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Vol. 22, No. 7, pp 1023-1027. 1992

RESEARCH NOTE APPARENT LACK OF GENETIC VARIATION WITHIN PELECITUS ROEMERI (NEMATODA: FILARIOIDEA) FROM THREE AUSTRALIAN SPECIES OF MACROPODID MARSUPIAL NEIL

B. CHILTON,*~

,$ Ross H. ANDREW@ and DAVID M. SPRATT~ IAN BEVERIDGE

* South Australian Museum, North Terrace, Adelaide, South Australia 5000, Australia 2 Department of Veterinary Science, University of Melbourne, Parkville, Victoria 3052, Australia 5 Department of Microbiology and Immunology, University of Adelaide, G.P.O. Box 498, Adelaide, South Australia 5001, Australia 7 Division of Wildlife and Ecology, CSIRO, P.O. Box 84, Lyneham, Australian Capital Territory 2602, Australia (Received 26 May 1992: accepted 9 August 1992)

Abstract-CHILTON N. B., BEVERIDGEI., ANDREWSR. H. and SPRATTD. M. 1992. Apparent lack of genetic variation within Pelecitus roemeri (Nematoda: Filarioidea) from three Australian species of macropodid marsupial. International Journal for Parasitology 22: 1023-1027. An electrophoretic study of Pelecitus roemeri from Macropus robustus, M. giganteus and Wallabia bicolor revealed no genetic differences at 23 enzyme loci. The genetic data support the existing morphological evidence that P. roemeri from these three hosts represents a single species. The data show no genetic variation between nematodes from the same or different host species collected in northern and southern Australia. This result is discussed briefly in relation to Price’s model of parasite speciation. INDEX KEY WORDS: genetic variation

Pelecitus roemeri; nematodes;

IN Australia, the filarioid nematode Pelecitus roemeri (formerly Dirojilaria roemeri, see Bartlett & Greiner, 1986) is transmitted by tabanid flies (Diptera) to a number of species of macropodid marsupial belonging to the genera Macropus, Wallabia, Onychogalea, Petrogale and Dendrolagus (see Spratt, Beveridge & Walter, 1991). Its prevalence differs between host species and geographical regions (Spratt, 1972, 1974, 1975; Beveridge & Arundel, 1979) which can in part be attributed to differences in the feeding behaviour of tabanid flies on different host species and the distribution of different species of tabanid (Spratt, 1974). Based on the reproductive status of female P. roemeri and the concentration of microfilariae in the peripheral circulation, it has been suggested that in southeastern Australia Macropus robustus is the usual host for this parasite and that it acts

as a reservoir

species

(e.g.

abnormal

of infection

M. giganteus,

hosts,

that

for other

macropodid

M. rufogriseus)

is, hosts

in which

which are there is

t Present address and address for all correspondence: of Veterinary Science, University of Department Melbourne, Parkville, Victoria 3052, Australia.

allozyme

electrophoresis;

macropodid

marsupials;

premature termination of the parasite’s life-cycle (Spratt, 1972, 1974, 1975). The parasite may also be able to complete its life-cycle in M. rufus which occurs in the arid interiors of Australia and may therefore also represent a source of infection for other macropodid species (Spratt, 1975). P. roemeri adults infecting M. robustus are morphologically indistinguishable from those infecting M. giganteus, yet they are generally much larger are several possible (Spratt, 1972). There explanations for the observed size differences. P. roemeri may represent a single variable species whose morphology is influenced by the host in which it develops. Alternatively, P. roemeri may consist of a series of sibling species, each infecting a different host species. The latter explanation would not be surprising given the results of recent electrophoretic studies on nematode and cestode parasites of Australian marsupials which have revealed the existence of several genetically distinct but morphologically cryptic species in the anocephalid cestode Progamotaenia ,festiva (see Baverstock, Adams & Beveridge, 1983, and in the strongyloid nematodes Hypodontus macropi (see Chilton,

1023

1024

N. B. CHILTON et al. TABLE

I-ENZYMES

USED IN THIS STUDY, THEIR

ENZYME

(E.C.)

COMMISSION

NUMBERS

AND THE

BUFFERS AND RUN TIMES

Enzyme

E.C.

Buffer*

Run time (h)

Aldolase Citrate synthase Enolase Fumarate hydratase Glyceraldehyde-3-phosphate dehydrogenase Aspartate aminotransferase Glucose-phosphate isomerase Glutamate-pyruvate transaminase Hexosaminidase Hexokinase Leucine aminopeptidase Malate dehydrogenase Malic enzyme Mannose-phosphate isomerase Nucleoside diphosphate kinase Peptidase valine-leucine Peptidase leucine-glycineglycine Phosphoglycerate mutase Phosphogluconate dehydrogenase Phosphoglucomutase Pyruvate kinase Triosephosphate isomerase

ALD CS ENOL FUM

4.1.2.13 4.1.3.7 4.2.1.11 4.2.1.2

1 4 2 1

2 2.5 2 2

GA3PD GOT GPI GPT HEX HK LAP MDH ME MPI NDPK PEPA PEPB PGAM 6PGD PGM PK TPI

1.2.1.12 2.6.1.1 5.3.1.9 2.6.1.2 3.2.1.30 2.7.1.1 3.4.11.1 1.1.1.37 1.1.1.40 5.3.1.X 2.7.4.6 3.4.13.11 3.4.11.4 5.4.2.1 1.1.1.44 5.4.2.2 2.7.1.40 5.3.1.1

4 1

2 2 2 2.5 2 2 2.5 2 2 2.5 2 2 2 2 2 2 2 2

1, 2? 3 4

1 2

1 1 1 3 2, 3 2 2, 3 1 3 4

I

* Buffers used: 1 = 0.02 M-phosphate, pH 7.0; 2 = 0.01 M-citrate-phosphate, 0.05 M-Tris-maleate, pH 7.8; 4 = 0.015 M-Tris-EDTA-borateMgCl,, pH 7.8. f Two buffers were used in combination to resolve electrophoretic patterns.

& Andrews, 1992), Paramacropostrongyh typicus (see Chilton, Beveridge & Andrews, in press) and Macropostrongyloides baylisi (see Beveridge, Chilton & Andrews, in press). This study reports the results of an electrophoretic analysis of P. roemeri which examined the null hypothesis that specimens from different host species and/or geographical localities represent a single species. Twenty-eight P. roemeri adults were removed from the subcutaneous and intermuscular connective tissues of the (stifle) knee region of one Wallabia &color (no. of nematodes = 7) and one Macropus giganteus (n = 4) from Bondo State Forest, New South Wales, one M. giganteus (n = 9) from Melmoth Station via Dingo, Queensland and one M. robustus (n = 8) from Warrawee Station via Charters Towers, Queensland. Nematodes were washed in normal saline, stored separately in microcentrifuge tubes and frozen live in liquid nitrogen. Blood samples from M. giganteus and M. robustus were also frozen for later use on all electrophoretic gels as host controls. All samples were subsequently stored at ~ 70°C until required for electrophoretic analysis. Beveridge

pH 6.4; 3 =

An equivalent volume of lysing solution (100 ~1 p mercaptoethanol, 10 mg nicotinamide-adenine dinucleotide phosphate and 100 ml distilled water) was added to each thawed sample prior to sonication and centrifugation at 5000 g for 10 min at 4°C. Electrophoresis was conducted on cellulose acetate, Milan) according to the ‘Cellogel’ (Chemetron, Richardson, general methods described by Baverstock & Adams (1986), but using modifications employed in our other genetic studies of parasites (Andrews & Beveridge, 1990; Chilton et al., 1992, in press; Andrews, Chilton, Beveridge, Spratt & Mayrhofer, 1992; Beveridge et al., in press). Each nematode sample was screened for a total of 48 enzymes, of which 22 gave sufficient staining intensity and resolution to allow reliable genetic interpretation. These enzymes, their Enzyme Commission (E.C.) numbers and the running conditions of gels (i.e. buffers and run times) are listed in Table 1. For 18 enzyme loci (AH, Cs, Enol, Fum, Ga3pd, Got, Gpt, Hex, Hk, Lap, Me, Mpi, Ndpk, Pep& Pgam, Pgm-I, Pgm-2 and Tpi) there was no evidence of contamination of the parasite

Research

detected between P. roemeri from different host species or localities, thus the null hypothesis that P. roemeri is a single species cannot be rejected. Furthermore, all nematodes had an identical allelic profile. The number of enzyme loci established herein for the identification of individual P. roemeri falls within the range of loci established for other nematodes (i.e. 11-38 loci; Nadler, 1990; Chilton et al., 1992, in press; Beveridge et al., in press). It is therefore unlikely that a sufficient level of fixed allelic differences (i.e. > 15% of enzyme loci for allopatric taxa; Richardson et al., 1986) would be detected to refute the null hypothesis if the P. roemeri samples were screened at a larger number of enzyme loci. Hence, genetic evidence from this study provides additional support to the morphological data (Spratt, 1974) which suggest that P. roemeri from M. robustus, M. giganteus and W. bicolor are indeed one species and that the size differences in nematodes from different hosts reported by Spratt (1972) are host-induced. It remains to be determined whether P. roemeri from the other host species (e.g. M. rufus, M. agilis,

GA3PD

1

PEPA

M. 6PGD --m-e

Qca

-e-e

Hl

1025

Note

H2

H3

Nl

N2

N3

N4

FIG. I. Diagrammatic representation of the electrophoretic patterns of the three host controls (W. bicolor, HI; M. robustus, H2; M. giganreus, H3) and P. roemeri samples from W. bicolor (lane Nl), M. robusrus (N2), M. giganteus in Queensland (N3) and M. giganteus in New South Wales (N4) at four enzyme loci. Note that for the enzymes GA3PD and PGM the electrophoretic bands of nematode samples do not comigrate with those of the host controls. Contaminations of parasite homogenates were detected for PEPA and 6PGD but the parasite-specific locus for each enzyme is easily distinguished from bands of host enzyme which are often weaker in staining intensity. homogenates with host enzyme. All of these enzymes were encoded by a single locus, except for PGM where all nematode samples expressed two loci (Fig. 1). For a further five enzymes (GPI, MDH, PEPA, 6PGD and PK), varying levels of host enzymes were detected within some or all of the parasite homogenates. Despite this, the parasite-specific enzyme locus was readily distinguished from the host enzyme as they did not comigrate (see Fig. 1). A comparison of P. roemeri from different host species was therefore made using 23 parasite-specific enzyme loci. No fixed genetic differences were

dorsalis,

M.

parryi,

rufogriseus,

M. fuliginosus,

persephone,

Onychogalea

M.

antilopinus,

Petrogale

fraenata,

0.

assimilis, unguifera

M. P.

and Dendrolagus lumholtzi, see Spratt et al., 1991) are the same nematode species. Given the lack of genetic differences between specimens in M. robustus, M. giganteus and W. bicolor, it is likely that specimens in these other hosts, which are sympatric over parts of their range with either M. robustus, M. giganteus or W. bicolor in areas where P. roemeri infection occurs, also represent the same widely distributed species. However, a curious feature of the anatomical location of P. roemeri in M. parryi and D. lumholtzi is that it is not found in the stifle region, as in other hosts, but rather in the subcutaneous and especially the intermuscular connective tissue of the rump region (Spratt, unpublished). This may represent a form of niche specialization in different hosts or could indicate that the parasite in M. parryi and D. lumholtzi represents a different species. The presence of P. roemeri in a given macropodid species in a particular region is determined by both the geographical distribution and the biting behaviour of those species of Tabanidae which are capable of serving as intermediate hosts (Spratt, 1974). Nine such species representing three genera (Dasybasis, Tabanus and Mesomyia) are known (Spratt, 1974) but it is likely that additional species and possibly genera are suitable intermediate hosts. Spratt (1972) suggested that the parasite may have had a long association with its hosts because macropodids and tabanid flies may have been

1026

N. B. CHILTON et al.

isolated together in Australia for 40-80 million years. However, in the present study, no genetic variation was detected at any of the 23 enzyme loci between populations of P. roemeri collected from widely disparate geographical localities in eastern Australia, suggesting a more recent association between host and parasite. Bartlett & Greiner (1986) proposed that the two species of Pelecitus from mammals, P. scupiceps in the tendons of the ankle region of North American leporids and P. roemeri which generally occurs near the insertion of the sartorius muscle in the knee region of Australian macropodids, were recently derived from filarioid nematodes occurring in the legs of birds. One species, P. fulicaeatrae has been described thus far from an Australian bird (Mawson, Angel & Edmonds, 1986). Consistent with this is the absence of other genera within the Dirofilariinae in Australian mammals (Spratt & Varughese, 1975) and the absence of P. roemeri in, marsupials on other continents. By contrast, the principal filarioid nematodes of Australian marsupials are species of Brie&a, Sprattia and Dipetalonema, genera within the Onchocercinae (Spratt & Varughese, 1975). Thus, the genetic evidence is consistent with the recent acquisition hypothesis. The lack of genetic variation detected between P. roemeri from different hosts and localities may also be indicative of cross-transmission of the parasite from one host species to another by relatively nonspecific biting flies. Four aspects of tabanid biology augment this; (I) tabanids are interrupted feeders, (2) there is a long season for potential transmission of the parasite (8 months in southeastern Queensland) at least in the northern half of the continent, (3) peak biting activity occurs during the middle of the day and is most pronounced during fine hot weather which corresponds with the peak in numbers of circulating microfilariae in the blood of those hosts considered normal (Spratt, 1972, 1974, 1975) and (4) tabanids generally are strong fliers so have the potential, although this has not been demonstrated to date, to disperse the parasite over wide geographical areas, thereby breaking down geographical barriers in the ranges of the host species. The apparent lack of genetic variation between populations of P. roemeri is in direct contrast to results from other Australian nematodes belonging to the Strongyloidea that infest macropodid marsupials (e.g. in Hypodontus macropi, Paramacropostrongylus typicus and Mucropostrongyloides bay&i) where within-species polymorphism has been detected at 447% of enzyme loci (Chilton et rd., 1992, in press; Beveridge et al., in press). For instance, one fixed genetic difference, as well as allele

frequency differences at several loci, was detected between the mainland and Tasmanian populations of a species of H. macropi in M. rufogriseus. The species of H. macropi in different subspecies of M. robustus also had a similar level of within-species genetic variation (Chilton et al., 1992). The data presented here also contrast sharply with studies on trichostrongyloid and ascaridoid nematodes. Withinspecies polymorphism was detected at 12 (37.5%) of 34 enzyme loci in an electrophoretic study of the trichostrongyloid nematode Teladorsagia circumcincta from sheep (Andrews & Beveridge, 1990) and in a limited number of electrophoretic studies on ascaridoid nematodes, there appears to be a general pattern of within-species polymorphism (Nadler, 1990). The lack of genetic variation between P. roemeri populations, unlike that for the other nematodes mentioned above, does not support Price’s (1977) hypothesis of extensive parasite radiations within host species. Price suggested that parasites would become genetically different from neighbouring populations more frequently than non-parasitic organisms because parasites are often small. dispersal between populations is low and the reproductive systems are geared for the production of large numbers of progeny in a relatively short time. However, low levels of genetic variation have been detected in other helminths. For example, Renaud, Gabrion & Pasteur (1986) found no polymorphism at any of the 1I enzyme loci they examined for Atlantic and Mediterranean populations of the pseudophyllidean cestode Bothriocephalus burhutus. There is also limited genetic variation among conspecific populations of three sibling species of the ascaridoid nematode Pseudoterranova decipiens collected from localities thousands of kilometres apart (Paggi, Nascetti, Cianchi, Orecchia, Mattiucci. D’Amelio, Berland, Brattey, Smith & Bullini, 1991). The lack of genetic variation between parasite populations is a phenomenon not restricted to hclminths because it also occurs in ticks (Bull, Andrews & Adams, 1984). Clearly. more extensive population studies on a variety of parasite groups are required in order to determine the general patterns of the extent of population sub-structuring and the mechanisms of parasite speciation. Acknowledgemmts~We wish to thank Drs D. Jenkins. L. Warner and R. Speare for assistance with the collection of specimens. This research was supported by a grant from the Australian Research Council.

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