Quantitative Trait Loci for Susceptibility to Tapeworm Infection in the Red Flour Beetle

Corresponding author: Guiyun Yan, Hoch 220, State University of New York, Buffalo, NY 14260., gyan{at}buffalo.edu (E-mail)

Communicating editor: J. A. BIRCHLER

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Parasites have profound effects on host ecology and evolution, and the effects of parasites on host ecology are often influenced by the magnitude of host susceptibility to parasites. Many parasites have complex life cycles that require intermediate hosts for their transmission, but little is known about the genetic basis of the intermediate host's susceptibility to these parasites. This study examined the genetic basis of susceptibility to a tapeworm (Hymenolepis diminuta) in the red flour beetle (Tribolium castaneum) that serves as an intermediate host in its transmission. Quantitative trait loci (QTL) mapping experiments were conducted with two independent segregating populations using amplified fragment length polymorphism (AFLP) markers and randomly amplified polymorphic DNA (RAPD) markers. A total of five QTL that significantly affected beetle susceptibility were identified in the two reciprocal crosses. Two common QTL on linkage groups 3 and 6 were identified in both crosses with similar effects on the phenotype, and three QTL were unique to each cross. In one cross, the three main QTL accounted for 29% of the total phenotypic variance and digenic epistasis explained 39% of the variance. In the second cross, the four main QTL explained 62% of the variance and digenic epistasis accounted for only 5% of the variance. The actions of these QTL were either overdominance or underdominance. Our results suggest that the polygenic nature of beetle susceptibility to the parasites and epistasis are important genetic mechanisms for the maintenance of variation within or among beetle strains in susceptibility to tapeworm infection.

POPULATION biologists have widely recognized the potential importance of parasites in host ecology and evolution. Parasites are intimately dependent on the host for survival, live at the expense of the host, and often have deleterious effects on host reproductive success. Thus, parasites can be important selective agents on hosts. Parasites have been invoked as causal agents in the maintenance of sex (JAENIKE 1978 ; BREMERMANN 1980 ; HAMILTON 1980 ; LIVELY 1987 ; READ 1988 ; CLAYTON 1991 ; POULIN and THOMAS 1999 ) and in the evolution of male secondary sexual traits (READ 1988 ; READ and HARVEY 1989 ; CLAYTON 1991 ). The ecological importance of parasites is reflected by their effects on host population abundance and species interactions (PARK 1948 ; ANDERSEN and MAY 1979 ; BREMERMANN 1980 ; GRIMALDI and JAENIKE 1984 ; HOLT and PICKERING 1985 ; PRICE et al. 1986 , PRICE et al. 1988 ; YAN et al. 1998 ). Parasites can alter host behaviors in ways that may increase transmission success or benefit the hosts (HOLMES and BETHEL 1972 ; MOORE 1984 ; MOORE and GOTELLI 1990 ; YAN et al. 1994 ).

In many cases, the effects of parasites on host ecology are influenced by the magnitude of host susceptibility to parasites. For example, host mortality and reduction in reproduction and parasite-induced behavioral changes are a function of infection intensity (the number of parasites in an infected individual; YAN et al. 1994 ; YAN 1997 ). On the other hand, infection heterogeneity promotes the coexistence of hosts and parasites and influences the dynamics of host and parasite populations (ANDERSON 1988 ). Previous studies have demonstrated considerable variation in susceptibility to parasite infection among host species (FREEHLING and MOORE 1993 ), among genetic strains within a species (YAN and NORMAN 1995 ), and among individuals within a population (DOBSON and HUDSON 1992 ). Also, many parasites, including some protozoans, nematodes, trematodes, and cestodes, as well as all acanthocephalans, have complex life cycles that require intermediate hosts, often transmitted through the food chain, to complete their life cycles. Indirectly transmitted parasites are important elements in the community and ecosystem (see review by MOORE and GOTELLI 1990 ); however, little is known about the genetic basis of the intermediate host's susceptibility to parasites transmitted through the food chain.

The tapeworm Hymenolepis diminuta has been extensively used in the laboratory for parasitological, physiological, and ecological research (GORDON and WHITFIELD 1985 ; SANGSTER and METTRICK 1987 ; SUKHDEO 1992 ; KEARNS et al. 1994 ; YAN 1997 ; STARKE and OAKS 2001 ). The adult parasite lives in nature in the lumen of the small intestine of rats, where the eggs are produced and passed out with the host's feces. Tapeworm eggs develop into cysticercoids when ingested by the proper intermediate host, often grain-infesting insects such as Tribolium and Tenebrio beetles. The vertebrate host may be infected after ingestion of infected intermediate hosts. Thus, H. diminuta is not transmissible from beetle to beetle horizontally or vertically. The life cycle of the tapeworm can be readily completed under laboratory conditions.

Most traits of economic and medical importance in human, animal, or plant species are polygenic. THODAY 1961 suggested that Mendelian markers can be used to partition complex quantitative traits into their underlying Mendelian components, termed quantitative trait loci (QTL) mapping. The availability of highly polymorphic DNA markers in many species allows the development of well-saturated genetic maps and consequently the genetic dissection of complex quantitative traits. Through the use of molecular linkage maps, a systematic search of entire genomes for QTL has revealed that a large proportion of the total phenotypic variance is often attributable to a few major QTL and several modifier genes (ALPERT and TANKSLEY 1996 ). For the red flour beetle, BEEMAN and BROWN 1999 constructed a linkage map on the basis of 123 randomly amplified polymorphic DNA (RAPD) markers, six identified genes, and five morphological markers. Genetic linkage mapping has proven to be a powerful tool for gene localization and isolation, marker-assisted selection, and evolutionary studies. Amplified fragment length polymorphism (AFLP) is a PCR-based multilocus fingerprinting technique (VOS et al. 1995 ). It offers improved reproducibility compared to the RAPD markers, but does not require prior information on the PCR primers for the study organism. We have developed an AFLP-based linkage map that includes 269 AFLP and 18 RAPD markers (ZHONG et al. 2004 ). The total map length is 573 cM, giving an average marker resolution of 2.0 cM and an average physical distance per genetic distance of 350 kb/cM. Development of the linkage maps provides the necessary tools to determine the genetic basis of beetles' susceptibility to tapeworm infection. Here, we report the results of QTL mapping on the infection intensity of the red flour beetle to H. diminuta. Using AFLP markers and reciprocal F₂ intercrosses between resistant and susceptible strains, we identified five QTL affecting the beetle's susceptibility to tapeworm.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Selection for highly susceptible and highly resistant beetle populations:
Two Tribolium castaneum strains, cSM and TIW1, were used in this study. The strain cSM was created by combining several laboratory strains in the 1970s (WADE 1977 ), and TIW1 was kindly provided by Dr. Richard Beeman of the Grain Marketing and Production Research Center, U.S. Department of Agriculture. Both of them are standard laboratory strains that have been used in genetic linkage mapping and other ecological and evolutionary genetics studies (e.g., WADE and GOODNIGHT 1991 ; GOODNIGHT and CRAIG 1996 ; YAN 1997 ; BEEMAN and BROWN 1999 ). TIW1 is less susceptible to tapeworm infection than is cSM, but there is considerable within-strain variability in beetle susceptibility to parasites. The two lab strains have been reared in the laboratory for >10 years, but they exhibited considerable within-strain variations in susceptibility to the tapeworm parasites. Therefore, we prescreened the two beetle strains for highly susceptible cSM individuals and highly resistant TIW1 individuals for pairwise mating to establish appropriate QTL mapping populations. Beetle maintenance and tapeworm infection followed the method of YAN and STEVENS 1995 . Briefly, fresh rat feces infected with H. diminuta eggs were obtained from Carolina Biological Supply Co. (Burlington, NC). A total of 100 adult virgin male and female beetles of each strain, 1-week postemergence, were exposed to 0.5 g of infected rat feces mixed in 0.3 ml distilled water on a 35-mm diameter filter paper per 20 beetles for 24 hr in a plastic petri dish. Fresh batches of infected rat feces were replaced for another 24 hr. The filter papers were removed, and beetles were transferred to vials with 5 g of flour medium and randomly paired. Eggs from each female were collected, and adult beetles were dissected. Offspring from three cSM pairs with the highest infection intensity (number of parasites in an infected individual) and three TIW1 pairs with no parasite infection were reared to adults, while the offspring from other pairs were discarded. We repeated this process for three generations and selected resistant TIW1 and susceptible cSM individuals for pairwise mating (see below). Throughout the experiments, all beetles were raised in 8-dram shell vials containing 5 g standard medium (95% by weight fine-sifted whole wheat flour and 5% dried powdered brewer's yeast). Experimental vials were placed in a dark incubator regulated at 29° and 70% relative humidity.

F₂ segregating populations and beetle susceptibility to tapeworm infection:
Two segregating populations were set up for QTL mapping. The first was generated from pairwise mating between a TIW1 male and a cSM female and F₁ intercross (hereafter referred to as cross 1). The second was from pairwise mating between a cSM male and a TIW1 female and F₁ intercross (hereafter referred to as cross 2). The parental TIW1 and cSM populations and F₁ and F₂ individuals from cross 1 and cross 2 were evaluated for tapeworm susceptibility using the method described above. All beetles in cross 1 (n = 123) and cross 2 (n = 120) groups were infected simultaneously; they were then dissected and the number of parasites was counted microscopically. Beetle carcasses were collected and used for the subsequent DNA analysis. Parasite tissues were discarded.

Molecular genotyping and linkage analysis:
Construction of a linkage map for T. castaneum using AFLP and RAPD markers was described in ZHONG et al. 2004 . Briefly, genomic DNA was prepared individually for parents and F₁ and F₂ individuals following the method of SEVERSON 1997 . The parents and F₁ individuals were used to establish the segregation pattern of the molecular markers. All beetles were genotyped using AFLP and RAPD markers. The AFLP genotyping method involved genomic DNA digestion with EcoRI and MseI. The DNA fragments were then ligated with EcoRI and MseI adaptors, generating template DNA for PCR amplification. Two primers used for PCR amplification were designed on the basis of the adaptor sequences and restriction site sequences. Selective nucleotide sequences were added to the 3' end of each primer. PCR amplification was conducted in two steps: preselective amplification and selective amplification. Preselective amplification used EcoRI primer (5'-GACTGCGTACCAATTC-3') and MseI primer (5'-GATGAGTCCTGAGTAA-3'). The EcoRI and MseI primers in the selective amplification used three additional nucleotides in the 3' end; therefore, each primer combination amplified different subsets of restriction fragments. To detect the PCR products with the Li-Cor automated DNA analyzer (Li-Cor, Lincoln, NE), the EcoRI primers were labeled with a fluorescent dye (infrared dye, IRD800). Polymorphism screening of AFLP products was conducted on the Li-Cor model 4200 automated DNA analyzer using a 6.5% polyacrylamide gel. The gel electrophoresis was maintained under a constant temperature of 45°. Allele sizes were determined using GENE IMAGIR computer software provided by the manufacturer (Li-Cor). RAPD markers were used to integrate the AFLP-based linkage maps with the RAPD-based linkage map of T. castaneum developed by BEEMAN and BROWN 1999 , following the protocol of BEEMAN and BROWN 1999 .

After screening 300 AFLP primer combinations and 50 RAPD primers, we selected 48 pairs of AFLP primer combinations and 12 RAPD primers for linkage mapping analysis on the basis of good reproducibility and the ability to obtain polymorphic fragments (Table 1). A linkage map was generated for each of the two independent segregating populations using MapMaker (version 3.0) computer software (LINCOLN et al. 1992 ). A composite map was developed using JoinMap computer software (VAN OOIJEN and VOORRIPS 2001 ). The composite map reported in ZHONG et al. 2004 includes 269 amplified AFLP markers and 10 previously mapped RAPD markers with a total length of 573 cM. The average physical distance per genetic distance was 350 kb/cM. This map was used for the subsequent QTL analysis.

QTL analysis:
QTL analyses were conducted separately for cross 1 and cross 2 to determine whether similar QTL may be identified in two crosses. Before the QTL analysis, the beetle susceptibility to tapeworm as measured by infection intensity was assessed for significant deviation from normality using the W-test (SHAPIRO and WILK 1965 ). When necessary, appropriate transformation was performed on the infection intensity trait (SAS INSTITUTE 1996). The computer software Mapmanager QTX (MANLY et al. 2001 ) was used to determine the QTL positions, the expected additive and dominance effects, and the phenotypic variance explained by individual QTL. The LOD threshold value for declaring the presence of a QTL was determined by permutation test (n = 1000; CHURCHILL and DOERGE 1994 ). Average levels of dominance were estimated using the ratio dominance/additive effects (i.e., h = d/a). The type of gene action for each QTL was determined on the basis of h: underdominance or recessive if h < 0, additive if h = 0–0.20, partial dominance if h = 0.21–0.80, dominance if h = 0.81–1.20, and overdominance if h > 1.20 (STUBER et al. 1987 ). Individual QTL designation has the following format: hds[n, y], where hds is the H. diminuta susceptibility, n is the linkage group number, and y is the AFLP marker closest to the QTL.

Detection of digenic epistasis:
The above analysis did not test the effect of epistasis on beetle infection intensity, but epistasis may be an especially important genetic basis for quantitative traits with low heritability (LI et al. 1997 ). Epistasis influencing the phenotype of interest may occur between the main QTL and other loci not linked with the main QTL. We tested the simplest case of epistasis-digenic epistasis using the following linear model in a two-way analysis of variance, a method that has been used in other studies (DEVICENTE and TANKSLEY 1993 ; GROOVER et al. 1994 ; XIAO et al. 1995 ; LI et al. 1997 ; SHOOK and JOHNSON 1999 ; HEO et al. 2001 ; PERIPATO et al. 2002 ),

(1)

where y_ijm is the phenotype of the mth F₂ individual with the digenic genotype at loci i and j, and _i and _j are the main effects, which include the additive and dominance effects associated with loci i and j, respectively. _ij represents the effects of interactions between alleles at loci i and j. _ijm is the random residual effects and is assumed to have a normal distribution and a mean of zero.

The Mapmanager QTX15 computer software (MANLY et al. 2001 ) was used to determine the effects of digenic epistatic QTL, following the method of LI et al. 2001 . Briefly, stepwise regression analyses were conducted to identify all markers significantly associated with beetle infection intensity across the genome with a threshold of P < 0.005. All putative main-effect and epistatic QTL were determined using the composite-interval mapping method. Each of the QTL included in the model were significant at a threshold of P < 0.002 and R² > 5% to minimize the probability of false positives (WANG et al. 1999 ). The effects and likelihood-ratio statistics were computed for significant main-effect and epistatic QTL at the positions of respective LOD peaks (WANG et al. 1999 ; LI et al. 2001 ).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Phenotypic variability in susceptibility to tapeworm parasites:
The tapeworm infection intensity distribution was examined in the two parental populations and in F₁ and F₂ populations that were exposed to the same amount of tapeworm egg-infected feces. Generally, strain cSM was significantly more susceptible than TIW1. The mean infection intensity and standard error in the parent cSM population were 6.2 ± 0.8 (n = 33) and 2.6 ± 0.7 (n = 17) for parental TIW1 population (ANOVA, F = 18.6, d.f. = 1, P < 0.0001). The individual beetles used for establishing the segregating population had been preselected for susceptibility to tapeworm: the cSM female had 9 parasites and the TIW1 male 0 parasites in cross 1 (cSM female x TIW1 male), and the cSM male had 2 parasites and the TIW1 female 0 parasites in cross 2 (cSM male x TIW1 female). The average infection intensity of F₁ populations was 4.7 ± 0.8 (n = 8) and 4.1 ± 0.7 (n = 10) for cross 1 and cross 2, respectively. The mean parasite number in F₂ populations was 3.7 ± 0.2 (n = 123) and 3.2 ± 0.2 (n = 120) for cross 1 and cross 2, respectively. The frequency distribution of infection intensity did not deviate significantly from normality (Fig 1). Therefore, infection intensity in F₂ was not transformed for the subsequent QTL analysis.

View larger version (35K): In this window In a new window Download PPT slide   Figure 1. Frequency distribution of number of tapeworms in two F2 segregating Tribolium castaneum populations derived from pairwise mating between TIW1 and cSM strains. Cross 1 represents an F2 segregating population from a cross between a TIW1 male and a cSM female, while cross 2 indicates a cross between a cSM male and a TIW1 female.

QTL analysis:
In the cross 1 group, we detected three QTL that significantly affect the susceptibility of the red flour beetle to tapeworm on linkage groups 3, 6, and 8, using the composite-interval mapping method (Fig 2). The LOD score plots for linkage groups with identified QTL provide a basis for identifying molecular markers most closely linked to the QTL (Fig 3). These three QTL are designated as hds[3, L1B1.69], hds[6, L1A16.141], and hds[8, L6B2.100]. Each QTL explained 8, 7, and 14% of the phenotypic variation in beetles' infection intensity (Table 2). The three QTL collectively explained 29% of the total phenotypic variation. The permutation tests indicated that all three QTL were statistically significant at the P < 0.01 level (Table 2). The three QTL had additive effects ranging from 0.62 to 1.17 parasites (Table 2). In particular, the QTL hds[6, L1A16.141] showed a positive additive regression coefficient (a = 0.62), suggesting that the susceptible strain cSM contributed alleles for increased infection intensity (Table 2). However, the negative dominance regression coefficient (d = -1.74) and negative ratio of additive regression coefficient to the dominance regression coefficient (h) indicate that the gene action at this QTL was primarily underdominance or recessive. In contrast, hds[3, L1B1.69] and hds[8, L6B2.100] exhibited negative additive and dominance regression coefficients and large positive h values, suggesting that the gene action at these two QTL was overdominance.

View larger version (40K): In this window In a new window Download PPT slide   Figure 2. AFLP-based linkage map of Tribolium castaneum and QTL locations for beetle susceptibility to tapeworm detected in two reciprocal F2 intercrosses. The numbers on the left side of each linkage group are genetic distances in Kosambi centimorgans. AFLP markers are designated by EcoRI primer name, MseI primer name, and molecular size of the fragment. For example, marker L1A1.200 represents the 200-bp fragment amplified by EcoRI primer L1 and MseI primer A1, whereas L2B1.97 represents the 97-bp fragment amplified by EcoRI primer L2 and MseI primer B1. RAPD marker designation has the form of A12.300, where the number to the right of the decimal is the size in base pairs, and the characters to the left of the decimal represent Operon Technology's 10-mer designation (BEEMAN and BROWN 1999 ). For example, X11.800 represents the 800-bp fragment amplified by primer OPX-11, whereas A1.1100 is the 1100-bp fragment amplified by primer OPX-A1.

View larger version (25K): In this window In a new window Download PPT slide   Figure 3. Composite-interval mapping of the susceptibility of the red flour beetle to the tapeworm. Cross 1 represents an F2 segregating population from a cross between a TIW1 male and a cSM female, while cross 2 is from a cross between a cSM male and a TIW1 female. Significance thresholds are indicated by dashed horizontal lines at LOD = 1.96 (genome-wide P < 0.05) and LOD = 3.82 (genome-wide P < 0.001), as determined by 1000 permutations of our mapping data (CHURCHILL and DOERGE 1994 ).

For cross 2, four QTL were identified (Fig 2), and they were designated as hds[1, L2A16.155], hds[3, L1B1.69], hds[6, L1A16.141], and hds[10, L3A18.82]. The four QTL were statistically significant at the level of P < 0.01 or less (Table 2). In addition to the two QTL detected in cross 1 (hds[3, L1B1.69] and hds[6, L1A16.141]), two new QTL (hds[1, L2A16.155] and hds[10, L3A18.82]) were found in cross 2 (Fig 3). The four QTL accounted for 8, 9, 38, and 7% of variance in infection intensity, respectively (Table 2). QTL hds[3, L1B1.69] and hds[6, L1A16.141] identified in cross 2 exhibited the same gene actions as in cross 1. QTL hds[1, L2A16.155] and hds[10, L3A18.82] had additive effects of 0.96 and 0.65 parasites, respectively (Table 2). The positive additive regression coefficients at these two QTL suggest that the susceptible cSM strain contributes alleles for increased infection intensity in the beetles. The gene action was underdominance or recessive at hds[1, L2A16.155] and hds[6, L1A16.141], but overdominance at hds[3, L1B1.69] and hds[10, L3A18.82], as evidenced by the large h values (Table 2).

Digenic epistasis:
Six digenic epistatic QTL in cross 1 and one in cross 2 were detected for beetle infection intensity (Table 3). All digenic epistatic QTL in the cross 1 group involved a marker on linkage group 3 (L1A16.131). One digenic epistasis involved a previously identified QTL marker, L6B2.100, and it accounted for 5.9% of the total phenotypic variation. The six digenic epistatic QTL in the cross 1 group accounted for a total of 39.0% of the phenotypic variance, but the one digenic epistatic QTL in the cross 2 group explained only 5.0% of the phenotypic variance.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

This study has demonstrated that susceptibility to tapeworm infection in red flour beetles is a polygenic trait. Using AFLP markers and the composite-interval mapping method, we determined that five chromosome regions significantly affected beetle susceptibility in two independent crosses. Two QTL on linkage groups 3 and 6 were identified in both crosses with similar effects on the phenotype. However, some unique QTL were identified in each cross, probably due to the fact that the beetle strains used for QTL mapping experiments were genetically heterogeneous and different QTL for beetle susceptibility to tapeworm parasites were present in the founding populations. The three QTL in one cross accounted for 29% of the total phenotypic variance while in another cross the four QTL accounted for 62% of the variance. In general, the effects of these QTL on infection intensity were small to medium, and three QTL exhibited overdominance effects and two were recessive or underdominant.

Because the main QTL accounted for a small proportion (29%) of the phenotypic variance in one cross, we extended the composite-interval mapping method to include digenic epistasis (WANG et al. 1999 ; LI et al. 2001 ). Epistasis, or interaction between loci, implies that the genotype at one locus has an effect on the expression of another locus. Although the role of epistasis in evolutionary and quantitative genetics has been of great interest, little is known about the importance of epistasis in the expression of quantitative traits due to the inherent difficulties of measuring epistatic genetic variance using classical quantitative genetics methods (CHEVERUD and ROUTMAN 1995 ). New analytical methods can provide a direct statistical estimation of epistatic interactions through whole-genome scan in segregating populations (LARK et al. 1995 ; YU et al. 1997 ). In this study, we detected six digenic epistatic QTL for infection intensity in one cross and one digenic epistatic QTL in another cross. The six digenic epistatic QTL in cross 1 accounted for a total of 39% of the phenotypic variance, while one digenic epistatic QTL in cross 2 explained only 5% of the total phenotypic variance. One reason that only one digenic QTL was detected in cross 2 may be that some epistatic loci in this cross were identified as main QTL due to allele fixation of these epistatic loci. Indeed, four main QTL were identified in cross 2, but only three main QTL in cross 1. The majority (six among seven digenic interactions) of the epistasis did not involve main QTL identified using composite-interval mapping. Thus, this result supports the hypothesis that the effect of epistasis on susceptibility to the tapeworm depends on the beetles' genetic background.

YAN and NORMAN 1995 examined variation in susceptibility to the tapeworm among 11 genetic strains of T. castaneum. The parasites generally exhibited aggregated distribution within a strain, but significant variation among genetic strains in infection intensity suggests that beetles' susceptibility to the parasite is genetically variable. Our data suggest two genetic mechanisms for the observed within- and among-strain variations in beetle susceptibility to the tapeworm infection. First, susceptibility to tapeworm infection is a polygenic trait, and polygenic traits are expected to show a large phenotypic variation (KLUG and CUMMINGS 1994 ). Second, beetle infection intensity is strongly affected by digenic epistasis. Epistasis constitutes a major source of genetic variance in infection intensity, but it does not contribute to the heritability of the trait. This result is consistent with our recent finding that infection intensity has a very low heritability (h² = 0.02; A. PAI and G. YAN, unpublished data). Therefore, the potential for the beetle population to respond to selection by the tapeworm is low.

Parasite susceptibility in the beetle hosts depends on various factors, including (1) number of tapeworm eggs ingested, (2) parasite egg hatchability within the beetle's gut, (3) the ability of young oncospheres to penetrate through the beetle gut, and (4) viability of oncospheres to develop into cysticercoids (YAN and NORMAN 1995 ). In the interaction of the Aedes aegypti mosquito and the filarial worm Brugia malayi, refractory mosquitoes ingested significantly fewer microfilariae than susceptible mosquitoes did and significantly fewer numbers of microfilariae penetrated through the refractory midgut as compared to the susceptible midgut (BEERNTSEN et al. 1995 ). It is unknown whether all of the above mechanisms are affected by the identified QTL in the Tribolium-Hymenolepis system. Discerning these factors requires further experimentation.

In summary, we have identified five QTL that affect beetle susceptibility to the tapeworm. These QTL exhibited small to medium effects on infection intensity, and the actions of these QTL were overdominance or underdominance. Digenic epistasis was a major source of variance for susceptibility, and its effects on susceptibility depended on the beetles' genetic background. Our results suggest that the polygenic nature of susceptibility to tapeworm and epistasis are important mechanisms for significant variation within or among beetle strains in infection intensity.

	ACKNOWLEDGMENTS

We thank Monica Karsay-Klein and Christina Costa for technical assistance. Two anonymous reviewers provided constructive criticisms and suggestions. This research was supported by a grant from National Science Foundation, DEB 0076106.

Manuscript received May 4, 2003; Accepted for publication July 25, 2003.

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