THE UPTAKE OF TRITIATED WATER BY A BEETLE,
Chilocorus cacti (Coleoptera; Coccinellidae),
FROM A MITE, Hemisarcoptes cooremani (Acari: Acariformes)
by AURAL! E. HOLTE, B.S.
A THESIS
IN
BIOLOGY
Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE
Approved
December, 1999 mr'-^'^-"""'^'" .——^—--— •« • "ji» • »j»«p»^"^i^»w
/^n^^l C>^i^f ACKNOWLEDGMENTS
I thank a number of people for their assistance and support in the completion of iS-f' 7^ this work. First, I would like to thank Dr. Marilyn Houck for her generous
encouragement, understanding and guidance, without which I would not have been able
to start or complete this project. I also thank the members of my committee. Dr. Nathan
Collie and Dr. Richard Deslippe who provided valuable comments and information
utilized for this research. Elizabeth Richards, Heather Roberts, and Qingtian Li were
encouraging and helpful colleagues in all my endeavors as a graduate student.
I also thank a number of people for personal support; foremost, Damon for
without his devoted love, constant support and immutable encouragement, I would have
not been able to accomplish this work. I v^sh to thank my family for all of the love and
understanding they have given me, especially my mother who guided me with her
example, demonstrating that I could do anything once I set my mind to it. I also would
like to acknowledge all of the other friends and family members who have given me
encouragement.
Finally, financial support for this research was provided by the Texas Tech
University Biology Department and the Bi-National Agricultural Research and
Development grants (#IS-1397-87 and #US-2359-93C to M. A. H.).
11
II III iiiiiiiir li y*"*''* * *••»• *''*^«*»*»"J^fcjt * *Mr^f t^^m m'm m w
TABLE OF CONTENTS
ACKNOWLEDGMENTS ii
ABSTRACT v
LIST OF TABLES vh
LIST OF FIGURES vm
CHAPTER
I. INTRODUCTION
Scale Insects 2
Hemisarcoptes
Chilocorus.
Biological Control of Scale Insects 6
Coevolutionary Relationship of Hemisarcoptes and Chilocorus 8
Research Objective 9
IL MATERIALS AND METHODS 11
Laboratory Monocultures 11
Experimental Design 11
Cohort Conditions 12
Treatment Condifions 12
Controls 13
Cohort Number 14
Data Analysis 15
III. RESULTS 18
111 ^^r-fl K ^^mimm MM ^
Tritium Levels 18
Effects of Mite Numbers on Tritium Levels 19
Comparisons 19
IV. DISCUSSION 48
Future Directions 50
LITERATURE CITED 52
APPENDIX 59
IV 3BB h^jja^m i_.>.r»iT.»w;iviir!iiTirii-Trfiinfi tv •—i-, .•-. JT,
ABSTRACT
Understanding the origin and evolution of symbiotic interactions between extant species plays an important role in comprehending the nature of their ecological relationships. The dynamics of such complex evolutionary relationships must be examined from multiple perspectives, using both field and laboratory methods.
Phoresy is a form of symbiotic interaction that results in dispersal. It is a coevolved interaction that benefits the dispersed organism but does not affect the phoretic host. In this thesis, I address a purportedly phoretic interaction between the deutonymphal stage (subadult) of a mite Hemisarcoptes cooremani (Astigmata:
Acariformes) and the adult stage of the beetle Chilocorus cacti (Coleoptera :
Coccinellidae), two potentially important biological control agents of scale insects.
Past radiolabeling studies indicated that the deutonymph of//, cooremani acquired tritiated water from adult C. cacti, challenging the validity of the paradigm that this relationship, and many like it among the Astigmata, is phoretic. Passage of materials from one organism to another can be indicative of a parasitic relationship. This conclusion may be an incomplete assessment of a fuller relationship in which materials are freely passed among symbionts (mutualism). To probe this Hemisarcoptes-
Chilocorus relationship, I conducted a tritiated radiolabel study, similar to the previous test, to determine whether materials were being passed from the deutonymphs to the beetles.
The explicit research hypothesis was that tritiated water could be passed from the deutonymphs to the beetles during the symbiotic interaction. To test this hypothesis, wr^arntmmnmK*-^. * •^'»j'?'a£iL^lQ3MMMW)ottiiaCT-iflin«PirTi nr itrrnramniiiriri ft^J^M»M"~T.
deutonymphs of//, cooremani were radiolabeled and then allowed to attach to C cacti.
After remaining attached for 48-Hours, the beetles were tested for tritium concentrations.
The results of this experiment demonstrated that beetles acquired tritium from the
deutonymphs. Both the elytra and the body of the beetle showed significantly higher
levels of tritium when mites were attached than when they were not. These results have
interesting implications for understanding the complex relationship between
Hemisarcoptes and Chilocorus. I propose that we now tentatively redefine this
relationship as mutualistic. However, this judgment must be held in reserve until verified
by examination of the potential fitness advantages conferred on both members of this
interaction.
VI
asaBSBSss esacs^^-, - iT.^ - .-* LIST OF TABLES 2.1 Full-Blocked Design of Experiment (Treatment and Controls) 16 2.2 Experimental Blocked Design of the Treatment and Controls 17 3.1 Summary of Tritium Concentrafion in Beetle Bodies at the End of the 48-Hour Experimental Exposure 22 3.2 Summary of Tritium Concentration in Beetle Elytra at the End of the 48-Hour Experimental Exposure 22 3.3 Number of Mites Attached to Each Beefie in the Treatment and Control #1 23 3.4 Results oft-test for All Pair-Wise Comparisons of Tritium Concentrations in the Beetle Bodies after the 48-Hour Experimental Exposure 24 3.5 Results oft-test for All Pair-Wise Comparisons of Tritium Concentrations in the Beetle Elytra after the 48-Hour Experimental Exposure 24 3.6 Results of Mann-Whitney test for All Pair-Wise Comparisons of Tritium Concentrations in the Beetle Bodies after the 48-Hour Experimental Exposure. 25 3.7 Results of Mann-Whitney test for All Pair-Wise Comparisons of Tritium Concentrations in the Beetle Elytra after the 48-Hour Experimental Exposure. 25 A. 1 Tritium Concentrations (dpm) from Beetle Bodies after the 48-Hour Experimental Exposure 60 A.2 Tritium Concentrations (dpm) from Beetle Bodies after the 48-Hour Experimental Exposure 61 Vll JB ^7^-.^. ^,. ,-^,>...... 1.-.., • ,».. ^.....^ •„„.^ LIST OF FIGURES 3.1 Trifium Concentrations from Beetle Body after 48-Hour Exposure of Treatment 26 3.2 Frequency Distribufion of Tritium Concentrafion in Beefie's Bodies after 48-Hour Exposure of Treatment 27 3.3 Tritium Concentrations from Beetle Elytra after 48-Hour Exposure of Treatment 28 3.4 Frequency Distribution of Tritium Concentration in Beetle's Elytra after 48-Hour Exposure of Treatment 29 3.5 Tritium Concentrations from Beetle Body after 48-Hour Exposure of Control #1 30 3.6 Frequency Distribution of Tritium Concentration in Beetle's Bodies after 48-Hour Exposure of Control #1 31 3.7 Tritium Concentrations from Beetle Elytra after 48-Hour Exposure of Control #1 32 3.8 Frequency Distribution of Tritium Concentration in Beetle's Elytra after 48-Hour Exposure of Control #1 33 3.9 Tritium Concentrations from Beetle Body after 48-Hour Exposure of Control #2 34 3.10 Frequency Distribution of Tritium Concentration in Beetle's Bodies after 48-Hour Exposure of Control #2 35 3.11 Tritium Concentrations from Beetle Elytra after 48-Hour Exposure of Control #2 36 3.12 Frequency Distribufion of TriUum Concenlration in BeeUe\ Elytra after 48-Hour Exposure of Control #2 37 3.13 Tritium Concentrations from Beetle Body after 48-Hour Exposure of Control #3 38 3.14 Frequency Distribufion of Trifium Concentration in Beefie's Bodies after 48-Hour Exposure of Control #3 39 viii 3.15 Tritium Concentrations from Beetle Elytra after 48-Hour Exposure of Control #3 40 3.16 Frequency Distribution of Tritium Concentration in Beetle" s Elytra after 48-Hour Exposure of Control #3 41 3.17 Correlation Between Number of Deutonymphs Attached to a Beetle and the Tritium Concentration from that Beetle's Body after the 48-Hour Exposure Period in the Treatment 42 3.18 Correlation Between Number of Deutonymphs Attached to a Beetle and the Tritium Concentration from that Beetle's Elytra after the 48-Hour Exposure Period in the Treatment 43 3.19 Correlation Between Number of Deutonymphs Attached to a Beetle and the Tritium Concentration from that Beetle's Body after the 48-Hour Exposure Period in Control #1 44 3.20 Correlation Between Number of Deutonymphs Attached to a Beetle and the Tritium Concentration from that Beetle's Elytra after the 48-Hour Exposure Period in Control #1 45 3.21 Summary Boxplots and Median Tritium Concentrations from the Beetle's Bodies for the Treatment and All Controls 46 3.22 Summary Boxplots and Median Tritium Concentrations from the Beetle's Elytra for the Treatment and All Controls 47 IX ^... < a»« V'J*^-J^^^•S•^' Ji' ^>*•• ••-!' CHAPTER I INTRODUCTION Coevolution has played a significant role in the adaptation of organisms to their environments. Coevolufion, reciprocal changes caused in interacting organisms over time (Brooks and McLennan, 1993), can be seen in many different types of interactions including phoresy, parasifism and mutualism. Phoresy can be defined as an interspecific relationship in which one organism attaches to another for the sole purpose of transportafion (Houck and OConnor, 1991). Phoretic relafionships can be ancient and can involve major morphological adaptations for attachment, including grasping appendages and sucker plates (Houck and OConnor, 1991; Houck, 1993). Although the goal of phoresy is simply dispersal, the relationships between the organisms involved can be complex (Krombein, 1962; Fain and Ide, 1976; Crawford, 1984; Houck and OConnor, 1991). Parasitism requires that nutritional benefit be obtained from the host, during association, without killing it (Vinson, 1974). The parasite typically adapts to the host's natural defenses while the host is selected to maintain strategies that limit the extent of the parasitic infection (Esch and Fernandez, 1993). Mutualism has been defined as a relationship between two species in which both species benefit from the interaction (Solomon, Berg and Martin, 1999) in a minor way or to the extent that the organisms are dependent upon each other for their survival. All of the above relationships can be complex. Some of the more complex natural relationships exist among the Astigmata and their insect hosts. One such relationship that has received much attention is that of the beetle Chilocorus (Coleoptera: Coccinellidae) and the mite Hemisarcoptes (Acari: Acariformes) (OConnor and Houck, 1989a; OConnor and Houck, 1989b; Houck, 1989; Gerson, OConnor and Houck, 1990; Houck and OConnor 1990; McCormick et al., 1994; Houck, 1994; Shi, Attygalle, Meinwald, Houck and Eisner, 1995; Houck and Cohen, 1995; Houck and OConnor, 1996; Houck, 1998; Houck and OConnor, 1998; Huang, Attygalle, Memwald, Houck and Eisner, 1998; Houck, 1999a; Luck, Jiang and Houck, 1999). Hemisarcoptes attaches to the underside of the elytra of Chilocorine beetles for transport from one food source to another. This interaction is especially notable because Chilocorus and Hemisarcoptes share a food source that is an important crop pest, armored scale insects (Diaspididae). Scale Insects The three most common families of scale insects are the armored scale (Diaspididae), the soft scale (Coccidae), and the mealybugs (Pseudococcidae) (Stimmel, 1996). The family Diaspididae includes the armored, or hard-scale, insects and are significant pests of many important perennial crops. Because of monoculture cultivation practices of finit crops and ornamental trees, scale insects have become major pests worldwide (Gulmahamad and Debach, 1978) and continue to cause massive destruction of many cash crops (Luck et al.. 1999), costing billions of dollars to nurseries and crop producers. Diaspidid scale insects infest ornamentals, commercial coniferous and deciduous groves as well as fruit and nut orchards (DeBoo and Weidhaas, 1976; Delfosse, 1993; Ji, Gerson and Izraylevich, 1994; Stimmel, 1996). In a typical grove, scales will infest all I iji I !• ja»waf above ground portions of the plant (Luck et al., 1999). At low densifies, scale causes cosmetic damage to the fiiiit making it unmarketable. As densities become higher, scales can effect leaves, twigs, branches and even boles of the trees. When unchecked for 2-3 years, this can lead to complete destruction of trees and even entire groves (Nalepa, Drea and Bryan, 1993). Many scale species are distributed worldwide and international commerce of nursery stocks has caused scale populations to be inadvertently imported to new areas where no natural control systems occur. Because of the unique lifestyle of scale insects, a soft body under a hard protective shell attached tightly to the host plant, chemical control has not been an effective option (Delfosse, 1993). Therefore, utilizing natural predators such as Hemisarcoptes and Chilocorus for biological control is an important avenue that requires further investigation. Hemisarcoptes Hemisarcoptes (Astigmata: Hemisarcoptidae) is a cosmopolitan genus of astigmatid mite that is associated with diaspidid scale insects (Gerson et al., 1990). The plesiomorphic life cycle of this genus includes an egg, prelarva, larva, protonymph, deutonymph, tritonymph and adult male and female. These mites go through an annual cycle peaking in mid-summer corresponding with the peak of adult scale insects (Gerson. 1967) and are able to adjust the sex rafio of their progeny when harsh condifions exist (Izraylevich and Gerson, 1995b; Izraylevich, Gerson and Wysoki. 1995). The demographic stages of Hemisarcoptes, with the exception of the deutonymph, are only slightly chitinized but have well-developed chelicerae (Gerson et al., 1990). Because of iimmrtratrMBiVMHirYi iTiii "- nan- i 'iiiiinyi inrr satgasaBBMiiii. » i I'l i their small size (<300pm), new species in this genus are still being discovered (Gerson. 1994; Fain and Ripka, 1998). The deutonymph (hypopode) differs in appearance from all of the other life stages. It is highly chifinized, lacks a mouth and chelicerae, and was originally described as lacking a funcfional gut (Gerson and Schneider, 1982). The deutonymph is a dispersal stage in the life cycle of Hemisarcoptes associated with beetles of the genus Chilocorus. For many years the origin and evolution of the distinctive hypopode, within a monomorphic lineage, has been hotly debated (Dugas, 1834; Hughes, 1955; Fain, Lukoschus, Louppen and Mendez, 1973; Pence and Webb, 1977: Athias-Binche, 1981; OConnor, 1982; Eickwort, 1990; Houck, 1993; Houck and Cohen, 1995) and its meaning as an insertion stage in the life cycle quesfioned. The primitive occurrence of the hypopode was acknowledged to have resulted in a tremendous radiation within the astigmatid clade. However, the reason for this radiation was unknown. Houck and OConnor (1991) developed a conceptual model accounting for the relevance of the hypopode to the diversification of the Astigmata into expanded coevolutionary roles (e.g., parasitism). Subsequently it was shown that the deutonymph of Hemisarcoptes does have a functional hindgut (Houck and Cohen, 1995), despite the fact that no oral opening or foregut exists, and that (like some other astigmatids) Hemisarcoptes does exhibit free living species and parasitic species within the same phylogenetic lineage. Accordingly, it has been suggested that phoresy somehow acts as an evolufionary transition stage between free living and parasitic life styles (Houck and Cohen, 1995). i*^ '" •'-— - -1 ^•«i6D(,Jii:<..jw?iffre*Bti'iiiMflMww.w •" " am BX= kuMi -- Hemisarcoptes was selected as the focus of this study for two reasons: it has an interesting ecological and evolutionary relationship with Chilocorus beetles that is still not fully resolved, and it has been given attention as an important predator of scale insects for many years (Ewing and Webster, 1912; Tothill, 1918; Gerson, 1971; Gerson and Schneider, 1981; Gerson and Izraylevich, 1994). Chilocorus Chilocorus (Coleoptera: Coccinellidae) is a large genus of beetles, found worldwide, that feeds on scale insects during all life stages (Muma, 1955; Gordon, 1985). These life stages include 4-5 larval stages and the adult stage. Members of this genus have life cycles of varying lengths, but most have 2-3 generations per year (Avidov and Yinon, 1969; Sakuratani and Ito, 1995). These beetles exhibit a behavior described as reflexive bleeding. Reflexive bleeding in Chilocorus consists of exuding hemolymph, which contains a suite of seven toxic alkaloids (Houck, 1999b), through weak sutures as a defense mechanism. Three of these seven compounds have recently been biochemically characterized (McCormick et al., 1994; Xiongwei, Attygalle, Meinwald, Houck and Eisner, 1995). The hemolymph is not only toxic but also crystallizes once exposed to air, causing mechanical problems to a predator that attempts to attack this beetle (Houck and Cohen, 1995). This predator-defense behavior is a strong selective force that probably adds to the fitness of this beetle. Chilocorus was chosen for this research for two reasons. First it has a unique association with Hemisarcoptes. Second, it is considered to be an important and voracious predator of diaspidid scale insects. ., ,. ,,,,. -pp^-- ^ g.„ ,..,, rr..-,...-^.».,n>,,^...... ,,|,^,^^|, , ,....1 «^^i.||n^^.. ,n,..^| , Biological Control of Scale Insects Diaspidid scale insects are significant crop pests, when introduced into habitats with no natural enemies. This is a common occurrence, with up to 40% of all insect pests in the United States being introduced from other parts of the world (Hoffmann and Frodsham, 1993). Scale control has tradifionally relied on broad-spectrum synthefic pesticides and dormant oil sprays (Gulmahamad and DeBach, 1978). However, biological control has become an increasingly important and well-studied avenue for defense against these global pests (Luck et al., 1999). Hemisarcoptes and Chilocorus have been recognized for many years as important natural predators of scale insects (Ewing and Webster, 1912; Tothill, 1918; Muma, 1955; Yinon, 1969). Both of these economically important predators are the subjects of study as significant biological control agents (Charles, Hill and Allan, 1995a; Houck and Cohen, 1995; Gerson and Izraylevich, 1997; Luck et al., 1999). One of the first important predators of scale insects to be noted in the literature was Hemisarcoptes, and it was the first mite applied to biological control in North America. Ewing and Webster (1912) recognized H. malus as an important predator of scale insects. Tothill (1918) published a paper stating that H malus was by far the most important factor in the natural control of diaspidid scale insects. Researchers noted that this predatory mite spends most of its life attached to, and feeding on, scale insects, significant!\ lowering their fecundity as well as causing mortality in some cases (Ewing and Webster. 1912; Tothill, 1918). Biological control studies have repeatedly noted that Hemisarcoptes is a highly effective predator against scale infestafions (Tothill, 1919: ;;^^.*» » ••'•.ar.*' *'»?*»<«^'»*ynw Gerson et al., 1990; Hill, Allen, Henderson and Charles, 1993; Ji et al.. 1994; Luck et al., 1999). In one notable case, when released in New Zealand from 1987 to 1992, Hemisarcoptes became established and effectively controlled kiwi scale (Hill et al., 1993). This mite remained resident and controlled scales without any additional inundafion release program (Charles, Hill and Allan, 1995b). Because of their importance as natural predators, recent research has focused on understanding the mechanisms involved in prey utilization by Hemisarcoptes (Izraylevich and Gerson, 1995a; Izraylevich and Gerson, 1995c; Izraylevich, Hasson and Gerson, 1995). Many tritrophic predator-prey issues must be considered, including host- plant resource type, scale species, scale morphology, demographics and dominant age- class of the scale colony, scale seasonality, and abundance and distribution of the host scale (Izraylevich and Gerson, 1995a; Izraylevich and Gerson, 1995b; Izraylevich, Hasson and Gerson, 1995). Although Hemisarcoptes has been noted as an efficient predator of scale insects, not all biological control efforts utilizing this mite have been equally successful (Hill et al., 1993). This unpredictability in application has been attributed to lack of knowledge about the biological and ecological requirements of the various species of Hemisarcoptes (Luck etal., 1999). Chilocorus has also been used in many successful biological control attempts (Kaufmann, 1977; Samways, 1989; Nalepa et al, 1993; Van Driesche, Idoine, Rose and Bryan, 1998). Chilocorus is an aggressive inundation predator, with each animal feeding on hundreds of individual scale insects (Yinon, 1969). Much research on this natural predator has been spent on determining the most effective field-release strategy for the different species of beetles in this genus (Applebaum, Kfir, Gerson and Tadmor, 1971; Podoler and Henen, 1986; Samways, 1989; Hattmgh and Samways, 1991; Ponsonby and Copland, 1996; Van Driesche et al., 1998). Also, reproductive behavior and biology of the different species has been a recent focus (Hattingh and Samways, 1995; Sakuratani and Ito, 1995). There is evidence that this beetle does have a major impact on scale insect populations when released alone (DeBoo and Weidhaas, 1976; Drea and Carlson, 1987; Van Driesche et al., 1998). However, as v^th Hemisarcoptes, some releases have been not been completely successful (Kaufmann, 1977). This has been attributed to the vagrant behavior of dispersing adults out of the area of release and to the fact that these cosmopolitan beetles are often introduced into environments with which they are not compatible. Other parasites and parasitoids of scale insects have been studied for potential use as biological control agents of scale insects (Prinsloo, 1996; Rodrigo, Troncho and Garcia-Mari, 1996). However, none of these predators have been as successful as Hemisarcoptes or Chilocorus. Because of evident past success, Hemisarcoptes and Chilocorus can be considered the most promising biological control agents for scale insects investigated thus far. Coevolutionary Relationship of Hemisarcoptes and Chilocorus Hemisarcoptes has a heteromorphic life stage, the deutonymph, which does not feed on scale insects, but instead attaches to the elytra of Chilocorus for dispersal. During fimes of scale depletion, or severe drought, this dispersal stage prevents termination of the mite lineage due to starvation. This dispersal relationship is interesting 8 h.'[ because the toxic hemolymph, expelled by Chilocorus appears to have no effect on the survival of the deutonymphs (Houck, 1994). It was hypothesized that this was due to natural selection for a tolerance to the hemolymph over millions of years (Houck and OConnor, 1991). Houck and Cohen (1995) quesfioned the theory that this relationship is purely phorefic and explored the relationship between Hemisarcoptes and Chilocorus in more depth. They determined that a transfer of materials from Chilocorus to Hemisarcoptes occurred. They resolved this by using a radiolabeled tracer in which they tracked tritiated water being passed from Chilocorus to Hemisarcoptes, showing that water (at least) was being passed from the beetle to the mite. Since a material is being passed from Chilocorus to Hemisarcoptes, this relationship would be considered potentially parasitic, or perhaps mutualistic. Research Obiective The objective of my research was to further define the Hemisarcoptes-Chilocorus relationship and discriminate whether it could be functionally defined as parasitic (one way flow of materials between organisms) or potentially mutualistic (bidirectional flow of materials between organisms). To test this hypothesis, I determined whether tritiated water could be detected in C. cacti as a consequence of its relationship with H cooremani. This finding would be important because it would help resolve the relationship between these two organisms, and contribute a critical element for improving the efficacy of the system for biological control. It would also contribute to a fuller understanding of the nature of phoresy in the evolution of parasitism in the Astigmata. i-^y.rr ZL'':—r-^— =aE ItJ • »»'»»>^l<«.IWi<<' •• KMW The working assumption for this research was that if materials do not pass from the deutonymph of// cooremani to C. cacti then the deutonymphal stage of the mite would be considered a facultative parasite of the beetle and would potentially have a negative influence on the bionomics of Chilocorus. If materials do pass from the deutonymph of// cooremani to C. cacti, then the deutonymphal stage of the mite would potentially be considered a mutualistic partner of the beetle and could possibly have a positive influence on the bionomics of Chilocorus. With either outcome there are important implications for the use of these two organisms as biological control agents of scale insects. A better understanding of this relationship will only add to the effective use of these already important predators. 10 •ar^srffjjn i>«j>''*j^'^ * 11 II unimiiniintiiiTiirBinniTifir ^ —^ Ha)iBaa*«M CHAPTER II MATERIALS AND METHODS Laboratory Monocultures Hemisarcoptes cooremani and Chilocorus cacti were originally collected in southern Texas, near Weslaco, from citrus trees and then reared in the laboratory at Texas Tech University. The resource base of Hemisarcoptes and Chilocorus consisted of scale monocultures (Diaspididae: Aspidiotus nerii) reared on russet potatoes at 22°C in a constant temperature chamber. The beetles and mites were reared separately on 10-12 potatoes covered with scales, in an enclosed plastic box, maintained at 23°C, in a walk-in constant temperature room. Beetles were periodically examined under a dissecting microscope, to assure that no mites had infested the beetle monoculture. Mite colonies were also examined to assure that no beetles had invaded them. All organisms, used in this study, were selected randomly from these long-standing laboratory monocultures. Experimental Design A treatment is defined here as an applied exposure of beetles to tritiated atmosphere and tritiated deutonymphs. An experiment is defined as a treatment and all the appropriate controls for that treatment. A control is a test against which the treatment effect is compared. A replicate (N=15) is defined as one cognate of a set (treatment or control) and is assumed to be a random representative for that set. A cohort is defined as three replicates run simultaneously. 11 Bf/^Tim "Ti <'. "W ••• rm i JTT T "«Ti Tl • »^< Cohort Condifions Physical parameters were held constant including plate size, time of day, light, and temperature, to standardize experimental conditions. Three beetles were randomly selected from the monoculture and carefully examined to ensure that no mites were present. Three beetles were used as a cohort, and confined to a common petri dish containing deutonymphs. This assured that sufficient quantities of deutonymphs were available for attachment to each beetle and provide trifiated-mite biomass (sufficient to overcome a collective minimum-count threshold) in the event that tritium was transferred from the mites to the beetles. Treatment Conditions At 1700hr. on day one of a treatment, deutonymphs of Hemisarcoptes were brushed with an artist's brush into a petri dish with a tight fitting lid. .\ vial of tritiated water (final tritium concentration 25pCi ml-i' >) was capped with cotton and placed into another vial that was covered \\ ith fine mesh so no mites or beetles could directly contact the tritium in the inner vial. This nested-vial set up was restrained on the bottom of the petri dish with plastic adhesive, and the lid to the plate was secured. The plate was left undisturbed for 43 hours, adequate time for the deutonymphs to acquire a recordable amount of tritium from the atmosphere. Then, at 1200hr. on the third day of the treatment, beetles from the monoculture (devoid of mites) were added to the trifiated chamber containing mites. The beetles remained in the chamber in contact with the mites unfil 1200hr. on the fifth day of the treatment (48hr.), sufficient time for the deutommphs to attach to the beetle and be in intimate contact with the beetles, but not long enough for 12 gtjpi"">^" '»•—**«" «!«-•••.'«i IJOM—If.li. . M.».i. .1 1«.,c».>M- the beetles to starve. At that point, the treatment exposure was terminated, and the following steps were immediately performed. First, the mites were removed from the underside of the beefie's elytra using a hooked minutm pin. The removed mites were counted and placed into a vial of Soluene 350 fissue solubilizer. The beetle's elytra were removed from the body and placed into separate vials of fissue solubilizer. After the fissues were dissolved in the solubilizer, scinfillation cocktail (Hionic Fluor, Packard Instruments) was added to all three types of treatment vials. Beetles and mites were surveyed, by scintillafion, for resident radiation in their tissues. The radioactivity (dpm) from these tissues was determined using a Beckman LS 6500 Scintillation Counter to determine whether tritiated water was mobilized from tissues of H. cooremani to C. cacti. Controls The full-blocked research design (Table 2.1) consisted of three qualitatively different controls to assess the validity of the treatment (Table 2.2). The treatment potentially provided the beetles with two sources of tritium: that acquired from the atmosphere and that acquired directly though association with the mites. A blocked design allowed the quantitative partitioning of the two potential contributions (i.e., atmosphere and mites) to a total tritium load in the beetle tissues. All four experimental petri dishes (i.e., 1 treatment + 3 controls) were standardized by receiving 43hr. of tritium exposure. In Controls #1 and #3. the trifium source was removed after 43hr. of exposure, and the lid of the petri dish was removed for 5 minutes to clear the chamber of tritium prior to the addition of the cohort of beetles. 13 iMinawMuraam E3Z3B J_U«JI.IJ-B.^".-^- The Treatment and Control #2 retained the tritium source for the entire experimental period. The purpose of Control #1 was to determine whether or not mites were capable of acquiring and retaining trifium in their tissues for the duration of the study (48hr.), and to test whether the mites were capable of transferring acquired trifium to beetles in the absence of atmospheric tritium. The purpose of Control #2 was to determine whether or not beefies were capable of acquiring atmospheric tritium, in the absence of mites. The purpose of Control #3 was to negate the possibility of endogenous background tritium in the beetle fissues, or potentially residual tritium attached to the sides of the petri dish following initial exposure to tritium. Cohort Number Many factors influenced the number of cohorts that were feasible for this study, including difficulties inherent in maintaining long-standing monocultures of the mites, beetles and scales. The scale colony could only be maintained at high enough numbers to provide food for one mite monoculture and one beetle monoculture. This limited the organisms available. Other dynamics limited the number of cohorts that were used during this study, including the cyclic nature of Hemisarcoptes in the monoculture. Because of the cycling of life stages, enough deutonymphs to run a cohort only became available about every eight weeks. The beetle colony also had a regular pulsed population cycle (many adults for a period of time followed by many larvae for a period of time). Because of this, many adults were not simultaneously available during the deutonymph surges. Finally, when running cohorts it was necessary to be cautious with 14 twwwwwwauimir ii.'inry;;-.gr--si the number of animals removed from each monoculture so that the colony would not be depleted. Therefore, five cohorts (15 beetles) for the treatment and all controls was the selected number for this study. Data Analysis Non-parametric statistics were applied to the data because a restricted number of replicates (N=15) were possible and there was a non-normal variance structure to the data. Non-parametric summary statistics (e.g., boxplots: medians, quartiles, and ranges) and the Mann-Whitney test of differences in the medians were determined using Minitab for Windows. A t-test, for differences between means, was also applied using Minitab®for Windows. Bivariate plots of the tritium levels, per replicate, and correlations were generated using Microsoft® Excel for Windows. 15 •~'zisapssaj^tBa\XMri\Mfftnvntm^\'MtKMi!t'.o^' • vtw^^- o-* Table 2.1. Full-Block Design of Experiment (Treatment and Controls). Atmospheric Tritium No Atmospheric Tritium Present Present Tritiated Mites Treatment Control #1 Present No Tritiated Control #2 Control #3 Mites Present 16 l.«ffiWA» ITITJUIWW taamaaaasattiittm •wni»Mwoaiir»iri.«inffig»i tr, .r. imxini"«. r. i T^i . • > t • am. Table 2.2. Experimental Block Design of the Treatment and Controls. Condifion of Petri Dish Tritium Final Prior to Addition of a Removed Prior Experimental Cohort of Beetles to Final Conditions (43hr. Exposure) Conditions (48hr. Exposure) Beetles 1 1 Mites Tritiated Mites Treatment Tritiated Atmosphere NO Tritiated Atmosphere Beetles Mites Tritiated Mites Control #1 Tritiated Atmosphere YES No Tritiated Atmosphere Beetles No Mites No Mites Control #2 Tritiated Atmosphere NO Tritiated Atmosphere Beetles No Mites No Mites Control #3 Tritiated Atmosphere YES No Tritiated Atmosphere 17 »'jiMMuuiM>(ii»rwin(i»iti>i<>ii..iiiO!>i.fi:,f iiriiniliiilii MlTtjiglUMllll CHAPTER III RESULTS Tritium Levels The tritium concentrations (dpm) of the tissues of the treatment beetle bodies and elytra, at the end of 48hr. of trifium exposure, were substanfially higher than the controls (Tables 3.1, 3.2, A. 1 and A.2). The measures were highly variable and were not normally distributed (Figures 3.1-3.4). Tritium concentrafions (dpm) of the fissues of the Control #1 beetle bodies and elytra, at the end of the 48-hour exposure to tritiated deutonymphs, were measurable (Tables 3.1, 3.2, A. 1 and A.2). The measures were also highly variable were not normally distributed (Figures 3.5-3.8). The tritium concentrafions (dpm) of the tissues of the Control #2 beetle bodies and elytra, at the end of 48-Hours of atmospheric tritium exposure, were substantially higher than the other Controls (Tables 3.1, 3.2, A. 1 and A.2). Like the treatment and Control #1, they were highly variable and were not normally distributed (Figures 3.9- 3.12). From the tritium concentrations (dpm) of Control #3 (Tables 3.1, 3.2, A. 1 and A.2), it can be noted that little residual trifium remained in the chamber after the five minute clearing process. The Control replicates were not normally distributed (Figures 3.13-3.16). 18 --.K-^Tr. i^nit^iu-t ^fimnwimmimKUAtm .i.K ii 0*1. aj'?»|iujujeonup-!•»>• • "if • • •' ^"• Effects of Mite Numbers on Tritium Levels It was necessary to exclude the possibility that the number of mites attached to the underside of the elytra determined the tritium levels that could be taken up (Table 3.3). To examine this possibility, trifium levels in the beefie's body and elytra were correlated with the number of mites attached to that beetle. There was no association between the number of deutonymphs attached to Chilocorus and the amount of tritium taken up by either the body or the elytra (Figures 3.17-3.20). Comparisons The results of the treatment and controls are summarized in Figure 3.21 and 3.22 as boxplots. All treatment and control pair-wise comparisons were made using t-tests and Mann-Whitney tests (Tables 3.4-3.7). These comparisons were done for beetle bodies and elytra separately. Comparison 1 (Treatment versus Control #2). The purpose of this comparison was to evaluate whether tritium was taken up by Chilocorus from the deutonymphs. It compared beetles simultaneously exposed to tritiated deutonymphs and tritiated atmosphere with beetles exposed only to tritiated atmosphere. The research hypothesis implied by this comparison was that the tritium counts from the treatment (tritium is taken up from the deutonymphs. as well as the atmosphere) were significantly greater than Control #2 (no deutonymphs). The null hypothesis for this comparison was that the tritium counts from the treatment and Control #2 were statistically equivalent (tritium is only taken up from the atmosphere, not from the deutonymphs). The results of the t-test for both the beetle body and beetle elytra were 19 LCJ-t gJ-TJ.-? g« mMn\.M*tP*. significanfiy different with the treatment being higher (p< 0.0026 and p< 0.0015, respectively; Tables 3.4, 3.5) leading to rejecfion of the null hypothesis. The results of the Mann-Whitney test were also significantly different (p< 0.0006 for the bodies and p< 0.0055 for the elytra; Tables 3.6, 3.7) again rejecting the null hypothesis. Comparison 2 (Control #1 versus Control #3V The purpose of this comparison was to evaluate Control #1 versus Control #3 as a second verification that beefies were acquiring tritiated water from the deutonymphs and to test whether deutonymphs were able to retain acquired trifium for 48 hr, in the absence of a tritiated atmosphere. This was necessary because no estimate of the time-course of water cycling in mite tissues was available from the literature. It compared replicates (Control #1) in which trifiated mites were present versus replicates (Control #3) in which no mites were present. The research hypothesis implied by this comparison was that the tritium counts from Control #1 was significantly greater than Control #3 and that tritium is taken up from the deutonymphs, which did not purge all tritium acquired during the time allotted for the trial. The null hypothesis for this comparison was that tritium counts from Control #1 and Control #3 are equivalent, indicating that tritium was purged prior to scintillation or that the beetles did not acquire tritium from the mites. There was a significant difference in mean values between Control #1 and Control #2 (p< 0.0019 for the bodies and p< 0.0009 for the elytra; Tables 3.4, 3.5) and thus the null hypothesis is rejected. The Mann-\Miitncy test also indicated that results were significantly different for the beetle bodies and elytra (p< 0.0000 and p< 0.0000, respectively: Tables 3.6. 3.7) again causing the rejecfion of the null hypothesis. 20 MBMKin •' 'i fft Y?ii'r, i-;r^CL' p^^nnv^WEWWv^ Comparison 3 (Treatment versus Control #1, and Control #2 versus Control #3). These comparisons verified that tritium could be acquired by the beetles directly from the atmosphere, by comparing replicates that had atmospheric tritium with replicates that did not. The research hypothesis was that the treatment will be significantly greater than Control #1 and that Control #2 will be significantly greater than Control #3. The null hypothesis was that the treatment counts were equivalent to the counts from Control #1, and that the counts from Control #2 were equivalent to Control #3. The t-tests and Mann-Whitney tests for both comparisons of the body and elytra indicated highly significant differences (p< 0.0000; Tables 3.4-3.7) causing the rejection of the null hypothesis. 21 mHBiuiviMBg VfB'wgiiiin nfcua'v A-JIM.:'.!VM," - • Table 3.1. Summary of Trifium Concentrafion in Beefie Bodies at the End of the 48-Hour Experimental Exposure. Treatment Control #1 Control #2 Control #3 Mean 35825 934 19952 95 Median 36292 454 18657 90 Standard 15914 849 8432 32 Deviation Standard 4109 219 2177 8 Error 95% Confidence ±8813 ±470 ±4670 ±18 Interval Table 3.2. Summary of Tritium Concentration in Beefie Elytra at the End of the 48-Hour Experimental Exposure. Treatment Control #1 Control #2 Control #3 Mean 5046 192 2995 65 Median 5232 141 3011 60 Standard 1963 117 800 12 Deviation Standard 507 30 207 3 Error 95% Confidence ±1087 ±65 ±443 ±7 Interval 22 vssaasKssssgR —r.'-'-TTffffr. Table 3.3. Number of Mites Attached to Each Beefie in the Treatment and Control #1 Beetle Number Number of Number of Mites/Beetle in the Mites/Beetle in the Treatment Control #1 1 18 22 2 13 41 3 10 25 4 23 10 5 28 13 6 20 14 7 26 23 8 22 18 9 20 20 10 17 22 11 25 25 12 22 21 13 20 28 14 21 35 15 30 22 Mean 21 22.6 Standard 5.3 8.0 Deviafion 23 PJTiM »riTHM.u •>. • ri I <•>-fI. ^>i;w» Table 3.4. Results oft-test for all Pair-Wise Comparisons of Trifium Concentrafions in the Beetle Bodies after the 48-Hour Experimental Exposure. t value Probability df Treatment vs. 8.48 p< 3.2X10-^ 28 Control #1 Treatment vs. 3.41 p<2X10-^ 28 Control #2 Treatment vs. 8.70 p< 1.9X10-^ 28 Control #3 Control #1 vs. -8.69 p< 1.9X10-^ 28 Control #2 Control #1 vs. 3.82 p< 6.8X10-^ 28 Control #3 Control #2 vs. 9.12 p<7X10"'° 28 Control #3 Table 3.5. Results oft-test for all Pair-Wise Comparisons of Tritium Concentrations in the Beetle Elytra after the 48-Hour Experimental Exposure. t value Probability df Treatment vs. 9.56 p<3X10-^° 28 Control #1 Treatment vs. 3.75 p< 8.2X10-^ 28 Control #2 Treatment vs. 9.83 p<1X10-^° 28 Control #3 Control #1 vs. -13.42 p<1 xio-'^ 28 Control #2 Control #1 vs. 4.17 p< 2.6X10-^ 28 Control #3 Control #2 vs. 14.18 p<1 xio-^^ 28 Control #3 24 •^^^W""-^^!*^ .•! .-•(•., ^VMiUMMWIWAVl'T^ ' :re~ I w»• g- •»" Table 3.6. Results of Mann-Whitoey test for all Pair-Wise Comparisons of Tritium Concentrations in the Beetle Bodies after the 48-Hour Experimental Exposure. Significance W Level Treatment vs. 345 p< 0.0000 Control #1 Treatment vs. 299 p< 0.0006 1 Control #2 Treatment vs. 345 p< 0.0000 Control #3 Control #1 vs. 345 p< 0.0000 Control #2 Control #1 vs. 345 p< 0.0000 Control #3 Control #2 vs. 345 p< 0.0000 Control #3 Table 3.7. Results of Mann-Whitney test for all Pair-Wise Comparisons of Tritium Concentrations in the Beetle Elytra after the 48-Hour Experimental Exposure. Significance W Level Treatment vs. 345 p< 0.0000 Control #1 Treatment vs. 300 p< 0.0055 Control #2 Treatment vs. 345 1 p< 0.0000 Control #3 Control #1 vs. 345 ' p< 0.0000 Control #2 Control #1 vs. 345 ' p< 0.0000 Control #3 1 Control #2 vs. I 345 1 p< 0.0000 , Control #3 3 keatti megaw^vm^Aimmmii&vm'nmuKx i A jmfx ir<•-mrt,jrnTft"w»I•jjii.u, ^I'^i IULL.. 80000 70000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Sample Number Figure 3.1. Tritium Concentrafions from Beefie Body after 48-Hour Exposure of Treatment (This data is NOT continuous) 26 'M.J^.. ^-ggiVIWgaTOTOBBWRyvr.lWlWUauwinffMf'Wii^i' 'i ir i-ri^r-ir-jB 6 c 0 i « •'0 '^ /• 4 / ^y * y S^ i Frequenc y ^ * 2 - / > * 1- t % i A. 10000-18500 18500-27000 27000-35500 35500-44000 44000-52500 52500-61000 61000-69500 Tritium Levels (dpm) Figure 3.2. Frequency Distribution of Tritium Concentrafion in Beetle's Bodies after 48-Hour Exposure of Treatment 27 'MmmoKOKOs^ mim^ immwiiwnrwwBaB 1000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Sample Number Figure 3.3. Tritium Concentrafions from Beefie Elytra after 48-Hour Exposure of Treatment (This data is NOT continuous) 28 i 'ii«mimmamHsmBBwmr^.\iMmuMamiinr nwif irjaaaaa ... ,:tm't. 4 - o.ti 3 \ Freque r I.O^ ^ X - 1 * •J* '/^i 0.& 4^ '• •>•'; •'. ^ 0 ' 2000-3100 3100-4200 4200-5300 5300-6400 6400-7500 7500-8600 8600-9700 Tritium Levels (dpm) Figure 3.4. Frequency Distribution of Trifium Concentration in Beetle's Elytra after 48-Hour Exposure of Treatment 29 aMMaMi ^viWTMi>WBg^'awflwwwawiijjiji.i'.!utiwiaTiiwT)tr niwwaie-jrwii 3000 Sample Number Figure 3.5. Tritium Concentrations from Beetle Body after 48-Hour Exposure of Control #1 (This data is NOT continuous) 30 KBfflWRSaBEKKss.--- 8- ' •//. • '/^' • •/ ^ •'' PLH ''?' ''' < -as-* I tSzA 200-550 550-900 900-1250 1250-1600 1600-1950 1950-2300 2300-2650 Tritium Levels (dpm) Figure 3.6. Frequency Distribution of Trifium Concentration in Beetle's Bodies after 48-Hour Exposure of Control #1. 31 8 wfl&iwmiwu'irparrrt' 500r 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Sample Number Figure 3.7. Tritium Concentrations from Beetle Elytra after 48-Hour Exposure of Control #1 (This data is NOT continuous) 32 roWBUgtfl»T1?i.ri vm^mnini .fcjryi JI- - --'.Av- 10r 9 8 O C 6 s^ 0^ 95-146 146-197 197-248 248-299 299-350 350-401 401-452 Tritium Levels (dpm) Figure 3.8. Frequency Distribution of Trifium Concentration in Beetle's Elytra after 48-Hour Exposure of Control #1. 33 f.'VJJHfflMWWHWVWPir Ti,1 ggqgcv I III »1»lll.H«^_ 45000 40000 35000 30000 E 25000 a. •o ^ 20000 E 15000 10000 5000 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Sample Number Figure 3.9. Tritium Concentrations from Beetle Body after 48-Hour Exposure of Control #2 (This data is NOT continuous) 34 '»T:g.T,Mniav,wji'j7y*^r^sy" p^i_»^»i»—jiij^iw ^_ 45 4 7 C — ----- 1 3 - • PH ! 1.5 1 c ^ '-•• J? 0.5 0 8000-12500 12500-17000 17000-22500 22500-27000 27000-31500 31500-36000 36000-40500 Tritium Levels (dpm) Figure 3.10. Frequency Distribution of Trifium Concentration in Beefie's Bodies after 48-Hour Exposure of Control #2. 35 ftWWWUiMinTi:i'ifli»iViiii'iniM"-rir«w.r 500G 6 7 8 9 10 11 12 13 14 15 Sample Number Figure 3.11. Tritium Concentrafions from Beetle Elytra after 48-Hour Exposure of Control #2 (This data is NOT continuous) 36 i i tmit^Kmm* i HJ >i. i '•»•,T'f ••?g«wirrt' 3.5- 3 ' 1 l.i - 1 nc y CD 2 (D VH i PLH f 1.J » .X ;:\ 1 i 1 1 —\ 1 i - O.J «3 0 1500-1900 1900-2300 2300-2700 2700-3100 3100-3500 3500-3900 3900-4300 Tritium Levels (dpm) Figure 3.12. Frequency Distribufion of Tritium Concentration in Beefie s Elytra after 48-Hour Exposure of Control #2. 37 *J»';»*»'wir?*.'W«^»-^ f • !3l r-,«.—,-,p,^ •^.,.,„.,^, ^.^ jqgy.-:y]^^^paBBpip Figure 3.13. Tritium Concentrafions from Beefie Body after 48-Hour Exposure of Control #3 (This data is NOT continuous) iBBSWSBSSCSS 5 4 - c VH < PH '1 2 i-K ^ > 1 1 ^1 0 ^ 50-65 65-80 80-95 95-110 110-125 125-140 140-155 Tritium Levels (dpm) Figure 3.14. Frequency Distribution of Tritium Concentrafion in Beetle's Bodies after 48-Hour Exposure of Control #3. I ••• "Vf.fi •^•vwtV\tH]Mtfnnaati^ lOOr ^rd Figure 3.15. Tritium Concentrafions from Beetle Elytra after 48-Hour Exposure of Control #3 (This data is NOT confinuous) 40 IIUMHM ir'jTfrrrtt. i.rii'T iir:c^T/:r: \>^\AJfV"VM," '=g: 5 4 '-- - - enc y cr 1 I 1 -^ • 0 .: 46-52 52-58 58-64 64-70 70-76 76-82 82-88 Tritium Levels (dpm) '-J.1 Figure 3.16. Frequency Distribution of Tritium Concentrafion in Beefie's Elytra after 48-Hour Exposure of Control #3. iiylF^MmMW*"'"''^''^™'™"™''^^ ' •«^g«r 8000& 10 13 17 18 20 20 20 21 22 22 23 25 26 28 30 Number of Mites Figure 3.17. Associafion Between Number of Deutonymphs Attached to a Beetle and the Trifium Concentration from that Beefie's Body after the 48-Hour Exposure Period in the Treatment. 42 jswn,.. t JTjnrT^JWWiV{.-MW>»;^iViwtnuf• •.•••• T- • -1 r 10 13 17 18 20 20 20 21 22 22 23 25 26 28 30 Number of Mites Figure 3.18. Association Between Number of Deutonymphs Attached to a Beefie and the Trifium Concentration from that Beetle's Elytra after the 48-Hour Exposure Period in the Treatment. 43 "' *IW MW WiWWWW 300Q 250C 200C es e u J 1500t^ E s L. H lOOC 500 «" Figure 3.19. Associafion Between Number of Deutonymphs Attached to a Beefie and the Trifium Concentration from fiiat Beetle's Body after the 48-Hour Exposure Period in Control #1. •S. 44 \-'f-*rsigmaisasasaamsmBS9fpms^9Si;^s;is^^ aaDBBSBas: tawpifc**jfji^'».^ 500 •/ Figure 3.20. Association Between Number of Deutonymphs Attached to a Beetle and the Tritium Concentration from that Beetle's Elytra after the 48-Hour Exposure Period in Control #1. JBf-r-f • !>•••• • ^ ' ^fm t»iMaKms^-- D KVV>i_'*rgr.jWIIW'iV«A'*. fAVTJ.V/. IV^":TVIV.VI"T I I IW 70000 60000 — 50000 _ E a. 40000 _ •o e _o '^ C8 30000 — e o U 20000 _ 186578 10000 _ 4S1.07 90^ 0 — Treatment Control #2 Control #1 Control #3 Figure 3.21. Summary Boxplots and Median Tritium Concentrafions from the Beetle's Bodies for the Treatment and All Controls 46 • •• n ' w •^"g" r^' y!SEXS» 10000 E o. e o es B 5000 _ w c o u 3011.02 140.7 60J9 0 _ Treatment Control #2 Control«1 Control «3 Figure 3.22. Summary Boxplots and Median Tritium Concentrations from the Beetle's Elytra for the Treatment and All Controls s 47 Broll^^aft.wl^.m< ijyAmff-f.,viijxj^ .%vjrjiT.w CHAPTER IV DISCUSSION Hypopodes, the unique insertion stage in the lineage of astigmatid mites, have been studied for many years (Dugas, 1834; Fain, Lukoschus, Louppen and Mendez, 1973; OConnor, 1982; Houck and Cohen, 1995) to determine their function as a life stage and to learn more about their biology. Many interesfing features of the biology of hypopodes have been explored in different astigmatid mite species. One such feature is that, although hypopodes lack fimctional mouth parts, they appear capable of imbibing materials from the host (Fain and Bafort, 1966; Fain, 1969; Fain et al., 1973). This observation led to important developments in exploring the relationship of Hemisarcoptes and its beetle host Chilocorus. For over 100 years, the hypopodes of Hemisarcoptes had been considered phoretic on beetles in the genus Chilocorus (Gerson, 1967; Gerson and Schneider, 1982). Houck and Cohen (1995) refuted the phoretic theory and determined that the relationship between Hemisarcoptes and Chilocorus could be more accurately defined as at least a unidirectional interaction (parasitism), but that mutualism could not be ruled out. Houck and OConnor (1991) stated that parasitism and mutualism are altemafive options, derived from the interface of free-living organisms that begin an ecological interaction that extends into an evolutionary interaction. Discriminating between mutualism and parasitism is not always easy and requires detailed information on the life-history characteristics and physiological dynamics of both members of the symbiotic pair. 48 IMilMH ^nfwriMi'^if !•» The quesfion remained from Houck and Cohen's research (1995) whether this symbiotic relationship could be further defined. It is clear in the case of Hemisarcoptes and Chilocorus that the deutonymph is gaining significant benefit from this relationship. It is not only gaining transportation to a potentially novel food source, but it also appears to be gaining nutrition from the beetle that allows it to remain attached for 5-21 days and still go through ecdysis (Houck and Cohen, 1995). What was unclear was whether Chilocorus was gaining any benefit from the interaction. Further definition of this relationship is given by the work reported here. It demonstrates that tritiated water is passed from H cooremani to C. cacti under experimental conditions. This study has many important implications for defining the relationship between Hemisarcoptes and Chilocorus and for the use of these animals as biological control agents. Because the beetles were actually acquiring materials (at least water) from the mites, this relationship must now be viewed in an entirely different way. The relationship between Hemisarcoptes and Chilocorus can no longer be discussed as phoretic (Houck and Cohen, 1995). But, the relationship may not be accurately described as parasitic either. As demonstrated by this study, some materials are being passed bidirectionally (both from the beetle to the mite and from the mite to the beetle). Therefore, mutualism may be a more appropriate descripfion of this relationship, if a selective advantage can be documented independently. For the relafionship to be mutualistic, both members must benefit from the interaction. Benefit could accrue if the beetles acquire materials from the mites that contribute to their selective advantage (fitness). The materials could range from metabolites or hormones to simply water. However, it cannot be ruled out at this point 49 r,it>(mimtaawMMMWmmn '•Mi:..mretrsmsifi i "^' •iiffia\-/?yn,ii? i raaaBB^^^ that the deutonymphs may be passively eliminating waste into the beetles through the connection between the two organisms. Further research is needed to elucidate whether there is a selective advantage for both parties in this relationship. Future Directions This study demonstrates that although there is a bidirectional flow of materials, the precise relationship between Hemisarcoptes and Chilocorus still need to be resolved. The work also raises new questions that could be answered by studies involving the mite, the beetle or the entire natural system. Studies are needed to determine the make up of the materials withdrawn by the mites and the beetles. Also, determining the method for the uptake of materials from the mite would be interesting. Another potential study involving Chilocorus would be to determine if the beefies exhibh a higher fitness when raised with or without mites. Understanding this relationship may have important implications for an integrated pest management system that includes both organisms. Potential future studies could include determining if beetles in the genus Chilocorus and mites in the genus Hemisarcoptes exhibit a higher reduction in scale density than either animal exhibits alone. It would be interesfing to determine if astigmatid hypopodes other than Hemisarcoptes are also mutualistic with their "phoretic" hosts. For example, Lardoglyphus, another astigmatid mite, has been observed not to survive without the presence of its phoretic host (Houck, 1999b). A study similar to that of Houck and Cohen (1995) could aid in determining if the relafionship is something besides phorefic. 50 !e!^^^5?F'gw?»sQe5«iiws=3!^<5==5r^=^^ saaBxawK'n ' •• Studies such as this one are critical to the understanding of how organisms make the transition from free-living forms to parasitism. Few systems exist where extant members in transition are available for study. This data adds significantly to our i understanding of the role of phoresy in the evolution of parashism. •tt 51 -^xsmmm LITERATURE CITED Applebaum, S. W., R. Kfir, U. Gerson, and U. Tadmor. 1971. Studies on the summer decline of Chilocorus bipustulatus in citrus groves of Israel. Entomophaga. 16:433-444. Athias-Binche, F. 1981. Observations on the genus Protodinychus Evans, 1957 (Acari: Mesostigmata) with descriptions of the male and phorefic deutonymph. Proceedings of the Royal Irish Academy. Section B-Biological, Geological and Chemical Science. 81:25-38. Avidov, Z. and U. Yinon. 1969. 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Beetle Treatment Control #1 Control #2 Control #3 Number 1 31119.2 2313.6 18657.8 136.4 2 56527.5 2609.1 17446.7 152.9 3 19748.0 2443.4 19444.9 154.5 4 51340.1 427.2 14936.7 82.6 5 39252.5 396.9 16144.9 94.1 6 36156.8 431.8 13189.8 89.3 7 17609.5 583.2 20779.7 90.4 8 14646.5 1572.8 39728.0 79.3 9 15885.4 835.5 28848.3 57.0 10 66688.2 454.1 31210.0 53.9 11 48911.1 261.8 13091.3 71.8 12 44377.7 561.5 8644.6 55.8 13 36292.1 358.3 24311.3 109.1 14 37360.5 346.3 22957.7 91.8 15 21460.2 407.3 9894.1 104.8 60 IIHJIJJWIl WtlWW I "•" ;.4.- f.issw»««aw«i'«^J .•.-5fgga*gTsarj: •C^n7::c:S^W¥;oKi'A^aB<«BU« >»ti>»ni.y.»**»»»''' >jrf»,. Table A.2. Tritium Concentrations (dpm) from Beetle Elytra after the 48-Hour Experimental Exposure. Beetle Treatment Control #1 Control #2 Control #3 Number 1 3531.1 238.7 3794.0 66.7 2 9309.3 416.8 3011.0 73.8 3 5082.8 448.7 3909.4 70.5 4 7636.6 100.8 2934.6 73.4 5 5547.3 254.7 2498.3 86.1 6 5231.9 104.0 2836.7 84.7 7 2483.6 143.9 3730.1 79.0 8 2587.7 312.6 4290.2 57.2 9 2247.9 175.9 3200.2 57.4 10 5656.8 140.7 3546.3 49.6 11 5822.5 100.9 1697.0 51.4 12 5170.5 107.6 1581.1 53.4 13 6466.2 106.7 3146.9 60.4 14 5537.0 132.6 2667.5 57.1 15 3372.8 99.0 2079.0 54.5 61 g!iUli.i.iUJJ!y8S» rrr visimB-^'jtBmmwiraywMiiWiwjri^ yyM^^\^j\-m' .•.•'.•"'.-j' ,• •••.T..yv.-.. Tn.iT>,. .IN^ PERMISSION TO COPY In presenting this thesis in partial fulfillment of the requirements for a master's degree at Texas Tech University or Texas Tech University Health Sciences Center, I agree that the Library and my major department shall make it freely available for research purposes. Permission to copy this thesis for scholarly purposes may be granted by the Director of the Library or my major professor. 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