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1 Department of Biology, University of California, Riverside, California 92521, USA
2 Department of Nematology, University of California, Riverside, California 92521, USA
3 Department of Neurosciences, Deaconess Billings Clinic, 2825 Eighth Avenue North, Billings, Montana 59107, USA
4 The Marine Mammal Center, Golden Gate National Recreation Area, Marin Headlands, Sausalito, California 94965, USA
5 Corresponding author (jelsonriggins{at}msubillings.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key words: Immunoglobulin, lungworm, nematode, northern elephant seal, Mirounga angustirostris, Otostrongylus circumlitus, Pacific harbor seal, Phoca vitulina richardsi.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Northern elephant seals appear to be less well adapted to the parasite than PHS, because the severity of disease in this species is not proportional to the intensity of infection, and mortality often occurs during the prepatent period, thus preventing parasite reproduction in the host (Gulland et al., 1997). Mortality of juvenile NES may result from pulmonary arteritis and disseminated intravascular coagulation (DIC) with as few as 20 parasites (Gulland et al., 1996, 1997), yet adult NES may have asymptomatic infections with parasites in the airways (Gulland, pers. obs.). Characteristics of infection in PHS are, however, typical of those in other host species and include bronchitis and pulmonary arteritis (Gulland et al., 1997; Measures 2001). Due to mortality during the pre-patent period, O. circumlitus infection is difficult to diagnose ante mortem in NES. The clinical signs are nonspecific in this host, and the presence of larvae in the feces cannot be used to diagnose infection. An effective diagnostic test for O. circumlitus in young NES is required so that appropriate treatment and prophylaxis can be developed and evaluated in captive animals and those being rehabilitated.
To investigate whether age was a factor in the unusual NES response to O. circumlitus, currently available techniques were used to detect immunoglobulin (Ig) to different tissues of O. circumlitus in serum of two different age classes of NES infected with O. circumlitus. The Ig binding patterns of these animals were then compared with the Ig binding pattern of PHS infected with O. circumlitus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Adult nematodes were obtained from stranded seals during postmortem examination at The Marine Mammal Center (TMMC), Sausalito, California, between May 1997 and July 2001. Some of these animals had been euthanized because the chances of survival were determined to be minimal. This was done by intravenous injection of pentobarbital (389 mg/ml; Anthony Products, Arcadia, California). The nematodes were then rinsed in 0.15 M saline and stored at –80 C.
Blood samples were taken from the extradural intravertebral sinus of live animals by the veterinary staff at TMMC and Sea World, San Diego, California. All animals at Sea World were alive. If an animal at TMMC had recently died, blood was obtained by cardiac puncture. Blood was collected in Vacutainers containing serum separation gel (Becton-Dickinson and Company, Franklin Lakes, New Jersey, USA) and centrifuged at 2,000 x G for 30 min to obtain serum. This was stored at –80 C until used. Serum was taken from NES between March 1997 and March 2001 and from PHS between May 1996 and August 1999.
Nematode extract preparation
Otostrongylus circumlitus were dissected in 0.15 M saline that had been chilled to 4 C. Intestine, excretory-secretory (ES) glands, male and female reproductive tract, and body wall were excised, rinsed in fresh saline, and stored at –80 C until extracts were prepared. Whole nematodes (O. circumlitus, Parafilaroides sp., Pseudoterranova sp., and Anisakis sp.) and O. circumlitus reproductive tracts were homogenized in phosphate buffered saline (PBS; Maizels et al., 1991) containing a general use pro-tease inhibitor cocktail (Sigma-Aldrich Corporation, St. Louis, Missouri, USA). Homogenization was performed on ice for 5 min in Eppendorf tubes using disposable grinders (Belart Products, Scienceware, Pequannock, New Jersey, USA). One microliter of each homogenate was placed on microscope slides and checked for full maceration by light microscopy. The homogenate was then centrifuged at 16,000 x G for 30 min at 4 C. The supernatant was removed and further centrifuged at 16,000 x G for 30 min at 4 C. This procedure was repeated until no more pellet was obtained. The supernatant was then stored at –80 C.
Extracts of O. circumlitus ES gland and intestine were prepared as above, except that they were homogenized in ripa buffer (150 mM NaCl, 50 mM trizma base, 5 mM ethylenedi-aminetetraacetic acid [EDTA], 1% nonidet P-40, 1% sodium deoxycholate, 0.1% sodium do-decyl sulfate [SDS], pH 7.5) containing the protease inhibitor mentioned above. Otostrongylus circumlitus body wall extracts were homogenized in PBS as above. However, the homogenate was then sonicated on ice at 50 W in a Virsonic 50 Disrupter (Virtis Company, Inc., Gardiner, New York, USA) for 2 min. This was further homogenized on ice for 5 min before centrifuging at 13,800 x G for 10 min at 4 C. The supernatant was labeled F1 and stored at –80 C. The pellet was boiled in PBS containing 1% SDS for 2 min. This was then centrifuged at 13,800 x G for 10 min at room temperature. This supernatant was labeled F2 and frozen at –80 C. The F2 pellet was then boiled in PBS containing 1% SDS and 5% ß-mercaptoethanol for 2 min. This was centrifuged at 13,800 x G for 10 min at room temperature. The supernatant was labeled F3 and stored at –80 C.
The protein content of each extract and serum sample was estimated by either Bio-Rad or Detergent Compatible (DC) protein assays (Bio-Rad, Hercules, California, USA) using a Benchmark microplate reader (Bio-Rad) with bovine serum albumin as a standard.
SDS-PAGE and immunoblotting
Protein extracts were boiled in Laemmli sample buffer (Laemmli, 1970) for 10 min and separated by SDS polyacrylamide gel electrophoresis (PAGE) on either 4% stacking and 12% separating gels (Doucet and Trifaró, 1988) or 4–20% tris-HCl precast gels (Bio-Rad). For immunoblotting, proteins were transferred to Millipore Immobilon-P transfer membranes (Millipore Corporation, Bedford, Massachusetts, USA) using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad), dried according to the manufacturer’s instructions and stored at –20 C until use. The membranes were then wetted with 100% methanol and rinsed first in nanopure water, then in TBT (20 mM trizma base, 500 mM NaCl, pH 7.5, with 0.05% Tween-20). They were then blocked with TBT containing 5% dry skim milk (Becton Dickinson and Company, Sparks, Maryland, USA) for 1 hr. After this treatment and each subsequent treatment the membranes were rinsed in TBT. They were incubated for 1 hr in antibody buffer (TBT containing 1% skim dry milk) containing serum of the appropriate concentration, which was determined in previous experiments. They were then incubated for 1 hr in horseradish peroxidase linked protein A (protein A-HRP; Amersham Pharmacia Bio-tech, Arlington Heights, Illinois, USA) diluted to 4 ng/ml in antibody buffer, and the proteins were detected by enhanced chemiluminescence (ECL; Amersham) according to the manufacturer’s instructions. The membranes were then incubated for 1 hr in 0.015 µg/ml HRP conjugated streptavidin (Pierce, Rockford, Illinois, USA) followed by ECL to detect the standards. Afterward, selected membranes were rinsed and stripped using a Re-Blot Plus Western Blot Recycling Kit (Chemicon International, Temecula, California, USA) according to the manufacturer’s instructions. These membranes were then blocked and incubated in serum and protein A-HRP before the detection processes described above.
Four micrograms of protein per well were loaded for immunoblots of extracts of O. circumlitus using Bio-Rad Mini-Protean II and Mini-Protean 3 electrophoresis cells (Bio-Rad) and 15 well combs. These blots were probed with serum at a concentration of 0.1 µg protein/µl for NES and 0.06 µg protein/µl for PHS. These sera concentrations were selected to optimize immunoblot conditions. Two micrograms of protein per well were loaded for the immunoblots testing different nematode genera and serum was used at a concentration of 0.2 µg/µl protein for NES and 0.12 µg/µl protein for PHS. Biotinylated SDS-PAGE standards (Bio-Rad; 0.26 µg protein/well) were used to determine molecular weights. Full range rainbow markers (Amersham) were also run on each gel to determine when to terminate electrophoresis. This marker lane and lanes not directly pertaining to the results were digitally removed from the figures.
Study design
The majority of animals were classed as infected with O. circumlitus if these nematodes were detected in airways, heart, or pulmonary arteries at necropsy. However, three of the yearling NES were diagnosed as infected with O. circumlitus by fecal examination for O. circumlitus larvae. Two of these animals were treated to remove nematodes, recovered from clinical signs of infection, and were subsequently returned to the wild. A seal was classed as uninfected if no O. circumlitus were detected at necropsy, no O. circumlitus larvae were detected in feces, and it had a feeding history consistent with lack of exposure (while in captivity seals were fed frozen fish assumed free of live nematodes). Parasites of species other than O. circumlitus were not detected in uninfected animals. However, infected animals had the following histories with respect to other parasites: of the 10 NES infected with O. circumlitus that were age class 2–9 mo, O. circumlitus alone was detected in five animals, Parafilaroides sp. in one, anisakids in four, tapeworms in three, and flukes in one; of the seven NES infected with O. circumlitus that were age class 1–2 yr, O. circumlitus alone was detected in two animals, Parafilaroides sp. in one, anisakids in three, tapeworms in two, flukes in five, and acanthocephalans in one; of the 12 PHS infected with O. circumlitus that were examined, O. circumlitus alone was detected in five animals, Parafilaroides sp. in three, anisakids in four, flukes in one, and acanthocephalans in five. Animals from TMMC were fed thawed previously frozen herring, and animals from Sea World were fed thawed previously frozen herring, sardines, and capelin.
The O. circumlitus extract gels were probed with serum from the following classes of NES infected with O. circumlitus (all from TMMC): nine animals 2–6 mo of age, one 9-mo-old animal, and seven 1- to 2-yr-old animals (Table 1
). The O. circumlitus extract gels were also probed with serum from four uninfected NES. Three of these uninfected seals were 2 mo old and had been in captivity prior to weaning. The fourth uninfected NES was taken into captivity at weaning age and died 4 days later. Gels were also probed with serum from 12 PHS infected with O. circumlitus, all from TMMC, ranging in age from 4 to 6 wk to one adult of over 4 yr of age. Serum from six uninfected PHS was also tested. Four were seals that had been born in captivity and raised at Sea World, and two were animals from TMMC. The animals from Sea World ranged from 6 mo to 18 yr of age. The uninfected PHS from TMMC had been taken into captivity prior to weaning and were at least 6 wk of age when the serum was taken.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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There was individual variation in intensity and location of bands on immunoblots among seals. Despite this variation, seals of different species and age classes had distinct patterns. The principle differences between patterns in these seal classes are summarized in Table 1
. Intensity of band staining for each species and age class was graded visually on a 5-point scale from 0 for no staining to 4 for very intense staining. This analysis was performed for each individual seal and the mean banding intensity for each species and age class indicated in the table.
Immunoblots using sera from infected NES that were 6 mo of age or younger had a strong band of about 28 kDa that was detected most intensely in the female reproductive tract of the nematode (Fig. 1a; Table 1). A band of this approximate size was also present to a lesser degree in all other tissues except body wall fraction F3. The 9-mo-old NES had a weakly stained band of this size. When a gradient gel was probed with serum from a 4- to 6-mo-old animal, this band was shown to be composed of a strongly staining band of 28 kDa and a band of 26 kDa that stained less strongly in all the aforementioned tissues, except the ES glands. The band in the ES glands was composed of a triplet of bands of 27, 26, and 24 kDa. All infected animals 
9 mo of age that had antibodies binding strongly to the female reproductive tract also had antibodies binding to all of the nematode tissues. However, no one tissue appeared to be more intensely stained than the others. None of the uninfected NES sera tested had Ig binding to the 28 kDa band (Table 1) or the 26 kDa band. Sera from two uninfected seals did not contain detectable antibodies recognizing any of the nematode tissues. The serum from the third uninfected seal produced faint smears on the gel without distinct bands, and that of the fourth uninfected seal had one strongly stained band of about 97 kDa in the lanes containing whole adult O. circumlitus and a few weakly stained bands in the body wall fractions. In 1 yr and older NES, intensity of the 28 kDa band varied from nonexistent to strong. All older animals contained antibodies to all O. circumlitus tissues; however, the ES glands appeared to be most strongly stained (Fig. 1b; Table 1). This tissue did not appear to be as strongly stained in the younger NES (Fig. 1a; Table 1).
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1-yr-old infected NES, eight of the remaining 10 infected PHS had strong binding to the ES glands (Table 1). Seven of these animals lacked the antibodies to the 28 kDa band in the female reproductive tract that were seen in young NES, but a very faint band in this position was detected in one animal. The antibody binding pattern of the two remaining PHS was similar to that of the young NES (a reasonably strong 28 kDa band in the female reproductive tract and binding to the ES glands at the same approximate strength as the other tissues). The banding pattern of the >4-yr-old animal was similar to that of the majority of the younger PHS. This PHS appeared to contain high levels of Ig that recognized the ES glands, but binding to the 28 kDa band of the female reproductive tract was not detected. Sera of the uninfected animals did not appear to contain high levels of Ig recognizing the ES glands, with the exception of one animal that had a strong band of about 28 kDa. In this animal Ig appeared to bind strongly to a band of this size in all tissues, except body wall fraction F3. Furthermore, the band was of similar intensity in the female reproductive tract, whole O. circumlitus, male reproductive tract, ES glands, and gut. Specifically, in the four uninfected controls from Sea World, binding was not detected for one animal, one had nonspecific faint binding, and one had very faint binding (equivalent to an animal infected with O. circumlitus that had Ig levels that were too low for analysis). The binding was so weak in these animals that they were scored as zero. The last animal, however, had the 28 kDa band previously mentioned and a band of about 60 kDa that was detected in all tissues except two of the body wall fractions. Several bands were detected in all tissues for one of the PHS from TMMC, but no 28 kDa band was seen. In the other uninfected PHS from TMMC, some high molecular weight bands were detected in the whole O. circumlitus and body wall fractions, and there were a few bands in the ES glands. Immunoblots of different nematode genera
Banding patterns in immunoblots using phocid sera against whole nematode extracts varied between species of nematode (Fig. 2a). Otostrongylus was easily distinguished from the other genera tested. Immunoblot patterns were quite different among the Parafilaroides sp. from different host species. Immunoblot patterns within the anisakid group were, however, quite similar, with Pseudoterranova sp. being the most distinct. When blots were probed with sera from juvenile PHS, the 28 kDa band was not seen for any of the nematode genera (Fig. 2a). In immunoblots using juvenile NES sera, however, Parafilaroides lanes showed antibodies to a protein of about the same molecular weight as the intensely staining 28 kDa protein in O. circumlitus. This band appeared to be strongest in the Parafilaroides sp. from NES. To test whether this is the same protein as the 28 kDa band of O. circumlitus detected with serum from juvenile NES infected with O. circumlitus, Parafilaroides sp. from NES was run on a gradient gel and compared with extracts of O. circumlitus whole nematodes and female reproductive tract by immunoblotting with serum from juvenile NES infected with O. circumlitus (Fig. 2b). Despite banding intensity being much weaker for Parafilaroides sp. than for O. circumlitus, the band was shown to be the same size in the two genera. No antibodies to extracts from the different genera were detected in the uninfected NES.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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9 mo of age that were infected with O. circumlitus had Ig binding at a similar strength for all O. circumlitus tissues. However, the host would be expected to respond most strongly to the tissues and secretions to which it is exposed. Immunoglobulin from infected animals was thus expected to bind strongly to the cuticle because it is the external surface of the nematode and is sloughed during molting. Also, adult O. circumlitus have large and highly active ES glands (Elson-Riggins et al., 2002); thus we expected these to bind Ig strongly in infected animals. However, only PHS and older NES had strong binding to these antigens. Binding of Ig to internal antigens was unexpected. Host response to internal antigens may indicate parasite death or molt. Kennedy et al. (1989) showed that immune responses to secreted and somatic antigens of parasitic nematodes occur. A 14 kDa protein that occurred in ES secretions and pseudocoelomic fluid (perienteric fluid as used by Kennedy et al., 1989) of Ascaris (Ascarida) had a homologue that occurred in the pseudocoelomic fluid only of Toxocara canis (Ascarida; Kennedy et al., 1989). This protein was immunoprecipitated from both Ascaris and T. canis using antiserum to Ascaris ES products and larvae, but not using antiserum to T. canis ES secretions or larvae. Further evidence that the host may not always be exposed to internal antigens comes from work on Anisakis simplex. Hosts infected with A. simplex larvae did not respond to the 14 kDa protein, but those exposed to A. simplex homogenate did (Kennedy et al., 1988). Anisakis simplex was shown to contain a homologue of the 14 kDa Ascaris protein, but the host did not have an IgG response. In addition, hosts infected with A. simplex had a stronger immune response to ES components than somatic materials.
In light of the current results and the fact that clinical signs and lesions associated with O. circumlitus infection can be severe in juvenile NES, it is likely that many of these parasites die and disintegrate in this host. From 1992 to 1995, 37% of NES weanling deaths and 12% of PHS deaths at TMMC were associated with O. circumlitus (Gulland et al., 1997). Juvenile NES often died before the female nematodes started shedding larvae in the feces. High numbers of dead and disintegrating parasites could result from the severe inflammatory response to migrating larvae observed in juvenile NES (Gulland et al., 1997). In contrast, yearling NES often shed O. circumlitus larvae in feces and rarely show clinical signs associated with the infection (Gulland, pers. obs.).
Although conclusions regarding the quantitative aspects of the humoral responses were limited by our techniques, this work suggested that NES 1 yr old infected with O. circumlitus and the majority of PHS infected with O. circumlitus had a stronger immune response to the ES glands of O. circumlitus than the other tissues. Thus, the different age classes of NES seem to respond differently to the ES glands. It is not possible, however, to determine from the current results whether the older NES are responding to additional proteins in the ES glands or whether they are producing a stronger immune response to the same proteins as the younger NES. Assays such as enzyme-linked immunosorbent assays (ELISA) are needed to quantify the response. A further limitation of this study is that the Ig detected was mostly IgG, rather than IgE. This is a consequence of the use of protein A to detect Ig binding. Protein A was used because it is a readily available reagent and has been previously used for phocid Ig detection. However, there are likely to be species differences in the amount and type of Ig detected by this method. Ideally, IgE should be investigated in future studies as this class of Ig is commonly involved in host reaction to parasites (Kennedy, 2000).
Four hypotheses may account for the difference in Ig binding patterns between the NES age classes. First, older NES may be better able to respond to the proteins secreted by the ES gland than younger NES, this response being protective. Onderka (1989) suggested that the peribronchial inflammatory reaction of ringed seals to O. circumlitus might be a response to secretions from the excretory pore. This author reported that the anterior 5–10 mm of the nematodes were embedded in the peribronchial tissue. Thus, since the excretory pore is usually 437–617 µm from the anterior of the nematode (Elson-Riggins et al., 2001), it is possible that secretions from the ES glands are responsible for the host response. A second explanation is that younger NES may have such a strong response to the parasites that the nematodes do not survive to the stage where the glands are actively secreting; that is, the majority of the parasites die and disintegrate in the host and the host usually dies from the infection. Thus, the older NES may have an increased Ig response to the ES glands because they are exposed to higher concentrations of these proteins as the worms mature. The younger NES would still be exposed to the contents of the ES glands when the parasites die and disintegrate, but these proteins would be present in lower concentrations, hence the lesser immune response. A third possibility is that there is a sequential recognition of ES antigens over time. This might occur if the parasite undergoes antigenic switching. Juvenile NES tend to be infected with smaller and more immature worms than PHS and NES 1 yr and older (Gulland et al., 1997). Thus, small nematodes may not be secreting the same ES antigens as larger nematodes. The fourth potential explanation is that there is individual variation in response to O. circumlitus antigens, irrespective of age. If individuals responding to a suite of antigens excluding the strong response to the ES glands died as juveniles, whereas individuals responding strongly to the ES glands survived infection, a change in response with age would be observed. Determination of which explanation is most likely will require longitudinal studies of the immune response of individual seals to O. circumlitus over time.
The majority of the infected PHS had an Ig binding pattern to O. circumlitus that appeared to be similar to that of the 1- to 2-yr-old NES. Immunoglobulin bound to all nematode tissues, but binding was strongest for the ES glands. Pacific harbor seals appear to be better adapted to O. circumlitus infection than juvenile NES (Gulland et al., 1997). Gulland et al. (1997) reported that PHS shed larvae in the feces much earlier than NES, beginning in June as compared with August for NES. Deaths took place much earlier in NES, occurring from April to August during the prepatent period, compared with June to December during the patent period for PHS. However, once NES reach approximately 1 yr of age, they are more likely to survive the infection.
A significant immune response to the parasite was not detected in two of the PHS that were infected with O. circumlitus. It is not known whether this caused them to succumb to the infection any faster than the other PHS. Interestingly, one of these animals had 10 O. circumlitus in the right ventricle and pulmonary artery and the other had 50 O. circumlitus in the pulmonary artery. A fecal examination on one of these animals was negative for O. circumlitus larvae. Mortality of PHS from O. circumlitus during the prepatent period is not typical (Gulland et al., 1997). Two other infected PHS had a similar Ig binding pattern to that of the juvenile NES. One of these two PHS succumbed to DIC as a result of the infection, which is a more typical host-parasite reaction in juvenile NES than in PHS (Gulland et al., 1997). It is therefore possible that this Ig binding pattern may be typical of disease during the prepatent period. This animal had 30 adult O. circumlitus in the vena cava. On necropsy, the second PHS had 24 adult O. circumlitus in the pulmonary artery. Arteritis of the colon was also evident. This may have resulted from aberrant parasite migration. Further work on individual cases is required to establish whether there is any link between the Ig binding pattern, location of the nematodes, prepatent period, and severity of the infection.
The response of uninfected PHS was much less uniform than the uninfected NES. However, strong Ig binding to the ES glands was not detected in any of these animals. No difference in the banding pattern was detected between the older infected PHS tested and the younger infected PHS tested. If the ES glands are an important source of antigens that are deleterious to the host, then there is probably no need for a shift in pattern with age since young PHS have a banding pattern similar to older NES. It would be of benefit, however, to test a greater number of older PHS.
It was possible to distinguish O. circumlitus from the other nematode genera tested by running extracts of whole nematodes on gels and probing with seal serum. Furthermore, all juvenile NES tested that were infected with O. circumlitus, unlike uninfected NES, had Ig that bound to a 28 kDa protein that was dominant in the female nematode reproductive tract. Immunoglobulins binding to this protein were also detected in the serum of some of the infected 1- to 2-yr-old NES and in serum of three infected PHS. In addition, one uninfected PHS also had Ig that bound to a protein of the same size. The intensity of the band using serum from this animal did not, however, appear to be greater for the female reproductive tract of O. circumlitus than the other nematode tissues. Because mortality of NES infected with O. circumlitus occurs during the pre-patent period, ante mortem diagnosis of infections is not currently possible in this host species. Due to the intensity of the 28 kDa band in the female reproductive tract of O. circumlitus in Western blots probed with serum from juvenile infected NES and its absence in uninfected NES, we suggest that this protein may be a suitable marker for an antibody-based diagnostic test for O. circumlitus in these animals. There was, however, a band of this size present in extracts of Parafilaroides sp. in blots probed with serum from infected juvenile NES. Further work is therefore required to determine whether this is the same protein in both genera and hence its suitability as a marker for O. circumlitus infection. However, in contrast to O. circumlitus infection, juvenile NES infected with Parafilaroides sp. shed larvae in their feces when clinical signs occur. Therefore, even if this protein proves to be the same in both nematode genera, it may be possible to use the 28 kDa band as a marker of O. circumlitus infection in conjunction with a fecal exam for Parafilaroides larvae in clinical infections.
In conclusion, there are marked differences in Ig binding patterns to antigens of O. circumlitus among NES of different age classes and between seal species. Further studies are required to quantify antibody binding and to determine association between differences in immune response and clinical signs and lesions.
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ACKNOWLEDGMENTS |
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Received for publication 9 February 2004.
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