Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus

Cells of Catharanthus roseus (L.) G. Don were genetically engineered to over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) and tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key steps in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultures established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic pathway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic activity through the pathway. Two such lines accumulated over 200 mg · L⁻¹ of the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, including ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation by highly productive transgenic lines showed considerable instability and was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High transgene-encoded TDC activity was not only unnecessary for increased productivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appeared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression of Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield positive results only in the presence of a favorable epigenetic environment. Received: 12 June 1997 / Accepted: 24 October 1997     

High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and β-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54–95% of the cotyledon explants on MXB selective medium containing 200 μg ml⁻¹ kanamycin and 500 μg ml⁻¹ carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4–5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode. Received: 13 July 1999 / Accepted: 8 August 1999     

Hairy roots cultures from different Solanaceous species have varying capacities to produce E. coli B-subunit heat-labile toxin antigen

The gene encoding enterotoxigenic Escherichia coli B-subunit heat-labile toxin (LTB) antigen was co-transformed into hairy root cultures of Nicotiana tabacum (tobacco), Solanum lycopersicum (tomato) and Petunia parodii (petunia) under the CaMV35S promoter. Tobacco and petunia roots contained ~65–70 μg LTB g⁻¹ tissue whilst hairy roots of tomato contained ~10 μg LTB g⁻¹. Antigen at ~600 ng ml⁻¹ was detected in growth medium of tobacco and petunia. Tobacco roots with higher LTB levels showed growth retardation of ~80% whereas petunia hairy roots with similar levels of LTB showed only ~35% growth retardation, relative to vector controls. Regeneration of plants from LTB-containing tobacco hairy roots was readily achieved and re-initiated hairy roots from greenhouse-grown plants showed similar growth and LTB production characteristics as the original hairy root cultures.     

Statistical medium optimization for enhanced azadirachtin production in hairy root culture of Azadirachta indica

Azadirachtin, a well-known biopesticide, is a secondary metabolite extracted from the seeds of Azadirachta indica. In the present study, azadirachtin was produced in hairy roots of A. indica, generated by Agrobacterium rhizogenes-mediated transformation of leaf explants. Liquid cultures of A. indica hairy roots were developed with a liquid-to-flask volume ratio of 0.15. The kinetics of growth and azadirachtin production were established in a basal plant growth medium containing MS medium major and minor salts, Gamborg’s medium vitamins, and 30 g l⁻¹ sucrose. The highest azadirachtin accumulation in the hairy roots (up to 3.3 mg g⁻¹) and azadirachtin production (∼44 mg l⁻¹) was obtained on Day 25 of the growth cycle, with a biomass production of 13.3 g l⁻¹ dry weight. To enhance the production of azadirachtin, a Plackett–Burman experimental design protocol was used to identify key medium nutrients and concentrations to support high root biomass production and azadirachtin accumulation in hairy roots. The optimal nutrients and concentrations were as follows: 40 g l⁻¹ sucrose, 0.19 g l⁻¹ potassium dihydrogen phosphate, 3.1 g l⁻¹ potassium nitrate, and 0.41 g l⁻¹ magnesium sulfate. Concentrations were determined by a central composite design protocol and verified in shake-flask cultivation. The optimized medium composition yielded a root biomass production of 14.2 g l⁻¹ and azadirachtin accumulation of 5.2 mg g⁻¹, which was equivalent to an overall azadirachtin production of 73.84 mg l⁻¹, 68% more than that obtained under non-optimized conditions.     

Genetic transformation of <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis> by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>: iridoid and phenylethanoid glycoside accumulation in hairy root cultures

A genetic transformation method using Agrobacterium rhizogenes was developed for Harpagophytum procumbens. The influence of three factors on hairy root formation was tested: bacterial strains (A4 and ATCC 15834), various types of explants and acetosyringone (AS) (200 μM). The highest frequency of transformation (over 50% of explants forming roots at the infected sites after 6 weeks of culture on Lloyd and McCown (WP) medium) was achieved using a combination of nodal stem explants and A. rhizogenes strain A4. The addition of 200 μM AS to root induction medium was found to enhance hairy root induction but its effect varied depending on bacterial strain and explant type. Three of the most vigorously growing hairy root clones of H. procumbens were chosen and analyzed for accumulation of iridoid and phenylethanoid glycosides. The transgenic nature of these root clones was confirmed by PCR amplification; they were positive for rolB and rolC genes. Harpagoside, verbascoside and isoverbascoside were identified by HPLC and LC–ESI-MS as the major compounds from all analyzed hairy root clones. The Hp-3 root clone showed the higher harpagoside content (0.32 mg g⁻¹ dry wt.) compared with other analyzed transformed and non-tuberized untransformed roots of H. procumbens. However, the level of the compound in the hairy root clone was lower than that detected in a sample of commercially available root tubers of H. procumbens. The Hp-3 root clone also produced high amounts of verbascoside and isoverbascoside (8.12 mg g⁻¹ dry wt. and 9.97 mg g⁻¹ dry wt., respectively) comparable to those found in root tubers.     

Micropropagation and hairy root culture of <Emphasis Type="Italic">Solanum Laciniatum</Emphasis> Ait.

Summary An efficient system for in vitro micropropagation of Solanum laciniatum Ait. has been established. Shoot induction on leaf explants was most successful on Murashige and Skoog (MS) medium supplemented with 10 μM N⁶-benzyladenine (BA) and 1 μM α-naphthaleneacetic acid (NAA). BA (13 μM) was optimal for further shoot multiplication, and rooting of separated shoots was achieved on medium without plant growth regulators. At each subculture, 20–25 shoots were obtained on each explant, from which six to eight were suitable for separation and further rooting. Leaf explants grown in vitro were successfully infected by Agrobacterium rhizogenes ATCC 15834. The established hairy root culture was, on the basis of dry weight, more productive when grown on half-strength MS medium than on full-strength MS (3% sucrose) and full-strength MS (6% sucrose) medium. The amount of solasodine-containing glycoalkaloids in hairy roots as measured by a colorimetric method was 0.3–1% of dry weight, which is higher than in the shoot culture (0.5% of dry weight) and lower than in leaves of in vivo-grown plants (1.1–1.4% of dry weight). The amount of solasodine-containing glycoalkaloids in leaves of in vivo-grown plants of S. laciniatum was similar to the related species Solanum aviculare Forst. Both species are morphologically similar, therefore we effectively distinguished them by flow cytometry. The genome size of S. laciniatum was determined as 4.03 pg and the genome size of S. aviculare as 1.69 pg.     

Nickel tolerance and hyperaccumulation in shoot cultures regenerated from hairy root cultures of <Emphasis Type="Italic">Alyssum murale</Emphasis> Waldst et Kit

Shoot cultures of nickel hyperaccumulating Alyssum murale were established from epicotyl explants of seedlings aseptically germinated on hormone-free MS medium. They were further maintained on media with 0–0.92 μM kinetin. Optimal shoot multiplication was at 0.46 μM kinetin. Inoculation by shoot wounding was performed with overnight suspension of A. rhizogenes A4M70GUS which contains GUS gene cointegrated in pRiA4. After 30 days hairy roots were produced at the wounding site in 31 explant (25% out of 124). Hairy roots were excised and further propagated on hormone-free medium as separate clones. In the first passage clones 3 and 6 could be distinguished by fast growth and spontaneous shoot regeneration. In other clones (12, 23 and 25) shoot regeneration required presence of cytokinins. The five shoot culture clones regenerated from hairy roots were further cultured on media with 0.46 μM kinetin. These shoots were characterized by good elongation and lateral shoot branching, short internodes, minute slightly curled leaves and well developed plagiotropic root system spreading over the surface of media. Thus all plants regenerated from hairy root cultures manifested the characteristic Ri syndrome phenotype. They all had a strong positive GUS reaction. PCR analysis confirmed presence of uidA sequence from the gus construct. They were also tolerant to nickel accumulating up to 24,700 μg g⁻¹ dry weight.     

Modulation of plumbagin production in Plumbago zeylanica using a single-chain variable fragment antibody against plumbagin

A single-chain variable fragment antibody (scFv) against plumbagin (PL) accumulated the PL production in the hairy roots of Plumbago zeylanica. Recombinant Agrobacterium rhizogenes (ATCC 15834) containing an scFv gene against PL (PL-scFv) were obtained through triparental mating and transformed into P. zeylanica to induce PL-scFv protein in the hairy roots. Up to 40 μg recombinant PL-scFv were expressed per milligram of soluble protein in transgenic P. zeylanica hairy root cultures. The mean PL content obtained from transgenic hairy roots (12.24 μg/100 mg dry weight) exhibited 2.2 times higher than those obtained from wild-type (5.48 μg/100 mg dry weight). The high correlation between the PL-scFv expression level and PL content of the recombinant plants suggested that the PL biosynthesis pathway had been modulated by the expression of PL-scFv protein in the hairy roots of P. zeylanica.     

Direct rhizogenesis and establishment of fast growing normal root organ culture of Withania somnifera Dunal

Direct rooting from leaf explants of Withania somnifera was achieved on half strength Murashige and Skoog’s medium supplemented with 15 g l⁻¹ sucrose, and different concentrations of growth regulators. Basal medium supplemented with 2.85 μM indoleacetic acid and 9.85 μM indolebutyric acid achieved maximum number of roots with 100% response. The roots were cultured on MS liquid medium for the establishment of root-organ culture with the same plant growth regulators and incubated on an orbital shaker at 80 rpm at 25 ± 2 °C. A root biomass of 6.15 ± 0.17 g was obtained after 5 weeks. When 1 g roots were inoculated to 2.5 l bubble column reactor, 47 g roots were obtained after 6 weeks. The concentration of alkaloids was increased as compared to field grown roots. The maximum concentration of withanolides (10 mg g⁻¹ dry weight) was obtained in the bioreactor.     

Improvement of artemisinin production by chitosan in hairy root cultures of <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Artemisinin production by hairy roots of Artemisia annua L. was increased 6-fold to 1.8 μg mg⁻¹ dry wt over 6 days by adding 150 mg chitosan l⁻¹. The increase was dose-dependent. Similar treatment of hairy roots with methyl jasmonate (0.2 mM) or yeast extract (2 mg ml⁻¹) increased artemisinin production to 1.5 and 0.9 μg mg⁻¹ dry wt, respectively.     

A comparison between hairy root cultures and wild plants of Saussurea involucrata in phenylpropanoids production

Saussurea involucrata is an important medicinal plant that produces a few bioactive secondary metabolites, such as hispidulin, rutin, and syringin. Previously, we established a hairy root culture system for this species through Agrobacterium-mediated transformation. The present study addressed the issue as how hairy root cultures perform in phenylpronoid accumulation. From the ethanolic extract of a hairy root culture established for Saussurea involucrata, syringin, rutin and hispidulin, were isolated and their chemical structures were confirmed by HPLC-ESI-MS. A quantitative study of the compounds showed great levels of syringin and hispidulin (being 43.5±1.13 and 0.34±0.023 mg g⁻¹ dry weight, respectively), about 40 and 3 times, respectively, higher than those from wild plants. But, the levels of rutin from hairy roots were much lower (0.71±0.043 vs. 6.59±0.56 mg g⁻¹ dry weight). Compared with untransformed root cultures, syringin and hispidulin levels were also higher. An experiment on culture media showed that MS was superior to others for phenylpropanoids accumulation in hairy roots, a 28-day culture produced 405 mg l⁻¹ syringin.     

Accumulation of Heavy Metals by Ectomycorrhizal Fungi Colonizing Birch Trees Growing in an Industrial Desert Soil

The results of analyses conducted to determine contents of Cd(II) and Pb(II) in mushrooms of mycorrhizal fungi and selected parts of birches growing in an industrial desert surrounding a nonferrous works are presented in this study. The fruiting bodies of fungi accumulated several times higher contents (up to 80 μg g⁻¹ dry weight) of Cd(II) compared to those found in the soil (20 μg g⁻¹ dry weight). In contrast, the mushroom contents of Pb(II) were only slightly increased (up to 895 μg g⁻¹ dry weight) than those present in the soil (500 μg g⁻¹ dry weight). However, fivefold higher concentrations of the metals were found in the mycorrhizal roots. Comparing the distribution of the metals analysed, the protective role of the ectomycorrhizae in relation to the host tree was indicated. Mycorrhizal fungi persistently fixed heavy metals, forming an efficient biological barrier that reduced movement of the metals in birch tissues.     

Zn biosorption by Rhizopus arrhizus and other fungi

Biosorption of zinc ions by inactivated fungal mycelia was studied. Of the six fungal species, Rhizopus arrhizus, Mucor racemosus, Mycotypha africana, Aspergillus nidulans, Aspergillus niger and Schizosaccharomyces pombe, R. arrhizus exhibited the highest capacity (Q ^max = 213 μmol g⁻¹ dry weight). Further experiments with different cellular fractions of R. arrhizus showed that Zn was predominantly bound to cell-wall chitin and chitosan (Q ^max = 312 μmol g⁻¹ dry weight). Adsorption data were best modelled by the Langmuir isotherm, although they can be modelled by the Freundlich equation as well at relatively low aqueous concentrations. Biosorption generally decreased with increase in biosorbent particle size and its concentration. Low pH reduced Zn sorption, because of the strong competition from hydrogen ions for binding sites on fungi. The presence of ligands reduced metal uptake, chiefly by forming metal complexes of a less biosorbable nature. Received: 2 November 1998 / Received revision: 12 January 1999 / Accepted: 17 January 1999     

Mercury and cadmium concentrations in the tissues of three species of southern albatrosses

Cadmium and mercury concentrations were measured in the tissues of 64 individual albatrosses [23 wandering albatrosses (Diomedea exulans), 9 royal albatrosses (Diomedea epomophora) and 32 shy albatrosses (Thalassarche cauta)] which were killed as by-catch in longline fishing activities between 1991 and 1994. Mercury concentrations were also determined for 33 shy albatross eggs (excluding shells). The birds were all sexed and assigned to one of two age classes (immature and adult). The three species exhibited differences both in overall concentrations of cadmium and mercury, and also in the pattern of accumulation of metals with age and sex. Wandering albatrosses exhibited the highest mercury concentrations with a mean concentration in adult liver samples of 920.0 ± 794.1 μg g⁻¹ dry weight. Shy albatrosses had the lowest mercury concentrations with mean concentrations in adult livers of 36.3 ± 21.4 mg g⁻¹ dry weight. The highest mercury concentration was 1800 μg g⁻¹ for an adult female wandering albatross. Cadmium concentrations were less variable, with adult royal albatrosses having the highest average concentrations (180.0 ± 165.0 in adult kidneys) and adult shy albatrosses the lowest (40.1 ± 20.0 in adult kidney). The highest individual cadmium concentration was 287 μg g⁻¹ for a juvenile wandering albatross. There was no evidence of increased accumulation of cadmium with age in any of the species, but wandering albatrosses showed higher mercury concentrations in adults than juveniles. Female wandering albatrosses also had significantly higher mercury concentrations than males. The mercury contents of the shy albatross eggs were very low, with a maximum concentration of 5.4 μg g⁻¹. The results of this study are consistent with the findings of previous work on albatrosses and support the notion that the life-history strategy of these species (i.e. long-lived with low reproductive output) may be an important determinant in the concentrations of some metals found in their tissues. Accepted: 15 February 1999     

Seasonal changes in growth rate, carrageenan yield and lectin content in the red alga Kappaphycus alvarezii cultivated in Camranh Bay, Vietnam

The three color morphotypes of the red alga Kappaphycus alvarezii (brown, red and green) were cultured in Camranh Bay, Vietnam, using the fixed off-bottom monoline culture method to evaluate the growth rate, carrageenan yield, 3,6-anhydrogalactose, gel strength and lectin content. The brown morphotype was cultivated over a 12-month period; the red and green morphotypes were over a 6-month period. At the 60-day culture timepoint, the brown morphotype showed a higher growth rate (3.5–4.6% day⁻¹) from September to February, and lower growth rate (1.6–2.8% day⁻¹) from March to August. Significant (P < 0.05) differences in growth rate between culture months were found with the brown morphotype. High growth rates for the red (3.6–4.4% day⁻¹) and green (3.7–4.2% day⁻¹) morphotypes were obtained from September to February. The carrageenan yield, 3,6-anhydrogalactose and gel strength of the three morphotypes showed little variation, with the highest values obtained in November–December. At the 30-day sampling point, the brown morphotype had a higher lectin content (167–302 μg g⁻¹ dry alga) from August to March and a lower lectin content (23–104 μg g⁻¹ dry alga) from April to July. High lectin contents were recorded for the red (139–338 μg g⁻¹ dry alga) and green (124–259 μg g⁻¹ dry alga) morphotypes from September to February. This study shows that the different morphotypes of K. alvarezii can be grown in the tropical waters of the Camranh during the northeast monsoon, and part of the southwest monsoon, especially the brown morphotype, which can be grown during any season.     

Camptothecin biosynthetic genes in hairy roots of Ophiorrhiza pumila: cloning,characterization and differential expression in tissues and by stress compounds

Camptothecin derivatives are clinically used anti-tumor compounds that biogenetically belong to a group of monoterpenoid indole alkaloids (TIA). We have already established a hairy root culture of Ophiorrhiza pumila (Rubiaceae) that produces camptothecin. The present study describes the cloning and characterization of cDNAs encoding strictosidine synthase (OpSTR; EC 4.3.3.2) and tryptophan decarboxylase (OpTDC; EC 4.1.1.28), two key enzymes in the biosynthesis of TIA from hairy roots of O. pumila. We also isolated the cDNA coding for NADPH:cytochrome P450 reductase (OpCPR; EC 1.6.2.4) that is presumed to be indirectly involved in camptothecin synthesis. The recombinant OpSTR and OpTDC proteins exhibit STR and TDC activities, respectively, when expressed in Escherichia coli. The tissue-specific and stress-inducible expression patterns of OpSTR and OpTDC were quite similar, unlike those of OpCPR. The high expression of OpSTR and OpTDC observed in hairy roots, roots and stems were closely correlated with STR protein accumulation as observed by immunoblot analysis. Plant stress compounds like salicylic acid repressed expression of OpSTR and OpTDC, suggesting coordinate regulation of these genes for camptothecin biosynthesis.     

Optimization of protoplast yields from the red algae <Emphasis Type="Italic">Gracilaria dura</Emphasis> (C. Agardh) J. Agardh and G. <Emphasis Type="Italic">verrucosa</Emphasis> (Huds.) Papenfuss

This study reports on the optimization of protoplast yield from two important tropical agarophytes Gracilaria dura and Gracilaria verrucosa using different cell-wall-degrading enzymes obtained from commercial sources. The conditions for achieving the highest protoplast yield was investigated by optimizing key parameters such as enzyme combinations and their concentrations, duration of enzyme treatment, enzyme pH, mannitol concentration, and temperature. The significance of each key parameter was also further validated using the statistical central composite design. The enzyme composition with 4% cellulase Onozuka R-10, 2% macerozyme R-10, 0.5% pectolyase, and 100 U agarase, 0.4 M mannitol in seawater (30‰) adjusted to pH 7.5 produced the highest protoplast yields of 3.7 ± 0.7 × 10⁶ cells g⁻¹ fresh wt for G. dura and 1.2 ± 0.78 × 10⁶ cells g⁻¹ fresh wt for G. verrucosa when incubated at 25°C for 4–6 h duration. The young growing tips maximally released the protoplasts having a size of 7–15 μm in G. dura and 15–25 μm in G. verrucosa, mostly from epidermal and upper cortical regions. A few large-size protoplasts of 25–35 μm, presumably from cortical region, were also observed in G. verrucosa.     

Effects of alkaloid precursor feeding on a Camptotheca acuminata cell line

To better understand the biosynthesis of Camptotheca acuminata alkaloids, the effect on camptothecin production of feeding with potential precursors of biosynthesis was studied (i.e., tryptamine and loganin combined, secologanin, and strictosidine). Two key enzymes in alkaloid biosynthesis 〚i.e., tryptophan decarboxylase (TDC; EC 4.1.1.28) and strictosidine synthase (STR; EC 4.3.3.2)〛 were also studied. The analyses were conducted using a C. acuminata CG1 cell line that does not produce alkaloids, which could be useful in better understanding the biosynthetic pathway and in identifying possible limiting factors. The activity of TDC was 5 pkat mg–1; the activity of STR was 1.1 pkat mg^–1. Feeding with strictosidine revealed that this precursor is easily biotransformed by two enzymes (i.e., a hydroxylase and a dehydrogenase) in hydroxystrictosidine and didehydrostrictosidine, but camptothecin was never detected. The indole pathway and the low level of STR activity could be limiting factors in the production of camptothecin in the cell line used.     

Adaptive Copper Tolerance in <Emphasis Type="Italic">Elsholtzia haichowensis</Emphasis> Involves Production of Cu-induced Thiol Peptides

Copper (Cu) accumulation and tolerance mechanisms in Elsholtzia haichowensis, an indicator plant of Cu mines, were investigated under hydroponics supplied with different concentrations (0.32, 50.0, 100.0 and 200.0 μM) of Cu for 8 days. Cu at 100 and 200 μM significantly decreased the root dry weight, but had no significant effect on shoot dry weight. The plants grown in the presence of 200 μM Cu accumulated 288 and 7626 μg g⁻¹ DW total Cu in the shoots and roots, respectively. A greater proportion of accumulated Cu was water-soluble accounting for 42–93% of the total Cu content in the shoots. The concentrations of reduced glutathione (GSH) and protein thiols were significantly enhanced under excess Cu supply. However, the concentrations of these compounds, particularly protein thiols, were much higher in the leaves than that in the roots. Three UV-absorbing peaks could be eluted out through gel filtration chromatography on Sephadex G-50. A large amount of Cu was detected in the UV-absorbing peaks in 40–50 and 70–90 ml elution fractions of the root extract, and in 40–50 and 120–140 ml elution fractions of the leaf extract. The results suggested that the adaptive Cu tolerance mechanism in E. haichowensis might involve the active participation of protein thiols which had a more important role in the leaves than in the roots.     

Feeding dynamics and respiration of the bottom-dwelling caridean shrimp Nauticaris marionis Bate, 1888 (Crustacea: Decapoda) in the vicinity of Marion Island (Southern Ocean)

The feeding dynamics and oxygen uptake of the bottom-dwelling caridean shrimp Nauticaris marionis were studied during the April/May 1984, 1996 and 1997 cruises to Marion Island (Prince Edward Islands, Southern Ocean). N. marionis is thought to have an opportunistic feeding mode. Prey composition varied considerably between the years and sites investigated. Overall, benthic (mainly hydrozoans and bottom-dwelling polychaetes) and, at times, pelagic (largely euphausiids and copepods) prey items dominated in the stomachs of N. marionis both by occurrence and by volume. Generally, pelagic prey contributed more to the diets of smaller shrimps, while benthic prey was a more important component in the guts of larger specimens. Wet, dry and ash-free dry weight were determined for specimens used in respiration experiments. The respiration rates of N. marionis females with carapace length 6.6–11.1 mm ranged from 80 to 250 μl O₂ individual⁻¹ · h⁻¹, or from 0.588 to 2.756 μl O₂ · mg⁻¹ dry weight h⁻¹. Regression analyses showed highly significant correlations between oxygen consumption and carapace length for N. marionis. Daily ingestion rates estimated using an in situ gut content analysis technique (4.4% of body dry weight) and an energy budget approach (average 4.7% of body dry weight, range 2.0–7.5%) showed good agreement with each other. Accepted: 29 July 1998