Effect of Tryptophan Dietary Content on Meagre, Argyrosomus regius, Juveniles Stress and Behavioral Response.

There are a high number of stressors present in aquaculture that can affect fish welfare and quality. One way of mitigating stress response is by increasing dietary tryptophan. In this study, three diets containing 0.5% (Tript1), 0.6% (Tript2), and 0.8% (Tript3) of tryptophan were tested in 32 g juvenile meagre for 56 days. At the end of the trial, survival, growth, and proximate composition were similar between treatments. Significant differences were found in the plasma parameters before and after a stress test consisting of 30 s of air exposure. Blood glucose levels were higher in the post-stress for all treatments (e.g., 63.9 and 76.7 mg/dL for Tript1 before and after the stress test), and the hemoglobin values were lower in the post-stress of Tript1 (1.9 g/dL compared to 3.0 and 2.4 g/dL for Tript2 and Tript3, respectively). In terms of behavior, three tests were carried out (novel tank diving and shoaling assays, and lateralization test), but no significant differences were found, except for the number of freezing episodes during the anxiety test (1.4 for Tript3 compared to 3.5 and 4.2 for the other treatments). This study suggests that supplementation with dietary tryptophan, particularly in higher dosage (0.8%), can reduce anxiety-like behavior in meagre exposure to acute stress (novel tank). Although the remaining results showed mild effects, they provide some clues as to the potential of this amino acid as a stress mitigator in aquaculture.

Effect of Dietary Incorporation of Yellow Mealworm as a Partial Fishmeal Replacer on Growth, Metabolism, and Intestinal Histomorphology in Juvenile Meagre (Argyrosomus regius).

Efforts have been made to find alternatives to fish meal (FM), as the sustainability of aquaculture depends on it. Insect meal (IM) is a potential candidate to partially replace FM, being more sustainable and economically viable. In this experimental trial, three diets were tested with different yellow mealworm incorporation: a control diet with no IM, a diet with an inclusion of 10% IM (Ins10), and a diet with an incorporation of 20% IM (Ins20). The diets were tested on 10.5 g meagre for 47 days. The results showed that an IM inclusion higher than 10% affected both growth (2.6 vs. 2.2) and FCR (1.5 vs. 1.9) of meagre juveniles. However, this reduction in growth did not result from lower protein retention or changes in muscle fibre area or density. Little differences were observed in the activity of pancreatic and intestinal enzymes except for aminopeptidase total activity which was higher in the control and Ins10 compared to Ins20 (3847 vs. 3540 mU/mg protein), suggesting no limitations in protein synthesis. Also, the alkaline phosphatase intestinal maturation index was higher in the control group compared to the IM groups (437 vs. 296). On the contrary, several differences were also found in the proteolytic activity in the hepatic and muscle tissues of meagre juveniles fed the Ins10 diet. The inclusion of IM had no impact on intestine histomorphology but changes were detected in the enterocytes of fish from control and Ins10 which showed hypervacuolization and nucleus misplacement compared to the Ins20 treatment. Nevertheless, a higher percentage of Vibrionaceae was recorded for meagre fed on the Ins20 diet. Since no signs of inflammation were observed in the distal intestine, this suggests IM incorporation could have had an important impact on intestinal health due to its antimicrobial properties. This is supported by an increase in the haematocrit in the treatments where IM was added (20 to 25%). In conclusion, incorporations of IM at percentages up to 10% do not seem to have a negative impact on meagre performance at this age but can enhance the fish immune system and protection against intestinal inflammation.

Distinguishing the Effects of Water Volumes versus Stocking Densities on the Skeletal Quality during the Pre-Ongrowing Phase of Gilthead Seabream (Sparus aurata).

Gilthead seabream (Sparus aurata) production is a highly valued aquaculture industry in Europe. The presence of skeletal deformities in farmed gilthead seabream represents a major bottleneck for the industry leading to economic losses, negative impacts on the consumers' perception of aquaculture, and animal welfare issues for the fish. Although past work has primarily focused on the hatchery phase to reduce the incidence of skeletal anomalies, this work targets the successive pre-ongrowing phase in which more severe anomalies affecting the external shape often arise. This work aimed to test the effects of: (i) larger and smaller tank volumes, stocked at the same density; and (ii) higher and lower stocking densities maintained in the same water volume, on the skeleton of gilthead seabream fingerlings reared for ~63 days at a pilot scale. Experimental rearing was conducted with gilthead seabream juveniles (~6.7 ± 2.5 g), which were selected as 'non-deformed' based on external inspection, stocked at three different densities (Low Density (LD): 5 kg/m3; Medium Density (MD): 10 kg/m3; High Density (HD): 20 kg/m3) in both 500 L and 1000 L tanks. Gilthead seabream were sampled for growth performance and radiographed to assess the skeletal elements at the beginning and end of the experimental trial. Results revealed that (i) LD fish were significantly longer than HD fish, although there were no differences in final weights, regardless of the water volume; (ii) an increase in the prevalence of seabream exhibiting cranial and vertebral axis anomalies was found to be associated with increased density. These results suggest that farmers can significantly reduce the presence of some cranial and axis anomalies affecting pre-ongrown gilthead seabream by reducing the stocking density.

Postprandial pattern of digestive enzymes and protein turnover in meagre (Argyrosomus regius) juveniles.

After a meal, a sequence of physiological changes occurs in animals in response to digestion, absorption and assimilation of the ingested nutrients. These processes are very important for the aquaculture sector since they will define the efficiency by which food is converted into growth, thus contributing to reduce the production costs and also undigested food in the effluents. Here we investigated the activity of digestive enzymes in the midgut and the protein degradation systems following a single meal to define postprandial patterns of action in hepatic and muscle tissues of meagre juveniles. Fish were fed with a single meal followed by a period of 24 h without feed. The activity of several digestive enzymes (α-amylase, trypsin, aminopeptidase, alkaline and acid phosphatases) plus the analysis of key players of the ubiquitin-proteasome (UPS) and autophagy-lysosomal (ALS) systems were examined just before feeding (0 h, basal point) and 2, 4, 6, 8 and 24 h after food ingestion. Digestion was activated around 4 h after food ingestion and nutrients available for protein degradation 2 h later. This work provided information about the short-term physiological effects induced by a single meal to support scientists' decision when planning a specific study involving digestion and protein degradation, and also to fish farmers on how to better manage feeding protocols when producing A. regius juveniles. Our results suggested that meagre juveniles, under the experimental conditions used, can be fed every 4 h, time when digestion starts, however further studies should be addressed to find the optimal feeding regime for this juvenile species.

Plant-based diets fed to juvenile meagre Argyrosomus regius with low methionine and taurine supplementation led to an overall reduction in fish performance and to an increase in muscle fibre recruitment.

Methionine and taurine are amino acids (AA) that are usually deficient when fish meal is replaced by plant proteins. In this study, three diets were tested in juvenile meagre (initial weight: 13.4 g) for 8 weeks. The D1 diet had 0.2% methionine and 1% taurine supplementation; the D2 and D3 diets had 0.6% methionine and 1% and 2% taurine supplementation, respectively. The results showed that meagre fed the D1 diet had lower specific growth rate (2.2 to 2.5), lower feed efficiency (0.9 to 1.2) and higher food conversion rate (FCR, 1.1 to 0.8) as well as a lower activity of the alanine aminotransferase (ALAT) enzyme. Furthermore, a higher recruitment of muscle fibres (46% compared to 36%) as well as a higher fibre density was observed (1019 compared to 870 fibres mm-2 ). This study shows that meagre requires a sufficient quantity of methionine in plant-based diets to avoid a reduction in fish performance. Furthermore, taurine supplementation in the D1 diet was not able to mitigate the effects of methionine deficiency. A higher taurine supplementation did not improve meagre performance.

Effects of steaming on health-valuable nutrients from fortified farmed fish: Gilthead seabream (Sparus aurata) and common carp (Cyprinus carpio) as case studies.

Fish fortification with iodine-rich macroalgae (Laminaria digitata) and Selenium-rich yeast is expected to promote nutritional added value of this crucial food item, contributing to a healthy and balanced diet for consumers. However, it is not known if steaming can affect these nutrient levels in fortified fish. The present study evaluates the effect of steaming on nutrients contents in fortified farmed gilthead seabream (Sparus aurata) and common carp (Cyprinus carpio). Fortified seabream presented enhanced I, Se and Fe contents, whereas fortified carp presented enhanced I, Se and Zn contents. Steaming resulted in increased I and Se contents in fortified seabream, and increased Fe and Zn levels in fortified carp, with higher elements true retention values (TRVs >90%). The consumption of 150 g of steamed fortified seabream contributes to a significant daily intake (DI) of I (up to 12%) and Se (up to >100%). On the other hand, steamed fortified carp contributes to 19-23% of I DI and 30%-71% of Se DI. These results demonstrate that steaming is a healthy cooking method, maintaining the enhanced nutritional quality of fortified fish. Moreover, the present fortification strategy is a promising solution to develop high-quality farmed fish products to overcome nutritional deficiencies.

Biofortified Diets Containing Algae and Selenised Yeast: Effects on Growth Performance, Nutrient Utilization, and Tissue Composition of Gilthead Seabream (Sparus aurata).

Efforts have been made to find natural, highly nutritious alternatives to replace fish meal (FM) and fish oil (FO), which can simultaneously promote fish health and improve the nutritional quality of filets for human consumption. This study evaluated the impact of biofortified diets containing microalgae (as replacement for FM and FO), macroalgae (as natural source of iodine) and selenised yeast (organic source of selenium) on gilthead seabream growth, nutrient utilization, tissue composition and gene expression. A control diet (CTRL) with 15% FM and 5.5% FO was compared with three experimental diets (AD1, AD2, and AD3), where a microalgae blend (Chlorella sp., Tetraselmis sp., and DHA-rich Schizochytrium sp.) replaced 33% of FM. Diet AD1 contained 20% less FO. Diets were supplemented with Laminaria digitata (0.4% AD1 and AD2; 0.8% AD3) and selenised yeast (0.02% AD1 and AD2; 0.04% AD3). After feeding the experimental diets for 12 weeks, growth was similar in fish fed AD1, AD2, and CTRL, indicating that microalgae meal can partially replace both FM and FO in diets for seabream. But AD3 suppressed fish growth, suggesting that L. digitata and selenised yeast supplementation should be kept under 0.8 and 0.04%, respectively. Despite lower lipid intake and decreased PUFAs bioavailability in fish fed AD3, compared to CTRL, hepatic elovl5 was upregulated resulting in a significant increase of muscle EPA + DHA. Indeed, filets of fish fed AD2 and AD3 provided the highest EPA + DHA contents (0.7 g 100 g-1), that are well above the minimum recommended values for human consumption. Fish consuming the AD diets had a higher retention and gain of selenium, while iodine gain remained similar among diets. Upregulation of selenoproteins (gpx1, selk, and dio2) was observed in liver of fish fed AD1, but diets had limited impact on fish antioxidant status. Overall, results indicate that the tested microalgae are good sources of protein and lipids, with their LC-PUFAs being effectively accumulated in seabream muscle. Selenised yeast is a good fortification vehicle to increase selenium levels in fish, but efforts should be placed to find new strategies to fortify fish in iodine.

Enriched feeds with iodine and selenium from natural and sustainable sources to modulate farmed gilthead seabream (Sparus aurata) and common carp (Cyprinus carpio) fillets elemental nutritional value.

Developing tailor-made fortified farmed fish is a promising solution to overcome nutritional deficiencies and increase consumer confidence in these products. This study evaluated the supplementation of three fortified diets with I-rich seaweed and selenised-yeast on essential and toxic elements levels in gilthead seabream (Sparus aurata) and common carp (Cyprinus carpio). Fortified diets resulted in increased I, Se and Fe in fish muscle. Biofortified seabream and carp revealed lower Cu and Br. The reduction of fishmeal and fish oil in fortified diets resulted in lower Hg and Cd in seabream muscle. Contrarily, fortified diets increased As and Hg in carp fillets. The consumption of 150 g of fortified seabream enabled a significantly higher contribution to the daily recommended intake (DRI) of I (10%) and Se (76%) than non-fortified fish, whereas fortified carp fulfilled 23% of I DRI and 91% of Se DRI. Moreover, the exposure to Pb decreased with the consumption of biofortified seabream (23-82% BMDL01) and carp (26-92% BMDL01). These results support the strategy of developing eco-innovative biofortified farmed fish using sustainable, natural, safe and high-quality ingredients in feeds, to enable consumers to overcome nutritional deficiencies without significantly increased feed costs.

Sound production in the Meagre, Argyrosomus regius (Asso, 1801): intraspecific variability associated with size, sex and context.

BACKGROUND: Many fish taxa produce sound in voluntary and in disturbance contexts but information on the full acoustic repertoire is lacking for most species. Yet, this knowledge is critical to enable monitoring fish populations in nature through acoustic monitoring. METHODS: In this study we characterized the sounds emitted during disturbance and voluntary contexts by juvenile and adult meagre, Argyrosomus regius, in laboratory conditions. Breeding sounds produced by captive adults were also compared with meagre sounds registered in the Tagus estuary (Lisbon, Portugal) from unseen fish during the breeding season. RESULTS: The present dataset demonstrates for the first time that in this species dominant frequency is inversely related to fish size, and that sounds vary according to sex, context and age. Sounds from captive breeding adults were similar to sounds recorded in the field. DISCUSSION: Our findings indicate that A. regius sound features carry information about size, sex, age and motivation. This variability could potentially be used to identify meagre in the field and to infer about ontogenetic phase (i.e., juveniles vs. adults, and variation with size) and motivation (e.g., spawning). Future studies should confirm sex differences and ascertain the influence of water temperature on acoustic features.

Effect of copper exposure and recovery period in reared Diplodus sargus.

The aquaculture growth can be followed by the occurrence of more and new pathogenic agents, since the production leads to higher fish densities in confined areas more appropriate to the appearance and propagation of pathologies. Copper sulfate has been widely used in preventing and controlling fish parasites. The objective of this study is to investigate the effects of copper treatments in the fish tissues (bioaccumulation and histological changes in different organs), mortality and evaluate what happens during the recovery period. White sea bream (Diplodus sargus) were exposed to copper sulfate (0.25 and 0.5 mg L-1) during 60 days followed with a 75-day recovery period. The results showed that the concentration of copper in fish liver was significantly higher in the 0.5 mg L-1 treatment than in the 0.25 mg L-1 treatment. Conversely, copper load in the muscle did not differ significantly between treatments and control. Copper levels in muscle, and especially in liver, increased during copper exposure (up to 60 days). In summary, at higher concentrations copper sulfate treatment (0.5 mg L-1) might be toxic to fish, which showed histological alterations and copper accumulation in their tissues, mainly in the liver. Nevertheless, individuals returned to their original state after a 75-day recovery period and the tested copper concentrations does not represents risk for food safety.

Unveiling the neurotoxicity of methylmercury in fish (Diplodus sargus) through a regional morphometric analysis of brain and swimming behavior assessment.

The current study aims to shed light on the neurotoxicity of MeHg in fish (white seabream - Diplodus sargus) by the combined assessment of: (i) MeHg toxicokinetics in the brain, (ii) brain morphometry (volume and number of neurons plus glial cells in specific brain regions) and (iii) fish swimming behavior (endpoints associated with the motor performance and the fear/anxiety-like status). Fish were surveyed for all the components after 7 (E7) and 14 (E14) days of dietary exposure to MeHg (8.7µgg-1), as well as after a post-exposure period of 28days (PE28). MeHg was accumulated in the brain of D. sargus after a short time (E7) and reached a maximum at the end of the exposure period (E14), suggesting an efficient transport of this toxicant into fish brain. Divalent inorganic Hg was also detected in fish brain along the experiment (indicating demethylation reactions), although levels were 100-200 times lower than MeHg, which pinpoints the organic counterpart as the great liable for the recorded effects. In this regard, a decreased number of cells in medial pallium and optic tectum, as well as an increased hypothalamic volume, occurred at E7. Such morphometric alterations were followed by an impairment of fish motor condition as evidenced by a decrease in the total swimming time, while the fear/anxiety-like status was not altered. Moreover, at E14 fish swam a greater distance, although no morphometric alterations were found in any of the brain areas, probably due to compensatory mechanisms. Additionally, although MeHg decreased almost two-fold in the brain during post-exposure, the levels were still high and led to a loss of cells in the optic tectum at PE28. This is an interesting result that highlights the optic tectum as particularly vulnerable to MeHg exposure in fish. Despite the morphometric alterations reported in the optic tectum at PE28, no significant changes were found in fish behavior. Globally, the effects of MeHg followed a multiphasic profile, where homeostatic mechanisms prevented circumstantially morphometric alterations in the brain and behavioral shifts. Although it has become clear the complexity of matching brain morphometric changes and behavioral shifts, motor-related alterations induced by MeHg seem to depend on a combination of disruptions in different brain regions.

Inorganic mercury accumulation in brain following waterborne exposure elicits a deficit on the number of brain cells and impairs swimming behavior in fish (white seabream-Diplodus sargus).

The current study contributes to fill the knowledge gap on the neurotoxicity of inorganic mercury (iHg) in fish through the implementation of a combined evaluation of brain morphometric alterations (volume and total number of neurons plus glial cells in specific regions of the brain) and swimming behavior (endpoints related with the motor activity and mood/anxiety-like status). White seabream (Diplodus sargus) was exposed to realistic levels of iHg in water (2µgL(-1)) during 7 (E7) and 14 days (E14). After that, fish were allowed to recover for 28 days (PE28) in order to evaluate brain regeneration and reversibility of behavioral syndromes. A significant reduction in the number of cells in hypothalamus, optic tectum and cerebellum was found at E7, accompanied by relevant changes on swimming behavior. Moreover, the decrease in the number of neurons and glia in the molecular layer of the cerebellum was followed by a contraction of its volume. This is the first time that a deficit on the number of cells is reported in fish brain after iHg exposure. Interestingly, a recovery of hypothalamus and cerebellum occurred at E14, as evidenced by the identical number of cells found in exposed and control fish, and volume of cerebellum, which might be associated with an adaptive phenomenon. After 28 days post-exposure, the optic tectum continued to show a decrease in the number of cells, pointing out a higher vulnerability of this region. These morphometric alterations coincided with numerous changes on swimming behavior, related both with fish motor function and mood/anxiety-like status. Overall, current data pointed out the iHg potential to induce brain morphometric alterations, emphasizing a long-lasting neurobehavioral hazard.

Comparative analysis on natural spawning of F1 meagre, Argyrosomus regius, with wild broodstock spawns in Portugal.

The purpose of this study was to describe and compare the reproductive success and egg and larvae quality between wild and first-generation (F1) breeders of Argyrosomus regius. Wild broodstock were adapted to captivity, and good-quality spawns were obtained in 2009-2010, after GnRH treatment. In 2012, the F1 meagre (3 years old) spawned naturally at IPMA's Aquaculture Research Station facilities. From each spawning event, the following parameters were determined: number of floating and non-floating eggs, egg size, hatching success and larval total length. Eggs size and percentage of hatching obtained from F1 breeders (1.04 ± 0.10 mm and 90.5 ± 6.4%) were significantly higher when compared with wild breeders (0.97 ± 0.13 mm and 17.0 ± 12.7%). Although wild breeder spawns exhibited 2.7 ± 0.2 mm for larval total length, F1 breeder spawns presented 2.6 ± 0.2 mm. The wild and F1 breeder spawns exhibit a good egg and larval quality, indicating a promising starting point for a successful meagre hatchery production.

A new page on the road book of inorganic mercury in fish body - tissue distribution and elimination following waterborne exposure and post-exposure periods.

There are several aspects of inorganic mercury (iHg) toxicokinetics in fish that remain undeveloped despite its environmental ubiquity, bioaccumulation capacity and toxicity. Thus, this study presents new information on the uptake, distribution and accumulation of iHg following water contamination by adopting a novel set of body compartments (gills, eye wall, lens, blood, liver, brain and bile) of the white sea bream (Diplodus sargus) over 14 days of exposure. Realistic levels of iHg in water (2 µg L(-1)) were adopted in order to engender reliable conclusions in the assessment of fish health. A depuration phase of 28 days was also considered with the purpose of clarifying iHg elimination. It was found that iHg was accumulated faster in the gills (within 1 day), which also had the highest accumulated levels among all the target tissues/organs. Moreover, iHg increased gradually with exposure time in all the tissues/organs, except for the lens that showed relatively unaltered levels throughout the experiment. After 14 days of exposure, lower values of Hg were recorded in the brain/eye wall compared to the liver, which is probably related with the presence of blood-organ protection barriers, which limit iHg influx. iHg reached the brain earlier than the eye wall (3 and 7 days, respectively) and, hence, higher accumulated levels were recorded in the former. A depuration period of 28 days did not allow the total elimination of iHg in any of the tissues/organs. Despite this, iHg was substantially eliminated in the gills, blood and liver, whereas the brain and eye wall were not able to eliminate iHg within this timeframe. The brain and eye wall are more "refractory" structures with regard to iHg elimination, and this could represent a risk for wild fish populations.

Hydroxybenzoate paralytic shellfish toxins induce transient GST activity depletion and chromosomal damage in white seabream (Diplodus sargus).

Fish are routinely exposed to harmful algal blooms that produce noxious compounds and impact the marine food web. This study investigates the role of phase I and II detoxification enzymes on metabolism of the novel paralytic shellfish toxins (PSTs), the hydroxybenzoate analogues recently discovered in Gymnodinium catenatum strains, in the liver of white seabream, assessing ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities. Additionally, the genotoxic potential of hydroxybenzoate PSTs was examined through the erythrocytic nuclear abnormality (ENA) assay. Fish were injected with hydroxybenzoate PSTs into the coelomic cavity and sacrificed 2 and 6 days later for biochemical and cytogenetic analyses. While the activity of EROD was unresponsive to toxins, a significant GST activity decrease was observed at 2 days after injection indicating an impairment of this line of the detoxification system. The genotoxic potential of PSTs was demonstrated by the induction of clastogenic/aneugenic effects at 2 days, as measured by the ENA assay. Overall, this study contributes to better understand the impact of toxins produced by G. catenatum blooms in fish, revealing effects that, even transitory, point out a risk associated to hydroxybenzoate analogues.

Biotransformation modulation and genotoxicity in white seabream upon exposure to paralytic shellfish toxins produced by Gymnodinium catenatum.

Fish are recurrently exposed to paralytic shellfish toxins (PSTs) produced by Gymnodinium catenatum. Nevertheless, the knowledge regarding metabolism of PSTs and their toxic effects in fish is scarce. Consequently, the current study aims to investigate the role of phase I and II detoxification enzymes on PST metabolism in the liver of white seabream (Diplodus sargus), assessing ethoxyresorufin-O-deethylase (EROD) and glutathione S-transferase (GST) activities. Moreover, the genotoxic potential of PSTs was examined through the erythrocytic nuclear abnormality (ENA) assay. Fish were intracoelomically (IC) injected with a nominal dose (expressed as saxitoxin equivalents) of 1.60 µg STXeq kg⁻¹ semipurified from a G. catenatum cell culture with previously determined toxin profile. Fish were sacrificed 2 and 6 days after IC injection. PST levels determined in fish liver were 15.2 and 12.2 µg STXeq kg⁻¹, respectively, at 2 and 6 days after the injection. Though several PSTs were administered, only dcSTX was detected in the liver after 2 and 6 days. This was regarded as an evidence that most of the N-sulfocarbamoyl and decarbamoyl toxins were rapidly biotransformed in D. sargus liver and/or eliminated. This was corroborated by a hepatic GST activity induction at 2 days after injection. Hepatic EROD activity was unresponsive to PSTs, suggesting that these toxins enter phase II of biotransformation directly. The genotoxic potential of PSTs was also demonstrated; these toxins were able to induce cytogenetic damage, such as chromosome (or chromatid) breaks or loss and segregational anomalies, measured by the ENA assay. Overall, this study pointed out the ecological risk associated with the contamination of fish with PSTs generated by G. catenatum blooms, providing the necessary first data for a proper interpretation of biomonitoring programs aiming to assess the impact of phytoplankton blooms in fish.

Distribution patterns of macrobenthic species in relation to organic enrichment within aquaculture earthen ponds.

The relationship between organic enrichment and macrobenthic colonization patterns was investigated during an 8-month period in Diplodus sargus (white seabream) production ponds. A stratified sampling design was applied and each pond was divided into three zones: water entrance (WE); central (C); and automatic feeder zones (AF). Generally, the number of species and Shannon-Wiener diversity increased from the WE to the AF zone. Abundance did not present a clear trend. The recently developed marine biotic index (AMBI) was applied and showed to be sufficiently robust to discriminate, within a relatively small area, differences in macrobenthic communities due to organic enrichment. Nevertheless, caution is advised when applying this index or others based on ecological group's assignment, as the classification of a certain area may differ when allocating a certain species to an unsuitable group. This is particularly evident when common species are involved.