Transcriptome profile in heat resilient Pacific oyster Crassostrea gigas families under thermal challenge.

Since the introduction of the Pacific oyster Crassostrea gigas in Baja California Sur, Mexico, its culture has faced environmental challenges, specifically increasing temperatures that result in high mortalities. The inter-tidal zone seawater temperature during a year at the Baja California Peninsula broadly ranges from 7 °C to 39 °C. Therefore, to understand how oysters respond to heat stress during daily temperature oscillations, heat-resistant (RR, father, and mother resistant) and heat-susceptible (SS, both parents susceptible) phenotypes families from a C. gigas breeding program were exposed to a thermal challenge. Based on a laboratory-simulated daily oscillatory thermal challenge (26 to 34 °C) for 30 days, RR phenotype presented differences compared to SS phenotype since the beginning (day 0) of the thermal challenge. Gene expression analyses revealed 1822 differentially expressed up-regulated transcripts in RR, related to functions of metabolic processes, biological regulation, and response to stimulus and signaling. At the end of the experiment (day 30), 2660 differentially expressed up-regulated transcripts were identified in RR. Functional analysis of the genes expressed indicates responses of regulation of biological processes and response to a stimulus. Additionally, 340 genes were differentially expressed among RR vs. SS from the beginning to the end of the thermal challenge, where 170 genes were up-regulated, and 170 were down-regulated. These transcriptomic profiles represent the first report to identify gene expression markers associated with RR phenotypes for the Pacific oyster to the future broodstock selection.

Photobiomodulation therapy in management of cancer therapy-induced side effects: WALT position paper 2022.

Disclaimer: This article is based on recommendations from the 12th WALT Congress, Nice, October 3-6, 2018, and a follow-up review of the existing data and the clinical observations of an international multidisciplinary panel of clinicians and researchers with expertise in the area of supportive care in cancer and/or PBM clinical application and dosimetry. This article is informational in nature. As with all clinical materials, this paper should be used with a clear understanding that continued research and practice could result in new insights and recommendations. The review reflects the collective opinion and, as such, does not necessarily represent the opinion of any individual author. In no event shall the authors be liable for any decision made or action taken in reliance on the proposed protocols. Objective: This position paper reviews the potential prophylactic and therapeutic effects of photobiomodulation (PBM) on side effects of cancer therapy, including chemotherapy (CT), radiation therapy (RT), and hematopoietic stem cell transplantation (HSCT). Background: There is a considerable body of evidence supporting the efficacy of PBM for preventing oral mucositis (OM) in patients undergoing RT for head and neck cancer (HNC), CT, or HSCT. This could enhance patients' quality of life, adherence to the prescribed cancer therapy, and treatment outcomes while reducing the cost of cancer care. Methods: A literature review on PBM effectiveness and dosimetry considerations for managing certain complications of cancer therapy were conducted. A systematic review was conducted when numerous randomized controlled trials were available. Results were presented and discussed at an international consensus meeting at the World Association of photobiomoduLation Therapy (WALT) meeting in 2018 that included world expert oncologists, radiation oncologists, oral oncologists, and oral medicine professionals, physicists, engineers, and oncology researchers. The potential mechanism of action of PBM and evidence of PBM efficacy through reported outcomes for individual indications were assessed. Results: There is a large body of evidence demonstrating the efficacy of PBM for preventing OM in certain cancer patient populations, as recently outlined by the Multinational Association for Supportive Care in Cancer/International Society of Oral Oncology (MASCC/ISOO). Building on these, the WALT group outlines evidence and prescribed PBM treatment parameters for prophylactic and therapeutic use in supportive care for radiodermatitis, dysphagia, xerostomia, dysgeusia, trismus, mucosal and bone necrosis, lymphedema, hand-foot syndrome, alopecia, oral and dermatologic chronic graft-versus-host disease, voice/speech alterations, peripheral neuropathy, and late fibrosis amongst cancer survivors. Conclusions: There is robust evidence for using PBM to prevent and treat a broad range of complications in cancer care. Specific clinical practice guidelines or evidence-based expert consensus recommendations are provided. These recommendations are aimed at improving the clinical utilization of PBM therapy in supportive cancer care and promoting research in this field. It is anticipated these guidelines will be revised periodically.

A natural antisense transcript of the fem-1 gene was found expressed in female gonads during the characterization, expression profile, and cellular localization of the fem-1 gene in Pacific white shrimp Penaeus vannamei.

The fem-1 gene in Caenorhabditis elegans is involved in sex differentiation; it is specifically required for all aspects of male development. In this study, the full-length cDNA of the fem-1 (Pvfem-1) gene was isolated from the Pacific whiteleg shrimp Penaeus vannamei. The Pvfem-1 transcript is 3778 nt long and encodes a putative protein (PvFEM-1) of 638 amino acids that presented eight ankyrin repeats. The translated protein showed a significant (P < 0.05) structural similitude by superposition with C. elegans FEM-1 protein. Pvfem-1 expression was evaluated by qPCR and in situ hybridization (ISH) during embryogenesis, larval development, and gonads of both genders in subadult and adult life stages. Pvfem-1 was found expressed in brain, intestine, hepatopancreas, and in the gonads of both genders in subadults and adults when quantified by RT-qPCR. A significant finding was the discovery of a natural antisense transcript (NAT) of Pvfem-1 by ISH. It was present in the oocyte nucleus of subadult female shrimp gonads but was not seen within oocytes from adult females, although it was detected in follicular cells, suggesting a possible post-transcriptional regulation of Pvfem-1 in female gonad. Conversely, in males, no NAT was observed, and Pvfem-1 was found expressed in spermatogonia of both, subadult and adult shrimps indicating a function in male sexual differentiation and gametes generation. This study represents the first step for future functional analysis that is expected to contribute to clarifying the role of Pvfem-1 in sex differentiation and determination.

Chromosomal and molecular characterization of 5S rRNA genes in the North American abalones Haliotis rufescens Swainson (red abalone) and H. fulgens Philippi (blue abalone).

Abalone is an extremely valuable food source derived from cultured and wild animals, the later from populations under intense fishing exploitation and of high conservation value. As part of a long-term study to characterize genes from abalone that can be used as markers for hybrids certification, we characterised 5S ribosomal DNA (5S rDNA) in red abalone (Haliotis rufescens) and blue abalone (H. fulgens). The 5S rDNA arrays occur to a single pair of metacentric chromosomes at interstitial positions in both species. Two types of 5S genes were found, named types I and II, each associated with different non-transcribed spacer (NTS) sequences. The structure of the 5S rRNA genes and the NTS indicate incomplete homogenisation of the 5S rDNA arrays. The divergence of the 5S genes between species provide polymorphisms which can be used to distinguish red from blue abalone in forensic analysis of commercial production.

Isolation of the sex-determining gene Sex-lethal (Sxl) in Penaeus (Litopenaeus) vannamei (Boone, 1931) and characterization of its embryogenic, gametogenic, and tissue-specific expression.

The Pacific white shrimp Penaeus vannamei is the most cultured shrimp species around the world. Because females grow larger than males, the culture of 'only females' is of great interest, but knowledge on sex determination and differentiation is required for producing only females. In an effort to obtain information associated with reproduction in P. vannamei, transcriptomic data from female gonads was generated, and partial sequences of a transcript were identified as Sex-lethal (Sxl). Its characterization indicated that, differently from other penaeids in which this gene has been isolated, there are six isoforms of the Sxl transcript in P. vannamei (PvanSxl 1-6). These isoforms result from alternative splicing at three splice sites (SS1, SS2, SS3). The first splice-site is unique to P. vannamei, as it has not been reported for other Arthropod species; the second splice-site (SS2) is common among crustaceans, and the third splice-site (SS3) is also unique to P. vannamei and when spliced-out, it is always together with SS2. All isoforms are expressed during embryogenesis as well as gametogenesis of both genders. The two shorter isoforms, PvanSxl-5 and PvanSxl-6, which result from the splicing of SS2 and SS3, were found mostly expressed in adult testis, but PvanSxl-6 was also expressed in oocytes during gametogenesis. During oogenesis, the second largest isoform, PvanSxl-2, which splices-out only SS1, and PvanSxl-4 that splices-out SS1 and SS2 were highly expressed. These two isoforms were also highly expressed during embryonic development. In situ hybridization allowed pinpointing more specifically the cells where the PvanSxl transcripts were expressed. During embryogenesis, hybridization was observed from the one-cell stage embryo to late gastrula. In the female gonad in previtellogenesis, hybridization occurred in the nucleus of oocytes, whereas in secondary vitellogenesis the transcript also hybridized cytoplasmic granules and cortical crypts. Finally, in situ hybridization corroborated the expression of PvanSxl also in the male gonad during spermatogenesis, mostly occurring in the cytoplasm from spermatogonia and spermatocytes.

Sex determination and differentiation genes in a functional hermaphrodite scallop, Nodipecten subnodosus.

The lion-paw, Nodipecten subnodosus is one of three scallop species commercially exploited on the west coast of the Peninsula of Baja California. Because nothing is known about sex determination and sexual differentiation in hermaphrodite scallops, in the present work, a global transcriptomic analysis was performed in two early developmental stages, settling eyed-larvae and spat, as well as in three tissues (undifferentiated gonad, digestive gland, and adductor muscle). Over 27 million Illumina paired-end reads were obtained through the MiSeq platform. After processing the reads a total of 243,774 transcripts were assembled with an N50 of 980 and an average length of 775nt. A total of 43,252 proteins were inferred and 36,103 transcripts had at least one homolog in the SwissProt database according to a blastx search. After differential expression analyses and GO annotations it was possible to identify several sex-related genes in the scallop, including one known to be involved in the sex determination pathway of the hermaphrodite model organism Caenorhabditis elegans, N. subnodosus-sex1 (Ns-sex1). Other interesting sex determination and differentiation genes were Ns-dmrta2, Ns-sox9, Ns-wnt4, Ns-doa, Ns-ovo, Ns-vir, among others. Most of these genes were mainly expressed in the testis region, suggesting their participation in male gonad region sex differentiation. These results represent the first available information on the genetics of sex determination and differentiation in a functional hermaphrodite scallop.

Transcriptomic information from Pacific white shrimp (Litopenaeus vannamei) ovary and eyestalk, and expression patterns for genes putatively involved in the reproductive process.

The increased use of massive sequencing technologies has enabled the identification of several genes known to be involved in different mechanisms associated with reproduction that so far have only been studied in vertebrates and other model invertebrate species. In order to further investigate the genes involved in Litopenaeus vannamei reproduction, cDNA and SSH libraries derived from female eyestalk and gonad were produced, allowing the identification of expressed sequences tags (ESTs) that potentially have a role in the regulation of gonadal maturation. In the present study, different transcripts involved in reproduction were identified and a number of them were characterized as full-length. These transcripts were evaluated in males and females in order to establish their tissue expression profiles during developmental stages (juvenile, subadult and adult), and in the case of females, their possible association with gonad maturation was assessed through expression analysis of vitellogenin. The results indicated that the expression of vitellogenin receptor (vtgr) and minichromosome maintenance (mcm) family members in the female gonad suggest an important role during previtellogenesis. Additionally, the expression profiles of genes such as famet, igfbp and gpcr in brain tissues suggest an interaction between the insulin/insulin-like growth factor signaling pathway (IIS) and methyl farnesoate (MF) biosynthesis for control of reproduction. Furthermore, the specific expression pattern of farnesoic acid O-methyltransferase suggests that final synthesis of MF is carried out in different target tissues, where it is regulated by esterase enzymes under a tissue-specific hormonal control. Finally, the presence of a vertebrate type steroid receptor in hepatopancreas and intestine besides being highly expressed in female gonads, suggest a role of that receptor during sexual maturation.

A novel CHH gene from the Pacific white shrimp Litopenaeus vannamei was characterized and found highly expressed in gut and less in eyestalk and other extra-eyestalk tissues.

The crustacean hyperglycemic hormone (CHH) family is an important group of neuropeptides involved in controlling growth, reproduction, and stress response in decapod species. In this study, a new gene containing 4 exons-3 introns flanked by canonical 5'-GT-AG-3' intron splice-site junctions was isolated from Litopenaeus vannamei. Two full length transcripts of this CHH were isolated from eyestalk and pericardial tissue of males and females using rapid amplification of cDNA ends (RACE). Transcripts sequences were 1578bp in length in males pericardial tissues and in males and females eyestalk with 100% identity, but the transcript isolated from females pericardial tissues was shorter (974bp). The differences in transcripts length is a result of two polyadenylation sites present in the 3'UTR resulting in two transcription termination signals. Transcript sequences encoded one unique protein that can be classified as type I CHH subfamily because of the 4 exons and 3 introns structure, although the CPRP region is not-well conserved and there is no amidation in the C-terminal of the deduced amino acid sequence. Furthermore, there is a glycine inserted in the mature peptide not at position 12 as in type II CHHs but after amino acid 31 and the phylogenetic analysis did not group the peptide within type I, but closer to type II CHHs. We demonstrated by endpoint-PCR, qPCR, and in situ hybridization (ISH), that this gene is expressed in neuroendocrine organs known to express CHHs in penaeid shrimp, including X-organ and optic nerve in eyestalk, supraesophageal ganglion (SoG), but it is also expressed in other organs as gill, gut, pericardial cavity, as well as in terminal ampoule or spermatophore and vas deferens of males.

Expression of the myostatin gene in the adductor muscle of the Pacific lion-paw scallop Nodipecten subnodosus in association with growth and environmental conditions.

The cDNA sequence of the myostatin gene in the Pacific lion-paw Nodipecten subnodosus (Ns-mstn) was characterized, and the temporal expression during grow-out was analyzed for the first time in a scallop. Ns-mstn encodes a 459-amino-acid protein in which two propeptide proteolytic sites were identified, the previously recognized (RSKR) and a second one at position 266-269 aa (RRKR). The alternative furin cleavage site could be related with post-translational processing, or it could be a tissue-specific mechanism for signaling activity. The Ns-mstn transcript was located by in situ hybridization in sarcomeres and around the nucleus of muscle fibers. The temporal expression analysis by qPCR in the adductor muscle showed that Ns-mstn expression was significantly different (P < 0.05) between months during the grow-out period, increasing largely during the summer months when both biomass and muscle weight did not increase or even decreased; muscle fiber size and number were found to decrease significantly. Exogenous and endogenous factors such as high temperature and low food availability, as well as gametogenesis and reproduction, can be associated with the growth pattern and Ns-mstn expression changes. Our results indicate that MSTN is involved in adductor muscle growth regulation in N. subnodosus as it occurs in vertebrate skeletal muscle although Ns-mstn expression in non-muscle organs/tissues suggests additional functions.

Identification of male gametogenesis expressed genes from the scallop Nodipecten subnodosus by suppressive subtraction hybridization and pyrosequencing.

Despite the great advances in sequencing technologies, genomic and transcriptomic information for marine non-model species with ecological, evolutionary, and economical interest is still scarce. In this work we aimed to identify genes expressed during spermatogenesis in the functional hermaphrodite scallop Nodipecten subnodosus (Mollusca: Bivalvia: Pectinidae), with the purpose of obtaining a panel of genes that would allow for the study of differentially transcribed genes between diploid and triploid scallops in the context of meiotic arrest and reproductive sterility. Because our aim was to isolate genes involved in meiosis and other testis maturation-related processes, we generated suppressive subtractive hybridization libraries of testis vs. inactive gonad. We obtained 352 and 177 ESTs by clone sequencing, and using pyrosequencing (454-Roche) we maximized the identified ESTs to 34,276 reads. A total of 1,153 genes from the testis library had a blastx hit and GO annotation, including genes specific for meiosis, spermatogenesis, sex-differentiation, and transposable elements. Some of the identified meiosis genes function in chromosome pairing (scp2, scp3), recombination and DNA repair (dmc1, rad51, ccnb1ip1/hei10), and meiotic checkpoints (rad1, hormad1, dtl/cdt2). Gene expression analyses in different gametogenic stages in both sexual regions of the gonad of meiosis genes confirmed that the expression was specific or increased towards the maturing testis. Spermatogenesis genes included known testis-specific ones (kelch-10, shippo1, adad1), with some of these known to be associated to sterility. Sex differentiation genes included one of the most conserved genes at the bottom of the sex-determination cascade (dmrt1). Transcript from transposable elements, reverse transcriptase, and transposases in this library evidenced that transposition is an active process during spermatogenesis in N. subnodosus. In relation to the inactive library, we identified 833 transcripts with functional annotation related to activation of the transcription and translation machinery, as well as to germline control and maintenance.

Identification of a tubulin-α gene specifically expressed in testis and adductor muscle during stable reference gene selection in the hermaphrodite gonad of the lion's paw scallop Nodipecten subnodosus.

For non-model species, as many used for aquaculture, with minimal or no genomic information, relative quantification of gene expression studies requires preliminary research including the isolation of potential reference genes and the identification of those stably expressed under the biological conditions of interest. Here we report on the isolation of five partial gene sequences from gonad tissue cDNA in the functional hermaphrodite scallop Nodipecten subnodosus to be evaluated as reference genes: 18S-rRNA, riboprotein l8 (rp-l8), actin-ß (act-ß), elongation factor 1α (ef-1α) and alpha-tubulin-α (tub-α). We found that 18S-rRNA was stably expressed independently of the priming method used to reverse transcribe RNA to cDNA, oligo-dT or random hexamer. Stability analysis for the five putative reference genes with geNorm and NormFinder indicated that 18S together with rp-l8 were the most stable genes for normalization of gene expression during gonad development in both, male and female sexual regions of the hermaphrodite N. subnodosus. The least stable gene was tub-α, showing a biased expression profile between sexual regions of the gonad, therefore this gene was analyzed thereafter as a target gene together with vitellogenin (vit) and a DEAD-box RNA helicase (dbx) gene. Relative expression, estimated by normalization with the combination of 18S and rp-l8 as reference genes, indicated that as gonad development advanced two of the target genes were up-regulated, tub-α in the male region and vit in the female region. Whereas an increased expression was expected during development for vit for its known role in vitellogenesis, the increased expression of tub-α in the male sexual region was unexpected, and pointed toward this gene being a testis-specific α-tubulin isotype. Further analyses of gene expression among tissues indicated that tub-α is specifically and highly expressed in the male gonad, although expression in adductor muscle was also observed at significantly lower levels. The existence of testis specific α- and ß-tubulins has been previously reported in other taxa, relating their function to sperm axoneme formation. Tissue-specific tubulin genes, particularly their promoters, have recently found an application as native promoters for transgene tissue-specific expression in research and reproductive control of insect plagues. The third target gene, a putative member of the DEAD-box RNA helicase family (dbx), showed no changes in expression during gonad development or between sexual regions, therefore it was chosen to discuss the different statistical inferences resulting from the arbitrary use of 'randomly chosen' reference genes when normalizing gene expression.

Allotriploid genotypic assignment in abalone larvae by detection of microsatellite-recombinant genotypes.

Abalone species are different from most mollusks utilized in aquaculture as they are known to hybridize in laboratory-induced matings. Allotriploidization of hybrid abalone has not yet been studied, and methodology useful in verifying the genotypic condition of such allotriploids do not exist. Genotypic verification of hybridization and allotriploidization in a cross of Haliotis fulgens and Haliotis rufescens was performed utilizing 6 crossamplifying microsatellite loci. Five H. rufescens spawns were used in this experiment, dividing each spawn into control and experimental hybrid groups and further into diploids and triploids. Two microsatellite loci developed for H. fulgens and H. rufescens allowed for the genotypic identification of hybrids within diploid and triploids. To further verify the percentage of allotriploids within the genotypic hybrids in the triploid hybrid groups, microsatellite loci originally developed in Haliotis corrugata and Haliotis kamtschatkana were tested for crossamplification in H. fulgens and H. rufescens. Of 21 loci, 4 were chosen for this study based on their crossamplification, heterozygosity in the females, and centromere recombination frequencies. Allotriploids in triploid-hybrid larvae were then detected by identifying larvae with recombinant genotypes at any of those loci. One family had low success verification associated with reduced recombination frequencies for all loci in that family. These results demonstrate that allotriploid verification at larval stages is feasible but depends on the number of loci available, their crossamplification in the species, and their recombination frequencies.

Genotype by environment interaction for adult body weights of shrimp Penaeus vannamei when grown at low and high densities.

Shrimp is one of few marine species cultured worldwide for which several selective breeding programs are being conducted. One environmental factor that can affect the response to selection in breeding programs is the density at which the shrimp are cultured (low-medium-high). Phenotypic plasticity in the growth response to different densities might be accompanied by a significant genotype by environment interaction, evidenced by a change in heritabilities between environments and by a genetic correlation less than one for a unique trait between environments. Our goal was to understand whether different growth densities affect estimates of those genetic parameters for adult body weight (BW) in the Pacific white shrimp (Penaeus vannamei). BW heritabilities were significantly different between environments, with the largest at high density. These differences resulted from both an increased additive genetic variance and a decreased environmental variance when grown at high density. The genetic correlation between BWs at the two environmental conditions was significantly less than one. Whereas these results might be suggestive for carrying out shrimp selective breeding for BW under high density conditions, further understanding of genetic correlations between growth and reproductive traits within a given environment is necessary, as there are indications of reduced reproductive fitness for shrimp grown at high densities.

Fluorescent in situ hybridization and characterization of the SalI family of satellite repeats in the Haliotis L. species (abalone) of the Northeast Pacific.

The SalI satellite repeat previously identified in Haliotis L. (abalone) was thought to be present in H. rufescens and absent in H. fulgens. However, we show here that SalI is also found in H. fulgens and is not useful for screening hybrid individuals bred in aquaculture or occurring naturally in the wild. SalI is a family of predominantly subtelomeric tandemly repeated sequences, and sequenced clones revealed clustering to species and little intraspecific variation. Analysis of SalI sequence divergence between Haliotis species of the Northeast Pacific revealed that evolutionary distances correlate well with bathymetric and latitudinal species distributions. Analysis of the structure of the tandem repeats revealed two regions of high sequence conservation that may contain conserved transcription factor binding sites, a surprise for an apparently "non-coding" tandem repeat. We speculate that these regions might be involved in heterochromatin silencing, perhaps mediated via transcriptional activity and RNA interference. The repeats show substantial differences in their chromosomal distributions, even between individuals of the same species, indicating a dynamic organization of repeats, perhaps mediated via sequence homogenization.

An induced mass spawn of the hermaphroditic lion-paw scallop, Nodipecten subnodosus: genetic assignment of maternal and paternal parentage.

The Pacific lion-paw scallop is commonly propagated for aquaculture by induced mass spawns of few individuals. Parentage of a mass spawn of this species has not been evaluated nor has the maternal and paternal contribution of each of these functional hermaphrodites to the progeny. Genotypes of 6 spawners and 374 resulting progeny at 6 microsatellite loci were coupled with mitochondrial DNA sequencing to assign maternal and paternal parentage. After the identification of a high proportion of null alleles (9.7%), microsatellite data revealed that 51.7% of the progenies were full siblings, with a significant, unequal contribution of the 6 spawners to the progeny. Three progenies were the result of self-fertilization. All spawners contributed paternally (though unequally); however, 2 spawners were the maternal parents of all but 7 progenies resulting in a variance effective population size of 3.52. DNA sequencing confirmed 4 microsatellite mutations within 4476 alleles scored, all in the paternal germ line. With minor exception, the loci conformed to Mendelian rules of segregation when null alleles were accounted for, and 2 loci were found to be linked. These results lend insight to the genetic composition of induced mass spawns and provide a basis for the development of more effective spawning techniques.

Genetic variability assessed by microsatellites in a breeding program of Pacific white shrimp (Litopenaeus vannamei).

Genetic diversity in a shrimp-breeding program was monitored for 2 generations by microsatellite DNA markers (Pvan1578 and Pvan1815) to establish levels of variation and proceed with a selection program. An increase in the number and frequencies of some alleles in both microsatellite loci from G0 to G2 was induced by foreign sire contributions. Most common alleles and high heterozygosities (around 70% in both loci) were maintained through the generations, indicating that there had not been a significant loss of genetic variability in the breeding program. However, when compared with variability in other wild and cultured stocks, the presence of 4 main alleles at both loci may be an indication that a certain reduction in variability already was present in the line used as founder stock (G0). Therefore, it is recommended that additional genetic variability be introduced to the breeding stock by crossing it with a different line.