Insights in Pharmaceutical Pollution: The Prospective Role of eDNA Metabarcoding.

Environmental pollution is a growing threat to natural ecosystems and one of the world's most pressing concerns. The increasing worldwide use of pharmaceuticals has elevated their status as significant emerging contaminants. Pharmaceuticals enter aquatic environments through multiple pathways related to anthropogenic activity. Their high consumption, insufficient waste treatment, and the incapacity of organisms to completely metabolize them contribute to their accumulation in aquatic environments, posing a threat to all life forms. Various analytical methods have been used to quantify pharmaceuticals. Biotechnology advancements based on next-generation sequencing (NGS) techniques, like eDNA metabarcoding, have enabled the development of new methods for assessing and monitoring the ecotoxicological effects of pharmaceuticals. eDNA metabarcoding is a valuable biomonitoring tool for pharmaceutical pollution because it (a) provides an efficient method to assess and predict pollution status, (b) identifies pollution sources, (c) tracks changes in pharmaceutical pollution levels over time, (d) assesses the ecological impact of pharmaceutical pollution, (e) helps prioritize cleanup and mitigation efforts, and (f) offers insights into the diversity and composition of microbial and other bioindicator communities. This review highlights the issue of aquatic pharmaceutical pollution while emphasizing the importance of using modern NGS-based biomonitoring actions to assess its environmental effects more consistently and effectively.

Balancing between Artemia and microdiet usage for normal skeletal development in zebrafish (Danio rerio).

Targeting in zebrafish fast growth, high survival rates and improved reproductive performance has led over the last years in variable feeding regimes between different facilities. Despite its significance on fish function and welfare, normal skeletal development has rarely been evaluated in establishing the best feeding practices for zebrafish. The aim of this study was to establish a protocol for normal skeletal development, growth and survival of zebrafish larvae through live feed-to-microdiet transition at an appropriate rate. Four feeding regimes including feeding exclusively on Artemia nauplii (A) or dry microdiet (D), and feeding on both Artemia and microdiet at two different transition rates (slow (B) or fast (C)) were applied from 5 to 24 dpf (days post-fertilization). Results demonstrated a significant effect of feeding regimes on the incidence of skeletal abnormalities (gill cover, fins and vertebral column, p < .05) in zebrafish larvae. The A and B experimental groups presented the highest (88 ± 3 and 84 ± 17%, respectively), but the C and D the lowest (18 ± 14 and 11 ± 2%, respectively), rates of normal fish (fish without any abnormality). Similarly, growth rate was comparatively elevated in A and B groups. No significant differences were observed in fish survival between A, B and C groups. However, D group presented a significantly lower survival rate. To our knowledge, this is the first study to show that the live feed-to-microdiet transition rate influences larval growth, survival and abnormality rates in a non-homogenous pattern.

Long lasting effects of early temperature exposure on the swimming performance and skeleton development of metamorphosing Gilthead seabream (Sparus aurata L.) larvae.

Temperatures experienced during early ontogeny significantly influence fish phenotypes, with clear consequences for the wild and reared stocks. We examined the effect of temperature (17, 20, or 23 °C) during the short embryonic and yolk-sac larval period, on the swimming performance and skeleton of metamorphosing Gilthead seabream larvae. In the following ontogenetic period, all fish were subjected to common temperature (20 °C). The critical swimming speed of metamorphosing larvae was significantly decreased from 9.7 ± 0.6 TL/s (total length per second) at 17 °C developmental temperature (DT) to 8.7 ± 0.6 and 8.8 ± 0.7 TL/s at 20 and 23 °C DT respectively (p < 0.05). Swimming performance was significantly correlated with fish body shape (p < 0.05). Compared with the rest groups, fish of 17 °C DT presented a slender body shape, longer caudal peduncle, terminal mouth and ventrally transposed pectoral fins. Moreover, DT significantly affected the relative depth of heart ventricle (VD/TL, p < 0.05), which was comparatively increased at 17 °C DT. Finally, the incidence of caudal-fin abnormalities significantly decreased (p < 0.05) with the increase of DT. To our knowledge, this is the first evidence for the significant effect of DT during the short embryonic and yolk-sac larval period on the swimming performance of the later stages.

3D heart morphological changes in response to developmental temperature in zebrafish: More than ventricle roundness.

A new, three-dimensional geometric morphometric approach was assessed to study the effect of developmental temperature on fish heart shape utilizing geometric morphometrics of three-dimensional landmarks captured on digitally reconstructed zebrafish hearts. This study reports the first three-dimensional analysis of the fish heart and demonstrates significant shape modifications occurring after three developmental temperature treatments (TD = 24, 28 or 32°C) at two distinct developmental stages (juvenile and adult fish). Elevation of TD induced ventricle roundness in juveniles, males and females. Furthermore, significant differences that have not been described so far in heart morphometric indices (i.e., ventricle sphericity, bulbus arteriosus elongation and relative location, heart asymmetry) were identified. Sex proved to be a significant regulating factor of heart shape plasticity in response to TD. This methodology offers unique benefits by providing a more precise representation of heart shape changes in response to developmental temperature that are otherwise not discernable with the previously described two-dimensional methods. Our work provides the first evidence of three-dimensional shape alterations of the zebrafish heart adding to the emerging rationale of temperature-driven plastic responses of global warming and seasonal temperature disturbances in wild fish populations and in other ectothermic vertebrates as well (amphibians and reptiles).

Differences in otolith shape and fluctuating-asymmetry between reared and wild gilthead seabream (Sparus aurata Linnaeus, 1758).

Otolith structure is a useful tool in discrimination among fish populations as it is a permanent record of the influence of endogenous and exogenous factors. In the present study we examined otolith morphology and fluctuating asymmetry (FA) for differences between wild-caught (by bottom trawl) and reared specimens of Gilthead seabream (Sparus aurata). Based on the frequency of regenerated scales (degree of scale regeneration, SRD) on each specimen, a threshold of 30% SRD was used to assign wild-caught fish individuals as wild (≤30% SRD, LR group) or as possible aquaculture escapees (>30% SRD, HR group). Based on the analysis of elliptic Fourier descriptors, significant differences were found in otolith shape between reared (Rr) and the wild-caught groups (LR, HR). Reared fish had otoliths with significantly larger perimeter (OP ) than wild-caught fish. Furthermore, FA was significantly higher in the Rr than the LR group for OP and all except one shape descriptors (harmonics 2-7). The HR group exhibited intermediate levels of FA between the low and high FA levels observed in the LR and Rr groups. Results are discussed in terms of the value of combining otolith and scale morphology for the identification of escapees in wild Gilthead seabream stocks.

Recovery of haemal lordosis in Gilthead seabream (Sparus aurata L.).

Haemal lordosis is a frequent abnormality of the vertebral column. It has been recorded to develop in different finfish species, during the hatchery rearing phase. Under certain conditions, this abnormality reaches a high prevalence and severity degree, with significant effects on the external morphology of the fish. We show that haemal lordosis recovers during the on-growing of Gilthead seabream in sea cages. At the end of the hatchery phase, 1700 seabream juveniles were tagged electronically and examined for the presence of haemal lordosis. Subsequently, their morphology was examined periodically up to the end of the on-growing period. We found that the prevalence of fish with a lordotic external morphology decreased during the studied period by approximately 50%. Interestingly, 27% of the recovered fish presented a completely normal vertebral column. Geometric morphometric analysis showed no significant differences in the body shape between the fish with a recovered normal phenotype and the fish that were normal since the beginning of the on-growing period. Our results provide the first evidence for the recovery of lordosis during the growth of fish. A mechanism with multiple levels of remodeling of abnormal bones is suggested.