A rapid and simple procedure for the whole-mount bone staining of small fish.

The cartilage and bone structure has provided insightful knowledge about evolution and ecology of fish, which is an important component of biological oceanography. However, the whole-body bone staining is a lengthy and complicated process that typically takes five days to several months, and the improvement of the conventional method has been one of the important issues in this field. Here we report a quick and easy whole-mount bone staining method for small fish, in which a newly designed fixative is applied. Compared to conventional methods, this novel protocol is a straightforward process that could be adopted for small estuarine fish and other small vertebrates.

Integration of Multi-Omics, Histological, and Biochemical Analysis Reveals the Toxic Responses of Nile Tilapia Liver to Chronic Microcystin-LR Exposure.

Microcystin-LR (MC-LR) is a cyanobacterial metabolite produced during cyanobacterial blooms and is toxic to aquatic animals, and the liver is the main targeted organ of MC-LR. To comprehensively understand the toxicity mechanism of chronic exposure to environmental levels of MC-LR on the liver of fish, juvenile Nile tilapia were exposed to 0 µg/L (control), 1 µg/L (M1), 3 µg/L (M3), 10 µg/L (M10), and 30 µg/L (M30) MC-LR for 60 days. Then, the liver hepatotoxicity induced by MC-LR exposure was systematically evaluated via histological and biochemical determinations, and the underlying mechanisms were explored through combining analysis of biochemical parameters, multi-omics (transcriptome and metabolome), and gene expression. The results exhibited that chronic MC-LR exposure caused slight liver minor structural damage and lipid accumulation in the M10 group, while resulting in serious histological damage and lipid accumulation in the M30 group, indicating obvious hepatotoxicity, which was confirmed by increased toxicity indexes (i.e., AST, ALT, and AKP). Transcriptomic and metabolomic analysis revealed that chronic MC-LR exposure induced extensive changes in gene expression and metabolites in six typical pathways, including oxidative stress, apoptosis, autophagy, amino acid metabolism, primary bile acid biosynthesis, and lipid metabolism. Taken together, chronic MC-LR exposure induced oxidative stress, apoptosis, and autophagy, inhibited primary bile acid biosynthesis, and caused fatty deposition in the liver of Nile tilapia.

Eicosapentaenoic acid (EPA) improves grass carp (Ctenopharyngodon idellus) muscle development and nutritive value by activating the mTOR signaling pathway.

Skeletal muscle is the mainly edible part of fish. Eicosapentaenoic acid (EPA) is a crucial nutrient for fish. This study investigated the effect of EPA on the muscle development of grass carp along with the potential molecular mechanisms in vivo and in vitro. Muscle cells treated with 50 µM EPA in vitro showed the elevated proliferation, and the expression of mammalian target of rapamycin (mTOR) signaling pathway-related genes was upregulated (P < 0.05). In vivo experiments, 270 grass carp (27.92 g) were fed with one of the three experimental diets for 56 days: control diet (CN), 0.3% EPA-supplement diet (EPA), and the diet supplemented with 0.3% EPA and 30 mg/kg rapamycin (EPA + Rap). Fish weight gain rate (WGR) was improved in EPA group (P < 0.05). There was no difference in the viscerosomatic index (VSI) and body height (BH) among all groups (P > 0.05), whereas the carcass ratio (CR) and body length in the EPA group were obviously higher than those of other groups (P < 0.05), indicating that the increase of WGR was due to muscle growth. In addition, both muscle fiber density and muscle crude protein also increased in EPA group (P < 0.05). The principal component analysis showed that total weight of muscle amino acid in EPA group ranked first. Dietary EPA also increased protein levels of the total mTOR, S6k1, Myhc, Myog, and Myod in muscle (P < 0.05). In conclusion, EPA promoted the muscle development and nutritive value via activating the mTOR signaling pathway.

Comparative digestive biology between the ponyfishes from the Pranburi River estuary, Thailand.

We investigated the digestive biology of two prevalent leiognathid species in Pranburi River estuary, Thailand: the decorated ponyfish (Nuchequula gerreoides) and the splendid polyfish (Eubleekeria splendens). A total of 632 samples collected from February to April and September to November 2017 were analysed using morphological and histological approaches. The overall structures were similar between the species: a short mucous-cell-rich oesophagus region, a well-developed gastric gland uniformly present across the stomach's mucosal layer, and three finger-like pyloric caeca between the stomach and intestine. However, there were marked differences in the mouth, gill raker, and intestinal coefficient (IC). N. gerreoides had a relatively longer mouth, smoother gill rakers, and an IC of 1.08 ± 0.01, similar to those of other carnivorous fish. In contrast, the gill raker of E. splendens had more villiform teeth that can filter-feed better, and their IC was 2.16 ± 0.02 (i.e., longer intestine). Although digestive structures were generally similar between the ponyfishes, these differences suggest that N. gerreoides is relatively carnivorous with stronger suction, whereas E. splendens may be an omnivorous or herbivorous filter-feeder.

Comprehensive mRNA and microRNA analysis revealed the effect and response strategy of freshwater fish, grass carp (Ctenopharyngodon idella) under geosmin exposure.

Geosmin is an environmental pollutant that causes off-flavor in water and aquatic products. The high occurrence of geosmin contamination in aquatic systems and aquaculture raises public awareness, however, few studies have investigated the response pathways of geosmin stress on freshwater fish. In this research, grass carp were exposed to 50 µg/L geosmin for 96 h, liver tissue was sequenced and validated using real-time qPCR. In total of 528 up-regulated genes and 488 down-regulated genes were observed, includes cytochrome P450 and uridine diphosphate (UDP)-glucuronosyltransferase related genes. KEGG analysis showed that chemical carcinogenesis-DNA adducts, metabolism of xenobiotics by cytochrome P450, drug metabolism-cytochrome P450 pathway was enriched. Common genes from the target genes of microRNAs and differential expression genes are enriched in metabolism of xenobiotics cytochrome P450 pathway. Two miRNAs (dre-miR-146a and miR-212-3p) down regulated their target genes (LOC127510138 and adh5, respectively) which are enriched cytochrome P450 related pathway. The results present that geosmin is genetoxic to grass carp and indicate that cytochrome P450 system and UDP-glucuronosyltransferase play essential roles in biotransformation of geosmin. MicroRNAs regulate the biotransformation of geosmin by targeting specific genes, which contributes to the development of strategies to manage its negative impacts in both natural and artificial environments.

Histological structure of the digestive tract, liver, and pancreas of Ambassis nalua (Hamilton, 1822) with ultrastructural details of the gastric gland.

The scalloped perchlet Ambassis nalua is one of the dominant fishes in the Estuarine Pranburi River, Thailand. It is suggested that this fish is in the secondary trophic level with a carnivorous nature. Studies on digestive system will help us further identify the niche of this species in the food web/food chain. The present study therefore aimed to report the detailed structure and ultrastructure of A. nalua digestive system. Fish samples (n = 30) with a total length of 5.7 ± 0.5 cm were obtained using beach seines from the Estuarine Pranburi River. Their digestive tract length and intestine coeficient were 3.6 ± 0.07 cm and 0.91, respectively. Light microscopic observation showed that the digestive wall comprised four layers, namely mucosa, submucosa, muscularis, and serosa. The prominent mucous-secreting cells were found in the mucosal oesophagus. The stomach had many gastric folds, with height and width being 649.76 ± 85.15 and 370.30 ± 68.56 µm, respectively. Gastric glands were found in the anterior stomach but not in the posterior stomach. Each gastric gland was made up of a single type of columnar cells. The gastric cells were ultrastructurally characterized by numerous mitochondria and well-developed secretory granules of varying sizes. A few small vacuoles were also identified in the apical area of the gastric cells. The intestine had two regions (anterior and posterior intestines), and pyloric caecum was absent. The density of the goblet cell was significantly higher in the posterior intestine. These results provide basic knowledge of the digestive system of A. nalua, and the low intestine coefficient and the absence of pyloric caecum suggest the carnivorous feeding habit of this species.

Docosahexaenoic acid (DHA) inhibits abdominal fat accumulation by promoting adipocyte apoptosis through PPARγ-LC3-BNIP3 pathway-mediated mitophagy.

Obesity has always been an overwhelming health concern worldwide. Docosahexaenoic acid (DHA) reduces abdominal fat accumulation by inducing adipocyte apoptosis, but the underlying mechanism remains unclear. Mitophagy, the process of maintaining mitochondrial homeostasis, has a double-edged sword effect that positively or negatively regulates apoptosis. In this study, grass carp (Ctenopharyngodon idellus) was used as an animal model to investigate the role of mitophagy in regulating apoptosis and the potential molecular mechanisms for DHA-induced mitophagy in vivo and in vitro. Firstly, we found that DHA induced the intrinsic apoptosis in grass carp adipocytes, accompanying by activating BNIP3/NIX-mediated mitophagy. Then, suppression of mitophagy alleviated apoptosis and eliminated the inhibition of lipid accumulation induced by DHA in vivo and in vitro. Mechanistically, the DHA-induced mitophagy was caused by activating PPARγ and its DNA binding capacity to the LC3 promoter, which promoted the interaction of BNIP3 (rather than NIX) with LC3. However, the inhibition of PPARγ in vitro significantly decreased the expression of autophagy-related genes (P < 0.05), reducing the colocalization of mitochondria and lysosomes while preventing BNIP3/NIX-mediated mitophagy-mediated apoptosis and subsequently alleviating the inhibition of lipid accumulation in adipocytes induced by DHA. For the first time, we demonstrated that DHA activates mitophagy by regulating the PPARγ-LC3-BNIP3 pathway, consequently inducing apoptosis, which decreases adipocytes, inhibiting lipid accumulation in grass carp. These findings provide new insight into the mechanism of DHA-induced apoptosis mediated by mitophagy as the potential therapeutic target of inhibiting abdominal fat accumulation in vertebrates.

Effects of thermal stress responses in goldfish (Carassius auratus): growth performance, total carotenoids and coloration, hematology, liver histology, and critical thermal maximum.

The present study aimed to investigate the effect of thermal stress on growth, feed utilization, coloration, hematology, liver histology, and critical thermal maximum (CTmax) in goldfish (Carassius auratus) cultured at three different acclimation temperatures including 27 °C, 30 °C, and 34 °C for 10 weeks. Goldfish were assigned randomly to tanks with a quadruplicate setup, accommodating 20 fish per tank. The result showed that fish acclimated to different temperatures did not significantly differ in weight gain (WG) and specific growth rate (SGR). However, increasing temperature significantly decreased feed efficiency ratio (FER), protein efficiency ratio (PER), and protein productive value (PPV), but significantly increased feed conversion ratio (FCR) (P < 0.05). The coloration parameters significantly decreased by high temperature in the trunk region with increasing temperature (L* and a* at week 5; L*, a*, and b* at week 10; P < 0.05). Total carotenoid contents in serum, fin, muscle, and skin also significantly decreased with increasing temperature (P < 0.05). Total protein, albumin, and globulin levels exhibited a notable decrease, while the albumin: globulin ratio showed a slight insignificant increase, with increasing temperature. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol, and triglycerides significantly increased with increasing temperature (P < 0.05). While, high-density lipoprotein cholesterol (HDL-c) decreased linearly (P < 0.05). Glucose and cortisol levels linearly increased with increasing temperature, the highest levels being observed in the 34 °C group. Liver histology showed swollen hepatocytes, nuclei displacement, and infiltration of inflammation in fish cultured at 34 °C. Goldfish acclimated to 34 °C displayed a higher CTmax of 43.83 °C compared to other groups. The present study showed that temperature should be kept below 34 °C for goldfish culture to prevent high FCR, fading coloration, and liver damages.

Genomic loss of the HSP70cA gene in the vertebrate lineage.

Metazoan 70 kDa heat shock protein (HSP70) genes have been classified into four lineages: cytosolic A (HSP70cA), cytosolic B (HSP70cB), endoplasmic reticulum (HSP70er), and mitochondria (HSP70m). Because previous studies have identified no HSP70cA genes in vertebrates, we hypothesized that this gene was lost on the evolutionary path to vertebrates. To test this hypothesis, the present study conducted a comprehensive database search followed by phylogenetic and synteny analyses. HSP70cA genes were found in invertebrates and in two of the three subphyla of Chordata, Cephalochordata (lancelets) and Tunicata (tunicates). However, no HSP70cA gene was found in the genomes of Craniata (another subphylum of Chordata; lamprey, hagfish, elephant shark, and coelacanth), suggesting the loss of the HSP70cA gene in the early period of vertebrate evolution. Synteny analysis using available genomic resources indicated that the synteny around the HSP70 genes was generally conserved between tunicates but was largely different between tunicates and lamprey. These results suggest the presence of dynamic chromosomal rearrangement in early vertebrates that possibly caused the loss of the HSP70cA gene in the vertebrate lineage.

Effects of heat stress on the chemical composition, oxidative stability, muscle metabolism, and meat quality of Nile tilapia (Oreochromis niloticus).

The present study investigated the effects of chronic heat stress (HS) on the chemical composition, oxidative stability, muscle metabolism, and meat quality of Nile tilapia (Oreochromis niloticus). Compared with the control (26 °C), chronic HS (32 °C) lowered growth performance, the contents of whole-body lipid, muscle protein, and muscle lipid. Also, HS significantly increased the contents of reactive oxygen species (ROS) and decreased antioxidative status, causing a decline in meat quality, including increased lipid and protein oxidation, the centrifugal water loss, and cooking loss as well as decreased the fragmentation index and pH at 24 h, which may be attributed to induced apoptosis by excessive ROS in Nile tilapia meat. Moreover, metabolomic analysis showed HS lowered flavor and nutritional value by affecting amino acid, lipid, and nucleotide metabolism. These results reveal that HS adversely affects oxidative stability, meat quality, flavor, and nutrition, warranting its recognition and prevention.

A genome-wide screening of the 70 kDa heat shock protein (HSP70) genes in the rotifer Brachionus plicatilis sensu stricto with a characterization of two heat-inducible HSP70 genes.

The 70 kDa heat shock proteins (HSP70s) and the constitutive members of the HSP70 family (heat shock cognates; HSC70s) play essential roles in various biological processes. The number of hsp70/hsc70 in the database is rapidly increasing because of their importance and the automatic annotation of newly sequenced genomes. However, accumulating evidence indicates that neither hsp70 nor hsc70 forms a monophyletic gene family, raising the need to reconsider the annotation strategy based on the traditional concept of the inducible HSP70 and constitutive HSC70s. The main aim of this study is to establish a systematic scheme to annotate hsp70-like genes taking the latest phylogenetic insights into account. We cloned two hsp70s from the rotifer Brachionus plicatilis sensu stricto (s.s.), an emerging model in evolutionary genetics, and conducted a genome-wide screening of B. plicatilis s.s. hsp70s using the two sequences as queries. A total of 15 hsp70-like genes were found, and 7 of them encoded distant members of the HSP70 family, the function of which largely remains unknown. Eight canonical hsp70s were annotated according to a recently proposed nomenclature based on the molecular evolution: e.g., HSP70cA1/B1 for the cytosolic lineage, HSP70er1 for the endoplasmic reticulum lineage, and HSP70m1 for the mitochondrial lineage. The two cloned hsp70s, HSP70cB1 and HSP70cB2, ubiquitously increased their mRNA levels up to 7.5 times after heat treatment as assessed by semi-quantitative PCR, real-time PCR, and in situ hybridization. This systematic screening incorporating a reasonable update to the annotation strategy would provide a useful example for future HSP70 studies, especially those in non-traditional model organisms.

Comprehensive Structure of the Female Marine Water-Strider Asclepios annandalei Distant, 1915 from Pranburi River Estuary, Thailand: New Information for the Genus Asclepios.

The objective of this study was to describe the structure and histochemistry of the systemic organs in the female marine water-strider Asclepios annandalei from Pranburi river estuary, Thailand. Results from this study revealed for the first time that the integumentary system of this species consisted of three layers including epicuticle, exocuticle and endocuticle. The muscular system apparently contained only skeletal muscle along the body. In the urinary system, we observed well-developed Malpighian tubules, each of which was covered with the simple cuboidal epitheliums. These epitheliums also contained the secretory granules that were reacted positively with periodic acid Schiff (PAS). The digestive system of this species was composed of three distinct parts including foregut, midgut and hindgut. The respiratory system was composed of the respiratory organ, which was rarely found near the integument system. This organ was lined with a simple squamous epithelium. Two regions of nervous system, i.e., frontal ganglion connected to the eye structure and ventral nerve cord, were found. Each ganglion basically consisted of two layers, outer cortex and inner medullae. The outer cortex contained three types of cells, including neurosecretory cells, neuroglial cells and neurons. The cytoplasmic inclusion of neurosecretory cells contained secretory granules, which reacted positively with PAS, indicating the presence the glycoprotein. The neuroglia and neuron were also observed in the inner medullae layer. The female reproductive system (the ovarian structure, the reproductive tract and the accessory organ) of this gerrid species was seen under histological sections. The well-developed integument system and Malpighian tubule as well as the abundant respiratory organ is a characteristic of this species, which might be useful for the adaption to the estuarine condition.

Phylogenetic annotation of Caenorhabditis elegans heat shock protein 70 genes.

Annotation of the 70 kDa heat shock proteins (Hsp70s) has been chaotic especially in invertebrates. In this study, we validated an emerging nomenclature of Hsp70s, which can be potentially applied to all metazoan Hsp70s, by conducting a genome-wide annotation of Caenorhabditis elegans Hsp70s. Using the phylogenetic annotation, the seven canonical C. elegans Hsp70s were successfully classified into four known lineages, cytosolic A, cytosolic B, endoplasmic reticulum, and mitochondria. Motifs specific to each lineage were all conserved in the C. elegans Hsp70s. From these results, we propose new aliases of C. elegans Hsp70s that should help future annotation of this important molecular chaperone.

Phylogenetic annotation of Drosophila melanogaster heat shock protein 70 genes.

The traditional classification of stress-inducible 70 kDa heat shock protein (Hsp70) and heat shock cognate (Hsc70) requires a revision because of a recent finding that neither of them constitutes a monophyletic gene family. Here we inferred a phylogenetic relationship among Drosophila melanogaster Hsp70 family members. D. melanogaster Hsp70 family members were separated into four known metazoan Hsp70 lineages: cytosolic A, cytosolic B, endoplasmic reticulum, and mitochondria. Hsc70s sporadically distributed in the phylogenetic tree, indicating their paraphyletic origin. Detailed sequence inspection found several motifs that support the phylogenetic analysis. Taken together, we propose new aliases of D. melanogaster Hsp70 family members based on their evolutionary history.

Effects of long-term exposure to high temperature on growth performance, chemical composition, hematological and histological changes, and physiological responses in hybrid catfish [♂Clarias gariepinus (Burchell, 1822) ×♀C. macrocephalus (Günther, 1864)].

The anthropogenic and climate-driven rise in water temperature is expected to have an effect on the physiological functions of ectothermic species. In the present study, hybrid catfish were subjected to three different temperatures (27, 32, and 37 °C) for 50 days to examine the effect of long-term exposure to high temperatures on growth and physiological parameters. The results showed that acclimation temperature improves growth and feed utilization with a quadratic effect (P < 0.05). The highest performance was observed at 32 °C, but fish acclimated at 37 °C decreased growth and feed utilization. In addition, skin darkening was observed in fish acclimated with increasing temperatures. Fat content of whole-body, liver, and dorsal muscle of fish was decreased by increasing temperatures (P < 0.05). Higher temperature levels significantly increased in all blood parameters (P < 0.05), except for high-density lipoprotein cholesterol, which was quadratically decreased (P = 0.004). Fish acclimated with increasing temperature also altered gill and liver histology such as gill shortening, hyperplasia and edema in the connective tissue, severe hyperplasia of epithelial cells, and desquamation, hepatocyte vacuolization, nuclei displacement, and pyknotic hepatic cells. While mucus cells were periphery distributed in the subcutaneous skin. In addition, cuboidal shape-like of club cells and melanophores were also observed in fish acclimated at 37 °C resulting in increased epithelial layer thickness. After fish subjected to increasing temperature exhibited an increase in the number of operculum movement and number of gasping for air (P < 0.05) in all acclimated groups. While fish challenged at 37 °C showed higher critical thermal maximum (CTmax, 41.33 °C) than those of the other groups. Overall, the maximum temperature (37 °C) may rick to hybrid catfish. To prevent physiological damage to the fish, as well as reduction of growth and productivity, the temperature in the aquaculture setting should be kept below 37 °C.

MicroRNA-dependent regulation of targeted mRNAs for improved muscle texture in crisp grass carp fed with broad bean.

Among legumes, broad bean (Vicia faba L.) has received particular attention due to its nutraceutical, functional and economic importance. The plant-derived microRNAs (miRNAs), as novel dietary functional components, are found to regulate the expression of endogenous mRNAs in vertebrates. To understand the role of broad bean miRNAs in the regulation of muscle texture, we investigated the miRNA-mRNA network in the established crisp grass carp model fed with broad bean. We identified various miRNAs that potentially improved muscle texture; miR-101b-3p activated Wnt signaling and satellite cell proliferation; miR-126-3p, miR-29a and miR-148b promoted hyperplasia by targeting muscle structure genes (tln2, TPM1, etc.); miR-152-5p and miR-185 regulated collagen expression via Smads signaling pathways; and miR-146a and miR-371-3p increased reactive oxygen species (ROS) by suppressing cat and prdx6. Among these changes, at least the Wnt signal activation was driven by broad bean-derived miR-101b-3p. This paper was conducted to investigate the cross-kingdom regulatory effects of broad bean miRNA on muscle structure and provide basic data for the development and application of broad bean.

An update on the evolutionary origin of aglomerular kidney with structural and ultrastructural descriptions of the kidney in three fish species.

The kidney of fish contains numerous nephrons, each of which is divided into the renal corpuscle and renal tubules. This glomerular structure is the filtration unit of the nephron and is important for the kidney function, but it has been reported that the renal corpuscle was lost in at least four independent linages of fish (i.e., aglomerular kidney). In this study, the authors newly described renal structures for three species by histological and ultrastructural observations: two aglomerular kidneys from a seahorse Hippocampus barbouri and a toadfish Allenbatrachus grunniens and a glomerular kidney from a snake eel Pisodonophis boro. The renal development of H. barbouri was also described during 1-35 days after birth. In all species tested, the anterior kidney was comprised of haematopoietic tissues and a few renal tubules, whereas the posterior kidney contained more renal tubules. Although the glomerular structure was present in P. boro, light microscopic observations identified no glomeruli in the kidney of H. barbouri and A. grunniens. Ultrastructurally, abundant deep basal infoldings with mitochondria in the renal tubules were observed in A. grunniens compared to H. barbouri and P. boro, suggesting the possible role of basal infoldings in maintaining the osmotic balance. By integrating the results from the three species and comprehensive literature search, the authors further showed that 56 species have been reported to be aglomerular, and that the aglomerular kidney has evolved at least eight times in bony fishes.

Impact of Pre-Mortem Factors on Meat Quality: An Update.

Meat quality is closely associated with the chemical composition of skeletal muscle and is therefore influenced by the pre-mortem metabolic state of skeletal muscle tissue [...].

Phylogenetic position of the Atlantic Gnomefish, Scombrops oculatus (Teleostei: Scombropidae), within the genus Scombrops, inferred from the sequences of complete mitochondrial genome and cytochrome c oxidase subunit I genes.

We determined the complete mitochondrial genome of the Atlantic Gnomefish, Scombrops oculatus (Scombropidae). The total length of mitochondrial DNA (mtDNA) was 16,515 bp and included 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and one control region. The gene arrangement of S. oculatus was identical to those of three Japanese scombropid species and those of other teleosts. The phylogenetic analysis using the whole mtDNA, excluding the control region, indicates the Atlantic species is distinct from the Japanese clade, whereas that using cytochrome c oxidase subunit I gene showed the Atlantic species is most closely related to the African species.

The complex evolution of the metazoan HSP70 gene family.

The metazoan 70-kDa heat shock protein (HSP70) family contains several members localized in different subcellular compartments. The cytosolic members have been classified into inducible HSP70s and constitutive heat shock cognates (HSC70s), but their distinction and evolutionary relationship remain unclear because of occasional reports of "constitutive HSP70s" and the lack of cross-phylum comparisons. Here we provide novel insights into the evolution of these important molecular chaperones. Phylogenetic analyses of 125 full-length HSP70s from a broad range of phyla revealed an ancient duplication that gave rise to two lineages from which all metazoan cytosolic HSP70s descend. One lineage (A) contains a relatively small number of genes from many invertebrate phyla, none of which have been shown to be constitutively expressed (i.e., either inducible or unknown). The other lineage (B) included both inducible and constitutive genes from diverse phyla. Species-specific duplications are present in both lineages, and Lineage B contains well-supported phylum-specific clades for Platyhelminthes, Rotifera, Nematoda, Porifera/Cnidaria, and Chordata. Some genes in Lineage B have likely independently acquired inducibility, which may explain the sporadic distribution of "HSP70" or "HSC70" in previous phylogenetic analyses. Consistent with the diversification history within each group, inducible members show lower purifying selection pressure compared to constitutive members. These results illustrate the evolutionary history of the HSP70 family, encouraging us to propose a new nomenclature: "HSP70 + subcellular localization + linage + copy number in the organism + inducible or constitutive, if known." e.g., HSP70cA1i for cytosolic Lineage A, copy 1, inducible.