Aggregation patterns of helminth populations in the introduced fish, Liza haematocheilus (Teleostei: Mugilidae): disentangling host–parasite relationships

A number of hypotheses exist to explain aggregated distributions, but they have seldom been used to investigate differences in parasite spatial distribution between native and introduced hosts. We applied two aggregation models, the negative binomial distribution and Taylor’s power law, to study the aggregation patterns of helminth populations from Liza haematocheilus across its native (Sea of Japan) and introduced (Sea of Azov) distribution ranges. In accordance with the enemy release hypothesis, we predicted that parasite populations in the introduced host range would be less aggregated than in the native host area, because aggregation is tightly constrained by abundance. Contrary to our expectation, aggregation of parasite populations was higher in the introduced host range. However, the analyses suggested that the effect of host introduction on parasite aggregation depends on whether parasite species, or higher level taxonomic groups, were acquired in or carried into the new area. The revealed similarity in the aggregation parameters of co-introduced monogeneans can be attributed to the repeatability and identity of the host–parasite systems. In contrast, the degree of aggregation differed markedly between regions for higher level taxa, which are represented by the native parasites in the Sea of Japan versus the acquired species in the Sea of Azov. We propose that the host species plays a crucial role in regulating infra-population sizes of acquired parasites due to the high rate of host-induced mortality. A large part of the introduced host population may remain uninfected due to their resistance to native naïve parasites. The core concept of our study is that the comparative analysis of aggregation patterns of parasites in communities and populations, and macroecological relationships, can provide a useful tool to reveal cryptic relationships in host–parasite systems of invasive hosts and their parasites.     

The paradox of the parasites: implications for biological invasion

The enemy-release hypothesis for biological invasions supposes that invasive species may be more successful in their introduced ranges than in their native ranges owing to the absence of coevolved natural enemies. Recent studies supporting this hypothesis have found that introduced plants and animals are less parasitized in their introduced ranges than in their native ranges. Expanding on this theory, I hypothesize that the role of enemy release may differ among the introduction, establishment and spread phases of an invasion. I present a simple model indicating that parasite release is unlikely to greatly affect the chance of establishment in populations with and without an immune subpopulation. The specific numerical relationship between the number of individuals introduced and the chance of establishment depends on a relationship between virulence, here conceptualized as the chance for the extinction of a lineage, and the fraction of the population infected at introduction. These results support the idea of a 'filter effect' in which different biological processes regulate the different phases of an invasion.     

Two tests of enemy release of commonly co-occurring bunchgrasses native in Europe and introduced in the United States

The popularly cited enemy release hypothesis, which states that non-native species are released from population control by their enemies, has not been adequately tested in plants. Many empirical studies have compared damage to native versus non-native invaders only in the invaded range, which can lead to erroneous conclusions regarding enemy release. Biogeographical studies that have compared natural enemies in native and introduced ranges have typically focused on a small area of the plants’ distributions in each range, only one plant species, and/or only one guild of natural enemies. To test enemy release, we first surveyed both pathogens and herbivores in multiple populations in both the native and naturalized ranges of three commonly co-occurring perennial bunchgrasses introduced to the United States from Europe. We then compared our field results to the number of fungal pathogens that have been documented on each species from published host-pathogen data compilations. Consistent with enemy release, our field survey showed less herbivory and denser populations in the naturalized range, but there was no evidence of release from pathogens. In contrast, the published host-pathogen data compilations produced evidence of enemy release from pathogens. The difference in results produced by the two approaches highlights the need for multiple approaches to testing mechanisms of invasions by introduced species, which can enable well supported theory to inform sound management practices.     

High parasite infection level in non‐native invasive species: it is just a matter of time

The enemy release hypothesis is often used to explain the success of non‐native species invasions. Growing evidence indicates that parasite or pathogen species richness increases over time in invasive non‐native species; however, this increase should not directly translate into release from enemy pressure as infection intensity of parasites (number of parasites per host) has a more profound impact on host fitness. The changes in intensity of parasitic infections in invasive non‐native species have not yet been thoroughly analysed in newly colonized areas. The goal of this study was to determine whether gastrointestinal parasite (nematode and trematode) infection intensity has increased with time since the populations of American mink Neovison vison were established and how host demographic parameters affect infection intensity. We tested the enemy release hypothesis by substituting space for time, evaluating parasite abundance in American mink at six sites along a chronosequence of mink invasion history. Nematode and trematode abundance increased with time since mink introduction, from a few parasites on average per mink after 16 yr, to 200–250 parasites per mink after 34 yr. The rate of increase in parasite abundance varied among demographic groups of mink (sex and age). Both nematodes and trematodes were more abundant in males than in females, and in subadults than in adults. Higher nematode abundance negatively affected body condition of mink. Our results provide evidence that non‐native species are released from enemy pressure only in the first phase of invasion, and that infection is modulated by host demographics and season. These results contribute to the evaluation of the long‐term patterns of parasite accumulation in invasive non‐native species after their colonization of new territories.     

Environmental,geographical and time-related impacts on avian malaria infections in native and introduced populations of house sparrows (Passer domesticus), a globally invasive species

Aim

The increasing spread of vector-borne diseases has resulted in severe health concerns for humans, domestic animals and wildlife, with changes in land use and the introduction of invasive species being among the main possible causes for this increase. We explored several ecological drivers potentially affecting the local prevalence and richness of avian malaria parasite lineages in native and introduced house sparrows (Passer domesticus) populations.

Location

Global.

Time period

2002–2019.

Major taxa studied

Avian Plasmodium parasites in house sparrows.

Methods

We analysed data from 2,220 samples from 69 localities across all continents, except Antarctica. The influence of environment (urbanization index and human density), geography (altitude, latitude, hemisphere) and time (bird breeding season and years since introduction) were analysed using generalized additive mixed models (GAMMs) and random forests.

Results

Overall, 670 sparrows (30.2%) were infected with 22 Plasmodium lineages. In native populations, parasite prevalence was positively related to urbanization index, with the highest prevalence values in areas with intermediate urbanization levels. Likewise, in introduced populations, prevalence was positively associated with urbanization index; however, higher infection occurred in areas with either extreme high or low levels of urbanization. In introduced populations, the number of parasite lineages increased with altitude and with the years elapsed since the establishment of sparrows in a new locality. Here, after a decline in the number of parasite lineages in the first 30 years, an increase from 40 years onwards was detected.

Main conclusions

Urbanization was related to parasite prevalence in both native and introduced bird populations. In invaded areas, altitude and time since bird introduction were related to the number of Plasmodium lineages found to be infecting sparrows.     

Resistance in introduced populations of a freshwater snail to native range parasites

Introduced species provide an opportunity to examine responses to novel ecological conditions, in particular to the absence of co-evolved enemies. Introduced populations could evolve lower investment in resistance or could down-regulate their immune system as a plastic response to enemy absence. The response might have consequences for the success of introduced species. Assuming a trade-off between resistance and traits related to demographic success, an evolved change or reallocation from resistance could increase the chances of invasions. On the other hand, introduced populations could have increased resistance as a correlate of greater vigour and competitive ability among successful invaders [Sampling Bias hypothesis (SBH)]. These hypotheses make different predictions about investment in resistance in introduced populations. Using a New Zealand clonal snail (Potamopyrgus antipodarum), we examined the resistance of three introduced genotypes (one from the US and two from Europe) to several populations of a native range parasite (Microphallus sp.). One genotype (Euro A) was resistant to all native range parasite populations, consistent with the SBH. However, two remaining genotypes (Euro C and US 1) were less susceptible to parasite populations that were allopatric to their source populations. Furthermore, resistance of one genotype (US 1) collected from the introduced range was indistinguishable from its resistance when collected from the range of the parasite. Hence, there was no evidence for decreased resistance in the absence of native enemies, which is inconsistent with hypotheses that envision reduced allocation to resistance or a trade-off between competitive ability and resistance.     

Release from natural enemies mitigates inbreeding depression in native and invasive Silene latifolia populations

Inbreeding and enemy infestation are common in plants and can synergistically reduce their performance. This inbreeding ×environment (I × E) interaction may be of particular importance for the success of plant invasions if introduced populations experience a release from attack by natural enemies relative to their native conspecifics. Here, we investigate whether inbreeding affects plant infestation damage, whether inbreeding depression in growth and reproduction is mitigated by enemy release, and whether this effect is more pronounced in invasive than native plant populations. We used the invader Silene latifolia and its natural enemies as a study system. We performed two generations of experimental out‐ and inbreeding within eight native (European) and eight invasive (North American) populations under controlled conditions using field‐collected seeds. Subsequently, we exposed the offspring to an enemy exclusion and inclusion treatment in a common garden in the species’ native range to assess the interactive effects of population origin (range), breeding treatment, and enemy treatment on infestation damage, growth, and reproduction. Inbreeding increased flower and leaf infestation damage in plants from both ranges, but had opposing effects on fruit damage in native versus invasive plants. Inbreeding significantly reduced plant fitness; whereby, inbreeding depression in fruit number was higher in enemy inclusions than exclusions. This effect was equally pronounced in populations from both distribution ranges. Moreover, the magnitude of inbreeding depression in fruit number was lower in invasive than native populations. These results support that inbreeding has the potential to reduce plant defenses in S. latifolia, which magnifies inbreeding depression in the presence of enemies. However, future studies are necessary to further explore whether enemy release in the invaded habitat has actually decreased inbreeding depression and thus facilitated the persistence of inbred founder populations and invasion success.     

Parasite-mediated enemy release and low biotic resistance may facilitate invasion of Atlantic coral reefs by Pacific red lionfish (<Emphasis Type="Italic">Pterois volitans</Emphasis>)

Successful invasions are largely explained by some combination of enemy release, where the invader escapes its natural enemies from its native range, and low biotic resistance, where native species in the introduced range fail to control the invader. We examined the extent to which parasites may mediate both release and resistance in the introduction of Pacific red lionfish (Pterois volitans) to Atlantic coral reefs. We found that fewer lionfish were parasitized at two regions in their introduced Atlantic range (The Bahamas and the Cayman Islands) than at two regions in their native Pacific range (the Northern Marianas Islands and the Philippines). This pattern was largely driven by relatively high infection rates of lionfish by didymozoan fluke worms and parasitic copepods (which may be host-specific to Pterois lionfishes) in the Marianas and the Philippines, respectively. When compared with sympatric, native fishes in the Atlantic, invasive lionfish were at least 18 times less likely to host a parasite in The Bahamas and at least 40 times less likely to host a parasite in the Cayman Islands. We found no indication that lionfish introduced Pacific parasites into the Atlantic. In conjunction with demographic signs of enemy release such as increased density, fish size, and growth of invasive lionfish, it is possible that escape from parasites may have contributed to the success of lionfish. This is especially true if future studies reveal that such a loss of parasites has led to more energy available for lionfish growth, reproduction, and/or immunity.     

The effect of enemy-release and climate conditions on invasive birds: a regional test using the rose-ringed parakeet (Psittacula krameri) as a case study

Aim Some invasive species succeed particularly well and manage to establish populations across a wide variety of regions and climatic conditions. Understanding how biotic and environmental factors facilitate their invasion success remains a challenge. Here, we assess the role of two major hypotheses explaining invasion success: (1) enemy‐release, which argues that invasive species are freed from their native predators and parasites in the new areas; and (2) climate‐matching, which argues that the climatic similarity between the exotic and native range determines the success of invasive populations. Location India, Israel and the UK. Methods We studied the reproductive success of one of the most successful avian invaders, the rose‐ringed parakeet (Psittacula krameri), in its native range (India) and in two introduced regions, varying in their climate conditions (Israel and the UK). We combined literature and field data to evaluate the role of predation pressure and climatic conditions in explaining the differences in reproductive success between the three regions. Results We found significant differences in reproductive success between regions. In accordance with the enemy‐release hypothesis, we discovered that while predation was the main factor responsible for the reduction of fecundity in India, it did not significantly affect the fecundities of parakeet populations in the two introduced regions. In accordance with the climate‐matching hypothesis, we found that in the colder temperate UK, egg infertility was high, resulting in lower fecundities. Populations in both the warmer Mediterranean climate of Israel and in the native Indian range had significantly lower egg infertility and higher fecundities than the UK populations. Main conclusions Our findings support both the enemy‐release and the climate‐matching hypotheses. While release from predators facilitates the reproductive success and therefore the invasiveness of parakeets in both the UK and in Israel, colder climate impedes reproduction and therefore the spread of parakeets in the UK.     

Phenotypic and genetic differentiation between native and introduced plant populations

Plant invasions often involve rapid evolutionary change. Founder effects, hybridization, and adaptation to novel environments cause genetic differentiation between native and introduced populations and may contribute to the success of invaders. An influential idea in this context has been the Evolution of Increased Competitive Ability (EICA) hypothesis. It proposes that after enemy release plants rapidly evolve to be less defended but more competitive, thereby increasing plant vigour in introduced populations. To detect evolutionary change in invaders, comparative studies of native versus introduced populations are needed. Here, we review the current empirical evidence from: (1) comparisons of phenotypic variation in natural populations; (2) comparisons of molecular variation with neutral genetic markers; (3) comparisons of quantitative genetic variation in a common environment; and (4) comparisons of phenotypic plasticity across different environments. Field data suggest that increased vigour and reduced herbivory are common in introduced plant populations. In molecular studies, the genetic diversity of introduced populations was not consistently different from that of native populations. Multiple introductions of invasive plants appear to be the rule rather than the exception. In tests of the EICA hypothesis in a common environment, several found increased growth or decreased resistance in introduced populations. However, few provided a full test of the EICA hypothesis by addressing growth and defence in the same species. Overall, there is reasonable empirical evidence to suggest that genetic differentiation through rapid evolutionary change is important in plant invasions. We discuss conceptual and methodological issues associated with cross-continental comparisons and make recommendations for future research. When testing for EICA, greater emphasis should be put on competitive ability and plant tolerance. Moreover, it is important to address evolutionary change in characteristics other than defence and growth that could play a role in plant invasions.     

Patterns of haemosporidian prevalence along a range expansion in introduced Kenyan house sparrows Passer domesticus

Avian haemosporidian infections (of the genera Haemoproteus, Plasmodium and Leucocytozoon) can regulate passerine populations. Thus, reduction in the number of avian haemosporidian infections in a population, for example in recently introduced hosts, may facilitate host establishment or spread (i.e. enemy release). Alternatively, colonizers could decrease competitive ability of native individuals in the novel range by increasing the prevalence of avian haemosporidians in that native passerine community (i.e. novel weapons). However, whether either or both of these phenomena will occur is difficult to predict because infection risk can be highly heterogeneous and dependent upon the interaction of biotic and abiotic factors at the microclimate level, especially because of the important role of vectors for these parasites. Here, we describe which factors best predicted avian haemosporidian prevalence in populations of house sparrows Passer domesticus introduced to Kenya. House sparrows inhabit an invasion gradient in Kenya; they were introduced via the eastern port city of Mombasa in ? 1950 and have since spread west‐ward across the country. This range expansion gave us the opportunity to examine how parasite prevalence changes over small spatiotemporal scales and what role is played by environmental and individual traits. Among all individuals, body mass was the strongest predictor of infection, with larger house sparrows being more likely to be infected. At the population level, capture month, precipitation (higher prevalence with more rainfall), and population age (increasing prevalence with increasing time since introduction) were important risk factors. Overall, haemosporidian prevalence in Kenyan house sparrows appears to be more strongly associated with individual characteristics rather than with time since introduction as was predicted, though this does not necessarily rule out a role for enemy release or novel weapons in this system.     

Invasive Eupatorium adenophorum suffers lower enemy impact on carbon assimilation than native congeners

Enemy release hypothesis predicts that alien plants that escape from their natural enemies suffer lower enemy regulation in their introduced ranges than in native ranges. An extension of this theory suggests that if enemy release plays a crucial role in invasive success, then in the introduced range, invasive plants should also suffer lower local enemy impact than native residents (local enemy release hypothesis, LERH). In order to test LERH, we compared invasive Eupatorium adenophorum with two native congeners (E. heterophyllum and E. japonicum) in terms of damage by leaf enemies at two natural field sites and two manipulated sites. We also determined enemy impact on carbon assimilation at two manipulated sites. In each site, E. adenophorum was only damaged by herbivores, while in native congeners, leaf scabs or (and) leaf rolls was found in addition to herbivory damage. In both manipulated sites, the total enemy impact on carbon assimilation was lower for E. adenophorum than for native congeners; this observation was consistent with LERH. The results of this study indicate that a short co-existence time with generalist enemies (behavior constraint) might be the main contributor to the lower enemy impact on E. adenophorum.     

Evidence for the enemy release hypothesis in <Emphasis Type="Italic">Hypericum perforatum</Emphasis>

The enemy release hypothesis (ERH), which has been the theoretical basis for classic biological control, predicts that the success of invaders in the introduced range is due to their release from co-evolved natural enemies (i.e. herbivores, pathogens and predators) left behind in the native range. We tested this prediction by comparing herbivore pressure on native European and introduced North American populations of Hypericum perforatum (St Johns Wort). We found that introduced populations occur at larger densities, are less damaged by insect herbivory and suffer less mortality than populations in the native range. However, overall population size was not significantly different between ranges. Moreover, on average plants were significantly smaller in the introduced range than in the native range. Our survey supports the contention that plants from the introduced range experience less herbivore damage than plants from the native range. While this may lead to denser populations, it does not result in larger plant size in the introduced versus native range as postulated by the ERH.     

For or against: the importance of variation in growth rate for testing the EICA hypothesis

The evolution of increased competitive ability (EICA) hypothesis proposes that invasive species evolve decreased defense and increased competitive ability following natural enemy release. Previous studies have found evidence both for and against EICA. The resource-enemy release hypothesis (R-ERH) suggests that fast-growing species may experience stronger enemy release than slow-growing species. On the basis of R-ERH, the prediction of EICA will be held true for slow-growing genotypes, i.e., the slow-growing genotypes from the introduced range will be less resistant to herbivory and grow faster than those from the home range; while the EICA will not be held for fast-growing genotypes, i.e., there will be no significant differences in growth and defense traits between the introduced and native fast-growing genotypes. We tested these predictions preliminarily using five populations of the invasive plant Alternanthera philoxeroides. This species has two varieties in its home range, which showed a distinct growth-defense strategy: the northern A. p. var. acutifolia (Apa) had higher growth rate but lower resistance, while the southern A. p. var. obtusifolia (Apo) had lower growth rate but higher resistance level. Our results suggest that the EICA hypothesis is consistent with the slow-growing Apo, but not with the fast-growing Apa. We suggest that evolutionary changes in growth or resistance following enemy release are influenced by variation in growth rate within an invasive alien plant. These findings have important implications for the EICA hypothesis, and may partially explain why previous studies have found evidence both for and against EICA.     

Immunological change in a parasite-impoverished environment: divergent signals from four island taxa

Dramatic declines of native Hawaiian avifauna due to the human-mediated emergence of avian malaria and pox prompted an examination of whether island taxa share a common altered immunological signature, potentially driven by reduced genetic diversity and reduced exposure to parasites. We tested this hypothesis by characterizing parasite prevalence, genetic diversity and three measures of immune response in two recently-introduced species (Neochmia temporalis and Zosterops lateralis) and two island endemics (Acrocephalus aequinoctialis and A. rimitarae) and then comparing the results to those observed in closely-related mainland counterparts. The prevalence of blood parasites was significantly lower in 3 of 4 island taxa, due in part to the absence of certain parasite lineages represented in mainland populations. Indices of genetic diversity were unchanged in the island population of N. temporalis; however, allelic richness was significantly lower in the island population of Z. lateralis while both allelic richness and heterozygosity were significantly reduced in the two island-endemic species examined. Although parasite prevalence and genetic diversity generally conformed to expectations for an island system, we did not find evidence for a pattern of uniformly altered immune responses in island taxa, even amongst endemic taxa with the longest residence times. The island population of Z. lateralis exhibited a significantly reduced inflammatory cell-mediated response while levels of natural antibodies remained unchanged for this and the other recently introduced island taxon. In contrast, the island endemic A. rimitarae exhibited a significantly increased inflammatory response as well as higher levels of natural antibodies and complement. These measures were unchanged or lower in A. aequinoctialis. We suggest that small differences in the pathogenic landscape and the stochastic history of mutation and genetic drift are likely to be important in shaping the unique immunological profiles of small isolated populations. Consequently, predicting the impact of introduced disease on the many other endemic faunas of the remote Pacific will remain a challenge.     

Origin,local experience,and the impact of biotic interactions on native and introduced <Emphasis Type="Italic">Senecio</Emphasis> species

A key gap in understanding the long-term success of invasive species is how biotic interactions change with the duration of experience in the introduced range. We examined biotic interactions using a common garden experiment with native, hybrid, and exotic Senecio species representing a range of experience in the UK. Introduced species had fewer aphids and pathogens and more root colonization by mycorrhizal fungi compared to natives; hybrids generally had intermediate levels of interactions. The duration of experience in the introduced range was reflected by an increasing degree of variability in enemy release. These findings support the enemy release hypothesis and indicate the potential for changes in enemy release as time and experience in the new range increase.     

飞机草入侵种群和原产地种群生长和数量型化学防御物质含量差异的比较研究

增强竞争能力的进化假说认为,在入侵地外来植物逃离了原产地天敌的控制,把原来用于防御的资源分配到生长、生殖等,从而提高竞争力。为探讨进化在恶性外来入侵植物飞机草（Chromolaena odorata）入侵中的作用,在同质种植园中的两个养分条件下比较研究了飞机草原产地和入侵地各8个种群叶片单宁含量,茎和叶片总酚、半纤维素和纤维素含量以及总生物量的差异。结果表明,在两个养分条件下,飞机草入侵种群和原产地种群总生物量差异均不显著,入侵种群茎和叶片半纤维素含量均低于原产地种群;在高养分条件下,飞机草入侵种群叶片纤维素含量低于原产地种群;在低养分条件下,入侵种群茎和叶片总酚含量高于原产地种群。由此,我们得出结论：在入侵地,飞机草未发生加快生长的进化,但数量型化学防御物质发生了遗传变化;降低的半纤维素和纤维素含量可能是对入侵地专性天敌缺乏做出进化响应的结果,提高的总酚含量有利于飞机草防御入侵地的广谱天敌。     

Do alien plants escape from natural enemies of congeneric residents? Yes but not from all

As predicted by the enemy release hypothesis, plants are supposedly less attacked by herbivores in their introduced range than in their native range. However, the nature of the natural enemies, in particular their degree of specificity may also affect the level of enemy escape. It is therefore expected that ectophagous invertebrate species, being generally considered as more generalists than endophagous species, are more prompt to colonise alien plants. In Swiss, Siberian and Russian Far East arboreta, we tested whether alien woody plants are less attacked by native herbivorous insects than native congeneric woody plant species. We also tested the hypothesis that leaf miners and gall makers show stronger preference for native woody plants than external leaf chewers. In all investigated regions, leaf miners and gall makers were more abundant and showed higher species richness on native woody plants than on congeneric alien plants. In contrast, external leaf chewers did not cause more damage to native plants than to alien plants, possibly because leaf chewers are, in general, less species specific than leaf miners and gall makers. These results, obtained over a very large number of plant-enemy systems, generally support the hypothesis that alien plants partly escape from phytophagous invertebrates but also show that different feeding guilds may react differently to the introduction of alien plants.     

Reduced inflammation in expanding populations of a neotropical bird species

The loss of regulating agents such as parasites is among the most important changes in biotic interactions experienced by populations established in newly colonized areas. Under a relaxed parasite pressure, individuals investing less in costly immune mechanisms might experience a selective advantage and become successful colonizers as they re‐allocate resources to other fitness‐related traits. Accordingly, a refinement of the evolution of increased competitive ability (EICA) hypothesis proposed that immunity of invasive populations has evolved toward a reduced investment in innate immunity, the most costly component of immunity, and an increased humoral immunity that is less costly. Biogeographical approaches comparing populations between native and expansion ranges are particularly relevant in exploring this issue, but remain very scarce. We conducted a biogeographical comparison between populations of Spectacled Thrush (Turdus nudigenis) from the native area (South America) and from the expansion range (Caribbean islands). First, we compared haemosporidian prevalence and circulating haptoglobin (an acute‐phase protein produced during inflammation). Second, we challenged captive birds from both ranges with Escherichia coli lipopolysaccharides (LPS) and measured postchallenge haptoglobin production and body mass change. Birds from the expansion range showed lower haemosporidian prevalence and lower levels of haptoglobin than birds from the native range. In addition, the inflammation elicited by LPS injection and its associated cost in terms of body mass loss were lower in birds from the expansion range than in birds from the native range. In accordance with the enemy release hypothesis, our results suggest that range expansion is associated with a reduced infection risk. Our study also supports the hypothesis that individuals from newly established populations have evolved mechanisms to dampen the inflammatory response and are in accordance with one prediction of the refined EICA hypothesis, proposed to understand biological invasions.