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The impact of Tomicobia tibialis (Hymenoptera : pteromalidae) on the pine engraver, Ips pini (Coleoptera… Senger, Susan Evelyn 1993

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The Impact of Tomicobia tibialis (Hymenoptera:Pteromalidae) on the Pine Engraver, lp s pini (Coleoptera: Scolytidae). by  Susan Evelyn Senger B.Sc., Simon Fraser University, 1991  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Plant Science  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA July 1993 © Susan Evelyn Senger, 1993  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  ^p i . r--;,/,/ T^SC -E  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  Ju'L/^)  ^7  ii  Abstract Augmentative release of biological control agents against pest insects is a management tool that has been largely ignored in forestry due to a lack of understanding and means of producing potential agents. I investigated the effects of Tomicobia tibialis Ashmead on the reproductive potential of adult Os pini (Say) by comparing the reproductive indices of parasitized and healthy adult beetles maintained together in different combinations in pine logs. From these experiments I determined that the parasitoid most greatly impacts female beetle reproductive performance resulting in shorter gallery lengths and decreased offspring production before the female dies. Male beetle reproductive success depended on the females in his harem; however, the parasitized male cannot go on to subsequent infestations as a healthy male may. Thus the overall reduction in beetle reproduction resulting from parasitism can be substantial. Laboratory rearing methods for T. tibialis using pine logs were also established, and an attempt to rear these parasitoids from beetles held on artificial medium was made. In sum, it is possible to rear this parasitoid and the impact on its host beetle is better understood. I feel further investigation into augmentation using T. tibialis against I. pini is warranted.  Table of Contents Abstract Table of Contents List of Tables List of Figures Acknowledgements Chapter One  Biological Control in Forestry Introduction Biology of the Host: 1ps pini Biology of the Parasitoid: Tomicobia Potential for Control  Chapter Two  Impact of Tomicobia tibialis on the Reproduction of 1ps pini^ .8 Introduction^ . • .8 ^ Methods and Materials 10 Colony Maintenance^ . . 10 Experiments^ . . 12 Experiment 1: Healthy Males with Healthy and/or Parasitized Females^.. 12 Experiment 2: Parasitized Males with Healthy and/or Parasitized Females ^. . 15 Experiment 3: Factorial Design^. . 15 Parameters^ • • 16 Results 16 Impact of Tomicobia tibialis on 1ps pini 16 Tomicobia tibialis Colony Maintenance 30 Healthy Male Residence Time 31 Location of Parasitized Beetle Remains 35 Discussion 35  Chapter Three  Laboratory Rearing of Tomicobia tibialis Introduction Wood Piece Studies Is Wood Necessary? How Much Wood is Needed? How Much Water is Needed? Diet Studies General Methodologies  .  .  .  1 1 4 5 7  .. 39 .. 39 .. 40 .. 40 .. 41 .. 43 .. 45 .. 45  iv Can Artificial Diet Work? ^.. How Much Diet is Needed?^.. Can Tomicobia tibialis be Reared?^.. Discussion^ ..  48 49 52 58  Chapter Four^Could Augmentative Release Work? ^ .. 61 Can Mass-rearing be Developed? ^.. 61 Is the Reduction Enough? ^ .. 61 Bibliography^  .. 64  Appendix 1^  .. 68  Appendix 2^  .. 69  List of Tables Chapter 2 Table 1: Treatments used in the comparison of reproductive parameters for all combinations of healthy (H) and parasitized (P) male and female 1. pini.  . . . . 13  Table 2: Reproductive performance for the healthy males in experiment 1. Each male has four females in his harem, where H=healthy and P= parasitized.  . . . . 17  Table 3: Reproductive performance for the female beetles in experiment 1. (H=healthy, P=parasitized).^  . . . . 19  Table 4: Reproductive performance for the parasitized males in experiment 2. Each male has four females in his harem, where H=healthy and P= parasitized.^  . . . . 22  Table 5: Reproductive performance of the female beetles in experiment 2. (H=healthy, P=parasitized).^  . . . . 23  Table 6: Reproductive performance for the healthy (H) and parasitized (P) males used in experiment 3. Each male has two females.^. . . . 27 Table 7: Reproductive performance for the healthy (H) and parasitized (P) females used in experiment 3. All parameters are measured with the value per female.  . . . . 29  Chapter 3 Table 8: Cumulative proportion of beetles dying at 1 and 2 weeks for the vial-rearing trial conducted on July 16, 1990. Treatments (fp= filter paper, t= tissue, and b= bark/phloem) were applied at 7 and at 14 days. Each treatment had 8 beetles for a total of 72 adult 1. pini used.  . . . . 42  Table 9: Cumulative weekly mortality rates for the vial rearing trial conducted on June 2, 1992. Different water regimes were tested using the treatments given below.  . . . . 46  Table 10: Feeding scores for the diet trial conducted Oct 27 to Nov 10, 1992. Four treatments were assessed: H/H (n=7), H/S (n=9), M/H (n=8), and M/S (n=8), where H=5-holed plastic lids, M=mesh lids and S=solid plastic lids, and the procedure involved changing lids after the first two days. [Feeding scores: 0=none, 1=slight, 2=  vi moderate, 3=moderate to heavy, 4=heavy, and 5=very heavy]. ^. . . . 50 Table 11: Feeding scores for the diet trials conducted from Dec 1 to Dec 15, 1992. Treatments are: H/H (n=16), H/S (n=16), M/H (n=16), and M/S (n=16), where H= 5-holed plastic lid, M= mesh lid and S= solid plastic lid, and lids are changed after the first 2 days. [Feeding scores: 0= none, 1=slight, 2=moderate, 3=moderate to heavy, 4=heavy, and 5=very . . . . 51 heavy].^ Table 12: Parasitoid production from the diet trial conducted Feb 3 to Mar 23, 1993, in which diet cups were either half (wt: 10.6 ± 0.8g, n=10) or quarter (wt: 6.3 ± 1.2g, n=10) full. ^  . . . . 57  Table 13: Parasitoid production from the diet trial conducted Feb 9 to Mar 23, 1993, in which two diet treatments: diet plus a filter paper disk, or diet alone, and two lid types: 5H= 5 hole lid, or 20H= 20 hole lid, were being assessed.^  . . . . 57  vii  List of Figures Chapter 2 Figure 1: Within-harem variation among offspring production for experiment 1. Females within a harem were ranked from 1 (highest) to 4 (lowest) according to the number of offspring they had, and the average value is graphed below.  .. 20  Figure 2: Within-harem variation among offspring production for experiment 2. Females within a harem were ranked from 1 (highest) to 4 (lowest) according to the number of offspring they had,and the average value is graphed below.  .. 24  Figure 3: Within-harem variation among offspring production for experiment 3. Females within a harem were ranked from 1 (highest) to 2 (lowest)^• according to the number of offspring they had,and the average value is graphed below.  .. 25  Figure 4: Frequency distribution of extra galleries dug by males in experiment 3. (healthy males n=27; parasitized males n=23). ^. 28 Figure 5: Male mass and number of days to re-emergence for the healthy males in experiment 1 with 4 female harems. Mean ± SE, n: Treatment 1: 10.5±2.5, 2; Treatment 2: 16.8±0.6, 4; Treatment 3: 10.0±1.6, 3. ANOVA: F=9.18, p=0.015.  .. 32  Figure 6: Male mass and number of days to re-emergence for the healthy males in experiment 3 with 2 female harems. Mean ± SE, n: Treatment 7: 12.8±1.1, 8; Treatment 9: 17.0±2.8, 8. ANOVA: F=2.09, p=0.170.  .. 33  Figure 7: Distribution of parasitized male and female beetle remains. Gallery structure was divided into quarters where 1= the quarter closest to the nuptial chamber and 4= the quarter at the tip of the female gallery. (Female n=44; Male n=23).  .. 34  Chapter 3 Figure 8: Cumulative mortalities for the wood size trial at 2 week and 4 week intervals. Treatments are: L L= large vial + large wood; L-S= large vial + small wood; M L= medium vial + large wood; and M-S= medium vial + small wood. (N=80). -  -  Figure 9: Feeding score response of beetles given one of two amount of diet, half- or quarter-full cups. [Feeding scale: 0= none, 1=  .. 44  viii  slight, 2= moderate, 3= moderate-heavy, 4= heavy, and 5= very heavy]. .. 53 Figure 10: a) Feeding score response of beetles given either diet alone or diet with a filter paper disk. [Feeding scale: 0= none, 1= slight, 2= moderate, 3= moderate-heavy, 4= heavy, and 5= very heavy].^  .. 54  Figure 10: b) The same graph showing the lid treatment breakdown, where 5H= 5-hole lid and 20H= 20-hole lid. [Feeding scale: 0= none, 1= slight, 2= moderate, 3= moderate-heavy, 4= heavy, and 5= very heavy]. .. 55  ix  Acknowledgements I wish to say a heartfelt thank you to my supervisor, Judy Myers, for her support over these months and her attention to detail. This thesis could not have been completed so rapidly or so well without her. The same is true of my advisory committee: Bernard Roitberg, John McLean, and Murray lsman, each of whom gave generously of their time and abilities. Their different perspectives on this topic gave me many angles from which to approach various problems I encountered. Don Elliott of Applied Bionomics provided useful insights and guidance with respect to the biological control industry and his inputs contributed to the depth of this thesis greatly. I am indebted to my fellow lab-mates, as well, each of whom collected beetles or moved logs for me at some time or other: Maynard Milks, Fion Horgan, Lorne Rothman, Alison Hunter, and Shiyou Li. An especially huge thank you and hug belong to Barbara Kukan, whose constant support, assistance and cinnamon buns saw me through even the worst chamber failures. I also wish to thank Nancy Brard and Tom Lowry from the Isman lab for their advice and friendship. Many people outside of UBC contributed greatly to the success of this thesis. PheroTech Inc. provided technical assistance. Mary Reid and Dan Miller offered expert information and council on 1ps behaviours. John Borden, and various members of his lab, gave helped me to keep my beetles from chewing through everything in sight. Lynne & Scott Merilees, Mark Heinrichs, my family, Ed's family, and Richard & Nancy Castro provided essential emotional support and helped me remain sane during my studies. But through everything, there was Ed Senger. For all those hours driving to Princeton, chain-sawing trees, loading and unloading logs, late dinners and long hours away . . . there is no replacement, or thanks great enough, for help and support such as this. This thesis belongs as much to him as it does to me. This thesis was supported by NSERC and the Science Council of British Columbia.  1  Chapter 1 Biological Control in Forestry  Introduction Natural control, or "the action of parasites, predators, or pathogens to maintain the density of an organism at a lower level than would occur without these natural enemies" (Andres, et al.,1979), has been demonstrated management agricultural pests on crop, orchard and greenhouse plants (King, et a1.,1985). However, natural control may not be sufficient to lower the damage to a level acceptable to producers and consumers. Modifications to the environment resulting from current management practices may necessitate boosting natural enemy populations to obtain adequate control. This manipulative technique might be achieved in two ways: 1) inundative release - where the individuals released, and not their progeny, exert control over the pest (Debach, 1964); and 2) inoculative release - where the progeny of the released individuals control the pest over subsequent generations (Debach, 1964). A more general term for both of these approaches is augmentative release and this will be used in this thesis to describe situations in which both the natural enemies released, and subsequent progeny, exert control on a pest population (Parrella, et a1.,1992). For decades, forestry has ignored the potential use of biological control in the management of forest pests (Moeck & Safranyik, 1984; Miller, et a1.,1987; Dahlsten & Whitmore, 1989; Carrow, 1990; Nealis, 1991; Parrella, et a1.,1992). Despite this past reluctance to the use of natural enemies, Nealis (1991) identifies five characteristics of the Canadian forest system which actually favour the use of such methods: 1) long rotation times require low-cost interventions on a per hectare basis; 2) extensive pest outbreaks restrict treatment to high-value stands while natural enemies are more likely be effective in all stands;  2 3) increasingly stricter standards on conventional tools to control pest outbreaks, such as pesticides and clear-cutting, require development of alternatives; 4) public opinions are changing and influencing forestry practices toward more natural control (also see Behan, 1990; Heilman, 1990); and 5) native insects cause the most damage in forest systems, indicating a need to manage natural enemies as well as pests. Precedents for successful use of biocontrol techniques in forestry has already been established both in Europe and Australia. In Europe, the native predator Rhizophagus grandis (Gyllenhal) (Coleoptera: Rhizophagidae) has been successfully mass-reared and reassociated with its host Dendroctonus micans (Kugelann) (Coleoptera: Scolytidae) to achieve control (see Dahlsten & Whitmore, 1989). In Australia, the biocontrol approach has been given precedence over silvicultural methods for controlling two introduced pests, the wood wasp Sirex noctilio Fabricius (Hymenoptera: Siricidae) and the 5-spined engraver fps grandicofiis Eichhoff (Coleoptera: Scolytidae), by introducing two natural predators and two parasitoids of these insects (Morgan, 1989). More recently in eastern Canada, the use of Trichogramma minutum Riley (Hymenoptera: Trichogrammatidae) against the eastern spruce budworm, Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae) has demonstrated the feasibility of using mass-rearing and inundative release techniques against a major forest pest (Carrow, 1990). The greatest barrier to implementing natural enemies in forestry pest control programs in Canada at present is the lack of information on the potential biocontrol agents (Miller, et a1.,1987; Dahlsten & Whitmore, 1989; Nealis, 1991), and a lack of rearing methodologies to facilitate research and release (Moeck & Safranyik, 1984; Carrow, 1990). Current control methods, and areas of control research, for 1ps and other bark beetles include: 1) aggregation pheromone trapping (Miller, et al.,1989; Borden, 1989), 2) pheromone repellents (Miller, et 41990; Borden, 1989), 3) competitive exclusion using other beetles  3 (Miller & Borden, 1985), 4) slash and debris management (Miller, et a1.,1987), and 5) traptrees (Miller, et a1.,1987). Pheromone traps used to capture aggregating populations only collect a portion of the aggregating beetles and do not influence beetle population dynamics, which is dictated by the availability of breeding substrates (Vite & Baader, 1990). Similarly, aggregation inhibitors and competition from other beetles may reduce attacks, but are unlikely to be successful at preventing damage on a large scale (Vite & Baader, 1990). Removal of slash and debris, although effective at reducing local beetle numbers, destroys habitat for natural enemies in the process and may further the imbalances between pests and biocontrol agents (Miller, et aL,1987). Lethal trap-trees designed to kill aggregating beetles also kill natural enemies who follow the beetles to their death when those trees are destroyed (Payne, 1989). Thus although an array of techniques for localized beetle control are currently available, none are useful on a large scale and several detrimentally impact the natural enemies that operate unaided in the system. The use of biocontrol agents has several advantages over these conventional methods, since a natural enemy is: mobile, host-specific and often stage-specific, produces sustained mortality in its host population, and is compatible with many conventional techniques (Nealis, 1991). One host-enemy system that lends itself to the study of biological control in a forest setting is that of the pine engraver, Ips pini (Say) (Coleoptera: Scolytidae), and its adult-stage-parasitoid Tomicobia tibialis Ashmead (Hymenoptera: Pteromalidae). Although I. pini is typically considered a secondary forest pest compared to the vast damage done by more aggressive bark beetles such as the Mountain Pine beetle, Dendroctonus ponderosae Hopkins  (Coleoptera: Scolytidae), Ips outbreaks in which the beetles attack and kill live trees can cause serious damage (Livingston, 1979). For example, pine mortality in northern Idaho due to I. pini outbreaks was approximately 15,000 trees in 1989 (Beckman, et al.,1990). With the  4 increasing number of second growth stands in British Columbia due to more intensive forestry practices, more trees will be in younger successional stages and thus will be vulnerable to Ips damage during stress periods (Thomas, 1961, Livingston, 1979). Although Ips and other bark beetles have been intensively studied (Borden, 1989; Vite & Baader, 1990), their parasitoids and predators have often only be considered secondarily (Moeck & Safranyik, 1984). This is the case with the parasitoid T. tibialis, whose characteristics make it a potential biocontrol agent, but for which little scientific data exists.  Biology of the Host: Ips pini The genus Ips De Geer is comprised of approximately 15 species of beetles, all of which construct galleries in the phloem-cambial regions of weakened and injured trees (Bright, Jr., 1976). Ips pini (Say) (Coleoptera: Scolytidae), found throughout the northern coniferous forest from Alaska to Newfoundland and in parts of eastern and western United States, predominantly attacks Pinus spp. (Bright, Jr., 1976). The biology of I. pini is outlined by Thomas (1961). Male beetles initiate a nuptial chamber in the phloem region and release aggregation pheromones to attract others, creating a mass-aggregation (Birch, 1984; Miller, et a1.,1989). The beetles are polygynous, with males typically having 3-4 females in a harem  resulting in a characteristic X or Y shaped gallery pattern radiating from the nuptial chamber. Eggs are laid singly in niches along the female's gallery and the larvae mine at right angles to this maternal gallery. Larvae go through three instars before pupating at the end of their own gallery. After approximately 10 days, the new adults feed for a variable amount of time before leaving the log in which they developed. Ips pini may have two generations per year in some parts of Canada and the United States, and overwinter in the forest duff as adults (Bright, Jr., 1976). They are capable of reproducing in slash and debris, often associated with  5 clear cuts and thinning operations, to produce large populations that can kill standing trees (Livingston, 1979).  Biology of the parasitoid: Tomicobia tibialis Tomicobia Ashmead is a holarctic genus that was last reported to contain five parasitic  species attacking either adult bark beetles (Ips) or adult curculionids (Otiorrynchus) (Hedqvist, 1959). The biology of T. tibialis Ashmead (Hymenoptera: Pteromalidae) was first detailed by Bedard (1965). Female parasitoids attack moving beetles by physically jumping on top of them. The female then orients herself perpendicular to the body axis of the host, usually over the beetle's thorax, and quickly deposits an egg through the host's integument. Beetles do not respond to the presence of the parasitoid until the female assumes its ovipositional stance. At this point, the beetle becomes highly active, running and trying to hide under flakes of bark. Both male and female beetles are attacked as they join the beetle aggregation. A single parasitoid larva develops within the body of the beetle, killing the adult within two weeks of the attack. By the pupal stage, the parasitoid completely fills the interior of the beetle and is anteriorly oriented towards the elytral declivity of the beetle. The new adult cuts a circular hole in the declivity and emerges through the beetle galleries, leaving the empty beetle remains behind. Bedard found that it took 30 to 35 days for adult parasitoids to emerge from the oviposition date at a laboratory temperature of 22° ± 2°C. There was little success in attempts to rear this parasitoid in the laboratory, and longevity of the lab-kept insects was poor. The first host selection studies on T. tibialis were conducted by Rice (1968). He confirmed the specificity of T. tibialis with Os species hosts and suggested three distinct steps to host selection: 1. olfactory attraction to Ips infestations, 2. visual attraction to moving  6 beetles, and 3. tactile or olfactory acceptance of a host. This was the first indication that beetle pheromones (kairomones) may be involved in attracting the parasitoids from a distance, and that other cues likely became important once a host source was found. In 1969, Rice confirmed this hypothesis by demonstrating that T. tibialis do in fact respond to the aggregating pheromone of Os species. This behaviour, of using the aggregating pheromone to locate hosts, makes T. tibialis rather unique among bark beetle natural enemies, most of whom aggregate when the beetle larvae are abundant (Payne, 1989). The presence of the parasitoid early in the beetle's aggregation cycle provides the maximum opportunities for the parasitoids to attack the adult beetles, further illustrating the coevolved nature of the Tomicobia-Ips relationship. Preliminary field studies that I conducted in the summers of 1989 and 1990 also supported Rice's hypothesis. Female T. tibialis were located in the field by finding infestations of I. pini. The females were seen moving over the log surfaces and were most often found near the entrances of newly arrived males. The parasitoids were observed attacking both female beetles, as they came to join the males, and male beetles, as they emerged from their entrance holes to admit females or remove frass. Several times, a female parasitoid was observed in the field entering a male beetle's nuptial chamber, driving him out, and then parasitizing him at the entrance. In the laboratory, female parasitoids placed in petri dishes-containing a pile of fresh beetle frass oriented towards these piles and often tried to clear away some frass with their wings. Females could be easily induced to attack beetles in these dishes. Although the basics of this system are apparent, much remains unknown. For instance, Bedard reports that T. tibialis overwinter as diapausing prepupae; however, Reid (1957) reported that the pupa is the overwintering stage. There are no clear reasons for this  7 discrepancy and no studies have been conducted. Similarly, Bedard speculated that parasitized Ips females produce few or no eggs whatsoever. A recent study by Senger and Roitberg (1991), however, demonstrated that female Ips do in fact produce eggs, but 50% fewer than healthy females. This initial study did not examine the impact of parasitism on male Ips, or the result when parasitized males and females occur in combination as is the case in nature.  Potential For Control All control programs are based on the premise that natural enemies exert a controlling influence on host populations (Nealis, 1991). Despite the coevolved relationship between T. tibialis and I. pini, more information is needed to determine if T. tibial's can exert a significant  influence on I. pini populations. To learn more about the impact of this parasitoid on beetle reproduction, I conducted three experiments to examine how parasitized beetles perform compared to unparasitized beetles. Furthermore, control programs rely on efficient rearing techniques in order to produce insects for research and release (Ochieng'-Odero; et al.,1991). Since no effective rearing methodologies exist for T. tibial's, I attempted to lab-rear this insect and later to develop a method of rearing which could facilitate future research.  8  Chapter 2 Impact of Tomicobia tibialis on the Reproduction of Ips pini  Introduction The aggregation of bark beetle populations, and their subsequent reproduction and damage to the forest, have been intensively studied over the past several decades (Birch, 1984; Moeck & Safranyik, 1984; Borden, 1989). However, the biology and impact of natural enemies in these systems has been largely ignored (Moeck & Safranyik, 1984; Carrow, 1990; Nealis, 1991; Parrella, et al.,1992). One critical point in the life cycle of bark beetles where control measures can be extremely effective, is during the aggregation phase of an infestation (Borden, 1989). Parasitoids and predators that use the kairomones produced by beetles during aggregation could substantially effect the magnitude of an infestation by: 1) rendering the adult host sterile, 2) reducing adult fecundity and/or 3) altering the selectivity of adults for mates and brooding sites. On the other hand, parasitized adults may show no ill-effects with respect to reproduction or survival. From a pest management perspective, complete or partial reduction in pest fecundity is the most desirable effect since this results in a two-fold impact on the pest population: 1) a reproducing pest adult has been eliminated and 2) the number of offspring, which are future adults, has been reduced. Unfortunately, few studies exist. One example of the impact of parasitism on the reproduction of an adult beetle host is that of Karpinskiella paratomicobia Hagen & Caltagirone (Hymentoptera: Pteromalidae) on its sole host Dendroctonus pseudotsugae Hopkins (Coleoptera: Scolytidae) (Furniss, 1968). Parasitization reduced female beetle  9 fecundity by two thirds in the first brood and killed the adult female, preventing subsequent broods. More commonly, however, only the reproductive behaviour of the parasitoid or predator, and not its impact, has been studied (Frazier, et al.,1981; Gerdin & Hedquist, 1984). The coevolved relationship between Tomicobia tibialis Ashmead (Hymentoptera: Pteromalidae) and its adult 1ps spp. (Coleoptera: Scolytidae) hosts offers a system in which the impact of parasitism on reproduction can be examined. 1ps pini (Say) is naturally polygynous with three females per male being the average harem size (Reid, 1991). Male I. pint produce pheromones during the aggregation phase of an infestation that attract both  female beetles and T. tibialis (Rice, 1969). The parasitoid attacks beetles as they join the aggregation prior to beetle reproduction, and kills the beetle within two weeks (Bedard, 1965). Early investigations suggested that female 1ps parasitized by T. tibialis produced few or no eggs (Bedard, 1965), however, no experiments were performed. The focus of this study is to determine if parasitism by T. tibialis alters the reproductive capacity of male and female I. pint.  Following the procedures of Senger and Roitberg (1991) in the first examination of the impact of parasitism on female 1. pini, three experiments were carried out to examine changes in beetle reproduction resulting from either or both sexes being parasitized. Based on Senger and Roitberg (1991), I predicted that the fecundity of parasitized females would be reduced— compared to healthy females. Parasitized males have never been studied, but reduced vigour of parasitized males could influence the reproductive output of their harems. Since both parasitized and unparasitized beetles are likely to occur in the same harem in nature, mixed harems were also examined. If competitive interactions occur between females in mixed harems, I predict that parasitized females in mixed harems will do less well than if in an all parasitized group, while the healthy females will do better than those in all healthy groups.  10 If these interactions exist, then the reproductive output of the harem will be similar to that of an all healthy harem, and males would not benefit from selecting against the parasitized females.  Methods and Materials Colony Maintenance Ips pini were collected in May of 1992 from naturally infested lodgepole pine, Pinus contorta Douglas var. latifolia Englem., logs from the Finnegan Creek watershed near Princeton, BC. This entire district has had intense Mountain Pine beetle and Ips infestations for the last 10-15 years and is currently being salvage logged by several logging companies. The "wild" logs collected were placed in cages in a temperature-controlled chamber (28°C, 40% relative humidity and 16h:8h light/dark cycle) and all insects were allowed to emerge. Emerging L piniwere collected and their sex determined. Emergent beetles were either used immediately to create a lab colony or were stored in glass jars, containing tissue paper and several drops of water, at 6°C in the refrigerator. Each jar was dated with the collection date and the sex of the beetles it contained. For colony maintenance, twenty to thirty male I. pini were allowed to initiate attacks on "clean", uninfested rOdgepole pine bolts placed in a cage in the chamber. Males were -  usually placed in the cage over two or three days to stagger the attack. Once males had created adequate nuptial chambers, as evidenced by the frass piles, female I. piniwere added over another two or three days. Generally enough females were added such that each male had two or three females in a harem. Cages were monitored daily and beetles that reemerged, the parental beetles, were collected. Brood or offspring beetles began emerging approximately 4 to 6 weeks after the attack was initiated. Brood beetles were used in the  11 experiments and to produce the next lab generation of I. pini. Tomicobia tibialis also emerged from the naturally infested logs brought in from  Princeton. Individuals were collected in gel capsules from the sides and roof of the cage. A male and female parasitoid were placed together in a gel capsule for up to an hour for mating. The pair was then kept in a mason jar inverted on a paper towel. A water wick, a small disk of raw honey moistened with water, and a small disk of hydrolysed yeast were placed inside the jar to provide food and water. Because the sex ratio was male biased, excess males were often kept together in jars grouped by their emergence date. The older males were used first when a new female emerged. Females were ready for oviposition two days after they had emerged and mated. To propagate T. tibialis, each female was placed in a 2 by 10 cm Petri dish containing a filter paper disk and some fresh beetle frass. Beetles collected from the colony were added and parasitism was identified according to the description by Bedard (1965). Females were generally given only two or three beetles a day to parasitize. Fecundity studies for the parasitoids proved impossible because limited rearing space prevented observation of the offspring production of individual females. Parasitized beetles were added to the I. pini lab colony during an infestation period. These cages were monitored for emergence of parasitoids and brood beetles. This process was repeated with every generation. Additional "wild" material was collected from Princeton on several dates and emergent insects were added to the existing colonies. Some "wild" logs were stored at 7°C in a cold chamber to slow beetle development. These logs were brought into the colony chamber at regular intervals to boost beetle colony numbers for experiments.  12 Experiments Three types of experiments were done as described below. Experiments 1 and 2 were run concurrently (July 10 to October 28, 1992) and Experiment 3 was performed subsequently (November 14, 1992 to February 11, 1993). A total of thirty-seven 50-cm lodgepole pine log sections from several different trees were used in these experiments to allow adult L pini to reproduce. Actual logs were used instead of bark sections to avoid any confounding effects resulting from manipulation of the bark and phloem (Furniss, 1968). The ends of each log were sealed with paraffin wax to reduce desiccation. The beetles and parasitoids used in these experiments were collected from the lab colonies as described above.  Experiment 1: Healthy Males with Healthy and/or Parasitized Females Fresh logs collected in May, 1992, were used in both experiments 1 and 2. The circumference of each of the 10 logs used for experiment 1 was divided into three equal sections such that one treatment occupied one third of the log surface area. Three unparasitized, "healthy", male beetles were randomly assigned to these log sections, and were seeded into a section of the log by confinement in a gel capsule (Borden, 1967). Paraffin wax was dripped around the edge of the gel capsule to seal the crack between the log and the capsule; males that escaped -by chewing out of the- capsule could then be detected. Escaped males were either replaced or the log was abandoned. Within each log, males were randomly assigned to receive one of the three treatment groups of females used in experiment 1 (Table 1): parasitized, unparasitized (healthy) or mixed harem. Treatments will be referred to in a standard format of male plus number of females, eg. H+4H meaning healthy male plus four healthy females in the harem. On the first day of the experimental set-up, teneral male beetles were  13 Table 1: Treatments used in the comparison of reproductive parameters for all combinations of healthy (H) and parasitized (P) male and female I. pini.  a) Experiment 1. GROUP^MALE^4-FEMALES  Treatment 1^H^H Treatment 2^H^P Treatment 3^H^2H + 2P  b) Experiment 2. GROUP^MALE^4-FEMALES  Treatment 4^P^H Treatment 5^P^P Treatment 6^P^2H + 2P  c) Experiment 3. GROUP^MALE^2-FEMALES^-  Treatment 7^H^H Treatment 8^P^H Treatment 9^H^P Treatment 10  14 collected from the lab colony and stored in a glass jar, containing tissue paper and some water, in a refrigerator at 6°C for 24 h. Males were warmed to room temperature, weighed on a Mettler AC100 balance, and placed in individual 6 ml vials containing a filter paper disc, tissue, and 1-drop of water. These vials were stopped with cork stoppers and left for 24 h at room temperature. This procedure was used to minimize the differences between individuals prior to the experiment. On the third day, males were seeded into their assigned sections by gel capsule confinement (Borden, 1967) as described above. Males were given 24 h to establish nuptial chambers before receiving their first female. Once the male was established, he received two females per day according to his assigned treatment. The first and second females of the harem were collected on day 2 of the experimental set-up and stored in the refrigerator as previously described. On day 3, the females were weighed and randomly assigned to be either healthy or parasitized. Healthy females were treated in the same manner as the healthy males, by being placed in vials for 24 h. Parasitized females were confined to a 2 by 10 cm Petri dish containing a filter paper disc, fresh beetle frass, and one T. tibialis female. Once parasitism was observed, the beetle was placed in a vial for 24 h. Thus on the morning of day 4, when the males had established their nuptial chambers, the first female was implanted ^with her assigned male. Approximately, four to six hours later the second female was also implanted and the logs were-left overnight. The third and fourth females of the harem were collected on day 3 of the experimental set-up and stored in the refrigerator for 24 h. On day 4, the females were treated in the exact manner as described above such that on the morning of day 5, each male received his third female. By the end of day 5, all males had complete harems of four females each. On day 6, each male entrance area was fitted with a Solo P125 (37.0 ml) plastic container (Solo Cup  15 Co., Urbana, Ill.) which had the central region of its bottom cut out. These cups were sealed to the log using an automotive windshield tape kit and glue. The lid of each container was punctured repeatedly with an insect pin to create approximately 25 air holes. The cups were used to capture any beetle re-emerging from the male entrance hole, thus allowing residence times to be established. The logs were then placed in a temperature controlled chamber which was maintained at 26 to 28°C and 40 to 60% relative humidity for the duration of the experiment. Cages were inspected daily and any beetles trapped in the cups or loose in the cage were collected. Six weeks after implantation, all parental beetles and parasitoid adults had emerged. Galleries were exposed by removing the bark and an acetate sheet was pinned across the galleries of each harem so that a trace could be produced. All measurements were taken directly from these traces. Because parasitized beetles could be identified by the empty beetle remains in the gallery, only those beetles for which parasitism was confirmed were used in the data analysis for the parasitized treatment groups.  Experiment 2: Parasitized Males with Healthy and/or Parasitized Females Experiment 2 was identical to experiment 1 except that parasitized males were used (Table 1). Eleven uninfested logs were used in this experiment: -Males were parasitized on— day 2 of the experimental set-up using the same method as described for parasitized females and were stored, as described above for the healthy males, in vials at room temperature for 24 h.  Experiment 3: Factorial Design In experiment 3, fourteen logs collected in September 1992 were used. Four males,  16 each with two females, were used per log instead of three, as in experiments 1 and 2. Combinations of healthy and parasitized individuals were used and treated as described above (Table 1). Since males only received two females each, both females were implanted with their assigned males on Day 4 of the experimental set-up. Male entrances were fitted with cups on day 5 and placed in the chamber. All other procedures remained the same as described above.  Parameters Gallery lengths were measured using a Koizumi ComCurve-5 digital curvi-meter. Values given are the average of 5 repeated measures per gallery. The number of larvae produced, measured as the number of larval niches radiating from the maternal gallery, was used as a direct measure of female reproductive potential. The potential effects on larval survival were obtained by subtracting the number of unsuccessful larvae, those for which the larval gallery terminated after only a few centimetres, from the number of larvae produced. Male reproductive potential was assessed using the total output of all females in the harem for each parameter examined. Data were analyzed using the Systat 5.1 computer program.  Results Impact of Tomicobla tibial's on Ips 071 In experiment 1, offspring production was reduced by 49% in harems with all parasitized females as compared to those with all healthy females (Table 2). While this reduction may be biologically significant, variation within harems was such that the difference was not quite statistically significant at the 5% level (Table 2). When the number of surviving larvae, or total offspring production less early larval mortality, is considered, healthy harems  17 Table 2: Reproductive performance for the healthy males in experiment 1. Each male has four females in his harem, where H=healthy and P=parasitized.  a) Means and standard errors for the total output per male. Treat Male Females  n  Total Offspring Mean ± SE  Surviving Larvae Mean ± SE  Gallery L.(cm) Mean ± SE  1  H  H  10  45.0± 9.2  41.1 ± 8.9  27.9 ± 3.5  2  H  P  5  23.0 ± 10.1  14.6± 9.2  25.0 ± 4.7  3  H  H+P  7  52.9 ± 15.9  47.3 ± 15.3  32.9 ± 4.8  F=1.30, p=0.296  F=1.77, p=0.198  F=0.75, p=0.484  ANOVA:  b) Independent t-tests were used to compare the harem totals for each treatment. Comparison Total Offspring  Treatment Effects Surviving Larvae  Gallery L.(cm)  1 vs 2  t=^1.63, p=0.134  t= 2.10, p=0.061  t= 0.49, p=0.637  1 vs 3  t= -0.43, p=0.676  t= -0.29, p=0.775  t= -0.85, p=0.416  2 vs 3  t= -1.59, p=0.144  t= -1.83, p=0.100  t= -1.16, p=0.276  c) Paired t-tests were used to compare the healthy and parasitized components of each male's harem in treatment 3. Health^Total Offspring^ Surviving Larvae^Gallery L.(cm) Mean±SE Effect Mean±SE Effect Mean±SE Effect H  29.7±9.2 t=0.911  26.4±9.0 t=0.885  17.0±2.6 t=0.549  P  23.1±8.2 p=0.397  20.9±7.7 p=0.410  15.9±2.6 p=0.603  18 do 2.8 times better than parasitized harems (Table 2a). No differences in total gallery lengths occur between healthy and parasitized harems. Interestingly, no significant difference was found between the mixed harems and either one of the pure harems when the male was healthy. The mixed harem (treatment 3) can be broken down such that male reproductive success is composed of two components: contributions from 2 healthy females and contributions from 2 parasitized females (identified by beetle remains in the gallery). However, this does not result in a significant difference for any parameter measured, although the mean values for parasitized females are consistently lower (Table 2c). Thus there is an indication of an impact of T. tibialis when an entire harem is parasitized, but this is not obvious when the harem is mixed. Comparison of individual females across replicates showed statistically significant variation (Table 3). Reproductive success of females in treatment 2 was reduced primarily by poor survival of offspring (Table 3): healthy females (treatment 1) produced 2.35 times as many surviving offspring than parasitized females (treatment 2) when the male was healthy, but there was no difference in gallery length (Table 3a). Females in mixed harems did not differ significantly in total offspring, before or after larval mortality, nor in gallery length (Table 3b). There were difficulties associated with using parasitized females in these experiments, including males rejecting parasitized females from the harems, emergence of a female for unknown reasons, or failure of the female to be parasitized at all. Although these difficulties arose in all treatments, getting complete parasitized harems for treatment 2, and treatments 5, 9, and 10 in later experiments, was the most difficult. The large amount of within-harem variation in this experiment makes interpretation of results difficult. Typically one or two females outproduced the other individuals in the harem (Figure 1). Because individuals could not be tracked, I was unable to link this result to order  19 Table 3: Reproductive performance for the female beetles in experiment 1. (H=healthy,  P=parasitized). a) Independent t-tests were used to compare females from treatments 1 (n=38) and 2 (n=16). # Offspring^Surviving Larvae Mean±SE Effect^Mean±SE Effect  Gallery L. (cm) Mean±SE Effect  1  11.8±1.8 t= 1.33^10.8±1.8 t= 2.23  7.3±0.6 t=-0.36  2  7.2±2.8 p= 0.19^4.6±2.0 p= 0.03  7.8±1.2 p= 0.72  Treat Females  b) Paired Nests were used to compare the healthy (H) and parasitized (P) females in the mixed harem of treatment 3 (n=14). Health  H  ^ Surviving Larvae # Offspring Mean±SE Effect^Mean±SE Effect  Gallery length Mean±SE Effect  14.9±3.7 t=0.66^13.2±3.7 t=1.15  8.5±1.1 t=1.04  11.6±3.4 p=0.52^10.4±3.4 p=0.27  7.9±1.5 p=0.32  20  Figure 1: Within-harem variation among offspring production for experiment 1. Females within a harem were ranked from 1 (highest) to 4 (lowest) according to the number of offspring they had, and the average value is graphed below.  u 50J I-U cc Lu CL  0 cC  20-'  u_ 0 it 100  '^  1  2^3^4  FEMALE RANK  MI 1: H+4H VINA 2: H+4P^13: H+2H/2P  21 of entrance into the harem (eg. first vs second female in) or to female weight/size. MannWhitney U-tests were performed on the number of offspring per female to determine if differences in production occurred between healthy and parasitized individuals regardless of their harem associations. There was a significant difference between the healthy and parasitized females in treatments 1 and 2 (U  406  . 381; a< 0.05), but no difference between  the healthy and parasitized females in treatment 3. Thus parasitism affected individual females in homogenous harems, but this effect appears to be altered in the mixed harem situation, perhaps through competitive interactions (see: Discussion). In experiment 2, variation in offspring production, survival, or gallery length was not significant when compared among males (total for all females in harem) (Table 4) or females (values for individual females) (Table 5). On average, healthy females (treatment 4) produced 2.0 times as many offspring than parasitized females (treatment 5) when the male was parasitized (Table 5a); and offspring survivorship showed the same trend. Gallery lengths did not differ. No significant differences were found between the healthy and parasitized females within the mixed harem (treatment 6) when either male or female reproductive success was considered (Table 4c, 5c). As in experiment 1, there was considerable within-harem variation (Figure 2). Mann-Whitney U-tests showed significant differences between the healthy and parasitized females of treatments 4 and 5 (U 297. 519; a< 0.001), but no difference was found within the mixed harem (treatment 6). The difficulty with these experiments lies in the large variation created by using four females per male (Figure 1 & 2). This variation obscures results which are believed to be biologically significant. Therefore, I designed a third experiment to reduce variation (Figure 3). The factorial design used in experiment 3 clearly demonstrates where T. fibialis effects  22 Table 4: Reproductive performance for the parasitized males in experiment 2. Each male has four females in his harem, where H=healthy and P=parasitized.  a) Means and standard errors for the total output per male. Treat Male  Females  n  Total Offspring Mean ± SE  Surviving Larvae Mean ± SE  Gallery L.(cm) Mean ± SE  4  P  H  8  44.1 ± 11.9  42.6 ± 12.2  25.2 ± 4.5  5  P  P  5  21.2 ± 8.0  20.0 ± 7.1  26.2 ± 4.9  6  P  H+P  7  46.0 ± 13.2  44.9 ± 12.7  32.1 ± 4.2  ANOVA:^F=1.08, p=0.362^F=1.13, p=0.347^F=1.17, p=0.211  b) Independent t-tests were used to compare the harem totals for each treatment.  Total Offs nn  Treatment Effects Survivin•Larvae  Galle^L. cm  4 vs 5  t=^1.59, p= 0.141  t=^1.60, p= 0.139  t= -0.15, p=0.885  4 vs 6  t= -0.11, p= 0.918  t= -0.13, p= 0.900  t= -1.09, p=0.295  5 vs 6  t= -1.60, p= 0.144  t= -1.69, p= 0.125  t= -0.91, p=0.387  Comparison  c) Paired t-tests were used to compare the healthy and parasitized components of each male's harem in treatment 6. Health^Total Offspring^Surviving Larvae^Gallery L. (cm) Mean±SE Effect^Mean±SE Effect^Mean±SE Effect H^25.6±7.1 t= 0.630^25.0±6.9 t= 0.617^15.7±2.4 t=-0.279 20.4±8.5 p= 0.552^20.0±8.2 p- 0.560^16.5±2.9 p= 0.7,90  ^  23 Table 5: Reproductive performance of the female beetles in experiment 2. (H=healthy, P=parasitized).  a) Independent t-tests were used to compare females from treatments 4 (n=29) and 5 (n=17). Treat Females^# Offspring Mean±SE Effect  Surviving Larvae^Gallery L. (cm) Mean±SE Effect^Mean±SE Effect  4^H^12.2±2.7 t=1.87^11.8±2.7 t=1.90^7.0±1.1 t=-0.55 5^P^6.2±1.6 p=0.07^5.9±1.6 p=0.06^7.7±0.8 p= 0.58  b) Paired t-tests were used to compare the healthy and parasitized females in the mixed harem of treatment 6 (n=14). Surviving Larvae # Offspring^ Mean±SE Effect^Mean±SE Effect  Gallery L.(cm) Mean±SE Effect  H  12.8±3.4 t= 0.68^12.5±3.4 t= 0.66  7.8±1.1 t=-0.34  P  10.2±3.4 p= 0.51^10.0±3.0 p= 0.52  8.2±1.1 p= 0.74  Health  24 Figure 2: Within-harem variation among offspring production for experiment 2. Females within a harem were ranked from 1 (highest) to 4 (lowest) according to the number of offspring they had, and the average value is graphed below.  25 Figure 3: Within-harem variation among offspring production for experiment 3. Females within a harem were ranked from 1 (highest) to 2 (lowest) according to the number of offspring they had, and the average value is graphed below.  50 ' -  w  2^-------------^-------------^---------------- — U-  ------------- -------------------- -------------^----------------- --  0  z  cc  -------------^  -----------------  -------------^  ---------------  -  LL  0 -  0  1  FEMALE RANK  MI 7: H+2H^8: P+2H^9: H+2P  2  ni 10: P+2P  26 I. pini reproduction. It becomes clear that male reproductive success is most greatly effected by whether his harem is parasitized or healthy, regardless of whether the male himself is parasitized (Table 6). Healthy males with healthy harems had on average 2.4 times as many offspring and 1.4 times as great total gallery length as either healthy males with parasitized harems (treatment 9) or the all parasitized group (treatment 10). There was no significant difference in offspring production or gallery length for parasitized males with healthy harems (treatment 8), but always a trend for less, as compared to healthy males with healthy harems (treatment 7). Parasitized males with healthy harems had 2.3 times as many offspring and 1.6 times as great total gallery length as those with parasitized harems (Table 6). Comparing treatments 8 and 9 produces several significant results which appear to be due mostly to the impact of parasitism on female beetles as opposed to parasitism of males. Male beetles in experiment 3 also demonstrated a behavioral difference associated with their parasitization status. About half the healthy males dug an "extra gallery" (Figure 4), a short gallery entrance radiating from the nuptial chamber which would be used by the next female to join the harem. However, only about a third of the parasitized males dug an extra gallery (Figure 4). Average lengths of extra galleries (healthy male: 2.2±0.3 cm; parasitized male: 1.7±0.4 cm) did not differ significantly (T-test = 1.6; p = 0.13). It is unclear whether this behaviour affects the probability of a male actually attracting another female for his harem (see: Discussion). Female I. pini reproductive success is most strongly influenced by whether the individual is parasitized or not (Table 7). There were no significant differences when comparing treatments in which the females were healthy, treatments 7 vs 8, or in which the females were parasitized, treatments 9 vs 10, despite the differences in the health of the males used. However, when females are compared according to their "health" status, significant differences result. Healthy females (treatment 7) produce 2.6 times as many  27 Table 6: Reproductive performance for the healthy (H) and parasitized (P) males used in experiment 3. Each male has two females.  a) Means and standard errors are given for the total output of the harem. Treat Male  Females  n  Total Offspring Mean ± SE  Surviving Larvae Mean ± SE  Gallery L. (cm) Mean ± SE  7  H  H  14  29.1 ± 4.5  28.0 ± 1.9  20.1 ± 1.9  8  P  H  12  23.8 ± 6.6  20.5 ± 5.2  24.0 ± 2.8  9  H  P  14  14.7 ± 1.1  9.3 ± 1.9  14.7 ± 1.9  10  P  P  11  10.4 ± 2.7  9.2 ± 2.7  14.6 ± 1.7  ANOVA:^F=5.39, p=0.003^F=6.18, p=0.001^F=5.70, p=0.002  b) Independent t-tests were used to compare the harem means of each treatment. Comparison Total Offspring  Treatment Effects Surviving Larvae  Gallery L (cm)  7 vs 8 7 vs 9 7 vs 10  t= 0.69, p= 0.501 t= 2.42, p= 0.024 t= 3.70, p= 0.001  t= 1.12, p= 0.275 t= 3.95, p= 0.001 t= 3.70, p= 0.001  t=-1.12, p= 0.276 t= 2.42, p= 0.024 t= 2.24, p= 0.035  8 vs 9 8 vs 10  t= 2.00, p= 0.067 t= 1.92, p= 0.076  t= 2.07, p= 0.058 t= 1.87, p= 0.080  t= 2.89, p= 0.011 t= 2.79, p= 0.013  9 vs 10  t=-0.07, p= 0.946  t=-0.20, p= 0.843  t= 0.07, p= 0.944  28 Figure 4: Frequency distribution of extra galleries dug by males in experiment 3. (healthy males n=27; parasitized males n=23).  1  0.75 ' -  0.5 ' -  0.25 ' -  0^  HEALTHY^PARASITIZED EXTRA GALLERY DUG BY MALE GALLERY I I NO GALLERY  (  29 Table 7: Reproductive performance for the healthy (H) and parasitized (P) females used in experiment 3. All parameters are measured with the value per female.  a) Means and standard errors are given for the females in each treatment. Treat Male Females  n  # Offspring Mean ± SE  Surviving Larvae Mean ± SE  Gallery L. (cm) Mean ± SE  7  H  H  28  14.6 ± 2.6  14.0 ± 2.6  10.0 ± 1.1  8  P  H  24  11.9 ± 2.9  10.2 ± 2.4  12.0 ± 1.5  9  H  P  25  5.7 ± 1.5  5.2 ± 1.0  8.3 ± 1.0  10  P  P  19  6.0 ± 1.3  5.7 ± 1.3  8.4 ± 0.9  F=5.44, p=0.002  F=3.02, p=0.034  ANOVA:  F=5.01, p=0.003  b) Independent t-tests were used to compare the means of each treatment. Comparison # Offspring  Treatment Effects Surviving Larvae  Gallery L. (cm)  7 vs 8 7 vs 9 7 vs 10  t= 0.69, p= 0.494 t= 3.02, p= 0.004 t= 2.93, p= 0.006  t= 1.05, p= 0.300 t= 3.02, p= 0.005 t= 2.83, p= 0.008  t=-1.03, p= 0.310 t= 1.34, p= 0.188 t= 1.15, p= 0.256  8 vs 9 8 vs 10  t= 1.98, p= 0.057 t= 1.89, p= 0.069  t= 1.86, p= 0.072 t= 1.65, p= 0.108  t= 2.30, p= 0.028 t= 2.13, p= 0.041  9 vs 10  t=-0.18, p= 0.856  t= -0.32, p= 0.754  t=-0.20, p= 0.842  30 offspring and 1.2 times as long galleries than parasitized females (treatment 9) when the male is healthy. When the male is parasitized, these differences change to healthy females (treatment 8) producing 2.0 times as many offspring and 1.4 times as long galleries than parasitized females (treatment 10). Thus being parasitized greatly reduces a female's current reproductive success. The significant differences seen when comparing treatments 8 and 9 appear to arise from the health or parasitism of the females and not of that of the males, as was discussed earlier.  Tomicobla tibialis Colony Maintenance  Sixteen continuous generations of T. tibialis were produced during this study. The first wild females emerged in the laboratory on September 22, 1991. The first generation of labreared T. tibialis began with a female on October 21, 1991. Parasitoids were maintained in the laboratory until March of 1993, with three inputs of wild T. tibialis into the lab colony following establishment in September, 1991. The first of these was in July, 1992, following a late June collection of attacked material from the field, in which 20 individuals (8 males and 12 females) were added. A second field collection of attacked material in August, 1992, resulted in 38 individuals (18 males and 20 females) being added in late August. Several of the logs from this field collection were stored at 7°C and later used to add wild stock to -both the beetle and parasitoid colonies. An addition 31 individuals (15 males and 16 females) were added in this manner from November, 1992 to February, 1993. Overall, female T. tibialis lived an average of 19 days (n=233, X±SE= 18.8 ± 0.4) in the laboratory, with a maximum lifespan of 43 days. Males lived an average 18 days (n=398, X±SE= 18.1 ± 0.4), with a maximum lifespan of 55 days. There was no difference between the longevity of wild (n=66, X±SE= 19.0 ± 0.81) and lab-reared (n=167, XtSE= 18.8 ± 0.52)  31 individuals (t-test: t= 0.19, p=0.852). The sex ratio of wild T. tibialis was decidedly femalebiased, ranging from 0.88 to 1.50 females per male, with an average of 1.14 (n=4, SE=0.14). Laboratory-reared parasitoids showed a marked male-biased sex ratio, ranging from 0.12 to 1.57 females per male, with an average of 0.42 (n=17, SE=0.08). It is unclear at this time why lab-reared ratios remained male-biased. The efficiency of the rearing technique improved over time as I became more familiar with the insects. In the first generation of lab-reared parasitoids, only 13% of the beetles attacked resulted in adult parasitoids. However, with improved efficiency in manipulating the females, that value increased to an average of 32% (n=7, SE=0.03) over 7 generations. During the reproductive experiments described in this chapter, efficiency ranged from 53% to 92%, with an average of 74% (n=10, X±SE= 73.6% ± 3.5). One problem which hampered parasitoid production was the presence of individuals that appeared to diapause, or delayed development, under the chamber conditions. Estimates from the reproductive experiments reveal that an average of 25% (n= 10, SE=2.5) of the parasitoids were delaying development. Thus determining the cause and correcting this problem could significantly increase parasitoid production. Temperature variation/fluctuation may be a key in resolving the diapause problem (see: Chapter 3).  Healthy Male Residence Time Healthy male beetles tended to stay longer in logs with parasitized females. This is most clearly seen in experiment 1 (Figure 5), while in experiment 3 (Figure 6) the average shows the same trend, but the data are biased by two males that were very slow to emerge. Female residence time was rarely assessed because individual females could not be tracked. The majority of females emerged away from the male entrance hole and thus were not  32 Figure 5: Male mass and number of days to re-emergence for the healthy males in experiment 1 with 4 female harems. Mean ± SE, n: Treatment 1: 10.5±2.5, 2; Treatment 2: 16.8±0.6, 4; Treatment 3: 10.0±1.6, 3. ANOVA: F=9.18, p=0.015.  20^ w 18 U^)1(^)1( Z 16-^ LLI )s( 0 cc 14^ Lu^ ^^• M 12w .L.1:il 10 -^ A < ^ M 8 i 6^  o  A^  a  u) ^ >- 4 0  2^  o  3  i 3.5^4^41.5^5^5 1.5^6^61.5^ MALE WEIGHT (mg) ^ 1: H+4H^* 2: H+4P^A 3: H+2H/2P  7  ■  7 5^8  33  Figure 6: Male mass and number of days to re-emergence for the healthy maes in experiment 3 with 2 female harems. Mean±SE, n: Treatment 7: 12.8±1.1, 8; Treatment 9: 17.0±2.8, 8. ANOVA: F=2.09, p=0.170.  30  x  Lu 25 0 Z w (.5 cr 20 ^ w 2 w A w LU 15^  A NE NE  NE^A^A  M  A^A A  ° I- 10^ CJ)  w  A  o 5^ 0^ 3  3.5^4^4.5^5^5.5^6^6.5^7^7.5 MALE WEIGHT (mg)  A  7: H+2H NE 9: H+2P  8  34 Figure 7: Distribution of parasitized male and female beetle remains. Gallery structure was divided into quarters where 1= the quarter closest to the nuptial chamber and 4. the quarter at the tip of the female gallery. (Female n=44; Male n=23).  0.75-'  0.5-'  0.25-'  ^ ^ 1 2 3 QUARTER OF GALLERY .111■•■•  .1•11■11•11/  FEMALE I  I  .1■1•11■11,1/1■  MALE  35 trapped in the cups. Therefore, it can not be determined which female emerged from the harem at a given time.  Location of Parasitized Beetle Remains When the galleries in old infested logs from the field are examined, dead adult beetles remaining in the galleries have traditionally been considered dead without dissections to look for parasitoid pupae (Thomas, 1961). However, because beetles parasitized by T. tibia/is do not re-emerge from logs, it may be possible to locate empty beetle remains and/or beetles containing parasitoid pupae, in the nuptial chambers and galleries of I. pini. From experiment 3 it was determined that the majority (61%) of parasitized male beetles died in the nuptial chamber itself, or within the first quarter of a female's gallery (Figure 7). No trend was seen with respect to male weight or whether the male had healthy or parasitized females in his harem. Parasitized female beetles, however, were discovered most frequently (66%) in the last quarter of their gallery (Figure 7). This information is useful for locating parasitized beetles in the field when studying natural infestations.  Discussion In nature, male I. pini initiate nuptial chambers and release aggregation pheromones to attract females for their harems (Birch, 1984). The mechanisms by which a female chooses an individual male within that aggregation are only now being investigated (Reid, 1991). Since larvae from adjacent female galleries may meet, usually resulting in combat and the death of the smaller larva (Schmitz, 1972; Kirkendall, 1989; Reid, 1991), it is to a female beetle's advantage to be the first to mate and lay eggs, providing a headstart over later arriving females (Byers, 1989; Kirkendall, 1989). It is unclear whether the male behaviour of  36 digging extra galleries, observed in experiment 3, affects the probability of a male attracting additional females to his harem. Female I. pin' do prefer recently arrived males that have few mates with short egg galleries (Reid, 1991). If females also preferred males who dug extra galleries, perhaps advertising space in the harem, then healthy males would have a greater probability of attracting full harems more quickly than parasitized males. However, the presence of T. tibialis females, drawn to ips infestations by the male's kairomones (Rice, 1969), complicates the mate choice decisions of female I. pin/. Tomicobia tibialis females have been observed both searching over the log surface for landing beetles  and waiting for arriving beetles near a male's entrance hole (personal observ.). Reid (1991) reported a parasitism rate of approximately 16% during field studies conducted in 1989. Thus in nature, it is possible to have healthy and parasitized males in combination with all healthy, all parasitized, and mixed female harems. Experiments 1 and 2 suggest that parasitism more greatly effects female reproductive success than male success. Individual females suffer marked reductions in offspring production as a result of parasitism. When those females occur together in the harem of a male, whether or not that male is parasitized, the male's reproductive success is much reduced. The curious result is that mixing healthy and parasitized females in the same harem appears to lessen the impact on parasitized females. I predicted that males with mixed harems would have similar reproductive success to males with all healthy harems due to the two healthy females having improved production in the mixed harem situation. While the results show that the mixed harems do not reduce male reproductive success, the suggested mechanism, of enhanced production by the healthy females, is not apparent. On the contrary, the healthy females respond normally and the parasitized individuals have increased reproductive output. Thus there is no difference in performance between healthy and  37 parasitized females in the mixed harem even though there is a difference if the harem is all healthy or all parasitized. Reasons for this are unclear. This result does not appear to be dependent on the health of the male, since the findings are similar whether the male is healthy or parasitized. Similarly, there is no evidence from the male residence time data to suggest that males with mixed harems spend more time in the galleries. Thus it appears that some positive interaction is occurring amongst the females themselves. One recent study suggests that Os acuminatus (Gyllenhal) females do not respond to the presence of neighbouring females as  measured by their gallery spacing or egg distribution, and females in larger harems then suffer from reduced reproductive success due to increased larval competition (Kirkendall, 1989). However, my results suggest that I. pini females can somehow respond to or affect other females within the harem, perhaps by some pheromonal influence on parasitoid development. This interaction apparently results in improved performance for the parasitized females in the presence of healthy females. Male residence time data also present some counterintuitive results. Healthy males with parasitized harems were shown to have lowered reproductive success (see: Impact results). Since male residence time is associated with harem performance (Reid, 1991), then it could be predicted that males with parasitized harems would abandon them sooner in favour — of future reproductive opportunities, if they acted so as to maximize their reproductive success. This does not appear to happen though. If healthy males spend more time with parasitized harems then they are investing more time for fewer offspring, and their spread to second infestations will be delayed. Since synchrony of the initial aggregation is critical in 1ps reproduction (Reid, 1991), this delay could further reduce the success of secondary attacks. This possibility illustrates how male and female I. pini are differentially affected by  38 parasitism. Experiment 3 clearly demonstrates that individual female beetles experience a dramatic reduction in reproductive success as a result of parasitism, but individual male reproductive success is dependent upon the health or parasitism of the females in the harem. However, this experimental design only measures the reproductive success resulting from the first mating opportunity, or the current reproductive success. Wild I. pini may have two or even three broods in their lifetime (Bright, Jr., 1976). Thus although current male reproductive success is not directly dependent on the male's health status, a parasitized male can not produce any more harems in future infestations whereas a healthy male might. The major impact of T. tibialis on I. pini males lies in lost future reproduction which is not accounted for in these experiments. Female I. pin!, however, suffer both from losses in current and future reproduction when they are parasitized. This shows up as the marked differences seen in the above experiments. What is needed now are field studies to examine the natural dynamics of this interaction. Two approaches could be used. On the one hand, unifested logs could be set out for Ips colonization in areas known to have T. tibialis. These logs could be used to determine the natural rate of percent parasitism and the probability of mixed and pure harems. Although this information is useful and needed for modelling of the system, it does little to further the understanding of how augmentation of the parasitoid would affect Ips populations.-Instead, field trials using released T. tibialis could be conducted to determine if localized releases impact Ips numbers. Mark/recapture procedures could be used to examine the spread of parasitoids from the release point, and examination of logs could still provide percent parasitism data. This field information is necessary for a better understanding of how T. tibialis and I. pin! interact, and will aid in the assessment of whether T. tibialis can exert a  controlling influence of its host.  39  Chapter 3 Laboratory Rearing of Tomicobia tibialis  Introduction The augmentative release of biological control agents is a practice which has seen much success in agriculture (King, et al.,1985). However, to achieve such success, an ample supply of parasitoids is necessary. Sometimes it is both economical and practical to collect wild agents from a densely populated region and release them at an infestation site to locally boost their numbers as needed (Haugen & Underdown, 1990). More commonly, the insects must be reared in the laboratory to obtain sufficient numbers for release (Ochieng'-Odero, at a1.,1991; Lawson & Morgan, 1992; Laing & Eden, 1990). Efficient lab-rearing procedures are  essential for the development of biocontrol programs. Thus a critical step in considering the role of T. tibialis in the biological control of 1. pini is the development of rearing methodologies for this parasitoid. Past attempts to rear T. tibialis in the laboratory were unsuccessful. Bedard (1965) reports that both field-collected and laboratory-reared parasitoids died within 5 days in the laboratory. As reported in chapter 2, the current method of "bolt-rearing", or seeding parasitized beetles into logs, has greatly improved the success of rearing parasitoids. However, this method requires a great deal of laboratory space for cages and log storage, and many live pine trees are required to fuel the mass production of parasitoids. Further research, and the exploration of the possibility of using T. tibialis as a biocontrol agent, require an efficient procedure for rearing beetles and parasitoids. The goals of this research were to: 1. find an alternative to using whole logs for host material, 2. reduce mortality due to fungi seen when using wood, 3. find a less time- and  40 space- consuming means of rearing individuals, and 4. develop a rearing method to facilitate future research on T. tibial's. The key in developing a successful rearing method is to focus on the trends that indicate biological significance, rather than statistical analyses and complex procedures that may not further the development effort.  Wood Piece Studies Is Wood Necessary? Because T. tibial's attacks adult beetles that do not grow following the attack, and only two weeks of beetle activity are required for larval development, it was unclear• if wood was essential for culturing the wasps. To determine if beetles require wood to survive for two weeks, all combinations of three materials were tested: filter paper - provides a surface for beetles to right themselves on; Kimwipe tissue - provides a surface for climbing and hiding; and bark/phloem - provides a food source. The filter paper was a disk cut to fit on the bottom of the vial and it was moistened with one drop of distilled water. The tissue was a 2.5 by 3.0 cm strip torn from a Kimwipe and added to the vial so that it touched a filter paper disk on the bottom, but not the cork stopper. The core of bark/phloem, obtained from a fresh, uninfested lodgepole pine log with a #4 corer (diameter: 0.7 cm), was placed so that the phloem side touched the filter paper disk. On July 16, 1990, a trial using all possible permutations was — begun with 72 newly emerged adult L pini. The vials were maintained in a temperaturecontrolled chamber, operating at 26°C, 16h/8h light/dark cycles, and a relative humidity of approximately 40%, and were monitored daily for two weeks. On the seventh day of the trial, old materials were removed and fresh materials were placed in all vials according to their assigned treatment. This study demonstrated that beetles need wood to survive a two week period at 26°C,  41 a temperature that allowed rapid parasitoid development. Beetle mortality for the different treatments are given in Table 8. Mortalities as high as 100% in several of the filter paper and tissue treatments illustrate how inappropriate these methods are, even though beetles can be maintained this way for month(s) at cooler temperatures. Clearly, the best treatment for beetle survival is treatment 7 in which the beetles have a piece of bark/phloem for the full 14 days of confinement in the vial.  How Much Wood is Needed? Since wood could not be eliminated  in the rearing process, steps to minimize its use  are necessary to reduce costs. On September 19, 1991, trials to test the influence of wood piece size and vial size were begun. Two sizes of vials: medium (15 ml) and large (30 ml), and two sizes of wood pieces: small (approximately 0.5 g) and large (approximately 1.3 g), were tested to determine which conditions were better for beetle survival. Eighty adult I. pini that had emerged from field collected wood were used in the experiment. Sheets composed of bark, phloem and a small portion of outer xylem were obtained from clean, uninfested lodgepole pine logs by stripping the logs with wood chisels. These sheets were then chopped into small rectangular pieces weighing approximately 0.5 or 1.3 grams, and were maintained on damp tissues until inserted into glass vials, each with a circle of moistened filter paper on -its bottom. Beetles were weighed and sexed before being assigned to one of the four treatments: medium vial + 0.5g wood (M/S), medium vial + 1.3g wood (M/L), large vial + 0.5g wood (L/S), or large vial + 1.3g wood (UL). Vials were corked and placed in a temperaturecontrolled chamber at 28°C, 16h/8h light/dark cycles, and a relative humidity of approximately 40%. Each week for four weeks, the vials were examined for the condition of the beetles and the wood pieces. If the wood appeared dry, a single drop of distilled water was added to the  42 Table 8: Cumulative proportion of beetles dying at 1 and 2 weeks for the vial-rearing trial conducted on July 16, 1990. Treatments (fp= filter paper, t= tissue, and b= bark/phloem) were applied at 7 and at 14 days. Each treatment had 8 beetles for a total of 72 adult I. pin! used.  Treatment (7-day + 7-day)  Mortality 1-week^2-week  1:  fp  + fp  0.38  1.00  2:  fp  +^t  0.38  1.00  3:  fp  +b  0.63  0.75  4:  t  +^t  0.63  0.88  5:  t  + fp  0.38  1.00  6:  t  +b  0.25  0.50  7:  b  +b  0.00  0.25  8:  b  + fp  0.25  0.50  9:  b  +^t  0.13  0.38  43 vial. Several parasitized beetles were placed in vials similar to those used in this experiment to determine how the parasitoids responded. Although no one treatment is vastly superior in this trial, the vials containing small pieces of wood have lower mortalities than those containing large pieces of wood (Figure 8). The large vials were found to be cumbersome and tended to break easily. The medium vials were more convenient to use, making the medium-sized vial with the small wood piece the best procedure overall. Several problems were encountered in this experiment. Phloem thickness varied between different areas of the same log, making it difficult to control the amount of phloem each beetle received. Maintaining an appropriate moisture level within the vials proved to be very difficult. Beetles seemed to die easily if the wood was kept too wet or too dry. Fungi grew quickly in some of the vials killing the beetles. In several cases, nematodes were found infesting a wood piece and the dead beetle. These difficulties are directly associated with the use of fresh pine wood. Several adult T. tibialis were successfully obtained from beetles held on wood pieces in vials, but the moisture problems were a great hinderance; many parasitized beetles became covered with fungus once the beetle died.  How Much Water Is Needed? In an attempt to control humidity problems, a trial was conducted on June 2, 1992, to determine how much water was needed to best maintain the beetles and wood over four weeks in the vials. Fifty newly emerged adult I. pini were randomly assigned to receive one of four water treatments: 1= control (no water added), 2= 1 drop water per week, 3= 2 drops of water per week, or 4= 1 drop per 2 weeks. Bark/phloem pieces of approximately the same weight were obtained as described in the previous experiment and were placed in 15 ml vials  44 Figure 8: Cumulative mortalities for the wood size trial at 2 week and 4 week intervals. Treatments are: L-L= large vial + large wood; LS= large vial + small wood; M-L= medium vial + large wood; and M-S= medium vial + small wood. (N=80).  0  0.25  0.5 MORTALITY  0.75  1 1 2-WEEK 1.011 4-WEEK -  ^(  1  45 on a filter paper disk. Vials were placed in a temperature-controlled chamber at 28°C, 16h/8h light/dark cycles, and a relative humidity of approximately 40% . Each week the vials were examined for the condition of the beetle and of the wood, and the water treatment was applied. Particular attention was paid to mortality resulting from fungus, wet wood, or dry wood. The amount of moisture in vials clearly was important. The lowest mortality of beetles occurred in vials receiving 1 drop per week for which 80% of beetles survived 4 weeks (Table 9); the incidence of fungus was also low (Table 9). I estimated that over 30% of the mortality occurring in the vials was associated with moisture and fungus growth. These factors are difficult to control using wood pieces which are not uniformly moist to begin with,. nor are they sterile. The difficulties encountered in these trials prompted the search for a sterile, woodcontaining medium on which to maintain the adult beetles for 2 to 4 weeks so that T. tibialis larvae could develop.  Diet Studies General Methodologies A modification of the white pine weevil diet developed by Zerillo and Odell (1972) was first tried in October, 1992. Details of the formula and preparation methods for the diet are given in Appendix 1. Various methods were used to determine an effective means of handling the medium so that the beetles would survive the two to three weeks needed to sustain a parasitoid larva. Initially, the medium was poured into large containers and then cut into squares to be used. With this method, a filter paper disk is required on the bottom of the cup to aid the beetle in  46 Table 9: Cumulative weekly mortality rates for the vial rearing trial conducted on June 2, 1992. Different water regimes were tested using the treatments given below.  Treatment (drops H 2 O/week)  (n)  1: 0/week  10  2: 1/week  # Fungus  Mortality 1-Week  2-Week  3-Week  1  0.01  0.30  0.60  0.70  15  0  0.00  0.00  0.13  0.20  3: 2/week  10  3  0.10  0.20  0.40  0.50  4: 0.5/week  15  1  0.07  0.20  0.33  0.40  4-Week  47 righting itself should it fall off the medium. Plastic cups, (Solo P125: 37.0 ml or Solo P100: 29.6 ml; Solo Cup Co., Urbana, Ill.) were most commonly used in the experiments. Later, the diet was poured directly into the cups, with amounts ranging from 1/4 to 1/2 full (quantities detailed with experiments). The diet in cups was always allowed to stand overnight, loosely covered, at room temperature prior to any beetles being placed on it. This allowed any condensation in the cups to evaporate which reduced mortality substantially. Various types of lids were tested to obtain better control of the humidity when the diet was fresh and also when drying. Solid plastic lids (S), fine mesh covers held on by elastic bands (M), and plastic lids with a number of holes punctured in them (H) were all tried. Treatments usually involved the changing of lids after two days. For example, mesh-to-holes (M/H) is a treatment where a mesh cover is used for two days and then replaced by a plastic lid with holes for the remainder of the experiment. Initially beetles were placed on the surface of the medium and allowed to move about freely. This usually resulted in little or no feeding, and the beetle often flipped over and died, since it was unable to right itself. A better method was to make a small hole in the centre of the diet with a #3 corer (diameter: 0.5 cm). A beetle was added to the hole and the core was replaced loosely over the hole to encourage the beetle to bore into the diet. Beetles did emerge regularly from the medium to walk about on the surface of the diet. This again resulted in death if the beetle fell over in the cup. To reduce this mortality, one end of the tray holding the cups was elevated so that the surface of the medium tilted somewhat. This proved sufficient to allow beetles to right themselves after falling over. Trays holding the diet cups were placed in a small temperature-controlled chamber that operated at 26°C, 16h/8h light/dark cycles, and had a relative humidity of approximately 40%. The chamber was later altered to a 26°C daytime- and 22°C nighttime-varied  48 temperature in an attempt to reduce or break the diapause of the parasitoids (see: Can Tomicobia tibialis be reared?)  The success of trials were scored on the basis of how much diet was fed upon by the beetles. The cups were examined at 2-day, 1-week, and 2-week intervals and the amount of feeding was determined as: 0= None, or beetle dead; 1= Slight (chewing around core hole obvious); 2= Moderate (one gallery-like trench); 3= Moderate to Heavy (two gallery-like trenches); 4= Heavy (three or more trenches and some frass accumulation); 5= Very Heavy (diet is chewed up and there is large amounts of frass). Following the trial, the average performance of beetles at different time intervals was compared by looking at the scores. Low scores indicate little feeding while high scores indicate heavy feeding.  Can an ArtifIcal Diet Work? In initial trials, condensation created by the diet in closed cups usually killed the beetles within days and little feeding occurred. On October 13, 1992, I conducted a small trial using 8 male and 8 female adults for which half the cups were covered with lids and the other half were first covered by mesh for 2 days and then lids for the remaining time. Beetles were maintained most successfully in the mesh-to-lid groups, with all 4 females and 2 of the males feeding and surviving for 15 days on the medium. This success encouraged me to experiment further with diet. On October 27 an experiment using 32 newly emerged beetles (16 males and 16 females) was set-up to evaluate four types of lid conditions: holes-to-holes (H/H), holes-tosolid (H/S), mesh-to-holes (M/H), and mesh-to-solid (M/S). For example, for a holes-to-holes treatment, the cup would first have a lid with 5 holes punched into it for two days followed by a similar lid for the remainder of the trial. To determine if the addition of beetle frass would  49 increase feeding by the beetles, frass was randomly assigned to 16 of the cups (8 male and 8 female). Results after two weeks indicated that: 1. treatments allowing more drying of the diet (H/H & M/H) appeared better than the treatments with solid lids (H/S & M/S) (Table 10); 2. addition of frass had no effect on the response of either males or females (Table 10); and 3. more replicates were needed to examine beetle response more closely. On December 1, 1992, an experiment using 64 male and 64 female beetles was conducted. This time the diet was weighed to ensure that both males and females had similar amounts in their cups (food weight: 5.5 g ± 0.9; n=64). The same four lid treatments were used as mentioned above. In this trial, the high mortality in the treatments using 5-hole lids first, which still traps moisture in the cups, clearly demonstrates that the diet must be allowed to dry initially (Table 11). Both males and females ate the diet, showing substantial amounts of feeding within one week (Table 11). Thus this artifical diet was able to sustain adult I. pini for a two week period. No fungus was encountered and each beetle received an equal amount of wood by diet weight, unlike in the fresh wood-vial trials. There were several problems however. The mesh lids held on by elastic bands were cumbersome to work with and often resulted in severe disturbances of the beetle in the cup. Having to change the lids on trays of cups after two days was labour intensive. It was wasteful and time consuming to cut round filter paper disks to line each cup and to cut equal sized squares of food. Pouring diet directly into cups and using lids with different numbers of holes were tried next to simplify procedures.  How Much Diet Is Needed? Having established the usefulness of the diet, it became important to determine how  50 Table 10: Feeding scores for the diet trial conducted Oct 27 to Nov 10, 1992. Four treatments were assessed: H/H (n=7), H/S (n=9), M/H (n=8), and M/S (n=8), where H=5-holed plastic lids, M=mesh lids and S=solid plastic lids, and the procedure involved changing lids after the first two days. [Feeding scores: 0=none, 1=slight, 2=moderate, 3=moderate to heavy, 4=heavy, and 5=very heavy].  a) Median and range of the feeding scores at 2-day, 1-week, and 2-week intervals. 2-day  Treatment  1-week  2-week  n  median range  n  median range  n  median range  H/H  7  1  4  7  2  3  7  4  3  H/S  7  1  2  6  2  3  6  2  5  WH  8  1.5  2  8  2  0  5  4  2  M/S  8  0.5  2  6  1.5  4  5  3  3  b) Median and range of the feeding scores at the 2-week interval, grouped by whether or not the beetles had frass added to their cups. Treatment^Male^ n  Median  Frass Added  8  1  No Frass  8 16  Total  Range  Female Range  n  Median  5  8  3  5  2  4  8  2  4  2  5  16  2  5  ^n  51 Table 11: Feeding scores for the diet trials conducted from Dec 1 to Dec 15, 1992. Treatments are: H/H (n=16), H/S (n=16), M/H (n=16), and M/S (n=16), where H= 5-holed plastic lid, M= mesh lid and S= solid plastic lid, and lids are changed after the first 2 days. [Feeding scores: 0=none, 1=slight, 2=moderate, 3=moderate to heavy, 4=heavy, and 5=very heavy].  a) Median and range of the feeding scores for male beetles. Treatment^2-day^ 1-week^2-week  median  range^n^median range^n median range  H/H^4^1.5^3^1^  1  H/S^6^1.5^3^0^  0  M/H^16^2^4^15^4^2^15^5^1 M/S^15^2^4^14^4^3^13^5^3  b) Median and range of the feeding scores for female beetles. Treatment^2-day^ 1-week^2-week ^n median range^n median range^n median range H/H  6  1  1  1  0  H/S  3  1  0  0  0  M/H  16  2  4  15  3  3  15  5  3  M/S  15  2  4  14  2.5  2  14  5  2  52 much diet was really needed to maintain beetles. On February 3, 1993, twenty beetles were placed on one of two treatments: cups that had been half filled with diet (food weight: 10.62 g ± 0.8; n=10) or cups that were only a quarter full (food weight: 6.25 g ± 1.2; n=10). Six of the 20 beetles used were parasitized, three in each treatment (see: Can T. tibialis be reared?). Although beetles had the same general feeding levels after two weeks in each treatment, results showed that the beetles responded faster (Figure 9) and survived better (Mortality: 1/4-full = 0%; 1/2-full = 50%) in the cups that had less diet. On February 9, 1993, an experiment to examine the role of adding filter paper to cups of directly-poured diet was set-up to determine if some wicking effect could be achieved to reduce moisture. There were two diet treatments: diet + a filter paper disk, or diet alone, and two lid treatments: 20-hole lids (20H) or 5-hole lids (5H), placed on from the beginning and left in place. Twenty beetles were used with 10 of those being parasitized (see: Can T. tibialis be Reared?). The diet alone treatment showed slightly better feeding (Figure 10a) and less mortality (Mortality: alone = 10%; diet+filter paper = 30%) after two weeks than the diet plus filter paper treatment. Instead of wicking moisture away as the diet dried, the filter paper trapped excess moisture beneath it, creating puddles that drowned beetles and an emerging T. tibialis female. Lid type did not seem to greatly affect the amount of feeding observed using either diet treatment (Figure 10b).  Can Tomicobla tibialis be Reared? To determine if parasitoids could develop inside beetles held on the medium, groups of parasitized beetles were placed on the diet during several experiments. On December 20, 1992, four female beetles were parasitized and stored at room temperature as described in the methods section of chapter 2. The next day they were placed on diet, using cups with  53  Figure 9: Feeding score response of beetles given one of two amounts of diet, half- or quarter-full cups. [Feeding scale: 0= none, 1= slight, 2= moderate, 3= moderate-heavy, 4= heavy, and 5= very heavy].  FEEDING SCALE  n 1-DAY ^ 4-DAYS 1/1 2-WEEKS  54 Figure 10: a) Feeding score response of beetles given either diet alone or diet with a filter paper disk. [Feeding scale: 0= none, 1= slight, 2= moderate, 3= moderate-heavy, 4= heavy, and 5= very heavy].  u) DIET ALONE  0 0 u_ DIET+PAPER  0^1^2^3^4 FEEDING SCALE  ni 1-WEEK Mg 2-WEEK  ^  5  55 Figure 10: b) The same graph showing the lid treatment breakdown, where 5H= 5-hole lid and 20H= 20-hole lid. [Feeding scale: 0= none, 1= slight, 2= moderate, 3= moderate-heavy, 4= heavy, and 5=very heavy].  (D) 5H  (D) 20H  4$*:;,5f..eeee'e  cc /"" (D+P) 5H  (D+P) 20H  ............  0  ^  ^IJ  1^2^3^4 FEEDING SCALE  ni 1-WEEK FM 2-WEEK  5  56 mesh lids, and placed in the chamber at 26°C. The mesh lids were replaced with solid lids the following day and the cups were monitored regularly. On December 23, 10 additional beetles were parasitized, held at room temperature, and then placed on diet the following day, this time using 5-hole lids. Beetles died within 2-3 weeks, but no parasitoids emerged from either set and so on February 8, 1993, the beetles were dissected. Two of the• four original females had live parasitoid pupae in them. Of the 10 beetles in the second group, three were dissected on February 8 to reveal one live pupa, one parasitoid that had died at an earlier instar and the third beetle had no obvious parasitoid present (indeterminate). The remaining cups were placed in the refrigerator at 6°C as a possible way of breaking the diapause of any other parasitoid pupae. Two of these 6 were removed from the refrigerator on March 8 and left at room temperature until March 29. The remaining 4 cups were left in the refrigerator until March 29. All 6 beetles were dissected March 29 to determine if diapause had been broken. Of the two beetles taken out on March 8 and left at room temperature, one contained a dead parasitoid pupa while the other contained a fully formed adult T. tibialis female. Of the four chilled until March 29, one contained a live pupa, one a dead pupa, and in the last two it could not be determined if they were parasitized (indeterminate). Thus T. tibialis larvae survived in beetles that were held on diet, but seemed to diapause in the pupal stage. Chilling possibly broke the diapause in one case, but the adult wasp that formed did not emerge from the beetle. In another attempt to prevent diapause of the parasitoids, I shifted the chamber temperature from a constant 26°C to the varied daytime 26°C and nighttime 22°C.' The first known T. tibialis to be reared using the wood-based medium emerged from the quarter-full cups used in the diet experiment begun on Feb.3, 1992. This was the only parasitoid of the six parasitized beetles to emerge (Table 12), although she was trapped in  57 Table 12: Parasitoid production from the diet trial conducted Feb 3 to Mar 23, 1993, in which diet cups were either half (wt: 10.6 ± 0.8g, n=10) or quarter (wt: 6.3 ± 1.2g, n=10) full.  Treatment^Output Half-full  n=3: 2 indeterminate 1 late instar, dead  Quarter-full  n=3: 1 indeterminate 1 pupa, dead 1 adult female, emerged  Table 13: Parasitoid production from the diet trial conducted Feb 9 to Mar 23, 1993, in which two diet treatments: diet plus a filter paper disk, or diet alone, and two lid types: 5H= 5 hole lid, or 20H= 20 hole lid, were being assessed.  Treatment^Output Diet+paper/20H  n=3: 2 indeterminate 1 pupa, live  Diet+paper/5 H  n=2:  1 pupa, dead 1 adult female, emerged  Diet alone/20H  n=3:  1 indeterminate 1 pupa, live 1 adult male, emerged  Diet alone/5H  n=2: 1 indeterminate 1 pupa, dead  58 the diet and died. Since none of the parasitized beetles in the half-full cups even reached pupal stage, the cups having less food, and therefore a drier medium, appear to provide the better environment for parasitoid development. Two more adult T. tibialis emerged from the diet used in the Feb. 9 experiment (Table 13); however, both were drowned or trapped in the diet. Although 60% of the parasitized beetles from Feb. 9 reached pupal stage or beyond (Table 13), the medium creates a problem for the emerging insects. Thus of twenty-nine attempts to rear T. tibialis on beetles held on medium, eighteen (62%) resulted in some parasitoid development, with 55% of those parasitoids reaching the pupal stage or beyond. Three adult T. tibia/is, one male and two females, emerged from the beetles held on diet, but died due to complications with the rearing methods. It would likely be more effective to remove the parasitized beetles from the medium once the beetle "dies" and place the beetle in a container that did not have any medium to allow parasitoid emergence. This would make watching for emerging parasitoids easier and would likely increase the success of this rearing method. The diapause of pupal parasitoids was an additional complication. Eleven parasitoids (38%) were found in the pupal stage during these experiments, over half of which were in pupal diapause. I believe that a modification of the rearing temperatures could overcome this problem. If these rearing procedures can be sufficiently streamlined, then the use of artificial medium provides a cost- and space-saving means of producing individual parasitoids for further research.  Discussion By the spring of 1993, a medium capable of sustaining adult I. pini for at least 4 weeks was developed. In order to improve on this diet, a number of modifications can be attempted. With the formula itself, it would be useful to conduct formal feeding trials in which the amount  59 of wood added to the diet was varied. This would prevent adding more wood than is needed for maintaining the beetles. The critical modifications still needed, however, are to perfect the rate of drying, and the temperature of rearing, so that parasitoid development is not hindered. One possibility is to alter the humidity in the chamber, rather than attempting to control diet moisture with lids. Lower or varying chamber temperatures may also be effective, but could prolong the turnover time between parasitoid generations. Although I suggested that the best approach to use this diet immediately would be to remove beetles once they died and allow parasitoid emergence elsewhere, finding a way to forego this would greatly enhance the efficiency of parasitoid production. This problem may be corrected in conjunction with solving the moisture content problem. Having a successful rearing technique would greatly facilitate future research on both the beetles and the parasitoids. The medium provides a sterile environment in which investigation of beetle disease could be conducted. Since the beetles consume the diet, they could be easily exposed to disease in a controlled manner. The medium also .provides the means to hold adult beetles for long periods of time without reduced vitality. This is useful when fresh insects are not readily available for research. This medium may also prove useful on other beetle species such as the Mountain Pine beetle, which has similar life-history traits to fps. For parasitoid research, the diet provides the opportunity to conduct detailed studies-on fecundity, survivorship, sex ratio, etc. which are currently difficult to perform due to laboratory space constraints. The response of parasitoids reared on diet to the beetles, to natural cues, and to their performance levels must be checked to ensure that the diet method creates high quality wasps. Also, if the diet can be modified for use with other beetle species, then new avenues of research exist for their associated parasitoids as well. A further benefit exists with this rearing method. If the diet can be successfully  60 adapted to mass-rearing of parasitoids, the medium provides an efficient means of producing T. tibialis since only dried bark and phloem are required to rear the parasitoids instead of whole logs. This is better than the vial-rearing method which requires fresh phloem for which there is no ready supply. This also means that the heart-wood section of the log is no longer being wasted in the process of parasitoid production, as with bolt-rearing, but can be used to its maximum, revenue-generating potential. Since lodgepole pine is debarked at mills as part of its processing, there is a ready source of bark for use in the diet. Milling waste-products could be used and no additional trees would be required once the system was perfected. Since the phloem is dried, large quantities of wood could be processed and stored easily. A cottage industry linked to the mill then becomes a plausible means of producing required agents for release. Thus the development of a more efficient rearing method increases the likelihood of T. tibialis, and perhaps other species of parasitoids, of being developed into biocontrol agents for use in forestry.  61  Chapter 4 Could Augmentative Release Work? Can Mass-rearing be Developed? Although scientists require the ability to track individuals throughout their lives to understand the influence of various parameters on parasitoid development, those who produce biocontrol agents for release require no such technology. Instead, the goal is to raise as many quality insects as possible while minimizing time, space and effort in order to make the product affordable to consumers and profitable to the industry. The artificial medium used in this thesis could provide an easy means of mass-rearing parasitoids. Artificial medium is easily adapted to automation for its production and packaging into containers. Depending on systems already in place, the beetles could be maintained either individually, as demonstrated, or in groups in larger containers since the beetles are naturally gregarious. Methods used to handle other parasitoids once they have emerged could be adapted for use with T. tibialis. The only stage in production which would require serious modification would be the parasitization of hosts. Tomicobia tibialis requires olfactory cues, such as frass, and a mobile beetle to stimulate attack. Too many beetles at one time (eg. 4 or 5 in a 2 by 10 cm Petri dish) can create confusion and lead to a reduction in the number of attacks observed. The most likely solution lies in current biocontrol production operations for other parasitoids that have similar stringent attack requirements.  Is the Reduction Enough? The experiments in Chapter 2 demonstrate that T. tibialis reduces I. pini offspring production anywhere from 18%, when only the male is parasitized, to as much as 64% when  62 the male and his harem are parasitized. But how these figures translate into an impact on a natural population need to be considered. A simple mathematical exercise (Appendix 2) reveals that I. pini offspring production could be reduced by 15% alone assuming: average offspring production per harem, equal probability of any of the six combinations of healthy and parasitized beetles and zero larval mortality. Obviously none of these assumptions are valid in the real world, but even under these conditions an impact is observed. The question to resolve is what proportion of harems fall into the lowest offspring production categories in nature. This is determined by a number of factors including: 1) the number of beetles, 2) how quickly the aggregation peaks, 3) the numbers and foraging ability of parasitoids, and 4) climatic factors. Clearly, an augmentation program for T. tibialis has the potential to alter the numbers of foraging parasitoids through releases and thereby influence beetle offspring production. This could be an effective means of treating stands in which conventional control measures are simply impractical (Nealis, 1991; Parrella, et aL,1992). However, the use of T. tibialis is not restricted to such cases. If released during or following a pheromone trapping program, which can only remove a portion of the aggregating beetle population (Vite & Baader, 1990), the impact of the parasitoid could be very dramatic since the ratio of hosts to parasitoids would be greatly reduced. Furthermore, since this parasitoid is a native insect, augmentation can be expected to have lasting effects through the progeny of released insects. The coevolved nature of this system ensures that the parasitoid will seek out all 1ps hosts in an attempt to reproduce, but will not impact non-target beetle populations. With the potential to reduce harem outputs by 64%, a rearing methodology adaptable to mass-production, and the successes seen using biocontrol in Europe, Australia, and Canada (see Dahlsten & Whitmore,  63 1989; Morgan, 1989; Carrow, 1990), I feel further investigation into augmentation of T. tibialls is warranted.  64  Bibliography  Andres, L.A., E.R. Oatman & R.G. Simpson. 1979. Re-examination of pest control practices, pp. 1-10. In D.W. Davis, S.C. Hoyt, J.A. McMurtry & M.T. AliNiazee [Eds.], Biological control and insect pest management. Division of Agricultural Sciences, University of California. Publication 4096, Oakland. Beckman, D., S. Gast, J. Schwandt, B. James, L. Livingston, A. Knapp, J. Weatherby, J. Hoffman, & R. Williams. 1990. Idaho forest pest conditions and program summary, 1990. Idaho Department of Lands. USDA Forest Service, Northern and Intermountain Range. Coeur d'Alene, Idaho 83814. Report No. 91-2. Bedard, W.D. 1965. The biology of Tomicobia tibialis (Hymenoptera: Pteromalidae) parasitizing Ips confusus (Coleoperta: Scolytidae) in California. Contrib. Boyce Thompson Inst. 23: 77-82. Behan, R.W. 1990. Multiresource forest management: a paradigmatic challenge to professional forestry. J. For. 88: 12-18. Birch, M.C. 1984. Aggregation in bark beetles, pp. 331-353./n W.J. Bell & R.T. Carde [Eds.], Chemical Ecology of Insects. Chapman & Hall Ltd. Borden, J.H. 1967. Factors influencing the response of Ips confusus (Coleoptera: Scolytidae) to male attractant. Can. Ent. 99: 1164-1193. Borden, J.H. 1989. Semiochemicals and bark beetle populations: Exploitation of natural phenomena by pest management strategists. Holarctic Ecol. 12: 501-510. Bright, Jr., D.E. 1976. The insects and arachnids of Canada, Part 2: The bark beetles of Canada and Alaska (Coleoptera: Scolytidae), pp. 14, 158-159. Biosystematics Research Institute Research Branch. Canada Department of Agriculture. Ottawa, Ontario, Canada. Publication 1576. Byers, J.A. 1989. Behavioral mechanisms involved in reducing competition in bark beetles. Holarctic Ecol. 12: 466-476. Carrow, J.R. 1990. Background and overview of project, pp. 5-9. In S.M. Smith, J.R. Carrow, & J.E. Laing [Eds.], Inundative Release of the Egg Parasitoid, Trichogramma minutum (Hymenoptera: Trichogrammatidae) Against Forest Insect Pests such as the Spruce Budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae): the Ontario Project 1982-1986. Memoirs Ent. Soc. Can., No. 153. Dahlsten, D.L. & M.C. Whitmore. 1989. Potential for biocontrol of Dendroctonus and Ips bark beetles: The case for and against the biocontrol of bark beetles. pp. 3-19 In Kulhavy, D.L. & N.C. Miller [Eds.], Potential for Biological Control of Dendroctonus and 1ps Bark  65 Beetles. Stephen F. Austin State University, Centre for Applied Studies, School of Forestry, Nacagdoches, TX. Debach, P. 1964. Biological control of insect pests and weeds. Chapman and Hall, London. Frazier, J.L., T.E. Nebeker, R.F. Mizell & W.H. Calvert. 1981. Predatory behaviour of the clerid beetle Thanasimus debius (Coleoptera: Cleridae) on the southern pine beetle (Coleoptera: Scolytidae). Can. Ent. 113: 35-43. Furniss, M.M. 1968. Notes on the biology and effectiveness of Karpinskiella paratomicobia parasitizing adults of Dendroctonus pseudotsugae. Ann. Ent. Soc. Am. 61(6): 13841389. Gerdin, S. & K-J Hedquist. 1984. Perilitus areolaris sp.n. (Hymenoptera: Braconidae), an imago-parasitoid of the large pine weevil, Hylobius abietis (Linnaeus) and its reproductive behaviour. Ent. Scand. 15: 363-369. Haugen, D.A. & M.G. Underdown. 1990. Release of parasitoids for Sirex noctilio control by transporting infested logs. Aust. For. 53: 266-270. Hedqvist, K. 1959. Notes on Chalcidoidea. IV. Genus Tomicobia Ashm. Opuscula Entomol. 24: 177-184. Heilman, P.E. 1990. Forest management challenged in the Pacific Northwest. Increasingly cynical public questions sustainability of current practices. J. For. 88: 16-23. King, E.G., K.R. Hopper & J.E. Powell. 1985. Analysis of systems for biological control of crop pests in the U.S. by augmentation of predators and parasites, pp. 201-227. In M.A. Hoy & D.C. Herzog [Eds.], Biological Control in Agricultural IPM Systems. Academic, Orlando, Florida. Kirkendall, L.R. 1989. Within-harem competition among Ips females, an overlooked component of density-dependent larval mortality. Holarct. Ecol. 12: 477-487. Laing, J.E. & G.M. Eden. 1990. Mass-production of Trichogramma minutum Riley on factitious host eggs, pp. 10-24. In S.M. Smith, J.R. Carrow, & J.E. Laing [Eds.], Inundative Release of the Egg Parasitoid, Trichogramma minutum (Hymenoptera: Trichogrammatidae) Against Forest Insect Pests such as the Spruce Budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae): the Ontario Project 1982-1986. Memoirs Ent. Soc. Can., No. 153. Lawson, S.A. & F.D. Morgan. 1992. Rearing of two predators, Thanasimus dubius and Temnochila virescens, for the biological control of Ips grandicollis in Australia. Entomol. Exp. Appl. 65: 225-233. Livingston, R.L. 1979. The pine engraver, 1ps pini (Say) in Idaho: Life history, habits and management recommendations. Idaho Department of Lands, Forest Insect and Disease Control. P.O. Box 670. Coeur d'Alene, Idaho 83814. Report 79.-3: 1-7.  66 Miller, D.R. & J.H. Borden. 1985. Life history and biology of Ips latidens (Leconte) (Coleoptera: Scolytidae). Can. Ent. 117: 859-871. Miller, D.R., J.H. Borden & K.N. Slessor. 1989. Inter-and intrapopulation variation of the pheromone Ipsdienol produced by male pine engravers, Ips pini (Say) *(Coleoptera: Scolytidae). J. Chem. Ecol. 15: 233-247. Miller, D.R., G. Gries, & J.H. Borden. 1990. E-Myrcenol: a new pheromone for the pine engraver, Ips pini (Say) (Coleoptera: Scolytidae). Can. Ent. 122: 401-406. Miller, M.C., J.C. Moser, M. McGregor, J-C. Gregoire, M. Baisier, D. L. Dahisten, & R.A. Werner. 1987. Potential for biological control of native North American Dendroctonus beetles (Coleoptera: Scolytidae). Ann. Entomol. Soc. Am. 80: 417-428. Moeck, H.A. & L. Safranyik. 1984. Assessment of predator and parasitoid control of bark beetles. Canadian Forestry Service, Pacific Forest Research Centre, Victoria, BC. Information Report BC-X-248 Morgan, F.D. 1989. Forty years of Sirex noctillo and Ips grandicollis in Australia. New Zealand J. For. Sci. 19: 198-209. Nealis, V.G. 1991. Natural enemies and forest pest management. Forestry Chronicle 67: 500505. Ochieng'-Odero, J.P.R., F.O. Onyango, J.T. Kilori, & M.D.O. Bungu. 1991. Insect rearing management as a prerequisite in the development of IPM for sustainable food production. Insect Science Applic. 12: 645-651. Parrella, M.P., K.M. Heinz, & L. Nunney. 1992. Biological control through augmentative release of natural enemies: a strategy whose time has come. Amer. Ent. 38: 172-179. Payne, T.L. 1989. Olfactory basis for insect enemies of allied species. pp. 55-69 In Kulhavy, D.L. & N.C. Miller [Eds.], Potential for Biological Control of Dendroctonus and Ips Bark Beetles. Stephen F. Austin State University, Centre for Applied Studies, School of Forestry, Nacagdoches, TX. Reid, M.L. 1991. Female mate choice and male parental care in a bark beetle (Ips pini, Coleoptera: Scolytidae). PhD thesis. Dept. of Biological Sciences, Simon Fraser University. Reid, R.W. 1957. The bark beetle complex associated with lodgepole pine slash in Alberta. Part II. Notes on the biologies of several Hymenopterous parasites. Can. Ent. 89: 5-8. Rice, R.E. 1968. Observations on host selection by Tomicobia tibialis Ashmead (Hymenoptera: Pteromalidae). Contrib. Boyce Thompson Inst. 24: 53-56.  67 Rice, R.E. 1968. Observations on host selection by Tomicobia tibialis Ashmead (Hymenoptera: Pteromalidae). Contrib. Boyce Thompson Inst. 24: 53-56. Rice, R.E. 1969. Response of some predators and parasites of fps confusus (lec.) (Coleopera: Scolytidae) to olfactory attractants. Contrib. Boyce Thompson Inst. 24: 189-194. Schmitz, R.F. 1972. Behaviour of Ips pini during mating, oviposition, and larval development (Coleoptera: Scolytidae). Can. Ent. 104: 1723-1728. Senger, S.E and Roitberg, B.D. 1991. Effects of parasitism by Tomicobia tibialis Ashmead (Hymenoptera: Pteromalidae) on reproductive parameters of female pine engravers, fps pini (Say). Can. Ent. 124: 509-513. Thomas, J.B. 1961. The life history of Ips pini (Say) (Coleopera: Scolytidae).Can. Ent.93: 384390. Vite, J.P. & E. Baader. 1990. Present and future use of semiochemicals in pest management of bark beetles. J. Chem. Ecol. 16: 3031-3041. Zerillo, R.T. & T.M. Odell. 1972. White pine weevil: A rearing procedure and artificial medium. J. Econ. Entomol. 66: 593-594.  68  Appendix 1 A modification of the white pine weevil diet developed by Zerillo and Odell (1972) was first tried in October, 1992. The recipe was altered periodically throughout the diet trials and thus results between trials are not directly comparable. The following recipe is the most successful one to date for maintaining adult I. pini:  Ingredients^  Amounts  Water, distilled^ 640.00 ml Pine bark and phloem (dry)^20.00 g 24.00 g Wheat germ^ 19.20 g Sucrose^ 4.80 g Starch^ 2.40 g Wesson Salt Mix^ 19.20 g Casein^ 0.40 g Cholesterol^ 16.00 g Sorbic Acid^ Ethanol (70%)^ 136.00 ml 0.32 ml Formalin^ Streptomycin sulfate^ 0.22 g Vanderzant Vitamix Mix^22.40 g  The outer layers of bark and phloem were stripped from lodgepole pine logs as described previously (see: How Much Wood is Needed?). The sheets were ripped Into thin ..■  strips and placed in an oven at approximately 100°C for an hour, or until brittle. The dried pieces were weighed, placed in a blender, and processed until only small pieces and dust remained (approximately 5 minutes). The remaining ingredients were added to the blender and the mixture was processed for 5 to 10 minutes to ensure adequate mixing. This mixture was added to 20g of Agar (and 600 ml distilled water), mixed again and then poured either directly into diet cups or into a large container for storage in the fridge at 6°C. Diet was  69 usually made in the afternoon and was allowed to stand overnight with a loose cover before being refrigerated the next morning. Diet left in blocks in the fridge could then be cut to the desired size when needed. Cups with diet poured in directly were also successfully stored in the fridge with tight lids; however, the cups had to be left out of the fridge overnight to allow some moisture to evaporate before they could be used.  Appendix 2 A simple mathematical exercise illustrates how T. tibialis may impact I. pini:  Suppose there are 10,000 female I. pini and that each male will have 4 females in a harem for a total of 2500 harems. Also suppose that each of these harems will produce the average total offspring seen in experiments 1 and 2 (Table 2 & 4), and there is an equal probability of having any combination of healthy and parasitized beetles. Therefore, 2500 harems divided by 6 combinations means that there are approximately 417 harems per combination.  Combination H + 2H/2P P + 2H/2P H + 4H P + 4H H + 4P P + 4P  Total Brood Output  Avg. Total Offsp. 52.9 46.0 45.0 41.1 23.0 21.2  *417  22059.3 19182.0 18765.0 17138.7 9591.0 8840.4 95,576.4  If 2500 harems had all been healthy then (2500*45.0)=112,500 brood beetles are expected. Therefore, there is a 15% reduction in offspring output as a result of parasitism.  

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