Estuarine, Coastal and Shelf Science 145 (2014) 1e8
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Portuguese native Artemia parthenogenetica resisting invasion by Artemia franciscana ee Assessing reproductive parameters under different environmental conditions Pedro M. Pinto a, *, Francisco Hontoria c, Natividade Vieira a, b, Ana Bio a a b c
CIMAR/CIIMAR e Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas, 289, 4050-123 Porto, Portugal Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal Instituto de Acuicultura de Torre de la Sal (IATS e CSIC), 12595 Ribera de Cabanes, Castellón, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 August 2013 Accepted 19 April 2014 Available online 28 April 2014
There is widespread interest in the conservation of native Artemia biodiversity. In Portugal, only two known populations of native Artemia remain: one in the Rio Maior salina, the other in the Aveiro salina complex, both of the diploid Artemia parthenogenetica species. All other Portuguese hypersaline environments where Artemia can be found have been invaded by Artemia franciscana, which has eradicated the native strains. Invasiveness and resilience of, respectively, exotic and indigenous species are thought to depend on strain-specific traits and adaptation to local conditions. This work evaluates the reproductive performance of the two Portuguese native strains and the invasive species exposed to different salinities, temperatures, photoperiods and food supplies. Reproduction periods, quantity and quality of offspring varied significantly, depending on both the Artemia strain and environmental conditions. A. parthenogenetica from Rio Maior reproduced better than A. franciscana at high salinity (150) and low food supply, which may reflect an adaptation to its biotope that aids its resistance to invasion. But A. parthenogenetica form Aveiro performed much worse than its invasive competitor, under most of the conditions tested. It is unlikely that A. franciscana has not been introduced in this salina by chance alone. Other biological traits of the local A. parthenogenetica or adaptation to unstudied local factors (e.g. pollution) are probably responsible for this strain’s survival. Further knowledge on specific local conditions and trait-specific tolerances to biotic and abiotic conditions are needed to understand (non-) invasion patterns and preserve the remaining native populations. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: brine shrimp reproduction introduced species intraspecific relationships environmental conditions Portugal
1. Introduction Artemia is a cosmopolitan genus (Pacios and Muñoz, 2010) and several species, with sexual or parthenogenetic reproduction (Browne and Bowen, 1991) have been identified, four of which with natural populations on the Iberian Peninsula (Amat et al., 1995, 2007; Pacios and Muñoz, 2010). But in recent decades, many native Artemia Iberian populations have been eradicated by their introduced congener Artemia franciscana. Because of this, there is a widespread interest in the conservation of native Artemia biodiversity and therefore also in understanding the mechanisms and factors influencing the success or failure of invasive species.
* Corresponding author. E-mail address:
[email protected] (P.M. Pinto). http://dx.doi.org/10.1016/j.ecss.2014.04.009 0272-7714/Ó 2014 Elsevier Ltd. All rights reserved.
Artemia franciscana is a native species from the American continent (Amat et al., 2005), but can currently be found on all continents where Artemia has been described, evidencing its invasive power. Several studies (e.g. Ruebhart et al., 2008) describe characteristics that give A. franciscana a competitive advantage in comparison to other Artemia species, favouring the development and spread of this invasive species. A. franciscana is very euryhaline and eurythermal, maintaining reproductive success at a variety of temperatures and salinities (Browne and Wanigasekera, 2000). Amat et al. (2007) compared the sexual fitness of several Artemia species from different places of the Mediterranean basin and found that, also under standard conditions, A. franciscana had the best reproductive performance. In Portugal, only two known populations of native Artemia are left: one in an inland salina (near Rio Maior), the other in a salina embedded in a coastal lagoon (near Aveiro); both belong to the diploid Artemia parthenogenetica species (Amat et al., 2007). All
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other Portuguese hypersaline environments where Artemia can be found have been invaded by A. franciscana (Amat et al., 2007; Pinto et al., 2013a), which has eradicated the native strains. Distinct physiological responses of different Artemia populations belonging to the same species are common in this genus (Browne and Bowen, 1991; Browne, 1992). These variations, which are possibly genetic, suggest local adaptations of the populations, in response to different selective pressures experienced in the most varied hypersaline environments they inhabit (Persoone and Sorgeloos, 1980; Vanhaecke et al., 1984). Pinto et al. (2013b) found significant differences in the survival of the Portuguese A. parthenogenetica populations and of the invasive A. franciscana, when exposed to variations in salinity, temperature, light and food supply. They suggested possible local adaptations of the native strains to specific characteristics their biotopes and identified local factors which potentially limit the invasive ability of A. franciscana. The present work tests the hypothesis that the two Portuguese native Artemia populations have strain-specific tolerance ranges for salinity, temperature, food supply and light exposure, and that this is reflected in their reproductive success. Therefore, reproductive features of the native strains and of the invasive species were assessed when exposed to variations in salinity, temperature, light and food availability. Pre-reproductive and reproductive periods, as well as the quantity, type and quality of reproduction were determined, as these factors decisively influence the biological fitness and lifetime of each of these species (Allan, 1976; Barata et al., 1995, 1996a,b; Browne et al., 1984, 1988, 1991), determining permanence or elimination of native strains in these hypersaline environments (Amat et al., 2007). 2. Material and methods 2.1. Biological material
40°0'N
45°0'N
Adult diploid A. parthenogenetica were collected at two sites: the Troncalhada salina (40 380 4000 N, 8 390 4600 W) located in the Ria de Aveiro lagoon, an artisanal, solar salina covering an area of 4.2 ha, and the salina of Rio Maior (39 2105000 N, 8 560 3800 W), an inland salina supplied by pumped up brine from naturally dissolved rock salt (more information about these salinas can be found in Pinto et al., 2013b) (Fig. 1). A. franciscana were hatched from a 10°0'W
5°0'W
±
Aveiro
Spain Rio Maior
commercial brand of Artemia cysts (Ocean NutritionÔ, Great Salt Lake). The animals were kept in the laboratory, separated per population, and allowed to acclimate to a salinity of 70 and a temperature of 24 C. The first 24 females of each Artemia strain to reach sexual maturity (or less, when fewer animals reached sexual maturity during the experiment, which happened for some extreme experimental conditions) were transferred immediately and individually (for A. parthenogenetica) or together with a male (for A. franciscana) to 50 ml Falcons tubes, maintaining the acclimatization culture conditions. Females were considered to be sexually mature after developing their ovisac and males when their antenna and penis were clearly observable. The following abbreviations are used throughout: AV: A. parthenogenetica form Aveiro, RM: A. parthenogenetica from Rio Maior, FR: A. franciscana. 2.2. Experimental setup We considered a salinity of 70 (the salinity was measured using the Practical Salinity Scale), 24 C water temperature, 12:12 h L:D photoperiod and 300,000 cells ml1 Tetraselmis suecica food supply as standard conditions. In each experiment one of these parameters was varied to assess its effect on reproduction; the remaining parameters were kept constant. Treatment conditions were: salinities of 70, 110 and 150 (prepared using natural sea water and Tropic Marin Sea SaltÒ and confirmed with a refractometer); temperatures of 24 C, 29 C and 34 C 1 C (maintained keeping the falcons in water baths, with temperatures regularly checked with a thermometer); photoperiods of 12:12 h L:D, constant light and constant darkness, and three levels of food supply, with 300,000 cells ml1, 150,000 cells ml1 and 37,500 cells ml1 of T. suecica respectively (densities were obtained through dilution, counting T. suecica cells in a Neubauer chamber). It is of note that supplied food concentrations were independent of the number of individuals present in each replicate (one female for parthenogenetic populations and a couple for the bisexual species), but the medium was renewed every second day. Each treatment condition was applied to up to 24 replicates (depending on the number of mature females obtained). For each replicate, the experiment was terminated with the death of the parthenogenetic individual or the A. franciscana female. If a male A. franciscana died before its replicate’s female, the male was replaced by another that had been kept under the same treatment conditions. Every second day, the animals were observed to determine the following life history features: pre-reproductive (PRP) and reproductive (RP) periods, in days; life span (LS), in days; broods per female (BPF); time between broods (TBB) in days; offspring per brood (OPB) and offspring per female (OPF); ovoviviparous offspring (number, percentage and quality); and oviparous offspring (number and apparent quality). Cyst quality was evaluated by its chorion appearance and floating capacity, considering good external appearance and floating capacity as indicators of good reproductive viability. The TBB was assessed considering only females that produced at least 3 broods during the experiment (seen as females with a consistent breeding period).
35°0'N
2.3. Data analysis
0 50 100
200 Km
Fig. 1. Location of the A. parthenogenetica sampling sites.
The variability and distribution of reproduction parameter values for each Artemia strain and experimental treatment was presented using Tukey’s boxplots (Chambers et al., 1983). The boxplots present the interquartile range (IQR), from the first to the third quartile, whiskers indicate the minimum and maximum values (excluding outliers, i.e. points that differ more than 1.5 times
P.M. Pinto et al. / Estuarine, Coastal and Shelf Science 145 (2014) 1e8
the IQR from the respective quartiles), and outliers are presented as separate circles. Data were tested for normality, using the ShapiroeWilk test for normality (Royston, 1982) and homogeneity of variance across groups was assessed using Levene’s test (Levene, 1960). Given that many treatment results failed the test for normality (with pvalue < 0.05), also when log transformed, differences between Artemia sources and between treatment effects were assessed using non-parametric tests. We used KruskaleWallis tests to determine whether any treatment group was different from the others, and post-hoc pairwise Wilcoxon rank sum tests, corrected for multiple testing (with p-value adjustment by the Holm method; Holm, 1979), to determine which groups differed (Hollander and Wolfe,1973). It is of note that these tests assume homogenous variances between compared groups. Since several groups of data failed Levene’s test (with p-value < 0.05), results have to be interpreted with care. Multivariate analyses were used to compare Artemia from different sources, considering all of the reproductive parameters (except for the time between broods, which was omitted to avoid eliminating from the data females that produced less than three broods). An Analysis of Similarity (ANOSIM), following a 2D NonMetric Multidimensional Scaling (NMDS) based on standardized and normalized Euclidian distances, was used to evaluate whether strains differ significantly in the multivariate space. NMDS and ANOSIM were computed using Primer 5 (Clarke and Warwick, 2001). All other statistical analyses were done in R (R Dev. Core R Development Core Team, 2009).
3
FR showed longer median RP than AV; the latter presented relative long PRP, but short RP and total LS (Fig. 2, Table 2; N.B. Test results have to be considered with care, as assumptions were not always met). An increase in salinity to 110 led to a significant increase of the median PRP for RM and to significant increases of the PRP, RP and LS for FR (Table 3). At a salinity of 100, only one AV survived, and at a salinity of 150 only RM was able to reproduce. An increase in temperature to 29 C significantly shortened the RP and LS of RM and FR, but not the RP of AV; the latter increased its PRP and LS at 29 C. At 29 C RM had significantly shorter RP than the other strains. RM and FR had significantly shorter LS and displayed shorter pre and post reproductive periods than AV. Halving the food supply shortened the RP of FR. Under extreme food shortage (37,500 cells ml1 of Tetraselmis suecica) AV did not survive, RM and FR showed very short RP and FR an increased LS. Continuous light caused shorter RP for RM and FR and continuous darkness also shorter RP for RM. The PRP increased with continuous light, for all strains, and increased with complete darkness for RM. The LS of RM decreased for both continuous light and darkness (Table 4). 3.3. Broods and offspring quantity
Not all of the experimental conditions led to the survival (see also Pinto et al., 2013b) and maturity of 24 females (Table 1). For instance, at 34 C temperature no animals of any of the studied Artemia strains reached maturity. And at 29 C, only 13 individuals from Rio Maior (RM) reached maturity and only one produced more than 2 broods. High salinities proved to be lethal for A. parthenogenetica from Aveiro (AV; only one individual matured at a salinity of 110, none at a salinity of 150) and A. franciscana (FR; no animal matured at a salinity of 150). RM, on the other hand, was more tolerant to high salinity. Food shortage affected mainly AV. The photoperiods studied hardly affected survival and maturation.
Under base conditions, there were significantly less BPF, OPF and OPB for AV than for the other two strains (Fig. 3, Table 2). FR had furthermore more OPB than AV and RM and a shorter TBB than RM. At a salinity of 110, FR produced significantly more OPF and OPB than RM, and more BPF and OPF than under standard conditions. At a salinity of 150, only RM was able to reproduce. An increase in temperature to 29 C greatly reduced the number of BPF, OPF and OPB in FR and RM. In general RM had the lowest reproduction at that temperature; AV showed more BPF but less OPF and OPB than FR. OPB decreased with diminishing food supply (Table 3). Halving the food supply furthermore reduced OPF in FR and AV. AV produced significantly less BPF, OPF and OPB than the other two strains. Under extreme food shortage, RM and FR hardly reproduced, with FR producing more BPF and OPF than RM. Changes in the photoperiod did not affect AV. Here, RM was the most affected strain, producing less BPF and OPF under both continuous light and continuous darkness. FR showed lower BPF and OPF in continuous light. There were few significant differences in the TBB, comparing Artemia strains or treatment levels. Under standard conditions and at a salinity of 110, FR had shorter TBB than RM, but both strains did not differ in TBB from AV. Under continuous light, FR had shorter TBB than the other strains.
3.2. Life time and reproductive periods
3.4. Offspring type and quality
Exposed to the base conditions, RM had the longest median PRP of all tested species; the shortest was obtained for FR. Both RM and
Under base conditions, the offspring from RM and AV was about half ovoviviparous, whereas FR produced mainly ovoviviparous
3. Results 3.1. Survival and maturation
Table 1 Total number of mature females (obtained from the initial 30) and number of females producing more than two broods obtained for the different treatment conditions. The first treatment constitutes the base treatment. Treatments used to make comparisons per treatment variable are marked in bold. Treatment
Salinity
Temperaturea
Light
Food supply
A. parthenogenetica females Rio Maior
1 2 3 4 5 6 7 8 a b
70 110 150 70 70 70 70 70
24 24 24 29 24 24 24 24
No animals reached maturity at 34 C. Too few samples to allow statistical analysis.
12 12 12 12 12 12 0 24
300,000 300,000 300,000 300,000 150,000 37,500 300,000 300,000
Aveiro
A. franciscana
Total
>2 broods
Total
>2 broods
Total
>2 broods
24 12 18 13 23 12 24 24
20 9 9 1b 16 0b 3 4
22 1b 0b 18 23 0b 23 23
11 1b 0b 11 4 0b 18 11
23 24 0b 22 24 14 21 24
21 23 0 9 20 3 19 12
4
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Pre-reproductive period
45
40
40
70
40
60
35
50
30
35
35
30 30
40
25
25
25
30
20
20
20
20
50
40
40
40
30
30
30
AV RM FR
50
AV RM FR
50
AV RM FR
60
AV RM FR
60
AV RM FR
AV RM FR
AV RM FR
AV RM FR
AV RM FR
AV RM FR
AV RM FR
AV RM FR
AV RM FR
60
80 60
0
0
80
60
80
50
60
40
40
110
Salinity
150
30 20
AV
20 AV av.24 RM RM rm.24 FR FR fr.24 AV x.24 AV RM av.29 RM FR rm.29 FR fr.29 29
AV
AV RM av.70 RM FR rm.70 FR fr.70 AV x.70 AV RM av.110 RM FR rm.110 FR fr.110 AV x.110 AV RM av.150 RM FR rm.150 FR fr.150 150 70
50
40
20
20
60
60
30
40
AV RM FR
70 80
24
29
Temperature (°C)
300000
150000
37500
Food supply (cells ml−1)
12
24
0
100
80
100
AV
70
RM AV av.300000 FR RM rm.300000 FR fr.300000 AV x.300000 RM AV av.150000 FR RM rm.150000 FR fr.150000 AV x.150000 RM AV av.37500 FR RM rm.37500 FR fr.37500 37500
120
AV RM FR
0
AV RM FR
0
AV RM FR
10
AV RM FR
20
10
AV RM FR
20
10
AV RM FR
20
20
AV RM AV av.12 FR RM rm.12 FR fr.12 AV x.12 RM AV av.24 FR RM rm.24 FR fr.24 AV x.24 RM av.0 AV FR rm.0 RM fr.0 FR
40
AV RM FR
Reproductive period (days)
100
140
Life span (days)
15
15
AV RM FR
15
0
Light (h)
Fig. 2. Pre-reproductive period, reproductive period and life span, depending on Artemia source and culture conditions (AV: A. parthenogenetica from Aveiro, RM: A. parthenogenetica from Rio Maior, FR: A. franciscana). The first set of bars in each plot of the same row refers to the base treatment condition. Notice the different y-axes.
offspring (Fig. 4). An increase in salinity (to 110), temperature (29 C) or light hours (24 h) led to predominantly ovoviviparous reproduction for AV strain, while food shortage caused more oviparous offspring. RM, on the other hand, reacted with predominantly oviparous reproduction to a salinity of 110 and to continuous light, but with ovoviviparous reproduction under food shortage and at 29 C (with many dead and abortive embryos). At a salinity of 150 only RM was able to reproduce, mostly through good quality ovoviviparous offspring. FR produced more cysts than the other strains at 29 C and under food shortage. Compared to standard conditions, continuous light or darkness, increased temperature and extreme food shortage caused losses in ovoviviparous offspring quality. Notice that food shortage may have been aggravated for FR because a pair of brine shrimp (instead of a single animal, as for the parthenogenetic strains) was kept in each tube. The evaluation of cyst quality showed that about half of the cysts from AV were of poor quality, for all treatments. RM and FR produced about 25% bad cysts under base conditions and halved food supply, but about 50% under continuous light. All cysts appeared to be unviable for AV at a salinity of 110, for AV and RM at 29 C, and for
FR under continuous light. Notice, however, that cyst evaluation was based solely on external appearance and floating capacity, and that the efficiency of this method has not been tested. 3.5. Multivariate analysis Analysis of similarity (ANOSIM) after 2D NMDS showed significant (p < 0.05) discrimination between Artemia sources, except for the very overlapping samples of RM and FR kept at a salinity of 110. There was often little separation between samples from different sources (R < 0.250). Reasonable separation was obtained between RM and the other two strains under continuous light (R > 0.500) and in food shortage conditions (R > 0.400), and between RM and AV kept in complete darkness (R > 0.400). 4. Discussion The present study confirms that the effect of environmental conditions on the reproductive parameters and, therefore, success, is strain-specific in Artemia. This suggests that different strains have
P.M. Pinto et al. / Estuarine, Coastal and Shelf Science 145 (2014) 1e8
5
Table 2 Pairwise Wilcoxon rank sum test results for the comparison of lifetable parameters between Artemia from different sources (AV: A. parthenogenetica from Aveiro, RM: A. parthenogenetica from Rio Maior, FR: A. franciscana), considering the different treatments, using the Holm (1979) adjustment for multiple comparisons (110 and 150 refer to the salinity; c./ml: cells ml1 of Tetraselmis food supply; 24 h and 0 h refer to hours light; d : not enough data for testing; significant differences are presented in bold, nonsignificant differences in italics; only females producing at least three broods were considered for the calculation of the time between broods). Treatment Source Pre-reproductive Reproductive Lifespan Broods Time Offspring Offspring Ovoviviparous Life Dead Abortive Oviparous per female between per female per brood embryos embryos embryos broods Base
110
150
29 C
150,000 c./ml 37,500 c./ml 24 h
0h
AV-RM AV-FR RM-FR AV-RM AV-FR RM-FR AV-RM AV-FR RM-FR AV-RM AV-FR RM-FR AV-RM AV-FR RM-FR AV-RM AV-FR RM-FR AV-RM AV-FR RM-FR AV-RM AV-FR RM-FR
0.0022 0.0000 0.0000 e e 0.0000 e e e 0.0000 0.0000 0.0001 0.0000 0.0000 0.0580 e e 0.0000 0.4200 0.0000 0.0000 0.0000 0.0010 0.0000
0.0009 0.0007 0.9745 e e 0.6200 e e e 0.0140 0.5200 0.0490 0.0003 0.0000 0.1534 e e 0.0006 0.1400 0.4200 0.1400 0.0003 0.1916 0.0000
0.0001 0.0265 0.0265 e e 0.7100 e e e 0.0000 0.0001 0.2800 0.0700 0.4900 0.2100 e e 0.2200 0.0087 0.0030 0.0030 0.0110 0.5250 0.0150
0.0008 0.0001 0.3003 e e 0.2700 e e e 0.0260 0.4850 0.0640 0.0002 0.0000 0.0821 e e 0.0006 0.0200 0.9910 0.0130 0.0002 0.0131 0.0000
0.2609 0.2609 0.0054 e e 0.0130 e e e 0.8800 0.8800 0.8800 0.0600 0.8200 0.0600 e e e 1.0000 0.0220 0.0230 0.5760 0.0670 0.4970
developed different tolerance ranges, probably due to adaptation to local conditions. The native parthenogenetic Artemia strains from Aveiro and Rio Maior showed very different reproductive performances, in terms of both quantity and quality, confirming the variability in diploid parthenogenetic Artemia strains (Browne, 1992; Pinto et al., 2013b).
0.0003 0.0000 0.0509 e e 0.0120 e e e 0.0140 0.1920 0.0140 0.0000 0.0000 0.0540 e e 0.0004 0.5231 0.1282 0.0057 0.0044 0.0001 0.0000
0.0006 0.0000 0.0019 e e 0.0038 e e e 0.1330 0.0120 0.0110 0.0000 0.0000 0.5100 e e 0.0590 0.0076 0.0061 0.1269 0.5025 0.0000 0.0002
0.0043 0.5218 0.0458 e e 0.0590 e e e 0.0920 0.6160 0.7550 0.0000 1.0000 0.0000 e e 0.8400 0.0000 0.0490 0.0000 0.0006 0.0000 0.0000
0.0015 0.7109 0.0432 e e 0.1300 e e e 0.0410 0.5740 0.8340 0.0000 0.4800 0.0000 e e 0.0009 0.0000 0.1900 0.0000 0.0001 0.0000 0.0008
0.3600 0.9000 0.3600 e e 0.7000 e e e 0.4349 0.0009 0.0817 0.5490 0.5490 0.0730 e e e 0.0082 0.4342 0.0020 0.3154 0.0047 0.0306
0.9100 0.4200 0.4200 e e 0.0049 e e e 0.7700 0.5600 0.7300 0.0517 0.3686 0.0069 e e 0.0170 0.0004 0.5272 0.0014 0.0690 0.0000 0.0000
0.0079 0.0003 0.0038 e e 0.7100 e e e 0.0730 0.0730 0.0100 0.0377 0.0000 0.0004 e e 0.0001 0.0000 0.0130 0.0000 0.0000 0.2800 0.0220
Their reproductive success under the experimental conditions was often worse than that of the potential invader A. franciscana, particularly for the strain from Aveiro. It is clear that the observed differences in tolerance to salinity, temperature, food and light conditions are insufficient to explain why the two studied salinas resist invasion by A. franciscana. While
Table 3 Pairwise Wilcoxon rank sum test results for the comparison of lifetable parameters between treatment levels, considering Artemia from different sources (AV: A. parthenogenetica from Aveiro, RM: A. parthenogenetica from Rio Maior, FR: A. franciscana), using the Holm (1979) adjustment for multiple comparisons (70, 110 and 150 refer to the salinity; c./ml: cells ml1 of Tetraselmis food supply; 12 h, 24 h and 0 h refer to hours light; d: not enough data for testing; significant differences are presented in bold, non-significant differences in italics; only females producing at least three broods were considered for the calculation of the time between broods). Source Parameter
PreReproductive Life reproductive span
Offspring Offspring Ovoviviparous Life Dead Abortive Oviparous Broods Time embryos embryos embryos between per female per brood per female broods
AV
0.6100 0.0000 0.0000 0.0010 0.0020 0.4550 0.0000 0.0040 0.3120 0.1500 0.0001 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.2100 0.0000 0.0000 0.0000 0.0010 0.0000 0.0930
0.3100 0.4500 0.1600 0.5470 0.0760 0.1210 0.9593 0.0045 0.2088 0.0000 0.0660 0.0000 0.0001 0.0000 0.0000 0.7500 0.0002 0.0001 0.0161 0.0005 0.0018 0.0001 0.8494 0.0001
RM
FR
70e110 24e29 C 300,000e150,000c./ml 12e24 h 12e0 h 24e0 h 70e110 70e150 110e150 24e29 C 300,000e150,000 c./ml 300,000e37,500 c./ml 150,000e37,500 c./ml 12e24 h 12e0 h 24e0 h 70e110 24e29 C 300,000e150,000 c./ml 300,000e37,500 c./ml 150,000e37,500 c./ml 12e24 h 12e0 h 24e0 h
0.2000 0.3800 0.1700 0.2760 0.0240 0.1920 0.6990 0.0120 0.1530 0.0000 0.0593 0.0000 0.0001 0.0000 0.0000 0.6100 0.0001 0.0005 0.0356 0.0030 0.0075 0.0001 0.5026 0.0002
0.2900 0.0001 0.0038 0.0277 0.0009 0.0383 0.3800 0.1300 0.1100 0.0000 0.3000 0.0000 0.3000 0.0300 0.0001 0.0000 0.0000 0.0035 0.7572 0.0001 0.0002 0.0087 0.0369 0.0003
0.3800 0.6700 0.3900 0.2900 0.1100 0.9300 0.0050 0.8310 0.0420 0.2100 d d d 1.0000 1.0000 1.0000 0.0000 0.1700 0.8300 0.1500 0.2200 0.2170 0.2170 0.0610
0.2400 0.6300 0.0150 0.4700 0.1100 0.1600 0.7630 0.0001 0.0150 0.0000 0.7100 0.0000 0.0000 0.0000 0.0000 0.7200 0.0004 0.0000 0.0000 0.0000 0.0000 0.0000 0.7690 0.0004
0.3200 0.0170 0.0014 0.6800 0.2800 0.6200 0.7500 0.0000 0.0000 0.0000 0.0290 0.0000 0.0000 0.4900 0.1200 0.3900 0.5800 0.0004 0.0000 0.0000 0.0000 0.2600 0.8500 0.4200
0.1100 0.3200 0.0005 0.0270 0.0000 0.0000 0.0390 0.0250 0.7350 0.0001 0.1280 0.0002 0.0002 0.0000 0.0018 0.0040 0.0006 0.9100 0.1700 0.5600 0.5600 0.0094 0.0024 0.2505
0.1100 0.2700 0.0002 0.0460 0.0000 0.0000 0.0310 0.0170 0.4720 0.0001 0.1447 0.0002 0.0010 0.0000 0.0002 0.0061 0.0073 0.9000 0.1190 0.0620 0.2310 0.0550 0.0530 0.6010
0.1300 0.0085 0.4700 0.3130 0.3290 0.0550 0.6000 0.4700 0.2500 0.9600 0.2865 0.0073 0.0309 0.0083 0.3309 0.3309 0.0710 0.3100 0.2200 0.1300 0.2900 0.0940 0.0580 0.4510
0.2900 0.1100 0.2000 0.0231 0.0879 0.0002 0.4180 0.0340 0.4180 0.1700 0.2010 0.1350 0.0460 0.3100 0.6700 0.3100 0.0000 0.1800 0.3685 0.0249 0.0057 0.0018 0.0000 0.0164
1.0000 0.8800 0.4100 0.6100 0.0001 0.0000 0.1903 0.0006 0.0173 0.0000 0.1600 0.0000 0.0000 0.9340 0.0140 0.0110 0.3900 0.0003 0.0144 0.0004 0.0000 0.0000 0.0190 0.0001
Table 4 Analysis of similarity results for the life table parameters of the different Artemia sources, depending on treatment conditions (AV: A. parthenogenetica from Aveiro, RM: A. parthenogenetica from Rio Maior, FR: A. franciscana, 100: salinity of 100, c./ml: cells ml1 of Tetraselmis food supply); ANOSIM R and respective p-values are given (d: not enough data for testing). Global
P
R
p
R
p
0.001 e 0.005 0.001 e 0.001 0.001
0.311 e 0.176 0.335 e 0.063 0.424
0.001 e 0.004 0.001 e 0.026 0.001
0.233 0.092 0.364 0.476 0.456 0.574 0.228
0.001 0.092 0.001 0.001 0.001 0.001 0.001
12
12
12
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0.12 0.10 0.16 0.17 0.10 0.14 0.13
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−1
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Fig. 3. Brood and offspring parameters depending on Artemia source and culture conditions (AV: A. parthenogenetica from Aveiro, RM: A. parthenogenetica from Rio Maior, FR: A. franciscana). The first set of bars in each plot of the same row refers to the base treatment condition. Only females producing at least three broods were considered for the calculation of the time between broods. Notice the different y-axes.
P.M. Pinto et al. / Estuarine, Coastal and Shelf Science 145 (2014) 1e8
100%
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cysts abortive embryos dead embryos
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life embryos
Fig. 4. Mean percentages of offspring per type, depending on Artemia source and culture conditions (AV: A. parthenogenetica from Aveiro, RM: A. parthenogenetica from Rio Maior, FR: A. franciscana). Only offspring from females producing at least three broods was considered.
some results point at an advantage for the Rio Maior strain due to site-specific adaptations, the Aveiro strain showed less reproductive success for all of the culture conditions considered, suggesting that other biological traits of the local A. parthenogenetica or unstudied local factors (e.g. pollution) are responsible for the strain’s survival. The present results agree with previous studies stating that A. franciscana is an eurythermal species (Wear and Haslett, 1986; Browne, 1988; Browne et al., 1991; Browne and Wanigasekera, 2000). FR showed much higher survival at 29 C than the native Portuguese parthenogenetic strains (Pinto et al., 2013b found even some survival at 34 C), but prefers a lower temperature. A. franciscana showed also more reproductive success in intermediate/high salinities than the Portuguese parthenogenetic strains (also in terms of survival; Pinto et al., 2013b). RM seems to be well adapted to the particular conditions of its biotope, the inland salina. It was the only strain that survived and produced viable offspring at a salinity of 150. This may be related to the salina’s source of saltwater, which is pumped up from a rock salt mine, with an output salinity of 150. RM also had better reproductive results at 24 C and worse at 29 C than its invasive competitor, which may be advantageous in the deep, less warmed up tanks. Furthermore, RM outperformed FR when food supply was halved, which may reflect an adaptation to low food abundance; the Rio Maior salina tanks are made of cement and cleaned at the end of every salt season production, showing little algae concentration during the salt production season. However, available food
density was the same for the single parthenogenetic Artemia and the couple of A. franciscana in each experiment, which favoured the Rio Maior strain. Despite this, we also found features that should enhance invasiveness of FR in the Rio Maior biotope: FR outcompeted RM at very low food concentrations, at higher temperatures and in complete darkness, which may constitute an advantage in the deep tanks of this salina with little light incidence. A possible reason why the Rio Maior salina has not been invaded (yet) may be related to its geographical inland location, which lays far from the main bird migration routes and is far from fish farming facilities and urban areas with aquaria, which are considered main invasive enhancers (Amat et al., 2007). Considering AV, possible site-specific adaptations to the environmental parameters studied cannot explain its resistance to invasion. It was outperformed under all conditions by FR, and also by RM, except under extreme photoperiod conditions. The reproduction of AV was not negatively affected by extreme photoperiods, unlike the reproduction of the other two strains. But these results are difficult to relate to natural conditions, where continuous light or darkness hardly occur, and their significance in the invasion context is unknown. The Aveiro salina complex is fed by water from the Ria de Aveiro lagoon, with a salinity of up to 35, which increases gradually from the inlet channel to the crystallisers, where a salinity of 300 can be reached (Vieira, 1989). Food is abundant, which could explain the weak performance of AV at low food concentrations (Vieira and Bio, 2011). Furthermore, this salina must be seriously threatened by invasion given that there are records of
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A. franciscana in a salina of the same complex, not too distant from the salina here studied, and given that the lagoon and salinas are inhabited by numerous bird species that could disseminate the invasive Artemia strain (Amat et al., 2007). The fact that, in spite of its poor survival (Pinto et al., 2013b), biological fitness and lifespan, A. parthenogenetica persists in this salina means that either A. franciscana has not yet reached that salina, which is very unlikely, or it did appear but was not able to establish itself in that biotope. The salina is an ‘urban’ salina lying at the city border, in a lagoon polluted with heavy metals and pesticides (e.g. Martins et al., 2010); these are contaminants to which AV is adapted which may hence be a reason why FR has not been able to establish itself in this particular salina (as also suggested by Pinto et al., 2013b). A broader assessment of strain-specific traits, considering additional environmental (e.g. pollution) and possibly also biological (e.g. predator preferences) variables is needed to understand competition, resilience and resistance of native Artemia strains in relation to the invasive A. franciscana. Future studies ought to analyse the specific characteristics of the European hypersaline biotopes, taking into account local conditions such as environmental problems, and relate them to the specific biological traits of the local Artemia strains we aim to preserve and protect against exotic invasive species. Acknowledgements This study was supported by the Science and Technology Foundation, Ministry of Education and Science (Portuguese Foundation for Science and Technology) and European funds (FEDER), through the project “Chemical Wars: the role of chemically mediated interactions in the invasiveness potential of non-native Artemia”, PTDC/MAR/108369/2008 (FCT). This research was partially supported by the European Regional Development Fund (ERDF) through the COMPETE e Operational Competitiveness Programme and national funds through FCT e Foundation for Science and Technology, under the project PEst-C/MAR/LA0015/2013. References Allan, D., 1976. Life history patterns in zooplankton. Am. Nat. 110, 165e180. Amat, F., Barata, C., Hontoria, F., Navarro, J.C., Varó, I., 1995. Biogeography of the genus Artemia (Crustacea, Branchiopoda, Anostraca) in Spain. Int. J. Salt Lake Res. 3, 175e190. Amat, F., Hontoria, F., Ruiz, O., Green, A., Sánchez, M., Figuerola, J., Hortas, F., 2005. The American brine shrimp as an exotic invasive species in the western Mediterranean. Biol. Invasion 7 (1), 37e47. Amat, F., Hontoria, F., Navarro, J.C., Vieira, N., Mura, G., 2007. Biodiversity loss in the genus Artemia in the Western Mediterranean region. Limnetica 26 (2), 387e 404. Barata, C., Hontoria, F., Amat, F., 1995. Life history, resting egg formation, and hatching may explain the temporal-geographical distribution of Artemia strains in the Mediterranean basin. Hydrobiologia 298, 295e305. Barata, C., Hontoria, F., Amat, F., Browne, R., 1996a. Competition between sexual and parthenogenetic Artemia: temperature and strain effects. J. Exp. Mar. Biol. Ecol. 196, 313e328.
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