Intraspecific variability in Karlodinium veneficum: Growth rates, mixotrophy, and lipid composition

We isolated eleven strains of the harmful algal bloom (HAB)-forming dinoflagellate Karlodinium veneficum during a bloom event in the NW Mediterranean coastal waters and we studied the inter-strain variability in several of their physiological and biochemical traits. These included autotrophic growth parameters, feeding capabilities (mixotrophy), lipid composition, and, in some cases, their responses to biotic and abiotic factors. The strains were found to differ in their growth rates (0.27–0.53 d⁻¹) and in the maximum cell concentrations achieved during stationary phase (6.1 × 10⁴–8.6 × 10⁴ cells mL⁻¹). Their ingestion performance, when offered Rhodomonas salina as prey, was also diverse (0.22–1.3 cells per K. veneficum per day; 8–52% of their daily ration). At least two strains survived for several months under strict heterotrophic conditions (no light, low inorganic nutrients availability, and R. salina as food source). These strains also showed very distinct fatty acid compositions, with very low contents of monounsaturated and polyunsaturated fatty acids. According to a Bray Curtis similarity analysis, three or four strain groups able to perform different roles in bloom development were identified. We further analyzed one strain from each of the two most distinct groups with respect to prey concentration, light intensity, nutrient availability, and we determined the functional responses (growth and feeding rates) to food concentration. Taken together, the results served to highlight the role of mixotrophy and clone variability in the formation of HABs.     

Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori

Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air–liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2–3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402.     

A novel low-temperature active alkaline pectate lyase from Klebsiella sp. Y1 with potential in textile industry

Alkaline pectate lyases are favorable for the textile industry. Here we report the cloning of a pectate lyase gene (pl A), from Klebsiella sp. Y1, and its heterologous expression in Escherichia coli. The full-length pl A consists of 1710 bp and encodes for a 569-amino acid polypeptide including a putative 22-residue signal peptide and a catalytic domain belonging to pectate lyase family 2. The recombinant enzyme (r-PL A) was purified to electrophoretic homogeneity by single-step Ni²⁺-NTA affinity chromatography and showed an apparent molecular weight of ∼60 kDa. The pH and temperature optima of r-PL A were found to be 9.0 and 30–50 °C, respectively. r-PL A was highly active at low temperatures, exhibiting >60% of the maximal activity at 20 °C and >20% activity even at 0 °C. The enzyme was stable in a broad alkaline pH range of 7.0–12.0 for 1 h at 37 °C. The values of K_m(app) and V_max(app) of r-PL A for polygalacturonic acid were 2.47 mg/ml and 11.94 μmol/min/mg, respectively. Compared with the commercial compound pectinase from Novozymes, purified r-PL A showed similar efficacy in reducing the intrinsic viscosity of polygalacturonic acid (68.8% vs. 67.1%) and in bioscouring of jute (7.38% vs. 7.58%). Thus r-PL A is a valuable material for the textile industry.     

The Serotonin 1A A Receptor: A Representative Member of the Serotonin Receptor Family

1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions.2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin_1A (5-HT_1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors.3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin_1A receptor and its interaction with G-proteins are discussed.4. The pharmacology of serotonin_1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides.5. Membrane biology of 5-HT_1A receptors is presented using the bovine hippocampal serotonin_1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.     

Phylogenetic affinity of arbuscular mycorrhizal symbionts in <Emphasis Type="Italic">Psilotum nudum</Emphasis>

Many lineages of land plants (from lycopsids to angiosperms) have non-photosynthetic life cycle phases that involve obligate mycoheterotrophic arbuscular mycorrhizal (AM) associations where the plant host gains organic carbon through glomalean symbionts. Our goal was to isolate and phylogenetically identify the AM fungi associated with both the autotrophic and underground mycoheterotrophic life cycle phases of Psilotum nudum. Phylogenetic analyses recovered 11 fungal phylotypes in four diverse clades of Glomus A that form AM associations with P. nudum mycoheterotrophic gametophytes and autotrophic sporophytes, and angiosperm roots found in the same greenhouse pots. The correspondence of identities of AM symbionts in P. nudum sporophytes, gametophytes and neighboring angiosperms provides compelling evidence that photosynthetic heterospecific and conspecific plants can serve as the ultimate sources of fixed carbon for mycoheterotrophic gametophytes of P. nudum, and that the transfer of carbon occurs via shared fungal networks. Moreover, broader phylogenetic analyses suggest greenhouse Psilotum populations, like field-surveyed populations of mycoheterotrophic plants, form AM associations with restricted clades of Glomus A. The phylogenetic affinities and distribution of Glomus A symbionts indicate that P. nudum greenhouse populations have the potential to be exploited as an experimental system to further study the physiology, ecology and evolution of mycoheterotrophic AM associations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evaluation of different species-specific PCR protocols for the detection of Vibrio tapetis

In this study the specificity and sensitivity of three primer pairs, Jvt1–Jvt2, VtF–VtR and VtKF–VtKR, for the detection of Vibrio tapetis were evaluated in parallel using 23 V. tapetis strains isolated from different mollusc and fish species and with different geographical origin, as well as 29 representatives of related Vibrio species. The three primer pairs amplified all the V. tapetis strains, regardless of their host or geographical origin. However, with primer sets VtF–VtR and VtKF–VtKR amplification products of the expected size were obtained from chromosomal DNA of some of the non-V. tapetis bacteria tested. The sensitivity of the three PCR detection methods was also different. The detection limit obtained with primer pairs Jvt1–Jvt2 and VtF–VtR was between 1 and 10 pg DNA/PCR tube (2–20 bacterial cells per reaction). The primer set VtKF–VtKR showed a reduction of sensitivity in at least one order of magnitude. The results were highly reproducible with all primer sets when using the same thermal cycler, although some differences were observed in the results obtained in different PCR machines. Based on the findings reported here, we propose the Jvt1–Jvt2 PCR protocol as the most adequate for an accurate detection of V. tapetis in diagnostic pathology as well as in epidemiological studies of this clam pathogen.     

A putative disease-associated haplotype within the SCN1A gene in Dravet syndrome

Dravet syndrome (DS), previously known as severe myoclonic epilepsy of infancy, is one of the most severe forms of childhood epilepsy. DS is caused by a mutation in the neuronal voltage-gated sodium-channel alpha-subunit gene (SCN1A). However, 25–30% of patients with DS are negative for the SCN1A mutation screening, suggesting that other molecular mechanisms may account for these disorders. Recently, the first case of DS caused by a mutation in the neuronal voltage-gated sodium-channel beta-subunit gene (SCN1B) was also reported. In this report we aim to make the molecular analysis of the SCN1A and SCN1B genes in two Tunisian patients affected with DS. The SCN1A and SCN1B genes were tested for mutations by direct sequencing. No mutation was revealed in the SCN1A and SCN1B genes by sequencing analyses. On the other hand, 11 known single nucleotide polymorphisms were identified in the SCN1A gene and composed a putative disease-associated haplotype in patients with DS phenotype. One of the two patients with putative disease-associated haplotype in SCN1A had also one known single nucleotide polymorphism in the SCN1B gene. The sequencing analyses of the SCN1A gene revealed the presence of a putative disease-associated haplotype in two patients affected with Dravet syndrome.     

Echinococcus granulosus: In vitro effectiveness of warm water on protoscolices

Hydatid disease is one of the most important helminthic diseases worldwide. Hydatid cysts may be found anywhere in the body. The most effective treatment of hydatid cyst is surgical operation. Spillage of live protoscolices during the operation is the major cause of recurrence. Instillation of scolicidal agent into hydatid cyst is the most commonly employed measure to prevent this complication. To date, many scolicidal agents have been used for inactivation of the hydatid cyst content, however, most common scolicidal agents may cause unacceptable side-effects, limiting their use. In this study the scolicidal effect of warm water (45, 50, 55, and 60 °C) at different exposure times (1, 2, 3, 4, 5, 6, 8, 10, 12, and 15 min) is investigated. Protoscolices were collected aseptically from sheep livers containing hydatid cyst. Viability of protoscolices was determined by 0.1% eosin staining. Even though the highest scolicidal activity of warm water at 45 °C was 40.4% at the end of 15 min, the best scolicidal effect (100%) of warm water at 50, 55, and 60 °C was obtained after 5, 2, and 1 min, respectively. The results of this in vitro study showed that warm water at 50–60 °C can be regarded as an effective scolicidal agent. Warm water is commonly available, easily prepared, and inexpensive. In vivo scolicidal activity of warm water and also the possible side effects need further investigation.     

Nine microsatellite markers for the Australian side-necked turtle Chelodina expansa (Chelidae) and cross species amplifications

Nine microsatellite DNA loci for the Australian broad-shelled freshwater turtle (Chelodina expansa) are presented. Markers were tailed with 20-mer oligonucleotides for use in four-colour fluorescent multiplex PCRs. The markers show high allelic richness (mean N_A = 10.9, range 2–38) and expected heretozygosity (mean H_E = 0.643; range 0.161–0.963) indicating that they will be valuable for population genetics studies in C. expansa. Cross-species amplification in three Australian freshwater turtle species further highlights the potential utility of these markers, particularly in the side-neck species C. longicollis and C. rugosa.     

Differences in shoot regeneration response from cotyledonary node explants in Asiatic Vigna species support genomic grouping within subgenus Ceratotropis (Piper) Verdc.

The efficiency of any plant regeneration system lies in part in its wide applicability to diverse genotypes. In Asiatic Vigna, cotyledon and cotyledonary node explants from 4-day-old seedlings of 27 genotypes were cultured in a medium consisting of MS salts, B5 vitamins, 3.0% sucrose and 1.0 mg l^-1 BA. Direct and efficient multiple shoot regeneration (80–100%) from the cotyledonary nodes was obtained in all epigeal species namely radiata, mungo, aconitifolia, subspecies radiata var. sublobata, mungo var. silvestris and in the hypogeal but allotetraploid glabrescens. In contrast, two other hypogeal species V. angularis and V. umbellata failed to initiate shoots from the nodes. However, adventititious shoots developed at the basipetal cut (hypocotyl) in 35–67% of V. angularis explants. These results provide evidence in support of the existing genomic grouping within subgenus Ceratotropis, which designates AA, A₁A₁ and A₁A₁/- to epigeal, hypogeal and the allotetraploid species, respectively. Mean shoot production ranged from 3.3 to 10.4 shoots per explant during the first subculture and varied significantly among the responsive genotypes within 4 species. Additional shoots were obtained in all genotypes after subsequent subculture. However, cotyledons were not as regenerable as cotyledonary node explants. Although significant differences in rooting were observed among the shoots of the 15 genotypes, the response was generally higher in MS basal medium (MSO) than in MS with 1.0 mg l^-1 IAA. Regenerated plants were successfully transferred to soil (50–100% survival rate) and all surviving plants were reproductively fertile. This revised version was published online in June 2006 with corrections to the Cover Date.     

Morphological redescriptions of four marine ciliates (Ciliophora: Cyrtophorida: Dysteriidae) from Qingdao, China

The morphology and infraciliature of four marine cyrtophorid ciliates isolated from Qingdao, China, were investigated. Based on the present work and on previous data, improved diagnoses for three rarely known species are provided: (1) Mirodysteria decora; small-sized marine Mirodysteria about 35–60 × 25–35 μm in vivo, oval in outline; body surface with two or three conspicuous dorsal spines and one caudal spine; three right kineties, the rightmost one extending dorso-apically; left frontal kineties reduced, each consisting of three basal bodies only; podite subcaudally positioned; two ventrally located contractile vacuoles. (2) Dysteria legumen; body oval with two longitudinal grooves on different plates; six right kineties, the rightmost two of which extend dorso-apically; two left frontal kineties and two ventrally located contractile vacuoles. (3) Dysteria proraefrons; body about 60 × 35 μm in vivo; six right kineties, the two rightmost of which extend dorso-apically and the leftmost one is considerably shortened; three left frontal kineties; two ventrally located contractile vacuoles. A population of D. derouxi with eight or nine right kineties is also briefly described. The current investigation further demonstrates high diversity and cosmopolitan distribution of this highly specialized group of benthic ciliates.     

Preliminary investigation of an integrated aquaculture–wetland ecosystem using tertiary-treated municipal wastewater in Los Angeles County, California

The objective of this study was to determine the feasibility of using a designed integrated aquaculture–wetland ecosystem (AWE) for experimental food production and inorganic nitrogen removal from tertiary-treated wastewater. The AWE connected polyculture aquaculture ponds with in-pond aquatic plant systems (water hyacinths, Eichhornia crassipes, and Chinese water spinach, Ipomea aquatica), a solar energy aeration system, and an artificial wetland. Ponds were stocked with hybrid tilapia (Oreochromis mossambicus×O. urolepis hornorum), common carp (Cyprinus carpio), mosquitofish (Gambusia affinis), and red swamp crayfish (Procambarus clarkii), and were flushed weekly with new wastewater at 20%. Fish were fed a 32% protein floating ration at 1% fish body weight per day, and wheat bran was added at 1 mg l⁻¹ when water conductivities exceeded 900 μmhos cm⁻¹. Plants were allowed to grow until they reached approximately 50% of the pond surface area, then maintained at this area by manual harvesting. Pond water quality (temperature, conductivity, pH, oxygen) was monitored twice daily, and weekly water samples were taken for analyses of inorganic nitrogen (ammonia and nitrate-N) in the ponds, wetland, and wetland discharge waters (n=30). Tilapia harvest from three ponds was 1134.5 kg. Fish standing crop biomass increased from 0.16 to 0.21 at stocking to 1.50–2.00 kg m⁻³ at harvest. Tilapia grew from an average stocking weight of 21 to 362–404 g at harvest but had poor survival (48–64%) due to heavy bird predation. Total food conversion ratios ranged 0.9–1.2. Approximately 70% of the tilapia were marketed live at $2.20 kg⁻¹. An estimated standing crop of 1.4 tons wet weight of Ipomea aquatica grew luxuriantly in one 200-m² polyculture pond which could be harvested sustainably at 20 kg week⁻¹. Water hyacinths removed approximately 90% of the ammonia and nitrate-N in wastewater, and the wetland removed an additional 7% (total removal was 97% of wastewater input concentrations). Overflow water exiting the wetland had less than 0.4 mg ammonia–nitrogen l⁻¹ and no detectable nitrate–nitrogen. The experimental AWE accomplished aquatic food production and almost complete removal of inorganic nitrogen from wastewater, functioning as a `quartenary' wastewater treatment/food production ecosystem. However, more rigorous experimentation is required to optimize fish- and plant-carrying capacities, nutrient cycles, and testing for bioaccumulation of metals in order for the AWE to be socially and economically relevant. The concept of using tertiary-treated wastewater for aquatic food production may be attractive in the peri-urban areas of many meagcities like Los Angeles, both for fish markets and to stem the growing discharges of wastewaters that are causing coastal pollution.     

Intersubgenomic heterosis in seed yield potential observed in a new type of Brassica napus introgressed with partial Brassica rapa genome

This paper reports the observation on the intersubgenomic heterosis for seed yield among hybrids between natural Brassica napus (AⁿAⁿCⁿCⁿ) and a new type of B. napus with introgressions of genomic components of Brassica rapa (A^rA^r). This B. napus was selected from the progeny of B. napus × B. rapa and (B. napus × B. rapa) × B. rapa based on extensive phenotypic and cytological observation. Among the 129 studied partial intersubgenomic hybrids, which were obtained by randomly crossing 13 lines of the new type of B. napus in F₃ or BC₁F₃ to 27 cultivars of B. napus from different regions as tester lines, about 90% of combinations exceeded the yield of their respective tester lines, whereas about 75% and 25% of combinations surpassed two elite Chinese cultivars, respectively. This strong heterosis was further confirmed by reevaluating 2 out of the 129 combinations in a successive year and by surveying hybrids between 20 lines of the new type of B. napus in BC₁F₅ and its parental B. napus in two locations. Some DNA segments from B. rapa were identified with significant effects on seed yield and yield components of the new type of B. napus in BC₁F₅ and intersubgenomic hybrids in positive or negative direction. It seems that the genomic components introgressed from B. rapa contributed to improvement of seed yield of rapeseed.     

Cation-π and π-π stacking interactions allow selective inhibition of butyrylcholinesterase by modified quinine and cinchonidine alkaloids

Scaffold varied quaternized quinine and cinchonidine alkaloid derivatives were evaluated for their selective butyrylcholinesterase (BChE) inhibitory potential. K_i values were between 0.4–260.5 μM (non-competitive inhibition) while corresponding K_ivalues to acetylcholinesterase (AChE) ranged from 7.0–400 μM exhibiting a 250-fold selectivity for BChE.Docking arrangements (GOLD, PLANT) revealed that the extended aromatic moieties and the quaternized nitrogen of the inhibitors were responsible for specific π–π stacking and π–cation interactions with the choline binding site and the peripheral anionic site of BChE’s active site.     

Detection and discrimination of Loa loa, Mansonella perstans and Wuchereria bancrofti by PCR–RFLP and nested-PCR of ribosomal DNA ITS1 region

The ribosomal deoxyribonucleic acid (DNA) internal transcribed spacer region (ITS1) of two filarial nematodes, Loa loa and Mansonella perstans, was amplified and further sequenced to develop an species-specific polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) protocol for the differentiation of both species from Wuchereria bancrofti, three filarial nematodes with blood circulating microfilariae. The ITS1–PCR product digested with the restriction endonuclease Ase I generated an specific diagnostic pattern for each of the three species. Moreover, three new specific nested-PCRs, targeting the ITS1 region, for differential detection of L. loa, M. perstans and W. bancrofti were developed and used when the ITS1–PCR products were insufficient for the Ase I enzymatic digestion. These filarial species-specific molecular protocols were evaluated in forty blood samples from African adult immigrants attending in the Hospital Insular of Gran Canaria, Canarias, Spain.     

Validation of the <Emphasis Type="Italic">VRN-H2</Emphasis>/<Emphasis Type="Italic">VRN-H1</Emphasis> epistatic model in barley reveals that intron length variation in <Emphasis Type="Italic">VRN-H1</Emphasis> may account for a continuum of vernalization sensitivity

The epistatic interaction of alleles at the VRN-H1 and VRN-H2 loci determines vernalization sensitivity in barley. To validate the current molecular model for the two-locus epistasis, we crossed homozygous vernalization-insensitive plants harboring a predicted “winter type” allele at either VRN-H1 (Dicktoo) or VRN-H2 (Oregon Wolfe Barley Dominant), or at both VRN-H (Calicuchima-sib) loci and measured the flowering time of unvernalized F₂ progeny under long-day photoperiod. We assessed whether the spring growth habit of Calicuchima-sib is an exception to the two-locus epistatic model or contains novel “spring” alleles at VRN-H1 (HvBM5A) and/or VRN-H2 (ZCCT-H) by determining allele sequence variants at these loci and their effects relative to growth habit. We found that (a) progeny with predicted “winter type” alleles at both VRN-H1 and VRN-H2 alleles exhibited an extremely delayed flowering (i.e. vernalization-sensitive) phenotype in two out of the three F₂ populations, (b) sequence flanking the vernalization critical region of HvBM5A intron 1 likely influences degree of vernalization sensitivity, (c) a winter habit is retained when ZCCT-Ha has been deleted, and (d) the ZCCT-H genes have higher levels of allelic polymorphism than other winterhardiness regulatory genes. Our results validate the model explaining the epistatic interaction of VRN-H2 and VRN-H1 under long-day conditions, demonstrate recovery of vernalization-sensitive progeny from crosses of vernalization-insensitive genotypes, show that intron length variation in VRN-H1 may account for a continuum of vernalization sensitivity, and provide molecular markers that are accurate predictors of “winter vs spring type” alleles at the VRN-H loci.     

DNA Sequence-Specific Ligands: XI.* The Synthesis and Binding to DNA of bis-Netropsins with the C-Ends of Their Netropsin Fragments Tethered by Tetra- or Pentamethylene Linkers

Bis-Netropsins with the C-ends of their netropsin fragments tethered via tetra- or pentamethylene linkers and with Gly or L-Lys-Gly residues on their N-ends were synthesized. The footprinting technique was used to study the specificity of bis-netropsin binding to the specially constructed DNA fragments containing various clusters of A · T pairs. It was found that the linker length affects the binding of bis-netropsins, with the tetramethylene linker providing better protection than the pentamethylene linker. It was shown that the newly synthesized bis-netropsins bind tighter to the 5"-A ₄ T ₄-3" sequence, whereas the bis-netropsin with a linker between the netropsin N-ends binds better to 5"-T ₄ A ₄-3" sequences.     

Brominated aliphatic hydrocarbons and sterols from the sponge Xestospongia testudinaria with their bioactivities

Four brominated aliphatic hydrocarbons (1–4), including a novel brominated ene-tetrahydrofuran named as mutafuran H (1), and five sterols (5–9) were isolated from the South China Sea sponge Xestospongia testudinaria. The structure of 1 was determined on the basis of NMR (¹H, ¹³C NMR, HSQC, HMBC, ¹H–¹H COSY, and NOESY), MS, and optical rotation analysis. Known compounds were identified by comparison of their NMR data with those reported in the literature. Compounds 1–4, and 6–9 were evaluated for their toxicity against Artemia salina larvae, and anti-acetylcholinesterase activity.     

Effect of cryopreservation on fish sperm subpopulations

The evaluation of the motility data obtained with a CASA system, applying a Two-Step Cluster analysis, identified in seabream sperm 3 different sperm subpopulations that correlated differently with embryo hatching rates. Hence, we designed an experiment to understand the effect of the application of different cryopreservation protocols in these sperm motility-based subpopulations. We analyzed Sparus aurata frozen/thawed semen motility 15, 30, 45 and 60 s after activation, using CASA software. Different protocols were applied for cryopreservation: three different cryoprotectants (Dimethyl Sulfoxide (Me₂SO), Ethylene Glycol (EG) and Propylene Glycol (PG)) each at two different concentrations and two packaging volumes (0.5 ml straws, and 1.8 ml cryovials) were tested. Different freezing rates were evaluated corresponding to 1, 2, 3, 4 and 8 cm above the liquid nitrogen surface for the straws and 1, 2 and 4 cm for the cryovials. Motility parameters rendered by CASA were treated with a Two-Step Cluster analysis. Three different subpopulations were obtained: SP1 – slow non-linear spermatozoa, SP2 – slow linear spermatozoa and SP3 – fast linear spermatozoa. We considered SP3 as the subpopulation of interest and focused further analyses on it. Generally, SP3 was the best represented subpopulation 15 s after activation and was also the one showing a greater decrease in time, being the least represented after 60 s. According to the applied univariate general linear model, samples frozen in straws with 5% Me₂SO and in cryovials with 10% Me₂SO at 2 and 1 cm from the LN_2, respectively, produced the best results (closer to the control). Clustering analysis allowed the detection of fish sperm subpopulations according to their motility pattern and showed that sperm composition in terms of subpopulations was differentially affected by the cryopreservation protocol depending on the cryoprotectant used, freezing rates and packaging systems.