water as adults were placed in 40-ml vials in groups of 10, preconditioned for 1 h and then released 1 m downwind from the odor source. All tests were conducted from 3 h prior to the onset of scotophase to 1 h after when the onset of scotophase would normally have occurred. The tunnel was maintained at 26 + 1°C, r.h. 34-67%. Percent beetle response to inoculation treatments was analyzed by a two-way analysis of variance (ANOVA) on arcsin(/y)-transformed values with substrates and inoculation treatments as factors. To compare substrate preference, all substrates, including the acorns, yeasts, and wood fungi, were analyzed by a GLM, and means were separated by LSD when a significant F-value occurred. Determination of Behavioral Differences Twelve S. octomaculata and 16 S. geminata were videorecorded as previously described (Blackmer & Phelan, in press) as they responded to aseptic banana placed in the center of the upwind end of the horizontal wind tunnel. Beetle tracks were transcribed onto acetate sheets using 71 the playback system on the video cassette recorder and calculations were made on rate of locomotion, rate of turning (>45° change in direction), distance traveled, number of pauses exceeding 5 s, and duration of pauses. Distance traveled was determined using an Inch Counter™, a hand-held instrument for measuring distances on maps. Each 1ocomotory tra it of walking S. octomaculata and S. geminata. or of flying versus walking S. geminata was compared using a paired t-te st (Minitab, 1989). The change in rate of locomotion as the beetles approached the source was analyzed by a GLM. 72 Results Diel Activity and Response to Light Quality in a Vertical Flight Chamber S. geminata displayed a similar diel pattern in its response to the two light sources (Fig. 13); however, the beetles overall flight propensity was significantly greater to the mercury-vapor lamp than to the high-pressure sodium lamp (F=4.68, P<0.04, df=l,66). A gradual, but steady increase in takeoff occurred with maximum response occurring from 6 to 0 h before the beginning of scotophase. S. geminata flight response was significantly greater during this 6 h period than during the preceding 6 h period (F=5.54, P<0.005, df=9,66). S. octomaculata never took flight regardless of the light source or the time of day. Die! Periodicity of Host Finding Neither S. geminata nor S. octomaculata displayed distinct periods of host orientation during the photophase (Fig. 14). S. geminata exhibited no significant differences in its activity levels during the diel period (F=2.09, P=0.07, df=9,27), and although S. octomaculata did display periods of greater activity (F=3.17, P=0.01, df=9,27), host orientation oscillated throughout the day. When the proportion of walking versus flying S. geminata was compared there was a significant negative correlation (r=-0.47, P=0.002, df=39). Host orientation in the form of walking was 73 Table 6. Reported hosts of Stelidota geminata and S. octomaculata Host ______Reference S. geminata: Decaying oranges Armstrong (1976)* Fresh tree wounds & oak wilt Dorsey & Leach (1956) Stored rice Douglas (1941)* Pig carrion Payne & King (1970)* Sapote & figs USDA (1959)* Sweet corn " (1963)* Tomato " (1967)* Fermenting apples & melons Vogt (1950-51)** Acer sp. flowers on ground Connell (1956)** Ouercus sp., weevil-infested acorns II Hibiscus sp., flowers II Podophyllum peltatum fruit II Maclura pomifera. fallen fruit It Strawberry II Peach II Grape (Vitis sp.) II Pea, under lodged vines II Vaccinium sp., flowers n Pear Gertz (1968) Cherry tl Dewberry II Raspberry Blackmer (unpubl.data) Cantelope II Fungi: Lactarius pjperatus Moennich (1939)* Panus strigosus (1941)* Polyporus chinoeus Weiss & West (1920)* Stelidota octomaculata: Red-oak acorns Winston (1956) Rotten apples Gibson & Carrillo-S. (1959)* * cited in Williams et a l. (1983) ** cited in Gertz (1968) Figure 13. Mean percent takeoff (+ s.e.) by Stelidota geminata in a vertical flight chamber, from 16 h prior to scotophase to 2 h after when the onset of scotophase would normally have occurred. 74 iue 13. Figure Mean % Takeoff 20 40 60 16 ie ro t Soohs (h) Scotophase to Prior Time 14 I—I. Mruyvpr lamp Mercury-vapor - - 12 ihpesr lamp High-pressure 10 8 6 4 2 0 2 75 Figure 14. Percentage (+ s.e.) of Stelidota geminata (solid line) and S. octomaculata (dashed line) flying and/or walking to the odor source from 16 h prior to scotophase to 2 h after the onset of scotophase. 76 iue 14. Figure Mean % Response 100 40 20 60 80 16 ie ro t Soohs (h) Scotophase to Prior Time 14 12 10 8 6 4 2 0 v 2 78 greatest at 14 h prior to the onset of scotophase and declined thereafter, whereas flight activity was greatest from 6-0 h prior to scotophase. Response to Host Odors Fungal inoculation of fruit and vegetable substrates did not significantly enhance upwind response by S. geminata (Fig. 15; F=2.29, P>0.10, df=2,81), or by S. octomaculata (Fig. 16; F=1.18, P>0.30, df=2,70). Furthermore, with the exception of banana versus 0. radicata and C. fagacearum for S. octomaculata, neither beetle species displayed a preference among food odors (Table 7; S. geminata F=1.93, P>0.05, df=10,42; S. octomaculata F=2.5, P<0.02, df=10,38). Determination of Behavioral Differences Typical tracts of walking S. octomaculata and S. geminata. and of flying S. geminata are illustrated in Fig. 17. In all cases, the responses were generally directed up the center of the odor plume, but the beetles often alternated from side-to-side of the center line. S. octomaculata walked upwind at a much slower rate than did walking S. geminata, and S. geminata flew upwind to the source at a significantly greater rate than did beetles that walked (Table 8). This difference in rate of locomotion was not influenced by distance traveled, as there were no significant differences in this variable (t=1.43, P=0.2, df=22, for walking S. geminata versus S. octomaculata and t=0.8, P=0.5, df=14, for walking versus flying S. geminata) . S. octomaculata turned more frequently than did S. geminata. and S. geminata that walked to the Figure 15. Percentage (+ s.e.) of Stelidota geminata that flew or walked to banana, strawberry, corn, tomato, fig, Candida krusei on YM-agar, Saccharomvces cerevisiae on YM-agar, Ceratocvstis faqacearum on PDA-agar, Oudemansiella radicata on PDA-agar, black-oak acorn or red-oak acorn. 79 iue 15. Figure Response to Source (%) Response to Source (%) 100 100 20 40 60 80 Aseptic inoculated C L kru sei - sei CLkru S. cerevisiae S. inoculated Figure 16. Percentage (+ s.e.) of Stelidota octomaculata that walked to banana, strawberry, corn, tomato, fig, Candida krusei on YM-agar, Saccharomvces cerevisiae on YM-agar, Ceratocvstis fagacearum on PDA-agar, Oudemansiella radicata on PDA-agar, black-oak acorn or red-oak acorn. 81 iue 16. Figure % Walking to Source % Walking to Source 100 100 IH Aseptic / / J r r J / / .kr ei- se ru k C. inocutated 4 ? * eeii e cerevisia . S inoculated 83 Table 7. Mean percentage (+ s.e.) of Stelidota aeminata and S. octomaculata responding to food odors. S. aeminata S. octomaculata Substrate: Banana 26.4 + 4.5 a 37.2 + 7.4 a Strawberry 27.9 + 5.5 a 32.9 + 8.4 ab Corn 19.5 + 4.8 a 31.9 + 4.8 ab Red-oak acorn 33.2 + 2.9 a 22.3 + 9.6 ab Tomato 20.8 + 3.9 a 23.8 + 4.9 ab Fig 18.6 + 3.3 a 23.4 + 5.6 ab Candida krusei 17.8 + 8.5 a 14.6 + 5.2 ab Saccharomvces cerevisiae 30.0 + 9.1 a 13.6 + 5.0 ab Black-oak acorn 10.0 + 5.8 a 12.7 + 4.5 ab Oudemansiella radicata 11.0 + 4.5 a 7.4 + 2.4 b Ceratocvstis faaacearum 12.1 + 5.1 a 7.0 + 4.4 b *Means within a column with the same letter are not significantly different, P>0.05, LSD Figure 17. Representative tracks for Stelidota octomaculata walking to aseptic banana (O.s) and S. qeminata a) walking and b) flying to aseptic banana located 50 cm upwind in a horizontal wind tunnel. 84 85 Ste/idota octomacu/ata Stef/dota geminata a Figure 17. Table 8. Mean (+ s.e.) rate of locomotion, distance traveled, number of turns > 45°, number of pauses > 5 s, and duration of pauses for Stelidota oeminata walking or flying and for S. octomaculata walking to aseptic banana located 50 cm upwind in a horizontal wind tunnel. S. aeminata S. octomaculata Parameters measured flvina fn=4) walkina fn=*12> walkina (n=121 Rate of locomotion (cm/s) 23.49 + 1.30° 0.54 + 0.02a 0.16 + 0.01s Distance traveled 71.75 + 8.65n‘s' 65.81 + 3.30 74.15 + 4.78 Number of turns (per cm) 0.18 + 0.04b 0.26 + 0.02b 0.39 + 0.04b Number of pauses (per cm) — 0.009 ± 0.004c 0.03 + 0.01c Duration of pauses (s) — 13.48 + 2.26n‘s" 13.45 + 2.63 at«13.46, P<0.005, df-23 for S. aeminata (walking) versus S. octomaculata and t=34.53, P<0.005, df=15 for S. aeminata walking versus flying bt=2.89, P<0.005, df=22 for S. aeminata (walking) versus S. octomaculata and t= l.83, P=0.09, df=14 for S. aeminata walking versus flying ct=1.9, P=0.07, df=21 for S. aeminata (walking) versus S. octomaculata 87 source tended to turn more frequently than did S. aeminata that flew to the source; however, the latter difference in turning was not statistically significant. Although S. octomaculata was more likely to pause on the Way to the odor source than was S. aeminata. the difference was not statistically significant (P=0.07), nor was the duration of these pauses different between the two species. There were no significant differences in rate of walking at different distances from the odor source (Fig. 18; F=2.01, P>0.1, df=4,25, S. octomaculata: F=0.99, P>0.4, df=4,31, S. aeminata) . Figure 18. Mean rate of walking (+ s.e.) for Stelidota geminata (dashed line) and S. octomaculata (solid line), 50 to 0 cm from the odor source. 88 iue 18. Figure Rate of Walking (cm/s) 0.00 0.20 0.40 0.60 0.80 40 -4 0 5 - itne rm ore (cm) Source from Distance 40-30 30-20 20-10 -0 0 1 89 Discussion S. aeminata displayed similar diel patterns of response to mercury-vapor and to high-pressure sodium lamps (with peaks in flight activity from 6-0 h before the onset of scotophase). However, the cumulative response of S. geminata over the diel period was greater to the shorter-wavelength mercury-vapor lamp, a response typical of most insects (Weiss, 1943). S. geminata displayed a pattern of phototactic activity during the diel period that is similar to that for C. hemipterus (Blackmer & Phelan, in press) and C. luqubris (Blackmer, unpublished data). Lewis & Taylor (1964) found that insects that exhibit this type of flight periodicity are often associated with decaying organic matter (e.g., mature to rotting fruits and vegetables), and indeed this is the case for all three of these nitidulid species. However, when the diel activity of host orientation was examined in S. geminata and S. octomaculata. the response observed was very different from the response of C. hemipterus and C. luqubris. Both Carpophilus species exhibited a distinct bimodal periodicity in host orientation (Blackmer & Phelan, submitted a), whereas neither Stelidota species displayed bimodality. Also, the response of S. geminata and S. octomaculata to fungal-inoculated substrates contrasts with the response of C. hemipterus and C. luqubris (Blackmer & Phelan, submitted a). Neither S. geminata or S. octomaculata responded preferentially to fungal-inoculated fruits and vegetables, whereas both 91 C. hemipterus and C. luqubris exhibited a significant preference to these substrates over aseptic substrates. As fungal inoculation appears to increase behavioral activity in C. hemipterus. primarily through an increased production of host volatiles (Phelan & Lin, in press), perhaps S. aeminata and S. octomaculata are more efficient in locating their hosts at lower odor concentrations (i.e., they have lower behavioral thresholds). Alternatively, these two species may be responding to a different blend of compounds, perhaps ones that vary l it t le following inoculation. The lack of preference among substrate odors demonstrated by S. octomacul ata was not expected because of the relatively small number of hosts it appears to use in nature. In fact, the response of S. octomaculata almost mirrored the response of the polyphagous S. geminata. Galford et a l. (1991) has recently demonstrated in the laboratory that S. octomaculata can survive and reproduce on a variety of nuts and seeds (e.g., three maple species, hickory, butternut, walnut, 12 oak species, beech, and ash). Many of these trees co-occur with the native hosts of S. octomaculata (red, white and black oak), however, this beetle has never been found on these other apparently suitable hosts in nature (see Table 6). The factors resulting in and maintaining host specialization have been debated much in the literature (e.g.,"Special Feature - Insect Host Range" Ecology. 1988), and will not be reviewed herein, but explanations can be divided into two main categories: i) adaptive - where specialization confers a selective advantage in terms of efficiency of host utilization; this mechanism often implies a 92 cost/benefit relationship, and ii) nonadaptive - where specialists are numerous because of an evolutionary predisposition to speciate rather than being ecologically superior in performance or competitive ability (Berenbaum, 1990). Many factors have probably influenced the host- special ization of S. octomacul ata in nature, but response to host volatiles is apparently not one of them. Neither species was able to distinguish between any of the substrates presented via an olfactory response. However, several behavioral differences were observed. S. geminata readily exhibited both phototactic and host-orienting flights, whereas in the more than 1,600 S. octomaculata examined in this study, none took flight. J. Gal ford (pers. comm.) has studied extensively the biology and economic impact of this species on oak-stand-regeneration projects, and has never seen it fly. Furthermore, when the upwind walking response of S. octomaculata and S. geminata was compared, S. octomaculata walked at a significantly slower rate, turned more frequently, and was more likely to pause as it progressed upwind to the food source. Based on these findings, it is probable that these two species use different tactics for locating their hosts. Bell (1990) defined a searching tactic as a group of related, often sequential behaviors that leads the organism to a resource. One important component of search orientation is "scanning"; a mechanism for obtaining information from the environment. Most animals scan for resources periodically, and to reduce noisy signals scanning is done while the animal is stationary. The way an insect scans is related to the kind of resource it seeks, its own perceptual abilities and movement patterns, and the structure of its appendages. The differences in host-orientating behaviors between S. geminata and S. octomacul ata probably are influenced partially by the type of resources they exploit and by the nature of the habitats in which each species occurs; perceptual abilities and morphological differences may also play an important role. S. geminata exploits a succession of ephemeral hosts throughout the growing season (i.e., strawberries, raspberries, cherries, peaches, corn, canteloupe, and tomatoes [Blackmer, unpublished data]), and these resources are widely dispersed in agricultural settings. It seems reasonable that this species needs to be quite efficient in moving between its resources and would be able to accomplish this best through flight. S. octomacul ata. on the other hand, exploits a reliable, stable resource (i.e., acorns), in a less disturbed environment (i.e ., mature woodlots). The breakdown of an acorn can take several years, and during that period its interior provides a favorable habitat for many organisms (Winston, 1956). The embryo and parenchyma provide a plentiful supply of food and the hard outer layer provides protection from predators and the environment (Winston, 1956; Moffett, 1989). It has been theorized that powers of dispersal are likely to be greater in those plants and animals utilizing unstable or marginal habitats (i.e., S. geminata) (Darlington, 1970). On the other hand, existence in relatively favorable and stable areas (i.e ., S. octomaculata) may lead to a loss of flight. This explanation of the low dispersal capabilities of S. octomaculata is consistent with the hypothesis that ecological isolation has been a more important determinant of speciation events in the Nitidulidae than has geographical isolation (Skalbeck, 1976). Certainly this particular species appears to be ecologically isolated. Galford (pers. comm.) has found that even within areas that support populations of S. octomaculata they have patchy distributions. I submit that both Stelidota species represent "olfactory generalists," and that the restricted host range of S. octomaculata compared to S. geminata is not mediated by differences in long-range response to host odors, but rather is due to differences in other behaviors such as reduced rate of locomotion and possibly ecological factors. CHAPTER IV EFFECT OF HOST VOLATILES, SEASONAL OCCURRENCE AND HABITAT ON HOST ORIENTATION AND SELECTION BY NITIDULIDAE Introduction The Nitidulidae is a diverse family of insects and in many regions, especially the tropics, they represent one of the dominant insect groups. These beetles feed on a variety of substrates from decaying fruits and vegetables, fungal mats and carrion, to pollen and flowers (Gazit et al., 1982; Gertz, 1968). This divergence in feeding habits (decaying organic matter versus pollen and flowers) has led to a dichotomy within the family; Cateretinae and Meligethinae feed and breed in flowers, and Carpophilinae, Cryptarchinae and Nitidulinae feed on fungi and decaying organic matter. The primitive feeding habit is believed to be the nitidulid association with wood and wood fungi, with more derived nitidulids feeding on herbaceous plants (Skalbeck, 1976); however, the most primitive group is thought to be the Cateretinae, flower inhabitants. Within the nitidulids, 1ittle is known about the behavioral mechanisms and ecological factors governing host location and selection. However, we now know that chemical cues are important for eliciting host-orientation behaviors (Blackmer & Phelan, submitted a & b), and that fungus inoculation of food substrates enhances host location in at least two species, C. hemipterus and C. luqubris 95 96 (Blackmer & Phelan, submitted a). When substrate preference was examined in the laboratory there was a great deal of overlap between species (with the exception of C. luqubris! . For S. octomaculata. we proposed that the apparent discrepancy between the beetles host range in nature and its olfactory response in the laboratory might be explained by its low dispersal capability, which could lead to ecological isolation (Blackmer & Phelan, submitted b). However, we were not able to satisfactorily explain why there was so much overlap in olfactory response for the other species. This latter observation combined with the apparent divergence in feeding habits within the family led me to believe that other dimensions might play a role in resource partitioning. In nature, a resource can be divided along at least three dimen sions: chemical, spatial, and/or temporal (Greenfield & Karandinos, 1979; Holbrook & Schmitt, 1989), and the present research was directed towards better understanding these dimensions of host selection in the Nitidulidae. The divergence in feeding habits has led to species occurrence in dissimilar ecotypes and as a result I submit that: 1) spatial (i.e., habitat preference) differences between nitidulid species will be the dominant force shaping community structure, while chemical or temporal differences will be important within the different habitats; and 2) since nitidulid beetles have been associated with forests for a longer period of time in comparison to agricultural settings, species diversity will be greater in the woodlots. 97 Materials and Methods In 1989, field studies were conducted at Moreland Fruit Farm located 11.2 km south of Wooster, Ohio. This farm measured approximately 100 ha and was selected because of the diversity of crops grown: strawberry, raspberry, blackberry, elderberry, cherry, peach, grape, cantaloupe, tomato, corn, green beans and pumpkins, and because it bordered an extensive woodlot. The dominant vegetation within the woodlot was pin oak (Quercus palustris). white oak (Quercus alba). American elm (Ulmus americana) . black cherry (Prunus serotina) and pin cherry (Prunus pensvlvanica) . Other less prevalent vegetation included shell bark hickory (Carya laciniosa). bur oak (Quercus macrocarpa), black ash (Fraxinus nigra). flowering dogwood (Cornus Florida), sassafras (Sassafras albidum), bigtooth aspen (Pooulus grandidentata) and sugar maple (Acer saccharum) . Common understory flora included pokeweed (Phytolacca americana). Rubus sp.. poison ivy (Rhus radicans). Rosa sp., fern (Pteridium sp.) and carrion flower (Smilax herbacea) . Temporal and Chemical Comparisons - 1989 Beginning in early June, traps consisting of a four-vaned yellow Trece® top and a 4.25-1 plastic jug, were placed in the agricultural setting. These traps were placed on 0.5-m metal rods and positioned 2 m apart in a line that ran perpendicular to the prevailing wind. The treatments included aseptic and Candida krusei-inoculated banana, 98 strawberry, corn and tomato (inoculation procedure previously described, Blackmer & Phelan submitted a). Whole wheat bread dough (WWBD) was included for comparison as this substrate is frequently used for trapping nitidulid beetles and blank traps were included as controls. Each substrate was placed inside 150-ml plastic containers and then a fine-mesh fabric was placed securely on top to prevent feeding. These smaller plastic containers were placed inside the 4.25- 1 jugs, which contained 2.5 cm of detergent water for trapping the insects as they responded to the host odors. Each treatment was replicated twice. Traps were collected, re-randomized, and substrates changed every other day from early June through early September. Beetles were returned to the laboratory and stored in 70% ethanol until they could be counted, sexed and species determined. All females were dissected to determine reproductive status, and voucher specimens were retained for species confirmation and deposited in the Ohio State University Collection of Insects and spiders. Spatial Comparisons - 1989 Beginning in early September and continuing through early October, a second experiment was set up to examine habitat preference among the endemic nitidulid population. Additional traps were placed in the adjacent woodlot and sampling protocol was as described previously; however, treatments were reduced to banana, WWBD (substrates found to be most attractive in the previous experiment) and controls, and were presented in a randomized complete block design (n=3) in each habitat type. 99 Spatial, Chemical and Temporal Comparisons - 1990 In 1990, beginning in early June and continuing through early November three different locations (all within 20 km of each other) were used to examine the spatial, chemical and temporal dimensions of resource partitioning in the Nitidulidae. All of these sites produced a diversity of crops and were adjacent to woodlots. Moreland Fruit Farm is described above. Ullom Farm, measuring 40.5 ha, is located 24.3 km southwest of Wooster, Ohio, and the major crops included: corn, strawberry, soybean, rye and wheat. In the adjacent woodlot the dominant vegetation was sassafras (S. albidum), black cherry (Prunus serotina) and red maple (Acer rubrum) . Lesser representatives of this community included American beech (Fagus grandifolia) . white oak (fl. alba), red oak (Quercus rubra). American elm (U. americana) , mockernut hickory (Carva tomentosa) , Prunus sp., and white ash (Fraxinus americana) . Understory components included stinging nettle (Urtica dioica) , carrion f1ower (Smilax herbacea), poison ivy (Rhus radicans) , Geranium sp ., pokeweed (Phytolacca americana). mayapple (Podophyllum peltatum) and Viola sp. The third site, Maurer Farm, measured 30.4 ha and is located 4.8 km southwest of Wooster, Ohio. The major crops included: strawberry, tomato, alfalfa, corn, green pepper, blackberry, raspberry, pumpkins, green beans, cabbage, broccoli, cauliflower, peas, squash, melons and green onions. In the adjacent woodlot the dominant vegetation was black cherry (Prunus serotina). sassafras (S. albidum). white oak (fi. alba) . and American elm (U. americana) . Lesser representatives in the community included red maple (A. rubrum), flowering dogwood (C. florida). and American beech (F. grandifolia) . 100 and the understory was represented by Jewelweed (Imoatiens capensis) ♦ pokeweed ( Phytolacca americana!. Rubus sp., carrion flower (Smilax herbacea) . poison ivy (Rhus radicans) and Rosa sp. Because aseptic and inoculated substrates caught equivalent numbers of nitidulids beetles in 1989, at least for the four dominant species, only aseptic treatments were used in 1990 and these included: corn, banana, WWBD and controls. All traps were placed on 0.3-m tall metal rods and were presented in a randomized complete block design (n=3). Traps were collected and changed every other day, twice per week on the 4th and 6th day of the week. Beetles were returned to the laboratory for determinations as in 1989; however, when the number of specimens for a particular species exceeded 40, subsamples of 20 individuals were used to determine sex ratio and reproductive status. In 1989, an analysis of variance (AN0VA) on transformed values (1og[x+1], in which x was the number of beetles) was used to determine if fungal inoculation, substrates and habitat type influenced trap catch during the season. In 1990, transformed (log[x+l]) data were analyzed as a split-plot design with location as replications and habitat type and substrates as the whole and subplot, respectively. I compared nitidulid communities (species total and abundance) for the two habitat types and three farms using cluster analysis by the single linkage method and Euclidian distance (Wilkinson, 1989), and I determined species diversity using Shannon-Wiener Index. 101 Results Temporal and Chemical Comparisons - 1989 Four species comprised approximately 95% of the nitidulid specimens collected: Stelidota geminata. Carpophilus luqubris. G1ischrochilus fasciatus and G1ischrochilus quadrisiqnatus. S. geminata and C. luqubris were first collected in mid- to late June (Fig. 19). Approximately 60% of the females from these early collections were gravid. Both species were present throughout the season, but were collected in greatest numbers in mid- to late July. S. geminata appeared to have at least three generations with the first emergence occurring in early July, the second in late July and the third in early August. In each case, the peaks in trap catch were preceded, at approximately 2-wk intervals, by a high percentage of gravid females (60-80%). C. luqubris showed only one major peak in, mid-July, and remained at a relatively constant level thereafter. Approximately 50% of the females were gravid at any time throughout the season for this species. G. fasciatus and G. quadrisiqnatus were firs t collected in early to mid-July, with the greatest numbers collected shortly thereafter in mid- to late July. None of the females for either species were found to be gravid during the trapping period. In all four species, males and females were collected with the same frequency (approx. 1:1). Figure 19. Total number per trapping period of a) Stelidota geminata. Carpophilus luqubris. G1ischrochilus fasciatus and b) G1ischrochilus Quadrisiqnatus collected at Moreland Fruit Farm, Moreland, Ohio, 1989. 102 103 3 00 S. gem inata ■ H C. iugubris o 240 <0 G. fasciatus o ° 180 < D £ k E 120 z3 CO o 60 I- V i i i i r i lV l 6/15 7/1 7/15 8/1 8/15 9/1 D a te 1500 G. quadrisignatus ■o *■* Figure 19. The response to host volatiles was generally broad (Table 9); only C. luqubris showed a significant preference for one substrate (WWBD) over all other substrates. G. quadrisiqnatus showed a preference for corn early in the season. However, a sharp decline in G. Quadrisiqnatus trapped by this substrate occurred later in the season and was correlated with corn maturation (milk-dough stage) in the field; trapping rates for other substrates remained about the same (Fig. 20). The greatest number of beetles was collected from banana, corn, and WWBD (Table 9). For the four dominant nitidul id species there were no significant differences between the number of beetles trapped by aseptic and C. krusei-inoculated substrates; however, greater numbers of C. hemipterus were collected from fungal-inoculated strawberry, tomato and banana than from the same aseptic substrates (88% versus 12%, n=29 beetles collected). Spatial Comparisons - 1989 Each of the four dominant species displayed well-defined habitat preferences (Fig. 21). C. luqubris was collected almost exclusively at the agricultural site (F=33.5, df=l,27, P<0.005) as was G. quadrisiqnatus (F=21.2, df=l,27, P<0.005). S. geminata and G. fasciatus on the other hand, were collected primarily in the woodlots (F=16.8, df=l,27, P<0.005; F=50.6, df=l,27, P<0.005, respectively). Temporal. Chemical and Spatial Comparisons - 1990 As in 1989, S. geminata. C. luqubris. G. fasciatus and G. quadrisiqnatus comprised from 95 to 99% of the nitidulid specimens Table 9. Mean (+ s.e.) number of beetles collected per 2-d trapping period with food substrates and control in 1989. S. geminata C. luqubris G. fasciatus G. Quadrisiqnatus Substrates: Banana 21 + 5 a1 3 + 0.6 b 6 + 2 ab 59 + 17 ab Corn 20 + 8 a 4 + 1 b 9 + 4 a 74 + 34 a Bread dough 18 + 3 a 18 + 5 a 9 + 2 a 75 + 15 a Strawberry 12 + 3 ab 2 + 0.5 b 3 + 1 ab 55 + 17 ab Tomato 12 + 4 ab 2 + 0.3 b 2 + 1 b 31 + 10 ab Control 0.7 + 0.3 b 0.3 + 0.1 b 0 b 0.8 + 0.3 b 1means within a column with the same letters are not significantly different; ANOVA followed by LSD (P<0.05). Figure 20. Cumulative number of 61ischrochilus Quadrisiqnatus collected with whole wheat bread dough, corn, banana, strawberry and tomato at Moreland Fruit Farm, Moreland, Ohio, 1989. 106 iue 20. Figure Cumulative Beetles Trapped 1000 2000 0 0 5 1 0 0 5 15 7/ 28 8/ 9 8/ 24 8/ /5 9 0 /3 8 4 /2 8 5 /1 8 /9 8 /3 8 8 /2 7 1 /2 7 5 /1 7 S- r r rry e b w tra -S S _ C -C orn orn -C C To ato om -T T dough d a re -B D B B -B an an a a an an -B 1 J * s - - - - - 1 ------' - 1 - - - - - / 1 ature m _ _ _ I _ _ _ I _ _ _ Date I _ _ _ I _ _ _ 1 I I I I l I I I I I I I I 1 t 107 Figure 21. Proportion of Stelidota aeminata (diagonally-slashed barsl. Carpophilus lugubris (solid bars), Glischrochilus fasciatus (cross-hatched bars) and G. ouadrisionatus (dotted bars) collected at the agricultural site and woodlot at Moreland Fruit Farm, Moreland, Ohio, September through October - 1989. 108 iue 21. Figure Proportion Collected 100 40 60 20 20 80 0 A gricultural gricultural A site site oodiot W &♦!♦!♦!&♦!« 109 110 collected and usually in a 1:1 male:female ratio (C. luoubris had a 1.4:1 ratio). S. oeminata and C. luoubris were both collected throughout the trapping period and S. oeminata displayed a similar pattern of activity at all three sites (Fig. 22); however, the activity of C. luoubris was more variable (Fig. 23). Trap catches were almost always greater at the agricultural sites for these two species. As in 1989, increases in trap catch usually followed (approx. 2-3 wk later) an increase in the proportion of gravid females. G. fasciatus and G. ouadrisionatus were firs t collected in early to mid-July and showed similar activity patterns at all three locations (Figs 24 & 25). As in 1989, G. fasciatus was usually collected more frequently in the woodlots and G. ouadrisionatus was trapped more frequently in the agricultural sites. Unlike 1989, gravid females (20-60%) were detected for both Glischrochilus species through late to early July, especially for G. fasciatus. Pitfall collections from a corn field in late April and early May indicated that nearly 100% of the Glischrochilus females were gravid (Blackmer, unpublished data); oviposition is believed to have begun shortly thereafter as a decline in the proportion of females with eggs was noted by the end of May. In 1990, the agricultural sites were significantly preferred by C. luqubris. S. oeminata and G. ouadrisionatus: G. fasciatus did not show a significant preference for either habitat type (Table 10). Although, when locations were analyzed individually, significantly more G. fasciatus were trapped in the woodlots at Morel and and Ullom Farms (Fig. 26). Figure 22. Total number per trapping period of Stelidota oeminata collected at Moreland, Ullom and Maurer Farms and adjacent woodlots, 1990. Ill Moreland Total Number Colleoted e a O so a O 3 Total Number Colleoted © o «s B C 1 a O © M o <9 o e o o © Total Number Colleoted Figure 2 2 . «/n e/str 7 /1® 7/30 8/27 «/2« Figure 23. Total number per trapping period of Carpophilus luoubris collected at Moreland, Ullom and Maurer Farms and adjacent woodlots, 1990. 113 to -s CD ro cu Total Number Colleoted Total Number Colleoted Total Number Colleoted © o Moreland Maurer o 3 *4 mS <4 © © to to M M Figure 24. Total number per trapping period of Glischrochilus fasciatus collected at Moreland, Ullom and Maurer Farms and adjacent woodlots, 1990. 115 Moreland Total Number Colleoted U o © <0 Total Number Colleoted © » o 09 ** H Ok M Total Number Colleoted O & N* o N Figure 24 9 > Figure 25. Total number per trapping period of G1ischrochilus ouadrisionatus collected at Moreland, Ullom and Maurer Farms and adjacent woodlots, 1990. 117 009 Total Number Colleoted CD © O r* o 3 Total Number Colleoted A © A O s 5 CD a o o M o © o © o o Total Number Colleoted Figure 2 5 . 6/13 6/27 7 / 16 7/30 8/27 9 /2 6 Table 10. Analysis for habitat preference (whole plot), substrate preference (subplot) and interaction for three locations: Moreland, Ullom and Maurer Farms, 1990. C. luoubris S. oeminata G. fasciatus G. ouadrisionatus Habitat: Agriculture-(Ag) 178 + 871 1985 + 7251 239 + 65 1025 + 2561 Woodlot-(Wd) 15 + 9 523 + 207 430 + 185 224 + 80 Substrates: Bread dough-(Bd) 350 + 1 a2 3681 +10 a 915 + 263 a 1114 + 283 a Banana-(Ban) 17 + 8 b 1170 + 423 b 341 + 60 b 888 + 371 a Corn 20 + lib 154 + 71 c 80 + 23 c 493 + 291 b Control-(Ct) 2 + 1 c 10 + 5 d 1 + 1 d 4 + 2 c Hab.*Subs. Bd-Ag 642 ± 153 a3 n.s. 451 + 88 b n.s. Bd-Wd 58 + 25 b 1379 + 351 a Corn-Ag 37 + 16 b 104 + 43 c Ban-Ag 32 + 9 b 399 + 104 b Ct-Ag 3 + 1 c 1 + 1 d Ban-Wd 2 + 1 cd 283 + 63 b Corn-Wd 2 + 1 cd 56 + 12 c Ct-Wd 0.3 + 0.3 d 0 d 1ANOVA, P<0.05 2Means for substrates within a column with the same letter are not significantly different, ANOVA followed by LSD (P<0.05) 3Means for Hab.*Subs. interaction within a column with the same letter are not significantly different, ANOVA followed by LSD (P<0.05) Figure 26. Proportion of Stelidota oeminata (diagonally-slashed bars). Carpophilus luoubris (solid bars), Glischrochilus fasciatus (cross-hatched bars) and Glischrochilus ouadrisionatus (dotted bars) collected in agricultural sites and woodlots at Moreland, Ullom and Maurer Farms, 1990. 120 Proportion Collected Proportion Collected Proportion Collected Whole wheat bread dough was significantly preferred over banana and corn by C. luoubris. S. oeminata and G. fasciatus: G. ouadrisionatus did not distinguish between bread dough and banana, but preferred them over corn (Table 10). A significant interaction between habitat type and substrate occurred with C. luoubris and G. fasciatus. When nitidulid species and abundance in the agricultural settings and the woodlot areas were compared using cluster analysis, the agricultural sites formed one cluster and the woodland sites formed a second cluster (Fig. 27). The woodlots were more similar as indicated by smaller Euclidian distances and the agricultural sites at Maurer and Moreland Farms were more similar to each other than to the agricultural site at the Ullom Farm. The nitidulid diversity of these two habitat types did not appear to differ greatly (Table 11), although the less common nitidulid fauna associated with each was quite distinct (Table Figure 27. Dendrogram comparing nitidulid communities in agricultural settings and woodlots; based on cluster analysis using the single linkage method and Euclidian distance. 123 iue 27. Figure Woodlots Agricultural Sites oreland M oreland M aurer M Ullom aurer M Ullom I I I I I lse Distance Cluster 1000 2000 124 125 Table 11. Nitidulid community description for agricultural settings (Ag) and woodlots (Wd) using species total (ST), species abundance (N), and Shannon-Wiener diversity index (H*). Location: Maurer Moreland Ullom ______Aa Wd Aa______Wd Aq Wd ST 13 12 12 10 13 9 N 14,156 4,230 11,991 5,195 20,021 7,818 1.14 0.94 0.89 1.12 1.10 1.09 126 Table 12. Other less common nitidulid species with numbers collected in each habitat, 1990. Species: Number Collected Agricultural Site ______Woodlots Carpophilus hemipterus 32 0 Carpophilus brachvoterus 318 3 Carpophilus marqinellus 8 0 Meligethes sp. 59 0 Crvotarcha amp!a 26 2 Lobiopa undulata 12 0 Colopterus sp. 2 5 Glischrochilus sanouinolentus 0 17 Eouraea sp. 0 12 Phenolia qrossa 0 6 127 Discussion The differences observed in abundance and occurrence between the dominant nitidulid species, along the temporal dimension, are most likely a result of differences in reproductive strategies. S. oeminata and C. luoubris are multivoltine species, whereas both Glischrochilus species are univoltine. The two multivoltine species were detected approximately 1 mo earlier than the univoltine species, and after the emergence of the two G1ischrochilus species, there was no noticeable decline in the abundance of the multivoltine species; I would have expected a decline if these species were partitioning their resources along the temporal dimension. The temporal dimension may play a more important role for the less common species; most of which were present only in the early part of the season, although C. hemipterus only occurred at the end of the trapping season (Blackmer, unpublished data). During the 1989 season, there was a great deal of overlap between species along the chemical dimension, except C. luoubris. which demonstrated a significant preference for MWBD, and G. ouadrisionatus. which exhibited a preference for corn early in the season. For the most part, these findings were consistent with our 1aboratory results (Blackmer & Phelan, submitted a & b). C. luoubris. however, appeared to be even more discriminating in the field as it rarely responded to any of the other substrates offered. The response of G. ouadrisionatus to corn during the early part of the 1989 season may represent an example of 'local specialization' (Fox & Morrow, 1981). This phenomenon occurs when an apparently 'generalist' herbivore exhibits a specialized response to a locally abundant host. There may be a correlation between this response by 6. ouadrisignatus as corn is believed to be the main oviposition site for this species (Foott & Timmins, 1971). The decline in trap catch by corn substrates corresponded with corn phenology, a decline correlated with sweet corn reaching the milk-dough stage; the remaining food substrates (strawberry, tomato and WWBD) continued to elicit similar levels of attractiveness throughout the season. This might suggest that a polymorphism for different hosts exists within the G. ouadrisionatus population. The 1990 results differed from 1989 in that WWBD was significantly preferred over corn and banana by all of the dominant species except G. ouadrisionatus. although the preference was much greater for C. luoubris. Climatic conditions differed considerably between 1989 and 1990: most notably rainfall in 1990 was 1.6 times greater than in 1989. The higher humidity in 1990 may have kept WWBD moist longer and thus extended the time the substrate was attractive during the 2-d trapping period, leading to higher trap catch in comparison to 1989 and to other substrates. A second anomaly between 1989 and 1990 was that the late-season decline in trap catch of G. ouadrisignatus by corn was not observed. Greater rainfall in 1990 may also have affected this outcome either directly by reducing trap catch early in the season or indirectly by delaying oviposition, ultimately 129 delaying the emergence of G. ouadrisignatus. This latter conclusion seems justified as gravid females were detected in 1990 through early July, whereas oviposition must have occurred prior to June in 1989 as evidenced by the lack of gravid females during the trapping period. As proposed, there was a strong preference for particular habitats. In both years, C. luoubris and G. ouadrisignatus exhibited a strong preference for agricultural sites, whereas G. fasciatus exhibited a preference for woodlots. S. oeminata was found more commonly in woodlots in 1989 and in agricultural sites in 1990. There are at least two possible explanations for this discrepancy. Climatic conditions in 1990 may have favored reproduction by S. oeminata in the agricultural sites over woodlots. Alternatively, as it is believed that this species returns to woodlots to overwinter (Gertz, 1968), it is possible that the 1989 collections sampled the overwintering population. If the distribution of S. oeminata in the two habitats is compared from mid-September through early November in 1990, this species was collected more frequently in the woodlots than in the agricultural sites, so migration is a possibility. Less common nitidulid species also showed strong preferences for particular habitats. I also postulated that woodlots would be more diverse in nitidulid species than would the agricultural sites. This hypothesis was based on the assumption that nitidulid beetles have had a longer association with forests than with agricultural settings. For several introduced plant species it has been demonstrated that the greater the time span between introduction to the present, the greater the number 130 of insects associated with the particular plant species (Strong et al., 1984); however, nitidul id diversity in woodlots was not greater than in the agricultural setting despite the apparently longer association. This lack of difference between habitats may have resulted because of the substantial plant diversity within the selected agricultural settings or it may have something to do with the native hosts of these beetles and the ease of transition between habitat types (e.g., wild strawberries to cultivated strawberries, pin and black cherry to cultivated cherry, hawthorne to apples and wild raspberries and blackcaps to cultivated raspberries and blackberries). Many of these native hosts are taxonomically, phenologically, biochemically and morphologically similar to the present-day cultivated crops. In summary, most of the endemic nitidulid species appear to be partitioning their resources on at least the spatial dimension, and in G. ouadrisignatus and C. luqubris there appears to be some separation on the chemical dimension as well; however, the temporal dimension does not appear to be particularly effective for species separation, at least not for the four dominant species. SUMMARY The information contained herein represents one of the first detailed investigations on nitidulid host location and selection, and the factors that affect these processes. In Chapter I, I examined the transition from phototactic to vegetative orientation in C. hemipterus by using a vertical flight chamber. This species readily exhibited phototactic flight in response to an overhead high-pressure sodium lamp and this response was especially strong in beetles 3-7 days old. The majority of these flights lasted less than 15 min, but ranged up to 100 min. A bimodal periodicity in flight propensity was recorded during the photophase with a small peak in activity occurring 14-10 h prior to scotophase and a large 4 h peak occurring from 3 h prior to scotophase to 1 h after when the onset of scotophase normally would have occurred; these peaks in flight activity corresponded with early to mid-morning and dusk. Bimodal flight periodicity has been found to be correlated with insect feeding guilds that are associated with decaying organic matter (Lewis & Taylor, 1964). Supposedly, this type of periodicity is a result of environmental factors (e.g., windspeed and humidity) interacting with the perception of host volatiles. Windspeed is generally lower and humidity usually higher at dawn and dusk, conditions that would probably enhance host-finding success. After C. hemipterus had exhibited a period of vertical flight, 131 132 photokinetic and phototactic response declined, and flight instability increased, as indicated by an overall decrease in the mean rate of climb, accompanied by an increase in the variability of this measure and an increase in horizontal displacement. When food odor (apple cider vinegar) was introduced the rate of climb dropped rapidly and beetles usually landed regardless of how long they had been in flight. When the food odor was removed, takeoff occurred and the beetle returned to its previous rate of climb. These results indicated that although flight in phototactic C. hemipterus was similar in many respects to flight behavior in aphids, its response to host cues was quite different. In aphids, conflicting behavioral systems (phototactic response versus host-orienting response) are resolved through a behavioral transition; initially during flight aphids exhibit a strong phototactic response and when a host cue is introduced, antagonistic induction occurs (as evidenced by an increase in the rate of climb), whereas if the host cue is presented later during the same flight, antagonistic depression occurs (as evidenced by a decrease in the rate of climb). C. hemipterus always displayed a reduction in the rate of climb with host-odor introduction and when the host odor was removed the beetle either reinitiated flight or returned to its previous rate of climb prior to food-odor introduction. Beetles that landed with food-odor introductions took off at an excelerated rate of climb in comparison to their rates of climb prior to food-odor introduction and this elevated rate of climb was most likely due to post-inhibitory rebound of the flight mechanism; non-landers returned to the previous rate of climb indicating that antagonistic induction 133 was not operating in this behavioral system. Using Kennedy's (1985) definition of "true migration" and "foraging," C. hemipterus flight falls readily into the latter category in which there is no evidence for a reciprocal interaction between phototactically modulated flight and host-finding flight, but rather where phototactic flight is inhibited by the presence of food odor irrespective of the beetles' behavioral history. In Chapter II and III, host orientation by C. hemipterus. C. luoubris. S. aeminata and S. octomaculata was investigated in a 2.5-m horizontal wind tunnel to ascertain if these species exhibited different responses to fungal inoculation and to various hosts, and to establish whether they differed in host-finding tactics. Both Carpophilus species demonstrated a significantly greater response to fungal-inoculated substrates in comparison to aseptic substrates; however, neither of the Stelidota species responded preferentially to fungal-inoculated substrates over aseptic substrates. Phelan & Lin (in press) have demonstrated that the increase in behavioral activity in response to fungal inoculation by C. hemipterus is due to an increased production of host volatiles and is not due to the presence of unique fungal metabolites. Therefore, I proposed that the difference between the response observed in the Carpophilus species and the Stelidota species had more to do with differences in these species' abilities to perceive host volatiles. Perhaps S. geminata and S. octomaculata are able to locate their hosts at lower concentrations (i.e., they may have a lower behavioral threshold), or maybe they are responding to different chemical blends, ones that vary 1 ittle following inoculation. 134 When host preference was examined three out of four species exhibited fairly broad responses. This was unexpected for S. octomaculata. which is narrowly restricted to acorns in nature. It appears that this restriction to acorns is not in response to strict nutritional requirements as any of a number of seeds can serve as reproductive sites, at least in the 1aboratory (Galford et a l., 1990). However, based on my behavioral study it appears that low dispersal capabilities hinder this particular species from expanding its host range. Among the other three species examined there also appears to be an association between host range and dispersal capabilities. C. hemipterus and S. qeminata both have a very broad host range and are very active dispersers, whereas C. luoubris has a narrower host range and, at least in the laboratory, rarely flies. Chapter IV addressed nitidulid host finding and selection in the field by examining the temporal, chemical and spatial dimensions of resource partitioning. The temporal dimension did not seem to be particularly effective in separating the dominant species, but could play a more important role in resource partitioning for the less common species as these species were seen either very early in the season or late in the season, before the dominant species increased in number. The chemical dimension appeared to play a slightly more important role in separating the dominant species; however, the spatial dimension, specifically habitat preference, was the most important 'one dimension' resulting in species separation. The underlying tenet believed to lead to resource partitioning is competition and it is probable that some form of competition has shaped present day host utilization in the 135 Nitidulidae, but the research contained herein did not directly address this concept. Many questions have been addressed herein and we now have a better understanding of the factors that affect host orientation and selection in the Nitidulidae (hopefully this information is germane to other insects); however, many questions still remain to be answered. Under natural conditions, a variety of factors can affect the catenary process of host location. It is likely that macro- and micro environmental differences influence this process (e.g., differences in light quality and quantity, humidity, windspeed and the structure of the odor plume). Each of these variables could be manipulated in the 1aboratory and the ensuing effects on the insects behavior examined. As each species has evolved under a different set of circumstances a study comparing woodland species versus species that occur in more open areas would be most informative. Another worthwhile area of research could focus on additional factors that have shaped the narrow host range of S. octomaculata. Although this species can reproduce on a variety of seeds and nuts in a no-choice situation, in nature, this does not occur; other factors must be influencing their choice (e.g., acorn availability during their major period of oviposition, parasite or predator pressures associated with certain hosts, or as this species is predominately a secondary pest of acorns it may actually be cuing in on odors associated with the feeding activities of the primary pests). 136 LIST OF REFERENCES Abbott, C.E., 1937, The necrophilous habit in Coleoptera. Bull. Brooklyn Entomol. Soc. 32: 202-204. Agrios, G.N., 1980. Insect involvement in the transmission of fungal pathogens. 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