Free-swimming Larvae Research Articles

Centriole Translational Planar Polarity in Monociliated Epithelia.

Ciliated epithelia are widespread in animals and play crucial roles in many developmental and physiological processes. Epithelia composed of multi-ciliated cells allow for directional fluid flow in the trachea, oviduct and brain cavities. Monociliated epithelia play crucial roles in vertebrate embryos, from the establishment of left-right asymmetry to the control of axis curvature via cerebrospinal flow motility in zebrafish. Cilia also have a central role in the motility and feeding of free-swimming larvae in a variety of marine organisms. These diverse functions rely on the coordinated orientation (rotational polarity) and asymmetric localization (translational polarity) of cilia and of their centriole-derived basal bodies across the epithelium, both being forms of planar cell polarity (PCP). Here, we review our current knowledge on the mechanisms of the translational polarity of basal bodies in vertebrate monociliated epithelia from the molecule to the whole organism. We highlight the importance of live imaging for understanding the dynamics of centriole polarization. We review the roles of core PCP pathways and of apicobasal polarity proteins, such as Par3, whose central function in this process has been recently uncovered. Finally, we emphasize the importance of the coordination between polarity proteins, the cytoskeleton and the basal body itself in this highly dynamic process.

Microbiome changes through the ontogeny of the marine sponge Crambe crambe

BackgroundPoriferans (sponges) are highly adaptable organisms that can thrive in diverse marine and freshwater environments due, in part, to their close associations with internal microbial communities. This sponge microbiome can be acquired from the surrounding environment (horizontal acquisition) or obtained from the parents during the reproductive process through a variety of mechanisms (vertical transfer), typically resulting in the presence of symbiotic microbes throughout all stages of sponge development. How and to what extent the different components of the microbiome are transferred to the developmental stages remain poorly understood. Here, we investigated the microbiome composition of a common, low-microbial-abundance, Atlantic-Mediterranean sponge, Crambe crambe, throughout its ontogeny, including adult individuals, brooded larvae, lecithotrophic free-swimming larvae, newly settled juveniles still lacking osculum, and juveniles with a functional osculum for filter feeding.ResultsUsing 16S rRNA gene analysis, we detected distinct microbiome compositions in each ontogenetic stage, with variations in composition, relative abundance, and diversity of microbial species. However, a particular dominant symbiont, Candidatus Beroebacter blanensis, previously described as the main symbiont of C. crambe, consistently occurred throughout all stages, an omnipresence that suggests vertical transmission from parents to offspring. This symbiont fluctuated in relative abundance across developmental stages, with pronounced prevalence in lecithotrophic stages. A major shift in microbial composition occurred as new settlers completed osculum formation and acquired filter-feeding capacity. Candidatus Beroebacter blanensis decreased significatively at this point. Microbial diversity peaked in filter-feeding stages, contrasting with the lower diversity of lecithotrophic stages. Furthermore, individual specific transmission patterns were detected, with greater microbial similarity between larvae and their respective parents compared to non-parental conspecifics.ConclusionsThese findings suggest a putative vertical transmission of the dominant symbiont, which could provide some metabolic advantage to non-filtering developmental stages of C. crambe. The increase in microbiome diversity with the onset of filter-feeding stages likely reflects enhanced interaction with environmental microbes, facilitating horizontal transmission. Conversely, lower microbiome diversity in lecithotrophic stages, prior to filter feeding, suggests incomplete symbiont transfer or potential symbiont digestion. This research provides novel information on the dynamics of the microbiome through sponge ontogeny, on the strategies for symbiont acquisition at each ontogenetic stage, and on the potential importance of symbionts during larval development.

Multi-ocean distribution of a brooding predator in the abyssal benthos

How far are species distributed on the abyssal plains? Spanning from 3000 to 6000 m below sea level, abyssal plains cover three-quarters of the ocean floor and are the largest but also least explored habitat on Earth. The question of vertical and horizontal distribution is central to understanding biogeographic and population genetic processes within species inhabiting the deep-sea benthos. Amphipod crustaceans are an important and dominant taxon in this ecosystem. As they are brooders, their dispersal capacities are more limited compared to species with free-swimming larvae, and with the exception of a few scavenging species deep-sea amphipods are restricted to a single ocean. Based on an integrative taxonomic approach (morphology, COI, 16S and 18S) we demonstrate the occurrence of a predatory amphipod species, Rhachotropis abyssalis, in three oceans: the Antarctic Ross Sea, the Northwest Pacific and the North Atlantic; regions more than 20,000 km apart. Although such extensive geographic distributions may represent a rare exception for brooding predators, these findings might also be no exception at all, but a reflection of the rare sampling and rare taxonomic investigation of invertebrate predators in the deep-sea. Our findings highlight our abysmal state of knowledge regarding biodiversity and biogeography on abyssal plains.

Monitoring the Reproduction and Development of Eggs in the Sea Hare Aplysia fasciata under Laboratory Conditions

This study looks at spawning patterns, egg mass shape, and embryo development to learn more about the reproductive biology of the A. fasciata species that lives in Libya's coastal waters. In June 2022, eight mature Aplysia fasciata collected from Tajura coast and kept into aquarium contain aerated seawater and marine algae (Ulva lactuca). After five days, a mating couple was seen in the aquarium, and, a mass of fertilized eggs were produced. The clusters eggs were transfer to new aquarium. After few hours, the eggs started development to embryo and at 10 day the egg masses colour changed to a brownish as the embryo developed to the trochophore stage and started to rotate inside capsules. After 16 day of spawning the trochophore broke the egg capsules to hatch as free swimming larvae (the veliger stage). Unfortunately, after 25 day of fertilized eggs all larvae died as veliger. Probably, the result of inadequate conditions for rearing or lack an availability of appropriate food. Whereas A. fasciata has never been recorded before in the Libyan waters.

Postmetamorphic retention of a lateral line system in African slippery frogs (Amphibia: Anura: Conraua)

Adult anurans normally lose their larval lateral-line (LL-)system during metamorphosis. Only some species with an extensive aquatic life style (e.g., Bombina bombina, all pipid frogs) are known to retain that system as adults. The aquatic African Slippery Frogs, genus Conraua, have never been studied for this system. We examined adults of all known Conraua species and tadpoles of six species from this genus for the occurrence of LL-systems. We detected a LL-system in all tadpoles studied, in adults of all West African species (C. alleni, C. derooi, C. kamancamarai, C. sagyimase, C. sp.), as well as in adults of the East African C. beccarii. In contrast we found no LL-systems in adult C. crassipes, C. goliath, and C. robusta from Central Africa. The tadpoles exhibited a LL-system typical for anurans with free-swimming larvae. In particular it was morphologically congruent to other stream tadpoles. Topography and histology of neuromasts in adults indicates their larval origin. Neuromast abundance, topography and histology, may allow Conraua a complex access to spatial information. However, although all species of the genus are aquatic, only two-thirds of them retain a postmetamorphic LL-system. In both major clades within the monophyletic genus we detected species with and without a LL-system. We hypothesize that the ancestor of current Conraua retained the larval LL-system, and that it was aquatic and lived in non or little torrent sections of forest rivers and streams. Those Central African species which live in particularly torrent streams, C. goliath and C. robusta, probably lost the LL-system. Conraua crassipes, which today lives in slow flowing streams, may have had ancestors with a torrenticulous lifestyle.

Tadpoles of Central Amazonia (Amphibia: Anura).

Amazonian lowland rainforests epitomize, as few other biomes, the terrestrial and freshwater biological diversity of our planet. We provide here a comprehensive description of the larval anurans of Central Amazonia, and their natural history. We base our analyses on fieldwork conducted in six terra-firme rainforest and two várzea floodplain sites during ~60 months between 1990 and 2013, complemented with an examination of museum specimens and a review of published literature. Ninety-nine species of anurans are known to occur in Central Amazonia. Of these, 84 species (85%) have a free-swimming exotrophic larva, six species (6%) have a terrestrial endotrophic larva, one species has a terrestrial exotrophic larva (1%) and another seven species (7%) have direct development of eggs into froglets. The life cycle of one species (1%) remains unknown. We formally describe and illustrate the larval stage of 68 species (i.e., 75% of the species with a known larval stage), five of them previously unkown and nineteen never described for Central Amazonia. We compile, review and update information on species natural history including reproductive modes, habitat use, phenology, and species interactions including diet, predators, competitors and parasites. Finally, we assessed the ecomorphological diversity in the region by the application of a system inspired in Altig & Johnston (1989), later updated by Altig & McDiarmid (1999), which recognizes tadpole guilds based on a combination of morphological and natural history data. Basic scientific information presented in this monograph provide the essential background for further studies on the ecological and evolutionary forces shaping anuran Amazonian assemblages, and their conservation.

Skeletal ontogeny of the Plainfin Midshipman, Porichthys notatus (Percomorphacea: Batrachoidiformes).

Batrachoidiformes are benthic fishes that utilize the undersides of rocks as spawning nests. Their larvae are attached to the nest and nourished by a large yolk sac. The evolutionary shift from feeding, free-swimming larvae to sedentary larvae that are reliant on their yolk sac for nutrition can lead to changes in skeletal development. Batrachoidiformes also have many morphological specializations, such as five pectoral-fin radials (versus four in other acanthomorphs) that are of uncertain homology, the determination of which may have phylogenetic implications. A larval series of Porichthys notatus was collected and its skeletal ontogeny is described. In P. notatus the ossification of the pharyngeal toothplates occurs relatively later than in percomorphs with free-swimming larvae. The posterior basibranchial copula cartilage (= fourth basibranchial) in Porichthys notatus has a unique development among fishes: it initially develops as a paired element at 6.8-7.1 mm NL before fusing posteriorly and forming single median cartilage at 7.4 mm SL. Cartilages of hypobranchial four are transitory, being observed in two specimens of 6.8 and 7.3 mm NL before fusing with ceratobranchial four. The previously identified dorsalmost pectoral radial is a bone formed by a hypertrophied propterygium that ossifies later in development. The earliest stages of P. notatus have three dorsal spines, but during late larval development, the growth of the third dorsal spine is interrupted. The development of P. notatus is compared and discussed in context to that of other acanthomorph.

Zebrafish embryos hatch early in response to chemical and mechanical indicators of predation risk, resulting in underdeveloped swimming ability of hatchling larvae.

Plasticity in hatching time allows embryos to maximize fitness by balancing the benefits and costs of remaining bound within the chorion against the benefits and costs of emerging as a free-swimming larva. Here, in the first experiment, we exposed zebrafish (Danio rerio) embryos to either chemical cues from crushed embryos (simulating egg predation) or to blank water control. Embryos exposed to alarm cues hatched sooner, and had shorter body lengths and underdeveloped fins, relative to larvae from the water treatment. Burst swimming speed was significantly slower for larvae that hatched from the alarm cue treatment than for larvae from the water treatment. In a second 2×2 experiment, we exposed zebrafish embryos to either chemical alarm cues from conspecific embryos, mechanical disturbance (magnetic stir bar) to simulate a predator probing the substrate for developing embryos, both chemical and mechanical indicators of risk, or neither (control). We found similar effects in terms of earlier time to hatch at an earlier stage of development and poorer swimming performance of hatchling larvae. In the second experiment, these effects occurred in response to mechanical disturbance with or without the presence of chemical alarm cues. Alarm cues alone produced no effects in the second experiment. Taken together, these data indicate that zebrafish embryos demonstrate a facultative trade-off between risk of predation acting on two stages of their life history.

Larvae of a marine gastropod and a marine bivalve share common gene expression signatures during metamorphic competence

Many aquatic invertebrates undergo an indirect development, a biphasic life cycle which encompasses the transformation of free-swimming larvae into benthic juveniles via settlement and metamorphosis. During this transition, metamorphic competence is a crucial developmental stage that allows larvae to swim and feed in the planktonic realm while retaining the ability to settle and metamorphose in response to environmental cues. Although there have been substantial efforts to decipher the molecular mechanisms underlying this event in several molluscan species, the conserved biological pathways that are crucial to enable this transition across species are not well understood. Here, we performed a comparative analysis of the developmental transcriptomes between bivalve Crassostrea gigas and gastropod Rapana venosa. We particularly explored the common gene expression signatures that may underlie their larval competence. We showed that, although the developmental transcriptomes differed remarkably between C. gigas and R. venosa, they likely shared a plethora of genes (n = 690) that exhibited similar expression signatures during their larval competence. Gene Ontology enrichment and expression analyses further indicated that competent larvae of both species exhibited up-regulation of pathways associated with response to stimuli, metal ion binding and transport, and neuronal development, but showed down-regulation of pathways that were mainly involved in cilium assembly and organ development. Using oyster and whelk as models, our study suggests that regulation of these conserved pathways is crucial for their subsequent settlement and metamorphosis and may represent a universal mechanism that enables the pelagic-to-benthic transition in a broader range of marine invertebrates.

Co-occurrence between the presence of epidermal giant cells and alarm chemical cues in tadpole skin homogenates: An ontogenetic and cross-species comparison analysis

In amphibians, intra- or interspecific chemical cues are an important source of information about possible predation risk. In anuran tadpoles, this information causes changes at different levels including behavior, morphology, and growth and development. It has been shown that chemical alarm cues trigger antipredator behaviors, such as decreased exploratory activity, in a wide variety of anuran species; however, the cellular origin of the chemical cues has not yet been confirmed by new evidence. Previous works have suggested that the alarm cues originate from a particular cell type in the skin in tadpoles of the family Bufonidae: the epidermal giant cells (GCs). Here, we confirm the presence of GCs in the epidermis of Rhinella arenarum larvae from developmental stages as early as G22, when free-swimming larvae show gregarious behavior. In addition, larval skin homogenates trigger antipredator behaviors in conspecifics from stage G22 onwards, but not at early stages (G19 and G21). This fact exposes experimental evidence for the coexistence between the appearance of GCs and the production of chemical alarm cues during the development of R. arenarum. Furthermore, the antipredator behavioral response of R. arenarum larvae triggered by skin preparations of other species that belong to the same family who also exhibit GCs allows us to speculate that chemical cues appear to be conserved among phylogenetically related species, allowing them to cross-respond to heterospecific cues. Our experimental approaches support the role of GCs as the source of alarm cues in anuran larvae of the family Bufonidae.

Host transmission dynamics of first- and third-stage Angiostrongylus cantonensis larvae in Bullastra lessoni.

Given the importance of angiostrongyliasis as an emerging infectious disease of humans, companion animals, and wildlife, the current study focused on the transmission dynamics of first- and third-stage larvae of the parasitic nematode, Angiostrongylus cantonensis. The migration of infective larvae and their subsequent distribution within the Lymnaeidae snail, Bullastra lessoni, were investigated over time using microscopic examination of histological sections and fresh tissue. Snails were divided into four anatomical regions: (i) anterior and (ii) posterior cephalopedal masses, (iii) mantle skirt and (iv) visceral mass. The viability of free-swimming third-stage larvae, after their release from snail tissues, was evaluated in vitro by propidium iodide staining and infectivity by in vivo infection of Wistar rats. Snails were sequentially dissected over time to assess the number and anatomical distribution of larvae within each snail and hence infer their migration pathway. Herein, ongoing larval migratory activity was detected over 28 days post-infection. A comparison of infection rates and the larval distribution within the four designated snail regions demonstrated a significant relationship between anatomical region and density of infective larvae, with larvae mostly distributed in the anterior cephalopedal mass (43.6 ± 10.8%) and the mantle skirt (33.0 ± 8.8%). Propidium iodide staining showed that free-swimming third-stage larvae retained viability for between 4 and 8 weeks when stored under laboratory conditions. In contrast to viability, larval infectivity in rats remained for up to 2 weeks only. Knowledge gained from the current work could provide information on the development of new approaches to controlling the transmission of this parasite.

Characterization of locomotor phenotypes in zebrafish larvae requires testing under both light and dark conditions.

Despite growing knowledge, much remains unknown regarding how signaling within neural networks translate into specific behaviors. To pursue this quest, we need better understanding of the behavioral output under different experimental conditions. Zebrafish is a key model to study the relationship between network and behavior and illumination is a factor known to influence behavioral output. By only assessing behavior under dark or light conditions, one might miss behavioral phenotypes exclusive to the neglected illumination setting. Here, we identified locomotor behavior, using different rearing regimes and experimental illumination settings, to showcase the need to assess behavior under both light and dark conditions. Characterization of free-swimming zebrafish larvae, housed under continuous darkness or a day/night cycle, did not reveal behavioral differences; larvae were most active during light conditions. However, larvae housed under a day/night cycle moved a shorter distance, had lower maximum velocity and maximum acceleration during the startle response under light conditions. Next, we explored if we could assess behavior under both dark and light conditions by presenting these conditions in sequence, using the same batch of larvae. Our experiments yielded similar results as observed for naïve larvae: higher activity during light conditions, regardless of order of illumination (i.e. dark-light or light-dark). Finally, we conducted these sequenced illumination conditions in an experimental setting by characterizing behavioral phenotypes in larvae following neuromast ablation. Depending on the illumination during testing, the behavioral phenotype following ablation was characterized differently. In addition, the results indicate that the order in which the light and dark conditions are presented has to be considered, as habituation may occur. Our study adds to existing literature on illumination-related differences in zebrafish behavior and emphasize the need to explore behavioral phenotypes under both light and dark condition to maximize our understanding of how experimental permutations affect behavior.

Chromosome-level genome assembly, annotation, and phylogenomics of the gooseneck barnacle Pollicipes pollicipes.

BackgroundThe barnacles are a group of >2,000 species that have fascinated biologists, including Darwin, for centuries. Their lifestyles are extremely diverse, from free-swimming larvae to sessile adults, and even root-like endoparasites. Barnacles also cause hundreds of millions of dollars of losses annually due to biofouling. However, genomic resources for crustaceans, and barnacles in particular, are lacking.ResultsUsing 62× Pacific Biosciences coverage, 189× Illumina whole-genome sequencing coverage, 203× HiC coverage, and 69× CHi-C coverage, we produced a chromosome-level genome assembly of the gooseneck barnacle Pollicipes pollicipes. The P. pollicipes genome is 770 Mb long and its assembly is one of the most contiguous and complete crustacean genomes available, with a scaffold N50 of 47 Mb and 90.5% of the BUSCO Arthropoda gene set. Using the genome annotation produced here along with transcriptomes of 13 other barnacle species, we completed phylogenomic analyses on a nearly 2 million amino acid alignment. Contrary to previous studies, our phylogenies suggest that the Pollicipedomorpha is monophyletic and sister to the Balanomorpha, which alters our understanding of barnacle larval evolution and suggests homoplasy in a number of naupliar characters. We also compared transcriptomes of P. pollicipes nauplius larvae and adults and found that nearly one-half of the genes in the genome are differentially expressed, highlighting the vastly different transcriptomes of larvae and adult gooseneck barnacles. Annotation of the genes with KEGG and GO terms reveals that these stages exhibit many differences including cuticle binding, chitin binding, microtubule motor activity, and membrane adhesion.ConclusionThis study provides high-quality genomic resources for a key group of crustaceans. This is especially valuable given the roles P. pollicipes plays in European fisheries, as a sentinel species for coastal ecosystems, and as a model for studying barnacle adhesion as well as its key position in the barnacle tree of life. A combination of genomic, phylogenetic, and transcriptomic analyses here provides valuable insights into the evolution and development of barnacles.

Natural history traits of the terrestrial breeding frog Batrachyla taeniata (Anura: Batrachylidae) in wet meadows of Patagonia

ABSTRACT Anurans have diverse reproductive modes, and some species of the austral forests of Patagonia have become specialized in terrestrial reproduction. Batrachyla taeniata is one of these species: it inhabits wetlands, lays its eggs during late summer and fall in terrestrial conditions, and has overwintering tadpoles. We studied a population of B. taeniata that breeds in wet meadows surrounded by native Patagonian forest and described its life cycle parameters (breeding phenology, egg development, and coexistence with other anurans) over two different years with contrasting hydroperiods. We also analyzed the effect of predatory insects on tadpole survival. Embryo development occurred in wet soil, which may be exposed to the sun or not, and took from 48 to 63 days to hatch. Free-swimming larvae overwintered in the wetland and coexisted in spring with pond-breeding anurans that varied in composition and abundance according to the wetland hydroperiod. The survivorship of B. taeniata tadpoles was significantly greater than that of P. thaul tadpoles when both were exposed to a free belostomatid predator. We consider that the terrestrial reproductive mode and life cycle of B. taeniata enable this species to survive in the variable biotic conditions determined by hydroperiod length.

Comparative Assessment of the Bioremediation Potential of Polychaetes (Lug worm-Arenicola marina sp and Syllidae Worm-Syllis prolifera sp) in Aquaculture Pond Sediment

Polychaetes play an important role in nutrient cycling and remediation of coastal ecosystems. Large quantities of organic matter that could lead to pollution of pond and coastal waters are generated by aquaculture waste. To assess the remediation prospects of Arenicola marina and Syllis prolifera species, laboratory sediment microcosm experiments were conducted where large size Arenicola marina and Syllis prolifera were introduced to sediment in microcosm A, large size Arenicola marina to sediment in microcosm B, large sized Syllis prolifera to sediment in microcosm C and no polychaetes to sediment in microcosm D. Microcosm A′, B′ and C′ as replicates for small size polychaetes were also set up, respectively. After 30 days, microcosm A, B and C had significant decrease in organic carbon levels with microcosm B being the highest (Total organic carbon (TOC); 27.87%; p< 0.05). Both large and small polychaetes promoted significant decrease in sulphur (S) content (mean=62.76±0.21; 62.81±0.21%) and iron(Fe) (mean=49.43±1.47; 36.28±5.90%) respectively. Increase in pH by 31.15±0.13% was found in the presence of large size polychaetes, most likely associated with the burrowing process involving oxidation of Fe to Fe2O3. Large size polychaetes had better survival (mean=92±0.82%) than their small size counterpart (mean=55±4.08%). The extent of biodegradation B>A>C>D observed revealed that large size Arenicola marina was a better bioremediator of organic matter (OM), Fe and S enriched aquaculture pond sediment, probably due to its biological characteristics, well suited for the aquaculture than other species of sea worms that produce free swimming larvae. Therefore, large size Arenicola marina significantly improved sediment quality as well as increased its pH without compromising their survival. As the search for a better bioremediator of organically enriched sediment continues, our result revealed large size Arenicola marina as a more promising candidate compared to other species documented elsewhere in the world. Hence, rearing of large size Arenicola marina sp is recommended as their feeding habits are well suited for aquaculture.

Evaluating the Death and Recovery of Lateral Line Hair Cells Following Repeated Neomycin Treatments.

Acute chemical ablation of lateral line hair cells is an important tool to understand lateral line-mediated behaviors in free-swimming fish larvae and adults. However, lateral line-mediated behaviors have not been described in fish larvae prior to swim bladder inflation, possibly because single doses of ototoxin do not effectively silence lateral line function at early developmental stages. To determine whether ototoxins can disrupt lateral line hair cells during early development, we repeatedly exposed zebrafish larvae to the ototoxin neomycin during a 36 h period from 3 to 4 days post-fertilization (dpf). We use simultaneous transgenic and vital dye labeling of hair cells to compare 6-h and 12-h repeated treatment timelines and neomycin concentrations between 0 and 400 µM in terms of larval survival, hair cell death, regeneration, and functional recovery. Following exposure to neomycin, we find that the emergence of newly functional hair cells outpaces cellular regeneration, likely due to the maturation of ototoxin-resistant hair cells that survive treatment. Furthermore, hair cells of 4 dpf larvae exhibit faster recovery compared to 3 dpf larvae. Our data suggest that the rapid functional maturation of ototoxin-resistant hair cells limits the effectiveness of chemical-based methods to disrupt lateral line function. Furthermore, we show that repeated neomycin treatments can continually ablate functional lateral line hair cells between 3 and 4 dpf in larval zebrafish.

Mechanical overstimulation causes acute injury and synapse loss followed by fast recovery in lateral-line neuromasts of larval zebrafish

Excess noise damages sensory hair cells, resulting in loss of synaptic connections with auditory nerves and, in some cases, hair-cell death. The cellular mechanisms underlying mechanically induced hair-cell damage and subsequent repair are not completely understood. Hair cells in neuromasts of larval zebrafish are structurally and functionally comparable to mammalian hair cells but undergo robust regeneration following ototoxic damage. We therefore developed a model for mechanically induced hair-cell damage in this highly tractable system. Free swimming larvae exposed to strong water wave stimulus for 2 hr displayed mechanical injury to neuromasts, including afferent neurite retraction, damaged hair bundles, and reduced mechanotransduction. Synapse loss was observed in apparently intact exposed neuromasts, and this loss was exacerbated by inhibiting glutamate uptake. Mechanical damage also elicited an inflammatory response and macrophage recruitment. Remarkably, neuromast hair-cell morphology and mechanotransduction recovered within hours following exposure, suggesting severely damaged neuromasts undergo repair. Our results indicate functional changes and synapse loss in mechanically damaged lateral-line neuromasts that share key features of damage observed in noise-exposed mammalian ear. Yet, unlike the mammalian ear, mechanical damage to neuromasts is rapidly reversible.

Spawning substrate preference and spawning behavior of chocolate mahseer, Neolissochilus hexagonolepis

Captive breeding programs for Neolissochilus hexagonolepis are essential for population restoration. To develop an efficacious method for enhancing N. hexagonolepis spawning in captivity, there was examination of: (1) different types of spawning substrate and (2) area of spawning in the substrate. The study was conducted to describe spawning behavior of males and females. There was a choice of three substrates in which to spawn: gravel, small cobble, and coarse sand. There was preferential choosing of gravel followed by cobble with there being no use of sand for spawning. Behavior of N. hexagonolepis included preparation of a spawning pit by females, a behavior that has not been previously ascertained for cyprinids. Males expressed courting behaviors, including chasing, nudging, and quivering. Courting males expressed aggressive behaviors towards other males. Results from the present study are the first on the volitional spawning of N. hexagonolepis in captivity using spawning substrate. It was further revealed that using a gravel substrate tray would also be a feasible approach for egg production. Mean total eggs per female and mean fertilized eggs collected were less when there was siphoning used for egg collections in the preference study. Hence, stripping was implemented to increase the egg collection when spawning behaviors were observed. Total eggs collected were 40,540 with 3685 eggs per female, 90.3% fertilization rate, 82.8% hatching rate, and 97.4% free-swimming larvae survival rate. The implications of this study could be beneficial for enhancing the natural population through environmental management and developing a viable egg production technique in captivity.

Fine structure of a tiny gymnophalloid miracidium (Digenea).

We elucidate the morphology of the miracidia with passive strategy of infection. In contrast to the well-studied "active" free-swimming larvae (e.g., those of Schistosoma, Fasciola, Echinostoma), "passive" miracidia do not search for their hosts in the external environment. The infection occurs only after the mollusk ingests the eggs with the larvae. The miracidia of this type are extremely miniaturized organisms whose somatic elements are reduced compared to the "active" forms. The details of the structure are unknown for the majority of taxa with "passive" larvae. Here, we provide the first description of a gymnophalloid miracidium based on ultrastructural data. The larva of Parvatrema affinis Jameson & Nicoll, 1913 consists of 21 cells. Its nervous and excretory systems are reduced to the extreme level. Its penetration apparatus includes two crystalloid gland-cells, unique among digeneans. The "true" epithelium of its body wall is a novelty never described for any other miracidium. We compare the structure of gymnophalloid and bucephaloid miracidia trying to determine possible ancestral features of the larvae in this digenean lineage and trends of their evolution.

Current Methods to Investigate Nociception and Pain in Zebrafish.

Pain is an unpleasant, negative emotion and its debilitating effects are complex to manage. Mammalian models have long dominated research on nociception and pain, but there is increasing evidence for comparable processes in fish. The need to improve existing pain models for drug research and the obligation for 3R refinement of fish procedures facilitated the development of numerous new assays of nociception and pain in fish. The zebrafish is already a well-established animal model in many other research areas like toxicity testing, as model for diseases or regeneration and has great potential in pain research, too. Methods of electrophysiology, molecular biology, analysis of reflexive or non-reflexive behavior and fluorescent imaging are routinely applied but it is the combination of these tools what makes the zebrafish model so powerful. Simultaneously, observing complex behavior in free-swimming larvae, as well as their neuronal activity at the cellular level, opens new avenues for pain research. This review aims to supply a toolbox for researchers by summarizing current methods to study nociception and pain in zebrafish. We identify treatments with the best algogenic potential, be it chemical, thermal or electric stimuli and discuss options of analgesia to counter effects of nociception and pain by opioids, non-steroidal anti-inflammatory drugs (NSAIDs) or local anesthetics. In addition, we critically evaluate these practices, identify gaps of knowledge and outline potential future developments.