Food-deprivation induces HSP70 and HSP90 protein expression in larval gilthead sea bream and rainbow trout

Heat shock proteins (HSPs) expression is commonly used as indicators of cellular stress in animals. However, very little is known about either the expression patterns of HSPs or their role in the stress-tolerance phenomenon in early life stages of fish. To this end, we examined the impact of food-deprivation (12 h), reduced oxygen levels (3.5 mg/L for 1 h) and heat shock (HS: + 5 °C for 1 h) on HSP70 and HSP90 protein expression in early life stages of the gilthead sea bream (Sparus aurata), a warm-water aquaculture species. Also, we investigated HSP70 and HSP90 response to food-deprivation (7 days) in early life stages of rainbow trout (Oncorhynchus mykiss), a cool-water aquaculture species, and the tolerance of this larvae to heat shock (either + 5 or + 10 °C for 1 h). Our results clearly demonstrate that food-deprivation enhances HSP70 and HSP90 protein expression in larvae of both species. In gilthead sea bream larvae, the stressors-induced HSP70 and HSP90 (only in the reduced oxygen group) protein expression returned to unstressed levels after 24 h recovery. In fed trout larvae, a + 5 °C heat shock did not elevate HSP70 and HSP90 expression, whereas 100% mortality was evident with a + 10 °C HS. However, food-deprived trout larvae, which had higher HSP70 and HSP90 protein content, survived HS and showed HS-dependent increases in HSP70, but not HSP90 expression. Overall, HSP70 and HSP90 protein expression in early life stages of fish have the potential to be used as markers of nutritional stress, while elevation of the tissue HSPs content may be used as a means to increase stress tolerance during larval rearing.     

Molecular characterization of heat shock protein 70 genes in the liver of three warm freshwater fishes with differential tolerance to microcystin-LR

Heat shock protein 70 (HSP70) protect cell from oxidative stress by preventing the irreversible loss of vital proteins and facilitating their subsequent regeneration. Silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idellus), and Nile tilapia (Oreochromis nilotica) are three warm freshwater fishes with differential tolerance to microcystin-LR (MC-LR). Full-length cDNAs encoding the HSP70 were cloned from the livers of the three fishes. The HSP70 cDNAs of silver carp, grass carp, and Nile tilapia were 2356, 2348, and 2242 bp in length and contained an open-reading frame of 1950 bp (encoding a polypeptide of 649 amino acids), 1950 bp (649 amino acids), and 1917 bp (638 amino acids), respectively. Like mammalian HSP70, the HSP70 of the three fish was also composed of an ATPase domain from residues 1 to 383 (44 kDa), substrate peptide binding domain from residues 384 to 544 (18 kDa), and a C-terminus domain from residues 545 to 649 (10 kDa). The relatively high conservation of HSP70 sequences among different vertebrates is consistent with their important role in fundamental cellular processes. Using beta-actin as an external control, RT-PCR within the exponential phase was conducted to determine the constitutive and inducible expression level of HSP70 gene among the three fishes (6-12 g) intraperitoneally injected with MC-LR (50 μg kg(-1) body weight). Both constitutive and inducible liver mRNA levels of the fish HSP70 genes showed positive relationships with their tolerance to MC-LR: highest in Nile tilapia, followed by silver carp, and lowest in grass carp. The differential expression pattern of liver HSP70 genes in the three fish indicated a potential role of HSP70 in the detoxification process of MC-LR.     

Using gravel for environmental enrichment in salmonid hatcheries: The effect of gravel size during egg incubation,endogenous and first feeding in rainbow trout

Environmental enrichment aims for a deliberate increase in structural complexity in otherwise plain rearing units, helping to reduce aberrant traits and promote welfare of fish kept in captivity. Before putting enrichment protocols into practice, however, practitioners like hatchery managers need clear guidelines on enrichment measures and on the substrates used. In the present study, we used rainbow trout as a model species for salmonid rearing and investigated the use of a single layer of three different gravel types, i.e., small (4–8 mm), medium (8–16 mm) and large (16–32 mm), for environmental enrichment during egg incubation, endogenous and first feeding of rainbow trout and compared this to a barren control. From the egg stage onwards, we determined mortality, fungal prevalence as well as growth of larvae and fingerlings. We found that gravel size significantly affected mortality and fungal prevalence with the smallest gravel size and the control showing the lowest incidents. Growth of larvae and fingerlings was not affected by gravel, both when compared between gravel types and to the barren control. When using gravel for environmental enrichment in salmonid hatcheries, a small gravel size should be used. Small gravel provides the fish with a more natural environment without compromising practical feasibility of enrichment in hatcheries, still allowing for easy visual inspection and manual control of the reared fish.     

Localization of constitutive heat shock proteins in developing ascidians

Heat shock proteins (HSP) are a group of highly conserved proteins that regulate protein folding and ameliorate the effects of environmental stress. In the present study, the question of whether or not ascidian oocytes, embryos and larvae constitutively synthesize HSP was studied using HSP 60 and HSP 70 antibodies. Developmental stages obtained from Boltenia villosa, Cnemidocarpa finmarkiensis, Styela montereyensis and Corella willmeriana were examined for HSP using indirect immunocytochemistry. Myoplasm in oocytes and unfertilized eggs reacted with HSP 60 and 70 antibodies. HSP signals dramatically moved into the vegetal egg cytoplasm during ooplasmic segregation and colocalized with the myoplasm. In cleavage-stage embryos, HSP signals were partitioned with the myoplasm into muscle progenitor blastomeres and HSP signals were evident in the tail muscle cells of larvae. Immunoblots of proteins extracted from oocytes, eggs, embryos and larvae indicate that anti-HSP 60 recognizes a single band having an estimated molecular weight of 60 kDa. Egg centrifugation experiments suggest that most of the ascidian myoplasmic HSP are mitochondrial proteins. These results raise an intriguing possibility that mitochondria associated with the myoplasm perform biochemical functions that are unique to the embryonic muscle cell lineage.     

Effects of heavy metals on insect immunocompetent cells

The influence of the following heavy metals, copper (Cu), zinc (Zn), cadmium (Cd) and lead (Pb), on haemocytes of the house fly Musca domestica L. was studied under laboratory conditions. House fly larvae were exposed to low or high, semi-lethal concentrations of metals. These particular metals were selected because they are present in polluted environments in Poland. In addition, we studied expression of the stress proteins HSP70 and HSP72 in haemocytes collected from larvae that had been exposed to heavy metal. The obtained results showed changes in haemocytes morphology and phagocytotic plasticity in the experimental flies in comparison to control. The number of prohaemocytes, regarded as stem cells, increased, while granulocytes, responsible for phagocytosis, decreased. However, we have not detected any clear changes in expression of HSP70 or HSP72 in flies treated with low or high concentrations of the heavy metals.     

The effect of substrate rearing on growth,aerobic scope and physiology of larval white sturgeon Acipenser transmontanus

The effect of substratum on growth and metabolic rate was assessed in larval white sturgeon Acipenser transmontanus. Yolk‐sac larvae (YSL) were reared in bare tanks or tanks with gravel as substratum from hatch until approximately 16 days post hatch (dph). The effect of an artificial substratum was also evaluated on growth alone. Substratum had a significant effect on mass, with larvae reared in gravel and artificial substrata being larger than those reared without substratum. Routine metabolic rates were significantly lower and relative aerobic scope (the difference between maximum and routine metabolic rate) was significantly higher for YSL and feeding larvae (FL) reared in gravel relative to those reared in bare tanks, particularly before fish started feeding exogenously. Furthermore, gravel‐reared larvae had higher whole‐body glycogen concentrations relative to bare‐tank‐reared larvae. Routine factorial scope (maximum metabolic rate divided by routine metabolic rate) was relatively low in all treatments (< 1·7) indicating a limited ability to elevate metabolic rate above routine early in development and mass exponents for metabolic rate exceeded 1. Taken together, these data indicate that YSL reared without substratum may divert more of their energy to non‐growth related processes impairing growth. This finding underscores the importance of adequate rearing substratum for growth of A. transmontanus and may provide support for habitat restoration and alternative hatchery rearing methods associated with sturgeon conservation.     

Behavioural response of Kootenai white sturgeon (Acipenser transmontanus,Richardson, 1836) early life stages to gravel,pebble, and rubble substrates: guidelines for rearing substrate size

Guidelines for creating rearing substrate for sturgeon early life stages are needed for restoration programmes creating habitats for spawning and rearing of early life stages. To determine the effects of rock size on motile early life stages, experiments were conducted in artificial streams to observe the behaviour of free embryos and larvae of Kootenai River white sturgeon (Acipenser transmontanus) relative to rock size. Most (≥90%) of the free embryos in replicate test streams with 100% gravel, 100% pebble, or 100% rubble hid under rocks, with few moving downstream. There was no difference in downstream movement of free embryos among rock treatments, therefore all rock types provided cover habitat. Similarly, in rock mixture tests, with a variable percentage of pebble, small rubble, or large rubble in different tanks, even fewer free embryos moved downstream. With increasing age, larvae increasingly used the open bottom and velocity refuges downstream of or alongside rocks of any size while drift feeding. Downstream movement of larvae in both rock regime tests was affected by rock size, with significantly reduced movement relative to increasing abundance of large rock (rubble). However, in all rock mixtures, free embryos (and later, larvae when they stopped dispersing) preferred the smallest rock size available (pebble; P = 0.0001). This suggests a strong innate preference of both life stages for small substrate that is likely related to increased survival. A rock mixture of 10% gravel (16–32 mm diameter) and 30–40% pebble (diameter, 30–60 mm) should provide adequate rearing substrate for free embryos and early‐larvae. The remaining 50–60% should be mixed rubble and boulders for spawning and egg rearing.     

Activation of a stress-induced gene by insecticides in the midge, Chironomus yoshimatsui

Stress proteins (heat shock proteins, HSPs) have been proposed as general biomarkers for environmental monitoring. In the present study, we evaluated the environmental stress-burden on the aquatic midge Chironomus yoshimatsui using hsp70 expression. Larvae collected from streams receiving polluted runoff (field strain) were resistant to the organophosphorus insecticide, fenitrothion (F), and the synthetic pyrethroid, ethofenprox (E), whereas a strain originally collected from an unpolluted area (susceptible strain) showed low resistance to insecticide exposure. To examine the expression of an HSP70 gene in C. yoshimatsui, an hsp70 cDNA probe was prepared using RNA obtained from the field strain larvae and used for Northern blot analyses. The expression of this HSP70 gene in larvae collected from two field sites in May about 1 week after insecticide spraying in the fields was 2.3 (p = 0.018) to 3.3 fold higher than that in the susceptible strain and was also 4.6 and 1.4 (p = 0.033) fold higher than those collected in November 3 months after the cessation of insecticide spraying. In order to identify potential inducers of the HSP70 gene of the field strain, larvae of the susceptible strain were exposed to F or E for 24 h and hsp70 mRNA levels determined. Exposures to F at 0.4 microg/L and E at 1.1 microg/L increased hsp70 mRNA levels 2.7 (p = 0.049) and 4.4 (p = 0.043) fold over controls, respectively. These results suggest that larvae collected from polluted areas are burdened by environmental stressors and the tested insecticides are potential inducers of HSP70. The results also support the suggestion that HSP70 gene expression is a sensitive indicator of low level (nonlethal) exposures to certain insecticides.     

美国白蛾热激蛋白70基因的鉴定及功能分析

[目的]探究热激蛋白70 (heat shock protein 70,HSP70)基因在美国白蛾Hyphantria cunea抵御高温胁迫过程中的作用,为揭示美国白蛾的扩散机制以及预测潜在分布区提供理论支撑.[方法]利用PCR技术克隆美国白蛾HSP70基因,并进行生物信息学分析;利用qPCR技术检测新蜕皮的美国白蛾...     

棉铃虫的紫云英-麦胚人工饲料

用不同人工饲料连续饲养棉铃虫Heliothis armtgera;通过试验比较,设计和发展了适合于大量饲养棉铃虫的紫云英-麦胚人工饲料。与自然饲料相比,用这种人工饲料饲养的棉铃虫发育快、存活率高、蛹大、成虫产卵多。已经用紫云英-麦胚人工饲料连续饲养棉铃虫16代,各代幼虫的存活率在85—97%之间,成虫平均产卵1,000多粒,孵化率不低于70%。6龄幼虫对这种人工饲料的利用和转化效率分别是26.7%和65.0%。     

Expression of the heat-shock protein HSP70 in Drosophila buzzatii lines selected for thermal resistance

The level of HSP70 expression induced by a non-lethal high temperature was examined in lines selected for increased thermal resistance and in corresponding control lines of Drosophila buzzatii, in order to test if selection for high temperature resistance leads to an increased level of HSP70 expression. The lines used were selected for up to 64 generations either as adults or through all larval stages. In adult selection lines, hard selection was implemented every second generation after mild heat hardening. In larval selection lines, larvae were exposed each generation to laboratory "natural" selection. Generally lines selected as adults showed a higher HSP70 expression than did controls, both in third instar larvae and in adults. A strong negative response to selection of HSP70 expression was found in all lines that were selected at cycling temperatures during larval development. The results suggests that a trade off between heat resistance in form of HSP70 expression and fecundity/fertility are responsible for the level of HSP70 expression. The effect of the different methods of selection on HSP70 expression suggests that heat resistance constitutes more than one trait.     

Effects of rearing and induction temperature on the temporal dynamics of heat shock protein 70 expression in a butterfly

The temporal dynamics of heat shock protein 70 (HSP70) expression in response to longer‐term acclimation and rapid hardening in the butterfly Lycaena tityrus is investigated. After a 1‐h exposure to 1 °C or 37 °C, HSP70 is quickly up‐regulated within 1 h and down‐regulated within 2 h. The fast dynamic of HSP70 expression is in contrast to the patterns found in organisms inhabiting more stable thermal environments, and is interpreted as an adaptation to the large and rapid temperature variation experienced by flying ectotherms. HSP70 expression is higher in males than in females, as well as in animals reared at 27 °C than at 20 °C, although it is very similar across the high and low induction temperatures. Animals reared at the higher temperature, however, respond less strongly to high‐temperature stress.     

Heat shock protein expression enhances heat tolerance of reptile embryos

The role of heat shock proteins (HSPs) in heat tolerance has been demonstrated in cultured cells and animal tissues, but rarely in whole organisms because of methodological difficulties associated with gene manipulation. By comparing HSP70 expression patterns among representative species of reptiles and birds, and by determining the effect of HSP70 overexpression on embryonic development and hatchling traits, we have identified the role of HSP70 in the heat tolerance of amniote embryos. Consistent with their thermal environment, and high incubation temperatures and heat tolerance, the embryos of birds have higher onset and maximum temperatures for induced HSP70 than do reptiles, and turtles have higher onset and maximum temperatures than do lizards. Interestingly, the trade-off between benefits and costs of HSP70 overexpression occurred between life-history stages: when turtle embryos developed at extreme high temperatures, HSP70 overexpression generated benefits by enhancing embryo heat tolerance and hatching success, but subsequently imposed costs by decreasing heat tolerance of surviving hatchlings. Taken together, the correlative and causal links between HSP70 and heat tolerance provide, to our knowledge, the first unequivocal evidence that HSP70 promotes thermal tolerance of embryos in oviparous amniotes.     

Quantitative RT-PCR analysis and immunohistochemical localization of HSP70 in sea bass Dicentrarchus labrax exposed to transport stress

In aquaculture, fish are exposed to stressful conditions, which cause an increased synthesis of heat shock proteins (HSPs) at the cellular level. In this work we considered the expression of the constitutive and inducible forms of HSP70 as an indicator of stress caused by transport, during development of the sea bass (Dicentrarchus labrax), a teleost fish of high value for aquaculture. Qualitative RT-PCR analysis revealed expression of inducible HSP70 gene in larvae and fry (25, 40 and 80 days) as well as in adult tissues (liver, brain, muscle, gills, kidney, gonads, heart, spleen and skin) of both control and stressed animals. Expression of inducible HSP70 mRNA examined in different adult tissues by Real-Time PCR, was significantly higher in skin and skeletal muscle of stressed animals than in controls. Immunolocalization of inducible and constitutive forms of heat shock protein 70 (HSP70 and HSC70), reported here for the first time, demonstrated an ubiquitous distribution of HSC70 protein in several tissues of both stressed and control animals (at all stages), while inducible HSP70 protein was found only in skeletal muscle of stressed animals. In all stressed animals, regardless of their developmental stage, cortisol levels were higher than in control animals.     

Developmental regulation of heat shock protein synthesis and HSP 70 RNA accumulation during postimplantation rat embryogenesis

Exposure of postimplantation rat embryos on days 9, 10, 11, and 12 of gestation to an in vitro heat shock of 43 degrees C for 30 min results in the induction of heat shock proteins (HSPs) in day 9 and 10 embryos, a severely attenuated response in day 11 embryos, and no detectable response in day 12 embryos. The heat shock response in day 9 embryos (presomite stage) is characterized by the synthesis of HSPs with molecular weights of 28-78 kDa. In heat shocked day 10 embryos, two additional HSPs are induced (34 and 82 kDa). In addition, two HSPs present on day 9 are absent on day 10. In day 11 heat shocked embryos, only three HSPs (31, 39, and 69 kDa) are induced, while in day 12 embryos no detectable HSPs are induced. Northern blot analysis of HSP 70 RNA levels indicates that the accumulation of this RNA, but not actin RNA, varies depending on developmental stage at the time of exposure to heat as well as the duration of the heat shock. Day 9 embryos exhibit the most pronounced accumulation of HSP 70 RNA while embryos on days 10-12 exhibit an increasingly attenuated accumulation of HSP 70 RNA, particularly after the more acute exposures (43 degrees C for 30 or 60 min). Thus, the ability to synthesize HSP 70 and to accumulate HSP 70 RNA changes dramatically as rat embryos develop from day 9 to day 12 (presomite to 31-35 somite stages).