Germ Cell Transplantation Using Sexually Competent Fish: An Approach for Rapid Propagation of Endangered and Valuable Germlines

The transplantation of germ cells into adult recipient gonads is a tool with wide applications in animal breeding and conservation of valuable and/or endangered species; it also provides a means for basic studies involving germ cell (GC) proliferation and differentiation. Here we describe the establishment of a working model for xenogeneic germ cell transplantation (GCT) in sexually competent fish. Spermatogonial cells isolated from juveniles of one species, the pejerrey Odontesthes bonariensis (Atherinopsidae), were surgically transplanted into the gonads of sexually mature Patagonian pejerrey O. hatcheri, which have been partially depleted of endogenous GCs by a combination of Busulfan (40 mg/kg) and high water temperature (25°C) treatments. The observation of the donor cells'' behavior showed that transplanted spermatogonial cells were able to recolonize the recipients'' gonads and resume spermatogenesis within 6 months from the GCT. The presence of donor-derived gametes was confirmed by PCR in 20% of the surrogate O. hatcheri fathers at 6 months and crosses with O. bonariensis mothers produced hybrids and pure O. bonariensis, with donor-derived germline transmission rates of 1.2–13.3%. These findings indicate that transplantation of spermatogonial cells into sexually competent fish can shorten considerably the production time of donor-derived gametes and offspring and could play a vital role in germline conservation and propagation of valued and/or endangered fish species.     

Surrogate Production of Eggs and Sperm by Intrapapillary Transplantation of Germ Cells in Cytoablated Adult Fish

Germ cell transplantation (GCT) is a promising assisted reproductive technology for the conservation and propagation of endangered and valuable genetic resources. In teleost fish, GCT in adult gonads has been achieved only in male recipients, limiting greatly the usefulness of this technique in situations where both sexes need equal and timely attention for conservation and/or propagation. Here we describe a simplified GCT approach that ultimately leads to production of donor-derived eggs and sperm in considerably short time. Donor germ cells isolated from young pejerrey Odontesthes bonariensis (Atherinopsidae) were transplanted non-surgically through the genital papilla into the sexually mature gonads of Patagonian pejerrey O. hatcheri recipients whose gonads have been depleted of endogenous GCs by heat (26°C) and chemical treatment (four doses of Busulfan at 30 mg/kg and 40 mg/kg for females and males, respectively). Transplanted spermatogonial and oogonial cells were able to recolonize the recipients'' gonads and produce functional donor origin eggs and sperm within 7 months from the GCT. We confirmed the presence of donor-derived gametes by PCR in 17% and 5% of the surrogate O. hatcheri fathers and mothers, respectively. The crosses between surrogate fathers and O. bonariensis mothers yielded 12.6–39.7% pure O. bonariensis and that between a surrogate mother and an O. bonariensis father yielded 52.2% pure O. bonariensis offspring. Our findings confirm that transplantation of germ cells into sexually competent adult fish by non-surgical methods allows the production of functional donor-derived eggs and sperm in a considerably short time. The methods described here could play a vital role in conservation and rapid propagation of endangered fish genetic resources.     

Effects of acclimation and egg-incubation temperature on selected temperature by hatchling western painted turtles (Chrysemys picta bellii)

The ability of hatchling turtles to detect environmental temperature differences and to effectively select preferred temperature is a function that critically impacts survival. In some turtle species, temperature preference may be influenced by embryonic and post-hatching conditions, such as egg-incubation and acclimation temperature. We tested for effects of embryonic incubation temperature (27.5 °C, 30 °C) and acclimation temperature (20 °C, 25 °C) on the selected temperature and movement patterns of 32 Chrysemys picta bellii (Reptilia: Emydidae) hatchlings in an aquatic thermal gradient of 14-34 °C and in single-temperature (20 °C, 25 °C) control tests. Among 10-11 month old hatchlings, acclimation temperature and egg-incubation temperature influenced temperature selection and movement patterns. Acclimation temperature affected activity and movement: in thermal gradient and single-temperature control tests, 25 °C-acclimated turtles relocated between chambers significantly more frequently than individuals acclimated to 20 °C. Acclimation temperature also affected temperature selection: 20 °C-acclimated turtles selected a specific temperature during gradient tests, but 25 °C-acclimated turtles did not. Among 20 °C-acclimated turtles, egg-incubation temperature was inversely related to selected temperature: hatchling turtles incubated at 27.5 °C selected the warmest temperature available (34 °C); individuals incubated at 30 °C selected the coldest temperature (14 °C). These results suggest that interactions of environmental conditions may influence post-hatching thermoregulatory behavior in C. picta bellii, a factor that ultimately affects fitness.     

The combined influence of sub-optimal temperature and salinity on the in vitro viability of Perkinsus marinus, a protistan parasite of the eastern oyster Crassostrea virginica

Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25 ppt) to 10 °C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 °C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7 ppt cultures acclimated to each temperature and then transferred to 3.5 ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30 days (3.5 ppt, 2 °C: 0% viability), 60 days (3.5 ppt, 10 °C: 0% viability) and 90 days (7 ppt, 2 °C: 0.6 ± 0.7%; 7 ppt, 10 °C: 0.2 ± 0.2%).     

Thermal response and reversibility of prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis undergoes prolonged larval diapause that is terminated by chilling and warming cycles. To examine the effects of warming temperatures and their duration on diapause termination, we exposed diapause larvae that had not been reactivated after chilling at 5 °C to 20 or 25 °C and chilled them again before incubation at 20 °C. With increasing warming duration at 20 °C, diapause termination after chilling increased and shorter chilling durations became effective. In contrast, few or no larvae warmed at 25 °C terminated diapause after chilling, irrespective of the warming duration. To investigate the effect of warming temperature on diapause intensity, larvae with diapause weakened by initial incubation at 20 °C after the first chilling were subsequently incubated at 15, 20, or 25 °C, then chilled at 5 °C before incubation at 20 °C. Diapause termination increased significantly after the larvae were treated at 15 or 20 °C but decreased significantly after they were treated at 25 °C. The intensification of prolonged diapause at 25 °C was reversed when the larvae were transferred to 20 °C. Diapause intensity in C. sikkimensis therefore decreases at 20 °C, increases at 25 °C, and can be reversed by alternately exposing diapause larvae to 20 and 25 °C. In C. sikkimensis, prolonged diapause does not always proceed in one direction, and its intensity fluctuates in response to ambient temperature conditions.     

The physiological response of reef corals to diel fluctuations in seawater temperature

An opportunity to explore the effects of fluctuating temperatures on tropical scleractinian corals arose when diurnal warming (as large as 4.7 °C) was detected over the rich coral communities found within the back reef of Moorea, French Polynesia. In April and May 2007, experiments were completed to determine the effects of fluctuating temperature on Pocillopora meandrina and Porites rus, and consecutive trials were used to expose them for 13 days to 26 °C, 28 °C (ambient conditions), 30 °C, or a fluctuating treatment ranging from 26 to 30 °C over 24 h. The multivariate response was assessed using maximum dark-adapted quantum yield of PSII (F_V/F_M), Symbiodinium density, chlorophyll-a content, and calcification. In trial 1, multivariate physiology of both species was significantly affected by treatments, with the fluctuating temperature resulting in a 17-45% decline in Symbiodinium density (relative to the ambient) matching that occurring at a constant 30 °C; F_V/F_M, chlorophyll-a content, and calcification, did not differ between the fluctuating and the steady treatments. In contrast, in trial 2 that utilized corals collected two weeks after those used in trial 1, the corals were unaffected by the treatments, likely due to an environment × trial interaction caused by seasonal declines in Symbiodinium density. Together, these results demonstrate that short transgressions to ecologically relevant high and low temperatures can elicit a potentially detrimental response equivalent to that occurring upon exposure to a constant high temperature. The dissimilar responses among dependent variables and consecutive trials underscore the importance of temporal replication and multivariate approaches in coral ecophysiology. It is likely that recent history has a stronger effect on the response of corals to treatments than is currently recognized.     

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

Phenotypic variation in hatchling Chinese ratsnakes (Zaocys dhumnades) from eggs incubated at constant temperatures

We incubated eggs of the Chinese ratsnake Zaocys dhumnades at four constant temperatures (24, 27, 30 and 30 °C) to examine the effects of incubation temperature on hatching success and hatchling phenotypes. Incubation length increased nonlinearly as temperature decreased, with the mean incubation length being 76.7 d at 24 °C, 57.4 d at 27 °C, 47.3 d at 30 °C, and 44.1 d at 33 °C. Hatching successes were lower at the two extreme temperatures (69% at 24 °C, and 44% at 33 °C) than at the other two moderate temperatures (96% at 27 °C, and 93% at 30 °C). Incubation temperature affected nearly all hatchling traits examined in this study. Incubation of Z. dhumnades eggs at 33 °C resulted in production of smaller hatchlings that characteristically had less-developed carcasses but contained more unutilized yolks. Hatchlings from eggs incubated at 27 and 30 °C did not differ in any examined traits. Taking the rate of embryonic development, hatching success and hatchling phenotypes into account, we conclude that the temperature range optimal for incubation of Z. dhumnades eggs is narrower than the range of 24−33 °C but should be wider than the range of 27−30 °C.     

Response of juvenile diamond-backed terrapins (Malaclemys terrapin) to an aquatic thermal gradient

In many ectotherms, selection of environmental thermal niches may positively affect growth, nutrient assimilation rates, immune system function, and ultimately survival. Temperature preference in some turtle species may be influenced by environmental conditions, including acclimation temperature. We tested for effects of acclimation temperature (22 °C, 27 °C) on the selected temperature and movement patterns of 14 juvenile Malaclemys terrapin (Reptilia: Emydidae) in an aquatic thermal gradient of 14–34 °C and in single-temperature (22 °C, 27 °C) control tests. Among 8–10 month old terrapins, acclimation temperature influenced activity and movement patterns but did not affect temperature selection. In thermal gradient and single-temperature control tests, turtles acclimated to 27 °C used more tank chambers and relocated between chambers significantly more frequently than individuals acclimated to 22 °C. However, acclimation temperature did not affect temperature selection: both 22- and 27 °C-acclimated turtles selected the warmest temperature (34 °C), and avoided the other temperatures available, during thermal gradient tests. These results suggest that young M. terrapin are capable of detecting small temperature increments and prefer warm temperatures that may positively influence growth and metabolism.     

Temperature-induced oviposition in the brachyuran crab Cancer setosus along a latitudinal cline: Aquaria experiments and analysis of field-data

Ovigerous females of Cancer setosus are present year-round throughout most of its wide range along the Peruvian/Chilean Pacific coast (2°S-46°S). However, their number of egg-masses produced per year remains speculative and as such has neither been considered in latitudinal comparisons of reproduction, nor for its fisheries management. In order to reveal the effect of temperature on egg-mass production and egg-development, female C. setosus were held in through-flow aquaria under natural seasonal temperature conditions (16-23 °C) in Antofagasta (23°S), Northern Chile (05/2005-03/2006; 10 months), and at three constant temperatures (12, 16, 19 °C) in Puerto Montt (41°S), Central Southern Chile (09/2006-02/2007; 5 months). Female crabs uniformly produced up to 3 viable egg-masses within 4 1/2 months in Antofagasta and in Puerto Montt (at 19 °C). The second egg-mass was observed 62.5 days (± 7.6; N = 7) after the oviposition of the first clutch and a third egg-mass followed 73.5 days (± 12.5; N = 11) later in Antofagasta (at 16-23 °C). Comparably, a second oviposition took place 64.4 days (± 9.8, N = 5) after the first clutch and a third, 67.0 days (± 2.8, N = 2), thereafter, at 19 °C in Puerto Montt. At the two lower temperatures (16 and 12 °C) in Puerto Montt a second egg-mass was extruded after 82.8 days (± 28.9; N = 4) and 137 days (N = 1), respectively. The duration of egg-development from oviposition until larval hatching decreased from 65 days at 12.5 °C to 22.7 days at the observed upper temperature threshold of 22 °C. Based on the derived relationship between temperature and the duration of egg-development (y = 239.3175e^{− 0.107x}; N = 21, r² = 0.83) and data on monthly percentages of ovigerous females from field studies, the annual number of egg-masses of C. setosus was calculated. This analysis revealed an annual output of about one egg-mass close to the species northern and southern distributional limits in Casma (9°S) and Ancud (43°S), respectively, while at Coquimbo (29°S) about two and in Concepción (36°S) more than 3 egg-masses are produced per year.     

Metabolism,thermoregulation and evaporative water loss in the Chaotung Vole (Eothenomys olitor) in Yunnan-Kweichow Plateau in summer

Proper adjustment of thermoregulatory mechanisms ensures the survival of mammals when they are subjected to seasonal changes in their natural environment. To understand the physiological and ecological adaptations of Eothenomys olitor, we measured their metabolic rate, thermal conductance, body temperature (T_b) and evaporative water loss at a temperature range of 5–30 °C in summer. The thermal neutral zone (TNZ) of E. olitor was 20–27.5 °C, and the mean body temperature was 35.81±0.15 °C. Basal metabolic rate (BMR) was 2.81±0.11 ml O₂/g h and mean minimum thermal conductance (C_m) was 0.18±0.01 ml O₂/g h °C. Evaporative water loss (EWL) in E. olitor increased when the ambient temperature increased. The maximal evaporative water loss was 6.74±0.19 mg H₂O/g h at 30 °C. These results indicated that E. olitor have relatively high BMR, low body temperature, low lower critical temperature, and normal thermal conductance. EWL plays an inportant role in temperature regulation. These characteristics are closely related to the living habitat of the species, and represent its adaptive strategy to the climate of the Yunnan-Kweichow Plateau, a low-latitude, high-altitude region where annual temperature fluctuations are small, but daily temperature fluctuations are greater.     

Evaporative water loss and energy metabolic in two small mammals,voles (Eothenomys miletus) and mice (Apodemus chevrieri), in Hengduan mountains region

Evaporative water loss (EWL) and energy metabolism were measured at different temperatures in Eothenomys miletus and Apodemus chevrieri in dry air. The thermal neutral zone (TNZ) of E. miletus was 22.5–30 °C and that of A. chevrieri was 20–27.5 °C. Mean body temperatures of the two species were 35.75±0.5 and 36.54±0.61 °C. Basal metabolic rates (BMR) were 1.92±0.17 and 2.7±0.5 ml O₂/g h, respectively. Average minimum thermal conductance (C_m) were 0.23±0.08 and 0.25±0.06 ml O₂/g h °C. EWL in E. miletus and A. chevrieri increased with the increase in temperature; the maximal EWL at 35 °C was 4.78±0.6 mg H₂O/g h in E. miletus, and 5.92±0.43 mg H₂O/g h in A. chevrieri. Percentage of evaporative heat loss to total heat production (EHL/HP) increased with the increase in temperature; the maximal EHL/HP was 22.45% at 30 °C in E. miletus, and in A. chevrieri it was 19.96% at 27.5 °C. The results may reflect features of small rodents in the Hengduan mountains region: both E. miletus and A. chevrieri have high levels of BMR and high levels of total thermal conductance, compared with the predicted values based on their body masses, while their body temperatures are relatively low. EWL plays an important role in temperature regulation.     

Duration tenacity: A method for assessing acclimatory capacity of the Antarctic limpet, Nacella concinna

The limpet, Nacella concinna, collected from the Antarctic Peninsula (67°S), was incubated at − 0.3 °C and 2.9 °C for 9 months to test if the previously reported absence of acclimation capacity in Antarctic marine ectotherms could be due to the extended time it takes for them to adjust their physiology to a new stable state. Acclimation was tested through acute measurements of upper lethal limit and a modified measure of tenacity, that tested muscle capacity by measuring the length of time that N. concinna were able to remain attached to the substratum at different temperatures. Both measures acclimated in response to incubation to the higher temperature. Lethal limits were elevated in N. concinna incubated at 2.9 °C (8.1 ± 0.3 °C) compared to those incubated at − 0.3 °C (6.9 ± 0.4 °C). 2.9 °C incubated N. concinna also had a maximum tenacity at 2.1 °C, a higher temperature than the maximum tenacity of those incubated at − 0.3 °C, which occurred at − 1.0 °C. This study is the first to show that the Antarctic limpet can acclimate its physiology, but that it requires a greater period of time for acclimation to occur than previous studies have allowed for.     

The use of small subcutaneous transponders for quantifying thermal biology and torpor in small mammals

Remote measurements of body temperature (T_b) in animals require implantation of relatively large temperature-sensitive radio-transmitters or data loggers, whereas rectal temperature (T_rec) measurements require handling and therefore may bias the results. We investigated whether ∼0.1 g temperature-sensitive subcutaneously implanted transponders can be reliably used to quantify thermal biology and torpor use in small mammals. We examined (i) the precision of transponder readings as a function of temperature and (ii) whether subcutaneous transponders can be used to remotely record subcutaneous temperature (T_sub). Five adult male dunnarts (Sminthopsis macroura, body mass 24 g) were implanted with subcutaneous transponders to determine T_sub as a function of time and ambient temperature (T_a), and in comparison to thermocouple readings of T_rec. Transponder temperature was highly correlated with water bath temperature (r²=0.96–0.99) over a range of approximately 10.0–40.0 °C. Transponders provided reliable data (±0.6 °C) over the T_sub of 21.4–36.9 °C and could be read from a distance of up to 5 cm. Below 21.4 °C, accuracy was reduced to ±2.8 °C, but individual transponder accuracy varied. Consequently, small subcutaneous transponders are useful to remotely quantify thermal physiology and torpor patterns without having to disturb the animal and disrupt torpor. Even at T_sub<21.4 °C where the accuracy of the temperature readings was reduced, transponders do provide reliable data on whether and when torpor is used.     

Thermoregulatory behavior and oxygen consumption of Octopus mimus paralarvae: The effect of age

Octopus mimus is an important cephalopod species in the coastal zone of Peru and Chile that is exposed to temperature variations from time to time due to El Niño/Southern Oscillation (ENSO) episodes when surface temperatures can reach 24 °C, 6 °C above typical temperatures in their habitat. The relationships between temperature and food availability are important factors that determine the recruitment of juveniles into the O. mimus population. The present study was to evaluate the relationship between thermoregulatory behavior and the age of paralarvae (summer population) to determine whether changes in this behavior occur during internal yolk consumption, making larvae more vulnerable to environmental temperature change. Oxygen consumption of paralarvae when 1–4 d old was determined to establish if respiration could be used to monitor the physiological changes that occur during yolk consumption. Horizontal thermal selection (17–30 °C), critical thermal maxima (CTMax), minima (CTMin), and oxygen consumption experiments were conducted with fasting paralarvae 1–4 d old at 20 °C. Preferred temperatures were dependent on the age of O. mimus paralarvae. One day old paralarvae selected a temperature 1.1 °C (23·4 °C) higher than 2 – 4 d old paralarvae (22·3 °C). The CTMax of paralarvae increased with age with values of 31·9±1.1 °C in 1-d-olds and 33·4±0.3 to 4-d-olds. CTMin also changed with age with low values in 2-d-old paralarvae (9.1±1·3 °C) and 11·9±0·9 °C in 4-d-old animals. The temperature tolerance range of paralarvae was age-dependent (TTD=difference between CTMax and CTMin) with higher values in 2 and 3 d old paralarvae (25–26 °C) as compared to 1 d old (23·1 °C) and 4 d old animals (22.7 °C). Oxygen consumption was not affected by the age of paralarvae, suggesting that mechanisms exist that compensate their metabloism until at least 4 d of age. The temperature tolerance range of a planktonic paralarvae of octopus species is presented for the first time. This range was dependent on the age of paralarvae, and so rendered the paralarvae more vunerable to a combination of high temperature and food deprivation during first days of life. Results in the present study provide evidence that O. mimus could be under ecological pressure if a climate change causes increased or decreased temperatures into their distribution range.     

Diapause pupal color diphenism induced by temperature and humidity conditions in Byasa alcinous (Lepidoptera: Papilionidae)

We investigated whether diapause pupae of Byasa alcinous exhibit pupal color diphenism (or polyphenism) similar to the diapause pupal color polyphenism shown by Papilio xuthus. All diapause pupae of B. alcinous observed in the field during winter showed pupal coloration of a dark-brown type. When larvae were reared and allowed to reach pupation under short-day conditions at 18 °C under a 60 ± 5% relative humidity, diapause pupae exhibited pupal color types of brown (33%), light-brown (25%), yellowish-brown (21%), diapause light-yellow (14%) and diapause yellow (7%). When mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C and 25 °C under a 60 ± 5% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 91.2, 8.8 and 0.0% at 10 °C, and 12.2, 48.8 and 39.0% at 25 °C, respectively. On the other hand, when mature larvae reared at 18 °C were transferred and allowed to reach pupation at 10 °C, 18 °C and 25 °C under an over 90% relative humidity after a gut purge, the developmental ratio of brown and light-brown, yellowish-brown, and diapause light-yellow and diapause yellow types was 79.8, 16.9 and 3.3% at 10 °C, 14.5, 26.9 and 58.6% at 18 °C, and 8.3, 21.2 and 70.5% at 25 °C, respectively. These results indicate that diapause pupae of brown types are induced by lower temperature and humidity conditions, whereas yellow types are induced by higher temperature and humidity conditions. The findings of this study show that diapause pupae of B. alcinous exhibit pupal color diphenism comprising brown and diapause yellow types, and suggest that temperature and humidity experienced after a gut purge are the main factors that affect the diapause pupal coloration of B. alcinous as environmental cues.     

Effects of water temperature change on immune function in surf clams, Mactra veneriformis (Bivalvia: Mactridae)

Surf clam, Mactra veneriformis is one of the crucial fishery resources in Korea. This study was performed to examine the immune functions of the surf clam under the stress of water temperature changes at 10 °C, 20 °C or 30 °C for 24 h. Viable bacterial counts (VBC), total haemocyte count (THC), phagocytic activity, lysozyme activity, NRR times and SOD activity were assessed in three different water temperature groups. Clams held at 10 °C decreased in THC, lysozyme activity and NRR times, but phagocytic activity was increased. The highest temperature (30 °C) significantly increased in THC, whereas it decreased in phagocytic activity, lysozyme activity and NRR times. In clams maintained at 20 °C, phagocytic activity, lysozyme activity and NRR times were increased whereas THC was somewhat decreased with respect to clams held at 30 °C. However, water temperature changes did not elicit any alteration of VBC and SOD activity. The present study demonstrates that acute water temperature change affects the haemocytic and haemolymphatic functions, reducing immunosurveillance in stressed surf clam, M. veneriformis.     

Predation activity of two winter-active spiders (Araneae: Anyphaenidae,Philodromidae)

Anyphaena accentuata and Philodromus spp. are cold adapted and winter-active spider species. Their predation activity was investigated at constant temperatures between –4 and 30 °C. The lower temperature threshold for Anyphaena was –3.7 °C, while that of Philodromus was –1.2 °C. At 1 °C the latency to capture and prey consumption was significantly shorter in Anyphaena than in Philodromus. The capture rate increased with temperature and was maximal at 15 °C in Anyphaena and at 30 °C in Philodromus. At 30 °C, the latency to the capture was significantly shorter in Philodromus than in Anyphaena whose mortality significantly increased.     

Thermal adaptability and disease association in common carp (Cyprinus carpio communis) acclimated to different (four) temperatures

The present study was designed to investigate the effect of temperature (20 °C, 24 °C, 28 °C and 32 °C) on the heamato-biochemical and histological alterations of Cyprinus carpio communis. Increase in the temperature showed significant decrease in the serum protein, while a reduced level of blood glucose at high temperature of 32 °C was observed leading to hypoglycemic conditions in the experimental fishes. A significant correlation (P<0.01) was observed between cholesterol (Cho) and triglycerides (TG) for different temperature treatments. Elevated blood urea nitrogen (BUN) at high temperatures was a good indicator of gill osmoregulatory failure. A variation of 86.40% and 38.33%, respectively, was noticed in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) at 32 °C over minimum experimental temperature of 20 °C. The increase in red blood cell (RBC) and Heamoglobin (Hb) concentration is associated with the decrease of mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), could be the reason for observed poikilo-anisocytosis. Histological studies of different organs of experimental fishes showed accumulation of MMC's (melanomacrophagic centers) and atrophy of the interrenal tissue on exposure to various levels of temperature. These changes were related to severity of thermal stress, being most marked when high temperature was prolonged during acclimatization. Some fishes were found infested by protozoan parasite at elevated temperature of 32 °C. Increased levels of certain biochemical and haemotological parameters studied were strongly correlated with disease in the Cyprinus carpio communis species.     

Effects of storage conditions on sprouting of microtubers of yam (Dioscorea cayenensis–D. rotundata complex)

The control of field tuber dormancy in the yam (Dioscorea cayenensis–D. rotundata complex) is poorly understood. Although studies have examined single environmental factors and chemical treatments that might prolong tuber dormancy and storage, only a few were focused on further tuber sprouting. The present study concerns microtubers obtained by in vitro culture. When microtubers were harvested (after 9 months of culture) and directly transferred on a new medium without hormones, the tubers rapidly sprouted in in vitro conditions. No dormancy was observed in this case. Harvested microtubers were also stored dry in jars in sterile conditions during 2 to 18 weeks before in vitro sprouting. In this case, microtubers stored during 18 weeks sprouted more rapidly than those stored 8 weeks. A constant “dormancy-like period” (storage duration + sprouting delay) was observed, between 20 and 28 weeks respectively for the more rapid and the slower microtubers. The size of the tubers used for the storage had great influence on further sprouting. The larger they were, the better they sprouted. Light during storage had no effect on the sprouting delay while a temperature of 25 °C permit a quicker sprouting than 18 °C. The medium used to obtain microtubers could also have an effect on sprouting rate.