Deleterious impacts of heat stress on steroidogenesis markers,immunity status and ovarian tissue of Nile tilapia (Oreochromis niloticus)

The water temperature of aquacultures is a primary factor of fish welfare, reproductive patterns, and immunity. To elucidate the molecular and biological processes of the temperature modulation of reproduction and immunity, female Nile tilapia (190 ± 10g) were allocated into five groups following acclimatization (150 females, three replicates, each n = 10). Each group was subjected to various temperatures (28 °C, 30 °C, 32 °C, 34 °C, and 37 °C), the group at 28 °C representing the control. Their serum levels of estradiol, cortisol, and vitellogenin were measured as well as serum triiodothyronine (T3) hormone, thyroxine (T4) hormone, and non-specific immunity (phagocytic and lysozyme activity). In addition, steroidogenic acute regulatory protein (STAR), vitellogenin gene receptor, and heat shock protein 70 (HSP70) gene expression were evaluated. The serum levels of estradiol, cortisol, and vitellogenin markedly declined (P < 0.05) in fish group at higher temperatures. In addition to T3, T4 was significantly affected (P < 0.05) in the control group. The expressions of the STAR gene (steroidogenesis) and vitellogenin receptors were also considerably down-regulated. The histopathological photomicrograph of fish subjected to high water temperature revealed injuries in ovary tissues, demonstrating its harmful effects. The experimental results verified the possible role of water temperature as a main stressor on Nile tilapia’ physiology through modulation of steroidogenesis-related gene expression and immunity.     

Antarctic marine molluscs do have an HSP70 heat shock response

The success of any organism depends not only on niche adaptation but also the ability to survive environmental perturbation from homeostasis, a situation generically described as stress. Although species-specific mechanisms to combat “stress” have been described, the production of heat shock proteins (HSPs), such as HSP70, is universally described across all taxa. Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) members, plus GRP78 (glucose-regulated protein, 78 kDa), a related HSP70 family member, were cloned using degenerate polymerase chain reaction (PCR) from two evolutionary divergent Antarctic marine molluscs (Laternula elliptica and Nacella concinna), a bivalve and a gastropod, respectively. The expression of the HSP70 family members was surveyed via quantitative PCR after an acute 2-h heat shock experiment. Both species demonstrated significant up-regulation of HSP70 gene expression in response to increased temperatures. However, the temperature level at which these responses were induced varied with the species (+6–8°C for L. elliptica and +8–10°C for N. concinna) compared to their natural environmental temperature). L. elliptica also showed tissue-specific expression of the genes under study. Previous work on Antarctic fish has shown that they lack the classical heat shock response, with the inducible form of HSP70 being permanently expressed with an expression not further induced under higher temperature regimes. This study shows that this is not the case for other Antarctic animals, with the two molluscs showing an inducible heat shock response, at a level probably set during their temperate evolutionary past.     

Changes in the Cellular and Organismal Stress Responses of the Subtropical Fish, the Indo-Pacific Sergeant, Abudefduf vaigiensis, due to the 1997–1998 El Niño/Southern Oscillation

There are accumulating reports showing that the El Niño/Southern Oscillation (ENSO) has a significant effect on the population dynamics of marine fishes. However, the influence of ENSO on the physiology of fishes, as possible components of those ecological changes in fish populations, is not fully understood. This study investigated the cellular, physiological, and organismal stress responses in a wild fish population under natural thermal stress. The sea surface temperature at the subtropical ocean of Okinawa, Japan, was the highest in the last 10 years (>32°C) during the summer of 1998 with a strong ENSO. To examine the effects of those unusually warm ocean temperatures on fish, we compared tissue 70-kDa heat shock protein (HSP70) levels and growth rates between the ENSO summer of 1998 and the normal summer of 1999 in a common fish species in Okinawa, the Indo-Pacific sergeant, Abudefduf vaigiensis. We also conducted a complementary heat shock experiment in the laboratory. The field collected Indo-Pacific sergeant had significantly higher muscle HSP70 levels in 1998 than 1999. Higher muscle HSP70 and plasma cortisol levels were observed at 32°C than at 28°C in the laboratory heat shock experiment, indicating that the highest summer ocean temperature in 1998 was sufficient for the fish to up-regulate the cellular and physiological stress responses. In support of this effect, otoliths showed slower growth rates of the fish during the summer of 1998; this may reflect the significant energetic cost of these stress responses.     

Temperature-dependent physiological and biochemical responses of the marine medaka Oryzias melastigma with consideration of both low and high thermal extremes

This study aimed to investigate temperature effect on physiological and biochemical responses of the marine medaka Oryzias melastigma larvae. The fish were subjected to a stepwise temperature change at a rate of 1 °C/h increasing or decreasing from 25 °C (the control) to six target temperatures (12, 13, 15, 20, 28 and 32 °C) respectively, followed by a 7-day thermal acclimation at each target temperature. The fish were fed ad libitum during the experiment. The results showed that cumulative mortalities were significantly increased at low temperatures (12 and 13 °C) and at the highest temperature (32 °C). For the survivors, their growth profile closely followed the left-skewed ‘thermal performance curve’. Routine oxygen consumption rates of fish larvae were significantly elevated at 32 °C but suppressed at 13 and 15 °C (due to a high mortality, larvae from 12 °C were not examined). Levels of heat shock proteins and activities of malate dehydrogenase and lactate dehydrogenase were also measured in fish larvae exposed at 15, 25 and 32 °C. The activities of both enzymes were significantly increased at both 15 and 32 °C, where the fish larvae probably suffered from thermal discomfort and increased anaerobic components so as to compensate the mismatch of energy demand and supply at these thermal extremes. Coincidently, heat shock proteins were also up-regulated at both 15 and 32 °C, enabling cellular protection. Moreover, the critical thermal maxima and minima of fish larvae increased significantly with increasing acclimation temperature, implying that the fish could develop some degrees of thermal tolerance through temperature acclimation.     

The time-profile of the PBMC HSP70 response to in vitro heat shock appears temperature-dependent

Summary. Heat shock proteins (HSPs) are synthesised by cells subsequent to a stress exposure and are known to confer protection to the cell in response to a second challenge. HSP induction and decay are correlated to thermotolerance and may therefore be used as a biomarker of thermal history. The current study tested the temperature-dependent nature of the heat shock response and characterised its time profile of induction. Whole blood from 6 healthy males (Age: 26 ± (SD) 2 yrs; Body mass 74.2 ± 3.8 kgs; VO_2max: 49.1 ± 4.0 ml·kg⁻¹·min⁻¹) were isolated and exposed to in vitro heat shock (HS) at 37, 38, 39, 40, and 41 °C for a period of 90 min. After HS the temperature was returned to 37 °C and intracellular HSP70 was quantified from the leukocytes at 0, 2, 4, and 6 h after heat treatment. The concentration of HSP70 was not different between temperatures (P > 0.05), but the time-profile of HSP70 synthesis appeared temperature-dependent. At control (37 °C) and lower temperatures (38–39 °C) the mean HSP70 concentration increased up to 4 h post HS (P < 0.05) and then returned towards baseline values by 6 h post HS. With in vitro hyperthermic conditions (40–41 °C), the time-profile was characterised by a sharp rise in HSP70 levels immediately after treatment (P < 0.05 for 40 °C at 0 h), followed by a progressive decline over time. The results suggest a temperature-dependent time-profile of HSP70 synthesis. In addition, the temperature at which HSP70 is inducted might be lower than 37 °C.     

Effect of heat shock on gene expression of Aedes albopictus cells infected with Mayaro virus

Three major Mayaro virus proteins of 62, 50 and 34 kDa were detected in Aedes albopictus cells after 48 h postinfection at 28°C. When the infected cells were shifted from 28 to 37°C for 90 min (heat shock conditions), the synthesis of two major heat shock proteins (HSP) 82 and 70 kDa was induced concomitantly with strong inhibition of virus and normal protein synthesis. Total cellular RNA was isolated from mock and infected cells incubated at 28°C or under heat shock. Northern blot analysis with HSP genomic probes from Drosophila sp showed that (1) the probe for HSP 82 hybridized with an RNA of 2.6 kb present only in heat-shocked cells, (2) the HSP 70 probe hybridized with RNA species of 2.5 kb, present only in RNA from heat-shocked cells. These results showed that Mayaro virus was not able to alter the reprogrammation of gene expression induced by heat shock in A. albopictus cells.     

Heat shock protein responses in thermally stressed bay scallops, Argopecten irradians, and sea scallops, Placopecten magellanicus

The effects of thermal stress on the induction of heat shock proteins (HSPs) were examined in northern bay scallops, Argopecten irradians irradians, a relatively heat tolerant estuarine species, and sea scallops, Placopecten magellanicus, a species residing in cooler, deeper water. Polyclonal antibodies used in this work for analysis of inducible HSP70 and HSP40 only recognized proteins of 72 and 40 kDa respectively from the mantles of both scallop species. Additionally, HSP quantification using the antibody to HSP70 was equally effective by either immunoprobing of western blots or ELISA, demonstrating that either approach could be successfully employed for analysis of thermal response in scallops. Sea scallop HSP70 and HSP40 did not change when animals were heat-shocked for 3 h by raising the temperature from 10 °C to 20 °C; however, a 24 h treatment of the same magnitude elicited a significant response. Conversely, bay scallops displayed rapid and prolonged HSP70 and HSP40 responses during the recovery period following a 3 h heat shock from 20 °C to 30 °C. Temperature reduction from 20 °C to 3 °C for 3 h also caused significant HSP70 and HSP40 increases in bay scallops; this represents the first time cold shock was shown to induce HSP synthesis in bivalve mollusks. The onset of the HSP40 response was more rapid than for HSP70, occurring at the end of the cold shock itself prior to transfer to a recovery temperature. Both proteins responded maximally during recovery at control temperature. HSP responses of sea and bay scallops to thermal stress may be related to their habitat in the natural environment and they suggest a differential capacity for adaptation to temperature change. This is an important consideration in assessing the response of these scallops to different culture conditions.     

Effect of thermal stress on HSP70 expression in dermal fibroblast of zebu (Tharparkar) and crossbred (Karan-Fries) cattle

The present studies were conducted to investigate the difference response of dermal fibroblasts to heat stress in Tharparkar and Karan-Fries cattle. Skin is the most important environmental interface providing a protective envelope to animals. In skin, dermal fibroblasts are the most regular cell constituent of dermis that is crucial for temperature homeostasis. The study aimed to examine the reactive oxygen species (ROS) formation, cytotoxicity (%) and heat shock protein 70 (HSP70) genes expression in dermal fibroblast of Tharparkar and Karan-Fries cattle and to assess whether resistance of dermal fibroblast to heat stress is breed specific. Dermal fibroblasts from ear pinna of Tharparkar and Karan-Fries cattle were exposed at 25 °C, 37 °C, 40 °C and 44 °C for 3 h to measure the ROS, cytotoxicity (%) and HSP 70 (HSPA1A, HSPA2 and HSPA8) genes’ expression. The results showed that ROS formation at low temperature (25 °C) decreased in both breeds as compared to control (37 °C) and the differences were significant (P<0.0001). Heat stress at 40 °C did not increase ROS formation significantly in Tharparkar but increased significantly (P<0.001) in Karan-Fries cattle. The overall cytotoxicity (%) was also found to be significantly different (P<0.001) between Tharparkar and Karan-Fries cattle, and on exposure to different temperatures (P<0.001). The cytotoxicity (%) in dermal fibroblast cells of Karan-fries cows was more than Tharparkar. The expression studies indicated that all HSP70 genes (HSPA8, HSPA1A and HSPA2) were up-regulated at different temperatures in both breeds. In Tharparkar, the relative mRNA expression of HSPA8 gene was higher but HSPA1A and HSPA2 genes were low as compared to Karan-Fries cattle. At 40 and 44 °C, the relative expressions of inducible HSP 70 genes (HSPA1A and HSPA2) were higher in Karan-Fries than Tharparkar. In summary, dermal fibroblast resistance to heat shock differed between breeds. Dermal fibroblasts of Tharparkar were observed to be more heat tolerant than crossbred Karan-Fries cattle. The study concludes that zebu cattle (Tharparkar) dermal fibroblasts are more adapted to tropical climatic condition than crossbreed cattle (Karan-Fries). Differences exist in dermal fibroblasts of heat adapted and non-adapted cattle.     

Lack of an HSP70 heat shock response in two Antarctic marine invertebrates

Members of the HSP70 gene family comprising the inducible (HSP70) genes and GRP78 (glucose-regulated protein 78 kDa) were identified in an Antarctic sea star (Odontaster validus) and an Antarctic gammarid (Paraceradocus gibber). These genes were surveyed for expression levels via Q-PCR after an acute 2-hour heat shock experiment in both animals and a time course assay in O. validus. No significant up-regulation was detected for any of the genes in either of the animals during the acute heat shock. The time course experiment in O. validus produced slightly different results with an initial down regulation in these genes at 2°C, but no significant up-regulation of the genes either at 2 or 6°C. Therefore, the classical heat shock response is absent in both species. The data is discussed in the context of the organisms’ thermal tolerance and the applicability of HSP70 to monitor thermal stress in Antarctic marine organisms.     

Changes in cell morphology and actin organization during heat shock in Dictyostelium discoideum: does HSP70 play a role in acquired thermotolerance?

In response to heat shock (34°C, 30 min), cell morphology and actin organization in Dictyostelium discoideum are drastically changed. Loss of pseudopodia and disappearance of F-actin-containing structures were observed by using fluorescence microscopy. These changes were paralleled by a rapid decrease of the F-actin content measured by a TRITC-phalloidin binding assay. The effects of heat shock on cell morphology and actin organization are transient: After heat shock (34°C) or during a long-term heat treatment (30°C), cell morphology, F-actin patterns and F-actin content recovered/adapted to a state which is characteristic for untreated cells. Because F-actin may be stabilized by increased amounts of heat shock proteins, their response and interaction with F-actin was analyzed. After a 1 h heat treatment (34°C), the major heat shock protein of D. discoideum (HSP70) showed maximally increased synthesis rates and levels. During recovery from a 34°C shock or during a continuous heat treatment at 30°C, the HSP70 content first increased and then declined slowly toward normal levels. Pre-treatment of cells with a short heat shock of 30 min at 34°C stabilized the F-actin content when the cells were exposed to a second heat shock. Furthermore, a transient colocalization of HSP70 and actin was observed at the beginning of heat treatment (30°C) using immunological detection of HSP70 in the cytoskeletal actin fraction.     

Behavioural and physiological responses of juvenile geoduck (Panopea zelandica) following acute thermal stress

Climate extremes, such as heatwaves, are expected to become more intense and of longer duration in the near future. These climatic conditions may have a significant impact on the prospects of establishing a new aquaculture industry for the endemic New Zealand geoduck, Panopea zelandica. This study focused on characterising animal behaviour, haemocytes , and heat shock protein (HSP70 & HSP90) mRNA expression following exposure to elevated temperatures, such as those encountered during marine heatwaves around 20 °C and an extreme scenario of 25 °C, contrasted to an ambient temperature of 17 °C. After 24 h of heat challenge, P. zelandica were found to be significantly influenced by the thermal changes, as there were differences recorded in all the responses examined. With increasing temperatures, juvenile geoduck were observed to fully emerge from the sediment a behaviour that has not previously been quantified nor associated with stress in this species. The ability of P. zelandica juveniles to re-bury still warrants further investigation, as adults are unable to do so. Haemocyte analyses revealed an increase in the abundance of granulocytes, cellular aggregations, and size of these aggregations at the highest temperature exposure. Increased expression of the hsp70 gene in the haemolymph after exposure at 25 °C for 24 h was detected and attributed to attempts to mitigate protein denaturation caused by thermal stress. The inter-individual variability in the response of heat shock proteins recorded could aid in future selective breeding programs if it is reflected in net thermotolerance. P. zelandica shows great potential for growing in subtidal habitats around New Zealand, and this study highlights the importance of temperature considerations when selecting potential farm and reseeding locations.     

Low heat shock thresholds in wild Antarctic inter-tidal limpets (Nacella concinna)

Heat shock proteins (HSPs) are a family of genes classically used to measure levels of organism stress. We have previously identified two HSP70 genes (HSP70A and HSP70B) in sub-tidal populations of the Antarctic limpet (Nacella concinna). These genes are up-regulated in response to increased seawater temperatures of 15°C or more during acute heat shock experiments, temperatures that have very little basis when considering the current Antarctic ecology of these animals. Therefore, the question was posed as to whether these animals could express HSP70 genes when subjected to more complex environmental conditions, such as those that occur in the inter-tidal. Inter-tidal limpets were collected on three occasions in different weather conditions at South Cove, Rothera Point, over a complete tidal cycle, and the expression levels of the HSP70 genes were measured. Both genes showed relative up-regulation of gene expression over the period of the tidal cycle. The average foot temperature of these animals was 3.3°C, far below that of the acute heat shock experiments. These experiments demonstrate that the temperature and expression levels of HSP production in wild animals cannot be accurately extrapolated from experimentally induced treatments, especially when considering the complexity of stressors in the natural environment. However, experimental manipulation can provide molecular markers for identifying stress in Antarctic molluscs, provided it is accompanied by environmental validation, as demonstrated here. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.