Intermediate predator naïveté and sex-skewed vulnerability predict the impact of an invasive higher predator

The spread of invasive species continues to reduce biodiversity across all regions and habitat types globally. However, invader impact prediction can be nebulous, and approaches often fail to integrate coupled direct and indirect invader effects. Here, we examine the ecological impacts of an invasive higher predator on lower trophic groups, further developing methodologies to more holistically quantify invader impact. We employ functional response (FR, resource use under different densities) and prey switching experiments to examine the trait- and density-mediated impacts of the invasive mosquitofish Gambusia affinis on an endemic intermediate predator Lovenula raynerae (Copepoda). Lovenula raynerae effectively consumed larval mosquitoes, but was naïve to mosquitofish cues, with attack rates and handling times of the intermediate predator unaffected by mosquitofish cue-treated water. Mosquitofish did not switch between male and female prey, consistently displaying a strong preference for female copepods. We thus demonstrate a lack of risk-reduction activity in the presence of invasive fish by L. raynerae and, in turn, high susceptibility of such intermediate trophic groups to invader impact. Further, we show that mosquitofish demonstrate sex-skewed predator selectivity towards intermediate predators of mosquito larvae, which may affect predator population demographics and, perversely, increase disease vector proliferations. We advocate the utility of FRs and prey switching combined to holistically quantify invasive species impact potential on native organisms at multiple trophic levels.

Invasive fishes have been especially damaging to freshwater ecosystems, driving extinctions of indigenous species 18 . Human-mediated introductions of fish into novel, previously fishless systems risk fundamentally altering species compositions and diversities through processes such as predation 19,20 . A key challenge therefore surrounds the quantification and prediction of invasive higher predator impacts on underlying trophic groups. These impacts can be profound 21 , and may manifest in trophic cascades driven by both consumptive, density-mediated indirect interactions (DMIIs 22,23 ), and non-consumptive, trait-mediated indirect interactions (TMIIs 23,24 ). Critically, TMII effects may be as impactful as those resulting from direct consumption [24][25][26] . These effects can, in turn, be dependent on coevolutionary histories between trophic groups, or 'adaptive lag' of native assemblages 27 , and aquatic systems present an ideal platform to examine indirect, TMII effects due to the prevalence and ease of manipulation of water-borne predator cues 28 . However, predicting impacts by invasive species on native prey can be complicated due to density-and context-dependencies, which may be non-additive in effect [29][30][31] .
Functional responses (FRs) have been used extensively in the quantification of consumer-resource interactions, and FRs can be powerful tools to quantify density-and context-dependencies of invader impact [32][33][34] . Indeed, FRs can be applied to examine multiple predator effects between interacting con-and interspecific invasive species 21,30 . In the context of predation, the FR encapsulates prey consumption by predators in relation to prey density, with both FR form and magnitude powerful indicators for the derivation of consumer impact strengths 34 . Three common forms of FRs have been categorised: (1) Type I FRs are regarded as filter feeder-specific, with intake increasing linearly with prey availability 35 ; (2) Type II FRs are characterised by a decelerating intake rate, which may be conducive to prey destabilisation as a result of high proportional consumption at low prey densities 34 ; (3) Type III FRs are, in turn, characterised by low intake rate at low prey densities, and are sigmoidal in form 33 , thus potentially imparting stability to prey populations by facilitating refugia for prey at low densities. The application of comparative FRs can not only be informative in terms of relative consumer impacts, but also directly enables the derivation of emergent context-dependencies that modulate consumer-resource interaction strengths 34,36 . These effects can be both abiotic (e.g. temperature/structural complexity 37 ) and biotic (e.g. higher predators 21 ). For instance, the detection of kairomones from a familiar higher predator can modify foraging intensity of intermediate predators towards basal prey 38 . This may manifest in modulations to the form and magnitude of FRs 28 . However, in cases where an intermediate predator is exposed to a novel threat, these responses may be nullified due to naïveté and, thus, predation vulnerability may not be alleviated.
Another classic concept within consumer-resource ecology surrounds prey switching, or frequency-dependence of predation 39 . Prey switching may be a powerful indicator when utilised alongside FRs to examine consumptive traits and impacts. However, prey switching has hitherto remained under-applied in invasion science, reducing our capacity to predict invader impacts (but see Cuthbert et al. 17 ). Characteristically, when consumers exhibit a prey switching propensity, disproportionately more of the abundant prey type are consumed whilst disproportionately fewer rare prey are consumed 39 . This can foster stability in diverse prey populations, enabling coexistence patterns to emerge. Indeed, prey switching can be a key driver of the sigmoidal, stabilising Type III FR 40 . Furthermore, switching between intraspecific prey types can have demographic implications, particularly if prey consumption is sex-skewed. In turn, this can lead to emergent inequalities in sex ratios which may affect the population persistence of lower trophic groups 41,42 . As such, quantifying prey switching propensities between intraspecific prey forms can elucidate likely demographic and density-mediated outcomes for prey species following novel higher predator introductions.
The mosquitofish, Gambusia affinis (Baird and Girard), is one of the most widespread fish globally, having been introduced extensively in mosquito control efforts in recent decades 43 . Further, it is regarded as one of the world's worst invasive species 44 , inducing negative impacts on native fish, amphibians and aquatic invertebrates 20,45,46 . The effectiveness of mosquitofish in biological control has been fundamentally questioned 47 , and their application has been recorded to, perversely, increase mosquito proliferations due to interguild predation upon intermediate trophic groups such as notonectids 48 . In turn, this has resulted in calls to cease the use of such non-native fish in biological control efforts 49 . Furthermore, mosquitoes have been shown to comprise less than 1% of the diet of G. affinis, whilst zooplankton compose a majority 50 , demonstrating generalist feeding strategies that reduce biological control efficacy of the mosquitofish. Yet, we currently lack holistic impact quantifications of such invasive species upon ecosystems outside of their native range.
In the present study, we thus use FR and prey switching experiments to quantify the impact of G. affinis on native trophic groups which are vulnerable to localised extinctions 19 . We examine the responsiveness of an intermediate predator, endemic to South Africa, the open-water calanoid copepod Lovenula raynerae Suárez-Morales, Wasserman and Dalu to water-borne mosquitofish cues, using mosquito larvae of the Culex pipiens complex as a basal prey. The C. pipiens mosquito complex is widespread globally, and colonises an extensive range of aquatic habitats, including temporary ponds. Calanoid copepods are also widespread and form an abundant and important component of freshwater ecosystems 51 . Lovenula raynerae is an ephemeral pond specialist species 52 , and thus has evolved within fishless aquatic systems. Given a limited distribution, this copepod is highly vulnerable to environmental change. Indeed, mosquitofish have been documented to invade ephemeral systems 53,54 , and L. raynerae have been detected in longstanding fishless systems where fish may persist if introduced (Wasserman pers. obs.). Thus, the potential for impact of mosquitofish on such vulnerable populations is high. Our approach examines responsiveness of L. raynerae consumption to visual and chemical mosquitofish cues and thus naïveté to predation by the novel invader. Additionally, we examine prey switching propensities of mosquitofish between female and male copepods, elucidating whether predation of L. raynerae by G. affinis will affect prey population viability through the establishment of sex-skewed ratios. Thus, we aim to illustrate the likely trait-and density-mediated impacts of the introduction of an invader on an intermediate predator and the cascade to its prey.
As first order terms were significantly negative in each experimental treatment (Table 1), we deemed all FRs to be categorically Type II. Attack rates of L. raynerae did not differ significantly between cue-free and G. affinis cue treatments (chemical cue: z = 0.63, p = 0.53; visual cue: z = 0.30, p = 0.76; both cues: z = 0.31, p = 0.76), and there was no significant difference within cue treatments (all p ≥ 0.44). Handling times of L. raynerae also did not vary significantly between cue-free and G. affinis-treated waters (chemical cue: z = 0.99, p = 0.32; visual cue: z = 0.32, p = 0.75; both cues: z = 0.20, p = 0.84), and there was, again, no significant difference within cue treatments (all p ≥ 0.24). Confidence intervals overlapped amongst all FRs across the entire spectrum of prey supplies, further illustrating similarities in attack rate, handling time and, inversely, maximum feeding rate parameters between different cue treatments ( Fig. 1).
In Experiment 2, mosquitofish displayed strong preference for female over male copepods at all prey proportions with the exception of extreme ratios (30:0, 0:30), wherein prey choice was necessarily restricted to one copepod sex (Table 2; Fig. 2). Thus, prey switching did not occur between male and female copepod prey, with preference for female copepods exhibited even when presented at relatively low proportions relative to males. Overall consumption was significantly greater for females than males (F 1,54 = 20.22, p < 0.001), and was significantly affected by the proportion of prey available (F 6,48 = 10.89, p < 0.001), with greater consumption for a specific prey type exhibited when it was available in higher proportions. There was no significant 'sex × proportion' interaction (F 6,42 = 1.01, p = 0.44), and thus this interaction was removed from the model. Manly's α preference indices were significantly greater for females, suggesting an overall preference for this prey type (χ 2 = 31.17, df = 1, p < 0.001; Table 2). Manly's α values were additionally significantly affected by the proportions of prey available (χ 2 = 58.82, df = 6, p < 0.001), and there was a significant 'sex × proportion' interaction (χ 2 = 15.08, df = 6, p = 0.02), with greater preference for females shown at intermediate prey ratios (Fig. 2).

Discussion
The identification of measures to understand and forecast invasive species impacts on recipient ecosystems is critical for biodiversity protection and developing proactive management approaches for invasions 34,55 . In our study system, we forecast trait-and density-mediated impacts of a widespread, invasive fish, the mosquitofish G. affinis, on an endemic intermediate predator, the calanoid copepod L. raynerae. We apply FR 32,33 and prey switching 42 approaches experimentally, showing firstly that the feeding magnitude of L. raynerae is not significantly affected by either chemical or visual cues of G. affinis. Secondly, our study highlights the much higher susceptibility of female over male L. raynerae copepods to G. affinis predation. Therefore, we show that the potential for invader impact is high, given that the invasive mosquitofish readily consumes and impacts populations of naïve intermediate predators of mosquito larvae, which may affect overall biotic resistance towards mosquito prey. In addition, invader impact may have implications for L. raynerae demographics as the copepod exhibits sex-skewed vulnerabilities to the invasive fish. These results are pertinent given that Wasserman et al. 41 showed, conversely, that natural predation on L. raynerae by common aquatic insects resulted in lower risk levels for females. Thus, augmented vertebrate predation through G. affinis introductions would likely have implications for L. raynerae population sex demographics in natural systems, having a further destabilising effect which may reduce population persistence of threatened endemic populations.
Predatory copepods, such as L. raynerae, often dominate small aquatic ecosystems which are of high importance for biodiversity in arid environments 56,57 . The small ecosystems which L. raynerae dominate function entirely differently to other aquatic systems, and are characterised by restricted higher trophic structuring 52 . Thus, populations within these habitats are especially vulnerable to augmented higher order predation through species introductions 19 . Given the orientation of this copepod to surface waters, vulnerabilities of the species to fish predation may be bolstered by indifferent foraging intensities in the presence of predator cues shown here, coupled with a pronounced association with the upper water column where mosquitofish forage 50 . Biotic contexts such as higher predator risk can have a substantial impact on predator-prey interaction strengths 21,47,58 , but can often be dependent on coevolutionary context 10,27,59,60 . Indeed, invertebrates have been found to be generally responsive to higher predator cues arising from different diets 26 . Such responses frequently reduce predatory impacts exerted upon basal prey by intermediate predators 28 61 and Cuthbert et al. 62,63 , where destabilising FRs of L. raynerae were also constrained with daphniids and culicids as a basal prey.
In addition to indirect interactions, selectivity by higher-order predators can have direct implications for the demographics of recipient ecosystems 41 . Higher male copepod vulnerability to predation has been recurrently hypothesised due to risks associated with mate-searching and copulation 64,65 . Indeed, Wasserman et al. 41 illustrated that predation of L. raynerae by native hexapods is selective towards males due to the processes of copulation. Here, however, we find the opposite in the presence of an invasive higher predator, with high, frequency-independent selectivity demonstrated towards females, which are larger and less motile than males (Cuthbert pers. obs.). The lack of prey switching exhibited here is indicative of an absence of prey refuge for female L. raynerae when available in lower proportions, which may have stark implications for demographics and the reproductive success in mature zooplankton populations following invasive fish introductions. The mechanisms of higher-order predatory pressure from fish operate entirely differently from invertebrates; where partial prey consumption is often exhibited by invertebrates, fish consume prey whole 41 . Therefore, the selective tendencies of higher-order fish predation towards females exhibited here may be compounded by the nullification of risk-evasion responses of females when copulating, with copulating pairs perceived, rather, as a single prey unit by fish. This is particularly relevant in light of the extended copulation period of L. raynerae and associated reduced instantaneous escape speed 41 . Thus, the introduction of invasive fish may fundamentally alter the demographics of prey populations in aquatic systems ecosystems previously dominated by invertebrates, potentially increasing extinction risk.

Conclusion
The spread of invasive species continues to circumvent biogeographical barriers and reduce biodiversity, and impacts on recipient communities can be intensified due to naïveté in recipient ecosystems 13,14 . Here, we illustrate, through the coupled use of experimental FR and prey switching approaches, that endemic intermediate predators in insular aquatic ecosystems are naïve to cues from the invasive mosquitofish G. affinis, and that selective predation by mosquitofish may affect the population structuring and persistence of native species. Furthermore, G. affinis will consume endemic intermediate predators of mosquito larvae that have themselves been suggested for use in mosquito biocontrol 62,63 . The frequency-independent preferences for female copepods demonstrated here by mosquitofish defies the selective preference for male copepods which has been typically posited 64,65 . Thus, the introduction of invasive mosquitofish for vector control could fundamentally shift the dynamics in recipient ecosystems, with effects on intermediate predators that potentially nullify or reverse attempts to control important vector mosquitoes through interguild predation 48 . We advocate that the use of FRs and prey switching offer robust and quantitative insights into the coupled direct and indirect impacts of invasive species on native populations. Prior examinations of such impacts could help to curtail damaging introductions, for instance through 'classical' biological control approaches which seek to release non-native agents into novel environments. Further research which incorporates multiple co-existing and interacting invaders alongside native biota would be of additional value in deciphering additive or non-additive trophic interactions within our framework.

Materials and Methods
Animal collection and maintenance. Ethical    80 mL water at five larval densities (2,4,8,16,32; n = 4 per density and treatment). The 80 mL inner experimental arenas were each placed within a larger opaque polypropylene outer arena of 16.5 cm diameter containing 800 mL water. We employed a fully factorial 2 × 2 experimental design with respect to predatory cues of G. affinis. Factor 1 comprised chemical cues (present/absent) and Factor 2 visual cues (present/absent). For chemical cues (Factor 1), a 2 L cue accumulation tank was established. In this tank, G. affinis were stocked at a density of 0.5 fish L −1 and left unfed for 48 h prior following the standard diet. The G. affinis treated water (cue water) was then used as the medium within the 80 mL experimental arenas. To implement visual cues (Factor 2), regular water was again used within the experimental arenas, but a single G. affinis was placed within the outer 800 mL arena and allowed to move freely, yet unable to consume the L. raynerae within the glass inner arena. Mosquito larvae and mosquitofish were added to the inner and outer arenas, respectively, two hours before the addition of the copepod predators and allowed to settle. Following their addition to the inner arena, copepods fed undisturbed for 6 h, after which they were removed and the remaining prey counted to derive those killed. Controls consisted of a replicate of all treatments in the absence of predators in order to constrain background mortality driven by processes outside of predation. In Experiment 2, adult female and male copepods (female, 4.8 ± 0.1 mm; male, 4.4 ± 0.1 mm) were supplied at seven different ratios (30:0, 25:5, 20:10, 15:15, 10:20, 5:25, 0:30 individuals; n = 4 per ratio) to G. affinis, which had been starved for 24 h. These ratios reflect the varying proportions of L. raynerae in aquatic ecosystems (see Wasserman et al. 41 ). Experiments were undertaken in arenas of 16.5 cm diameter containing 2 L water from a continuously aerated source. Once introduced, fish fed undisturbed for 3 h, after which they were removed and remaining living copepods counted and sexed. Controls consisted of a replicate at all treatments in the absence of predators.

Statistical analyses.
All statistical analyses were undertaken in R v3.4.2 66 . In Experiment 1, generalised linear models (GLMs) assuming a Poisson error distribution were used to analyse overall prey consumption with respect to 'chemical cue' , 'visual cue' and 'prey supply' , and their interactions. Non-significant terms and interactions were removed stepwise from the model to facilitate parsimony, with χ 2 used for model simplification via analysis of deviance 67 . Functional response (FR) analyses were undertaken using the 'frair' package in R 68 . Logistic regression of the proportion of prey consumed as a function of prey density was used to infer FR types. A Type II FR is characterised by a significantly negative first order term, whilst a Type III FR is characterised by a significantly positive first order term followed by a significantly negative second order term 32,33,69,70 . As prey were not replaced as they were consumed, we applied Rogers' random predator equation for depleting prey densities 69,70 : where N e is the number of prey eaten, N 0 is the initial density of prey, a is the attack constant, h is the handling time and T is the total experimental period. The Lambert W function was used to enable model fitting 71 . We used the 'difference method' 70 to compare attack rate and handling time parameters generated from FRs across treatments. To account for multiplicity, we compared coefficients against Bonferroni-adjusted p-values. Functional responses were non-parametrically bootstrapped (n = 2000) to generate confidence intervals, allowing the FRs to be considered in population terms 68 . In Experiment 2, as residuals were overdispersed, GLMs assuming a quasi-Poisson error distribution were used to compare overall prey consumption with respect to 'sex' and 'proportion' , with F-tests used for model simplification. Again, non-significant terms and interactions were removed stepwise 67 . Manly's α 72,73 assuming no prey replacement was used to determine prey preferences between prey across the varying provision ratios: n r n n r n (ln(( )/ ))/ (ln(( ) / )) (2) where a i is Manly's selectivity index for prey type i, n i0 is the number of prey type i available at the start of the experiment, r i is the number of prey type i consumed, m the number of prey types, n j0 the number of prey type j available at the start of the experiment and r j is the number of prey type j consumed. The value of α i ranges from 0 to 1, with 0 indicating complete avoidance and 1 indicating complete positive selection. In a two-prey system, values of 0.5 are indicative of null preference. Manly's α indices were transformed to reduce extremes 74 (0 s, 1 s) prior to analysis: where α t is the transformed output and n is the sample size. Beta regression using the 'betareg' package 75 in R was used to compare Manly's α values between 'sex' and 'proportion' , and their interactions. Akaike's Information Criterion was used to confirm that models minimised information loss (lower values indicate a better fit).

Data Availability
Raw functional response and prey switching data are available in the electronic supplementary material.