The role of macromolecular antifreeze in the darkling beetle,Meracantha contracta

Summary Macromolecular antifreeze solutes are present in the hemolymph of the overwintering larvae of the darkling beetle,Meracantha contracta. These antifreeze solutes produce a thermal hysteresis in the hemolymph of overwinteringMeracantha larvae whereby the freezing point of the hemolymph may be 3–4 °C below the melting point. This thermal hysteresis is very similar to that produced by proteinaceous and glycoproteinaceous antifreezes which are used by many cold water, marine teleost fishes to prevent freezing. One function of the macromolecular antifreeze inMeracantha may be to hinder inoculative freezing which might otherwise occur because of the dampness of the hibernaculae. A probably more important function is to depress the supercooling point of the frost susceptibleMeracantha larvae, thereby preventing lethal ice formation in the larvae's body fluids down to temperatures of approximately –11 °C.     

Physiological adaptations to low temperature and brine exposure in the circumpolar amphipod Gammarus wilkitzkii

Summary The amphipod Gammarus wilkitzkii does not survive being frozen totally into solid sea ice. When the animals are cooled in air or freezing seawater, they will freeze and die at a temperature of about-4° C. However, during sea ice growth, the amphipods may tolerate to stay in the vicinity of the ice by conforming to the ambient brine in a salinity range of 34 ppt to about 60 ppt. A passive relationship between the concentrations of the haemolymph and seawater Na⁺ and Cl^-, lowers the melting point of the body fluids of the animals, thus preventing internal ice formation at low temperatures.     

The effect of environmental variables on the structure and function of hemocyanin fromCallianassa californiensis

Summary Callianassa, an estuarine animal, can experience changes in salinity which reduce the level of divalent cations in the blood, as well as changes in temperature and variations in protein concentration in the hemolymph. The hemocyanin in whole hemolymph shows evidence of dissociation ( 12%) after 3 days acclimation to 40% seawater. Using purified hemocyanin the dissociation from 39 S to 17 S hemocyanin was investigated for the three variables, salinity, temperature and protein concentration. An equation was developed to define the amount of dissociation which should be observed under physiological conditions.     

Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode Globodera rostochiensis

Perry R. N. and Wharton D. A. 1985. Cold tolerance of hatched and unhatched second stage juveniles of the potato cyst-nematode, Globodera rostochiensis. International Journal for Parasitology15: 441–445. Hatched second-stage juveniles of Globodera rostochiensis can survive sub-zero temperatures by supercooling when not in contact with water. When frozen in water free juveniles cannot survive ice seeding across the cuticle and concomitant freezing of their body contents. Unhatched juveniles can survive in water, probably because the egg-shell protects the juvenile from ice seeding across from the medium; in this state juveniles survive by supercooling.     

Salinity tolerance and osmoregulation of the arctic marine amphipods Onisimus litoralis (Kroyer) and Anonyx nugax (Phipps)

Summary The gammarid amphipod Onisimus litoralis, which inhabits arctic and subarctic intertidal and under-ice habitats, is a euryhaline hyperosmotic regulator. It survives 10 d exposures to salinities from 5 to 55 ppt. It hyperregulates its hemolymph osmolality during 3 h exposures to dilutions of 33 ppt seawater and remains hyperosmotic for at least 2 w. The hemolymph is isosmotic to the medium after 12 h exposures to salinities higher than 33 ppt. The gammarid amphipod Anonyx nugax, which inhabits arctic and subarctic subtidal areas, tolerates salinities from 23 to 45 ppt with little mortality. Unlike Onisimus, however, it is an osmoconformer and its hemolymph becomes isosmotic to all dilute salinities within its tolerance range after 12 h and to concentrated media after 3 h. The salinity tolerances and osmoregulatory abilities of both species are reflected in their distributions in the field.     

Isolation and identification of an ice-nucleating bacterium from the gills of the intertidal bivalve mollusc Geukensia demissa

In the fall, freeze tolerant intertidal invertebrates usually produce ice-nucleating proteins that are secreted into the hemolymph. These proteins help protect against freeze damage by insuring that ice formation is limited to extracellular spaces. Geukensia demissa, a freeze tolerant, salt marsh bivalve mollusc was examined for the presence of ice nucleating proteins. The ice-nucleating temperature (INT) of the hemolymph was not significantly different from artificial seawater of the same salinity indicating the lack of an ice nucleating protein in the hemolymph. The palial fluid did have an elevated INT, indicating the presence of an ice nucleator. The INT of the palial fluid was significantly reduced by boiling and filtration through a 0.45-&mgr;m filter. High INT was also observed in the seawater associated with the bivalves, and was demonstrated in water samples collected from salt marshes but not sand and pebble beaches. Moreover, the INT of water samples collected from a salt marsh decreased in the summer. All of these data suggest that the ice-nucleating agents in the hemolymph and the seawater are ice-nucleating bacteria. One species of ice-nucleating bacteria, Pseudomonas fulva was isolated from the gills of Geukensia. These bacteria could perform the same function as hemolymph ice-nucleating proteins by limiting ice formation to extracellular compartments.     

Changes in the chemical composition of the common shore crab,Carcinus maenas, during the molting cycle

Juvenile and young adult specimens ofCarcinus maenas were kept in the laboratory under controlled conditions. The main organic constituents and their variations during the molt cycle were quantitatively determined.

Effect of salinity on the osmotic,chloride, total protein and calcium concentrations in the hemolymph of the prawn Peneaus monodon (fabricius)

• 1.1. Osmolality and chloride concentrations in the hemolymph of Penaeus monodon became stable 1 day after molting in 32 ppt, while total protein and calcium concentrations remained stable throughout the molting cycle. When intermolt (≥ 36 hr postmolt) animals were transferred from control (32 ppt) to experimental (8–40 ppt) salinities, osmolality, chloride and total protein, but not calcium, concentrations in the hemolymph achieved steady state values 24–48 hr after transfer.
• 2.2. The hemolymph osmolality was a linear function (slope = 0.28) of medium osmolality at salinities between 8 and 40 ppt. It was isosmotic to seawater at 698 mOsm (10 g prawns) and 752 mOsm (30 g), and was hyperosmotic to the medium below isosmotic concentrations, and hypoosmotic to those above.
• 3.3. Hemolymph chloride concentration was isoionic to seawater at 334 mM, and was hyperregulated below isoionic concentrations, and hyporegulated to those above.
• 4.4. P. monodon maintained its hemolymph calcium concentration between 6.4 and 10 mM when medium salinities increased from 8 to 40 ppt.
• 5.5. Total protein concentration in the hemolymph was independent of medium salinity (8–40 ppt) and hemolymph osmolality (540–850 mOsm).

     

Salinity tolerance of adult and juvenile red king crabs Paralithodes camtschatica

• 1.1. Juvenile king crabs were more tolerant of reduced salinities than adult crab; juvenile crab were better volume regulators at reduced salinities than adult crab.
• 2.2. Adult female king crab hemolymph was hyperosmotic to full seawater (30 ppt) and isosmotic to dilute seawater. Juvenile king crab (2 years old) were hypoosmotic at the same concentrations.
• 3.3. Lower osmotic concentration of juvenile hemolymph is at least partially due to lower sodium concentration.
• 4.4. Juvenile king crab can tolerate some dilution and survive for short periods in the reduced salinity of the lower intertidal zone.

     

Hydrographic patterns in McMurdo Sound,Antarctica and their relationship to local benthic communities

Summary Measurements of hydrographic parameters (temperature, salinity, nitrate, nitrite, phosphate, chlorophyll a, phaeophytin, and oxygen) in McMurdo Sound, Antarctica during spring, 1984, before the regional phytoplankton bloom, and summer, 1984, after the peak of the bloom, indicate the several processes contribute to changes in the vertical and horizontal structure of the water column. Regional variation in the source of water masses within the Sound, ice cover patterns, and meltwater from the Ross Ice Shelf and nearby continental glaciers result in east-west and north-south gradients in the thermohaline, nutrient, and productivity characteristics of the Sound. These patterns are also related to the extremely variable structure and productivity of shallow water benthic macrofaunal communities in McMurdo Sound. Hydrographic patterns during Spring (November) were indicative of conditions at the end of winter prior to the spring phytoplankton bloom. The water column was nearly isothermal with temperatures near or below the surface freezing point of seawater with only a slight salinity increase with depth. Salinity was lower in the west Sound than in the east, probably in response to glacial meltwater input from the Ross Ice Shelf and/or terrestrial sources. Nutrient levels were high and nearly homogenous throughout the Sound. Chlorophyll a was low (<1.0 g/l) throughout most of the Sound, but was lowest in the western sound, as expected from the circulation pattern (Barry and Dayton 1988). Oxygen was uniformly low during spring. The summer hydrographic distributions, estimated from samples collected during the decline of the regional plankton bloom, were dramatically different than in during spring. Both the salinity and temperature were vertically stratified at all sites, particularly in the west Sound. Temperatures near the surface were well above the freezing point and occasionally near or above 0°C. Near surface salinity in the western Sound was nearly fresh (0.4 ppt) at some locations in the southwestern Sound. Chlorophyll a was high throughout the Sound relative to spring concentrations, and nutrient levels (NO₃, PO₄) were strongly depressed near the surface, due mainly to phytoplankton uptake rather than by dilution. Primary productivity estimates based on the summer nitrate and phosphate deficits over 90 days were 1.96–2.02 and 0.39–1.02 gCm^-2d^-1 for the east and west sound, respectively. Nutrient ratios indicated that glacial meltwater from the Ross Ice Shelf and/or nearby terrestrial sources may be an important component of the summer meltwater input to the western Sound. Enhanced water column stability due to this input may prolong the maintenance of high water column stability as this water mass flows northward and result in particularly high productivity in northern McMurdo Sound.     

Effects of Antarctic sea ice biota on seeding as studied in aquarium experiments

Summary The potential seeding impact of sea ice microbial communities was studied during late austral winter early spring 1988 in the Weddell Sea, Antarctica. Experiments were performed in seawater aquariums with natural seawater and seawater enriched with crushed ice. Algal, protozoan and bacterial cell numbers were followed, as well as nutrients and DOC levels. The results showed a potential seeding effect of sea ice communities to the water column. However, the type of ice communities differed greatly from each other and the effect of such seeding will be patchy. In our experiments seeding of seawater by ice rich in algae, flagellates and/or particulate organic carbon lead to the development of communities dominated either by diatoms or bacteria.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Catecholamines and related o-diphenols in cockroach hemolymph and cuticle during sclerotization and melanization: comparative studies on the order Dictyoptera

Summary Catecholamines and related o-diphenols extracted from the cuticle and hemolymph of adult cockroaches during sclerotization and pigmentation of the cuticle were analyzed by reverse phase HPLC with electrochemical detection. At ecdysis, dopamine (DA) oconjugates predominated in the hemolymph of Periplaneta americana, P. australasiae, P. fuliginosa, P. brunnea, and Blatta orientalis (Blattidae); Blattella germanica (Blattellidae); and Gromphadorhina portentosa and Blaberus craniifer (Blaberidae). n-Acetyldopamine (NADA) conjugates were second in abundance in these species, but were major in the hemolymph of the other blaberoid species, Leucophaea maderae and Nauphoeta cinerea. After ecdysis NADA became the major hemolymph catecholamine in all species as DA decreased rapidly. n--Alanyldopamine (NBAD) concentrations in the hemolymph remained low in all species, although NBAD and its metabolite, n--alanylnorepinephrine (NBANE), were generally the major catecholamines in tanning cuticle. Catechol (1,2-dihydroxybenzene) occurred mainly as a conjugate(s) at high levels in the hemolymph of nymphs and adults of all blattid species. Only trace amounts were detected in B. germanica and Cryptocercus punctulatus (Cryptocercidae), and none was found in any of the blaberoid species. High concentrations of NBANE and NBAD accumulated in tanning cuticle of B. germanica, G. portentosa, and all blattid species, whereas NADA and DA predominated in cuticle from the other blaberoid species, particularly L. maderae and N. cinerea. However, cockroaches as a group appear to utilize both the n-acetyl and n--alanyl catecholamines for stabilization of the exoskeleton. The Blattidae differed most from the other families in having considerably higher concentrations of catecholamines in hemolymph and cuticle, as well as the large amounts of catechol conjugates in the hemolymph.Abbreviations AMD -methlydopa - DA dopamine - DOBA 3,4-dihydroxybenzoic acid - DOPA 3,4-dihydroxyphenylalanine - DOPAC 3,4-dihydroxyphenylacetic acid - DOPET 3,4-dibydroxyphenylethanol - DOPKET 3,4-dihydroxyphenylketoethanol - HPLC high performance liquid chromatography - NADA n-acetyldopamine - NANE n-acetylnorepinephrine - NBAD n--alanyldopamine - NBANE n--alanylnorepinephrine - NE notepinephrine Contribution No. 90-88-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS. Cooperative investigation between the KAES and the ARS, USDA. K.J. Kramer is a research chemist and Adjunct Professor at the U.S. Grain Marketing Research Laboratory and Kansas State University, respectively. Address reprint requests to T.L. Hopkins     

Growth ofEnteromorpha linza in sewage effluent and sewage effluent-seawater mixtures

Enteromorpha linza (L.) J. Ag. was grown in full strength sewage effluent, various combinations of sewage effluent and seawater, and in natural seawater. It was found that full strength sewage effluent with a salinity of 14 supported best growth of the alga. After a 12 day cultivation period, growth ofE. linza in full strength sewage effluent and 75% sewage effluent- seawater mixture showed 3.5-fold and 2-fold increase in fresh weight over that grown in natural seawater; respectively. Uptake of PO _inf4 ^sup3– -P, NH₃-N and NO _inf3 ^sup– -N by cells ofE. linza was extremely efficient in all tested media. Data obtained from the experiments indicated that inorganic nitrogen rather than phosphorus was the limiting nutrient factor for growth ofE. linza in full strength sewage effluent and in other sewage effluent- seawater mixtures. NH₃-N at concentrations above 4.5 ppm was found to inhibit uptake of NO _inf3 ^sup– -N in the same culture medium by the algal cells. The fact that sewage grownE. linza contained comparatively much higher protein content (30.2% dry weight) than that grown in natural seawater (12.5% dry weight) leads to the conclusion that sewage grownE. linza could serve as an economically feasible feed for livestock in Hong Kong where the sewage is characterized by having a salinity of approximately 14. It is proposed that this multicellular green alga is a suitable algal species to serve the dual function of wastewater purification through the production of algal protein from sewage effluent having high salinities.     

In vivo development of the entomogeneous hyphomycetePaecilomyces farinosus in hostSpodoptera exigua (beet armyworm) larvae

The in vivo development of the entomogenous hyphomycetePaecilomyces farinosus inSpodoptera exigua (beet armyworm) larvae was examined using light and electron microscopic techniques. Blastospores injected into larval hemocoels (500 blastospores/larva) were immediately ingested by phagocytic hemocytes, and no fungal cells were detected in the hemolymph until 36 h post-injection. As indicated by immunocytochemical methods, the in vivo-produced blastosopres, in contrast to in vitro blastospores, lacked a galacto-mannan surface layer required for opsonization by aS. exigua humoral lectin. Therefore, these in vivo cells were not recognized by phagocytic granulocytes and were freely-circulating in the hemolymph. Hyphae differentiating from the blastospores were recognized by the hemocytes and induced formation of multicellular hemocytic nodules. By 72 h post-injection, mycelia were observed emerging from the nodules and by 96 h, larvae had become mummified due to extensive proliferation of the fungus throughout host tissues. Neither phagocytosis of the initially injected in vitro-produced blastospores nor nodule formation around hyphal cells later in the infection process was effective in stopping fungal growth. The in vivo development ofP. farinosus was similar to that of another hyphomycete,Beauveria bassiana except that in the latter case, extensive nodule formation was inhibited by the production of fungal metabolites.     

Aglomerularism in Harpagifer bispinis: a subantarctic notothenioid fish living at reduced salinity

We investigated the renal morphology, histology and ultrastructure of Harpagifer bispinis, as a first step toward understanding the morpho-functional basis of its adaptation to potentially freezing brackish seawater. Fish were separated into two groups of ten individuals each, and acclimated to 2 and 38 salinity. A study of complete serial sections of the kidney revealed that the nephrons were aglomerular. At the highly convoluted proximal segment two different regions were evident, a feature that has not been previously reported for other aglomerular species. In electron photomicrographs we distinguished light and dark cells in the proximal tubule epithelium, with highly infolded basolateral membranes and closely associated mitochondria. The dark cells also had a large number of mitochondria in the apical region. The intercellular spaces at the epithelium of the proximal tubule were larger in fish acclimated at 2 salinity, a modification that might facilitate urine secretion, thus contributing to the survival of an aglomerular fish in a hyposmotic medium.     

Size-selective predation by fourhorn sculpin,Myoxocephalus quadricornis (L.) (Pisces) on Mesidotea entomon (L.) (Crustacea,Isopoda)

During three different seasons Mesidotea entomon specimens from fourhorn sculpin stomachs were analyzed, and compared with the M. entomon population in the field. The field samplings were carried out in the northern Bothnian Sea. Feeding experiments revealed fourhorn sculpins to be highly selective when feeding on M. entomon.The fourhorn sculpin most preferably selected large M. entomon in the field as well as in the laboratory experiments. The preference for large M. entomon remained after correction for availability of differently sized M. entomon. In summer the actual sizes of M. entomon eaten by fourhorn sculpin were smaller than in autumn and winter. During all three seasons the oldest M. entomon were the ones most preferred. Ultimate effects of predation by fourhorn sculpin on the life-history of M. entomon are discussed.     

Acclimation of Antarctic bottom-ice algal communities to lowered salinities during melting

Sea-ice brine algal communities were exposed to salinities between 30 and 10 during melting. There was a progressive decline in maximum quantum yield, relative electron transfer rate (rETR_max) and photosynthetic efficiency () with decreasing salinity of the final melted sample. While all species showed a drop in these parameters, Fragilariopsis curta and Entomoneis kjellmannii showed the least inhibition. There was a steady increase in rETR_max and over 5 days after melting, especially in the samples melted into the highest salinities. In addition, the samples melted from the ice without added filtered seawater showed no photosynthetic activity after 2 days. Our results suggest that for experimental work using sea-ice microalgae, the final salinity of the melted sample should be greater than 28 (i.e. the ratio of sea ice to filtered seawater should be at least 1:2).     

Hemolymph ion composition and volume changes in the supralittoral isopod Ligia pallasii Brandt, during molt

We analyzed ion composition and volume of the hemolymph of Ligia pallasii in four different stages of the molt cycle using capillary electrophoresis and ³H-inulin. The main ions in the hemolymph were Na⁺, K⁺, Mg²⁺, Ca²⁺, and Cl⁻. The Ca²⁺concentration increased significantly during the molt by 47% from intermolt to intramolt and by 37% from intermolt to postmolt, probably due to resorption of Ca²⁺ from the cuticle and sternal CaCO₃ deposits. The K⁺ concentration increased significantly by 20% during molt. The hemolymph volume normalized to the dry mass of the animals decreased by 36% from intermolt to late premolt. This was due to a reduction in the hemolymph volume and to an increase in dry mass of the animals during premolt. A sudden increase in the hemolymph volume occurring between late premolt and intramolt served to expand the cuticle. Since the Na⁺, K⁺, Mg²⁺, and Cl⁻ concentrations did not change significantly from late premolt to intramolt, the increase in hemolymph volume suggests an uptake of seawater rather than freshwater. Accepted: 7 March 2000     

Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone

Summary WhenManduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both -MDH and -fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 g/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.Abbreviations -MDH L-3-(3,4 dihydroxyphenyl)-2-hydrazine-methylpropionic acid - -FM-dopa R-S--fluoromethyl-dopa - DCC dopa decarboxylase - 20-HE 20-hydroxyecdysone - JH juvenile hormone - HCS head capsule slippage     

The combined effect of temperature and salinity changes on osmoregulation and haemocyanin concentration in Saduria entomon (Linnaeus, 1758)

Global warming is having an impact on the temperature and salinity of Baltic Sea waters. Therefore, it is important to determine the conditions in which animals can exist and how these changes may influence their functioning. Hence, the purpose of this research was to determine the broad tolerance limits of temperature and salinity of the glacial relict Saduria entomon by studying its behaviour, osmoregulatory ability and haemocyanin concentration. This effect of temperature was confirmed in the laboratory for individuals acclimated to different salinity and temperature regimes. Changes in the physiological parameters of S. entomon at various temperatures (5.5–21.5°C) and salinity levels (1–15 PSU) were recorded. There were statistically significant differences in haemolymph osmotic pressure under the influence of salinity and temperature. The mean haemolymph osmotic pressures were the lowest at 1 PSU at all the temperatures examined and the highest at 15 PSU and high temperatures 16.5 and 21.5°C. The haemocyanin concentration decreased significantly with increasing temperature at 1 PSU. There was a significant difference in haemocyanin concentration due to salinity at temperatures of 5.5 and 10.0°C (the haemocyanin concentration decreased with increasing salinity). The results showed that, although S. entomon is classified as a cold-water animal, it can survive at high temperatures above 16.5°C at least for a short time, as it is capable of osmoregulation. The tolerance to temperature changes was better than expected.