The agrochemical S-metolachlor disrupts molecular mediators and morphology of the swim bladder: Implications for locomotor activity in zebrafish (Danio rerio).

Metolachlor herbicides are derived from the chloroacetamide chemical family of which there are the S- and R-metolachlor isomers. S-metolachlor is a selective herbicide that inhibits cell division and mitosis via enzyme interference. The herbicide is used globally in agriculture and studies report adverse effects in aquatic organisms; however, there are no studies investigating sub-lethal effects of S-metolachlor on swim bladder formation, mitochondrial ATP production, nor light-dark preference behaviors in fish. These endpoints are relevant for larval locomotor activity and metabolism. To address these knowledge gaps, we exposed zebrafish embryos/larvae to various concentrations of S-metolachlor (0.5-50 µM) over early development. S-metolachlor affected survival, hatching percentage, and increased developmental deformities at concentrations of 50 µM and above. Exposure levels as high as 200 µM for 24 and 48 h did not alter oxygen consumption rates in zebrafish, and there were no changes detected in endpoints related to mitochondrial oxidative phosphorylation. We observed impairment of swim bladder inflation at 50 µM in 6 dpf larvae. To elucidate mechanisms related to this, we measured relative transcript abundance for genes associated with the swim bladder (smooth muscle alpha (α)-2 actin, annexin A5, pre-B-cell leukemia homeobox 1a). Smooth muscle alpha (α)-2 actin mRNA levels were reduced in fish exposed to 50 µM while annexin A5 mRNA levels were increased in abundance, corresponding to reduced swim bladder size in larvae. A visual motor response test revealed that larval zebrafish exhibited some hyperactivity in the light with exposure to the herbicide and only the highest dose tested (50 µM) resulted in hypoactivity in the dark cycle. Regression analysis indicated that there was a positive relationship between surface area of the swim bladder and distance traveled, and the size of the swim bladder explained ~10-14% in the variation for total distance moved. Lastly, we tested larvae in a light dark preference test, and we did not detect any altered behavioral response to any concentration tested. Here we present new data on sublethal endpoints associated with exposure to the herbicide S-metolachlor and demonstrate that this chemical may disrupt transcripts associated with swim bladder formation and morphology, which could ultimately affect larval zebrafish activity. These data are expected to contribute to further risk assessment guidelines for S-metolachlor in aquatic ecosystems.

The effects of exposure to crude oil or PAHs on fish swim bladder development and function.

The failure of the swim bladder to inflate during fish development is a common and sensitive response to exposure to petrochemicals. Here, we review potential mechanisms by which petrochemicals or their toxic components (polycyclic aromatic hydrocarbons; PAHs) may affect swim bladder inflation, particularly during early life stages. Surface films formed by oil can cause a physical barrier to primary inflation by air gulping, and are likely important during oil spills. The act of swimming to the surface for primary inflation can be arduous for some species, and may prevent inflation if this behavior is limited by toxic effects on vision or musculature. Some studies have noted altered gene expression in the swim bladder in response to PAHs, and Cytochrome P450 1A (CYP1A) can be induced in swim bladder or rete mirabile tissue, suggesting that PAHs can have direct effects on swim bladder development. Swim bladder inflation failure can also occur secondarily to the failure of other systems; cardiovascular impairment is the best elucidated of these mechanisms, but other mechanisms might include non-inflation as a sequela of disruption to thyroid signaling or cholesterol metabolism. Failed swim bladder inflation has the potential to lead to chronic sublethal effects that are as yet unstudied.

Histological development and integration of the Zebrafish Weberian apparatus.

BACKGROUND: The Weberian apparatus enhances hearing in otophysan fishes, including Zebrafish (Danio rerio). Several studies have examined aspects of morphological development of the Weberian apparatus and hearing ability in Zebrafish. A comprehensive developmental description including both hard and soft tissues is lacking. This information is critical for both interpretation of genetic developmental analyses and to better understand the role of morphogenesis and integration on changes in hearing ability. RESULTS: Histological development of hard and soft tissues of the Weberian apparatus, including ossicles, ear, swim bladder, and ligaments are described from early larval stages (3.8 mm notochord length) through adult. Results show a strong relationship in developmental timing and maturation across all regions. All required auditory elements are present and morphologically integrated early, by 6.5 mm SL. Dynamic ossification patterns and changes in shape continue throughout the examined developmental period. CONCLUSIONS: This study provides the first comprehensive histological description of Weberian apparatus development in Zebrafish. Morphological integration was found early, before increases in hearing ability were detected in functional studies (>10 mm total length), suggesting morphological integration precedes functional integration. Further research is needed to examine the nature of the functional delay, and how maturation of the Weberian apparatus influences functionality.

The synthetic pyrethroid deltamethrin impairs zebrafish (Danio rerio) swim bladder development.

Deltamethrin (DM) is a widely used insecticide and reveals neural, cardiovascular and reproductive toxicity to various aquatic organisms. It has been known that DM negatively affects motion of zebrafish (Danio rerio). However, little is known in relation to the impacts of DM on development of swim bladder, which is a key organ for motion. In the present study, zebrafish embryos were exposed to 20 and 40 µg/L DM. The changes of swim bladder morphology were observed and transcription levels of key genes were compared between DM treatments and the control. The results showed that DM treatments significantly blocked the formation of progenitor and tissue layers in swim bladder of zebrafish embryos, leading to failed inflation of swim bladder. Compared with the control, the key genes (pbx1, foxA3, mnx1, has2, anxa5b, hprt1l and elovl1a) responsible for swim bladder development also showed decreased levels in response to DM treatments, suggesting that DM might specifically affect swim bladder development. Moreover, transcription levels of genes in the Wnt (wnt5b, tcf3a, wnt1, wnt9b, fzd1, fzd3 and fzd5) and Hedgehog (ihhb, ptc1 and ptc2) signaling pathways all decreased significantly in response to DM treatments, compared with the control. Considering the importance of Wnt and Hedgehog pathways in development of swim bladder, these results suggested that DM might affect swim bladder development through inhibiting the Wnt and Hedgehog pathways. Overall, the present study reported that swim bladder might be a potential target organ of DM toxicity in zebrafish, which contributed more information to the evaluation of DM's environmental risks.

Slc25a17 acts as a peroxisomal coenzyme A transporter and regulates multiorgan development in zebrafish.

Slc25a17 is known as a peroxisomal solute carrier, but the in vivo role of the protein has not been demonstrated. We found that the zebrafish genome contains two slc25a17 genes that function redundantly, but additively. Notably, peroxisome function in slc25a17 knockdown embryos is severely compromised, resulting in an altered lipid composition. Along the defects found in peroxisome-associated phenotypic presentations, we highlighted that development of the swim bladder is also highly dependent on Slc25a17 function. As Slc25a17 showed substrate specificity towards coenzyme A (CoA), injecting CoA, but not NAD+ , rescued the defective swim bladder induced by slc25a17 knockdown. These results indicated that Slc25a17 acts as a CoA transporter, involved in the maintenance of functional peroxisomes that are essential for the development of multiple organs during zebrafish embryogenesis. Given high homology in protein sequences, the role of zebrafish Slc25a17 may also be applicable to the mammalian system.

JAK/STAT signaling is involved in air sac primordium development of Drosophila melanogaster.

The dorsal thoracic air sacs in fruit flies (Drosophila melanogaster) are functionally and developmentally comparable to human lungs. The progenitors of these structures, air sac primordia (ASPs), invasively propagate into wing imaginal disks, employing mechanisms similar to those that promote metastasis in malignant tumors. We investigated whether Janus kinase/signal transducer and activator of transcription JAK/STAT signaling plays a role in the directed morphogenesis of ASPs. We find that JAK/STAT signaling occurs in ASP tip cells and misexpression of core components in the JAK/STAT signaling cascade significantly impedes ASP development. We further identify Upd2 as an activating ligand for JAK/STAT activity in the ASP. Together, these data constitute a considerable step forward in understanding the role of JAK/STAT signaling in ASPs and similar structures in mammalian models.

The Air Sac Primordium of Drosophila: A Model for Invasive Development.

The acquisition of invasive properties preceding tumor metastasis is critical for cancer progression. This phenomenon may result from mutagenic disruption of typical cell function, but recent evidence suggests that cancer cells frequently co-opt normal developmental programs to facilitate invasion as well. The signaling cascades that have been implicated present an obstacle to identifying effective therapeutic targets because of their complex nature and modulatory capacity through crosstalk with other pathways. Substantial efforts have been made to study invasive behavior during organogenesis in several organisms, but another model found in Drosophilamelanogaster has not been thoroughly explored. The air sac primordium (ASP) appears to be a suitable candidate for investigating the genes and morphogens required for invasion due to the distinct overlap in the events that occur during its normal growth and the development of metastatic tumor cells. Among these events are the conversion of larval cells in the trachea into a population of mitotically active cells, reduced cell⁻cell contact along the leading edge of the ASP, and remodeling of the extracellular matrix (ECM) that surrounds the structure. Here, we summarize the development of ASPs and invasive behavior observed therein.

Ontogeny and morphometrics of the gills and swim bladder of air-breathing striped catfish Pangasianodon hypophthalmus.

The air-breathing fish Pangasianodon hypophthalmus has been shown to have highly plastic branchial surfaces whose area (SA) increases with temperature and aquatic hypoxia. This modulation occurs through development of inter-lamellar cell mass (ILCM). Paradoxically, in conditions where this fish has been shown capable of covering its entire aerobic scope from the water phase, it has been shown to have a very small branchial SA. To address this paradox, we measured the SA, harmonic mean diffusion distance (τh) and calculated the anatomic diffusion factor (ADF) of the branchial and swim bladder surfaces in fish ranging from 3 to 1900 g at 27°C in normoxia. Since the lamellae were distinguishable from the ILCM, we measured the actual SA as well as the potential SA if ILCM were lost. As a result of low τh, P. hypophthalmus has a high capacity for branchial oxygen uptake with or without ILCM. Actual and potential gill ADF were 361 and 1002 cm2 µm-1 kg-1, respectively, for a 100 g fish and the ADF of the swim bladder was found to be 308 cm2 µm-1 kg-1 By swimming fish to exhaustion at different temperatures, we show that modulation of this SA is rapid, indicating that the apparent paradox between previous studies is eliminated. Regression analysis of log-log plots of respiratory SA in relation to body mass shows that the gill scales with mass similarly to the SA in active water-breathing fish, whereas the swim bladder scales with mass more like the mammalian lung does. This fish presents a combination of respiratory surfaces not previously seen in air-breathing fish.

The Drosophila DOCK family protein Sponge is required for development of the air sac primordium.

Dedicator of cytokinesis (DOCK) family genes are known as DOCK1-DOCK11 in mammals. DOCK family proteins mainly regulate actin filament polymerization and/or depolymerization and are GEF proteins, which contribute to cellular signaling events by activating small G proteins. Sponge (Spg) is a Drosophila counterpart to mammalian DOCK3/DOCK4, and plays a role in embryonic central nervous system development, R7 photoreceptor cell differentiation, and adult thorax development. In order to conduct further functional analyses on Spg in vivo, we examined its localization in third instar larval wing imaginal discs. Immunostaining with purified anti-Spg IgG revealed that Spg mainly localized in the air sac primordium (ASP) in wing imaginal discs. Spg is therefore predicted to play an important role in the ASP. The specific knockdown of Spg by the breathless-GAL4 driver in tracheal cells induced lethality accompanied with a defect in ASP development and the induction of apoptosis. The monitoring of ERK signaling activity in wing imaginal discs by immunostaining with anti-diphospho-ERK IgG revealed reductions in the ERK signal cascade in Spg knockdown clones. Furthermore, the overexpression of D-raf suppressed defects in survival and the proliferation of cells in the ASP induced by the knockdown of Spg. Collectively, these results indicate that Spg plays a critical role in ASP development and tracheal cell viability that is mediated by the ERK signaling pathway.

Cells must express components of the planar cell polarity system and extracellular matrix to support cytonemes.

Drosophila dorsal air sac development depends on Decapentaplegic (Dpp) and Fibroblast growth factor (FGF) proteins produced by the wing imaginal disc and transported by cytonemes to the air sac primordium (ASP). Dpp and FGF signaling in the ASP was dependent on components of the planar cell polarity (PCP) system in the disc, and neither Dpp- nor FGF-receiving cytonemes extended over mutant disc cells that lacked them. ASP cytonemes normally navigate through extracellular matrix (ECM) composed of collagen, laminin, Dally and Dally-like (Dlp) proteins that are stratified in layers over the disc cells. However, ECM over PCP mutant cells had reduced levels of laminin, Dally and Dlp, and whereas Dpp-receiving ASP cytonemes navigated in the Dally layer and required Dally (but not Dlp), FGF-receiving ASP cytonemes navigated in the Dlp layer, requiring Dlp (but not Dally). These findings suggest that cytonemes interact directly and specifically with proteins in the stratified ECM.

The Effect of Hypoxia and Hyperoxia on Growth and Expression of Hypoxia-Related Genes and Proteins in Spotted Gar Lepisosteus oculatus Larvae and Juveniles.

We studied the molecular responses to different water oxygen levels in gills and swim bladder of spotted gar (Lepisosteus oculatus), a bimodal breather. Fish at swim-up stage were exposed for 71 days to normoxic, hypoxic, and hyperoxic water conditions. Then, all aquaria were switched to normoxic conditions for recovery until the end of the experiment (120 days). Fish were sampled at the beginning of the experiment, and then at 71 days of exposure and at 8 days of recovery. We first cloned three hypoxia-related genes, hypoxia-inducible factor 2α (HIF-2α), Na(+) /H(+) exchanger 1 (NHE-1), and NHE-3, and uploaded their cDNA sequences in the GeneBank database. We then used One Step Taqman® real-time PCR to quantify the mRNA copies of target genes in gills and swim bladder of fish exposed to different water O2 levels. We also determined the protein expression of HIF-2α and neuronal nitric oxide synthase (nNOS) in the swim bladder by using confocal immunofluorescence. Hypoxic stress for 71 days significantly increased the mRNA copies of HIF-2α and NHE-1 in gills and swim bladder, whereas normoxic recovery for 8 days decreased the HIF-2α mRNA copies to control values in both tissues. We did not found significant changes in mRNA copies of the NHE-3 gene in either gills or swim bladder in response to hypoxia and hyperoxia. Unlike in normoxic swim bladder, double immunohistochemical staining in hypoxic and hyperoxic swim bladder using nNOS/HIF-2α showed extensive bundles of HIF-2α-positive nerve fibers in the trabecular musculature associated with a few varicose nNOS immunoreactive nerve terminals.

FGF coordinates air sac development by activation of the EGF ligand Vein through the transcription factor PntP2.

How several signaling pathways are coordinated to generate complex organs through regulation of tissue growth and patterning is a fundamental question in developmental biology. The larval trachea of Drosophila is composed of differentiated functional cells and groups of imaginal tracheoblasts that build the adult trachea during metamorphosis. Air sac primordium cells (ASP) are tracheal imaginal cells that form the dorsal air sacs that supply oxygen to the flight muscles of the Drosophila adult. The ASP emerges from the tracheal branch that connects to the wing disc by the activation of both Bnl-FGF/Btl and EGFR signaling pathways. Together, these pathways promote cell migration and proliferation. In this study we demonstrate that Vein (vn) is the EGF ligand responsible for the activation of the EGFR pathway in the ASP. We also find that the Bnl-FGF/Btl pathway regulates the expression of vn through the transcription factor PointedP2 (PntP2). Furthermore, we show that the FGF target gene escargot (esg) attenuates EGFR signaling at the tip cells of the developing ASP, reducing their mitotic rate to allow proper migration. Altogether, our results reveal a link between Bnl-FGF/Btl and EGFR signaling and provide novel insight into how the crosstalk of these pathways regulates migration and growth.

A Cathepsin-L is required for invasive behavior during Air Sac Primordium development in Drosophila melanogaster.

The Drosophila Air Sac Primordium (ASP) has emerged as an important structure where cellular, genetic and molecular events responsible for invasive behavior and branching morphogenesis can be studied. In this report we present data which demonstrate that a Cathepsin-L encoded by the gene CP1 in Drosophila is necessary for invasive behavior during ASP development. We find that CP1 is expressed in ASP and knockdown of CP1 results in suppression of migratory and invasive behavior observed during ASP development. We further show that CP1 possibly regulates invasive behavior by promoting degradation of Basement Membrane. Our data provide clues to the possible role of Cathepsin L in human lung development and tumor invasion, especially, given the similarities between human lung and Drosophila ASP development.

Myoblast cytonemes mediate Wg signaling from the wing imaginal disc and Delta-Notch signaling to the air sac primordium.

The flight muscles, dorsal air sacs, wing blades, and thoracic cuticle of the Drosophila adult function in concert, and their progenitor cells develop together in the wing imaginal disc. The wing disc orchestrates dorsal air sac development by producing decapentaplegic and fibroblast growth factor that travel via specific cytonemes in order to signal to the air sac primordium (ASP). Here, we report that cytonemes also link flight muscle progenitors (myoblasts) to disc cells and to the ASP, enabling myoblasts to relay signaling between the disc and the ASP. Frizzled (Fz)-containing myoblast cytonemes take up Wingless (Wg) from the disc, and Delta (Dl)-containing myoblast cytonemes contribute to Notch activation in the ASP. Wg signaling negatively regulates Dl expression in the myoblasts. These results reveal an essential role for cytonemes in Wg and Notch signaling and for a signal relay system in the myoblasts.

Development of seahorse (Hippocampus reidi, Ginsburg 1933): histological and histochemical study.

Biological aspects and global demand for aquarium promote seahorses as new species with high potential for commercial purposes; however, the low newborn survival rate represents the main bottleneck of seahorses farming. In this study, the organogenesis of the Hippocampus reidi was analysed from release until the 30th day after birth, using histological and histochemical approaches. To study the stages of their early life, 360 individuals were killed, sectioned, and stained with haematoxylin and eosin, periodic acid-Schiff, and Sudan Black B techniques. At birth, mouth and anus were open, the swim bladder inflated, and the visual system highly developed. Among the results, it was emphasized the presence of the yolk sac until the 2nd day after birth, the loops of the intestine to accommodate its elongation, and the ability of the larvae to absorb lipids in the anterior and posterior tract of the intestine. A short time (7/8 days) between reabsorption of yolk sac and formation of gonads was registered, with primordial follicles visible from the 10th day after birth. For the first time, organogenesis in H. reidi was described in detail; seahorses underwent a marked metamorphosis, and the indirect development observed in this species lead up to reconsider the term "juvenile" used for H. reidi during this period.

Ontogeny of body density and the swimbladder in yellowtail kingfish Seriola lalandi larvae.

The ontogeny of larval body density and the morphological and histological events during swimbladder development were investigated in two cohorts of yellowtail kingfish Seriola lalandi larvae to understand the relationship between larval morphology and body density. Larvae <3 days post hatch (dph) were positively buoyant with a mean ± s.d. body density of 1.023 ± 0.001 g cm(-3). Histological evidence demonstrated that S. lalandi larvae are initially transient physostomes with the primordial swimbladder derived from the evagination of the gut ventral to the notochord and seen at 2 dph. A pneumatic duct connected the swimbladder to the oesophagus, but degenerated after 5 dph. Initial swimbladder (SB) inflation occurred on 3 dph, and the inflation window was 3-5 dph when the pneumatic duct was still connected to the gut. The swimbladder volume increased with larval age and the epithelial lining on the swimbladder became flattened squamous cells after initial inflation. Seriola lalandi developed into a physoclist with the formation of the rete mirabile and the gas-secreting gland comprised low-columnar epithelial cells. Larvae with successfully inflated swimbladders remained positively buoyant, whereas larvae without SB inflation became negatively buoyant and their body density gradually reached 1.030 ± 0.001 g cm(-3) by 10 dph. Diel density changes were observed after 5 dph, owing to day time deflation and night-time inflation of the swimbladder. These results show that SB inflation has a direct effect on body density in larval S. lalandi and environmental factors should be further investigated to enhance the rate of SB inflation to prevent the sinking death syndrome in the early life stage of the fish larvae.

Expression of a lung developmental cassette in the adult and developing zebrafish swimbladder.

The presence of an air-filled organ (AO), either lungs or a swimbladder, is a defining character of the Osteichthyes (bony vertebrates, including tetrapods). Despite the functional and structural diversity of AOs, it was not previously known whether the same group of developmental regulatory genes are involved in the early development of both lungs and swimbladders. This study demonstrates that a suite of genes (Nkx2.1, FoxA2, Wnt7b, GATA6), previously reported to be co-expressed only in the tetrapod lung, is also co-expressed in the zebrafish swimbladder. We document the expression pattern of these genes in the adult and developing zebrafish swimbladder and compare the expression patterns to those in the mouse lung. Early-acting genes involved in endoderm specification are expressed in the same relative location and stage of AO development in both taxa (FoxA2 and GATA6), but the order of onset and location of expression are not completely conserved for the later acting genes (Nkx2.1 and Wnt7b). Co-expression of this suite of genes in both tetrapod lungs and swimbladders of ray-finned fishes is more likely due to common ancestry than independent co-option, because these genes are not known to be co-expressed anywhere except in the AOs of Osteichthyes. Any conserved gene product interactions may comprise a character identity network (ChIN) for the osteichthyan AO.

Early development of New Zealand hapuku Polyprion oxygeneios eggs and larvae.

This study describes for the first time the normal development of New Zealand hapuku Polyprion oxygeneios embryos and larvae reared from fertilization to 11 days post-hatch (dph) at a constant temperature. Fertilized eggs were obtained from natural spawnings from communally reared captive wild broodstock. Eggs averaged 2 mm in diameter and had single or multiple oil globules. Embryos developed following the main fish embryological stages and required an average of 1859·50 degree hours post-fertilization (dhpf) to hatch. The newly hatched larvae (4·86 mm mean total length, L(T) ) were undifferentiated, with unpigmented eyes, a single and simple alimentary tube and a finfold that covered the entire body. Larvae relied on the energy from the yolk-sac reserves until 11 dph (7·33 mm mean L(T) ), when yolk-sac reabsorption was almost completed. Some of the major developmental stages from hatching to yolk-sac reabsorption were eye pigmentation (5 dph), upper jaw formation (7 dph), lower jaw formation (8 dph) and mouth opening (8-9 dph). By 9 dph, the digestive system consisted of pancreas, liver, primordial stomach, anterior and posterior gut; therefore, P. oxygeneios larvae would be capable of feeding on live prey. The developmental, morphological and histological data described constitutes essential baseline information on P. oxygeneios biology and normal development.

Knock-down of cathepsin D affects the retinal pigment epithelium, impairs swim-bladder ontogenesis and causes premature death in zebrafish.

The lysosomal aspartic protease Cathepsin D (CD) is ubiquitously expressed in eukaryotic organisms. CD activity is essential to accomplish the acid-dependent extensive or partial proteolysis of protein substrates within endosomal and lysosomal compartments therein delivered via endocytosis, phagocytosis or autophagocytosis. CD may also act at physiological pH on small-size substrates in the cytosol and in the extracellular milieu. Mouse and fruit fly CD knock-out models have highlighted the multi-pathophysiological roles of CD in tissue homeostasis and organ development. Here we report the first phenotypic description of the lack of CD expression during zebrafish (Danio rerio) development obtained by morpholino-mediated knock-down of CD mRNA. Since the un-fertilized eggs were shown to be supplied with maternal CD mRNA, only a morpholino targeting a sequence containing the starting ATG codon was effective. The main phenotypic alterations produced by CD knock-down in zebrafish were: 1. abnormal development of the eye and of retinal pigment epithelium; 2. absence of the swim-bladder; 3. skin hyper-pigmentation; 4. reduced growth and premature death. Rescue experiments confirmed the involvement of CD in the developmental processes leading to these phenotypic alterations. Our findings add to the list of CD functions in organ development and patho-physiology in vertebrates.

Effects of simulated microgravity on the development of the swimbladder and buoyancy control in larval zebrafish (Danio rerio).

The gas-filled swimbladder of teleost fishes provides hydrodynamic lift which counteracts the high density of other body tissues, and thereby allows the fish to achieve neutral buoyancy with minimal energy expenditure. In this study, we examined whether the absence of a constant direction gravitational vector affects the ontogeny of the swimbladder and buoyancy control in zebrafish (Danio rerio). We exposed fertilized eggs to simulated microgravity (SMG) in a closed rotating wall vessel with control eggs placed in a similar but nonrotating container. All eggs hatched in both groups. At 96 hr of postfertilization (hpf), all larvae were removed from the experimental and control vessels. At this point, 62% of the control larvae, but only 14% of SMG-exposed larvae, were observed to have inflated their swimbladder. In addition, the mean volume of the inflated swimbladders was significantly greater in the control larvae compared with larvae raised in SMG. After transfer to open stationary observation tanks, larvae with uninflated swimbladders in both groups swam to the surface to complete inflation, but this process was significantly delayed in larvae exposed to SMG. Initial differences in swimbladder inflation and volume between groups disappeared by 144 hpf. Furthermore, there were no apparent changes in patterns of development and maturation of swimbladder musculature, vasculature, or innervation resulting from SMG exposure at later stages of ontogeny. These data indicate that, despite a transient delay in swimbladder inflation in zebrafish larvae exposed to SMG, subsequent swimbladder development in these animals proceeded similarly to that in normal larvae.