Effect of tumor necrosis factor-alpha on intracellular Staphylococcus aureus in vascular endothelial cells.

Although tumor necrosis factor-alpha (TNF-alpha) is an important host factor against intracellular bacteria, little is known about the effect of TNF-alpha on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-alpha influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-alpha, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10(6) CFU well(-1)) into HUVECs for 1 h. TNF-alpha (1 ng mL(-1)) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-alpha plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-alpha preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-alpha helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-alpha preferentially induces apoptosis of endothelial cells infected by S. aureus.     

Interferon-γ, interleukin-1 and tumour necrosis factor-α synthesis during experimental murine staphylococcal infection

Abstract Several exotoxins of Staphylococcus aureus were shown to modulate the host immune system by stimulation of monokine release. BALB/c mice infected intravenously (i.v.) with live cells if S. aureus , strain Cowan 1, had a detectable serum level of TNF-α at 3, 4 and 5 h after injection. When S. epidermidis (strain F3380, clinical isolate) was used to infect mice, the level of TNF-α was lower (the detection limit of the cytotoxicity assay with WEHI cells was 40 pg ml⁻). Kinetics of TNF synthesis was different from that observed in experimental infections caused by Gram-negative bacteria. Similarly to TNF-α, IL-1α appears in a measureable level at 3 h after i.v. injection of bacteria. The highest serum level of IFN-γ was observed 12 h after infection with both S. aureus and S. epidermidis . A quantity ten times more of S. epedermidis than of S. aureus cells was required to induce similar levels of TNF-α and IFN-γ administered in vivo in four daily doses followed by infection of S. aureus resulted in increased elimination of bacteria from the spleen, liver and peritoneal cavity of mice.     

The Vi-capsule prevents Toll-like receptor 4 recognition of Salmonella

The viaB locus enables Salmonella enterica serotype Typhi to reduce Toll-like receptor (TLR) dependent cytokine production in tissue culture models. This DNA region contains genes involved in the regulation ( tviA ), biosynthesis ( tviBCDE ) and export ( vexABCDE ) of the Vi capsule. Expression of the Vi capsule in S.  Typhimurium, but not expression of the TviA regulatory protein, reduced tumour necrosis factor-alpha (TNF-α) and IL-6 production by murine bone-marrow derived macrophages. Production of TNF-α and IL-6 was dependent on expression of TLR4 as stimulation of macrophages from TLR4^−/− mice with S.  Typhimurium did not result in expression of these cytokines. Intraperitoneal infection of mice with S.  Typhimurium induced expression of TNF-α and inducible nitric oxide synthase (iNOS) in the liver. Introduction of the cloned viaB region into S.  Typhimurium reduced TNF-α and iNOS expression to levels observed after infection with a S.  Typhimurium msbB mutant. In contrast, no differences in TNF-α expression between the S.  Typhimurium wild type and strains expressing the Vi-capsule or carrying a mutation in msbB were observed after infection of TLR4^−/− mice. We conclude that the Vi capsule prevents both in vitro and in vivo recognition of S.  Typhimurium lipopolysaccharide by TLR4.     

Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice

A recently developed model for enterocolitis in mice involves pre-treatment with the antibiotic streptomycin prior to infection with Salmonella enterica serovar Typhimurium ( S.  Typhimurium). The contribution of Nramp1/Slc11a1 protein, a critical host defence mechanism against S.  Typhimurium, to the development of inflammation in this model has not been studied. Here, we analysed the impact of Nramp1 expression on the early development of colitis using isogenic Nramp1^+/+ and Nramp1^−/− mice. We hypothesized that Nramp1 acts by rapidly inducing an inflammatory response in the gut mucosa creating an antibacterial environment and limiting spread of S.  Typhimurium to systemic sites. We observed that Nramp1^+/+ mice showed lower numbers of S.  Typhimurium in the caecum compared with Nramp1^−/− mice at all times analysed. Acute inflammation was much more pronounced in Nramp1^+/+ mice 1 day after infection. The effect of Nramp1 on development of colitis was characterized by higher secretion of the pro-inflammatory cytokines IFN-γ, TNF-α and MIP-1α and a massive infiltration of neutrophils and macrophages, compared with Nramp1^−/− animals. These data show that an early and rapid inflammatory response results in protection against pathological effects of S.  Typhimurium infection in Nramp1^+/+ mice.     

Legionella pneumophila EnhC is required for efficient replication in tumour necrosis factor alpha-stimulated macrophages

Legionella pneumophila enhC ^- mutants were originally identified as being defective for uptake into host cells. In this work, we found that the absence of EnhC resulted in defective intracellular growth when dissemination of intracellular bacteria to neighbouring cells was expected to occur. No such defect was observed during growth within the amoeba Dictyostelium discoideum. Culture supernatants containing the secreted products of infected macrophages added to host cells restricted the growth of the Δ enhC strain, while tumour necrosis factor α (TNF-α), at concentrations similar to those found in macrophage culture supernatants, could reproduce the growth restriction exerted by culture supernatants on L. pneumophila Δ enhC . The absence of EnhC also caused defective trafficking of the Legionella- containing vacuole in TNF-α-treated macrophages. EnhC was shown to be an envelope-associated protein largely localized to the periplasm, with its expression induced in post-exponential phase, as is true for many virulence-associated proteins. Furthermore, the absence of EnhC appeared to affect survival under stress conditions, as the Δ enhC mutant was more susceptible to H₂O₂ treatment than the wild-type strain. EnhC therefore is a unique virulence factor that is required for growth specifically when macrophages have heightened potential to restrict microbial replication.     

Role of tumor necrosis factor-α and glucocorticoid on lipopolysaccharide (LPS)-induced apoptosis of thymocytes

Abstract Administration of bacterial lipopolysaccharide (LPS) into mice markedly induced the apoptosis of CD4⁺8⁺ thymocytes. The injection of anti-tumor necrosis factor (TNF)-α antibody or RU38486, a glucocorticoid receptor antagonist, into mice definitely inhibited LPS-induced apoptosis of thymocytes. Addition of the sera 1 h after injection of LPS into in vitro cultures of thymocytes caused thymocyte apoptosis. It was also prevented by either anti-TNF-α antibody or RU38486. Further, recombinant TNF-α and hydrocortisone collaborated in induction of the thymocyte apoptosis in vitro. The in vivo phenomenon of LPS-induced apoptosis of thymocytes was reproducible by the in vitro experimental system. It was therefore suggested that both TNF-α and glucocorticoid participate and collaborate as effector molecules in LPS-induced apoptosis of thymocytes.     

Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria–host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp-2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6⁺, F1⁺) and JRS145 (M6⁻, F1⁺ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6⁺, F1⁻ mutant) or SAM2 (M6⁻, F1⁻ mutant) infected HEp-2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4 h of infection. Western blot analyses showed that the amounts of Bcl-2 and Bcl-x_L were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase-9 inhibitor (Ac-LEHD-CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS-induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.     

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF-α/IL-1β. The inhibitory effect of ET on TNF-α/IL-1β-stimulated iNOS expression appears to be mediated by ET_B receptors, because (1) both ET-1 and ET-3 inhibited the effects of TNF-α/IL-1β on iNOS expression and nitrite accumulation, (2) a selective ET_B receptor agonist, Suc-[Glu⁹,Ala^11,15]-ET-1 (8–21) (IRL1620), decreased the effects of TNF-α/IL-1β, and (3) a selective ET_B receptor antagonist, N-cis -2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET-3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP-elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF-α/IL-1β-induced and LPS-induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF-α/IL-1β-induced and LPS-induced iNOS expression via ET_B receptors and that cyclic AMP may be involved in this process.     

α-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

Parkinson's disease (PD) is characterized in part by the presence of α-synuclein (α-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α-synuclein gene ( SNCA ) are associated with familial PD. Since Ca²⁺ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca²⁺ homeostasis in cells stably transfected with either wild-type α-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca²⁺ influx evoked by exposure of cells to 50 mM K⁺ was enhanced in cells expressing all three forms of α-syn, an effect which was due specifically to increased Ca²⁺ entry via L-type Ca²⁺ channels. Mobilization of Ca²⁺ by muscarine was not strikingly modified by any of the α-syn forms, but they all reduced capacitative Ca²⁺ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca²⁺]_i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca²⁺ content was unaffected by any form of α-synuclein. However, only WT α-syn transfected cells displayed significantly impaired viability. Our findings suggest that α-syn regulates Ca²⁺ entry pathways and, consequently, that abnormal α-syn levels may promote neuronal damage through dysregulation of Ca²⁺ homeostasis.     

IL-17 is involved in bone resorption in mouse periapical lesions

Periapical lesions are induced by bacterial infection of the dental pulp and result in destruction of the surrounding alveolar bone. Although various immunological studies concerning periapical bone resorption have been reported, the role of cytokines in the formation of periapical lesions remains unclear. In this study, the role of IL-17A in periapical lesions in mice was investigated. Normal C57BL/6, IFN-γ^−/−, TNF-α^−/−, and IL-17A^−/− mice were subjected to pulp exposure and infected with Prevotella intermedia (ATCC25611) and Porphyromonas gingivalis (ATCC33277) in the mandibular first molar. Periapical lesions were determined by μCT on day 21 after infection, and 3D visual construction was performed using 3D picture quantification software. The expression of IL-17A mRNA in periapical lesions was determined by the RT-PCR and real-time RT-PCR method. Periapical lesions developed in wild-type, IFN-γ^−/−, and TNF-α^−/− mice after infection with P. intermedia and P. gingivalis . However, periapical lesions were not observed in IL-17A^−/− mice. The expression of IL-17A mRNA was significantly induced in periapical lesions of wild-type mice after infection. These results suggest that IL-17A, but not IFN-γ or TNF-α, plays an important role in the formation of periapical lesions.     

Cellular prion protein coupling to TACE-dependent TNF-α shedding controls neurotransmitter catabolism in neuronal cells

Despite considerable efforts to unravel the role of cellular prion protein (PrP^C) in neuronal functions, the mechanisms by which PrP^C takes part in the homeostasis of a defined neuronal phenotype remain poorly characterized. By taking advantage of a neuroectodermal cell line (1C11) endowed with the capacity to differentiate into serotonergic (1C11^5-HT) or noradrenergic (1C11^NE) neurons, we assessed the contribution of PrP^C to bioaminergic cell functions. We established that in 1C11-derived neuronal cells antibody-mediated PrP^C ligation triggered tumor necrosis factor (TNF)-α release, through recruitement of the metalloproteinase TNF-α converting enzyme (TACE). TNF-α shed in response to PrP^C acts as a second message signal, eliciting serotonin (5-HT) or norepinephrine (NE) degradation in 1C11^5-HT or 1C11^NE cells, respectively. Our data thus introduced TNF-α as a PrP^C-dependent modulator of neuronal metabolism. Of note, we previously reported on a control of neurotransmitter catabolism by 5-HT_2B or α_1D autoreceptors in 1C11 bioaminergic neurons, via the same TACE/TNF-α pathway (Ann. N Y Acad. Sci. 1091, 123). Here, we show that combined stimulation of PrP^C and these two bioaminergic receptors add their effects on neurotransmitter degradation. Overall, these observations unveil a novel contribution of PrP^C to the control of neuronal functions and may have implications regarding dysfunction of the bioaminergic systems in prion diseases.     

Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Abstract: Tumor necrosis factor-a (TNF-α), interferon-γ (IFN-7), and interleukin-6 (IL-6), but not TNF-β, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose-dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF-α and IFN-γ can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, l -N^G-monomethylarginine. In contrast, the differentiation of N103 cells by IL-6 was not affected by l -N^G-monomethylarginine. These results indicate that TNF-α and IFN-γ, but not IL-6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture super- natants of N103 cells induced by TNF-α and IFN-γ, but not that by IL-6, contained high levels of NO₂⁻, the production of which was inhibited by l - N ^G-monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose-dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF-α and IFN-γ and that neuronal cells, in addition to the macrophagelike brain cells, can be induced by immunological stimuli to produce large quantities of NO.     

Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1

The ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and difficulties in treatment. Here, we show cellular invasion as a novel function for an S. aureus adhesin, previously implicated solely in attachment. S. aureus , but not S. epidermidis , invaded epithelial 293 cells in a temperature- and F-actin-dependent manner. Formaldehyde-fixed and live bacteria were equally invasive, suggesting that no active bacterial process was involved. All clinical S. aureus isolates analysed, but only a subset of laboratory strains, were invasive. Fibronectin-binding proteins (FnBPs) acted as S. aureus invasins, because: (i) FnBP deletion mutants of invasive laboratory strains lost invasiveness; (ii) expression of FnBPs in non-invasive strains conferred invasiveness; and (iii) the soluble isolated fibronectin-binding domain of FnBP (D1–D4) completely blocked invasion. Integrin α₅β₁ served as host cell receptor, which interacted with staphylococcal FnBPs through cellular or soluble fibronectin. FnBP-deficient mutants lost invasiveness for epithelial cells, endothelial cells and fibroblasts. Thus, fibronectin-dependent bridging between S. aureus FnBPs and host cell integrin α₅β₁ is a conserved mechanism for S. aureus invasion of human cells. This may prove useful in developing new therapeutic and vaccine strategies for S. aureus infections.     

Nramp1 expression by dendritic cells modulates inflammatory responses during Salmonella Typhimurium infection

Host resistance against Salmonella enterica serovar Typhimurium ( S . Typhimurium) is mediated by natural resistance-associated macrophage protein 1 (Nramp1/Slc11a1). Nramp1 is critical to host defence, as mice lacking Nramp1 fail to control bacterial replication and succumb to low doses of S . Typhimurium. Despite this crucial role, the mechanisms underlying Nramp1's protective effects are unclear. Dendritic cells (DCs) that sample the intestinal lumen are among the first cells encountered by S. Typhimurium following oral infection and act as a conduit for S. Typhimurium to cross the intestinal epithelial barrier. We report that DCs, including intestinal, splenic and bone marrow-derived DCs (BMDCs), express Nramp1 protein. In the small intestine, Nramp1 expression is greater in a subset of DCs (CD11c⁺CD103^-) characterized by the elevated expression of pro-inflammatory cytokines in response to bacterial products. While Nramp1 expression did not affect S. Typhimurium replication in BMDCs, infected Nramp1+/+ BMDCs and intestinal CD11c⁺CD103^- DCs secreted more inflammatory cytokines (IL-6, IL-12 and TNF-α) than Nramp1−/−, suggesting that Nramp1 expression may promote a more rapid inflammatory response following infection. Collectively, these findings reveal a new role for DCs and Nramp1 in modulating the host inflammatory response to S. Typhimurium.     

Muscle precursor cells isolated from aged rats exhibit an increased tumor necrosis factor-α response

Improving muscle precursor cell (MPC, muscle-specific stem cells) function during aging has been implicated as a key therapeutic target for improving age-related skeletal muscle loss. MPC dysfunction during aging can be attributed to both the aging MPC population and the changing environment in skeletal muscle. Previous reports have identified elevated levels of tumor necrosis factor-α (TNF-α) in aging, both circulating and locally in skeletal muscle. The purpose of the present study was to determine if age-related differences exist between TNF-α-induced nuclear factor-kappa B (NF-κB) activation and expression of apoptotic gene targets. MPCs isolated from 32-month-old animals exhibited an increased NF-κB activation in response to 1, 5, and 20 ng mL⁻¹ TNF-α, compared to MPCs isolated from 3-month-old animals. No age differences were observed in the rapid canonical signaling events leading to NF-κB activation or in the increase in mRNA levels for TNF receptor 1, TNF receptor 2, TNF receptor-associated factor 2 (TRAF2), or Fas (CD95) observed after 2 h of TNF-α stimulation. Interestingly, mRNA levels for TRAF2 and the cell death-inducing receptor, Fas (CD95), were persistently upregulated in response to 24 h TNF-α treatment in MPCs isolated from 32-month-old animals, compared to 3-month-old animals. Our data indicate that age-related differences may exist in the regulatory mechanisms responsible for NF-κB inactivation, which may have an effect on TNF-α-induced apoptotic signaling. These findings improve our understanding of the interaction between aged MPCs and the changing environment associated with age, which is critical for the development of potential clinical interventions aimed at improving MPC function with age.     

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F_2α-induced phosphoinositide hydrolysis and elevation of intracellular Ca²⁺ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF_2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC₅₀ value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF_2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF_2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF_2α at 1 μ M transiently increased astrocytic intracellular Ca²⁺ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF_2α receptor linked to phospholipase C in astrocytes.     

Cloning, molecular characterization and heterologous expression of AMY1, an α-amylase gene from Cryptococcus flavus

A Cryptococcus flavus gene ( AMY1 ) encoding an extracellular α-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) ¹⁴⁴DVVVNH¹⁴⁹, (II) ²³⁵GLRIDSLQQ²⁴³, (III) ²⁶³GEVFN²⁶⁷, (IV) ³²⁷FLENQD³³², placed the enzyme in the GH13 α-amylase family. Furthermore, sequence comparison suggests that the C. flavus α-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae . The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL⁻¹) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme ( c . 67 kDa), part of which was due to N-glycosylation.     

Microbial colonization of an in vitro model of a tissue engineered human skin equivalent – a novel approach

This was a preliminary investigation to define the conditions of colonization of a human skin equivalent (SE) model with cutaneous microorganisms. SEs of 24 mm diameter were constructed with a dermal matrix of fibrin containing fibroblasts and a stratified epidermis. Microbial colonization of the SEs was carried out in a dry environment, comparable to ' in vivo ' skin, using a blotting technique to remove inoculation fluid. The microbial communities were sampled by scrub washing and viable cells enumerated on selective growth medium. Staphylococcus epidermidis , Propionibacterium acnes and Malassezia furfur (human skin commensals) and Staphylococcus aureus (transient pathogen) were colonized at inoculum densities of 10²–10⁶ CFU SE⁻¹ on the surface of replicate SEs. Growth of all species was supported for upto 72–120 h, with recovery densities of between 10⁴–10⁹ CFU SE⁻¹. A novel, real-time growth monitoring method was also developed, using S. aureus containing a lux cassette. Light output increased from 20 to 95 h, and colonization increased from 10² to 10⁸ CFU SE⁻¹, as confirmed by conventional recovery. Thus, the SE model has potential to investigate interactions between resident and transient microbial communities with themselves and their habitat, and for testing treatments to control pathogen colonization of human skin.     

Longevity of the freshwater anostracan Streptocephalus mackini (Crustacean: Anostraca) in relation to food (Chlorella vulgaris) concentration

SUMMARY 1. Temporary ponds are inhabited by a variety of invertebrates, of which anostracans are an important group. We studied the lifetables of male and female anostracan Streptocephalus mackini at 3 algal concentrations (0.5 × 10⁶, 1.0 × 10⁶ and 1.5 × 10⁶ cells mL⁻¹).
2. Regardless of sex, S. mackini showed better survivorship at lower food levels. The longest average lifespan observed was 85 ± 2 days for males fed Chlorella at 0.5 × 10⁶ cells mL⁻¹.
3. Both net reproductive rate and generation time decreased with increasing food level. The highest net reproductive rate was about 120 cysts per female. The longest generation time of about 40 days, observed at 0.5 × 10⁶ cells mL⁻¹, was more than three times that at 1.5 × 10⁶ cells mL⁻¹.
4. The rate of population increase ( r ) was nearly the same (0.31 ± 0.06) at high (1.5 × 10⁶ cells mL⁻¹) and intermediate (1.0 × 10⁶ cells mL⁻¹) food levels. The r -value at low food level (0.5 × 10⁶ cells mL⁻¹ of Chlorella ) was 0.20 ± 0.01 per day.