The Feeding Tube of Cyst Nematodes: Characterisation of Protein Exclusion

Plant parasitic nematodes comprise several groups; the most economically damaging of these are the sedentary endoparasites. Sedentary endoparasitic nematodes are obligate biotrophs and modify host root tissue, using a suite of effector proteins, to create a feeding site that is their sole source of nutrition. They feed by withdrawing host cell assimilate from the feeding site though a structure known as the feeding tube. The function, composition and molecular characteristics of feeding tubes are poorly characterised. It is hypothesised that the feeding tube facilitates uptake of host cell assimilate by acting as a molecular sieve. Several studies, using molecular mass as the sole indicator of protein size, have given contradictory results about the exclusion limits of the cyst nematode feeding tube. In this study we propose a method to predict protein size, based on protein database coordinates in silico. We tested the validity of these predictions using travelling wave ion mobility spectrometry – mass spectrometry, where predictions and measured values were within approximately 6%. We used the predictions, coupled with mass spectrometry, analytical ultracentrifugation and protein electrophoresis, to resolve previous conflicts and define the exclusion characteristics of the cyst nematode feeding tube. Heterogeneity was tested in the liquid, solid and gas phase to provide a comprehensive evaluation of three proteins of particular interest to feeding tube size exclusion, GFP, mRFP and Dual PI. The data and procedures described here could be applied to the design of plant expressed defence compounds intended for uptake into cyst nematodes. We also highlight the need to assess protein heterogeneity when creating novel fusion proteins.     

Effects of Microbial and Other Antagonistic Organism and Competition on Entomopathogenic Nematodes

Antagonistic factors, broadly identified as antibiosis, competition and natural enemies, impact on entomopathogenic nematodes. Antibiosis can occur through the release of plant chemicals from the roots into the soil, which may adversely affect the host-finding behavior of the infective stage nematode, or the presence of these chemicals in the host insect may negatively affect nematode reproduction. In laboratory studies, intra-specific and inter-specific competition reduces nematode fitness, and inter-specific competition can cause local extinction of a nematode species. For example, after concomitant infection of a host, a steinernematid species usually excludes a heterorhabditid species. The mechanism for the steinernematid superiority has been postulated to be a bacteriocin(s) produced by Xenorhabdus, the symbiotic bacterium of the steinernematid, which prevents Photorhabdus, the symbiotic bacterium of the heterorhabditid, from multiplying. Inter-specific competition between two steinernematid species shows that both can co-exist in a host, but one species will eventually prevail in the environment. By having different foraging strategies, however, both steinermatid species may co-exist in the same habitat. An important issue is whether the introduction of an exotic entomopathogenic nematode species will competitively displace an indigenous nematode species. Although the environmental risks are small, the recommended policy is that the introduction of exotic nematodes be regulated. With other pathogens, entomopathogenic nematodes can out-compete entomopathogenic fungi, but not Bacillus thuringiensis, for the same host individual when both the nematode and entomopathogen are applied simultaneously. The best studied natural enemy is the nematophagous fungus, Hirsutella rhossiliensis, which causes higher mortality in Steinernema glaseri compared with Heterorhabditis bacteriorphora. Differential susceptibility to the fungus may be associated with the retention of the second-stage cuticle by H. bacteriophora. Invertebrate predators including mites and collembolans feed on entomopathogenic nematodes. Although a number of studies have been conducted with antagonists, there is a dearth of field data. We suggest that long-term research plots be established where natural populations of entomopathogenic nematodes occur and include antagonists as a component of such studies.     

The bacterial community of entomophilic nematodes and host beetles

Insects form the most species‐rich lineage of Eukaryotes and each is a potential host for organisms from multiple phyla, including fungi, protozoa, mites, bacteria and nematodes. In particular, beetles are known to be associated with distinct bacterial communities and entomophilic nematodes. While entomopathogenic nematodes require symbiotic bacteria to kill and reproduce inside their insect hosts, the microbial ecology that facilitates other types of nematode–insect associations is largely unknown. To illuminate detailed patterns of the tritrophic beetle–nematode–bacteria relationship, we surveyed the nematode infestation profiles of scarab beetles in the greater Los Angeles area over a five‐year period and found distinct nematode infestation patterns for certain beetle hosts. Over a single season, we characterized the bacterial communities of beetles and their associated nematodes using high‐throughput sequencing of the 16S rRNA gene. We found significant differences in bacterial community composition among the five prevalent beetle host species, independent of geographical origin. Anaerobes Synergistaceae and sulphate‐reducing Desulfovibrionaceae were most abundant in Amblonoxia beetles, while Enterobacteriaceae and Lachnospiraceae were common in Cyclocephala beetles. Unlike entomopathogenic nematodes that carry bacterial symbionts, insect‐associated nematodes do not alter the beetles' native bacterial communities, nor do their microbiomes differ according to nematode or beetle host species. The conservation of Diplogastrid nematodes associations with Melolonthinae beetles and sulphate‐reducing bacteria suggests a possible link between beetle–bacterial communities and their associated nematodes. Our results establish a starting point towards understanding the dynamic interactions between soil macroinvertebrates and their microbiota in a highly accessible urban environment.     

Magnetic and electric fields induce directional responses in Steinernema carpocapsae

Entomopathogenic nematode species respond directionally to various cues including electrical stimuli. For example, in prior research Steinernema carpocapsae was shown to be attracted to an electrical current that was applied to an agar dish. Thus, we hypothesised that these nematodes may use electromagnetic reception to assist in navigating through the soil and finding a host. In this study we discovered that S. carpocapsae also responds to electrical fields (without current) and to magnetic fields; to our knowledge this is the first report of nematode directional movement in response to a magnetic field. Our research expands on the range of known stimuli that entomopathogenic nematodes respond to. The findings may have implications for foraging behavior.     

Microbial control of plant-parasitic nematodes: a five-party interaction

Plant-parasitic nematodes cause significant economic losses to a wide variety of crops. Chemical control is a widely used option for plant-parasitic nematode management. However, chemical nematicides are now being reappraised in respect of environmental hazard, high costs, limited availability in many developing countries or their diminished effectiveness following repeated applications. This review presents progress made in the field of microbial antagonists of plant-parasitic nematodes, including nematophagous fungi, endophytic fungi, actinomycetes and bacteria. A wide variety of microorganisms are capable of repelling, inhibiting or killing plant-parasitic nematodes, but the commercialisation of these microorganisms lags far behind their resource investigation. One limiting factor is their inconsistent performance in the field. No matter how well suited a nematode antagonist is to a target nematode in a laboratory test, rational management decision can be made only by analysing the interactions naturally occurring among “host plant–nematode target–soil–microbial control agent (MCA)–environment”. As we begin to develop a better understanding of the complex interactions, microbial control of nematodes will be more fine-tuned. Multidisciplinary collaboration and integration of biological control with other control methods will␣also contribute to more successful control practices.     

Parasitism genes and host range disparities in biotrophic nematodes: the conundrum of polyphagy versus specialisation

This essay considers biotrophic cyst and root-knot nematodes in relation to their biology, host-parasite interactions and molecular genetics. These nematodes have to face the biological consequences of the physical constraints imposed by the soil environment in which they live while their hosts inhabit both above and below ground environments. The two groups of nematodes appear to have adopted radically different solutions to these problems with the result that one group is a host specialist and reproduces sexually while the other has an enormous host range and reproduces by mitotic parthenogenesis. We consider what is known about the modes of parasitism used by these nematodes and how it relates to their host range, including the surprising finding that parasitism genes in both nematode groups have been recruited from bacteria. The nuclear and mitochondrial genomes of these two nematode groups are very different and we consider how these findings relate to the biology of the organisms.     

Optimal time to patency in parasitic nematodes: host mortality matters

We develop an optimality model based on classical epidemiological models to investigate the optimal time to patency in parasitic nematodes in relation to host mortality and parasite mortality. We found that the optimal time to patency depends on both host longevity and prepatent mortality of nematodes. We tested our models using a comparative analysis of the relationships between nematode time to patency, nematode mortality and host mortality. Although we confirmed the importance of prepatent mortality, we also found a significant positive influence of host mortality. Host mortality rate affects parasite survivorship and life history strategies in the same way that habitat-specific mortality regimes drive the evolution of life histories in free-living organisms.     

Population dynamics of a host-specific root-feeding cyst nematode and resource quantity in the root zone of a clonal grass

Recent studies have suggested that root-feeding nematodes influence plant community dynamics, but few studies have investigated the population dynamics of the nematodes. In coastal foredunes, feeding-specialist cyst nematodes ( Heterodera spp.) are dominant in the soil nematode community and greenhouse studies pointed at bottom-up control by their host plant. Here we examine the population dynamics of H. arenaria in the field in relation to resource quantity in the root zone of Ammophila arenaria (marram grass).
Ammophila arenaria is a pioneer grass in mobile coastal foredunes of northwestern and Mediterranean Europe. Every year, the plant expands clonally into freshly deposited layers of wind-blown sand, followed by dispersal and build-up of the nematode population into the new root zone. In a newly developing root zone the first H. arenaria cysts were observed one month after the first new roots were detected, indicating that nematode dispersal is not limiting the initial establishment of new populations. Throughout the growth season, the numbers of cysts, as well as the numbers of eggs and juveniles within cysts, were related to the quantity of roots. However, cyst density varied between years. Therefore, we conclude that in new root layers of A. arenaria cyst nematodes are bottom-up controlled by resource quantity, but that other factors, for example resource quality, influence the relation between nematode abundance and resource quantity.
In deeper root zones the nematode abundance declines over time. Here, numbers of cysts were not related to root biomass, while numbers of eggs and juveniles inside the cysts were weakly related. This points at other factors than resource quantity, for example the quality of the roots or unsuitable abiotic environmental conditions that have a stronger influence on cyst numbers than resource quantity. We discuss how bottom-up control of cyst nematodes may indirectly protect the plant against harmful root knot nematodes.     

The Natural Host Range of Steinernema and Heterorhabditis spp. and Their Impact on Insect Populations

The natural host range of entomopathogenic nematodes of the genera Steinernema and Heterorhabditis can be defined as the range of insects which indigenous nematode populations use for propagation. Information on the natural host range is rare. However, based on records of insects found to be naturally infected with nematodes, some conclusions regarding the natural host range of some Steinernema spp. and Heterorhabditis spp. are presented. Reports of indigenous nematode populations impacting on insect populations can be divided between relatively balanced, long-lasting nematode-host associations and unbalanced, short-lasting epizootics. Examples of the augmentation and inoculative introduction of nematodes in agriculture and forestry ecosystems are presented. Based on current knowledge, nematode reproduction strategies are discussed and indications of the risk involved in the release of non-indigenous nematodes are given.     

Ecology in the service of biological control: the case of entomopathogenic nematodes

 Biological control manipulations of natural enemies to reduce pest populations represent large-scale ecological experiments that have both benefited from and contributed to various areas of modern ecology. Unfortunately, economic expediency and the need for rapid implementation often require that biological control programs be based more on trial and error than on sound ecological theory and testing. This approach has led to some remarkable successes but it has also produced dismal failures. This point is particularly well illustrated in the historical development and use of entomopathogenic nematodes for the biological control of insect pests. Intense effort has focused on developing these natural enemies as alternatives to chemical insecticides, in part because laboratory assays indicated that these nematodes possess a broad host range. This illusory attribute launched hundreds of field releases, many of which failed due to ecological barriers to infection that are not apparent from laboratory exposures, where conditions are optimal and host-parasite contact assured. For example, the entomopathogenic nematode Steinernema carpocapsae is a poor choice to control scarab larvae because this nematode uses an ambusher foraging strategy near the soil surface whereas the equally sedentary scarab remains within the soil profile, shows a weak host recognition response to scarabs, has difficulty overcoming the scarab immune response, and has low reproduction in this host. Conversely, two other nematodes, Heterorhabditis bacteriophora and S. glaseri, are highly adapted to parasitize scarabs: they use a cruising foraging strategy, respond strongly to scarabs, easily overcome the immune response, and reproduce well in these hosts. Increased understanding of the ecology of entomopathogenic nematodes has enabled better matches between parasites and hosts, and more accurate predictions of field performance. These results underline the importance of a strong partnership between basic and applied ecology in the area of biological control. Received: 15 July 1996 / Accepted: 5 November 1996     

Effect of neonicotinoid synergists on entomopathogenic nematode fitness

In previous greenhouse and field studies, the neonicotinoid insecticide imidacloprid interacted synergistically with five entomopathogenic nematode species against five scarab species. Two other neonicotinoids, thiamethoxam and acetamiprid, showed a weaker interaction with nematodes in scarab larvae. Entomopathogenic nematodes have the potential to recycle in hosts after inundative applications, thereby increasing the persistence of nematodes and insect control. Thus we investigated the effect of neonicotinoids on nematode fitness after tank mixing and after combined applications. Tank mixing only had a negative effect on nematode survival and infectivity in a few nematode–insecticide combinations and only if both insecticide concentration and exposure time were several times higher than typical for field applications. Combined application of nematodes with imidacloprid generally had no negative effect on the percentage of scarab cadavers producing progeny or the number of nematode progeny emerging per cadaver. In experiments with a synergistic increase in scarab mortality, the total number of progeny in combination treatments was up to four times higher than in nematodes only treatments. Similarly, nematode populations in soil from combination treatments were 13.2 times greater than for nematodes only treatments at 28 days after treatment. Combined imidacloprid–nematode applications did not affect the pathogenicity or infectivity of the nematode progeny. Combining thiamethoxam with nematodes had no negative effects on nematode reproduction in the majority of treatments. However, due to the weaker interaction of thiamethoxam and nematodes on scarab mortality, the total number of nematode progeny per treatment generally did not increase compared with nematodes only treatments. The demonstrated tank mix compatibility of imidacloprid and nematodes improves the feasibility of combining these agents for curative white grub control. The positive effect of imidacloprid on nematode reproduction after combined application may increase the likelihood of infection of white grubs by subsequent generations of nematodes, thereby improving their field persistence and biological control potential.     

Functionality of resistance gene Hero, which controls plant root-infecting potato cyst nematodes, in leaves of tomato

The expression of host genomes is modified locally by root endoparasitic nematode secretions to induce the development of complex cellular structures referred as feeding sites. In compatible interactions, the feeding sites provide the environment and nutrients for the completion of the nematode's life cycle, whereas in an incompatible (resistant) interaction, the host immune system triggers a plant cell death programme, often in the form of a hypersensitive reaction, which restricts nematode reproduction. These processes have been studied in great detail in organ tissues normally infected by these nematodes: the roots. Here we show that host leaves can support a similar set of programmed developmental events in the potato cyst nematode Globodera rostochiensis life cycle that are typical of the root-invading nematodes. We also show that a gene-for-gene type specific disease resistance that is effective against potato cyst nematodes (PCN) in roots also operates in leaves: the expression of the resistance (R) gene Hero and members of its gene family in leaves correlates with the elicitation of a hypersensitive response only during the incompatible interaction. These findings, and the ability to isolate RNA from relevant parasitic stages of the nematode, may have significant implications for the identification of nematode factors involved in incompatible interactions.     

A novel ascaroside controls the parasitic life cycle of the entomopathogenic nematode Heterorhabditis bacteriophora

Entomopathogenic nematodes survive in the soil as stress-resistant infective juveniles that seek out and infect insect hosts. Upon sensing internal host cues, the infective juveniles regurgitate bacterial pathogens from their gut that ultimately kill the host. Inside the host, the nematode develops into a reproductive adult and multiplies until unknown cues trigger the accumulation of infective juveniles. Here, we show that the entomopathogenic nematode Heterorhabditis bacteriophora uses a small-molecule pheromone to control infective juvenile development. The pheromone is structurally related to the dauer pheromone ascarosides that the free-living nematode Caenorhabditis elegans uses to control its development. However, none of the C. elegans ascarosides are effective in H. bacteriophora, suggesting that there is a high degree of species specificity. Our report is the first to show that ascarosides are important regulators of development in a parasitic nematode species. An understanding of chemical signaling in parasitic nematodes may enable the development of chemical tools to control these species.     

Population biology and biological control of nematodes.

We studied the population biology of the nematophagous fungus Hirsutella rhossiliensis to understand its potential as a biological control agent. Because the fungus is an infectious and transmissible parasite, we framed our study within an epidemiological context. Field observations, theory, and experiments demonstrated that (i) parasitism of nematodes by H. rhossiliensis is dependent on nematode density, (ii) local populations of the fungus will go extinct unless supplied with some minimum number of nematodes (the host threshold density), and (iii) natural epidemics of this fungus in populations of nematodes develop slowly and only after long periods of high host density. Additional in-depth research on population biology is needed to explain other biological control systems and to guide future research. The most effective research will combine field observation, theory, and experimentation.     

The effect of manganese deficiency and cereal cyst nematode infection on the growth of barley

Summary Two factorial pot experiments, each with four rates of soil-applied manganese (Mn), four levels of cereal cyst nematodes (CCN) and four replicates, were conducted under controlled environmental conditions to investigate the interaction of these factors on growth of barley. The experiments were harvested sequentially to count numbers of nematodes at two developmental stages. Development of the nematodes was independent of the Mn status of the host, indicating no effect of Mn on host resistance. In the absence of CCN, decreased Mn had a small effect on vegetative growth, not significantly decreasing dry weight of tops but tolerance at the higher densities of CCN was considerably reduced at low Mn supply, both vegetative growth and grain yield being depressed. Increasing CCN density exerted little effect on the growth of Mn sufficient hosts. Two field surveys supported conclusions from the pot experiments in that increasing nematode densities were correlated with decreasing plant size only at the site where plants were low in Mn. An implication for the field is that crops infected with the nematode and also low in Mn may yield less than expected because the two factors interact to reduce growth and retard development.     

Identification of genes involved in Meloidogyne incognita-induced gall formation processes in Arabidopsis thaliana

Root-knot nematodes (RKN; Meloidogyne incognita) are phytoparasitic nematodes that cause significant damage to crop plants worldwide. Recent studies have revealed that RKNs disrupt various physiological processes in host plant cells to induce gall formation. However, little is known about the molecular mechanisms of gall formation induced by nematodes. We have previously found that RNA expression levels of some of genes related to micro-RNA, cell division, membrane traffic, vascular formation, and meristem maintenance system were modified by nematode infection. Here we evaluated these genes importance during nematode infection by using Arabidopsis mutants and/or β-glucronidase (GUS) marker genes, particularly after inoculation with nematodes, to identify the genes involved in successful nematode infection. Our results provide new insights not only for the basic biology of plant–nematode interactions but also to improve nematode control in an agricultural setting.     

Colorado potato beetle control by application of the entomopathogenic nematode Heterorhabditis marelata and potato plant alkaloid manipulation

Control of the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), with the entomopathogenic nematode Heterorhabditis marelata Liu and Berry (Nematoda: Heterorhabditidae) was examined in the laboratory and in potato fields in north central Oregon. This research tested the hypothesis that varying nitrogen fertilizer levels would affect foliar alkaloid levels, which would stress the host, and allow increased nematode reproduction and long‐term control of the CPB. Laboratory results indicated that nematodes tended to reproduce more readily in CPB fed on potato plants with high levels of fertilizer. Field trials tested CPB population responses to four treatments: application of nematodes vs. no nematodes, with application of low vs. high rates of nitrogen fertilizer. The higher nitrogen application rate increased field foliar levels of the alkaloids solanine by 35%, and chaconine by 41% over the season. Nematodes were applied twice during the season, causing a 50% reduction in adult CPB populations, and producing six times as many dead prepupae in nematode‐treated soil samples as in the untreated samples. However, no reproducing nematodes were found in the 303 dead prepupae and pupae collected from nematode‐treated plots. Nitrogen fertilizer levels, and their related alkaloid levels, did not affect nematode infection rates or reproduction in the field. Foliar alkaloid levels of plants from the growth chamber were 3–6‐fold as high as those in the field, which may explain the variation in nematode response to nitrogen applications to host plants of the CPB. Heterorhabditis marelata is effective for controlling CPB in the field, and does not have negative non‐target effects on one of the most common endemic CPB control agents, Myiopharus doryphorae (Riley) (Diptera: Tachinidae), but the low rate of nematode reproduction cannot be manipulated through alkaloid stress to the beetle. Until H. marelata can be mass‐produced in an inexpensive manner, it will not be a commercially viable control for CPB.     

Profiling Nematode Communities in Unmanaged Flowerbed and Agricultural Field Soils in Japan by DNA Barcode Sequencing

Soil nematodes play crucial roles in the soil food web and are a suitable indicator for assessing soil environments and ecosystems. Previous nematode community analyses based on nematode morphology classification have been shown to be useful for assessing various soil environments. Here we have conducted DNA barcode analysis for soil nematode community analyses in Japanese soils. We isolated nematodes from two different environmental soils of an unmanaged flowerbed and an agricultural field using the improved flotation-sieving method. Small subunit (SSU) rDNA fragments were directly amplified from each of 68 (flowerbed samples) and 48 (field samples) isolated nematodes to determine the nucleotide sequence. Sixteen and thirteen operational taxonomic units (OTUs) were obtained by multiple sequence alignment from the flowerbed and agricultural field nematodes, respectively. All 29 SSU rDNA-derived OTUs (rOTUs) were further mapped onto a phylogenetic tree with 107 known nematode species. Interestingly, the two nematode communities examined were clearly distinct from each other in terms of trophic groups: Animal predators and plant feeders were markedly abundant in the flowerbed soils, in contrast, bacterial feeders were dominantly observed in the agricultural field soils. The data from the flowerbed nematodes suggests a possible food web among two different trophic nematode groups and plants (weeds) in the closed soil environment. Finally, DNA sequences derived from the mitochondrial cytochrome oxidase c subunit 1 (COI) gene were determined as a DNA barcode from 43 agricultural field soil nematodes. These nematodes were assigned to 13 rDNA-derived OTUs, but in the COI gene analysis were assigned to 23 COI gene-derived OTUs (cOTUs), indicating that COI gene-based barcoding may provide higher taxonomic resolution than conventional SSU rDNA-barcoding in soil nematode community analysis.     

An emerging example of tritrophic coevolution between flies (Diptera: Fergusoninidae) and nematodes (Nematoda: Neotylenchidae) on Myrtaceae host plants

A unique obligate mutualism occurs between species of Fergusonina Malloch flies (Diptera: Fergusoninidae) and nematodes of the genus Fergusobia Currie (Nematoda: Neotylenchidae). These mutualists together form different types of galls on Myrtaceae, mainly in Australia. The galling association is species‐specific, and each mutualism in turn displays host specificity. This tritrophic system represents a compelling arena to test hypotheses about coevolution between the host plants, parasitic nematodes and the fergusoninid flies, and the evolution of these intimate mutualisms. We have a basic knowledge of the interactions between the host plant, fly and nematode in this system, but a more sophisticated understanding will require a much more intensive and coordinated research effort. Summaries of the known Fergusonina/Fergusobia species associations and gall type terminology are presented. This paper identifies the key advantages of the system and questions to be addressed, and proposes a number of predictions about the evolutionary dynamics of the system given our understanding of the biology of the mutualists. Future research will profitably focus on (1) gall cecidogenesis and phenology, (2) the interaction between the fly larva and the nematode in the gall, and between the adult female fly and the parasitic nematode, (3) the means by which the fly and nematode life cycles are coordinated, (4) a targeted search of groups in the plant family Myrtaceae that have not yet been identified as gall hosts, and (5) establishment and comparison of the phylogenetic relationships of the host plants, fly species and nematodes. Recently derived phylogenies and divergence time estimation studies of the Diptera and the Myrtaceae show that the fly family Fergusoninidae is less than half the age of the Myrtaceae, discounting the hypothesis of cospeciation and coradiation of the fly/nematode mutualism and the plants at the broadest levels. However, cospeciation may have occurred at shallower levels in the phylogeny, following the establishment of the fly/nematode mutualism on the Myrtaceae. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 699–718.     

NemChR-DB: a database of parasitic nematode chemosensory G-protein coupled receptors

Nematode Chemosensory G-Protein Coupled Receptors have expanded within nematodes, where they play important roles in foraging and host-seeking behaviour. Nematode Chemosensory G-Protein Coupled Receptors are most highly expressed during free-living stages when chemosensory signalling is required for host detection and nematode activation in various parasitic nematodes, and therefore position Nematode Chemosensory G-Protein Coupled Receptors at the transition from infective to parasitic stages, making them important regulators to study in terms of host-seeking and host specificity. To facilitate the analysis of Nematode Chemosensory G-Protein Coupled Receptors, here we describe an integrative database of nematode chemoreceptors called NemChR-DB. This database enables users to study diverse parasitic nematode chemoreceptors, functionally explore sequence entries through structural and literature-based annotations, and perform cross-species comparisons.