Heredity 61(1988) 379—387 The Genetical Society of Great Britain Received 21 December 1987

Genetics of carbohydrate metabolism and growth in Eisenia foetida (Oligochaeta: Lumbricidae)

Walter J. Diehi Department of Biological Sciences, Mississippi State University, P.O. Drawer GY, Mississippi State, MS 39762.

The heterozygosity of 13 polymorphic loci, encoding 10 from several pathways in carbohydrate metabolism, was tested for its effect on growth in juvenile manure worms Eisenia foetida (n =169),raised under stressful (limited food, low moisture) followed by non-stressful (abundant food, high moisture) conditions. The predictive value of heterozygosity on growth was greatly improved by treating each locus separately rather than summing heterozygosity across all loci (multilocus heterozygosity). Under non-stressful conditions, heterozygosity among loci treated separately had no effect on growth rate (F =1.60;df =13,155; NS); whereas under stressful conditions, heterozygosity among loci treated separately had significant and differential effects on growth rate (F =234;df =13,155; P <0.01) with 7 loci contributing to a positive heterozygosity-growth rate correlation (r =0235;P <0.005) and 6 loci contributing to a negative heterozygosity-growth rate correlation (r =—0•202,P <0.01). Implicated loci could not be easily grouped into specific metabolic pathways. The negative contribution by some loci may arise as a consequence of heterozygote inferiority, linkage disequilibria with positively-contributing loci, or strong selection at that locus.

INTRODUCTION tion more slowly than homozygous individuals. Diehl et aL (1986) showed that reduced metabolic Multilocusheterozygosity, measured cumulatively costs in heterozygous Mytilus edulis were associ- for a small number of electrophoretically detect- ated more with resistance to weight loss than with able loci, has been correlated with fitness-related capacity for weight gain. Thus the advantages con- parameters in a variety of experimental organisms ferred by heterozygosity appear to be of primary (see reviews by Mitton and Grant, 1984; Zouros benefit when the environment challenges the and Foltz, 1987). Briefly, multilocus heterozygosity energy balance of the individual. In M. edulis this is often positively correlated with age-specific may be manifest as improved survivorship of more growth, survivorship, and fecundity in large pan- heterozygous individuals during the energetically- mictic populations. The physiological basis for this stressful fall and winter months (Diehl and Koehn, phenomenon is postulated to be economy of stan- 1985). dard metabolism arising from efficiency of protein Until recently, the contributions of single loci synthesis and turnover (Koehn and Shumway, to the heterozygosity-growth phenomenon were 1982; Hawkins et a!., 1986). thought to be uniformly small, often undetectable, The effects of multilocus heterozygosity on a conclusion resulting from the numerous studies fitness-related parameters may be exaggerated, employing limited sample sizes of both individuals thus more easily visualized, under conditions of and polymorphic loci (e.g., Koehn and Gaffney, environmental stress. Koehn and Shumway (1982) 1984). It is now obvious from studies incorporating showed that metabolic costs, measured as weight- relatively large numbers of individuals and loci specific consumption, increased dispropor- that single-locus effects are, in fact, not uniform tionately in homozygous Crassostrea virginica (Bush et a!., 1987; Koehn et a!., 1988). Further, compared with heterozygous individuals when differential contributions by loci argue forcefully stressed by both high temperature and low salinity. for a causal role in determining growth rate (Bush Rodhouse and Gaffney (1984) showed that et a!., 1987), particularly if "important" loci can heterozygous C. virginica lost weight during starva- be grouped into specific metabolic pathways which 380 W. J. DIEHL

provide energy or raw materials for growth. Koehn prior to weighing, individuals were rinsed briefly et a!. (1988) implicated enzymes catalyzing re- in distilled water then blotted. Stressful conditions actions of protein catabolism, preglycolysis and (limited food, low soil moisture) consisted of 20 g glycolysis as contributing more to the multilocus dry culture medium containing autoclaved, com- heterozygosity-growth relationship in Mulinia posted cow manure: peat moss : CaCO3 ::20:4: 1, lateralis than enzymes involved in other pathways plus distilled water to bring moisture content to (e.g., pentose shunt, detoxification). Rates of 40 per cent; no additional food was provided. preglycolytic and glycolytic reactions may poten- Non-stressful conditions (abundant food, high soil tially impact a variety of energetic conditions of moisture) consisted of 20 g dry culture medium an individual including ATP levels and energy plus distilled water to 70 per cent moisture content; charge, redox balance, glycogen synthesis and commercially prepared earthworm food (Carolina utilization. Biological Supply) was provided ad libitum. These The present experiment was undertaken to conditions had been shown to be stressful and determine the contribution of heterozygosity non-stressful, respectively, in pilot experiments as among loci encoding enzymes of carbohydrate determined by differences in individual growth. metabolism to growth in the manure worm Eisenia This experimental design is analogous to those foetida(Oligochaeta: Lumbricidae) cultured studies in which growth is inferred or measured under stressful vs. non-stressful conditions. To the of individuals collected in the field and then re- extent examined, lumbricid earthworms possess a measured of the same individuals under specific full complement of glycolytic, pentose shunt and conditions in the laboratory (e.g., Koehn et a!., citric acid cycle enzymes (Dastolli, 1964). Thus 1988). far, polymorphisms have been identified for only Moisture content was monitored daily with an a few of the loci encoding these enzymes in E. Instamatic® moisture meter; distilled water was foetida (Jaenike, 1982; Robotti, 1982). In the cur- added to bring moisture content to the desired rent experiment however, 13 polymorphic loci en- level after overnight evaporation. Soil pH, coding ten enzymes are identified from a variety monitored daily with a Brookstone® soilpH meter, of relevant pathways including preglycolysis, remained at 7 for the duration of the experiment. glycolysis, pentose shunt, citric acid cycle, redox Ambient temperature was 23—25°C. Only aclitellate balance, and glycerol synthesis. Thus in the study juveniles were used in the experiment; individuals of the heterozygosity-growth phenomenon, this which attained sexual maturity during the course experiment represents the first attempt to evaluate of the experiment, determined by the development loci encoding enzymes from a unified set of inter- of a clitellum, were not considered further. During related metabolic pathways. the experiment, mortality was less than 2 per cent; the final sample size was 169 individuals. The genotypes of 13 polymorphic loci (ten METHODSAND MATERIALS enzymes) in carbohydrate metabolism were resol- ved by horizontal starch gel electrophoresis Approximately750 E.foetida were purchased from according to Koehn et a!. (1976). Approximately a commercial bait farm in West Point, Mississippi, 100 mg of the posterior region of each earthworm U.S.A. Individuals were taken from concrete was homogenized with 100 l grinding buffer bunkers filled with damp peat moss (surface area = (0.05 M Tris/HCI, 20 per cent glycerol, 02 per 22m2; depth =04m); each bunker was estimated cent /3-mercaptoethanol) using a motorized coni- to contain 40,000 large, adult individuals plus cal teflon tissue homogenizer in a 15 ml microcen- numerous but uncounted small adults and trifuge tube. Homogenates were centrifuged at juveniles. These earthworms were originally 1O,000g for 20 mm in an Eppendorf model 5415 derived from a mixture of commercial stocks in microcentrifuge; the supernatant was used as the Mississippi and Texas and have been maintained source. The following enzymes (with E.C. locally for six years. number; locus abbreviation; metabolic function) In the laboratory, juveniles were separated were resolved: hexokinase (EC 2.7.1.1; Hkl, Hk2; from adults and were raised singly in 200 ml plastic glycolysis), mannose-6-phosphateisomerase cups for one week under stressful conditions fol- (EC 5.3.1.8; Mpi; preglycolysis), L-iditol dehy- lowed by one week under non-stressful conditions drogenase (EC 1.1.1.14; Iddh 1; preglycolysis), (n =218).The specific ages of these individuals glucose dehydrogenase (EC 1.1.1.47; Glcdh; pen- were unknown. Whole-animal fresh weights were tose shunt), phosphoglucomutase (EC 5.4.2.2; measured before and after each growth period; Pgm; preglycolysis), glycerol-3-phosphate dehy- GENETICS OF CARBOHYDRATE METABOLISM IN EISENIA 381 drogenase (EC 1.1.1.8; Gpdhl, Gpdh2; glycerol RESULTS metabolism), phosphogluconate dehydrogenase (EC 1.1.1.44; Pgdh; pentose shunt), L-lactate dehy- InE. foetida, the average heterozygosity among 13 drogenase (EC 1.1.1.27; Ldhl, Ldh2; redox bal- polymorphic loci in carbohydrate metabolism was ance), strombine dehydrogenase (EC 1.5.1.-; Stdh; 0397. Of these loci, Iddhl, Ldhl, Ldh2, Stdh redox balance), malate dehydrogenase showed significant deviations from Hardy-Wein- (EC 1.1.1.37; Md/i; citric acid cycle). berg equilibrium (table 1). In the case of Iddhl, Electromorphs of various loci were separated Ldhl, and Ldh2, this deviation represented a using buffer systems described by Selander and heterozygote deficiency; in the case of Stdh, this Yang (1969). Hkl, Hk2, Mdh, Gpdhl, Gpdh2, and represented a heterozygote excess. The overall Pgm were resolved on a Tris-borate-EDTA (TBE) heterozygote deficiency (D ={Ho-He]/He;Selan- buffer system; Pgdh, Mpi, Ldhl, and Ldh2 were der, 1970) was only —0046, reflecting a general resolved on a Tris-maleate (TM) buffer system; lack of inbreeding. Linkage disequilibrium was Stdh, Glcdh, and Iddh 1 were resolved on a dis- tested among all possible pairs of loci by Pearson's continuous Tris-citrate (Poulik) buffer system. x2testfor independent association among The appropriate (NADP or NAD) was genotypes. Di-locus genotypes represented by added to the TBE and TM gels prior to degassing fewer than five individuals were pooled into a (final concentration: 02 mM) to improve resolu- single category. Of the 78 possible combinations, tion of the dehydrogenases. Activities of HK, only five pairs of loci showed significant deviations IDDH, LDH, MDH, PGM, PGDH were detected from independent association (table 2). Of course according to Shaw and Prasad (1970); activities at a =005,approximately 4 significant deviations of GPDH, MPI, G1cDH were detected accord- from independent association are expected on the ing to Harris and Hopkinson (1976); activity of basis of chance alone; thus the extent of true StDH was detected according to Dando et a!. linkage disequilibrium may be quite small. (1981). Nevertheless, three possible linkage groups were The data were analyzed on an IBMPC® with identified: Hkl -Pgdh-Iddh 1, Ldh 1 -Ldh2-Stdh, a 30 megabyte Flashcard®. Statistical programs and Gpdh 1 -Mdh. included the PROC FREQ and PROC GLM pro- Added weight was a positive function of cedures from the Statistical Analysis System soft- individual weight prior to a growth period under ware for personal computers (SAS Institute, Inc., both stressful and non-stressful conditions; Cary, NC). Other statistics were from Sokal and however, the growth trajectories differed sig- Rohlf (1981). nificantly between experimental regimes (F = The effect of multilocus heterozygosity on 4F90;df= 1, 334; P<0001; fig. 1). As expected, growth rate was tested according to two models. weight added under non-stressful conditions was Model A, consistent with traditional approaches significantly greater than weight added under to this phenomenon (Zouros et a!., 1980; Koehn stressful conditions (paired t=3188; df= 168; and Gaffney, 1984), assumed that the effect of individual loci on growth rate was equal and addi- tive among loci. For each locus, individuals were 0) scored 0 if homozygous or 1 if heterozygous. Multi- E locus heterozygosity was calculated as the sum of 0 0 I— 0 0 0 I o 0 these scores over n loci; effect of multilocus 0O 000O heterozygosity was then evaluated by simple linear w 00 regression or -moment correlation. Model :°'8 0 0 0 0 B allowed the effects of individual loci to be 0 0 0LIJ unequal and non-additive (Bush et aL,. 1987; 0 we..1••#• S. S Koehn et a!., 1988). Individuals were scored as 4 . above but the effects of heterozygosity among n .5 ._ loci on growth were evaluated by multiple 200 300 400 regression in which loci occurred as independent INITIALWEIGHT (mg) variables. Each model was evaluated for predicting Figure 1 Correlation between added weight and initial weight the effects of heterozygosity on growth measured for both stressful (closed circles; r—0592; df= 167; P< 0.001) and non-stressful (open circles; r=0699;df=167; as initial weight, as weight added under stressful P <0.001) growth conditions. Initial weight is defined as conditions, and as weight added under non- the weight of an individual prior to the respective growth stressful conditions. period. 382 W. J. DIEHL

Table 1Allele frequencies, observed heterozygosity (Ho), heterozygote-deficiency (D) for the polymorphic loci of carbohydrate metabolism in Eiseniafoetida. Goodnessoffit to Hardy- Weinberg proportions was tested by x2; genotypes represented by less than five individuals were pooled into an adjacent category (Hedrick, 1985). n =169for each locus

Locus allele freq Ho D x2(df) P

Hkl 110 0240 0698 0067 2986 (3) ns 105 0370 100 0391 Hk2 110 0050 0•089 —0066 nt 100 0950 Mpi 110 0361 0461 00004 0001 (1) ns 100 0639 Iddhl 105 0391 0396 —0398 59048 (4) <0001 100 0414 95 0098 90 0098 Glcdh 110 0192 0562 —0085 5i86(3) ns 100 0509 90 0299 Pgm 110 0281 0420 O•040 0.018 (1) ns 100 0719 Gpdhl 100 0917 0166 0088 nt 90 0083 Gpdh2 110 0459 0462 —0071 1.149 (1) ns 100 0541 Pgdh 110 0240 0396 0087 1305(1) ns 100 0760

Ldh 1 110 0275 0290 —0273 12.606 (1) <0001 100 0725 Ldh2 110 0062 0320 —0185 6781 (2) <0.05 100 0754 90 0183 Sidh 100 0784 0402 0188 4921 (1) <005 90 0216 Mdh 100 0518 0503 0007 0.161 (1) ns 90 0482 nt =Hardy-Weinbergnot tested due to insufficient sample size. ns =notsignificant.

P<0001; table 3); yet the coefficient of variation Table 2Pairs of loci whose genotypes are not associated independently, tested by the Pearson x2 (a 0.05). Di- for added weight was greater under the latter con- locus genotypes represented by less than five individuals dition. were pooled into a single category Contrary to the results of previous studies, multilocus heterozygosity (heterozygosity summed Di-locus pair x2 df P across loci; model A) was not significantly corre- Hkl,Pgdh 39594 10 <0001 lated with initial weight (r= 0045;df= 167; NS); Pgdh,Iddhl 24265 11 <005 with weight added under stressful conditions (r

Ldhl, Ldh2 17247 9 <005 OO5O; df= 167; NS) or with weight added under Ldh2,Stdh 26410 4 <0001 non-stressful conditions (r =—0058;df= 167; NS; table 4). A significant correlation was detected only Gpdhl,Mdh 9823 2 <001 between heterozygosity among loci treated separ- GENETICS OF CARBOHYDRATE METABOLISM IN EISENIA 383

Table 3 Effect of supplemental food and moisture content on Table 5 Rank of single loci contributing to the relationship added fresh weight (mg) in Eisenia foetida. Where between added weight and heterozygosity among loci for appropriate, data are presented as means (standard error). Eisenia foetida under stressful conditions (limited food, n =169 low moisture). Effects of initial weight have been taken into account by multiple regression Week 1 Week 2 (stressful) (non-stressful) Partial regression F Supplemental food none ad libitum Locus SS coefficient r2 (1, 154) P Soil moisture content 3O-40% 60-70% Added fresh weight 276 (1.13) 1054 (2.86) Gpdh2 000081 00046 0-025 6327 <0O5 Coefficient of variation 534 (364) 353 (214) Hk2 000060 00070 0019 4738 <0-05 Ldhl 000030 00031 0009 2328 ns Pgm 000019 00023 0006 1459 ns Mdh 000016 00021 0-005 1285 ns Ldh2 0-00006 00014 0002 0459 ns Glcdh 000002 00006 0•001 0120 ns Table 4 Coefficients of determination (r2 or R2) between two 0869 ns models predicting the effects of heterozygosity on 3 Hkl 000011—00018 0003 Gpdhl 0.00015 —0-0026 0.005 1207 ns measures of growth. Model A assumes individual loci —00025 0008 1888 ns contribute similarly to growth rate and that effects are Mpi 0-00024 —00027 0009 2190 ns additive; model B assumes individual loci contribute Pgdh 000028 Stdh 000037 —0-0034 0012 2919 ns differentially and that effects are not additive. Significance Iddhl 000057—00040 0018 4492 <0-05 tested by ANOVA; model A, linear regression, df= 1, 167; model B, multiple regression, df= 13, 155 Dependent variable Model A Model B coefficients. Thus sampling smaller subsets of poly- morphic loci, as is typical of most studies, may Initial weight 0002 0082 F=0342,ns F=1071,ns reveal starkly different results depending on the Weight added 0002 0164 loci chosen. For example, if loci which showed (stressful env.) F=0416, ns F=2344, P<0-01 only a positive effect were arbitrarily sampled, the Weight added 0003 0118 equal and additive model would not be refuted F = F = ns (non-stressful env.) 0-350,ns 1600, and multilocus heterozygosity would explain about five per cent of the variation in growth (fig. 2). Interestingly, if negatively-contributing loci were ately (model B) and weight added under stressful arbitrarily sampled, multilocus homozygosity conditions (R =O4O5;F =2344;df= 13, 155; P < would explain about four per cent of the variation OO1; table 4). Indeed heterozygosity among loci in growth. In contrast to the study of Koehn et al treated separately explained more than 15 per cent (1988), it is not possible to associate single-locus of the variation in added weight under these condi- contributions to growth in E. foetida with obvious tions but showed no significant effect on either metabolic functions in carbohydrate metabolism. initial weight (R =O287;F =1O71; df= 13, 155; This is particularly evident for enzymes such as NS) or weight added under non-stressful condi- HK and GPDH both of which are encoded by two tions (R=O344; F=1600; df=13, 155; NS). loci: one contributing positively, the other con- Clearly these data support a model which assumes tributing negatively. differential, possibly non-additive effects among The existence of both positively and negatively loci. Moreover, this genetic effect is better visual- contributing loci suggests that the effects of ized under conditions of environmental stress. heterozygosity may not always be additive among The contribution of heterozygosity at single loci loci, possibly due to interactions among loci: phy- toward explaining variation in growth under stress- sical (linkage), functional (epistasis) or both. ful conditions was evaluated by ranking the partial Differences in growth among genotypes Df single sums of squares from a multiple regression while loci were detected only for Gpdh2 (F =477;df= considering the sign of the partial regression 3, 165; P

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