External Fertilization Research Articles

Zebrafish Models for Skeletal and Extraskeletal Osteogenesis Imperfecta Features: Unveiling Pathophysiology and Paving the Way for Drug Discovery.

In the last decades, the easy genetic manipulation, the external fertilization, the high percentage of homology with human genes and the reduced husbandry costs compared to rodents, made zebrafish a valid model for studying human diseases and for developing new therapeutical strategies. Since zebrafish shares with mammals the same bone cells and ossification types, it became widely used to dissect mechanisms and possible new therapeutic approaches in the field of common and rare bone diseases, such as osteoporosis and osteogenesis imperfecta (OI), respectively. OI is a heritable skeletal disorder caused by defects in gene encoding collagen I or proteins/enzymes necessary for collagen I synthesis and secretion. Nevertheless, OI patients can be also characterized by extraskeletal manifestations such as dentinogenesis imperfecta, muscle weakness, cardiac valve and pulmonary abnormalities and skin laxity. In this review, we provide an overview of the available zebrafish models for both dominant and recessive forms of OI. An updated description of all the main similarities and differences between zebrafish and mammal skeleton, muscle, heart and skin, will be also discussed. Finally, a list of high- and low-throughput techniques available to exploit both larvae and adult OI zebrafish models as unique tools for the discovery of new therapeutic approaches will be presented.

Sperm performance in the race for fertilization, the influence of female reproductive fluid.

In studies of sperm competition, particularly in external fertilizers, the importance of the fertilization environment on the paternity share among rival males often goes overlooked. The female reproductive fluid (FRF), produced and released by females, creates the microenvironment that sperm encounter on their quest for fertilization and can generate paternity biases by affecting key traits in sperm competition. Yet, whether there is a direct link between FRF effects on sperm traits and its effect on competitive fertilization dynamics remains to be explored. Here, using the zebrafish Danio rerio, we compare within-female paternity share among two competing males and predictors of fertilization success (i.e. sperm traits) in the presence/absence of FRF. Our results unequivocally reveal a direct link between the direction and magnitude of the effect of FRF on sperm traits and the change in the competitive fertilization success of each male. This study demonstrates that the FRF directly mediates post-mating female control through its differential effect on sperm performance and that the FRF's effect on sperm quality alone is sufficient to predict the magnitude of the fitness effects. These findings highlight the need to consider the role of FRF in fertilization, avoiding biases resulting from an exclusive focus on male intrinsic sperm quality.

Comparative sperm morphology of 15 species of limpet (Mollusca: Patellogastropoda: Lottioidea) and correlation with current systematics and phylogenies

ABSTRACT The spermatozoa of 14 species (four genera) of Lottiidae and one species of Acmaeidae are described by transmission electron microscopy. All have ect-aquasperm, which are relatively small, morphologically simple, free-swimming sperm produced by species with external fertilization. Based on the size, shape and structure of the nucleus and acrosome, as well as features of the mid-piece, several morphological sperm ‘types’ are recognized within the species of Lottioidea described by the present and several previous studies. Sperm morphology largely supports current taxonomy as well as systematic and phylogenetic relationships. However, the morphology of the sperm of Lottia dorsuosa suggests that its generic status should be re-examined. The sperm morphology of Niveotectura pallida (formerly Acmaea pallida) is more similar to that of species of Tectura than of Acmaea mitra, supporting the current placement of N. pallida in the Lottiidae. This result is at odds with the close relationship between N. pallida and A. mitra indicated by recent phylogenies based on molecular data.

Assessing the temporal isolation barrier between two closely related species of the genus Fissurella off the southwestern coast of South America

ABSTRACT Knowledge of the reproductive isolation mechanisms that prevent gene flow between species is essential for understanding the process of speciation. In this study, we investigate the spawning periods in Fissurella cumingi and F. latimarginata, two closely related sympatric species of keyhole limpets with external fertilization, as a potential prezygotic barrier between them. The reproductive cycle was assessed in two distinct locations in northern Chile using the gonadosomatic index (GSI) and gonadal histology. Species, locality, and sex were designated as predictive variables, while average GSI values, as well as those of partial and total spawning, were considered dependent variables. Both species exhibit a continuous gametogenic cycle throughout the year, characterized by multiple spawning events. A generalized linear model (GLM) factorial ANOVA design revealed that neither species, locality nor sex were significant factors with respect to the GSI and partial spawning scores. However, sex was found to be the only significant factor related to total spawning. No significant interactions were observed among predictive factors. These findings suggest that reproductive periods may not serve as effective barriers to gamete encounters between species. Our study prompts questions about how closely-related externally-fertilizing species maintain reproductive isolation.

Investigating the timing of spawning of two sea urchins (Tripneustes gratilla and Toxopneustes pileolus)

AbstractMany marine organisms broadcast gametes for external fertilization, thus conspecifics need to concentrate the time of gamete release through synchronization with environmental factors. We investigated the timing of spawning in two sea urchin species, Tripneustes gratilla and Toxopneustes pileolus, by assessing the spawning potential of individuals using both surveys and experiments in southern Taiwan from March to November 2010. We developed a new method to observe the spawning potential without sacrificing any individuals, defining those individuals‐initiated spawning within 30 min after 10 s of exposure to air as having the spawning potential. We discovered that the percentage of individuals with spawning potential, that is, the spawning ratios of the population, varied greatly from 0% to 80% in To. pileolus and from 0% to 50% in Tr. gratilla within the reproductive seasons. We evaluated the possibility of a lunar/semilunar rhythm of spawning ratios of the population and tested three environmental factors, that is, light intensity, tide, and temperature that could potentially affect the spawning potential of the sea urchins by experiments. Individuals with spawning potential were neither randomly nor evenly distributed among days but were aggregated on certain days; however, no lunar/semilunar rhythm of the spawning ratios was detected within the reproductive season. The possible role of the tidal factor within a day was not found in experiments in either species. However, when water temperature was >27°C in our surveys, significantly higher spawning ratios were observed in To. pileolus, but not in Tr. gratilla in the reproductive season. A further temperature experiment in To. pileolus supported the positive role of high temperature on the spawning ratios of individuals. In addition, To. pileolus was more likely to spawn during the day than at night, and the light/dark experiments suggested that the spawning potential was controlled by an endogenous biological clock rather than by the ambient light conditions directly. The lack of rhythmic pattern and yet concentrated spawning of these sea urchins may be adaptive to avoid predation.

Evolutionary trade-offs between testes size and parenting in Neotropical glassfrogs.

In males, large testes size signifies high sperm production and is commonly linked to heightened sperm competition levels. It may also evolve as a response to an elevated risk of sperm depletion due to multiple mating or large clutch sizes. Conversely, weapons, mate or clutch guarding may allow individuals to monopolize mating events and preclude sperm competition, thereby reducing the selection of large testes. Herein, we examined how paternal care, sexual size dimorphism (SSD), weaponry and female fecundity are linked to testes size in glassfrogs. We found that paternal care was associated with a reduction in relative testes size, suggesting an evolutionary trade-off between testes size and parenting. Although females were slightly larger than males and species with paternal care tended to have larger clutches, there was no significant relationship between SSD, clutch size and relative testes size. These findings suggest that the evolution of testes size in glassfrogs is influenced by sperm competition risk, rather than sperm depletion risk. We infer that clutch guarding precludes the risk of fertilization by other males and consequently diminishes selective pressure for larger testes. Our study highlights the prominent role of paternal care in the evolution of testes size in species with external fertilization.

Hardly Venus’s servant—morphological adaptations of Veneriserva to an endoparasitic lifestyle and its phylogenetic position within Dorvilleidae (Annelida)

Endoparasitic annelids living inside another annelid host are known, particularly with regard to Oenonidae, but in general are poorly studied. The dorvilleid Veneriserva pygoclava is known from southern California, and its genus name (Latin = Venus’s servant) alludes to the close association with the host aphroditid scaleworm Aphrodita longipalpa. Little is known on fundamental questions on the biology of Veneriserva pygoclava. What is its mode of reproduction? How do they feed? How do they penetrate the host? We have studied multiple parasitized hosts and V. pygoclava specimens, using an integrative approach, combining µCT, histology, and electron microscopy. 3D reconstructions from µCT data of a parasitized Aphrodita show the exact position of the parasites in their natural condition within the host’s coelomic cavity. Ultrastructural investigations of the parasites revealed interesting adaptations to their lifestyle such as the complete reduction of their gut, despite the presence of a functional jaw apparatus and a modified epidermis enabling nutrient uptake from the host’s coelomic fluid. In addition to these, we also investigated spermatogenesis and oogenesis in V. pygoclava. Sperm morphology indicates an external fertilization of eggs within the coelomic cavity of the host. Mature male and female parasites living inside the same mature host and the presence of juvenile V. pygoclava within juveniles of Aphrodita suggest an obligate form of parasitism with a very early penetration of the hosts. In addition to our detailed morphological investigation, we conducted a phylogenetic analysis showing the position of Veneriserva within Dorvilleidae and its position was recovered nested among taxa of the Iphitime. Our phylogenetic analyses also show that the taxation Ophryotrocha puerilis siberti should be given full species rank and referred to as Ophryotrocha siberti. Finally, we publish here the full mitochondrial genome of V. pygoclava and discuss its novel gene order with reference to other annelids.

Screening suitable legumes for living mulches to support nitrogen dynamics and weed control in a durum wheat-forage sorghum crop sequence

Relay intercropping of service legumes (living mulch) is a valuable agroecological practice to support nutrient availability, yield and non-chemical weed control in a crops sequence, if suitable legumes are chosen. This study tested the suitability of eight legume living mulches for relay intercropping in durum wheat (Triticum durum Desf.) evaluating their effects on the subsequent forage sorghum (Sorghum bicolor L. Moench) in a Mediterranean low-input cereal-based cropping system. Legumes include perennial (Medicago sativa, Medicago lupulina, Trifolium repens, Hedysarum coronarium), annual (Trifolium incarnatum, Trifolium resupinatum) and annual self-seeding (Trifolium subterraneum, Medicago polymorpha) species. A plot experiment repeating two times a wheat-sorghum crop sequence, was carried out in Central Italy to assess the effects of relay intercropping of legumes on agronomical and economic performance of the associated wheat and subsequent sorghum. The legumes were undersown in the already developed durum wheat in late winter. They were maintained after wheat harvest and used as green manure by biomass chopping and plowing in the following spring at sorghum sowing time. None of the intercropped legumes affected N uptake or grain protein content of the companion wheat with respect to the control (wheat sole crop). In the spring before sorghum sowing, N input from legume biomass residues was assessed and it sensibly changed according to legume species, ranging from 1.2 (T. incarnatum) to 182 kg ha−1 (H. coronarium). The legume treatments significantly affected the N uptake in the subsequent sorghum and it ranged from 20.2 to 172.6 kg ha−1. Without the use of additional external nitrogen input, average sorghum dry biomass production preceded by H. coronarium (13.3 t ha−1), T. repens (11.8 t ha−1), T. subterraneum (11.0 t ha−1) and M. sativa (9.3 t ha−1) was in line with the productive level under conventional conditions. Biomass residues of M. sativa significantly reduced the total weed biomass in sorghum by 65% compared with the control. In sorghum, preceding legumes such as T. resupinatum and T. incarnatum promoted dicotyledonous weed growth. Hedysarum coronarium and T. repens were the best legumes for relay intercropping in the low-input wheat-sorghum crop sequence under Mediterranean conditions of this study according to agronomic and economic evaluation. These legumes were able to increase soil nitrogen content allowing to significantly reduce external nitrogen fertilization while optimizing sorghum production. When gross income is calculated at cropping system level, most legumes provide a positive economic balance.

Feasibility of In Vivo Semen Collection and Description of the Morphology and Ultrastructure of the Spermatozoa of Arapaima gigas (Schinz, 1822)

The pirarucu, Arapaima gigas, is an emblematic endangered Amazon osteoglossid with high potential for aquaculture development. In this study, we report the in vivo collection of semen from pirarucu for the first-time and a complete description of the spermatozoa ultrastructure using scanning and transmission electron microscopy (SEM and TEM). The spermatozoon of pirarucu is biflagellate with no acrosomal cell, comprising a spherical head (8.26 ± 2.19 µm2), a short intermediate piece region and two long flagella (68.34 ± 5.69 µm). The nucleus is spherical and electron-dense, and at its base, there are two nuclear fossae where the centrioles are found. The anterior part of the flagellum is positioned at the lateral base of the nucleus, forming an intermediate piece. Lateral to the intermediate piece, an abundant number of mitochondria are present and occupy most of the cytoplasmic volume. The two flagella were found with a 9 + 2 axonemal structure (nine peripheral doublets and two central microtubules). The central microtubules are aligned with two side fins formed laterally by the axoneme plasma membrane. Side fins were observed from the anterior region to the posterior end. The results show that the spermatozoa of A. gigas are biflagellate, mitochondria-rich and robust, corroborating the external fertilization characteristic of the species. The novel description of sperm morphology presented herein will contribute to advancing the knowledge of and aiding future research on the reproductive biology of this species.

Intra-specific correlations between ejaculate traits and competitive fertilization success: a meta-analysis across species and fertilization modes.

Understanding of how selection can act on traits that improve competitiveness and subsequent paternity has advanced, including the idea that internal and external fertilization presents different environments that may select differentially on ejaculate traits. However, no studies have quantitatively synthesized the intra-specific relationships between these traits and paternity. Therefore, we conducted a meta-analysis across 52 papers to determine which ejaculate traits positively correlate with paternity share and how these correlations vary with fertilization mode. Overall, most ejaculate traits were positively associated with paternity, with the notable exception of sperm length. Sub-analyses on sperm number, sperm length, and sperm velocity revealed no statistical differences between fertilization modes in the relationship between traits and paternity when all effect sizes across species were combined. However, in a sub-analysis on fish species only, we found evidence that sperm velocity may be more important in external fertilizers. We also observed differences in the importance of phylogenetic relatedness and some species-specific differences. Our results suggest that while most ejaculate traits should be under positive directional selection in both internal and external fertilizers, sperm length may be subject to more nuanced selection pressures. Overall, we highlight important patterns of intra-specific relationships between ejaculate traits and competitive fertilization success.

The reaction-diffusion basis of animated patterns in eukaryotic flagella

The flagellar beat of bull spermatozoa and C. Reinhardtii are modelled by a minimal, geometrically exact, reaction-diffusion system. Spatio-temporal animated patterns describe flagellar waves, analogous to chemical-patterns from classical reaction-diffusion systems, with sliding-controlled molecular motor reaction-kinetics. The reaction-diffusion system is derived from first principles as a consequence of the high-internal dissipation by the flagellum relative to the external hydrodynamic dissipation. Quantitative comparison with nonlinear, large-amplitude simulations shows that animated reaction-diffusion patterns account for the experimental beating of both bull sperm and C. Reinhardtii. Our results suggest that a unified mechanism may exist for motors controlled by sliding, without requiring curvature-sensing, and uninfluenced by hydrodynamics. High-internal dissipation instigates autonomous travelling waves independently of the external fluid, enabling progressive swimming, otherwise not possible, in low viscosity environments, potentially critical for external fertilizers and aquatic microorganisms. The reaction-diffusion system may prove a powerful tool for studying pattern formation of movement on animated structures.

Unveiling the impact of environmental microplastics on mussel spermatozoa: First evidence of prothymosin-α detection in invertebrate’s male gametes

Mytilus galloprovincialis mussels, like many other marine invertebrates, employ external fertilization as a mating strategy, exposing their gametes to various contaminants upon release into seawater. Environmental microplastics (EMP) are prevalent marine pollutants that pose a significant threat to aquatic biota. In this regard, our study aimed to investigate the potential effects of exposing mussels’ male gametes to increasing concentrations of EMP (1, 10, 50, and 100 μg/l) collected from a Mediterranean sandy beach. We focused on assessing gamete quality by analysing physiological parameters such as viability, mitochondrial membrane potential, oxidative status, and motility. Additionally, we evaluated DNA integrity and activation of apoptosis. Furthermore, our study aimed to detect the presence of the prothymosin-α (PTMA) protein, which has never been previously investigated in invertebrate spermatozoa. Our data revealed that exposure of mussel spermatozoa to EMPs altered their oxidative status and mitochondrial membrane potential, induced a decrease in motility, DNA integrity, and an increased apoptotic occurrence, leading to a decline in overall viability. The localization of PTMA into the head and flagellum of spermatozoa further supported its presence and susceptibility to the effects of microplastics. These findings raise concerns about the reproductive capacity of mussels under environmental microplastic pollution and highlight potential long-term threats to population sustainability.

Fertilization Mode Covaries with Body Size.

AbstractThe evolution of internal fertilization has occurred repeatedly and independently across the tree of life. As it has evolved, internal fertilization has reshaped sexual selection and the covariances among sexual traits, such as testes size, and gamete traits. But it is unclear whether fertilization mode also shows evolutionary associations with traits other than primary sex traits. Theory predicts that fertilization mode and body size should covary, but formal tests with phylogenetic control are lacking. We used a phylogenetically controlled approach to test the covariance between fertilization mode and adult body size (while accounting for latitude, offspring size, and offspring developmental mode) among 1,232 species of marine invertebrates from three phyla. Within all phyla, external fertilizers are consistently larger than internal fertilizers: the consequences of fertilization mode extend to traits that are only indirectly related to reproduction. We suspect that other traits may also coevolve with fertilization mode in ways that remain unexplored.

A Review on the Genus Paramacrobiotus (Tardigrada) with a New Diagnostic Key

Paramacrobiotus species have been described in almost every corner of the world. To date, 45 species have been reported from this genus. Among which, 13 belong to the areolatus group (without a microplacoid) and 32 belong to the richtersi group (with a microplacoid). The species’ presence in different climatic conditions and habitats provides evidence of their adaptation to various harsh environments. The species of the genus are both bisexual (diploid) and parthenogenetic (triploid). The bisexual species have external fertilization. And they are omnivorous whose diet consists of certain cyanobacteria, algae, fungi, rotifers, nematodes and juvenile tardigrades. The life history of species from this genus varies from species to species. Because the species has a strong predilection for cryptobiosis, numerous investigations involving anhydrobiosis have been conducted utilizing specimens from varied Paramacrobiotus species to date. In this review, we provide a concise summary of changes observed due to various cryptobiotic conditions in many species of this genus, the geographical distribution of all the species, feeding behaviour, life history, microbiome community, Wolbachia endosymbiont identification, reproduction, phylogeny and general taxonomy of the species from the genus Paramacrobiotus. Furthermore, we provide a new diagnostic key to the genus Paramacrobiotus based on the morphological and morphometric characters of adults and eggs.

TMEM16A activation for the fast block to polyspermy in the African clawed frog does not require conventional activation of egg PLCs.

Fertilization of an egg by more than one sperm, a condition known as polyspermy, leads to gross chromosomal abnormalities and is embryonic lethal for most animals. Consequently, eggs have evolved multiple processes to stop supernumerary sperm from entering the nascent zygote. For external fertilizers, such as frogs and sea urchins, fertilization signals a depolarization of the egg membrane, which serves as the fast block to polyspermy. Sperm can bind to, but will not enter, depolarized eggs. In eggs from the African clawed frog, Xenopus laevis, the fast block depolarization is mediated by the Ca2+-activated Cl- channel TMEM16A. To do so, fertilization activates phospholipase C, which generates IP3 to signal a Ca2+ release from the ER. Currently, the signaling pathway by which fertilization activates PLC during the fast block remains unknown. Here, we sought to uncover this pathway by targeting the canonical activation of the PLC isoforms present in the X. laevis egg: PLCγ and PLCβ. We observed no changes to the fast block in X. laevis eggs inseminated in inhibitors of tyrosine phosphorylation, used to stop activation of PLCγ, or inhibitors of Gαq/11 pathways, used to stop activation of PLCβ. These data suggest that the PLC that signals the fast block depolarization in X. laevis is activated by a novel mechanism.

Animal models of silicosis: fishing for new therapeutic targets and treatments.

Silicosis as an occupational lung disease has been present in our lives for centuries. Research studies have already developed and implemented many animal models to study the pathogenesis and molecular basis of the disease and enabled the search for treatments. As all experimental animal models used to date have their advantages and disadvantages, there is a continuous search for a better model, which will not only accelerate basic research, but also contribute to clinical aspects and drug development. We review here, for the first time, the main animal models developed to date to study silicosis and the unique advantages of the zebrafish model that make it an optimal complement to other models. Among the main advantages of zebrafish for modelling human diseases are its ease of husbandry, low maintenance cost, external fertilisation and development, its transparency from early life, and its amenability to chemical and genetic screening. We discuss the use of zebrafish as a model of silicosis, its similarities to other animal models and the characteristics of patients at molecular and clinical levels, and show the current state of the art of inflammatory and fibrotic zebrafish models that could be used in silicosis research.

Female reproductive fluid concentrations affect sperm performance of alternative male phenotypes in an external fertilizer.

There is growing evidence that the female reproductive fluid (FRF) plays an important role in cryptic female choice through its differential effect on the performance of sperm from different males. In a natural spawning event, the male(s) may release ejaculate closer or further away from the spawning female. If the relative spatial proximity of competing males reflects the female pre-mating preference towards those males, then favoured males will encounter higher concentrations of FRF than unpreferred males. Despite this being a common situation in many external fertilizers, whether different concentrations of FRF can differentially influence the sperm performance of distinct male phenotypes (favoured and unfavoured by the female) remains to be elucidated. Here, we tested this hypothesis using the grass goby (Zosterisessor ophiocephalus), a fish with distinct territorial-sneaker reproductive tactics and female pre-mating preference towards territorial males, that consequently mate in an advantaged position and whose sperm experience higher concentrations of FRF. Our findings revealed a differential concentration-dependent effect of FRF over sneaker and territorial sperm motility only at low concentrations (i.e. at the distance where sneakers typically ejaculate), with increasing FRF concentrations (i.e. close to the eggs) similarly boosting the sperm performance of both sneaker and territorial males. The ability to release sperm close to the eggs is a prerogative of territorials, but FRF can likewise advantage the sperm of those sneakers that are able to get closer, allowing flexibility in the direction of female post-mating choice.

Flat mount preparation for whole-mount fluorescent imaging of zebrafish embryos.

The zebrafish is a widely used model organism for biomedical research due to its ease of maintenance, external fertilization of embryos, rapid embryonic development, and availability of established genetic tools. One notable advantage of using zebrafish is the transparency of the embryos, which enables high-resolution imaging of specific cells, tissues, and structures through the use of transgenic and knock-in lines. However, as the embryo develops, multiple layers of tissue wrap around the lipid-enriched yolk, which can create a challenge to image tissues located deep within the embryo. While various methods are available, such as two-photon imaging, cryosectioning, vibratome sectioning, and micro-surgery, each of these has limitations. In this study, we present a novel deyolking method that allows for high-quality imaging of tissues that are obscured by other tissues and the yolk. Embryos are lightly fixed in 1% PFA to remove the yolk without damaging embryonic tissues and are then refixed in 4% PFA and mounted on custom-made bridged slides. This method offers a simple way to prepare imaging samples that can be subjected to further preparation, such as immunostaining. Furthermore, the bridged slides described in this study can be used for imaging tissue and organ preparations from various model organisms.

Histological Investigation of the Female Gonads of Chiropsalmus quadrumanus (Cubozoa: Cnidaria) Suggests Iteroparous Reproduction

The box jellyfish Chiropsalmus quadrumanus (Chirodropida: Cubozoa: Cnidaria) is common in warm waters. Although it is assumed that external fertilization is a characteristic of Chirodropida, the life history of C. quadrumanus is not yet known since its reproductive behavior has never been described, nor has the polyp has been found in nature. As a result, in the absence of documentation of reproductive behavior, we sought to test the hypothesis of external fertilization through a histological analysis of the female gonads. Herein, we analyze ten females collected in São Paulo and Rio de Janeiro (Brazil) and describe the gonadal organization and pattern of oocyte development. The discovery of four distinct stages of oocyte differentiation augments the scant existing reports of the structural and functional maturation of sex cells in Cubozoa species. Furthermore, the gonads of mature females comprise both mature (average diameter of 122 µm) and immature oocytes, suggesting that C. quadrumanus is iteroparous and exhibits multiple reproductive cycles during its life. Medusa bell size was not found to correlate with maturity state as even small females possessed a high percentage of oocytes in late vitellogenesis, suggesting that sexual maturation occurs rapidly in C. quadrumanus females.

Toxicity of microplastics and triclosan, alone and in combination, to the fertilisation success of a broadcast spawning bivalve Tegillarca granosa

Since most marine invertebrates adopted external fertilisation, their fertilisation process is particularly vulnerable to aquatic pollutants. Both antimicrobial ingredients and microplastics (MPs) are ubiquitous in aquatic environments; however, their synergistic effects on the fertilisation of marine invertebrates remain unclear. Therefore, in this study, the fertilisation toxicity of MPs and triclosan (TCS), alone and in combination, was investigated in the broadcast spawning bivalve Tegillarca granosa. Results showed that MPs and TCS significantly suppressed the fertilisation success of T. granosa. As the fertilisation success of broadcast spawning invertebrates depends on successful gamete collisions, gamete fusion, and egg activation, sperm swimming velocity, viability, gamete collision probability, ATP status, and ion-transport enzyme activities were also analysed to further ascertain the underlying toxicity mechanisms. In summary, our findings indicate that the presence of MPs may enhance the fertilisation toxicity of TCS by hampering sperm-egg collision probability, reducing gamete fusion efficiency, and restricting Ca2+ oscillation formation.