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Salinity limits mosquitofish invasiveness by altering female activity during mate choice

September 2024

DOI:10.3389/frish.2024.1455775

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CC BY 4.0

Authors:

Sara Pirroni

Royal Holloway

Francesca Leggieri

Linnaeus University

Jessica Cuccuru

Linnaeus University

Paolo Domenici

Italian National Research Council

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Biological invasions of freshwater habitats are of increasing biological and economical concern, and both, salinity and parasites are considered to be key contributors to invasion success. Salinity, for example, influences the distribution of invasive mosquitofish ( Gambusia holbrooki ) and native killifish ( Aphanius fasciatus ) in Europe, with the latter now predominantly confined to high-salinity habitats. Here, we examined how salinity might affect female activity and preference for large and non-parasitized males in multiple populations of mosquitofish and killifish in Sardinia, Italy. We predicted that (1) females of both species would associate preferentially with larger and uninfected males, and that (2) female behavior in both species would be significantly influenced by salinity. We used dichotomous choice tests, in which we presented focal females with video animations of photos of the same male but differing in body size and presence/absence of an ectoparasite ( Lernaea cyprinacea ). We calculated female preference based on association time and quantified female inactivity as time spent in the central neutral zone during trials. Contrary to prediction 1, females did not prefer the large or uninfected male stimuli over their counterparts in any of the populations. However, while salinity did not significantly affect female preferences, it did significantly affect their activity, with mosquitofish becoming more inactive at higher salinities and killifish exhibiting the opposite pattern, matching prediction 2. These results suggest that salinity limits mosquitofish invasiveness by reducing their activity and thus provides a refuge for the Mediterranean killifish.

FIGURE E Schematic overview of the experimental set-up.

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TYPE Original Research

PUBLISHED 25 September 2024

DOI 10.3389/frish.2024.1455775

OPEN ACCESS

EDITED BY

Sébastien Alfonso,

Université de Nice Sophia Antipolis, France

REVIEWED BY

Tatiana Colchen,

Université d’Angers, France

Ming-Yih Leu,

National Dong Hwa University, Taiwan

*CORRESPONDENCE

R¨

udiger Riesch

Rudiger.Riesch@rhul.ac.uk

†These authors share senior authorship

RECEIVED 27 June 2024

ACCEPTED 06 September 2024

PUBLISHED 25 September 2024

CITATION

Pirroni S, Leggieri F, Cuccuru J, Domenici P,

Brown MJF, Marras S and Riesch R (2024)

Salinity limits mosquitoﬁsh invasiveness by

altering female activity during mate choice.

Front. Fish Sci. 2:1455775.

doi: 10.3389/frish.2024.1455775

Brown, Marras and Riesch. This is an

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reproduction in other forums is permitted,

provided the original author(s) and the

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permitted which does not comply with these

terms.

Salinity limits mosquitoﬁsh

invasiveness by altering female

activity during mate choice

Sara Pirroni1, Francesca Leggieri2, Jessica Cuccuru2,

Paolo Domenici2,3, Mark J. F. Brown1, Stefano Marras2† and

R¨

udiger Riesch1*†

1Department of Biological Sciences, Centre for Ecology, Evolution and Behaviour, School of Life

Sciences and the Environment, Royal Holloway University of London, Egham, United Kingdom,

2Consiglio Nazionale delle Ricerche, Istituto per lo studio degli Impatti Antropici e Sostenibilità in

ambiente marino (CNR-IAS), Oristano, Italy, 3Consiglio Nazionale delle Ricerche, Istituto d Bioﬁsica

(CNR-IBF), Pisa, Italy

Biological invasions of freshwater habitats are of increasing biological and

economical concern, and both, salinity and parasites are considered to be key

contributors to invasion success. Salinity, for example, inﬂuences the distribution

of invasive mosquitoﬁsh (Gambusia holbrooki) and native killiﬁsh (Aphanius

fasciatus) in Europe, with the latter now predominantly conﬁned to high-

salinity habitats. Here, we examined how salinity might aect female activity

and preference for large and non-parasitized males in multiple populations

of mosquitoﬁsh and killiﬁsh in Sardinia, Italy. We predicted that (1) females of

both species would associate preferentially with larger and uninfected males,

and that (2) female behavior in both species would be signiﬁcantly inﬂuenced

by salinity. We used dichotomous choice tests, in which we presented focal

females with video animations of photos of the same male but diering in

body size and presence/absence of an ectoparasite (Lernaea cyprinacea). We

calculated female preference based on association time and quantiﬁed female

inactivity as time spent in the central neutral zone during trials. Contrary to

prediction 1, females did not prefer the large or uninfected male stimuli over

their counterparts in any of the populations. However, while salinity did not

signiﬁcantly aect female preferences, it did signiﬁcantly aect their activity, with

mosquitoﬁsh becoming more inactive at higher salinities and killiﬁsh exhibiting

the opposite pattern, matching prediction 2. These results suggest that salinity

limits mosquitoﬁsh invasiveness by reducing their activity and thus provides a

refuge for the Mediterranean killiﬁsh.

KEYWORDS

video animations, female mate choice, sexual selection, parasites, Gambusia holbrooki,

Aphanius fasciatus

1 Introduction

Mounting evidence documents the negative impacts of human activities on

biodiversity and ecosystem functioning (1,2). For example, biological invasions, which

are often caused by human action, are considered one of the main threats to global

biodiversity, ranking third behind habitat fragmentation and habitat loss (3,4). The

introduction of invasive species can be damaging for several reasons, including that they

may aﬀect native species through predation, competition for resources and transmission

of parasites (5,6). Freshwater ecosystems are deemed particularly susceptible to the

establishment of invading species (3). Additionally, the invasive success of an introduced

non-native species may depend on factors such as the level of parasitism in those

species, and their personality and behavioral traits, including mate choice (7,8).

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Pirroni et al. 10.3389/frish.2024.1455775

Sexual selection is widely recognized as one of the main

evolutionary forces driving the development and reﬁnement of

traits that inﬂuence mating success (9,10), and sexual selection

can drive diﬀerences in sexual traits within and between native

and invasive populations of the same species (11,12). Mate choice

is a multi-phase process that can occur before, during and after

mating (13,14). It typically involves the detection of signals and

cues, their evaluation, decisions on who to mate with and diﬀerent

ﬁtness consequences arising from mating (13,14). For instance,

choosers’ preferences are partially dependent on their ability to

process multiple cues and signals, including, but not limited to,

visual [e.g., body size and coloration, (15,16)] and olfactory stimuli

[e.g., as often observed in insects; e.g., (17)]. Moreover, there is

evidence that other cues such as parasite load can also directly

aﬀect mate choice, with choosers often preferring to associate with

non-parasitized mating partners (13,18,19).

Mating preferences can vary among individuals, species and

populations (14,20,21), and the strength and direction of

these preferences are often shaped by environmental factors (22–

24). These, including factors inﬂuenced by human activities, can

aﬀect the production and expression of traits relevant to mate

choice, the transmission of information between potential mates,

and the reception and processing of received information (25);

for example, in ﬁshes, pollution with synthetic hormones and

hormone analogs has been shown to reduce the motivation to

make informed mate choice decisions (26), turbidity has been

shown to interfere with mate choice (27) and higher salinity has

been demonstrated to negatively aﬀect the desire to mate, activity

levels during mate choice, and mating preferences [(24); for a

negative inﬂuence of salinity on other aspects of ﬁsh activity,

such as maximum swimming speed, see (28), for example]. Thus,

by altering mating preferences and activity levels, environmental

factors may inﬂuence the population viability and survival of a

species (20,29). Moreover, freshwater habitats around the world are

getting saltier, a process termed freshwater salinization syndrome

[e.g., (30,31)]. How might salinization (and other human induced

environmental change) inﬂuence mate choice and activity levels

during mate choice? Since invasive species have often been found

to be bolder and show more exploratory behavior compared to

non-invasive species [e.g., (32,33)], behavioral inactivity could

inﬂuence pathways of species introductions, the likelihood of

successful establishment in novel habitats and the level of impact

on native organisms and ecosystems (34,35)? Knowledge of these

patterns is crucial to eﬀectively predict to what extent climate

change will impact species invasiveness and implement correct

management protocols.

As a result of their broad distribution and capacity to tolerate

a wide range of environmental conditions, eastern mosquitoﬁsh

(Gambusia holbrooki) provide excellent model organisms for

investigating environmental eﬀects on female mate choice. They

are native to North America but are, and have been, intentionally

introduced worldwide as mosquito biocontrol agents, making

them one of the most widely introduced aquatic species globally

(36). Several studies have documented G. holbrooki introductions

being responsible of the displacement and decline of native biota

[reviewed in (37)], and competition from mosquitoﬁsh has been

proposed as one of the main causes for the displacement of many

Mediterranean ﬁsh species, like the Mediterranean banded killiﬁsh

Aphanius fasciatus (38,39). This cyprinodont ﬁsh is endemic to

the central-eastern Mediterranean and is listed as a protected

fauna species in the Annex II of the European Habitat Directive

(92/43/CEE) and Annexes II and III of the Convention on the

Conservation of European Wildlife and Natural Habitats. Like

mosquitoﬁsh, killiﬁsh are capable of tolerating a wide range of

temperatures and salinities [e.g., from freshwater to >60‰; (39)].

Nonetheless, their distribution is now mostly conﬁned to higher

salinity waters due to the successful establishment of mosquitoﬁsh

(39,40). Therefore, it has been proposed that salinity may limit the

invasive success of mosquitoﬁsh and that high salinity may act as a

refuge for the native A. fasciatus (39,40).

Both A. fasciatus and G. holbrooki display reverse sexual size

dimorphism, with females being larger than males, but while males

of A. fasciatus also diﬀer from females in body and ﬁn coloration

(i.e., being generally more colorful), coloration of male and female

G. holbrooki is very similar (41,42). Mating behavior in A. fasciatus

involves male courtship displays (43,44) and the likelihood of

successful mating has been proposed to be determined by both,

aggressive interactions between males and female choice [e.g.,

through control on the latency to spawn; (45)]. Nonetheless, to

our knowledge, no research has yet investigated female mate choice

in this species. By contrast, mating in G. holbrooki is dominated

by male coercion, where males approach females from behind and

thrust their gonopodium (i.e., modiﬁed anal ﬁn) into the females’

genital pore (46–48). Although female cooperation is not necessary

in this mating system, females can inﬂuence the likelihood that

forced copulation attempts are successful by, for example, selecting

a particular male and actively staying close to it, thus facilitating

copulation and increasing the future reproductive success of the

oﬀspring (47,49). Most studies on G. holbrooki have focused on

evaluating the inﬂuence of male body size on female preference and

females have often been reported to prefer larger males [e.g., (50,

51); but see (52,53)]. However, while a recent study of Zhou et al.

(24) has investigated the eﬀects of salinity on female preference

for larger males in a close relative, the western mosquitoﬁsh

Gambusia aﬃnis, no research has yet investigated the inﬂuence

of parasites and salinity on female mating decisions in Gambusia

holbrooki, despite these factors being documented as contributors

to their invasiveness (39,40,54,55). Thus, investigating how

factors such as male body size and the presence of parasites

inﬂuence female-mate choice in both species will substantially

increase our understanding of their mating systems. Moreover,

understanding how salinity inﬂuence mate-choice interactions in

invasive mosquitoﬁsh and native killiﬁsh could provide more

insight into mosquitoﬁsh invasiveness and establish whether or not

salinity is a key determinant of successful mosquitoﬁsh invasions

and their impact on killiﬁsh.

We examined how male body size and parasitism aﬀect female

mate preferences and activity patterns in G. holbrooki and the

co-occurring native A. fasciatus, and how salinity inﬂuences the

strength and direction of these behaviors. To do this, we sampled

female G. holbrooki from four and female A. fasciatus from three

distinct, allopatric populations in Sardinia, Italy. We investigated

female mate preference using dichotomous choice tests, in which

we presented focal females with computer animations of pictures

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of the same male, but diﬀering in body size (i.e., large vs. small)

and presence of parasites (i.e., infected vs. uninfected). Thus, we

aimed to assess: (a) variation in female preferences and activity

within and between populations of each species and between the

two species and (b) the potential inﬂuence of changing salinity on

female preferences and activity. Based on previous research, which

we outlined above (24,50,51), and given that both species are

known to withstand a wide range of salinities (36,39), we predicted

that: (i) during baseline experiments (i.e., trials examining initial

female preferences within the context of each population’s natural

salinity) females of both species would associate preferentially with

larger and non-parasitized males, and (ii) female activity and mate

choice in both species would be signiﬁcantly inﬂuenced by salinity,

with mosquitoﬁsh showing decreased activity in higher salinity and

killiﬁsh exhibiting the opposite pattern.

2 Materials and methods

2.1 Field sampling and housing conditions

Fieldwork was performed in September and October of 2021 in

the central-western part of Sardinia, Italy. Adult specimens of G.

holbrooki and A. fasciatus were collected with dip nets (∼1–2 mm

mesh size) from four and three sites, respectively (hereafter G1-G4

for sampling sites of mosquitoﬁsh and K1-K3 for sampling sites

of killiﬁsh; Table 1;Figure 1; due to logistical constraints, an even

sampling scheme was not possible). We measured the following

parameters three to four times across consecutive days in situ at

each site: Water temperature (◦C) was measured using a Handy

Polaris Probe (OxyGuard R

, Denmark) and salinity (ppt) with a

Handy Salinity Probe (OxyGuard R

, Denmark).

Immediately upon capture, ﬁsh were transported to the

experimental facilities at CNR-IAS in Torregrande (Sardinia, Italy).

In the laboratory, specimens of each species and population were

sexed based on their respective sexual characteristics [modiﬁed anal

ﬁn in Gambusia, (48); bar patterning in Aphanius, (44)]. Specimens

from each population and species were housed separately in

mixed-sex groups (134.89 ±57.86 total ﬁsh per tank) in large

well-oxygenated housing tanks of ∼148 L (44.4 ×37 ×90 cm)

for a week. The tanks were provided with artiﬁcial vegetation

to resemble their natural environments and ensure the animals’

welfare. Furthermore, each tank was maintained at 25 ±0.7◦C

and at a 9:15 h light:dark cycle photoperiod. Tanks were checked

daily for the presence of ammonium, nitrate and nitrite, but values

never reached thresholds of concern (i.e., ammonium was never

above 0 ppm; nitrate was never >25 mg/L and nitrite was never

>0.2 mg/L). The salinity of each tank was kept as close as possible

to the environmental values measured in the ﬁeld. However, this

was not possible for the housing tank with killiﬁsh from site K1

given that this site was characterized by extremely high salinity

levels (>60 ppt). As artiﬁcial sea salt could not be obtained,

these extremely high salinity levels were impossible to replicate

in laboratory settings, and we, therefore, housed this populations

in marine water (∼36 ppt). All wild-caught specimens were daily

fed ad libitum with commercial food ﬂakes (Tetra) during the

acclimation period. During this time, baseline mate-choice trials

(i.e., trials performed at natural salinity levels for each population

prior to salinity manipulation) were performed on nine females of

each population of each species (i.e., 36 female G. holbrooki and 27

female A. fasciatus) at the same salinity level as their housing tanks.

These females were subsequently removed from the setup and were

not reused in any other part of the study.

2.2 Salinity acclimation protocol

After the initial acclimation period described above, we

investigated salinity-dependent mate choice in two populations of

each species (i.e., mosquitoﬁsh: G1 and G2; killiﬁsh: K1 and K2);

logistical constraints again prevented us from doing this for all

populations. Before the experiments, female and male specimens

from these populations underwent a salinity acclimatization

protocol, which consisted of an initial gradual adjustment of

salinity in the housing tanks until the two desired experimental

salinities were reached (i.e., 15 and 30 ppt), followed by 2 weeks of

acclimation at those salinities in circulating systems. This protocol

was not applied to all populations simultaneously but was done in

two blocks. Speciﬁcally, the ﬁrst block consisted of mosquitoﬁsh

population G1 and killiﬁsh population K1, and the second block

of mosquitoﬁsh population G2 and killiﬁsh population K2 (see

details below).

2.2.1 Populations G1 and K1

Because ﬁsh from G1 and K1 were captured at sites with

divergent levels of salinity (Site G1 was a river with a mean salinity

of 0.3 ppt, while site K1 was a lagoon with a mean salinity of

62.2 ppt), the protocol consisted of reducing or increasing the

salinity of each housing tank by 15 ppt each day until the two

desired experimental salinities (i.e., 15 and 30 ppt, ±0.7 ppt) were

reached. These experimental conditions and adaptation protocols

were chosen given that both mosquitoﬁsh and killiﬁsh have been

reported to withstand signiﬁcant spatial-temporal ﬂuctuations in

salinity (41,56), and that similar salinity changes with these and

similar species, had not revealed any potential problems in previous

settings (R. Riesch, personal observation). Once experimental

salinities were obtained, ﬁsh were transferred into four independent

indoor recirculating systems. Speciﬁcally, three replicates per

species and salinity (15 and 30 ppt) were set up, each tank holding

40 L (26 ×30.7 ×50.5 cm) and 24 ﬁsh each (12 females and

12 males). Fish of each species were kept in these tanks, and

under these conditions, for 2 weeks prior to the start of behavioral

assays. Water temperature in the systems was maintained at 25

±0.7◦C given the temperatures observed during sampling. Water

temperature and salinity were monitored daily using a Handy

Salinity Probe (OxyGuard R

, Denmark). Furthermore, commercial

food ﬂakes were supplied ad libitum as a food source each day. Due

to high mortality (90%) in G. holbrooki at 30 ppt within the ﬁrst few

minutes, female mosquitoﬁsh’ mate choice preference and activity

at 30 ppt could not be investigated.

2.2.2 Populations G2 and K2

Due to the high mortality of G1 mosquitoﬁsh at 30 ppt in

block 1, the acclimation protocol for the ﬁsh for block 2 was

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TABLE 1 List of Gambusia holbrooki and Aphanius fasciatus sampling sites, with site location (latitude and longitude), number of ﬁsh caught (F, females;

M, males), number of tested females along with their standard length (SL), and mean values ±SD of water temperature and salinity measured in situ.

Population Location Latitude Longitude Temperature

(◦C) Salinity

(ppt) No. of ﬁsh

caught (F/M) No. tested

females SL

(mm)

Gambusia holbrooki

G1 Tirso river 39◦56′2′′ N 8◦40′49′′ E 27.63 ±1.42 0.30 ±0 232 (99/134) 18 22.8 ±4.6

G2 Channel Bau

Mannu

39◦54′42′′ N 8◦30′43′′ E 26.40 ±3.68 23.30 ±7.26 339 (249/90) 27 21.0 ±6.9

G3 S’Ena Arrubia

lagoon

39◦48′56′′ N 8◦33′35′′ E 24.17 ±1.50 8.50 ±11.95 58 (28/30) 9 25.7 ±5.5

G4 Santa Giusta

pond

39◦52′12′′ N 8◦36′31′′ E 23.80 ±1.82 27.70 ±7.75 82 (63/19) 9 21.5 ±2.3

Aphanius fasciatus

K1 Mistras lagoon 39◦54′27′′ N 8◦28′57′′ E 26.80 ±2.60 62.17 ±3.04 382 (257/125) 23 21.8 ±4.4

K2 Santa Giusta

canal

39◦52′54′′ N 8◦35′11′′ E 27.25 ±2.79 33.15 ±4.14 194 (105/89) 27 22.4 ±6.5

K3 Is Benas pond 40◦2′21′′ N 8◦27′19′′ E 21.47 ±1.36 39.87 ±1.25 60 (30/30) 9 26.0 ±9.8

FIGURE 1

Map of the sampling sites of invasive mosquitoﬁsh (Gambusia holbrooki; i.e., G1–G4) and native killiﬁsh (Aphanius fasciatus; i.e., K1–K3) in Sardinia,

Italy, with representative photos of each sample site at the day of sampling. The Map was generated using QGIS 3.2 (https://www.qgis.org/en/site/);

photos taken by S. Pirroni and R. Riesch.

slightly modiﬁed. Similar to block 1, ﬁsh from these populations

were found in habitats with diﬀerent levels of salinity (Site

G2 was a canal with a mean salinity of 23.3 ppt, while site

K2 was a lagoon with a mean salinity level of 33.15 ppt).

Therefore, to avoid salinity stress (57), we adopted a protocol

consisting of the gradual increasing or decreasing of salinity

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Pirroni et al. 10.3389/frish.2024.1455775

by 5 ppt every 2 days until the experimental conditions were

reached. Fish of both species were then transferred into the 12

aquaria described above for block 1. Killiﬁsh mortality in the

acclimatization tanks was ∼14% whereas mosquitoﬁsh mortality

was <5%.

2.3 Mate-choice experiments

2.3.1 Video animations

We investigated female preference for large and non-

parasitized males in each sampled population of each species

(G1-G4 and K1-K3) under diﬀerent salinity settings. Speciﬁcally,

we investigated female preference in nine females for each

population within each testing condition. Female preference

was assessed using 2D computer video animations as stimuli.

The use of video animations is a validated technique to study

female mate choice, and it has been previously applied in mate-

choice studies on Gambusia spp. and other poecilids [e.g., (58–

62)]. This technique allowed us to control and manipulate

single male traits (i.e., male body size and parasitism) and

test their eﬀects on female preferences while keeping potential

confounding factors (e.g., males’ activity levels, boldness and

length of gonopodium) constant. To generate animations, digital

photographs were taken of individual males swimming in a narrow

glass tank (20 ×20 ×3 cm) using a Nikon D70 digital camera.

We took pictures of about three males from each population

of mosquitoﬁsh. By contrast, because male killiﬁsh showed a

high level of stress when transferred into the photo setup, we

used only one picture of an original male taken in the lab

and two pictures taken from the Web for this species. All ﬁsh

photographed for video animations were not used in any of the

experiments. Each resulting picture was then imported in Gimp

[v2.10.24, (63)] to remove the background and only keep the

image of the ﬁsh. Male size was manipulated at a later stage

(see below), but to create parasitized males, the picture of an

anchor worm (Lernaea cyprinacea) was pasted around the anal

pore of a copy of each male’s image. This freshwater parasite was

chosen because it is a widely distributed common ﬁsh parasite

(64,65), it provides a clear visual cue and it was found on

many collected specimens of G. holbrooki at our study sites

(Supplementary Figure S1).

The resulting images were then animated using Adobe

Animate 2021 (Adobe): The background of each picture was

changed to white and the male size on the screen was digitally

adjusted. Speciﬁcally, we created two pairs of animations: one

pair showing two identical large males diﬀering only in the

presence or absence of L. cyprinacea and the other pair

showing two identical males diﬀering only in their size [i.e.,

one small (killiﬁsh SL: 18 ±1.2 mm; mosquitoﬁsh SL: 14

±0.9 mm) and one large male (killiﬁsh SL: 27 ±0.24 mm;

mosquitoﬁsh SL: 19.6 ±2.1 mm)]. The created animations

were then converted into an mp4 ﬁle using Adobe Media

Encoder 2021 (Adobe). Each animation was 130 s in duration

and consisted of a male swimming vertically and horizontally,

with invisible turns of 1 s. AVI video playbacks with inﬁnite

FIGURE 2

Schematic overview of the experimental set-up.

loops of these animations were created with Windows Video

Editor (Microsoft).

2.3.2 Experimental procedure

All mate-choice experiments were performed using the same

experimental setup consisting of a 31L tank (50 ×30.4 ×30.2 cm

ﬁlled to a depth of 20.5 cm), covered with white opaque material

on all sides to reduce external disturbance. Water temperature was

again maintained at 25 ±0.7◦C while salinity matched salinity of

the tank of origin for the tested ﬁsh (i.e., baseline salinity, 15 or 30

ppt). During each mate-choice trial, a randomly chosen female was

placed at the center of the experimental arena and two CHUWI

10.1-inch tablets were placed on either side of the tank (Figure 2).

After a 5-min acclimation period, during which the ﬁsh could swim

freely and explore the test tank, the screens started playing the

video animations of the stimulus males (e.g., one screen playing

videos of a stimulus male “infected with L. cyprinacea” and the

opposite screen playing videos of the same individual, but without

L. cyprinacea).

Female mating behavior was recorded using a GoPro Hero9

(60 fps), mounted above the tank in a position not visible to the

animals and the time spent by the focal female in the compartment

near each of the monitors (i.e., preference zone) was measured

for an observation period of 5 min. To detect side biases, the

video playbacks were then switched oﬀ and after 1 min, they were

switched on again but now with the location of each video being

reversed. The behavior of the focal female was again recorded for

5 min. This procedure was repeated twice for each ﬁsh (i.e., one

time with the screens displaying parasitized and non-parasitized

males and another time with screens displaying animations of

larger vs. smaller males). Mate-choice preference was calculated

based on the association time in seconds near each of the screens

(the bottom of the experimental arena was divided into three equal-

sized zones using VSDC Video Editor: one central neutral zone and

two peripheral preference zones near the screens; Figure 2).

In addition to estimating female preference, female behavioral

inactivity was recorded as the time the focal female spent in

the central neutral zone during trials. After behavioral assays,

the standard length (SL in mm), total length (TL in mm) and

body mass (g) of each experimental subject were measured

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(Supplementary Table S1). Tested females were transferred into

a new tank and were not re-used for any other procedure. To

further reduce the risk of a tank eﬀect, we used three replicates

for each salinity treatment (i.e., three tanks at each level of salinity

for each species) and tested a total of three females from each

replicate (i.e., three females per tank) to investigate whether salinity

inﬂuenced female mate choice preference. Furthermore, water in

the experimental tank was replaced after every trial to avoid any

potential confounding eﬀects of chemical cues on ﬁsh behavior.

After the completion of the experiments, all A. fasciatus were

released back into the wild (depending on the opinion of the

veterinarian) where they had been captured, while invasive G.

holbrooki were sacriﬁced by percussive stunning in accordance with

local guidelines (Art. 2, paragraph b of Legislative Decree No. 26 of

04/03/2014), and stored in 95% ethanol for potential future studies.

2.4 Statistical analyses

2.4.1 Do females exhibit a preference for large

and non-parasitized males?

Following previous studies [e.g., (61,66)] we excluded all trials

from our analyses in which females exhibited a side bias; i.e., in

which the focal females spent more than 85% of her total time

during both 5-min trials in the same preference zone irrespective

of which stimulus male was shown there. Trials in which females

spent >50% of the total time in the preference zones were also

discarded as females were considered not motivated to choose.

For experiments with animations of large vs. small males, side

biases occurred in 27 out of 122 total trials and only one trial

was discarded due to low female response. For experiments with

animations of parasitized vs. non-parasitized males, side biases

occurred instead in 31 out of 122 trials and one trial was eliminated

from the analyses due to low response. Furthermore, due to data

loss from a faulty hard drive we could only analyse ﬁve out of nine

females from K1 at 15 ppt.

First, we examined female preference separately for each

population and testing condition (i.e., baseline, 15 and 30 ppt). This

was done by comparing association times near both types of stimuli

(i.e., large vs. small and parasitized vs. non-parasitized male) using

paired t-tests.

Then, we investigated within- and between-species diﬀerences

and the inﬂuence of salinity on the strength of preference (SOP).

Each focal female’s SOP for large vs. small and non-parasitized vs.

parasitized male was calculated with the following equation:

SOP =time spent near large (or non −parasitised) male −time spent near small (or parasitised) male

time spent near both stimuli

SOP values ranged between from −1 (female spent all her time

near small and parasitised males) to 1 (female spent all her time

near large-bodied and non-parasitized males).

Analyses were conducted in two separate steps and once each

for our two choice scenarios (i.e., once for large vs. small and

once for parasitized vs. non-parasitized). First, because populations

originated from habitats with drastically diﬀerent salinities, we

tested for diﬀerences in SOP between populations and species

within the baseline salinity treatments. This would help us identify

if the baseline salinity already had a signiﬁcant inﬂuence on

ﬁsh behavior. We therefore used SOP as the dependent variable

in a two-way ANCOVA, for which species (A. fasciatus vs.

G. holbrooki) and “population-nested-within-species” [hereafter:

population(species)] were included as independent variables

along with the interaction term of “SL-by-species”, and log10-

transformed standard length (SL) was included as the covariate. If

the interaction term had a p-value >0.1, then it was subsequently

removed from the ﬁnal model. One ANCOVA model tested SOP

within the context of a choice between a large and a small male,

while a second ANCOVA model tested SOP within the context of

a choice between a parasitized and a non-parasitized male of the

same size.

The outcome of the above tests then determined how we

planned to analyse mate choice quantiﬁed via SOP in the two

experimental salinity treatments of 15 and 30 ppt. If we did not

ﬁnd any signiﬁcant eﬀects of “species” or “population(species)” in

the baseline comparisons, then we constructed another ANCOVA

with SOP as the dependent variable, SL as the covariate, and the

independent variables were now “species”, “population(species)”

and “salinity treatment” (15 vs. 30 ppt). We also initially added

the interactions terms of “SL-by-species” and “salinity treatment-

by-species”, but those were removed from the ﬁnal model if p>

0.1. However, if we uncovered a signiﬁcant eﬀect in the initial

(i.e., baseline) ANCOVA, we then implemented a mixed-eﬀect

ANCOVA on SOP with SL as the covariate, the independent

variables now being “species” and “salinity treatment” (15 vs.

30 ppt), and the random eﬀect being “baseline salinity”. We

again initially added the interactions terms of “SL-by-species” and

“salinity treatment-by-species”, but removed them from the ﬁnal

model if p>0.1.

2.4.2 Does salinity inﬂuence female activity

during mate choice?

To examine potential diﬀerences in female behavioral activity

within and between species, and the potential inﬂuence of salinity,

we applied the same statistical approach as outlined above for SOP

(Section 2.4.1) also to inactivity time (i.e., time the focal female

spent in the central neutral zone). Speciﬁcally, we again conducted

these in two steps, because baseline salinities diﬀered between

species and populations. We therefore used “inactivity time” as the

dependent variable in a two-way ANCOVA, for which “species”

and “population(species)” were included as independent variables,

and log10-transformed standard length (SL) was included as the

covariate. The interaction term of “SL-by-species” was removed

from the models when it was associated with a p-value >0.1 and

ﬁnal models were reﬁtted with the remaining parameters. One

ANCOVA model tested inactivity within the context of a choice

between a large and a small male, while a second ANCOVA model

tested inactivity within the context of a choice between a parasitized

and a non-parasitized male of the same size.

The outcome of the above tests then determined how we

planned to analyse inactivity in the two experimental salinity

treatments of 15 and 30 ppt. If we did not ﬁnd any signiﬁcant eﬀects

in the baseline comparisons, we constructed another ANCOVA

with SOP as the dependent variable, SL as the covariate, and the

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independent variables were now “species”, “population(species)”

and “salinity treatment” (15 vs. 30 ppt). We also initially added

the interactions terms of “SL-by-species” and “salinity treatment-

by-species”, but those were removed from the ﬁnal model if p>

0.1. However, if we uncovered a signiﬁcant eﬀect of species in the

initial ANCOVA, we then implemented a mixed-eﬀect ANCOVA

on inactivity, with SL as the covariate, the independent variables

now being “species” and “salinity treatment” (15 vs. 30 ppt), and

the random eﬀect being “baseline salinity”. We again initially added

the interactions terms of “SL-by-species” and “salinity treatment-

by-species”, but those were removed from the ﬁnal model if p

>0.1.

All t-tests were performed using the software R x60 3.5.1 (67),

while all other analyses were performed using IBM SPSS Statistics

VS 28.0.1.1 (IBM Corporation). Each model was applied after

having checked model validation. Diagnostic plots were used to

validate all models prior to consideration of estimated parameters.

3 Results

3.1 No female preference for large males

and no inﬂuence of salinity

We did not ﬁnd a signiﬁcant diﬀerence in the amount of time

females spent associating with the large vs. small male stimuli in

any of the populations and treatment combinations for each species

(all P≥0.080; Table 2). However, when not considering testing

conditions (i.e., baseline, 15 and 30 ppt treatment) separately,

there was substantial individual variation among females within

populations, with some females spending considerably more time

near the large male stimulus while others exhibited either no

preference or an opposite pattern (>65% of the total time

considered as an indicator of a preference; Figure 3). For instance,

of the total number of female mosquitoﬁsh from population G3,

29% exhibited a preference for the small male whereas 14% showed

a preference for the opposite male stimulus, and 57% did not exhibit

a preference for any of the stimuli (Figure 3).

When only considering the baseline treatments, our nested

ANCOVA testing for diﬀerences in SOP found no signiﬁcant eﬀects

[e.g., SL: P=0.802; population(species): P=0.599], but indicated

two non-signiﬁcant trends, one for species (P=0.056) and one for

the interaction eﬀect of “SL-by-species” (P=0.078; Table 3A).

When testing females’ responses to 15 and 30 ppt, the

interaction terms of “SL-by-species” (P=0.875) and “salinity

treatment-by-species” (P=0.262) were removed in a stepwise

fashion from the nested ANCOVA. In the resulting ﬁnal model, no

term had a signiﬁcant eﬀect on SOP (species: P=0.724; salinity

treatment: P=0.765; SL: P=0.887; Table 4A).

3.2 No female preference for

non-parasitized males and no inﬂuence of

salinity

There was no eﬀect of non-parasitized or parasitized male

stimuli on female preference within any of the populations or

treatment combinations for each species (Table 5). Again, there

was large variance between individuals in association time within

populations, with females exhibiting a preference for one of the

stimuli on some occasions (Figure 3). For example, 33% of the

female killiﬁsh from population K3 showed a preference for the

non-parasitized male while 33% exhibited a preference for the

parasitized male and 33% showed no preference (Figure 3).

When comparing the baseline treatments with each other,

female SOP for non-parasitized males did not signiﬁcantly diﬀer

between species (P=0.692), between diﬀerent populations of the

same species [population(species): P=0.695], and also not as a

function of SL (P=0.307; Table 3B). Furthermore, the interaction

term “SL-by-species” had also not been signiﬁcant (P=0.235) and

had been removed from the ﬁnal model.

When testing females’ SOP in 15 and 30 ppt, both interaction

terms (“SL-by-species”: P=0.887; “salinity treatment-by-species”:

P=0.762) were removed in a stepwise fashion from the nested

ANCOVA. Nonetheless, no term had a signiﬁcant eﬀect on SOP

in the ﬁnal model (species: P=0.078; salinity treatment: P=0.762;

SL: P=0.145; Table 4B).

3.3 Salinity signiﬁcantly aects female

activity levels during some aspects of mate

choice

Our nested ANCOVA on inactivity during baseline trials and

when given a choice between a large and a small male did not reveal

any signiﬁcant eﬀects (species: P=0.340; population(species): P=

0.597; SL: P=0.646; Table 3C), and the interaction term of “SL-by-

species” had also been removed from the initial model due to lack

of eﬀect (P=0.674).

We therefore applied a nested ANCOVA also to the inactivity

data from females tested in 15 and 30 ppt. Again, the two

interactions “SL-by-species” (P=0.671) and “salinity treatment-

by-species” (P=0.219) were removed from the ﬁnal model. In

that ﬁnal model, no signiﬁcant eﬀects were discovered [species: P

=0.271; population(species): P=0.760; SL: P=0.707; Table 4C].

Our nested ANCOVA on inactivity during baseline trials and

when given a choice between a parasitized and a non-parasitized

male, however, did reveal signiﬁcant eﬀects of species (P=

0.012) and “population(species)” (P=0.019), while SL was not

signiﬁcant (P=0.616; Table 3D), and the interaction term of “SL-

by-species” had been removed from the ﬁnal model (P=0.835).

Species diﬀerences were due to mosquitoﬁsh spending a greater

amount of time inactive (estimated marginal means ±standard

error: 73.89 ±8.38 s) than killiﬁsh (37.14 ±11.09 s). The eﬀect

highlighted by the nested term “population(species)” was driven by

signiﬁcant diﬀerences between some populations of mosquitoﬁsh,

but not killiﬁsh. Speciﬁcally, G1 (estimated marginal means ±

standard error: 29.92 ±15.97 s) diﬀered signiﬁcantly in inactivity

from G3 (113.37 ±17.06 s; post-hoc Bonferroni-corrected pairwise

comparison: P=0.004) and G4 (94.77 ±17.23 s; P=0.040), while

all other comparisons were not signiﬁcant (P≥0.108 in all cases).

However, when we attempted to therefore implement a mixed-

eﬀect ANCOVA as outlined in our methods, the model failed to

reach convergence. We therefore applied another nested ANCOVA

also to this inactivity data (qualitatively, both models, the mixed-

eﬀect and the nested ANCOVA, provided the same results).

This model revealed signiﬁcant eﬀects on inactivity of “salinity

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TABLE 2 Parameter estimates of paired t-tests investigating dierences in association times near large and small males.

Population Treatment NTime (s) spent near the

large male (mean ±SD) Time (s) spent near the

small male (mean ±SD) T-value df p-value

Gambusia holbrooki

G1 0 ppt (baseline) 5 292.40 ±61.04 244.00 ±56.19 0.963 4 0.390

15 ppt 9 302.78 ±55.64 232.33 ±58.57 2.006 8 0.080

G2 15 ppt (baseline) 7 300.00 ±144.74 249.57 ±51.06 0.481 6 0.648

15 ppt 8 205.75 ±106.37 353.88 ±112.89 −1.927 7 0.095

30 ppt 7 236.00 ±103.22 269.86 ±129.51 −0.389 6 0.710

G3 0 ppt (baseline) 7 220.57 ±110.17 319.29 ±120.09 −1.139 6 0.298

G4 20 ppt (baseline) 9 250.11 ±128.02 281.89 ±100.51 −0.424 8 0.683

Aphanius fasciatus

K1 35.5 ppt (baseline) 7 254.43 ±127.81 277.86 ±131.70 −0.241 6 0.817

15 ppt 4 209.75 ±67.11 268.25 ±85.49 −0.942 3 0.416

30 ppt 7 261.43 ±98.40 254.71 ±77.43 0.105 6 0.920

K2 30 ppt (baseline) 3 440.33 ±129.28 134.33 ±108.21 2.232 2 0.155

15 ppt 9 320.00 ±129.00 216.89 ±125.02 1.232 8 0.253

30 ppt 8 251.13 ±110.71 244.50 ±1124.93 0.082 7 0.937

K3 35.5 ppt (baseline) 4 224.50 ±101.95 224.25 ±103.01 0.998 3 0.392

“Treatment” indicates the salinity level, which was either held as close as possible to the natural salinity of the habitat of origin (indicated by “Baseline”) or was experimentally adjusted to 15

and 30 ppt (only populations G1, G2, K1, and K2). Nrefers to the number of ﬁsh tested. All p-values are two-tailed, and times are provided in seconds.

treatment-by-species” (P=0.004), “SL-by-species” (P=0.002),

species (P=0.002), salinity treatment (P<0.001), and SL (P

=0.016), while “population(species)” was not signiﬁcant (P=

0.981; Table 4D). Speciﬁcally, while there were general diﬀerences

between Gambusia and Aphanius, with the former spending more

time inactive than the latter (estimated marginal means ±standard

error: 127.32 ±12.99 s and 74.07 ±9.26 s, respectively), and general

diﬀerences in inactivity between salinity treatments (i.e., 71.29 ±

8.96 s at 15 ppt compared to 130.10 ±12.59 s at 30 ppt), the way

of how the salinity treatment aﬀected inactivity, diﬀered between

the species with Gambusia showing a much greater increase in

inactivity compared to Aphanius (Figure 4A). Similarly, while there

was an overall decrease in inactivity with SL (Figure 4B), this

pattern only held true for Aphanius, while in Gambusia, larger

females increased their inactivity (Figure 4C).

4 Discussion

We investigated how male body size and parasitism inﬂuence

female behavioral activity and mating preferences in multiple

populations of invasive eastern mosquitoﬁsh and native killiﬁsh.

Furthermore, we examined how salinity inﬂuences the strength and

direction of these behaviors. We did not ﬁnd signiﬁcant female

preferences, signiﬁcant eﬀects of salinity on female preferences,

or signiﬁcant variation in preference between and within species.

However, our analyses did reveal that salinity aﬀected female

activity during mate choice trials, with mosquitoﬁsh becoming less

active at high salinities and killiﬁsh exhibiting the opposite pattern.

4.1 Female mosquitoﬁsh and killiﬁsh do not

prefer to associate with larger males

Male body size is often considered an indirect signal of male

dominance and a critical component of male ﬁtness that can have

direct beneﬁts (e.g., increased fecundity and reduced predation

risk) to the choosing female and/or confers indirect beneﬁts to

its oﬀspring ﬁtness and viability (13). Our ﬁrst prediction stated

that females of both species would exhibit a preference for large

males. However, our analyses did not reveal a signiﬁcant preference

by females for larger males in either species. To our knowledge,

this is the ﬁrst study to investigate female mating decisions in

Aphanius fasciatus. However, across mosquitoﬁsh more broadly,

our ﬁndings contrast with those reported by several previous

studies on mosquitoﬁsh [e.g., (51,68)[ and other poeciliids [e.g.,

in Heterandria formosa: (69); in Xiphophorus nigrensis: (70)], but

are congruent with a previous study of Bisazza and Marin (52) on

another population of invasive G. holbrooki from Italy.

Our results, thus, suggest indiﬀerence of female mosquitoﬁsh

and killiﬁsh toward male stimuli diﬀering in body size but also

that male body size might play only a small role in sexual

selection for these species. In many poeciliids, large males court

females while small males rely on sneaking to copulate (46).

Such sexual harassment by small males can be highly costly to

females [e.g., reduce their foraging eﬃciency; (71)], hence, resulting

in females preferring to associate with large males to avoid the

costs of harassment (46). In G. holbrooki, however, as males do

not court females, but males of all sizes try to force copulation,

the cost of associating with a large male could be equal to

the cost of associating with a smaller male. This may result in

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FIGURE 3

Individual-level variability in female choice (blue: large vs. small male; green: non-parasitized vs. parasitized male) for each mosquitoﬁsh (Gambusia

holbrooki: G1–G3) and killiﬁsh (Aphanius fasciatus: K1–K3) population without considering testing conditions separately. Note that in these charts

>65% of the total time spent associated with a male stimulus is considered indicative of a preference for that male. The name of the locations where

these populations were sampled from is provided in brackets.

females not exhibiting any preference, but rather associating with

males apparently at random (i.e., potentially based on individual

circumstances that might change from day to day). Furthermore,

this could also explain why we found strong individual variability

in mating preference within mosquitoﬁsh populations in our study.

Such variation among females in their choosiness has also been

documented before [e.g., (72–74)], indicating that it is important to

distinguish between population- and individual-level preferences

when interpreting the mating behavior of a species. With respect

to A.fasciatus, a large male body size alone may not be a strong

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TABLE 3 Parameter estimates of nested ANCOVAs on baseline (i.e., natural salinity) treatments investigating the eects of species (Aphanius fasciatus vs.

Gambusia holbrooki), “population nested within species” [population(species)], standard length (SL) and the interaction SL-by-species on female

strength of preference (SOP) for (A) larger males and (B) non-parasitized males; and the eects of species, population(species), SL and the interaction

SL-by-species on female inactivity during mate choice trials investigating preferences for (C) larger males and (D) non-parasitized males.

Variable df Sum of squares F-value P-value

(A) Female SOP during trials “large vs. small”

SL 1, 33 0.010 0.064 0.802

Species 1, 33 0.593 3.641 0.065

Population(species) 5, 33 0.603 0.740 0.599

SL ×species 1, 33 0.538 3.304 0.078

(B) Female SOP during trials “non-parasitized vs. parasitized”

SL 1, 34 0.307 2.075 0.307

Species 1, 34 0.046 0.160 0.692

Population(species) 5, 34 0.869 0.608 0.694

SL xspecies 1, 33 0.412 1.461 0.235

SL 1, 34 311.482 0.215 0.646

Species 1, 34 1,357.893 0.937 0.340

Population(species) 5, 34 5,380.893 0.743 0.597

SL ×species 1,33 267.342 0.180 0.674

(D) Female inactivity during trials “non-parasitized vs. parasitized”

SL 1, 34 447.536 0.256 0.161

Species 1, 34 12,263.582 7.026 0.012

Population(species) 5, 34 27,443.619 3.145 0.019

SL ×species 1,33 78.760 0.044 0.835

Interaction terms in gray were removed from the ﬁnal model to increase statistical power if P>0.1 and signiﬁcant P-values are in bold.

indicator of male dominance and beneﬁts for the female but other

traits such as number and span of the bars along the body ﬂank

could have a stronger inﬂuence in male mating success as suggested

by Malavasi et al. (45) and observed in swordtails (75).

Furthermore, we cannot exclude that multiple-interacting

factors drove the observed patterns. Personality traits also play an

important role in female mating decisions and often aﬀect male

body size eﬀects on female preferences (68,74). For instance,

Chen et al. (68) found an increasing female preference for

larger males with increasing male boldness and activity levels

in western mosquitoﬁsh, and other studies found female mating

decisions to be inﬂuenced by social context (62,76). Thus,

we cannot exclude that our results may have been due to

our speciﬁc setup (i.e., no additional conspeciﬁcs or cues on

male personality).

We used computer animations to examine female mate-choice,

therefore, it is possible that the lack of female preference for larger

males in both species was the result of females not being able

to discriminate animated male stimuli. However, we think this

to be unlikely for two reasons. First, the use of animations for

investigating mate-choice has been validated in multiple systems,

including Poeciliidae [e.g., swordtails: (59,77); guppies: (78);

mollies: (72,79); western mosquitoﬁsh: (24,68)]. Second, we

conducted several trial runs of this experimental setup using our

laboratory stocks of G. holbrooki (an invasive population from

southern Italy) at Royal Holloway, University of London, in the

summer of 2021. These trial runs resulted in strong trends for

preferences for (a) large males [t(8) =1.095, p=0.093] and (b) non-

parasitized males [t(8) =2.055, p=0.070]. Third, while computer

animations have not yet been applied to test mating decisions

in A. fasciatus, the ﬁnding of high visual acuity in the congener

species, A. sirhani (80), suggest that A. fasciatus should be able

to discriminate between animated potential partners. Nonetheless,

we acknowledge that a computer image may not allow size- or

parasitization-perception as clearly as a real conspeciﬁc would be

and so this is a potential limitation of the employed methods.

Additionally, we used testing protocols with 2 ×5 min observation

periods and video animations that are well-established for poeciliid

ﬁshes [e.g.,: (81–83); including previous studies used in our lab,

such as (72,84)], but we cannot completely rule out that this is not

long enough, or that the rather uniform movement of the animated

ﬁsh is not stimulating enough, to elicit a response for A. fasciatus.

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TABLE 4 Parameter estimates of nested ANCOVAs on experimental salinity (i.e., 15 and 30 ppt) treatments investigating the eects of species (Aphanius

fasciatus vs. Gambusia holbrooki), “population nested within species” [population(species)], salinity treatment, standard lengh (SL) and the interactions

SL-by-species and salinity treatment-by-species on female strength of preference (SOP) for (A) larger males and (B) non-parasitized males; and the

eects of species, population(species), salinity treatment, SL and the interactions SL-by-species and salinity treatment-by-species on female inactivity

during mate choice trials investigating preferences for (C) larger males and (D) non-parasitized males.

Variable df Sum of squares F-value P-value

(A) Female SOP during trials “large vs. small”

SL 1, 46 0.003 0.020 0.887

Species 1, 46 0.018 0.126 0.724

Population(species) 2, 46 0.613 2.196 0.123

Salinity treatment 1, 46 0.013 0.091 0.765

SL ×species 1,44 0.004 0.025 0.875

Salinity treatment ×species 1,45 0.179 1.290 0.262

(B) Female SOP during trials “non-parasitized vs. parasitized”

SL 1, 42 0.409 2.206 0.145

Species 1, 42 0.606 3.270 0.078

Population(species) 2, 42 0.062 0.168 0.846

Salinity treatment 1,42 0.017 0.093 0.762

SL ×species 1,40 0.004 0.020 0.887

Salinity treatment ×species 1,41 0.018 0.093 0.762

SL 1, 46 384.363 0.143 0.707

Species 1, 46 3,333.004 1.239 0.271

Population(species) 2, 46 1,488.461 0.277 0.760

Salinity treatment 1,46 7,211.325 2.680 0.108

SL ×species 1,44 494.777 0.183 0.975

Salinity treatment ×species 1,45 4,139.493 1.557 0.219

(D) Female inactivity during trials “non-parasitized vs. parasitized”

SL 1, 40 12,975.464 6.299 0.016

Species 1, 40 21,587.625 10.480 0.002

Population(species) 2, 40 77.122 0.019 0.981

Salinity treatment 1,40 31,939.083 15.505 <0.001

SL ×species 1,40 23,888.434 11.597 0.002

Salinity treatment ×species 1,40 19,348.800 9.393 0.004

Interaction terms in gray were removed from the ﬁnal model to increase statistical power if P>0.1 and signiﬁcant P-values are in bold.

4.2 Female mosquitoﬁsh and killiﬁsh do not

prefer to associate with non-parasitized

males

In addition to mating with large males, mating with non-

parasitized males is also thought to have ﬁtness beneﬁts for

choosing females (79). Thus, we predicted females of both species to

exhibit a preference for non-parasitized males. However, contrary

to our expectation, we did not ﬁnd any population-level preference

for non-parasitized vs. parasitized male stimuli. While we know of

no other studies that have examined the inﬂuence of parasites on

female mating preferences in both Aphanius and Gambusia [but

see (18) for male mate choice in G. aﬃnis], our results contrast with

several previous studies in other Poeciliids (79,85), where females

preferred to mate with males that had no (or few) parasites over

(more heavily) parasitized males.

Here, we digitally “infected” males with the ectoparasitic

copepod (also called anchor worm), Lernaea cyprinacea. This

parasite has a direct life cycle consisting of adult females releasing

eggs onto the sediment, which hatch into non-parasitic nauplii

that molt into parasitic copepods, attach to several parts of a ﬁsh

host and undergo further metamorphosis. While attached to the

host, this parasite penetrates ﬁsh skin and causes inﬂammation

and lesions that might become necrotic or lead to secondary

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TABLE 5 Parameter estimates of paired t-tests investigating dierences in female association times near parasitized and non-parasitized males.

Population Treatment NTime (s) spent near the

non-parasitized male

(mean ±SD)

Time (s) spent near the

parasitized male

(mean ±SD)

T-value df p-value

Gambusia holbrooki

G1 0 ppt (baseline) 7 188.33 ±168.80 384.33 ±165.85 −1.438 5 0.210

15 ppt 7 266.14 ±98.49 249.71 ±129.00 0.193 6 0.853

G2 15 ppt (baseline) 6 301.33 ±79.40 245.83 ±75.98 0.966 5 0.378

15 ppt 8 305.50 ±101.16 243.88 ±86.26 0.898 7 0.399

30 ppt 7 254.57 ±121.79 205.71 ±24.07 0.727 6 0.495

G3 0 ppt (baseline) 6 268.22 ±120.56 244.33 ±151.16 0.27 8 0.794

G4 20 ppt (baseline) 6 277.22 ±110.37 247 ±121.35 0.399 8 0.700

Aphanius fasciatus

K1 35.5 ppt (baseline) 8 259.50 ±213.11 305.38 ±211.99 −0.306 7 0.769

15 ppt 5 159.40 ±122.19 357.20 ±121.91 −1.853 4 0.137

30 ppt 6 260.33 ±134.59 268 ±130.29 −0.071 5 0.946

K2 30 ppt (baseline) 3 267.33 ±160.13 297.33 ±186.83 −0.15 2 0.895

15 ppt 7 278.66 ±95.78 269.43 ±86.71 0.138 6 0.895

30 ppt 8 204.70 ±175.34 326.00 ±158.08 −1.168 9 0.273

K3 35.5 ppt (baseline) 6 272.00 ±177.38 285.00 ±155.37 −0.096 5 0.927

“Treatment” indicates the salinity level, which was either held as close as possible to the natural salinity of the habitat of origin (indicated by “baseline”) or was experimentally adjusted to 15 and

30 ppt (only populations G1, G2, K1, and K2). Nrefers to the number of ﬁsh tested. All p-values are two-tailed, and times are provided in seconds.

infections (65). Moreover, infection by this parasite often leads

to a reduction in ﬁsh growth, fecundity and swimming abilities

(64,86). Fish in the wild are often able to reject these parasites

even after penetration has occurred (87), so the observed lack of

female responsiveness toward male stimuli diﬀering in the parasites

could be due to the fact females did not perceive this parasite to

aﬀect male reproductive state. Alternatively, we cannot rule out that

females showed no preference because they did not observe any

secondary infections (e.g., fungal infection) or other characteristics

such as reduced swimming performance on the infected males in

our video animations.

Furthermore, salinity has been documented to aﬀect how well

this parasite reproduces, with direct infection being signiﬁcantly

reduced at high salinities (65). This could explain why we did not

ﬁnd this parasite in any of the female specimens of A. fasciatus

captured for this study (killiﬁsh were found in habitats often

characterized by salinities >30 ppt) and a female preference for

non-parasitized males in this species. However, we found anchor

worms in almost all mosquitoﬁsh populations and a preference

for uninfected males was not found even in the mosquitoﬁsh

populations sampled in freshwater habitats (i.e., G1 and G3).

4.3 Salinity does not inﬂuence female

preferences but activity levels during

choice

In partial contrast with our prediction 2 (i.e., salinity eﬀects

on both female activity and mate-choice), our analyses did not

reveal a signiﬁcant eﬀect of salinity on the strength and direction

of female preferences in both species. However, congruent with

this prediction, salinity signiﬁcantly aﬀected female activity levels,

with mosquitoﬁsh being more active at lower salinities and killiﬁsh

showing the opposite pattern. To our knowledge, this is the

ﬁrst study to investigate salinity eﬀects on female preference and

activity levels during mate choice in these species. Our results

align with those of a recent study by Zhou et al. (24), who found

female western mosquitoﬁsh (G. aﬃnis) reduced their activity

levels with increasing salinity. However, that study also found

female G. aﬃnis to prefer larger males only under freshwater

and low-salinity conditions. Such context-dependent reduction in

female activity during mate-choice has also been documented in

invasive guppies, Poecilia reticulata (88). Speciﬁcally, the authors

uncovered a reduction in female sexual activity and preference

for male stimuli under predation threat. Together with our

results, this suggests that the levels of female sexual activity in a

species can be highly dependent on local habitat characteristics

and environmental factors. In invasive species, changes in sexual

activity may potentially be a way to adapt to novel environments.

In this context, it is important to note that we only found a

signiﬁcant eﬀect of salinity on inactivity in one of our two choice

scenarios (i.e., when given the choice between a parasitized and a

non-parasitized male) but that the underlying patterns of inactivity

were the same in both settings (i.e., when choosing between

a large and a small male, estimated marginal mean inactivity

±standard error for G. holbrooki at 15 ppt: 52.12 ±12.66 s;

at 30 ppt: 105.22 ±23.54 s; A. fasciatus, at 15 ppt: 84.31 ±

14.94 s; 30 ppt: 95.83 ±13.49 s). Nonetheless, we only measured

inactivity within the context of mate choice and therefore call on

future studies to investigate if this pattern holds also across other

behavioral contexts.

Frontiers in Fish Science 12 frontiersin.org

Pirroni et al. 10.3389/frish.2024.1455775

FIGURE 4

Visualization of signiﬁcant eects from the ANCOVA on behavioral

inactivity (i.e., time in the middle compartment) during mate-choice

trials investigating female preference for non-parasitized males in

mosquitoﬁsh (Gambusia holbrooki) and killiﬁsh (Aphanius fasciatus)

populations; (A) the interaction eect of salinity

treatment-by-species with ﬁlled circles and solid line representing

G. holbrooki and empty squares and dotted line representing A.

fasciatus; estimated marginal means +/−standard error (B) the

eects of standard length (logSL); and (C) the interaction eect of

“SL-by-species” with ﬁlled circles and solid line representing G.

holbrooki and empty squares and dotted line representing A.

fasciatus.

Moreover, in our comparison of inactivity during baseline trials

when females were given a choice between a parasitized and a

non-parasitized male, we had found G. holbrooki to spend more

time inactive than A. fasciatus, and that the G. holbrooki from G1

(habitat/baseline salinity of 0 ppt) spent less time being inactive

than G. holbrooki from G3 (0 ppt) and G4 (20 ppt). Since baseline

salinities between G. holbrooki and A. fasciatus were consistently

greater in A. fasciatus, we cannot tease apart whether the species

diﬀerence is based on taxon-speciﬁc diﬀerences or rather based on

the salinity diﬀerences between the sampled habitats. However, the

second eﬀect on population diﬀerences in G. holbrooki partially

supports the interpretation that salinity aﬀects patterns of inactivity

at least in G. holbrooki. However, since we also found signiﬁcant

diﬀerences between two habitats of the same salinity (i.e., 0 ppt),

this also highlights the importance of testing multiple population

replicates for certain environmental conditions, when trying to

identify the impact of that environmental factor on certain

organismal traits.

Observed salinity eﬀects on female behavioral activity in our

study species alone do not help explain mosquitoﬁsh invasiveness

in Europe. However, taking these results together with those of

another study we performed on the same populations in Sardinia

(all authors, unpublished data) and a study of Alcaraz et al.

(40), where mosquitoﬁsh food consumption and aggressiveness

were signiﬁcantly reduced at high salinities while killiﬁsh showed

opposite patterns, this maps onto the current distribution patterns

of invasive mosquitoﬁsh and native killiﬁsh in the Mediterranean.

Hence, this suggests that salinity may limit the negative eﬀects of

invasive mosquitoﬁsh and that high-salinity habitats may act as a

refuge for native killiﬁsh.

5 Conclusion

In conclusion, the results of this study suggest that male body

size and parasitization with Lernea may play little role in the sexual

decisions of invasive mosquitoﬁsh and native killiﬁsh females in

Sardinia. In contrast, salinity appears to profoundly alter female

sexual activity in both species and these eﬀects also help explain

their distribution patterns in other parts of Europe. Speciﬁcally,

our ﬁndings suggest that while increasing salinization of freshwater

habitats poses a serious global threat to ecosystem health and

biodiversity (31), it may decrease the potential for freshwater

invasive species such as G. holbrooki to spread in aquatic systems.

Hence, salinization may reduce their impacts on native biota such

as A. fasciatus (24).

Data availability statement

The datasets presented in this study can be found in

online repositories. The names of the repository/repositories

and accession number(s) can be found below: The datasets

generated and analyzed for this study can be found on Figshare

(doi: 10.17637/rh.26067916).

Ethics statement

The animal study was approved by the Animal Welfare Ethical

Review Board at Royal Holloway, University of London (RHUL-

NRR-0038-2020). The study was conducted in accordance with the

local legislation and institutional requirements.

Author contributions

SP: Conceptualization, Data curation, Formal analysis,

Methodology, Visualization, Writing – original draft, Writing –

review & editing. FL: Investigation, Writing – review & editing.

Frontiers in Fish Science 13 frontiersin.org

Pirroni et al. 10.3389/frish.2024.1455775

JC: Investigation, Writing – review & editing. PD: Methodology,

Resources, Writing – review & editing. MB: Methodology,

Supervision, Writing – review & editing. SM: Funding acquisition,

Methodology, Project administration, Resources, Supervision,

Writing – review & editing. RR: Conceptualization, Funding

acquisition, Project administration, Resources, Supervision,

Writing – review & editing.

Funding

The author(s) declare ﬁnancial support was received for the

research, authorship, and/or publication of this article. Funding was

provided by Royal Holloway University of London.

Acknowledgments

We thank A. Dunn and R. Thomas, who commented on

the thesis version of this manuscript, and we thank the Regione

Autonoma della Sardegna, Assessorato dell’Agricoltura e Riforma

Agropastorale - Divisione Pesca e Acquacoltura for issuing a permit

for ﬁsh collection (Autorizzazione alla pesca scientiﬁca—Prot. No.

0018256 del 27/09/2021) and the Ministero della Salute, Direzione

generale della sanità animale e dei farmaci veterinari for issuing the

permit for the experiments (Autorizzazione alla sperimentazione

Animale No. 138/2022-PR) in Sardinia.

Conﬂict of interest

The authors declare that the research was conducted in the

absence of any commercial or ﬁnancial relationships that could be

construed as a potential conﬂict of interest.

The author(s) declared that they were an editorial

board member of Frontiers, at the time of submission.

This had no impact on the peer review process and the

ﬁnal decision.

Publisher’s note

All claims expressed in this article are solely those of the

authors and do not necessarily represent those of their aﬃliated

organizations, or those of the publisher, the editors and the

reviewers. Any product that may be evaluated in this article, or

claim that may be made by its manufacturer, is not guaranteed or

endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found

online at: https://www.frontiersin.org/articles/10.3389/frish.2024.

1455775/full#supplementary-material

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The Mediterranean Killifish Aphanius fasciatus (Valenciennes, 1821) (Teleostei: Cyprinodontidae) as a Sentinel Species for Protection of the Quality of Transitional Water Environments: Literature, Insights, and Perspectives

Article

Full-text available

Jul 2023

Transitional waters are fragile ecosystems with high ecological values, representing the breeding and resting sites for rare and threatened species. They warrant particular attention in regards to protection, as they experience numerous anthropogenic threats. The present review aims to analyze the recent literature on Aphanius fasciatus, currently considered one of the most strictly estuarine-dependent fish species, thus affected by the degradation of lagoon habitats, and to discuss its suitability as a sentinel species for protection of the quality of transitional water environments. The analysis and discussion highlight the potential applicability of the molecular, cellular, and physiological responses of this species as diagnostic tools for detecting the subtle effects induced by environmental pollution on the biota in transitional water environments. Moreover, the suitability of the responses of this species is suggested in the wider framework of the One Health perspective, which considers human and animal health and the environmental state to be highly interconnected, sharing common aspects. To date, omics technologies show great potential in reacquiring novel knowledge on the responses of the organisms to environmental changes and to the alterations of the environmental health status. Therefore, considering the relevant potential of this organism as a sentinel species, many efforts are required in the near future to improve the quantity and quality of the omics tools that refer to A. fasciatus.

Biodiversity-ecosystem function relationships change in sign and magnitude across the Hill diversity spectrum

Article

Full-text available

May 2023

Motivated by accelerating anthropogenic extinctions, decades of biodiversity–ecosystem function (BEF) experiments show that ecosystem function declines with species loss from local communities. Yet, at the local scale, changes in species' total and relative abundances are more common than species loss. The consensus best biodiversity measures are Hill numbers, which use a scaling parameter, ℓ , to emphasize rarer versus more common species. Shifting that emphasis captures distinct, function-relevant biodiversity gradients beyond species richness. Here, we hypothesized that Hill numbers that emphasize rare species more than richness does may distinguish large, complex and presumably higher-functioning assemblages from smaller and simpler ones. In this study, we tested which values of ℓ produce the strongest BEF relationships in community datasets of ecosystem functions provided by wild, free-living organisms. We found that ℓ values that emphasized rare species more than richness does most often correlated most strongly with ecosystem functions. As emphasis shifted to more common species, BEF correlations were often weak and/or negative. We argue that unconventional Hill diversities that shift emphasis towards rarer species may be useful for describing biodiversity change, and that employing a wide spectrum of Hill numbers can clarify mechanisms underlying BEF relationships. This article is part of the theme issue ‘Detecting and attributing the causes of biodiversity change: needs, gaps and solutions’.

Looking back on biodiversity change: lessons for the road ahead

Article

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May 2023

Estimating biodiversity change across the planet in the context of widespread human modification is a critical challenge. Here, we review how biodiversity has changed in recent decades across scales and taxonomic groups, focusing on four diversity metrics: species richness, temporal turnover, spatial beta-diversity and abundance. At local scales, change across all metrics includes many examples of both increases and declines and tends to be centred around zero, but with higher prevalence of declining trends in beta-diversity (increasing similarity in composition across space or biotic homogenization) and abundance. The exception to this pattern is temporal turnover, with changes in species composition through time observed in most local assemblages. Less is known about change at regional scales, although several studies suggest that increases in richness are more prevalent than declines. Change at the global scale is the hardest to estimate accurately, but most studies suggest extinction rates are probably outpacing speciation rates, although both are elevated. Recognizing this variability is essential to accurately portray how biodiversity change is unfolding, and highlights how much remains unknown about the magnitude and direction of multiple biodiversity metrics at different scales. Reducing these blind spots is essential to allow appropriate management actions to be deployed. This article is part of the theme issue ‘Detecting and attributing the causes of biodiversity change: needs, gaps and solutions’.

Can female guppies learn to like male colours? A test of the role of associative learning in originating sexual preferences

Article

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Magdalena Herdegen-Radwan

How do female sexual preferences for male ornamental traits arise? The developmental origins of female preferences are still an understudied area, with most explanations pointing to genetic mechanisms. One intriguing, little-explored, alternative focuses on the role of associative learning in driving this process. According to this hypothesis, a preference learned in an ecological context can be transferred into a sexual context, resulting in changes in mating preferences as a by-product. I tested this hypothesis by first training female guppies to associate either orange or black colour with food delivery; I then presented videos of males with computer-manipulated coloured spots and measured female preference towards them. I also allowed females from both treatments to mate with males differing in their ratio of orange-to-black spots and measured the males' reproductive success. After training, female sexual preferences significantly diverged among treatments in the expected direction. In addition, orange males sired a greater proportion of offspring with females food-conditioned on orange compared to those conditioned on black. These results show that mating preferences can arise as a by-product of associative learning, which, via translation into variation in male fitness, can become associated with indirect genetic benefits, potentially leading to further evolution.

Freshwater salinisation: a research agenda for a saltier world

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Jan 2022
TRENDS ECOL EVOL

The widespread salinisation of freshwater ecosystems poses a major threat to the biodiversity, functioning, and services that they provide. Human activities promote freshwater salinisation through multiple drivers (e.g., agriculture, resource extraction, urbanisation) that are amplified by climate change. Due to its complexity, we are still far from fully understanding the ecological and evolutionary consequences of freshwater salinisation. Here, we assess current research gaps and present a research agenda to guide future studies. We identified different gaps in taxonomic groups, levels of biological organisation, and geographic regions. We suggest focusing on global- and landscape-scale processes, functional approaches, genetic and molecular levels, and eco-evolutionary dynamics as key future avenues to predict the consequences of freshwater salinisation for ecosystems and human societies.

Pushed to the edge: Environmental factors drive ecological responses of Aphanius fasciatus when in sympatry with invasive Gambusia holbrooki

Article

Full-text available

May 2021

• Interspecific competition may be particularly exacerbated when occurring between native and invasive alien species, with environmental conditions affecting drivers of natural selection and shaping spatio‐temporal dynamics of animal communities. The effects of invaders are particularly evident in aquatic ecosystems with delicate and complex balances, such as Mediterranean coastal wetlands, characterized by unpredictable water flows and variable reserves. Understanding how native species respond to human‐induced biological invasions is of paramount importance for planning management measures aiming at conservation purposes. • This study investigated whether the occurrence of a competing invader species resulted in changes in the ecological adaptations of a native species by comparing two coastal wetland fish species: the invasive eastern mosquitofish Gambusia holbrooki and the native Mediterranean killifish Aphanius fasciatus of conservation concern. The hypothesis was tested by sampling fishes at a coastal Ramsar site in central Italy, over 2 years. • Generalized linear mixed models were used to examine the occurrence of killifish in relation to water parameters and the presence of its putative competitor. The aim was to determine whether salinity, temperature, and water depth influenced the presence and abundance of killifish when in sympatry with mosquitofish. • Both the probability of presence and abundance of killifish increased with water salinity, but this increase was stronger when mosquitofish was present. Increasing water temperature and water depth were associated with a higher probability of presence and abundance of killifish respectively, although the effect of water depth was small. Both salinity and the presence of the invader acted synergistically in characterizing and shaping the population structure of the native species. • Living in the most extreme and stressed environments, the killifish is potentially at higher risk of extinction. These results highlight the importance of considering the interplay between multiple abiotic stressors combined with comparative data between invasive and native species as increasingly critical to aid in conservation efforts.

Male Choice, Female Competition, and Female Ornaments in Sexual Selection

Book

Jul 2021

Ingo Schlupp

When Darwin first proposed sexual selection theory he suggested two mechanisms: competition among males and choice by females. There is no doubt that these mechanisms are immensely important, but their mirror images have been largely underappreciated so far. In fact, males choose as well and females compete. Males choose based on female quality, often selecting mating partners that are more fecund. But male choice is also associated with changes in the sex ratio of a population and males can be choosy when they are rare. Furthermore, males sometimes invest heavily into reproduction and that too can be associated with male choice. That females compete with another, although less often with open aggression, is another understudied phenomenon. Finally, we now know that females are often ornamented, but are these ornaments under sexual selection by males? This book tries to review what we know and point to what we don’t know while pointing out the connections between male mate choice and female competition for a more complete view of sexual selection.

Increasing salinization of freshwater limits invasiveness of a live-bearing fish: Insights from behavioral and life-history traits

Article

Sep 2022
ENVIRON POLLUT

Biological invasions and continued salinization of freshwater are two global issues with largely serious ecological consequences. Increasing salinity in freshwater systems, as an environmental stressor, may negatively affect normal life activities in fish. It has been documented that salinity limits the invasive success of alien species by mediating physiological and life-history performances, however, there are few studies on how salinity affects its invasive process via altered behaviors. Using wild-caught invasive western mosquitofish (Gambusia affinis) as animal model, in this study, we asked whether gradual increasing salinity affects behaviors (personality and mate choice decision here), life-history traits, as well as the correlation between them by exposing G. affiins to three levels salinity (freshwater, 10 and 20‰). Results showed that, with increased salinity, male tended to be shyer, less active, less sociable, and reduced desire to mate, and female tended to be shyer, less active and lost preferences for the larger male. Furthermore, across salinity treatments, male exhibited reduced body fat content and rising reproduction allocation, however, pregnant female revealed diametrically opposed trends. In addition, the correlation between life-history traits and behaviors was only identified in pregnant female. It seems that either salinity or life-history traits directly affects mosquitofish behaviors. In summary, our results partially emphasize the harmful consequences of salinity on both life-history traits and behavioral performances. These findings provide a novel perspective on how salinity potentially affect fish fitness via altering personalities, mate choice decisions, as well as body condition, and hence supports the idea that salinity could affect the spread of invasive mosquitofish.

Sexual selection and the ascent of women: Mate choice research since Darwin

Article

Jan 2022
SCIENCE

Darwin's theory of sexual selection fundamentally changed how we think about sex and evolution. The struggle over mating and fertilization is a powerful driver of diversification within and among species. Contemporaries dismissed Darwin's conjecture of a "taste for the beautiful" as favoring particular mates over others, but there is now overwhelming evidence for a primary role of both male and female mate choice in sexual selection. Darwin's misogyny precluded much analysis of the "taste"; an increasing focus on mate choice mechanisms before, during, and after mating reveals that these often evolve in response to selection pressures that have little to do with sexual selection on chosen traits. Where traits and preferences do coevolve, they can do so whether fitness effects on choosers are positive, neutral, or negative. The spectrum of selection on traits and preferences, and how traits and preferences respond to social effects, determine how sexual selection and mate choice influence broader-scale processes like reproductive isolation and population responses to environmental change.

Testing measures of boldness and exploratory activity in native versus invasive species: geckos as a model system

Article

Jun 2021

Keywords: behavioural syndrome exploratory activity lizards movement open-field test personality traits Invasive species often possess a suite of behaviours that increase their likelihood of establishing populations in novel environments. These suites often include combinations of high motility, exploration, aggression, boldness or plasticity in resource use. In Australia, invasive house geckos, Hemidactylus fre-natus, have continued to expand their range since the 1960s, spreading from predominantly human-disturbed areas to peri-urban regions. Here, we used open-field tests to investigate whether invasive house geckos showed greater exploratory activity and boldness than native Australian house geckos, Gehyra dubia. We expected that, like many other successful invasive species, invasive house geckos would be bolder and show greater exploratory activity than native house geckos. Consistent with our expectations , invasive house geckos showed significantly higher levels of exploratory activity, travelling on average 1.6 times further than native house geckos. However, none of our boldness tests indicated that invasive house geckos were bolder than native house geckos. In addition, we found no influence of sex on exploratory activity or boldness in either species. While invasive house geckos were not bolder than native house geckos, their increased exploratory activity may have contributed to their successful expansion into the natural environments surrounding urbanized areas.