Population parameters and conservation implications for one of the world's rarest marine fishes, the red handfish (Thymichthys politus).

Population estimates are required for effective conservation of many rare marine species, but can be difficult to obtain. The critically endangered red handfish (Thymichthys politus) is a coastal anglerfish known only from two fragmented populations in southeast Tasmania, Australia. It is at a high risk of extinction due to low numbers, loss of habitat, and the impacts of climate change. To aid conservation efforts, we provide the first empirical population size estimates of red handfish and investigate other important aspects of the species' life history, such as growth, habitat association, and movement. We surveyed both red handfish local populations via underwater visual census on scuba over 3 years and used photographic mark-recapture techniques to estimate biological parameters. In 2020, the local adult population size was estimated to be 94 (95% confidence interval [CI] 40-231) adults at one site, and 7 (95% CI 5-10) at the other site, suggesting an estimated global population of 101 adults. Movement of individuals was extremely limited at 48.5 m (± 77.7 S.D.) per year. We also found evidence of declining fish density, a declining proportion of juveniles, and increasing average fish size during the study. These results provide a serious warning that red handfish are likely sliding toward extinction, and highlight the urgent need to expand efforts for ex situ captive breeding to bolster numbers in the wild and maintain captive insurance populations, and to protect vital habitat to safeguard the species' ongoing survival in the wild.

Minor class splicing shapes the zebrafish transcriptome during development.

Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human pre-mRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11- and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.

Autophagy induction is a Tor- and Tp53-independent cell survival response in a zebrafish model of disrupted ribosome biogenesis.

Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (tti(s450)), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In tti(s450), the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in tti(s450) larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in tti(s450) larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.

Development of a two-current choice flume behavioural bioassay for juvenile Panulirus ornatus response to moulting cues.

Characterising crustacean behaviour in response to conspecific chemical cues contributes to our evolving knowledge of the drivers of their social behaviour. There is particular interest in understanding the chemical and behavioural mechanisms contributing to cannibalism at ecdysis, as this behaviour substantially limits culture productivity of several commercially important crustaceans. Before investigating the role of chemoreception in cannibalism of moulting crustaceans, we must investigate its role in detecting moulting conspecifics. Here we use a two-current choice flume to observe juvenile tropical rock lobster (Panulirus ornatus) behavioural response to conspecific moulting cues and identifying attracted and avoidant behaviours correlating to moult stage and social relationship. Observed cue preferences show inter-moult juveniles are attracted to the moulting cues of lobsters to which they are socially naïve. In contrast, post-moult and inter-moult juveniles avoid the moulting cues of individuals whom they are socially familiar with. Average speed and total distance travelled by lobsters increases in response to conspecific moulting cues. This study demonstrates the suitability of a two-current choice flume for behavioural assays in P. ornatus and characterises clear behavioural patterns in juveniles exposed to conspecific moulting cues. This provides important framework for understanding the role of chemical communication in eliciting cannibalism.

The Effect of Feed Frequency on Growth, Survival and Behaviour of Juvenile Spiny Lobster (Panulirus ornatus).

Spiny lobsters have a range of complex chemical communication pathways that contribute to feeding behaviour. Feed intake is modulated by feed availability and feed characteristics, such as attractiveness and palatability, with behavioural factors, such as social competition and circadian rhythm, providing an extra layer of complexity. In this study, we investigated the effect of feed frequency on survival and growth of early-stage (instar 2-6) juvenile Palunirus ornatus. In addition, we investigated the interactive effect of feed frequency and circadian rhythm on lobster feed response. Lobsters were fed a set ration at a frequency of either one, two, four, eight, sixteen or thirty-two times per day over 49 days. The effect of feed frequency on growth and survival was determined. Circadian feeding activity under these feeding treatments was assessed by time-lapse photography. Increased feed frequency from one to sixteen feeds daily improved growth by increasing apparent feed intake (AFI) and feed attraction, as confirmed by the increased presence of lobsters in the feeding area. The rapid leaching of feed attractant, particularly free amino acid, suggests a beneficial effect of multiple feeding frequencies on feed intake and growth. However, more than sixteen feeds per day resulted in decreased feed intake and a subsequent reduction in growth. The decrease in feed intake is thought to be associated with saturation of the culture environment with attractants, resulting in a reduced behavioural response to feed supplies. This may indicate the need for depletion of attractants to retrigger a feeding response. As lobsters were grown communally, faster growth at sixteen rations per day was also coupled with increased cannibalism, likely driven by increased vulnerability with the occurrence of more frequent ecdysis events. Whereas circadian rhythm indicated more activity at night, an interaction between daytime activity and feed frequency was not observed.

Abnormal nuclear pore formation triggers apoptosis in the intestinal epithelium of elys-deficient zebrafish.

BACKGROUND & AIMS: Zebrafish mutants generated by ethylnitrosourea-mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, "flotte lotte" (flo), displays striking defects in intestinal, liver, pancreas, and eye formation at 78 hours postfertilization (hpf). In this study, we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype. METHODS: Positional cloning was employed to map the flo mutation. Subcellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, and confocal and electron microscopy. RESULTS: The molecular lesion in flo is a nonsense mutation in the elys (embryonic large molecule derived from yolk sac) gene, which encodes a severely truncated protein lacking the Elys C-terminal AT-hook DNA binding domain. Recently, the human ELYS protein has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Although elys messenger RNA (mRNA) is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally expressed elys mRNA until 24 hpf. From 72 hpf, elys mRNA expression is restricted to proliferating tissues, including the intestinal epithelium, pancreas, liver, and eye. Cells in these tissues display disrupted nuclear pore formation; ultimately, intestinal epithelial cells undergo apoptosis. CONCLUSIONS: Our results demonstrate that Elys regulates digestive organ formation.

Aquimarina sp. Associated With a Cuticular Disease of Cultured Larval Palinurid and Scyllarid Lobsters.

Shell (cuticular) disease manifests in various forms and affects many crustaceans, including lobsters. Outbreaks of white leg disease (WLD) with distinct signs of pereiopod tissue whitening and death have been observed in cultured larvae (phyllosomas) of ornate spiny lobster Panulirus ornatus, eastern rock lobster Sagmariasus verreauxi, and slipper lobster Thenus australiensis. This study aimed to characterise and identify the causative agent of WLD through morphological and molecular (16S rRNA gene and whole genome sequencing) analysis, experimental infection of damaged/undamaged P. ornatus and T. australiensis phyllosomas, and bacterial community analysis (16S rRNA gene amplicon sequencing) of P. ornatus phyllosomas presenting with WLD during an outbreak. Bacterial communities of WLD-affected pereiopods showed low bacterial diversity and dominant abundance of Aquimarina spp. compared to healthy pereiopods, which were more diverse and enriched with Sulfitobacter spp. 16S rRNA gene Sanger sequencing of cultures from disease outbreaks identified the dominant bacterial isolate (TRL1) as a Gram-negative, long non-flagellated rod with 100% sequence identity to Aquimarina hainanensis. Aquimarina sp. TRL1 was demonstrated through comparative genome analysis (99.99% OrthoANIu) as the bacterium reisolated from experimentally infected phyllosomas presenting with typical signs of WLD. Pereiopod damage was a major predisposing factor to WLD. Histopathological examination of WLD-affected pereiopods showed masses of internalised bacteria and loss of structural integrity, suggesting that Aquimarina sp. TRL1 could enter the circulatory system and cause death by septicaemia. Aquimarina sp. TRL1 appears to have important genomic traits (e.g., tissue-degrading enzymes, gliding motility, and aggregate-promoting factors) implicated in the pathogenicity of this bacterium. We have shown that Aquimarina sp. TRL1 is the aetiological agent of WLD in cultured Palinurid and Scyllarid phyllosomas and that damaged pereiopods are a predisposing factor to WLD.

Morphologic analysis of the zebrafish digestive system.

The zebrafish provides an ideal model for the study of vertebrate organogenesis, including the formation of the digestive tract and its associated organs. Despite optical transparency of embryos, the internal position of the developing digestive system and its close juxtaposition with the yolk initially made morphological analysis relatively challenging, particularly during the first 3 d of development. However, methodologies have been successfully developed to address these problems and comprehensive morphologic analysis of the developing digestive system has now been achieved using a combination of light and fluorescence microscope approaches-including confocal analysis-to visualize wholemount and histological preparations of zebrafish embryos. Furthermore, the expanding number of antibodies that cross-react with zebrafish proteins and the generation of tissue-specific transgenic green fluorescent protein reporter lines that mark specific cell and tissue compartments have greatly enhanced our ability to successfully image the developing zebrafish digestive system.

Speciation in fractured rock landforms: towards understanding the diversity of subterranean cockroaches (Dictyoptera: Nocticolidae: Nocticola) in Western Australia.

Three new species of subterranean cockroach of the genus Nocticola from the Pilbara region of Western Australia are described on morphological characters of males. Nocticola quartermainei n. sp., Nocticola cockingi n. sp. and Nocticola currani n. sp. occur in fractured rock landforms and have varying degrees of troglomorphies. Sequence divergence of mitochondrial cytochrome c oxidase subunit I (COXI) clearly demonstrated populations are reproductively isolated over very short distances for the highly troglomorphic Nocticola cockingi n. sp. and Nocticola currani n. sp. and conversely, there is less isolation within the same landforms for the less troglomorphic Nocticola quartermainei n. sp.

Modified RV short axis series--a new method for cardiac MRI measurement of right ventricular volumes.

PURPOSE: The current standard image orientation employed in the MRI assessment of right ventricular volumes uses a series of short axis cine acquisitions located with respect to a horizontal long axis view with the first slice placed across the atrio-ventricular valve plane at end diastole. Inherent inaccuracies are encountered with the use of this image orientation due to difficulty in defining the tricuspid valve and the border between atrium and ventricle on the resultant images. Our experience indicates that because the tricuspid valve is usually not in-plane in the slice the atrio-ventricular margin is difficult to distinguish. This leads to inaccuracies in measurements at the base of the RV and miscalculation of the RV volume. The purpose of this study was to assess an alternative method of image orientation aimed at increasing the accuracy of RV volume measurements using current commercially available CMRI sequences. This technique, the modified RV short axis series, is oriented to the outflow tract of the right ventricle. METHOD: We undertook a prospective study of 50 post cardiac transplant patients. A series of LV short axis multi-slice cine acquisition FIESTA images was acquired using the current standard technique. From this data set, LV and RV stroke volumes were derived on an Advantage Windows workstation using planimetry of the endocardial and epicardial borders in end systole and end diastole. Our new technique involved obtaining a set of multi-slice cine acquisition FIESTA images in a plane perpendicular to a line from the centre of the pulmonary valve to the apex of the RV. Planimetry of the RV was then performed and a stroke volume calculated using the same method of analysis. RV stroke volumes obtained from both techniques were compared with LV stroke volumes. Three operators independently derived RV data sets. RESULTS: On the images acquired with the new technique, the tricuspid valve was easier to define leading to more accurate and reproducible planimetry of ventricular borders. RV stroke volumes calculated from the new method showed better agreement with LV stroke volumes than with the current method. These results were consistent across the three operators. CONCLUSIONS: This new method improves visualisation of the tricuspid valve and makes analysis easier and less prone to operator error than the current standard technique for MRI assessment of RV volumes.