- Email: [email protected]
Acetylesterase pattern in the earthworm genus Eisenia (Oligochaeta, Lumbricidae): Implications for laboratory use and taxonomic status
Soil Biol.Biockm. Vol. 23. No. 3. pp. 243247. Prinwd in Great Britam.All nghtr reserved
0038-0717:91 53.00 + 0.00
1991
copyright % 1991PergamonPma plc
ACETYLESTERASE PATTERN IN THE EARTHWORM GENUS EISEZVIA (OLIGOCHAETA, LUMBRICIDAE): IMPLICATIONS FOR LABORATORY USE AND TAXONOMIC STATUS FREDDYENGELSTAD’and JQRGEN STENERSEN Biological Institute. Division of Molecular Cell Biology. University of Oslo. P.O. Box 1050. Blindem, N-0316 Oslo 3. Norway (Accepted 25 September 1990) Summary-Polyacrylamide gel electrophoresis has been used to describe acetylesterases (EC 3.1.1.6) in the species Ei.renia andrei, E. fitida and &. renelo. Four bands were common in E. undrei and E. /elido. whereas the three bands found in E. cenefo were unique for this species. Two of the electromotphs described are gut enzymes. of which one is unique to E. firida. Analysis for this enzyme proved to be a reliable method to distinguish the two species E. undrei and E.fitida. The frequencies of the acetylesterase electromorph were very different in two populations of E. venetu. which reflects great variability of acetylesterase alleles. Indications of E. firida and E. rjenefa hybridization were found. Short-term starvation had little influence on the reliability of the method.
INTWODUCl’ION
earthworms as a test organism. to have a proper way of verifying as to what spccics an individual belongs. Elcctrophorcsis is a tcchniquc that dcmonstratcs gcncs or gcnc products and in this way a suitable tool to prove individual-, population- and spccics diffcrcnccs. In this work. clcctrophorcsis was used to verify spccics with absolute certainty, to describe the pattern of acetylesterascs (EC 3. I. I .6) and to evaluate the reliability of this method.
Earthworms in the genus Eiseniu arc’ widcsprcad and well known both as laboratory test organisms (OECD. 1984) and for compost production. In the
litcraturc
the grcatcst emphasis is laid on
“Eismicr
fiwridu”. but as this has proved to bc the two spccics Eiseniu undrei and E./etidu (Jacnickc. 1982; 0icn and
Stcncrscn. 1984). doubt may be raised as to which of the species arc dcscribcd. In a study of the .growth and reproductive ditTcrenccs bctwcen the species E. feridu and E. undrri it was ncccssary to establish pure culture of the two spccics. External taxonomic characters arc similar for the two species, but the pigmentation is ditfcrcnt: E. undrei is more uniformly reddish in contrast to E. firidu, which has wider yellow intcrsegmcntal furrows that give a crossstriped pattern. This has been known since Andre (1963) described the two colour variants as subspecies. This small difference in coloration is not sufficient for a sure distinction between the two species. which is necessary when pure cultures are to be cstablishcd. Eismiu cenetu (Rosa) is another spccics used in waste conversion cxpcrimcnts (LofsHolmin. 1985, 1986). This was earlier classified as a Dendrobuenu (Easton, 1983). but is today rcgardcd as an Eiseniu spccics (Sims and Gerard, 1985). Two colour variants have been dcscribcd. Dendrobuenu I’L’IWU typicu and D. ~‘etw~u :ehru (Gerard. 1964). They arc considcrcd to bc phcnotypic variants and taxonomically indistinguishable. The colour pattern of the E. wnetu zehru variant is very similar to E. fetida. but the position of the sctac is difTcrcnt so there arc no questions of the taxonomic status rclativc to E.ferirfu. To the incxpcricnccd this diffcrcnce is not evident. It is important to pcoplc working with
MATEWIAIS AND METIIODS
Eurhvorms
Specimens of Eiseniu undrei were obtained from a commercial earthworm grower in Norway, and wild type specimens of E. undrei were collcctcd from two sites in the vicinity of Oslo. E. fetidu were collected at the goat barn of the Agricultural University of Norway, As. The source of E. uenetu was a commercial Swedish stock. which here will be designated pop I. The origin of this population is not known. Wild type specimens of E. venetu were obtained from Dr K. Sylvia Richards at the University of Keelc, England (pop 2). All populations were kept in culture in the laboratory. A mixture of horse manure and commercially available “standard soil” consisting of 84% peat, 10% sand and 6% clay was used as medium. Electrophoresis
For the clcctrophorcsis analysis the Protan II slab ccl1 from Bio-Rad. California. and Pharmacia model EPS 500/400 power supply were used. The following were the sources of the major reagents used: acrylamide. bisacrylamidc, ammonium pcrsulfatc and TEMED from Bio-Rad. Richmond, U.S.A.; naphtylacetat, naphtylbutyrate, Fast Blue B Salt, Tris base and glycinc from Sigma, St Louis, U.S.A. Dicthyl 4-nitrophcnyl phosphate (paraoxon)
*Present address: Centre for Soil and Environmental Reseach. P.O. BOX 9. N-1432 AS-NLH.
Norway. 243
244
FREDDY ESGEL.YTADand JCJRGEN SIWERSEN
was obtained from Bayer AG. Leverkusen. Germany. AI1 reagents were of analytical grade. Worms were washed in tap water, placed on a cleaning paper and weighed. They were homogenized in Tris-glycine buffer, 5 rnr+i of Tris, pH = 8.5 with I.2 M glycerol (1 + 3 w/v) on ice. The samples were centrifuged for 5 min at 3500g and the supematants were then frozen at -2O’C. Specimens for dissection were frozen in liquid N2, and the partition was carried out while thawing. The tissue samples were washed in electrode buffer. Samples of 20~1 were applied after addition of bromophenolblue (0.01%). Electrophoresis was carried out according to Laemmli (1970). Voltage was set to 300 V and the gels were cooled with tap water until the marker (bromphenol blue) reached the lower end of the gel. After electrophoresis the gels were placed in a 4 x 10-r M paraoxon solution made up in 50 mM Tris-HCI buffer (pH = 8) in order to inhibit the carboxyland cholincsterases. The gels were then transferred to freshly-made staining solution: which consisted of Tris-HCI (50 mM. pH = 8). /Inaphtylacctatc (IO mg 100 ml-’ buffer) dissolved in I ml acctonc and Fast Blue B salt (50 mg 100 ml-’ buffer). Bands dcvclopcd within 15 min and the gels wcrc then washed in tap water and placed in 7% acetic acid solution.
Stenersen (1984). Four bands (E4, E5, E6 and E7) were indistinguishable in E. andrei and E. fetida, whereas one band was present in E. fetido only (E3) (Fig. 1). Three bands were present in E. ceneta (EVl, EV2 and EV3) which were unique to this species (Fig. 1). Figure 2 shows a composite picture of authentic gels, demonstrating frequently observed phenotypes. The electromorph frequencies of the three species are shown in Table I. Noteworthy from the result is that the electromorph frequencies in E. undrei and E. fetidu is usually close to one. and when the bands are missing no other electromorph appears. The electromorph frequencies in E. reneta differ greatly between the two populations (Table I). Of the 43 individuals tested from pop 1, electromorph EV3 was the most common with a frequency of 0.884, EVI had a frequency of 0.658 and the intermediate EV2 was present with a frequency of only 0.211. From pop 2 the EVI was the most common, and appeared with a frequency of 0.821. EV2 had a frequency of 0.679 and EV3 only 0.286 0ien and Stenersen (1984) found that the acetylesterases were mainly present in the anterior part of the animals. This was further investigated by analyzing tissue from frozen and dissected individuals in the following categories: facces. lumen. gut cpithelian tissue, coelomic fluid and muscle tissue. Samples from thcsc catcgorics. cxccpt for facccs. wcrc taken from the middlo section of the animals. The results HKSl!I.TS from this investigation (Table 2) arc inconsistent concerning cxprcssion or activity of the enzymes. It was possible to distinguish bctwccn eight bands or elcctromorphs. The bands arc dcsignatcd accordIt indicates ncvcrthclcss that the E3 and E4 ing to Fig. I. Five bands were prcscnt in E. f~~~iclu bands arc conncctcd to the gut. whcrcas the other bands originate from enzymes found in other and four in E. andrei. DitTercnccs in elcctromorphs tissues. The E3 and E4 bands were present only bctwccn the populations of E. mulrcv’ were not dctected. The mobility of electromorph E4 is set to bc occasionally in faeccs, but when prcscnt they wcrc highly active. I to fncilimtc comparison with the work of 0icn and ELECIXOMORH I
E.
EV3
EV2
EVI
E7
+
E6
-
ES
-
+
E3
Fig.
I.
Pullcrn
ol acctylestcrascs
from E.
onhi.
&. /(*Mu
and E. rm~ra.
Ei.ienia acetylesterase pattern
1
2
3
4
5
7
6
9
9
10
11
Fig. 2. Composite picture of acetylcsterase pattern in the Ek-niu species. E. a&rui (lane I, (lane 3, 4. I?); E. L’PIICIU(lane 5. 6. 8. IO, II). Lane 7 is overloaded.
To reveal a possible relation bctwccn the nutritional status of the worms and the presence of the E3 and E4 clcctromorphs a new experiment was carried out. Specimens of E. ancfrreiand E.fefidu (IO of each), were placed individual,ly in horse manure to gain weight without competnron. After 4 wk five of each species were starved for 4 days by keeping them on moistened filter paper in boxes. Starved and not starved animals were homogenized and analyzed by elcctrophorcsis. The E4 band was present in all animals indcpcndcntly of trsatmcnt. The E3 band was also prcscnt in all E. ftUtc individuals. Thcrc was however. a reduction in the intensity of the E4 band in E. crrrc/rtGindividuals who had hccn dcprivod of food.
The cnzymcs WC have been working with have the characteristics of acctylesterasr (EC 3. I. I .h) (e.g. Barman, 1969) as shown by 0icn and Stcnerscn (19X4), hut the pattern of electromorphs found in this investigation is not identical with that described earlier whcrc five acctylesterase bands in E. fetidu and E. cmhi wcrc dcscribcd. However, no individuals has more than two clearly distinguishable bands. WC used an electrophoretie method with bcttcr separating ability and found up to five bands present in individuals of E. fc4rlcl and four in E. utrclrei (Fig. 2). Thcrcforc a direct comparison bctwccn the present and curl& results is difficult. Howcvcr. WC think that the isozymcs forming EL2, EL4 and EL5 (Oicn and Stcncrsen. 198-I) wcrc further resolved in our
Relatitc mohdrt) Elcc~romorph
(1)
--....
--.--
E. cmcbci
M.lS
Xlm
2.7,~); E.fpti&
investigation. E3, E4 and E6 compare best to those originally described (EL2, EL4 and ELS). ELI and EL3 were present at very low frequencies in the work of 0ien and Stenersen (1984) and no bands in this investigation corresponded to them. Small sample size may be the cause of this. No alternative electromorph appears when one of the E3-E6 bands was missing. The reason may be the lack of not-functional all& products, or because the amount of enzyme synthcsizcd was too low. Dcnaturation of only one or two of the clcctromorphs is less likely as the acctylestcrascs have good stability. In E. IYN~I(I the three clcctromorphs found arc assumed to bc allele products from one locus. The frequencies of the clcctromorphs (Table I), and their corresponding alleles (Table 3). were quite different in the two populations tested. T’hc all& coding for the EVI band is designated p* the EV2 = y and EV3 = r, In both populations all combinations except homozygotcr for the y allclc wcrc present (Table 4). The y allele appeared with a frequency of 0.093 in pop 1 and 0.339 in pop 2 even though no homozygotcs were found. The population difference was significant (P < 0.05) as tested by a x2 test (xr = 6.9 with 2 d.f.). A similar test on the actual numbers of each allele combination also gave a significant difference (P < 0.01) between the two populations (x’= 32.4 with 4 d.f.). Based on the calculated allele frequencies the expected number of individuals in the categories are estimated in the two populations according to the Hardy-Weinberg theorem (Table 4). Thcrc are dcviations from the observed numbers but the difrerence
Frcqucnclcs -.-__-__-
R;Il-lgC
” = 60
of occurrc”ce .___. E. ff
Iida
n = 3’)
E. L’ww,t, --.__-I-_
P’P 1
pop2
” n-t?
n 5 3
WI
0792
0.X07
0
0
0.XX-l
EV’
0.X34
0.x51-0.X22
0
0
0.21
EV;
0 XYI
0.907-O.XX?
0
0
0.6%
0321
E7
0 923
0.935
O.Yn.8
I
I
0
0
O.VJ?
I
0.93
0
0
0.957
0.X46
0
0
0.719
E6
0.951
O.VhV
ES
0.977
0.9R5-0.96X
EJ
1.000
E3
I.0Z.r
1.035-1.017
12
0.2X6
I
0.67Y
o.xvv
0.7ia
0
0
0
0.974
0
0
&I’ EJ. E5 ix E3, EC?.El ND ND ‘HI& aclrvily when present. but okn ‘Low activity.
E3’. E4: E3. EJ. ES‘ ES. E6. E? E9, E6’. LF ES. E6. E7 missing.
Table 3. Frequencies of alleles in the two 6 rmwta populations. Actuaf number in pmtheses Pop 1. Sweden Pop. 2. England (3 zz NJ t2n = 561
p (EVI) q (Eva I (EV3)
0.337 (?I) 0.093 (8) 0.570 (49)
0.500 CX) 0.339(19! O.lfil (9)
is not significant as tested by the ,$ method. For pop I xzr3.42 with 2 d.f., 0. I < P < 0.25, and for pop 2 )[?= 5.22 with 2 d.f. 0.05 < P < 0. I. Due to patchincss of their natural habitat and anthropogcnic ciispersai it would have been most rcmarkahfc if gcn frcqucnoics wcrc cyuaf in so distant po~ufiitions. Culturing ~l)r~~~it~~lns in the Swedish popufittion may ~~11 have cittlscd I scltaion prcssurc ditfia-iny from wild populations resulting in diffcrcnt aflclc frcqucncics. The fact that no homozygous indiviciuols for ths q-allclc wcrc found in cithsr of the populations indicates that this may Icad to rcduccd viability. f fybri~li~~ti~3n htwccn E. rrfrcfm+ and E. frIi&r is &scribed in the work uf Oicn and Stenerssn (19X4), bum at very low frsqucnsy in wild populations They hybridize casicr in cultured populations, but produce &spring at lower nrtc. Some individuals in the Swedish population (pop I) had bands that corresponded both to E jid& and E. twm (Fig. 3). According to Mufdal (1952) and Qmodeo (1952) there ari: morphological diflcrcncos in the nucleus of the species and the chromosome numbers arc 22 in E. fc*tic/tr and 36 in E. twtvtt. Viablr hybrids bctwosn thcsc two species arc thcrcforc improbable. Caution was taken to avoid cross-contamination of samples during this work and breeding expcrimtnts are ncccsairy to find out if hybridi~~~tion may indeed occur. The staining is depondcnt on cnzymc activity and consequently the band intensity varied markedly. Thcrc was always high activity in the samples from lurncn. whcrcas the bands from the gut cpithcli:d tissues gave less dcvelopod bands. fn f~scs h:mds detelopcd onfy five times of the IX samples tested. This indicates that the E3 and W bonds represent digestive c’storascs synthcsizcd in the cpithelia lining of the put wall behind the giaard and sccrctcd into
1
2
Fig. 3. Picture of an authentic gel showing nn individual with both E. _krirla and E. r~wc~ubands. Lane I: possible hybrid E. wnen~la x E. f&i&; Lane 2: E. wnctt~. the lumen. The enzytnrs arc’ gr;tduitfly denatured during the passage through the gut. Thus, the samples from the LICCCS somctimcs arc active and sometimes not. The prcscncc of the cfcctromorphs E5, E6 and E7 in the coclomic fluid and the muscfc Inyor in E. fvrirkt indicates tfW lhcir onsymc products arc normal ~~~ipori~~~t~ of cells. A more llnifornl distr~butioll within the organism is thcrcforc expcctcd. The result of the starvation cxpcrimcnt shows that clcctrophorcsis is a rcliahle method cvcn if animals have been starved or have lived ou scanty food for some cfays. The gut transit time is found to be 7 h at 15°C and 2.5 h at 25 C in E. _/hi& (ii~rt~nst~in et d.. 1981) and after 4 days the synthesis of digestive cstcrasc enzymes still conlinucs. However. animals with another physiologial status. cxamplificd by an individual in a quiescent state due to drought or cold, may have ceased to synthcsizr: this enzyme. For laboratory use this is LI minor problem. but if animals are taken from the ftctd they should have proper time to reach an Ltctivc st;ttc b&rc analysis of CStCraSCS. In laboratories whcrc arthworms arc used as test organisms, it is possible to anAyzc the cstcr;Lscs in the posterior part of the animal by cutting this off. This will rcgcncratc and the inrlividu;tls sre fully capable oT p~op~~~tin~. The rcfkbility of the CSICTISC pattern tfrscribed here suggests that USC of the cfcctrophorctic method is a pr;lctical way to establish pure cultures or to verify to whicfl spcciss an individual belongs.
Eirenia acetyiesterase pattern Acknowledgements-This work b ken 6nancially supported from The Norwegian Agricultural Research Council. We thank Dr K. Sylvia Richards, University of Kccic. for providing specimens of E. vencfa.
REFERENCES
And+ F. (1963) Contribution a I’analyr exp&imentaie de la reproduction dcs iombricicns. Bulletin Biologique dc la Frame et dc la fkfgique 97, 3401. Barman T. E. (1969) Enzyme Handbook, Vol. 11. Springer, Berlin. ___..~.. Easton E. G. (1983) A guide to the valid names of Lumbricidae (Oiiaochaeta). Jn Earthworm Ecoloav. From Darwin ro Ver&.&ure (J.’ E. SatchelI. Ed.), pp.475-485. Chapman & Hall, London. Gerard 9. M. (1964) Lumbricidae (Ann&da) with keys and descriptions. Synopses ofthe British Fauna, 2nd Edn. Vol. 6. The Linnean S&i& of London. Hartenstein F.. Hartcnsttin E. and Hartenstein R. 11981) Gut load and transit time in the earthworm &.se& foerida. PedobiOrogia22, S-20. Jaenickc J. (t 982) “Eisenia fwtida” is two biological species. Megadrilogica 4, 6-8.
247
Lacmmb U. K. (1970) Cicavagc of structural proteins during the assembly of the head of bacteriophap T4. Narure 227,680-685. Lofs-Holmin A. (1985) Present knowledge of earthworm farming-a summary of rcceut literature. Swedish University of Agricuiturai Sciences, Dcpt of Ecoiogy and En~~o~~~ Reseat& Report No. 20. -Lofs-Hoimin A. (1986) Processina of municioal dudan through carthwdm (Dendrobae& uenera). Siedt3h J&r~1 of Agricultural Research 16,67-l I. Muidal S. (1952) The chromosomes of the carthwotms 1. The evolution of poiypioidy. Heredity 6, U-76. OECD (1984) OECD Gui&lines for Tes!ing of Chemicafs. ~gani~tion for Economic Co-operation and Dcvciopment, Paris. Omodeo P. (1952) Cariologia dei Lumbricidae. Curyofogiu 4, 173-275. 0ien N. and Stcnerscn J. (1984) Estetascs of carthworms111.Elcctrophornis reveals that ELseniajerida (Savigny) is two species. Comparative Biockemissry and Physiology 78C, 277-282. Sims R. W. and Gerard 9. M. (1985) Earthworms. Synopses of tke British Fauna (New Series) (D. M. Kenack and R. S. K. Barnes. Eds). E. J. Briil & Dr W. Brackhuys. London.
0038-0717:91 53.00 + 0.00
1991
copyright % 1991PergamonPma plc
ACETYLESTERASE PATTERN IN THE EARTHWORM GENUS EISEZVIA (OLIGOCHAETA, LUMBRICIDAE): IMPLICATIONS FOR LABORATORY USE AND TAXONOMIC STATUS FREDDYENGELSTAD’and JQRGEN STENERSEN Biological Institute. Division of Molecular Cell Biology. University of Oslo. P.O. Box 1050. Blindem, N-0316 Oslo 3. Norway (Accepted 25 September 1990) Summary-Polyacrylamide gel electrophoresis has been used to describe acetylesterases (EC 3.1.1.6) in the species Ei.renia andrei, E. fitida and &. renelo. Four bands were common in E. undrei and E. /elido. whereas the three bands found in E. cenefo were unique for this species. Two of the electromotphs described are gut enzymes. of which one is unique to E. firida. Analysis for this enzyme proved to be a reliable method to distinguish the two species E. undrei and E.fitida. The frequencies of the acetylesterase electromorph were very different in two populations of E. venetu. which reflects great variability of acetylesterase alleles. Indications of E. firida and E. rjenefa hybridization were found. Short-term starvation had little influence on the reliability of the method.
INTWODUCl’ION
earthworms as a test organism. to have a proper way of verifying as to what spccics an individual belongs. Elcctrophorcsis is a tcchniquc that dcmonstratcs gcncs or gcnc products and in this way a suitable tool to prove individual-, population- and spccics diffcrcnccs. In this work. clcctrophorcsis was used to verify spccics with absolute certainty, to describe the pattern of acetylesterascs (EC 3. I. I .6) and to evaluate the reliability of this method.
Earthworms in the genus Eiseniu arc’ widcsprcad and well known both as laboratory test organisms (OECD. 1984) and for compost production. In the
litcraturc
the grcatcst emphasis is laid on
“Eismicr
fiwridu”. but as this has proved to bc the two spccics Eiseniu undrei and E./etidu (Jacnickc. 1982; 0icn and
Stcncrscn. 1984). doubt may be raised as to which of the species arc dcscribcd. In a study of the .growth and reproductive ditTcrenccs bctwcen the species E. feridu and E. undrri it was ncccssary to establish pure culture of the two spccics. External taxonomic characters arc similar for the two species, but the pigmentation is ditfcrcnt: E. undrei is more uniformly reddish in contrast to E. firidu, which has wider yellow intcrsegmcntal furrows that give a crossstriped pattern. This has been known since Andre (1963) described the two colour variants as subspecies. This small difference in coloration is not sufficient for a sure distinction between the two species. which is necessary when pure cultures are to be cstablishcd. Eismiu cenetu (Rosa) is another spccics used in waste conversion cxpcrimcnts (LofsHolmin. 1985, 1986). This was earlier classified as a Dendrobuenu (Easton, 1983). but is today rcgardcd as an Eiseniu spccics (Sims and Gerard, 1985). Two colour variants have been dcscribcd. Dendrobuenu I’L’IWU typicu and D. ~‘etw~u :ehru (Gerard. 1964). They arc considcrcd to bc phcnotypic variants and taxonomically indistinguishable. The colour pattern of the E. wnetu zehru variant is very similar to E. fetida. but the position of the sctac is difTcrcnt so there arc no questions of the taxonomic status rclativc to E.ferirfu. To the incxpcricnccd this diffcrcnce is not evident. It is important to pcoplc working with
MATEWIAIS AND METIIODS
Eurhvorms
Specimens of Eiseniu undrei were obtained from a commercial earthworm grower in Norway, and wild type specimens of E. undrei were collcctcd from two sites in the vicinity of Oslo. E. fetidu were collected at the goat barn of the Agricultural University of Norway, As. The source of E. uenetu was a commercial Swedish stock. which here will be designated pop I. The origin of this population is not known. Wild type specimens of E. venetu were obtained from Dr K. Sylvia Richards at the University of Keelc, England (pop 2). All populations were kept in culture in the laboratory. A mixture of horse manure and commercially available “standard soil” consisting of 84% peat, 10% sand and 6% clay was used as medium. Electrophoresis
For the clcctrophorcsis analysis the Protan II slab ccl1 from Bio-Rad. California. and Pharmacia model EPS 500/400 power supply were used. The following were the sources of the major reagents used: acrylamide. bisacrylamidc, ammonium pcrsulfatc and TEMED from Bio-Rad. Richmond, U.S.A.; naphtylacetat, naphtylbutyrate, Fast Blue B Salt, Tris base and glycinc from Sigma, St Louis, U.S.A. Dicthyl 4-nitrophcnyl phosphate (paraoxon)
*Present address: Centre for Soil and Environmental Reseach. P.O. BOX 9. N-1432 AS-NLH.
Norway. 243
244
FREDDY ESGEL.YTADand JCJRGEN SIWERSEN
was obtained from Bayer AG. Leverkusen. Germany. AI1 reagents were of analytical grade. Worms were washed in tap water, placed on a cleaning paper and weighed. They were homogenized in Tris-glycine buffer, 5 rnr+i of Tris, pH = 8.5 with I.2 M glycerol (1 + 3 w/v) on ice. The samples were centrifuged for 5 min at 3500g and the supematants were then frozen at -2O’C. Specimens for dissection were frozen in liquid N2, and the partition was carried out while thawing. The tissue samples were washed in electrode buffer. Samples of 20~1 were applied after addition of bromophenolblue (0.01%). Electrophoresis was carried out according to Laemmli (1970). Voltage was set to 300 V and the gels were cooled with tap water until the marker (bromphenol blue) reached the lower end of the gel. After electrophoresis the gels were placed in a 4 x 10-r M paraoxon solution made up in 50 mM Tris-HCI buffer (pH = 8) in order to inhibit the carboxyland cholincsterases. The gels were then transferred to freshly-made staining solution: which consisted of Tris-HCI (50 mM. pH = 8). /Inaphtylacctatc (IO mg 100 ml-’ buffer) dissolved in I ml acctonc and Fast Blue B salt (50 mg 100 ml-’ buffer). Bands dcvclopcd within 15 min and the gels wcrc then washed in tap water and placed in 7% acetic acid solution.
Stenersen (1984). Four bands (E4, E5, E6 and E7) were indistinguishable in E. andrei and E. fetida, whereas one band was present in E. fetido only (E3) (Fig. 1). Three bands were present in E. ceneta (EVl, EV2 and EV3) which were unique to this species (Fig. 1). Figure 2 shows a composite picture of authentic gels, demonstrating frequently observed phenotypes. The electromorph frequencies of the three species are shown in Table I. Noteworthy from the result is that the electromorph frequencies in E. undrei and E. fetidu is usually close to one. and when the bands are missing no other electromorph appears. The electromorph frequencies in E. reneta differ greatly between the two populations (Table I). Of the 43 individuals tested from pop 1, electromorph EV3 was the most common with a frequency of 0.884, EVI had a frequency of 0.658 and the intermediate EV2 was present with a frequency of only 0.211. From pop 2 the EVI was the most common, and appeared with a frequency of 0.821. EV2 had a frequency of 0.679 and EV3 only 0.286 0ien and Stenersen (1984) found that the acetylesterases were mainly present in the anterior part of the animals. This was further investigated by analyzing tissue from frozen and dissected individuals in the following categories: facces. lumen. gut cpithelian tissue, coelomic fluid and muscle tissue. Samples from thcsc catcgorics. cxccpt for facccs. wcrc taken from the middlo section of the animals. The results HKSl!I.TS from this investigation (Table 2) arc inconsistent concerning cxprcssion or activity of the enzymes. It was possible to distinguish bctwccn eight bands or elcctromorphs. The bands arc dcsignatcd accordIt indicates ncvcrthclcss that the E3 and E4 ing to Fig. I. Five bands were prcscnt in E. f~~~iclu bands arc conncctcd to the gut. whcrcas the other bands originate from enzymes found in other and four in E. andrei. DitTercnccs in elcctromorphs tissues. The E3 and E4 bands were present only bctwccn the populations of E. mulrcv’ were not dctected. The mobility of electromorph E4 is set to bc occasionally in faeccs, but when prcscnt they wcrc highly active. I to fncilimtc comparison with the work of 0icn and ELECIXOMORH I
E.
EV3
EV2
EVI
E7
+
E6
-
ES
-
+
E3
Fig.
I.
Pullcrn
ol acctylestcrascs
from E.
onhi.
&. /(*Mu
and E. rm~ra.
Ei.ienia acetylesterase pattern
1
2
3
4
5
7
6
9
9
10
11
Fig. 2. Composite picture of acetylcsterase pattern in the Ek-niu species. E. a&rui (lane I, (lane 3, 4. I?); E. L’PIICIU(lane 5. 6. 8. IO, II). Lane 7 is overloaded.
To reveal a possible relation bctwccn the nutritional status of the worms and the presence of the E3 and E4 clcctromorphs a new experiment was carried out. Specimens of E. ancfrreiand E.fefidu (IO of each), were placed individual,ly in horse manure to gain weight without competnron. After 4 wk five of each species were starved for 4 days by keeping them on moistened filter paper in boxes. Starved and not starved animals were homogenized and analyzed by elcctrophorcsis. The E4 band was present in all animals indcpcndcntly of trsatmcnt. The E3 band was also prcscnt in all E. ftUtc individuals. Thcrc was however. a reduction in the intensity of the E4 band in E. crrrc/rtGindividuals who had hccn dcprivod of food.
The cnzymcs WC have been working with have the characteristics of acctylesterasr (EC 3. I. I .h) (e.g. Barman, 1969) as shown by 0icn and Stcnerscn (19X4), hut the pattern of electromorphs found in this investigation is not identical with that described earlier whcrc five acctylesterase bands in E. fetidu and E. cmhi wcrc dcscribcd. However, no individuals has more than two clearly distinguishable bands. WC used an electrophoretie method with bcttcr separating ability and found up to five bands present in individuals of E. fc4rlcl and four in E. utrclrei (Fig. 2). Thcrcforc a direct comparison bctwccn the present and curl& results is difficult. Howcvcr. WC think that the isozymcs forming EL2, EL4 and EL5 (Oicn and Stcncrsen. 198-I) wcrc further resolved in our
Relatitc mohdrt) Elcc~romorph
(1)
--....
--.--
E. cmcbci
M.lS
Xlm
2.7,~); E.fpti&
investigation. E3, E4 and E6 compare best to those originally described (EL2, EL4 and ELS). ELI and EL3 were present at very low frequencies in the work of 0ien and Stenersen (1984) and no bands in this investigation corresponded to them. Small sample size may be the cause of this. No alternative electromorph appears when one of the E3-E6 bands was missing. The reason may be the lack of not-functional all& products, or because the amount of enzyme synthcsizcd was too low. Dcnaturation of only one or two of the clcctromorphs is less likely as the acctylestcrascs have good stability. In E. IYN~I(I the three clcctromorphs found arc assumed to bc allele products from one locus. The frequencies of the clcctromorphs (Table I), and their corresponding alleles (Table 3). were quite different in the two populations tested. T’hc all& coding for the EVI band is designated p* the EV2 = y and EV3 = r, In both populations all combinations except homozygotcr for the y allclc wcrc present (Table 4). The y allele appeared with a frequency of 0.093 in pop 1 and 0.339 in pop 2 even though no homozygotcs were found. The population difference was significant (P < 0.05) as tested by a x2 test (xr = 6.9 with 2 d.f.). A similar test on the actual numbers of each allele combination also gave a significant difference (P < 0.01) between the two populations (x’= 32.4 with 4 d.f.). Based on the calculated allele frequencies the expected number of individuals in the categories are estimated in the two populations according to the Hardy-Weinberg theorem (Table 4). Thcrc are dcviations from the observed numbers but the difrerence
Frcqucnclcs -.-__-__-
R;Il-lgC
” = 60
of occurrc”ce .___. E. ff
Iida
n = 3’)
E. L’ww,t, --.__-I-_
P’P 1
pop2
” n-t?
n 5 3
WI
0792
0.X07
0
0
0.XX-l
EV’
0.X34
0.x51-0.X22
0
0
0.21
EV;
0 XYI
0.907-O.XX?
0
0
0.6%
0321
E7
0 923
0.935
O.Yn.8
I
I
0
0
O.VJ?
I
0.93
0
0
0.957
0.X46
0
0
0.719
E6
0.951
O.VhV
ES
0.977
0.9R5-0.96X
EJ
1.000
E3
I.0Z.r
1.035-1.017
12
0.2X6
I
0.67Y
o.xvv
0.7ia
0
0
0
0.974
0
0
&I’ EJ. E5 ix E3, EC?.El ND ND ‘HI& aclrvily when present. but okn ‘Low activity.
E3’. E4: E3. EJ. ES‘ ES. E6. E? E9, E6’. LF ES. E6. E7 missing.
Table 3. Frequencies of alleles in the two 6 rmwta populations. Actuaf number in pmtheses Pop 1. Sweden Pop. 2. England (3 zz NJ t2n = 561
p (EVI) q (Eva I (EV3)
0.337 (?I) 0.093 (8) 0.570 (49)
0.500 CX) 0.339(19! O.lfil (9)
is not significant as tested by the ,$ method. For pop I xzr3.42 with 2 d.f., 0. I < P < 0.25, and for pop 2 )[?= 5.22 with 2 d.f. 0.05 < P < 0. I. Due to patchincss of their natural habitat and anthropogcnic ciispersai it would have been most rcmarkahfc if gcn frcqucnoics wcrc cyuaf in so distant po~ufiitions. Culturing ~l)r~~~it~~lns in the Swedish popufittion may ~~11 have cittlscd I scltaion prcssurc ditfia-iny from wild populations resulting in diffcrcnt aflclc frcqucncics. The fact that no homozygous indiviciuols for ths q-allclc wcrc found in cithsr of the populations indicates that this may Icad to rcduccd viability. f fybri~li~~ti~3n htwccn E. rrfrcfm+ and E. frIi&r is &scribed in the work uf Oicn and Stenerssn (19X4), bum at very low frsqucnsy in wild populations They hybridize casicr in cultured populations, but produce &spring at lower nrtc. Some individuals in the Swedish population (pop I) had bands that corresponded both to E jid& and E. twm (Fig. 3). According to Mufdal (1952) and Qmodeo (1952) there ari: morphological diflcrcncos in the nucleus of the species and the chromosome numbers arc 22 in E. fc*tic/tr and 36 in E. twtvtt. Viablr hybrids bctwosn thcsc two species arc thcrcforc improbable. Caution was taken to avoid cross-contamination of samples during this work and breeding expcrimtnts are ncccsairy to find out if hybridi~~~tion may indeed occur. The staining is depondcnt on cnzymc activity and consequently the band intensity varied markedly. Thcrc was always high activity in the samples from lurncn. whcrcas the bands from the gut cpithcli:d tissues gave less dcvelopod bands. fn f~scs h:mds detelopcd onfy five times of the IX samples tested. This indicates that the E3 and W bonds represent digestive c’storascs synthcsizcd in the cpithelia lining of the put wall behind the giaard and sccrctcd into
1
2
Fig. 3. Picture of an authentic gel showing nn individual with both E. _krirla and E. r~wc~ubands. Lane I: possible hybrid E. wnen~la x E. f&i&; Lane 2: E. wnctt~. the lumen. The enzytnrs arc’ gr;tduitfly denatured during the passage through the gut. Thus, the samples from the LICCCS somctimcs arc active and sometimes not. The prcscncc of the cfcctromorphs E5, E6 and E7 in the coclomic fluid and the muscfc Inyor in E. fvrirkt indicates tfW lhcir onsymc products arc normal ~~~ipori~~~t~ of cells. A more llnifornl distr~butioll within the organism is thcrcforc expcctcd. The result of the starvation cxpcrimcnt shows that clcctrophorcsis is a rcliahle method cvcn if animals have been starved or have lived ou scanty food for some cfays. The gut transit time is found to be 7 h at 15°C and 2.5 h at 25 C in E. _/hi& (ii~rt~nst~in et d.. 1981) and after 4 days the synthesis of digestive cstcrasc enzymes still conlinucs. However. animals with another physiologial status. cxamplificd by an individual in a quiescent state due to drought or cold, may have ceased to synthcsizr: this enzyme. For laboratory use this is LI minor problem. but if animals are taken from the ftctd they should have proper time to reach an Ltctivc st;ttc b&rc analysis of CStCraSCS. In laboratories whcrc arthworms arc used as test organisms, it is possible to anAyzc the cstcr;Lscs in the posterior part of the animal by cutting this off. This will rcgcncratc and the inrlividu;tls sre fully capable oT p~op~~~tin~. The rcfkbility of the CSICTISC pattern tfrscribed here suggests that USC of the cfcctrophorctic method is a pr;lctical way to establish pure cultures or to verify to whicfl spcciss an individual belongs.
Eirenia acetyiesterase pattern Acknowledgements-This work b ken 6nancially supported from The Norwegian Agricultural Research Council. We thank Dr K. Sylvia Richards, University of Kccic. for providing specimens of E. vencfa.
REFERENCES
And+ F. (1963) Contribution a I’analyr exp&imentaie de la reproduction dcs iombricicns. Bulletin Biologique dc la Frame et dc la fkfgique 97, 3401. Barman T. E. (1969) Enzyme Handbook, Vol. 11. Springer, Berlin. ___..~.. Easton E. G. (1983) A guide to the valid names of Lumbricidae (Oiiaochaeta). Jn Earthworm Ecoloav. From Darwin ro Ver&.&ure (J.’ E. SatchelI. Ed.), pp.475-485. Chapman & Hall, London. Gerard 9. M. (1964) Lumbricidae (Ann&da) with keys and descriptions. Synopses ofthe British Fauna, 2nd Edn. Vol. 6. The Linnean S&i& of London. Hartenstein F.. Hartcnsttin E. and Hartenstein R. 11981) Gut load and transit time in the earthworm &.se& foerida. PedobiOrogia22, S-20. Jaenickc J. (t 982) “Eisenia fwtida” is two biological species. Megadrilogica 4, 6-8.
247
Lacmmb U. K. (1970) Cicavagc of structural proteins during the assembly of the head of bacteriophap T4. Narure 227,680-685. Lofs-Holmin A. (1985) Present knowledge of earthworm farming-a summary of rcceut literature. Swedish University of Agricuiturai Sciences, Dcpt of Ecoiogy and En~~o~~~ Reseat& Report No. 20. -Lofs-Hoimin A. (1986) Processina of municioal dudan through carthwdm (Dendrobae& uenera). Siedt3h J&r~1 of Agricultural Research 16,67-l I. Muidal S. (1952) The chromosomes of the carthwotms 1. The evolution of poiypioidy. Heredity 6, U-76. OECD (1984) OECD Gui&lines for Tes!ing of Chemicafs. ~gani~tion for Economic Co-operation and Dcvciopment, Paris. Omodeo P. (1952) Cariologia dei Lumbricidae. Curyofogiu 4, 173-275. 0ien N. and Stcnerscn J. (1984) Estetascs of carthworms111.Elcctrophornis reveals that ELseniajerida (Savigny) is two species. Comparative Biockemissry and Physiology 78C, 277-282. Sims R. W. and Gerard 9. M. (1985) Earthworms. Synopses of tke British Fauna (New Series) (D. M. Kenack and R. S. K. Barnes. Eds). E. J. Briil & Dr W. Brackhuys. London.