Reduced Eye Size Research Articles

Knockdown of best1 Gene in Zebrafish Caused Abnormal Neuronal and Skeletal Development - A Subtype of Craniovertebral Junction Malformation?

To investigate the developmental defects caused by knockdown of best1 gene in zebrafish as a model for a subtype of craniovertebral junction (CVJ) malformation. Two antisense morpholinos (MOs) were designed targeting zebrafish best1 to block translation (ATG-MO) or to disrupt splicing (I3E4-MO). MOs were microinjected into fertilized one-cell embryos. Efficacy of splicing MO was confirmed by reverse transcription-polymerase chain reaction. Phenotypes were analyzed and quantified by microscopy at multiple developmental stages. Neuronal outgrowth was assessed in transgenic zebrafish expressing green fluorescent protein in neurons. Skeletal ossification was visualized by Calcein staining. Knockdown of best1 resulted in zebrafish embryos with shorter body length, curved axis, low survival rate, microcephaly, reduced eye size, smaller head and brain, impaired neuronal outgrowth, and reduced ossification of craniofacial and vertebral bone. Best1 gene plays critical roles in ophthalmologic, neurological and skeletal development in zebrafish. A patient with a premature stop codon in BEST1 gene exhibited similar phenotypes, implying a subtype of CVJ malformation.

Morphological, teratogenic and behavioral evaluations of Gelidium spinosum methanol extract on zebrafish embryos

Gelidium spinosum is an edible red seaweed from the family Gelidiaceae with possibility to be developed. The potential medical benefits of G. spinosum have been established, yet adequate empirical research on its toxicity is still lacking. Hence, the present work was aimed to examine the toxicity of G. spinosum methanol extract (GsME) on zebrafish (Danio rerio) embryos and to identify the phytoconstituents using gas chromatography-mass spectrometry (GC-MS) analysis. Results of this study showed that GsME induced morphological defects in zebrafish embryos, including reduction in eye size and body length. Moreover, yolk sac size and mortality were increased in zebrafish embryos exposed to GsME in a dose-dependent manner. GsME at high concentrations triggered teratogenic effects in zebrafish embryos such as decrease in heartbeat/minute, lack of pigmentation, lack of somite, structural deformity, pericardial oedema, and yolk oedema. The LC50 and EC50 of GsME were < 100 mg/L, which classified as a harmful category. The teratogenic index of GsME was found to be > 1, indicating its teratogenic attribute. Additionally, GsME exerted behavioral effects i.e. significantly lower total distance of movement and slower swimming speed of zebrafish embryos. GC-MS analysis of GsME was confirmed the presence of amino acid, phenolics, carboxylic acids, reducing sugars, saturated fatty acids and brominated saturated fatty acid in the extract. It suggesting that compounds of 13-bromotetradecanoic acid, palmitic acid, and stearic acid containing in GsME were contributed to the toxic effects.

A metabolomics approach to reveal the mechanism of developmental toxicity in zebrafish embryos exposed to 6-propyl-2-thiouracil

A crucial component of a substance registration and regulation is the evaluation of human prenatal developmental toxicity. Current toxicological tests are based on mammalian models, but these are costly, time consuming and may pose ethical concerns. The zebrafish embryo has evolved as a promising alternative model to study developmental toxicity. However, the implementation of the zebrafish embryotoxicity test is challenged by lacking information on the relevance of observed morphological alterations in fish for human developmental toxicity. Elucidating the mechanism of toxicity could help to overcome this limitation. Through LC-MS/MS and GC-MS metabolomics, we investigated whether changes to the endogenous metabolites can indicate pathways associated with developmental toxicity. To this aim, zebrafish embryos were exposed to different concentrations of 6-propyl-2-thiouracil (PTU), a compound known to induce developmental toxicity. The reproducibility and the concentration-dependence of the metabolome response and its association with morphological alterations were studied. Major morphological findings were reduced eye size, and other craniofacial anomalies; major metabolic changes included increased tyrosine, pipecolic acid and lysophosphatidylcholine levels, decreased methionine levels, and disturbance of the ‘Phenylalanine, tyrosine and tryptophan biosynthesis’ pathway. This pathway, and the changes in tyrosine and pipecolic acid levels could be linked to the mode of action of PTU, i.e., inhibition of thyroid peroxidase (TPO). The other findings suggested neurodevelopmental impairments. This proof-of-concept study demonstrated that metabolite changes in zebrafish embryos are robust and provide mechanistic information associated with the mode of action of PTU.

Antioxidant/protective effects of carob pod (Ceratonia siliqua L.) water extract against deltamethrin-induced oxidative stress/toxicity in zebrafish larvae

In our study, the antioxidant capacity of carob pods water extract (CPWE) against deltamethrin (DM)-induced oxidative stress, a widely used pesticide around the world, was investigated in vitro and in vivo in a zebrafish model. The in vitro antioxidant capacity of the obtained extract was evaluated with different methods using trolox, BHA and BHT standard antioxidants. For in vivo experiments, 4hpf zebrafish embryos were exposed to 10 ppb and 25 ppb DM for 120 h and the larvae were treated with 1–10 and 100 ppm CPWE for 4 h at 72th hours. According to the results obtained, it has been determined that the exposure of zebrafish to DM during the developmental period causes important body malformations, decrease in survival rate, reduction in eye size, shortening in body length and decrease in locomotor activity in the dark period. In addition, according to the results of whole-mount staining, it was determined that DM caused a significant increase in the amount of free oxygen radicals and apoptotic cells. It was also confirmed by metabolome analysis that CPWE application for 4 h reduced DM-induced toxicity and oxidative stress. As a result, it can be said that CPWE has an important antioxidant capacity in eliminating DM-induced oxidative stress.

The world’s largest cave fish from Meghalaya, Northeast India, is a new species, Neolissochilus pnar (Cyprinidae, Torinae)

The world’s largest subterranean fish was discovered in 2019, and was tentatively identified as a troglomorphic form of the golden mahseer, Tor putitora. Detailed analyses of its morphometric and meristic data, and results from molecular analyses now reveal that it is a new species of the genus Neolissochilus, the sister taxon of Tor. We formally describe the new species as Neolissochilus pnar, honouring the tribal communities of East Jaintia hills in Meghalaya, Northeast India, from where it was discovered. Neolissochilus pnar possesses a number of characters unique among species of Neolissochilus, with the exception of the similarly subterranean N. subterraneus from Thailand. The unique characters that diagnose N. pnar from all epigean congeners comprise highly reduced eye size to complete absence of externally visible eyes, complete lack of pigmentation, long maxillary barbels, long pectoral-fin rays, and scalation pattern. Neolissochilus pnar is distinguished from the hypogean N. subterraneus, the type locality of which is a limestone cave ~2000 kms away in Central Thailand, by a lesser pre-pelvic length (47.8–49.4 vs. 50.5–55.3 %SL), a shorter caudal peduncle (16.1–16.8 vs. 17.8–23.7 %SL), and shorter dorsal fin (17.4–20.8 vs. 21.5–26.3 %SL). In addition, Neolissochilus pnar is also genetically and morphologically distinct from its close congeners with a raw genetic divergence of 1.1–2.7% in the COI gene with putative topotype of N. hexastichus and 2.1–2.6% with putative topotype of N. hexagonolepis.

Thyroid hormone disruption by bis-(2-ethylhexyl) phthalate (DEHP) and bis-(2-ethylhexyl) adipate (DEHA) in Japanese medaka Oryzias latipes

Pollution of water bodies with plasticizers is a serious environmental problem worldwide. In this study, we investigated the effects of plasticizers bis-(2-ethylhexyl) phthalate (DEHP) and bis-(2-ethylhexyl) adipate (DEHA) in Japanese medaka (Oryzias latipes). DEHP significantly increased the expression of all the genes tested: thyroid stimulating hormone beta subunit (tshβ-like), tshβ, deiodinase 1 (dio1), deiodinase 2 (dio2), and thyroid hormone receptor alpha (trα) and beta (trβ). However, DEHA only significantly increased tshβ at 7.4 µg/L but significantly decreased dio2 expression at 25.8, 111.1, and 412.6 4 µg/L, while other genes were not significantly affected. Both chemicals reduced eye size and total body length, but did not affect embryo development, hatching time and rate, and swimming performance. DEHA alone affected swim bladder inflation and not DEHP. This is the first report that not only DEHP but also DEHA disrupt thyroid hormone activity in fish. DEHP contamination (13.2 μg/L) was detected in tap water from Kobe, Japan; thus, tap water itself may disrupt thyroid hormone activity in Japanese medaka. Importantly, the effective concentration of DEHP for thyroid hormone–related gene expression and growth was close to or lower than DEHP concentrations reported in surface water elsewhere, indicating that DEHP contamination is a serious aquatic pollution.

Domestication-induced reduction in eye size revealed in multiple common garden experiments: The case of Atlantic salmon (Salmo salar L.).

Domestication leads to changes in traits that are under directional selection in breeding programmes, though unintentional changes in nonproduction traits can also arise. In offspring of escaping fish and any hybrid progeny, such unintentionally altered traits may reduce fitness in the wild. Atlantic salmon breeding programmes were established in the early 1970s, resulting in genetic changes in multiple traits. However, the impact of domestication on eye size has not been studied. We measured body size corrected eye size in 4000 salmon from six common garden experiments conducted under artificial and natural conditions, in freshwater and saltwater environments, in two countries. Within these common gardens, offspring of domesticated and wild parents were crossed to produce 11 strains, with varying genetic backgrounds (wild, domesticated, F1 hybrids, F2 hybrids and backcrosses). Size‐adjusted eye size was influenced by both genetic and environmental factors. Domesticated fish reared under artificial conditions had smaller adjusted eye size when compared to wild fish reared under identical conditions, in both the freshwater and marine environments, and in both Irish and Norwegian experiments. However, in parr that had been introduced into a river environment shortly after hatching and sampled at the end of their first summer, differences in adjusted eye size observed among genetic groups were of a reduced magnitude and were nonsignificant in 2‐year‐old sea migrating smolts sampled in the river immediately prior to sea entry. Collectively, our findings could suggest that where natural selection is present, individuals with reduced eye size are maladapted and consequently have reduced fitness, building on our understanding of the mechanisms that underlie a well‐documented reduction in the fitness of the progeny of domesticated salmon, including hybrid progeny, in the wild.

Exome sequencing and functional studies in zebrafish identify WDR8 as the causative gene for isolated Microspherophakia in Indian families.

Isolated Microspherophakia (MSP) is an autosomal recessive disorder characterized by a smaller than normal spherical lens. Till date, LTBP2 is the only gene shown to cause MSP. We used homozygosity mapping and whole-exome sequencing and identified a homozygous mutation, c.1148C > T (p.Pro383Leu), in the WDR8 (or WRAP73) gene in two Indian MSP families. In vitro experiments showed that the missense mutation renders the protein unstable. WDR8 is a centriolar protein that has important roles in centrosomal assembly, spindle pole formation and ciliogenesis. Co-immunoprecipitation experiments from HeLa cells indicated that the mutation interferes with the interaction of WDR8 with its binding partners. In zebrafish, both morpholino-mediated knockdown and CRISPR/Cas knockout of wdr8 resulted in decreased eye and lens size. The lack of wdr8 affected cell cycle progression in the retinal cells, causing a reduction in cell numbers in the retina and lens. The reduction in eye size and the cell cycle defects were rescued by exogenous expression of the human wild-type WDR8. However, the human mutant WDR8 (p.Pro383Leu) was unable to rescue the eye defects, indicating that the missense mutation abrogates WDR8 protein function. Thus, our zebrafish results suggested that WDR8 is the causative gene for MSP in these Indian families.

EPHA2 Segregates with Microphthalmia and Congenital Cataracts in Two Unrelated Families.

EPHA2 is a transmembrane tyrosine kinase receptor that, when disrupted, causes congenital and age-related cataracts. Cat-Map reports 22 pathogenic EPHA2 variants associated with congenital cataracts, variable microcornea, and lenticonus, but no previous association with microphthalmia (small, underdeveloped eye, ≥2 standard deviations below normal axial length). Microphthalmia arises from ocular maldevelopment with >90 monogenic causes, and can include a complex ocular phenotype. In this paper, we report two pathogenic EPHA2 variants in unrelated families presenting with bilateral microphthalmia and congenital cataracts. Whole genome sequencing through the 100,000 Genomes Project and cataract-related targeted gene panel testing identified autosomal dominant heterozygous mutations segregating with the disease: (i) missense c.1751C>T, p.(Pro584Leu) and (ii) splice site c.2826-9G>A. To functionally validate pathogenicity, morpholino knockdown of epha2a/epha2b in zebrafish resulted in significantly reduced eye size ± cataract formation. Misexpression of N-cadherin and retained fibre cell nuclei were observed in the developing lens of the epha2b knockdown morphant fish by 3 days post-fertilisation, which indicated a putative mechanism for microphthalmia pathogenesis through disruption of cadherin-mediated adherens junctions, preventing lens maturation and the critical signals stimulating eye growth. This study demonstrates a novel association of EPHA2 with microphthalmia, suggesting further analysis of pathogenic variants in unsolved microphthalmia cohorts may increase molecular diagnostic rates.

Intimate functional interactions betweenTGS1 and the Smncomplex revealed by an analysis of the Drosophila eye development.

Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to understand the molecular mechanisms underlying SMA pathogenesis and devise new therapies for this genetic disease.

Zika virus non-structural protein NS4A restricts eye growth in Drosophila through regulation of JAK/STAT signaling.

ABSTRACTTo gain a comprehensive view of the changes in host gene expression underlying Zika virus (ZIKV) pathogenesis, we performed whole-genome RNA sequencing (RNA-seq) of ZIKV-infected Drosophila adult flies. RNA-seq analysis revealed that ZIKV infection alters several and diverse biological processes, including stress, locomotion, lipid metabolism, imaginal disc morphogenesis and regulation of JAK/STAT signaling. To explore the interaction between ZIKV infection and JAK/STAT signaling regulation, we generated genetic constructs overexpressing ZIKV-specific non-structural proteins NS2A, NS2B, NS4A and NS4B. We found that ectopic expression of non-structural proteins in the developing Drosophila eye significantly restricts growth of the larval and adult eye and correlates with considerable repression of the in vivo JAK/STAT reporter, 10XStat92E-GFP. At the cellular level, eye growth defects are associated with reduced rate of proliferation without affecting the overall rate of apoptosis. In addition, ZIKV NS4A genetically interacts with the JAK/STAT signaling components; co-expression of NS4A along with the dominant-negative form of domeless or StatRNAi results in aggravated reduction in eye size, while co-expression of NS4A in HopTuml (also known as hopTum) mutant background partially rescues the hop-induced eye overgrowth phenotype. The function of ZIKV NS4A in regulating growth is maintained in the wing, where ZIKV NS4A overexpression in the pouch domain results in reduced growth linked with diminished expression of Notch targets, Wingless (Wg) and Cut, and the Notch reporter, NRE-GFP. Thus, our study provides evidence that ZIKV infection in Drosophila results in restricted growth of the developing eye and wing, wherein eye phenotype is induced through regulation of JAK/STAT signaling, whereas restricted wing growth is induced through regulation of Notch signaling. The interaction of ZIKV non-structural proteins with the conserved host signaling pathways further advance our understanding of ZIKV-induced pathogenesis.This article has an associated First Person interview with the first author of the paper.

Offshore Crude Oil Disrupts Retinoid Signaling and Eye Development in Larval Atlantic Haddock

The aim of this study was to examine the impact of dispersed crude oil on retinoid metabolism and signaling in developing haddock larvae (Melanogrammus aeglefinus). Retinoids and its active metabolites are involved in the regulation of several developmental processes as cell differentiation, growth and limb patterning. Thus, disruption of retinoid signaling, especially during early developmental stages, may have detrimental effects on the organism. In the present study, crude oil exposure during embryonic development and early larvae development disrupted vitamin A signaling by affecting retinoid levels and genes involved in retinoid metabolism. The disruption of retinoid signaling was also evident when looking at the gene expression pattern at several time-points during and after exposure, demonstrating the complexity and temporal factor of retinoid signaling disruption. Exposure during the embryonic period (pre hatch) resulted in reduced eye size and increased incidence of abnormal eye morphology. The observed changes in expression of retinoid metabolic genes related to the visual cycle indicates that there is a link between the modulated retinoid levels (all-trans retinoic acid and retinol) and increased incidence of abnormal eye development as a result of crude oil exposure. This is the first study to demonstrate disrupted retinoid signaling in fish following crude oil exposure at environmental relevant levels, giving novel insight in to the mechanism of toxicity.

QDPR homologues in Danio rerio regulate melanin synthesis, early gliogenesis, and glutamine homeostasis.

Dihydropteridine reductase (QDPR) catalyzes the recycling of tetrahydrobiopterin (BH4), a cofactor in dopamine, serotonin, and phenylalanine metabolism. QDPR-deficient patients develop neurological symptoms including hypokinesia, truncal hypotonia, intellectual disability and seizures. The underlying pathomechanisms are poorly understood. We established a zebrafish model for QDPR deficiency and analyzed the expression as well as function of all zebrafish QDPR homologues during embryonic development. The homologues qdpra is essential for pigmentation and phenylalanine metabolism. Qdprb1 is expressed in the proliferative zones of the optic tectum and eye. Knockdown of qdprb1 leads to up-regulation of pro-proliferative genes and increased number of phospho-histone3 positive mitotic cells. Expression of neuronal and astroglial marker genes is concomitantly decreased. Qdprb1 hypomorphic embryos develop microcephaly and reduced eye size indicating a role for qdprb1 in the transition from cell proliferation to differentiation. Glutamine accumulation biochemically accompanies the developmental changes. Our findings provide novel insights into the neuropathogenesis of QDPR deficiency.

Regulation of Notch signaling in the developing Drosophila eye by a T-box containing transcription factor, Dorsocross.

Owing to a multitude of functions, there is barely a tissue or a cellular process that is not being regulated by Notch signaling. To allow the Notch signal to be deployed in numerous contexts, many different mechanisms have evolved to regulate the level, duration and spatial distribution of Notch activity. To identify novel effectors of Notch signaling in Drosophila melanogaster, we analyzed the whole transcriptome of the wing and eye imaginal discs in which an activated form of Notch was overexpressed. Selected candidate genes from the transcriptome analysis were subjected to genetic interaction experiments with Notch pathway components. Among the candidate genes, T-box encoding gene, Dorsocross (Doc) showed strong genetic interaction with Notch ligand, Delta. Genetic interaction between them resulted in reduction of eye size, loss of cone cells, and cell death, which represent prominent Notch loss of function phenotypes. Immunocytochemical analysis in Df(3L)DocA/Dl 5f trans-heterozygous eye discs showed accumulation of Notch at the membrane. This accumulation led to decreased Notch signaling activity as we found downregulation of Atonal, a Notch target and reduction in the rate of Notch-mediated cell proliferation. Doc mutant clones generated by FLP-FRT system showed depletion in the expression of Delta and subsequent reduction in the Notch signaling activity. Similarly, Doc overexpression in the eye discs led to modification of Delta expression, loss of Atonal expression and absence of eye structure in pharate adults. Taken together, our results suggest that Doc regulates the expression of Delta and influence the outcome of Notch signaling in the eye discs.

Hepatocyte growth factor promotes the proliferation of human embryonic stem cell derived retinal pigment epithelial cells.

Research that pertains to the molecular mechanisms involved in retinal pigment epithelial (RPE) development can significantly contribute to cell therapy studies. The effects of periocular mesenchymal cells on the expansion of RPE cells remain elusive. We have examined the possible proliferative role of hepatocyte growth factor (HGF) as a mesenchymal cell secretory factor against human embryonic stem cell derived RPE (hESC-RPE). We found that the conditioned medium of human mesenchymal stem cells from apical papilla and/or exogenous HGF promoted proliferation of the hESC-RPE cells as single cells and cell sheets, in addition to rabbit RPE sheets in vitro. Blockage of HGF signaling by HGF receptor inhibitor, PHA-665752, inhibited proliferation of hESC-RPE cells. However, differentiation of hESCs and human-induced pluripotent stem cells to a rostral fate and eye-field specification was unaffected by HGF. Our in vivo analysis showed HGF expression in periocular mesenchymal cells after optic cup formation in chicken embryos. Administration of HGF receptor inhibitor at this developmental stage in chicken embryos led to reduced eye size and disorganization of the RPE sheet. These findings suggested that HGF administration could be beneficial for obtaining higher numbers of hESC-RPE cells in human preclinical and clinical trials.

Mutation in the intracellular chloride channel CLCC1 associated with autosomal recessive retinitis pigmentosa.

We identified a homozygous missense alteration (c.75C>A, p.D25E) in CLCC1, encoding a presumptive intracellular chloride channel highly expressed in the retina, associated with autosomal recessive retinitis pigmentosa (arRP) in eight consanguineous families of Pakistani descent. The p.D25E alteration decreased CLCC1 channel function accompanied by accumulation of mutant protein in granules within the ER lumen, while siRNA knockdown of CLCC1 mRNA induced apoptosis in cultured ARPE-19 cells. TALEN KO in zebrafish was lethal 11 days post fertilization. The depressed electroretinogram (ERG) cone response and cone spectral sensitivity of 5 dpf KO zebrafish and reduced eye size, retinal thickness, and expression of rod and cone opsins could be rescued by injection of wild type CLCC1 mRNA. Clcc1+/- KO mice showed decreased ERGs and photoreceptor number. Together these results strongly suggest that intracellular chloride transport by CLCC1 is a critical process in maintaining retinal integrity, and CLCC1 is crucial for survival and function of retinal cells.

The Second True TroglobiontHeteropodaSpecies from a Limestone Cave System in Palawan, Philippines (Araneae: Sparassidae: Heteropodinae)

Abstract A new cave-dwelling huntsman spider species from Palawan, Philippines is described: Heteropoda simoneallmannae sp. nov. (female) from the Puerto Princesa Underground River Cave system. It represents, besides H. steineri Bayer & Jager, 2009 from Laos, the second species within the genus with true cave adaptations like reduction of eye size and pigments.

Visual impairment and progressive phthisis bulbi caused by recessive pathogenic variant in MARK3.

Developmental eye defects often severely reduce vision. Despite extensive efforts, for a substantial fraction of these cases the molecular causes are unknown. Recessive eye disorders are frequent in consanguineous populations and such large families with multiple affected individuals provide an opportunity to identify recessive causative genes. We studied a Pakistani consanguineous family with three affected individuals with congenital vision loss and progressive eye degeneration. The family was analyzed by exome sequencing of one affected individual and genotyping of all family members. We have identified a non-synonymous homozygous variant (NM_001128918.2: c.1708C > G: p.Arg570Gly) in the MARK3 gene as the likely cause of the phenotype. Given that MARK3 is highly conserved in flies (I: 55%; S: 67%) we knocked down the MARK3 homologue, par-1, in the eye during development. This leads to a significant reduction in eye size, a severe loss of photoreceptors and loss of vision based on electroretinogram (ERG) recordings. Expression of the par-1 p.Arg792Gly mutation (equivalent to the MARK3 variant found in patients) in developing fly eyes also induces loss of eye tissue and reduces the ERG signals. The data in flies and human indicate that the MARK3 variant corresponds to a loss of function. We conclude that the identified mutation in MARK3 establishes a new gene-disease link, since it likely causes structural abnormalities during eye development and visual impairment in humans, and that the function of MARK3/par-1 is evolutionarily conserved in eye development.

Development and sexual dimorphism of the sonic system in three deep-sea neobythitine fishes and comparisons between upper mid and lower continental slope

Based on morphology, NB Marshall identified cusk-eels (family Ophidiidae) as one of the chief sound-producing groups on the continental slope. Due to food scarcity, we hypothesized that sonic systems will be reduced at great depths despite their potential importance in sexual reproduction. We examined this hypothesis in the cusk-eel subfamily Neobythitinae by comparing sonic morphology in Atlantic species from the upper-mid (Dicrolene intronigra) and deeper continental slope (Porogadus miles and Bathyonus pectoralis) with three Taiwanese species previously described from the upper slope (Hoplobrotula armatus, Neobythites longipes and N. unimaculatus). In all six species, medial muscles are heavier in males than in females. Dicrolene has four pairs of sonic muscles similar to the shallow Pacific species, suggesting neobythitine sonic anatomy is conservative and sufficient food exists to maintain a well-developed system at depths exceeding 1km. The sonic system in Porogadus and Bathyonus was reduced to a single pair of ventral medial muscles that connects to a smaller and thinner swimbladder via a long tendon. Small muscle fiber diameters, a likely indicator of rapid contraction, were present in males of five of the species. However, in Bathyonus, the deepest species (pale coloration, reduced eye size, shorter sonic muscles and longer tendons), muscle fibers were larger suggesting an adaptation to facilitate rapid bladder movement for sound production while using slower contractions and less metabolic energy. The six species separate into three groups in length-weight regressions: the three upper slope species have the greatest weights per unit length, Dicrolene is lower, and the two deep species are further reduced consistent with the hypothesis that food limitation affects sonic anatomy at great depths.

Balligratus, new genus of wingless ground beetles from equatorial Andean montane forest (Coleoptera: Carabidae: Lachnophorini).

A new carabid beetle genus, Balligratus gen. nov., belonging to the tribe Lachnophorini, is described. It is geographically restricted to the equatorial Andes, and ecologically linked to the montane pluvial forest ecosystem, at elevations ranging from 1,200 to 3,600 m. As other carabid lineages that have radiated in such environments, Balligratus gen. nov. is a wingless clade, characterized by the loss of flight wings associated with metathoracic reduction, constriction of the elytral base, and reduced eye size. This evolution is unique among Lachnophorini. Four new species are described, all of them from Ecuador: Balligratus brevis sp. nov., Balligratus globosus sp. nov., Balligratus gracilis sp. nov. and Balligratus humerangulus sp. nov.