Development of eggs and nauplii of Euphausia superba

Summary The development of eggs and nauplii was studied over 19 days. At 1.5°C nauplii hatched after 5 days. Abdominal spines changed their appearance successively during nauplius development, but no moulting was observed in the course of naupliar development. Moulting occurred between the nauplius and the metanauplius stage 13 days after spawning. Swimming activity was weak in the nauplius, while it was vigorous in the metanauplius. A model is presented for the depth distribution of eggs and larval stages. It is also suggested that present collection techniques may inadequately sample eggs and nauplii based on experiments that show that eggs are extremely sensitive to sieving.     

Relationship Between N:P Ratio and Growth Rate During the Life Cycle of Calanoid Copepods: An in situ Measurement

The mean nitrogen and phosphorus contents of the calanoid copepodMixodiaptomus laciniatus Lilljeborg were measured to the stagelevel throughout the ice-free period of a high mountain lake.Our results indicate large intraspecific variations in calanoidelemental composition. While mean N as dry weight increasedfrom 3.0 ± 1.3 in nauplii to 6.0 ± 2.1 in copepodites,mean P content showed the opposite trend, varying intraspecificallyfrom 0.98 ± 0.26% in nauplii, to 0.87 ± 0.21%in copepodites and 0.51 ± 0.16% in adults. Thus, themean N:P ratio increased ontogenetically from 3.3 in naupliito 13.3 in copepodites and 24.6 in adults. Two ontogenetic parameters,the growth rate and body size, were associated with zooplanktonstoichiometry. Among all 11 copepod stages, growth rate waspositively related to %P and negatively related to %N and N:Pratio. A two-part analysis of these relationships, before andafter metamorphosis, showed that the nauplii growth rate explainednearly all the variance in naupliar P content. A high P contentin nauplii may reflect a high content of RNA, translating intorapid growth rates. Overall, these results tend to support thehypothesis linking specific growth rate with P content for copepods,but these results also suggest that the validity of this hypothesisis robustness when differences in the life history of copepodsas a consequence of metamorphosis are accounted for. We suggestthat the intra-stage variation in P content is associated withpeaks of intensive metabolic activity during the process ofmolting in copepods, and we emphasize the importance of newempirical evidence to examine this hypothesis further.     

Glasgow Lake: an early-warning sentinel of lake acidification in Cape Breton Highlands National Park (Nova Scotia,Canada)

In contrast to other lakes studied in Cape Breton Highlands National Park (Nova Scotia, Canada), our paleolimnological results indicated that Glasgow Lake has been impacted by acidic deposition starting in the early 1900s. Based on analysis of diatom assemblages, the lake experienced a decrease in diatom-inferred lakewater pH from a pre-industrial pH of ~5.8 to a current pH of 5.3 (2000–2002 measured mean pH = 5.0) as well as a decrease in diatom-inferred Gran-alkalinity. In this study, diatom-based paleolimnological techniques were used in conjunction with a dynamic biogeochemical model (MAGIC) to assess both the timing and extent of the acidification trend, as well as determine a probable explanation as to why this lake, and none of the other 15 Cape Breton Highlands lakes studied for paleolimnology thus far, acidified under a peak non-marine sulphate deposition load of 43.6 mmol_c m⁻² year⁻¹ in the mid-1970s. Steady-state models estimate that Glasgow Lake had the lowest buffering capacity of six study lakes and estimated critical sulphate loading of <1 mmol_c m⁻² year⁻¹. MAGIC also estimated a loss of charge balance alkalinity from a pre-1850 value of 38 μmol_c l⁻¹ to a low of 12 μmol_c l⁻¹. While no evidence of biological recovery has been recorded, MAGIC estimates an increase in charge balance alkalinity to 27 μmol_c l⁻¹ in 2002 in response to decreased SO₂ emissions. Of the five other lakes that were modelled, all showed trends towards more acidic states and subsequent increases in charge balance alkalinity; however, the empirical paleo-diatom approach applied to these lakes showed no evidence of acidification. Thus, Glasgow Lake has the lowest buffering capacity among the Cape Breton Highland study lakes and serves as a sentinel of potential acidification trends and recovery in this region. Handling editor: J. Saros     

Acid and aluminum effects on osmoregulation and survival of the freshwater copepod Skistodiaptomus oregonensis

The freshwater copepod Skistodiaptomus oregonensis was exposed,in laboratory bioassays, to a 3 x 3 factorial array of pH andaluminum treatments (pH 4.5, 6.0 and 7.5, and 0, 100 or 200µg l^–1 Al). After 12 and 24 h, whole-body Na contentand survivals were determined for groups of 10 animals per treatment.Declines in bodily Na were taken as evidence of osmoregulatorydysfunction. Both Na content and survival were reduced at pH6.0 and especially at pH 4.5. The magnitude of responses dependedon the Al concentration. Exposure to the highest Al level tendedto reduce Na content and survival at pH 6.0, but tended to enhanceNa content and survival at pH 4.5. The results suggest thatextinction of large copepods during lake acidification is duein part to effects of acid stress on osmoregulation and thatAl toxicity may be a contributing factor, since population declinesin nature occur at pH near 6.0.     

A novel α-galactosidase from <Emphasis Type="Italic">Βifidobacterium bifidum</Emphasis> with transgalactosylating properties: gene molecular cloning and heterologous expression

A genomic library of Bifidobacterium bifidum (NCIMB 41171) DNA was constructed in Escherichia coli RA11r (melA⁻B⁺) and one α-galactosidase encoding gene was isolated. Conceptual translation combined with insertional mutagenesis analysis indicated an open reading frame (ORF) of 759 amino acid (aa) residues encoding an α-galactosidase (named as MelA) of 82.8 kDa. Partial purification and characterisation showed that the enzyme had an apparent native molecular mass of ≈243 kDa and a subunit size of ≈85 kDa. The enzyme belongs to glycosyl hydrolases 36 family with high aa sequence similarities (≈73%) to other known α-galactosidases of bifidobacterial origin. Under optimum pH conditions for activity (pH 6.0) and high melibiose concentration (40% w/v), the enzyme was able to form oligosaccharides with degree of polymerisation (DP) ≥3 at higher concentration than DP = 2, with a total yield of 20.5% (w/w).     

Variation in calanoid copepod resting egg abundance among lakes with different acidification histories

The maintenance of species and genetic diversity within zooplankton egg banks may be crucial to the re-establishment of zooplankton communities following historical disturbance, such as anthropogenic acidification which globally caused widespread damage to ecological communities. Despite this, no other study has described basic characteristics of zooplankton egg banks among lakes with different acidification histories, such as variation in resting egg concentration. Theoretically, habitats with frequent periods of harsh environmental conditions are expected to select for resting egg production or prolonged dormancy in zooplankton, which would increase the size of the resting egg bank in lake sediments. In this study, we compared abundances of viable and inviable calanoid copepod resting eggs among three freshwater lakes with different acidification histories. While Swan Lake underwent major chemical and biological changes from acid and metal deposition, Teardrop and Bat lakes were relatively unaffected by historical acidification and had comparatively constant, but different pH over time. We also tested the effect of age on the viability of resting eggs. As predicted, higher numbers of viable resting eggs were found in recent sediments from acid-recovering Swan Lake compared to study lakes with relatively temporally constant environments (Teardrop and Bat lakes) when the total number of eggs was held as a covariate. We detected this result in spite of similar pelagic abundances of Leptodiaptomus minutus, the dominant species in zooplankton communities of these lakes. This pattern did not necessarily hold for inviable egg concentrations since these eggs were more abundant in both Swan and Bat lakes compared to Teardrop Lake in older sediments (1939–1951, 1800s). Within study lakes, the abundance of viable resting eggs declined with increased egg age. Further study is required to test mechanisms underlying these patterns. Handling editor: S. I. Dodson     

Mesenterial filaments of the black coral <Emphasis Type="Italic">Cirrhipathes</Emphasis> cfr. <Emphasis Type="Italic">anguina</Emphasis> provide a home to developing nauplii

Inspection of two female colonies of the monopodial black coral Cirrhipathes cfr. anguina from the coral reef of the Marine Park of Bunaken (Indonesia) revealed the occurrence of crustacean developing eggs within the mesenterial filaments of the polyps. Egg diameter, which in the smallest gametes was about 50–60 μm, increased in tandem with embryo development, reaching the value of 170 μm, at the nauplius stage. The attribution to the crustacean taxon was derived from morphological investigations carried out in light and electron microscopy (TEM, SEM) on the eggs and on the embryos removed from them. The final stage of nauplius was characterised by three pairs of appendages: uniramouse antennulae, biramouse antennae and manidibulae. In addition, naupliar eye and caudal setae were also evident. These nauplii were ascribed to the larval stage of an unidentified species. Coral/copepod association could represent a reproductive strategy, put into action by some marine copepods. Incubation within an appropriate host prevents predation by planktotrophic organisms, thus reducing population depletion.     

Copepod nauplii use phosphorus from bacteria,creating a short circuit in the microbial loop

In subtropical oceans phytoplankton carbon: phosphorus (C : P) ratios are high, and these ratios are predicted to increase further with rising ocean temperatures and stratification. Prey stoichiometry may pose a problem for copepod zooplankton nauplii, which have high phosphorus demands due to rapid growth. We hypothesised that nauplii meet this demand by consuming bacteria. Naupliar bacterial and phytoplankton carbon and phosphorus ingestion, assimilation and incorporation were traced using ³³P and ¹⁴C radioisotopes. Bacterial carbon was incorporated four times less efficiently into biomass than phytoplankton carbon. In contrast, bacterial and phytoplankton phosphorus were incorporated at similar efficiencies, and bacteria could meet a substantial amount of naupliar phosphorus requirements. As parts of the ocean become more oligotrophic, bacteria could help sustain naupliar growth and survival under suboptimal stoichiometric conditions. Thus, nauplii may be a shortcut for phosphorus from the microbial loop to the classical food web.     

Egg production and hatching success in the peri-Antarctic copepod Calanus simillimus

In situ rates of egg production and hatching success are reportedfor Calanus simillimus, one of the most abundant calanoid speciesin peri-Antarctic regions, during the Italian ‘Italics’cruise in the Straits of Magellan in March-April 1995. Low fecundity(8.6 eggs female^–1 day^–1) and fecal pellet production(0.8 fecal pellets female^–1 day^–1) in this periodindicate that the species was feeding very poorly. Sixty-sixof the 126 females sampled did not produce eggs and 80 of thesedid not produce any fecal pellets during the 24 h period ofincubation. Striking abnormal naupliar and embryonic developmentwas recorded in 81.8% of the eggs spawned. Aberrant eggs didnot undergo normal cleavage, and failed to develop to hatching.Deformed nauplii were asymmetrical and presented strong anatomicalanomalies in the total body length and number of swimming appendages.These results are discussed in the light of recent findingson the causes of low hatching success in copepods.     

Rapid evolution of arginine deiminase for improved anti-tumor activity

Arginine deiminase (ADI), an arginine-degrading enzyme, has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas. Based on our preliminary results, it was noticed that the optimum pH of ADI from Pseudomonas plecoglossicida (PpADI) was 6.0, and less than 10% of the activity was retained at pH 7.4 (pH of human plasma). Additionally, the K _m value for wild-type ADI (WT-ADI) was 2.88 mM (pH 6.0), which is over 20 times of the serum arginine level (100–120 μM). These are two major limitations for PpADI as a potential anti-cancer drug. A highly sensitive and efficient high-throughput screening strategy based on a modified diacetylmonoxime–thiosemicarbazide method was established to isolate ADI mutants with higher activity and lower K _m under physiological pH. Three improved mutants was selected from 650 variants after one round of ep-PCR, among which mutant 314 (M314: A128T, H404R, I410L) exhibiting the highest activity. Interestingly, sequence alignment shows that three amino acid substitutes in M314 are coincident with corresponding residues in ADI from Mycoplasma arginini. The specific activity of M314 (9.02 U/mg) is over 20-fold higher than that of WT-ADI (0.44 U/mg) at pH 7.4, and the K _m value was reduced to 0.65 mM (pH 7.4). Noticeably, the pH optimum was shifted from 6.0 to 6.5 in M314. Homology model of M314 was constructed to understand the molecular basis of the improved enzymatic properties. This work could provide promising drug candidate for curing arginine-auxotrophic cancers.     

Molecular and biochemical characterization of a new alkaline β-propeller phytase from the insect symbiotic bacterium <Emphasis Type="Italic">Janthinobacterium</Emphasis> sp. TN115

A phytase-encoding gene (phyA115) was cloned from Janthinobacterium sp. TN115, a symbiotic bacterial strain isolated from the gut contents of Batocera horsfieldi larvae (Coleoptera: Cerambycidae), and expressed in Escherichia coli. The 1,884-bp full-length gene encodes a 28-residue putative signal peptide and a 599-residue mature protein with a calculated mass of 64 kDa. The deduced PhyA115 shares low identity with known sequences (47% at most) and contains an N-terminal incomplete domain (residues 29–297; domain N) and a typical β-propeller phytase domain at the C terminus (residues 298–627; domain C). Distinct from other β-propeller phytases that have neutral pH optima (pH 6.0–7.5), purified recombinant PhyA115 exhibits maximal activity at pH 8.5 and 45°C in the presence of 1 mM Ca²⁺ and is highly active over a wider pH range (pH 6.0–9.0). These results indicate that PhyA115 is a β-propeller phytase that has application potential in aquaculture feed. To our knowledge, this is the first report of cloning of a phytase gene from the symbiotic microbes of an insect digestive tract and from the genus Janthinobacterium. The N-terminal incomplete domain is found to have no phytase activity but can influence the pH property of PhyA115.     

Acidification of small mountain lakes in the High Tatra Mountains,Poland

A hydrochemical investigation was carried out on eight small High Tatra mountain lakes (Poland). When comparing recent data with those from the period 1935–1965, a constant process of acidification of the lakes is found. The average pH of precipitation is 4.8 in the study area, but the lakes are in two stages of acidification: weak (pH 6.0–6.5) and intermediate (pH 5.5–5.8). The differences are due to differences in water sources.     

Reproduction,grazing, and development of the large subarctic calanoid <Emphasis Type="Italic">Eucalanus bungii</Emphasis>: is the spring diatom bloom the key to controlling their recruitment?

The response of large calanoid, Eucalanus bungii, to environmental fluctuation, particularly in relation to the spring diatom bloom in the Oyashio region, western subarctic Pacific Ocean, was examined by investigating egg production, grazing, development and starvation tolerance. Mean in situ egg production rate increased with ambient chlorophyll-a concentration, ranging from 0 to 47 eggs female⁻¹ d⁻¹, while no diurnal synchronous spawning behavior was observed. Under the spring bloom condition, E. bungii showed prey preference for less mobile and larger-sized prey (≥30 μm ESD) and bloom-forming diatom Thalassiosira spp. accounted for >80% of ingested carbon. In the laboratory, E. bungii was successfully reared from newly hatched nauplii to adult with the diatom, Thalassiosira nordenskioldi, as a food resource. Nauplii newly hatched from eggs reached the adult stage in ca. 150 days (5°C) with a sigmoidal developmental pattern and no sexual difference in development pattern. Starvation experiments indicated that the starved copepodids (C1–C4) became more vulnerable to high temperature with the progression of developmental stage, suggesting that the post-bloom condition with low food availability and increased temperature is harsh for their copepodids. The results of this study in conjunction with previous findings suggest that E. bungii is well adapted to utilize large-sized phytoplankton, such as a bloom-forming diatoms and, therefore, their recruitment processes, including egg production, development and mortality would be strongly affected by the duration and intensity of the spring bloom.     

Homeostatic responses in the gut of Porcellio scaber (Isopoda: Oniscidea) optimize litter degradation

In order to examine the influence of differences in food conditions on gut characteristics in Porcellio scaber, pH-manipulated and microbially inoculated leaf litter from three different tree species were offered. Microbial activity was clearly influenced by the pH levels of the leaves. Analyses of the pH levels in the gut indicated the ability of P. scaber to buffer the pH value in the intestinal tract to about 5.5–6.0 in the anterior hindgut, and to about 6.0–6.5 in the posterior hindgut. The pH levels of the gut sections remained in this range, within a range of food pH from 4.0 to 7.5, no matter what kind of leaves the animals were fed. Homeostatic responses to changes in food pH guarantee optimized digestion of leaf litter. However, when the pH level of the litter dropped below 3.5, P. scaber was not able to maintain the pH conditions in the gut. Furthermore, microorganisms colonizing the litter biased the pH level in the anterior hindgut where digestive processes mainly take place. These results indicate a decline of litter quality with regard to the nutrition of terrestrial isopods, caused by acidification and consequently reduced microbial activity. Accepted: 19 July 1997     

Necator americanus: temperature, pH, light, and larval development, longevity, and desiccation tolerance

The effect of incubation temperature and pH on the hatch rate of eggs of Necator americanus, and the desiccation tolerance of the resulting infective stage-3 larvae were investigated in the laboratory under controlled conditions. Hatching did not occur below 15 C and above 35 C. A 21% hatch rate was obtained at 15 C while a 10.6% hatch rate was obtained at 35 C. The highest hatch rate (93.7%) was obtained at 30 C. The optimum pH for hatching was 6.0, but the larvae did not reach the infective stage. Incubation temperature of the eggs affected the longevity and desiccation tolerance of resultant infective larvae. Larvae hatched at 30 C and maintained at 26 C under bright fluorescent light had a 50% survival time (S50) of 4 days. In the dark or shade, the S50 for larvae raised at 30 C was 5 weeks, while that of larvae hatched at 20 C was 7 weeks. Incubation temperature also affected the desiccation tolerance of larvae. Larvae developed at 20 C were more resistant to desiccation at various relative humidity values than larvae hatched at 30 C.     

Demographic characteristics of the copepod <Emphasis Type="Italic">Acanthocyclops americanus</Emphasis> (Sars, 1863) (Copepoda: Cyclopoida) fed mixed algal (<Emphasis Type="Italic">Scenedesmus acutus</Emphasis>)-rotifer (<Emphasis Type="Italic">Brachionus havanaensis</Emphasis>) diet

The predatory copepod Acanthocyclops americanus is commonly found in shallow ponds and lakes in Mexico. We tested the survivorship and reproduction-related variables of this copepod, isolated from Lake Huetzalin (Xochimilco, Mexico City), on four mixed diets comprising two algal concentrations (0.8 × 10⁶ and 1.6 × 10⁶ cells ml⁻¹ of Scenedesmus acutus) and two rotifer densities (1 and 8 ind. ml⁻¹ of Brachionus havanaensis). Survivorship patterns of naupliar and adult stages of A. americanus were evaluated at 24 ± 1°C. The demography experiments were initiated with five females and five males (1:1 ratio, 5th copepodites) into each of the 16 test [= 4 diet combinations × 4 replicates (cohorts)] jars containing 50 ml medium and with one of the chosen algal-rotifer diets. Data on the survival of naupliar and copepodites (up to V) showed a high death rate (80%) in A. americanus by the time nauplii reached the adult stage. The duration of developmental time for nauplii and the copepodite V (C-V) was about 28 days by which time A. americanus became adults. Food combination had a significant effect on both survival and duration of the naupliar and C-V but not on the development time in C-I, C-II, C-III, and C-IV. Naupliar stages showed highest life expectancy at low food levels (rotifers or alga). Adult male and female copepods showed better survival under higher availability of rotifers in the medium, while higher algal density resulted in steep mortality during the first days. Average adult lifespan and life expectancy of A. americanus varied from 19 to 23 days, depending on the diet combination. Age-specific reproductive output (m _x ) of female A. americanus showed low reproduction (<10 offspring per female on any given day) when raised on low rotifer density, regardless of the algal concentration. On the other hand, enhanced offspring production (average of about 50 nauplii per female) was recorded when the rotifer density was higher (i.e., 8 ind. ml⁻¹). Gross and net reproductive rates varied from 48 to 438 and 27 to 318 offspring per female, respectively. In general, higher availability of rotifers in the medium resulted in higher (8–10 times as compared to those in low rotifer density) gross and net reproductive rates. There was no significant difference in the generation time (13–14 days) in relation to the diet combination. The rate of population increase (r) (range: 0.29–0.69 per day) increased with greater abundance of B. havanaensis in the medium. Thus, our study showed that inclusion of animal protein in the diet is necessary for high population growth rates of A. americanus.     

Factors Influencing the Formation and Stability of Filled Hydrogel Particles Fabricated by Protein/Polysaccharide Phase Separation and Enzymatic Cross-Linking

Filled hydrogel particles can be used to encapsulate, protect, and deliver lipophilic components. In this study, we investigated the influence of preparation conditions on the size of filled hydrogel particles created using biopolymer phase separation and enzymatic cross-linking. We then investigated the stability of these particles to external stresses: pH (pH 2–8); heat (40°–90 °C, 20 min); sodium chloride (0–500 mM); and calcium chloride (0–8 mM). Filled hydrogel particles were fabricated as follows: (i) high methoxy pectin, sodium caseinate, and caseinate-coated lipid droplets were mixed at pH 7 under conditions where phase separation due to thermodynamic incompatibility occurred; (ii) this mixture was acidified (pH 5) to induce adsorption of anionic pectin molecules around lipid-filled caseinate-rich particles; (iii) the caseinate within the particles was enzymatically cross-linked using transglutaminase. Three mixing conditions (0, 100, and 1,000 rpm) were tested during particle acidification. Particle size measurements indicated that larger particles were formed at 0 and 100 rpm than at 1,000 rpm. Under high pH conditions (pH 6–8), particles cross-linked with transglutaminase remained intact while control particles (not cross-linked) disintegrated. The addition of calcium to both control and cross-linked particles resulted in system gelation above 4 mM calcium chloride. Control and cross-linked particles remained stable to heating and to the addition of sodium chloride. Results from this study demonstrate the versatility and robustness of this delivery system for lipophilic bioactives.     

Effect of cold storage on survival, reproduction and development of adults and eggs of Franklinothrips vespiformis (Crawford)

Cold storage effects on both female adults and eggs of the predatory thrips Franklinothrips vespiformis (Crawford) (Thysanoptera: Aeolothripidae) were investigated. The effect of low temperatures (5.5, 7.0, 8.5, 10.0 and 12.5 °C) on survival of F. vespiformis adults was firstly recorded. Survival times were significantly reduced at the lower temperatures tested, whereas storage at 10.0 and 12.5 °C provided the longest survival. Life-history consequences of exposing adults to moderately low temperatures were examined in terms of pre-oviposition period, oviposition rate, egg viability and survival after storage. Adults stored at 7.0 °C showed longer pre-oviposition period and shorter longevity than unstored females but other reproductive attributes were not significantly affected by storage regime. Low temperature and storage period affected egg viability and subsequent development of pre-imaginal stages. No eggs hatched after a 20-day period of storage at 5.5 and 7.0 °C, whereas eggs stored at 12.5 °C hatched significantly faster than ones stored at 10.0 °C and unstored eggs. Increasing the egg storage period from 10 to 20 and 30 days decreased the oviposition rate of adults and egg viability. An essential component in the successful mass rearing and distribution of these predators is the development of a reliable storage schedule of eggs and adults. Long-term storage was unsatisfactory, however their short-term storage (3.5 weeks at 10.0 and 12.5 °C for adults and 4–5 weeks at 12.5 °C for eggs) gave satisfactory results, which suggest the efficacy of such storage during the mass production of the biocontrol agent.     

Long-term lake acidification trends in high- and low-sulphate deposition regions from Nova Scotia,Canada

Diatom-based paleolimnological techniques were used to study 14 lakes from two regions of Nova Scotia which represent regions of high and low sulphate deposition. Using decadal scale intervals, changes in diatom assemblages and diatom-inferred pH were tracked in relation to deposition of anthropogenic-sourced strong acids. Eight study lakes were located in Kejimkujik National Park in the southwestern part of the province, which receives an annual sulphate deposition (2000–2002) of ~10.5 kg ha⁻¹ yr⁻¹. These lakes showed significant changes in diatom assemblages with overall diatom-inferred acidification of ~0.5 pH units starting between 1925 and 1940, with the timing of acidification related to pre-industrial (or pre−1850) lakewater pH. Six study lakes were located in Cape Breton Highlands National Park, in northern Nova Scotia, a region of lower sulphate deposition. These lakes did not show any consistent trends in diatom assemblages or inferred pH values consistent with recent acidic deposition, but rather variations that may be related to climatic influences. Nova Scotia lakes that have been most impacted by acidic deposition had the lowest pre-industrial lakewater pH values and were in an area of relatively high sulphate deposition. Handling editor: K. Martens     

Aluminium toxicity and nodulation ofTrifolium repens

Summary Effects of aluminium on theTrifolium repens var Huia-Rhizobium trifolii strain HP3 symbiosis were studied using an axenic solution-culture system. With, 10 μM phosphate, 50 μM aluminium reduced or inhibited root elongation at pH<5.0, root hair formation at pH< 5.0–5.5, and Rhizobium multiplication in the rhizosphere and nodule formation at pH<6.0. In the absence of aluminium, root elongation and root hair formation were reduced at pH<4.3, and Rhizobium multiplication and nodule formation were inhibited at pH<5.0. Root hair formation was more sensitive to aluminium at pH<5 than was root elongation. No effect of aluminium on Rhizobium multiplication and nodule formation at pH<5 was detected because both were sensitive to pH alone. At pH 5.5 most of the aluminium changed immediately to a form which was susceptible to low-speed centrifugation, but which was detected by the aluminon method of analysis, and after 24 h a precipitate formed. the concentration of phosphate was reduced also, to approximately 1μM. Toxicity was overcome by either increasing the phosphate concentration from 10 to 50 μM, or by increasing the pH to 6.0 and the calcium, concentration to 1000μM.