The DIC carbon isotope evolutions during CO2 bubbling: implications for ocean acidification laboratory culture

Ocean acidification increases pCO2 and decreases pH of seawater and its impact on marine organisms has emerged as a key research focus. In addition to directly measured variables such as growth or calcification rate, stable isotopic tracers such as carbon isotopes have also been used to more completely understand the physiological processes contributing to the response of organisms to ocean acidification. To simulate ocean acidification in laboratory cultures, direct bubbling of seawater with CO2 has been a preferred method because it adjusts pCO2 and pH without altering total alkalinity. Unfortunately, the carbon isotope equilibrium between seawater and CO2 gas has been largely ignored so far. Frequently, the dissolved inorganic carbon (DIC) in the initial seawater culture has a distinct 13C/12C ratio which is far from the equilibrium expected with the isotopic composition of the bubbled CO2. To evaluate the consequences of this type of experiment for isotopic work, we measured the carbon isotope evolutions in two chemostats during CO2 bubbling and composed a numerical model to simulate this process. The isotopic model can predict well the carbon isotope ratio of dissolved inorganic carbon evolutions during bubbling. With help of this model, the carbon isotope evolution during a batch and continuous culture can be traced dynamically improving the accuracy of fractionation results from laboratory culture. Our simulations show that if not properly accounted for in experimental or sampling design, many typical culture configurations involving CO2 bubbling can lead to large errors in estimated carbon isotope fractionation between seawater and biomass or biominerals, consequently affecting interpretations and hampering comparisons among different experiments. Therefore, we describe the best practices on future studies working with isotope fingerprinting in the ocean acidification background.

Morphological ontogeny of Neoseiulus zwoelferi (Acari: Phytoseiidae)

The morphological ontogeny of Neoseiulus zwoelferi (Dosse, 1957) (Acari: Phytoseiidae) was studied based on specimens from a laboratory culture originally collected from the leaves of Xanthium sibiricum Patrin ex Widder (Asteraceae) in Taigu County, Shanxi province, China. All life stages including larva, protonymph, deutonymphal female and male, adult female and male were described and illustrated. The morphological characters of Neoseiulus species with both immature and adult stages are discussed. Neoseiulus subreticulatus (Wu, 1987) is considered a junior synonym of Neoseiulus zwoelferi (Dosse, 1957).

B Chromosomes in Free-Living Flatworms of the Genus Macrostomum (Platyhelminthes, Macrostomorpha)

B chromosomes (Bs) or supernumerary chromosomes are extra chromosomes in the species karyotype that can vary in its copy number. Bs are widespread in eukaryotes. Usually, the Bs of specimens collected from natural populations are the object of the B chromosome studies. We applied another approach analyzing the Bs in animals maintained under the laboratory conditions as lines and cultures. In this study, three species of the Macrostomum genus that underwent a recent whole-genome duplication (WGD) were involved. In laboratory lines of M. lignano and M. janickei, the frequency of Bs was less than 1%, while in the laboratory culture of M. mirumnovem, it was nearer 30%. Their number in specimens of the culture varied from 1 to 14. Mosaicism on Bs was discovered in parts of these animals. We analyzed the distribution of Bs among the worms of the laboratory cultures during long-term cultivation, the transmission rates of Bs in the progeny obtained from crosses of worms with different numbers of Bs, and from self-fertilized isolated worms. The DNA content of the Bs in M. mirumnovem was analyzed with the chromosomal in situ suppression (CISS) hybridization of microdissected DNA probes derived from A chromosomes (As). Bs mainly consisted of repetitive DNA. The cytogenetic analysis also revealed the divergence and high variation in large metacentric chromosomes (LMs) containing numerous regions enriched for repeats. The possible mechanisms of the appearance and evolution of Bs and LMs in species of the Macrostomum genus were also discussed.

Increased Pathogenicity of the Nematophagous Fungus Drechmeria coniospora Following Long-Term Laboratory Culture

Domestication provides a window into adaptive change. Over the course of 2 decades of laboratory culture, a strain of the nematode-specific fungus Drechmeria coniospora became more virulent during its infection of Caenorhabditis elegans. Through a close comparative examination of the genome sequences of the original strain and its more pathogenic derivative, we identified a small number of non-synonymous mutations in protein-coding genes. In one case, the mutation was predicted to affect a gene involved in hypoxia resistance and we provide direct corroborative evidence for such an effect. The mutated genes with functional annotation were all predicted to impact the general physiology of the fungus and this was reflected in an increased in vitro growth, even in the absence of C. elegans. While most cases involved single nucleotide substitutions predicted to lead to a loss of function, we also observed a predicted restoration of gene function through deletion of an extraneous tandem repeat. This latter change affected the regulatory subunit of a cAMP-dependent protein kinase. Remarkably, we also found a mutation in a gene for a second protein of the same, protein kinase A, pathway. Together, we predict that they result in a stronger repression of the pathway for given levels of ATP and adenylate cyclase activity. Finally, we also identified mutations in a few lineage-specific genes of unknown function that are candidates for factors that influence virulence in a more direct manner.

A simple method to produce fragment seedstock for aquaculture of Pterocladiella capillacea (Gelidiales, Rhodophyta)

Raw material of gelidioid red algae yielding high-quality agar has been in short supply due to overharvesting, but in situ farming of gelidioids has not been practical due to their slow growth. To produce vegetative seedstock of a cosmopolitan species, Pterocladiella capillacea, we investigated the number and length of regenerated branches arising from sectioned fragments during 3 weeks of laboratory culture at 10, 15, 20, and 25°C. All sectioned fragments formed axis-like branches mostly from the upper cut edge and stolon-like branches mostly from the lower cut edge, showing a high capacity of regeneration and intrinsic bipolarity. At 20°C, the number of regenerated branches increased to 2.74 ± 1.29 on the upper cut edge and 4.26 ± 2.66 on the lower cut edge. Our study reveals that the use of fragments bearing regenerated branches as seedstock can be a simple method to initiate fast propagation for mass cultivation in the sea or outdoor tank.

The Lesser Pacific Striped Octopus, Octopus chierchiae: An Emerging Laboratory Model

Cephalopods have the potential to become useful experimental models in various fields of science, particularly in neuroscience, physiology, and behavior. Their complex nervous systems, intricate color- and texture-changing body patterns, and problem-solving abilities have attracted the attention of the biological research community, while the high growth rates and short life cycles of some species render them suitable for laboratory culture. Octopus chierchiae is a small octopus native to the central Pacific coast of North America whose predictable reproduction, short time to maturity, small adult size, and ability to lay multiple egg clutches (iteroparity) make this species ideally suited to laboratory culture. Here we describe novel methods for multigenerational culture of O. chierchiae, with emphasis on enclosure designs, feeding regimes, and breeding management. O. chierchiae bred in the laboratory grow from a 3.5 mm mantle length at hatching to an adult mantle length of approximately 20–30 mm in 250–300 days, with 15 and 14% survivorship to over 400 days of age in first and second generations, respectively. O. chierchiae sexually matures at around 6 months of age and, unlike most octopus species, can lay multiple clutches of large, direct-developing eggs every ∼30–90 days. Based on these results, we propose that O. chierchiae possesses both the practical and biological features needed for a model octopus that can be cultured repeatedly to address a wide range of biological questions.

B Chromosomes in Free-Living Flatworms of the Genus Macrostomum (Platyhelminthes, Macrostomorpha)

B chromosomes (Bs) or supernumerary chromosomes are extra chromosomes in the species karyotype that can vary in their copy number. Bs are widespread. Usually, the Bs of specimens collected from natural populations are involved in studies. We applied another approach analyzing the Bs in animals of laboratory cultures. In this study, three species of the Macrostomum genus that underwent a recent whole-genome duplication (WGD) were involved. In laboratory lines of M. lignano and M. janickei, the frequency of Bs was less than 1%, while in the laboratory culture of M. mirumnovem, it was nearer 30%. Their number in specimens of the culture varied from 1 to 14. Mosaicism on Bs was discovered in parts of these animals. We analyzed the distribution of Bs among the worms of the laboratory cultures during long-term cultivation, the transmission rates of Bs in the progeny obtained from crosses of worms with different numbers of Bs, and from self-fertilized isolated worms. The DNA content of the Bs in M. mirumnovem was analyzed with the chromosomal in situ suppression (CISS) hybridization of microdissected DNA probes derived from A chromosomes (As). Bs mainly consisted of repetitive DNA. The cytogenetic analysis also revealed the divergence and high variation in large metacentric chromosomes (LMs) containing numerous regions enriched for repeats. The possible mechanisms of the appearance and evolution of Bs and LMs in species of the Macrostomum genus were also discussed.

Role and Regulation of Poly-3-Hydroxybutyrate in Nitrogen Fixation in Azorhizobium caulinodans

An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR native RBS is increased approximately sixfold in the phaC mutant background, suggesting that the level of PhaR is controlled by PHB. Translation from the phaR mutated RBS (RBS*) of the suppressor mutant strain (OPS0921) is locked at a low basal rate and unaffected by the phaC mutation, suggesting that RBS* renders the level of PhaR insensitive to regulation by PHB. In the original phaC mutant (OPS0865), the lack of nitrogen fixation and poor growth on many carbon sources is likely to be due to increased levels of PhaR causing dysregulation of its complex regulon, because PHB formation, per se, is not required for effective nitrogen fixation in A. caulinodans. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Culturomics revealed the bacterial constituents of the microbiota of a 10-year-old laboratory culture of planarian species S. mediterranea

The planarian species Schmidtea mediterranea is a flatworm living in freshwater that is used in the research laboratory as a model to study developmental and regeneration mechanisms, as well as antibacterial mechanisms. However, the cultivable microbial repertoire of the microbes comprising its microbiota remains unknown. Here, we characterized the bacterial constituents of a 10-year-old laboratory culture of planarian species S. mediterranea via culturomics analysis. We isolated 40 cultivable bacterial species, including 1 unidentifiable species. The predominant phylum is Proteobacteria, and the most common genus is Pseudomonas. We discovered that parts of the bacterial flora of the planarian S. mediterranea can be classified as fish pathogens and opportunistic human pathogens.