Divergent requirements for fibroblast growth factor signaling in zebrafish maxillary barbel and caudal fin regeneration

The zebrafish maxillary barbel is an integumentary organ containing skin, glands, pigment cells, taste buds, nerves, and endothelial vessels. The maxillary barbel can regenerate (LeClair & Topczewski 2010); however, little is known about its molecular regulation. We have studied fibroblast growth factor (FGF) pathway molecules during barbel regeneration, comparing this system to a well‐known regenerating appendage, the zebrafish caudal fin. Multiple FGF ligands (fgf20a, fgf24), receptors (fgfr1‐4) and downstream targets (pea3, il17d) are expressed in normal and regenerating barbel tissue, confirming FGF activation. To test if specific FGF pathways were required for barbel regeneration, we performed simultaneous barbel and caudal fin amputations in two temperature‐dependent zebrafish lines. Zebrafish homozygous for a point mutation in fgf20a, a factor essential for caudal fin blastema formation, regrew maxillary barbels normally, indicating that the requirement for this ligand is appendage‐specific. Global overexpression of a dominant negative FGF receptor, Tg(hsp70l:dn‐fgfr1:EGFP)^pd1 completely blocked fin outgrowth but only partially inhibited barbel outgrowth, suggesting reduced requirements for FGFs in barbel tissue. Maxillary barbels expressing dn‐fgfr1 regenerated peripheral nerves, dermal connective tissue, endothelial tubes, and a glandular epithelium; in contrast to a recent report in which dn‐fgfr1 overexpression blocks pharyngeal taste bud formation in zebrafish larvae (Kapsimali et al. 2011), we observed robust formation of calretinin‐positive tastebuds. These are the first experiments to explore the molecular mechanisms of maxillary barbel regeneration. Our results suggest heterogeneous requirements for FGF signaling in the regeneration of different zebrafish appendages (caudal fin versus maxillary barbel) and taste buds of different embryonic origin (pharyngeal endoderm versus barbel ectoderm).     

Effects of a flow-gauging weir on the migratory behaviour of adult barbel, a riverine cyprinid

Barbel Barbus barbus exhibited substantial movements in the River Nidd between March and July 1994, with some individuals moving nearly 20 km upstream, although there was considerable individual variation. Most upstream movement occurred during May. Fifteen of the 23 fish tracked attempted to pass the weir, and of these six were successful. Barbel tended to approach the weir around dusk and dawn, reflecting observed patterns of localized activity, or at night, but crossed the weir only at night. Those fish which crossed the weir moved substantial distances upstream to spawn, while those that were unsuccessful moved down-stream. The weir delayed the net upstream progress of all adult barbel. Successful traversal was not closely related to flow or water temperature conditions. The study emphasizes the impact of relatively minor obstructions on the natural migrations of barbel.     

Bibarba bibarba: A new genus and species of Cobitinae (Pisces: Cypriniformes: Cobitidae) from Guangxi Province (China)

A new genus of Cobitinae, Bibarba gen. n., and a new species, B. bibarba sp. n., were discovered and are described for the Chengjiang River, a tributary of the Hongshuihe River in Guangxi Province of southern China. This river region is characterized by a Karst landscape, and the river that is inhabited by the new genus is a slowly moving stream with arenaceous and cobblestone beds. The new genus resembles Cobitis Linnaeus, 1758 (subfamily Cobitinae) in the shape and pigmentation pattern of their body, the absence of scales on their head, and the presence of a suborbital spine, but differs from it by a single Lamina circularis on the third pectoral fin ray instead of on the base of the second pectoral fin ray; two pairs of barbels (one rostral pair and one maxillo-mandibular pair) instead of three pairs of barbels (one rostral pair, one maxillary pair, and one maxillo-mandibular pair); a relatively thick and short suborbital spine with a strong medio-lateral process instead of a suborbital spine without or with a weakly formed medio-lateral process as in Cobitis; and the lack of a black stripe extending from the occiput through the eye to the insertion of the rostral barbel. The first two characters have not been reported in any other genus of the subfamily Cobitinae. A morphometric character analysis based on PCA reveals differences between B. bibarba and C. sinensis in body size, barbel length, interorbital width, pectoral fin length in males, and the position of the dorsal and ventral fins. Type specimens of the new species are kept in the Freshwater Fishes Museum of the Institute of Hydrobiology at the Chinese Academy of Sciences in Wuhan, Hubei Province.     

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.     

Regulation of hypocretin (orexin) expression in embryonic zebrafish

Hypocretins/orexins are neuropeptides involved in the regulation of sleep and energy balance in mammals. Conservation of gene sequence, hypothalamic localization of cell bodies, and projection patterns in adult zebrafish suggest that the architecture and function of the hypocretin system are conserved in fish. We report on the complete genomic structure of the zebrafish and Tetraodon hypocretin genes and the complete predicted hypocretin protein sequences from five teleosts. Using whole mount in situ hybridization, we have traced the development of hypocretin cells in zebrafish from onset of expression at 22 h post-fertilization through the first week of development. Promoter elements of similar size from zebrafish and Tetraodon were capable of driving efficient and specific expression of enhanced green fluorescent protein in developing zebrafish embryos, thus defining a minimal promoter region able to accurately mimic the native hypocretin pattern. This enhanced green fluorescent protein expression also revealed a complex pattern of projections within the hypothalamus, to the midbrain, and to the spinal cord. To further analyze the promoter, a series of deletion and substitution constructs were injected into embryos, and resulting promoter activity was monitored in the first week of development. A critical region of 250 base pairs was identified containing a core 13-base pair element essential for hypocretin expression.     

Seasonal Shifts in Day-Time Resource Use of 0+ Barbel, Barbus Barbus

Habitat preference and diet of 0+ barbel were studied both on a meso- and a microhabitat scale in the River Sieg (Germany) between May 1993 and January 1995. Changes in mesohabitat use were observed. Barbel moved from shallow bays (larvae and step 1 juveniles) to gravel banks, and subsequently, to riffles (step 2 juveniles). Size-dependent shifts in microhabitat-use were observed during the second juvenile step. These juveniles left the shoreline and preferred microhabitats with stronger current velocities. 0+ barbel in riffles fed on Chironomidae and Ephemeroptera. No remarkable shifts in diet were detected between larvae and juveniles. We suggest that the observed ontogenetic shifts to habitats with high food supply and low predation pressure might contribute to the high abundances of barbel in the River Sieg.     

Analysis of mouthpart movements during feeding of Frankliniella occidentalis (pergande) and F. schultzei trybom (Thysanoptera : Thripidae)

Scanning electron microscopy was used to elucidate the morphology and sequential movement of thrips feeding structures in the 2 species, Frankliniella occidentalis and F. schultzei, (Thysanoptera : Thripidae). The mouthcone consists of paired paraglossae, and fringed labral pad, a single, apically fused mandible and a pair of interlocking maxillae. The maxillae are open apically and form a feeding tube. Ten pairs of sensory pegs of 3 distinct morphological types (sensilla basiconica with a cuticular collar, sensilla basiconica without a cuticular collar, and sensilla trichoidea) were found on the paraglossae. The possible function of these sensory structures in host finding and choice are discussed. No structure for rasping the leaf's surface was found on the mouthcone or the ventral surface of the insect. Live specimens were observed feeding through Parafilm on artificial media (sucrose solution) and lettuce leaf tissue. These observations support earlier findings that thrips feed by piercing leaf cells with the mandible and ingesting cell contents through the feeding tube formed by the maxillary stylets. Based on these findings, we suggest that thrips be classified as piercing-sucking rather than rasping-sucking insects.     

Morphology of the immature stages of Adoretus tenuimaculatus Waterhouse, 1875 (Coleoptera: Scarabaeidae: Rutelinae: Adoretini)

The immature stages of the brown chafers Adoretus tenuimaculatus Waterhouse, 1875 were investigated morphologically using scanning electron microscopy. The scarabaeiform larvae of A. tenuimaculatus are peculiar for bearing a pair of eye spots on the head capsule, devoid of palidium but numerous hamate setae on the raster. The larvae possess a crown-like apical sensory area on each antenna, maxillary and labial palp. The pupa possesses dorsally six pairs of dioneiform organs on the intersegment regions between the first seven abdominal segments. This study provides the first ultra morphological study of the larvae in Adoretini. Both advantages and disadvantages of the scanning electron microscopy in the larval taxonomy of Scarabaeidae were briefly discussed.     

Feeding ecology of the Iberian cyprinid Barbus bocagei Steindachner, 1865 in a lowland river

Patterns of food resource utilization by Barbus bocagei in a lowland river in the central part of the Iberian Peninsula are presented and discussed. Focal points include feeding activity, diet composition and seasonal and ontogenetic diet shifts. Striking features of overall feeding pattern of barbel are its continuous feeding activity, bottom habits, high consumption of detritus and dipteran larvae and narrow diet breadth. Seasonal changes in food resource availability elicited changes in food utilization patterns, revealing a trend to more generalist feeding during spring. Barbel increase consumption of plant material and large prey during ontogeny, but overlap in prey resource utilization between size classes is high. The versatility of barbel foraging behaviour may be an important feature in its exploitation of Iberian lotic systems.     

Aminoglycoside-Induced Hair Cell Death of Inner Ear Organs Causes Functional Deficits in Adult Zebrafish (Danio rerio)

Aminoglycoside antibiotics, like gentamicin, kill inner ear sensory hair cells in a variety of species including chickens, mice, and humans. The zebrafish (Danio rerio) has been used to study hair cell cytotoxicity in the lateral line organs of larval and adult animals. Little is known about whether aminoglycosides kill the hair cells within the inner ear of adult zebrafish. We report here the ototoxic effects of gentamicin on hair cells in the saccule, the putative hearing organ, and utricle of zebrafish. First, adult zebrafish received a single 30 mg/kg intraperitoneal injection of fluorescently-tagged gentamicin (GTTR) to determine the distribution of gentamicin within inner ear sensory epithelia. After 4 hours, GTTR was observed in hair cells throughout the saccular and utriclar sensory epithelia. To assess the ototoxic effects of gentamicin, adult zebrafish received a single 250 mg/kg intraperitoneal injection of gentamicin and, 24 hours later, auditory evoked potential recordings (AEPs) revealed significant shifts in auditory thresholds compared to untreated controls. Zebrafish were then euthanized, the inner ear fixed, and labeled for apoptotic cells (TUNEL reaction), and the stereociliary bundles of hair cells labeled with fluorescently-tagged phalloidin. Whole mounts of the saccule and utricle were imaged and cells counted. There were significantly more TUNEL-labeled cells found in both organs 4 hours after gentamicin injection compared to vehicle-injected controls. As expected, significantly fewer hair cell bundles were found along the rostral-caudal axis of the saccule and in the extrastriolar and striolar regions of the utricle in gentamicin-treated animals compared to untreated controls. Therefore, as in other species, gentamicin causes significant inner ear sensory hair cell death and auditory dysfunction in zebrafish.     

Zebrafish type XVII collagen: Gene structures,expression profiles,and morpholino “knock-down” phenotypes

The human COL17A1 gene encodes type XVII collagen (also known as the 180-kDa bullous pemphigoid antigen), an integral component of hemidesmosomes, attachment complexes providing integrity to the dermal–epidermal junction. Zebrafish, a useful model system to study skin development, displays fully developed hemidesmosomes at approximately 5 days post-fertilization (dpf). We have identified two COL17A1 orthologues in the zebrafish genome, col17a1a and col17a1b, which are expressed in the skin and the neural system, respectively. The proteins coded by these genes have structural module organizations homologous to the human type XVII collagen. “Knock-down” of the expression of col17a1a with a specific morpholino targeting the 5′ UTR of the gene resulted in a blistering phenotype and in perturbations in the basement membrane zone. “Knock-down” of col17a1b expression resulted in ablation or in marked reduction of neuromasts in the lateral line. Thus, zebrafish has two COL17A1 orthologues which may have evolved tissue-specific functions during vertebrate development. Collectively, zebrafish provides a model system to study the molecular aspects of skin development and offers insights into the corresponding human diseases.     

The 'goatee' of goatfish: innervation of taste buds in the barbels and their representation in the brain

Goatfish use a pair of large chin barbels to probe the sea bottom to detect buried prey. The barbels are studded with taste buds but little else is known about the neural organization of this system. We found that the taste buds of the barbel are innervated in a strict orthogonal fashion. The barbel is innervated by a main nerve trunk running in the core of the barbel. A longitudinal nerve bundle originates from the main trunk and, after running a short distance distally, divides into two circumferential nerve bundles (CNB) extending respectively, medially and laterally around the barbel. Approximately 15 CNBs innervate each 1 mm length of barbel. At each transverse level, the CNB innervates two clusters of taste buds, each containing 14 end-organs. The primary taste centre in the brain is similarly extraordinary. The sensory inputs from the barbel terminate in a derived dorsal facial lobe, which has a highly convoluted surface forming a multitude of tubercles. Electrophysiological mapping experiments show that the entire barbel is somatotopically represented in a recurved elongate tubular fashion within the dorsal facial lobe.     

Embryonic substances induce alarm response in adult zebrafish (Danio rerio)

Many aquatic animals rely on chemicals released by injured individuals of the same species to assess predation risk. Among these chemical cues, alarm substances released from the injured skin of ostariophysan fishes have been extensively examined. In most fish species examined, these cues appear to be released by all injured individuals (including larvae, juveniles and adults) and elicit alarm responses in conspecifics. Adult alarm cues also affect development and physiology of embryos. Nonetheless, whether embryos produce alarm cues that affect adults is not known. This study reports that extracts of zebrafish (Danio rerio) embryos at 36 h post-fertilization or later induce antipredator behaviours reminiscent of those induced by skin alarm substances. At an equivalent of 10⁻⁶ g embryo per millilitre, the extract induced bottom-dwelling and freezing in adults. These behaviours are consistent with those induced by adult alarm substances. This study concludes that zebrafish embryos produce alarm substances.     

The annual cycle of intraovarian egg size and fecundity of the barbel Barbus barbus in the River Severn

SUMMARY. The annual cycle of intraovarian egg size in 124 mature barbel caught during a 3-year period was investigated. Storage methods, subsampling and measuring procedures were assessed and found to be reliable. Estimates of fecundity were made on thirty-two barbel containing developing ripening eggs. A new egg separation and counting technique was used modified from the volumetric ratio method described by Bagenal & Braum (1968). Fecundity estimates were found to be accurate by comparison with manual counts. Barbel ovaries contained two egg groups throughout the year. A third large, ripening egg group developed prior to spawning. The diameter of the ripening eggs, measured in arbitrary optical units, was significantly correlated with fork length. Large fish spawned bigger eggs than small fish. Fecundity was logarithmically related to fork length and ovary weight and linearly related to somatic weight. The mean diameter of the ripening intraovarian egg group was also logarithmically related to ovary weight. Heavier ovaries contained proportionally fewer, but larger, ripening eggs than small ovaries.     

Antibiotic Toxicity and Absorption in Zebrafish Using Liquid Chromatography-Tandem Mass Spectrometry

Evaluation of drug toxicity is necessary for drug safety, but in vivo drug absorption is varied; therefore, a rapid, sensitive and reliable method for measuring drugs is needed. Zebrafish are acceptable drug toxicity screening models; we used these animals with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring mode to quantify drug uptake in zebrafish to better estimate drug toxicity. Analytes were recovered from zebrafish homogenate by collecting supernatant. Measurements were confirmed for drugs in the range of 10–1,000 ng/mL. Four antibiotics with different polarities were tested to explore any correlation of drug polarity, absorption, and toxicity. Zebrafish at 3 days post-fertilization (dpf) absorbed more drug than those at 6 h post-fertilization (hpf), and different developmental periods appeared to be differentially sensitive to the same compound. By observing abnormal embryos and LD₅₀ values, zebrafish embryos at 6 hpf were considered to be suitable for evaluating embryotoxicity. Also, larvae at 3 dpf were adapted to measure acute drug toxicity in adult mammals. Thus, we can exploit zebrafish to study drug toxicity and can reliably quantify drug uptake with LC-MS/MS. This approach will be helpful for future studies of toxicology in zebrafish.     

α- and β-adrenoceptors of zebrafish in melanosome movement: a comparative study between embryo and adult melanophores

Zebrafish, like other teleosts, display rapid skin color change in response to the background through sympathetic nerves. Here, the α- and β-adrenoceptors of melanophores were studied pharmacologically both in zebrafish embryo and adult scale. In vitro experiments on adult scale melanophores demonstrated that both α₁- and α₂-adrenoceptors are functional in melanosome aggregation, the α₂ subtype being predominant. Most melanophores in zebrafish embryos were able to concentrate melanosomes to α₂-adrenergic agonist α-methylnorepinephrine when they first appeared. This ability increased at least in the following 48 h, showing melanophores at these stages have developed functional adrenoceptors and these receptors increase independently before sympathetic innervation. However, even high concentration (10⁻³ M) of α₁-adrenoceptor agonist phenylephrine was not able to evoke any paling of the embryos. In adult scales, propranolol enhanced the melanosome-aggregating response of epinephrine and isoproterenol, but not norepinephrine, indicating β-adrenoceptor mediates melanosome-dispersing response in adult zebrafish. Similar response was not observed in embryos until 60 h post-fertilization (hpf). The melanophore adrenoceptor blocking effects of phentolamine and propranolol in embryos were much lower than that in adult zebrafish, suggesting these adrenoceptors in developing melanophores are less sensitive to the classical antagonists.