Transcriptome Responses to Different Salinity Conditions in Litoditis marina, Revealed by Long-Read Sequencing.

The marine nematode Litoditis marina is widely distributed in intertidal zones around the globe, yet the mechanisms underlying its broad adaptation to salinity remain elusive. In this study, we applied ONT long-read sequencing technology to unravel the transcriptome responses to different salinity conditions in L. marina. Through ONT sequencing under 3‱, 30‱ and 60‱ salinity environments, we obtained 131.78 G clean data and 26,647 non-redundant long-read transcripts, including 6464 novel transcripts. The DEGs obtained from the current ONT lrRNA-seq were highly correlated with those identified in our previously reported Illumina short-read RNA sequencing data. When we compared the 30‱ to the 3‱ salinity condition, we found that GO terms such as oxidoreductase activity, cation transmembrane transport and ion transmembrane transport were shared between the ONT lrRNA-seq and Illumina data. Similarly, GO terms including extracellular space, structural constituents of cuticle, substrate-specific channel activity, ion transport and substrate-specific transmembrane transporter activity were shared between the ONT and Illumina data under 60‱ compared to 30‱ salinity. In addition, we found that 79 genes significantly increased, while 119 genes significantly decreased, as the salinity increased. Furthermore, through the GO enrichment analysis of 214 genes containing DAS, in 30‱ compared to 3‱ salinity, we found that GO terms such as cellular component assembly and coenzyme biosynthetic process were enriched. Additionally, we observed that GO terms such as cellular component assembly and coenzyme biosynthetic process were also enriched in 60‱ compared to 30‱ salinity. Moreover, we found that 86, 125, and 81 genes that contained DAS were also DEGs, in comparisons between 30‱ and 3‱, 60‱ and 30‱, and 60‱ and 3‱ salinity, respectively. In addition, we demonstrated the landscape of alternative polyadenylation in marine nematode under different salinity conditions This report provides several novel insights for the further study of the mechanisms by which euryhalinity formed and evolved, and it might also contribute to the investigation of salinity dynamics induced by global climate change.

EAT-2 attenuates C. elegans development via metabolic remodeling in a chemically defined food environment.

Dietary intake and nutrient composition regulate animal growth and development; however, the underlying mechanisms remain elusive. Our previous study has shown that either the mammalian deafness homolog gene tmc-1 or its downstream acetylcholine receptor gene eat-2 attenuates Caenorhabditis elegans development in a chemically defined food CeMM (C. elegans maintenance medium) environment, but the underpinning mechanisms are not well-understood. Here, we found that, in CeMM food environment, for both eat-2 and tmc-1 fast-growing mutants, several fatty acid synthesis and elongation genes were highly expressed, while many fatty acid ß-oxidation genes were repressed. Accordingly, dietary supplementation of individual fatty acids, such as monomethyl branch chain fatty acid C17ISO, palmitic acid and stearic acid significantly promoted wild-type animal development on CeMM, and mutations in either C17ISO synthesis gene elo-5 or elo-6 slowed the rapid growth of eat-2 mutant. Tissue-specific rescue experiments showed that elo-6 promoted animal development mainly in the intestine. Furthermore, transcriptome and metabolome analyses revealed that elo-6/C17ISO regulation of C. elegans development may be correlated with up-regulating expression of cuticle synthetic and hedgehog signaling genes, as well as promoting biosynthesis of amino acids, amino acid derivatives and vitamins. Correspondingly, we found that amino acid derivative S-adenosylmethionine and its upstream metabolite methionine sulfoxide significantly promoted C. elegans development on CeMM. This study demonstrated that C17ISO, palmitic acid, stearic acid, S-adenosylmethionine and methionine sulfoxide inhibited or bypassed the TMC-1 and EAT-2-mediated attenuation of development via metabolic remodeling, and allowed the animals to adapt to the new nutritional niche.

Transcriptomic and Proteomic Analysis of Marine Nematode Litoditis marina Acclimated to Different Salinities.

Salinity is a critical abiotic factor for all living organisms. The ability to adapt to different salinity environments determines an organism's survival and ecological niches. Litoditis marina is a euryhaline marine nematode widely distributed in coastal ecosystems all over the world, although numerous genes involved in its salinity response have been reported, the adaptive mechanisms underlying its euryhalinity remain unexplored. Here, we utilized worms which have been acclimated to either low-salinity or high-salinity conditions and evaluated their basal gene expression at both transcriptomic and proteomic levels. We found that several conserved regulators, including osmolytes biosynthesis genes, transthyretin-like family genes, V-type H+-transporting ATPase and potassium channel genes, were involved in both short-term salinity stress response and long-term acclimation processes. In addition, we identified genes related to cell volume regulation, such as actin regulatory genes, Rho family small GTPases and diverse ion transporters, which might contribute to hyposaline acclimation, while the glycerol biosynthesis genes gpdh-1 and gpdh-2 accompanied hypersaline acclimation in L. marina. This study paves the way for further in-depth exploration of the adaptive mechanisms underlying euryhalinity and may also contribute to the study of healthy ecosystems in the context of global climate change.

Genome-Wide Transcriptional Responses of Marine Nematode Litoditis marina to Hyposaline and Hypersaline Stresses.

Maintenance of osmotic homeostasis is essential for all organisms, especially for marine animals in the ocean with 3% salinity or higher. However, the underlying molecular mechanisms that how marine animals adapt to high salinity environment compared to their terrestrial relatives, remain elusive. Here, we investigated marine animal's genome-wide transcriptional responses to salinity stresses using an emerging marine nematode model Litoditis marina. We found that the transthyretin-like family genes were significantly increased in both hyposaline and hypersaline conditions, while multiple neurotransmitter receptor and ion transporter genes were down-regulated in both conditions, suggesting the existence of conserved strategies for response to stressful salinity environments in L. marina. Unsaturated fatty acids biosynthesis related genes, neuronal related tubulins and intraflagellar transport genes were specifically up-regulated in hyposaline treated worms. By contrast, cuticle related collagen genes were enriched and up-regulated for hypersaline response. Given a wide range of salinity tolerance of the marine nematodes, this study and further genetic analysis of key gene(s) of osmoregulation in L. marina will likely provide important insights into biological evolution and environmental adaptation mechanisms in nematodes and other invertebrate animals in general.

Biodiversity-based development and evolution: the emerging research systems in model and non-model organisms.

Evolutionary developmental biology, or Evo-Devo for short, has become an established field that, broadly speaking, seeks to understand how changes in development drive major transitions and innovation in organismal evolution. It does so via integrating the principles and methods of many subdisciplines of biology. Although we have gained unprecedented knowledge from the studies on model organisms in the past decades, many fundamental and crucially essential processes remain a mystery. Considering the tremendous biodiversity of our planet, the current model organisms seem insufficient for us to understand the evolutionary and physiological processes of life and its adaptation to exterior environments. The currently increasing genomic data and the recently available gene-editing tools make it possible to extend our studies to non-model organisms. In this review, we review the recent work on the regulatory signaling of developmental and regeneration processes, environmental adaptation, and evolutionary mechanisms using both the existing model animals such as zebrafish and Drosophila, and the emerging nonstandard model organisms including amphioxus, ascidian, ciliates, single-celled phytoplankton, and marine nematode. In addition, the challenging questions and new directions in these systems are outlined as well.

Transcriptome Analysis of the Nematode Caenorhabditis elegans in Acidic Stress Environments.

Ocean acidification and acid rain, caused by modern industries' fossil fuel burning, lead to a decrease in the living environmental pH, which results in a series of negative effects on many organisms. However, the underlying mechanisms of animals' response to acidic pH stress are largely unknown. In this study, we used the nematode Caenorhabditis elegans as an animal model to explore the regulatory mechanisms of organisms' response to pH decline. Two major stress-responsive pathways were found through transcriptome analysis in acidic stress environments. First, when the pH dropped from 6.33 to 4.33, the worms responded to the pH stress by upregulation of the col, nas, and dpy genes, which are required for cuticle synthesis and structure integrity. Second, when the pH continued to decrease from 4.33, the metabolism of xenobiotics by cytochrome P450 pathway genes (cyp, gst, ugt, and ABC transporters) played a major role in protecting the nematodes from the toxic substances probably produced by the more acidic environment. At the same time, the slowing down of cuticle synthesis might be due to its insufficient protective ability. Moreover, the systematic regulation pattern we found in nematodes might also be applied to other invertebrate and vertebrate animals to survive in the changing pH environments. Thus, our data might lay the foundation to identify the master gene(s) responding and adapting to acidic pH stress in further studies, and might also provide new solutions to improve assessment and monitoring of ecological restoration outcomes, or generate novel genotypes via genome editing for restoring in challenging environments especially in the context of acidic stress through global climate change.

Succinate Dehydrogenase-Regulated Phosphoenolpyruvate Carboxykinase Sustains Copulation Fitness in Aging C. elegans Males.

Dysregulated metabolism accelerates reduced decision-making and locomotor ability during aging. To identify mechanisms for delaying behavioral decline, we investigated how C. elegans males sustain their copulatory behavior during early to mid-adulthood. We found that in mid-aged males, gluco-/glyceroneogenesis, promoted by phosphoenolpyruvate carboxykinase (PEPCK), sustains competitive reproductive behavior. C. elegans' PEPCK paralogs, pck-1 and pck-2, increase in expression during the first 2 days of adulthood. Insufficient PEPCK expression correlates with reduced egl-2-encoded ether-a-go-go K+ channel expression and premature hyper-excitability of copulatory circuits. For copulation, pck-1 is required in neurons, whereas pck-2 is required in the epidermis. However, PCK-2 is more essential, because we found that epidermal PCK-2 likely supplements the copulation circuitry with fuel. We identified the subunit A of succinate dehydrogenase SDHA-1 as a potent modulator of PEPCK expression. We postulate that during mid-adulthood, reduction in mitochondrial physiology signals the upregulation of cytosolic PEPCK to sustain the male's energy demands.

TMC-1 attenuates C. elegans development and sexual behaviour in a chemically defined food environment.

Although diet affects growth and behaviour, the adaptive mechanisms that coordinate these processes in non-optimal food sources are unclear. Here we show that the C. elegans tmc-1 channel, which is homologous to the mammalian tmc deafness genes, attenuates development and inhibits sexual behaviour in non-optimal food, the synthetic CeMM medium. In CeMM medium, signalling from the pharyngeal MC neurons and body wall muscles slows larval development. However, in the non-standard diet, mutation in tmc-1 accelerates development, by impairing the excitability of these cells. The tmc-1 larva can immediately generate ATP when fed CeMM, and their fast development requires insulin signalling. Our findings suggest that the tmc-1 channel indirectly affects metabolism in wild-type animals. In addition to regulating the development, we show that mutating tmc-1 can relax diet-induced inhibition of male sexual behaviour, thus indicating that a single regulator can be genetically modified to promote growth rate and reproductive success in new environments.

Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis.

Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene (Fclectin) was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey (Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have 11 amino acids residues, which are relatively invariant in animals' C-type lectin CRDs. Five residues at Ca2+ binding site 1 are conserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control.

A genetic linkage map of Pacific white shrimp (Litopenaeus vannamei): sex-linked microsatellite markers and high recombination rates.

Pacific white shrimp (Litopenaeus vannamei) is the leading species farmed in the Western Hemisphere and an economically important aquaculture species in China. In this project, a genetic linkage map was constructed using amplified fragment length polymorphism (AFLP) and microsatellite markers. One hundred and eight select AFLP primer combinations and 30 polymorphic microsatellite markers produced 2071 markers that were polymorphic in either of the parents and segregated in the progeny. Of these segregating markers, 319 were mapped to 45 linkage groups of the female framework map, covering a total of 4134.4 cM; and 267 markers were assigned to 45 linkage groups of the male map, covering a total of 3220.9 cM. High recombination rates were found in both parental maps. A sex-linked microsatellite marker was mapped on the female map with 6.6 cM to sex and a LOD of 17.8, two other microsatellite markers were also linked with both 8.6 cM to sex and LOD score of 14.3 and 16.4. The genetic maps presented here will serve as a basis for the construction of a high-resolution genetic map, quantitative trait loci (QTLs) detection, marker-assisted selection (MAS) and comparative genome mapping.

[The application of microsatellite markers for parentage determination in selective breeding of Pacific white shrimp (Litopenaeus vannamei)].

Used simulations and controlled mating to examine the potential of microsatellite markers in assigning parentage to Pacific white shrimp progeny. Cervus simulations demonstrated that the theoretical expectations for parentage exclusion of 10 microsatellite loci and six most polymorphic of the 10 loci were both 0.99, and the assignment success rate of the 6 most polymorphic microsatellite loci set was nearly to 0.97 with 95% confidence. Based on this information, offspring from 10 crosses where parents were known were genotyped by the 6 microsatellite loci and used for parentage analysis. The result showed that assignment success of the progeny to their 'true' mother and father was 88% and 78% respectively, which were lower than predicted by the Cervus simulations. This could be explained by the existence of null or mutant alleles and by Taq DNA Polymerase slippage in the microsatellite loci.

[A preliminary study on the inheritance of microsatellite in two selective breeding families of shrimp (Litopenaeus vannamei)].

Two full-sib families of Litopenaeus vannamei were used to study the inheritance of allelic variation at 10 microsatellite loci. Of the 20 genotypic ratios observed (10 loci x 2 crosses), 17 ratios conformed to Mendelian segregation. When null alleles were considered, one loci (TUMXLv8.220) confirmed Mendelian expectations in all families. While one loci (TUMXLv3.1) showed deviation from family 06. Three loci (TUMXLv5.66,TUMXLv7.74,TUMXLv8.224)were monomorphic in both controlled crosses; 3 loci were polymorphic and confirmed to Mendelian ratios in all families, can be used for parental analysis and population genetic studies. These results indicated the need to test the inheritance pattern for microsatellite markers in shrimp before using them for population genetic or kinship analysis.